Comparative clinical and molecular landscape of primary and secondary myelofibrosis: Superior performance of MIPSS70+ v2.0 over MYSEC-PM.

Comparative clinical characteristics, molecular landscape and prognosis scoring for primary (PMF) and secondary myelofibrosis (SMF).

Distinct clinico-molecular arterial and venous thrombosis scores for myeloproliferative neoplasms risk stratification.

Current recommended risk scores to predict thrombotic events associated with myeloproliferative neoplasms (MPN) do not discriminate between arterial and venous thrombosis despite their different physiopathology. To define novel stratification systems, we delineated a comprehensive landscape of MPN associated thrombosis across a large long-term follow-up MPN cohort. Prior arterial thrombosis, age >60 years, cardiovascular risk factors and presence of TET2 or DNMT3A mutations were independently associated with arterial thrombosis in multivariable analysis. ARTS, an ARterial Thrombosis Score, based on these four factors, defined low- (0.37% patients-year) and high-risk (1.19% patients-year) patients. ARTS performance was superior to the two-tiered conventional risk stratification in our training cohort, across all MPN subtypes, as well as in two external validation cohorts. Prior venous thrombosis and presence of a JAK2V617F mutation with a variant allelic frequency ≥50% were independently associated with venous thrombosis. The discrimination potential of VETS, a VEnous Thrombosis Score based on these two factors, was poor, similar to the two-tiered conventional risk stratification. Our study pinpoints arterial and venous thrombosis clinico-molecular differences and proposes an arterial risk score for more accurate patients' stratification. Further improvement of venous risk scores, accounting for additional factors and considering venous thrombosis as a heterogeneous entity is warranted.

Recent advances in therapies for primary myelofibrosis.

Primary myelofibrosis (PMF), polycythemia vera (PV) and essential thrombocythemia (ET) form the classical BCR-ABL1-negative myeloproliferative neoplasms (MPNs) that are driven by a constitutive activation of JAK2 signaling. PMF as well as secondary MF (post-ET and post-PV MF) are the most aggressive MPNs. Presently, there is no curative treatment, except allogenic hematopoietic stem cell transplantation. JAK inhibitors, essentially ruxolitinib, are the therapy of reference for intermediate and high-risk MF. However, presently the current JAK inhibitors behave mainly as anti-inflammatory drugs, improving general symptoms and spleen size without major impact on disease progression. A better understanding of the genetics of MF, the biology of its leukemic stem cells (LSCs), the mechanisms of fibrosis and of cytopenia and the role of inflammatory cytokines has led to new approaches with the development of numerous therapeutic agents that target epigenetic regulation, telomerase, apoptosis, cell cycle, cytokines and signaling. Furthermore, the use of a new less toxic form of interferon-α has been revived, as it is presently one of the only molecules that targets the mutated clone. These new approaches have different aims: (a) to provide alternative therapy to JAK inhibition; (b) to correct cytopenia; and (c) to inhibit fibrosis development. However, the main important goal is to find new disease modifier treatments, which will profoundly modify the progression of the disease without major toxicity. Presently the most promising approaches consist of the inhibition of telomerase and the combination of JAK2 inhibitors (ruxolitinib) with either a BCL2/BCL-xL or BET inhibitor. Yet, the most straightforward future approaches can be considered to be the development of and/or selective inhibition of JAK2V617F and the targeting MPL and calreticulin mutants by immunotherapy. It can be expected that the therapy of MF will be significantly improved in the coming years.

Single-cell multi-omics identifies chronic inflammation as a driver of TP53-mutant leukemic evolution.

Understanding the genetic and nongenetic determinants of tumor protein 53 (TP53)-mutation-driven clonal evolution and subsequent transformation is a crucial step toward the design of rational therapeutic strategies. Here we carry out allelic resolution single-cell multi-omic analysis of hematopoietic stem/progenitor cells (HSPCs) from patients with a myeloproliferative neoplasm who transform to TP53-mutant secondary acute myeloid leukemia (sAML). All patients showed dominant TP53 'multihit' HSPC clones at transformation, with a leukemia stem cell transcriptional signature strongly predictive of adverse outcomes in independent cohorts, across both TP53-mutant and wild-type (WT) AML. Through analysis of serial samples, antecedent TP53-heterozygous clones and in vivo perturbations, we demonstrate a hitherto unrecognized effect of chronic inflammation, which suppressed TP53 WT HSPCs while enhancing the fitness advantage of TP53-mutant cells and promoted genetic evolution. Our findings will facilitate the development of risk-stratification, early detection and treatment strategies for TP53-mutant leukemia, and are of broad relevance to other cancer types.

SRSF2-P95H decreases JAK/STAT signaling in hematopoietic cells and delays myelofibrosis development in mice.

