Abstract 2653: Integrative analysis of proteomic alterations in human PBMC’s treated by HPK1 inhibitor FB849 reveals enriched pathways from various immune cells leading to potential novel biomarker discovery

Abstract HPK1, also known as MAP4K1, is a critical signaling kinase molecule as a negative feedback loop in various immune cells. Despite the advancement of multiple clinical programs for therapeutic inhibition of HPK1, there is an urgent need to understand HPK1 biology in genome-wide signaling networks not only from lymphocytes but also from monocytes. FB849 is designed as a potent and highly selective inhibitor of HPK1 and is currently in phase I clinical trial with advanced cancer patients (NCT05761223). We aim to generate proteomic and phosphoproteomic profiles to reveal modes of action of FB849 in the broad spectrum of cancer immunity. Human PBMCs were treated with FB849 under conditions with or without CD3/CD28 stimulation. Utilizing an antibody array comprising 1,930 antibodies, the expression of 1,438 distinct proteins was assessed. The antibody arrays in human PBMCs identified a total of 85 and 33 phospho-proteins showing differential expression in response to FB849 treatment compared to the control in the presence or absence of CD3/CD28 stimulations, respectively (fold change > 2, adj. p-value < 0.01). KEGG pathway enrichment analysis demonstrated that the differentially expressed proteins by FB849 in the presence of CD3/CD28 stimulation were significantly enriched in immune-related biological processes, including 'pathways in cancer', 'PI3K-Akt signaling pathway', 'Focal adhesion', 'cytokine-cytokine receptor interaction', 'MAPK signaling pathway', 'JAK-STAT signaling pathway', and 'Th1 and Th2 cell differentiation'. Reactome biology pathway analysis also showed significant enrichment in ‘immune system’, ‘homeostasis’, and ‘cytokine signaling in immune cells’. In addition, we performed LC-MS/MS-based phosphoproteomics in PBMC samples pooled from CT26 mouse syngeneic models treated with 25mg/kg and 50mg/kg of FB849 and its derivative. The proteomics identified 1239 unique phosphorylated proteins and 915 distinct phosphorylation proteins, while 68 and 39 phosphorylated proteins were identified as differentially expressed proteins in FB849 or its derivative treated groups compared with control groups, respectively. The several phosphoproteins exhibited consistent decreases or increases in both FB849 and its derivative. Together, this work reveals genome-wide alterations in the proteomic and phosphoproteomic landscape by FB849 in both mouse PBMC’s and human PBMC’s with clear pathway enrichment potentially from various immune cell lineages. Further exploration of the differential expression of each protein and/or phosphoproteins especially in T cell-, B cell-, and monocyte-enriched biomarkers which link to HPK1-relevant signal transduction is needed to find out their clinical implication and to highlight new understanding of HPK1 signaling network in cancer immunity. Citation Format: Seungmook Lim, Sooryeonhwa Jin, A Yeong Park, Hyekyoung Kim, Seongkon Kim, Jamie Jae Eun Kim, Jinhwa Lee. Integrative analysis of proteomic alterations in human PBMC’s treated by HPK1 inhibitor FB849 reveals enriched pathways from various immune cells leading to potential novel biomarker discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2653.