The Role of Glutamine Metabolism in Monocyte-Derived Dendritic Cell Differentiation in the Soft-Tissue Sarcoma Microenvironment

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PROJECT SUMMARY Soft-tissue sarcomas (STS) are a diverse and often fatal set of malignancies arising from connective tissue with a 16% five-year survival rate for metastatic disease, reflecting the need for novel therapeutic strategies. One approach showing promise against multiple cancers is immune checkpoint blockade. However, clinical trials of immunotherapy in STS have produced disappointing results, likely due to immunosuppressive microenvironments characteristic of these diseases. The STS microenvironment is dominated by tumor- associated macrophages, which largely differentiate from tumor-infiltrating monocytes. These can also differentiate into anti-tumor monocyte-derived dendritic cells (Mo-DCs) in inflammatory conditions, but Mo-DC differentiation is inhibited in STS by as-yet unknown factors. We have recently shown that enhanced Mo-DC differentiation leads to synergy with immune checkpoint blockade. It is therefore critical to discover additional processes that regulate Mo-DC differentiation in order to improve the efficacy of immunotherapy against STS. One such mechanism may depend on glutamine metabolism. Glutamine is utilized as a metabolic fuel by several immune cell types, as it is involved in generating biosynthetic products via the rate-limiting enzyme glutaminase, glycosylation of proteins, and activation of mammalian target of rapamycin complex 1 (mTORC1) mediated signaling pathways. Additionally, recent work has shown that blocking glutamine metabolism can modulate the anti-tumor activity of tumor-infiltrating immune cells. Using an in vitro model of Mo-DC differentiation, we found that glutamine deprivation blocked Mo-DC differentiation, surprisingly independent of glutaminase activity. This finding suggests Mo-DC differentiation requires glutamine flux through separate pathway(s), such as the hexosamine biosynthetic pathway (HBP) or glutamine-leucine antiport leading to mTORC1 activation. I hypothesize that glutamine metabolism regulates monocyte-derived dendritic cell differentiation in the soft tissue sarcoma microenvironment through either the hexosamine biosynthetic pathway or leucine-dependent mTORC1 activation. Aim 1 will identify the mechanisms by which glutamine metabolism regulates the differentiation of monocytes into Mo-DCs. Isotopic labeling and mass spectrometry will be used to assess the incorporation of glutamine into downstream metabolites of glutaminase as well as HBP intermediates and export from the cell. Conditional knockout mice will be used to assess the contributions of HBP flux and mTORC1 activation to Mo- DC differentiation. Aim 2 will test the impact of glutamine availability and inhibition of glutamine metabolism on Mo-DC differentiation and function in the STS microenvironment, as well as synergy with immune checkpoint blockade. Together, these approaches will elucidate the role of glutamine metabolism in Mo-DC differentiation and function in the STS microenvironment.