Search

Background Fever during neutropenia and after neutrophil engraftment (post-engraftment fever) occurs commonly during autologous transplantation (ASCT), but infections are infrequently identified. Tests that reliably exclude infection may reduce the cost and toxicity of unnecessary diagnostic testing and empiric treatment. We assessed whether serum levels of inflammatory cytokines could distinguish infectious from non-infectious causes of fever in patients undergoing ASCT. Methods Serum levels of IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12(p70), TNF-α and IFN-γ were measured by sandwich ELISA at multiple pre-determined times and at the onset of the first fever during neutropenia and after neutrophil engraftment in patients with hematologic malignancies undergoing ASCT. Standard clinical criteria were used to assess for the presence of infection. Results Seventy-two febrile episodes occurred in 54 of 65 enrolled patients; 29 (40%) of the episodes occurred after neutrophil engraftment. Infections were identified as the cause of 28% and 24% of the neutropenic and post-engraftment febrile episodes, respectively. The level of IL-12 decreased and that of IL-6 increased significantly during fever because of infection, such that the IL-12:IL-6 ratio accurately excluded infection. The area under the ROC curve for the IL-12:IL-6 ratio was 0.88 (95% CI 0.79–0.97). The sensitivity, specificity, positive predictive and negative predictive values associated with a cut-off ratio of 4.1 were 95%, 75%, 60%, and 97%, respectively. Discussion The IL-12:IL-6 ratio effectively discriminates infectious from non-infectious causes of fever during ASCT. It may be useful in assessing the probability of infection in patients with post-engraftment fever. [ABSTRACT FROM AUTHOR]

Myelodysplastic neoplasms/syndromes (MDS) are a heterogeneous group of biologically distinct entities characterized by variable degrees of ineffective hematopoiesis. Recently, 2 classification systems (the 5th edition of the World Health Organization Classification of Haematolymphoid tTumours and the International Consensus Classification) further subcharacterized MDS into morphologically and genetically defined groups. Accurate diagnosis and subclassification of MDS require a multistep systemic approach. The International Consortium for MDS (icMDS) summarizes a contemporary, practical, and multimodal approach to MDS diagnosis and classification., (Copyright © 2024 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.)

The WHO and International Consensus Classification 2022 classifications of myelodysplastic syndromes enhance diagnostic precision and refine decision-making processes in these diseases. However, some discrepancies still exist and potentially cause inconsistency in their adoption in a clinical setting. We adopted a data-driven approach to provide a harmonisation between these two classification systems. We investigated the importance of genomic features and their effect on the cluster assignment process to define harmonised entity labels. A panel of expert haematologists, haematopathologists, and data scientists who are members of the International Consortium for Myelodysplastic Syndromes was formed and a modified Delphi consensus process was adopted to harmonise morphologically defined categories without a distinct genomic profile. The panel held regular online meetings and participated in a two-round survey using an online voting tool. We identified nine clusters with distinct genomic features. The cluster of highest hierarchical importance was characterised by biallelic TP53 inactivation. Cluster assignment was irrespective of blast count. Individuals with monoallelic TP53 inactivation were assigned to other clusters. Hierarchically, the second most important group included myelodysplastic syndromes with del(5q). Isolated del(5q) and less than 5% of blast cells in the bone marrow were the most relevant label-defining features. The third most important cluster included myelodysplastic syndromes with mutated SF3B1. The absence of isolated del(5q), del(7q)/-7, abn3q26.2, complex karyotype, RUNX1 mutations, or biallelic TP53 were the basis for a harmonised label of this category. Morphologically defined myelodysplastic syndrome entities showed large genomic heterogeneity that was not efficiently captured by single-lineage versus multilineage dysplasia, marrow blasts, hypocellularity, or fibrosis. We investigated the biological continuum between myelodysplastic syndromes with more than 10% bone marrow blasts and acute myeloid leukaemia, and found only a partial overlap in genetic features. After the survey, myelodysplastic syndromes with low blasts (ie, less than 5%) and myelodysplastic syndromes with increased blasts (ie, 5% or more) were recognised as disease entities. Our data-driven approach can efficiently harmonise current classifications of myelodysplastic syndromes and provide a reference for patient management in a real-world setting., Competing Interests: Declaration of interests UG reports speaker honoraria from Novartis, AbbVie, and BMS; and institutional research support from BMS, AbbVie, and Jazz Pharmaceuticals. FE reports consultancy or advisory roles for AbbVie, Incyte, Syros, Novartis, and Jazz Pharmaceuticals. SH reports research support from STORM Therapeutics and AstraZeneca. EAG reports honoraria from AAMDS, MedscapeLIVE!, MediCom Worldwide, MJH Life Sciences, ASH, MDS International Foundation, and Physicians’ Education Resource; consulting fees from AbbVie, Alexion, Apellis, Takeda Oncology, Astex/Taiho Oncology, Alexion/AstraZeneca Rare Disease, Celgene/BMS, CTI BioPharma, Novartis, Partner Therapeutics, Picnic Health, and Servier; and research funding from Alexion, Apellis, Astex /Otsuka/Taiho Oncology, Blueprint Medicines, Celldex Therapeutics, Genentech, and NextCure. MAS reports participation on advisory boards for BMS, Kurome, Schrödinger, and Karyopharm. MD-C reports participation on a data safety monitoring board or advisory board for BMS, Novartis, Blueprint Medicines, GSK, Agios, Hemavan, Syros, Keros, Curis, and Astex/Otsuka; and payment or honoraria for lectures, presentations, speakers’ bureaus, manuscript writing, or educational events for BMS, Novartis, and Keros. UP reports research support and honoraria from BMS, Geron, Curis, AbbVie, and Janssen. RBe reports employment or equity from Aptose Biosciences; participation on advisory boards for BMS, Servier, NeoGenomics, and Geron; being Data Monitoring Committee Chair for Gilead, Ipsen, and Keros; and consultancy for TenSixteen. YM reports honoraria from Nippon Shinyaku, BMS, Novartis, Sumitomo Pharma, Kyowa Kirin, AbbVie, Daiichi Sankyo, Takeda, Janssen, Astellas, Pfizer, Eisai, and Otsuka; and research funding from Chugai. AED reports participation on advisory boards, consultancy, or honoraria from Celgene/BMS, Agios, Novartis, Astellas, and Gilead; and participation on clinical trial committees or data safety monitoring boards for Novartis, AbbVie, Kura, Geron, Servier, Keros, and Celgene/BMS. DPS reports employment by Ajax Therapeutics; former employment by Novartis; and minor equity in Arrowhead and Bluebird. TKK reports consultancy for Agenus and ImmunoBiome. AK reports research support from Novartis and BMS; consulting fees from Alexion, Novartis, Amgen, Agios, Pfizer, Samsung, Celgene, F Hoffmann-La Roche, and Sobi; honoraria from Alexion, Novartis, Pfizer, Amgen, Samsung, Celgene, F Hoffmann-La Roche, BMS, Sobi, and Silence Therapeutics; and speakers fees from Alexion, Novartis, Amgen, Pfizer, Celgene, F Hoffmann-La Roche, and Sobi. All other authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved, including those for text and data mining, AI training, and similar technologies.)

