Combining CRISPR/Cas9 mutagenesis and a small-molecule inhibitor to probe the function of MELK in cancer

The Maternal Embryonic Leucine Zipper Kinase (MELK) has been identified as a promising therapeutic target in multiple cancer types. MELK over-expression is associated with aggressive disease, and MELK has been implicated in numerous cancer-related processes, including chemotherapy resistance, stem cell renewal, and tumor growth. On the basis of these findings, a MELK inhibitor is currently being tested in several clinical trials. Here, we report that cancer cell lines harboring CRISPR/Cas9-induced null mutations in MELK exhibit wild-type growth in vitro, under environmental stress, in the presence of multiple chemotherapy agents, and in vivo. By combining our MELK-knockout clones with a recently-described, highly-specific MELK inhibitor, we further demonstrate that the acute inhibition of MELK results in no specific anti-proliferative phenotype. Analysis of gene expression data from cohorts of cancer patients identifies MELK expression as a correlate of tumor mitotic activity, explaining its association with poor clinical prognosis. In total, our results demonstrate the power of CRISPR/Cas9-based genetic approaches to investigate cancer drug targets, and call into question the rationale for treating patients with anti-MELK monotherapies.


Introduction
Cancer cells require the expression of certain genes, called "addictions" or "genetic dependencies", that encode proteins necessary for tumor growth. Silencing the expression of these genes or blocking the activity of the proteins that they encode can trigger cell death and durable tumor regression (1). Identifying and characterizing cancer dependencies is therefore a key goal of pre-clinical cancer research.
Moreover, knockdown of MELK using RNA interference (RNAi) has been reported to block cancer cell proliferation and trigger cell cycle arrest or mitotic catastrophe (3,4,(10)(11)(12)(13). On the basis of these pre-clinical results, several companies have developed small-molecule MELK inhibitors, and one MELK inhibitor (OTS167) is currently being tested in multiple clinical trials (14).
In contrast to these results, we recently reported that triple-negative breast cancer cells harboring CRISPR/Cas9-induced loss of function mutations in MELK proliferate at wild-type levels in vitro (15). Additionally, we demonstrated that the MELK inhibitor OTS167 remained effective against MELK-mutant cells, suggesting that OTS167 kills cells through an off-target mechanism. These results have been replicated by an independent group, who further demonstrated that the shRNA vectors commonly used to study MELK also kill cells in a MELKindependent manner (16). The off-target effects of both the small-molecule MELK inhibitor and the MELK-targeting shRNAs provide a potential explanation for certain previous results obtained studying this potential drug target.
Despite the conflicting in vitro data, MELK expression remains one of the strongest predictors of patient mortality in diverse cancer types (17). Additionally, MELK has been implicated in several other cancer-related processes, including cancer stem cell maintenance, chemotherapy resistance, anchorage-independent growth, and reactive oxygen species (ROS)signaling (4,5,13,(18)(19)(20)(21)(22). These processes may not be challenged by the routine in vitro growth assays that have been performed in MELK-knockout (MELK-KO) cells to date. Several of these studies were conducted using distinct RNAi constructs and small-molecule inhibitors, raising the possibility that they reflect true functions of this kinase. Moreover, the overexpression of MELK has been reported to transform cells, suggesting that in addition to MELK's putative role as a cancer dependency, it may also function as a driver oncogene (4).
While RNA interference is susceptible to off-target interactions that confound experimental interpretation (23,24), CRISPR/Cas9 mutagenesis is also prone to several important limitations. In particular, clonal cell lines harboring Cas9-induced modifications must be expanded from a single cell with a mutation of interest to several million cells. This intense pressure may select for secondary mutations that blunt any anti-proliferative consequences of the original mutation (25). In a therapeutic context, the immediate inhibition of a particular target achieved with a small-molecule drug may induce a more severe phenotype than observed in a CRISPR-modified cell line subjected to evolutionary pressure over the course of days or weeks.
To investigate the role of MELK in cancer-related processes beyond cell proliferation, and to assess the therapeutic potential of immediate MELK inhibition, we performed assays combining CRISPR-knockout cell lines with a recently-described, highly-specific MELK inhibitor (16). In a variety of in vitro and in vivo challenges, we found that cells lacking MELK behave indistinguishably from wild-type cells. Moreover, through a close analysis of gene expression data, we report that MELK levels strongly correlate with mitotic activity in human tumors, suggesting that MELK may function as an indirect proxy for aggressive growth. In total, these results cast doubt on the possibility that MELK-specific inhibition will serve as a useful monotherapy in cancer.

