Abstract P2-06-01: Parp inhibition sensitizes brca deficient cancer cell lines and tumors to clinical x-ray and proton irradiation
Authors: Kacem Mariam Ben , Bright Scott J. , Turner Broderick X , Flint David B , Manandhar Mandira , Martinus David , Sawakuchi Gabriel O , Shaitelman Simona F 
Published: March 2022

Purpose Triple negative breast cancers (TNBC) are more often found among BRCA gene mutation carriers; even among non-mutation carriers, TNBCs are thought to carry a phenotype of BRCAness. Inhibitors of Poly(adenosine diphosphate-ribose) polymerase (PARPi) have improved survival outcomes for both advanced and early stage breast cancer associated with a BRCA mutation, as well as BRCA mutated ovarian and prostate cancers. Radiotherapy (RT) is a widely used treatment to locally control cancers, but it is unclear how different types of RT, including clinical x-rays and protons, interact with PARPi and how these affect antitumor immunity. Protons induce more clustered DNA lesions, including clustered double and single strand breaks (DSBs and SSBs) and clustered base damages, than x-rays due to their higher ionization density. If not properly repaired, DSBs can generate micronuclei (MN), which activate the cyclic GMP-AMP synthase (cGAS) and stimulator of interferon (IFN) genes (STING) (cGAS-STING) pathway, leading to antitumor immunity. Residual DNA damage also induces cellular senescence, which is a known tumor suppressor mechanism. In this study we investigated the effects of combining a PARPi with x-rays or protons on clonogenic cell survival, residual DNA damage, senescence, cGAS co-localization with MN and delay in tumor growth in the context of BRCA1 mutation. Methods In vitro, BRCA1 deficient (HCC1937 and MDA-MB-436) and proficient (HCC1937-BRCA and MDA-MB-436-BRCA) breast cancer cell lines were treated with a PARPi (Olaparib, 0.1 - 5.0 μM) and irradiated with 6 MV x-rays or protons (9.9 keV/um) with doses from 0.5 to 5 Gy. HCC1937-BRCA was gifted from Dr. Stecklein (The University of Kansas Cancer Center) and MDA-MB-436-BRCA was gifted from Dr. Johnson (Fox Chase Cancer Center). We then quantified clonogenic cell survival, gH2AX and 53BP1 foci at 24 h after irradiation, MN number and MN-cGAS co-localization (cGAS+ MN) at 24 and 74 h after irradiation, and RT-induced senescence. In vivo, we used a preclinical TNBC mouse model (4T1 tumors on the leg of BALB/c mice) to assess survival and tumor growth delay on animals treated with DMSO, PARPi alone (Olaparib, 100 mg/kg), DMSO+x-rays, and PARPi+x-rays. Experiments with protons are ongoing. PARPi was administrated via oral gavage 2 h prior to irradiation (1 × 11 Gy). Results Survival fraction was lower after protons compared to x-rays (RBED10%&gt;1) in HCC1937-BRCA, HCC1937 and MDA-MB-436 cells. Regardless of the radiation type, survival fraction was lower for HCC1937 compared to HCC1937-BRCA cells. PARPi treatment appears to result in greater radiosensitization of cells exposed to protons than those exposed to x-rays as assessed by clonogenic cell survival. The number of MN per nucleus was higher in HCC1937 than HCC1937-BRCA at 24 and 72 h after protons and x-rays at both 2 and 5 Gy, and this effect that was amplified with PARPi vs. without PARPi. Moreover, the number of cGAS+ MN was greater after protons than x-rays for HCC1937 but not HCC1937-BRCA at 24 and 72 h. The proportion of senescent cells for HCC1937-BRCA was higher (1.4 fold) after PARPi+protons compared to PARPi+x-rays. In vivo, x-rays lead to a delay in tumor growth, which was higher for the PARPi+x-rays group compared to PARPi alone or DMSO+x-rays groups. Conclusion In vitro, a PARPi treatment in BRCA1 mutated cell lines leads to a greater sensitivity to radiation compared to their counterparts with recovered BRCA1 function. This sensitivity is higher after protons compared to x-rays (survival fraction and number of micronuclei). In vivo, PARPi treatment combined with x-rays lead to higher delay in tumor growth compared to x-rays alone. These preliminary data are promising results on the effect of radiation combined with PARPi on BRCA1 mutated models.

Citation Format: Mariam Ben Kacem, Scott J. Bright, Broderick X Turner, David B Flint, Mandira Manandhar, David Martinus, Gabriel O Sawakuchi, Simona F Shaitelman. Parp inhibition sensitizes brca deficient cancer cell lines and tumors to clinical x-ray and proton irradiation [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-06-01.