Abstract 2539: Discovery of degradation pathway for maternal embryonic leucine zipper kinase (MELK): Implications for breast cancer therapy
Authors: Nyati Shyam , Chandler Benjamin , Michmerhuizen Anna , Pesch Andrea , Ritter Cassandra , Moubadder Leah , Liu Meilan , Cameron Meleah , Olsson Eric , Wilder-Romans Kari , Ray Dipankar , Lawrence Theodore S. , Feng Felix Y. , Pierce Lori J. , Speers Corey 
Published: August 2019

Background: Despite previous studies suggesting that MELK is not involved in proliferation in cancer, many groups have shown that MELK is a critical mediator of radioresistance and may be an ideal target for radiosensitization. Furthermore, additional studies suggest MELK functions to regulate the cell-cycle and that its mRNA and protein levels are regulated during cell-cycle, although the mechanisms of this regulation are currently unknown. We sought to identify mediators of MELK protein regulation, determine whether it was ubiquitin-ligase mediated, and if so, which E3-ubiquitin ligase machinery degrades MELK during cell-cycle progression.

Methods: Sequence alignments were carried out to identify degron motifs in MELK. Site directed mutagenesis were performed to mutate degrons in MELK. CDC20 and CDH1 were over-expressed to identify the co-activators of the E3-ubiquitin ligase complex. Cells were synchronized by double thymidine block or by nocodazole. CDC27/APC3 and CDC20 antibodies were used to pull-down the Anaphase Promoting Complex/Cyclosome (APC/C) and Mitotic Checkpoint Complex (MCC), respectively. Resulting pellets were probed with antibodies against BubR1/Mad3, Bub3, CDC20, CDC27, MELK, Cyclin B1, CDK1, BUB1, and Mad2. A role for MELK kinase activity on its half-life and formation of APC/C and MCC was evaluated using kinetically inactive (MELK-KD) mutant or OTSSP167. siRNA against MELK was used to confirm a requirement of MELK for formation of APC/C and MCC. The effect of MELK degron mutant on radiosensitization will be evaluated in MELK CRISPR TNBC cell lines.

Results: In this study we provide evidence that MELK has two D-boxes and a KEN box which are largely conserved from C. elegance to H. sapiens. We identified that MELK is degraded by the E3-ubiquitin ligase APC/C with the coactivator being CDC20. Rescue with MG132 confirms that MELK is degraded by a proteosomal-mediated pathway. CO-IP studies show that MELK is required for APC/C and MCC formation and that MELK interacts with CDC20, CDK1, BUB1, and BUBR1. Cycloheximide chase studies show D-Box1 and KEN box mutants of MELK are more stable while D-BOX2 mutants are least stable. The KEN and D-Box mutant MELK show impairment in APC/C and MCC formation, altered Cyclin B1-CDK1 and Cyclin B1-CDC20 interactions. MELK KD and OTSSP prove that kinase activity of MELK plays a role in its stability. We anticipate that the least stable MELK mutant will result in higher radiosensitivity compared to MELK-WT.

Conclusions: MELK is regulated by APC/CCDC20 through its D-Box1, KEN and D-Box2. Degron mutant and MELK KD show altered protein stability which leads to attenuated APC/C and MCC formation. These results provide proof that MELK is a cell-cycle checkpoint kinase and support the rationale for developing clinical strategies to degrade and inhibit MELK in combination with radiation therapy.

Citation Format: Shyam Nyati, Benjamin Chandler, Anna Michmerhuizen, Andrea Pesch, Cassandra Ritter, Leah Moubadder, Meilan Liu, Meleah Cameron, Eric Olsson, Kari Wilder-Romans, Dipankar Ray, Theodore S. Lawrence, Felix Y. Feng, Lori J. Pierce, Corey Speers. Discovery of degradation pathway for maternal embryonic leucine zipper kinase (MELK): Implications for breast cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2539.