Targeting Gcn2 Kinase-Driven Stress Response Inactivation By Orally Available Small Molecules To Restore Immune Tumor Microenvironment In Prostate Cancers.

Abstract In patients with metastatic castration-resistant prostate cancers (mCRPC) who have previously been treated with abiraterone or enzalutamide, the median overall survival is only 14 months. Unlike many other cancers, immunotherapies have had limited successes, due to the fact that there are very few T cells in the tumor microenvironment of prostate cancer (PC) patients. Identifying ways to boost immunotherapy responses could change the paradigm of mCRPC, a disease still difficult to treat. The highly proliferative nature of tumor cells, along with infiltration of myeloid cells into the tumors, leads to depletion of nutrients such as functional/natural amino acids. This metabolically stressful milieu causes activation of nutrient stress pathways, autophagy, and repressed immune responses. A key meditator of this nutrient stress pathway is a cytoplasmic Ser/Thr protein kinase called General Control Nonderepressible 2 (GCN2), also called EIF2AK4. GCN2 switches on by a reduction of amino acids, and its activity results in T cell inactivation, T cell death, regulatory T cell expansion, and the potentiation of myeloid-derived suppressor cells (MDSCs). We have developed and synthesized a series of novel small molecule immunotherapeutic agents that reversibly bind to GCN2 kinase, competitively block the ATP site, and elicit pharmacological responses in immune cells. GCN2 cell-free kinase binding, kinome selectivity, pGCN2, pEIF2α, ATF-4 phosphorylation inhibition assays were performed and confirmed it's on-target efficacy and potency of lead GCN2 inhibitor HCI-1046. In these studies, our lead GCN2 kinase inhibitor HCI-1046 demonstrated potent activity with an IC50 of 36 nM in inhibiting GCN2. GCN2 expression has been detected in PC-3, DU-145, and LNCap cell lines, and HCI-1046 exhibited cellular efficacy with an IC50 of 0.9 to 8 μM range, reduced phosphorylation of GCN2, ATF-4 significantly and its downstream target eIF2α. HCI-1046 inhibited pGCN2 and pEIF2α in human prostate cancer clinical patient samples derived MDSC cultured in amino-acid starved conditions. HCI-1046, in a dose-dependent fashion, restored CD8+ T cell proliferation and function in clinical samples of PC patients. Our preliminary results support the hypothesis that inhibition of GCN2 reinstates anti-tumor immunity and blocks tumor progression in PC cellular models. In vivo PK studies of HCI-1046 in rodent species showed excellent PK properties; 55% oral bioavailability, low clearance, and >5 hours half-life. HCI-1046 is nominated as a pre-clinical agent. Additional 3D cellular efficacy studies, FACS for cellular apoptosis, cell migration, live PC cells including immuno-ELISA, ELISpot experimental results for MDSCs suppressive function of T cells, and restoration by HCI-1046 will be presented at the conference. PC mouse model efficacy studies will also be discussed. Citation Format: Kyle Medley, Zhaoliang Li, Dongqing Yan, Umang Swami, Neeraj Agarwal, Hariprasad Vankayalapati. Targeting GCN2 kinase-driven stress response inactivation by orally available small molecules to restore immune tumor microenvironment in prostate cancers [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 LB136.