TET2 converts 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC) in DNA and is frequently mutated in myeloid malignancies, including myeloproliferative neoplasms. Here we show that the level of ...5-hmC is decreased in granulocyte DNA from myeloproliferative neoplasm patients with TET2 mutations compared with granulocyte DNA from healthy patients. Inhibition of TET2 by RNA interference decreases 5-hmC levels in both human leukemia cell lines and cord blood CD34+ cells. These results confirm the enzymatic function of TET2 in human hematopoietic cells. Knockdown of TET2 in cord blood CD34+ cells skews progenitor differentiation toward the granulomonocytic lineage at the expense of lymphoid and erythroid lineages. In addition, by monitoring in vitro granulomonocytic development we found a decreased granulocytic differentiation and an increase in monocytic cells. Our results indicate that TET2 disruption affects 5-hmC levels in human myeloid cells and participates in the pathogenesis of myeloid malignancies through the disturbance of myeloid differentiation.
Oncogenic pathways underlying in the development of myelodysplastic syndromes (MDS) remain poorly characterized, but mutations of the ten-eleven translocation 2 (TET2) gene are frequently observed. ...In the present work, we evaluated the prognostic impact of TET2 mutations in MDS. Frameshift, nonsense, missense mutations, or defects in gene structure were identified in 22 (22.9%) of 96 patients (95% confidence interval CI, 14.5-31.3 patients). Mutated and unmutated patients did not significantly differ in initial clinical or hematologic parameters. The 5-year OS was 76.9% (95% CI, 49.2%-91.3%) in mutated versus 18.3% (95% CI, 4.2%-41.1%) in unmutated patients (P = .005). The 3-year leukemia-free survival was 89.3% (95% CI, 63.1%-97.0%) in mutated versus 63.7% (95% CI, 48.2%-75.4%) in unmutated patients (P = .035). In univariate analysis (Cox proportional hazard model), the absence of TET2 mutation was associated with a 4.1-fold (95% CI, 1.4-12.0-fold) increased risk of death (P = .009). In multivariate analysis adjusted for age, International Prognostic Scoring System, and transfusion requirement, the presence of TET2 mutation remained an independent factor of favorable prognosis (hazard ratio, 5.2; 95% CI, 1.6-16.3; P = .005). These results indicate that TET2 mutations observed in approximately 20% of patients, irrespective of the World Health Organization or French-American-British subtype, represent a molecular marker for good prognosis in MDS.
Megakaryocyte polyploidy is characterized by cytokinesis failure resulting from defects in contractile forces at the cleavage furrow. Although immature megakaryocytes express 2 nonmuscle myosin II ...isoforms (MYH9 NMIIA and MYH10 NMIIB), only NMIIB localizes at the cleavage furrow, and its subsequent absence contributes to polyploidy. In this study, we tried to understand why the abundant NMIIA does not localize at the furrow by focusing on the RhoA/ROCK pathway that has a low activity in polyploid megakaryocytes. We observed that under low RhoA activity, NMII isoforms presented different activity that determined their localization. Inhibition of RhoA/ROCK signaling abolished the localization of NMIIB, whereas constitutively active RhoA induced NMIIA at the cleavage furrow. Thus, although high RhoA activity favored the localization of both the isoforms, only NMIIB could localize at the furrow at low RhoA activity. This was further confirmed in erythroblasts that have a higher basal RhoA activity than megakaryocytes and express both NMIIA and NMIIB at the cleavage furrow. Decreased RhoA activity in erythroblasts abolished localization of NMIIA but not of NMIIB from the furrow. This differential localization was related to differences in actin turnover. Megakaryocytes had a higher actin turnover compared with erythroblasts. Strikingly, inhibition of actin polymerization was found to be sufficient to recapitulate the effects of inhibition of RhoA/ROCK pathway on NMII isoform localization; thus, cytokinesis failure in megakaryocytes is the consequence of both the absence of NMIIB and a low RhoA activity that impairs NMIIA localization at the cleavage furrow through increased actin turnover.
•RhoA-dependent differential activity of NMII isoforms determines its localization at the cleavage furrow of megakaryocytes.•Perturbation of the actin cytoskeleton was sufficient to induce the differential localization of NMII isoforms.
