Development Of Bone Marrow Fibrosis Research Articles

Mutational Analysis of the Genome Stability Factor BOD1L1 in Myeloproliferative Neoplasm

The myeloproliferative neoplasms (MPN) are a group of clonal hematopoietic stem cell disorders that include essential thrombocythemia (ET), polycythemia vera (PV) and myelofibrosis (MF) which arise from somatic mutations in JAK2, MPL or CALR, but which can evolve to acute myeloid leukemia (AML) by the acquisition of additional somatic mutations. To investigate the contribution that mutations might make to the pathogenesis or clinical course of MPNs, 261 genes recurrently mutated in myeloid malignancies were sequenced in DNA from germline and blood samples from patients with AML, ET and MF enrolled in studies of the LSD1 inhibitor bomedemstat. Earlier work identified somatic mutations inthegene BOD1L1 in isolated megakaryocytes in 6 of 12 patients with MPN with mutations suggestive of loss-of-function (Guo et al., 2017). BOD1L1 plays a key role in multiple pathways of cellular DNA repair in conjunction with the histone methyltransferase SETD1A, which regulates transcription and DNA repair (Higgs et al. 2018; Hoshii et al. 2018. We previously demonstrated that BOD1L1 prevents uncontrolled resection of damaged DNA forks under conditions of replication stress, and that its loss leads to severe genome instability (Higgs et al., 2015, 2018). In addition, BOD1L1 promotes repair of DNA double-strand breaks after ionizing radiation through its interaction with the DNA repair protein RIF1 (Bayley et al., 2022). This sequencing-based analysis identified specific germline mutations in BOD1L1 that were 3-6 times more prevalent in MPN patients compared to a control population (gnomAD). This enrichment of BOD1L1 mutations was confirmed in whole-exome sequencing from an independent data set (EGA) of 183 patients with MPNs. We predicted these highly deleterious mutations, or at least a subset, may compromise the DNA repair functions of BOD1L1 and potentially influence the development and/or progression of MPN. To investigate this prediction, we disrupted BOD1L1 using a doxycycline-inducible CRISPR-Cas9 system in HeLa cells and complemented them with gRNA-resistant wild-type BOD1L1,or one of six mutants distributed throughout the protein. None of the mutants affected BOD1L1 protein expression; however, two of the six variants were defective for RIF1 binding by co-immunoprecipitation/proximity ligation, failed to recruit this key DNA repair factor to radiation-induced double-strand breaks, and compromised repair of these breaks. Moreover, single molecule DNA fiber analyses revealed that these same two mutants were unable to protect stalled replication forks against resection, leading to elevated levels of genomic damage. To explore the functional consequences of BOD1L1 loss-of-function on megakaryocytes, the gene was silenced using shRNA in human megakaryoblastic JAK2 V617F SET-2 cells, differentiated with thrombopoietin, and analyzed by flow cytometry. These cells were CD41-positive, consistent with megakaryocytic differentiation, and showed increased DNA damage (γH2AX) compared to controls. Depletion of BOD1L1 also lead to a greater number of cells with 2n nuclear content, and fewer with ≥8n, compared with controls, suggesting that the loss of BOD1L1 led to impaired polyploidization likely due to unrepaired DNA damage and subsequent cell death. In summary, we have shown that MPN patients have a high prevalence of mutations in the key genome instability factor BOD1L1 and that a subset of these mutations compromised its DNA repair functions and increased genome instability. The presence of BOD1L1 somatic mutations in megakaryocytes in MF patients may deregulate polyploidization, altering megakaryocyte function that may contribute to the development of bone marrow fibrosis. That no patients with BOD1L1 germline mutations progressed to AML in these clinical studies, coupled with the fact that these variants were not enriched in patients with AML, invites the possibility that BOD1L1 mutations may be synthetically lethal with other somatic mutations that result in genome instability and, hence, protect against progression to AML.

SRSF2 mutation reduces polycythemia and impairs hematopoietic progenitor functions in JAK2V617F-driven myeloproliferative neoplasm

SRSF2 mutations are found in association with JAK2V617F in myeloproliferative neoplasms (MPN), most frequently in myelofibrosis (MF). However, the contribution of SRSF2 mutation in JAK2V617F-driven MPN remains elusive. To investigate the consequences of SRSF2P95H and JAK2V617F mutations in MPN, we generated Cre-inducible Srsf2P95H/+Jak2V617F/+ knock-in mice. We show that co-expression of Srsf2P95H mutant reduced red blood cell, neutrophil, and platelet counts, attenuated splenomegaly but did not induce bone marrow fibrosis in Jak2V617F/+ mice. Furthermore, co-expression of Srsf2P95H diminished the competitiveness of Jak2V617F mutant hematopoietic stem/progenitor cells. We found that Srsf2P95H mutant reduced the TGF-β levels but increased the expression of S100A8 and S100A9 in Jak2V617F/+ mice. Furthermore, enforced expression of S100A9 in Jak2V617F/+ mice bone marrow significantly reduced the red blood cell, hemoglobin, and hematocrit levels. Overall, these data suggest that concurrent expression of Srsf2P95H and Jak2V617F mutants reduces erythropoiesis but does not promote the development of bone marrow fibrosis in mice.

