Neutrophils and monocytes are persistently elevated in sickle cell anemia (SCA), yet the intrinsic mechanisms driving pathological myelopoiesis and inflammation remain poorly defined. Through single-cell RNA sequencing and functional assays, we demonstrate that hematopoietic stem and multipotent progenitor cells (HSPCs) in SCA are transcriptionally reprogrammed toward myeloid differentiation. This process is orchestrated by aberrant activation of type I interferon (IFN-I) signaling, which promotes premature myeloid commitment of hematopoietic stem cells. SCA progenitors further exhibit unexpected responsiveness to granulocyte colony-stimulating factor (G-CSF) through upregulation of CSF3R, resulting in skewed myelopoiesis toward the monocytic lineage. Importantly, hydroxyurea treatment attenuates IFN-I signaling in neutrophils, consistent with its therapeutic role in reducing excessive inflammation and granulopoiesis. Collectively, these findings uncover IFN-I-driven remodeling of hematopoiesis as a fundamental mechanism of leukocytosis and chronic inflammation in SCA, and establish a tractable therapeutic axis to mitigate innate immunity activation in this disease.

Abstract TP53 mutations are found in over 50% of tumor types, including myeloproliferative neoplasms (MPNs). MPNs are characterized by a chronic phase, which may progress to secondary acute myeloid leukemia (sAML). Here, we discuss the physiological functions of p53 in hematopoiesis and its deregulation in MPNs. Additionally, we explore the mechanisms underlying TP53 mutations in the leukemic transformation of MPNs, including clonal evolution to multihit status and the role of inflammation and therapy. Finally, recent findings on the clinical impact of multihit TP53 mutations and potential strategies for targeting the p53 pathway in MPNs and sAML are presented.

Classic myeloproliferative neoplasms, including essential thrombocythemia, polycythemia vera, and primary myelofibrosis, are chronic, clonal hematopoietic stem-cell disorders. These disorders are driven by gain-of-function mutations in the genes Janus kinase 2 (JAK2), calreticulin (CALR), or the thrombopoietin receptor (MPL) that activate cytokine signaling. These mutations arise decades before clinical disease develops and confer a clonal advantage that is further shaped by comutations in epigenetic, splicing, or signaling genes. Inflammation enhances clonal dominance, favoring the development of myelofibrosis and thrombotic complications. Disease evolution may culminate in secondary acute myeloid leukemia, which has a poor prognosis. Current therapies primarily aim to control symptoms, thrombosis, and splenomegaly, but they have limited disease-modifying effects, except for pegylated interferon alfa and JAK2 inhibitors in some patients. Emerging therapies that selectively target mutant CALR and JAK2 V617F using immunotherapy and selective inhibitors could be a breakthrough in the treatment of persons with myeloproliferative neoplasms, with the expectation of achieving durable disease modification and potentially clonal eradication.

Erythropoiesis and iron metabolism are closely interconnected, under both physiological and pathological conditions. Polycythemia vera (PV) is a myeloproliferative neoplasm (MPN) caused by a Janus kinase-2 (JAK2) mutation, resulting in uncontrolled red blood cell production and elevated hemoglobin and hematocrit levels. In PV, iron deficiency is common and may be due to several factors, including chronic gastrointestinal bleeding, chronic inflammation, dysregulated hepcidin signaling, and therapeutic phlebotomy. Many patients exhibit low serum ferritin and low to normal hepcidin levels despite erythroid proliferation, suggesting a maladaptive iron-restricted state. This functional iron deficiency may limit erythropoiesis to some extent, but also contributes to burdensome symptoms such as fatigue, cognitive impairment, and restless leg syndrome. Novel therapeutic approaches, including hepcidin mimetics or ferroportin inhibitors, aim to restore iron homeostasis, improving quality of life and potentially reducing the need for cytoreductive therapy (drugs used to treat MPNs by reducing blood cell production) in low-risk PV patients. In secondary forms of erythrocytosis (both congenital and acquired), iron homeostasis has been less often investigated. However, exploring it may be of great interest since in these affections, iron overload could act as a hidden driver of erythrocytosis by stimulating erythroblast proliferation. Hereditary hemochromatosis (HH) is a well-known cause of iron overload. While its association with erythrocytosis has been a subject of interest, it remains incompletely understood. In this review, we will explore the complex relationship between iron (deficiency or overload, including HH) and erythrocytosis (including PV), discussing underlying mechanisms and potential therapeutic applications.