SETD2 is the only enzyme responsible for transcription-coupled histone H3 lysine 36 trimethylation (H3K36me3). Mutations in SETD2 cause human diseases including cancer and developmental defects. In mice, Setd2 is essential for embryonic vascular remodeling. Given that many epigenetic modifiers have recently been found to possess noncatalytic functions, it is unknown whether the major function(s) of Setd2 is dependent on its catalytic activity or not. Here, we established a site-specific knockin mouse model harboring a cancer patient-derived catalytically dead Setd2 (Setd2-CD). We found that the essentiality of Setd2 in mouse development is dependent on its methyltransferase activity, as the Setd2 CD/CD and Setd2 -/- mice showed similar embryonic lethal phenotypes and largely comparable gene expression patterns. However, compared with Setd2 -/- , the Setd2 CD/CD mice showed less severe defects in allantois development, and single-cell RNA-seq analysis revealed differentially regulated allantois-specific 5' Hoxa cluster genes in these two models. Collectively, this study clarifies the importance of Setd2 catalytic activity in mouse development and provides a new model for comparative study of previously unrecognized Setd2 functions., (© 2024. Higher Education Press.)

SF3B1 mutations frequently occur in cancer yet lack targeted therapies. Clinical trials of XPO1 inhibitors, selinexor and eltanexor, in high-risk myelodysplastic neoplasms (MDS) revealed responders were enriched with SF3B1 mutations. Given that XPO1 (Exportin-1) is a nuclear exporter responsible for the export of proteins and multiple RNA species, this led to the hypothesis that SF3B1-mutant cells are sensitive to XPO1 inhibition, potentially due to altered splicing. Subsequent RNA sequencing after XPO1 inhibition in SF3B1 wildtype and mutant cells showed increased nuclear retention of RNA transcripts and increased alternative splicing in the SF3B1 mutant cells particularly of genes that impact apoptotic pathways. To identify novel drug combinations that synergize with XPO1 inhibition, a forward genetic screen was performed with eltanexor treatment implicating anti-apoptotic targets BCL2 and BCLXL, which were validated by functional testing in vitro and in vivo. These targets were tested in vivo using Sf3b1 K700E conditional knock-in mice, which showed that the combination of eltanexor and venetoclax (BCL2 inhibitor) had a preferential sensitivity for SF3B1 mutant cells without excessive toxicity. In this study, we unveil the mechanisms underlying sensitization to XPO1 inhibition in SF3B1-mutant MDS and preclinically rationalize the combination of eltanexor and venetoclax for high-risk MDS., (© 2024. The Author(s).)

Notable treatment advances have been made in recent years for patients with myelodysplastic syndromes/neoplasms (MDS), and several new drugs are under development. For example, the emerging availability of oral MDS therapies holds the promise of improving patients' health-related quality of life (HRQoL). Within this rapidly evolving landscape, the inclusion of HRQoL and other patient-reported outcomes (PROs) is critical to inform the benefit/risk assessment of new therapies or to assess whether patients live longer and better, for what will likely remain a largely incurable disease. We provide practical considerations to support investigators in generating high-quality PRO data in future MDS trials. We first describe several challenges that are to be thoughtfully considered when designing an MDS-focused clinical trial with a PRO endpoint. We then discuss aspects related to the design of the study, including PRO assessment strategies. We also discuss statistical approaches illustrating the potential value of time-to-event analyses and their implications within the estimand framework. Finally, based on a literature review of MDS randomized controlled trials with a PRO endpoint, we note the PRO items that deserve special attention when reporting future MDS trial results. We hope these practical considerations will facilitate the generation of rigorous PRO data that can robustly inform MDS patient care and support treatment decision-making for this patient population., Competing Interests: Fabio Efficace had consultancy or advisory role for AbbVie, Incyte, Syros, Novartis, and JAZZ Pharmaceuticals outside the submitted work. Rena Buckstein: Research funding and honoraria for advisory boards and speaking engagements from BMS, TAIHO, and Abbvie. Gregory A. Abel has consulted for Novartis and Geron outside the submitted work. Pierre Fenaux received research funding from BMS, Abbvie, Jazz Pharmaceuticals, Novartis, and Janssen; and had a consultancy with and received honoraria from BMS, Abbvie, Jazz Pharmaceuticals, and Novartis. Andrew M. Brunner received consulting or advisory board honoraria from Novartis, Acceleron, Agios, Abbvie, Takeda, Celgene/BMS, Keros Therapeutics, Taiho, Gilead; and has research support from the NIH SPORE in Myeloid Malignancies, and from the Edward P. Evans Foundation. Rafael Bejar owns equity in and is employed by Aptose Biosciences; he has served as an advisor to BMS, Servier, Gilead, and Ipsen; he is on the SAB for NeoGenomics. Amy E DeZern participated in advisory boards, and/or had a consultancy with and received honoraria from Celgene/BMS, Agios, Regenergon, Sobi, Novartis, Astellas, and Gilead. Amy E. DeZern served on clinical trial committees for Novartis, Abbvie, Kura, Geron, and Celgene/BMS. Gail J. Roboz: Consultancy: Abbvie, Amgen, Astra Zeneca, Bristol‐Myers Squibb, Caribou Biosciences, Celgene, Daiichi Sankyo, Ellipses Pharma, Genoptix, Geron, GlaxoSmithKline, Janssen, Jasper Pharmaceuticals, Jazz Pharmaceuticals, Molecular Partners, Novartis, Pfizer, Oncoverity, OncoPrecision, Rigel, Roche, Syndax, Takeda (IRC Chair), Telix Pharma; and research support: Janssen. Michael R. Savona: Membership on a board or advisory committee: Bristol Myers Squibb, CTI, Forma, Geron, GSK, Karyopharm, Rigel Ryvu, Taiho, Takeda, Treadwell; patents and royalties: Boehringer Ingelheim, Empath Biosciences; research funding: ALX Oncology, Astex, Incyte, Takeda, TG Therapeutics; equity ownership: Empath Biosciences, Karyopharm, Ryvu; consultancy: Forma, Geron, Karyopharm, Ryvu. Rami Komrokji: Abbvie: Speaker Bureau, Advisory board; BMS: Research grant, Advisory board; DSI: Advisory board; Geron: Consultancy; Janssen: Consultancy; Jazz: Speaker Bureau, Advisory board; Pharma Essentia: Speaker Bureau, Advisory board; Rigel: Speaker Bureau, Advisory board; Servio: Speaker Bureau, Advisory board; Sobi: Speaker Bureau, Advisory board; Sumitomo Pharma: consultancy, Advisory board. David A. Sallman served on the advisory board or panel for Agios, Avencell, BlueBird Bio, BMS, Dark Blue, Jasper Therapeutics, Kite, Magenta Therapeutics, NKARTA, Novartis, Rigel Shattuck Labs, Servier, Syndax, Syros; and had a consultancy with AbbBie, Gilead, Molecular Partners AG, Takeda. Guillermo Sanz received honoraria, advisory board membership, or consultation fees from AbbVie, BMS, ExCellThera, Novartis, Roche, and Takeda and participated in sponsored speaker's bureau for BMS, Novartis, and Takeda. Hetty E. Carraway: Consultancy: Abbvie, Amgen, Bristol‐Myers Squibb, Celgene, Daiichi Sankyo, Jazz Pharmaceuticals, Novartis, Rigel, Syndax, Servier; and research support: Celgene. Maximilian Stahl consulted for Curis Oncology and Boston Consulting; served on the advisory board for Novartis and Kymera, GSK, Rigel, and Sierra Oncology; and participated in GME activity for Novartis, Curis Oncology, Haymarket Media, and Clinical care options (CCO). Mikkael A. Sekeres has served on advisory boards for BMS, Novartis, Kurome, and Gilead. Amer M. Zeidan received research funding (institutional) from Celgene/BMS, Abbvie, Astex, Pfizer, Medimmune/Astra Zeneca, Boehringer‐Ingelheim, Cardiff oncology, Incyte, Takeda, Novartis, Aprea, and ADC Therapeutics. He participated in advisory boards, and/or had a consultancy with and received honoraria from AbbVie, Otsuka, Pfizer, Celgene/BMS, Jazz, Incyte, Agios, Boehringer‐Ingelheim, Novartis, Acceleron, Astellas, Daiichi Sankyo, Cardinal Health, Taiho, Seattle Genetics, BeyondSpring, Cardiff Oncology, Takeda, Ionis, Amgen, Janssen, Epizyme, Syndax, Gilead, Kura, Chiesi, ALX Oncology, BioCryst, Notable, Orum, and Tyme. He served on clinical trial committees for Novartis, Abbvie, Gilead, BioCryst, Abbvie, ALX Oncology, Geron, and Celgene/BMS. Fabio Efficace, Rena Buckstein, and Gregory A. Abel report being involved in the development and validation of the QUALMS, one of the PROs discussed in this paper. The QUALMS is copyrighted by Dana‐Farber Cancer Institute (DFCI) and the Children's Hospital of Eastern Ontario (CHEO). The remaining authors declare no conflict of interest., (© 2024 The Authors. HemaSphere published by John Wiley & Sons Ltd on behalf of European Hematology Association.)