MELK over-expression fails to transform immortalized cell lines
The over-expression of driver oncogenes allows immortalized but non-transformed cell lines to form colonies when grown in soft agar, a phenotype that is tightly linked with in vivo tumorigenicity (26)(27)(28). It has previously been reported that the over-expression of MELK was sufficient to induce anchorage-independent growth in several cell lines, including Rat1 fibroblasts expressing dominant-negative p53 (p53dd) and the human mammary epithelial cell line MCF10A (4). We attempted to replicate these results using Rat1-p53dd and MCF10A cells, as well as the immortalized 3T3 mouse fibroblast cell line. To accomplish this, we stably transduced each cell line with a retroviral vector encoding either the mouse or the human MELK protein. Western blot analysis confirmed that full-length mouse or human MELK was overexpressed in all six cell lines that we generated relative to the level of MELK in vectortransduced control cell lines ( Figure 1A,D). Additionally, as positive controls in this assay, we transduced each cell line with an allele of H-Ras known to function as a strong driver oncogene (H-Ras G12V ) (29), and with an allele of EGFR that weakly transforms cells (EGFR L858R ) (30). We then assessed whether cell lines over-expressing each gene would proliferate when suspended in soft agar. As expected, cells that had been transduced with an empty vector exhibited minimal anchorage-independent growth, while cells transduced with H-Ras G12V or EGFR L858R formed numerous colonies (Figure 1B-C and E-F). However, in multiple independent experiments, we failed to detect an increase in anchorage-independent growth in any of the six cell lines over-expressing either mouse or human MELK. We conclude that, under the conditions tested, the over-expression of MELK fails to transform cells.

MELK is dispensable for growth in vitro and in vivo
We previously showed that MELK was not required for cell division in two triple negative breast cancer cell lines, Cal51 and MDA-MB-231 (15). However, MELK has been reported to 6 support growth in several other cancer types, including colorectal cancer and melanoma (6,10,20). To examine the role of MELK in other cancer types, we used CRISPR/Cas9 to generate multiple MELK-knockout (MELK-KO) clones in A375, a melanoma cell line, and DLD1, a colorectal cancer cell line (Supplemental Figure 1). As controls, we also derived clones of A375 and DLD1 harboring guide RNAs that targeted the non-essential Rosa26 locus. MELK mutagenesis was verified by sequencing the sites targeted by the gRNA, and loss of the MELK protein was verified by western blot (Supplemental Figure 1A-C). MELK-KO melanoma and colorectal cancer clones grew at wild-type levels in vitro, demonstrating that MELK is dispensable for proliferation in these cancer types as well (Supplemental Figure 1D-E). We previously reported that OTS167, a putative MELK inhibitor in clinical trials, kills breast cancer cells in a MELK-independent manner (15). Consistent with these observations, MELK-KO and Rosa26 clones were equally sensitive to OTS167, verifying that this drug kills cells via an offtarget effect across cancer types (Supplemental Figure 1F).
Many genes that are non-essential for cell division in vitro may still play crucial roles in cancer by supporting other processes, including stem-cell renewal, resistance to anoikis, and angiogenesis (31)(32)(33)(34). We therefore subjected our MELK-KO and Rosa26 clones to various in vitro and in vivo assays to assess whether MELK loss impairs any cancer-related phenotypes.
Although all MELK-KO cells grow well when seeded at high density in proliferation assays (15, 16), plating cells at low density can challenge a cell's colony-forming ability and replicative lifespan (35). To test whether MELK loss confers a defect in colony growth, MELK-KO and Rosa26 DLD1, A375, Cal51, and MDA-MB-231 clones were serially-diluted and allowed to grow at varying cell densities. Crystal violet staining of these plates revealed that the loss of MELK failed to impair colony growth relative to the control Rosa26 cell lines (Figure 2A). In fact, one MELK-KO clone (MDA-MB-231 c1) grew consistently better than either control clone in this assay. Significant variation in proliferative capacity has previously been described among 7 independent clones of the MDA-MB-231 cell line (36), and may arise due to heterogeneity in the parental population or secondary mutations acquired during cell line derivation.
To further investigate the impact of MELK loss on anoikis and tumorigenicity, MELK-KO and Rosa26 cells were plated in soft agar. However, we observed no significant difference in anchorage-independent growth between MELK-KO cells and Rosa26 cells in every cell line tested ( Figure 2B-C). MELK has previously been implicated in the maintenance of breast cancer stem cells (5,21,37). We therefore tested whether loss of MELK impairs mammosphere formation, a phenotype tightly linked with breast cancer stem cell activity (38). While we observed inter-clonal variability in mammosphere growth, all MELK-KO clones were capable of forming mammospheres, and two MELK-KO clones exhibited consistently greater mammosphere formation than their wild-type controls ( Figure 2D-E). We conclude that MELK is dispensable for growth as a mammosphere.
Finally, we sought to determine whether MELK was required for tumor formation in vivo.