Poly(ADP-ribose) polymerase inhibitors (PARPi) have profoundly transformed the management of patients with cancer, particularly ovarian cancer patients. However, their use is associated with hematologic toxicities, ranging from transient cytopenias to myeloid neoplasia post-cytotoxic therapy (MN-pCT). Transient cytopenias, mainly anemia, occur early after treatment initiation and display a variable profile depending on the PARPi, with a central role of PARP2 inhibition in erythropoietic abnormalities. More rarely, MN-pCT develop after prolonged exposure to PARPi and are associated with an unfavorable prognosis, characterized by complex cytogenetic abnormalities and high-risk mutations, particularly TP53. Current data suggest a mechanism of selection of pre-existing clonal hematopoiesis under the selective pressure of cytotoxic therapies followed by PARPi. A better understanding of the pathophysiological mechanisms and early identification of high-risk patients could enable optimized monitoring puis des PARPi. Une meilleure compr & eacute;hension des m & eacute;canismes physiopathologiques et l'identification pr & eacute;coce des patients & agrave; risque pourraient permettre d'optimiser la surveillance et, potentiellement, de d & eacute;velopper des approches pr & eacute;ventives visant & agrave; limiter ces complications h & eacute;matologiques. and, potentially, the development of preventive approaches to limit the development of MN-pCT.

INCB160058 is a first-in-class, orally bioavailable Janus kinase 2 (JAK2) V617F mutant-selective inhibitor, currently being studied in phase 1 clinical trials (eg, NCT06313593) in patients with myeloproliferative neoplasms (MPNs) harboring JAK2V617F mutation. Previously, INCB160058 was shown to bind the pseudokinase domain (JH2) of JAK2. This unique binding results in a novel mechanism of action where INCB160058 selectively inhibits ligand-independent oncogenic JAK2V617F homodimers associated with class I cytokine receptors (eg, thrombopoietin receptor), while preserving physiological, cytokine-dependent signaling. Aberrant JAK2/STAT5 signaling and proliferation in JAK2V617F+ cells are inhibited with minimal impact on signaling and growth of wild-type counterparts (Stubbs. Blood 2023;142[Suppl 1]:860). This study evaluated the activity of INCB160058 in JAK2V617F+ cells possessing different secondary mutations or having previously been treated with MPN-directed therapy, simulating possible clinical scenarios. We evaluated the effect of INCB160058 on the clonogenic potential of JAK2V617F⁺ CD34⁺ hematopoietic stem and progenitor cells using colony-forming unit (CFU) assays and assessed JAK2V617F-dependent erythroid and megakaryocyte differentiation potential through liquid culture assays. In CFU assays, INCB160058 inhibited colony formation by JAK2V617F+ CD34+ cells, irrespective of additional mutations, including high-risk variants such as ASXL1 or EZH2, while exhibiting minimal impact on normal CD34+ cells. These findings demonstrate that INCB160058 effectively inhibits the aberrant proliferation of MPN CD34⁺ cells irrespective of additional mutations, highlighting that their oncogenic growth remains primarily dependent on the JAK2V617F mutation. In addition, in liquid culture assays, INCB160058 selectively impaired cytokine-independent JAK2V617F-driven erythroid and megakaryocytic maturation of CD34+ cells from JAK2V617F+ polycythemia vera, essential thrombocythemia, and myelofibrosis patient samples, while sparing erythroid and megakaryocyte maturation of CD34+ cells from healthy donors. Importantly, both acute and chronic INCB160058 treatment reduced oncogenic signaling pathways (pSTAT5, pERK, and pAKT), JAK2/STAT5 target gene expression (eg, PIM1, ID1, CISH) as well as cytokine production (eg, IL8, MCP1, and MIP-1B) in JAK2V617F+ CD34+ cells, with minimal effects on normal CD34+ cells. We also assessed the in vitro efficacy of INCB160058 on cells exhibiting persistence to ruxolitinib, which was established by exposing JAK2V617F-dependent SET2 cells to increasing concentrations of ruxolitinib, resulting in a population capable of proliferating despite high-dose ruxolitinib treatment. Notably, treatment with INCB160058 inhibited STAT5 phosphorylation and suppressed proliferation to a similar extent in both ruxolitinib-persistent and inhibitor-naive JAK2V617F-dependent SET2 cells, suggesting potential benefit in ruxolitinib-resistant or refractory MPNs. Furthermore, currently approved JAK2 inhibitors for MPNs induce paradoxical hyperphosphorylation of the JAK2 activation loop (Y1007/1008) associated with withdrawal syndrome; in contrast, INCB160058 reduced activation loop phosphorylation, consistent with its binding to the JH2 domain of JAK2V617F. These findings support the potential of INCB160058 to overcome ruxolitinib resistance and mitigate withdrawal-related signaling effects in clinical settings. In conclusion, our findings demonstrate that INCB160058 selectively targets JAK2V617F-mediated pathways and inhibits constitutive differentiation of CD34+ cells driven by the mutation, while preserving cytokine-dependent signaling for normal hematopoietic differentiation across numerous clinically relevant in vitro and ex vivo MPN models.

