MELK is not necessary for the proliferation of basal-like breast cancer cells

Abstract

Thorough preclinical target validation is essential for the success of drug discovery efforts. In this study, we combined chemical and genetic perturbants, including the development of a novel selective maternal embryonic leucine zipper kinase (MELK) inhibitor HTH-01-091, CRISPR/Cas9-mediated MELK knockout, a novel chemical-induced protein degradation strategy, RNA interference and CRISPR interference to validate MELK as a therapeutic target in basal-like breast cancers (BBC). In common culture conditions, we found that small molecule inhibition, genetic deletion, or acute depletion of MELK did not significantly affect cellular growth. This discrepancy to previous findings illuminated selectivity issues of the widely used MELK inhibitor OTSSP167, and potential off-target effects of MELK-targeting short hairpins. The different genetic and chemical tools developed here allow for the identification and validation of any causal roles MELK may play in cancer biology, which will be required to guide future MELK drug discovery efforts. Furthermore, our study provides a general framework for preclinical target validation.

YY1 Is a Structural Regulator of Enhancer-Promoter Loops

“…We used an inducible degradation system (Erb et al, 2017; Huang et al, 2017; Winter et al, 2015) to fully deplete YY1 protein levels and measured the impact on gene expression in mESCs genome-wide through RNA sequencing (RNA-seq) analysis (Figures 5A and 5B). Depletion of YY1 led to significant (adjusted p value <0.05) changes in expression of 8,234 genes, divided almost equally between genes with increased expression and genes with decreased expression (Figure 5C; Table S2; Table S3).…”

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

“…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.…”

“…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.…”

“…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 We first sought to identify a concentration at which HTH-01-091 inhibited MELK in our cells of interest.…”

“…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.…”