Inhibition of ERK1/2 signaling prevents bone marrow fibrosis by reducing osteopontin plasma levels in a myelofibrosis mouse model

Clonal myeloproliferation and development of bone marrow (BM) fibrosis are the major pathogenetic events in myelofibrosis (MF). The identification of novel antifibrotic strategies is of utmost importance since the effectiveness of current therapies in reverting BM fibrosis is debated. We previously demonstrated that osteopontin (OPN) has a profibrotic role in MF by promoting mesenchymal stromal cells proliferation and collagen production. Moreover, increased plasma OPN correlated with higher BM fibrosis grade and inferior overall survival in MF patients. To understand whether OPN is a druggable target in MF, we assessed putative inhibitors of OPN expression in vitro and identified ERK1/2 as a major regulator of OPN production. Increased OPN plasma levels were associated with BM fibrosis development in the Romiplostim-induced MF mouse model. Moreover, ERK1/2 inhibition led to a remarkable reduction of OPN production and BM fibrosis in Romiplostim-treated mice. Strikingly, the antifibrotic effect of ERK1/2 inhibition can be mainly ascribed to the reduced OPN production since it could be recapitulated through the administration of anti-OPN neutralizing antibody. Our results demonstrate that OPN is a novel druggable target in MF and pave the way to antifibrotic therapies based on the inhibition of ERK1/2-driven OPN production or the neutralization of OPN activity.

Interleukin-1 contributes to clonal expansion and progression of bone marrow fibrosis in JAK2V617F-induced myeloproliferative neoplasm

Chronic inflammation is frequently associated with myeloproliferative neoplasms (MPN), but the role of inflammation in the pathogenesis of MPN remains unclear. Expression of the proinflammatory cytokine interleukin-1 (IL-1) is elevated in patients with MPN as well as in Jak2V617F knock-in mice. Here, we show that genetic deletion of IL-1 receptor 1 (IL-1R1) normalizes peripheral blood counts, reduces splenomegaly and ameliorates bone marrow fibrosis in homozygous Jak2V617F mouse model of myelofibrosis. Deletion of IL-1R1 also significantly reduces Jak2V617F mutant hematopoietic stem/progenitor cells. Exogenous administration of IL-1β enhances myeloid cell expansion and accelerates the development of bone marrow fibrosis in heterozygous Jak2V617F mice. Furthermore, treatment with anti-IL-1R1 antibodies significantly reduces leukocytosis and splenomegaly, and ameliorates bone marrow fibrosis in homozygous Jak2V617F mice. Collectively, these results suggest that IL-1 signaling plays a pathogenic role in MPN disease progression, and targeting of IL-1R1 could be a useful strategy for the treatment of myelofibrosis.

SF3B1 mutations in the Driver Clone Increase the Risk of Evolution to Myelofibrosis in Patients with Myeloproliferative Neoplasms (MPN)

Introduction: Next generation sequencing (NGS) studies identified additional somatic mutations impacting disease evolution and prognosis in MPN. SF3B1, a component of the U2 small nuclear ribonucleoprotein splicing complex, is frequently mutated in myelodysplastic syndromes where it has been proposed to define a new entity (Malcovati et. al. Blood 2020). In MPN, SF3B1 is mutated in approximately 10% of patients with primary myelofibrosis (PMF) and 3-5% with polycytemia vera or essential thrombocytemia (ET). Recent reports suggested that spliceosome mutations may adversely affect myelofibrosis free survival (MFS) in ET (Tefferi et. al. Br J Haematol. 2020). The main objective of this study was to evaluate the impact of the concomitant presence of driver (JAK2, MPLor CALR) and SF3B1 clonal or sub-clonal mutations on MPN phenotype and evolution in a large single center cohort of MPN patients. Methods: A total of 1243 consecutive patients were diagnosed with MPN according to WHO criteria between January 2011 and May 2020 in our center, of whom 707 had molecular analysis by NGS targeting a panel of 36 myeloid genes performed at diagnosis and/or during follow-up. Significant variants were retained with a sensitivity of 0.5%. Patients were grouped according to variant allele frequencies (VAF) determined by NGS as "driver" SF3B1mutated patients when driver and SF3B1 mutations VAF were similar (double mutated clone), and as "non-driver" SF3B1 mutants when SF3B1VAF was lower than that of the driver mutation, suggestive of a sub-clone. 4 patients with SF3B1 mutations but no driver mutation were excluded. We then compared the characteristics and outcomes of 3 groups of patients according to their SF3B1 mutational status: wild type (WT), driver and non-driver SF3B1 mutations. Results: A total of 39/703 (5.6%) patients had SF3B1 mutations, of whom 11/39 (28.2%) and 28/39 (71.8%) harbored driver and non-driver SF3B1mutations, respectively. Driver SF3B1 mutations were associated with PMF (OR 6.1, 95%CI [1.1; 33.6], p= 0.039) and MPN unclassified (OR 15.6,95%CI [1.1; 116.5], p= 0.007) subtypes, presence of immature myeloid cells ≥ 2% (OR 9.3, CI [2.6; 32.8], p= 0.001) and peripheral blasts ≥ 1% (OR 5.0,95%CI [1.0; 24.7], p= 0.047) at diagnosis (Figure A). Other variables were not significantly different between patients with driver, non-driver and WT SF3B1, including age, driver mutation type, MPN-related symptoms, cytogenetics and high molecular risk mutations (ASXL1, EZH2, SRSF2, IDH1/2or U2AF1). There was no significant difference in the response to therapy: complete hematological response was seen in 5/11 (45.5%), 10/28 (35.7%) and 327/664 (49.3%) of patients with driver, non-driver and WT SF3B1 respectively. After a median follow-up of 103.7 months IQR [47.2; 175.6], evolution to myelofibrosis occurred in 5/7 (71.4%), 6/20 (30.0%) and 99/564 (17.6%) of patients with driver, non-driver and WT SF3B1 respectively. Interestingly, driver SF3B1 but not non-driver SF3B1 mutational status adversely impacted MFS (OR 7.56,95%CI[2.95; 19.38], p<0.001)(Figure B). Other variables independently associated with adverse MFS in multivariate COX regression analysis included age at MPN diagnosis (OR 1.02, 95%CI[1.00; 1.04], p=0.003), JAK2V617F allele burden (OR 1.03, 95%CI[1.02; 1.04], p<0.001), MPL (OR 13.94, 95%CI[4.90; 39.70], p<0.001) and CALR (OR 7.06, 95%CI[3.69; 13.51], p<0.001) mutations. SF3B1 mutational status had no impact on overall survival, transformation to MDS/AML or thrombotic/hemorrhagic events free survival. Conclusion: This study in a large cohort of MPN patients highlights for the first time to our knowledge the adverse impact on MFS of SF3B1 and MPN-driver co-mutated clones. In contrast, presence of an SF3B1 mutation at sub-clonal level didn't increase the risk of MF development. In line with our findings, a recent study reported an association between rapid progression to myelofibrosis and SF3B1 mutations in patients with age-related clonal hematopoiesis (Bartels et al. Leukemia 2020). Further studies are warranted to confirm our results on independent cohorts and to investigate the mechanisms of bone marrow fibrosis development in patients with SF3B1 and MPN-drivermutations. Disclosures Rea: Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Kiladjian:BMS: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; AOP Orphan: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees. Benajiba:Gilead Foundation: Research Funding.