ABSTRACT:Most calreticulin (CALR) mutations in myeloproliferative neoplasms are classified as either type 1, a 52-base pair deletion (CALRdel52); or type 2, a 5-base pair insertion (CALRins5). Both are gain-of-function (GOF) mutations that generate an identical mutant C-terminal tail, which mediates the binding to, and activation of, the thrombopoietin receptor myeloproliferative leukemia protein (MPL). We recently reported that despite this shared GOF, CALRdel52 but not CALRins5 mutations cause loss of calcium binding function, leading to activation of, and dependency on, the inositol-requiring enzyme 1/X-box binding protein 1 pathway of the unfolded protein response (UPR). This led us to ask whether CALRins5 mutations activate and depend on a different UPR pathway, and whether this is likewise mediated by a mutation type-specific loss-of-function (LOF). Here, we show that CALRins5 mutations lead to activation of the activating transcription factor 6 (ATF6) pathway of the UPR due to loss of CALR chaperone function. This LOF is caused by interference of the CALRins5 mutant C terminus with key chaperone residue H170. Furthermore, we show that CALRins5 cells are partially dependent on ATF6 for cytokine-independent growth, and identify B-cell lymphoma extra large as a transcriptional target of ATF6 that promotes type 2 CALR-mutant cell survival.

Mastocytosis, a clonal disorder characterized by the accumulation of mast cells in various tissues, affects both adults and children. Adults frequently exhibit KIT activating mutations, usually in the phospho-transferase domain (PTD KIT mutations). Our previous findings revealed that children also harbor oncogenic KIT activating mutations, but more commonly within the extra-cellular domain (non-PTD KIT mutations). While the disease persists chronically in adults, it often regresses spontaneously in children through an unknown mechanism. Here, we report that tumor senescence in childhood mastocytosis may be triggered by significantly shortened telomeres in mast cells harboring non-PTD KIT mutations compared to those with PTD KIT mutations. In vitro models further demonstrated a senescent phenotype associated with shorter telomeres for the non-PTD KIT mutant compared to the PTD KIT mutant. Mechanistically, we found that telomere shortening in mast cells from children with non-PTD KIT mutations is linked with increased p38 MAP-kinase activation, resulting in lower TRF2 occupancy on telomeres. Thus, non-PTD KIT mutations trigger distinct signaling pathways leading to telomere shortening and cellular senescence, providing mechanistic insights into the differing outcomes between childhood- and adult-onset mastocytosis.

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.

