JAK2V617F-positive Myeloproliferative Neoplasms Research Articles

Decoding Endothelial MPL and JAK2V617F Mutation: Insight Into Cardiovascular Dysfunction in Myeloproliferative Neoplasms.

Patients with JAK2V617F-positive myeloproliferative neoplasms (MPNs) and clonal hematopoiesis of indeterminate potential face a significantly elevated risk of cardiovascular diseases. Endothelial cells carrying the JAK2V617F mutation have been detected in many patients with MPN. In this study, we investigated the molecular basis for the high incidence of cardiovascular complications in patients with MPN. We investigated the impact of endothelial JAK2V617F mutation on cardiovascular disease development using both transgenic murine models and MPN patient-derived induced pluripotent stem cell lines. Our investigations revealed that JAK2V617F mutant endothelial cells promote cardiovascular diseases under stress, which is associated with endothelial-to-mesenchymal transition and endothelial dysfunction. Importantly, we discovered that inhibiting the endothelial TPO (thrombopoietin) receptor MPL suppressed JAK2V617F-induced endothelial-to-mesenchymal transition and prevented cardiovascular dysfunction caused by mutant endothelial cells. Notably, the endothelial MPL receptor is not essential for the normal physiological regulation of blood cell counts and cardiac function. JAK2V617F mutant endothelial cells play a critical role in the development of cardiovascular diseases in JAK2V617F-positive MPNs, and endothelial MPL could be a promising therapeutic target for preventing or ameliorating cardiovascular complications in these patients.

Loss of Dnmt3a increases self-renewal and resistance to pegIFN-α in JAK2-V617F–positive myeloproliferative neoplasms

AbstractPegylated interferon alfa (pegIFN-α) can induce molecular remissions in patients with JAK2-V617F–positive myeloproliferative neoplasms (MPNs) by targeting long-term hematopoietic stem cells (LT-HSCs). Additional somatic mutations in genes regulating LT-HSC self-renewal, such as DNMT3A, have been reported to have poorer responses to pegIFN-α. We investigated whether DNMT3A loss leads to alterations in JAK2-V617F LT-HSC functions conferring resistance to pegIFN-α treatment in a mouse model of MPN and in hematopoietic progenitors from patients with MPN. Long-term treatment with pegIFN-α normalized blood parameters and reduced splenomegaly and JAK2-V617F chimerism in single-mutant JAK2-V617F (VF) mice. However, pegIFN-α in VF;Dnmt3aΔ/Δ (VF;DmΔ/Δ) mice worsened splenomegaly and failed to reduce JAK2-V617F chimerism. Furthermore, LT-HSCs from VF;DmΔ/Δ mice compared with VF were less prone to accumulate DNA damage and exit dormancy upon pegIFN-α treatment. RNA sequencing showed that IFN-α induced stronger upregulation of inflammatory pathways in LT-HSCs from VF;DmΔ/Δ than from VF mice, indicating that the resistance of VF;DmΔ/Δ LT-HSC was not due to failure in IFN-α signaling. Transplantations of bone marrow from pegIFN-α–treated VF;DmΔ/Δ mice gave rise to more aggressive disease in secondary and tertiary recipients. Liquid cultures of hematopoietic progenitors from patients with MPN with JAK2-V617F and DNMT3A mutation showed increased percentages of JAK2-V617F–positive colonies upon IFN-α exposure, whereas in patients with JAK2-V617F alone, the percentages of JAK2-V617F–positive colonies decreased or remained unchanged. PegIFN-α combined with 5-azacytidine only partially overcame resistance in VF;DmΔ/Δ mice. However, this combination strongly decreased the JAK2-mutant allele burden in mice carrying VF mutation only, showing potential to inflict substantial damage preferentially to the JAK2-mutant clone.

Exploiting Synthetic Lethality between Germline BRCA1 Haploinsufficiency and PARP Inhibition in JAK2V617F-Positive Myeloproliferative Neoplasms.

Myeloproliferative neoplasms (MPN) are rare hematologic disorders characterized by clonal hematopoiesis. Familial clustering is observed in a subset of cases, with a notable proportion exhibiting heterozygous germline mutations in DNA double-strand break repair genes (e.g., BRCA1). We investigated the therapeutic potential of targeting BRCA1 haploinsufficiency alongside the JAK2V617F driver mutation. We assessed the efficacy of combining the PARP inhibitor olaparib with interferon-alpha (IFNα) in CRISPR/Cas9-engineered Brca1+/- Jak2V617F-positive 32D cells. Olaparib treatment induced a higher number of DNA double-strand breaks, as demonstrated by γH2AX analysis through Western blot (p = 0.024), flow cytometry (p = 0.013), and confocal microscopy (p = 0.071). RAD51 foci formation was impaired in Brca1+/- cells compared to Brca1+/+ cells, indicating impaired homologous recombination repair due to Brca1 haploinsufficiency. Importantly, olaparib enhanced apoptosis while diminishing cell proliferation and viability in Brca1+/- cells compared to Brca1+/+ cells. These effects were further potentiated by IFNα. Olaparib induced interferon-stimulated genes and increased endogenous production of IFNα in Brca1+/- cells. These responses were abrogated by STING inhibition. In conclusion, our findings suggest that the combination of olaparib and IFNα presents a promising therapeutic strategy for MPN patients by exploiting the synthetic lethality between germline BRCA1 mutations and the JAK2V617F MPN driver mutation.

