Abstract 3453: First disclosure of a highly potent and selective HPK1 inhibitor that rescues T cell exhaustion
Authors: Mele Deanna A. , Grimster Neil , Bommakanti Gayathri , Shields Jason , Morrill Lucas , Giblin Kathryn , SanMartin Maryann , Wang Yanjun , Ye Minwei , KarapaReddy Iswarya , Song Kun , Proia Theresa , Hariparsad Niresh , Richards Ryan , Luheshi Nadia , Barry Simon T. , Fawell Stephen 
Published: April 2023

Abstract
Immune checkpoint blockade (ICB) has revolutionized cancer therapy demonstrating durable responses in multiple tumor types, however only a subset of patients respond, in part due to mechanisms that drive T cell dysfunction. HPK1 is a key inhibitory signaling node associated with T cell exhaustion that is activated in response to T cell receptor engagement (signal 1). HPK1 is thus distinct from inhibitory checkpoint receptors which require surface receptor expression, antigen recognition (signal 1) and costimulatory engagement (signal 2) for activity. HPK1 therefore has potential for broader activity across T cell subsets independent of checkpoint engagement. Selectivity is a critical parameter for the optimal profile of an HPK1 inhibitor because many kinases are positive regulators of T cell signaling and can be antagonistic if inhibited. Utilizing a structure-based drug design approach, we identified compound 1 a highly potent (pSLP76 IC50=55nM) inhibitor of HPK1, which displayed no functional antagonistic effects in human T cells up to 30μM. We attributed this its highly selective nature, possessing &gt;80-fold selectivity against other MAP4K family members, and &gt;25-fold selectivity against all other kinases tested. We demonstrated an inverse correlation between inhibition of pSLP76 and increase in IL-2, IFNg and proliferation in human T cells. We report a novel role for HPK1 in regulating T cell exhaustion in a human ex vivo assay. Compound 1 treatment prevents T cell exhaustion at &gt; 0.1uM and can restore aspects of already exhausted T cells, most notably rescue of cytotoxicity. RNAseq and nuclear localization assays reveal for the first time that HPK1i mechanistically restores AP-1 signaling which enables heterodimerization with NFAT restoring human T cell functionality. We present data demonstrating that our HPK1 inhibitor elevates anti-tumor immunity in multiple syngeneic mouse models including MCA205, CT26 and EMT6. In vitro IC50 correlates with in vivo decrease of pSLP76 in tumors and blood of syngeneic models associated with efficacy. Immunophenotyping from syngeneic tumors treated with compound 1 shows a two-fold increase in CD8+ T cells and most significantly enhanced cytokine production in T cells. In patient NSCLC samples Compound 1 has single agent and combination activity with Durvalumab restoring cytokine production. These results thus reveal the role of AP-1/NFAT signaling in HPK1 biology and highlight the importance of HPK1 in T cell exhaustion in both healthy donor and NSCLC patient tumor T cells, potentially limiting responses to ICB.
Citation Format: Deanna A. Mele, Neil Grimster, Gayathri Bommakanti, Jason Shields, Lucas Morrill, Kathryn Giblin, Maryann SanMartin, Yanjun Wang, Minwei Ye, Iswarya KarapaReddy, Kun Song, Theresa Proia, Niresh Hariparsad, Ryan Richards, Nadia Luheshi, Simon T. Barry, Stephen Fawell. First disclosure of a highly potent and selective HPK1 inhibitor that rescues T cell exhaustion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3453.