BACKGROUND Secondary erythrocytosis often results from conditions that cause tissue hypoxia or an improper increase in erythropoietin (EPO) production. EPO, the major regulator of erythropoiesis, has a complex and tightly regulated expression during development, with a liver-to-kidney switch shortly after birth. METHODS We identified six families with erythrocytosis that was associated with circulating EPO levels within the normal range and characterized as a novel molecular and functional entity. We investigated the effect of the identified pathogenic variants using EPO promoter-driven luciferase reporter genes. Induced pluripotent stem cells (iPSCs) were generated from patient cells and differentiated into hepatocyte-like EPO-producing cells. Samples of circulating EPO from patients with hereditary erythrocytosis and from healthy newborns were analyzed by means of isoelectric focusing, and EPO activity was assessed. RESULTS Three novel variants were identified in the noncoding regions of EPO. Experiments with reporter assays and iPSC-derived hepatocyte-like cells showed that the variants targeted previously uncharacterized regulatory elements of the gene, which, when the variants were present, showed high responsiveness to hypoxia. EPO samples from all the patients showed a modified isoelectric-focusing profile, identical to hepatic EPO that is expressed in premature neonates and in patients with acquired erythrocytosis associated with liver diseases. EPO that was purified from patient plasma and umbilical-cord blood samples showed enhanced EPO receptor signaling activity in vitro, which suggests a potential gain of function linked to the liver-type glycosylation of EPO. CONCLUSIONS We found that secondary erythrocytosis can be related to variants in EPO that lead to the production of hepatic-like EPO with an atypical glycosylation pattern and increased activity.

Bernard Soulier syndrome (BSS) is a severe bleeding disorder with moderate to severe thrombocytopenia, giant platelets, and platelet dysfunction, caused by biallelic mutations in GP1BA, GP1BB, or GP9 genes. We generated induced pluripotent stem cells (iPSC) from a BSS patient with a novel heterozygous GP1BA p.N103D mutation, resulting in moderate macrothrombocytopenia. The mutation does not affect megakaryocyte (MK) differentiation or GPIb-GPIX complex expression but reduces affinity to von Willebrand factor (VWF). It induces increased signaling independent of VWF and αIIbβ3-mediated outside-in signaling, causing a profound defect in proplatelet formation after adhesion on fibrinogen. Pre-activation of αIIbβ3 integrin and heightened stress fiber formation linked to RhoA pathway overactivation were observed, likely due to increased phosphorylation of SRC at Y419 downstream of GPIbα. Dasatinib, a SRC inhibitor, restored stress fiber formation. Using a 3D bone marrow model to mimic platelet release under flow, we demonstrated that the ROCK1/2 inhibitor Y27632 increased platelet number and restored platelet size in GPIbαN103D MK, as well as in MK from two other patients with heterozygous GP1BA mutations (p.L160P and p.N150S). However, Y27632 had no additional effect on platelet generation from MK of two patients with biallelic BSS, suggesting a distinct molecular mechanism in biallelic cases.

Congenital neutropenia (CN) comprises a heterogeneous group of rare genetic disorders. While some CN cases present only with neutropenia, others present with additional extra-hematological manifestations. The most common cause of CN is variants in ELANE; however, approximately 30 other genes have been implicated. Despite this, the genetic basis remains unknown in roughly 30% of cases. The clinical and genetic heterogeneity of CN makes diagnosis particularly challenging. To address this, we conducted exome or genome sequencing of 60 patients with a suspected diagnosis of CN that remained unresolved following targeted sequencing. A genetic diagnosis was established in 25 patients (42%). Variants were identified in 15 different genes. Half of these cases involved genes traditionally associated with hereditary immunodeficiencies (GINS4, CARD11, ADA2, GINS1, LCP1, SASH3, and WAS). One-third of the cases carried variants in genes linked to syndromic disorders (VPS13B, TAFAZZIN, CLPB, and TONSL), demonstrating variable penetrance of extra-hematological phenotypes. A smaller subset (15%) harbored variants in genes associated with inherited bone marrow failure syndromes (BLM, RPL18, SAMD9, and SRP72), identified incidentally due to atypical presentations. Compared to patients with ELANE-CN, these individuals were diagnosed later, had fewer severe bacterial infections and gingivitis, exhibited less profound neutropenia, lacked monocytosis, and had a granulocytic maturation arrest, often beyond the promyelocytic stage. A shared feature among these cases was a tendency toward reduced lymphocyte subsets, particularly NK cells. This study highlights the significant contribution of exome and genome sequencing in diagnosing CN, given the phenotypic overlap, genetic heterogeneity, and variable penetrance of immunological and extra-hematological features.