An important epigenetic component of tyrosine kinase signaling is the phosphorylation of histones, and epigenetic readers, writers, and erasers. Phosphorylation of protein arginine methyltransferases (PRMTs), have been shown to enhance and impair their enzymatic activity. In this study, we show that the hyperactivation of Janus kinase 2 (JAK2) by the V617F mutation phosphorylates tyrosine residues (Y149 and Y334) in coactivator-associated arginine methyltransferase 1 (CARM1), an important target in hematologic malignancies, increasing its methyltransferase activity and altering its target specificity. While non-phosphorylatable CARM1 methylates some established substrates (e.g. BAF155 and PABP1), only phospho-CARM1 methylates the RUNX1 transcription factor, on R223 and R319. Furthermore, cells expressing non-phosphorylatable CARM1 have impaired cell-cycle progression and increased apoptosis, compared to cells expressing phosphorylatable, wild-type CARM1, with reduced expression of genes associated with G2/M cell cycle progression and anti-apoptosis. The presence of the JAK2-V617F mutant kinase renders acute myeloid leukemia (AML) cells less sensitive to CARM1 inhibition, and we show that the dual targeting of JAK2 and CARM1 is more effective than monotherapy in AML cells expressing phospho-CARM1. Thus, the phosphorylation of CARM1 by hyperactivated JAK2 regulates its methyltransferase activity, helps select its substrates, and is required for the maximal proliferation of malignant myeloid cells., (© 2024. The Author(s).)

The development of dendritic cells (DCs), including antigen-presenting conventional DCs (cDCs) and cytokine-producing plasmacytoid DCs (pDCs), is controlled by the growth factor Flt3 ligand (Flt3L) and its receptor Flt3. We genetically dissected Flt3L-driven DC differentiation using CRISPR-Cas9-based screening. Genome-wide screening identified multiple regulators of DC differentiation including subunits of TSC and GATOR1 complexes, which restricted progenitor growth but enabled DC differentiation by inhibiting mTOR signaling. An orthogonal screen identified the transcriptional repressor Trim33 (TIF-1γ) as a regulator of DC differentiation. Conditional targeting in vivo revealed an essential role of Trim33 in the development of all DCs, but not of monocytes or granulocytes. In particular, deletion of Trim33 caused rapid loss of DC progenitors, pDCs, and the cross-presenting cDC1 subset. Trim33-deficient Flt3 + progenitors up-regulated pro-inflammatory and macrophage-specific genes but failed to induce the DC differentiation program. Collectively, these data elucidate mechanisms that control Flt3L-driven differentiation of the entire DC lineage and identify Trim33 as its essential regulator.

ASXL1 mutation frequently occurs in all forms of myeloid malignancies and is associated with aggressive disease and poor prognosis. ASXL1 recruits Polycomb repressive complex 2 (PRC2) to specific gene loci to repress transcription through trimethylation of histone H3 on lysine 27 (H3K27me3). ASXL1 alterations reduce H3K27me3 levels, which results in leukemogenic gene expression and the development of myeloid malignancies. Standard therapies for myeloid malignancies have limited efficacy when mutated ASXL1 is present. We discovered upregulation of lysine demethylase 6B (KDM6B), a demethylase for H3K27me3, in ASXL1-mutant leukemic cells, which further reduces H3K27me3 levels and facilitates myeloid transformation. Here, we demonstrated that heterozygous deletion of Kdm6b restored H3K27me3 levels and normalized dysregulated gene expression in Asxl1Y588XTg hematopoietic stem/progenitor cells (HSPCs). Furthermore, heterozygous deletion of Kdm6b decreased the HSPC pool, restored their self-renewal capacity, prevented biased myeloid differentiation, and abrogated progression to myeloid malignancies in Asxl1Y588XTg mice. Importantly, administration of GSK-J4, a KDM6B inhibitor, not only restored H3K27me3 levels but also reduced the disease burden in NSG mice xenografted with human ASXL1-mutant leukemic cells in vivo. This preclinical finding provides compelling evidence that targeting KDM6B may be a therapeutic strategy for myeloid malignancies with ASXL1 mutations.

