Number Of Megakaryocytes Research Articles

Palbociclib Prevents CDK4/6 Dependent-Myeloproliferation and Myelofibrosis

Introduction Primary and secondary myelofibrosis (MF) present the worst prognosis in the group of BCR-ABL-negative myeloproliferative neoplasms. Presently there is no really efficient therapy of myelofibrosis, except allogenic bone marrow transplantation. In PMF patients, the JAK inhibitors ruxolitinib and fedratinib do not significantly reduce the MF itself or the natural progression of the disease. This could be due to the absence of direct link between JAK2 signaling level and the development of MF. However, ruxolitinib only partially inhibits JAK2 signaling and a reactivation of JAK/STAT signaling through heterodimerization of JAK2 with other JAK may occur (Koppikar et al, Nature, 2012). Complete inhibition of JAK2 signaling would likely induce a profound anemia and thrombocytopenia and at long-term an aplastic anemia. It therefore remains of great interest to identify and inhibit specific downstream targets dysregulated by JAK2 activation that may be responsible of the MF to circumvent the absence of a specific JAK2V617F inhibitor. JAK2V617F mutation leads to the autonomous activation of homodimeric type 1 cytokine receptors and their downstream signaling that induces G1/S cell cycle transition by activating the cyclin-dependent kinases CDK4/6. Cdk6 loss in Jak2V617F KI mice delays the development of the MPN and increases mouse survival (Uras et al, Blood, 2019). We hypothesized that intensifying the JAK2V617F-mediated CDK4/6 increased activity could potentially accelerate the MF development, which is a late event in the Jak2V617F KI model. Our study aims to explore the role of CDK4/6 overactivation or inhibition in the disease and MF development. Methods Since p19INK4D is a cyclin dependent kinase inhibitor, which inhibits CDK4 and to a lesser extent CDK6 and which specifically regulate megakaryocyte (MK) cell cycle and the arrest of endomitosis, we established a pre-clinical MF model by crossing p19Ink4d knock-out (KO) mice with heterozygous inducible Jak2V617F KI mice to obtain p19Ink4d-/-/Jak2FLEXV617F/+SCL-Cre+/-mice called here KOKI mice. With this model, we studied the development of the myeloproliferative disorder. To prevent a MF development in this model, a CDK4/6 inhibitor Palbociclib was administered from 2 to 4 weeks post-Jak2V617F induction. Results In contrast to Jak2V617F (KI) mice that developed a severe MF, in both bone marrow and spleen, at 24 weeks post-Jak2V617F induction, all KOKI mice developed a grade I MF as early as 3 weeks after Jak2V617F induction that progressed to severe fibrosis at 8 weeks (grade III). KOKI mice were characterized by the rapid onset of an extramedullary hematopoiesis (EMH) and megakaryocytic hyperplasia in bone marrow and spleen. As MK are known to be a major source of TGF-β1 in the development of MF, we further focused on the MK lineage. At 1 week post-Jak2V617F induction, prior to MF development, the numbers of MK progenitors (MK-P) were markedly increased in the spleen of KOKI mice (11-fold) while only 3.9-fold increase was observed for KI mice as compared to WT littermates. A significant 2.6-fold increase in the number of MK in the spleen was only visible in KOKI mice, in accordance with splenomegaly and EMH. The analysis of mean ploidy level (>4N) revealed that it increased to 34.8N in KI, 43.7N in KO p19 and 59.4N in KOKI mice as compared to 19N in WT mice. Total TGF-β1 bone marrow levels were moderately elevated in KOKI mice, with a higher proportion of activated TGF-β1. Treatment of KOKI mice with a CDK4/6 inhibitor, Palbociclib, prevented the development of MF and markedly decreased the EMH, the abnormalities of the megakaryocyte differentiation including the ploidy level and the total and activated TGF-β1 levels. Conclusions Altogether, using a KOKI mouse model we show that the deregulation of CDK4/6 mainly in megakaryocytes is sufficient to induce a severe MF strongly suggesting that CDK4/6 inhibitors could be a very promising therapeutic issue for MF patients (Figure). Disclosure of Prior Presentation/Publication : The abstract will not have been presented and published between January 1, 2020 and December 5, 2020. Figure 1 Disclosures No relevant conflicts of interest to declare.

Co-Expression of JAK2-V617F and Calr-del52 In Vivo Enhances Myeloproliferative Phenotype in Mice and Does Not Influence Competitive Fitness of Hematopoietic Stem Cells

Myeloproliferative neoplasms (MPNs) are a group of hematopoietic stem cell disorders driven by mutations that constitutively activate physiologic signal transduction pathways essential for hematopoiesis. The majority of patients with classical MPNs harbor mutations within the Janus activated kinase 2 (JAK2), calreticulin (CALR), or thrombopoietin receptor (MPL) genes. The occurrence of driver mutations among patients is mutually exclusive but rare double positive cases have been reported. Employment of targeted sequencing methods for diagnostics revealed more double positive cases and reviewing published studies we estimate the CALR and JAK2 double positive MPN frequency to be about 0.5% in all MPNs and 2% in essential thrombocythemia. However, the mutual exclusivity of CALR and JAK2 mutations in double positive cases was confirmed at single cell level in few studies where clonogenic assays were performed with subsequent genotyping of colonies. In our MPN biobank of over 800 samples, we identified one case diagnosed with PMF, carrying both in JAK2 and CALR, with allelic burdens of 8% and 41%, respectively. Using a clonogenic assay, we confirmed mutual exclusivity of the mutations at CFU level confirming previous findings. Mutations can be mutually exclusive due to their synthetic lethal interaction. Such synthetic lethal interaction has been recently described in splicing factor mutated MDS, showing that SF3B1 and SRSF2 double mutant hematopoietic cells (HSC) have reduced fitness in vivo providing explanation why such patients are never observed. In this study, we tested the hypothesis that JAK2-V617F and CALR-del52 mutations are synthetic lethal if they occur in the same HSC. We have generated mice that co-expresses both JAK2-V617F and CALR-del52 mutations in hematopoietic lineages and analyzed their phenotype. First, we co-expressed JAK2-V617F and CALR-del52 on the Vav1-Cre backgound in which Cre recombinase activates the floxed transgenes in embryonic HSC. Double positive offspring were born at expected Mendelian frequency compared to single positive littermates, suggesting no signs of synthetic lethality in utero. The phenotype of the JAK2-V617F and CALR-del52 double positive mice was significantly more severe compared to single mutant mice. More specifically, double positive mice showed more pronounced splenomegaly, higher white blood cell, lymphocyte, granulocyte, monocyte, and platelet counts in peripheral blood. In the bone marrow, double positive mice had more prominent megakaryocyte dyspoiesis and altered myeloid to erythroid ratios, without evident myelofibrosis as observed in histological sections. This increase in megakaryocyte numbers was also confirmed by FACS. In addition, double positive mice had more obscured follicular architecture and more signs of enhanced extramedullary hematopoiesis in the spleen, and more pronounced megakaryocytic sequestration in the lungs when compared to the JAK2-V617F histology findings. These mice also had lower overall survival compared to the JAK2-V617F and CALR-del52 mice. Next, we performed competitive bone marrow transplantation (BMT) to examine HSC fitness in primary and secondary transplants. Wild type bone marrow (BM) derived from F1 hybrid CD45.1/CD45.2 mice was mixed with BM form either mice bearing single mutation or double mutations (CD45.2), and ingrafted into CD45.1 recipients. The changes in chimerism were followed in peripheral blood by FACS. Double positive BM engrafted recipients equally well as JAK2-V617F or CALR-del52 cells suggesting no functional defect at HSC level. Same results were seen also in secondary BMT. In summary, double positive mice have an enhanced MPN phenotype with lower overall survival compared to single positive JAK2-V617F and CALR-del52 animals. Our results suggest that the mutual exclusivity of MPN driver mutations JAK2-V617F and CALR-del52 is not due to synthetic lethality or loss of HSC fitness. It is possible that once the second mutation is acquired, JAK2-V617F and CALR-del52 double positive cells do not gain additional competitive advantage over single positive HSCs, and therefore, do not grow out into a significant population. Another reason why we do not observe JAK2-V617F and CALR-del52 double positive colonies in patients is the very low likelihood of such HSC arising. Our data shows that such MPN patients may be found and very likely will have more severe MPN. Disclosures No relevant conflicts of interest to declare.

