Tuesday, February 4, 2025
2/4/2025
ABSTRACT DNA double strand breaks (DSBs) are the most mutagenic and cytotoxic insults to the genome. DSBs are repaired through homology directed recombination (HDR), which is predominant in S and G2 phases, and also by error prone non-homologous end joining (NHEJ), which is active in all cell cycle phases. In cancer cells, heightened activity of these repair pathways has been linked to radioresistance. The transcriptional repressor ZEB1 is a well-established driver of the epithelial-to-mesenchymal transition (EMT) in both normal development and tumor progression. Recent studies suggest that ZEB1 plays new roles in the regulation of the DNA damage response (DDR), via enhancing the stability of the checkpoint kinase CHK1, and in regulating the repair of DSBs through the transcriptional silencing of polymerase theta, a major driver of microhomology- mediated alternative NHEJ (alt-NHEJ). We have discovered that ZEB1 is rapidly recruited to DSBs induced selectively in euchromatic genomic regions in a DNA-PK-dependent manner, and is essential for the recruitment 53BP1 to these sites. The recruitment of ZEB1 to these break sites was associated with local chromatin modifications permissive for NHEJ repair. Consequently, depletion or deletion of ZEB1 suppressed canonical NHEJ in cell-based DSB repair assays, and significantly increased both DSB-associated hyper- resection and HDR, This correlated with ATM-independent increases in chromosomal aberrations and enhanced sensitivity to IR. On the other hand, loss of ZEB1 was associated with decreased sensitivity of BRCA-deficient cells to PARP inhibitors (PARPi), indicative of restoration of HDR in these cell population, presumably through inhibiting 53BP1 recruitment to DSBs and stimulation of hyper-resection. Importantly, we found that ZEB1-dependent recruitment of 53BP1 to DSB is significantly amplified in hypoxia. Based on these novel observations, we hypothesize that ZEB1 promotes DSB repair via both direct and indirect mechanisms and this is critical for promoting therapeutic resistance to IR and for conferring sensitivity of BRCA-deficient cells to PARPi; on one hand, ZEB1 directly promotes c-NHEJ through the recruitment of the anti-resection and c-NHEJ repair protein 53BP1 to euchromatin-bound DSBs. On the other hand, ZEB1 facilitates the repair of DSBs through augmenting the DDR and through indirectly enhancing c-NHEJ repair through its chromatin- modifying as well as transcriptional silencing activity. Aim 1 of this proposal focuses on understanding the mechanism(s) by which ZEB1 regulates DSB repair. In Aim 2, we will determine the impact of ZEB1 on the therapeutic response of breast cancer to IR and PARP inhibitors in a panel of breast cancer cell lines and in xenograft mouse models of breast cancer. In Aim 3, we will determine how ZEB1 modulates the therapeutic response of hypoxic breast tumors to IR and PARPi. Understanding how the ZEB1/53BP1 axis regulates DSB repair in normoxia and hypoxia will allow us to exploit this axis as a biomarker to predict IR/PARPi sensitivity and well as to target the axis to decrease therapeutic resistance.
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