The guidelines for classification, prognostication, and response assessment of myelodysplastic syndromes/neoplasms (MDS) have all recently been updated. In this report on behalf of the International Consortium for MDS (icMDS) we summarize these developments. We first critically examine the updated World Health Organization (WHO) classification and the International Consensus Classification (ICC) of MDS. We then compare traditional and molecularly based risk MDS risk assessment tools. Lastly, we discuss limitations of criteria in measuring therapeutic benefit and highlight how the International Working Group (IWG) 2018 and 2023 response criteria addressed these deficiencies and are endorsed by the icMDS. We also address the importance of patient centered care by discussing the value of quality-of-life assessment. We hope that the reader of this review will have a better understanding of how to classify MDS, predict clinical outcomes and evaluate therapeutic outcomes., Competing Interests: Declaration of Competing Interest Maximilian Stahl consulted for Curis Oncology and Boston Consulting; served on the advisory board for Novartis and Kymera, GSK, Rigel, Sierra Oncology; and participated in GME activity for Novartis, Curis Oncology, Haymarket Media and Clinical care options (CCO). Elizabeth A. Griffiths has received honoraria for advisory board membership from AbbVie, Alexion Pharmaceuticals, Apellis, Celgene/BMS, CTI Biopharma, Genentech, Novartis, Picnic Health, Takeda Oncology, Taiho Oncology. EAG has received research funding from Astex Pharmaceuticals, AstraZeneca Rare Disease, Alexion Pharmaceuticals, Apellis Pharmaceuticals, Blueprint Medicines, Genentech Inc., and honoraria for CME activities from Physicians Educational Resource, MediComWorldwide, American Society of Hematology, AAMDS International Foundation. Ravindra Majeti is on the Advisory Boards of Kodikaz Therapeutic Solutions, Syros Pharmaceuticals, TenSixteen Bio, Roche, and Cullgen Inc. and is an inventor on several patents related to CD47 cancer immunotherapy licensed to Gilead Sciences. R.M. receives research support from Gilead Sciences. Ravindra Majeti. is a co-founder and equity holder of Pheast Therapeutics, MyeloGene, and Orbital Therapeutics. Stephanie Halene consulted for Forma Therapeutics. Daniel T. Starczynowski is a consultant and received research funding from Kymera Therapeutics, Kurome Therapeutics, Captor Therapeutics, and Tolero Therapeutics. Daniel T. Starczynowski has equity in Kurome Therapeutics. Gail Roboz: Consultancy: Abbvie, Amgen, Argenx, Astra Zeneca, Bluebird Bio, Blueprint Medicines, Bristol-Myers Squibb, Caribou Biosciences, Celgene, Daiichi Sankyo, Ellipses Pharma, GlaxoSmithKline, Janssen, Jasper Pharmaceuticals, Jazz Pharmaceuticals, Molecular Partners, Novartis, Pfizer, Rigel, Roche, Syndax, Takeda (IRC Chair), Telix Pharma Research support: Janssen. David A. Sallman served on the advisory board of Aprea, AvenCell, BlueBird Bio, BMS, Intellia, Kite, Novartis, Shattuck Labs, Servier, Syndax. David A. Sallman served as a consultant for AbbVie, Magenta, Molecular Partners AG, Takeda and on the speakers' bureau for BMS, Incyte, Servier; David A. Sallman received research funding from Aprea, Jazz. Mrinal Patnaik received research funding from Kura Oncology and StemLine Pharmaceuticals. Andrew Brunner received consulting or advisory board honoraria from Novartis, Acceleron, Agios, Abbvie, Takeda, Celgene/BMS, Keros Therapeutics, Taiho, Gilead. Andrew Brunner has research support from the NIH SPORE in Myeloid Malignancies, and from the Edward P. Evans Foundation. Tae Kon Kim received research funding from Nextcure and is a consultant for Agenus. Alan List is employed by and has equity in Precision BioSciences, and has served as a consultant for Halia Therapeutics, CTI Biopharma, Aileron. Naval Daver has received research funding from Daiichi-Sankyo, Bristol-Myers Squibb, Pfizer, Gilead, Sevier, Genentech, Astellas, Daiichi-Sankyo, Abbvie, Hanmi, Trovagene, FATE therapeutics, Amgen, Novimmune, Glycomimetics, Trillium, and ImmunoGen and has served in a consulting or advisory role for Daiichi-Sankyo, Bristol-Myers Squibb, Arog, Pfizer, Novartis, Jazz, Celgene, AbbVie, Astellas, Genentech, Immunogen, Servier, Syndax, Trillium, Gilead, Amgen, Shattuck labs, and Agios. Guillermo Sanz received honoraria, advisory board membership or consultation fees from AbbVie, BMS, ExCellThera, Novartis, Roche, and Takeda and participated in sponsored speaker's bureau for BMS, Novartis, and Takeda. Mikkael A. Sekeres has served on advisory boards for BMS, Novartis, Kurome, and Gilead. Pierre Fenaux received research funding from BMS, Abbvie, Jazz Pharmaceuticals, Novartis, and Janssen. Pierre Fenaux had a consultancy with and received honoraria from BMS, Abbvie, Jazz Pharmaceuticals, and Novartis. Fabio Efficace had a consultancy or advisory role for AbbVie, Incyte, Janssen, and Syros, outside the submitted work. Omar Abdel-Wahab has served as a consultant for H3B Biomedicine, Foundation Medicine Inc., Merck, Prelude Therapeutics, and Janssen, and is on the Scientific Advisory Board of Envisagenics Inc., AIChemy, Harmonic Discovery Inc., and Pfizer Boulder; Omar Abdel-Wahab has received prior research funding from H3B Biomedicine and LOXO Oncology unrelated to the current manuscript. Andrew H.Wei has served on advisory boards for Novartis, Astra Zeneca, Astellas, Janssen, Jazz, Amgen, Roche, Pfizer, Abbvie, Servier, Gilead, BMS, Shoreline, Macrogenics, Novartis and Agios; receives research funding to the Institution from Novartis, Abbvie, Servier, Janssen, BMS, Syndax, Astex, Astra Zeneca, Amgen; serves on speaker's bureaus for Abbvie, Novartis, BMS, Servier, Astellas; Andrew H.Wei is an employee of the Walter and Eliza Hall Institute (WEHI). WEHI receives milestone and royalty payments related to the development of Venetoclax. Current and past employees of Walter and Eliza Hall Institute may be eligible for financial benefits related to these payments. Andrew H. Wei receives such a financial benefit. Amer M. Zeidan received research funding (institutional) from Celgene/BMS, Abbvie, Astex, Pfizer, Medimmune/AstraZeneca, Boehringer-Ingelheim, Cardiff oncology, Incyte, Takeda, Novartis, Aprea, and ADC Therapeutics. AMZ participated in advisory boards, and/or had a consultancy with and received honoraria from AbbVie, Otsuka, Pfizer, Celgene/BMS, Jazz, Incyte, Agios, Boehringer-Ingelheim, Novartis, Acceleron, Astellas, Daiichi Sankyo, Cardinal Health, Taiho, Seattle Genetics, BeyondSpring, Cardiff Oncology, Takeda, Ionis, Amgen, Janssen, Epizyme, Syndax, Gilead, Kura, Chiesi, ALX Oncology, BioCryst, Notable, Orum, and Tyme. AMZ served on clinical trial committees for Novartis, Abbvie, Gilead, BioCryst, Abbvie, ALX Oncology, Geron and Celgene/BMS. The National Heart, Lung, and Blood Institute receives research funding for the laboratory of Dr. Hourigan from Sellas and from the Foundation of the NIH AML MRD Biomarkers Consortium. All other authors have no conflicts of interest to disclose., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Bromodomain-containing protein 4 (BRD4) is overexpressed and functionally implicated in various myeloid malignancies. However, the role of BRD4 in normal hematopoiesis remains largely unknown. Here, utilizing an inducible Brd4 knockout mouse model, we find that deletion of Brd4 (Brd4 Δ/Δ ) in the hematopoietic system impairs hematopoietic stem cell (HSC) self-renewal and differentiation, which associates with cell cycle arrest and senescence. ATAC-seq analysis shows increased chromatin accessibility in Brd4 Δ/Δ hematopoietic stem/progenitor cells (HSC/HPCs). Genome-wide mapping with cleavage under target and release using nuclease (CUT&RUN) assays demonstrate that increased global enrichment of H3K122ac and H3K4me3 in Brd4 Δ/Δ HSC/HPCs is associated with the upregulation of senescence-specific genes. Interestingly, Brd4 deletion increases clipped H3 (cH3) which correlates with the upregulation of senescence-specific genes and results in a higher frequency of senescent HSC/HPCs. Re-expression of BRD4 reduces cH3 levels and rescues the senescence rate in Brd4 Δ/Δ HSC/HPCs. This study unveils an important role of BRD4 in HSC/HPC function by preventing H3 clipping and suppressing senescence gene expression., (© 2023 The Authors.)