MELK is dispensable for cell growth under exogenous stress
Developing tumors must survive in hypoxic and nutrient-poor conditions (43), and MELK has been implicated in glucose signaling and in the detection of ROS (19,44). We therefore considered the possibility that MELK expression is necessary to support growth under metabolic or environmental stress. To generate ROS stress, we cultured cells in varying concentrations of H 2 O 2 , but we observed no difference in sensitivity between MELK-KO and control Rosa26 clones ( Figure 3A). MELK has been suggested to contribute to ROS signaling by phosphorylating ASK1 (44); however, this protein remained phosphorylated at normal levels in cells lacking MELK (Supplemental Figure 2F). MELK-KO clones also exhibited wild-type levels of growth when cultured under hypoxic, serum-deprived, or glucose-limited conditions ( Figure   3B-D). We conclude that MELK is dispensable for proliferation under common metabolic stresses.
As high MELK expression is associated with poor patient prognosis, we wondered if MELK could promote resistance to cytotoxic chemotherapies. Indeed, it has been previously reported that knocking down or inhibiting MELK sensitizes cells to DNA damage (13,18,20,37). To test whether MELK has a role in chemotherapy resistance, we performed drug sensitivity assays using a variety of DNA-damaging or anti-mitotic agents. However, we observed no significant difference in viability between MELK-KO and Rosa26 cell lines treated 9 with five common chemotherapies ( Figure 3E-I). In total, these results demonstrate that MELK is dispensable for cell survival under metabolic and cytotoxic stress.

Acute inhibition of MELK fails to block proliferation
Deriving CRISPR-knockout clones from single cells selects for a population of cells that are capable of surviving clonal expansion. Since our MELK-KO cell lines were generated from single cells, we considered the possibility that MELK plays an important role supporting proliferation, but the clones we generated had evolved to tolerate the loss of MELK. To assess this possibility, we performed an epistasis experiment combining our MELK-knockout clones with a recently-described, highly-specific small molecule MELK inhibitor, HTH-01-091 (16). We reasoned that treating Rosa26 clones with HTH-01-091 would reveal the consequences of the acute loss of MELK. However, if such phenotype(s) were also present in MELK-KO cell lines treated with HTH-01-091, then the phenotype(s) could be attributed to an off-target effect of the drug.
We first sought to identify a concentration at which HTH-01-091 inhibited MELK in our cells of interest. However, as described in this manuscript, we lacked a verified MELK substrate whose phosphorylation status could be monitored to confirm MELK inhibition. Nonetheless, it has been reported that a by-product of MELK inhibition is the degradation of MELK protein (13,16). Therefore, to determine an effective concentration of HTH-01-091, we monitored the level of MELK protein after drug treatment by western blot. We found that 1µM HTH-01-091 triggered near-complete MELK degradation in MDA-MB-231, DLD1, and Cal51 cells ( Figure 4A). The loss of MELK was not an indirect effect of cell cycle arrest, as these cells maintained high levels of the mitotic marker cyclin B. This concentration was therefore used in subsequent assays.
To test whether the acute inhibition of MELK affected clonogenicity or anchorageindependent growth, we grew MELK-KO and Rosa26 cells on plastic or in soft agar in the presence of DMSO or 1µM HTH-01-091. Neither MELK-KO nor Rosa26 clones were affected by HTH-01-091 treatment, verifying that MELK is dispensable for colony formation and anchorageindependent growth in these cells ( Figure 4B-C). Indeed, a drug sensitivity assay revealed that HTH-01-091 exhibited significant anti-proliferative effects only at concentrations above ~5µM ( Figure 4D). This toxicity is likely a consequence of an off-target effect, as these drug concentrations were found to affect Rosa26 and MELK-KO cells equivalently.
We next sought to test whether acute MELK inhibition sensitized cells to chemotherapy.
To accomplish this, we treated MELK-KO and Rosa26 clones with various chemotherapy drugs in the presence of HTH-01-091. Consistent with our previous results, HTH-01-091 treatment failed to sensitize the Rosa26 clones to 5-florouracil or paclitaxel treatment ( Figure 4E). Finally, we assessed the effect of HTH-01-091 treatment on eIF4B phosphorylation and MCL1 expression, and found that MELK inhibition failed to affect either target (Supplemental Figure   2G-H). In total, these results demonstrate that the acute loss of MELK results in no significant defect in cell viability, proliferation, or drug resistance, and suggest that our knockout clones have not acquired mutations that tolerize cells to the loss of MELK.

