2622 - Overcoming Resistance to PARP Inhibitors in BRCA-Mutated Cell Lines Using a Rucaparib-Derived Auger-Emitting PARP1 Radioligand

Purpose/Objective(s): Hereditary mutations in BRCA1 and BRCA2 are strongly associated with an increased risk of developing breast and ovarian cancer, as well as other cancer types. Cells lacking functional BRCA proteins are deficient in homologous recombination (HR), a process that ensures faithful repair of double stranded breaks (DSBs). This HR deficiency creates unique therapeutic vulnerabilities in BRCA-deficient cells, which have been exploited by poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi). Despite the promise of PARPi in treating BRCA-mutated tumors, acquired resistance remains a critical challenge. Herein, we assess the capacity of a rucaparib-derived, Auger electron-emitting PARP1 radioligand (known as [⁷⁷Br]Br-WC-DZ or [⁷⁷Br]Br-RD1) to overcome acquired resistance in BRCA-mutated cell lines. We hypothesize that by delivering medium LET radiation directly at sites of PARP1 binding, [⁷⁷Br]Br-WC-DZ will efficiently and rapidly induce DSBs that will overwhelm acquired resistance mechanisms.

Materials/Methods: We performed in vitro cytotoxicity and characterization of DNA damage signaling in a panel of ovarian cell lines that includes the parent BRCA-deficient, PARPi sensitive cell line (UWB1), the BRCA-complemented version of the cell line (UWB1+BRCA1), and two PARPi-resistant clones that remain BRCA-deficient (SYR12 and SYR14). For cytotoxicity assessment, we plated cells at low density and treated them with increasing concentrations of [⁷⁷Br]Br-WC-DZ or rucaparib and assessed viability after 6 days using an ATP-based luminescent viability assay (CellTiterGlo). We concurrently collected lysates for immunoblotting to assess DNA damage signaling.

Results: Cytotoxicity assessment after rucaparib treatment confirmed PARPi resistance of UWB1+BRCA1 and PARPi-resistant (SYR12 and SYR14) cell lines compared to parent UWB1 cells. Treatment with [⁷⁷Br]Br-WC-DZ resulted in greater toxicity in all four cell lines compared to rucaparib with an IC₅₀ in the low nanomolar range. Consistent with rapid onset of cytotoxicity, we observed diminished cell survival at 6 days even when [⁷⁷Br]Br-WC-DZ was removed after 4 hours of treatment. Moreover, DNA damage signaling differed between [⁷⁷Br]Br-WC-DZ and rucaparib. Rucaparib treated cells demonstrated markers of replication stress including increased phosphorylation of CHK1 at S345, whereas [⁷⁷Br]Br-WC-DZ treated cells rapidly demonstrated increases in ?-H2AX, a marker of DSBs.

Conclusion: [⁷⁷Br]Br-WC-DZ is significantly more potent than rucaparib, including in PARPi-resistant cell lines, and acts through a unique mechanism separate from traditional PARPi.