During emergency hematopoiesis, hematopoietic stem cells (HSCs) rapidly proliferate to produce myeloid and lymphoid effector cells, a response that is critical against infection or tissue injury. If unresolved, this process leads to sustained inflammation, which can cause life-threatening diseases and cancer. Here, we identify a role of double PHD fingers 2 (DPF2) in modulating inflammation. DPF2 is a defining subunit of the hematopoiesis-specific BAF (SWI/SNF) chromatin-remodeling complex, and it is mutated in multiple cancers and neurological disorders. We uncovered that hematopoiesis-specific Dpf2-KO mice developed leukopenia, severe anemia, and lethal systemic inflammation characterized by histiocytic and fibrotic tissue infiltration resembling a clinical hyperinflammatory state. Dpf2 loss impaired the polarization of macrophages responsible for tissue repair, induced the unrestrained activation of Th cells, and generated an emergency-like state of HSC hyperproliferation and myeloid cell-biased differentiation. Mechanistically, Dpf2 deficiency resulted in the loss of the BAF catalytic subunit BRG1 from nuclear factor erythroid 2-like 2-controlled (NRF2-controlled) enhancers, impairing the antioxidant and antiinflammatory transcriptional response needed to modulate inflammation. Finally, pharmacological reactivation of NRF2 suppressed the inflammation-mediated phenotypes and lethality of Dpf2Δ/Δ mice. Our work establishes an essential role of the DPF2-BAF complex in licensing NRF2-dependent gene expression in HSCs and immune effector cells to prevent chronic inflammation.

Over the course of the COVID-19 pandemic, SARS-CoV-2 variants of concern (VOCs) with increased transmissibility and immune escape capabilities, such as Delta and Omicron, have triggered waves of new COVID-19 infections worldwide, and Omicron subvariants continue to represent a global health concern. Tracking the prevalence and dynamics of VOCs has clinical and epidemiological significance and is essential for modeling the progression and evolution of the COVID-19 pandemic. Next generation sequencing (NGS) is recognized as the gold standard for genomic characterization of SARS-CoV-2 variants, but it is labor and cost intensive and not amenable to rapid lineage identification. Here we describe a two-pronged approach for rapid, cost-effective surveillance of SARS-CoV-2 VOCs by combining reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) and periodic NGS with the ARTIC sequencing method. Variant surveillance by RT-qPCR included the commercially available TaqPath COVID-19 Combo Kit to track S-gene target failure (SGTF) associated with the spike protein deletion H69-V70, as well as two internally designed and validated RT-qPCR assays targeting two N-terminal-domain (NTD) spike gene deletions, NTD156-7 and NTD25-7. The NTD156-7 RT-qPCR assay facilitated tracking of the Delta variant, while the NTD25-7 RT-qPCR assay was used for tracking Omicron variants, including the BA.2, BA.4, and BA.5 lineages. In silico validation of the NTD156-7 and NTD25-7 primers and probes compared with publicly available SARS-CoV-2 genome databases showed low variability in regions corresponding to oligonucleotide binding sites. Similarly, in vitro validation with NGS-confirmed samples showed excellent correlation. RT-qPCR assays allow for near-real-time monitoring of circulating and emerging variants allowing for ongoing surveillance of variant dynamics in a local population. By performing periodic sequencing of variant surveillance by RT-qPCR methods, we were able to provide ongoing validation of the results obtained by RT-qPCR screening. Rapid SARS-CoV-2 variant identification and surveillance by this combined approach served to inform clinical decisions in a timely manner and permitted better utilization of sequencing resources.