JAK (Janus Kinase) inhibitors, such as ruxolitinib, were introduced a decade ago for treatment of myeloproliferative neoplasms (MPN). To evaluate ruxolitinib's impact on MPN clonal evolution, we interrogate a myelofibrosis patient cohort with longitudinal molecular evaluation and discover that ruxolitinib is associated with clonal outgrowth of RAS pathway mutations. Single-cell DNA sequencing combined with ex vivo treatment of RAS mutated CD34+ primary patient cells, demonstrates that ruxolitinib induces RAS clonal selection both in a JAK/STAT wild-type and hyper-activated context. RAS mutations are associated with decreased transformation-free and overall survival only in patients treated with ruxolitinib. In vitro and in vivo competition assays demonstrate increased cellular fitness of RAS-mutated cells under ruxolitinib or JAK2 knock-down, consistent with an on-target effect. MAPK pathway activation is associated with JAK2 downregulation resulting in enhanced oncogenic potential of RAS mutations. Our results prompt screening for pre-existing RAS mutations in JAK inhibitor treated patients with MPN.

Myeloid malignancies, including myeloproliferative neoplasms (MPN), myelodysplastic syndromes, and acute myeloid leukemia (AML), are predominantly sporadic diseases caused by the progressive accumulation of somatic mutations. However, rare inherited predispositions have been identified and account for familial clustering. We previously identified a germline tandem duplication at 14q32 (CNV) involving ATG2B and GSKIP in a very large family from West Indies (>60 cases). This CNV predisposes heterozygous carriers to a broad spectrum of myeloid hematologic malignancies with a nearly complete penetrance. The CNV is associated with early-onset clonal hematopoiesis, frequently involving TET2 or JAK2V617F mutations, and follows distinct evolutionary trajectories: either progressing from essential thrombocythemia to myelofibrosis and AML or transforming directly into AML. While ATG2B is a key regulator of autophagy and mitochondrial homeostasis, and GSKIP modulates GSK3β and PKA signaling pathways that intersect with metabolism, proliferation, and stem cell maintenance, the precise mechanisms by which their overexpression drives hematopoietic transformation remain unclear. To dissect the functional impact of the CNV on hematopoietic stem cells (HSCs), we combined in vitro and in vivo approaches. We overexpressed ATG2B/GSKIP in human hematopoietic cell lines and employed induced pluripotent stem cells (iPSCs) derived from CNV carriers. In parallel, we generated a knock-in (KI) mouse model mimicking the CNV by trans-allelic targeted meiotic recombination and assessed its effects on hematopoiesis, both alone and in combination with Jak2V617F or Tet2 knockout (KO). Overexpression of ATG2B/GSKIP in hematopoietic cell lines led to mildly reduced proliferation. Transmission electron microscopy revealed marked mitochondrial abnormalities, including increased mitochondrial size and number, highly developed endoplasmic reticulum, and aberrant lipid droplets. Consistently, ATG2B/GSKIP overexpression increased mitochondrial mass and impaired mitochondrial fission, as indicated by elevated TOM20 levels and increased [S637] DRP1 phosphorylation. Seahorse assays demonstrated a 2-fold reduction in basal and maximal respiration, glycolytic reserve, and mitochondrial ATP production, while glycolytic ATP production was preserved. LC-MS/MS metabolomics revealed a selective and profound effect on α-ketoglutarate (αKG) levels, which led to activation of TET family demethylases, suggesting a link between metabolic reprogramming and epigenetic dysregulation. Notably, similar mitochondrial defects and impaired megakaryocyte differentiation were observed in megakaryocytes derived from human iPSCs carrying the CNV. To assess the in vivo impact of the 14q32 CNV on hematopoiesis, we analyzed KI mice harboring the CNV, which showed mild leukocytosis without overt hematological abnormalities. Lin- progenitors displayed mitochondrial abnormalities and impaired HSC cell cycle entry, as assessed by Ki67/DAPI staining. Transcriptomic analysis of Lin- progenitors confirmed overexpression of Atg2b and Gskip and revealed signatures of mitochondrial dysfunction,along with suppression of key proliferative pathways including cell cycle, translation, RAS, and JAK/STAT signaling, hallmarks of metabolically repressed HSC state. In both competitive and serial bone marrow transplantation assays, CNV HSCs showed reduced repopulation capacity and impaired fitness. We next examined cooperation between the CNV and MPN drivers. The CNV significantly altered the phenotypes of Jak2V617F and Tet2 KO models: polycythemia was attenuated in CNV× Jak2V617F mice, while platelet and leukocyte counts increased, and thrombocytopenia was alleviated in CNV×Tet2 KO animals. Notably, despite intrinsic fitness defects, CNV×Jak2V617F HSCs displayed enhanced long-term clonal expansion in competitive transplantation with CNV hematopoietic cells only, demonstrating that Jak2V617F confers a strong clonal advantage to HSC in a CNV context. Overall, our findings indicate that the 14q32 CNV disrupts HSC homeostasis through mitochondrial dysfunction, inhibition of JAK/STAT signaling, and αKG-driven epigenetic remodeling. The ensuing HSC dysfunction may create a permissive state for leukemogenesis by promoting the selection and expansion of clones with both signaling (JAK2V617F, CALR, MPL, RAS) and epigenetic (TET2 and IDH1/2) mutations.

