Recent genomic studies have identified novel recurrent somatic mutations in patients with myeloid malignancies, including myeloproliferative neoplasms (MPNs), myelodysplastic syndrome (MDS) and acute ...myeloid leukaemia (AML). In some cases these mutations occur in genes with known roles in regulating chromatin and/or methylation states in haematopoietic progenitors, and in other cases genetic and functional studies have elucidated a role for specific mutations in altering epigenetic patterning in myeloid malignancies. In this Review we discuss recent genetic and functional data implicating mutations in epigenetic modifiers, including tet methylcytosine dioxygenase 2 (TET2), isocitrate dehydrogenase 1 (IDH1), IDH2, additional sex combs-like 1 (ASXL1), enhancer of zeste homologue 2 (EZH2) and DNA methyltransferase 3A (DNMT3A), in the pathogenesis of MPN, MDS and AML, and discuss how this knowledge is leading to novel clinical, biological and therapeutic insights.
We present a highly sensitive and selective chemical labeling and capture approach for genome-wide profiling of 5-hydroxylmethylcytosine (5hmC) using DNA isolated from ∼1,000 cells (nano-hmC-Seal). ...Using this technology, we assessed 5hmC occupancy and dynamics across different stages of hematopoietic differentiation. Nano-hmC-Seal profiling of purified Tet2-mutant acute myeloid leukemia (AML) murine stem cells allowed us to identify leukemia-specific, differentially hydroxymethylated regions that harbor known and candidate disease-specific target genes with differential 5hmC peaks compared to normal stem cells. The change of 5hmC patterns in AML strongly correlates with differential gene expression, demonstrating the importance of dynamic alterations of 5hmC in regulating transcription in AML. Together, covalent 5hmC labeling offers an effective approach to study and detect DNA methylation dynamics in in vivo disease models and in limited clinical samples.
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•Chemical labeling enables highly sensitive profiling of 5hmC using limited input DNA•Genome-wide 5hmC distributions obtained for hematopoietic stem cell differentiation•5hmC changes correlate with differential gene expression in an AML model•Redistributions of 5hmC in the Tet2-deficient leukemia stem cells
Han et al. developed a highly sensitive and robust 5hmC sequencing approach using genomic DNA isolated from ∼1,000 cells. Using their approach, 5hmC maps across different stages of mouse hematopoietic differentiation were obtained and insights into roles of 5hmC in tumor-initiating cells in an AML mouse model were gained.
Recurrent somatic ASXL1 mutations occur in patients with myelodysplastic syndrome, myeloproliferative neoplasms, and acute myeloid leukemia, and are associated with adverse outcome. Despite the ...genetic and clinical data implicating ASXL1 mutations in myeloid malignancies, the mechanisms of transformation by ASXL1 mutations are not understood. Here, we identify that ASXL1 mutations result in loss of polycomb repressive complex 2 (PRC2)-mediated histone H3 lysine 27 (H3K27) tri-methylation. Through integration of microarray data with genome-wide histone modification ChIP-Seq data, we identify targets of ASXL1 repression, including the posterior HOXA cluster that is known to contribute to myeloid transformation. We demonstrate that ASXL1 associates with the PRC2, and that loss of ASXL1 in vivo collaborates with NRASG12D to promote myeloid leukemogenesis.
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► ASXL1 mutations are loss-of-function mutations ► ASXL1 loss results in a genome-wide reduction in H3K27me3 occupancy ► ASXL1 interacts with the PRC2 complex and is important for PRC2 recruitment ► ASXL1 collaborates with co-occurring oncogenes in vivo to promote leukemogenesis
Specific combinations of acute myeloid leukemia (AML) disease alleles, including FLT3 and TET2 mutations, confer distinct biologic features and adverse outcome. We generated mice with mutations in ...Tet2 and Flt3, which resulted in fully penetrant, lethal AML. Multipotent Tet2−/−;Flt3ITD progenitors (LSK CD48+CD150−) propagate disease in secondary recipients and were refractory to standard AML chemotherapy and FLT3-targeted therapy. Flt3ITD mutations and Tet2 loss cooperatively remodeled DNA methylation and gene expression to an extent not seen with either mutant allele alone, including at the Gata2 locus. Re-expression of Gata2 induced differentiation in AML stem cells and attenuated leukemogenesis. TET2 and FLT3 mutations cooperatively induce AML, with a defined leukemia stem cell population characterized by site-specific changes in DNA methylation and gene expression.
