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The classical BCR::ABL-negative myeloproliferative neoplasms (MPN) include Polycythemia Vera (PV), Essential Thrombocytemia (ET), and Primary Myelofibrosis (PMF). They are acquired clonal disorders of the hematopoietic stem cells (HSC) leading to hyperplasia of one or several myeloid lineages. MPN are caused by three main recurrent mutations, JAK2V617F and mutations in the calreticulin (CALR) and the thrombopoietin receptor (MPL) genes. Here, we review the general diagnosis, the complications, and the management of MPN. Second, we explain the physiopathology of the natural disease development and its regulation, which contributes to MPN heterogeneity. Thirdly, we describe the new paradigm of the MPN development highlighting the early origin of driver mutations decades before the onset of symptoms and the consequence on early detection of MPN cases in the general population for early diagnosis and better medical management. Finally, we present the interferon alpha (IFNα) therapy as a potential early disease-modifying drug after reporting its good hematological and molecular efficacies in ET, PV and early MF in clinical trials as well as its mechanism of action in pre-clinical studies. As a result, we may expect that, in the future, MPN patients will be diagnosed very early during the course of disease and that new selective therapies under development, such as IFNα, JAK2V617F inhibitors and CALRmut monoclonal antibodies, would be able to intercept the mutated clones.

Abstract The Janus kinase 2 (JAK2)-driven myeloproliferative neoplasms (MPNs) are chronic malignancies associated with high-risk complications and suboptimal responses to JAK inhibitors such as ruxolitinib. A better understanding of cellular changes induced by ruxolitinib is required to develop new combinatory therapies to improve treatment efficacy. Here, we demonstrate that ruxolitinib induced autophagy in JAK2V617F cell lines and primary MPN patient cells through the activation of protein phosphatase 2A (PP2A). Inhibition of autophagy or PP2A activity along with ruxolitinib treatment reduced proliferation and increased the death of JAK2V617F cells. Accordingly, proliferation and clonogenic potential of JAK2V617F-driven primary MPN patient cells, but not of normal hematopoietic cells, were markedly impaired by ruxolitinib treatment with autophagy or PP2A inhibitor. Finally, preventing ruxolitinib-induced autophagy with a novel potent autophagy inhibitor Lys05 improved leukemia burden reduction and significantly prolonged the mice’s overall survival compared with ruxolitinib alone. This study demonstrates that PP2A-dependent autophagy mediated by JAK2 activity inhibition contributes to resistance to ruxolitinib. Altogether, our data support that targeting autophagy or its identified regulator PP2A could enhance sensitivity to ruxolitinib of JAK2V617F MPN cells and improve MPN patient care.

Acute megakaryoblastic leukemia of Down syndrome (DS-AMKL) is a model of clonal evolution from a preleukemic transient myeloproliferative disorder requiring both a trisomy 21 (T21) and a GATA1s mutation to a leukemia driven by additional driver mutations. We modeled the megakaryocyte differentiation defect through stepwise gene editing of GATA1s, SMC3+/–, and MPLW515K, providing 20 different T21 or disomy 21 (D21) induced pluripotent stem cell (iPSC) clones. GATA1s profoundly reshaped iPSC-derived hematopoietic architecture with gradual myeloid-to-megakaryocyte shift and megakaryocyte differentiation alteration upon addition of SMC3 and MPL mutations. Transcriptional, chromatin accessibility, and GATA1-binding data showed alteration of essential megakaryocyte differentiation genes, including NFE2 downregulation that was associated with loss of GATA1s binding and functionally involved in megakaryocyte differentiation blockage. T21 enhanced the proliferative phenotype, reproducing the cellular and molecular abnormalities of DS-AMKL. Our study provides an array of human cell–based models revealing individual contributions of different mutations to DS-AMKL differentiation blockage, a major determinant of leukemic progression.

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.

Wang et al. use transcriptomic analysis to show that disruption of the transcription factor Lyl-1 in primitive macrophage progenitors results in defective differentiation that impacts on embryonic patterning and neurodevelopment. They also show that Lyl-1 disruption leads to a reduction in mature microglia, thus revealing its role in macrophage and microglial development

Calreticulin del52 and ins5 mutations induce two phenotypically distinct myeloproliferative neoplasms in patients. Here the authors show that modeling these mutations in knock-in mice recapitulate the two diseases and highlight how they impact the different hematopoietic compartments.