The association between MELK expression and cancer lethality is due to its correlation with mitotic activity
Our in vitro and in vivo experiments failed to reveal any cancer-related phenotypes affected by either the deletion or over-expression of MELK. Yet, MELK is up-regulated in many cancer types (10), and high levels of MELK expression have been reported to confer a dismal clinical prognosis (4,(7)(8)(9). If MELK plays no overt role in cancer biology, then why would MELK expression be linked with death from cancer? We note that MELK expression is cell cycleregulated, peaking in mitosis (4,45), and gene signatures that capture mitotic activity have been found to be prognostic in multiple cancer types (17,46,47). We therefore considered the possibility that, rather than functioning as an oncogene or a cancer dependency, MELK expression could report cell division within a tumor. To assess the link between MELK expression and cell proliferation, we analyzed gene expression data from different sets of cells and tissues. In normal human tissue, MELK expression was the lowest in non-proliferative organs, including the heart and skeletal muscle, while MELK expression was the highest in organs with on-going mitotic activity, including the bone marrow and testes ( Figure 5A). Indeed, across 32 tissue types, MELK levels were highly correlated with the expression of the proliferation marker MKI67 (R = 0.93). Similarly, in human fibroblasts cultured until senescence, MELK expression decreased up to 11-fold between proliferating and arrested populations, while stimulating lymphocytes to divide increased MELK expression 20-fold ( Figure 5B-C). These data suggest that MELK levels reflect mitotic activity in diverse cell types.
To examine the link between MELK and cell division in cancer, we compared the levels of MELK expression with 5 well-characterized proliferation markers: MKI67, PCNA, CCNB1, MCM2, and TOP2A (48). In cohorts of patients with tumor types in which MELK levels are associated with advanced disease, MELK expression was significantly correlated with each of the proliferation genes (median correlation = 0.82; Figure 5D). We then sought to determine whether the correlation between MELK expression and proliferation could explain the prognostic significance of MELK. To test this, we collected 15 breast cancer microarray datasets from patients with known clinical outcomes. We calculated Z scores for univariate Cox models for each patient cohort, and found that MELK expression was associated with poor outcome in 14 of 15 datasets (Table S3). Similarly, a proliferation meta-gene derived by combining MKI67, PCNA, CCNB1, MCM2, and TOP2A was prognostic in 13 of 15 datasets (Table S3). Strikingly, we found that the Z scores generated by our univariate proliferation model were highly correlated with the Z scores generated by the univariate MELK model ( Figure 5E; R = 0.78, P < .001), suggesting that these two models capture very similar clinical information. To confirm this, we constructed bivariate Cox models that considered both MELK expression and the proliferation meta-gene (Table S4). In the bivariate models, MELK was significantly associated with patient outcome in only 2 of 15 datasets, demonstrating that considering tumor cell 12 proliferation ablated its clinical utility ( Figure 5E). Thus, when tumors are stratified according to their proliferation level, MELK expression is no longer prognostic ( Figure 5F). In total, these results suggest that the observed pattern of MELK expression in cancer can be explained by the fact that MELK is up-regulated in rapidly-dividing cells.