Enhanced engraftment of human myelofibrosis stem and progenitor cells in MISTRG mice

AbstractThe engraftment potential of myeloproliferative neoplasms in immunodeficient mice is low. We hypothesized that the physiological expression of human cytokines (macrophage colony-stimulating factor, interleukin-3, granulocyte-macrophage colony-stimulating factor, and thrombopoietin) combined with human signal regulatory protein α expression in Rag2−/−Il2rγ−/− (MISTRG) mice might provide a supportive microenvironment for the development and maintenance of hematopoietic stem and progenitor cells (HSPC) from patients with primary, post–polycythemia or post–essential thrombocythemia myelofibrosis (MF). We show that MISTRG mice, in contrast to standard immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ and Rag2−/−Il2rγ−/− mice, supported engraftment of all patient samples investigated independent of MF disease stage or risk category. Moreover, MISTRG mice exhibited significantly higher human MF engraftment levels in the bone marrow, peripheral blood, and spleen and supported secondary repopulation. Bone marrow fibrosis development was limited to 3 of 14 patient samples investigated in MISTRG mice. Disease-driving mutations were identified in all xenografts, and targeted sequencing revealed maintenance of the primary patient sample clonal composition in 7 of 8 cases. Treatment of engrafted mice with the current standard-of-care Janus kinase inhibitor ruxolitinib led to a reduction in human chimerism. In conclusion, the established MF patient-derived xenograft model supports robust engraftment of MF HSPCs and maintains the genetic complexity observed in patients. The model is suited for further testing of novel therapeutic agents to expedite their transition into clinical trials.

Megakaryocyte Ablation Ameliorates Bone Marrow Fibrosis By Reducing Myofibroblast Differentiation in a Mouse Model of Primary Myelofibrosis

Primary Myelofibrosis (PMF) is a chronic myeloproliferative disorder characterized by excessive bone marrow (BM) fibrosis that can lead to ineffective hematopoiesis and reduced survival. PMF patients develop several megakaryocyte (MK) abnormalities including increased proliferation, abnormal morphology and upregulated expression of pro-fibrotic genes. Although several studies support the role of MK in BM fibrosis, the significance of MK in the development of BM fibrosis has not been demonstrated in vivo. Here, we investigated the in vivo role of MK in the pathogenesis of BM fibrosis. First, we reproduced an established bone marrow transplantation (BMT) mouse model of PMF. To achieve this, lethally irradiated wild-type mice were transplanted with wild-type hematopoietic stem cells (HSC) transduced with either MPLW515L gene construct (MPLW515L BMT) or MIGR1 empty vector (MIGR1 BMT). Consistent with previous studies, starting at day 14 post-BMT, MPLW515L BMT mice gradually developed increased number of leukocytes and platelets, increased proliferation of abnormal megakaryocytes in the BM and spleen, hepatosplenomegaly with extra-medullary hematopoiesis, and reticulin fibrosis in the BM. Notably, robust fibrosis was also seen in the spleen and liver. Next, to study the significance of MK in the development of BM fibrosis, we developed a MPLW515L BMT mouse model in which MK lineage could be selectively ablated. We accomplished this using PF4-Cre inducible diphtheria toxin receptor transgenic mice (iDTR+/-/PF4-Cre) as BMT donors. HSCs from iDTR+/-/PF4-Cre mice were transduced with MPLW515L or MIGR1 empty vector and transplanted as described above. Beginning day 14 post-BMT, recipient mice were injected with diphtheria toxin (DT) or water every 48 hours. At day 20 and day 29, DT injection significantly depleted MK in both MPLW515L BMT and MIGR1 BMT groups compared to water injection. Importantly, DT-injected MPLW515L BMT mice displayed attenuated fibrosis in the BM, spleen, and liver compared to water-injected MPLW515L BMT mice. In addition, DT treatment decreased the level of alpha smooth muscle actin in both BM and spleen of MPLW515L BMT mice, which suggests that MKs are critical for myofibroblast differentiation. MK ablation did not leukocytosis, thrombocytosis or hepatosplenomegaly. Together, our study show that successful MK ablation in vivo reduces fibrosis development in the BM, spleen, and liver of the MPLW515L mouse model of PMF. In summary, these results support the essential role of MK in the pathogenesis of BM fibrosis in PMF. Further studies are underway to elucidate the mechanisms by which MK contribute to fibrosis in PMF. Disclosures No relevant conflicts of interest to declare.

