Targeting the DNA damage response sensor replication protein A for first in class cancer therapy

The DNA damage response (DDR) and replication stress response (RSR) are validated pathways for targeted cancer therapy, particularly through the exploitation of synthetic lethal interactions. We have pursued the discovery and development of DDR and RSR sensor protein inhibitors to disrupt these dysregulated pathways in cancer. We have demonstrated the therapeutic efficacy of the novel Replication Protein A inhibitor (RPAi), NERx-329, which disrupts the RPA-DNA interaction, induces chemical exhaustion of RPA function, and exhibits potent anticancer activity in vivo. We identified a series of synthetic lethal interactions that revealed the RPAi mechanism of action and therapeutic efficacy is primarily mediated by molecular events at the replication fork in response to replication stress (RS). These studies showed that RPAi exacerbates both genetically and pharmacologically induced RS via chemical RPA exhaustion resulting in further genomic instability, replication catastrophe, and cell death of cancer cells, with minimal toxicity in vivo. Data presented demonstrate the development of a combined treatment regimen targeting RPA and PARP that abrogates cancer growth in an in vivo BRCA1-deficient breast cancer model and ovarian cancer cell line. To assess markers of genome instability, we performed metaphase spreads on single agent- and combination agent-treated TNBC MDA-MB-436 cells. Single-agent RPAi treatment had no observable effect on the chromosome structure, whereas olaparib treatment increased chromosome pulverization. However, the combination strikingly induced chromosome pulverization. Taken together, NERx-329 chemically exhausts RPA such that olaparib-induced ssDNA gaps are degraded, and chromosomal integrity is compromised. Considering the induction of chromosome pulverization, we assessed the generation of micronuclei (MN) following RPAi treatment in combination with olaparib and the PARP1-specific PARPi saruparib. The data revealed that PARPi treatment resulted in a significant increase in MN, whereas treatment with RPAi alone did not affect MN formation. The combination of RPAi-PARPi treatment did not significantly alter MN occurrence. These results indicate that MN formation is not required for the enhanced cell killing effect of the combination treatment. Overall, these data support a model in which loss of BRCA1 and its prevention of ssDNA gaps during replication is further exacerbated by PARP inhibition. This data suggests that RPAi-PARPi combination enhances therapeutic efficacy and offers a strategy to overcome PARPi resistance by targeting gap protection mechanisms.