Investigating molecular targets of the DNA damage response in triple negative breast cancer

Abstract

Triple negative breast cancer (TNBC) represents 10-20% of all breast cancers and is defined by the absence of estrogen receptors, progesterone receptors and absence of human epidermal growth factor receptor 2 amplification. TNBC is the most aggressive of the subtypes with the poorest prognosis and highest rates of recurrence within 5 years of diagnosis. The standard of care treatment for TNBC is systemic chemotherapy. The development of chemotherapy resistance is a common problem, with relapse rates up to 38% with less than 6 months median survival. Relapse is often associated with aggressive metastatic disease that no longer responds to chemotherapy.RAD51 recombinase is an evolutionarily conserved protein that plays a critical role the homologous recombination (HR) pathway, which faithfully repairs DNA double-strand breaks and damaged replication forks. In normal cells RAD51 expression is tightly regulated and its activity promotes high fidelity repair and genome integrity. However, dysregulation and overexpression of RAD51 is reported in many human malignancies, including TNBC, and is implicated in resistance to DNA damaging radiotherapy, chemotherapy and PARP inhibition and the promotion of tumour progression and metastasis. Wiegmans et al. (2014) identified that RAD51 is required for spontaneous metastasis in TNBC and that RAD51 expression level differentially regulates the expression of several CEBPβ target genes implicated in tumour progression and metastasis. Furthermore, co-immunoprecipitation identified that RAD51 and CEBPβ interact in situ. To elucidate a possible new non-canonical role for RAD51 in transcriptional regulation we set out to gene edit RAD51 with the aim of generating TNBC model cell lines expressing; (1) HR deficient, RAD51 K133R knock-in mutation, (2) RAD51 knockout and (3) CEBPβ knockout. This was a novel approach as RAD51 is required for CRISPR-Cas9 editing and there are no published studies of RAD51 gene editing. RAD51 knock-out was initially successful in MDA-MB-231 cells but could not be stably maintained by cells in culture, likely due to RAD51 being a core fitness gene in this cell line. CEBPβ was successfully knocked out in MDA-MB-231 cells, however difficulty culturing cells from a single cell or at low density prevented isolation of pure clones with CEBPβ knock-out in other TNBC cell lines. We concluded that as an essential cancer gene RAD51 modulation by small molecule inhibition or transient transfection would be more suitable methods for studying RAD51 function in TNBC.To identify a novel small molecule RAD51 inhibitor we evaluated a library of quinazolinone derivatives and analysed structure activity relationships. Among these compounds we identified compound 17, which binds directly to RAD51. We hypothesized that inhibiting RAD51 in TNBC cell lines would block repair by HR, thereby sensitising tumour cells to DNA damaging irradiation and chemotherapy. We found that compound 17 inhibits HR in MDA-MB-231 cells by ~7-fold. Compared to the base compound (B02), compound 17 exhibited up to ~8-fold improved growth inhibition in a panel of TNBC cell lines and 2.5-fold increased inhibition of DNA damage-induced RAD51 foci formation. Additionally, compound 17 significantly enhanced sensitivity to DNA damaging radiotherapy and chemotherapies, suggesting a potentially targeted therapy for TNBC. A known mechanism by which cancer cells acquire chemoresistance is by deregulation of the DNA damage response, a process that can also create dependencies on specific DNA repair pathways. The clinical success of PARP inhibitors in BRCA mutant TNBC highlights the potential of targeting these dependencies therapeutically to induce synthetic lethality. Understanding DNA repair deficiencies is therefore vital for appropriate therapeutic choices. This is clinically utilised with homologous recombination deficiency (HRD) scoring based on TNBC mutational load, with a positive HRD status predicting PARP inhibition-mediated synthetic lethality. While genetic instability provides a snapshot of deregulated DNA damage response that drives chemotherapy resistance, we have analysed the functional consequences of repair deregulation. Here we show adaption to frontline TNBC chemotherapy combination doxorubicin and docetaxel. We find that chemoresistance results in enhanced genome instability, reliance upon DNA repair mediated by RAD51 and changes in gene expression profile guided by c-ABL and p73 and loss of p53 and BRCA1. Further we find that targeting RAD51 with our small molecule inhibitor can resensitize cells to docetaxel and doxorubicin and overcome DNA damage induced chemoresistance.