Unlocking the Role of Endothelial MPL Receptor and JAK2V617F Mutation: Insights into Cardiovascular Dysfunction in MPNs and Chip

Introduction Patients with JAK2V617F-positive myeloproliferative neoplasms (MPNs) and clonal hematopoiesis of indeterminate potential (CHIP) are at a significantly higher risk of cardiovascular diseases (CVDs). Vascular endothelial cells (ECs) carrying the JAK2V617F mutation can be detected in many MPN patients. The thrombopoietin receptor MPL is expressed on vascular ECs, though its role in endothelial function is unclear. In this study, we investigated the mechanisms through which the JAK2V617F mutation induces endothelial dysfunction to accelerate CVDs, shedding light on endothelial MPL as a potential therapeutic target for preventing or mitigating cardiovascular complications in JAK2V617F-positive MPNs. Methods JAK2V617F Flip-Flop mice (FF1; provided by Dr. Radek Skoda) were crossed with VEcadherin-creERT2 mice (CDH5) to generate CDH5 +/-FF1 +/- mice, which can be induced by tamoxifen to express human JAK2V617F specifically in vascular ECs but not in blood cells. MPL fl mice (provided by Dr. Warren Alexander) were crossed with CDH5 +FF1 + mice to generate CDH5 +FF1 +MPL fl/fl mice. To validate findings in transgenic murine models in human cells, we utilized a pair of isogenic induced pluripotent stem (iPS) cell lines derived from the same MPN patient, one carrying the JAK2V617F mutation (PVB1.4) and the other being JAK2 wild-type (PVB1.11). These iPS cell lines were differentiated into human ECs using a modified iPS differentiation protocol. Results CDH5 +FF1 + mice exhibited mild thrombocytosis but no cardiac dysfunction. However, when challenged with a high-fat diet, these mice developed dilated cardiomyopathy with significant decreases in LV ejection fraction and increases in LV end-diastolic and end-systolic volumes. Pathological evaluation revealed the presence of thromboses in segment pulmonary arteries and scattered coronary capillaries. There was intimal thickening, smooth muscle cell thickening, and perivascular fibrosis of scattered coronary arterioles in CDH5 +FF1 + mice compared to control mice. These findings, along with impaired cardiac EC function (measured by angiogenesis in vitro) and loss of EC markers, as well as significant enrichment of gene expression signatures associated with inflammatory response and epithelial-mesenchymal transition in JAK2V617F mutant cardiac ECs, suggest that the JAK2V617F mutation induces endothelial-to-mesenchymal transition (EndMT) in cardiac ECs from the CDH5 +FF1 + mice. Similarly, JAK2 wild-type ECs derived from PVB1.11 iPS cell line maintained the expression of EC markers and the characteristic cobblestone endothelial morphology throughout serial passages in vitro; in contrast, JAK2V617F mutant ECs derived from PVB1.4 exhibited a gradual loss of EC marker expression, decreased EC function, and underwent a phenotypical transition toward a fibroblastoid morphology, providing compelling evidence of EndMT induction by the JAK2V617F mutation in human ECs. We noticed that the thrombopoietin receptor MPL is abundantly expressed in the heart's vascular endothelium, compared to other tissues (e.g., lung, marrow, spleen). MPL expression is upregulated in JAK2V617F mutant ECs compared to wild-type ECs, and Inhibiting JAK-STAT signaling using Ruxolitinib abolished this MPL upregulation. To investigate the role of endothelial TPO/MPL signaling in JAK2V617F-induced cardiovascular dysfunction, we generated CDH5 +FF1 +MPL fl/fl mice to knock down MPL in JAK2V617F-bearing vascular ECs. The CDH5 +FF1 +MPL fl/fl mice maintained normal blood counts and cardiac function even after a high-fat diet challenge. In JAK2V617F mutant human ECs derived from PVB1.4 iPS cell line, treatment with AMM2, an anti-MPL neutralizing antibody, preserved EC markers and endothelial morphology compared to untreated mutant ECs. These results demonstrated that inhibiting the endothelial MPL can suppress JAK2V617F-induced EndMT and prevent cardiovascular dysfunction caused by mutant ECs. Notably, the endothelial MPL receptor is not indispensable for the normal physiological regulation of blood cell counts and cardiac function. Conclusion Our study provides valuable insights into the increased risk of CVDs observed in patients with JAK2V617F-positive CHIP and MPNs. Further investigations into the role of EndMT in other CHIP-associated mutations may shed light on the diverse CVD risks associated with different CHIP mutations.

Proteomic Profiling and Cytokine Analysis Reveals Molecular Signatures and Inflammatory Factors Associated with Thrombosis in JAK2-V617F Positive Myeloproliferative Neoplasm (MPN) Cohorts

