PROJECT SUMMARY/ABSTRACT Dr. Salehi-Rad is a Staff Pulmonologist at the VA Greater Los Angeles Healthcare System (VA GLAHS) with a clinical and research interest in lung cancer, the leading cause of cancer death among U.S. Veterans. In applying for the VA Career Development Award (CDA-2), Dr. Salehi-Rad’s goal is to establish an independent translational research program at the VA GLAHS, focused on improving our understanding of the immunopathogenesis of lung cancer for the development of novel approaches for cancer immunotherapy. He is supported by Steven Dubinett, MD (Primary-Mentor), a renowned VA Merit-funded physician-scientist and a leading expert in lung cancer, Antoni Ribas, MD, PhD (Co-Mentor), an internationally recognized authority in cancer immunology, and Paul Boutros, PhD (Co-mentor), a distinguished data scientist. Mentors were identified based on their complementary scientific expertise for the proposed research and their extensive experience in mentoring academic physician-scientists. Through UCLA Clinical and Translational Science Institute (CTSI), Dr. Salehi-Rad will have access to numerous career development seminars that address such topics as grant writing, manuscript preparation, and ethical research. He will also take graduate courses to obtain further training in immunology and bioinformatics. Dr. Salehi-Rad will have the full institutional support of both the VA and UCLA Health Systems to carry out his research. Dr. Salehi-Rad has established clinically relevant murine models of NSCLC with increased mutational burden and identified a novel targetable mechanism of resistance to immunotherapy in LKB1-deficient NSCLC. Utilizing these murine models, Dr. Salehi-Rad has shown that in situ vaccination (ISV) with elite antigen cross- presenting conventional type 1 DCs that are gene-modified to secrete FMS-like tyrosine kinase 3 ligand (FLT3L- cDC1), a cytokine that promotes DC viability and expansion, sensitize immune refractory NSCLC to immune checkpoint inhibition (ICI). In this proposal, Dr. Salehi-Rad aims to study the immune mechanisms of DC ISV. Aim 1.1 builds on preliminary in vitro data indicating enhanced viability of FLT3L-cDC1 compared to cDC1 and seeks to determine the molecular mechanisms that result in increased survival of FTL3L-cDC1. Aim 1.2 & 1.3 utilize various murine models to determine the vaccine and endogenous DC viability, antigen trafficking and antigen-specific T cell priming following DC ISV. Aim 2 of the proposal focuses on elucidating the immune determinants of response to DC ISV as monotherapy or as a combination therapy with ICI. Aim 2.1 & 2.2 combine single cell immunophenotyping by flow cytometry and single cell RNA-sequencing (scRNA-seq) with spatial analysis by multiplex immunofluorescence (MIF) to develop a comprehensive understanding of the local and systemic immune responses induced by DC ISV. Aim 2.3 utilizes antibody depletion studies to evaluate the dependency of DC ISV on T cells and natural killer cells. Aim 3 will determine the evolution of T cell repertoires by TCR-β CDR3 sequencing and tumor-neoantigen profiles by whole-exome sequencing (WES) to assess whether DC ISV induces the expansion of the TCR repertoire and promotes tumor immunoediting. Improved understanding of the nature of immunosuppression in NSCLC and the immunostimulatory mechanisms of DC ISV will represent a significant contribution to the field of lung cancer immunology and could facilitate the clinical translation of FLT3L-cDC1 ISV as an innovative therapeutic strategy for this devastating disease that affects many of our Veterans.

PROJECT NARRATIVE Lung cancer is the leading cause of cancer-related death amongst U.S. Veterans. Despite recent advances in cancer immunotherapy, many patients with NSCLC do not respond to immune checkpoint inhibitors (ICI). Therefore, there is a critical need for the development of novel strategies that enhance the efficacy of immunotherapy. One approach is to utilize in situ vaccination (ISV) with rationally engineered cytokine-secreting dendritic cells (DCs) to generate broad tumor-specific T cell responses. In preclinical studies, we have shown that ISV with FLT3L-secreting conventional type 1 DCs (FLT3L-cDC1) synergize with ICI to eradicate highly aggressive immune-resistant NSCLC. This proposal seeks to elucidate the molecular mechanisms of ISV and DC immunobiology in murine NSCLC models to facilitate the development of novel vaccine therapies for Veterans with lung cancer.