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Emerging roles of DYRK2 in cancer

Open AccessPublished:January 06, 2021DOI:https://doi.org/10.1074/jbc.REV120.015217
      Over the last decade, the CMGC kinase DYRK2 has been reported as a tumor suppressor across various cancers triggering major antitumor and proapoptotic signals in breast, colon, liver, ovary, brain, and lung cancers, with lower DYRK2 expression correlated with poorer prognosis in patients. Contrary to this, various medicinal chemistry studies reported robust antiproliferative properties of DYRK2 inhibitors, whereas unbiased ‘omics’ and genome-wide association study-based studies identified DYRK2 as a highly overexpressed kinase in various patient tumor samples. A major paradigm shift occurred in the last 4 years when DYRK2 was found to regulate proteostasis in cancer via a two-pronged mechanism. DYRK2 phosphorylated and activated the 26S proteasome to enhance degradation of misfolded/tumor-suppressor proteins while also promoting the nuclear stability and transcriptional activity of its substrate, heat-shock factor 1 triggering protein folding. Together, DYRK2 regulates proteostasis and promotes protumorigenic survival for specific cancers. Indeed, potent and selective small-molecule inhibitors of DYRK2 exhibit in vitro and in vivo anti-tumor activity in triple-negative breast cancer and myeloma models. However, with conflicting and contradictory reports across different cancers, the overarching role of DYRK2 remains enigmatic. Specific cancer (sub)types coupled to spatiotemporal interactions with substrates could decide the procancer or anticancer role of DYRK2. The current review aims to provide a balanced and critical appreciation of the literature to date, highlighting top substrates such as p53, c-Myc, c-Jun, heat-shock factor 1, proteasome, or NOTCH1, to discuss DYRK2 inhibitors available to the scientific community and to shed light on this duality of protumorigenic and antitumorigenic roles of DYRK2.

      Keywords

      Abbreviations:

      CMGC (Cyclin-dependent kinases, Mitogen-activated protein kinases, Glycogen synthase kinases, and CDC-like kinases), CML (chronic myeloid leukemia), DYRK (Dual-specificity tYrosine phosphorylation–Regulated Kinase), EMT (epithelial–mesenchymal transition), GEMMs (genetically engineered mouse models), HIPK2 (homeodomain-interacting protein kinase), HSF1 (heat-shock factor 1), IFN (interferon), MM (multiple myeloma), NAPA (N-terminal autophosphorylation accessory), NOTCH1 (neurogenic locus notch homolog protein 1), NSCLC (non–small-cell lung cancer), PEST (Pro-Glu-Ser-Thr), Ser62 (serine62), Ser727 (serine727), STAT3 (signal transducer and activator of transcription 3), TBK1 (TANK-binding kinase 1), TCGA (The Cancer Genome Atlas), Thr58 (Threonine58), TNBC (triple-negative breast cancer)
      Protein kinase DYRK2 is a member of the Dual-specificity tYrosine phosphorylation–Regulated Kinase (DYRK) family, which in turn belongs to the Cyclin-dependent kinases, Mitogen-activated protein kinases, Glycogen synthase kinases, and CDC-like kinases (CMGC) superfamily within the kinase complement of the human genome (
      • Manning G.
      • Whyte D.B.
      • Martinez R.
      • Hunter T.
      • Sudarsanam S.
      The protein kinase complement of the human genome.
      ). The DYRK family consists of 5 members divided into two classes: Class I is comprised of DYRK1A and DYRK1B, whereas class II is comprised of DYRK2, DYRK3, and DYRK4 (Fig. 1A). DYRK2 is a class II DYRK that exhibits various structural features such as the NAPA or N-terminal autophosphorylation accessory domains (yellow/orange), DYRK-homology domain (green), activation loop segment (purple), nuclear localization sequence (red), the CMGC family–specific insert domain (gray) (Fig. 1B) most of which are conserved across the DYRK family (
      • Soundararajan M.
      • Roos A.K.
      • Savitsky P.
      • Filippakopoulos P.
      • Kettenbach A.N.
      • Olsen J.V.
      • Gerber S.A.
      • Eswaran J.
      • Knapp S.
      • Elkins J.M.
      Structures of down syndrome kinases, DYRKs, reveal mechanisms of kinase activation and substrate recognition.
      ). In DYRK2, specific loss-of-function mutations have been reported in cancer (Fig. 1B), which affect either the activity of the kinase or impede its ability to form functional complexes with interactors (
      • Mehnert M.
      • Ciuffa R.
      • Frommelt F.
      • Uliana F.
      • van Drogen A.
      • Ruminski K.
      • Gstaiger M.
      • Aebersold R.
      Multi-layered proteomic analyses decode compositional and functional effects of cancer mutations on kinase complexes.
      ). In fact, phosphoproteomics studies show that these cancer mutations significantly alter substrate specificity of DYRK2 in cells (
      • Mehnert M.
      • Ciuffa R.
      • Frommelt F.
      • Uliana F.
      • van Drogen A.
      • Ruminski K.
      • Gstaiger M.
      • Aebersold R.
      Multi-layered proteomic analyses decode compositional and functional effects of cancer mutations on kinase complexes.
      ). Class I DYRKs exhibit two distinct nuclear localization sequences and a stretch of polyserine and polyproline (PEST, Pro-Glu-Ser-Thr) domain with no distinct NAPA domains as in class II paralogues (Fig. 1C). Despite subtle structural differences between class I and II members, all DYRK isoforms exhibit a highly conserved autophosphorylation-mediated activation mechanism (
      • Lochhead P.A.
      • Sibbet G.
      • Morrice N.
      • Cleghon V.
      Activation-loop autophosphorylation is mediated by a novel transitional intermediate form of DYRKs.
      ). During translation, hydroxylation of a highly conserved proline residue (proline441 for hDYRK2) on the inert/nascent kinase domain of DYRKs triggers a tyrosine autophosphorylation event within the activation loop (tyrosine382 for hDYRK2), which leads to conversion of the inactive to the active conformation of the kinase (
      • Lee S.B.
      • Ko A.
      • Oh Y.T.
      • Shi P.
      • D'Angelo F.
      • Frangaj B.
      • Koller A.
      • Chen E.I.
      • Cardozo T.
      • Iavarone A.
      • Lasorella A.
      Proline hydroxylation primes protein kinases for autophosphorylation and activation.
      ). In the fully active form, the DYRK transition from a tyrosine-phosphorylating kinase to a serine-/threonine-directed kinase, thus acquiring the label ‘dual specificity’ (
      • Soundararajan M.
      • Roos A.K.
      • Savitsky P.
      • Filippakopoulos P.
      • Kettenbach A.N.
      • Olsen J.V.
      • Gerber S.A.
      • Eswaran J.
      • Knapp S.
      • Elkins J.M.
      Structures of down syndrome kinases, DYRKs, reveal mechanisms of kinase activation and substrate recognition.
      ,
      • Lochhead P.A.
      • Sibbet G.
      • Morrice N.
      • Cleghon V.
      Activation-loop autophosphorylation is mediated by a novel transitional intermediate form of DYRKs.
      ). The NAPA and DYRK-homology domain domains are thought to promote the structural integrity of the nascent kinase enough to execute the indispensable autophosphorylation event (
      • Soundararajan M.
      • Roos A.K.
      • Savitsky P.
      • Filippakopoulos P.
      • Kettenbach A.N.
      • Olsen J.V.
      • Gerber S.A.
      • Eswaran J.
      • Knapp S.
      • Elkins J.M.
      Structures of down syndrome kinases, DYRKs, reveal mechanisms of kinase activation and substrate recognition.
      ). The CMGC-specific insert is conserved across the CMGC kinase superfamily and is proposed to play important roles in stabilization of the tertiary structure of the kinase and promoting complex formation with interactors/substrates (
      • Kannan N.
      • Neuwald A.F.
      Evolutionary constraints associated with functional specificity of the CMGC protein kinases MAPK, CDK, GSK, SRPK, DYRK, and CK2alpha.
      ).
      Figure thumbnail gr1
      Figure 1DYRK2 belongs to the DYRK family within the CMGC superfamily and is mutated in cancer. A, DYRK2 is a class II DYRK on the CMGC superfamily branch of the kinome. B, structure of DYRK2 indicating the major structural domains and cancer-associated mutations (derived from PDB ID: 3K2L) with a hypothetical effect on DYRK2 structure/function. C, the domain diagram providing a 2D comparative image of the domains of class II DYRK2 and class I DYRK1B. Class I DYRKs exhibit two NLS sequences, a C-terminal PEST domain and a lack of NAPA domain characteristic of class II. The autophosphorylation of Tyr and hydroxylated Pro Y382/P441 (DYRK2) and Y273/P332 (DYRK1B) are shown. CMGC, Cyclin-dependent kinases, Mitogen-activated protein kinases, Glycogen synthase kinases, and CDC-like kinases; DYRK, Dual-specificity tYrosine phosphorylation–Regulated Kinase; NAPA, N-terminal autophosphorylation accessory; NLS, nuclear localization sequence; PEST, Pro-Glu-Ser-Thr.
      Like most CMGC kinases, DYRKs have an amino acid motif of preference on their substrates. DYRKs prefer an arginine (R) at the −3 position of the phosphoserine/threonine residue along with a strong preference for a proline (P) at +1: Rxx(pS/T)P motif (
      • Campbell L.E.
      • Proud C.G.
      Differing substrate specificities of members of the DYRK family of arginine-directed protein kinases.
      ). Being a preferred motif across all members, redundancies have been observed wherein multiple CMGC kinases phosphorylate the same site on the substrate (reviewed in Boni et al. [
      • Boni J.
      • Rubio-Perez C.
      • López-Bigas N.
      • Fillat C.
      • de la Luna S.
      The DYRK family of kinases in cancer: Molecular functions and therapeutic opportunities.
      ]). Although both the −3 R and +1 P are strongly preferred, some DYRK substrates lack the +1 P such as histone H3 for DYRK1A (
      • Himpel S.
      • Tegge W.
      • Frank R.
      • Leder S.
      • Joost H.G.
      • Becker W.
      Specificity determinants of substrate recognition by the protein kinase DYRK1A.
      ), 26S proteasome regulatory subunit 6B RPT3 (
      • Guo X.
      • Wang X.
      • Wang Z.
      • Banerjee S.
      • Yang J.
      • Huang L.
      • Dixon J.E.
      Site-specific proteasome phosphorylation controls cell proliferation and tumorigenesis.
      ), and heat-shock factor 1 (HSF1) (
      • Moreno Dorta R.
      • Banerjee S.
      • Jackson A.
      • Quinn J.
      • Baillie G.
      • Dixon J.E.
      • Dinkova-Kostova A.
      • Edwards J.
      • de la Vega L.
      The stress-responsive kinase DYRK2 activates heat shock factor 1 promoting resistance to proteotoxic stress.
      ) for DYRK2, whereas the −3 R is lacking on multiple DYRK2 substrates such as p53 (
      • Taira N.
      • Nihira K.
      • Yamaguchi T.
      • Miki Y.
      • Yoshida K.
      DYRK2 is targeted to the nucleus and controls p53 via Ser46 phosphorylation in the apoptotic response to DNA damage.
      ,
      • Taira N.
      • Yamamoto H.
      • Yamaguchi T.
      • Miki Y.
      • Yoshida K.
      ATM augments nuclear stabilization of DYRK2 by inhibiting MDM2 in the apoptotic response to DNA damage.
      ), c-Jun, c-Myc (
      • Taira N.
      • Mimoto R.
      • Kurata M.
      • Yamaguchi T.
      • Kitagawa M.
      • Miki Y.
      • Yoshida K.
      DYRK2 priming phosphorylation of c-Jun and c-Myc modulates cell cycle progression in human cancer cells.
      ), and SIAH2 (
      • Pérez M.
      • García-Limones C.
      • Zapico I.
      • Marina A.
      • Schmitz M.L.
      • Muñoz E.
      • Calzado M.A.
      Mutual regulation between SIAH2 and DYRK2 controls hypoxic and genotoxic signaling pathways.
      ). For those lacking the +1 P, the substrates have exhibited no redundant kinases within the related CMGC superfamily thus far (
      • Guo X.
      • Wang X.
      • Wang Z.
      • Banerjee S.
      • Yang J.
      • Huang L.
      • Dixon J.E.
      Site-specific proteasome phosphorylation controls cell proliferation and tumorigenesis.
      ). Among the DYRKs, DYRK2 often functions in tandem with related CMGC kinase, GSK3, in sequentially phosphorylating various substrates (
      • Campbell L.E.
      • Proud C.G.
      Differing substrate specificities of members of the DYRK family of arginine-directed protein kinases.
      ,
      • Himpel S.
      • Tegge W.
      • Frank R.
      • Leder S.
      • Joost H.G.
      • Becker W.
      Specificity determinants of substrate recognition by the protein kinase DYRK1A.
      ,
      • Woods Y.L.
      • Cohen P.
      • Becker W.
      • Jakes R.
      • Goedert M.
      • Wang X.
      • Proud C.G.
      The kinase DYRK phosphorylates protein-synthesis initiation factor eIF2Bepsilon at Ser539 and the microtubule-associated protein tau at Thr212: Potential role for DYRK as a glycogen synthase kinase 3-priming kinase.
      ,
      • Cole A.R.
      • Causeret F.
      • Yadirgi G.
      • Hastie C.J.
      • McLauchlan H.
      • McManus E.J.
      • Hernandez F.
      • Eickholt B.J.
      • Nikolic M.
      • Sutherland C.
      Distinct priming kinases contribute to differential regulation of collapsin response mediator proteins by glycogen synthase kinase-3 in vivo.
      ). DYRK2 provides a priming phosphorylation for further GSK3 activity (
      • Campbell L.E.
      • Proud C.G.
      Differing substrate specificities of members of the DYRK family of arginine-directed protein kinases.
      ,
      • Himpel S.
      • Tegge W.
      • Frank R.
      • Leder S.
      • Joost H.G.
      • Becker W.
      Specificity determinants of substrate recognition by the protein kinase DYRK1A.
      ,
      • Woods Y.L.
      • Cohen P.
      • Becker W.
      • Jakes R.
      • Goedert M.
      • Wang X.
      • Proud C.G.
      The kinase DYRK phosphorylates protein-synthesis initiation factor eIF2Bepsilon at Ser539 and the microtubule-associated protein tau at Thr212: Potential role for DYRK as a glycogen synthase kinase 3-priming kinase.
      ,
      • Cole A.R.
      • Causeret F.
      • Yadirgi G.
      • Hastie C.J.
      • McLauchlan H.
      • McManus E.J.
      • Hernandez F.
      • Eickholt B.J.
      • Nikolic M.
      • Sutherland C.
      Distinct priming kinases contribute to differential regulation of collapsin response mediator proteins by glycogen synthase kinase-3 in vivo.
      ). DYRK2 has been identified in all eukaryotes (
      • Guo X.
      • Wang X.
      • Wang Z.
      • Banerjee S.
      • Yang J.
      • Huang L.
      • Dixon J.E.
      Site-specific proteasome phosphorylation controls cell proliferation and tumorigenesis.
      ,
      • Aranda S.
      • Laguna A.
      • Luna S.d. l.
      DYRK family of protein kinases: Evolutionary relationships, biochemical properties, and functional roles.
      ), and interestingly across all orthologues, the conserved biological function of the DYRK2 isoform is regulation of cell division and/or tissue development (
      • Guo X.
      • Wang X.
      • Wang Z.
      • Banerjee S.
      • Yang J.
      • Huang L.
      • Dixon J.E.
      Site-specific proteasome phosphorylation controls cell proliferation and tumorigenesis.
      ,
      • Aranda S.
      • Laguna A.
      • Luna S.d. l.
      DYRK family of protein kinases: Evolutionary relationships, biochemical properties, and functional roles.
      ). A recent work has shown that DYRK2 is an essential kinase during embryogenesis, and mouse embryos with homozygous deletion of DYRK2 exhibit stunted development and pups die just before birth (
      • Yoshida S.
      • Aoki K.
      • Fujiwara K.
      • Nakakura T.
      • Kawamura A.
      • Yamada K.
      • Ono M.
      • Yogosawa S.
      • Yoshida K.
      The novel ciliogenesis regulator DYRK2 governs Hedgehog signaling during mouse embryogenesis.
      ).
      Of all the DYRK isoforms, DYRK2 is the only member that functions as a kinase activity–independent scaffold for an E3 ubiquitin ligase complex (
      • Hossain D.
      • Ferreira Barbosa J.A.
      • Cohen E.A.
      • Tsang W.Y.
      HIV-1 Vpr hijacks EDD-DYRK2-DDB1(DCAF1) to disrupt centrosome homeostasis.
      ,
      • Hossain D.
      • Javadi Esfehani Y.
      • Das A.
      • Tsang W.Y.
      Cep78 controls centrosome homeostasis by inhibiting EDD-DYRK2-DDB1(Vpr)(BP).
      ,
      • Jung H.Y.
      • Wang X.
      • Jun S.
      • Park J.I.
      Dyrk2-associated EDD-DDB1-VprBP E3 ligase inhibits telomerase by TERT degradation.
      ,
      • Maddika S.
      • Chen J.
      Protein kinase DYRK2 is a scaffold that facilitates assembly of an E3 ligase.
      ,
      • Wang X.
      • Singh S.
      • Jung H.Y.
      • Yang G.
      • Jun S.
      • Sastry K.J.
      • Park J.I.
      HIV-1 Vpr protein inhibits telomerase activity via the EDD-DDB1-VPRBP E3 ligase complex.
      ). DYRK2 is an integral part of the EDVP (EDD [ubiquitin protein ligase] + DDB1 [damage-specific DNA-binding protein] + VPRBP [HIV-1 Vpr-binding protein]) E3 ubiquitin ligase complex that carries out phosphorylation-mediated degradation of various cell cycle components to ensure smooth transition of G2/M stages of cell cycle (
      • Hossain D.
      • Ferreira Barbosa J.A.
      • Cohen E.A.
      • Tsang W.Y.
      HIV-1 Vpr hijacks EDD-DYRK2-DDB1(DCAF1) to disrupt centrosome homeostasis.
      ,
      • Hossain D.
      • Javadi Esfehani Y.
      • Das A.
      • Tsang W.Y.
      Cep78 controls centrosome homeostasis by inhibiting EDD-DYRK2-DDB1(Vpr)(BP).
      ,
      • Jung H.Y.
      • Wang X.
      • Jun S.
      • Park J.I.
      Dyrk2-associated EDD-DDB1-VprBP E3 ligase inhibits telomerase by TERT degradation.
      ,
      • Maddika S.
      • Chen J.
      Protein kinase DYRK2 is a scaffold that facilitates assembly of an E3 ligase.
      ,
      • Wang X.
      • Singh S.
      • Jung H.Y.
      • Yang G.
      • Jun S.
      • Sastry K.J.
      • Park J.I.
      HIV-1 Vpr protein inhibits telomerase activity via the EDD-DDB1-VPRBP E3 ligase complex.
      ). Some of the cancer mutations in Figure 1B are thought to affect efficient EDVP complex formation (
      • Mehnert M.
      • Ciuffa R.
      • Frommelt F.
      • Uliana F.
      • van Drogen A.
      • Ruminski K.
      • Gstaiger M.
      • Aebersold R.
      Multi-layered proteomic analyses decode compositional and functional effects of cancer mutations on kinase complexes.
      ). Thus, over the past few decades, many groups have identified various molecular mechanism and substrates for DYRK2 playing diverse roles in cellular growth, proliferation, and developmental processes with a focal point being its role in cancer (
      • Guo X.
      • Wang X.
      • Wang Z.
      • Banerjee S.
      • Yang J.
      • Huang L.
      • Dixon J.E.
      Site-specific proteasome phosphorylation controls cell proliferation and tumorigenesis.
      ,
      • Banerjee S.
      • Ji C.
      • Mayfield J.E.
      • Goel A.
      • Xiao J.
      • Dixon J.E.
      • Guo X.
      Ancient drug curcumin impedes 26S proteasome activity by direct inhibition of dual-specificity tyrosine-regulated kinase 2.
      ,
      • Banerjee S.
      • Wei T.
      • Wang J.
      • Lee J.J.
      • Gutierrez H.L.
      • Chapman O.
      • Wiley S.E.
      • Mayfield J.E.
      • Tandon V.
      • Juarez E.F.
      • Chavez L.
      • Liang R.
      • Sah R.L.
      • Costello C.
      • Mesirov J.P.
      • et al.
      Inhibition of dual-specificity tyrosine phosphorylation-regulated kinase 2 perturbs 26S proteasome-addicted neoplastic progression.
      ,
      • Yoshida S.
      • Yoshida K.
      Multiple functions of DYRK2 in cancer and tissue development.
      ,
      • Correa-Sáez A.
      • Jiménez-Izquierdo R.
      • Garrido-Rodríguez M.
      • Morrugares R.
      • Muñoz E.
      • Calzado M.A.
      Updating dual-specificity tyrosine-phosphorylation-regulated kinase 2 (DYRK2): Molecular basis, functions and role in diseases.
      ).
      Besides DYRK2, the other DYRK isoforms, especially the class I's, have a long history in the field of cancer. Although DYRK1B has an overall protumorigenic role specifically in pancreatic and ovarian cancers, DYRK1A exhibits a more controversial role with reports of both protumorigenic and antitumorigenic mechanism in different cancers (reviewed in Boni et al. [
      • Boni J.
      • Rubio-Perez C.
      • López-Bigas N.
      • Fillat C.
      • de la Luna S.
      The DYRK family of kinases in cancer: Molecular functions and therapeutic opportunities.
      ]). Within the class II DYRKs, very little is known about DYRK3 and DYRK4 with limited literature pointing to a more protumorigenic role for both (
      • Wippich F.
      • Bodenmiller B.
      • Trajkovska M.G.
      • Wanka S.
      • Aebersold R.
      • Pelkmans L.
      Dual specificity kinase DYRK3 couples stress granule condensation/dissolution to mTORC1 signaling.
      ,
      • Owusu M.
      • Bannauer P.
      • Ferreira da Silva J.
      • Mourikis T.P.
      • Jones A.
      • Májek P.
      • Caldera M.
      • Wiedner M.
      • Lardeau C.-H.
      • Mueller A.C.
      • Menche J.
      • Kubicek S.
      • Ciccarelli F.D.
      • Loizou J.I.
      Mapping the human kinome in response to DNA damage.
      ). DYRK2, on the other hand, is the most extensively studied class II isoform, and the high-profile substrates reported, such as p53 (
      • Taira N.
      • Nihira K.
      • Yamaguchi T.
      • Miki Y.
      • Yoshida K.
      DYRK2 is targeted to the nucleus and controls p53 via Ser46 phosphorylation in the apoptotic response to DNA damage.
      ,
      • Taira N.
      • Yamamoto H.
      • Yamaguchi T.
      • Miki Y.
      • Yoshida K.
      ATM augments nuclear stabilization of DYRK2 by inhibiting MDM2 in the apoptotic response to DNA damage.
      ), c-Jun (
      • Taira N.
      • Mimoto R.
      • Kurata M.
      • Yamaguchi T.
      • Kitagawa M.
      • Miki Y.
      • Yoshida K.
      DYRK2 priming phosphorylation of c-Jun and c-Myc modulates cell cycle progression in human cancer cells.
      ), c-Myc (
      • Taira N.
      • Mimoto R.
      • Kurata M.
      • Yamaguchi T.
      • Kitagawa M.
      • Miki Y.
      • Yoshida K.
      DYRK2 priming phosphorylation of c-Jun and c-Myc modulates cell cycle progression in human cancer cells.
      ), NOTCH1 (
      • Morrugares R.
      • Correa-Sáez A.
      • Moreno R.
      • Garrido-Rodríguez M.
      • Muñoz E.
      • de la Vega L.
      • Calzado M.A.
      Phosphorylation-dependent regulation of the NOTCH1 intracellular domain by dual-specificity tyrosine-regulated kinase 2.
      ), HSF1 (
      • Moreno Dorta R.
      • Banerjee S.
      • Jackson A.
      • Quinn J.
      • Baillie G.
      • Dixon J.E.
      • Dinkova-Kostova A.
      • Edwards J.
      • de la Vega L.
      The stress-responsive kinase DYRK2 activates heat shock factor 1 promoting resistance to proteotoxic stress.
      ), 26S proteasome (
      • Guo X.
      • Wang X.
      • Wang Z.
      • Banerjee S.
      • Yang J.
      • Huang L.
      • Dixon J.E.
      Site-specific proteasome phosphorylation controls cell proliferation and tumorigenesis.
      ,
      • Banerjee S.
      • Ji C.
      • Mayfield J.E.
      • Goel A.
      • Xiao J.
      • Dixon J.E.
      • Guo X.
      Ancient drug curcumin impedes 26S proteasome activity by direct inhibition of dual-specificity tyrosine-regulated kinase 2.
      ,
      • Banerjee S.
      • Wei T.
      • Wang J.
      • Lee J.J.
      • Gutierrez H.L.
      • Chapman O.
      • Wiley S.E.
      • Mayfield J.E.
      • Tandon V.
      • Juarez E.F.
      • Chavez L.
      • Liang R.
      • Sah R.L.
      • Costello C.
      • Mesirov J.P.
      • et al.
      Inhibition of dual-specificity tyrosine phosphorylation-regulated kinase 2 perturbs 26S proteasome-addicted neoplastic progression.
      ), and SIAH2 (
      • Pérez M.
      • García-Limones C.
      • Zapico I.
      • Marina A.
      • Schmitz M.L.
      • Muñoz E.
      • Calzado M.A.
      Mutual regulation between SIAH2 and DYRK2 controls hypoxic and genotoxic signaling pathways.
      ,
      • Morrugares R.
      • Correa-Sáez A.
      • Moreno R.
      • Garrido-Rodríguez M.
      • Muñoz E.
      • de la Vega L.
      • Calzado M.A.
      Phosphorylation-dependent regulation of the NOTCH1 intracellular domain by dual-specificity tyrosine-regulated kinase 2.
      ), have brought the kinase to the forefront of oncology research. For the past 2 decades, multiple studies have reported an overarching tumor suppressor role of DYRK2 across various cancers (reviewed in Yoshida and Yoshida [
      • Yoshida S.
      • Yoshida K.
      Multiple functions of DYRK2 in cancer and tissue development.
      ]), with antitumorigenic roles including regulation of cell cycle, apoptosis, epithelial–mesenchymal transition (EMT), cancer stemness, and antimetastatic roles (reviewed in Yoshida and Yoshida [
      • Yoshida S.
      • Yoshida K.
      Multiple functions of DYRK2 in cancer and tissue development.
      ]). On the other hand, since 2016, multiple studies report major protumorigenic roles of DYRK2 (
      • Guo X.
      • Wang X.
      • Wang Z.
      • Banerjee S.
      • Yang J.
      • Huang L.
      • Dixon J.E.
      Site-specific proteasome phosphorylation controls cell proliferation and tumorigenesis.
      ,
      • Moreno Dorta R.
      • Banerjee S.
      • Jackson A.
      • Quinn J.
      • Baillie G.
      • Dixon J.E.
      • Dinkova-Kostova A.
      • Edwards J.
      • de la Vega L.
      The stress-responsive kinase DYRK2 activates heat shock factor 1 promoting resistance to proteotoxic stress.
      ,
      • Banerjee S.
      • Ji C.
      • Mayfield J.E.
      • Goel A.
      • Xiao J.
      • Dixon J.E.
      • Guo X.
      Ancient drug curcumin impedes 26S proteasome activity by direct inhibition of dual-specificity tyrosine-regulated kinase 2.
      ,
      • Banerjee S.
      • Wei T.
      • Wang J.
      • Lee J.J.
      • Gutierrez H.L.
      • Chapman O.
      • Wiley S.E.
      • Mayfield J.E.
      • Tandon V.
      • Juarez E.F.
      • Chavez L.
      • Liang R.
      • Sah R.L.
      • Costello C.
      • Mesirov J.P.
      • et al.
      Inhibition of dual-specificity tyrosine phosphorylation-regulated kinase 2 perturbs 26S proteasome-addicted neoplastic progression.
      ), and a few studies have identified DYRK2 as a possible cancer driver (
      • Gorringe K.L.
      • Boussioutas A.
      • Bowtell D.D.
      Novel regions of chromosomal amplification at 6p21, 5p13, and 12q14 in gastric cancer identified by array comparative genomic hybridization.
      ,
      • Miller C.T.
      • Aggarwal S.
      • Lin T.K.
      • Dagenais S.L.
      • Contreras J.I.
      • Orringer M.B.
      • Glover T.W.
      • Beer D.G.
      • Lin L.
      Amplification and overexpression of the dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2) gene in esophageal and lung adenocarcinomas.
      ,
      • Wang H.
      • Diaz A.K.
      • Shaw T.I.
      • Li Y.
      • Niu M.
      • Cho J.-H.
      • Paugh B.S.
      • Zhang Y.
      • Sifford J.
      • Bai B.
      • Wu Z.
      • Tan H.
      • Zhou S.
      • Hover L.D.
      • Tillman H.S.
      • et al.
      Deep multiomics profiling of brain tumors identifies signaling networks downstream of cancer driver genes.
      ). Furthermore, mRNA expression analyses from The Cancer Genome Atlas (TCGA) tumors along with matched normal controls reveal that the majority of cancers have higher median expression of DYRK2 than adjacent normal tissues (
      • Banerjee S.
      • Wei T.
      • Wang J.
      • Lee J.J.
      • Gutierrez H.L.
      • Chapman O.
      • Wiley S.E.
      • Mayfield J.E.
      • Tandon V.
      • Juarez E.F.
      • Chavez L.
      • Liang R.
      • Sah R.L.
      • Costello C.
      • Mesirov J.P.
      • et al.
      Inhibition of dual-specificity tyrosine phosphorylation-regulated kinase 2 perturbs 26S proteasome-addicted neoplastic progression.
      ), and a similar pattern has been shown for DYRK2 protein levels in some tumor types (
      • Moreno Dorta R.
      • Banerjee S.
      • Jackson A.
      • Quinn J.
      • Baillie G.
      • Dixon J.E.
      • Dinkova-Kostova A.
      • Edwards J.
      • de la Vega L.
      The stress-responsive kinase DYRK2 activates heat shock factor 1 promoting resistance to proteotoxic stress.
      ,
      • Banerjee S.
      • Wei T.
      • Wang J.
      • Lee J.J.
      • Gutierrez H.L.
      • Chapman O.
      • Wiley S.E.
      • Mayfield J.E.
      • Tandon V.
      • Juarez E.F.
      • Chavez L.
      • Liang R.
      • Sah R.L.
      • Costello C.
      • Mesirov J.P.
      • et al.
      Inhibition of dual-specificity tyrosine phosphorylation-regulated kinase 2 perturbs 26S proteasome-addicted neoplastic progression.
      ). All of these data suggest that DYRK2 might be an excellent potential drug target. With such high profile, oncology-related substrates, could the function of DYRK2 differ based on cancer type or cell type? To shed some light onto this question, this review will re-examine the current literature on the role of DYRK2 in cancer and follow up with existing knowledge of small-molecule inhibitors developed to target DYRK2.

