Novel DNA damage response therapeutics targeting replication protein A Abstract The DNA damage response (DDR) is now considered a tractable pathway to target for cancer therapy. The DDR and DNA repair pathways are also amenable to personalized therapies by exploiting synthetic lethal interactions as evidenced by the clinical success of PARP inhibitors in homologous recombination (HR) deficient cancers. The DDR is initiated by engagement of the PI3 kinase-related kinases ATM, ATR, and DNA- PK. These kinases and the downstream checkpoint kinases CHK1 and CHK2, are clinically validated targets being actively investigated in multiple clinical trials. A novel target in the DDR pathway is the human single stranded DNA binding protein, replication protein A (RPA) which plays a critical role in the DDR to detect replication stress (RS) and signal to the ATR kinase. RS is a common feature in cancer cells and provides the therapeutic window for the anticancer activity of DDR targeted drugs. RS coupled with a DDR blockade results in replication catastrophe (RC) and eventually cell death. RPA is a critical protector from RC and depletion of RPA or “RPA exhaustion” can elicit RC and cell death when there is insufficient single-strand DNA binding capacity to protect the genomic DNA. We have identified a potent and selective small molecule RPA inhibitor (RPAi), NERx 329, which possesses biochemical RPA inhibition and cellular engagement of RPA. NERx 329 also displays no overt toxicity in vivo and possesses anticancer activity alone and in combination with DNA damaging chemotherapeutics and certain DDR targeted agents. While the anticancer activity could be the result of inhibiting RPA’s role in individual repair or replication pathways, our published and preliminary data point to a more global mechanism of action. Our overarching hypothesis is that chemical inhibition of RPA can mimic RPA exhaustion to inhibit the DDR and provide selective anticancer activity via novel genetic interactions common in lung and ovarian cancer. To address this hypothesis, we will elucidate the mechanisms and determinants of the cellular anticancer activity of our novel RPAi. We will focus on two cancers types, high grade serous epithelial ovarian cancer (EOC) and non-small cell lung cancer (NSCLC), as our analysis of clinical survival data reveals an important role for RPA in these cancers. In Aim 1 we will interrogate how chemical exhaustion of RPA impacts RS and the DDR in lung and ovarian cancer. Aim 2 will focus on elucidating the genetic determinants of sensitivity to RPAi’s. We will identify novel chemical-genetic interactions in a DDR focused CRISPR screen assessing RPAi activity. Clinically relevant genetic interactions will be identified in a unique series of PD-EOC spheroid lines we have developed. Whole genome sequencing will identify genetic alterations and chromosomal rearrangements which may correlate with response to RPAi therapy. From these studies, specific genetic alterations that impact RPAi sensitivity will be validated with in vivo PDX models of lung and ovarian cancer and DDR targeted developmental therapeutic agents. Completion of these aims will define RPAi mechanism of action and the genetic interactions that dictate RPAi sensitivity. These data are crucial towards developing novel DDR targeted cancer therapeutics and identifying the relevant genetics characteristics to maximize patient response and efficacy.

Health relevance The research described in this new application has direct relevance to human health in that we are investigating the mechanism of action of novel therapeutic agents to be used in the treatment of human cancer. We will conduct research exploiting a novel mechanism to impact the DNA damage response pathway to allow more effective treatment of human cancer.