Background: Myeloproliferative neoplasms (MPNs), a hematological malignancy commonly associated with the JAK2-V617F mutation, exhibit a considerable burden of comorbidities resulting from inflammation-mediated processes. The JAK2-V617F malignant clones instigate systemic inflammation by upregulating various pro-inflammatory cytokines. This leads to dysregulated immune and inflammatory responses, thereby driving a process called thrombo-inflammation. Neutrophils play a crucial role as inflammatory mediators and effectors of pro-thrombotic activity in MPNs. We, therefore, hypothesize that perturbations in the neutrophil proteome may alter the molecular communication networks within neutrophils, thereby potentially contributing to the pathological thrombus formation in veins and arteries. Aim: The study was aimed to elucidate (i) the pathological significance of dysregulated protein expression profile of neutrophils carrying the JAK2-V617F mutation, and (ii) the impact of pro-inflammatory cytokines on neutrophils during thrombo-inflammation. Methods: In this study, which was conducted at the University Hospital Magdeburg, Germany, 11 patients carrying the JAK2-V617F mutation (6 with thrombosis; 5 without thrombosis) and 9 healthy volunteers were enrolled. Neutrophils were isolated from freshly collected whole blood using a negative selection kit. The isolated neutrophils were then lysed, and protein extraction was performed. High-resolution liquid chromatography - mass spectrometry (LC-MS/MS) was employed to investigate the protein inventory of neutrophils. Furthermore, platelet-poor plasma was isolated from the same cohort to analyse plasma cytokine/chemokine levels using a bead-based multiplex assay. Results: LC-MS/MS identified 2,006 proteins in neutrophils, which were then used for quantitative comparative analysis. Amongst these, 28 and 26 proteins exhibited significant up-regulation in the JAK2-V617F positive patients ‘with thrombosis’ and ‘without thrombosis’, respectively. Notably, ATP2A3 and ROCK1 were significantly upregulated in the JAK2-V617F thrombotic group, ranking among the top 10-upregulated proteins. Of note, ROCK1 activation has previously been implicated in cell proliferation, survival and cell cycle control. Functional annotation analysis using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that upregulated proteins in the JAK2-V617F cohorts were significantly enriched for proteins involved in the calcium and TGF-ß signaling pathways. Furthermore, we assessed the levels of 60 cytokines/chemokines and soluble adhesion molecules in the same cohort, wherein significantly increased levels of ALCAM, ICAM-2 and VCAM-1 were observed in the JAK2-V617F thrombotic group. These molecules are known to play a critical role in vasculature-related inflammation and endothelial cell activation. Additionally, the JAK2-V617F thrombotic group exhibited significant upregulation of free active TGF-ß and pro-inflammatory cytokines such as IL-6 and IL-8 compared to both healthy subjects and JAK2-V617F non-thrombotic patients, indicating their contribution to the thrombo-inflammatory response. Moreover, the thrombotic cohort displayed elevated plasma levels of CXC chemokines and myeloperoxidase (MPO), a key enzyme linked to NETosis for neutrophil extracellular trap (NET) formation. Conclusions: The proteomic profile of neutrophils in MPN patients exhibit significant alterations, particularly between JAK2-V617F patients with and without thrombosis. The observed elevated expression of markers related to NETosis and calcium signaling pathways point towards the underlying mechanisms that could potentially contribute to the differential behavior of neutrophils, thereby facilitating the development of a thrombotic state in MPN patients.

ITGB3 Promotes the Progression of JAK2V617F-Positive Myeloproliferative Neoplams By Regulating Calreticulin to Inhibit Cellular Autophagy

O bjectives: Integrins are integral cell-surface proteins composed of an alpha chain and a beta chain, of which integrin beta 3 (ITGB3) was known to participate in cell adhesion as well as cell-surface mediated signaling and plays vital roles in different tumors. However, the role of ITGB3 in JAK2V617F-positive classical myeloproliferative neoplasms (cMPNs) remians unclear, and the aim of this study was to explore the effect of ITGB3 on cellular autophagy in JAK2V617F-positive cMPN cell line and its underlying mechanisms. Methods: ITGB3 expression in bone marrows from JAK2V617F-positive cMPN patients compared to normal controls were detected by quantitative reverse-transcription PCR (qPCR) and further validated by western blotting. Cell models of cMPNs were constructed using human HEL cell lines carrying JAK2V617F mutation and cytokine-dependent wild type BaF3 cells (Jak2 V617F BaF3 cells) with ectopic expression of Jak2 V617F seperately to perform phenotype and mechanism investigation. The putative targets of STAT5a on the promoters of ITGB3 were disclosed by public databases and a dual-luciferase reporter assay and chromatin immunoprecipitation assay (ChIP) was used to analyze the binding of STAT5a to ITGB3 promoter region. To observe the role of ITGB3 played in cMPN progression, stable-transfected HEL cell line with ITGB3 knockdown or overexpression were established by lentivirus packaging and cell transfection. Cell phenotype including cell proliferation, apoptosis and cell cycle distribution were detected by soft agar colony formation assay, BrdU staining, flow cytometry and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays. The variations of autophagic flux after ITGB3 dysregulation in HEL cells was analyzed using mRFP-GFP-LC3 adenovirus transfection and fluorescence microscopy, and LC3-II/I ratio as well as autophagy-related protein were detected by western blotting. Transmission electron microscopy was used to observe the formation of intracellular autophagosomes and autophagic lysosomes. RNA sequencing and GSEA analysis were applied to predict the downstream pathway and putative targets of ITGB3. Western blotting, immunofluorescence and qPCR were performed to detect the transcription and expression levels of genes. Results: 1.Western blotting and qRT-PCR detection showed increased expression of ITGB3 in bone marrow of JAK2V617F-positive cMPN patients compared to normal controls (A-B). Also, the evaluation of ITGB3 in a variety of myeloid tumor cell lines (HEL, HL60, K562, KG-1a) and wild-type BaF3 cells as well as Jak2 V617F BaF3 cells revealed that ITGB3 protein level is the highest in HEL (which harboring the JAK2V617F) and Jak2 V617F BaF3 cells (C-F). 2. Dual-luciferase reporter assay showed that STAT5a significantly induced the transcription activities of promoters of ITGB3 and inhibition of STAT5a reduced the transcription activities of promoters of ITGB3 (G-H). ITGB3 knockdown (I) in HEL cells reduced the proliferation capacity (J-L), promoted early apoptosis (M-O) and caused cell cycle arrest (P-Q). While ITGB3 overexpression in HEL cells (R) reduced the cell cycle arrest (S) and apoptosis (T-U). 3. Autophagic flux analysis showed that ITGB3 knockdown promoted the autophagic flux in HEL cells (V-X). Inhibition of ITGB3 increases the synthesis and degradation of autophagosomes and accelerates the process of cellular autophagy (Y-Z). Further identification of DEGs in ITGB3-dysregulated HEL cells (AA-AB) reveals that ITGB3 inhibits the expression of calreticulin (AC-AE), and ITGB3 might be involved in the regulation of autophagy in HEL cells through the regulation of calreticulin. Conclusions: 1. ITGB3 is significantly activated in HEL cell lines carrying the JAK2V617F mutation. 2. ITGB3 protein acts as a pro-oncogene in JAK2V617F-positive myeloproliferative neoplams progression by regulating calreticulin to inhibit autophagy, which deserves more in-depth study in JAK2V617F mutation-positive cMPNs.