Mutations in the calreticulin (CALR) gene are seen in about 30% of essential thrombocythemia and primary myelofibrosis patients. To address the contribution of the human CALR mutants to the pathogenesis of myeloproliferative neoplasms (MPNs) in an endogenous context, we modeled the CALRdel52 and CALRins5 mutants by induced pluripotent stem cell (iPSC) technology using CD34+ progenitors from 4 patients. We describe here the generation of several clones of iPSC carrying heterozygous CALRdel52 or CALRins5 mutations. We showed that CALRdel52 induces a stronger increase in progenitors than CALRins5 and that both CALRdel52 and CALRins5 mutants favor an expansion of the megakaryocytic lineage. Moreover, we found that both CALRdel52 and CALRins5 mutants rendered colony forming unit–megakaryocyte (CFU-MK) independent from thrombopoietin (TPO), and promoted a mild constitutive activation level of signal transducer and activator of transcription 3 in megakaryocytes. Unexpectedly, a mild increase in the sensitivity of colony forming unit-granulocyte (CFU-G) to granulocyte-colony stimulating factor was also observed in iPSC CALRdel52 and CALRins5 compared with control iPSC. Moreover, CALRdel52-induced megakaryocytic spontaneous growth is more dependent on Janus kinase 2/phosphoinositide 3-kinase/extracellular signal-regulated kinase than TPO-mediated growth and opens a therapeutic window for treatments in CALR-mutated MPN. The iPSC models described here represent an interesting platform for testing newly developed inhibitors. Altogether, this study shows that CALR-mutated iPSC recapitulate MPN phenotypes in vitro and may be used for drug screening.

High-throughput single-cell methods have uncovered substantial heterogeneity in the pool of hematopoietic stem and progenitor cells (HSPCs), but how much instruction is inherited by offspring from their heterogeneous ancestors remains unanswered. Using a method that enables simultaneous determination of common ancestor, division number, and differentiation status of a large collection of single cells, our data revealed that murine cells that derived from a common ancestor had significant similarities in their division progression and differentiation outcomes. Although each family diversifies, the overall collection of cell types observed is composed of homogeneous families. Heterogeneity between families could be explained, in part, by differences in ancestral expression of cell surface markers. Our analyses demonstrate that fate decisions of cells are largely inherited from ancestor cells, indicating the importance of common ancestor effects. These results may have ramifications for bone marrow transplantation and leukemia, where substantial heterogeneity in HSPC behavior is observed.

The discovery of the Ten-Eleven Translocation (TET) protein family was initiated by the identification of the MLL partner TET1, and of mutations in the TET2 gene in hematological malignancies including myeloproliferative neoplasms (MPN). TET1, 2 and 3 proteins hydroxylate 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC) and further oxidize 5-hmC into 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). Previous studies highlight the involvement of TET proteins in somatic cells reprogramming into induced pluripotent stem cells (iPSC), particularly Tet1 and 2 in mouse and TET1 in human. Here, we asked whether endogenous TET2 knockdown also displays this function. Using different shRNA against TET2, we provide evidence that TET2 strongly decreases the reprogramming of human hematopoietic progenitor cells into iPSC. Importantly, using 2 MPN patients, we observed that TET2 mutations affecting catalytic domain allowed iPSC generation. Instead, using another TET2 and TET3-mutated patient, we could only reprogram IPSC with TET3 mutation alone, suggesting that the type of TET2 mutation and/or the cooperation with TET3 mutations may alter the reprogramming activity. Altogether, this work highlights the importance of endogenous TET in the reprogramming process of human hematopoietic progenitors. Keywords: TET2, 5-hmC, iPSC, Reprogramming

Increased exposure of calreticulin (CALR) on malignant cells is associated with therapy-relevant adaptive immune responses and superior therapeutic outcome in solid tumors and haemato-oncological diseases, because surface-exposed CALR acts as an ‘eat-me’ signal facilitating the phagocytosis of stressed and dying cancer cells by immature dendritic cells, thus favoring antitumor immune responses. On the contrary, mutations of the CALR gene that cause the omission of the C-terminal KDEL endoplasmic reticulum retention motif from CALR protein, resulting in its secretion from cells, act as oncogenic drivers in myeloproliferative neoplasms via the autocrine activation of the thrombopoietin receptor. We recently showed that soluble CALR inhibited the phagocytosis of cancer cells by dendritic cells, thus dampening anticancer immune responses. Furthermore, systemic elevations of soluble CALR that is secreted from tumors or that is artificially supplied by injection of the recombinant protein decreased the efficacy of immunotherapy. Thus, depending on its location, CALR can have immunostimulatory or immunosuppressive functions.