Elevated circulating levels of calprotectin (CAL), the S100A8/A9 heterodimer, are biomarkers of severe systemic inflammation. Here, we investigate the effects of CAL on early human hematopoiesis. CAL demonstrates limited impact on gene expression in stem and progenitor cells, in contrast with interleukin-6 (IL6), which promotes the expression of the S100A8 and S100A9 genes in hematopoietic progenitors and the generation of monocytes that release CAL. The main target of CAL is an erythroid-megakaryocyte progenitor (EMP) subset. CAL prevents both erythropoietin-driven differentiation of healthy progenitors and JAK2-V617F-driven erythropoiesis. In the context of JAK2-V617F, CAL also promotes the expression of S100A8 and S100A9 genes in monocytes. The signature of CAL effects is detected in the bone marrow progenitors of patients with myeloid malignancy or severe infection. These results position CAL as a mediator of IL6 effects on triggering anemia during inflammation, an effect that is amplified in the context of JAK2-V617F-driven hematopoiesis.

Myelofibrosis (MF) is a severe hematologic malignancy characterized by clonal stem cell-derived myeloproliferation, splenomegaly, and progressive bone marrow fibrosis, ultimately leading to cytopenias1. While, JAK inhibitors provide meaningful clinical benefits, primarily through spleen volume reduction and symptom relief, their impact on bone marrow fibrosis remains limited2. Fibronectin (FN), a core component of interstitial extracellular matrix (ECM), plays a crucial role in ECM assembly by serving as a scaffold for other matrix proteins and regulating diverse cellular functions. FN and its spliced isoforms are aberrantly expressed in the fibrotic matrix of MF and contribute to disease progression. Elevated expression of EDA containing-FN has been shown to drive megakaryocytic hyperplasia, fibrosis, and inflammation3. Furthermore, increased expression of FN receptors and enhanced FN-binding capacity have been documented in JAK2V617F-mutant hematopoietic progenitors and megakaryocytes4. Despite these insights, effective therapies that directly disrupt ECM remodeling and its contribution to fibrosis remain a significant unmet clinical need in MF. Here, we present a novel antisense oligonucleotide (ASO) designed to suppress the expression of all FN isoforms as a direct anti-fibrotic strategy. We developed a chemically modified 2′-O-(2-methoxyethyl) phosphorothioate ASO targeting a highly conserved sequence shared between human and murine genomes, effectively silencing all cellular FN splice variants. In tissue culture, this ASO induced a sequence- and concentration-dependent reduction in FN mRNAs, including transcripts encoding the profibrotic EDA isoform. In vitro, FN ASO treatment of human bone marrow-derived mesenchymal stromal cells attenuated the acquisition of a myofibroblast phenotype induced by Transforming Growth Factor-β1 (TGF-β1), as indicated by reduced expression of α-smooth muscle actin (α-SMA) and impaired cell migratory capacity. In vivo, systemic subcutaneous administration of FN ASO in C57BL/6 mice (50 mg/kg twice weekly for two weeks) led to a robust reduction of FN mRNA and protein in the liver, along with an approximately 90% decrease in circulating plasma FN levels, as measured by ELISA. Biodistribution studies confirmed efficient targeting of tissue FN, including in the bone marrow and spleen, with approximately 50% reduction in FN protein levels. Importantly, no significant alterations were observed in blood markers of liver or kidney function, indicating a favorable safety profile. In a preclinical Romiplostim-induced mouse model of MF, FN ASO treatment significantly reduced reticulin deposition in the bone marrow, attenuated megakaryocyte expansion, and decreased expression of fibrogenic cytokines such as TGF-β1 and IL-6. While spleen weights remained unchanged, FN ASO-treated mice exhibited marked reversal of reticulin fibers, reduced type III collagen content, and diminished accumulation of α-SMA positive cells, consistent with decreased myofibroblast activity. These findings establish FN ASO as a first-in-class therapeutic approach that directly targets fibrosis in MF, with promising potential for broader application across fibrotic diseases. References D. A. Arber et al., The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood 127, 2391-2405 (2016). A. Tefferi, Primary myelofibrosis: 2023 update on diagnosis, risk-stratification, and management. Am J Hematol 98, 801-821 (2023). A. Malara et al., EDA fibronectin-TLR4 axis sustains megakaryocyte expansion and inflammation in bone marrow fibrosis. J Exp Med 216, 587-604 (2019). S. Matsuura et al., Adhesion to fibronectin via α5β1 integrin supports expansion of the megakaryocyte lineage in primary myelofibrosis. Blood. 135, 2286-2291 (2020).