Sustained ANKRD26 expression associated with germline ANKRD26 mutations causes thrombocytopenia 2 (THC2), an inherited platelet disorder associated with a predisposition to leukemia. Some patients ...also present with erythrocytosis and/or leukocytosis. Using multiple human-relevant in vitro models (cell lines, primary patients' cells and patient-derived induced pluripotent stem cells) we demonstrate for the first time that ANKRD26 is expressed during the early steps of erythroid, megakaryocyte and granulocyte differentiation, and is necessary for progenitor cell proliferation. As differentiation progresses, ANKRD26 expression is progressively silenced, to complete the cellular maturation of the three myeloid lineages. In primary cells, abnormal ANKRD26 expression in committed progenitors directly affects the proliferation/differentiation balance for the three cell types. We show that ANKRD26 interacts with and crucially modulates the activity of MPL, EPOR and G-CSFR, three homodimeric type I cytokine receptors that regulate blood cell production. Higher than normal levels of ANKRD26 prevent the receptor internalization that leads to increased signaling and cytokine hypersensitivity. These findings afford evidence how ANKRD26 overexpression or the absence of its silencing during differentiation is responsible for myeloid blood cell abnormalities in patients with THC2.
Thrombocytopenia is a major side effect of a new class of anticancer agents that target histone deacetylase (HDAC). Their mechanism is poorly understood. Here, we show that HDAC6 inhibition and ...genetic knockdown lead to a strong decrease in human proplatelet formation (PPF). Unexpectedly, HDAC6 inhibition-induced tubulin hyperacetylation has no effect on PPF. The PPF decrease induced by HDAC6 inhibition is related to cortactin (CTTN) hyperacetylation associated with actin disorganization inducing important changes in the distribution of megakaryocyte (MK) organelles. CTTN silencing in human MKs phenocopies HDAC6 inactivation and knockdown leads to a strong PPF defect. This is rescued by forced expression of a deacetylated CTTN mimetic. Unexpectedly, unlike human-derived MKs, HDAC6 and CTTN are shown to be dispensable for mouse PPF in vitro and platelet production in vivo. Our results highlight an unexpected function of HDAC6-CTTN axis as a positive regulator of human but not mouse MK maturation.
The functional diversity of cells that compose myeloid malignancies, i.e., the respective roles of genetic and epigenetic heterogeneity in this diversity, remains poorly understood. This question is ...addressed in chronic myelomonocytic leukemia, a myeloid neoplasm in which clinical diversity contrasts with limited genetic heterogeneity. To generate induced pluripotent stem cell clones, we reprogrammed CD34
cells collected from a patient with a chronic myelomonocytic leukemia in which whole exome sequencing of peripheral blood monocyte DNA had identified 12 gene mutations, including a mutation in
and two heterozygous mutations in
in the founding clone and a secondary
(G12D) mutation. CD34
cells from an age-matched healthy donor were also reprogrammed. We captured a part of the genetic heterogeneity observed in the patient, i.e. we analyzed five clones with two genetic backgrounds, without and with the
(G12D) mutation. Hematopoietic differentiation of these clones recapitulated the main features of the patient's disease, including overproduction of granulomonocytes and dysmegakaryopoiesis. These analyses also disclosed significant discrepancies in the behavior of hematopoietic cells derived from induced pluripotent stem cell clones with similar genetic background, correlating with limited epigenetic changes. These analyses suggest that, beyond the coding mutations, several levels of intraclonal heterogeneity may participate in the yet unexplained clinical heterogeneity of the disease.
Macrothrombocytopenias are the most important subgroup of inherited thrombocytopenias. This subgroup is particularly heterogeneous because the affected genes are involved in various functions such as ...cell signaling, cytoskeleton organization, and gene expression. Herein we describe the clinical and hematological features of a consanguineous family with a severe autosomal recessive macrothrombocytopenia associated with a thrombocytopathy inducing a bleeding tendency in the homozygous mutated patients. Platelet activation and cytoskeleton reorganization were impaired in these homozygous patients. Exome sequencing identified a c.222C>G mutation (missense p.74Ile>Met) in PRKACG, a gene encoding the γ-catalytic subunit of the cyclic adenosine monophosphate-dependent protein kinase, the mutated allele cosegregating with the macrothrombocytopenia. We demonstrate that the p.74Ile>Met PRKACG mutation is associated with a marked defect in proplatelet formation and a low level in filamin A in megakaryocytes (MKs). The defect in proplatelet formation was rescued in vitro by lentiviral vector-mediated overexpression of wild-type PRKACG in patient MKs. We thus conclude that PRKACG is a new central actor in platelet biogenesis and a new gene involved in inherited thrombocytopenia with giant platelets associated with a thrombocytopathy.
