Tumor and Immune Cell Dynamics during Immunotherapy and Cancer Progression

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PROJECT SUMMARY Treatment of cancer has been transformed by immunotherapies that aim to reactivate tumor-specific immune cell responses, in particular checkpoint blockade therapies that target inhibitory receptors on T cells. Although these therapeutics have achieved durable clinical responses, many patients do not respond or shortly relapse. Rationale design of effective immunotherapy strategies requires a detailed understanding of the dynamics between tumors and the immune system. My goal is to decode these dynamic interactions using a combination of in vivo models, genomic technologies, and large-scale analyses of patient data to gain novel insights into the biological processes that underlie cancer progression and response to immunotherapy. In the F99 phase, I aim to identify to origin of immune cells that respond to checkpoint blockade. An outstanding question is whether the T cell response to checkpoint blockade relies on reinvigoration of pre-existing tumor-infiltrating lymphocytes or on recruitment of novel T cells. In my dissertation work so far, I have used single cell RNA and T cell receptor (TCR) sequencing of site-matched tumor biopsies before and after PD-1 blockade to profile clonal T cell dynamics in response to immunotherapy. I found that T cells that clonally expand in response to PD-1 blockade are enriched for novel clones that have not been previously observed in the same tumor, a phenomenon we term clonal replacement. However, the source of novel T cells as well as the role of other immune cell populations in this process remain unclear. I hypothesize that novel T cells are derived from secondary lymphoid organs and that overcoming deficiencies in T cell priming by antigen presenting cells is required for clonal replacement following PD-1 blockade. I will use a combination of flow cytometry, single cell sequencing, and bulk TCR sequencing of in vivo syngeneic mouse tumor models to determine the origin of T cells that respond to PD-1 and CTLA-4 blockade. Further, I will use genetic mouse models to determine the role of PD-L1 expression on antigen presenting cells, in particular conventional type 1 dendritic cells, on PD-1 blockade efficiency. In the K00 phase, I aim to elucidate the relationship between extrachromosomal DNA amplification, innate immune signaling, and the efficacy of checkpoint blockade. Copy number alterations leading to oncogene amplification frequently occurs on circular extrachromosomal DNA (ecDNA), which are commonly found in the cytoplasm due to lack to centromeres and associate with loss of cytosolic DNA sensing through the cGAS-STING pathway. Further, innate immune signaling through cGAS-STING has been shown to improve the efficacy of checkpoint blockade. I propose that loss of cytosolic DNA sensing is permissive for extrachromosomal DNA production, which promotes tumor progression not only through oncogene amplification but also through impaired innate immune signaling, limiting immune surveillance and the efficacy of checkpoint blockade. Together, this work will provide novel insights into immune and tumor dynamics that underlie cancer progression and response to immunotherapy.