Activation of JAK/STAT Signaling in Megakaryocytes Sustains Myeloproliferation In Vivo.

The myeloproliferative neoplasms (MPN), including polycythemia vera, essential thrombocythemia, and primary myelofibrosis, are characterized by the expansion of the erythroid, megakaryocytic, and granulocytic lineages. A common feature of these disorders is the presence of abnormal megakaryocytes, which have been implicated as causative agents in the development of bone marrow fibrosis. However, the specific contributions of megakaryocytes to MPN pathogenesis remain unclear. We used Pf4-Cre transgenic mice to drive expression of JAK2V617F in megakaryocyte lineage-committed hematopoietic cells. We also assessed the critical role of mutant megakaryocytes in MPN maintenance through cell ablation studies in JAK2V617F and MPLW515L BMT models of MPN. JAK2V617F -mutant presence in megakaryocytes was sufficient to induce enhanced erythropoiesis and promote fibrosis, which leads to a myeloproliferative state with expansion of mutant and nonmutant hematopoietic cells. The increased erythropoiesis was associated with elevated IL6 level, which was also required for aberrant erythropoiesis in vivo. Furthermore, depletion of megakaryocytes in the JAK2V617F and MPLW515L BMT models ameliorated polycythemia and leukocytosis in addition to expected effects on megakaryopoiesis. Our observations reveal that JAK/STAT pathway activation in megakaryocytes induces myeloproliferation and is necessary for MPN maintenance in vivo. These observations indicate that MPN clone can influence the behavior of the wild-type hematopoietic milieu, at least, in part, via altered production of proinflammatory cytokines and chemokines. Our findings resonate with patients who present with a clinical MPN and a low JAK2V617F allele burden, and support the development of MPN therapies aimed at targeting megakaryocytes.

Developmental low-dose exposure to bisphenol A induces chronic inflammation, bone marrow fibrosis and reduces bone stiffness in female rat offspring only

BackgroundDevelopmental exposure to low doses of the endocrine disruptor bisphenol A (BPA) is known to alter bone tissue in young rodents, although how bone tissue is affected in aged animals is not well known. We have recently shown that low-dose developmental exposure to BPA increases procollagen type I N-terminal propeptide (P1NP) levels, a peptide formed during type 1 collagen synthesis, in plasma of 5-week-old female rat offspring while male offspring showed reduced bone size. ObjectiveTo analyze offspring bone phenotype at 52 weeks of age and clarify whether the BPA-induced increase in P1NP levels at 5 weeks is an early sign of bone marrow fibrosis development. MethodsAs in our 5-week study, pregnant Fischer 344 rats were exposed to BPA via drinking water corresponding to 0.5 μg/kg BW/day (BPA0.5), which is in the range of human daily exposure, or 50 μg/kg BW/day (BPA50) from gestational day 3.5 until postnatal day 22. Controls were given only vehicle. The offspring were sacrificed at 52 weeks of age. Bone effects were analyzed using peripheral quantitative and micro-computed tomography (microCT), 3-point bending test, plasma markers and histological examination. ResultsCompared to a smaller bone size at 5 weeks, at the age of 52 weeks, femur size in male offspring had been normalized in developmentally BPA-exposed rats. The 52-week-old female offspring showed, like the 5-week-old siblings, higher plasma P1NP levels compared to controls but no general increasing bone growth or strength. However, 2 out of 14 BPA-exposed female offspring bones developed extremely thick cortices later in life, discovered by systematic in vivo microCT scanning during the study. This was not observed in male offspring or in female controls. Biomechanical testing revealed that both doses of developmental BPA exposure reduced femur stiffness only in female offspring. In addition, histological analysis showed an increased number of fibrotic lesions only in the bone marrow of female rat offspring developmentally exposed to BPA. In line with this, plasma markers of inflammation, Tnf (in BPA0.5) and Timp1 (in BPA50) were increased exclusively in female offspring. ConclusionsDevelopmental BPA exposure at an environmentally relevant concentration resulted in female-specific effects on bone as well as on plasma biomarkers of collagen synthesis and inflammation. Even a dose approximately eight times lower than the current temporary EFSA human tolerable daily intake of 4 μg/kg BW/day, appeared to induce bone stiffness reduction, bone marrow fibrosis and chronic inflammation in female rat offspring later in life.