Discussion
Pre-clinical cancer research efforts apply different genetic and chemical tools (RNA interference, CRISPR, and small-molecule inhibitors) to a variety of artificial assays (in vitro proliferation, xenograft growth, etc.) in order to discover targets that will have clinical efficacy when inhibited in human patients. No single assay perfectly mimics the behavior of a tumor in a human cancer patient, and no single chemical or genetic tool exhibits absolute specificity.
Nonetheless, we believe that by using multiple orthogonal approaches and assays, we can gain insight into the role that certain genes play in human malignancies. In this current manuscript, we report that combining CRISPR and a small-molecule inhibitor in a variety of assays failed to reveal a role for MELK in several cancer-related processes. These results suggest that anti-MELK monotherapies are unlikely to be effective cancer treatments.
Our previous work demonstrated that MELK is dispensable for the proliferation of triplenegative breast cancer cells in vitro (15). Nonetheless, multiple cellular functions are dispensable for in vitro proliferation but required for tumor progression, including stem cell renewal, oxygen sensing, and chemotherapy resistance (31)(32)(33)(34). Though MELK has been implicated in each of these processes, our results demonstrate that MELK-knockout cancer cell lines grow at wild-type levels in a variety of assays designed to test these pathways. We speculate that, as has previously been reported for one MELK inhibitor and one set of MELKtargeting shRNA's (16), several previous studies of MELK function may have been compromised by off-target activity of the constructs and inhibitors that were used.
To assess MELK function in cancer, we combined clonal cell lines harboring null mutations in MELK with a specific MELK inhibitor. Of note, we observed some variability between clonal lines, underscoring the importance of assessing multiple independent clones in CRISPR experiments. To rule out the possibility that our MELK-KO clones had evolved to tolerate the loss of MELK, we performed "epistasis" experiments by treating these knockout clones with a MELK inhibitor. We reasoned that specific consequences of MELK inhibition 14 would be detectable upon drug treatment in MELK-WT but not MELK-KO clones, while nonspecific consequences of drug treatment would affect both genotypes equally. In all experiments conducted thus far, no phenotypes have been observed only in MELK-WT cells after HTH-01-091 treatment, further verifying that MELK is dispensable for cancer cell growth. We suggest that these "epistasis" experiments can be widely applied to assess the on-target consequences of acutely inhibiting potential cancer drug targets.
Initial interest in blocking MELK function in cancer stemmed from the discovery that it was over-expressed across cancer types (10). Further research revealed that patients whose tumors expressed the highest levels of MELK had the worst clinical outcomes (4, 7-9, 11). As we observed no role for MELK as either an oncogene or a cancer dependency, we sought to instead investigate whether its cell cycle-dependent expression pattern could explain its prognostic value. Consistent with this hypothesis, we discovered that MELK expression closely mirrors cell and tumor mitotic activity. As actively-growing tumors appear to up-regulate thousands of genes involved in cell cycle progression when compared to quiescent normal tissue (48), this observation may explain why MELK is commonly over-expressed in cancer.
Moreover, rapid cell division in tumors is indicative of tissue de-differentiation and aggressive disease; therefore, most cell cycle-regulated genes are also associated with clinical outcome (17,46,47). We found that controlling for cell proliferation ablates the significance of MELK expression, suggesting that this link may explain its prognostic role in cancer.
While many cell cycle genes are indeed suitable cancer drug targets (e.g., CDK4 and CDK6), other genes may be up-regulated during normal cell division but dispensable for this process (46). We suggest that MELK is an example of the latter. We believe that, if MELK does play a role in cancer, it may be detectable only in very limited circumstances, and likely in vivo.

Soft agar assays
To assay anchorage-independent growth, all cell lines except MCF10A were suspended at a cell count of 10,000 cells in a 0.35% Difco Agar Noble (VWR Scientific; Cat. No. 90000-772) solution in a six-well plate. MCF10A cell lines were suspended at a cell count of 20,000 cells.
The mixture was plated over a 0.5% Difco Agar Noble solution. Plates were allowed to solidify at room temperature for 2 hours and then placed in a 37˚C incubator overnight. 1 mL of normal growth media was added to each well the next day and every 3 days after (50). After 14 days, colony formation was quantified under 20x magnification.

CRISPR plasmid construction and virus generation
Guide RNAs were previously described in Lin et al. (15). In short, oligonucleotides were cloned into the LRG 2.1 vector [a gift from Jun-Wei Shi (University of Pennsylvania) and Chris Vakoc (Cold Spring Harbor Laboratory)] using a BsmBI digestion (52). To produce virus, HEK293T cells were transfected using the calcium-phosphate method. Supernatant was harvested 48 to 72 hours post-transfection, filtered through a 0.45μm syringe, and then applied to cells with 4μg/mL polybrene (49). Guide RNAs in each clone are listed in Table S1.

Analysis of CRISPR-mediated mutagenesis
Single cells isolated via fluorescence-activated cell sorting (FACS) were grown into clonal populations. Genomic DNA was extracted from these populations with the QIAmp DNA Mini kit (Qiagen; Cat. No. 51304). The cut site regions targeted by the guide RNAs were amplified using the primers listed in Table S2. PCR products were then sequenced with the forward and reverse primer at the Cold Spring Harbor Laboratory sequencing facility.

Analysis of published gene expression data
Data from normal human tissue was acquired from (53). Data from senescent fibroblasts was acquired from (54). Data from stimulated lymphocytes was acquired from (55). Cancer patient cohorts and probeset definitions were downloaded from the Gene Expression Omnibus as described in Table S3 (56). Probesets annotated to the same gene were collapsed by averaging. Data were cleaned and processed using python's pandas library, and cox proportional hazard models were constructed using the survival library in R and as described in (17). Several additional python packages were used for clarity, conciseness, and correctness 21 including numpy, getopt and rpy2. Source code is available on github (https://github.com/joansmith/survival-analysis-scripts).            Expression MELK expression in normal tissue