Abstract 519: Combined inhibition of WEE1 and PARP1 radiosensitizes KRAS mutant non-small cell lung cancers via inhibition of DSB repair

Abstract Mutant KRAS is found in approximately 30% of non-small cell lung cancers (NSCLC) and is associated with poor prognosis. Despite the use of radiation (RT) therapy for the treatment of locally advanced disease, local recurrence remains an issue. The observation that mutations in KRAS lead to both replication stress and DNA double-strand breaks (DSBs) suggests these cancers may have a greater reliance on DNA damage response pathways and therefore may be preferentially radiosensitized by therapies targeting DNA repair. In this study, we investigated the combined inhibition of WEE1 and PARP1 using the small molecule inhibitors, AZD1775 and olaparib, respectively as a radiosensitizing strategy in KRAS mutant NSCLC. We began by comparing radiosensitization by AZD1775+olaparib, in KRAS isogenic H1703 lung cancer cells and found that KRAS mutant cells were preferentially radiosensitized (ER, enhancement ratio 1.8) compared to KRAS wild type cells (ER 1.4). Combined WEE1 and PARP1 inhibition also radiosensitized KRAS mutant Calu-6 and NCI-H23 lung cancer cells (ER 1.9 and 1.5, respectively). These findings were further confirmed in vivo: Calu-6 tumor xenografts were significantly radiosensitized by AZD1775+olaparib, as evidenced by an 11 day delay in tumor volume doubling time relative to RT treatment. Given that WEE1 and PARP1 function to prevent and manage replication stress, respectively, we hypothesized that radiosensitization by AZD1775+olaparib results from persistent replication stress. While replication stress did contribute to AZD1775-mediated radiosensitization in Calu-6 cells, as evidenced by pan-nuclear γH2AX staining, and the ability of exogenous nucleosides to protect cells from radiosensitization by AZD1775 alone, nucleosides had little effect on radiosensitization by AZD1775+olaparib. These results suggest that replication stress is not required for radiosensitization by AZD1775+olaparib. As WEE1 and PARP1 both promote repair of radiation-induced DNA damage, we hypothesized that radiosensitization by AZD1775+olaparib results from persistent, unrepaired DSBs. Assessment of the kinetics of DSB repair by γH2AX flow cytometry demonstrated that while total γH2AX levels in cells treated with RT alone had returned to control levels within 24 h, AZD1775+olaparib treatment significantly delayed the resolution of γH2AX following RT, with 44.8% or 46.2% of Calu-6 or KRAS mutant H1703 cells, respectively remaining γH2AX-positive 24 h post-RT. This delay corresponded with the inhibition of radiation-induced RAD51 foci by AZD1775, indicating inhibition of homologous recombination repair. Taken together these data demonstrate the efficacy of combined inhibition of WEE1 and PARP1 with radiation in KRAS mutant lung cancer. Furthermore, these results suggest that although replication stress occurs in response to AZD1775 and olaparib, persistent DSBs are the cause of radiosensitization. Citation Format: Leslie A. Parsels, David Karnak, Joshua D. Parsels, Zachery Reichert, Jonathan Maybaum, Theodore S. Lawrence, Meredith Ann Morgan. Combined inhibition of WEE1 and PARP1 radiosensitizes KRAS mutant non-small cell lung cancers via inhibition of DSB repair. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 519.