The genetic landscape of classical myeloproliferative neoplasm (MPN) is in large part elucidated. The MPN-restricted driver mutations, including those in JAK2, calreticulin (CALR), and ...myeloproliferative leukemia virus (MPL), abnormally activate the cytokine receptor/JAK2 pathway and their downstream effectors, more particularly the STATs. The most frequent mutation, JAK2V617F, activates the 3 main myeloid cytokine receptors (erythropoietin receptor, granulocyte colony-stimulating factor receptor, and MPL) whereas CALR or MPL mutants are restricted to MPL activation. This explains why JAK2V617F is associated with polycythemia vera, essential thrombocythemia (ET), and primary myelofibrosis (PMF) whereas CALR and MPL mutants are found in ET and PMF. Other mutations in genes involved in epigenetic regulation, splicing, and signaling cooperate with the 3 MPN drivers and play a key role in the PMF pathogenesis. Mutations in epigenetic regulators TET2 and DNMT3A are involved in disease initiation and may precede the acquisition of JAK2V617F. Other mutations in epigenetic regulators such as EZH2 and ASXL1 also play a role in disease initiation and disease progression. Mutations in the splicing machinery are predominantly found in PMF and are implicated in the development of anemia or pancytopenia. Both heterogeneity of classical MPNs and prognosis are determined by a specific genomic landscape, that is, type of MPN driver mutations, association with other mutations, and their order of acquisition. However, factors other than somatic mutations play an important role in disease initiation as well as disease progression such as germ line predisposition, inflammation, and aging. Delineation of these environmental factors will be important to better understand the precise pathogenesis of MPN.
Mammal megakaryocytes (MK) undergo polyploidization during their differentiation. This process leads to a marked increase in the MK size and of their cytoplasm. Contrary to division by classical ...mitosis, ploidization allows an economical manner to produce platelets as they arise from the fragmentation of the MK cytoplasm. The platelet production in vivo correlates to the entire MK cytoplasm mass that depends both upon the number of MKs and their size. Polyploidization occurs by several rounds of DNA replication with at the end of each round an aborted mitosis at late phase of cytokinesis. As there is also a defect in karyokinesis, MKs are giant cells with a single polylobulated nucleus with a 2
x
N ploidy. However, polyploidization per se does not increase platelet production because it requires a parallel development of MK organelles such as mitochondria, granules and the demarcation membrane system. MK polyploidization is regulated by extrinsic factors, more particularly by thrombopoietin (TPO), which during a platelet stress increases first polyploidization before enhancing the MK number and by transcription factors such as RUNX1, GATA1, and FLI1 that regulate MK differentiation explaining why polyploidization and cytoplasmic maturation are intermingled. MK polyploidization is ontogenically regulated and is markedly altered in malignant myeloid disorders such as acute megakaryoblastic leukemia and myeloproliferative disorders as well as in hereditary thrombocytopenia, more particularly those involving transcription factors or signaling pathways. In addition, MKs arising from progenitors in vitro have a much lower ploidy in vitro than in vivo leading to a low yield of platelet production in vitro. Thus, it is tempting to find approaches to increase MK polyploidization in vitro. However, these approaches require molecules that are able to simultaneously increase MK polyploidization and to induce terminal differentiation. Here, we will focus on the regulation by extrinsic and intrinsic factors of MK polyploidization during development and pathological conditions.
Myeloproliferative neoplasms (MPNs) are clonal disorders characterized by excessive production of mature blood cells. In the majority of classic MPN—polycythemia vera, essential thrombocythemia, and ...primitive myelofibrosis—driver oncogenic mutations affecting Janus kinase 2 (JAK2) or MPL lead to constitutive activation of cytokine-regulated intracellular signaling pathways. LNK, c-CBL, or SOCSs (all negative regulators of signaling pathways), although infrequently targeted, may either drive the disease or synergize with JAK2 and MPL mutations. IZF1 deletions or TP53 mutations are mainly found at transformation phases and are present at greater frequency than in de novo acute myeloid leukemias. Loss-of-function mutations in 3 genes involved in epigenetic regulation, TET2, ASXL1, and EZH2, may be early events preceding JAK2V617F but may also occur late during disease progression. They are more frequently observed in PMF than PV and ET and are also present in other types of malignant myeloid diseases. A likely hypothesis is that they facilitate clonal selection, allowing the dominance of the JAK2V617F subclone during the chronic phase and, together with cooperating mutations, promote blast crisis. Their precise roles in hematopoiesis and in the pathogenesis of MPN, as well as their prognostic impact and potential as a therapeutic target, are currently under investigation.