•We identify a new type of autosomal recessive macrothrombocytopenia associated with a mutation in PRKACG, coding the PKA catalytic subunit.•The homozygous PRKACG mutation leads to a deep defect in proplatelet formation that was restored by the overexpression of wild-type PRKACG.
Fusion oncogenes are prevalent in several pediatric cancers, yet little is known about the specific associations between age and phenotype. We observed that fusion oncogenes, such as
, are associated ...with acute megakaryoblastic or other myeloid leukemia subtypes in an age-dependent manner. Analysis of a novel inducible transgenic mouse model showed that
expression in fetal hematopoietic stem cells induced rapid megakaryoblastic leukemia whereas expression in adult bone marrow hematopoietic stem cells resulted in a shift toward myeloid transformation with a strikingly delayed
leukemogenic potential. Chromatin accessibility and single-cell transcriptome analyses indicate ontogeny-dependent intrinsic and
-induced differences in the activities of key transcription factors, including ERG, SPI1, GATA1, and CEBPA. Importantly, switching off the fusion oncogene restored terminal differentiation of the leukemic blasts. Together, these data show that aggressiveness and phenotypes in pediatric acute myeloid leukemia result from an ontogeny-related differential susceptibility to transformation by fusion oncogenes. SIGNIFICANCE: This work demonstrates that the clinical phenotype of pediatric acute myeloid leukemia is determined by ontogeny-dependent susceptibility for transformation by oncogenic fusion genes. The phenotype is maintained by potentially reversible alteration of key transcription factors, indicating that targeting of the fusions may overcome the differentiation blockage and revert the leukemic state.
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The JAK2V617F mutation is frequently observed in classical myeloproliferative disorders, and disease progression is associated with a biallelic acquisition of the mutation occurring by mitotic ...recombination. In this study, we examined whether JAK2 activation could lead to increased homologous recombination (HR) and genetic instability. In a Ba/F3 cell line expressing the erythropoietin (EPO) receptor, mutant JAK2V617F and, to a lesser extent, wild-type (wt) JAK2 induced an increase in HR activity in the presence of EPO without modifying nonhomologous end-joining efficiency. Moreover, a marked augmentation in HR activity was found in CD34+-derived cells isolated from patients with polycythemia vera or primitive myelofibrosis compared with control samples. This increase was associated with a spontaneous RAD51 foci formation. As a result, sister chromatid exchange was 50% augmented in JAK2V617F Ba/F3 cells compared with JAK2wt cells. Moreover, JAK2 activation increased centrosome and ploidy abnormalities. Finally, in JAK2V617F Ba/F3 cells, we found a 100-fold and 10-fold increase in mutagenesis at the HPRT and Na/K ATPase loci, respectively. Together, this work highlights a new molecular mechanism for HR regulation mediated by JAK2 and more efficiently by JAK2V617F. Our study might provide some keys to understand how a single mutation can give rise to different pathologies.
Megakaryopoiesis is an original and complex cell process which leads to the formation of platelets. The homeostatic production of platelets is mainly regulated and controlled by thrombopoietin (TPO) ...and the TPO receptor (MPL)/JAK2 axis. Therefore, any hereditary or acquired abnormality affecting this signaling axis can result in thrombocytosis or thrombocytopenia. Thrombocytosis can be due to genetic alterations that affect either the intrinsic MPL signaling through gain-of-function (GOF) activity (
) and loss-of-function (LOF) activity of negative regulators (
) or the extrinsic MPL signaling by
GOF mutations leading to increased TPO synthesis. Alternatively, thrombocytosis may paradoxically result from mutations of
leading to an abnormal MPL trafficking, inducing increased TPO levels by alteration of its clearance. In contrast, thrombocytopenia can also result from LOF
or
mutations, which cause a complete defect in MPL trafficking to the cell membrane, impaired MPL signaling or stability, defects in the TPO/MPL interaction, or an absence of TPO production.
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