Involvement of MAF/SPP1 axis in the development of bone marrow fibrosis in PMF patients

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by hyperplastic megakaryopoiesis and myelofibrosis. We recently described the upregulation of MAF (v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog) in PMF CD34+ hematopoietic progenitor cells (HPCs) compared to healthy donor. Here we demonstrated that MAF is also upregulated in PMF compared with the essential thrombocytemia (ET) and polycytemia vera (PV) HPCs. MAF overexpression and knockdown experiments shed some light into the role of MAF in PMF pathogenesis, by demonstrating that MAF favors the megakaryocyte and monocyte/macrophage commitment of HPCs and leads to the increased expression of proinflammatory and profibrotic mediators. Among them, we focused our further studies on SPP1 and LGALS3. We assessed SPP1 and LGALS3 protein levels in 115 PMF, 47 ET and 24 PV patients plasma samples and we found that SPP1 plasma levels are significantly higher in PMF compared with ET and PV patients. Furthermore, in vitro assays demonstrated that SPP1 promotes fibroblasts and mesenchymal stromal cells proliferation and collagen production. Strikingly, clinical correlation analyses uncovered that higher SPP1 plasma levels in PMF patients correlate with a more severe fibrosis degree and a shorter overall survival. Collectively our data unveil that MAF overexpression contributes to PMF pathogenesis by driving the deranged production of the profibrotic mediator SPP1.

Fibroblast dynamics as an in vitro screening platform for anti-fibrotic drugs in primary myelofibrosis.

Although the cause for bone marrow fibrosis in patients with myelofibrosis remains controversial, it has been hypothesized that it is caused by extensive fibroblast proliferation under the influence of cytokines generated by the malignant megakaryocytes. Moreover, there is no known drug therapy which could reverse the process. We studied the fibroblasts in a novel system using the hanging drop method, evaluated whether the fibroblasts obtain from patients are part of the malignant clone of not and, using this system, we screen a large library of FDA-approved drugs to identify potential drugs candidates that might be useful in the treatment of this disease, specifically which would inhibit fibroblast proliferation and the development of bone marrow fibrosis. We have found that the BM fibroblasts are not part of the malignant clone, as previously suspected and two immunosuppressive medications-cyclosporine and mycophenolate mophetil, as most potent suppressors of the fibroblast collagen production thus potentially inhibitors of bone marrow fibrosis production in myelofibrosis.

HSC Hierarchy in Primary Myelofibrosis

Primary Myelofibrosis (PMF) is a Myeloproliferating Neoplasm (MPN) of hematopoietic stem cell origin, characterized by expansion of aberrant myeloid progenitors in the chronic phase that can lead to bone marrow fibrosis development and/or osteosclerosis. In 20-25% of PMF cases transformation to acute myeloid leukemia (AML) is observed. Identification of multiple molecular lesions suggests complex clone dynamics that indicates exceeding sub-clone dominance as PMF progresses. Our aim is to determine the HSC hierarchy orchestrating initiation and development of PMF.In our previous studies we reported a CD133+ HSC population in PMF peripheral blood that represents the aberrant long-term stem cell fraction responsible for PMF reproduction of chronic and acute phases in vivo. Molecular analysis of PMF xenografts indicates sustenance of genetically different HSC clones exhibiting variation in both their engraftment potential and reproduction of PMF parameters in the first mouse model of the disease.To further characterize the succession of molecular lesions determining HSC clone propagation we performed targeted exon sequencing of PMF HSC from 100 PMF patients for 54 genes. Mutations in the epigenetic regulators ASXL1 and EZH2 were detected in 38% and 15% of PMF patients respectively. HSC clonal evolution was determined by single cell molecular and phenotypic analysis in vitro and graft analysis in vivo.Mutations detected in the epigenetic regulators ASXL1 and EZH2 represent founding molecular lesions at the top of PMF HSC hierarchy. ASXL1 mutations precede and are connected with sustenance of clonal hematopoiesis without any significant influence on the HSC differentiation potential. EZH2 mutations are connected with impaired erythropoiesis in vitro and anemia, high engraftment and expansion of pre-leukemic clones in vivo. Occurrence of JAK2 and CALR mutations in the background of mutated epigenetic regulator genes is connected with expansion of HSC subclones and reproduction of chronic phase PMF as atypical myelopoiesis, splenomegaly and induction of fibrosis.Our results indicate genetic heterogeneity of PMF neoplastic HSC is comprised of three different mutational clusters. Mutations in epigenetic regulator genes (Group 1) precede and shape the epigenetic landscape conferring genetic instability to sustain the expansion of pre-leukemic clones (Group 3 mutations). JAK2 and CALR mutations (Group 2) occur later on and are connected with aberrant myelopoiesis of chronic phase disease. HSC clonal dynamics reflect genotype related phenotypes as determinants of chronic and acute phases of PMF. DisclosuresNo relevant conflicts of interest to declare.