Evaluation of Bone Marrow Fibrosis and Lymphocyte Subsets in Adult Patients with Primary Immune Thrombocytopenia and Correlation with Clinical Findings

Immune thrombocytopenia (ITP) is a common autoimmune disorder characterized by low platelet counts that can lead to bleeding. Diagnosis of ITP is generally made by review of peripheral smear and evaluation of history and examination of the patient. Bone marrow examination is not diagnostic in patients with ITP and is performed only in those with other hematologic abnormalities and in those who do not have an adequate response to treatment.Bone marrow examinations in ITP have been investigated in studies dating back several decades. Some investigators have found megakaryocyte numbers to be increased . Meanwhile,other study have shown reticulin levels among adult patients with primary ITP may be elevated with respect to the general adult population. Morphological changes in ITP MKs include cytoplasmic vacuolization, reduction of granules, and smoothing of plasma membrane associated with increased number of MKs in BM. However, fibrosis appears to be no correlation with disease progression and the correlation with clinical outcome remains equivocal at best.The current study aimed to understand the marrow levels of reticulin and/or collagen fibres among adult patients-the majority with clinical features of primary ITP-at the time of diagnosis and to evaluate any correlation between fibrosis and primary ITP specific clinical features. Results 1. Of the 282 newly diagnosedand chronic patients from October 2015 to November 2017 were enrolled into the study which 102 patients were newly diagnosed and 182 patients were chronic.Median age was 46 yr (range 18-85), with 108 males and 174 females. At diagnosis 229 (79%) patients were age 60 or younger and 62 (81.8%) were women. There were more females than males with no significant difference in age between genders. CD41+megakaryocyte numbers on bone marrow aspirates did not meet statistical significance, a trend that chronic group likely have more megakaryocyte numbers189(IQR 68-340) than newly diagnosed group143(IQR 70-309). The prevalence of a bone marrow grading greater than 0 among our sample of newly diagnosed and chronic ITP patients were 23.5% and 14.4%, but the difference was not statistically significant(P=0.063). 2. Comparing cases with BMF> 0 with those without (MF=0),the former showed increased bone marrow cellularity (P=0.022).In terms of changes in lymphocyte subsets, the percentage of CD19+B Lymphocyte and CD3-CD56+ NKLymphocyte in patients with bone marrow grading > 0 were obviously lower than those in bone marrow grading = 0[8 (4.1-12.4) vs 5.0 (3.6-8.8), P=0.008].The bone marrow fibrosis grade were inversely correlated with the percentage of CD3-CD56+ NK Lymphocyte(r=0.307, P=0.004). In a further analysis, a significant inverse correlation was present between the CD3-CD56+ NK Lymphocyte and bone marrow cellularity(r=0.432, P=0.008). 3. In newly diagnosed patients who had achieved R, the median (range) TTR was 5 days (6-14.5 days) in the bone marrow grading =0 group compared with 6 days (6-18 days) in bone marrow grading > 0 group (P =0.624). Notably, in chronic ITP patients ,MF> 0 cases more frequently required more therapies compared with bone marrow grading = 0 group 2 (range, 1-4) vs 4((range, 4-5) during the first year after diagnosis (P<0.01). twenty-seven (37.5%) of the newly diagnosed 72 patients were long-term responders. 4. Factors affecting response were assessed by univariate logistic regression, which indicated that CD3-CD56+NK Lymphocyte was independently associated with a higher response (P = 0.010; hazard ratio (HR)0.515, 95% CI 0.311-0.855). As for the factors affecting LTR, univariate logistic regression results in Table 4 showed that MF> 0 , CD19+B Lymphocyte and gender is a predictive factor for achieving LTR(0.009; hazard ratio (HR)0.789 , 95% CI 0.561-0.762). As a conclusion, BM fibrosis is not rare in ITP patients,its incidence is actually higher than expected, as stated in literature. In this study, there were differences between ITP-associated MF and without MF in terms of number of re-treatment ,LTR, lymphocyte subsets in ITP. However, the TTR were no differences between ITP-associated MF and without MF patients. Disclosures No relevant conflicts of interest to declare.