Increase in Total Protein Level of Bruton's Tyrosine Kinase in Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), are characterized by atherosclerosis and cardiovascular risk. The mechanisms that connect MPNs with atherothrombosis remain elusive. It is known that atherosclerotic plaque causes Glycoprotein VI (GPVI)-dependent platelet activation and hence activation of bruton kinase (Btk) and that Btk inhibitors in vivo and ex vivo suppress GPVI-mediated paltelet activation. Aim of this study is to measure the levels of GPVI and Btk. In addition, we measured the CXCL12 as marker of atherothrombosis. We studied 25 JAK2V617F positive MPNs patients (WHO criteria) (mean age 58 years) including PV 12, ET 8, and PMF 5. We measured platelet count by automated analyzer, GPVI by flow cytometry, total Btk (T-Btk) protein by immunoblot and total CXCL12 (T-CXCL12) protein by ELISA. All patients had thrombocytosis (630±40x109/L) and allele burden ≥ 50%. We found high GPVI expression (mean fluorescence intensity (MFI) 20±5.8) and high T-Btk (mean value 1.0) as well as high T-CXCL12 (15±2 μg/ml). A positive correlation there was between GPVI and Btk. We further investigated Btk mRNA tran script levels using RT-PCR assay in triplicate from each samples and found mean values ≥ 1.0. On the basis of these results the use of GPVI- and Btk- inhibiting agents as atherothrombosis-focused antiplatelet drugs is conceivable. Therefore, clinical trials are eagerly awaited.

PPIL2 Is a Target of the JAK2-STAT5 Pathway and Mediates p53 Polyubiquitination and Degradation

The activated JAK2-STAT pathway is characteristic of myeloproliferative neoplasms (MPNs). We previously revealed that Plek2, a downstream target of the JAK2/STAT5 pathway, functions as a scaffold protein to activate Akt and plays an important role in the pathogenesis of JAK2 V617F-induced MPNs. To further explore the mechanisms of the Plek2 signalosome, we performed a comprehensive proteomic analysis of Plek2 interacting proteins and found peptidylprolyl isomerase-like 2 (PPIL2) as a potential novel effector of the signalosome. As a U-box E3 ubiquitin ligase, PPIL2 was reported to be involved in cancer metastasis. However, its role in normal and malignant hematopoiesis remains unknown. We found that PPIL2 is required for human and murine erythropoiesis in vitro. Knockout of PPIL2 through CRISPR/Cas9 significantly reduced cell proliferation and differentiation and led to marked apoptosis. Mechanistically, PPIL2 interacts with and catalyzes p53 polyubiquitination at lysine 24 to promote proteosome-mediated p53 degradation, thereby preventing p53-dependent cell cycle inhibition and apoptosis. We further revealed that PPIL2 is a downstream target of the JAK2/STAT5 pathway and is upregulated in a JAK2 V617F-positive MPN mouse model and in patients with MPNs. Loss of Ppil2 ameliorated JAK2 V617F-induced myeloproliferative phenotypes including erythrocytosis, neutrophilia, thrombocytosis, and splenomegaly in JAK2 V617F-knockin mice. The same findings were also observed when the JAK2 V617F-knockin mice were treated with cyclosporin A, an inhibitor of PPIL2. Our findings reveal PPIL2 as an effector of the JAK2/STAT5 pathway and Plek2 signalosome. PPIL2 plays an important role in normal erythropoiesis and the pathogenesis of JAK2 V617F-induced MPNs by degrading p53, pointing to PPIL2 as a potential therapeutic target for the treatment of MPNs.

Detection and quantification of JAK2V617F copy number by droplet digital PCR versus real-time PCR.

Myeloproliferative neoplasm (MPN) usually has an adverse prognosis, progressing to acute leukemia or splanchnic vein thromboses (SVTs). Therefore, early diagnosis and intervention are significantly important. Clinically, the burden of JAK2V617F is a vital diagnostic basis, which can be detected during the early stage of MPN. Thus, an accurate and rapid detective technique is urgently required. In recent years, real-time quantitative PCR (qPCR) has primarily been applied to detect the copies of JAK2V617F, whereas droplet digital PCR (ddPCR), a novel and promising detective tool, can conduct precise and repeatable quantification of nucleic acid copies without relying on the standard curve. In our study, both qPCR and ddPCR are used to evaluate the mutation burden of JAK2V617F in a series of gradient diluted standards and clinical JAK2V617F-positive MPN patients' bone marrow samples collected, while using next-generation sequencing technology (NGS) as a contrast. With the help of statistical methods, our study concluded that ddPCR had a better performance in accuracy, sensitivity, and stability, especially in a low burden. Regarding the accuracy, ddPCR showed a better linearity (Pearson R2 = 0.9926; P < 0.0001) than qPCR (Pearson R2 = 0.9772; P < 0.0001). What is more, ddPCR showed lower intra-assay and inter-assay CVs and the limit of detection (LOD) for the series of diluted standards than qPCR, demonstrating better stability and lower LOD. In a nutshell, ddPCR is a more promising technique for the detection and quantification of JAK2V617F.