Imatinib Mesylate Reduces Bone Marrow Fibrosis and Overwhelms the Adverse Prognostic Impact of Reticulin Formation in Patients with Chronic Myeloid Leukemia

Introduction: Imatinib mesylate (IM) is the first tyrosine kinase inhibitor (TKI) licensed for the treatment of chronic myeloid leukemia (CML). Severe bone marrow fibrosis (BMF) has been reported in excess of 40% of the patients with CML at diagnosis. Before TKIs became available, BMF which emerged at diagnosis and/or in the late periods of the disease was defined to be a poor prognostic factor, and it contributed significantly to morbidity and mortality from 10% to 30% in patients with CML. The relationship between BMF and both disease progression and prognosis has been the subject of re-evaluation after the introduction of IM therapy. In patients with CML, it has not been clearly demonstrated yet, whether IM improves the poor prognostic effect of fibrosis, and prevents the new BMF development or not.Aim: The purpose of this study was to evaluate the effects of IM therapy on BMF formation, and the prognostic significance of BMF in patients with CML.Material and Methods: One hundred and thirty-five CML patients were enrolled in the study. Patients' demographics, Sokal risk scores, molecular and cytogenetic responses and follow-up periods were noted from the patients' files retrospectively. All pre- and post-IM bone marrow biopsy samples, which were stained with hematoxylin and eosin, were re-evaluated for the current analysis. Grading of BMF was according to the European consensus decisions, graded as 0-III. The term "last bone marrow biopsy" (LBMB) is referred to a biopsy, which was performed at 18th months or later on during IM treatment.Results: The median age was 44 years (range, 18-92 years), and 78 patients (58%) were male. One hundred and twenty-eight patients (95%) were in chronic phase [CP], 4 patients (3%) were in accelerated phase [AP], and 3 patients (2%) were in blast crisis [BC] at the time of IM initiation. Out of 128 CML-CP patients, one hundred and twenty patients (93%) were in early CP, whereas 8 (7%) were in late CP. The percentage of low, intermediate, and high Sokal risk scores were 35%, 43%, and 22%, respectively. Before IM was initiated, thirty-one patients had received previous treatment modalities (hydroxyurea (HU) in twenty-one, and 10 patients had received interferon plus HU. he median duration of IM treatment was 45 months (range, 2-106 months). The rates of complete hematological response (CHR) at 3rd month, complete cytogenetic response (CCyR) at 12th month, and major molecular response (MMR) at 18th month were 92.4%, 71.6%, and 67.6%, respectively. BMF before the initiation of IM therapy was grade 0 in 52 patients (39%), grade I in 39 patients (29%), grade II in 28 patients (21%), and grade III in 16 patients (12%). There was a positive correlation between the Sokal risk scores and the grades of BMF at diagnosis (r:0.313, p <0.01), and as the Sokal risk scores increase, the grades of BMF also increase and the difference was significant (p =0.025). Also the spleen size significantly differ according to the grade of BMF, and the patients with a higher grade of BMF also had a larger spleen (p <0.001). The control bone marrow biopsies at the 12th month of IM were available in one hundred and four patients, and 70 (67%) of them have grade 0, 26 (25%) of them have grade I, 7 (7%) of them have grade II, and 1 (1%) of them has grade III BMF. After 12 months of IM treatment, the grades of BMF have significantly decreased when compared to the pre-treatment values (p =0.001), and BMF grades in LBMB has shown to be significantly decreased when compared to the pre-treatment values (p <0.001) (Table 1 and Figures 1&2). There was no significant difference regarding the grades of BMF, between the biopsy at 12th months and LBMB (p =0.703).The CCyR rates at 12th month did not differ according to the pre-imatinib BMF grades (p =0.127). There was no significant difference between patients with or without CCyR at 12 months of IM regarding grades of BMF (p =0.785). The MMR rates at 18 months did not differ according to pre-treatment grades of BMF (p =0.112). There was no significantly difference in overall survival rates between initial BMF mild (grade 0-I) and severe (grade II-III) groups (p =0.278).Conclusion: IM can reduce BMF after a long period of follow-up, independently from the molecular and cytogenetic responses. The BMF grades at diagnosis does not have a negative impact on the response to IM treatment. Therefore, the adverse prognostic impact of the marrow fibrosis among CML patients seems to disappear in the era of the TKIs. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Gray platelet syndrome: proinflammatory megakaryocytes and α-granule loss cause myelofibrosis and confer metastasis resistance in mice

AbstractNBEAL2 encodes a multidomain scaffolding protein with a putative role in granule ontogeny in human platelets. Mutations in NBEAL2 underlie gray platelet syndrome (GPS), a rare inherited bleeding disorder characterized by a lack of α-granules within blood platelets and progressive bone marrow fibrosis. We present here a novel Nbeal2−/− murine model of GPS and demonstrate that the lack of α-granules is due to their loss from platelets/mature megakaryocytes (MKs), and not by initial impaired formation. We show that the lack of Nbeal2 confers a proinflammatory phenotype to the bone marrow MKs, which in combination with the loss of proteins from α-granules drives the development of bone marrow fibrosis. In addition, we demonstrate that α-granule deficiency impairs platelet function beyond their purely hemostatic role and that Nbeal2 deficiency has a protective effect against cancer metastasis.