Modeling Acute Megakaryoblastic Leukemia of Down Syndrome Using Induced Pluripotent Stem Cells

Introduction Development of Acute megakaryoblastic leukemia in Down syndrome children (DS-AMKL) is a multi-step process. Acquired GATA1s mutation during fetal hematopoiesis is responsible of a transient myeloproliferative disorder (TMD) characterized by an accumulation of megakaryoblasts. Although most of TMD regress around birth, some TMD can progress from the initial GATA1s clone to AMKL through the acquisition of additional mutations, including in (i) the cohesin complex (i.e: SMC3), (ii) the JAK/STAT signaling pathway, such as MPL and (iii) the polycomb repressive complex 2 (EZH2). How these mutations cooperate to deregulate megakaryocyte (MK) differentiation and to induce a full-blown AMKL, along with the precise role of trisomy 21 (T21) during this transformation process remain unclear. Because modeling of DS-AMKL is particularly difficult in mice, we performed a step-wise introduction of GATA1s, a gain of function mutation of MPL (MPLW515K) and a heterozygous loss of function mutation in a cohesin (SMC3), separately or in combination, in T21 and isogenic disomic 21 (Dis21) human induced Pluripotent Stem Cells (iPSCs). Methods Trisomy 21 iPSCs were kindly provided by M. Weiss (Memphis, TN). CRISPR/Cas 9 genome editing of GATA1 or SMC3 allowed the generation of GATA1s T21, SMC3+/- T21 and GATA1s SMC3+/- T21 iPSC clones. CRISPR/Cas9-mediated knock-in of MPLW515K was performed in T21 GATA1s iPSCs. The subsequent T21 GATA1sMPLW515K/W515Kclones were selected as well as a revertant Dis21 GATA1sMPLW515K/W515Kclone. Finally, SMC3 insertion/deletion were obtained in isogenic T21 and Dis21 GATA1s MPLW515K/W515K SMC3+/-iPSCs clones. Hematopoietic differentiation was induced in 2D cultures in presence of a matrix and a cocktail of cytokines followed by a MK differentiation with SCF and TPO. MK differentiation was studied by clonogenic assays, flow cytometry, confocal microscopy and ultrastructural studies. Gene expression analyses were performed by RNA-seq on highly purified MK from all genotypes. Results GATA1s alone blocked MK maturation characterized by a persistent CD34 expression, an accumulation of abnormal large granules, a defect in the development of demarcation membranes (DMS), and a marked decrease in proplatelet formation. The typical GATA1s MK were large megakaryoblasts with numerous large granules and rare DMS. However, GATA1s alone had no effect on the clonogenic activity in CFU-MK assays and MK numbers. The introduction of the MPLW515K mutation did not modify this phenotype either in Dis21 or T21 GATA1s MK, but induced a complete TPO independence. SMC3+/- alone enhanced the MK maturation allowing the generation of a higher number of proplatelets-generating MK. Importantly, the combination of GATA1s and SMC3+/- mutations had a marked cooperative effect that worsened the MK maturation defect, led to the generation of abnormal megakaryoblasts with only a pre-DMS and resulted in enhanced proliferation and ploidization both in Dis21 and T21 iPSCs. Interestingly, the proliferation was markedly higher in T21 clones compared to Dis21 counterparts. RNA-seq and GSEA analyses showed that T21 GATA1s SMC3+/- mutant MK exhibited transcriptional signatures consistent with a dramatic decrease in the expression of maturation genes, including GATA1 target genes, while DNA replication gene markers were increased compared with GATA1s alone. T21 GATA1s MPLW515K/W515K SMC3+/- MK were enriched for AMKL signatures as compared to isogenic Dis21 GATA1sMPLW515K/W515K SMC3+/- MK. Ongoing ATAC-seq analyses will define the consequence of the different mutations on chromatin accessibility. Conclusion Using iPSC modeling, we analyzed in a human cell-context the consequences of the different combination of mutations associated with DS-AMKL that would be difficult to model using human primary cells. Our data demonstrate that GATA1s expression cooperates with SMC3+/- to enhance proliferation of megakaryoblasts from T21 iPSCs and isogenic Dis21 iPSCs hence reproducing the abnormalities observed in DS-AMKL. T21 is not directly involved in the MK differentiation defects but rather give a proliferative advantage supporting its role in leukemia development. Disclosures No relevant conflicts of interest to declare.

Ifnα Attenuates the Disease Phenotype and Extends Survival in Mouse Models of MPN

The curative potential of Type I interferons for patients suffering from Myeloproliferative Neoplasms (MPNs) has been reported and these are the only class of drugs that can lead to reduction of the mutant allelic burden in patients. However, modelling IFN treatment in mice has been challenging. Here, we report the use of murine pegylated IFNα (murine ropeginterferon-a, mRopeg) developed by PharmaEssentia (Taipei, Taiwan) to model IFN treatment in transgenic MPN mouse models. We started treating JAK2V617Ff/+;vavCre and control vavCre mice (n=6-8) with PBS or mRopeg (600 ng/mouse/week), by subcutaneous injections from the time they were 4 weeks old. The mice were bled every 2 weeks from the facial vein and the blood parameters were monitored. We observed significant normalization of platelet and WBC counts in Jak2-V617F fl/+ vavCre mice to wild type levels. No effect on hematocrit and hemoglobin level was observed in the Jak2-V617F fl/+ vavCre mice. VavCre control animals showed no sign of negative effect such as cytopenia during the entire treatment course. We observed a highly significant prolongation of the survival of mRopeg treated JAK2V617Ff/+;vavCre mice over a duration of 80 days of treatment. While all the PBS treated JAK2V617Ff/+;vavCre mice died within 60 days, all the mRopeg treated mice were still alive till the end of the treatment duration. We also generated a novel transgenic mouse model that conditionally expresses hybrid mutant CALR protein (murine exons 1-8 and human CALR del52 exon9) from the endogenous murine Calr locus. We bred them into vavCre background (in both heterozyhous and homozygous states) to induce expression of CALR-del52 in hematopoietic cells. Upon Cre recombinase expression, the endogenous murine exon 9 is replaced by the human del52 exon 9 and the expression of the humanized Calr-del52 oncoprotein is detectable by Western blot analysis using mutant CALR specific antibodies. Calr-del52 animals develop an essential thrombocythemia (ET) like phenotype when expressed in a heterozygous state with elevated number of hematopoietic stem cells and megakaryocytes in the bone marrow. In the homozygous state, the thrombocythemia is more severe with splenomegaly and older animals show anemia with increased WBC. Bone marrow histology shows megakaryocytic hyperplasia with no sign of fibrosis up to age of one year. We treated a cohort of animals with 600 ng mRopeg/PBS once a week for 4 weeks. Peripheral blood counts were determined at baseline and at regular intervals during treatment. At the end of treatment, mice were sacrificed, and splenic and bone marrow cells were immunophenotyped and quantified by FACS. We observed correction of thrombocythemia in the homozygous Calr-del52 mice but no unspecific decrease of platelet count in the vavCre mRopeg treated animals. We observed significant specific reduction of the long-term hematopoietic stem cells (LT-HSCs/fraction A) in homozygous CALR-del52 mice. In conclusion, Type I IFN treatment significantly reduces platelet counts to normal levels in both JAK2 and CALR mutant driven MPN mouse models. The prolongation of survival of JAK2V617F transgenic mice upon Type I IFN treatment is particularly remarkable; as no survival data is reported until now in any clinical trials or other animal models. Further experiments are required to understand the mechanism of action of this phenomenon. Disclosures No relevant conflicts of interest to declare.