      DYRK2 regulates proteostasis: an oncogenic role

      DYRK2 maintains proteostasis of cancer cells by regulating two major players of the proteotoxic response pathway, which promotes the proper folding and/or degradation of proteins (Fig. 2). More than 90% of all solid human tumors carry numerous aberrations in chromosomes, referred to as aneuploidy (
      • Weaver B.A.
      • Cleveland D.W.
      Does aneuploidy cause cancer?.
      ). As a result of their severe aneuploidy, cancer cells are exposed to proteotoxic stress that increases the amount of toxic, unfolded proteins in the cell (
      • Donnelly N.
      • Storchová Z.
      Causes and consequences of protein folding stress in aneuploid cells.
      ,
      • Ohashi A.
      • Ohori M.
      • Iwai K.
      • Nakayama Y.
      • Nambu T.
      • Morishita D.
      • Kawamoto T.
      • Miyamoto M.
      • Hirayama T.
      • Okaniwa M.
      • Banno H.
      • Ishikawa T.
      • Kandori H.
      • Iwata K.
      Aneuploidy generates proteotoxic stress and DNA damage concurrently with p53-mediated post-mitotic apoptosis in SAC-impaired cells.
      ). To survive proteotoxic stress, cancer cells can either increase protein folding capacity (controlled by the transcription factor HSF1) or increase the degradation of the misfolded/aggregated proteins (via the 26S proteasome and/or autophagy). DYRK2 phosphorylates and activates both HSF1 and the 26S proteasome and thereby activates the proteotoxic stress pathway promoting tumorigenesis in cancers such as triple-negative breast cancer (TNBC) and multiple myeloma (MM).
      Figure thumbnail gr2
      Figure 2DYRK2 regulates proteostasis via a two-pronged mechanism. DYRK2 phosphorylates and upregulates the activity of the 26S proteasome, which reduces proteotoxic stress by degrading misfolded/unfolded proteins. In parallel, DYRK2 triggers phosphorylation-mediated activation of HSF1, which promotes transcriptional upregulation of chaperones that promotes folding of misfolded/unfolded proteins. Proteasome inhibitors (PIs) such as bortezomib, carfilzomib, and ixazomib inhibit the proteasome and result in enhanced proteotoxic stress because of toxic protein aggregates. Proteasome inhibition by PIs triggers indirect activation of the HSF1 pathway to compensate for the loss of proteasome activity thereby decoupling the proteasome dependence of cancer. HSF1, heat-shock factor 1.