PDGFB hypomethylation is a favourable prognostic biomarker in primary myelofibrosis

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterised by the clonal proliferation of the haematopoietic precursors together with the progressive development of bone marrow fibrosis. This stromal alteration is an important clinical issue and specific prognostic markers are not currently available. In bone marrow biopsies from 58 PMF patients, we explored the methylation pattern of genes encoding cytokines involved in the stromal reaction, namely platelet-derived growth factor-beta (PDGFB), transforming growth factor-beta (TGFB) and basic fibroblast growth factor (FGF2). We also evaluated the methylation profile of the Long Interspersed Nucleotide Element 1 (LINE-1). PDGFB, FGF2 and LINE-1, but not TGFB, were significantly differently methylated in PMF compared to controls. Significantly, PDGFB hypomethylation (<16%) was correlated with a favourable PMF prognosis (grade of marrow fibrosis, p=0.03; International Prognostic Scoring Systems p=0.01 and Dynamic International Prognostic Scoring Systems, p=0.02). Although the basis of the association of PDGFB hypomethylation with favourable prognosis remains to be clarified, we speculate that hypomethylation in PMF could represent the effect of acquired somatic mutations in genes involved in epigenetic regulation of the genome.

Pluripotent Fibrosis Initiating Mesenchymal Stromal Cell Linked To Cytopenias In LGL Leukemia Through Genetic Reprogramming

BackgroundLarge Granular Lymphocyte (LGL) leukemia is associated with chronic lymphoproliferation in association with unexplained cytopenias. Discovery of somatic mutations in STAT3 provides new insights into the molecular basis of LGL leukemia, but the precise mechanism leading to cytopenias remains unresolved. To gain insight into this mechanism, bone marrow fibrosis (Fig. 1A) was found to occur in 88% of cases and is significantly associated with splenomegaly, the presence of cytopenias, and co- existence of autoimmune diseases. In this study, we are reporting the identification of the fibrosis initiating cell population. [Display omitted] MethodsPrimary mesenchymal stromal cultures (MSCs) from LGL leukemia BM were isolated and grown under reduced oxygen conditions using undifferentiated and differentiation-specific culture conditions (Fig. 1B). These cultured cells were confirmed to deposit greater amounts of fibrillar Type I, III and V collagen matrix consistent with the heavy reticulin and trichrome positive stains observed in bone marrow biopsies (Fig. 1C). This collagen-producing cell population failed to support normal hematopoietic progenitor proliferation and colony formation in co-culture assays. Microarray analysis of the collagen-producing mesenchymal cell population was conducted and pathway analysis was performed using the GeneGo analysis platform from MetaCore™. Each list of genes was individually grouped and evaluated for membership on the set of GeneGo® pathway maps under the functional ontology enrichment tool. Calculation of p-values using hypergeometric distribution was used to determine pathway statistical significance. Flow cytometry was conducted to further evaluate the mesenchymal lineage. ResultsSupervised hierarchical clustering was performed to determine the overall similarity of LGL MSCs to publicly available, platform-compatible, microarrays from cells of mesenchymal lineage. Clustering showed that both patient and control MSCs display expression patterns distinct from committed mesenchymal lineages including osteoblasts, chondrocytes, and adipocytes. Mesenchymal-derived fibroblast differentiation can be distinguished by down-regulated CD29, CD44, CD105, CD106, CD117, bone morphogenetic protein receptor, and Sca-1 expression, and by up-regulated collagen type I, collagen type III, tenacin C, fibronectin, matrix metalloproteinase 1, fibroblast-specific protein 1, and vimentin. By microarray analysis and flow cytometry, we find no evidence that the cells are differentiated cells aside from collagen over expression. In order to define their pluripotent potential, sub-confluent cultures were grown in three types of differentiating media. Primary MSCs from both LGL leukemia patients and healthy controls were similar in their capacity to undergo trilineage differentiation into osteoblasts (stained with Alizarin Red S), adipocytes (Oil red O), and chondrocytes (Alican blue) (Fig. 1D). Next, pathway analyses revealed significant re-programming of the pluripotent MSCs. ECM production pathways were increased while proliferation pathways were down-regulated. Proliferation of MSCs is governed by production of autocrine growth factors. Both leukemia inhibitor factor (LIF) and basic fibroblast growth factor (FGFb) were significantly reduced compared to control MSCs (p<0.001) and the proliferative capacity of the LGL MSCs was severely diminished. Exogenous FGFb, but not LIF, restored LGLL MSC proliferation comparable to normal MSCs (Fig. 1E), normalized their spindle-shaped morphology, and maintained MSC plasticity. Interestingly, treatment with exogenous FGFb normalized collagen deposition (Fig. 1F) and restored hematopoietic supporting function. ConclusionSTAT-activated LGL leukemia cells, like myeloproliferative neoplasms, share a propensity for the development of bone marrow fibrosis. These results indicate that MSCs from LGL leukemia are pluripotent, but are re-programmed to deposit excessive amounts of collagen. Restoration of FGFb maintains the pluripotency of these MSCs while reversing the fibrosis phenotype. Our data suggests that FGFb-cultured MSCs may be used to reverse cytopenias in LGL leukemia. Disclosures:No relevant conflicts of interest to declare.