Preclinical Rationale for the Use of Crizanlizumab (SEG101) in Myelofibrosis

Myelofibrosis (MF), the end stage of Philadelphia-negative Myeloproliferative Neoplasms, is still an unmet clinical need. Treatment with the JAK1/2 inhibitor ruxolitinib ameliorates symptoms and improves overall survival but it is unclear whether it is effective in improving the clinical progression of these patients. The bone marrow (BM) and spleen from MF patients contain great numbers of impaired megakaryocytes (MK) which have been hypothesized to be responsible for their clinical manifestation by expressing on their surface high levels of the adhesion receptor P-selectin which triggers a process of pathological neutrophil emperipolesis leading to increased bioavailability of the pro-fibrotic transforming growth factor-β (TGF-β) in the microenvironment1,2. We have peviously reported that ablation of P-selectin cures myelofibrosis in Gata1low mice, a bona fide model for MF3. Using these data as a foundation, we tested whether Crizanlizumab, an antibody targeting human P-selectin that effectively prevents pain crisis in patients with Sickle Cell Disease4, represents an effective option to treat MF. To this aim, we treated Gata1low mice with RB40.34, an antibody against murine P-selectin used in the pre-clinical study that supported the clinical trial with crizanlizumab5, alone or in combination with ruxolitinib. Twenty-four 11-months old Gata1low mice were microchipped, randomly separated in four groups (3 males and 3 females/group) and treated as follow: Group 1: vehicle; Group 2: biotinylated RB40.34 (30 μg/mouse/day per 3 days/week by tail vein injection); Group 3: ruxolitinib (45mg/Kg twice per day by gavage); Group 4: biotinylated RB40.34 and ruxolitinib in combination. Mice were treated for 2 cycles of 5 consecutive days with two days of rest in between and sacrificed on day 5 (I end-point) and 12 (II end-point). End-points: Day 5: all mice were weighed and bled for blood counts determinations and presence of labeled-RB34.40 on platelets. Males were sacrificed for signaling studies and determination of the presence of RB40.34 on MK from BM and spleen. Day 12: females were weighed, bled for blood counts determinations and sacrificed for histopathology analyses. None of the treatments induced changes in body weight that remained similar to those observed before treatment, suggesting that overall all the treatments were well tolerated. None of the treatments rescued the platelet deficiency of Gata1low mice, which remained thrombocytopenic, nor induced anemia by day 12, inspite they had been bleed 1 week earlier. This lack of anemia indicates that the treatments do not impair the ability of the mice to recover from erythroid stress. Atday 5, high levels of RB40.34 were observed on the platelets and on the MK of mice treated with the antibody, alone or with ruxolitinib. However, only the two drugs in combination significantly normalized the TGF-β signaling in BM by reducing the abnormally high content of TFGβRII and SMAD2/3 and the phosphorylation levels of ERK in this organ. These results indicate that when used in combination, the two drugs are effective in rescuing the abnormal TGF-β signaling of BM from these mice. Byday 12, high levels of RB40.34 were observed on BM and spleen of mice treated with the antibody alone or with ruxolitinib but again only treatment with the two drugs in combination greatly improved the cellularity of the BM medulla (Hematoxilin-eosin staining), which is typically reduced in Gata1low mice, and reduced the massive fibrosis (Gomory staining) and high TGF-β content (immunostaining) observed in the diaphysis area of the long bones (Fig. 1). Treatment with the two drugs in combination also reduced fibrosis and TGF-β content in spleen restoring the architecture of this organ which regained a normal appearance with well recognizable red and white pulp zones. When used alone, neither RB40.34 nor ruxolitinib had any effect of the MF phenotype of this mouse model. In conclusion, RB40.34 in combination with ruxolitinib rescues the MF phenotype of Gata1low mice with limited toxicity providing pre-clinical evidence that treatment with crizanlizumab in combination with ruxolitinib may be more effective than that with ruxolitinib alone to cure MF patients.

Tuberculosis with Immune Thrombocytopenia an Unusual Association

Introduction Tuberculosis (TB) and is the leading infectious cause of death in adult's worldwide.TB can have a wide variety of presentations manifestation. Hematological manifestations of tuberculosis have different forms and one of the rare associated is TB with isolated thrombocytopenia. ITP is relatively common autoimmune bleeding disorder.. However, ITP is very rarely associated with TB with only a few cases reported. Isolated Thrombocytopenia in TB is multifactorial and could be explained by different mechanisms as follows Immune-mediated platelet destruction through antiplatelet antibodies as mycobacterium TB may share antibodies with platelets or platelet-associated immunoglobulin, Tuberculosis-induced hemophagocytosis, Defective platelet production due to bone marrow (BM) infiltration, Hypersplenism, intravascular coagulation, anti-tuberculous treatment (ATT) induced thrombocytopenia. Methodology We reviewed the literature using PubMed, google scholar and English language articles only. We have included all the reported cases of tuberculosis and immune thrombocytopenia if it is associations or initial presentations using the keyword: tuberculosis. Pulmonary tuberculosis, immune thrombocytopenia, Idiopathic thrombocytopenia purpura and we excluded drug induced thrombocytopenia. Our review identified forty-two cases in total, in our review Baseline and clinical characteristics are provided. Results Of the forty-two cases identified, more than one quarter of cases (n=12) were from India; the remainder of the reported cases from thirteen different countries. Gender was nearly equally distributed (males n= 21, females n=20, one case unknown); age ranged from 4 to 74 years. The most reported sites of TB as follows; lung (n=20), disseminated (n=8), lymph nodes (n=6), abdomen (n=4), mediastinum (n=2), spleen (n=1) and knee (n=1). Bone marrow aspiration and biopsy done in 27 cases, three of them (7%) found granuloma and 14 (32%) found to have normal to hyprecellular marrow with increased number of megakaryocytes. Of the forty-two cases, ITP was the first presentation of TB with or without typical TB symptoms and signs in more than three quarters of the cases (n=33), while ITP was discovered in TB patients in routine investigations without obvious bleeding symptoms or signs in only four cases. Hemorrhagic symptoms were reported in most of the cases (n=31), skin manifestations (petechiae/purpura/ecchymosis) being the most common (n=28). The therapeutic approach to TB-associated ITP showed significant heterogeneity with Anti tuberculous treatment and first- and/or second-line treatment for ITP given with or without platelets transfusion. Most of the cases (n=36) achieved full recovery with Anti-tuberculous treatment(ATT) and IVIG ± Steroids. Of the 42 cases, six cases were treated with blood and platelet transfusion for thrombocytopenia which did not normalize the platelet count as thrombocytopenia as expected as this is most likely an immune mediated phenomenon. Conclusion Unfortunately, there are no clear guidelines for management of TB-associated ITP and is anecdotal or observational at best. This highlights the importance of conducting prospective and standardized studies in the future. We found that Anti tuberculous treatment could have play pivotal role in management of ITP in TB Patients. Figure Disclosures No relevant conflicts of interest to declare.