OC 46.3 JAK2V617F-Positive Myeloproliferative Neoplasms are Associated with Impaired Fibrinolysis Both in Mice and Humans

OC 46.3 JAK2V617F-Positive Myeloproliferative Neoplasms are Associated with Impaired Fibrinolysis Both in Mice and Humans

Modulation of human thrombopoietin receptor conformations uncouples JAK2 V617F-driven activation from cytokine-induced stimulation

AbstractThe thrombopoietin receptor (TpoR) plays a central role in myeloproliferative neoplasms (MPNs). Mutations in JAK2, calreticulin, or TpoR itself drive the constitutive activation of TpoR and uncontrolled proliferation and differentiation of hematopoietic stem cells and progenitors. The JAK2 V617F mutation is responsible for most MPNs, and all driver mutants induce pathologic TpoR activation. Existing therapeutic strategies have focused on JAK2 kinase inhibitors that are unable to differentiate between the mutated MPN clone and healthy cells. Surprisingly, the targeting of TpoR itself has remained poorly explored despite its central role in pathology. Here, we performed a comprehensive characterization of human TpoR activation under physiological and pathological conditions, focusing on the JAK2 V617F mutant. Using a system of controlled dimerization of the transmembrane and cytosolic domains of TpoR, we discovered that human TpoR (hTpoR) adopts different dimeric conformations upon Tpo-induced vs JAK2 V617F–mediated activation. We identified the amino acids and specific dimeric conformation of hTpoR responsible for activation in complex with JAK2 V617F and confirmed our findings in the full-length receptor context in hematopoietic cell lines and primary bone marrow cells. Remarkably, we found that the modulation of hTpoR conformations by point mutations allowed for specific inhibition of JAK2 V617F–driven activation without affecting Tpo-induced signaling. Our results demonstrate that modulation of the hTpoR conformation is a viable therapeutic strategy for JAK2 V617F–positive MPNs and set the path for novel drug development by identifying precise residues of hTpoR involved in JAK2 V617F–specific activation.

Polycythemia Vera Associated with Diffuse Large B-Cell Lymphoma: A Case Report

Diffuse Large B-cell Lymphoma (DLBCL) is the most common type of non-Hodgkin’s lymphoma, accounting for more than 30% of cases, particularly in developing countries. Polycythemia Vera (PV) is a chronic myeloproliferative disorder characterized by an increase in the number of red blood cells, and often leukocytes and peripheral blood platelets. PV is most commonly diagnosed in individuals around the age of 60, and slightly more common in women, the clinical course is long and can lead to thrombotic complications, which may be the first symptom of the disease. In approximately 10% of patients, PV may progress to Myelodysplastic Syndrome (MDS) or Acute Myeloid Leukemia (AML). The co-occurrence of DLBCL with a JAK2V617F-positive Myeloproliferative Neoplasm (MPN) is rarely documented in the literature. Here we describe a 64-year-old patient diagnosed with both DIFFUSE LARGE B-CELL LYMPHOMA (DLBCL) and polycythemia, with a particularly short time between identification of these conditions.

Effect of hypoxia on HIF-1α and NOS3 expressions in CD34+ cells of JAK2V617F-positive myeloproliferative neoplasms

PurposeMyeloproliferative neoplasms (MPN) are a heterogeneous group of hematopoietic stem-cell diseases with excessive proliferation of one or more blood cell lines. In this study, we evaluated the effect of different oxygen concentrations on HIF-1α and NOS3 gene expression to determine the effect of the bone marrow microenvironment on JAK2V617F positive Philadelphia chromosome negative (Ph−) MPNs. Patients and methodsPeripheral blood mononuclear cells (MNC) of 12 patients with Ph− MPN were collected. The presence of JAK2V617F allele status was determined with allele-specific nested PCR analysis. MPN CD34+ and CD34depleted populations were isolated from MNC by magnetic beads. Separate cell cultures of CD34+/depleted populations were managed at different oxygen concentrations including anoxia (∼0%), hypoxia (∼3%), and normoxia (∼20%) conditions for 24 ​h. HIF-1α and NOS3 gene expression changes were examined in each population related to JAK2V617F status with real time RT-PCR. ResultIt was revealed that relative HIF-1α and NOS3 expressions were significantly increased in response to decreased oxygen concentration in all samples. Relative HIF-1α and NOS3 expressions were found to be higher especially in CD34+ and CD34depleted populations carrying JAK2V617F mutations compared to MPN patients carrying wild-type JAK2. ConclusionJAK2V617F might have specific role in HIF-1α and NOS3 regulations with respect to low oxygen concentrations in Ph− MPN. Further evaluations might reveal the effect of JAK2V617F on Ph− MPN pathogenesis in bone marrow microenvironment.

Exploration of the role of NKG2D ligands MICA and MICB in JAK2 V617F-positive myeloproliferative neoplasms.

JAK2 V617F-driven myeloproliferative neoplasms (MPNs) can escape immune surveillance through PD-L1 up-regulation and HLA class I pathway down-regulation. To complement these data we assessed the role of major histocompatibility complex class I-related genes (MICA and MICB) in JAK2 V617F+ MPNs. Using high resolution genotyping we identified two protective alleles, MICA*008:01 and MICA*016. MPN patients had significantly higher levels of soluble sMICA molecules. Peripheral blood JAK2 V617F+ granulocytes had higher surface expression of MICB but did not differ in the amount of MICA and MICB transcripts from normal granulocytes. MICA and MICB genes were significantly down-regulated in JAK2 V617F+ CD34+ cells from primary myelofibrosis patients in comparison to normal CD34+ hematopoietic stem cells. These data suggest minor but significant role of MICA and MICB genes in the pathogenesis of MPNs. It is also possible that MICA targeting approaches could be of clinical benefit for some of those patients.

