Maternal Embryonic Leucine Zipper Kinase (MELK) Reduces Replication Stress in Glioblastoma Cells*

Background: Protein kinase MELK is expressed at very high levels in glioblastomas, but it is not understood how this benefits tumor growth. Results: A deficiency of MELK causes replication stress and is associated with cell cycle arrest and senescence. Conclusion: MELK is required for progression through unperturbed S phase. Significance: The inhibition of MELK emerges as an attractive cancer therapy. Maternal embryonic leucine zipper kinase (MELK) belongs to the subfamily of AMP-activated Ser/Thr protein kinases. The expression of MELK is very high in glioblastoma-type brain tumors, but it is not clear how this contributes to tumor growth. Here we show that the siRNA-mediated loss of MELK in U87 MG glioblastoma cells causes a G1/S phase cell cycle arrest accompanied by cell death or a senescence-like phenotype that can be rescued by the expression of siRNA-resistant MELK. This cell cycle arrest is mediated by an increased expression of p21WAF1/CIP1, an inhibitor of cyclin-dependent kinases, and is associated with the hypophosphorylation of the retinoblastoma protein and the down-regulation of E2F target genes. The increased expression of p21 can be explained by the consecutive activation of ATM (ataxia telangiectasia mutated), Chk2, and p53. Intriguingly, the activation of p53 in MELK-deficient cells is not due to an increased stability of p53 but stems from the loss of MDMX (mouse double minute-X), an inhibitor of p53 transactivation. The activation of the ATM-Chk2 pathway in MELK-deficient cells is associated with the accumulation of DNA double-strand breaks during replication, as demonstrated by the appearance of γH2AX foci. Replication stress in these cells is also illustrated by an increased number of stalled replication forks and a reduced fork progression speed. Our data indicate that glioblastoma cells have elevated MELK protein levels to better cope with replication stress during unperturbed S phase. Hence, MELK inhibitors hold great potential for the treatment of glioblastomas as such or in combination with DNA-damaging therapies.

T-loop phosphorylation by protein kinase LKB1 (1). Instead, MELK is activated by autophosphorylation (2), but it is not known what triggers this autoactivation. In addition, MELK is regulated through phosphorylation by cyclin-dependent kinases (Cdks) and MAP kinases (3). It has also been established that the level of MELK is strictly controlled. The MELK-encoding gene is an E2F target that is induced in S phase (4). The concentration of MELK reaches a maximum in the G 2 /M phases of the cell cycle but drops again at the mitotic exit (5).
Because it is not known how the activity of MELK can be acutely modulated in intact cells, most functional studies have relied on knockdown or overexpression approaches. These studies have implicated MELK in various cellular processes, including (a)symmetric cell division (6 -10), transcription (11), pre-mRNA splicing (12), DNA repair (13), and apoptosis (14 -15). However, with some exceptions (15)(16)(17), the relevant MELK substrates are unknown, and a unifying hypothesis on the biological function of MELK is missing. It can be argued that at least some of the cellular effects of the loss or overexpression of MELK are indirect and stem from the activation of checkpoints, such as the replication, intra-S phase, or spindleassembly checkpoint, that result in a cell cycle arrest or apoptosis (18,19). A further complication is that at least some of the reported outcomes of a knockdown of MELK might result from siRNA off-target effects. 3 MELK is overexpressed in various cancers, including glioblastomas, which are notoriously resistant to chemo-and radiotherapy (8, 20 -22). The high expression level of MELK clearly gives a proliferative advantage to tumor cells. This has been explained by the ability of MELK to inhibit apoptosis (14) or regulate p53 (17). In addition, MELK has been implicated in DNA damage response pathways (13,23). Consistent with this notion, an elevated MELK protein level increases the resistance * This work was supported by National Science Foundation Flanders Grant to DNA-damaging treatments (24). MELK has also been suggested to contribute to cell cycle progression, but, remarkably, both the knockdown and overexpression of MELK were reported to cause a G 2 /M arrest (20). Other data are indicative for a role of MELK in G 1 /S transition (8).
We have re-examined the role of MELK in cell cycle progression using U87 MG glioblastoma cells and strictly controlled MELK knockdown and rescue experiments. Our results disclose a key role for MELK in progression through unperturbed S phase and show that MELK is needed to cope with replication stress, in particular DNA double-strand breaks. Thus, MELK has potential as a therapeutic target for the treatment of glioblastomas.