Refractory Thrombocytopenia in SLE with Evans Syndrome : A Case Report

Background: Evans syndrome (ES) is a rare autoimmune disorder of unknown etiology. It is characterized by the occurrence of two or more hematologic immune cytopenias, most often immune thrombocytopenia and autoimmune hemolytic anemia (AIHA). In this study we present a rare case of a 9-year-old girl, who was diagnosed with secondary ES associated with active systemic lupus erythematosus (SLE). Case presentation: On November 16th, 2018, a 9-year old girl, presented for the first time with recent onset of gum bleeding and decreased level of consciousness for two weeks. On examination, she was conscious but not oriented, unable to walk, showed scanty hair with oral and gum bleeding, and had multiple bruises and massive hepatosplenomegaly. No parental consanguinity, and no similar history in the family was noted. She had low platelet count (1×103 /μL), with high mean platelet volume (19 fl), anemia (HB 5.5 g/dL), with normal white blood cell count, positive direct Coombs test, and hemolysis with no malignant cells on a peripheral blood smear. Bone marrow evaluation showed hypercellularity, and increased number of megakaryocytes, with no other significant abnormalities. Her flowcytometry study was normal. Brain computed tomography (CT) revealed left subdural hematoma with elements of old brain atrophy (Photo 1), with significant dilatation of ventricular system. Chest X-ray showed mild right pleural effusion. Abdominal ultrasonography showed hepatosplenomegaly with no focal lesions, and mild-to-moderate ascites. Both antinuclear antibody and anti-phospholipid antibody tests showed positive results and C3 was low. Hepatitis and human immunodeficiency virus (HIV) serology were negative.Autoimmune lymphoproliferative syndrome gene sequence analysis was negative. The patient was diagnosed with SLE, after she fulfilled the American Rheumatology Association criteria, associated with Evans syndrome. Any planned surgery was postponed until thrombocytopenia correction, and their aim was to increase the platelet count to a level of above 100.The patient received packed red blood cells (RBCs) and platelets several times. To control her condition, several medications were tried. These medications included intravenous immunoglobulin (1 gm/kg) for two successive days, prednisolone (2 mg/kg/day); however, no improvement was observed after 10 days. Therefore, all of these medications were replaced with methylprednisolone pulse therapy (30 mg/kg for three days), with one dose of anti-D (75 mcg/kg IV). Our next choice was mycophenolate mofetil, which was stopped after finishing the course of five weeks, but the patient did not show any improvement. Moreover, platelets failed to respond to four doses of Rituximab (370 mg/m2/dose) for four weeks and to two cycles of high dose of dexamethasone 20mg/m2 for 4 days every 4 weeks. On 12/29/2018, chloroquine (4 mg/kg/day) was administered for two weeks in conjunction with cyclosporine (4 mg/kg/day). Splenectomy was an option but was not performed because her platelet levels never recovered. Her anemia started to improve on 1/9/2019 with combination of prednisolone and cyclosporine and Coombs test results were negative. On 1/23/2019, the patient was started on Eltrombopag, in a dose-escalated manner starting with 25 mg/kg/day up to 75 mg/kg/day in addition to corticosteroids. One week later, her platelet counts markedly improved (Photo 2). No major side effects were reported. On 2/12/2019, the patient underwent left side burr-hole evacuation of subdural hematoma, but her consciousness was never regained. She was maintained in intensive care unit and ventilated, but she developed multiple postoperative complication, including sepsis with high inflammatory markers and spiking fever. Her platelet count was continuously high, so the dose of Eltrombopag was decreased to 50 mg/kg/day to just keep the level between 50 and 100. Despite all of therapeutic interventions, she passed away one month later, on 3/13/2019, due to respiratory failure. Conclusion: The incidence of ES is quite rare, especially in children. Eltrombopag is a safe and effective drug for management of refractory thrombocytopenia in cases of ES associated with SLE. Figure Disclosures No relevant conflicts of interest to declare.

Combined Blockage of E-Selectin and CXCR4 (GMI-1359) Enhances Anti-Leukemia Effect of FLT3 Inhibition (Sorafenib) and Protects Hematopoiesis in Pre-Clinical AML Models