Megakaryopoiesis is primarily regulated by the interaction between thrombopoietin (THPO) and its receptor (MPL) located on the surface of cells, ranging from hematopoietic stem cells to megakaryocytes and platelets. MPL signaling is mediated by the JAK2-STAT5 pathway. THPO is constitutively produced by the liver, and its concentration is regulated by clearance through platelet mass. Genetic abnormalities affecting THPO/MPL/JAK2 axis can result in either thrombocytosis or thrombocytopenia. Specifically, somatic gain-of-function mutations in JAK2 and MPL genes are identified in myeloproliferative neoplasms. Conversely, constitutional mutations within THPO, MPL, or JAK2 genes can trigger either hereditary thrombocytosis or thrombocytopenia, depending on whether the mutation is gain or loss of function, respectively.1 Here, we report two cases of severe thrombocytosis in children from two unrelated families with the original association of two heterozygous germline mutations in the MPL gene previously described alone and leading when homozygous to a thrombocytosis for the first one (K39N) and to a thrombocytopenia for the second one (R102P). The first case was a 4-year-old child who presented with isolated thrombocytosis (981×109/L) discovered during an episode of angina with severe hyperthermia. The thrombocytosis persisted at 823×109/L after the resolution of the inflammatory syndrome, with no other reactive cause identified. Molecular screening for myeloproliferative neoplasms was performed, revealing no mutation in JAK2V617F, MPLW515, or CALR genes and absence of BCR::ABL1 transcript. Given this triple-negative thrombocytosis, sequencing of all coding regions of MPL gene and measurement of plasma THPO were performed. Two heterozygous mutations, MPL K39N (Baltimore variant) in exon 2 and MPL R102P in exon 3, were identified. The plasma THPO level was highly increased to 247 pg/mL (normal range < 40 pg/mL). A familial study was conducted showing normal platelet counts in parents and sister (Figure 1A). The MPL K39N mutation was found to be heterozygous in the mother and the young sister, accompanied by a slightly increase of THPO levels. The MPL R102P heterozygous mutation was identified in the father, correlating with a normal THPO level (Figure 1A). These results demonstrated that both variants were located on different alleles and thus were acting in trans. Consequently, the first child received the MPL K39N variant from his mother and the MPL R102P mutation from his father. A second case with a similar molecular pattern was observed in an unrelated family. A premature child (32-week gestation) exhibited high platelet levels at birth (499 × 109/L), which progressively worsened during follow-up to 1069×109/L at 3 months of age. Initial screening for an MPN driver gene mutation was negative, and a next-generation sequencing (NGS) was performed using a custom myeloid panel of 77 genes, revealing the association of MPL K39N and MPL R102P heterozygous variants without additional mutation. The plasma THPO concentration was also increased at 261 pg/mL, mirroring the first case (Figure 1A). The parents had normal platelets counts, and familial genetic study showed MPL K39N heterozygous variant in the mother and MPL R102P heterozygous variant in the father. Plasma THPO levels were not measured in the parents (Figure 1A). The MPL Baltimore (K39N) mutation is found heterozygous in around 9% of African and Afro-American population (gnomAD data) and is known to cause severe thrombocytosis when in a homozygous state (around 800×109/L) but results in mild thrombocytosis when heterozygous (around 500×109/L). In the present families, none of the relatives carrying the heterozygous MPL K39N mutation exhibited thrombocytosis, although plasma THPO levels were slightly increased. The K39N substitution is located in the extracellular domain of MPL, and the mechanism is related to abnormalities in MPL glycosylation leading to partial retention in the Golgi apparatus and subsequent reduced expression of the receptor on the cell membrane. The decreased expression of MPL on mature platelets in the