Epigenetic silencing of the oncogenic miR-17-92 cluster during PU.1-directed macrophage differentiationThis study unravels an epigenetic mechanism for the regulation of the oncogenic miRNA cluster 17-92, involving the master hematopoietic transcription factor PU.1/Egr-2 and Jarid1b.

IntroductionTranscriptional regulation of major hematopoietic oncogenes and tumor suppressor genes represents a critical step in tumor formation, tumor aggressiveness, and therapy resistance. [1][2][3] In addition, a posttranscriptional inhibitory mechanism involving microRNA (miRNA) binding to the 3Ј-untranslated region of target mRNAs causes transcript degradation or interferes with the translation initiation and has been linked to tumorigenesis. 4,5 Under physiologic conditions, miRNAs regulate developmental processes and cell fate decisions, and tight regulation of their levels represents an important factor in cell and tissue homeostasis. 6 MiR-155, a well-studied miRNA, regulates hematopoietic cell development as documented by murine gene targeting experiments and also by other studies describing its function during immune B-and T-cell response, in production of cytokines and antibodies and in antigen presentation. 7,8 Next, transgenic miR-155 overexpression in the mouse stimulates B-cell proliferation and frequent development of lymphomas. 9 In humans, miR-155 up-regulation has been repeatedly reported in chronic B-cell lymphocytic leukemia (B-CLL), in its solid indolent form of a small lymphocytic lymphoma [10][11][12] and also in aggressive types, including non-Hodgkin 10,13,14 and Hodgkin lymphomas. 13,15 Deregulation of several microRNAs was repeatedly described in B-CLL. 16,17 B-CLL, the most common adult leukemia, is characterized by clonal accumulation of B celllike mature-appearing elements (Ͼ 5000/L) 18 typically coexpressing the CD5, CD19, CD20, and CD23 surface markers. B-CLL represents a heterogeneous disease, the outcome of which may be predicted by the levels of surface protein CD38, intracellular tyrosine kinase ZAP70, or by a status of IgV H somatic hypermutation. 18,19 Cytogenetic alterations of 2 loci that contain the p53 gene (deletion of 17p) and the ATM gene (deletion of 11q) are associated with poor prognosis, shorter duration of remission, and shortest overall survival, 20 whereas normal karyotype or trisomy 12 is considered intermediate risk and the 13q14 deletion is considered a favorable mark. Subsets of B-CLL patients may progress to non-Hodgkin diffuse large B-cell lymphoma by a mechanism that remains largely unknown. Taken together, miR-155 appears to play a central role in B-cell function, and its up-regulation in lymphoproliferative disorders, including B-CLL, may lead to a block of differentiation and accumulation of lymphoid-like cells.Recent studies brought evidence of a context-dependent transcriptional regulation of the MIR155HG. First, oncogenic properties of miR-155 have been demonstrated in breast cancer cells where MIR155HG is up-regulated by transforming growth factor-␤/ Smad pathway involving a Smad response element at the position Ϫ454 nt from the transcription start site (TSS). 21 This regulatory pathway becomes disabled on inhibition of miR-155, resulting in derepression of miR-155 targets (including the RhoA protein) and in decreased cell migration and invasio...

Epigenetic 5-azacitidine (AZA) therapy of high-risk myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML) represents a promising, albeit not fully understood, approach. Hematopoietic transcription factor PU.1 is dynamically regulated by upstream regulatory element (URE), whose deletion causes downregulation of PU.1 leading to AML in mouse. In this study a significant group of the high-risk MDS patients, as well as MDS cell lines, displayed downregulation of PU.1 expression within CD34 þ cells, which was associated with DNA methylation of the URE. AZA treatment in vitro significantly demethylated URE, leading to upregulation of PU.1 followed by derepression of its transcriptional targets and onset of myeloid differentiation. Addition of colony-stimulating factors (CSFs; granulocyte-CSF, granulocyte --macrophage-CSF and macrophage-CSF) modulated AZA-mediated effects on reprogramming of histone modifications at the URE and cell differentiation outcome. Our data collectively support the importance of modifying the URE chromatin structure as a regulatory mechanism of AZA-mediated activation of PU.1 and induction of the myeloid program in MDS.

PU.1 downregulation within hematopoietic stem and progenitor cells (HSPCs) is the primary mechanism for the development of acute myeloid leukemia (AML) in mice with homozygous deletion of the upstream regulatory element (URE) of PU.1 gene. p53 is a well-known tumor suppressor that is often mutated in human hematologic malignancies including AML and adds to their aggressiveness; however, its genetic deletion does not cause AML in mouse. Deletion of p53 in the PU.1(ure/ure) mice (PU.1(ure/ure)p53(-/-)) results in more aggressive AML with shortened overall survival. PU.1(ure/ure)p53(-/-) progenitors express significantly lower PU.1 levels. In addition to URE deletion we searched for other mechanisms that in the absence of p53 contribute to decreased PU.1 levels in PU.1(ure/ure)p53(-/-) mice. We found involvement of Myb and miR-155 in downregulation of PU.1 in aggressive murine AML. Upon inhibition of either Myb or miR-155 in vitro the AML progenitors restore PU.1 levels and lose leukemic cell growth similarly to PU.1 rescue. The MYB/miR-155/PU.1 axis is a target of p53 and is activated early after p53 loss as indicated by transient p53 knockdown. Furthermore, deregulation of both MYB and miR-155 coupled with PU.1 downregulation was observed in human AML, suggesting that MYB/miR-155/PU.1 mechanism may be involved in the pathogenesis of AML and its aggressiveness characterized by p53 mutation.

The transcription factor PU.1 and its inhibitory microRNA-155 (miR-155) are important regulators of B-cell differentiation. PU.1 downregulation coupled with oncogenic miR-155 upregulation has been reported in lymphoid malignancies; however, these data have not been studied across different subtypes in relation to clinical outcomes. We studied expression of miR-155 and PU.1 in the six most prevalent human B-cell lymphomas (n = 131) including aggressive (DLBCL, HL, MCL) and indolent (B-CLL/SLL, MZL, FL) types. Levels of miR-155 and PU.1 inversely correlated in DLBCL, B-CLL/SLL, and FL tumor tissues. In HL tissues, an exceptionally high level of miR-155 was found in patients with unfavorable responses to first-line therapy and those who had shorter survival times. PU.1 downregulation was noted in B-CLL/SLL samples positive for the adverse prognostic markers CD38 and ZAP-70. Upregulation of miR-155 and downregulation of PU.1 expression are integral aspects of lymphoma biology that could mark aggressive behavior of some, but not all, lymphoma types.