Transfections were done using a commercial kit (Jet-Prime, Polyplus), according to the instructions of the manufacturer. Briefly, for a knockdown/rescue experiment, 600 l of Jet-Prime buffer (1ϫ), 20 l of Jet-Prime reagent, 3.5 g of vector-DNA construct, and 6 l of siRNA (from 20 M stock) was used per 15-cm plate.
For FACS analysis, cells were synchronized by overnight serum deprivation or a double thymidine block (2 mM thymidine for 16 h, 7-h release, 2 mM thymidine for 16 h). Next, cells were washed twice with PBS, released in complete medium, and harvested at the indicated time points. After fixation in 70% ethanol, cells were stained with propidium iodide (50 g/ml, Sigma) as explained by Shapiro (25). DNA content was analyzed by flow cytometric analysis (FAC-SCanto), and quantification was performed by using the FACSDiva software.
BrdU incorporation assays were performed as described in Ref. 26. Briefly, 3 h after release from a thymidine block, the cells were pulsed for 1 h with 10 M BrdU. After trypsinization and fixation in 70% ethanol, the lysates were denatured in 2 M HCl and neutralized with 0.1 M sodium borate. Following staining with the primary (M0744, Dako) and secondary (A21202, Invitrogen) antibodies, DNA staining with propidium iodide was performed, and fluorescence was measured for green (BrdU) or red (propidium iodide) signal by FACSCanto and quantified by FACSDiva software.
Trypan blue exclusion viability tests were performed as described in Ref. 29. Briefly, the trypsinized cell suspensions were washed and resuspended in PBS. Trypan blue (0.4%) was added to the cell suspensions (1:1) and incubated at room temperature for 3 min. A drop of the cell suspensions was applied to a hemocytometer and counted. The calculations were done as described (29).
Immunofluorescence Microscopy-Cells were grown on glass coverslips in 24-well plates. The cells were washed with PBS (pH 7.2-7.4) and fixed using the following steps: 15 min in 1.5 M HCl/4% paraformaldehyde/PBS at room temperature, rinsing three times in PBS, 15 min at Ϫ20°C in 100% methanol, and 10 min at room temperature in PBS/0.5% Nonidet P-40. Subsequently, the samples were blocked for 1 h at room temperature in PBS/1% BSA and incubated for 1 h at room temperature with the primary antibodies, namely BrdU (1:300, DakoCytomation M0744) or H2AX-S139ph (1:500, Upstate, catalog no. 05-636). Following incubation for 1 h at room temperature with the secondary antibodies, DAPI staining was performed, and coverslips were mounted in MowOil onto the microscope slides.
Single Cell DNA Fiber Analysis-Following a double thymidine block and a release for 2 h, the cells were sequentially labeled with 25 M IdU and 250 M CldU for 30 min each. DNA fiber spreads were prepared as described previously, with minor modifications (30). Briefly, after trypsinization, the cells were resuspended in PBS at ϳ1 ϫ 10 6 cells/ml, spotted onto a microscope slide (3 l), and lysed with 7 l of lysis buffer containing 200 mM Tris-HCl at pH 7.5, 50 mM EDTA, and 0.5% SDS. After 2-5 min, the slides were tilted 15°to allow lysates to slowly move down the slide. The resulting DNA spreads were air-dried and fixed for 10 min in 3:1 methanol/acetic acid. The slides were then treated with 2.5 M HCl for 80 min, washed, and stored in PBS (1ϫ) at 4°C overnight. For the stainings, the slides were first incubated for 30 min at room temperature in a humid chamber in blocking buffer (5% BSA/PBS) and then for 60 min with 1:400 rat anti-BrdU antibody (Abcam, catalog no. 6326, to detect CldU) plus 1:25 mouse anti-BrdU (BD Biosciences, catalog no. 347580, to detect IdU). After washing in PBS, the samples were blocked for 20 min in blocking buffer and then incubated in the secondary antibody solution (1:400, Alexa Fluor 488 goat anti-rat and 1:500, Alexa Fluor 555 donkey antimouse, Invitrogen) for 1 h. Slides were mounted in MowOil, and microscopy was carried out using a Leica SPE confocal unit on DMI 4000B. The fiber lengths were measured using Leica (Las AF Lite) software. The fork progression rate and the frequency of the stalled forks were calculated as explained previously (31,32).
Statistics-The results are presented as mean Ϯ S.E. for at least three independent experiments. The statistical significance between the control and the experimental groups was tested using Student's t test.