      DYRK2 regulates 26S proteasome function

      In 2016, an RNAi kinase screen identified DYRK2 as a kinase-regulating 26S proteasome activity (
      • Guo X.
      • Wang X.
      • Wang Z.
      • Banerjee S.
      • Yang J.
      • Huang L.
      • Dixon J.E.
      Site-specific proteasome phosphorylation controls cell proliferation and tumorigenesis.
      ). The study showed that DYRK2 depletion either by si/shRNA or CRISPR/Cas9 KO led to a 30 to 40% decrease in proteasome activity (
      • Guo X.
      • Wang X.
      • Wang Z.
      • Banerjee S.
      • Yang J.
      • Huang L.
      • Dixon J.E.
      Site-specific proteasome phosphorylation controls cell proliferation and tumorigenesis.
      ). The mature 26S proteasome is a complex of more than 30 distinct subunits that catalyzes 80% of eukaryotic protein degradation and harbors three distinct peptidase activities in the core subunit (chymotryptic, tryptic, and caspase-like) (
      • Collins G.A.
      • Goldberg A.L.
      The logic of the 26S proteasome.
      • Coux O.
      • Tanaka K.
      • Goldberg A.L.
      Structure and functions of the 20S and 26S proteasomes.
      ). Besides the core of the proteasome, the complex also consists of the 19S regulatory subunit that binds to ubiquitylated proteins, whereas a six-membered ATPase ring hydrolyzes the protein into a polypeptide chain for entry into the peptidase core for degradation (
      • Besche H.C.
      • Peth A.
      • Goldberg A.L.
      Getting to first base in proteasome assembly.
      ). Interestingly, DYRK2 phosphorylates the Rpt3 subunit on the ATPase ring of the 19S subunit of the proteasome on an evolutionarily conserved Thr25 site (
      • Guo X.
      • Wang X.
      • Wang Z.
      • Banerjee S.
      • Yang J.
      • Huang L.
      • Dixon J.E.
      Site-specific proteasome phosphorylation controls cell proliferation and tumorigenesis.
      ). Rpt3 pT25 had been previously reported by Steve Gygi's group in their 2008 work on quantitative phosphoproteomics of mitosis (
      • Dephoure N.
      • Zhou C.
      • Villen J.
      • Beausoleil S.A.
      • Bakalarski C.E.
      • Elledge S.J.
      • Gygi S.P.
      A quantitative atlas of mitotic phosphorylation.
      ), but the function of the phosphorylation was not known. A phospho-specific antibody generated against pT25 Rpt3 showed that the site is dynamically upregulated during G2/M stage of the cell cycle and that serum starvation leads to loss of Thr25 phosphorylation (
      • Guo X.
      • Wang X.
      • Wang Z.
      • Banerjee S.
      • Yang J.
      • Huang L.
      • Dixon J.E.
      Site-specific proteasome phosphorylation controls cell proliferation and tumorigenesis.
      ). Furthermore, CRISPR/Cas9 knock-in of a phospho-deficient Thr25Ala on Rpt3 mimics the DYRK2 KO phenotypes in cells wherein there is a delay in mitotic progression, slower cell proliferation rates, and inhibition of all three peptidase activities of the 26S proteasome (
      • Guo X.
      • Wang X.
      • Wang Z.
      • Banerjee S.
      • Yang J.
      • Huang L.
      • Dixon J.E.
      Site-specific proteasome phosphorylation controls cell proliferation and tumorigenesis.
      ). The 26S proteasome degrades nearly 80% of all eukaryotic proteins, and hence, a 30% loss in activity leads to significant proteotoxic stress and consequent cell death in breast cancer cells (
      • Guo X.
      • Wang X.
      • Wang Z.
      • Banerjee S.
      • Yang J.
      • Huang L.
      • Dixon J.E.
      Site-specific proteasome phosphorylation controls cell proliferation and tumorigenesis.
      ). Intriguingly, DYRK2 KO cells were significantly more sensitive to the proteasome inhibitor, bortezomib, suggesting DYRK2 could be a possible therapeutic target for treatment of cancer (
      • Guo X.
      • Wang X.
      • Wang Z.
      • Banerjee S.
      • Yang J.
      • Huang L.
      • Dixon J.E.
      Site-specific proteasome phosphorylation controls cell proliferation and tumorigenesis.
      ). Indeed, in an ectopic nude mouse xenograft model, DYRK2 KO and T25A Rpt3 knock-in cells were less efficient in generating a tumor as compared to parental cells (
      • Guo X.
      • Wang X.
      • Wang Z.
      • Banerjee S.
      • Yang J.
      • Huang L.
      • Dixon J.E.
      Site-specific proteasome phosphorylation controls cell proliferation and tumorigenesis.
      ). This study further established the DYRK2-proteasome axis as potentially tumor promoting because higher expression of DYRK2 significantly correlated with higher mortality and poorer relapse-free survival in patients with breast cancer (
      • Guo X.
      • Wang X.
      • Wang Z.
      • Banerjee S.
      • Yang J.
      • Huang L.
      • Dixon J.E.
      Site-specific proteasome phosphorylation controls cell proliferation and tumorigenesis.
      ). In fact, inhibition or genetic depletion of DYRK2 tipped the scales of proteostasis in TNBC and MM cells. DYRK2 mRNA levels are higher in newly diagnosed and relapsed MM than normal donors (
      • Banerjee S.
      • Wei T.
      • Wang J.
      • Lee J.J.
      • Gutierrez H.L.
      • Chapman O.
      • Wiley S.E.
      • Mayfield J.E.
      • Tandon V.
      • Juarez E.F.
      • Chavez L.
      • Liang R.
      • Sah R.L.
      • Costello C.
      • Mesirov J.P.
      • et al.
      Inhibition of dual-specificity tyrosine phosphorylation-regulated kinase 2 perturbs 26S proteasome-addicted neoplastic progression.
      ). In fact, mice bearing syngrafted/xenografted myeloma cells with genetic depletion of DYRK2 exhibit significantly slower myeloma disease progression and reduced bone degeneration (
      • Banerjee S.
      • Wei T.
      • Wang J.
      • Lee J.J.
      • Gutierrez H.L.
      • Chapman O.
      • Wiley S.E.
      • Mayfield J.E.
      • Tandon V.
      • Juarez E.F.
      • Chavez L.
      • Liang R.
      • Sah R.L.
      • Costello C.
      • Mesirov J.P.
      • et al.
      Inhibition of dual-specificity tyrosine phosphorylation-regulated kinase 2 perturbs 26S proteasome-addicted neoplastic progression.
      ). Furthermore, bortezomib-resistant RPMI8226 myeloma cells express higher protein levels of DYRK2 than nonresistant RPMI8226 (
      • Banerjee S.
      • Wei T.
      • Wang J.
      • Lee J.J.
      • Gutierrez H.L.
      • Chapman O.
      • Wiley S.E.
      • Mayfield J.E.
      • Tandon V.
      • Juarez E.F.
      • Chavez L.
      • Liang R.
      • Sah R.L.
      • Costello C.
      • Mesirov J.P.
      • et al.
      Inhibition of dual-specificity tyrosine phosphorylation-regulated kinase 2 perturbs 26S proteasome-addicted neoplastic progression.
      ), suggesting that DYRK2 might play a role in driving drug resistance in some myeloma cases. The potent and selective DYRK2 inhibitor, LDN192960, induces cytotoxicity in myeloma cells both in vitro and in vivo with minimal off-target effects (
      • Banerjee S.
      • Wei T.
      • Wang J.
      • Lee J.J.
      • Gutierrez H.L.
      • Chapman O.
      • Wiley S.E.
      • Mayfield J.E.
      • Tandon V.
      • Juarez E.F.
      • Chavez L.
      • Liang R.
      • Sah R.L.
      • Costello C.
      • Mesirov J.P.
      • et al.
      Inhibition of dual-specificity tyrosine phosphorylation-regulated kinase 2 perturbs 26S proteasome-addicted neoplastic progression.
      ). The fact that the DYRK2 inhibitor alleviates myeloma burden in vivo suggests DYRK2 could indeed be a viable in vivo target for myeloma therapeutics. Resistance to proteasome inhibitors have been reported in patients, and this is either brought about by cancer mutations in the proteasome core or via upregulation of HSF1-mediated proteotoxic response pathway.

      DYRK2 phosphorylates HSF1 and modulates proteotoxic response

      The transcription factor HSF1 is the master regulator of proteotoxic stress responses and supports oncogenesis by helping cancer cells cope with the proteotoxic stress associated with both aneuploidy and oncogenic mutations. This has been demonstrated by the reduced susceptibility of Hsf1-KO mice to tumor formation driven either by Ras/p53 mutations or by chemical carcinogens (
      • Dai C.
      • Whitesell L.
      • Rogers A.B.
      • Lindquist S.
      Heat shock factor 1 is a powerful multifaceted modifier of carcinogenesis.
      ,
      • Meng L.
      • Gabai V.L.
      • Sherman M.Y.
      Heat-shock transcription factor HSF1 has a critical role in human epidermal growth factor receptor-2-induced cellular transformation and tumorigenesis.
      ). Furthermore, high levels of HSF1 expression associate with poor outcome of various cancers (
      • Mendillo M.L.
      • Santagata S.
      • Koeva M.
      • Bell G.W.
      • Hu R.
      • Tamimi R.M.
      • Fraenkel E.
      • Ince T.A.
      • Whitesell L.
      • Lindquist S.
      HSF1 drives a transcriptional program distinct from heat shock to support highly malignant human cancers.
      ). Upon proteotoxic stress, HSF1 is activated, translocates to the nucleus (
      • Baler R.
      • Dahl G.
      • Voellmy R.
      Activation of human heat shock genes is accompanied by oligomerization, modification, and rapid translocation of heat shock transcription factor HSF1.
      ), and initiates the transcription of heat-shock proteins. Heat-shock proteins then function as molecular chaperones, protecting cells against proteotoxic stress by assisting in protein folding (
      • Jolly C.
      • Morimoto R.I.
      Role of the heat shock response and molecular chaperones in oncogenesis and cell death.
      ). HSF1 activity and stability are tightly controlled by multiple post-translational modifications (
      • Dayalan Naidu S.
      • Dinkova-Kostova A.T.
      Regulation of the mammalian heat shock factor 1.
      ). Among these, phosphorylation of serine 320 and serine 326 is associated with stability and nuclear accumulation followed by enhanced transcriptional activity of HSF1 (
      • Guettouche T.
      • Boellmann F.
      • Lane W.S.
      • Voellmy R.
      Analysis of phosphorylation of human heat shock factor 1 in cells experiencing a stress.
      ,
      • Murshid A.
      • Chou S.D.
      • Prince T.
      • Zhang Y.
      • Bharti A.
      • Calderwood S.K.
      Protein kinase A binds and activates heat shock factor 1.
      ,
      • Tang Z.
      • Dai S.
      • He Y.
      • Doty R.A.
      • Shultz L.D.
      • Sampson S.B.
      • Dai C.
      MEK guards proteome stability and inhibits tumor-suppressive amyloidogenesis via HSF1.
      ). DYRK2 positively regulates HSF1 nuclear stability and activity, by phosphorylating it at Ser320 and Ser326 in TNBC cells (
      • Moreno Dorta R.
      • Banerjee S.
      • Jackson A.
      • Quinn J.
      • Baillie G.
      • Dixon J.E.
      • Dinkova-Kostova A.
      • Edwards J.
      • de la Vega L.
      The stress-responsive kinase DYRK2 activates heat shock factor 1 promoting resistance to proteotoxic stress.
      ). Indeed, DYRK2-depleted TNBC cells were far more sensitive to heat shock–mediated proteotoxic stress than parental cells, thus corroborating that DYRK2 plays a major role in maintaining proteostasis in TNBC cells. This link between DYRK2 and HSF1 is also observed in TNBC tumor samples, wherein a marked correlation was observed between high DYRK2 levels and high nuclear HSF1 levels.
      The HSF1 pathway and the proteasome are not just two of the main pathways maintaining cell proteostasis, but they are interconnected and can compensate for each other. As mentioned before, proteasome inhibitors lead to the activation of HSF1 (Fig. 2) in an effort to protect the cell against the accumulation of toxic proteins (
      • Pirkkala L.
      • Alastalo T.-P.
      • Zuo X.
      • Benjamin I.J.
      • Sistonen L.
      Disruption of heat shock factor 1 reveals an essential role in the ubiquitin proteolytic pathway.
      ,
      • Shah S.P.
      • Lonial S.
      • Boise L.H.
      When cancer fights back: Multiple myeloma, proteasome inhibition, and the heat-shock response.
      ). The cytoprotective response mediated by HSF1 counteracts the cytotoxic effect of proteasome inhibitors (
      • Pirkkala L.
      • Alastalo T.-P.
      • Zuo X.
      • Benjamin I.J.
      • Sistonen L.
      Disruption of heat shock factor 1 reveals an essential role in the ubiquitin proteolytic pathway.
      ,
      • Shah S.P.
      • Lonial S.
      • Boise L.H.
      When cancer fights back: Multiple myeloma, proteasome inhibition, and the heat-shock response.
      ,
      • Sharma C.
      • Seo Y.H.
      Small molecule inhibitors of HSF1-activated pathways as potential next-generation anticancer therapeutics.
      ), and thus, HSF1 inhibition might be effective to overcome proteasome inhibitor resistance in cancer cells. In that sense, a DYRK2 inhibitor induced cytotoxicity even in MM cells resistant to proteasome inhibitors (
      • Banerjee S.
      • Ji C.
      • Mayfield J.E.
      • Goel A.
      • Xiao J.
      • Dixon J.E.
      • Guo X.
      Ancient drug curcumin impedes 26S proteasome activity by direct inhibition of dual-specificity tyrosine-regulated kinase 2.
      ,
      • Banerjee S.
      • Wei T.
      • Wang J.
      • Lee J.J.
      • Gutierrez H.L.
      • Chapman O.
      • Wiley S.E.
      • Mayfield J.E.
      • Tandon V.
      • Juarez E.F.
      • Chavez L.
      • Liang R.
      • Sah R.L.
      • Costello C.
      • Mesirov J.P.
      • et al.
      Inhibition of dual-specificity tyrosine phosphorylation-regulated kinase 2 perturbs 26S proteasome-addicted neoplastic progression.
      ), suggesting that in fact DYRK2 inhibition might be targeting different complementary pathways. This observation was further echoed by a recent study showing that MM cells were extremely sensitive to increased temperatures and heat shock (
      • Sha Z.
      • Goldberg A.L.
      Multiple myeloma cells are exceptionally sensitive to heat shock, which overwhelms their proteostasis network and induces apoptosis.
      ). In fact, combining heat shock with proteasome inhibitors led to higher accumulation of misfolded proteins leading to acute proteotoxic stress and apoptosis in the myeloma cells (
      • Sha Z.
      • Goldberg A.L.
      Multiple myeloma cells are exceptionally sensitive to heat shock, which overwhelms their proteostasis network and induces apoptosis.
      ). Because cancer cells harbor significantly higher misfolded proteins than normal cells, targeting DYRK2 could indeed tip the scales for proteostasis in malignant cells and provide a significant therapeutic window for targeting specific cancers. This is indeed the case because normal/noncancerous cells were far more resistant to DYRK2 inhibitors (
      • Banerjee S.
      • Ji C.
      • Mayfield J.E.
      • Goel A.
      • Xiao J.
      • Dixon J.E.
      • Guo X.
      Ancient drug curcumin impedes 26S proteasome activity by direct inhibition of dual-specificity tyrosine-regulated kinase 2.
      ,
      • Banerjee S.
      • Wei T.
      • Wang J.
      • Lee J.J.
      • Gutierrez H.L.
      • Chapman O.
      • Wiley S.E.
      • Mayfield J.E.
      • Tandon V.
      • Juarez E.F.
      • Chavez L.
      • Liang R.
      • Sah R.L.
      • Costello C.
      • Mesirov J.P.
      • et al.
      Inhibition of dual-specificity tyrosine phosphorylation-regulated kinase 2 perturbs 26S proteasome-addicted neoplastic progression.
      ). Thus, targeting DYRK2 can significantly affect proteostasis (Fig. 2) via perturbation of both HSF1 and 26S proteasome activity leading to cancer cell death.
      Hence, in the context of TNBC and MM, DYRK2 plays an overarching role as an oncogenic kinase and a potential therapeutic target.

