Project III. Engineering immunogenic cell death in melanoma and renal cell carcinoma Experimental Lead: Cheng Computational Lead: Leslie Experimental Co-Investigator: Li PROJECT SUMMARY Recent approval of immune checkpoint blockade (ICB) in multiple cancer types has revolutionized cancer care, but only a small fraction of patients achieve a durable complete response. Hence, there is an urgent need for novel immunotherapy-based therapeutic strategies to enhance response and offer long-term survival benefits. We hypothesize that induction of immunogenic cancer cell death is one of such strategy. Necroptosis and pyroptosis, two forms of programmed necrosis, are pro-inflammatory and probably immunogenic. By contrast, mitochondrial apoptosis is generally considered immunologically silent. The BCL-2 family proteins are central regulators of mitochondrial apoptosis. BAX and BAK, once activated by BH3-only molecules, trigger mitochondrial outer membrane permeabilization (MOMP), which releases cytochrome c to activate the apoptosome and caspases. Recent paradigm-shifting discoveries have shown that BAX/BAK activation in the absence of caspases can trigger the release of mitochondrial DNA to the cytosol through a process called “mitochondrial inner membrane permeabilization” (MIMP), which activates the cGAS/STING pathway and type I interferon response. These findings indicate that this form of BAX/BAK-dependent, caspase-independent cell death, which we termed “mimptosis”, is highly inflammatory. Here, we engineer models of inducible cell death in murine melanoma and genetically engineered mouse models (GEMMs) of clear cell renal cell carcinoma (ccRCC) to compare the immune response to apoptosis, mimptosis, pyroptosis, and necroptosis in vivo. Our goal is to identify the most immunogenic cell death that not only kills cancer cells but also instructs the immune system to enhance ICB response and elucidate the underlying molecular mechanisms using a comprehensive systems biology approach. In Aim 1, we will characterize the impact of different kinds of engineered immunogenic cell death (ICD) on tumor-immune ecosystem dynamics melanoma using integrated single-cell multiome, paired single-cell RNA and T cell receptor (TCR) sequencing, and multiplexed immunofluorescence. Computational modeling will define immune cell gene expression and regulatory programs in response to ICD to inform therapeutic strategies to improve ICB. In Aim 2, we will determine whether and how immunogenic cell abrogates tumor progression and metastasis and generates immunological memory against tumor rechallenge in GEMMs of ccRCC. A comprehensive multidimensional assessment and computational modeling of tumor- immune ecosystem evolution, T cell epigenetic and functional states, and TCR repertoire in response to different types of cell death will unravel mechanisms that promote anti-tumor immunity. TCGA analysis revealed that low expression of apoptosome components, APAF1 and Caspase-9, is associated with better survival outcomes, suggesting that caspase-independent mimptosis may activate anti-tumor immunity and confer survival benefits in ccRCC. We will assess this hypothesis by performing single-nucleus multiome and MxIF in patient ccRCC samples to characterize tumor-immune cross-talk comparing ccRCC with low vs high apoptosome expression.