A loss of MELK Causes a Cell Cycle Delay and Senescence-
To delineate the contribution of MELK to cell cycle progression in non-synchronized U87 glioblastoma cells, we first performed a FACS analysis 48 h after the RNAi-mediated knockdown of MELK with 2 different siRNAs (Fig. 1A). The loss of MELK protein was associated with a significantly increased number of cells in G 1 and a corresponding decrease in the S plus G 2 /M phases (Fig. 1B). However, the overexpression of MELK had no significant effect. Consistent with a delay in cell cycle progression, the knockdown of MELK for 7 days also showed a reduced proliferation rate, as detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays ( Fig. 1, C and D). Moreover, a prolonged knockdown of MELK eventually resulted in an enlarged and flattened cell morphology, and the accumulation of the cell senescence marker ␤-galactosidase ( Fig. 1, E and F). Also, the number of viable cells, as measured by trypan blue exclusion, was reduced significantly 96 h after transfection of the siRNA (Fig. 1G). Similar data were obtained with MCF-7 breast carcinoma cells (data not shown). Thus, a loss of MELK results in a G 1 /S delay that, during subsequent cell cycles, gradually culminates in a cell cycle arrest that results in either cell death or a senescence-like phenotype.
The cell cycle delay associated with the loss of MELK can be due to a deficiency in either G 1 or early S phase. Because MELK is an established E2F target and shows a dramatically increased expression at the G 1 /S transition (4), we reasoned that MELK is likely to play a role in the G 1 /S transition or in early S phase. To explore this hypothesis, U87 cells were first synchronized in early S phase with a thymidine block and then released for 0 -4 h. A FACS analysis did not show an effect of a MELK knockdown at the beginning of the release but revealed a significantly delayed progression through S phase after 4 h (Fig. 2, A and B). Consistent with this notion, the number of BrdU-positive cells was decreased after a knockdown of MELK (Fig. 2, A and C). This effect was rescued by the expression of a siRNA-resistant FLAG-tagged version of MELK, attesting to the specificity of the effect. The ectopic expression of a kinase-dead mutant of MELK (MELK-D150A) had no significant effect on cell cycle progression in U87 cells, indicating that it did not act as a dominant negative mutant. (Fig. 1, A-C). The knockdown of endogenous MELK and ectopic expression of the tagged versions of MELK was verified by immunoblotting (Fig. 2D).
Exploration of the Upstream Signaling Pathways-Cell cycle arrests are often mediated by the stress-induced expression of inhibitors of cyclin-dependent protein kinases. One of these Cdk-inhibitory proteins is p21 WAF1/CIP1 , which inhibits the G 1 /S transition, the progression through S phase, as well as the G 2 /M transition (33). The up-regulation of p21 in response to stress conditions, such as DNA damage, is also known to induce senescence (33,34). We found that the knockdown of MELK in U87 cells resulted in an increased expression of p21 (Fig. 3, A  and B). This effect was obtained with three different siRNAs (Fig. 3C) and was rescued by the ectopic expression of siRNA-resistant EGFP-tagged MELK (A and B). The increased expression of p21 in U87 cells correlated with the hypophosphorylation of the retinoblastoma protein (Rb) at Ser-807/811 (Ser-807/811ph). Rb phosphorylation by G 1 and S phase Cdks serves to disrupt its inhibitory interaction with E2F-type transcription factors, which promote the expression of genes that are important for S phase (33). As expected from the presence of hypophosphorylated Rb in MELK-deficient cells, the level of E2F-regulated Cdk2 and its associated cyclins (cyclins A and E) was decreased in this condition (Fig. 3, A and B). These effects could be rescued by the expression of siRNA-resistant MELK.
We have subsequently investigated whether the increased expression of p21 after the knockdown of MELK is due to the phosphorylation and activation of the transcription factor p53, a key regulator of p21 transcription (34). For that purpose, we compared the effects of a knockdown of MELK in U87 and U251 glioblastoma cells, which express wild-type p53 or an inactive but stable p53 mutant (35), respectively. No significant up-regulation of p21 was detected in MELK-deficient U251 cells (Fig. 4, A and B), hinting at an essential role of p53 in the up-regulation of p21. In accordance with this interpretation, a knockdown of Melk also resulted in an increased level of p21 in p53-positive MCF7 cells but not in p53-negative HeLa cells (Fig. 4, C and D).