Hematopoietic stem cells (HSCs) residing in the bone marrow (BM) accumulate during aging but are functionally impaired. However, the role of HSC-intrinsic and -extrinsic aging mechanisms remains ...debated. Megakaryocytes promote quiescence of neighboring HSCs. Nonetheless, whether megakaryocyte-HSC interactions change during pathological/natural aging is unclear. Premature aging in Hutchinson-Gilford progeria syndrome recapitulates physiological aging features, but whether these arise from altered stem or niche cells is unknown. Here, we show that the BM microenvironment promotes myelopoiesis in premature/physiological aging. During physiological aging, HSC-supporting niches decrease near bone but expand further from bone. Increased BM noradrenergic innervation promotes β2-adrenergic-receptor(AR)-interleukin-6-dependent megakaryopoiesis. Reduced β3-AR-Nos1 activity correlates with decreased endosteal niches and megakaryocyte apposition to sinusoids. However, chronic treatment of progeroid mice with β3-AR agonist decreases premature myeloid and HSC expansion and restores the proximal association of HSCs to megakaryocytes. Therefore, normal/premature aging of BM niches promotes myeloid expansion and can be improved by targeting the microenvironment.
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•Reduction of endosteal BM and expansion of non-endosteal BM occurs with age•β2/β3-ARs exhibit opposite and niche-dependent regulation of myelopoiesis•β2-AR overriding β3-AR promotes myeloid expansion during physiological aging•Premature HSC aging in HGPS can be improved by targeting the microenvironment
Recent studies have suggested a microenvironmental contribution to stem-cell aging, but the mechanisms are largely unexplored. Méndez-Ferrer et al. report anatomical remodeling of blood-stem-cell-supporting niches and functional switch of β adrenergic signals, leading to myeloid expansion during aging. Targeting the microenvironment can improve pathological, premature, niche-dependent hematopoietic aging in mice.
Blood platelets have important roles in haemostasis, where they quickly stop bleeding in response to vascular damage. They have also recognised functions in thrombosis, immunity, antimicrobal ...defense, cancer growth and metastasis, tumour angiogenesis, lymphangiogenesis, inflammatory diseases, wound healing, liver regeneration and neurodegeneration. Their brief life span in circulation is strictly controlled by intrinsic apoptosis, where the prosurvival Bcl-2 family protein, Bcl-xL, has a major role. Blood platelets are produced by large polyploid precursor cells, megakaryocytes, residing mainly in the bone marrow. Together with Mcl-1, Bcl-xL regulates megakaryocyte survival. This review describes megakaryocyte maturation and survival, platelet production, platelet life span and diseases of abnormal platelet number with a focus on the role of Bcl-xL during these processes.
Mutations in the calreticulin gene (CALR) represented by deletions and insertions in exon 9 inducing a −1/+2 frameshift are associated with a significant fraction of myeloproliferative neoplasms ...(MPNs). The mechanisms by which CALR mutants induce MPN are unknown. Here, we show by transcriptional, proliferation, biochemical, and primary cell assays that the pathogenic CALR mutants specifically activate the thrombopoietin receptor (TpoR/MPL). No activation is detected with a battery of type I and II cytokine receptors, except granulocyte colony-stimulating factor receptor, which supported only transient and weak activation. CALR mutants induce ligand-independent activation of JAK2/STAT/phosphatydylinositol-3′-kinase (PI3-K) and mitogen-activated protein (MAP) kinase pathways via TpoR, and autonomous growth in Ba/F3 cells. In these transformed cells, no synergy is observed between JAK2 and PI3-K inhibitors in inhibiting cytokine-independent proliferation, thus showing a major difference from JAK2V617F cells where such synergy is strong. TpoR activation was dependent on its extracellular domain and its N-glycosylation, especially at N117. The glycan binding site and the novel C-terminal tail of the mutant CALR proteins were required for TpoR activation. A soluble form of TpoR was able to prevent activation of full-length TpoR provided that it was N-glycosylated. By confocal microscopy and subcellular fractionation, CALR mutants exhibit different intracellular localization from that of wild-type CALR. Finally, knocking down either MPL/TpoR or JAK2 in megakaryocytic progenitors from patients carrying CALR mutations inhibited cytokine-independent megakaryocytic colony formation. Taken together, our study provides a novel signaling paradigm, whereby a mutated chaperone constitutively activates cytokine receptor signaling.
•Calreticulin mutants responsible for myeloproliferative neoplasms specifically activate the thrombopoietin receptor and in turn JAK2.•Activation of the thrombopoietin receptor requires the glycan binding site and a novel C-terminal tail of the mutant calreticulin.
Calreticulin (CALR) +1 frameshift mutations in exon 9 are prevalent in myeloproliferative neoplasms. Mutant CALRs possess a new C-terminal sequence rich in positively charged amino acids, leading to ...activation of the thrombopoietin receptor (TpoR/MPL). We show that the new sequence endows the mutant CALR with rogue chaperone activity, stabilizing a dimeric state and transporting TpoR and mutants thereof to the cell surface in states that would not pass quality control; this function is absolutely required for oncogenic transformation. Mutant CALRs determine traffic via the secretory pathway of partially immature TpoR, as they protect N117-linked glycans from further processing in the Golgi apparatus. A number of engineered or disease-associated TpoRs such as TpoR/MPL R102P, which causes congenital thrombocytopenia, are rescued for traffic and function by mutant CALRs, which can also overcome endoplasmic reticulum retention signals on TpoR. In addition to requiring N-glycosylation of TpoR, mutant CALRs require a hydrophobic patch located in the extracellular domain of TpoR to induce TpoR thermal stability and initial intracellular activation, whereas full activation requires cell surface localization of TpoR. Thus, mutant CALRs are rogue chaperones for TpoR and traffic-defective TpoR mutants, a function required for the oncogenic effects.
