Abstract 3218: Maternal embryonic leucine zipper kinase (MELK) confers radioresistance in triple-negative breast cancers (TNBC) through a nonhomologous end joining (NHEJ)-mediated pathway

Abstract

Abstract Background: Increased rates of locoregional recurrence have been observed in TNBC despite the use of radiation (RT); therefore, approaches that result in radiosensitizaton in TNBC may improve clinical outcomes. Despite some controversy as to the role of MELK kinase as an oncogene, we previously established its clear role in conferring radioresistance in TNBC. Here we extend those studies to understand the mechanism of conferred radioresistance. Methods: Clonogenic survival assays were used to quantify the degree of radiosensitivity after MELK inhibition. Mass spectrometry was utilized to identify proteins associated with MELK. NHEJ assays were performed using linearized pEYFP vector and quantitated by FACS. qRT-PCR was used to determine MELK RNA expression. Rad51 and γH2AX foci counting were performed to evaluate homologous recombination (HR). Neutral comet assay was used to quantitate dsDNA break repair. Laser microirradiation studies were performed to determine the location and kinetics of protein shuttle to and from sites of dsDNA breaks, including MELK, Ku70 and Ku80 proteins. Fluorescent microscopy was performed for localization of endogenous MELK protein post RT. To study the effect of MELK kinase activity on Ku70-Ku80 complex formation and recruitment at the DNA damage site, cells were lysed and immunoprecipitation was performed using MELK, Ku70 or Ku80-specific antibodies. Results: We previously demonstrated that genomic or pharmacologic inhibition of MELK confers significant radiosensitization. Mechanistic studies were undertaken to understand the pathways critical to MELK-mediated radioresistance. When MELK is genomically or pharmacologically inhibited, NHEJ and HR reporter assays demonstrate that this radiosensitization is driven through impaired NHEJ at 4, 16, and 24 hrs after RT and not mediated by HR in multiple TNBC cell lines. Tandem mass spectrometry studies of tagged MELK protein identify Ku70 and Ku80 as direct interactors with MELK protein, which is confirmed by Co-IP. Laser microirradiation studies confirm that MELK, Ku70 and Ku80 co-localize to sites of dsDNA breaks, and that MELK kinase function is required to stabilize the Ku70/Ku80 complex at these sites. Comet assay confirms that rapid dissolution of the Ku70/Ku80 complex when MELK is inhibited or functionally dead is not a result of accelerated dsDNA break repair. Conclusion: MELK kinase function is critical in conferring radioresistance in TNBC and inhibition of function confers radiosensitivity through an NHEJ-mediated pathway. MELK also stabilizes Ku70/Ku80 proteins at sites of dsDNA breaks and allows for more efficient repair of breaks induced by ionizing RT. These results further support the rationale for developing clinical strategies to inhibit MELK in combination with RT treatment as a novel radiosensitizing strategy in TNBC. Citation Format: Shyam Nyati, Ben Chandler, Eric Olsen, Leah Moubadder, Meilan Liu, Meleah Cameron, Kari Wilder-Romans, Theodore S. Lawrence, Powel H. Brown, Fellix Y. Fang, Lori J. Pierce, Corey Speers. Maternal embryonic leucine zipper kinase (MELK) confers radioresistance in triple-negative breast cancers (TNBC) through a nonhomologous end joining (NHEJ)-mediated pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3218.