The activation of p53 as a transcription factor is often correlated with a phosphorylation at Ser-15, which prevents its MDM2-mediated ubiquitination and degradation (36). How-ever, we did not see a significant effect of a MELK knockdown on the phosphorylation of p53 at Ser-15 (Fig. 5, A and B). Neither was the level of p53 affected. Thus, the increased expression of p21 in MELK-deficient cells is p53-dependent but cannot be explained by an increased steady-state level of p53 or an increased phosphorylation at Ser-15.
To get more insights into the mechanism of the p53 dependence of the increased p21 expression, we examined the effects of a knockdown of MELK on the level of MDMX, an established p53 inhibitor that acts by binding to the transactivation domain of p53 (37). MDMX was down-regulated in MELK-deficient cells, and this effect could be rescued by expression of siRNA-resistant MELK (Fig. 5, A and B). Moreover, the 14-3-3 protein, which promotes the degradation of MDMX (38), was significantly up-regulated after the knockdown of MELK (Fig.  5, A and B). These data indicate that the increased level of p21 in MELK-deficient cells can, at least partially, be explained by the activation of p53 because of a loss of the inhibitor MDMX. This is in agreement with a study in MCF7 cells showing that the depletion of MDMX increases the expression of p21 without a significant increase in p53 levels (39).
MDMX is down-regulated through phosphorylation by protein kinase Chk2, resulting in its ubiquitination and subsequent degradation (34,40). Moreover, 14-3-3 collaborates with Chk2 to stimulate MDMX ubiquitination (40). These data prompted us to examine whether a loss of MELK is associated with an activation of Chk2. Indeed, we found that the knockdown of MELK was associated with a significant increase of the priming phosphorylation of Chk2 at Thr-68 (Fig. 5, C and D), which leads to oligomerization, autophosphorylation, and activation of kinase activity (41). Moreover, the inhibition of the Chk2 kinases ATM/ATR with CGK733 (42) prevented both the phosphorylation of Chk2 and the accumulation of p21 in MELK-deficient cells. Also, an accumulation of p21 in MELK-  deficient cells was not detected after the prior knockdown of Chk2 (Fig. 5, E and F). Collectively, these data suggest that the accumulation of p21 in MELK-deficient cells can be explained by the consecutive activation of ATM/ATR, Chk2, and p53. In general, Chk2 is activated by ATM, whereas ATR phosphorylates and activates Chk1. However, the knockdown of MELK did not cause the activatory phosphorylation of Chk1 at Ser-317 (Fig. 5, G and H), indicating that the loss of MELK is associated with the selective activation of the ATM/Chk2 pathway.
The Depletion of MELK Induces Persistent DNA Doublestrand Breaks in Unperturbed S Phase-Various types of DNA damage, including double-strand breaks (DSBs), are a common feature of unperturbed replication (43). DSBs trigger the activation of the ATM-Chk2 pathway. One of the earliest responses to DSBs is the Chk2-mediated phosphorylation of the histone variant H2AX at Ser-139 (␥H2AX), which accumulates at DSBs and triggers the recruitment of DNA repair proteins (43).
Because a loss of MELK is associated with an activation of Chk2, we examined whether this activation is correlated with an accumulation of ␥H2AX. The knockdown of MELK in U87 cells that were arrested in G 0 by serum starvation did not affect the number of cells with ␥H2AX foci (Fig. 6, A and B). However, a MELK knockdown significantly increased the formation of foci in asynchronized cells (Fig. 6, C and D) and in cells that were synchronized in S phase by a double thymidine block (Fig. 6, E and F), and this effect was rescued by the expression of siRNA-resistant EGFP-MELK. A simultaneous staining for BrdU incorporation confirmed that the increased accumulation of ␥H2AX was only detected in replicating cells. Fig. 6G shows the quantification and statistics of the ␥H2AX data.
A Loss of MELK Results in the Accumulation of Stalled Replication Forks-To define the nature of the damage that results in the accumulation of ␥H2AX, we monitored the progression of individual replication forks by the successive labeling of newly synthesized DNA with IdU and CldU, as visualized by red and green immunofluorescence analysis, respectively. The sequential incorporation of the two nucleotide analogues is required for the correct identification of firing sites and direction of their progression (30,31,44). U87 cells were first synchronized with a double thymidine block and then released for 2 h (Fig. 7A). Subsequently, DNA synthesis was followed for two consecutive periods of 30 min each in the presence of CldU and IdU, respectively. The knockdown of MELK resulted in a 30% decrease of the average length of the fibers that were labeled with CldU, reflecting an average decreased fork progression speed (Fig. 7, B and C). This effect was rescued by the expression of siRNA-resistant MELK. Because fork velocities vary over a wide range of values (31,44), we have also presented the distribution of frequency of fork velocities in a histogram (Fig.  7D). These data revealed that the loss of MELK causes a shift in the distribution of the frequencies, with a dramatic reduction in the number of fast-progressing forks and an increased number of slowly progressing forks.
