Abstract LB-83: Transcriptional mechanisms of NR4A orphan nuclear receptor tumor suppression in AML
Authors: Duren Ryan P. , Conneely Orla M. 
Published: May 2015

Abstract
Acute myeloid leukemias are a highly heterogeneous group of diseases associated with corruption of myeloid cell differentiation, accumulation of immature blasts, and emergence of a transformed leukemia initiating cell (LIC) capable of sustaining leukemic expansion. Genetic and phenotypic properties of LICs are highly variable among AML patients as a consequence of variable cytogenetics, stem/progenitor cell of origin and stage of disease progression. The orphan nuclear receptors NR4A1 and NR4A3 are potent AML tumor suppressors whose deletion in mice leads to rapid AML development. They are also silenced in cytogenetically diverse human LICs, and their anti-leukemic effect is associated with acute transcriptional repression of c-MYC and reprogramming of a core MYC oncogenic gene signature that is common to LICs of distinct cytogenetic backgrounds. c-MYC transcription is controlled at the step of transcriptional elongation, and high levels of c-MYC expression are maintained in many cancers, including AML, by establishment of cancer cell-selective super enhancers that are marked by high levels of the BET bromodomain protein, BRD4, a key regulator of c-MYC transcription elongation together with BRD4 associated components of the Mediator coactivator complex. BRD4 promotes transcription elongation of c-MYC by recruiting the positive transcription elongation factor, P-TEFb, a catalytic component of the super elongation complex (SEC) which phosphorylates RNA PolII at serine 2 to enable release of promoter paused RNA PolII. Here we investigated the transcriptional mechanisms of NR4A1-dependent c-MYC repression. Interrogation of genome-wide NR4A1 binding in Kasumi-1 AML-ETO positive human AML cells revealed NR4A1 occupancy within the 5’ proximal regions of the c-MYC gene and at a downstream enhancer region previously identified as a c-MYC super enhancer in the RN2 mouse model of MLL-AF9/NrasG12D driven AML. Consistent with its classification as a super enhancer, we found strong enrichment of BRD4 and the MED1 component of the Mediator complex at the c-MYC enhancer in Kasumi-1 AML cells in the absence of NR4A1. Interrogation of the effects of acute rescue of NR4A1 expression on the enhancer status of this region and transcriptional elongation of c-MYC revealed that NR4A1 represses c-MYC transcription elongation by interacting directly with the enhancer locus to inhibit recruitment of Mediator complex and BRD4, leading to loss of P-TEFb recruitment to both the enhancer and c-MYC gene and a loss of elongation competent RNA PolII. Together, these data indicate the NR4A1 negatively regulates transcriptional elongation at the c-MYC locus by interfering with factors required for super enhancer maintenance and AML selective overexpression of c-MYC. Since AML cells which rely on c-MYC overexpression are extremely sensitive to BRD4 inhibition, our results predict that strategies directed toward NR4A activation in AML cells may provide an alternative means of targeting c-MYC driven AMLs.
Citation Format: Ryan P. Duren, Orla M. Conneely. Transcriptional mechanisms of NR4A orphan nuclear receptor tumor suppression in AML. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-83. doi:10.1158/1538-7445.AM2014-LB-83