Myelofibrosis is a chronic degenerative disorder characterized by progressive bone marrow fibrosis. Here, the authors show that the chaperone HSP27 contributes to myelofibrosis via regulation of the JAK2/STAT5 pathway, and that antisense oligonucleotides targeting HSP27 are effective in two mouse models of the disease

Over 95% of Polycythemia Vera (PV) patients carry the V617F mutation in the tyrosine kinase Janus kinase 2 (JAK2), resulting in uncontrolled erythroid proliferation and a high risk of thrombosis. Using mass spectrometry, we analyzed the RBC membrane proteome and showed elevated levels of multiple Ca2+ binding proteins as well as endoplasmic-reticulum-residing proteins in PV RBC membranes compared with RBC membranes from healthy individuals. In this study, we investigated the impact of JAK2V617F on (1) calcium homeostasis and RBC ion channel activity and (2) protein expression and sorting during terminal erythroid differentiation. Our data from automated patch-clamp show modified calcium homeostasis in PV RBCs and cell lines expressing JAK2V617F, with a functional impact on the activity of the Gárdos channel that could contribute to cellular dehydration. We show that JAK2V617F could play a role in organelle retention during the enucleation step of erythroid differentiation, resulting in modified whole cell proteome in reticulocytes and RBCs in PV patients. Given the central role that calcium plays in the regulation of signaling pathways, our study opens new perspectives to exploring the relationship between JAK2V617F, calcium homeostasis, and cellular abnormalities in myeloproliferative neoplasms, including cellular interactions in the bloodstream in relation to thrombotic events.

Histone deacetylase (HDAC) inhibitors, a class of cancer therapeutics, cause thrombocytopenia via an unknown mechanism. Here, the authors show that HDAC6 inhibition impairs proplatelet formation in human megakaryocytes, and show that this is linked to hyperacetylation of the actin-binding protein cortactin.

Summary: Hematopoiesis is particularly sensitive to DNA damage. Myeloid tumor incidence increases in patients with DNA repair defects and after chemotherapy. It is not known why hematopoietic cells are highly vulnerable to DNA damage. Addressing this question is complicated by the paucity of mouse models of hematopoietic malignancies due to defective DNA repair. We show that DNA repair-deficient Mcm8- and Mcm9-knockout mice develop myeloid tumors, phenocopying prevalent myelodysplastic syndromes. We demonstrate that these tumors are preceded by a lifelong DNA damage burden in bone marrow and that they acquire proliferative capacity by suppressing signaling of the tumor suppressor and cell cycle controller RB, as often seen in patients. Finally, we found that absence of MCM9 and the tumor suppressor Tp53 switches tumorigenesis to lymphoid tumors without precedent myeloid malignancy. Our results demonstrate that MCM8/9 deficiency drives myeloid tumor development and establishes a DNA damage burdened mouse model for hematopoietic malignancies. : Lutzmann et al. show that MCM8- or MCM9-deficient mice suffer chronic DNA damage, causing myeloid tumors, resembling human myelodysplastic syndromes, during aging. These tumors lose RB-mediated cell cycle control, cause splenomegaly, and preclude progressively normal hematopoiesis. Additional loss of the tumor suppressor Tp53 switches tumor development to T cell lymphoma. Keywords: MCM8, MCM9, DNA damage, DNA repair, myelodysplastic syndrome, cancer, homologous recombination, hematopoiesis

