Abstract 2677: ROCK inhibitors synergize with the BCL2 inhibitor venetoclax for treatment of AML
Authors: Golla Upendarrao , Patel Satyam , Tukaramrao Diwakar B. , Hengst Jeremy , Restrepo Juliana , Amin Shantu , Desai Dhimant , Dovat Sinisa , Claxton David , Sharma Arati 
Published: April 2023

Abstract
Acute Myeloid Leukemia (AML) is a rare but catastrophic hematological cancer with a poor prognosis, high mortality, and relapse rates. Several new agents targeting specific genes or cell survival pathways have been recently approved by the FDA. AML treatment with single agents often results in resistance; therefore, combination therapies may provide more durable and effective responses. Rho kinases (ROCK1/2) are aberrantly expressed in AML patient cells, and ROCK1 has emerged lately as a potential therapeutic target for AML treatment. Previously, our group developed a new ROCK inhibitor DJ4 as an effective inducer of AML cell death. Our initial screening of DJ4 with AML standard therapy (cytarabine, daunorubicin, azacytidine, venetoclax) showed promising synergy between DJ4 and the BCL2 (B-cell lymphoma-2) inhibitor venetoclax (VEN). Enrichment of ROCK pathway in recent CRISPR drop-out screening after VEN treatment in MOLM-13 cells further validates the synergistic cooperation between ROCK and BCL2 pathways. Though VEN is widely used in hematological malignancies, its clinical efficacy is limited due to the development of resistance. Thus, we sought to investigate the novel strategy of combining VEN with ROCK inhibitors (ROCKi) for AML treatment. The combination of VEN with ROCKi (Fasudil, Y-27632, GSK269962A, DJ4) exhibited synergetic cytotoxicity in naïve (MOLM-13, MV4-11) and VEN-resistant (MV4-11/VENR, HL-60, OCI-AML3, U937) human AML cell lines. Flow cytometric analysis of Annexin V and cellular ROS levels in MV4-11 (naïve and VENR) cells upon co-treatment with sub-lethal doses of VEN and ROCKi demonstrated a significant (p&lt;0.05) increase in the apoptosis and oxidative stress compared to single agents. Treatment of MV4-11 cells with VEN+ROCKi enhanced the activity of several caspases in the MultiCaspase assay. Furthermore, the colony-forming ability of AML cell lines and primary AML patient cells was significantly reduced with the combination treatment, while single agents exhibited minimal effects. MV4-11 (naïve and VENR) cells displayed alterations in the levels of critical apoptosis regulators (Bcl2, Bax, Mcl1, cleaved caspase-3) upon co-treatment in immunoblotting analysis. Additionally, MV4-11-Luc xenograft mice exhibited a significant reduction in the average leukemic burden after combination therapy relative to single agents (p&lt;0.05). Altogether, our in vitro and in vivo findings established a synergetic interaction between VEN and ROCKi to effectively inhibit AML cell progression and overcome the VEN resistance (inherent or acquired). These results indicate that therapy co-targeting BCL2 and ROCK pathways exerted antileukemic activity by inducing apoptosis and may be particularly effective for AML treatment. Further studies are ongoing to explore the precise molecular mechanism of action of the VEN+ROCKi combination and its efficacy in pre-clinical animal models.
Citation Format: Upendarrao Golla, Satyam Patel, Diwakar B. Tukaramrao, Jeremy Hengst, Juliana Restrepo, Shantu Amin, Dhimant Desai, Sinisa Dovat, David Claxton, Arati Sharma. ROCK inhibitors synergize with the BCL2 inhibitor venetoclax for treatment of AML [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 2677.