Heterozygous mutation targeting proline 95 in Serine/Arginine-rich Splicing Factor 2 (SRSF2) is associated with V617F mutation in Janus Activated Kinase 2 (JAK2) in some myeloproliferative neoplasms (MPNs), most commonly primary myelofibrosis. To explore the interaction of Srsf2P95H with Jak2V617F, we generated Cre-inducible knock-in mice expressing these mutants under control of the stem cell leukemia (Scl) gene promoter. In transplantation experiments, Srsf2P95H unexpectedly delayed myelofibrosis induced by Jak2V617F and decreased TGFß1 serum level. Srsf2P95H reduced the competitiveness of transplanted Jak2V617F hematopoietic stem cells while preventing their exhaustion. RNA sequencing of sorted megakaryocytes identified an increased number of splicing events when the two mutations were combined. Focusing on JAK/STAT pathway, Jak2 exon 14 skipping was promoted by Srsf2P95H, an event detected in patients with JAK2V617F and SRSF2P95 co-mutation. The skipping event generates a truncated inactive JAK2 protein. Accordingly, Srsf2P95H delays myelofibrosis induced by the thrombopoietin receptor agonist Romiplostim in Jak2 wild-type animals. These results unveil JAK2 exon 14 skipping promotion as a strategy to reduce JAK/STAT signaling in pathological conditions.

ANKRD26 is a new regulator of type I cytokine receptor signaling in normal and pathological hematopoiesis.

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.

Biological drivers of clinical phenotype in myelofibrosis.

Myelofibrosis (MF) is a myeloproliferative disorder that exhibits considerable biological and clinical heterogeneity. At the two ends of the disease spectrum are the myelodepletive or cytopenic phenotype and the myeloproliferative phenotype. The cytopenic phenotype has a high prevalence in primary MF (PMF) and is characterized by low blood counts. The myeloproliferative phenotype is typically associated with secondary MF (SMF), mild anemia, minimal need for transfusion support, and normal to mild thrombocytopenia. Differences in somatic driver mutations and allelic burden, as well as the acquisition of non-driver mutations further influences these phenotypic differences, prognosis, and response to therapies such as JAK2 inhibitors. The outcome of patients with the cytopenic phenotype are comparatively worse and frequently pose a challenge to treat given the inherent exacerbation of cytopenias. Recent data indicate that an innate immune deregulated state that hinges on the myddosome-IRAK-NFκB axis favors the cytopenic myelofibrosis phenotype and offers opportunity for novel treatment approaches. We will review the biological and clinical features of the MF disease spectrum and associated treatment considerations.

Secreted mutant calreticulins as rogue cytokines in myeloproliferative neoplasms.

Mutant calreticulin (CALR) proteins resulting from a -1/+2 frameshifting mutation of the CALR exon 9 carry a novel C-terminal amino acid sequence and drive the development of myeloproliferative neoplasms (MPNs). Mutant CALRs were shown to interact with and activate the thrombopoietin receptor (TpoR/MPL) in the same cell. We report that mutant CALR proteins are secreted and can be found in patient plasma at levels up to 160 ng/mL, with a mean of 25.64 ng/mL. Plasma mutant CALR is found in complex with soluble transferrin receptor 1 (sTFR1) that acts as a carrier protein and increases mutant CALR half-life. Recombinant mutant CALR proteins bound and activated the TpoR in cell lines and primary megakaryocytic progenitors from patients with mutated CALR in which they drive thrombopoietin-independent colony formation. Importantly, the CALR-sTFR1 complex remains functional for TpoR activation. By bioluminescence resonance energy transfer assay, we show that mutant CALR proteins produced in 1 cell can specifically interact in trans with the TpoR on a target cell. In comparison with cells that only carry TpoR, cells that carry both TpoR and mutant CALR are hypersensitive to exogenous mutant CALR proteins and respond to levels of mutant CALR proteins similar to those in patient plasma. This is consistent with CALR-mutated cells that expose TpoR carrying immature N-linked sugars at the cell surface. Thus, secreted mutant CALR proteins will act more specifically on the MPN clone. In conclusion, a chaperone, CALR, can turn into a rogue cytokine through somatic mutation of its encoding gene.

Inferring the initiation and development of myeloproliferative neoplasms.

The developmental history of blood cancer begins with mutation acquisition and the resulting malignant clone expansion. The two most prevalent driver mutations found in myeloproliferative neoplasms-JAK2V617F and CALRm-occur in hematopoietic stem cells, which are highly complex to observe in vivo. To circumvent this difficulty, we propose a method relying on mathematical modeling and statistical inference to determine disease initiation and dynamics. Our findings suggest that CALRm mutations tend to occur later in life than JAK2V617F. Our results confirm the higher proliferative advantage of the CALRm malignant clone compared to JAK2V617F. Furthermore, we illustrate how mathematical modeling and Bayesian inference can be used for setting up early screening strategies.