Primary familial and congenital polycythemia is characterized by erythropoietin hypersensitivity of erythroid progenitors due to germline nonsense or frameshift mutations in the erythropoietin receptor gene. All mutations so far described lead to the truncation of the C-terminal receptor sequence that contains negative regulatory domains. Their removal is presented as sufficient to cause the erythropoietin hypersensitivity phenotype. Here we provide evidence for a new mechanism whereby the presence of novel sequences generated by frameshift mutations is required for the phenotype rather than just extensive truncation resulting from nonsense mutations. We show that the erythropoietin hypersensitivity induced by a new erythropoietin receptor mutant, p.Gln434Profs*11, could not be explained by the loss of negative signaling and of the internalization domains, but rather by the appearance of a new C-terminal tail. The latter, by increasing erythropoietin receptor dimerization, stability and cell-surface localization, causes pre-activation of erythropoietin receptor and JAK2, constitutive signaling and hypersensitivity to erythropoietin. Similar results were obtained with another mutant, p.Pro438Metfs*6, which shares the same last five amino acid residues (MDTVP) with erythropoietin receptor p.Gln434Profs*11, confirming the involvement of the new peptide sequence in the erythropoietin hypersensitivity phenotype. These results suggest a new mechanism that might be common to erythropoietin receptor frameshift mutations. In summary, we show that primary familial and congenital polycythemia is more complex than expected since distinct mechanisms are involved in the erythropoietin hypersensitivity phenotype, according to the type of erythropoietin receptor mutation.

JAK inhibitors have been developed following the discovery of the JAK2V617F in 2005 as the driver mutation of the majority of non-BCR-ABL1 myeloproliferative neoplasms (MPNs). Subsequently, the search for JAK2 inhibitors continued with the discovery that the other driver mutations (CALR and MPL) also exhibited persistent JAK2 activation. Several type I ATP-competitive JAK inhibitors with different specificities were assessed in clinical trials and exhibited minimal hematologic toxicity. Interestingly, these JAK inhibitors display potent anti-inflammatory activity. Thus, JAK inhibitors targeting preferentially JAK1 and JAK3 have been developed to treat inflammation, autoimmune diseases, and graft-versus-host disease. Ten years after the beginning of clinical trials, only two drugs have been approved by the US Food and Drug Administration: one JAK2/JAK1 inhibitor (ruxolitinib) in intermediate-2 and high-risk myelofibrosis and hydroxyurea-resistant or -intolerant polycythemia vera and one JAK1/JAK3 inhibitor (tofacitinib) in methotrexate-resistant rheumatoid arthritis. The non-approved compounds exhibited many off-target effects leading to neurological and gastrointestinal toxicities, as seen in clinical trials for MPNs. Ruxolitinib is a well-tolerated drug with mostly anti-inflammatory properties. Despite a weak effect on the cause of the disease itself in MPNs, it improves the clinical state of patients and increases survival in myelofibrosis. This limited effect is related to the fact that ruxolitinib, like the other type I JAK2 inhibitors, inhibits equally mutated and wild-type JAK2 (JAK2WT) and also the JAK2 oncogenic activation. Thus, other approaches need to be developed and could be based on either (1) the development of new inhibitors specifically targeting JAK2V617F or (2) the combination of the actual JAK2 inhibitors with other therapies, in particular with molecules targeting pathways downstream of JAK2 activation or the stability of JAK2 molecule. In contrast, the strong anti-inflammatory effects of the JAK inhibitors appear as a very promising therapeutic approach for many inflammatory and auto-immune diseases.

Hematopoietic stem cells (HSCs) are characterized by the capacity for self-renewal and the ability to reconstitute the entire hematopoietic compartment. Thrombopoietin maintains adult HSCs in a quiescent state through the induction of cell cycle inhibitors p57Kip2 and p19INK4d. Using the p19INK4d−/− mouse model, we investigated the role of p19INK4d in basal and stress-induced hematopoiesis. We demonstrate that p19INK4d is involved in the regulation of HSC quiescence by inhibition of the G0/G1 cell cycle transition. Under genotoxic stress conditions, the absence of p19INK4d in HSCs leads to accelerated cell cycle exit, accumulation of DNA double-strand breaks, and apoptosis when cells progress to the S/G2-M stages of the cell cycle. Moreover, p19INK4d controls the HSC microenvironment through negative regulation of megakaryopoiesis. Deletion of p19INK4d results in megakaryocyte hyperproliferation and increased transforming growth factor β1 secretion. This leads to fibrosis in the bone marrow and spleen, followed by loss of HSCs during aging.

