DJ4 Targets Rho-associated Protein Kinase Pathway and Attenuates Disease Progression in Pre-clinical Murine Models of Acute Myeloid Leukemia

Introduction: The poor prognosis of acute myeloid leukemia (AML) and the highly heterogenous nature of the disease motivates targeted gene therapeutic investigations. Rho-associated protein kinases (ROCKs) are crucial for various actin cytoskeletal changes, which have established malignant consequences in various cancers, yet are still not being successfully utilized clinically towards cancer treatment. ROCK 1 and 2 overexpression has been linked to AML cell lines and overall survival of AML patients. This work reports the considerable therapeutic efficacy of the ROCK inhibitor DJ4 in both in vitro and in vivo preclinical models of AML to highlight the potential of this class of inhibitors. Experimental Design: The cytotoxic and pro-apoptotic activities of DJ4 for primary human AML samples and human AML cell lines was determined by cell viability (MTS), colony forming assays, and Annexin V assays. Immunoblot analysis was used to detect phosphorylation of downstream substrates of ROCK. To assess the preclinical therapeutic efficacy, the luciferase-expressing human AML cell line OCIAML-3-YFP-Luc was injected subcutaneously (SC) and intravenously (IV) into NRG-S mice. Mice were treated through an intraperitoneal (IP) injection with either vehicle control or DJ4 (10 mg/kg) for 3 weeks. Modified AML cell lines, OCI-AML3-YFP-Luc and MV4-11-Luc2-EGFP, were also treated with their respective IC50 dose of DJ4 or with vehicle control for 24 h and then administered via IV into NRG-S mice and the survival advantage was monitored over time without further treatment. Disease progression was tracked in mice studies by flow cytometry analysis and examining the survival, bioluminescent signal, tumor volume, and tumor weights of the animals over time. Results: DJ4 induced cytotoxic and pro-apoptotic effects, within the micromolar range and in a dose-dependent manner, in human AML cell lines (IC50: 0.05-1.68 μM) and primary patient cells (IC50: 0.264-13.43 μM) that harbored various mutations; however, normal hematopoietic cells are largely spared (Figures A-C). Treatment of DJ4 demonstrated ~5-fold selectivity towards AML patient samples relative to the CB-MNCs of healthy donors (IC50= 25 μM, Figure B). Representative flow cytometry plots of the Annexin V assay with AML primary cells (Figure C) depicted the increase in the apoptotic populations as a result of treatment with increasing concentrations of DJ4. ROCK inhibition by DJ4 disrupts the phosphorylation of downstream ta