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Interleukin-2-inducible T-cell Kinase (ITK) Targeting by BMS-509744 Does Not Affect Cell Viability in T-cell Prolymphocytic Leukemia (T-PLL)

  • Sabine Dondorf
    Affiliations
    Laboratory of Lymphocyte Signaling and Oncoproteome, Department I of Internal Medicine, Center for Integrated Oncology (CIO) Köln-Bonn, and Excellence Cluster for Cellular Stress Response and Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany
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  • Alexandra Schrader
    Correspondence
    Email:
    Affiliations
    Laboratory of Lymphocyte Signaling and Oncoproteome, Department I of Internal Medicine, Center for Integrated Oncology (CIO) Köln-Bonn, and Excellence Cluster for Cellular Stress Response and Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany
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  • Marco Herling
    Affiliations
    Laboratory of Lymphocyte Signaling and Oncoproteome, Department I of Internal Medicine, Center for Integrated Oncology (CIO) Köln-Bonn, and Excellence Cluster for Cellular Stress Response and Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany
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Open AccessPublished:April 17, 2015DOI:https://doi.org/10.1074/jbc.L115.644641
      Zhong et al. (
      • Zhong Y.
      • Dong S.
      • Strattan E.
      • Ren L.
      • Butchar J.P.
      • Thornton K.
      • Mishra A.
      • Porcu P.
      • Bradshaw J.M.
      • Bisconte A.
      • Owens T.D.
      • Verner E.
      • Brameld K.A.
      • Funk J.O.
      • Hill R.J.
      • Johnson A.J.
      • Dubovsky J.A.
      Targeting interleukin-2-inducible T-cell kinase (ITK) and resting lymphocyte kinase (RLK) using a novel covalent inhibitor PRN694.
      ) characterize PRN694, a dual inhibitor of ITK and resting lymphocyte kinase (RLK). They demonstrate activity against T-cell receptor (TCR) signaling in T-PLL and suggest a therapeutic application. However, an actual TCR dependence of T-PLL is not established (
      • Herling M.
      • Patel K.A.
      • Teitell M.A.
      • Konopleva M.
      • Ravandi F.
      • Kobayashi R.
      • Jones D.
      High TCL1 expression and intact T-cell receptor signaling define a hyperproliferative subset of T-cell prolymphocytic leukemia.
      ), and cytotoxic consequences of inhibited kinase activation in T-PLL cells by PRN694 (
      • Zhong Y.
      • Dong S.
      • Strattan E.
      • Ren L.
      • Butchar J.P.
      • Thornton K.
      • Mishra A.
      • Porcu P.
      • Bradshaw J.M.
      • Bisconte A.
      • Owens T.D.
      • Verner E.
      • Brameld K.A.
      • Funk J.O.
      • Hill R.J.
      • Johnson A.J.
      • Dubovsky J.A.
      Targeting interleukin-2-inducible T-cell kinase (ITK) and resting lymphocyte kinase (RLK) using a novel covalent inhibitor PRN694.
      ) or various other ITK inhibitors (
      • Vargas L.
      • Hamasy A.
      • Nore B.F.
      • Smith C. I.E.
      Inhibitors of BTK and ITK: state of the new drugs for cancer, autoimmunity and inflammatory diseases.
      ) are not reported.
      In a comparative meta-analysis of expression profiling data across mature T-cell leukemias/lymphomas, we characterize T-PLL as a category with a prominent signature of TCR signaling components, including ITK (
      • Warner K.
      • Weit N.
      • Crispatzu G.
      • Admirand J.
      • Jones D.
      • Herling M.
      Cell receptor signaling in peripheral T-cell lymphoma - a review of patterns of alterations in a central growth regulatory pathway.
      ). However, we describe here the surprisingly low in vitro efficacy of the ITK (not RLK) inhibitor BMS-509744 in T-cell leukemia lines (Fig. 1, A–D) and primary T-PLL cells (Fig. 1E). The assessment of target mRNA levels of 71 T-PLL compared with normal T-cells revealed that ITK was down-regulated; RLK expression was largely unaltered (Fig. 1F).
      Figure thumbnail gr1
      FIGURE 1BMS-509744 does not affect the viability of malignant T-cell lines and primary T-PLL cells. A, HH cells (2 independent experiments) were treated with BMS-509744 for 24 and 48 h. Apoptosis was measured using AnnV/7AAD flow cytometry. HH cells did only respond to BMS-509744 by apoptosis at high (micromolar) concentrations. The percentage of viable cells relative to vehicle control is shown. B, immunoblots. HH cells were treated for 48 h with different dosages of BMS-509744. Although the basal phosphorylation levels of ERK1/2 remain unaffected, the phosphorylation of PLCγ was decreased with higher dosages of BMS-509744. C, Jurkat cells (2 independent experiments) were treated with BMS-509744 for 24 h. Apoptosis was measured using AnnV/7AAD flow cytometry. Jurkat cells did not undergo cell death at marked degrees after BMS-509744 treatment. D, Jurkat cells were treated for 24 h with different dosages of BMS-509744. Comparable to HH cells, the phosphorylation of PLCγ was decreased only with higher dosages of BMS-509744. E, primary T-PLL cells (n = 5 cases) were treated ex vivo with BMS-509744 for 24 and 48 h. Apoptosis was measured using AnnV/7AAD staining by flow cytometry. BMS-509744 did not affect tumor cell viability. F, gene expression levels of ITK (left) and RLK (right) in CD3 positive pan T-cells, enriched from 10 healthy donors, were compared with those in 71 T-PLL cases (>97% tumor cell purity) using HumanHT-12 v4 Expression BeadChip arrays. Log2 expression levels are shown.
      Although we could not perform a head-to-head comparison of both inhibitors, evaluations of the potency of such ITK targeting have to consider the effects of individual drug selectivities (
      • Charrier J.D.
      • Knegtel R. M.A.
      Advances in the design of ITK inhibitors.
      ). The affinity of PRN694 to ITK is reported to be higher than for BMS-509744 (IC50 values: 0.3 nm versus 15 nm) (
      • Charrier J.D.
      • Knegtel R. M.A.
      Advances in the design of ITK inhibitors.
      ). Addressing potential cross-kinase stand-in functions, e.g. of RLK for ITK, might be another beneficial property of PRN694. Moreover, PRN694 targets additional driver kinases of T-PLL, including JAK3 (IC50 = 30 nm) (
      • Kiel M.J.
      • Velusamy T.
      • Rolland D.
      • Sahasrabuddhe A.A.
      • Chung F.
      • Bailey N.G.
      • Schrader A.
      • Li B.
      • Li J.Z.
      • Ozel A.B.
      • Betz B.L.
      • Miranda R.N.
      • Medeiros L.J.
      • Zhao L.
      • Herling M.
      • Lim M.S.
      • Elenitoba-Johnson K. S.J.
      Integrated genomic sequencing reveals mutational landscape of T-cell prolymphocytic leukemia.
      ).
      Overall, the success of BTK inhibition, as in B-cell chronic lymphocytic leukemia, is likely not easily translatable to ITK targeting in T-PLL. However, the limited therapeutic options in the notoriously refractory T-PLL warrant intensified approaches that address the unique biology of the transformed T-cell, i.e. TCR-specific kinases.

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