The molecular basis of hereditary thrombocytosis is germline mutations affecting the thrombopoietin (TPO)/TPO receptor (MPL)/JAK2 signaling axis. Here, we report one family presenting two cases with a mild thrombocytosis. By sequencing JAK2 and MPL coding exons, we identified a germline MPL R102P heterozygous mutation in the proband and his daughter. Concomitantly, we detected high TPO levels in the serum of these two patients. The mutation was not found in three other unaffected cases from the family except in another proband’s daughter who did not present thrombocytosis but had a high TPO level. The MPL R102P mutation was first described in congenital amegakaryocytic thrombocytopenia in a homozygous state with a loss-of-function activity. It was previously shown that MPL R102P was blocked in the endoplasmic reticulum without being able to translocate to the plasma membrane. Thus, this case report identifies for the first time that MPL R102P mutation can differently impact megakaryopoiesis: thrombocytosis or thrombocytopenia depending on the presence of the heterozygous or homozygous state, respectively. The paradoxical effect associated with heterozygous MPL R102P may be due to subnormal cell-surface expression of wild-type MPL in platelets inducing a defective TPO clearance. As a consequence, increased TPO levels may activate megakaryocyte progenitors that express a lower, but still sufficient level of MPL for the induction of proliferation.

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 (MPL, JAK2, CALR) and loss-of-function (LOF) activity of negative regulators (CBL, LNK) or the extrinsic MPL signaling by THPO GOF mutations leading to increased TPO synthesis. Alternatively, thrombocytosis may paradoxically result from mutations of MPL leading to an abnormal MPL trafficking, inducing increased TPO levels by alteration of its clearance. In contrast, thrombocytopenia can also result from LOF THPO or MPL 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.

Megakaryocytes are naturally polyploid cells that increase their ploidy by endomitosis. However, very little is known regarding the mechanism by which they escape the tetraploid checkpoint to become polyploid. Recently, it has been shown that the tetraploid checkpoint was regulated by the Hippo-p53 pathway in response to a downregulation of Rho activity. We therefore analyzed the role of Hippo-p53 pathway in the regulation of human megakaryocyte polyploidy. Our results revealed that Hippo-p53 signaling pathway proteins are present and are functional in megakaryocytes. Although this pathway responds to the genotoxic stress agent etoposide, it is not activated in tetraploid or polyploid megakaryocytes. Furthermore, Hippo pathway was observed to be uncoupled from Rho activity. Additionally, polyploid megakaryocytes showed increased expression of YAP target genes when compared to diploid and tetraploid megakaryocytes. Although p53 knockdown increased both modal ploidy and proplatelet formation in megakaryocytes, YAP knockdown caused no significant change in ploidy while moderately affecting proplatelet formation. Interestingly, YAP knockdown reduced the mitochondrial mass in polyploid megakaryocytes and decreased expression of PGC1α, an important mitochondrial biogenesis regulator. Thus, the Hippo pathway is functional in megakaryocytes, but is not induced by tetraploidy. Additionally, YAP regulates the mitochondrial mass in polyploid megakaryocytes.

The classic BCR-ABL-negative myeloproliferative neoplasms (MPNs), a form of chronic malignant hemopathies, have been classified into polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). ET and PMF are two similar disorders in their pathogenesis, which is marked by a key role of the megakaryocyte (MK) lineage. Whereas ET is characterized by MK proliferation, PMF is also associated with aberrant MK differentiation (myelodysplasia), leading to the release of cytokines in the marrow environment, which causes the development of myelofibrosis. Thus, PMF is associated with both myeloproliferation and different levels of myelodysplastic features. MPNs are mostly driven by mutated genes called MPN drivers, which abnormally activate the cytokine receptor/JAK2 pathway and their downstream effectors. The recent discovery of CALR mutations has closed a gap in our knowledge and has shown that this mutated endoplasmic reticulum chaperone activates the thrombopoietin receptor MPL and JAK2. These genetic studies have shown that there are two main types of MPNs: JAK2V617F-MPNs, including ET, PV, and PMF, and the MPL-/CALR-MPNs, which include only ET and PMF. These MPN driver mutations are associated with additional mutations in genes involved in epigenetics, splicing, and signaling, which can precede or follow the acquisition of MPN driver mutations. They are involved in clonal expansion or phenotypic changes or both, leading to myelofibrosis or leukemic transformation or both. Only a few patients with ET exhibit mutations in non-MPN drivers, whereas the great majority of patients with PMF harbor one or several mutations in these genes. However, the entire pathogenesis of ET and PMF may also depend on other factors, such as the patient’s constitutional genetics, the bone marrow microenvironment, the inflammatory response, and age. Recent advances allowed a better stratification of these diseases and new therapeutic approaches with the development of JAK2 inhibitors.