TET2 haploinsufficiency is a driving event in myeloid cancers and is associated with a worse prognosis in patients with acute myeloid leukemia (AML). Enhancing residual TET2 activity using vitamin C increases oxidized 5-methylcytosine (mC) formation and promotes active DNA demethylation via base excision repair (BER), which slows leukemia progression. We utilize genetic and compound library screening approaches to identify rational combination treatment strategies to improve use of vitamin C as an adjuvant therapy for AML. In addition to increasing the efficacy of several US Food and Drug Administration (FDA)-approved drugs, vitamin C treatment with poly-ADP-ribosyl polymerase inhibitors (PARPis) elicits a strong synergistic effect to block AML self-renewal in murine and human AML models. Vitamin-C-mediated TET activation combined with PARPis causes enrichment of chromatin-bound PARP1 at oxidized mCs and γH2AX accumulation during mid-S phase, leading to cell cycle stalling and differentiation. Given that most AML subtypes maintain residual TET2 expression, vitamin C could elicit broad efficacy as a PARPi therapeutic adjuvant., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Innate immune cells participate in the detection of tumor cells via complex signaling pathways mediated by pattern-recognition receptors, such as Toll-like receptors and nucleotide-binding and oligomerization domain-like receptors. These pathways are finely tuned via multiple mechanisms, including epigenetic regulation. It is well established that hematopoietic progenitors generate innate immune cells that can regulate cancer cell behavior, and the disruption of normal hematopoiesis in pathologic states may lead to altered immunity and the development of cancer. In this review, we discuss the epigenetic and transcriptional mechanisms that underlie the initiation and amplification of innate immune signaling in cancer. We also discuss new targeting possibilities for cancer control that exploit innate immune cells and signaling molecules, potentially heralding the next generation of immunotherapy., (©2022 The Authors; Published by the American Association for Cancer Research.)

Objectives: Patient education resources that address barriers to health literacy to improve understanding and outcomes in myelodysplastic syndromes (MDS) are limited. The aim of this study was to evaluate the impact and outcomes benefits of An Animated Patient's Guide to Myelodysplastic Syndromes (MDS) cancer educational modules (which includes the 'You and MDS' website and YouTube hosted resources) related to MDS education, awareness, understanding and health outcomes., Methods: This was a retrospective study of learner feedback, metrics, and utilization data from July 2018 to August 2021. We evaluated audience reach (number of visit sessions, unique visitors, page views) and calculated top views by media type (animation, expert video, patient video, and slide show) and top retention videos from the modules. We also assessed the educational impact and utilization through learner feedback surveys., Results: During the study period, 'You and MDS' had 233,743 views worldwide of which 104,214 were unique visitors and 78,161 (or 76% unique visitors) were from the United States. Of these, 61% were patients; 29% family members or caregivers; 5% were healthcare providers and 5% represented other groups. Most popular topics viewed among the animations were "Understanding Myelodysplastic Syndromes (MDS)" (40,219 views), "Managing and Treating MDS" (19,240 views), "Understanding Erythropoiesis" (17,564 views.) The most popular expert videos viewed were "What is iron overload, and how it is treated?" (20,310 views), "How serious a cancer is MDS? What is the prognosis for MDS?" (8,327 views), "What is MDS?" (3,157 views). Of participants who completed the online feedback survey, ≥ 95% reported improved knowledge gains and commitments to change., Conclusions: MDS patients using 'You and MDS - An Animated Patient's Guide to MDS' and its visual formats of learning represented a wide U.S. and global learner audience. This MDS educational resource had a significant impact on improved understanding among patients, families, and caregivers. Continued efforts should be made to provide patient-effective resources that address health literacy, improve patient understanding, and address educational needs that respond to the concerns of patients to achieve better quality of life and improved health outcomes in MDS., Competing Interests: No conflict of interest in relation to this manuscript. No funding was requested or received for the development of this manuscript. You and MDS: An Animated Patient's Guide to Myelodysplastic Syndromes is supported by unrestricted education grants from Acceleron Pharma, Bristol-Myers Squibb, Celgene Corporation, Jazz Pharmaceuticals, Novartis, and Takeda Oncology. Mechanisms in Medicine, the developers of this resource, declare no conflict of interest in the development of the program., (© 2022 The Authors. Published by Elsevier Ltd.)

Myelodysplastic syndromes (MDS) are hematopoietic stem and progenitor cell (HSPC) malignancies characterized by ineffective hematopoiesis and an increased risk of leukemia transformation. Epigenetic regulators are recurrently mutated in MDS, directly implicating epigenetic dysregulation in MDS pathogenesis. Here, we identified a tumor suppressor role of the acetyltransferase p300 in clinically relevant MDS models driven by mutations in the epigenetic regulators TET2, ASXL1, and SRSF2. The loss of p300 enhanced the proliferation and self-renewal capacity of Tet2-deficient HSPCs, resulting in an increased HSPC pool and leukemogenicity in primary and transplantation mouse models. Mechanistically, the loss of p300 in Tet2-deficient HSPCs altered enhancer accessibility and the expression of genes associated with differentiation, proliferation, and leukemia development. Particularly, p300 loss led to an increased expression of Myb, and the depletion of Myb attenuated the proliferation of HSPCs and improved the survival of leukemia-bearing mice. Additionally, we show that chemical inhibition of p300 acetyltransferase activity phenocopied Ep300 deletion in Tet2-deficient HSPCs, whereas activation of p300 activity with a small molecule impaired the self-renewal and leukemogenicity of Tet2-deficient cells. This suggests a potential therapeutic application of p300 activators in the treatment of MDS with TET2 inactivating mutations.

The activity of Rho family GTPase protein, RAC1, which plays important normal physiological functions, is dysregulated in multiple cancers. RAC1 is expressed in both estrogen receptor alpha (ER)-positive and ER-negative breast cancer (BC) cells. However, ER-positive BC is more sensitive to RAC1 inhibition. We have determined that reducing RAC1 activity, using siRNA or EHT 1864 (a small molecule Rac inhibitor), leads to rapid ER protein degradation. RAC1 interacts with ER within the ER complex and RAC1 localizes to chromatin binding sites for ER upon estrogen treatment. RAC1 activity is important for RNA Pol II function at both promoters and enhancers of ER target genes and ER-regulated gene transcription is blocked by EHT 1864, in a dose-dependent manner. Having identified that RAC1 is an essential ER cofactor for ER protein stability and ER transcriptional activity, we report that RAC1 inhibition could be an effective therapeutic approach for ER-positive BC., (© 2021. The Author(s).)

INTS11, the catalytic subunit of the Integrator (INT) complex, is crucial for the biogenesis of small nuclear RNAs and enhancer RNAs. However, the role of INTS11 in hematopoietic stem and progenitor cell (HSPC) biology is unknown. Here, we report that INTS11 is required for normal hematopoiesis and hematopoietic-specific genetic deletion of Ints11 leads to cell cycle arrest and impairment of fetal and adult HSPCs. We identified a novel INTS11-interacting protein complex, Polycomb repressive complex 2 (PRC2), that maintains HSPC functions. Loss of INTS11 destabilizes the PRC2 complex, decreases the level of histone H3 lysine 27 trimethylation (H3K27me3), and derepresses PRC2 target genes. Reexpression of INTS11 or PRC2 proteins in Ints11 -deficient HSPCs restores the levels of PRC2 and H3K27me3 as well as HSPC functions. Collectively, our data demonstrate that INTS11 is an essential regulator of HSPC homeostasis through the INTS11-PRC2 axis.

