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

Abstract

Introduction: The poor prognosis of acute myeloid leukemia (AML) and the highly heterogenous nature of the diseasemotivates targeted gene therapeutic investigations. Rho-associated protein kinases (ROCKs) are crucial for variousactin cytoskeletal changes, which have established malignant consequences in various cancers, yet are still not beingsuccessfully utilized clinically towards cancer treatment. ROCK 1 and 2 overexpression has been linked to AML celllines and overall survival of AML patients. This work reports the considerable therapeutic efficacy of the ROCKinhibitor 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 humanAML cell lines was determined by cell viability (MTS), colony forming assays, and Annexin V assays. Immunoblotanalysis was used to detect phosphorylation of downstream substrates of ROCK. To assess the preclinical therapeuticefficacy, the luciferase-expressing human AML cell line OCIAML-3-YFP-Luc was injected subcutaneously (SC) andintravenously (IV) into NRG-S mice. Mice were treated through an intraperitoneal (IP) injection with either vehiclecontrol or DJ4 (10 mg/kg) for 3 weeks. Modified AML cell lines, OCI-AML3-YFP-Luc and MV4-11-Luc2-EGFP, werealso treated with their respective IC50 dose of DJ4 or with vehicle control for 24 h and then administered via IV intoNRG-S mice and the survival advantage was monitored over time without further treatment. Disease progression wastracked 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-dependentmanner, in human AML cell lines (IC50: 0.05-1.68 μM) and primary patient cells (IC50: 0.264-13.43 μM) that harboredvarious mutations; however, normal hematopoietic cells are largely spared (Figures A-C). Treatment of DJ4demonstrated ~5-fold selectivity towards AML patient samples relative to the CB-MNCs of healthy donors (IC50= 25μM, Figure B). Representative flow cytometry plots of the Annexin V assay with AML primary cells (Figure C) depictedthe increase in the apoptotic populations as a result of treatment with increasing concentrations of DJ4. ROCKinhibition by DJ4 disrupts the phosphorylation of downstream targets, myosin light chain (MLC2), and myosin bindingsubunit of MLC phosphatase (MYPT) in OCI-AML3 and MV4-11, yielding a potent yet selective treatment response atmicromolar concentrations, 0.02 to 1 μM (Figure D). Analysis of mice treated with DJ4 via IP for 2.5 weeks indicatedthat DJ4 was well-tolerated with comparable CBC with differential and chemistry values and tissue morphology fromcross-sections of the spleen, liver, lung, and kidney relative to the vehicle treated group. Mice that were IV or SCinjected with OCIAML3-YFP-Luc and treated via IP with DJ4 exhibited an increase in overall survival and reductionin disease progression relative to the vehicle treated control mice (Figure E). Towards the end of the study, theOCIAML3-YFP-Luc SC injected mice exhibited at least a 3-fold reduction in the bioluminescent signal, ~4-fold decreasein tumor volume, and the tumor weight was significantly reduced by ~3-fold among the DJ4 treated group relative tothe control (Figure E). Mice that were IV administered DJ4 pretreated AML cells also exhibited a decreasedbioluminescent signal, increased survival of 10 and 20 days, and a significant reduction in the percent of human CD45cells in the bone marrow or spleen, indicating a diminished tumor burden (Figure F).Conclusion:The observed potency of DJ4 towards various AML cell lines and primary samples with a diverse set of mutationssuggests that this is a promising candidate to be incorporated into the standard AML regimen to help many differentsubsets of AML patients. Therapeutics that induce apoptosis have been shown to be promising candidates towardovercoming chemoresistance and to work well with appropriate combinations of standard of care drugs. This workhighlights the potential of targeting the Rho-ROCK pathway to improve the prognosis of AML and the need for thefuture development of chemotherapeutics that are both less toxic and more effective. [Display omitted] DisclosuresClaxton: Astellas: Other: Clinical Trial; Novartis: Research Funding; Astex: Research Funding; Cyclacel: Research Funding; Daiichi Sankyo: Research Funding; Incyte: Research Funding.