      DYRK2-p53 tumor suppressor link

      A major molecular mechanism by which DYRK2 has been reported to exhibit the antitumorigenic role is via phosphorylation of tumor suppressor p53 on serine46 (Ser46). Upon genotoxic stress, energy stress, or heat shock, multiple CMGC kinases such as homeodomain-interacting protein kinase (HIPK2), mitogen-activated protein kinase p38α, and DYRK2 can phosphorylate p53 on Ser46, which triggers transcription of proapoptotic genes leading to cell death or cell senescence (reviewed in Liebl and Hofmann [
      • Liebl M.C.
      • Hofmann T.G.
      Cell fate regulation upon DNA damage: p53 serine 46 kinases pave the cell death road.
      ]). Upon DNA damage, DYRK2 is phosphorylated by ataxia-telangiectasia mutated kinase, which protects DYRK2 from proteasomal degradation leading to its nuclear accumulation where it phosphorylates p53 on Ser46 and promotes its transcriptional tumor suppressor activity (
      • Taira N.
      • Nihira K.
      • Yamaguchi T.
      • Miki Y.
      • Yoshida K.
      DYRK2 is targeted to the nucleus and controls p53 via Ser46 phosphorylation in the apoptotic response to DNA damage.
      ,
      • Taira N.
      • Yamamoto H.
      • Yamaguchi T.
      • Miki Y.
      • Yoshida K.
      ATM augments nuclear stabilization of DYRK2 by inhibiting MDM2 in the apoptotic response to DNA damage.
      ). Although phosphorylated Ser46 on p53 is indeed a marker for its tumor suppressor role, DYRK2 by no means is the exclusive kinase here. With multiple kinases including PKCδ, HIPK2, ataxia-telangiectasia mutated kinase, and p38α phosphorylating Ser46 upon genotoxic stress (
      • Liebl M.C.
      • Hofmann T.G.
      Cell fate regulation upon DNA damage: p53 serine 46 kinases pave the cell death road.
      ), it is hard to decipher to what extent DYRK2 contributes to this tumor suppressor role. Furthermore, p53 is mutated or truncated in a vast number of solid tumors and cancer patients with altered p53 exhibit significantly poorer survival (
      • Donehower L.A.
      • Soussi T.
      • Korkut A.
      • Liu Y.
      • Schultz A.
      • Cardenas M.
      • Li X.
      • Babur O.
      • Hsu T.-K.
      • Lichtarge O.
      • Weinstein J.N.
      • Akbani R.
      • Wheeler D.A.
      Integrated analysis of TP53 gene and pathway alterations in the cancer genome atlas.
      ,
      • Kandoth C.
      • McLellan M.D.
      • Vandin F.
      • Ye K.
      • Niu B.
      • Lu C.
      • Xie M.
      • Zhang Q.
      • McMichael J.F.
      • Wyczalkowski M.A.
      • Leiserson M.D.M.
      • Miller C.A.
      • Welch J.S.
      • Walter M.J.
      • Wendl M.C.
      • et al.
      Mutational landscape and significance across 12 major cancer types.
      ). Mutated p53 often exhibits stoichiometrically lower phosphoSer46 (
      • Fanucchi S.
      • Veale R.B.
      Role of p53/FAK association and p53Ser46 phosphorylation in staurosporine-mediated apoptosis: Wild type versus mutant p53-R175H.
      ) and has been reported to trigger pro-oncogenic functions upon phosphorylation (
      • Muller P.A.J.
      • Vousden K.H.
      Mutant p53 in cancer: New functions and therapeutic opportunities.
      ). This suggests that p53 phosphorylated on Ser46 serves as a tumor suppressor only in the few percentage of cancers containing WT p53 where patients exhibit better chances of survival.
      Multiple publications carrying out sequencing or immunohistochemistry to study mRNA/protein levels of DYRK2 have suggested that DYRK2 is a tumor suppressor in colorectal (
      • Leiszter K.
      • Galamb O.
      • Sipos F.
      • Krenács T.
      • Veres G.
      • Wichmann B.
      • Kalmár A.
      • Patai Á V.
      • Tóth K.
      • Valcz G.
      • Molnár B.
      • Tulassay Z.
      Sporadic colorectal cancer development shows rejuvenescence regarding epithelial proliferation and apoptosis.
      ,
      • Yan H.
      • Hu K.
      • Wu W.
      • Li Y.
      • Tian H.
      • Chu Z.
      • Koeffler H.P.
      • Yin D.
      Low expression of DYRK2 (dual specificity tyrosine phosphorylation regulated kinase 2) correlates with poor prognosis in colorectal cancer.
      ,
      • Ito D.
      • Yogosawa S.
      • Mimoto R.
      • Hirooka S.
      • Horiuchi T.
      • Eto K.
      • Yanaga K.
      • Yoshida K.
      Dual-specificity tyrosine-regulated kinase 2 is a suppressor and potential prognostic marker for liver metastasis of colorectal cancer.
      ,
      • Wang Y.
      • Sun J.
      • Wei X.
      • Luan L.
      • Zeng X.
      • Wang C.
      • Zhao W.
      Decrease of miR-622 expression suppresses migration and invasion by targeting regulation of DYRK2 in colorectal cancer cells.
      ), liver (
      • Zhang X.
      • Xu P.
      • Ni W.
      • Fan H.
      • Xu J.
      • Chen Y.
      • Huang W.
      • Lu S.
      • Liang L.
      • Liu J.
      • Chen B.
      • Shi W.
      Downregulated DYRK2 expression is associated with poor prognosis and Oxaliplatin resistance in hepatocellular carcinoma.
      ), brain (
      • Shen Y.
      • Zhang L.
      • Wang D.
      • Bao Y.
      • Liu C.
      • Xu Z.
      • Huang W.
      • Cheng C.
      Regulation of glioma cells migration by DYRK2.
      ), and lung cancers (
      • Yamashita S.
      • Chujo M.
      • Tokuishi K.
      • Anami K.
      • Miyawaki M.
      • Yamamoto S.
      • Kawahara K.
      Expression of dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 2 (DYRK2) can be a favorable prognostic marker in pulmonary adenocarcinoma.
      ,
      • Yamashita S.
      • Chujo M.
      • Moroga T.
      • Anami K.
      • Tokuishi K.
      • Miyawaki M.
      • Kawano Y.
      • Takeno S.
      • Yamamoto S.
      • Kawahara K.
      DYRK2 expression may be a predictive marker for chemotherapy in non-small cell lung cancer.
      ) and that the kinase promotes chemosensitivity in ovarian cancer (
      • Yamaguchi N.
      • Mimoto R.
      • Yanaihara N.
      • Imawari Y.
      • Hirooka S.
      • Okamoto A.
      • Yoshida K.
      DYRK2 regulates epithelial-mesenchymal-transition and chemosensitivity through Snail degradation in ovarian serous adenocarcinoma.
      ). However, ovarian, liver, brain, lung, and colorectal cancers exhibit some of the highest mutations and variant allele frequencies in p53 compared with other cancer types (
      • Donehower L.A.
      • Soussi T.
      • Korkut A.
      • Liu Y.
      • Schultz A.
      • Cardenas M.
      • Li X.
      • Babur O.
      • Hsu T.-K.
      • Lichtarge O.
      • Weinstein J.N.
      • Akbani R.
      • Wheeler D.A.
      Integrated analysis of TP53 gene and pathway alterations in the cancer genome atlas.
      ,
      • Kandoth C.
      • McLellan M.D.
      • Vandin F.
      • Ye K.
      • Niu B.
      • Lu C.
      • Xie M.
      • Zhang Q.
      • McMichael J.F.
      • Wyczalkowski M.A.
      • Leiserson M.D.M.
      • Miller C.A.
      • Welch J.S.
      • Walter M.J.
      • Wendl M.C.
      • et al.
      Mutational landscape and significance across 12 major cancer types.
      ,
      • Hussain S.P.
      • Schwank J.
      • Staib F.
      • Wang X.W.
      • Harris C.C.
      TP53 mutations and hepatocellular carcinoma: Insights into the etiology and pathogenesis of liver cancer.
      ). Thus, it is unclear to what extent DYRK2's phosphorylation of p53 could play as a tumor-suppressive role in these solid tumors exhibiting p53 mutation or loss. Furthermore, in endothelial cells, the pan-DYRK inhibitor, harmine (albeit with possible off-target effects), promotes p53 phosphorylation on Ser15, Ser20, and Ser37 (
      • Dai F.
      • Chen Y.
      • Song Y.
      • Huang L.
      • Zhai D.
      • Dong Y.
      • Lai L.
      • Zhang T.
      • Li D.
      • Pang X.
      • Liu M.
      • Yi Z.
      A natural small molecule harmine inhibits angiogenesis and suppresses tumour growth through activation of p53 in endothelial cells.
      ), leading to higher p53 protein levels upon DNA damage (
      • Dai F.
      • Chen Y.
      • Song Y.
      • Huang L.
      • Zhai D.
      • Dong Y.
      • Lai L.
      • Zhang T.
      • Li D.
      • Pang X.
      • Liu M.
      • Yi Z.
      A natural small molecule harmine inhibits angiogenesis and suppresses tumour growth through activation of p53 in endothelial cells.
      ,
      • Chehab N.H.
      • Malikzay A.
      • Stavridi E.S.
      • Halazonetis T.D.
      Phosphorylation of Ser-20 mediates stabilization of human p53 in response to DNA damage.
      ). Seemingly, in this case, DYRK2 inhibition led to tumor suppression. Hence, it is also important to decipher the molecular functions of DYRK2 in noncancer models or as a potential cancer driver. A recent unbiased deep multiomics study looking at the proteome, phosphoproteome, and transcriptome of murine high-grade brain cancer glioma model reported 41 kinases including DYRK2 exhibiting higher activity and rewired substrate signaling (
      • Wang H.
      • Diaz A.K.
      • Shaw T.I.
      • Li Y.
      • Niu M.
      • Cho J.-H.
      • Paugh B.S.
      • Zhang Y.
      • Sifford J.
      • Bai B.
      • Wu Z.
      • Tan H.
      • Zhou S.
      • Hover L.D.
      • Tillman H.S.
      • et al.
      Deep multiomics profiling of brain tumors identifies signaling networks downstream of cancer driver genes.
      ). Furthermore, the glioma murine model was generated by intracranial implantation of genetically engineered p53 null astrocytes, thus making the tumor-suppressor role of DYRK2-p53 axis highly untenable in this model.
      Besides solid tumors, chronic myeloid leukemia (CML) cell lines exhibit significantly lower protein levels of DYRK2 than other hematological cancer cell lines (
      • Park C.S.
      • Lewis A.H.
      • Chen T.J.
      • Bridges C.S.
      • Shen Y.
      • Suppipat K.
      • Puppi M.
      • Tomolonis J.A.
      • Pang P.D.
      • Mistretta T.A.
      • Ma L.Y.
      • Green M.R.
      • Rau R.
      • Lacorazza H.D.
      A KLF4-DYRK2-mediated pathway regulating self-renewal in CML stem cells.
      ). Interestingly, transcriptional upregulation of DYRK2 inhibits survival and self-renewal of CML stem/progenitor cells via c-Myc depletion and p53 activation (
      • Park C.S.
      • Lewis A.H.
      • Chen T.J.
      • Bridges C.S.
      • Shen Y.
      • Suppipat K.
      • Puppi M.
      • Tomolonis J.A.
      • Pang P.D.
      • Mistretta T.A.
      • Ma L.Y.
      • Green M.R.
      • Rau R.
      • Lacorazza H.D.
      A KLF4-DYRK2-mediated pathway regulating self-renewal in CML stem cells.
      ). This tumor-suppressor role of DYRK2 seems to be CML specific because all other leukemia subtypes tested exhibited naturally elevated protein levels of DYRK2 at basal conditions (
      • Park C.S.
      • Lewis A.H.
      • Chen T.J.
      • Bridges C.S.
      • Shen Y.
      • Suppipat K.
      • Puppi M.
      • Tomolonis J.A.
      • Pang P.D.
      • Mistretta T.A.
      • Ma L.Y.
      • Green M.R.
      • Rau R.
      • Lacorazza H.D.
      A KLF4-DYRK2-mediated pathway regulating self-renewal in CML stem cells.
      ), suggesting alternate driving mechanisms for tumorigenesis. Hematological malignancies exhibit fewer p53 mutations/loss (
      • Donehower L.A.
      • Soussi T.
      • Korkut A.
      • Liu Y.
      • Schultz A.
      • Cardenas M.
      • Li X.
      • Babur O.
      • Hsu T.-K.
      • Lichtarge O.
      • Weinstein J.N.
      • Akbani R.
      • Wheeler D.A.
      Integrated analysis of TP53 gene and pathway alterations in the cancer genome atlas.
      ,
      • Kandoth C.
      • McLellan M.D.
      • Vandin F.
      • Ye K.
      • Niu B.
      • Lu C.
      • Xie M.
      • Zhang Q.
      • McMichael J.F.
      • Wyczalkowski M.A.
      • Leiserson M.D.M.
      • Miller C.A.
      • Welch J.S.
      • Walter M.J.
      • Wendl M.C.
      • et al.
      Mutational landscape and significance across 12 major cancer types.
      ), and hence, DYRK2 could indeed be a tumor suppressor in specific subtypes such as CML. On a similar note, silencing DYRK2 has been reported to increase cell proliferation and reverse cell adhesion–mediated drug resistance in non-Hodgkin's lymphoma cell lines (
      • Wang Y.
      • Wu Y.
      • Miao X.
      • Zhu X.
      • Miao X.
      • He Y.
      • Zhong F.
      • Ding L.
      • Liu J.
      • Tang J.
      • Huang Y.
      • Xu X.
      • He S.
      Silencing of DYRK2 increases cell proliferation but reverses CAM-DR in Non-Hodgkin's Lymphoma.
      ). Intriguingly, MM cells are highly sensitive to DYRK2 inhibition irrespective of p53 status (
      • Banerjee S.
      • Ji C.
      • Mayfield J.E.
      • Goel A.
      • Xiao J.
      • Dixon J.E.
      • Guo X.
      Ancient drug curcumin impedes 26S proteasome activity by direct inhibition of dual-specificity tyrosine-regulated kinase 2.
      ,
      • Banerjee S.
      • Wei T.
      • Wang J.
      • Lee J.J.
      • Gutierrez H.L.
      • Chapman O.
      • Wiley S.E.
      • Mayfield J.E.
      • Tandon V.
      • Juarez E.F.
      • Chavez L.
      • Liang R.
      • Sah R.L.
      • Costello C.
      • Mesirov J.P.
      • et al.
      Inhibition of dual-specificity tyrosine phosphorylation-regulated kinase 2 perturbs 26S proteasome-addicted neoplastic progression.
      ). Because DYRK2 inhibition in myeloma tips the balance of proteotoxic stress (
      • Banerjee S.
      • Wei T.
      • Wang J.
      • Lee J.J.
      • Gutierrez H.L.
      • Chapman O.
      • Wiley S.E.
      • Mayfield J.E.
      • Tandon V.
      • Juarez E.F.
      • Chavez L.
      • Liang R.
      • Sah R.L.
      • Costello C.
      • Mesirov J.P.
      • et al.
      Inhibition of dual-specificity tyrosine phosphorylation-regulated kinase 2 perturbs 26S proteasome-addicted neoplastic progression.
      • Sha Z.
      • Goldberg A.L.
      Multiple myeloma cells are exceptionally sensitive to heat shock, which overwhelms their proteostasis network and induces apoptosis.
      ), all cells whether p53 WT (such as cell line MM.1S) or mutated (such as cell lines RPMI8226 and U266B1) die (
      • Banerjee S.
      • Wei T.
      • Wang J.
      • Lee J.J.
      • Gutierrez H.L.
      • Chapman O.
      • Wiley S.E.
      • Mayfield J.E.
      • Tandon V.
      • Juarez E.F.
      • Chavez L.
      • Liang R.
      • Sah R.L.
      • Costello C.
      • Mesirov J.P.
      • et al.
      Inhibition of dual-specificity tyrosine phosphorylation-regulated kinase 2 perturbs 26S proteasome-addicted neoplastic progression.
      ). This suggests that in myeloma, the role of DYRK2 as an oncogenic driver probably plays a far greater role than its tumor-suppressor function potentiated by p53 phosphorylation. Hence, stratification of cancer subtypes before assigning molecular functions to DYRK2 is important. However, DYRK2 has tumor-suppressor mechanisms beyond p53 involvement, and it is important to investigate the diverse mechanisms at play to derive a larger perspective (Fig. 3).
      Figure thumbnail gr3
      Figure 3Overall summary of DYRK2 in neoplasia. The figure provides a holistic view of the various reported roles of DYRK2 in different forms of cancers. For each cancer, the various interactors/substrates/effectors of DYRK2 are highlighted either in green (tumor-suppressor role) or in red (protumorigenic role). The cancer models/tools (cell-based, mouse models, patient samples, DYRK2 inhibitor) used to derive the respective conclusions are also shown. Direct DYRK2 substrates are shown with the added (P) phosphate, and conclusions based on sequencing or immunohistochemistry are also highlighted. Gray arrows indicate those cancers where controversial or conflicting reports have been documented.

      Other molecular mechanisms linking DYRK2 and cancer

      Various p53-independent tumor-suppressor mechanisms have been reported for DYRK2, while other substrates point to an oncogenic role. Each mechanism focuses on specific cancer types and subtypes (Fig. 3 and Table 1). The main substrates and mechanisms are critically presented below.
      Table 1DYRK2 molecular mechanism and substrates/partners listed along with reported phosphorylation sites, overarching role, and the unanswered questions raised by each study
      CancerMolecular partnerPhosphositeReported rolePending question/issue
      Breast26S proteasome/RPT3 (
      • Guo X.
      • Wang X.
      • Wang Z.
      • Banerjee S.
      • Yang J.
      • Huang L.
      • Dixon J.E.
      Site-specific proteasome phosphorylation controls cell proliferation and tumorigenesis.
      ,
      • Banerjee S.
      • Ji C.
      • Mayfield J.E.
      • Goel A.
      • Xiao J.
      • Dixon J.E.
      • Guo X.
      Ancient drug curcumin impedes 26S proteasome activity by direct inhibition of dual-specificity tyrosine-regulated kinase 2.
      ,
      • Banerjee S.
      • Wei T.
      • Wang J.
      • Lee J.J.
      • Gutierrez H.L.
      • Chapman O.
      • Wiley S.E.
      • Mayfield J.E.
      • Tandon V.
      • Juarez E.F.
      • Chavez L.
      • Liang R.
      • Sah R.L.
      • Costello C.
      • Mesirov J.P.
      • et al.
      Inhibition of dual-specificity tyrosine phosphorylation-regulated kinase 2 perturbs 26S proteasome-addicted neoplastic progression.
      )
      Thr25Oncogenic; regulates proteostasisData specific for triple-negative breast cancer subtype.
      HSF1 (
      • Moreno Dorta R.
      • Banerjee S.
      • Jackson A.
      • Quinn J.
      • Baillie G.
      • Dixon J.E.
      • Dinkova-Kostova A.
      • Edwards J.
      • de la Vega L.
      The stress-responsive kinase DYRK2 activates heat shock factor 1 promoting resistance to proteotoxic stress.
      )
      Ser320, Ser326Data specific for triple-negative breast cancer subtype; possible redundancy with other kinases.
      SNAIL (
      • Mimoto R.
      • Taira N.
      • Takahashi H.
      • Yamaguchi T.
      • Okabe M.
      • Uchida K.
      • Miki Y.
      • Yoshida K.
      DYRK2 controls the epithelial-mesenchymal transition in breast cancer by degrading Snail.
      )
      Ser104Tumor suppressor; EMT downregulationData heavily reliant on ectopic overexpression; possible redundancies with other kinases
      NOTCH1 (
      • Morrugares R.
      • Correa-Sáez A.
      • Moreno R.
      • Garrido-Rodríguez M.
      • Muñoz E.
      • de la Vega L.
      • Calzado M.A.
      Phosphorylation-dependent regulation of the NOTCH1 intracellular domain by dual-specificity tyrosine-regulated kinase 2.
      )
      Thr2512Tumor suppressor; reduces invasionRedundancies with other kinases; C-Myc pSer62 can be oncogenic; redundancies with other kinases; small sample sizes used for in vivo work.
      C-Myc (
      • Taira N.
      • Mimoto R.
      • Kurata M.
      • Yamaguchi T.
      • Kitagawa M.
      • Miki Y.
      • Yoshida K.
      DYRK2 priming phosphorylation of c-Jun and c-Myc modulates cell cycle progression in human cancer cells.
      )
      Ser62
      C-Jun (
      • Taira N.
      • Mimoto R.
      • Kurata M.
      • Yamaguchi T.
      • Kitagawa M.
      • Miki Y.
      • Yoshida K.
      DYRK2 priming phosphorylation of c-Jun and c-Myc modulates cell cycle progression in human cancer cells.
      )
      Ser243Possible redundancies with other DYRK family kinases
      p53 (
      • Taira N.
      • Nihira K.
      • Yamaguchi T.
      • Miki Y.
      • Yoshida K.
      DYRK2 is targeted to the nucleus and controls p53 via Ser46 phosphorylation in the apoptotic response to DNA damage.
      ,
      • Taira N.
      • Yamamoto H.
      • Yamaguchi T.
      • Miki Y.
      • Yoshida K.
      ATM augments nuclear stabilization of DYRK2 by inhibiting MDM2 in the apoptotic response to DNA damage.
      )
      Ser46Tumor suppressor; proapoptotic upon genotoxic stressMost tumors are p53 mutated/null and mutated p53 becomes oncogenic; DYRK2 mRNA strongly overexpressed in breast cancer overall.
      CDK14 (
      • Imawari Y.
      • Mimoto R.
      • Hirooka S.
      • Morikawa T.
      • Takeyama H.
      • Yoshida K.
      Downregulation of dual-specificity tyrosine-regulated kinase 2 promotes tumor cell proliferation and invasion by enhancing cyclin-dependent kinase 14 expression in breast cancer.
      )
      n/aTumor suppressor; reduces invasion and proliferationNo specific mechanism reported on how DYRK2 regulates CDK14 transcription.
      LungSIAH2 (
      • Moreno P.
      • Lara-Chica M.
      • Soler-Torronteras R.
      • Caro T.
      • Medina M.
      • Álvarez A.
      • Salvatierra Á.
      • Muñoz E.
      • Calzado M.A.
      The expression of the ubiquitin ligase SIAH2 (seven in Absentia homolog 2) is increased in human lung cancer.
      )
      Ser16, Thr26, Ser28, Ser68, Thr119Tumor suppressor; modulates hypoxia response pathwaysDYRK2 is strongly overexpressed in lung adenocarcinoma; redundancies with other kinases
      OvarianWith EDVP/EDD only (
      • Matsuura K.
      • Huang N.J.
      • Cocce K.
      • Zhang L.
      • Kornbluth S.
      Downregulation of the proapoptotic protein MOAP-1 by the UBR5 ubiquitin ligase and its role in ovarian cancer resistance to cisplatin.
      )
      n/aOncogenic; degrades proapoptotic MOAP1; chemoresistancePhosphorylated substrate (if any) not established.
      SNAIL (
      • Yamaguchi N.
      • Mimoto R.
      • Yanaihara N.
      • Imawari Y.
      • Hirooka S.
      • Okamoto A.
      • Yoshida K.
      DYRK2 regulates epithelial-mesenchymal-transition and chemosensitivity through Snail degradation in ovarian serous adenocarcinoma.
      )
      Ser104Tumor-suppressor; EMT downregulation; chemoresistanceData heavily reliant on ectopic overexpression; possible redundancies with other CMGC kinases; 2 cell lines used only.
      BrainPI3K/AKT/GSK3β (
      • Shen Y.
      • Zhang L.
      • Wang D.
      • Bao Y.
      • Liu C.
      • Xu Z.
      • Huang W.
      • Cheng C.
      Regulation of glioma cells migration by DYRK2.
      )
      n/aTumor-suppressor; EMT downregulationDYRK2 mRNA is strongly overexpressed in glioma; DYRK inhibitors kill glioma cells.
      Unknown (unbiased multiomics) (
      • Wang H.
      • Diaz A.K.
      • Shaw T.I.
      • Li Y.
      • Niu M.
      • Cho J.-H.
      • Paugh B.S.
      • Zhang Y.
      • Sifford J.
      • Bai B.
      • Wu Z.
      • Tan H.
      • Zhou S.
      • Hover L.D.
      • Tillman H.S.
      • et al.
      Deep multiomics profiling of brain tumors identifies signaling networks downstream of cancer driver genes.
      )
      Oncogenic; DYRK2 increased activity and rewired signalingMultiomics data derived from p53 null murine glioma models; no direct mechanism studied.
      Multiple myeloma26S proteasome/RPT3 (
      • Banerjee S.
      • Ji C.
      • Mayfield J.E.
      • Goel A.
      • Xiao J.
      • Dixon J.E.
      • Guo X.
      Ancient drug curcumin impedes 26S proteasome activity by direct inhibition of dual-specificity tyrosine-regulated kinase 2.
      ,
      • Banerjee S.
      • Wei T.
      • Wang J.
      • Lee J.J.
      • Gutierrez H.L.
      • Chapman O.
      • Wiley S.E.
      • Mayfield J.E.
      • Tandon V.
      • Juarez E.F.
      • Chavez L.
      • Liang R.
      • Sah R.L.
      • Costello C.
      • Mesirov J.P.
      • et al.
      Inhibition of dual-specificity tyrosine phosphorylation-regulated kinase 2 perturbs 26S proteasome-addicted neoplastic progression.
      )
      Thr25Oncogenic; regulates proteostasisn/a
      Leukemiap53/c-myc/KLF4 (
      • Park C.S.
      • Lewis A.H.
      • Chen T.J.
      • Bridges C.S.
      • Shen Y.
      • Suppipat K.
      • Puppi M.
      • Tomolonis J.A.
      • Pang P.D.
      • Mistretta T.A.
      • Ma L.Y.
      • Green M.R.
      • Rau R.
      • Lacorazza H.D.
      A KLF4-DYRK2-mediated pathway regulating self-renewal in CML stem cells.
      )
      n/aTumor suppressor; reduces cancer stemnessData specific for chronic myeloid leukemia subtype.
      Liverp53/c-myc (
      • Ito D.
      • Yogosawa S.
      • Mimoto R.
      • Hirooka S.
      • Horiuchi T.
      • Eto K.
      • Yanaga K.
      • Yoshida K.
      Dual-specificity tyrosine-regulated kinase 2 is a suppressor and potential prognostic marker for liver metastasis of colorectal cancer.
      ,
      • Zhang X.
      • Xu P.
      • Ni W.
      • Fan H.
      • Xu J.
      • Chen Y.
      • Huang W.
      • Lu S.
      • Liang L.
      • Liu J.
      • Chen B.
      • Shi W.
      Downregulated DYRK2 expression is associated with poor prognosis and Oxaliplatin resistance in hepatocellular carcinoma.
      ,
      • Yokoyama-Mashima S.
      • Yogosawa S.
      • Kanegae Y.
      • Hirooka S.
      • Yoshida S.
      • Horiuchi T.
      • Ohashi T.
      • Yanaga K.
      • Saruta M.
      • Oikawa T.
      • Yoshida K.
      Forced expression of DYRK2 exerts anti-tumor effects via apoptotic induction in liver cancer.
      )
      n/aTumor suppressor; EMT downregulation; reduces invasion; chemoresistancep53 and c-Myc have extensive oncogenic mutations reported in liver cancer.
      LNC-HC/hsa-miR-183-5p (
      • Lan X.
      • Wu N.
      • Wu L.
      • Qu K.
      • Osoro E.K.
      • Guan D.
      • Du X.
      • Wang B.
      • Chen S.
      • Miao J.
      • Ren J.
      • Liu L.
      • Li H.
      • Ning Q.
      • Li D.
      • et al.
      The human novel gene LNC-HC inhibits hepatocellular carcinoma cell proliferation by sequestering hsa-miR-183-5p.
      )
      Tumor suppressor; transcriptional upregulation of DYRK2Multiple tumor suppressors upregulated including DYRK2; no specific DYRK2 mechanism reported.
      Colorectalp53 (
      • Taira N.
      • Nihira K.
      • Yamaguchi T.
      • Miki Y.
      • Yoshida K.
      DYRK2 is targeted to the nucleus and controls p53 via Ser46 phosphorylation in the apoptotic response to DNA damage.
      )
      Ser46Tumor suppressor; proapoptotic upon genotoxic stressMost colorectal tumors are p53 mutated/null, and mutated p53 becomes oncogenic; DYRK inhibitor promotes p53 phosphorylation.
      DNMT1 (
      • Kumamoto T.
      • Yamada K.
      • Yoshida S.
      • Aoki K.
      • Hirooka S.
      • Eto K.
      • Yanaga K.
      • Yoshida K.
      Impairment of DYRK2 by DNMT1-mediated transcription augments carcinogenesis in human colorectal cancer.
      )
      n/aTumor suppressor; epigenetic downregulation of DYRK2No specific DYRK2 mechanism reported.
      miR-622 (
      • Wang Y.
      • Sun J.
      • Wei X.
      • Luan L.
      • Zeng X.
      • Wang C.
      • Zhao W.
      Decrease of miR-622 expression suppresses migration and invasion by targeting regulation of DYRK2 in colorectal cancer cells.
      )
      Tumor suppressor; EMT downregulation; reduces invasion
      LymphomaCDK2/p27Kip1 (
      • Wang Y.
      • Wu Y.
      • Miao X.
      • Zhu X.
      • Miao X.
      • He Y.
      • Zhong F.
      • Ding L.
      • Liu J.
      • Tang J.
      • Huang Y.
      • Xu X.
      • He S.
      Silencing of DYRK2 increases cell proliferation but reverses CAM-DR in Non-Hodgkin's Lymphoma.
      )
      n/aTumor suppressor; EMT downregulation; chemoresistanceData specific for non-Hodgkin's lymphoma subtype.
      CMGC, Cyclin-dependent kinases, Mitogen-activated protein kinases, Glycogen synthase kinases, and CDC-like kinases; DYRK, Dual-specificity tYrosine phosphorylation–Regulated Kinase; EMT, epithelial–mesenchymal transition; HSF1, heat-shock factor 1; MOAP, modulator of apoptosis protein 1; NOTCH1, neurogenic locus notch homolog protein 1; n/a, not directly reported.
      Also, refer Figure 3.