Mitogen-activated protein kinases (MAPKs) are inactivated by dual-specificity phosphatases (DUSPs), the activities of which are tightly regulated during cell differentiation. Using knockdown screening and single-cell transcriptional analysis, we demonstrate that DUSP4 is the phosphatase that specifically inactivates p38 kinase to promote megakaryocyte (Mk) differentiation. Mechanistically, PRMT1-mediated methylation of DUSP4 triggers its ubiquitinylation by an E3 ligase HUWE1. Interestingly, the mechanistic axis of the DUSP4 degradation and p38 activation is also associated with a transcriptional signature of immune activation in Mk cells. In the context of thrombocytopenia observed in myelodysplastic syndrome (MDS), we demonstrate that high levels of p38 MAPK and PRMT1 are associated with low platelet counts and adverse prognosis, while pharmacological inhibition of p38 MAPK or PRMT1 stimulates megakaryopoiesis. These findings provide mechanistic insights into the role of the PRMT1-DUSP4-p38 axis on Mk differentiation and present a strategy for treatment of thrombocytopenia associated with MDS., Competing Interests: Declaration of interests M.L. has served on the Scientific Advisory Board for Epi One. A.V. has received research funding from GlaxoSmithKline, Incyte, MedPacto, Novartis, Curis, and Eli Lilly and Company; has received compensation as a scientific advisor to Novartis, Stelexis Therapeutics, Acceleron Pharma, and Celgene; and has equity ownership in Stelexis Therapeutics. The remaining authors declare no competing interests. Array BioPharma provided the p38 inhibitor pexmetinib (ARRY614) and participated in its phase I study (ClinicalTrials.gov: NCT01496495)., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Nucleophosmin (NPM1) is the most commonly mutated gene in acute myeloid leukemia (AML) resulting in aberrant cytoplasmic translocation of the encoded nucleolar protein (NPM1c + ). NPM1c + maintains a unique leukemic gene expression program, characterized by activation of HOXA/B clusters and MEIS1 oncogene to facilitate leukemogenesis. However, the mechanisms by which NPM1c + controls such gene expression patterns to promote leukemogenesis remain largely unknown. Here, we show that the activation of HOXBLINC, a HOXB locus-associated long non-coding RNA (lncRNA), is a critical downstream mediator of NPM1c + -associated leukemic transcription program and leukemogenesis. HOXBLINC loss attenuates NPM1c + -driven leukemogenesis by rectifying the signature of NPM1c + leukemic transcription programs. Furthermore, overexpression of HoxBlinc (HoxBlincTg) in mice enhances HSC self-renewal and expands myelopoiesis, leading to the development of AML-like disease, reminiscent of the phenotypes seen in the Npm1 mutant knock-in (Npm1 c/+ ) mice. HoxBlincTg and Npm1 c/+ HSPCs share significantly overlapped transcriptome and chromatin structure. Mechanistically, HoxBlinc binds to the promoter regions of NPM1c + signature genes to control their activation in HoxBlincTg HSPCs, via MLL1 recruitment and promoter H3K4me3 modification. Our study reveals that HOXBLINC lncRNA activation plays an essential oncogenic role in NPM1c + leukemia. HOXBLINC and its partner MLL1 are potential therapeutic targets for NPM1c + AML.

When South Florida became a hot spot for COVID-19 disease in March 2020, we faced an urgent need to develop test capability to detect SARS-CoV-2 infection. We assembled a transdisciplinary team of knowledgeable and dedicated physicians, scientists, technologists, and administrators who rapidly built a multiplatform, polymerase chain reaction- and serology-based detection program, established drive-through facilities, and drafted and implemented guidelines that enabled efficient testing of our patients and employees. This process was extremely complex, due to the limited availability of needed reagents, but outreach to our research scientists and multiple diagnostic laboratory companies, and government officials enabled us to implement both Food and Drug Administration authorized and laboratory-developed testing-based testing protocols. We analyzed our workforce needs and created teams of appropriately skilled and certified workers to safely process patient samples and conduct SARS-CoV-2 testing and contact tracing. We initiated smart test ordering, interfaced all testing platforms with our electronic medical record, and went from zero testing capacity to testing hundreds of health care workers and patients daily, within 3 weeks. We believe our experience can inform the efforts of others when faced with a crisis situation., Competing Interests: Declaration of Conflicting Interests: The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article. The University of Miami entered into a management agreement with Lab Corp on May 3, 2020, with Lab Corp agreeing to provide management for a broad range of clinical laboratory tests conducted on UM in-patients. M.M. is a member of the Viracor Eurofins medical advisory board., (© The Author(s) 2020.)

SETD2, the histone H3 lysine 36 methyltransferase, previously identified by us, plays an important role in the pathogenesis of hematologic malignancies, but its role in myelodysplastic syndromes (MDSs) has been unclear. In this study, low expression of SETD2 correlated with shortened survival in patients with MDS, and the SETD2 levels in CD34+ bone marrow cells of those patients were increased by decitabine. We knocked out Setd2 in NUP98-HOXD13 (NHD13) transgenic mice, which phenocopies human MDS, and found that loss of Setd2 accelerated the transformation of MDS into acute myeloid leukemia (AML). Loss of Setd2 enhanced the ability of NHD13+ hematopoietic stem and progenitor cells (HSPCs) to self-renew, with increased symmetric self-renewal division and decreased differentiation and cell death. The growth of MDS-associated leukemia cells was inhibited though increasing the H3K36me3 level by using epigenetic modifying drugs. Furthermore, Setd2 deficiency upregulated hematopoietic stem cell signaling and downregulated myeloid differentiation pathways in the NHD13+ HSPCs. Our RNA-seq and chromatin immunoprecipitation-seq analysis indicated that S100a9, the S100 calcium-binding protein, is a target gene of Setd2 and that the addition of recombinant S100a9 weakens the effect of Setd2 deficiency in the NHD13+ HSPCs. In contrast, downregulation of S100a9 leads to decreases of its downstream targets, including Ikba and Jnk, which influence the self-renewal and differentiation of HSPCs. Therefore, our results demonstrated that SETD2 deficiency predicts poor prognosis in MDS and promotes the transformation of MDS into AML, which provides a potential therapeutic target for MDS-associated acute leukemia., (© 2020 by The American Society of Hematology.)