A reduced fork velocity can be due to a slowdown of DNA polymerase or to an increased number of (transiently) blocked forks. When the progression of DNA polymerase is reduced, the two forks emanating from a single origin move more slowly but bidirectionally with the same speed, forming symmetric replication bubbles. In contrast, fork blocks result in the accumulation of asymmetrical forks (30,31,44).We found that the knockdown of MELK was associated with a significantly increased frequency (Ϸ 30%) of stalled forks, an effect that was rescued by the ectopic expression of siRNA-resistant MELK (Fig. 7, E and F). This increased incidence of stalled forks cannot be explained by mechanically broken DNA fibers because for the quantitation assays we considered only forks with the two halogenated labels.
MELK Levels Are Increased during Replication Stress-The above data indicate that MELK may be required to cope with replication stress. Accordingly, MELK expression levels were increased markedly after a preincubation with the topoisomerase I inhibitor camptothecin (45) (Fig. 8A), the replication inhibitor hydroxyurea (46) (B), or the DNA-damaging agent bleomycin (47) (C).

A Loss of MELK Induces a p21-mediated Cell Cycle Arrest-
The role of MELK in cell cycle progression is understood poorly, and the literature on this topic is rather confusing. Some of the previous studies may have led to erroneous conclusions because they were performed on the basis of improperly controlled siRNA experiments. We noted that 2 of 5 tested MELK siRNAs caused off-target effects (not shown), which prompted us to adopt a rather rigorous approach and to include a rescue condition with ectopically expressed siRNA-resistant MELK in all critical experiments. This has enabled us to demonstrate unequivocally that a deficiency of MELK in U87 glioblastoma or MCF-7 breast carcinoma cells is associated with a delayed transition through G 1 -S. In accordance with recent data from Gu et al. (48), this delay was correlated with an increased expression of p21 WAF1/CIP1 , which mainly acts by inhibiting S phase Cdks, i.e. cyclin E-Cdk2 and cyclin A-Cdk2 (33). The increased level of p21 can also explain why MELK-deficient cells show a decreased proliferation rate and, eventually, develop a cell cycle arrest and a senescence-like phenotype (34). The expression of p21 is regulated by p53, which binds to p53 response elements in the promoter of p21 and enhances transcription (34). The induction of p21 in MELK-deficient cells is, indeed, p53-dependent because it was absent in cells that lack functional p53. Under basal conditions, the level of p53 is kept low because it is degraded rapidly following ubiquitination by the associated ubiquitin ligase, MDM2. In specific stress conditions, p53 is stabilized through phosphorylation at Ser-15, which prevents the recruitment of MDM2 (49,50). However, the steady-state levels of p53 and its phosphorylation at Ser-15 in U87 cells were not affected by the knockdown of MELK, suggesting that p53 is activated in this condition by a distinct mechanism. Indeed, we found that a loss of MELK correlated with a decreased level of the p53 inhibitor MDMX and the activation of Chk2, which phosphorylates MDMX and targets it for MDM2-mediated degradation. In addition, the knockdown of MELK resulted in the up-regulation of the p53 target 14-3-3, which is well known to promote the ubiquitination and degradation of MDMX (37). Chk2 knockdown experiments confirmed that this kinase is required for the induction of p21 in MELK-deficient cells. Moreover, the inhibition of the upstream Chk2 kinase ATM prevented both the activation of Chk2 and the induction of p21. In contrast, a deficiency of MELK was not associated with a hyperphosphorylation of Chk1, indicating that the ATR/Chk1 pathway is not activated. Collectively, these data suggest that the induction of p21 in MELK-deficient cells stems from the consecutive activation of ATM, Chk2, and p53. The increased level of p21 inhibits Cdks, which explains the hypophosphorylation of Rb and the decreased expression of E2F targets (Fig. 9).
