Abstract 1281: Development and characterization of small molecule HPK1 inhibitors

Abstract

Abstract Background: Hematopoietic progenitor kinase 1 (HPK1, MAP4K1) is a member of the serine/threonine MAP4K family predominantly expressed in hematopoietic cell lineages which plays a pivotal role in the negative regulation of the signaling cascade triggered by T cell receptor engagement. Inhibition of its activity promotes secretion of IL-2, T cell maturation, and proliferation. It has been also suggested that inhibition of HPK1 may hyperactivate both B cells and dendritic cells via hampering HPK1- mediated negative feedback mechanisms involved in BCR regulation and enhancement of antigen presenting capability of dendritic cells. Suppression of the TCR inhibitory mechanisms with small molecule HPK1 inhibitors might constitute a novel approach in tumor immunotherapy which enhances neoantigen recognition and boosts immune responses of T and B lymphocytes against cancer cells. Thus, small molecule HPK1 inhibitors may provide additional benefit for patients subjected to existing immunotherapies. Methods: Inhibition of HPK1 was assessed by biochemical assay with recombinant human and mouse protein. Small molecule inhibitors were tested in biochemical assay on selected anti- and off-targets and profiled against a broad kinase panel. Phosphorylation of serine 376 on SLP-76 adaptor protein and specific TCR activation-related markers upon HPK1 inhibition was monitored by either Western Blotting or flow cytometry in human and murine T-cells. IL-2 release was measured in human PBMCs and mouse splenic T cells. PBMC cells were activated and exposed to compounds in the presence of PGE-2, followed by IL-2 release measurement. Mice were challenged with the compounds and pharmacodynamic biomarkers were evaluated through flow cytometry and AlphaLisa. Results: Small molecule Ryvu HPK1 inhibitors block kinase activity of recombinant mouse and human protein with sub-nanomolar IC50 values. Ryvu compounds selectively engage downstream biomarkers in human and murine T cells. While inhibiting phosphorylation of serine 376, Ryvu HPK1 inhibitors do not affect activatory phosphorylation of specific phosphotyrosine residues of SLP-76 in human or mouse CD3+ T cells. Ryvu compounds overcome PGE-2 induced resistance following TCR activation in human PBMCs, and mouse CD3+ T cells, inducing IL-2 release. Ryvu compounds have good physicochemical properties and good overall selectivity. In vivo activity and target engagement have been confirmed in the model with anti-CD3 antibody infusion. Conclusion: Pharmacological inhibition of HPK1 kinase activity has strong potential to become a novel immunomodulatory approach for cancer treatment. Citation Format: Maciej Kujawa, Eliza Zimoląg, Michał Gałęzowski, Paweł Guzik, Agata Dudek, Andrzej Gondela, Marcin Nowogródzki, Marianna Girardi, Kostiantyn Krolenko, Marta Bugaj, Sylwia Sudoł, Agnieszka Gibas, Joanna Szeremeta-Spisak, Aneta Bobowska, Magdalena Zastawna, Przemysław Wyrębek, Nicolas Boutard, Aleksandra Więckowska, Wojciech Jasnosz, Wojciech Schonemann, Karol Zuchowicz, David Synak, Urszula Kulesza, Oleksandr Leventes, Mateusz Świrski, Sujit Sasmal, Karolina Gluza, Patryk Kret, Mateusz Ogórek, Kinga Michalik, Katarzyna Banaszak, Adrian Podkowa, Katarzyna Wnuk-Lipińska, Monika Dobrzańska, Peter Littlewood, Krzysztof Brzózka, Anna Bartosik, Stefan Chmielewski. Development and characterization of small molecule HPK1 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1281.