Calreticulin Mutations in Myeloproliferative Neoplasms Patients Diagnosed in UKM Medical Centre

Introduction: Calreticulin (CALR) mutations are one of the molecular markers that has been incorporated for the diagnosis of myeloproliferative neoplasms (MPN) in the revised 2017 WHO Classification of Haematopoietic and Lymphoid Tumors. This study was performed to determine the prevalence of CALR mutations in patients with MPN diagnosed in UKMMC and to compare their demographics plus laboratory features with other MPN patients. Meth- ods: A total of 59 MPN patients who tested negative for JAK2V617Fmutation were selected and 21 MPN patients positive for JAK2V617F were included as controls. Screening for CALR exon 9 was done by multiplex polymerase chain reaction (PCR) followed by Sanger sequencing. Results: A total of six JAK2 V617F negative MPN samples were found to be positive for CALR mutations. Out of these six, three patients with CALR mutations were of type I mutation, two were type II while one was a mutation in the stretch III region. None of the twenty one JAK2 V617F positive MPN samples were positive for CALR mutation. Clinical phenotypes for those positive for CALR were restricted to Essential Thrombocythemia (ET), Primary Myelofibrosis (PMF) and one case of atypical Chronic Myeloid Leukaemia (CML). Conclusion: CALR mutations constituted 10.16% from the MPN patients who were negative for JAK2V617F mutation with no significant differences in platelet counts, hemoglobin (Hb), hematocrit and white cell counts as compared to MPN patients with JAK2 V617F mutations. Testing for CALR mutations among those who are negative for JAK2V617F within Malaysian population maybe worthwhile and require larger scale studies.

The relevance of HLA class II genes in JAK2 V617F-positive myeloproliferative neoplasms

In the present study we analyzed the relevance of HLA class II in JAK2 V617F-positive (JAK2 V617F+) myeloproliferative neoplasms (MPNs) focusing on genotype diversity, associations with specific alleles and haplotypes and the level of gene expression. One hundred and thirty-nine JAK2 V617F+ MPN patients and 1083 healthy controls, typed by Next generation sequencing (NGS) were included in the study. Multivariate generalized linear models with age as a covariate were applied for analysis of HLA-II allele and haplotype associations. Publicly available gene expression datasets were used to analyze HLA-II pathway genes expression in CD34+ stem cells (SCs) from MPN patients and healthy controls. We did not observe differences in HLA evolutionary divergence (HED) between JAK2 V617F+ MPNs and healthy controls. Two alleles: HLA-DPB1*03:01, DQB1*04:02 and 4 haplotypes: DPB1*02:01-DQA1*05:05-DQB1*03:01-DRB1*11:01, DPB1*04:02-DQA1*05:05-DQB1*03:01-DRB1*11:03, DPB1*02:01-DQA1*01:04-DQB1*05:03-DRB1*14:04, and DPB1*04:01-DQA1*03:01-DQB1*03:02-DRB1*04:01 had significantly lower frequency in MPN patients compared to controls. Additionally, we observed HLA-II alleles and haplotypes with statistically higher frequencies in JAK2 V617F+ patients. Differential gene expression analysis showed down-regulation of HLA-DRB1, -DRA, -DMA, -DMB, -DOA,-DRB4, CIITA, and CD74 genes in JAK2 V617F+ MPN CD34+ SCs as compared to normal CD34 + SCs. In conclusion, this study provides evidence for the pleiotropic effects of HLA-II genes in JAK2 V617F-driven MPNs.

Effects of Ruxolitinib on Myeloproliferative Neoplasms via the Negative Regulators.

We evaluated the JAK2V617F mutation and p-JAK2, SOCS-1, SHP-1 expression in JAK2V617F positive myeloproliferative neoplasms (MPNs) patients and the role of JAK/STAT pathway in human erythroleukemia (HEL) cells, which had JAK2V617F mutation. Protein expression of p-JAK2, SOCS-1, SHP-1 in bone marrow biopsies (BMBs) were detected by immunohistochemical staining methods. Cell apoptosis and cell cycle were detected by flow cytometry and Caspase 3/7 assay kits. 1. The p-JAK2, SOCS-1, and SHP-1 expressions were significantly different between JAK2V617F positive MPN and control patients (p < 0.01); 2. After being treated for 3 months, the p-JAK2, SOCS-1, and SHP-1 expressions were significantly different compared with newly diagnosed patients (p < 0.01). 3. HEL cell viabilities were significantly different after being treated with different concentrations of ruxolitinib. Ruxolitinib had a significant effect on the cell apoptosis, viability, and the protein activity of caspase-3 and -7 of HEL cells. 3. The mRNA and protein expressions of JAK2 and the protein expression of p-JAK2 were gradually decreased (p ＜ 0.01, p ＜ 0.05), while the mRNA and protein expressions of SOCS1 and SHP1 were gradually increased (all p ＜ 0.01).

The Thrombopoietin Receptor, MPL, Is Critical for the Development of JAK2V617F-Induced Cardiovascular Disease in a Murine Model of Myeloproliferative Neoplasm

