Abstract 721: CHD4 mediates genotoxic sensitivity and tumor initiation of AML cells through both MBD2-NuRD and MBD3-NuRD co-repressor complexes

Abstract

Abstract Despite several advances in therapy, the 5-year survival of AML, estimated at 40-45% for the young and 10% for the elderly, remains dismal. New modalities of therapy are thus clearly needed. Research in our laboratory has identified CHD4, an ATPase chromatin re-modeling component of the NuRD co-repressor complex, as an important mediator of genotoxic sensitivity to standard chemotherapy in AML cell lines and primary AML samples. Knockdown of CHD4 also markedly reduced colonies in soft agar. Interestingly, normal hematopoietic progenitor cells did not show these phenotypic alterations. Classical NuRD complex contains either MBD2 or MBD3, and at least four other proteins. We sought to determine if the previously observed CHD4 effects are through MBD2-NuRD or MBD3-NuRD. MBD2 and MBD3 are thought to link DNA to the GATAD2/CHD4 NuRD sub-components through coiled-coil domains of both proteins interacting with those of GATAD2A/B. It was thus hypothesized that if the observed effects of CHD4 are mediated through canonical NuRD, knockdown of either MBD2 or MBD3 should be able to replicate these. In order pursue this aim, we first knocked down MBD2 in U937 cell lines and performed colony forming assays. Persistent efforts to knockdown MBD3, however, were unsuccessful. Therefore, MBD3 was knocked out using CRISPR-Cas9. Single cell cloning to generate a completely knocked out cell line, however, introduces a potential intrinsic bias in colony forming assays by selecting for cells with an inherently increased colony forming potential. A guide RNA with an ~80% overall knockout efficiency was thus selected and the assay performed on the bulk population of U937 cells prior to limiting dilution and plating. U937 cells with CHD4 knocked down were used as positive controls. Neither MBD2, nor MBD3, when knocked down by 90% or knocked out, respectively, showed any effect on colony formation in vitro using the soft agar colony forming assay. However, knocking down MBD2 in U937 cells with MBD3 knocked out, resulted a significant reduction in the colony forming potential. A similar result was obtained in the genotoxic sensitivity assay, whereby cells were treated with cytosine arabinoside (AraC) and the percentage apoptosis determined by flow cytometry through 7-AAD staining as well as caspase cleavage assays. MBD2 knockdown did not increase the genotoxic sensitivity of U937 cells, while MBD3 knockout increased it only marginally. MBD2 knockdown in MBD3 knockout U937 cell lines, however, did markedly increase the sensitivity of these cells to AraC. We conclude that in terms of mediation of AML colony forming potential and sensitivity to standard chemotherapy agents, MBD2-NuRD and MBD3-NuRD may be redundant. Future therapeutic strategies aimed at exploiting the potent effects of disrupting the NuRD complex on AML cell sensitivity to chemotherapy and tumor initiating activity need to take this into account. Citation Format: Javeria Aijaz, Justin Sperlazza, Gordon D. Ginder. CHD4 mediates genotoxic sensitivity and tumor initiation of AML cells through both MBD2-NuRD and MBD3-NuRD co-repressor complexes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 721.