Abstract 1367: HPK1 inhibition enhances T cell activation and relieves the immunosuppressive phenotype of inhibitory signals found in the tumor microenvironment
Authors: Singh Rajesh K. , Rayaji Rameshwari , Patel Bindi , Zhang Kristen , Marubayashi Sachie , Cho Soonweng , Garrido-Shaqfeh Stefan , Kulusich Joseph , Meleza Cesar , Tibrewal Nidhi , Thomas Joice , Nareddy Pradeep , Sharif Ehesan , Zhao Sharon , Green David , Leleti Manmohan R. , Powers Jay P. , DiRenzo Daniel , Walters Matthew J. 
Published: June 2022

Abstract
Introduction: T cell activation is critical in the initiation and potentiation of anti-tumor immune responses. Hematopoietic Progenitor Kinase 1 (HPK1/MAP4K1) is a member of the MAP4K family whose activity has been demonstrated to restrain T cell activation through phosphorylation of SLP-76 at Serine 376 leading to TCR disassembly. Mediators generated in the tumor microenvironment (TME) such as adenosine, PGE2 and TGFβ can dampen T cell activity and present a significant barrier to cancer therapy. HPK1 inhibition may allow for enhanced T cell activation under such suppressive conditions. Herein, we describe studies to assess the effects of HPK1 inhibition on T cell activation and in alleviating suppression. We also test the ability of HPK1 inhibition in combination with adenosine receptor antagonism to further amplify T cell activation.
Methods: CD8+ T cells were isolated from human blood and RNA was isolated for qPCR analysis of MAP4K1-7 expression. Cells were activated using CD3/28 stimulation and supernatants were assayed for IL-2, IFNɣ and TNFα using CBA. CRISPR knockout of HPK1 in CD8+ T cells and reference or in-house HPK1 inhibitors used to characterize HPK1 function. We used a flow cytometry-based assay to quantify activation markers CD69 and CD25, and phospho-SLP-76 in isolated CD8+ T cells and in human and mouse whole blood.
Results: Using publicly available gene expression databases, we identified that HPK1 is primarily expressed in immune cells, with highest expression in T cells, B cells, and dendritic cells. In-house gene expression analyses demonstrated that HPK1 is the predominant MAP4K family member in human T cells. CRISPR knockout in primary human CD8+ T cells was used to demonstrate that HPK1 but not MAP4K3 and MAP4K4 negatively regulates T cell activation. Consistent with this, reference and in-house HPK1 kinase inhibitors reduced phosphorylation of SLP-76 in both isolated T cells and human and mouse whole blood. In human T cells, HPK1 inhibition dose-dependently increased IL-2, IFNɣ and TNFα secretion, highlighting the negative role of HPK1 in T cell activation. Using adenosine, PGE2 and TGFβ, expression of CD69 and T cell cytokine secretion is diminished. HPK1 inhibition restored T cell activation to unsuppressed levels. Consistent with these results, HPK1KO CD8+ T cells displayed significant resistance to adenosine, PGE2 and TGFβ-induced immunosuppression. Finally, upon suppression using adenosine, combining HPK1 inhibition with adenosine receptor antagonism further elevated T cell activation above individual inhibitor treatments alone.
Conclusion: These data demonstrate that HPK1 plays a significant role in dampening T cell activation and provide a strong therapeutic rationale for targeting HPK1 to relieve T cell immunosuppression in the TME and amplify anti-tumor immune responses.
Citation Format: Rajesh K. Singh, Rameshwari Rayaji, Bindi Patel, Kristen Zhang, Sachie Marubayashi, Soonweng Cho, Stefan Garrido-Shaqfeh, Joseph Kulusich, Cesar Meleza, Nidhi Tibrewal, Joice Thomas, Pradeep Nareddy, Ehesan Sharif, Sharon Zhao, David Green, Manmohan R. Leleti, Jay P. Powers, Daniel DiRenzo, Matthew J. Walters. HPK1 inhibition enhances T cell activation and relieves the immunosuppressive phenotype of inhibitory signals found in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1367.