Introduction Vascular endothelial cells (ECs) carrying the JAK2V617F mutation can be detected in many patients with myeloproliferative neoplasms (MPNs). Previously we reported that a JAK2V617F-positive murine model of MPN, in which the mutation is expressed in both blood cells and ECs, developed spontaneous heart failure with a thrombosis and vasculopathy phenotype, and mutant ECs were required to develop this phenotype. In this study, we tested the hypothesis that the JAK2V617F mutation induces endothelial dysfunction to promote cardiovascular diseases (CVDs), and the TPO/MPL signaling plays a critical role in JAK2V617F-induced CVDs. Methods JAK2V617F Flip-Flop (FF1) mice (from Radek Skoda, Switzerland) and Cdh5-CreERT2 mice were crossed to generate Cdh5-CreERT2+/-; FF1+/- mice, in which human JAK2V617F is expressed in vascular ECs after tamoxifen induction (ECJAK2V617F). The ECJAK2V617F mice were further crossed with MPLfl/fl mice (from Warren Alexander, Australia) to knock down the MPL protein in JAK2V617F-bearing ECs (Cdh5-CreERT2+/-; FF1+/-; Mpl fl/fl, or ECJAK2V617F;MPL-/-). Results As determined by RT-PCR, human JAK2V617F is expressed in vascular ECs (CD45-CD31+) from the ECJAK2V617Fmice after tamoxifen induction; no JAK2V617F expression is detected in blood cells from these mice. At 16wk of age, mild thrombocytosis (platelet 689 vs. 478 x 109/L, p=0.0003) and erythrocytosis (red blood cell 11 vs. 10 x 109/L, p=0.017) were observed in ECJAK2V617F mice compared to age-matched wild-type control mice. Serial transthoracic echocardiography did not reveal any cardiac dysfunction in the ECJAK2V617F mice during a 9-month follow-up, suggesting that mutant EC alone is not sufficient to develop the CVD. To test whether JAK2V617F mutant ECs promote the development of CVD in response to stressors, both ECJAK2V617F mice and control mice (with JAK2 wild-type ECs) were fed a high-fat/high-cholesterol diet. After 6-12wks of diet treatment, there were significant decreases in left ventricular ejection fraction and increases in left ventricular end-diastolic and end-systolic volumes in the ECJAK2V617F mice compared to control mice, indicative of cardiac dysfunction. Pathological evaluation revealed (1) increased heart weight; (2) presence of thrombosis in segment pulmonary arteries and scattered coronary capillaries; (3) evidence of intimal thickening, smooth muscle cell thickening, and perivascular fibrosis of scattered coronary arterioles; and (4) enlarged cardiomyocytes and increased reticulin fibers in the ECJAK2V617F mice after high-fat diet challenge. No atherosclerotic lesions were observed in the ECJAK2V617F mice. The TPO receptor MPL is expressed on several types of vascular ECs and TPO/MPL signaling can affect cardiovascular function. We found that MPL expression was upregulated in JAK2V617F mutant ECs compared to wild-type ECs, and this upregulation was completely blunted by Ruxolitinib, a clinically available JAK2 inhibitor. These observations prompted us to test whether deregulated TPO/MPL signaling contributes to the increased risks of CVDs in JAK2V617F-positive MPNs. First, we crossed the Cdh5-CreERT2 mice with the MPLfl mice to knock down the MPL protein in the vascular ECs (ECMPL-/-). The ECMPL-/- mice maintained normal blood cell counts and normal cardiac function (on serial echocardiography measurements) during a 6-mo follow-up. These results suggest that endothelial MPL receptor is not required to maintain normal blood count or cardiac function. Next, we crossed the ECJAK2V617Fmice with the MPLfl mice to knock down the MPL protein in JAK2V617F-bearing vascular ECs (ECJAK2V617F;MPL-/-). Serial echocardiography revealed normal cardiac function in the ECJAK2V617F;MPL-/- mice even after a high-fat diet challenge. Histology examination did not reveal any thrombosis or vasculopathy in these mice. Last, we found that JAK2V617F mutant ECs had increased senescence and decreased VEGFR2 and VE-cadherin expression compared to wild-type ECs after the high-fat diet challenge, which was rescued by MPL knockdown in the mutant ECs. Conclusions Our findings indicate that the JAK2V617F mutation induces endothelial dysfunction to promote CVDs, especially when challenged with additional stressors (e.g., high-fat diet). We also showed that inhibiting endothelial TPO/MPL signaling can prevent the development of CVDs in JAK2V617F-positive MPNs.

Ropegiterferon Alfa-2b-Njft for Treatment Myeloproliferative Neoplasms: A Single Institution Experience

Background: While interferon alfa has been used for decades as a cytoreductive agent in myeloproliferative neoplasms (MPNs), tolerability and need for frequent parenteral administration have limited its widespread adoption. Ropeginterferon Alfa-2b-njft (ropeg) is monopegylated extended half-life interferon alpha product that received FDA approval for treatment of polycythemia vera (PV) in November 2021. A phase III clinical trial has demonstrated efficacy and tolerability of this novel agent as compared to hydroxyurea (HU) in PV patients with indication for cytoreductive therapy. Real-world experience (RWE) assessment of a newly available pharmaceutical can yield important insights regarding barriers to access, clinical feasibility, and usage in populations not yet evaluated in clinical trial. In this single institution cohort, we report the first RWE using ropeg for patients with MPNs, including essential thrombocythemia (ET), in the United States. Methods: Patients presenting to an MPN clinic at an academic medical center were evaluated for initiation of ropeg based individualized assessment, with patients engaged in shared decision-making regarding risks and benefits. Patients were screened for autoimmune, neuropsychiatric, or endocrine disorder on basis of history and limited laboratory investigation as appropriate, including thyroid stimulating hormone, triglycerides, and anti-nuclear antibody testing. The treating physician and pharmacist provided pre-treatment counseling including review of potential toxicities, administration teaching, and financial considerations. Patients on HU were started on a dose 50mcg every 2 weeks via subcutaneous injection with subsequent HU tapering; patients not on HU were started 100mcg every 2 weeks. Ropeg dosing was increased to target hematologic response. Complete blood counts and comprehensive metabolic panels were ordered every 2 weeks until hematologic response achieved, with less frequent monitoring thereafter. Results: Between December 2021 to June 2022, 13 patients with JAK2 V617F positive MPNs have been started on ropeg. Four patients were unable to start ropeg due to payor denial of coverage and ineligibility for manufacturer patient assistance programs. Patients were predominantly female (10/13, 77%) with average age of 60.9 years (range 36-73 years). Seven patients had PV (transformed from ET in 2 cases), 5 patients had ET, and 1 patient had post-ET myelofibrosis. Mean pre-treatment hematocrit, platelet count, and WBC were 42.5% (range 34.3% - 51.5%), 630 *109/L (range 127-2065*109/L), and 7.0*109/L (range 2.5 - 13*109/L) respectively. At treatment start, 7 patients were on HU (either alone or in combination with therapeutic phlebotomy (TP)), 1 was on TP alone, 1 was on ruxolitinib, 1 was on peginterferon alfa-2a, and 3 were on no cytoreductive treatment. Reasons for switching to ropeg included poor tolerance or efficacy of prior therapy (6/13, 46%), patient interest in potential disease modifying properties of interferon (6/13, 46%), and, in the case of patient on peginterferon alfa-2a, ease of administration (1/13, 7.6%). Patients received medication through insurance approval with copay assistance (11/13, 85%) or through the manufacturer patient assistance program (2/13, 15%). Average time to receipt of medication from prescription submission was 25 days. At mean follow-up time of 12.9 weeks, all patients remain on treatment. One patient required treatment interruption due to severe fatigue, two patients deferred scheduled doses due to cold-like symptoms. While efficacy data is immature, one patient with PV and 2 patients with ET obtained a hematologic response (hematocrit <45% without phlebotomy in 3 months and platelet count <450), with mean response time 6.9 weeks (range 5.7 -7.7). Two patients experienced rebound in hematocrit after HU stop and are requiring TP as bridging therapy. Conclusions: Medication access, patient counseling, and symptom and laboratory monitoring must be addressed in translating clinical trial findings to clinical practice with ongoing evaluation of drug efficacy and safety. In a single institution analysis, 13 patients with MPNs, including 5 patients with ET, were started on ropeg with good initial tolerance at mean follow-up of 12.9 weeks. Lengthy insurance approval process and payor coverage denials limit medication access.

