HDAC Inhibition by SNDX-275 (Entinostat) Restores Expression of Silenced Leukemia-Associated Transcription Factors Nur77 and Nor1 and of Key Pro-Apoptotic Proteins in AML

Orphan nuclear receptors Nur77 & Nor1 are highly conserved members of the nuclear receptor family 4. They are involved in various physiological and cellular functions. Both proteins are products of immediate early genes triggered by growth factors or chemokines. Induction and activation of Nor-1 and/or Nur77 have been implicated in apoptosis for negative selection of T-lymphocytes and activation induced cell death of macrophages. In a previous study (Nat Med. 2007 Jun; 13(6):730–5) we reported that double knockout of Nur77 and Nor1 in mice resulted in the rapid development of AML. The disease was characterized with quick death of mice (2–4 weeks), abnormal expansion of hematopoietic stem cells (HSCs) and myeloid progenitors, hepatosplenomegaly, and lymphadenopathy. Molecular analysis revealed that the absence of both Nur77 and Nor1 leads to the downregulation of AP1 transcription factors c-Jun and JunB, and extrinsic apoptosis mediators TRAIL and FasL. Real- time RT-PCR analysis of AML patient samples (n=16) revealed the universal down-regulation of Nur77 and Nor1 transcripts in AML progenitor and stem cells (CD34+38−). These findings indicate that Nur77 and Nor1 play roles in maintaining hematopoiesis and silencing of Nur77 and Nor1 is a critical step in AML development. To investigate the role of Nur77/Nor1 in AML, we hypothesized that silenced gene expression of Nur7 and Nor1 is mediated by histone deacetylases (HDACs). HDACs are enzymes involved in chromatin modifications that play an important role in the epigenetic regulation of gene expression and mediate abnormal epigenetic alterations associated with cancer, which make HDAC inhibitors promising anti-cancer drugs. SNDX-275, a benzamide HDAC inhibitor that preferentially inhibits activities of nuclear HDACs, including HDAC1, HDAC2, and HDAC3, has been shown to induce extensive apoptosis in various cancer types and has progressed to clinical phase I and II trials. We demonstrated that HDAC inhibition with SNDX-275 restored the expression of Nur77 and Nor1 in AML cell lines and primary AML samples (n=5) to levels comparable to those found in normal bone marrows. In the knockout mouse model, TRAIL and AP-1 transcription factors (c-Jun and JunB) were identified as downstream target genes of Nur77 and Nor1. In our present study, we observed strong upregulation by SNDX-275 of TRAIL, c-Jun and JunB in leukemia cell lines and primary AML blasts. As is common with HDAC inihibitors, apoptosis induced by SNDX-275 was associated with dramatic H3 and H4 acetylation. We further exploited the lack of Nur77 and Nor1 in leukemic stem cells (CD34+/38−) and demonstrated that SNDX-275 restored their expression resulting in apoptotic cell death of leukemia stem cells. Besides, pro-apoptotic Bim and Noxa were transcriptionally upregulated by SNDX-275 in AML cell lines, primary AML samples and also leukemia stem cells. Altogether, our results suggest a novel mechanism of HDAC inhibitor-induced apoptosis in AML which involves the restoration of the silenced nuclear receptors Nur77 and Nor1 and the activation of their downstream target genes, in addition to affecting apoptotic machinery. These findings also characterize HDAC-mediated transcriptional silencing of nuclear receptors as one of the key mechanisms driving the pathogenesis of AML.