Leukemia stem cells (LSCs) are believed to have more distinct vulnerabilities than the bulk acute myeloid leukemia (AML) cells, but their rarity and the lack of universal markers for their prospective isolation hamper their study. We report that genetically clonal induced pluripotent stem cells (iPSCs) derived from an AML patient and characterized by exceptionally high engraftment potential give rise, upon hematopoietic differentiation, to a phenotypic hierarchy. Through fate-tracking experiments, xenotransplantation, and single-cell transcriptomics, we identify a cell fraction (iLSC) that can be isolated prospectively by means of adherent in vitro growth that resides on the apex of this hierarchy and fulfills the hallmark features of LSCs. Through integrative genomic studies of the iLSC transcriptome and chromatin landscape, we derive an LSC gene signature that predicts patient survival and uncovers a dependency of LSCs, across AML genotypes, on the RUNX1 transcription factor. These findings can empower efforts to therapeutically target AML LSCs., Competing Interests: Declaration of Interests E.P.P. has received honoraria from Celgene and Merck and research support from Incyte. M.G.K. has received consultant fees from Acent Therapeutics and research support from 28-7. These disclosures are not directly related to the present study., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The emergence of drug resistance is a major obstacle for the success of targeted therapy in melanoma. Additionally, conventional chemotherapy has not been effective as drug-resistant cells escape lethal DNA damage effects by inducing growth arrest commonly referred to as cellular dormancy. We present a therapeutic strategy termed "targeted chemotherapy" by depleting protein phosphatase 2A (PP2A) or its inhibition using a small molecule inhibitor (1,10-phenanthroline-5,6-dione [phendione]) in drug-resistant melanoma. Targeted chemotherapy induces the DNA damage response without causing DNA breaks or allowing cellular dormancy. Phendione treatment reduces tumor growth of BRAF V600E -driven melanoma patient-derived xenografts (PDX) and diminishes growth of NRAS Q61R -driven melanoma, a cancer with no effective therapy. Remarkably, phendione treatment inhibits the acquisition of resistance to BRAF inhibition in BRAF V600E PDX highlighting its effectiveness in combating the advent of drug resistance., (© 2020 Yue et al.; Published by Cold Spring Harbor Laboratory Press.)

Protein arginine methyltransferase 5 (PRMT5) catalyzes the symmetric di-methylation of arginine residues in histones H3 and H4, marks that are generally associated with transcriptional repression. However, we found that PRMT5 inhibition or depletion led to more genes being downregulated than upregulated, indicating that PRMT5 can also act as a transcriptional activator. Indeed, the global level of histone H3K27me3 increases in PRMT5 deficient cells. Although PRMT5 does not directly affect PRC2 enzymatic activity, methylation of histone H3 by PRMT5 abrogates its subsequent methylation by PRC2. Treating AML cells with an EZH2 inhibitor partially restored the expression of approximately 50% of the genes that are initially downregulated by PRMT5 inhibition, suggesting that the increased H3K27me3 could directly or indirectly contribute to the transcription repression of these genes. Indeed, ChIP-sequencing analysis confirmed an increase in the H3K27me3 level at the promoter region of a quarter of these genes in PRMT5-inhibited cells. Interestingly, the anti-proliferative effect of PRMT5 inhibition was also partially rescued by treatment with an EZH2 inhibitor in several leukemia cell lines. Thus, PRMT5-mediated crosstalk between histone marks contributes to its functional effects., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The AML1-ETO fusion protein, generated by the t(8;21) chromosomal translocation, is causally involved in nearly 20% of acute myeloid leukemia (AML) cases. In leukemic cells, AML1-ETO resides in and functions through a stable protein complex, AML1-ETO-containing transcription factor complex (AETFC), that contains multiple transcription (co)factors. Among these AETFC components, HEB and E2A, two members of the ubiquitously expressed E proteins, directly interact with AML1-ETO, confer new DNA-binding capacity to AETFC, and are essential for leukemogenesis. However, the third E protein, E2-2, is specifically silenced in AML1-ETO-expressing leukemic cells, suggesting E2-2 as a negative factor of leukemogenesis. Indeed, ectopic expression of E2-2 selectively inhibits the growth of AML1-ETO-expressing leukemic cells, and this inhibition requires the bHLH DNA-binding domain. RNA-seq and ChIP-seq analyses reveal that, despite some overlap, the three E proteins differentially regulate many target genes. In particular, studies show that E2-2 both redistributes AETFC to, and activates, some genes associated with dendritic cell differentiation and represses MYC target genes. In AML patients, the expression of E2-2 is relatively lower in the t(8;21) subtype, and an E2-2 target gene, THPO , is identified as a potential predictor of relapse. In a mouse model of human t(8;21) leukemia, E2-2 suppression accelerates leukemogenesis. Taken together, these results reveal that, in contrast to HEB and E2A, which facilitate AML1-ETO-mediated leukemogenesis, E2-2 compromises the function of AETFC and negatively regulates leukemogenesis. The three E proteins thus define a heterogeneity of AETFC, which improves our understanding of the precise mechanism of leukemogenesis and assists development of diagnostic/therapeutic strategies., Competing Interests: Conflict of interest statement: S.D.N. has coauthored papers with J.L. and O.A.-W. but has not collaborated directly with them. R.G.R. and J.L. are both investigators on a program grant but on separate projects.

ASXL1 is frequently mutated in myeloid malignancies and is known to co-occur with other gene mutations. However, the molecular mechanisms underlying the leukemogenesis associated with ASXL1 and cooperating mutations remain to be elucidated. Here, we report that Asxl1 loss cooperated with haploinsufficiency of Nf1, a negative regulator of the RAS signaling pathway, to accelerate the development of myeloid leukemia in mice. Loss of Asxl1 and Nf1 in hematopoietic stem and progenitor cells resulted in a gain-of-function transcriptional activation of multiple pathways such as MYC, NRAS, and BRD4 that are critical for leukemogenesis. The hyperactive MYC and BRD9 transcription programs were correlated with elevated H3K4 trimethylation at the promoter regions of genes involving these pathways. Furthermore, pharmacological inhibition of both the MAPK pathway and BET bromodomain prevented leukemia initiation and inhibited disease progression in Asxl1Δ/Δ Nf1Δ/Δ mice. Concomitant mutations of ASXL1 and RAS pathway genes were associated with aggressive progression of myeloid malignancies in patients. This study sheds light on the effect of cooperation between epigenetic alterations and signaling pathways on accelerating the progression of myeloid malignancies and provides a rational therapeutic strategy for the treatment of myeloid malignancies with ASXL1 and RAS pathway gene mutations.