      c-Myc

      DYRK2 has been reported to exhibit a p53-independent tumor-suppressor role by phosphorylating c-Myc on serine62 (Ser62) (
      • Taira N.
      • Mimoto R.
      • Kurata M.
      • Yamaguchi T.
      • Kitagawa M.
      • Miki Y.
      • Yoshida K.
      DYRK2 priming phosphorylation of c-Jun and c-Myc modulates cell cycle progression in human cancer cells.
      ). c-Myc is a major proto-oncogenic transcription factor known to be overexpressed and mutated in various cancers (
      • Wolf E.
      • Eilers M.
      Targeting MYC proteins for tumor therapy.
      ). Post-translational modifications of c-Myc have been a topic of much debate over the past 30 years in which sequential phosphorylation of Ser62 and Threonine58 (Thr58) seems to play major roles in c-Myc transactivation (
      • Hann S.R.
      Role of post-translational modifications in regulating c-Myc proteolysis, transcriptional activity and biological function.
      ,
      • Lutterbach B.
      • Hann S.R.
      Overexpression of c-Myc and cell immortalization alters c-Myc phosphorylation.
      ). The consensus in the field is that Thr58 is a GSK3-phosphorylation site while Ser62 seems to be the priming site for GSK3 activity, and similar to phosphorylation of p53 at Ser46, various CMGC kinases have been proposed (
      • Hann S.R.
      Role of post-translational modifications in regulating c-Myc proteolysis, transcriptional activity and biological function.
      ), including DYRK2 to phosphorylate c-Myc. Dual phosphorylation of c-Myc on Thr58 and Ser62 triggers binding to an E3 ubiquitin ligase SCF-Fbxw7 (Skp-Cullen-F-box) and consequent proteasomal degradation of c-Myc (
      • Taira N.
      • Mimoto R.
      • Kurata M.
      • Yamaguchi T.
      • Kitagawa M.
      • Miki Y.
      • Yoshida K.
      DYRK2 priming phosphorylation of c-Jun and c-Myc modulates cell cycle progression in human cancer cells.
      ), thus leading to the proposed tumor suppressor role of DYRK2. As stated previously, transcriptional upregulation of DYRK2 in CML promotes c-Myc degradation (
      • Park C.S.
      • Lewis A.H.
      • Chen T.J.
      • Bridges C.S.
      • Shen Y.
      • Suppipat K.
      • Puppi M.
      • Tomolonis J.A.
      • Pang P.D.
      • Mistretta T.A.
      • Ma L.Y.
      • Green M.R.
      • Rau R.
      • Lacorazza H.D.
      A KLF4-DYRK2-mediated pathway regulating self-renewal in CML stem cells.
      ). In Burkitt lymphoma, nearly 60% of patients exhibit mutation of the GSK3 site, Thr58 (
      • Bhatia K.
      • Huppi K.
      • Spangler G.
      • Siwarski D.
      • Iyer R.
      • Magrath I.
      Point mutations in the c–Myc transactivation domain are common in Burkitt's lymphoma and mouse plasmacytomas.
      ), whereas primary cells exhibit lower levels of Thr58 phosphorylation (
      • Lutterbach B.
      • Hann S.R.
      Overexpression of c-Myc and cell immortalization alters c-Myc phosphorylation.
      ), thus suggesting a tumor-suppressor role of GSK3 in this context. However, increased Ser62 phosphorylation has been observed in immortalized cells compared with primary cells (
      • Lutterbach B.
      • Hann S.R.
      Overexpression of c-Myc and cell immortalization alters c-Myc phosphorylation.
      ), and monophosphorylation of Ser62 has been linked to c-Myc stabilization and higher transcriptional activity in multiple studies (
      • Sears R.
      • Nuckolls F.
      • Haura E.
      • Taya Y.
      • Tamai K.
      • Nevins J.R.
      Multiple Ras-dependent phosphorylation pathways regulate Myc protein stability.
      ,
      • Seo H.R.
      • Kim J.
      • Bae S.
      • Soh J.-W.
      • Lee Y.-S.
      Cdk5-mediated phosphorylation of c-Myc on Ser-62 is essential in transcriptional activation of cyclin B1 by cyclin G1.
      ). This indicates a similar conundrum as observed with the p53 Ser46 site wherein multiple kinases and diverse cancer subtypes exhibit altered mechanisms of action of major cancer-associated genes such as p53 and c-Myc.

      c-Jun

      A similar story is observed in case of c-Jun wherein two phosphorylation sites serine249 (Ser249: a bona fide GSK3 site) and Ser243 (reported to be phosphorylated by DYRK2) have been reported (
      • Taira N.
      • Mimoto R.
      • Kurata M.
      • Yamaguchi T.
      • Kitagawa M.
      • Miki Y.
      • Yoshida K.
      DYRK2 priming phosphorylation of c-Jun and c-Myc modulates cell cycle progression in human cancer cells.
      ,
      • Morton S.
      • Davis R.J.
      • McLaren A.
      • Cohen P.
      A reinvestigation of the multisite phosphorylation of the transcription factor c-Jun.
      ). c-Jun is a transcription factor with established oncogenic roles (
      • Morton S.
      • Davis R.J.
      • McLaren A.
      • Cohen P.
      A reinvestigation of the multisite phosphorylation of the transcription factor c-Jun.
      ). Similar to c-Myc, the E3 ubiquitin ligase SCF-Fbxw7 degrades c-Jun upon dual phosphorylation of Ser249 and Ser243 (
      • Wei W.
      • Jin J.
      • Schlisio S.
      • Harper J.W.
      • Kaelin W.G.
      The v-Jun point mutation allows c-Jun to escape GSK3-dependent recognition and destruction by the Fbw7 ubiquitin ligase.
      ). Unlike c-Myc, Ser243 on c-Jun could be a DYRK-specific site because a previous study has elegantly ruled out most of the other CMGC kinase families (
      • Morton S.
      • Davis R.J.
      • McLaren A.
      • Cohen P.
      A reinvestigation of the multisite phosphorylation of the transcription factor c-Jun.
      ). The same study did, however, observe redundancies between DYRK1A and DYRK2 for Ser243 on c-Jun in vitro (
      • Morton S.
      • Davis R.J.
      • McLaren A.
      • Cohen P.
      A reinvestigation of the multisite phosphorylation of the transcription factor c-Jun.
      ), which is not surprising because the site is a +1P. In fact, dephosphorylation of Ser243 enhances c-Jun transcriptional activity in patients with cervical cancer exhibiting lower phosphoSer243 c-Jun in their tumors (
      • Huang C.C.
      • Wang J.M.
      • Kikkawa U.
      • Mukai H.
      • Shen M.R.
      • Morita I.
      • Chen B.K.
      • Chang W.C.
      Calcineurin-mediated dephosphorylation of c-Jun Ser-243 is required for c-Jun protein stability and cell transformation.
      ). Although the question of intra-DYRK redundancy remains, phosphoSer243 on c-Jun could indeed be a tumor-suppressor marker in specific cancers.

      SNAIL

      DYRK2 has also been reported to phosphorylate the zinc finger domain containing protein SNAIL that plays essential roles during development by triggering EMT (
      • Yamaguchi N.
      • Mimoto R.
      • Yanaihara N.
      • Imawari Y.
      • Hirooka S.
      • Okamoto A.
      • Yoshida K.
      DYRK2 regulates epithelial-mesenchymal-transition and chemosensitivity through Snail degradation in ovarian serous adenocarcinoma.
      ,
      • Mimoto R.
      • Taira N.
      • Takahashi H.
      • Yamaguchi T.
      • Okabe M.
      • Uchida K.
      • Miki Y.
      • Yoshida K.
      DYRK2 controls the epithelial-mesenchymal transition in breast cancer by degrading Snail.
      ). DYRK2 knockdown led to upregulation of mesenchymal markers with consequent downregulation of epithelial E-cadherin mRNA in colon cancer (
      • Yan H.
      • Hu K.
      • Wu W.
      • Li Y.
      • Tian H.
      • Chu Z.
      • Koeffler H.P.
      • Yin D.
      Low expression of DYRK2 (dual specificity tyrosine phosphorylation regulated kinase 2) correlates with poor prognosis in colorectal cancer.
      ) and promotion of proliferation and migration of glioma cells (
      • Shen Y.
      • Zhang L.
      • Wang D.
      • Bao Y.
      • Liu C.
      • Xu Z.
      • Huang W.
      • Cheng C.
      Regulation of glioma cells migration by DYRK2.
      ). This observation was consistent with other studies reporting downregulation of DYRK2 in metastatic colorectal secondary tumors found in the liver (
      • Ito D.
      • Yogosawa S.
      • Mimoto R.
      • Hirooka S.
      • Horiuchi T.
      • Eto K.
      • Yanaga K.
      • Yoshida K.
      Dual-specificity tyrosine-regulated kinase 2 is a suppressor and potential prognostic marker for liver metastasis of colorectal cancer.
      ). SNAIL has been reported to be overexpressed in specific cancers and promote oncogenic progression by promoting EMT, invasion, and metastasis (
      • Mimoto R.
      • Taira N.
      • Takahashi H.
      • Yamaguchi T.
      • Okabe M.
      • Uchida K.
      • Miki Y.
      • Yoshida K.
      DYRK2 controls the epithelial-mesenchymal transition in breast cancer by degrading Snail.
      ). DYRK2 phosphorylates serine104 on SNAIL that provides a priming site for GSK3, triggering the phosphorylation-mediated degradation of SNAIL (
      • Yamaguchi N.
      • Mimoto R.
      • Yanaihara N.
      • Imawari Y.
      • Hirooka S.
      • Okamoto A.
      • Yoshida K.
      DYRK2 regulates epithelial-mesenchymal-transition and chemosensitivity through Snail degradation in ovarian serous adenocarcinoma.
      ,
      • Mimoto R.
      • Taira N.
      • Takahashi H.
      • Yamaguchi T.
      • Okabe M.
      • Uchida K.
      • Miki Y.
      • Yoshida K.
      DYRK2 controls the epithelial-mesenchymal transition in breast cancer by degrading Snail.
      ). This mechanism of antitumorigenic activity by DYRK2 is thought to promote chemosensitivity for ovarian cancer cells (
      • Yamaguchi N.
      • Mimoto R.
      • Yanaihara N.
      • Imawari Y.
      • Hirooka S.
      • Okamoto A.
      • Yoshida K.
      DYRK2 regulates epithelial-mesenchymal-transition and chemosensitivity through Snail degradation in ovarian serous adenocarcinoma.
      ). A follow-up study reports that the DYRK2-mediated degradation of SNAIL is in fact reversed by p38α kinase (
      • Ryu K.J.
      • Park S.M.
      • Park S.H.
      • Kim I.K.
      • Han H.
      • Kim H.J.
      • Kim S.H.
      • Hong K.S.
      • Kim H.
      • Kim M.
      • Yoon S.J.
      • Asaithambi K.
      • Lee K.H.
      • Park J.Y.
      • Hah Y.S.
      • et al.
      p38 stabilizes snail by suppressing DYRK2-mediated phosphorylation that is required for GSK3β-βTrCP-induced snail degradation.
      ). Although an interesting molecular mechanism, in both studies, DYRK2 ectopic overexpression has been carried out to justify the phosphorylation. Overexpression of CMGC kinases often leads to nonphysiological false-positive subcellular localizations and substrate identifications because of redundancy and high affinity for +1 P sites and hence further tools need to be used to confirm the DYRK2–SNAIL mechanism.

