Abstract 4938: DP-9024, an investigational small molecule modulator of the integrated stress response kinase GCN2, synergizes with asparaginase therapy in leukemic tumors
Authors: Al-Ani Gada , Elliott Kristin M. , Groer Qi , Rudeen Aaron J. , Kearney Patrick C. , Zwicker Jeffery D. , Ahn Yu Mi , Bulfer Stacie L. , Heiniger Cale L. , Hood Molly M. , Javed Salim , Large Joshua W. , Petty Max D. , Stoltz Kristen L. , Bourdonnec Bertrand Le , Smith Bryan D. , Flynn Daniel L. 
Published: April 2023

Abstract
Background: The integrated stress response (ISR) is a major adaptive pathway stress response pathway and plays an important role in cell fate determination in response to stress. Oncogene addicted tumors are under high levels of stress, both extrinsic as well as intrinsic, and are dependent on a well-balanced ISR to cope with the high metabolic demands for accelerated growth. The ISR is a double edge sword of survival and cell death, and depending on context, activation of the ISR kinase GCN2 can have either cytoprotective or cytotoxic effects. Activation of GCN2 was identified as a resistance mechanism to asparaginase (ASNase) in asparagine synthetase (ASNS)-low leukemic cells and MAPK-driven solid tumors.1-3 The inhibition of GCN2 in the context of ASNase-resistant leukemic cells can be pharmacologically leveraged to induce anti-tumoral effects.
Methods: Modulation of ISR kinases was characterized using enzymatic assays. Kinome selectivity profiling was determined using enzymatic and cellular assays. ISR pathway modulation was assessed using cellular assays of phospho-GCN2 and ATF4 by Western blot or ELISA under basal, ASNase-treated, or amino acid starvation conditions. Re-sensitization of leukemic cells to ASNase was tested in cell proliferation assays in vitro. In vivo compound-mediated reversal of ASNase-induced upregulation of tumoral ATF4 was determined in a leukemia PK/PD xenograft model. Inhibition of tumor growth was determined in leukemia xenograft models in vivo.
Results: DP-9024 was designed as a selective and potent modulator of PERK and GCN2. DP-9024 was found to reverse ASNase-mediated upregulation of ISR readouts (phospho-GCN2 and ATF4) in leukemic cells in vitro. Additionally, DP-9024-mediated inhibition of GCN2 was found to re-sensitize leukemic cells to ASNase in vitro. In vivo asparagine depletion by Leunase (L-asparaginase) led to upregulation of ATF4 in leukemia tumors in a PK/PD xenograft model and oral dosing of DP-9024 reversed Leunase-mediated upregulation of tumoral ATF4 down to basal levels. Importantly, we found that DP-9024-mediated inhibition of GCN2 strongly synergized with ASNase in vivo, leading to potent inhibition of tumor growth in leukemic xenografts and causing a reversal of ASNase resistance in leukemic tumors in vivo.
Conclusions: The ISR kinase GCN2 was identified as a resistance mechanism to ASNase in ASNS-low leukemic cells. Inhibition of the ISR pathway through the potent and selective small molecule modulator of GCN2, DP-9024, synergized with ASNase and re-sensitized leukemic cells to amino acid withdrawal in vitro as well as in leukemic xenograft models in vivo.
Re*****ferences:
Nakamura et al. 2018. PNAS 115(33):E7776-E7785
Apfel et al. 2021. ACS Pharamacology and Trans Sci 4(1):327-337
Gwin et al. 2018. Cancer Cell 33(1):91-107
Citation Format: Gada Al-Ani, Kristin M. Elliott, Qi Groer, Aaron J. Rudeen, Patrick C. Kearney, Jeffery D. Zwicker, Yu Mi Ahn, Stacie L. Bulfer, Cale L. Heiniger, Molly M. Hood, Salim Javed, Joshua W. Large, Max D. Petty, Kristen L. Stoltz, Bertrand Le Bourdonnec, Bryan D. Smith, Daniel L. Flynn. DP-9024, an investigational small molecule modulator of the integrated stress response kinase GCN2, synergizes with asparaginase therapy in leukemic tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4938.