Abstract A30: Activation of GCN2 by HC-7366 results in significant anti-tumor efficacy as monotherapy and in combination with Venetoclax in AML models
Authors: Tameire Feven , Wojnarowicz Paulina M. , Fujisawa Sho , Huang Sharon , Reilly Owen , Dudgeon Crissy , Collette Nicholas , Drees Jeremy , Bieging-Rolett Kathryn , Kangas Takashi O , Zhang Weiyu , Fumagalli Maria , Dewji Iman , Li Yunfang , Chan Anissa SH , Qiu Xiaohong , Harrison Ben , LaCayo Ashley , Staschke Kirk A. , Rigby Alan C. , Ramurthy Savithri , Lightcap Eric S. , Surguladze David , Bose Nandita 
Published: May 2023

Abstract
The integrated stress response (ISR) is an adaptive signaling pathway that cells utilize to respond to a wide range of extrinsic and intrinsic stresses, many of which are important for tumorigenesis. Activation of ISR plays a dual role in cell fate decisions; while the ISR promotes survival, prolonged activation of ISR induces apoptosis. Activation of General Control Nonderepressible 2 (GCN2), an ISR kinase that senses and responds to nutrient stress conditions results in anti-tumor activity. We are developing HC-7366, a first-in-class, first-in-human GCN2 activator, and are currently evaluating it in a phase 1 clinical trial in solid tumors (NCT05121948). Here we present a series of studies characterizing the antitumor effects of HC-7366 in acute myeloid leukemia (AML). Higher expression of GCN2 and ISR markers such as ATF4 has been observed in primitive or minimally differentiated AML cells, suggesting that AML may be particularly sensitive to HC-7366. Encouragingly, in vivo efficacy studies in MOLM-16 and KG-1 tumor models showed 100% complete response and 100% tumor growth inhibition, respectively. Analysis of tumors from treated mice by IHC demonstrated activation of ISR as evidenced by increased expression of the ATF4 targets, ASNS and PSAT1, confirming that HC-7366 is functioning as a GCN2 activator in vivo. In the MV4-11 model, a differentiated subtype of AML that shows limited response to venetoclax, the combination of HC-7366 and venetoclax produced strong benefit resulting in 26% tumor regression. Enhanced activation of ISR pathway was again observed when HC-7366 was combined with venetoclax. HC-7366 treatment also impacted possible venetoclax resistance mechanisms by increasing the levels of PUMA while reducing levels of S100A8/A9 proteins. To investigate the effects of the compound on primary AML, we performed an ex vivo screen using cells from AML patients and treatment with HC-7366 showed a remarkable decrease in cell proliferation. Furthermore, in a xenotransplantable model of patient-derived AML, we found that HC-7366 significantly reduced mature myeloid (CD33+) AML cells in the bone marrow as compared to standard of care (SOC) agents, including venetoclax. We confirmed that activation of ISR, reduction in cell viability, and inhibition of protein synthesis following treatment with HC-7366 was dependent on GCN2 using CRISPR knockout cells. In addition, HC-7366 reduced mitochondrial respiration in MOLM-16 cells, suggesting effects on cellular bioenergetics. Metabolomics analyses of AML xenograft tumors showed that HC-7366 significantly altered metabolites associated with amino acid metabolism, urea cycle, and oxidative stress. Together, our in vitro and in vivo results demonstrate that HC-7366 is a potent GCN2 activator with strong antitumor activity in AML as a single agent and in combination with venetoclax, supporting its investigation in clinical trials in patients with AML.
Citation Format: Feven Tameire, Paulina M. Wojnarowicz, Sho Fujisawa, Sharon Huang, Owen Reilly, Crissy Dudgeon, Nicholas Collette, Jeremy Drees, Kathryn Bieging-Rolett, Takashi O Kangas, Weiyu Zhang, Maria Fumagalli, Iman Dewji, Yunfang Li, Anissa SH Chan, Xiaohong Qiu, Ben Harrison, Ashley LaCayo, Kirk A. Staschke, Alan C. Rigby, Savithri Ramurthy, Eric S. Lightcap, David Surguladze, Nandita Bose. Activation of GCN2 by HC-7366 results in significant anti-tumor efficacy as monotherapy and in combination with Venetoclax in AML models [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A30.