      SIAH2

      Seven In Absentia Homolog 2 or SIAH2 is an E3 ubiquitin ligase that plays a major role in targeted degradation of various proteins playing essential roles in regulating hypoxia (
      • Ma B.
      • Chen Y.
      • Chen L.
      • Cheng H.
      • Mu C.
      • Li J.
      • Gao R.
      • Zhou C.
      • Cao L.
      • Liu J.
      • Zhu Y.
      • Chen Q.
      • Wu S.
      Hypoxia regulates Hippo signalling through the SIAH2 ubiquitin E3 ligase.
      ). SIAH2 specifically regulates hypoxic tumor microenvironment by downregulation of key kinases in the Hippo signaling pathway (
      • Ma B.
      • Chen Y.
      • Chen L.
      • Cheng H.
      • Mu C.
      • Li J.
      • Gao R.
      • Zhou C.
      • Cao L.
      • Liu J.
      • Zhu Y.
      • Chen Q.
      • Wu S.
      Hypoxia regulates Hippo signalling through the SIAH2 ubiquitin E3 ligase.
      ). Furthermore, higher expression of SIAH2 is observed in lung cancer (
      • Moreno P.
      • Lara-Chica M.
      • Soler-Torronteras R.
      • Caro T.
      • Medina M.
      • Álvarez A.
      • Salvatierra Á.
      • Muñoz E.
      • Calzado M.A.
      The expression of the ubiquitin ligase SIAH2 (seven in Absentia homolog 2) is increased in human lung cancer.
      ) and it plays oncogenic roles in castration-resistant prostate cancer (
      • Qi J.
      • Tripathi M.
      • Mishra R.
      • Sahgal N.
      • Fazli L.
      • Ettinger S.
      • Placzek W.J.
      • Claps G.
      • Chung L.W.
      • Bowtell D.
      • Gleave M.
      • Bhowmick N.
      • Ronai Z.A.
      The E3 ubiquitin ligase Siah2 contributes to castration-resistant prostate cancer by regulation of androgen receptor transcriptional activity.
      ). Interestingly, DYRK2 phosphorylates SIAH2 on 5 residues Ser16, Thr26, Ser28, Ser68, and Thr119. These modifications alter its subcellular localization thereby rewiring SIAH2 substrate specificity (
      • Pérez M.
      • García-Limones C.
      • Zapico I.
      • Marina A.
      • Schmitz M.L.
      • Muñoz E.
      • Calzado M.A.
      Mutual regulation between SIAH2 and DYRK2 controls hypoxic and genotoxic signaling pathways.
      ). SIAH2, in turn, is capable of degrading DYRK2 in specific cancers thereby triggering a protumorigenic hypoxic microenvironment (
      • Pérez M.
      • García-Limones C.
      • Zapico I.
      • Marina A.
      • Schmitz M.L.
      • Muñoz E.
      • Calzado M.A.
      Mutual regulation between SIAH2 and DYRK2 controls hypoxic and genotoxic signaling pathways.
      ,
      • Ma B.
      • Chen Y.
      • Chen L.
      • Cheng H.
      • Mu C.
      • Li J.
      • Gao R.
      • Zhou C.
      • Cao L.
      • Liu J.
      • Zhu Y.
      • Chen Q.
      • Wu S.
      Hypoxia regulates Hippo signalling through the SIAH2 ubiquitin E3 ligase.
      ,
      • Moreno P.
      • Lara-Chica M.
      • Soler-Torronteras R.
      • Caro T.
      • Medina M.
      • Álvarez A.
      • Salvatierra Á.
      • Muñoz E.
      • Calzado M.A.
      The expression of the ubiquitin ligase SIAH2 (seven in Absentia homolog 2) is increased in human lung cancer.
      ). Some kinase redundancy has been observed wherein p38α kinase is capable of phosphorylating SIAH2 on same sites as DYRK2 (
      • Khurana A.
      • Nakayama K.
      • Williams S.
      • Davis R.J.
      • Mustelin T.
      • Ronai Z.e.
      Regulation of the ring finger E3 ligase Siah2 by p38 MAPK.
      ); however, the DYRK2–SIAH2 link points to an interesting interplay between a kinase and a ubiquitin ligase regulating each other and thereby balancing protumorigenic and antitumorigenic roles.

      EDVP E3 ubiquitin ligase

      As stated previously, DYRK2 forms a kinase-independent scaffold for the EDVP E3 ligase complex and a recent study has reported loss-of-function point mutations of DYRK2 in cancer, which largely alters the interactome and substrate specificity of DYRK2 (
      • Mehnert M.
      • Ciuffa R.
      • Frommelt F.
      • Uliana F.
      • van Drogen A.
      • Ruminski K.
      • Gstaiger M.
      • Aebersold R.
      Multi-layered proteomic analyses decode compositional and functional effects of cancer mutations on kinase complexes.
      ). The recurrent mutations (Fig. 1B) are thought to alter activity and/or formation of the EDVP complex (
      • Mehnert M.
      • Ciuffa R.
      • Frommelt F.
      • Uliana F.
      • van Drogen A.
      • Ruminski K.
      • Gstaiger M.
      • Aebersold R.
      Multi-layered proteomic analyses decode compositional and functional effects of cancer mutations on kinase complexes.
      ). As part of the EDVP complex, DYRK2 phosphorylates and triggers degradation of multiple substrates such as katanin p60 (KATNA1) (
      • Maddika S.
      • Chen J.
      Protein kinase DYRK2 is a scaffold that facilitates assembly of an E3 ligase.
      ), telomerase reverse transcriptase (TERT) (
      • Jung H.Y.
      • Wang X.
      • Jun S.
      • Park J.I.
      Dyrk2-associated EDD-DDB1-VprBP E3 ligase inhibits telomerase by TERT degradation.
      ), and centrosome protein 110 (CP110) (
      • Hossain D.
      • Javadi Esfehani Y.
      • Das A.
      • Tsang W.Y.
      Cep78 controls centrosome homeostasis by inhibiting EDD-DYRK2-DDB1(Vpr)(BP).
      ). Phosphorylation-mediated degradation of these substrates are required for proper cell cycle transitions especially the G2/M stage. Cancer mutations could result in incomplete EDVP complex formation, and incomplete EDVP can exhibit oncogenic prosurvival role because DYRK2+EDD alone degrades the proapoptotic factor modulator of apoptosis protein 1 independently of DDB1 and VPRBP in ovarian cancer (
      • Matsuura K.
      • Huang N.J.
      • Cocce K.
      • Zhang L.
      • Kornbluth S.
      Downregulation of the proapoptotic protein MOAP-1 by the UBR5 ubiquitin ligase and its role in ovarian cancer resistance to cisplatin.
      ). The substrates of DYRK2–EDVP exhibit both protumorigenic and antitumorigenic roles in various cancers thus adding further complexity. Ovarian cancer patients with higher levels of KATNA1 exhibit better overall survival (
      • Schiewek J.
      • Schumacher U.
      • Lange T.
      • Joosse S.A.
      • Wikman H.
      • Pantel K.
      • Mikhaylova M.
      • Kneussel M.
      • Linder S.
      • Schmalfeldt B.
      • Oliveira-Ferrer L.
      • Windhorst S.
      Clinical relevance of cytoskeleton associated proteins for ovarian cancer.
      ); higher CP110 can decrease breast cancer cell invasion (
      • Bijnsdorp I.V.
      • Hodzic J.
      • Lagerweij T.
      • Westerman B.
      • Krijgsman O.
      • Broeke J.
      • Verweij F.
      • Nilsson R.J.
      • Rozendaal L.
      • van Beusechem V.W.
      • van Moorselaar J.A.
      • Wurdinger T.
      • Geldof A.A.
      miR-129-3p controls centrosome number in metastatic prostate cancer cells by repressing CP110.
      ), yet lung cancer tissue expresses higher CP110 than the normal lung (
      • Hu S.
      • Danilov A.V.
      • Godek K.
      • Orr B.
      • Tafe L.J.
      • Rodriguez-Canales J.
      • Behrens C.
      • Mino B.
      • Moran C.A.
      • Memoli V.A.
      • Mustachio L.M.
      • Galimberti F.
      • Ravi S.
      • DeCastro A.
      • Lu Y.
      • et al.
      CDK2 inhibition causes anaphase catastrophe in lung cancer through the centrosomal protein CP110.
      ), while TERT is largely oncogenic (
      • Colebatch A.J.
      • Dobrovic A.
      • Cooper W.A.
      TERT gene: Its function and dysregulation in cancer.
      ). Thus, DYRK2–EDVP functions are tumor specific.

      STAT3

      DYRK2 has been reported to phosphorylate signal transducer and activator of transcription 3 (STAT3) in vitro (
      • Matsuo R.
      • Ochiai W.
      • Nakashima K.
      • Taga T.
      A new expression cloning strategy for isolation of substrate-specific kinases by using phosphorylation site-specific antibody.
      ). STAT3 is a transcription factor with both oncogenic and tumor-suppressor roles including regulation of tumor microenvironments (reviewed in Galoczova et al. [
      • Galoczova M.
      • Coates P.
      • Vojtesek B.
      STAT3, stem cells, cancer stem cells and p63.
      ]). STAT3 is phosphorylated on various residues upon interleukin/cytokine stimulation, and the phosphorylation on serine727 (Ser727) is thought to be an oncogenic biomarker in some subtypes of breast cancer (
      • Yeh Y.T.
      • Ou-Yang F.
      • Chen I.F.
      • Yang S.F.
      • Wang Y.Y.
      • Chuang H.Y.
      • Su J.H.
      • Hou M.F.
      • Yuan S.S.
      STAT3 ser727 phosphorylation and its association with negative estrogen receptor status in breast infiltrating ductal carcinoma.
      ). Although Ser727 is thought to promote the transcriptional activity of STAT3 (
      • Galoczova M.
      • Coates P.
      • Vojtesek B.
      STAT3, stem cells, cancer stem cells and p63.
      ), various kinases (CMGC family and beyond) have been reported to target Ser727 which is a +1P site (
      • Matsuo R.
      • Ochiai W.
      • Nakashima K.
      • Taga T.
      A new expression cloning strategy for isolation of substrate-specific kinases by using phosphorylation site-specific antibody.
      ). Thus, it is very difficult to dissect the importance of DYRK2 alone in driving phosphoSer727-mediated STAT3 activity.

      TBK1

      TANK-binding kinase 1 (TBK1) is an important upstream regulator of innate immune transcription pathways triggering type I interferon (IFN) translation and signaling in response to pathogens (
      • An T.
      • Li S.
      • Pan W.
      • Tien P.
      • Zhong B.
      • Shu H.-B.
      • Wu S.
      DYRK2 negatively regulates type I interferon induction by promoting TBK1 degradation via Ser527 phosphorylation.
      ). DYRK2 phosphorylates TBK1 at serine527, which leads to phosphorylation-mediated degradation of TBK1 and downregulation of type I IFN signaling upon viral infection (
      • An T.
      • Li S.
      • Pan W.
      • Tien P.
      • Zhong B.
      • Shu H.-B.
      • Wu S.
      DYRK2 negatively regulates type I interferon induction by promoting TBK1 degradation via Ser527 phosphorylation.
      ). Besides infections, the elevated presence of type I IFN correlates with a favorable prognosis in patients with different cancers (
      • Katlinski K.V.
      • Gui J.
      • Katlinskaya Y.V.
      • Ortiz A.
      • Chakraborty R.
      • Bhattacharya S.
      • Carbone C.J.
      • Beiting D.P.
      • Girondo M.A.
      • Peck A.R.
      • Puré E.
      • Chatterji P.
      • Rustgi A.K.
      • Diehl J.A.
      • Koumenis C.
      • et al.
      Inactivation of interferon receptor promotes the establishment of immune privileged tumor microenvironment.
      ,
      • Zitvogel L.
      • Galluzzi L.
      • Kepp O.
      • Smyth M.J.
      • Kroemer G.
      Type I interferons in anticancer immunity.
      ). In fact, reduced IFN-related gene expression leads to an immunosuppressive tumor microenvironment resulting in immunotherapy resistance in many solid tumors (
      • Zitvogel L.
      • Galluzzi L.
      • Kepp O.
      • Smyth M.J.
      • Kroemer G.
      Type I interferons in anticancer immunity.
      ). Thus, DYRK2-mediated downregulation of IFN signaling could play a major oncogenic role in triggering immunotherapy resistance in various cancers. However, the study reporting DYRK2 as the upstream kinase of TBK1 relies on ectopic overexpression of DYRK2 to demonstrate direct phosphorylation of a canonical +1P motif (
      • An T.
      • Li S.
      • Pan W.
      • Tien P.
      • Zhong B.
      • Shu H.-B.
      • Wu S.
      DYRK2 negatively regulates type I interferon induction by promoting TBK1 degradation via Ser527 phosphorylation.
      ). There could be redundancies with other CMGC kinases at that site which needs to be addressed more thoroughly.

      NOTCH1

      In response to chemotherapeutic agents, DYRK2 facilitates phosphorylation-mediated degradation of neurogenic locus notch homolog protein 1 (NOTCH1), which acts as an antiproliferative mechanism in breast cancer cells (
      • Morrugares R.
      • Correa-Sáez A.
      • Moreno R.
      • Garrido-Rodríguez M.
      • Muñoz E.
      • de la Vega L.
      • Calzado M.A.
      Phosphorylation-dependent regulation of the NOTCH1 intracellular domain by dual-specificity tyrosine-regulated kinase 2.
      ). NOTCH1 is a single transmembrane receptor and triggers intracellular signaling via binding to specific ligands (
      • Morrugares R.
      • Correa-Sáez A.
      • Moreno R.
      • Garrido-Rodríguez M.
      • Muñoz E.
      • de la Vega L.
      • Calzado M.A.
      Phosphorylation-dependent regulation of the NOTCH1 intracellular domain by dual-specificity tyrosine-regulated kinase 2.
      ). DYRK2 phosphorylates NOTCH1 on threonine2512 (Thr2512), which is a +1P site. However, NOTCH1 exhibits both tumor suppressor and oncogenic roles on a cancer-type basis (
      • Dotto G.P.
      Notch tumor suppressor function.
      ). Interestingly, Thr2512 lies in the intracellular carboxy-terminal region of NOTCH1 that exhibits a PEST domain. The PEST region is the target of multiple CMGC kinases such as DYRK1A, HIPK2, CDKs, and GSK3, which triggers hyperphosphorylation and proteasomal degradation of NOTCH1 (reviewed in Lee et al. [
      • Lee H.-J.
      • Kim M.-Y.
      • Park H.-S.
      Phosphorylation-dependent regulation of Notch1 signaling: The fulcrum of Notch1 signaling.
      ]). Thus, the redundancy conundrum remains to be solved to understand the function of NOTCH1's phosphorylation by DYRK2.

      Transcriptional/epigenetic mechanisms

      Besides modulating substrate phosphorylations, transcriptional and epigenetic mechanisms of DYRK2 regulation have also been proposed for some cancers. Specifically, the downregulation of DYRK2's gene expression has been linked to increased stemness in breast cancer (
      • Mimoto R.
      • Imawari Y.
      • Hirooka S.
      • Takeyama H.
      • Yoshida K.
      Impairment of DYRK2 augments stem-like traits by promoting KLF4 expression in breast cancer.
      ) and CML (
      • Park C.S.
      • Lewis A.H.
      • Chen T.J.
      • Bridges C.S.
      • Shen Y.
      • Suppipat K.
      • Puppi M.
      • Tomolonis J.A.
      • Pang P.D.
      • Mistretta T.A.
      • Ma L.Y.
      • Green M.R.
      • Rau R.
      • Lacorazza H.D.
      A KLF4-DYRK2-mediated pathway regulating self-renewal in CML stem cells.
      ) via upregulation of transcription factor Krüppel-like factor 4. DYRK2 expression was also downregulated transcriptionally by DNA methyltransferase 1 in colon cancer (
      • Kumamoto T.
      • Yamada K.
      • Yoshida S.
      • Aoki K.
      • Hirooka S.
      • Eto K.
      • Yanaga K.
      • Yoshida K.
      Impairment of DYRK2 by DNMT1-mediated transcription augments carcinogenesis in human colorectal cancer.
      ). The DYRK2 promoter region exhibited a higher level of methylation in cancer tissues than healthy tissues while treatment of cells with hypomethylating drug 5-azacytidine increased DYRK2 mRNA and protein levels (
      • Kumamoto T.
      • Yamada K.
      • Yoshida S.
      • Aoki K.
      • Hirooka S.
      • Eto K.
      • Yanaga K.
      • Yoshida K.
      Impairment of DYRK2 by DNMT1-mediated transcription augments carcinogenesis in human colorectal cancer.
      ). Furthermore, DYRK2 was reported to downregulate oncogenic miR-622 expression and reverse invasion of cancer cells (
      • Wang Y.
      • Sun J.
      • Wei X.
      • Luan L.
      • Zeng X.
      • Wang C.
      • Zhao W.
      Decrease of miR-622 expression suppresses migration and invasion by targeting regulation of DYRK2 in colorectal cancer cells.
      ), whereas long noncoding RNA long noncoding RNA derived from hepatocytes inhibits the proliferation of liver cancer cells by rescuing the expression of DYRK2 (
      • Lan X.
      • Wu N.
      • Wu L.
      • Qu K.
      • Osoro E.K.
      • Guan D.
      • Du X.
      • Wang B.
      • Chen S.
      • Miao J.
      • Ren J.
      • Liu L.
      • Li H.
      • Ning Q.
      • Li D.
      • et al.
      The human novel gene LNC-HC inhibits hepatocellular carcinoma cell proliferation by sequestering hsa-miR-183-5p.
      ).
      To reiterate, multiple molecular mechanisms have been proposed for DYRK2, and each mechanism is cancer-type or subtype specific (Fig. 3 and Table 1). The controversial role of DYRK2 is best highlighted in breast and lung cancers.