The relationship of the active and latent forms of TGF-β1 with marrow fibrosis in essential thrombocythemia and primary myelofibrosis

The aim of this study was to perform an immunohistochemical analysis from 100 megakaryocytes per sample, analyzing positivity and intensity levels of anti-LAP human TGF-β1 (or Latent TGF-β1) and anti-TGF-β1 (or Active TGF-β1) antibodies from 18 essential thrombocythemia (ET) and 38 primary myelofibrosis (PMF) patients (being 19 pre-fibrotic and 19 fibrotic). Six bone marrow donor biopsies were used as controls. Fibrosis in bone marrow biopsies (BMB) was evaluated according to the European Consensus. The average fibrosis grade differed between each group (P=0.001 or P=0.003). Latent TGF-β1 values differed significantly between pre-fibrotic (P=0.018) and fibrotic (P=0.031) groups when compared with the control group. The high immunoexpression level of Latent TGF-β1 in the megakaryocytes from patients with myelofibrosis, which was not observed in patients with essential thrombocythemia, may be associated with the development of bone marrow fibrosis.

Monocyte IL-2Rα expression is associated with thrombosis and the JAK2V617F mutation in myeloproliferative neoplasms

The development of bone marrow fibrosis and thrombosis are main causes of morbidity in essential thrombocythemia (ET). Monocyte activation has been associated to the production of fibrosis-related cytokines and pro-thrombotic factors. The aim of this study was to identify new markers of monocyte activation in Phi-negative myeloproliferative neoplasms and to search for their relationship with clinical features. Forty-five patients comprising 30 ET, eight myelofibrosis and seven polycythemia vera were included. We evaluated the α subunit of IL-2 receptor (CD25) on monocytes, basal and LPS-induced IL-1β release from mononuclear cells, and monocyte TGF-β mRNA content. Patients who had thrombotic events displayed higher monocyte CD25 levels (6.2%) than those without symptoms (1.3%) and controls (2.6%), p = 0.0006. JAK2V617F-positive patients had higher monocyte CD25 expression levels (4.7%), than JAK2V617F-negative (2.6%), p = 0.0213. Patients with myeloproliferative neoplasms had similar monocyte CD25 expression than controls, both, in basal conditions and after cell adhesion. IL-1β release and TGF-β mRNA levels were normal. In conclusion, increased monocyte CD25 expression is associated with history of thrombosis and is also up-regulated in patients harboring JAK2V617F mutation. The finding of increased CD25 levels together with normal IL-1β and TGF-β production reveals a selective monocyte activation profile in myeloproliferative neoplasms.

Adenoviral-mediated TGF-β1 inhibition in a mouse model of myelofibrosis inhibit bone marrow fibrosis development

Myelofibrosis is characterized by excessive deposits of extracellular matrix proteins, which occur as a marrow microenvironment reactive response to cytokines released from the clonal malignant myeloproliferation. The observation that mice exposed to high systemic levels of thrombopoietin (TPO) invariably developing myelofibrosis has allowed demonstration of the crucial role of transforming growth factor (TGF)-beta1 released by hematopoietic cells in the onset of myelofibrosis. The purpose of this study was to investigate whether TGF-beta1 inhibition could directly inhibit fibrosis development in a curative approach of this mice model. An adenovirus encoding for TGF-beta1 soluble receptor (TGF-beta-RII-Fc) was injected either shortly after transplantation (preventive) or 30 days post-transplantation (curative). Mice were transplanted with syngenic bone marrow cells transduced with a retrovirus encoding for murine TPO. All mice developed a myeloproliferative syndrome. TGF-beta-RII-Fc was detected in the blood of all treated mice, leading to a dramatic decrease in TGF-beta1 level. Histological analysis show that the two approaches (curative or preventive) were successful enough to inhibit bone marrow and spleen fibrosis development in this model. However, lethality of TPO overexpression was not decreased after treatment, indicating that in this mice model, myeloproliferation rather than fibrosis was probably responsible for the lethality induced by the disorder.

Monocyte/Macrophage Dysfunctions Do Not Impair the Promotion of Myelofibrosis by High Levels of Thrombopoietin

Several lines of evidence indicate that the megakaryocyte/platelet lineage is crucial in myelofibrosis induction. The demonstration that NOD/SCID mice with functionally deficient monocytes do not develop fibrotic changes when exposed to thrombopoietin (TPO) also suggests an important role for monocyte/macrophages. However, in this animal model, the development of myelofibrosis is dependent on the level of TPO. This study was conducted to investigate whether NOD/SCID mice exposed to high TPO levels mediated by a retroviral vector would be refractory to the development of bone marrow fibrosis. We show that TPO and TGF-beta1 in plasma from NOD/SCID and SCID mice engrafted with TPO-overexpressing hemopoietic cells reach levels similar to the ones reached in immunocompetent mice, and all animals develop a myeloproliferative disease associated with a dense myelofibrosis at 8 wk posttransplantation. Monocytes in NOD/SCID mice are functionally deficient to secrete cytokines such as IL-1alpha in response to stimuli, even under TPO expression. Surprisingly, the plasma of these mice displays high levels of IL-alpha, which was demonstrated to originate from platelets. Together, these data suggest that completely functional monocytes are not required to develop myelofibrosis and that platelets are able, under TPO stimulation, to synthesize inflammatory cytokines, which may be involved in the pathogenesis of myelofibrosis and osteosclerosis.