Tumor hypoxia induces DNA repair vulnerabilities through contextual "loss-of-heterozygosity"

Introduction: Intratumoral hypoxia leads to decreased expression in DNA damage response (DDR) and repair pathways. Given the “two-hit” model for loss of gene function, we hypothesize that hypoxia-mediated down-regulation of gene expression and function, coupled with an already inactive allele may ultimately give rise to an exploitable contextual “loss-of-heterozygosity” phenotype. Method: To interrogate this relationship, isogenic DLD-1 cells heterozygous and homozygous null for BRCA2, were placed under normoxic (21% O 2 ) or hypoxic (0.2% O 2 ) conditions for 72 hours. Hypoxia-mediated changes in DDR response and DNA repair were evaluated by cell proliferation, cell cycle analysis, western blots, qPCR, immunofluorescence, clonogenic assays and repair assays. Results: No differences in proliferation and cell survival were observed between oxic and hypoxic cells (72 hours - 0.2% O 2 ). Under chronic hypoxic conditions, confirmed by the up-regulation of VEGF and HIF1α, mRNA and protein expression of key homologous recombination (HR) genes (BRCA1, BRCA2, RAD51) were down-regulated. Functionally, BRCA2 (-/-) null cells proved unable to recruit Rad51 foci and resultantly presented a profound sensitivity to PARP inhibition. Conversely, heterozygote BRCA2 (+/-) cells retained the ability to recruit Rad51 foci under both oxic and hypoxic conditions, however, exposure to chronic hypoxia resulted in a reduction in the number of foci formed. Chronically hypoxic BRCA2 (+/-) cells exhibited a 30 to 40% increase in sensitivity to PARP inhibition when compared to their oxic counterparts. Preliminary data shows a similar synthetically lethal relationship in genetic and tumor microenvironment induced HR deficient cells, when challenged with DNA damage response (DDR) kinase inhibitors ATRi and DNAPKi. Conclusions: Herein we illustrate through a novel mechanism of contextual “loss-of heterozygosity”, which marries the tumor microenvironment and innate genetic alterations, increased sensitivity to DDR kinase inhibitors and PARPi. Citation Format: Osman Mahamud, Melvin L.K Chua, Winnie Lo, Gaetano Zafarana, Robert G. Bristow. Tumor hypoxia induces DNA repair vulnerabilities through contextual “loss-of- heterozygosity” [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B39.