In:
Blood, American Society of Hematology, Vol. 138, No. Supplement 1 ( 2021-11-05), p. 3350-3350
Abstract:
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 targets, myosin light chain (MLC2), and myosin binding subunit of MLC phosphatase (MYPT) in OCI-AML3 and MV4-11, yielding a potent yet selective treatment response at micromolar concentrations, 0.02 to 1 μM (Figure D). Analysis of mice treated with DJ4 via IP for 2.5 weeks indicated that DJ4 was well-tolerated with comparable CBC with differential and chemistry values and tissue morphology from cross-sections of the spleen, liver, lung, and kidney relative to the vehicle treated group. Mice that were IV or SC injected with OCIAML3-YFP-Luc and treated via IP with DJ4 exhibited an increase in overall survival and reduction in disease progression relative to the vehicle treated control mice (Figure E). Towards the end of the study, the OCIAML3-YFP-Luc SC injected mice exhibited at least a 3-fold reduction in the bioluminescent signal, ~4-fold decrease in tumor volume, and the tumor weight was significantly reduced by ~3-fold among the DJ4 treated group relative to the control (Figure E). Mice that were IV administered DJ4 pretreated AML cells also exhibited a decreased bioluminescent signal, increased survival of 10 and 20 days, and a significant reduction in the percent of human CD45 cells 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 mutations suggests that this is a promising candidate to be incorporated into the standard AML regimen to help many different subsets of AML patients. Therapeutics that induce apoptosis have been shown to be promising candidates toward overcoming chemoresistance and to work well with appropriate combinations of standard of care drugs. This work highlights the potential of targeting the Rho-ROCK pathway to improve the prognosis of AML and the need for the future development of chemotherapeutics that are both less toxic and more effective. Figure 1 Figure 1. Disclosures Claxton: Astellas: Other: Clinical Trial; Novartis: Research Funding; Astex: Research Funding; Cyclacel: Research Funding; Daiichi Sankyo: Research Funding; Incyte: Research Funding.
Type of Medium:
Online Resource
ISSN:
0006-4971
,
1528-0020
DOI:
10.1182/blood-2021-154542
Language:
English
Publisher:
American Society of Hematology
Publication Date:
2021
detail.hit.zdb_id:
1468538-3
detail.hit.zdb_id:
80069-7
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