Strategic Modulation of Liver Phenylalanine Metabolism and Neutrophil Kainate Receptor to Influence the Tumor Macroenvironment: Mechanisms and Preclinical Strategies - Project Summary Immune checkpoint inhibitors (ICIs) have transformed melanoma care, but durable benefit is constrained by poor initial response to first-line regimens. Beyond tumor microenvironment determinants, systemic metabolism, particularly phenylalanine (Phe), appears to shape early efficacy: variants at the phenylalanine hydroxylase (PAH) locus associate with melanoma risk, and elevated plasma Phe correlates with immune impairment, progression, resistance, and worse survival, while dietary Phe restriction suppresses melanoma growth and improves therapy sensitivity. The causal mechanism linking high Phe to blunted ICI activity has remained undefined. Our data show that ICIs induce hepatic PAH degradation, raising circulating Phe, which activates GRIK1-CaMKIV-STING signaling in tumor-associated neutrophils (TANs) and drives CD8+ T cell dysfunction and exhaustion. We hypothesize that ICI-triggered disruption of hepatic Phe metabolism causes systemic Phe accumulation that, via GRIK1 in TANs, suppresses antitumor T cell immunity. Aim 1 establishes how systemic Phe dysregulation impairs antitumor immunity, using Phe-rich, Phe-free, and aspartame-containing diets in humanized melanoma models and paired serum-tumor analysis from 562 advanced melanoma patients. Aim 2 dissects the Phe-GRIK1-CaMKIV-STING-type I IFN axis in neutrophils using PahR408W/R408W, neutrophil-restricted Grik1 deletion, and Pmel-1 TCR transgenic models with CD8+ T cell functional and mitochondrial metabolic assays. Aim 3 tests two translational interventions, hepatocyte-targeted LNP HULC RNA mimetics that stabilize PAH (source correction) and the FDA-approved GRIK1 inhibitor Topiramate (sensor blockade), alone and with anti-(PD-1+CTLA-4) therapy, for efficacy, immunometabolic remodeling, and safety. Human organoids and tumor-immune co-cultures are valuable for cell-intrinsic and short-range interactions, but they cannot reconstitute the systemic, inter-organ circuit central to this proposal, in which checkpoint blockade drives hepatic PAH loss, elevates circulating Phe, and reprograms continuously bonemarrow-replenished TANs to suppress CD8+ T cells, because they lack a liver that sets systemic Phe, a circulation to convey it, and a renewing myeloid compartment to sustain neutrophils. Establishing this liver-totumor causality, modeling diet-driven systemic Phe modulation, and testing hepatocyte-targeted LNP delivery with whole-body efficacy and safety therefore require intact humanized MISTRG mice and the cell-type-specific genetic models above. This work converts Phe from a correlative signal into a predictive, actionable driver, delivers combinable metabolic interventions, and provides a scalable framework for metabolite-guided immunotherapy across cancers.
