Introduction: HPK1, a member of the MAP4K family of protein serine/threonine kinases, is involved in regulating signal transduction cascades in cells of hematopoietic lineage. Recent data from HPK1 knockout animals and kinase-inactive knock-in animals underscores the role of HPK1 in negatively regulating lymphocyte activation. This negative-feedback role of HPK1 downstream of lymphocyte activation and function combined with its restricted expression in cells of hematopoietic origin make it an ideal drug target for enhancing anti-tumor immunity. Experimental Procedures: A structure-based drug design approach was used to identify potent and selective inhibitors of HPK1. Various biochemical and biophysical assays, as well as a primary in vitro T cell activation assay, were utilized for multiple rounds of structure-activity relationship (SAR) studies. In vivo target engagement and pharmacodynamic data were generated using an anti-CD3 mouse model. Results: In vitro, HPK1 small molecule inhibition resulted in enhanced IL-2 production in primary mouse T cells and in purified human T cells stimulated with a suboptimal dose of anti-CD3/anti-CD28. Increased selectivity of HPK1 inhibitors relative to T cell-specific kinases and within the MAP4K family was responsible for further enhancing the IL-2 response in activated T cells. In vivo, qd oral dosing of an HPK1 inhibitor completely abrogated phosphorylated SLP-76, induced by administration of anti-CD3. Furthermore, inflammatory cytokine production was enhanced in vivo upon HPK1 inhibition. Conclusion: Pharmacological blockade of HPK1 kinase activity represents a novel and powerful immunomodulatory approach for anti-tumor immunity. Citation Format: David Ciccone, Jennifer Rocnik, Vad Lazari, Ian Linney, Michael Briggs, Alan Collis, Christine Loh, Mark Ashwell, John Montana, Peter Tummino, Neelu Kaila. HPK1, hematopoietic progenitor kinase 1, is a promising therapeutic target for cancer immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 942.

Background Colon immune dysregulation results in a chronic inflammatory response typical of IBD and mainly driven by T-cell response to microbial antigens. Interestingly, stress can trigger or modulate inflammation and even modify the clinical course of IBD. Sphingosine 1-phosphate (S1P) increase in tissues is involved in T-cell recruitment and different compounds acting on S1P signaling are currently under clinical trials to test their ability to impact IBD progression, including sphingosine kinase 2 (Sphk2) inhibitors. Methods Male C57BL/6NJ and Sphk2-/- mice were randomly assigned to 4 experimental groups: Control WT (n=5), Control Sphk2-/- (n=5), Stress WT (n=8), and Stress Sphk2-/- (n=8). A sub-chronic stress mixed model based on immobilization and ultrasound exposure for 2h during 4 days was used. A set of tissue and biochemical assays was performed to evaluate stress and immune responses, S1P pathways, and epithelial barrier integrity. Results Stress caused weight loss and corticosterone upregulation regardless of the genotype. S1P was increased in the colon of stressed mice due to a decrease in its degradation enzymes and Sphk2, leading to an immune dysregulation reflected by an upregulation of TLR4 pathway, an inhibition of anti-inflammatory mechanisms – 15-lipoxygenase, N-formyl-peptide receptor 2 and Liver X Receptor – a decrease in IgA+ and a decrease in IgM+ B-cells and plasmablasts, and a Th17 polarization. Sphk2 deletion did not affect inflammatory processes but could interfere with Th17 response. Moreover, Sphk2-/- mice showed lower expression levels of claudins 3, 4, 5, 7, and 8 that could be related to structural abnormalities relevant to IBD. Stress exposure also decreased some of these claudins and increased intestinal permeability, but a synergistic effect between stress and genotype for permeability was not detected. Conclusion Sub-chronic stress induced colon S1P increase, immune dysregulation and increased intestinal permeability. Sphk2 deletion is involved in structural abnormalities and Th17 response but did not aggravate the inflammatory processes exerted by stress.