•CALR mutants rescue cell surface localization of traffic-deficient TpoR, including R102P that causes congenital thrombocytopenia.•Oncogenic TpoR activation by CALR mutants requires interaction, stabilization, and cell surface localization of the TpoR-CALR complex.
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Several prognostic scoring systems have been proposed for chronic myelomonocytic leukemia (CMML), a disease in which some gene mutations-including ASXL1-have been associated with poor prognosis in ...univariable analyses. We developed and validated a prognostic score for overall survival (OS) based on mutational status and standard clinical variables.
We genotyped ASXL1 and up to 18 other genes including epigenetic (TET2, EZH2, IDH1, IDH2, DNMT3A), splicing (SF3B1, SRSF2, ZRSF2, U2AF1), transcription (RUNX1, NPM1, TP53), and signaling (NRAS, KRAS, CBL, JAK2, FLT3) regulators in 312 patients with CMML. Genotypes and clinical variables were included in a multivariable Cox model of OS validated by bootstrapping. A scoring system was developed using regression coefficients from this model.
ASXL1 mutations (P < .0001) and, to a lesser extent, SRSF2 (P = .03), CBL (P = .003), and IDH2 (P = .03) mutations predicted inferior OS in univariable analysis. The retained independent prognostic factors included ASXL1 mutations, age older than 65 years, WBC count greater than 15 ×10(9)/L, platelet count less than 100 ×10(9)/L, and anemia (hemoglobin < 10 g/dL in female patients, < 11g/dL in male patients). The resulting five-parameter prognostic score delineated three groups of patients with median OS not reached, 38.5 months, and 14.4 months, respectively (P < .0001), and was validated in an independent cohort of 165 patients (P < .0001).
A new prognostic score including ASXL1 status, age, hemoglobin, WBC, and platelet counts defines three groups of CMML patients with distinct outcomes. Based on concordance analysis, this score appears more discriminative than those based solely on clinical parameters.
Subtle caspase activation is associated with the differentiation of several myeloid lineages. A tightly orchestrated dance between caspase-3 activation and the chaperone HSP70 that migrates to the ...nucleus to protect the master regulator GATA-1 from cleavage transiently occurs in basophilic erythroblasts and may prepare nucleus and organelle expel that occurs at the terminal phase of erythroid differentiation. A spatially restricted activation of caspase-3 occurs in maturing megakaryocytes to promote proplatelet maturation and platelet shedding in the bloodstream. In a situation of acute platelet need, caspase-3 could be activated in response to IL-1α and promote megakaryocyte rupture. In peripheral blood monocytes, colony-stimulating factor-1 provokes the formation of a molecular platform in which caspase-8 is activated, which downregulates nuclear factor-kappa B (NF-κB) activity and activates downstream caspases whose target fragments such as those generated by nucleophosmin (NPM1) cleavage contribute to the generation of resting macrophages. Human monocytes secrete mature IL-1β in response to lipopolysaccharide through an alternative inflammasome activation that involves caspase-8, a pathway that does not lead to cell death. Finally, active caspase-3 is part of the proteases contained in secretory granules of mast cells. Many questions remain on how these proteases are activated in myeloid cell lineages, which target proteins are cleaved, whereas other are protected from proteolysis, the precise role of cleaved proteins in cell differentiation and functions, and the link between these non-apoptotic functions of caspases and the death of these diverse cell types. Better understanding of these functions may generate therapeutic strategies to control cytopenias or modulate myeloid cell functions in various pathological situations.
Point mutations in the 5' UTR of ankyrin repeat domain 26 (ANKRD26) are associated with familial thrombocytopenia 2 (THC2) and a predisposition to leukemia. Here, we identified underlying mechanisms ...of ANKRD26-associated thrombocytopenia. Using megakaryocytes (MK) isolated from THC2 patients and healthy subjects, we demonstrated that THC2-associated mutations in the 5' UTR of ANKRD26 resulted in loss of runt-related transcription factor 1 (RUNX1) and friend leukemia integration 1 transcription factor (FLI1) binding. RUNX1 and FLI1 binding at the 5' UTR from healthy subjects led to ANKRD26 silencing during the late stages of megakaryopoiesis and blood platelet development. We showed that persistent ANKRD26 expression in isolated MKs increased signaling via the thrombopoietin/myeloproliferative leukemia virus oncogene (MPL) pathway and impaired proplatelet formation by MKs. Importantly, we demonstrated that ERK inhibition completely rescued the in vitro proplatelet formation defect. Our data identify a mechanism for development of the familial thrombocytopenia THC2 that is related to abnormal MAPK signaling.
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