Abstract 2816: BUB1 inhibition radiosensitizes triple-negative breast cancer by targeting the DNA-damage repair pathways

Abstract

Triple-negative breast cancer (TNBC) is the most lethal type of breast cancer (BC). TNBC has a substantial risk of locoregional recurrence and is difficult to cure due to absence of molecular targets or intrinsic heterogeneity. As radiotherapy (RT) is crucial in the treatment of BC, there is a need to identify novel molecular targets for increasing the efficacy of radiation therapy. BUB1 (Budding uninhibited by benzimidazoles 1) is a serine/threonine kinase implicated in chromosomal segregation during mitosis. However, it is still uncertain how BUB1 contributes to radioresistance in TNBC. By performing expression analysis (Oncomine), we observed that BUB1 is overexpressed in BC and even more in TNBC, indicating that this overexpression could be a prognostic factor for TNBC. MTT assay in several TNBC cell lines demonstrated that pharmacological (BAY1816032) or genomic (CRISPR) ablation of BUB1 was cytotoxic. By performing clonogenic survival assays, we observed that BUB1 ablation led to clinically meaningful radiosensitization in TNBC cell lines (SUM159, MDA-MB-231, MDA-MB-468) with radiation enhancement ratios ranging from 1.1 - 1.38. In addition, we showed that inhibition of BUB1 kinase function is essential for the radiosensitization phenotype using siRNAs and CRISPR knockouts by depleting endogenous BUB1 and reintroducing wild-type (WT) or kinase-dead (KD) BUB1. We observed significant increase in tumor doubling time in SUM159 xenografts (mammary fat pads in CB17/SCID mice) when BUB1i was combined with RT (DT=29.6 vs 7.9 days). A reduction in Ki67 was observed in these tumors further confirming the effectiveness of combination treatment. The effect of BUB1 depletion on DNA double strand break repair was assessed using γ-H2AX foci formation assay, which revealed sustained increase in γ-H2AX foci following radiation in BUB1i-treated cells. Further, BLRR assays confirmed that BUB1i reduces non-homologous end joining (NHEJ) efficiency. Biochemical analyses showed that BUB1i increases and stabilizes radiation-induced DNAPKcs phosphorylation (S2056). To evaluate the recruitment of DDR proteins to the chromatin following irradiation (IR), soluble nuclear and the chromatin-enriched fractionation was performed which demonstrated that cells pretreated with BUB1i had increased recruitment of DDR proteins to chromatin suggesting that BUB1 may play a role in the initial recruitment of NHEJ proteins to DSBs. Our findings demonstrate that both genetic and pharmacological inhibition of BUB1 sensitizes TNBC cells to RT and that BUB1 mediated radiosensitization may occur through NHEJ pathway. Our results suggest that BUB1 may represent a novel molecular target for radiosensitization in women with TNBC. Citation Format: Sushmitha Sriramulu, Shivani Thoidingjam, Pin Li, Stephen L. Brown, Farzan Siddiqui, Benjamin Movsas, Michael Green, Corey Speers, Shyam Nyati. BUB1 inhibition radiosensitizes triple-negative breast cancer by targeting the DNA-damage repair pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2816.

BUB1 Inhibition Sensitizes TNBC Cell Lines to Chemotherapy and Radiotherapy

“…Molecularly targeted agents can enhance chemoradiation sensitivity [13]. Given BUB1's strong correlation to aggressiveness and different classes of drugs [23] and our observation that BUB1 inhibition sensitizes TNBC to radiation and lung cancers to chemoradiation [27,[42][43][44], we rationalized that combining BUB1 inhibitor would provide strong chemoradiation sensitization in TNBC. Although the effectiveness of BUB1 inhibitor BAY1816032 was evaluated with PARPi, cisplatin and paclitaxel in a prior study [30], the combination with cisplatin resulted in antagonistic effects and BUB1ʹs potential role in improving the efficacy of chemoradiation in TNBC was not assessed.…”

BUB1 inhibition sensitizes lung cancer cell lines to radiotherapy and chemoradiotherapy

“…BUB1 has been recognized as a potential target in radiation sensitization (25), and is reported to play an integral role in DNA damage response to radiation (9, 26–29). Our results established BAY1816032 radiosensitizes NSCLC ( Figures 6A-C ).…”