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•Tet2−/− and Flt3ITD alleles cooperate to induce AML•LSK MPP cells propagate the disease and are resistant to anti-leukemic therapies•Tet2−/− and Flt3ITD cooperatively induce DNA hypermethylation and expression changes•Reactivation of Gata2 induces differentiation and inhibits leukemogenesis
Shih et al. show that combined mutations in Tet2 and Flt3 alter DNA methylation and gene expression to an extent not seen with either mutation alone and cause fully penetrant, lethal acute myeloid leukemia (AML) in mice. Similar characteristics are observed in human AML with combined TET2 and FLT3 mutations.
TET proteins convert 5‐methylcytosine to 5‐hydroxymethylcytosine, an emerging dynamic epigenetic state of DNA that can influence transcription. Evidence has linked TET1 function to epigenetic ...repression complexes, yet mechanistic information, especially for the TET2 and TET3 proteins, remains limited. Here, we show a direct interaction of TET2 and TET3 with O‐GlcNAc transferase (OGT). OGT does not appear to influence hmC activity, rather TET2 and TET3 promote OGT activity. TET2/3–OGT co‐localize on chromatin at active promoters enriched for H3K4me3 and reduction of either TET2/3 or OGT activity results in a direct decrease in H3K4me3 and concomitant decreased transcription. Further, we show that Host Cell Factor 1 (HCF1), a component of the H3K4 methyltransferase SET1/COMPASS complex, is a specific GlcNAcylation target of TET2/3–OGT, and modification of HCF1 is important for the integrity of SET1/COMPASS. Additionally, we find both TET proteins and OGT activity promote binding of the SET1/COMPASS H3K4 methyltransferase, SETD1A, to chromatin. Finally, studies in Tet2 knockout mouse bone marrow tissue extend and support the data as decreases are observed of global GlcNAcylation and also of H3K4me3, notably at several key regulators of haematopoiesis. Together, our results unveil a step‐wise model, involving TET–OGT interactions, promotion of GlcNAcylation, and influence on H3K4me3 via SET1/COMPASS, highlighting a novel means by which TETs may induce transcriptional activation.
This paper identifies the N‐acetylglucosamine transferase OGT as binding partner for TET2/3 proteins. Their genome‐wide chromatin binding and the characterization of the Set1/COMPASS complex as OGT target implies coordinated gene regulation.
Somatic Addition of Sex Combs Like 1 (ASXL1) mutations occur in 10-30% of patients with myeloid malignancies, most commonly in myelodysplastic syndromes (MDSs), and are associated with adverse ...outcome. Germline ASXL1 mutations occur in patients with Bohring-Opitz syndrome. Here, we show that constitutive loss of Asxl1 results in developmental abnormalities, including anophthalmia, microcephaly, cleft palates, and mandibular malformations. In contrast, hematopoietic-specific deletion of Asxl1 results in progressive, multilineage cytopenias and dysplasia in the context of increased numbers of hematopoietic stem/progenitor cells, characteristic features of human MDS. Serial transplantation of Asxl1-null hematopoietic cells results in a lethal myeloid disorder at a shorter latency than primary Asxl1 knockout (KO) mice. Asxl1 deletion reduces hematopoietic stem cell self-renewal, which is restored by concomitant deletion of Tet2, a gene commonly co-mutated with ASXL1 in MDS patients. Moreover, compound Asxl1/Tet2 deletion results in an MDS phenotype with hastened death compared with single-gene KO mice. Asxl1 loss results in a global reduction of H3K27 trimethylation and dysregulated expression of known regulators of hematopoiesis. RNA-Seq/ChIP-Seq analyses of Asxl1 in hematopoietic cells identify a subset of differentially expressed genes as direct targets of Asxl1. These findings underscore the importance of Asxl1 in Polycomb group function, development, and hematopoiesis.
Recurrent mutations at R140 and R172 in isocitrate dehydrogenase 2 (IDH2) occur in many cancers, including ∼12% of acute myeloid leukemia (AML). In preclinical models these mutations cause ...accumulation of the oncogenic metabolite R-2-hydroxyglutarate (2-HG) and induce hematopoietic differentiation block. Single-agent enasidenib (AG-221/CC-90007), a selective mutant IDH2 (mIDH2) inhibitor, produced an overall response rate of 40.3% in relapsed/refractory AML (rrAML) patients with mIDH2 in a phase 1 trial. However, its mechanism of action and biomarkers associated with response remain unclear. Here, we measured 2-HG, mIDH2 allele burden, and co-occurring somatic mutations in sequential patient samples from the clinical trial and correlated these with clinical response. Furthermore, we used flow cytometry to assess inhibition of mIDH2 on hematopoietic differentiation. We observed potent 2-HG suppression in both R140 and R172 mIDH2 AML subtypes, with different kinetics, which preceded clinical response. Suppression of 2-HG alone did not predict response, because most nonresponding patients also exhibited 2-HG suppression. Complete remission (CR) with persistence of mIDH2 and normalization of hematopoietic stem and progenitor compartments with emergence of functional mIDH2 neutrophils were observed. In a subset of CR patients, mIDH2 allele burden was reduced and remained undetectable with response. Co-occurring mutations in NRAS and other MAPK pathway effectors were enriched in nonresponding patients, consistent with RAS signaling contributing to primary therapeutic resistance. Together, these data support differentiation as the main mechanism of enasidenib efficacy in relapsed/refractory AML patients and provide insight into resistance mechanisms to inform future mechanism-based combination treatment studies.
