Molecular dissecting and targeting YAP1 mediated cancer stemness and immune suppression in advanced gastric adenocarcinoma

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Project Abstract The main objectives of this proposal are to elucidate the mechanisms of YAP1-mediated metastatic niche and immunosuppression in gastric adenocarcinoma (GAC) with peritoneal carcinomatosis (PC) for novel therapeutic discoveries. GAC is a major health burden in the US and worldwide. PC is common affecting ~45% of GAC patients. GAC patients with PC have short survival and treatments are ineffective. Molecular understanding for PC is limited. To address this unmet clinical challenge, we have established PC banking infrastructure aiming to utilize these patient-derived specimens to discover and validate our novel targets. Our preliminary RNAseq profiling of PC specimens revealed an enrichment of unique immune suppressive molecules, including TIM3 and it’s ligand Galectin-9 (Gal-9), TGF-β and VISTA, but less expression of PD-1/PDL-1 and CTLA-4. YAP1 has been implicated in human development, lineage plasticity, and upregulated in many tumor types. However, its role in mediating PC metastases and immune suppression in tumor microenvironment (TME) remain unclear. Our preliminary data suggest that YAP1 is highly expressed in primary and metastatic tumor cells of GAC patients and is significantly associated with poor survival. Genetic knockout (KO) YAP1 significantly decreased cancer stemness traits, tumor formation and PC in mice. Further, depletion of YAP1 in tumor cells consistently increased CD3 and CD8 T cell responses from GAC. Through a single cell RNAseq (scRNAseq) of PC samples and validation using immunofluorescent staining, we noticed that YAP1, TIM3 ligand Gal-9 and DKK1 are highly expressed in tumor cells of PC; while TIM3 is enriched in immune cells. RNAseq from YAP1high and YAP1 KO patient-derived tumor cells revealed that Gal-9 and DKK1 were significantly decreased upon depletion of YAP1 and these factors are associated with poor survival of patients. We hypothesize that YAP1high PC cells are metastasis-initiating cells that orchestrate a niche by conferring cancer stemness attributes to the tumor cells and promote tumor immunosuppression through activating TIM3/Gal-9 axis and increasing paracrine of DKK1 in PC TME. Therefore, simultaneously targeting Hippo/YAP1 and immune checkpoint (TIM3/Gal-9) could be an enhanced strategy. To test our hypothesis, we propose three Specific Aims: Aim 1. Determine the functional relevance of YAP1 in PC stemness and metastases in tumor cells using novel stem cell cloning technology and PDX/PDO models in vivo. Aim 2. To investigate the mechanisms whereby YAP1 mediates immunosuppression in TME of PC. Aim 3. Elucidating efficacy of inhibition of YAP1 alone or in combination with TIM3 inhibition using the PDO models, KP-Luc2 syngeneic mouse model, GEMM, and ongoing YAP1 clinical trial. By utilizing patient- derived PC cells, we will uncover functional importance of YAP1-mediated PC and elucidate the mechanisms by which YAP1 mediated immunosuppression for novel therapeutic strategies. Upon completion of this study, we will have a strong rationale for a novel combination that could overcome the shortcomings we experience in the clinics today.