Acute myelogenous leukemia (AML) is characterized by an accumulation of abnormal white blood cells. Internal tandem duplications in the fms-like tyrosine kinase 3 (FLT3-ITD) account for 30% of adult AML cases and confer poor prognosis (Nakao et al., Leukemia 1996). FLT3 inhibitors like sorafenib efficiently eliminate circulating leukemia blasts, but frequently not in the bone marrow (BM), which suggests a protective effect of the BM niche for leukemic stem cell survival (Zhang et al., JNCI 2008). The homing of AML cells in BM is mediated chiefly by the adhesion to E-selectin on endothelial cells (ECs) and by CXCR4-directed cellular migration to stromal CXCL12 (SDF1) sources (Chien et al., Blood 2013; Peled and Tavor, Theranostics 2013). In many respects, BM homing signals are shared between leukemia and hematopoietic stem cells (HSCs). Our previous study demonstrated that targeting E-selectin/CXCR4 with the dual E-selectin/CXCR4 antagonist GMI-1359 markedly reduced leukemia cell adhesion to ECs and mesenchymal stem cells, reduced the BM-mediated protection of leukemic cells during FLT3-targeted therapy in vitro, and effectively reduced leukemia cellularity in the BM in vivo (Zhang et al., Can Res suppl 2016). Further, GMI-1359 combined with cytarabine/daunorubicin provided a profound survival benefit in mice with FLT3-mutated leukemia (Zhang et al., Blood suppl 2015). In the present study, we sought to evaluate dual E-selectin/CXCR4 blockage in the context of FLT3 inhibition by sorafenib in vivo, and to better understand the underlying mechanism. We compared expression levels of E-selectin ligands and CXCR4 in FLT3 inhibitor-sensitive Ba/F3-FLT3-ITD cells and their inhibitor-resistant counterparts Ba/F3-FLT3-ITD+D835Y and Ba/F3-FLT3-ITD+F691L. Resistant cells expressed 1.7 to ~5.6-fold higher levels of total E-selectin ligand detected by a soluble E-selectin reagent, and 10-fold higher levels of CXCR4. In addition, BM-mimetic hypoxia culture profoundly upregulated the cell surface expression of E-selectin ligands and CXCR4 on leukemia cells. We evaluated anti-leukemia effects of co-targeting E-selectin/CXCR4 and FLT3 with GMI-1359 and sorafenib in a patient-derived AML xenograft (PDX) model harboring FLT3-ITD and WT1 mutations. We observed that addition of GMI-1359 to sorafenib greatly reduced leukemia cellularity compared to sorafenib alone, and as much as by 92%, 82%, 69% and 45% in, respectively, liver, lung, spleen and BM (Fig. 1) as compared with vehicle-treated mice (p < 0.05). As expected, the number of circulating leukemia cells transiently increased. The GMI-1359/sorafenib combination improved mouse survival (median survival 138.5 versus 109, 87 and 126 days for the GMI-1359/sorafenib versus vehicle, GMI-1359 and sorafenib, respectively, p < 0.001). Using intravital 2-photon microscopy, we observed AML cell behavior in calvarial BM and their response to acute GMI-1359 bolus infusion. Remarkably, AML cell mobility began to increase in the BM microenvironment as soon as 20 min after treatment (Fig. 2), followed by intravasation and cellular outflow through the BM capillary vasculature over the next 2-4 hours. Moreover, although BM homing signals are thought to be shared between leukemia and HSCs, the combination therapy improved hematopoiesis parameters compared to sorafenib alone. In particular, this important effect was associated with increased numbers of megakaryocytes (2.1-fold), myelocytes (2.1-fold), and erythrocytes (7.1-fold) in BM (p < 0.01). The underlying mechanism(s) of hematopoiesis protection by GMI-1359 are under investigation. Conclusion: Co-inhibition of E-selectin/CXCR4 enhances the anti-leukemia efficacy of FLT3 inhibition and preserves hematopoiesis in the BM in a PDX model of AML. Disclosures Fogler: GlycoMimetics: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Magnani:GlycoMimetics, Inc.: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Zal:Daiichi-Sankyo: Research Funding; Moleculin Biotech, Inc.: Research Funding. Andreeff:Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy.

Rationale for and Results of a Phase I Study of the TGF-β 1/3 Inhibitor AVID200 in Subjects with Myelofibrosis: MPN-RC 118 Trial

Rationale for and Results of a Phase I Study of the TGF-β 1/3 Inhibitor AVID200 in Subjects with Myelofibrosis: MPN-RC 118 Trial

The BET Inhibitor, CPI-0610, Promotes Myeloid Differentiation in Myelofibrosis Patient Bone Marrow and Peripheral CD34+ Hematopoietic Stem Cells

The BET Inhibitor, CPI-0610, Promotes Myeloid Differentiation in Myelofibrosis Patient Bone Marrow and Peripheral CD34+ Hematopoietic Stem Cells

RGS10 and RGS18 differentially limit platelet activation, promote platelet production, and prolong platelet survival

AbstractG protein–coupled receptors are critical mediators of platelet activation whose signaling can be modulated by members of the regulator of G protein signaling (RGS) family. The 2 most abundant RGS proteins in human and mouse platelets are RGS10 and RGS18. While each has been studied individually, critical questions remain about the overall impact of this mode of regulation in platelets. Here, we report that mice missing both proteins show reduced platelet survival and a 40% decrease in platelet count that can be partially reversed with aspirin and a P2Y12 antagonist. Their platelets have increased basal (TREM)-like transcript-1 expression, a leftward shift in the dose/response for a thrombin receptor–activating peptide, an increased maximum response to adenosine 5′-diphosphate and TxA2, and a greatly exaggerated response to penetrating injuries in vivo. Neither of the individual knockouts displays this constellation of findings. RGS10−/− platelets have an enhanced response to agonists in vitro, but platelet count and survival are normal. RGS18−/− mice have a 15% reduction in platelet count that is not affected by antiplatelet agents, nearly normal responses to platelet agonists, and normal platelet survival. Megakaryocyte number and ploidy are normal in all 3 mouse lines, but platelet recovery from severe acute thrombocytopenia is slower in RGS18−/− and RGS10−/−18−/− mice. Collectively, these results show that RGS10 and RGS18 have complementary roles in platelets. Removing both at the same time discloses the extent to which this regulatory mechanism normally controls platelet reactivity in vivo, modulates the hemostatic response to injury, promotes platelet production, and prolongs platelet survival.

Platelet-derived microparticles enhance megakaryocyte differentiation and platelet generation via miR-1915-3p

Thrombosis leads to platelet activation and subsequent degradation; therefore, replenishment of platelets from hematopoietic stem/progenitor cells (HSPCs) is needed to maintain the physiological level of circulating platelets. Platelet-derived microparticles (PMPs) are protein- and RNA-containing vesicles released from activated platelets. We hypothesized that factors carried by PMPs might influence the production of platelets from HSPCs, in a positive feedback fashion. Here we show that, during mouse acute liver injury, the density of megakaryocyte in the bone marrow increases following an increase in circulating PMPs, but without thrombopoietin (TPO) upregulation. In vitro, PMPs are internalized by HSPCs and drive them toward a megakaryocytic fate. Mechanistically, miR-1915-3p, a miRNA highly enriched in PMPs, is transported to target cells and suppresses the expression levels of Rho GTPase family member B, thereby inducing megakaryopoiesis. In addition, direct injection of PMPs into irradiated mice increases the number of megakaryocytes and platelets without affecting TPO levels. In conclusion, our data reveal that PMPs have a role in promoting megakaryocytic differentiation and platelet production.