      DYRK2 and breast cancer: a major controversy

      TNBC

      Various studies have focused on the role of DYRK2 in TNBC (
      • Guo X.
      • Wang X.
      • Wang Z.
      • Banerjee S.
      • Yang J.
      • Huang L.
      • Dixon J.E.
      Site-specific proteasome phosphorylation controls cell proliferation and tumorigenesis.
      ,
      • Moreno Dorta R.
      • Banerjee S.
      • Jackson A.
      • Quinn J.
      • Baillie G.
      • Dixon J.E.
      • Dinkova-Kostova A.
      • Edwards J.
      • de la Vega L.
      The stress-responsive kinase DYRK2 activates heat shock factor 1 promoting resistance to proteotoxic stress.
      ,
      • Banerjee S.
      • Ji C.
      • Mayfield J.E.
      • Goel A.
      • Xiao J.
      • Dixon J.E.
      • Guo X.
      Ancient drug curcumin impedes 26S proteasome activity by direct inhibition of dual-specificity tyrosine-regulated kinase 2.
      ,
      • Banerjee S.
      • Wei T.
      • Wang J.
      • Lee J.J.
      • Gutierrez H.L.
      • Chapman O.
      • Wiley S.E.
      • Mayfield J.E.
      • Tandon V.
      • Juarez E.F.
      • Chavez L.
      • Liang R.
      • Sah R.L.
      • Costello C.
      • Mesirov J.P.
      • et al.
      Inhibition of dual-specificity tyrosine phosphorylation-regulated kinase 2 perturbs 26S proteasome-addicted neoplastic progression.
      ). These studies revealed that both mRNA and protein levels of DYRK2 were higher in TNBC tumors than adjacent normal breast tissues (
      • Banerjee S.
      • Wei T.
      • Wang J.
      • Lee J.J.
      • Gutierrez H.L.
      • Chapman O.
      • Wiley S.E.
      • Mayfield J.E.
      • Tandon V.
      • Juarez E.F.
      • Chavez L.
      • Liang R.
      • Sah R.L.
      • Costello C.
      • Mesirov J.P.
      • et al.
      Inhibition of dual-specificity tyrosine phosphorylation-regulated kinase 2 perturbs 26S proteasome-addicted neoplastic progression.
      ). Complementing this information, a recent study with 715 samples of patients with breast cancer have shown that high protein levels of nuclear DYRK2 were associated with significantly reduced cancer survival and a shorter time to recurrence specifically within the TNBC subtype cohort (
      • Moreno Dorta R.
      • Banerjee S.
      • Jackson A.
      • Quinn J.
      • Baillie G.
      • Dixon J.E.
      • Dinkova-Kostova A.
      • Edwards J.
      • de la Vega L.
      The stress-responsive kinase DYRK2 activates heat shock factor 1 promoting resistance to proteotoxic stress.
      ). To test the potential therapeutic value of targeting DYRK2 in TNBC, three studies have compared the ability of parental and DYRK2-deficient TNBC cell lines to produce tumors in vivo (
      • Guo X.
      • Wang X.
      • Wang Z.
      • Banerjee S.
      • Yang J.
      • Huang L.
      • Dixon J.E.
      Site-specific proteasome phosphorylation controls cell proliferation and tumorigenesis.
      ,
      • Banerjee S.
      • Ji C.
      • Mayfield J.E.
      • Goel A.
      • Xiao J.
      • Dixon J.E.
      • Guo X.
      Ancient drug curcumin impedes 26S proteasome activity by direct inhibition of dual-specificity tyrosine-regulated kinase 2.
      ,
      • Banerjee S.
      • Wei T.
      • Wang J.
      • Lee J.J.
      • Gutierrez H.L.
      • Chapman O.
      • Wiley S.E.
      • Mayfield J.E.
      • Tandon V.
      • Juarez E.F.
      • Chavez L.
      • Liang R.
      • Sah R.L.
      • Costello C.
      • Mesirov J.P.
      • et al.
      Inhibition of dual-specificity tyrosine phosphorylation-regulated kinase 2 perturbs 26S proteasome-addicted neoplastic progression.
      ). Crispr/Cas9-mediated DYRK2 deletion in MDA-MB-231 or MDA-MB-468 cells showed that tumors derived from TNBC–DYRK2–deficient cells had significantly slower growth rates and lower tumor burden than those derived from their parental cells. Importantly, two studies have shown that treatment with the DYRK2 inhibitors, curcumin and LDN192960, impaired growth of established TNBC tumors (
      • Banerjee S.
      • Ji C.
      • Mayfield J.E.
      • Goel A.
      • Xiao J.
      • Dixon J.E.
      • Guo X.
      Ancient drug curcumin impedes 26S proteasome activity by direct inhibition of dual-specificity tyrosine-regulated kinase 2.
      ,
      • Banerjee S.
      • Wei T.
      • Wang J.
      • Lee J.J.
      • Gutierrez H.L.
      • Chapman O.
      • Wiley S.E.
      • Mayfield J.E.
      • Tandon V.
      • Juarez E.F.
      • Chavez L.
      • Liang R.
      • Sah R.L.
      • Costello C.
      • Mesirov J.P.
      • et al.
      Inhibition of dual-specificity tyrosine phosphorylation-regulated kinase 2 perturbs 26S proteasome-addicted neoplastic progression.
      ). In contrast with these findings, other studies have used MDA-MB-231–derived xenografts and reported DYRK2 control EMT by degrading SNAIL (
      • Mimoto R.
      • Taira N.
      • Takahashi H.
      • Yamaguchi T.
      • Okabe M.
      • Uchida K.
      • Miki Y.
      • Yoshida K.
      DYRK2 controls the epithelial-mesenchymal transition in breast cancer by degrading Snail.
      ) and promoting transcription factor Krüppel-like factor 4 expression (
      • Mimoto R.
      • Imawari Y.
      • Hirooka S.
      • Takeyama H.
      • Yoshida K.
      Impairment of DYRK2 augments stem-like traits by promoting KLF4 expression in breast cancer.
      ), thereby functioning as a tumor suppressor. Both the studies used a DYRK2 overexpression system to show that higher DYRK2 decreased tumor formation. One study reported that mice xenografted with DYRK2-overexpressing MDA-MB-231 cells showed few metastatic lesions and a prolonged survival compared with those injected with control cells (
      • Mimoto R.
      • Taira N.
      • Takahashi H.
      • Yamaguchi T.
      • Okabe M.
      • Uchida K.
      • Miki Y.
      • Yoshida K.
      DYRK2 controls the epithelial-mesenchymal transition in breast cancer by degrading Snail.
      ). In a second study, the authors compared the number of tumors produced by injecting increasing numbers of MDA-MB-231 cells with or without overexpressed DYRK2 (
      • Mimoto R.
      • Imawari Y.
      • Hirooka S.
      • Takeyama H.
      • Yoshida K.
      Impairment of DYRK2 augments stem-like traits by promoting KLF4 expression in breast cancer.
      ). The authors used a sample size of n = 6 mice per condition and show that the total number of tumors derived from DYRK2-overexpressing cells was marginally lower than controls (
      • Mimoto R.
      • Imawari Y.
      • Hirooka S.
      • Takeyama H.
      • Yoshida K.
      Impairment of DYRK2 augments stem-like traits by promoting KLF4 expression in breast cancer.
      ). This is in sharp contrast to others reporting DYRK2 depletion reduces proliferation and tumor formation potential of MDA-MB-231 cells (
      • Guo X.
      • Wang X.
      • Wang Z.
      • Banerjee S.
      • Yang J.
      • Huang L.
      • Dixon J.E.
      Site-specific proteasome phosphorylation controls cell proliferation and tumorigenesis.
      ,
      • Moreno Dorta R.
      • Banerjee S.
      • Jackson A.
      • Quinn J.
      • Baillie G.
      • Dixon J.E.
      • Dinkova-Kostova A.
      • Edwards J.
      • de la Vega L.
      The stress-responsive kinase DYRK2 activates heat shock factor 1 promoting resistance to proteotoxic stress.
      ,
      • Banerjee S.
      • Ji C.
      • Mayfield J.E.
      • Goel A.
      • Xiao J.
      • Dixon J.E.
      • Guo X.
      Ancient drug curcumin impedes 26S proteasome activity by direct inhibition of dual-specificity tyrosine-regulated kinase 2.
      ,
      • Banerjee S.
      • Wei T.
      • Wang J.
      • Lee J.J.
      • Gutierrez H.L.
      • Chapman O.
      • Wiley S.E.
      • Mayfield J.E.
      • Tandon V.
      • Juarez E.F.
      • Chavez L.
      • Liang R.
      • Sah R.L.
      • Costello C.
      • Mesirov J.P.
      • et al.
      Inhibition of dual-specificity tyrosine phosphorylation-regulated kinase 2 perturbs 26S proteasome-addicted neoplastic progression.
      ,
      • Wu X.
      • Zahari M.S.
      • Ma B.
      • Liu R.
      • Renuse S.
      • Sahasrabuddhe N.A.
      • Chen L.
      • Chaerkady R.
      • Kim M.S.
      • Zhong J.
      • Jelinek C.
      • Barbhuiya M.A.
      • Leal-Rojas P.
      • Yang Y.
      • Kashyap M.K.
      • et al.
      Global phosphotyrosine survey in triple-negative breast cancer reveals activation of multiple tyrosine kinase signaling pathways.
      ). Some of these discrepancies might be due to the differential approaches used (DYRK2 knockdown/KO versus overexpression systems) or due to underpowered sample sizes. Furthermore, a phosphotyrosine proteomics study in TNBC cells reported that DYRK2 was among the top 5 phosphorylated proteins observed in aggressive basal-like TNBC cells (
      • Wu X.
      • Zahari M.S.
      • Ma B.
      • Liu R.
      • Renuse S.
      • Sahasrabuddhe N.A.
      • Chen L.
      • Chaerkady R.
      • Kim M.S.
      • Zhong J.
      • Jelinek C.
      • Barbhuiya M.A.
      • Leal-Rojas P.
      • Yang Y.
      • Kashyap M.K.
      • et al.
      Global phosphotyrosine survey in triple-negative breast cancer reveals activation of multiple tyrosine kinase signaling pathways.
      ). Because there is no evidence of the activation loop tyrosine exhibiting altered stoichiometric phosphorylation, the high levels of phosphorylation observed could be due to higher DYRK2 protein levels. In fact, siRNA knockdown of DYRK2 in basal-like TNBC MDA-MB-231 and HCC1395 cells lead to reduced proliferation, invasion, and colony formation potential of the cells (
      • Wu X.
      • Zahari M.S.
      • Ma B.
      • Liu R.
      • Renuse S.
      • Sahasrabuddhe N.A.
      • Chen L.
      • Chaerkady R.
      • Kim M.S.
      • Zhong J.
      • Jelinek C.
      • Barbhuiya M.A.
      • Leal-Rojas P.
      • Yang Y.
      • Kashyap M.K.
      • et al.
      Global phosphotyrosine survey in triple-negative breast cancer reveals activation of multiple tyrosine kinase signaling pathways.
      ).

      Other breast cancer subtypes

      Multiple studies looking at the role of DYRK2 in breast cancer have used the hormone receptor–positive and HER2-negative MCF7 cell line for xenograft studies. In the main study that supports the tumor-suppressor role of DYRK2 in breast cancer, the group identified DYRK2 as a priming kinase for c-Jun and c-Myc (
      • Taira N.
      • Mimoto R.
      • Kurata M.
      • Yamaguchi T.
      • Kitagawa M.
      • Miki Y.
      • Yoshida K.
      DYRK2 priming phosphorylation of c-Jun and c-Myc modulates cell cycle progression in human cancer cells.
      ). In this study, the authors used a sample size of n = 3 mice per condition and carried out an orthotopic mammary-fat-pad breast cancer xenograft comparing MCF7 control cells and stable DYRK2 knockdown cells to investigate their ability to produce tumors (
      • Taira N.
      • Mimoto R.
      • Kurata M.
      • Yamaguchi T.
      • Kitagawa M.
      • Miki Y.
      • Yoshida K.
      DYRK2 priming phosphorylation of c-Jun and c-Myc modulates cell cycle progression in human cancer cells.
      ). They found that DYRK2 knockdown cells clearly produce bigger tumors. Furthermore, DYRK2 knockdown cells showed higher invasion potential in vivo in an intracardiac injection model (n = 6 mice per condition). The same shRNA DYRK2 depleted cells were used in other studies as well to report the various tumor-suppressor roles of DYRK2 (
      • Mimoto R.
      • Imawari Y.
      • Hirooka S.
      • Takeyama H.
      • Yoshida K.
      Impairment of DYRK2 augments stem-like traits by promoting KLF4 expression in breast cancer.
      ,
      • Kumamoto T.
      • Yamada K.
      • Yoshida S.
      • Aoki K.
      • Yanaga K.
      • Yoshida K.
      DNA methylation of dual-specificity tyrosine-regulated kinase 2 (DYRK2) promoter regulates proliferation of human colorectal cancer.
      ). From the study with 715 samples of patients with breast cancer, no correlation was observed between DYRK2 expression and poor outcome in any of the receptor-positive breast cancer subtypes (
      • Moreno Dorta R.
      • Banerjee S.
      • Jackson A.
      • Quinn J.
      • Baillie G.
      • Dixon J.E.
      • Dinkova-Kostova A.
      • Edwards J.
      • de la Vega L.
      The stress-responsive kinase DYRK2 activates heat shock factor 1 promoting resistance to proteotoxic stress.
      ). However, TCGA data suggest that mRNA expression of DYRK2 is higher in breast invasive carcinoma and that higher DYRK2 expression correlates with poor survival in overall patients with breast cancer (
      • Boni J.
      • Rubio-Perez C.
      • López-Bigas N.
      • Fillat C.
      • de la Luna S.
      The DYRK family of kinases in cancer: Molecular functions and therapeutic opportunities.
      ,
      • Guo X.
      • Wang X.
      • Wang Z.
      • Banerjee S.
      • Yang J.
      • Huang L.
      • Dixon J.E.
      Site-specific proteasome phosphorylation controls cell proliferation and tumorigenesis.
      ,
      • Banerjee S.
      • Wei T.
      • Wang J.
      • Lee J.J.
      • Gutierrez H.L.
      • Chapman O.
      • Wiley S.E.
      • Mayfield J.E.
      • Tandon V.
      • Juarez E.F.
      • Chavez L.
      • Liang R.
      • Sah R.L.
      • Costello C.
      • Mesirov J.P.
      • et al.
      Inhibition of dual-specificity tyrosine phosphorylation-regulated kinase 2 perturbs 26S proteasome-addicted neoplastic progression.
      ). Because mRNA and protein levels sometimes do not correlate, larger analysis looking at DYRK2 protein levels are needed to reach a finite conclusion.
      The best way forward is to generate a conditional lox-cre mouse model for DYRK2 and generate hemizygous/homozygous deletion of DYRK2 in different subtypes of breast cancer genetically engineered mouse models (GEMMs) (
      • Sakamoto K.
      • Schmidt J.W.
      • Wagner K.-U.
      Mouse models of breast cancer.
      ). Comparative tumor growth in the DYRK2 null versus parental GEMM over different subtypes would be a good way of addressing the pending questions on role of DYRK2 in breast cancer.

      DYRK2 in lung cancer: unresolved issues

      In 2003, the chromosome 12 region 12q13-14 was found to be amplified in adenocarcinomas of the lung and esophagus, and one of the resident genes, DYRK2, was significantly overexpressed in tumor samples as compared with normal tissues (
      • Miller C.T.
      • Aggarwal S.
      • Lin T.K.
      • Dagenais S.L.
      • Contreras J.I.
      • Orringer M.B.
      • Glover T.W.
      • Beer D.G.
      • Lin L.
      Amplification and overexpression of the dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2) gene in esophageal and lung adenocarcinomas.
      ). In fact, DYRK2 exhibited the highest mRNA overexpression and highest copy numbers in tumors compared with normal tissue and other genes located in the 12q13-14 chromosomal region, suggesting that the overexpression of DYRK2 is the driving force behind the amplicon (
      • Miller C.T.
      • Aggarwal S.
      • Lin T.K.
      • Dagenais S.L.
      • Contreras J.I.
      • Orringer M.B.
      • Glover T.W.
      • Beer D.G.
      • Lin L.
      Amplification and overexpression of the dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2) gene in esophageal and lung adenocarcinomas.
      ). This is reiterated in the TCGA lung adenocarcinoma and esophageal cancer cohort wherein tumor samples expressed higher DYRK2 mRNA than normal tissue (
      • Boni J.
      • Rubio-Perez C.
      • López-Bigas N.
      • Fillat C.
      • de la Luna S.
      The DYRK family of kinases in cancer: Molecular functions and therapeutic opportunities.
      ). However, two independent studies report that higher protein or mRNA expression of DYRK2 is a favorable marker in pulmonary adenocarcinoma (
      • Yamashita S.
      • Chujo M.
      • Tokuishi K.
      • Anami K.
      • Miyawaki M.
      • Yamamoto S.
      • Kawahara K.
      Expression of dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 2 (DYRK2) can be a favorable prognostic marker in pulmonary adenocarcinoma.
      ) and non–small-cell lung cancer (NSCLC) (
      • Yamashita S.
      • Chujo M.
      • Moroga T.
      • Anami K.
      • Tokuishi K.
      • Miyawaki M.
      • Kawano Y.
      • Takeno S.
      • Yamamoto S.
      • Kawahara K.
      DYRK2 expression may be a predictive marker for chemotherapy in non-small cell lung cancer.
      ). In fact, pulmonary adenocarcinoma patients with higher DYRK2 expression exhibited a substantially higher 5-year survival than the group with lower DYRK2 expression. The higher DYRK2 levels associating with negative lymphatic invasion (
      • Yamashita S.
      • Chujo M.
      • Tokuishi K.
      • Anami K.
      • Miyawaki M.
      • Yamamoto S.
      • Kawahara K.
      Expression of dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 2 (DYRK2) can be a favorable prognostic marker in pulmonary adenocarcinoma.
      ). Although the response rates to chemotherapy between the DYRK2-positive and DYRK2-negative patients were not different, patients with DYRK2+ tumors in recurrent NSCLC were suggested to have better outcome with chemotherapy (
      • Yamashita S.
      • Chujo M.
      • Moroga T.
      • Anami K.
      • Tokuishi K.
      • Miyawaki M.
      • Kawano Y.
      • Takeno S.
      • Yamamoto S.
      • Kawahara K.
      DYRK2 expression may be a predictive marker for chemotherapy in non-small cell lung cancer.
      ). Mechanistically, in lung adenocarcinoma and squamous-cell lung cancer, E3 ubiquitin ligase SIAH2 targets DYRK2 for proteasomal degradation (
      • Moreno P.
      • Lara-Chica M.
      • Soler-Torronteras R.
      • Caro T.
      • Medina M.
      • Álvarez A.
      • Salvatierra Á.
      • Muñoz E.
      • Calzado M.A.
      The expression of the ubiquitin ligase SIAH2 (seven in Absentia homolog 2) is increased in human lung cancer.
      ). SIAH2 protein and mRNA levels were found to be higher in samples of patients with lung cancer and exhibited a negative correlation with DYRK2 expression (
      • Moreno P.
      • Lara-Chica M.
      • Soler-Torronteras R.
      • Caro T.
      • Medina M.
      • Álvarez A.
      • Salvatierra Á.
      • Muñoz E.
      • Calzado M.A.
      The expression of the ubiquitin ligase SIAH2 (seven in Absentia homolog 2) is increased in human lung cancer.
      ). Overall, the exact role of DYRK2 in lung neoplasia is still up for debate. Hence, using a similar strategy as suggested previously to generate conditional DYRK2 depletion in genetically engineered lung cancer mouse models for NSCLC, squamous-cell lung cancer, and other subtypes (
      • Kwon M.-c.
      • Berns A.
      Mouse models for lung cancer.
      ) could provide more clarity to this debate.
      As reported previously, various global unbiased studies in various cancers have reported DYRK2 as a potential cancer driver with increased copy numbers, overexpression, and higher activity (
      • Gorringe K.L.
      • Boussioutas A.
      • Bowtell D.D.
      Novel regions of chromosomal amplification at 6p21, 5p13, and 12q14 in gastric cancer identified by array comparative genomic hybridization.
      ,
      • Miller C.T.
      • Aggarwal S.
      • Lin T.K.
      • Dagenais S.L.
      • Contreras J.I.
      • Orringer M.B.
      • Glover T.W.
      • Beer D.G.
      • Lin L.
      Amplification and overexpression of the dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2) gene in esophageal and lung adenocarcinomas.
      ,
      • Wang H.
      • Diaz A.K.
      • Shaw T.I.
      • Li Y.
      • Niu M.
      • Cho J.-H.
      • Paugh B.S.
      • Zhang Y.
      • Sifford J.
      • Bai B.
      • Wu Z.
      • Tan H.
      • Zhou S.
      • Hover L.D.
      • Tillman H.S.
      • et al.
      Deep multiomics profiling of brain tumors identifies signaling networks downstream of cancer driver genes.
      ). On a similar note, a study using integrated high-resolution microarray analysis of gene copy number and expression in head and neck squamous-cell carcinoma cells reported that DYRK2 had the highest copy number and clear overexpression when compared with other genes in the 12q chromosomal amplicon (
      • Järvinen A.K.
      • Autio R.
      • Haapa-Paananen S.
      • Wolf M.
      • Saarela M.
      • Grénman R.
      • Leivo I.
      • Kallioniemi O.
      • Mäkitie A.A.
      • Monni O.
      Identification of target genes in laryngeal squamous cell carcinoma by high-resolution copy number and gene expression microarray analyses.
      ). Furthermore, transcriptomics of blood identified DYRK2 as 1 of 10 potential prognostic biomarkers elevated in high-grade precancerous cervical lesions (
      • Zou C.
      • Lyu Y.
      • Jiang J.
      • Cao Y.
      • Wang M.
      • Sang C.
      • Zhang R.
      • Li H.
      • Liew C.-C.
      • Cheng C.
      • Zhao S.
      Use of peripheral blood transcriptomic biomarkers to distinguish high-grade cervical squamous intraepithelial lesions from low-grade lesions.
      ). Thus, unbiased identification of DYRK2 as a protein/kinase involved in potential protumorigenic role along with its substrates such as p53, c-Myc, and c-Jun further fuels the need to stratify cancers into subtypes before embarking on DYRK2 molecular studies. This duality of protumorigenic and antitumorigenic roles has been reported for the paralogue DYRK1A as well (
      • Fernández-Martínez P.
      • Zahonero C.
      • Sánchez-Gómez P.
      DYRK1A: The double-edged kinase as a protagonist in cell growth and tumorigenesis.
      ,
      • Laham A.J.
      • Saber-Ayad M.
      • El-Awady R.
      DYRK1A: A down syndrome-related dual protein kinase with a versatile role in tumorigenesis.
      ) (Fig. 3 and Table 1), and hence, there is a clear precedence for such controversial roles in the DYRK family. One way of deconvoluting cancer-type and cell-type functions of a controversial kinase is by generating further tools such as potent and specific small-molecule kinase inhibitors.

