Transcription factors regulate gene expression by binding regulatory DNA. Understanding the rules governing such binding is an essential step in describing the network of regulatory interactions, and its pathological alterations. We show that describing regulatory regions in terms of their profile of total binding affinities for transcription factors leads to increased predictive power compared to methods based on the identification of discrete binding sites. This applies both to the prediction of transcription factor binding as revealed by ChIP-seq experiments and to the prediction of gene expression through RNA-seq. Further significant improvements in predictive power are obtained when regulatory regions are defined based on chromatin states inferred from histone modification data.

Long non-coding RNAs (lncRNAs) can affect multiple layers of gene expression to control crucial cellular functions. We have previously demonstrated that the lncRNA EPR, by controlling gene expression at different levels, affects cell proliferation and migration in cultured mammary gland cells and impairs breast tumor formation in an orthotopic transplant model in mice. Here, we used ChIRP-Seq to identify EPR binding sites on chromatin of NMuMG mammary gland cells overexpressing EPR and identified its trans binding sites in the genome. Then, with the purpose of relating EPR/chromatin interactions to the reshaping of the epitranscriptome landscape, we profiled histone activation marks at promoter/enhancer regions by ChIP-Seq. Finally, we integrated data derived from ChIRP-Seq, ChIP-Seq as well as RNA-Seq in a comprehensive analysis and we selected a group of bona fide direct transcriptional targets of EPR. Among them, we identified a subset of EPR targets whose expression is controlled by TGF-β with one of them—Arrdc3—being able to modulate Epithelial to Mesenchymal Transition. This experimental framework allowed us to correlate lncRNA/chromatin interactions with the real outcome of gene expression and to start defining the gene network regulated by EPR as a component of the TGF-β pathway.

EPR is a long non-coding RNA (lncRNA) that controls cell proliferation in mammary gland cells by regulating gene transcription. Here, we report on Mettl7a1 as a direct target of EPR. We show that EPR induces Mettl7a1 transcription by rewiring three-dimensional chromatin interactions at the Mettl7a1 locus. Our data indicate that METTL7A1 contributes to EPR-dependent inhibition of TGF-β signaling. METTL7A1 is absent in tumorigenic murine mammary gland cells and its human ortholog (METTL7A) is downregulated in breast cancers. Importantly, re-expression of METTL7A1 in 4T1 tumorigenic cells attenuates their transformation potential, with the putative methyltransferase activity of METTL7A1 being dispensable for its biological functions. We found that METTL7A1 localizes in the cytoplasm whereby it interacts with factors implicated in the early steps of mRNA translation, associates with ribosomes, and affects the levels of target proteins without altering mRNA abundance. Overall, our data indicates that METTL7A1—a transcriptional target of EPR—modulates translation of select transcripts.

Patients with chronic lymphocytic leukemia (CLL) relapsing on ibrutinib are often treated with the Bcl-2 inhibitor venetoclax. However, the transition from one agent to another poses some clinical challenges due to disease flares sometimes occurring right after ibrutinib interruption. Here, we describe three clinical vignettes highlighting two distinct patterns of ibrutinib-to-venetoclax transition. While patients following the favorable pattern transited to venetoclax without experiencing disease flare, the one patient who took the unfavorable path showed rapid disease rebound, with large-cell transformation occurring one week after ibrutinib interruption. A high burden of BTK and PLCG2 mutations was found only in patients with the favorable transition pattern, suggesting that removing BTK inhibition might be particularly harmful if CLL cells are progressing through mechanisms external to the BTK axis.

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Differentiation arrest along the myeloid lineage is a defining feature of acute myeloid leukemia (AML), but its molecular determinants remain poorly defined. Pharmacological removal of the differentiation block leads to leukemia eradication in acute promyelocytic leukemia (APL) patients but has not yet been successfully translated to non-APL AMLs. Here, by investigating the function of hypoxia-inducible transcription factors HIF1α and HIF2α, we identify HIF2α as a new regulator of the AML differentiation block. Mechanistically, HIF2α cooperates with EZH2, the catalytic subunit of polycomb repressive complex 2, to promote deposition of the repressive H3K27me3 histone mark at the regulatory regions of myeloid differentiation genes. Thus, inhibiting HIF2α releases an epigenetic break to leukemic blasts maturation and cooperates with the pro-differentiation agent all-trans retinoic acid to trigger AML differentiation. We propose that HIF2α inhibition may open new therapeutic avenues for AML treatment by licensing blasts maturation and forcing leukemia debulking.

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Acute myeloid leukemia (AML) is characterized by epigenetic silencing of differentiation genes and accumulation of aberrant myeloid cells arrested at different stages of myeloid development. The combination of epigenetic and differentiation therapies represents an attractive opportunity for AML patients by promoting chromatin remodeling at differentiation genes and induction of terminal maturation and leukemia debulking. The aim of our work is to define the function of the hypoxia inducible transcription factor HIF2alpha in the pathogenesis of AML. By its genetic inactivation and pharmacological inhibition in AML cell lines and patient-derived xenograft models, we found that HIF2alpha acts as a novel regulator of the AML differentiation block and promotes leukemia progression. Gene expression profiling of HIF2alpha-dependent transcriptome revealed that HIF2alpha mainly promotes the expression of genes involved in transcriptional modulation and epigenetic modifications, whilst inhibiting genes of myeloid maturation and activation. Mechanistically, inhibition of HIF2alpha results in decreased global levels of the heterochromatin marker H3K27me3, which is specifically deposited by the catalytic subunit EZH2 of the Polycomb repressive complex 2. Intriguingly, we found that HIF2alpha and EZH2 cooperate at favoring EZH2-mediated deposition of H3K27me3 at reverse hypoxia responsive elements in the regulatory regions of myeloid maturation genes. Additionally, we demonstrate that HIF2alpha is positively regulated by the pro-differentiation agent all-trans retinoic acid (ATRA), and its inhibition cooperates with ATRA in triggering AML cell differentiation. In conclusion, we provide new mechanistic insights into the role of HIF2alpha in the pathogenesis of AML, by promoting an undifferentiated state via EZH2-mediated epigenetic silencing of myeloid differentiation genes. Importantly, small molecule inhibitors of HIF2alpha have been recently generated and are tested for solid cancers. Therefore, HIF2alpha inhibition may open new therapeutic avenues for AML patients and add therapeutic value to ATRA-based therapies by interrupting HIF2alpha upregulation and promoting an epigenetic state permissive to maturation of leukemic blasts and leukemia exhaustion. Citation Format: Daniela Magliulo, Carolina Caserta, Serena Belluschi, Cristina Fracassi, Kety Giannetti, Raffaella Pini, Ettore Zapparoli, Stefano Beretta, Eleonora Draghi, Marco J. Morelli, Raffaella Di Micco, Bernhard Gentner, Luca Vago, Rosa Bernardi. The transcription factor HIF2alpha partakes in the differentiation block of acute myeloid leukemia. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B027.