Increased number of pulmonary megakaryocytes in COVID-19 patients with diffuse alveolar damage: an autopsy study with clinical correlation and review of the literature.

Pulmonary megakaryocytes participate in the pathogenesis of lung damage, particularly in acute lung injury. Although megakaryocytes are not mentioned as a characteristic histologic finding associated to pulmonary injury, a few studies reveal that their number is increased in diffuse alveolar damage (DAD). In this autopsy study, we have observed a relevant number of pulmonary megakaryocytes in COVID-19 patients dying with acute lung injury (7.61 ± 5.59 megakaryocytes per 25 high-power fields vs. 1.14 ± 0.86 for the control group, p < 0.05). We analyzed samples of 18 patients, most of whom died after prolonged disease and use of mechanical ventilation. Most patients showed advanced DAD and abnormal coagulation parameters with high levels of fibrinogen, D-dimers, and variable thrombocytopenia. For comparison, pulmonary samples from a group of 14 non-COVID-19 patients dying with DAD were reviewed. They showed similar pulmonary histopathologic findings and an increase in the number of megakaryocytes (4 ± 4.17 vs. 1.14 ± 0.86 for the control group, p < 0.05). Megakaryocyte count in the COVID-19 group was greater but did not reach statistical significance (7.61 ± 5.59 vs. 4 ± 4.17, p = 0.063). Regardless of the cause, pulmonary megakaryocytes are increased in patients with DAD. Their high number seen in COVID-19 patients suggests a relation with the thrombotic events so often seen these patients. Since the lung is considered an active site of megakaryopoiesis, a prothrombotic status leading to platelet activation, aggregation and consumption may trigger a compensatory pulmonary response.Electronic supplementary materialThe online version of this article (10.1007/s00428-020-02926-1) contains supplementary material, which is available to authorized users.

Abstract 6038: Combined targeting of E-selectin/CXCR4 and FLT3 by GMI-1359 and sorafenib effectively reduces leukemia cell burden and protects normal hematopoiesis in a patient-derived AML xenograft model

Abstract Acute myelogenous leukemia (AML) is characterized by an accumulation of abnormal white blood cells. Internal tandem duplications in the Fms-like tyrosine kinase 3 (FLT3-ITD) account for 30% of adult AML cases and confer poor prognosis (Kottaridis et al., 2003; Thiede et al., 2002). FLT3 inhibitors like sorafenib efficiently eliminate leukemia blast in the peripheral blood (PB), but frequently not in the bone marrow (BM) (Zhang et al., 2008). This suggests a protective effect of the BM on leukemic stem cells, which is mediated by E-selectin and CXCL12 expression in endothelial cells (ECs) and mesenchymal stem cells (MSCs) in the BM vascular and endosteal niches (Horacek et al., 2013; Peled and Tavor, 2013). Our previous study demonstrated that targeting E-selectin/CXCR4 with the dual E-selectin/CXCR4 antagonist GMI-1359 markedly reduced leukemia cell adhesion to ECs and MSCs and eliminated the BM-mediated protection of leukemic cells during FLT3-targeted therapy in vitro, and effectively reduced leukemia cell in the BM in vivo (Zhang et al., 2016). Further, GMI-1359 combined with cytarabine/daunorubicin provided a profound survival benefit in FLT3-mutated leukemia cell MV4-11-bearing mice (Zhang et al., 2015). In the preset study, we compared expression levels of E-selectin ligands and CXCR4 in FLT3 inhibitor-sensitive Ba/F3-FLT3-ITD cells and their inhibitor-resistant counterparts Ba/F3-FLT3-ITD+D835Y and Ba/F3-FLT3-ITD+F691L. The resistant cells expressed 1.7 to ~5.6-fold higher E-selectin ligand and 10-fold higher CXCR4 levels. In addition, BM-mimetic hypoxia culture profoundly upregulated P-selectin glycoprotein ligand-1 (PSGL-1) and CXCR4 levels on leukemia cells. PSGL-1 is also a high-efficiency ligand for E-selectin. We then evaluated anti-leukemia effects of co-targeting E-selectin/CXCR4 and FLT3 with GMI-1359 and sorafenib in a patient-derived AML xenograft model (harboring FLT3-ITD and WT1 mutations). Leukemia cell engraftment was evaluated using FACS and immunohistochemistry by hCD45 staining. We observed that GMI-1359 efficiently mobilized leukemic cells into PB and in combination with sorafenib enhanced the anti-leukemia activity and significantly reduced leukemia cell infiltration by 92%, 82%, 69% and 45% in liver, lung, spleen and BM, respectively, as compared with vehicle-treated mice (p &amp;lt; 0.05). The GMI-1359/sorafenib combination improved mouse survival (median survival were 109, 87, 126 and 138.5 days for the vehicle, GMI-1359, sorafenib and combination groups, respectively). Importantly, we observed that the combination protected normal hematopoiesis by increasing the number of BM megakaryocytes, myelocytes, and erythrocytes. The underlying mechanism(s) for this effect is under investigation. Our findings suggest that co-targeting E-selectin/CXCR4/FLT3 exerts remarkable protection of normal hematopoiesis in addition to more efficiently reducing leukemia cells. Citation Format: Weiguo Zhang, Kyung Hee Chang, Mahesh Basyal, Yannan Jia, Lauren Ostermann, William E. Fogler, John L. Magnani, Michael Andreeff. Combined targeting of E-selectin/CXCR4 and FLT3 by GMI-1359 and sorafenib effectively reduces leukemia cell burden and protects normal hematopoiesis in a patient-derived AML xenograft model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6038.

Clinical Characteristics and Bone Marrow Histopathology Features in Essential Thrombocythaemia Patients with Different Gene Mutation in China

To investigate the clinical characteristics, laboratorial and bone marrow pathological features of primary thrombocytopenia (ET) patients with different mutations of CALR, JAK2 and MPL genes. The chinical data of 120 cases of ET in Jiangsu provincial people's hospital/ The First Affiliated Hospital of Nanjing Medical University from January 2015 to December 2017 were collected and analyzed, including 76 cases with JAK2 gene mutation, 40 cases with CALR gene mutation, 2 cases with MPL gene mutations, 2 cases without gene mutation. Among the ET patients, compared with the JAK2 gene mutation, CALR gene mutation showed statistically significant deareament of white blood cells and hemoglobin (P=0.001, P=0.01) and the male platelets in CALR group showed significant increament (P=0.04). Fourthermore, the average number of megakaryocytes and its cluster numbers in each hight power field of vision showed statistically significant decreament in CALR group as compared with JAK2 group (P=0.001, P=0.001), and thrombotic events in CALR group were signicantly lower than those in JAK2 group (7.5% vs 18.4%) (P=0.03). Mutations of CALR, JAK2 have different clinical characteristics and blood pathological changes of Chinese ET patients, and their clinical significance is worth to explore.

