Project 2: Determinants of Response and Resistance to DNA-Damaging Radiation Plus Immunotherapy Combinations in Triple Negative Breast Cancer – PROJECT SUMMARY/ABSTRACT Anti-PD1 (aPD1) immune checkpoint inhibition combined with four chemotherapeutic agents is the current standard-of-care for most patients with early-stage triple negative breast cancer (TNBC). However, treatment resistance and treatment-related toxicities persist as dual challenges that must be overcome with new strategies to guide personalized chemo-immunotherapy combinations. Data from our group and others have established the association between DNA damage-induced necroptosis (DDIN) and promotion of anti-tumor immune responses. Project 2 will investigate the role of DDIN as a determinant of response to RT plus anti- PD1 combination therapy using preclinical TNBC genetically engineered mouse models (GEMM) and correlative biomarker analyses of pre/post-treatment primary tumor, metastatic lymph node, and blood specimens from an ongoing clinical trial of preoperative anti-PD1 therapy, with or without RT, in early-stage TNBC with lymph node metastasis. In Aim 1, we will develop a gene signature of necroptosis proficiency and evaluate it as a predictive biomarker of RT/aPD1-induced spatial reprogramming of the tumor microenvironment. In Aim 2, we will determine whether RT/aPD1 can elicit systemic neoantigen-directed T and B cell responses, and whether neoantigen vaccination can overcome RT/aPD1 resistance induced by necroptosis deficiency. Project 2 will be supported in bioinformatics and statistical analyses by Core B and in spatial transcriptomics and clinical trial biospecimen analyses by Core C. The proposed studies will identify predictive biomarkers to guide future clinical trials of RT plus anti-PD1 combination therapy in early-stage TNBC—including testing strategies to reduce treatment toxicity by minimizing chemotherapy in patients expected to respond to RT/aPD1—and establish contexts where neoantigen vaccination may help to overcome radio-immunotherapy resistance.