Stepwise GATA1 and SMC3 mutations alter megakaryocyte differentiation in a Down syndrome leukemia model.

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.

In Vivo Monitoring of Polycythemia Vera Development Reveals Carbonic Anhydrase 1 as a Potent Therapeutic Target.

Current murine models of myeloproliferative neoplasms (MPNs) cannot examine how MPNs progress from a single bone marrow source to the entire hematopoietic system. Thus, using transplantation of knock-in JAK2V617F hematopoietic cells into a single irradiated leg, we show development of polycythemia vera (PV) from a single anatomic site in immunocompetent mice. Barcode experiments reveal that grafted JAK2V617F stem/progenitor cells migrate from the irradiated leg to nonirradiated organs such as the contralateral leg and spleen, which is strictly required for development of PV. Mutant cells colonizing the nonirradiated leg efficiently induce PV in nonconditioned recipient mice and contain JAK2V617F hematopoietic stem/progenitor cells that express high levels of carbonic anhydrase 1 (CA1), a peculiar feature also found in CD34+ cells from patients with PV. Finally, genetic and pharmacologic inhibition of CA1 efficiently suppresses PV development and progression in mice and decreases PV patients' erythroid progenitors, strengthening CA1 as a potent therapeutic target for PV. SIGNIFICANCE: Follow-up of hematopoietic malignancies from their initiating anatomic site is crucial for understanding their development and discovering new therapeutic avenues. We developed such an approach, used it to characterize PV progression, and identified CA1 as a promising therapeutic target of PV. This article is highlighted in the In This Issue feature, p. 265.

An inherited gain-of-function risk allele in EPOR predisposes to familial JAK2V617F myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPN) are mainly sporadic but inherited variants have been associated with higher risk development. Here, we identified an EPOR variant (EPORP488S ) in a large family diagnosed with JAK2V617F -positive polycythaemia vera (PV) or essential thrombocytosis (ET). We investigated its functional impact on JAK2V617F clonal amplification in patients and found that the variant allele fraction (VAF) was low in PV progenitors but increase strongly in mature cells. Moreover, we observed that EPORP488S alone induced a constitutive phosphorylation of STAT5 in cell lines or primary cells. Overall, this study points for searching inherited-risk alleles affecting the JAK2/STAT pathway in MPN.

Macrophage migration inhibitory factor is overproduced through EGR1 in TET2low resting monocytes.

Somatic mutation in TET2 gene is one of the most common clonal genetic events detected in age-related clonal hematopoiesis as well as in chronic myelomonocytic leukemia (CMML). In addition to being a pre-malignant state, TET2 mutated clones are associated with an increased risk of death from cardiovascular disease, which could involve cytokine/chemokine overproduction by monocytic cells. Here, we show in mice and in human cells that, in the absence of any inflammatory challenge, TET2 downregulation promotes the production of MIF (macrophage migration inhibitory factor), a pivotal mediator of atherosclerotic lesion formation. In healthy monocytes, TET2 is recruited to MIF promoter and interacts with the transcription factor EGR1 and histone deacetylases. Disruption of these interactions as a consequence of TET2-decreased expression favors EGR1-driven transcription of MIF gene and its secretion. MIF favors monocytic differentiation of myeloid progenitors. These results designate MIF as a chronically overproduced chemokine and a potential therapeutic target in patients with clonal TET2 downregulation in myeloid cells.

Germline ATG2B/GSKIP-containing 14q32 duplication predisposes to early clonal hematopoiesis leading to myeloid neoplasms.

The germline predisposition associated with the autosomal dominant inheritance of the 14q32 duplication implicating ATG2B/GSKIP genes is characterized by a wide clinical spectrum of myeloid neoplasms. We analyzed 12 asymptomatic carriers and 52 patients aged 18-74 years from six families, by targeted sequencing of 41 genes commonly mutated in myeloid malignancies. We found that 75% of healthy carriers displayed early clonal hematopoiesis mainly driven by TET2 mutations. Molecular landscapes of patients revealed two distinct routes of clonal expansion and leukemogenesis. The first route is characterized by the clonal dominance of myeloproliferative neoplasms (MPN)-driver events associated with TET2 mutations in half of cases and mutations affecting splicing and/or the RAS pathway in one-third of cases, leading to the early development of MPN, mostly essential thrombocythemia, with a high risk of transformation (50% after 10 years). The second route is distinguished by the absence of MPN-driver mutations and leads to AML without prior MPN. These patients mostly harbored a genomic landscape specific to acute myeloid leukemia secondary to myelodysplastic syndrome. An unexpected result was the total absence of DNMT3A mutations in this cohort. Our results suggest that the germline duplication constitutively mimics hematopoiesis aging by favoring TET2 clonal hematopoiesis.