      Small-molecule inhibitors of DYRK2

      Over the past three decades, various studies have been carried out to identify small-molecule inhibitors of kinases leading to the development of worldwide clinical trials and highly successful therapeutic targets and treatment options (
      • Cohen P.
      Protein kinases — the major drug targets of the twenty-first century?.
      ,
      • Cohen P.
      • Alessi D.R.
      Kinase drug discovery--what's next in the field?.
      ). For the DYRKs, more than 60 reported small-molecule inhibitors have been published or are available in the public domain. ChEMBL (https://www.ebi.ac.uk/chembl) predicts that there are >1500 potential small molecules that can bind and possibly inhibit DYRK2, including established anticancer drugs sunitinib, erlotinib, afatinib, ruxolitinib, and crizotinib. A significant effort has been focused on development of DYRK1A small-molecule inhibitors because DYRK1A has established roles in neurodegenerative disorders. Consequently, early on the only available DYRK2 inhibitors were those targeting DYRK1A with off-target activity on DYRK2. DYRKs are canonical CMGC kinases and broad-spectrum ATP-competitive kinase inhibitors such as staurosporine and its derivatives inhibit DYRK2 at low nanomolar concentrations (https://www.kinase-screen.mrc.ac.uk/kinase-inhibitors). Although there is a high degree of conservation between the kinase domains of class I and class II DYRKs, structural studies indicated that subtle amino acid substitutions in the hydrophobic inhibitor–docking pocket between DYRK1A and DYRK2 could confer significant degrees of inhibitor specificity (
      • Soundararajan M.
      • Roos A.K.
      • Savitsky P.
      • Filippakopoulos P.
      • Kettenbach A.N.
      • Olsen J.V.
      • Gerber S.A.
      • Eswaran J.
      • Knapp S.
      • Elkins J.M.
      Structures of down syndrome kinases, DYRKs, reveal mechanisms of kinase activation and substrate recognition.
      ). Interestingly, these amino acid substitutions contributed to the development and identification of various class-specific and often isoform-specific inhibitors for the DYRKs. Indeed, compound 5j that exhibited more than 100-fold sensitivity for DYRK1A over DYRK1B has no activity for class 2 DYRKs (
      • Falke H.
      • Chaikuad A.
      • Becker A.
      • Loaëc N.
      • Lozach O.
      • Abu Jhaisha S.
      • Becker W.
      • Jones P.G.
      • Preu L.
      • Baumann K.
      • Knapp S.
      • Meijer L.
      • Kunick C.
      10-Iodo-11H-indolo[3,2-c]quinoline-6-carboxylic acids are selective inhibitors of DYRK1A.
      ). Cocrystallization studies revealed that specific isoleucine to valine replacements in the docking site of curcumin resulted in a larger pocket in the class I DYRKs and thus reduced the shape complementarity to the inhibitor (
      • Banerjee S.
      • Ji C.
      • Mayfield J.E.
      • Goel A.
      • Xiao J.
      • Dixon J.E.
      • Guo X.
      Ancient drug curcumin impedes 26S proteasome activity by direct inhibition of dual-specificity tyrosine-regulated kinase 2.
      ). Similarly, ID-8 an indole derivative exhibits an IC50 of <100 nM for class 1 DYRKs but >10 μM for class 2 DYRKs, suggesting significant room for developing specific inhibitors for the kinases (
      • Bellmaine S.F.
      • Ovchinnikov D.A.
      • Manallack D.T.
      • Cuddy C.E.
      • Elefanty A.G.
      • Stanley E.G.
      • Wolvetang E.J.
      • Williams S.J.
      • Pera M.
      Inhibition of DYRK1A disrupts neural lineage specificationin human pluripotent stem cells.
      ). Similarly, β-carboline derivatives such as harmine or AnnH75 exhibit more in vivo and in vitro potency for class I than class II DYRKs (Table 2). However, the benzimidazole derivatives such as INDY, TG003, and DYR219 exhibit a pan-DYRK activity in vitro and in vivo (Table 2) and have been reported to trigger degradation of DYRK proteins when treated in cells (
      • Sonamoto R.
      • Kii I.
      • Koike Y.
      • Sumida Y.
      • Kato-Sumida T.
      • Okuno Y.
      • Hosoya T.
      • Hagiwara M.
      Identification of a DYRK1A inhibitor that induces degradation of the target kinase using Co-chaperone CDC37 fused with luciferase nanoKAZ.
      ,
      • Velazquez R.
      • Meechoovet B.
      • Ow A.
      • Foley C.
      • Shaw A.
      • Smith B.
      • Oddo S.
      • Hulme C.
      • Dunckley T.
      Chronic Dyrk1 inhibition delays the onset of AD-like pathology in 3xTg-AD mice.
      ). This might explain some of the pronounced in vivo efficacy compared with in vitro observations for DYRK inhibitors wherein prolonged treatment leads to inhibition + degradation of the DYRK target, leading to a significant phenotype. Some promiscuous casein kinase inhibitors derived from benzimidazole potently inhibited DYRK1A and DYRK2 in vitro (
      • Cozza G.
      • Sarno S.
      • Ruzzene M.
      • Girardi C.
      • Orzeszko A.
      • Kazimierczuk Z.
      • Zagotto G.
      • Bonaiuto E.
      • Di Paolo M.L.
      • Pinna L.A.
      Exploiting the repertoire of CK2 inhibitors to target DYRK and PIM kinases.
      ,
      • Pagano M.A.
      • Bain J.
      • Kazimierczuk Z.
      • Sarno S.
      • Ruzzene M.
      • Di Maira G.
      • Elliott M.
      • Orzeszko A.
      • Cozza G.
      • Meggio F.
      • Pinna L.A.
      The selectivity of inhibitors of protein kinase CK2: An update.
      ). Silmitasertib (CX-4945), a potent and selective inhibitor of CK2 (with IC50 of 1 nM in vitro), is an orally bioavailable drug currently in phase 1/2 of clinical trials for cancer (
      • Siddiqui-Jain A.
      • Drygin D.
      • Streiner N.
      • Chua P.
      • Pierre F.
      • O'Brien S.E.
      • Bliesath J.
      • Omori M.
      • Huser N.
      • Ho C.
      • Proffitt C.
      • Schwaebe M.K.
      • Ryckman D.M.
      • Rice W.G.
      • Anderes K.
      CX-4945, an orally bioavailable selective inhibitor of protein kinase CK2, inhibits prosurvival and angiogenic signaling and exhibits antitumor efficacy.
      ). Intriguingly, silmitasertib potently inhibits both class I and II DYRKs (
      • Kim H.
      • Lee K.S.
      • Kim A.K.
      • Choi M.
      • Choi K.
      • Kang M.
      • Chi S.W.
      • Lee M.S.
      • Lee J.S.
      • Lee S.Y.
      • Song W.J.
      • Yu K.
      • Cho S.
      A chemical with proven clinical safety rescues Down-syndrome-related phenotypes in through DYRK1A inhibition.
      ). The group did not report the IC50 for DYRK2; however, DYRK3 IC50 was reported to be 18 nM (
      • Kim H.
      • Lee K.S.
      • Kim A.K.
      • Choi M.
      • Choi K.
      • Kang M.
      • Chi S.W.
      • Lee M.S.
      • Lee J.S.
      • Lee S.Y.
      • Song W.J.
      • Yu K.
      • Cho S.
      A chemical with proven clinical safety rescues Down-syndrome-related phenotypes in through DYRK1A inhibition.
      ). Because the kinase domains of DYRK2 and DYRK3 are >90% similar at the amino acid level, there is a good chance that silmitasertib could indeed be a potent DYRK2 inhibitor as well. Silmitasertib exhibits blood–brain barrier penetrance similar to brain-penetrant DYR219 (
      • Velazquez R.
      • Meechoovet B.
      • Ow A.
      • Foley C.
      • Shaw A.
      • Smith B.
      • Oddo S.
      • Hulme C.
      • Dunckley T.
      Chronic Dyrk1 inhibition delays the onset of AD-like pathology in 3xTg-AD mice.
      ) and SM07883 (
      • Melchior B.
      • Mittapalli G.K.
      • Lai C.
      • Duong-Polk K.
      • Stewart J.
      • Guner B.
      • Hofilena B.
      • Tjitro A.
      • Anderson S.D.
      • Herman D.S.
      • Dellamary L.
      • Swearingen C.J.
      • Sunil K.C.
      • Yazici Y.
      Tau pathology reduction with SM07883, a novel, potent, and selective oral DYRK1A inhibitor: A potential therapeutic for Alzheimer's disease.
      ) and could therefore potentially pharmacologically target the DYRKs in the brain.
      Table 2The published DYRK2 inhibitors currently available to the scientific community
      CompoundStructure%Inhibition/IC50Other kinase targetsReferences
      1. Established potent and selective cell-permeable DYRK2 inhibitors
       LDN19296013 nMHaspin (10 nM); PIM3 (10 nM) PIM1/2; DYRK1A (122 nM) DYRK1B; DYRK3 (<3 nM)(
      • Banerjee S.
      • Wei T.
      • Wang J.
      • Lee J.J.
      • Gutierrez H.L.
      • Chapman O.
      • Wiley S.E.
      • Mayfield J.E.
      • Tandon V.
      • Juarez E.F.
      • Chavez L.
      • Liang R.
      • Sah R.L.
      • Costello C.
      • Mesirov J.P.
      • et al.
      Inhibition of dual-specificity tyrosine phosphorylation-regulated kinase 2 perturbs 26S proteasome-addicted neoplastic progression.
      ,
      • Cuny G.D.
      • Robin M.
      • Ulyanova N.P.
      • Patnaik D.
      • Pique V.
      • Casano G.
      • Liu J.F.
      • Lin X.
      • Xian J.
      • Glicksman M.A.
      • Stein R.L.
      • Higgins J.M.
      Structure-activity relationship study of acridine analogs as haspin and DYRK2 kinase inhibitors.
      ,
      • Cuny G.D.
      • Ulyanova N.P.
      • Patnaik D.
      • Liu J.F.
      • Lin X.
      • Auerbach K.
      • Ray S.S.
      • Xian J.
      • Glicksman M.A.
      • Stein R.L.
      • Higgins J.M.
      Structure-activity relationship study of beta-carboline derivatives as haspin kinase inhibitors.
      )
       GSK626616<1 nMDYRK3 (0.7 nM); DYRK1A/B; CK(
      • Erickson-Miller C.L.
      • Creasy C.
      • Chadderton A.
      • Hopson C.B.
      • Valoret E.I.
      • Gorczyca M.
      • Elefante L.
      • Wojchowski D.M.
      • Chomo M.
      • Fitch D.M.
      • Duffy K.J.
      GSK626616: A DYRK3 inhibitor as a potential new therapy for the treatment of anemia.
      )
       Leucettine L4135 nMCLK1 (15 nM)

      CLK 2

      DYRK1A (40 nM)

      GSK3 (410 nM)
      (
      • Debdab M.
      • Carreaux F.
      • Renault S.
      • Soundararajan M.
      • Fedorov O.
      • Filippakopoulos P.
      • Lozach O.
      • Babault L.
      • Tahtouh T.
      • Baratte B.
      • Ogawa Y.
      • Hagiwara M.
      • Eisenreich A.
      • Rauch U.
      • Knapp S.
      • et al.
      Leucettines, a class of potent inhibitors of cdc2-like kinases and dual specificity, tyrosine phosphorylation regulated kinases derived from the marine sponge leucettamine B: Modulation of alternative pre-RNA splicing.
      )
       EHT 537210.8 nMDYRK1A (0.22 nM)

      DYRK1B (0.28 nM); CLK1 (22.8 nM)

      CLK2 (88.8 nM); DYRK3 (93.2 nM); GSK3alpha (7.44 nM)

      GSK3Beta (221 nM)
      (
      • Coutadeur S.
      • Benyamine H.
      • Delalonde L.
      • de Oliveira C.
      • Leblond B.
      • Foucourt A.
      • Besson T.
      • Casagrande A.-S.
      • Taverne T.
      • Girard A.
      • Pando M.P.
      • Désiré L.
      A novel DYRK1A (Dual specificity tyrosine phosphorylation-regulated kinase 1A) inhibitor for the treatment of Alzheimer's disease: Effect on Tau and amyloid pathologies in vitro.
      )
       EHT 16103.16 nMDYRK1A (0.36 nM)

      DYRK1B (0.59 nM)

      CLK1 (11 nM)

      CLK2 (32.5 nM)

      CLK3 (1420 nM)

      DYRK3 (21.1 nM)

      GSK3 (9.11 nM)
      (
      • Chaikuad A.
      • Diharce J.
      • Schröder M.
      • Foucourt A.
      • Leblond B.
      • Casagrande A.-S.
      • Désiré L.
      • Bonnet P.
      • Knapp S.
      • Besson T.
      An unusual binding model of the methyl 9-Anilinothiazolo[5,4-f] quinazoline-2-carbimidates (EHT 1610 and EHT 5372) confers high selectivity for dual-specificity tyrosine phosphorylation-regulated kinases.
      )
       TG00380% inhibition at 1000 nMDYRK1A (24.01 nM)

      DYRK1B (34.39 nM)

      CLK1/2/3

      DYRK3
      https://www.kinase-screen.mrc.ac.uk/kinase-inhibitors (
      • Coutadeur S.
      • Benyamine H.
      • Delalonde L.
      • de Oliveira C.
      • Leblond B.
      • Foucourt A.
      • Besson T.
      • Casagrande A.-S.
      • Taverne T.
      • Girard A.
      • Pando M.P.
      • Désiré L.
      A novel DYRK1A (Dual specificity tyrosine phosphorylation-regulated kinase 1A) inhibitor for the treatment of Alzheimer's disease: Effect on Tau and amyloid pathologies in vitro.
      )
       INDY27.7 nMDYRK1A (139 nM)

      DYRK1B (69.2 nM)

      CLK1/2/3; DYRK3; CK
      (
      • Ogawa Y.
      • Nonaka Y.
      • Goto T.
      • Ohnishi E.
      • Hiramatsu T.
      • Kii I.
      • Yoshida M.
      • Ikura T.
      • Onogi H.
      • Shibuya H.
      • Hosoya T.
      • Ito N.
      • Hagiwara M.
      Development of a novel selective inhibitor of the Down syndrome-related kinase Dyrk1A.
      ,
      • Kii I.
      • Sumida Y.
      • Goto T.
      • Sonamoto R.
      • Okuno Y.
      • Yoshida S.
      • Kato-Sumida T.
      • Koike Y.
      • Abe M.
      • Nonaka Y.
      • Ikura T.
      • Ito N.
      • Shibuya H.
      • Hosoya T.
      • Hagiwara M.
      Selective inhibition of the kinase DYRK1A by targeting its folding process.
      )
      2. Brain-penetrant DYRK2 inhibitors
       DYR219benzimidazole analog90% inhibition at 10,000 nMDYRK1A (34 nM)

      DYRK1B (29 nM)

      GSK3; CDK5
      (
      • Velazquez R.
      • Meechoovet B.
      • Ow A.
      • Foley C.
      • Shaw A.
      • Smith B.
      • Oddo S.
      • Hulme C.
      • Dunckley T.
      Chronic Dyrk1 inhibition delays the onset of AD-like pathology in 3xTg-AD mice.