•Enasidenib inhibits mIDH2, leading to leukemic cell differentiation with emergence of functional mIDH2 neutrophils in rrAML patients.•RAS pathway mutations and increased mutational burden overall are associated with a decreased response rate to mIDH2 inhibition.
Myeloproliferative neoplasms, such as polycythemia vera, essential thrombocythemia, and primary myelofibrosis, are bone marrow disorders that result in the overproduction of mature clonal myeloid ...elements. Identification of recurrent genetic mutations has been described and aid in diagnosis and prognostic determination. Mouse models of these mutations have confirmed the biologic significance of these mutations in myeloproliferative neoplasm disease biology and provided greater insights on the pathways that are dysregulated with each mutation. The models are useful tools that have led to preclinical testing and provided data as validation for future myeloproliferative neoplasm clinical trials.
Although clinically tested JAK inhibitors reduce splenomegaly and systemic symptoms, molecular responses are not observed in most myeloproliferative neoplasm (MPN) patients. We previously ...demonstrated that MPN cells become persistent to type I JAK inhibitors that bind the active conformation of JAK2. We investigated whether CHZ868, a type II JAK inhibitor, would demonstrate activity in JAK inhibitor persistent cells, murine MPN models, and MPN patient samples. JAK2 and MPL mutant cell lines were sensitive to CHZ868, including type I JAK inhibitor persistent cells. CHZ868 showed significant activity in murine MPN models and induced reductions in mutant allele burden not observed with type I JAK inhibitors. These data demonstrate that type II JAK inhibition is a viable therapeutic approach for MPN patients.
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•CHZ868, a type II JAK inhibitor, reverses type I JAK inhibitor persistence•Type II JAK inhibition with CHZ868 preferentially inhibits V617F mutant versus WT JAK2•Type II JAK inhibition shows increased efficacy in preclinical MPN models•Type II JAK inhibition reduces mutant allele burden in JAK2-/MPL mutant MPN models
The current clinical JAK2 inhibitors are type I kinase inhibitors that provide clinical benefits to myeloproliferative neoplasm (MPN) patients but do not reduce mutant allele burden. Meyer et al. report a type II JAK2 inhibitor that shows activity against relevant MPN models and reduces mutant allele burden.
Intact cholesterol homeostasis helps to maintain hematopoietic stem and multipotential progenitor cell (HSPC) quiescence. Mice with defects in cholesterol efflux pathways due to deficiencies of the ...ATP binding cassette transporters ABCA1 and ABCG1 displayed a dramatic increase in HSPC mobilization and extramedullary hematopoiesis. Increased extramedullary hematopoiesis was associated with elevated serum levels of G-CSF due to generation of IL-23 by splenic macrophages and dendritic cells. This favored hematopoietic lineage decisions toward granulocytes rather than macrophages in the bone marrow leading to impaired support for osteoblasts and decreased Cxcl12/SDF-1 production by mesenchymal progenitors. Greater HSPC mobilization and extramedullary hematopoiesis were reversed by raising HDL levels in Abca1−/−Abcg1−/− and Apoe−/− mice or in a mouse model of myeloproliferative neoplasm mediated by Flt3-ITD mutation. Our data identify a role of cholesterol efflux pathways in the control of HSPC mobilization. This may translate into therapeutic strategies for atherosclerosis and hematologic malignancies.
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► Cholesterol efflux in splenic DCs and macrophages regulate HSPC mobilization ► Cholesterol efflux causes reduced G-CSF production and alters the osteoblastic niche ► Cholesterol efflux prevents HSPC mobilization and extramedullary hematopoiesis
Cholesterol homeostasis is an important checkpoint for maintaining HSC quiescence. When cholesterol efflux is impaired, splenic secretion of IL-23 raises G-CSF levels, which change the osteoblastic niche to favor HSC mobilization and extramedullary hematopoiesis.
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