Despite the approval of PARP inhibitors for the treatment of advanced BRCA-mutated breast cancer, progres- sion-free survival times are still quite short at only 7 months. This is a significant problem because once PARP inhibitors fail, few systemic treatment options remain and the disease cannot be cured, leading to the need for alternative treatment strategies. One strategy to enhance the immune response to tumors is radiotherapy (RT), which stimulates innate and adaptive immune responses through the release of antigens and immunostimula- tory mediators. However, relatively little is known about the physical mechanisms of RT that elicit immunostim- ulatory signals and how they can be harnessed in the context of DNA damage and DNA repair defects. We propose to investigate how immunostimulatory signals are modulated in the context of RT-induced DNA dam- age and DNA repair defects, particularly BRCA1 mutation. Our preliminary data indicate that BRCA1 mutation in combination with proton RT, a form of RT that induces relatively more clustered DNA lesions compared to photon RT, potentiates higher levels of micronuclei?a precursor of immunostimulation involving the cycling GMP-AMP synthase (cGAS) and stimulator of interferon (IFN) genes (STING) pathways. We hypothesize that DNA repair deficiency in combination with high-LET RT enhances an immune response through the cGAS- STING pathway. To test this we propose to: 1. Determine whether high-LET RT (protons) vs. low-LET RT (photons) influence the immunogenicity of cells with BRCA1 defects via the cGAS-STING pathway. In this aim, we will determine whether protons enhance cGAS-STING sensing pathway mediated anti-tumor im- mune signaling in BRCA1-mutant and PARP inhibited tumors relative to photons. 2. Determine in a BRCA1- defective murine mammary tumor model whether-high LET RT (protons) vs. low-LET RT (photons) in- fluence immunogenicity, alone and in the setting of PARP and PDL1 inhibitors. We will use syngeneic mouse tumor models with and without functional BRCA1 to evaluate the effect of protons vs. photons alone and with PARP and PD1 inhibitors. We will evaluate the differential activation of the cGAS-STING pathway by assessing micronuclei levels and activation of STING, TBK1, IRF3 and IFN-I and by quantifying inflammatory cytokines and alterations in tumor infiltrating immune cell components after RT. Successful completion of this project will generate innovative preclinical data to directly link the quality of RT and activation of cGAS-STING mediated anti-tumor immune signaling. Our research has the potential to define high LET RT as a way to aug- ment the immune response for patients with aggressive, BRCA1-mutated tumors and possibly for other tumors with DNA repair deficiency. Our proposed work is innovative in that it aims to define the effects of clustered DNA lesions generated by high LET RT on BRCA1 mutated tumors in the context of anti-tumor immune re- sponse, findings which may lead to new opportunities for harnessing unique physical properties of RT as a tool to activate the immune system to combat cancer.
There is an urgent, unmet need to substantively improve clinical outcomes in patients with metastatic, BRCA1 mutated breast cancers, for whom progression free survival after best systemic therapies remains < 8 months. Our preliminary data demonstrate that distinct forms of radiation based on their linear energy transfer (LET) levels differ in their abilities to cause clustered DNA damage, which result in micronuclei formation, an effect that is augmented in the presence of a BRCA1 mutation, a deficiency in homologous-recombination-based DNA repair. In our proposal, we will explore the biologically distinct effects of high versus low LET radiation, investigating if radiation-induced micronuclei enhance upregulation of the cGAS-STING innate immune response in the setting of a BRCA1 mutation in the presence of drug therapy, with the ultimate goal of using optimized radiation as a biological tool to activate the immune system to improve patient outcomes.
