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The transcription factor NFE2 is overexpressed in most patients with myeloproliferative neoplasms (MPN). Moreover, mutations in NFE2, found in a subset of MPN patients, strongly predispose for transformation to acute leukemia. Transgenic mice overexpressing NFE2 as well as mice harboring NFE2 mutations display an MPN phenotype and spontaneously develop leukemia. However, the molecular mechanisms effecting NFE2-driven leukemic transformation remain incompletely understood. Here we show that the pro-leukemic histone demethylase JMJD2C constitutes a novel NFE2 target gene. JMJD2C expression is elevated in MPN patients as well as in NFE2 transgenic mice. Moreover, we show that loss of JMJD2C selectively impairs proliferation of JAK2V617F mutated cells. Our data suggest that JMJD2C represents a promising drug target in MPN and provide a rationale for further investigation in preclinical and clinical settings.

Acute myeloid leukemia (AML) carries a 10–100 fold lower mutational burden than other neoplastic entities. Mechanistic explanations for why a low number of mutations suffice to induce leukemogenesis are therefore required. Here we demonstrate that transgenic overexpression of the wild type sphingosine-1-phosphate receptor 3 (S1P3) in murine hematopoietic stem cells is sufficient to induce a transplantable myeloid leukemia. In contrast, S1P3 expression in more mature compartments does not cause malignant transformation. Treatment with the sphingosine phosphate receptor modulator Fingolimod, which prevents receptor signaling, normalized peripheral blood cell counts and reduced spleen sizes in S1P3 expressing mice. Gene expression analyses in AML patients revealed elevated S1P3 expression specifically in two molecular subclasses. Our data suggest a previously unrecognized contribution of wild type S1P3 signaling to leukemogenesis that warrants the exploration of S1P3 antagonists in preclinical AML models.

In acute myeloid leukemia (AML), acquired genetic aberrations carry prognostic implications and guide therapeutic decisions. Clinical algorithms have been improved by the incorporation of novel aberrations. Here, we report the presence and functional characterization of mutations in the transcription factor NFE2 in patients with AML and in a patient with myelosarcoma. We previously described NFE2 mutations in patients with myeloproliferative neoplasms and demonstrated that expression of mutant NFE2 in mice causes a myeloproliferative phenotype. Now, we show that, during follow-up, 34% of these mice transform to leukemia presenting with or without concomitant myelosarcomas, or develop isolated myelosarcomas. These myelosarcomas and leukemias acquired AML-specific alterations, including the murine equivalent of trisomy 8, loss of the AML commonly deleted region on chromosome 5q, and mutations in the tumor suppressor Trp53. Our data show that mutations in NFE2 predispose to the acquisition of secondary changes promoting the development of myelosarcoma and/or AML.

Activity of the transcription factor NFE2 is elevated in the majority of patients with myeloproliferative neoplasms (MPNs), either by overexpression of the wild-type alleles or by the presence of an activating mutation. In murine models, enhanced NFE2 activity causes an MPN phenotype with spontaneous transformation to acute leukemia. However, little is known about the downstream target genes activated by augmented NFE2 levels. Here, we describe that NFE2 regulates expression of the hematopoietic master regulators GATA2 and SCL/TAL1, which are in turn overexpressed in primary MPN cells, suggesting that concomitant aberrant activation of several transcription factors coordinately contributes to the cellular expansion characteristic of these disorders.

The transcription factor "nuclear factor erythroid 2" (NFE2) is overexpressed in the majority of patients with myeloproliferative neoplasms (MPNs). In murine models, elevated NFE2 levels cause an MPN phenotype with spontaneous leukemic transformation. However, both the molecular mechanisms leading to NFE2 overexpression and its downstream targets remain incompletely understood. Here, we show that the histone demethylase JMJD1C constitutes a novel NFE2 target gene. JMJD1C levels are significantly elevated in polycythemia vera (PV) and primary myelofibrosis patients; concomitantly, global H3K9me1 and H3K9me2 levels are significantly decreased. JMJD1C binding to the NFE2 promoter is increased in PV patients, decreasing both H3K9me2 levels and binding of the repressive heterochromatin protein-1α (HP1α). Hence, JMJD1C and NFE2 participate in a novel autoregulatory loop. Depleting JMJD1C expression significantly reduced cytokine-independent growth of an MPN cell line. Independently, NFE2 is regulated through the epigenetic JAK2 pathway by phosphorylation of H3Y41. This likewise inhibits HP1α binding. Treatment with decitabine lowered H3Y41ph and augmented H3K9me2 levels at the NFE2 locus in HEL cells, thereby increasing HP1α binding, which normalized NFE2 expression selectively in JAK2V617F-positive cell lines.