bloodstream leads to increased plasma THPO levels, thereby stimulating megakaryopoiesis.2 The MPL R102P mutation is associated with congenital amegakaryocytosis thrombocytopenia (CAMT) in homozygous state3 and has an allele frequency of 0.03% in the general population (gnomAD data). This mutation is also located in the extracellular domain of MPL. Thrombocytopenia manifests due to receptor retention in the endoplasmic reticulum, causing a complete deficiency in its expression on the cellular membrane in homozygous mutations.4 Thrombocytosis was previously observed in two patients from a same family carrying the MPL R102P heterozygous mutation and exhibiting high levels of THPO.5 Herein, none of the relatives displayed thrombocytosis at diagnosis, suggesting either incomplete penetrance of this described association or the involvement of another variant in the prior report. Familial investigations in the two present cases confirmed that the two mutations were present on different alleles in a trans-configuration. This trans-association of R102P and K39N variants of MPL resulted in the complete retention of the MPL-R102P protein in the endoplasmic reticulum and only the expression of MPL-K39N on the cell surface, as represented in the model in Supplemental Figure S1. Thus, this genotype mimicked an MPL K39N homozygous status, leading to the observed severe thrombocytosis. An earlier association between MPL Baltimore variant and a nonsense MPL mutation has been previously described in a 44-year-old female with thrombocytosis.6 We performed western blot for MPL on purified platelets from the first family (mother, father, and child) (Figure 1B). A reduction in the expression of the MPL protein was found in all three individuals, but only the child with the K39N/R102P association exhibited the presence of an isoform with retarded electrophoretic mobility consistent with incomplete glycosylation. A similar pattern is found in patients homozygous for the MPL K39N mutation, as well as in a cell line engineered with the K39N mutation.2 The absence of such pattern in the mother heterozygous for K39N could be due to an exclusive dimerization of WT isoforms. While there are limited data in the literature, individuals with homozygous MPL K39N mutation typically do not exhibit thrombosis or bleeding syndromes. The two children mentioned in this report underwent annual blood count monitoring without requiring any specific treatment. In conclusion, we report here for the first time the germline association between MPL K39N Baltimore and MPL R102P heterozygous mutations on two different alleles in two children with severe thrombocytosis. The association between pro-thrombocytosis and pro-thrombocytopenia mutations is uncommon but possibly underestimated due to the frequency of these variants in the general population. Finally, this report highlights the importance of sequencing the entire of MPL gene for unexplained thrombocytosis and to consider the trans-association of mutations for biological interpretation. EV collected and analyzed the data and wrote the manuscript. ABo, Abe, and SG performed and analyzed genetics. IP and LD performed the analysis of MPL expression on platelets. VU reviewed the manuscript. JFB took care of patients and supervised the study. DLP supervised the study and wrote the manuscript. All authors declare no competing financial interests related to this work. The data that support the findings of this study are available on reasonable request from the corresponding author. Figure S1. Suggested mechanism to explain thrombocytosis in these patients. the left panel presents a wild type (WT) cell, with normal MPL receptor expression to cell membrane and normal THPO fixation. The right panel shows patient cases with thrombocytosis and the combination of MPLK39N (MPL Baltimore) and MPLR102P mutations: the R102P protein were not expressed in cell surface while the K39N protein is only expressed in few number leading to an increase of THPO level resulting in thrombocytosis. ER, endoplasmic reticulum; THPO, thrombopoietin. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.