Lyl-1 regulates primitive macrophages and microglia development.

During ontogeny, macrophage populations emerge in the Yolk Sac (YS) via two distinct progenitor waves, prior to hematopoietic stem cell development. Macrophage progenitors from the primitive/"early EMP" and transient-definitive/"late EMP" waves both contribute to various resident primitive macrophage populations in the developing embryonic organs. Identifying factors that modulates early stages of macrophage progenitor development may lead to a better understanding of defective function of specific resident macrophage subsets. Here we show that YS primitive macrophage progenitors express Lyl-1, a bHLH transcription factor related to SCL/Tal-1. Transcriptomic analysis of YS macrophage progenitors indicate that primitive macrophage progenitors present at embryonic day 9 are clearly distinct from those present at later stages. Disruption of Lyl-1 basic helix-loop-helix domain leads initially to an increased emergence of primitive macrophage progenitors, and later to their defective differentiation. These defects are associated with a disrupted expression of gene sets related to embryonic patterning and neurodevelopment. Lyl-1-deficiency also induce a reduced production of mature macrophages/microglia in the early brain, as well as a transient reduction of the microglia pool at midgestation and in the newborn. We thus identify Lyl-1 as a critical regulator of primitive macrophages and microglia development, which disruption may impair resident-macrophage function during organogenesis.

Inferring the dynamics of mutated hematopoietic stem and progenitor cells induced by IFNα in myeloproliferative neoplasms.

Classical BCR-ABL-negative myeloproliferative neoplasms (MPNs) are clonal disorders of hematopoietic stem cells (HSCs) caused mainly by recurrent mutations in genes encoding JAK2 (JAK2), calreticulin (CALR), or the thrombopoietin receptor (MPL). Interferon α (IFNα) has demonstrated some efficacy in inducing molecular remission in MPNs. To determine factors that influence molecular response rate, we evaluated the long-term molecular efficacy of IFNα in patients with MPN by monitoring the fate of cells carrying driver mutations in a prospective observational and longitudinal study of 48 patients over more than 5 years. We measured the clonal architecture of early and late hematopoietic progenitors (84 845 measurements) and the global variant allele frequency in mature cells (409 measurements) several times per year. Using mathematical modeling and hierarchical Bayesian inference, we further inferred the dynamics of IFNα-targeted mutated HSCs. Our data support the hypothesis that IFNα targets JAK2V617F HSCs by inducing their exit from quiescence and differentiation into progenitors. Our observations indicate that treatment efficacy is higher in homozygous than heterozygous JAK2V617F HSCs and increases with high IFNα dose in heterozygous JAK2V617F HSCs. We also found that the molecular responses of CALRm HSCs to IFNα were heterogeneous, varying between type 1 and type 2 CALRm, and a high dose of IFNα correlates with worse outcomes. Our work indicates that the long-term molecular efficacy of IFNα implies an HSC exhaustion mechanism and depends on both the driver mutation type and IFNα dose.

Functional Consequences of Mutations in Myeloproliferative Neoplasms.

Driver mutations occur in Janus kinase 2 (JAK2), thrombopoietin receptor (MPL), and calreticulin (CALR) in BCR-ABL1 negative myeloproliferative neoplasms (MPNs). From mutations leading to one amino acid substitution in JAK2 or MPL, to frameshift mutations in CALR resulting in a protein with a different C-terminus, all the mutated proteins lead to pathologic and persistent JAK2-STAT5 activation. The most prevalent mutation, JAK2 V617F, is associated with the 3 entities polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis (MF), while CALR and MPL mutations are associated only with ET and MF. Triple negative ET and MF patients may harbor noncanonical mutations in JAK2 or MPL. One major fundamental question is whether the conformations of JAK2 V617F, MPL W515K/L/A, or CALR mutants differ from those of their wild type counterparts so that a specific treatment could target the clone carrying the mutated driver and spare physiological hematopoiesis. Of great interest, a set of epigenetic mutations can co-exist with the phenotypic driver mutations in 35%-40% of MPNs. These epigenetic mutations, such as TET2, EZH2, ASXL1, or DNMT3A mutations, promote clonal hematopoiesis and increased fitness of aged hematopoietic stem cells in both clonal hematopoiesis of indeterminate potential (CHIP) and MPNs. Importantly, the main MPN driver mutation JAK2 V617F is also associated with CHIP. Accumulation of several epigenetic and splicing mutations favors progression of MPNs to secondary acute myeloid leukemia. Another major fundamental question is how epigenetic rewiring due to these mutations interacts with persistent JAK2-STAT5 signaling. Answers to these questions are required for better therapeutic interventions aimed at preventing progression of ET and PV to MF, and transformation of these MPNs in secondary acute myeloid leukemia.