MELK Is Required for the Repair of DSBs in Unperturbed Replication-The ATM-Chk2 pathway is activated by DNA double-strand breaks that recruit proteins, including ␥H2AX, that are needed for repair by homologous recombination or nonhomologous end joining. The resulting repair foci can be visualized with phospho-epitope-specific ␥H2AX antibodies. Double-strand breaks can be generated by extrinsic factors (e.g. ionizing radiation) but often also represent a key step in repairing replication forks that collapse during an unperturbed S phase. DNA damage, including DSBs, activate the intra-S phase checkpoint, which delays S phase progression until the damage is repaired. Our data suggest that the repair of DSBs during unperturbed S phase is hampered in MELK-deficient cells, as illustrated by the activation of the ATM/Chk2 pathway and the accumulation of ␥H2AX foci. This deficient repair is associated with the accumulation of slow or stalled replication forks, hinting at the activation of the intra-S phase checkpoint. However, MELK-deficient cells can still progress to G 2 /M and even the next G 1 , indicating that the induced replication stress is relatively mild. The progression to the next G 1 is not unexpected, given that the G 2 /M checkpoint is "imperfect" and can allow mitosis without complete repair (51,52).
The exact role of MELK in the DSB repair pathway remains elusive. We speculate that MELK is implicated in the recruitment of repair proteins or the (non)-homologous repair mechanism itself. In addition, it is possible that MELK is not involved in the repair process per se but is a key factor in setting the signaling threshold for the repair of DSBs. In any case, our findings are consistent with previous data that identified MELK as a critical player of the DNA damage response pathway (13,23).
Our data are also in accordance with the well established correlation between the increased expression of MELK in glioblastoma cells and their increased resistance to ionizing radiation (24,53).
In conclusion, MELK is emerging as a key regulator of DSB repair both in unperturbed S phase (this work) and after the extrinsic induction of DSBs (24,53). This function may be very important for cancer cells, in particular glioblastoma cells, which are under constant replication stress and have adapted by increasing the expression of MELK. Our data suggest that FIGURE 7. MELK affects replication fork dynamics. A, schematic adopted to study replication fork dynamics in U87 cells. O/N, overnight. B, visualization of replication fork progression in U87 cells 48 h after transfection with the indicated plasmids. Shown are representative confocal images of signals derived from the successive pulse labeling with IdU (red) and CldU (green). Measurements were made with Leica (Las Af Lite) software. C, quantitation of the fork progression rate in the indicated conditions, expressed as kb/min. At least 500 fibers were counted for each condition in three independent experiments. The results are expressed as mean Ϯ S.E. **, p Ͻ 0.001. D, distribution of the fork rates among the fiber populations in the indicated conditions. At least 500 fibers were counted for each condition in three independent experiments. The results are expressed as mean Ϯ S.E. E, confocal images showing stalled forks, identified as asymmetrical signals within a replication unit. Fork arrest events are indicated with white arrows. F, quantitation of stalled forks (ratio of stalled forks/number of ongoing forks ϫ 100). At least 70 replication units were counted for each condition in three independent experiments. The results are expressed as mean Ϯ S.E. *, p Ͻ 0.05. FIGURE 8. MELK is up-regulated during replication stress. A, thymidine-blocked U87 cells were released for 1 h and subsequently incubated for an additional hour without (-) or with (ϩ) 10 M camptothecin (CPT). Shown are immunoblot analyses of the lysates. B, same as in A but incubation without (-) or with (ϩ) 1 mM hydroxyurea (HU) for 1 h. C, asynchronous U87 cells were incubated without (-) or with (ϩ) bleomycin (10 g/ml) for 2 h. Shown are immunoblot analyses of the lysates. FIGURE 9. Model of the signaling pathway that is activated in response to a knockdown of MELK. A loss of MELK results in the accumulation of doublestrand breaks during S phase. This leads to the activation of the ATM-Chk2-p53 pathway, which causes a cell cycle delay through p21-mediated inhibition of S phase Cdks and, eventually, a cell cycle arrest and a senescence-like phenotype.
MELK is an attractive target for a targeted therapy to treat glioblastoma multiforme. Notably, the targeting of MELK with small-molecule inhibitors could be used to sensitize glioblastoma tumors or metastasis to DNA-damaging agents or radiation therapy. The recent reports that the thiazole antibiotic Siomycin A, which reduces the expression level of MELK, inhibits glioblastoma tumor growth in vivo when combined with radiotherapy or chemotherapy (treatment with the alkylating agent Temozolomide) fully supports this notion (53,54).