Background: Expression of the transcription factor and epigenetic modulator “nuclear factor erythroid-2” (NF-E2) is aberrantly elevated in patients with Myeloproliferative Neoplasms (MPN). We have shown that NF-E2 overexpression in a murine model causes a MPN phenotype. In addition, we have identified in-del mutations in the NF-E2 gene in MPN patients, which, despite having lost the ability to bind DNA, nonetheless increase the activity of wild type NF-E2. These mutants, which are acquired secondary to the JAK2V617F mutation, confer a proliferative advantage beyond that provided by the JAK2V617F alone. In all patients analyzed, cells carrying both mutations displayed clonal dominance, vastly outnumbering those carrying JAK2V617Falone. Mutant NF-E2 causes an increase in the number of cells entering S-phase mediated by increased expression of the cell cycle regulators CDK4, CDK6 and CyclinD3 that promote the G1-S transition. However, it is not known whether these cell cycle regulators constitute direct NF-E2 target genes or whether the proliferative effect is indirect. Moreover, additional downstream pathways by which this augmented transcription factor activity exerts its effects remain unknown. Aims: We therefore investigated whether CDK4, CDK6 and Cyclin D3 constitute direct NF-E2 target genes. Moreover, we identified additional novel NF-E2 targets in an effort to delineate the molecular mechanism by which increased NF-E2 activity contributes to MPN pathophysiology. Methods: We used ChIP and luciferase reporter assays as well as qRT-PCR and Western Blot to identify and characterize novel NF-E2 targets. Results: Our data show that both the cell cycle regulators CDK4, CDK6 and CyclinD3 as well as the histone methyltransferases MLL2 and MLL4 constitute direct NF-E2 target genes, as the transcription factor binds these genes in vivo. ChIP analysis of HEL cells shows NF-E2 binding to a site in the CDK4 promoter, four sites in the CDK6 and the Cyclin D3 locus each, to the MLL4 promoter as well as to three sites in the MLL2 locus, establishing these five genes as direct NF-E2 targets. Activity of NF-E2 on the MLL4 promoter was confirmed by reporter gene assays. Expression of both the cell cycle regulators and the methyltransferases was statistically significantly elevated in bone marrow from NF-E2 overexpressing mice. Most importantly, quantitative RT-PCR showed significantly increased mRNA expression of all five target genes in primary cells of patients with polycythemia vera (PV) compared to healthy controls. Hence, the expression of the cell cycle regulators and the epigenetically active enzymes is augmented in PV patients. Corresponding to the increased methyltransferase activity, primary cells from PV patients showed a significant elevation in global H3K4m1 levels, a chromatin mark conferred by the MLL proteins. The MLL2 protein has been previously shown to interact with NF-E2. NF-E2 serves as a scaffold and is required both for recruitment of MLL2 to the beta-globin locus as well as for chromatin remodeling at these sites. Here we show that MLL2 binds the CDK6 locus at the same sites as NF-E2, strongly suggesting that NF-E2 likewise acts to direct MLL2 to the CDK6 gene. Our data establish a novel interaction network where NF-E2 both regulates the expression levels of MLL2 while at the same time modulating its epigenetic activity at NF-E2 target genes, which include critical cell cycle regulators. Summary / Conclusions: Here we show that the cell cycle regulators CDK4, CDK6 and Cyclin D3 as well as the histone methyltransferases MLL2 and MLL4 constitute novel NF-E2 target genes. We propose that elevated NF-E2 levels in MPN patients contribute to disease pathophysiology by participating in a novel regulatory loop, which both enhances transcription and alteres histone methylation leading to increased proliferation. These data provide a molecular basis for pre-clinical investigation into the effects of CDK 4/6 inhibitors as well as of histone methyltransferase inhibitors on MPN cell biology. Disclosures No relevant conflicts of interest to declare.