A Comparative Study of Bone Marrow Squash and Wedge Aspiration Smears

Bone marrow examination entails study of aspirate smears, touch imprints and trephine biopsy. Bone marrow aspirate smears can be prepared by the squash (crush) or wedge method. Both techniques have their own advantages and disadvantages. There is paucity of studies that have compared these smear types. This study was conducted over a period of one year. Two hundred and five bone marrow aspirates were included. Both squash and wedge smears were made. Blinded slide review was done. Bone marrow cellularity, megakaryocyte number, myeloid to erythroid (M:E) ratio, morphology and final diagnosis on each smear type was compared. Chi square test, t-test and Kappa were applied to study the agreement between the wedge and squash smears. Among the 205 patients studied, squash smears showed significant over estimation of cellularity and megakaryocyte number (p < 0.05). There was no significant difference (p > 0.05) in the M:E ratio and morphological scores. Most patients (188/205 [91.7%]) showed complete diagnostic concordance while 17 (8.3%) patients had discrepancy in diagnosis between the squash and wedge smears. In 8 (3.9%) of these, major discrepancies were seen while 9 (4.4%) patients had minor discrepancies. Bone marrow cellularity and megakaryocyte numbers were underestimated in wedge smears with no differences in M:E ratio or morphology. Acceptable agreement for diagnosis was seen for patients with most disorders. Major diagnostic discrepancies were seen in patients with lesions known to have focal distribution-lymphoma, myeloma and tuberculosis (granulomas). Both squash and wedge smears should be studied for bone marrow examination.

Morphological Differential Diagnosis of Primary Myelofibrosis and Essential Thrombocythemia with Computer Cluster Analysis of a Megakaryocytic Lineage in Myeloid Tissue

The DBSCAN clustering algorithm, an in-house method of unsupervised machine learning, was used to explore specific histotopographical features of the megakaryocytic lineage in the bone marrow biopsies of patients with JAK2- or CALR-mutated essential thrombocythemia and prefibrotic primary myelofibrosis. Ninety-five bone marrow biopsies of patients with essential thrombocythemia and primary myelofibrosis were investigated to assess the histotopography of megakaryocytes, specifically, the mean number of megakaryocytes in one cluster, as well as the mean number of clusters and megakaryocytes per 1 mm2 of section area. The logistic regression model was statistically significant: χ2 = 14.703, p = 0.023, Nagelkerke R2 = 19.6%. Analysis of the histotopographical features of megakaryocytes allowed correct differentiation between essential thrombocythemia and primary myelofibrosis in 71.6% cases. The differences in the histotopographical features of megakaryocytes in the bone marrow of with JAK2- or CALR-mutated essential thrombocythemia and primary myelofibrosis revealed by the DBSCAN clustering algorithm indicate that a relationship exists between the disease and the pattern of megakaryocytic lineage development that can be used to create a logistic regression model for differentiating these diseases.

Adhesion to fibronectin via α5β1 integrin supports expansion of the megakaryocyte lineage in primary myelofibrosis

AbstractExcessive accumulation of extracellular matrix (ECM) is a hallmark of bone marrow (BM) milieu in primary myelofibrosis (PMF). Because cells have the ability to adhere to the surrounding ECM through integrin receptors, we examined the hypothesis that an abnormal ECM-integrin receptor axis contributes to BM megakaryocytosis in JAK2V617F+ PMF. Secretion of ECM protein fibronectin (FN) by BM stromal cells from PMF patients correlates with fibrosis and disease severity. Here, we show that Vav1-hJAK2V617F transgenic mice (JAK2V617F+) have high BM FN content associated with megakaryocytosis and fibrosis. Further, megakaryocytes from JAK2V617F+ mice have increased cell surface expression of the α5 subunit of the α5β1 integrin, the major FN receptor in megakaryocytes, and augmented adhesion to FN compared with wild-type controls. Reducing adhesion to FN by an inhibitory antibody to the α5 subunit effectively reduces the percentage of CD41+ JAK2V617F+ megakaryocytes in vitro and in vivo. Corroborating our findings in mice, JAK2V617F+ megakaryocytes from patients showed elevated expression of α5 subunit, and a neutralizing antibody to α5 subunit reduced adhesion to FN and megakaryocyte number derived from CD34+ cells. Our findings reveal a previously unappreciated contribution of FN-α5β1 integrin to megakaryocytosis in JAK2V617F+ PMF.

Neoplastic fibrocytes play an essential role in bone marrow fibrosis in Jak2V617F-induced primary myelofibrosis mice

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) characterized by clonal myeloproliferation, progressive bone marrow (BM) fibrosis, splenomegaly, and anemia. BM fibrosis was previously thought to be a reactive phenomenon induced by mesenchymal stromal cells that are stimulated by the overproduction of cytokines such as transforming growth factor (TGF)-β1. However, the involvement of neoplastic fibrocytes in BM fibrosis was recently reported. In this study, we showed that the vast majority of collagen- and fibronectin-producing cells in the BM and spleens of Jak2V617F-induced myelofibrosis (MF) mice were fibrocytes derived from neoplastic hematopoietic cells. Neoplastic monocyte depletion eliminated collagen- and fibronectin-producing fibrocytes in BM and spleen, and ameliorated most characteristic MF features in Jak2V617F transgenic mice, including BM fibrosis, anemia, and splenomegaly, while had little effect on the elevated numbers of megakaryocytes and stem cells in BM, and leukothrombocytosis in peripheral blood. TGF-β1, which was produced by hematopoietic cells including fibrocytes, promoted the differentiation of neoplastic monocytes to fibrocytes, and elevated plasma TGF-β1 levels were normalized by monocyte depletion. Collectively, our data suggest that neoplastic fibrocytes are the major contributor to BM fibrosis in PMF, and TGF-β1 is required for their differentiation.