Abstract 1947: Development and characterization of small molecule HPK1 inhibitors

• Published in: Cancer research (Chicago, Ill.) Ill.), 2020-08, Vol.80 (16_Supplement), p.1947-1947
• Main Authors: Chmielewski, Stefan, Kujawa, Maciej, Zimoląg, Eliza, Guzik, Paweł, Dudek, Agata, Topolnicki, Grzegorz, Sudoł, Sylwia, Gibas, Agnieszka, Bugaj, Marta, Krolenko, Kostiantyn, Nowogródzki, Marcin, Janiga, Anita, Wyrębek, Przemysław, Pięta, Jakub, Brzdonkiewicz, Aleksandra, Wilkowski, Grzegorz, Walczak, Marcin, Maciejewska, Katarzyna, Radzimierski, Adam, Jasnosz, Wojciech, Mahajan, Tushar, Bartolotta, Roberta, Gluza, Karolina, Kret, Patryk, Rutkowska, Ewelina, Michalik, Kinga, Banaszak, Katarzyna, Podkowa, Adrian, Gołas, Aniela, Wnuk-Lipińska, Katarzyna, Fabritius, Charles, Stasi, Luigi, Littlewood, Peter, Brzózka, Krzysztof, Bartosik, Anna, Dobrzańska, Monika
• Format: Article
• Language: English
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/1538-7445.AM2020-1947

Background: Hematopoietic progenitor kinase 1 (HPK1, MAP4K1) is predominantly expressed in hematopoietic cell linages and serves as a negative regulator of T cells and dendritic cells (DC). Alteration of ERK/MAPK pathway by HPK1 in T-cells and dendritic cells is an inhibitory mechanism that negatively regulates TCR-induced IL-2 gene transcription, T cell maturation and proliferation. Inhibiting kinase activity of HPK1 results in activation of antigen presenting properties of dendritic cells and stimulates maturation and proliferation of T cells. Therefore, small molecule inhibitors of HPK1 could serve as a novel agent to transform cold, resistant tumors into sensitive hot cancers and provide additional benefit in combination with 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 other MAP4Ks and in addition profiled against broad kinase panel. Phosphorylation of Serine 376 and Tyrosine 128 of SLP-76 adaptor protein upon HPK1 inhibition was monitored by Western Blotting in human and murine T-cells. IL-2 release was monitored in total human PBMC, human CD3+ T cells and mouse CD3+ splenocytes. Human CD3+ T cells were isolated from PBMC, activated with plate-bound anti-CD3/anti-CD28 and exposed to compounds in the presence of PGE-2, followed by IL-2 release measurement, viability and proliferation assessment using flow cytometry. Mouse CD3+ splenocytes were isolated from Balb/c mice, activated with plate-bound anti-CD3/anti-CD28 and exposed to compounds in the presence of PGE-2, followed by IL-2 release assessment. Results: Small molecule Ryvu HPK1 inhibitors block kinase activity of recombinant mouse and human protein with nanomolar IC50 values. Ryvu compounds show broad kinome selectivity. Ryvu HPK1 inhibitors selectively engage downstream biomarkers in human and murine T cells. While inhibiting phosphorylation of Serine 376, Ryvu compounds do not affect activatory phosphorylation of Tyrosine 128 of SLP-76 in human or mouse CD3+ T cells. Ryvu HPK1 inhibitors overcome PGE-2 induced resistance following TCR activation in human PBMCs, CD3+ T-cells and mouse CD3+ T cells, inducing IL-2 release. Compounds have good druglike physicochemical properties. Conclusion: Ryvu HPK1 inhibitors promote activation of in-vitro immunostimulatory properties of both mouse and human immune cells, overcoming immunosuppression. The chemical s