Micheliolide Exerts Therapeutic Effect in Myeloproliferative Neoplasms through Binding to STAT3 and STAT5 Proteins and Inhibiting Their Activities

The JAK1/2 inhibitor ruxolitinib is a cornerstone of management for some subsets of BCR-ABL negative myeloproliferative neoplasms (MPN). While many patients benefit with reduced splenomegaly and symptoms, some patients respond suboptimally to ruxolitinib, and even patients with a good initial response often develop resistance to ruxolitinib after several years of therapy. Here, we evaluated the efficacy of micheliolide (MCL), a natural guaianolide sesquiterpene lactone, in the JAK2V617F positive MPN entities. We firstly evaluated the effect of MCL treatment on cell growth and survival in human MPN cell lines expressing the JAK2V617F mutation. MCL alone reduced cell viability and induced apoptosis of UKE1 and SET2 cells in a dose-dependent manner, and obviously improved the effect of ruxolitinib as monotherapy. Importantly, treatment with MCL also induced significant apoptosis of UKE1 cells which were selected for resistance against ruxolitinib. We further explored the therapeutic potential of MCL in vivo using a Jak2V617F knock-in (Jak2VF) mouse model, and found that DMAMCL, an orally available derivative of MCL, significantly increased ruxolotinib efficacy in Jak2VF mice, manifested as amplified hematological and spleen responses. What's more, DMAMCL, whereas not ruxolitinib, contributed to reduce the in vivo Jak2V617F mutant allele burden reflected by the percentage of mutant hematopoietic cells in peripheral blood and bone marrow. To determine the molecular mechanisms by which MCL exerts efficacy in MPN, we firstly performed western blot (WB) anlyses to evaluate the effect of MCL on hyper-activated JAK-STAT signaling, which is the cornerstone of MPN pathogenesis. We observed a dose-dependent inhibition of STAT3 and STAT5 phosphorylation caused by MCL treatment in UKE1 and SET2 cells (Figure 1A), and as expected, combination MCL with ruxolitinib provided an enhanced inhibitory effect. By next performing the affinity pull-down experiment, we confirmed that MCL suppressed the activation of STAT3 and STAT5 mainly through directly binding to the proteins (Figure 1B). We further conducted RNA-sequencing analysis in UKE1 cells treated with MCL and/or ruxolitinib to gain insights into mechanisms involved in MCL effect. Gene set enrichment analysis (GSEA) showed that genes related to TNF-α-NF-κB signaling and inflammatory response pathway were significantly downregulated upon treatment with MCL, that were verified by WB analyses and real-time quantitative PCR. Additionally, GSEA also revealed significant upregulation of genes related to reactive oxygen species (ROS) and unfolded protein response (UPR) pathway, respectively. Both flow cytometry and fluorescent microscopy imaging confirmed the ROS accumulation by MCL treatment. We also determined the activation of PERK branch of UPR pathway caused by MCL, as assessed by increased phosphorylation of eIF2α, and upregulation of pro-apoptotic genes, including ATF4, CHOP and GADD34. Finally, by addition of antioxidant agent, the activation of UPR pathway and increase of apoptosis in UKE1 cells induced by MCL were significantly rescued, indicating that MCL exerts cytotoxic effects mainly through triggering the oxidative stress in MPN cells. In summary, our results demonstrate that MCL is an effective agent with multiple biological activities in MPN, especially inhibition of STAT3/5 activity via directly binding to the proteins, and may be a promising drug combined with ruxolitinib to benefit patients who have suboptimal response or acquire resistance to ruxolitinib. Figure 1. MCL inhibits STAT3 and STAT5 phosphorylation through binding to the proteins directly. (A) Western blot analyses are shown for phosphorylated-STAT3 (p-STAT3), STAT3, phosphorylated-STAT5 (p-STAT5) and STAT5 levels in total cell extracts from UKE1 and SET2 cells treated with MCL (5, 10, 20μM) for 3 hours. (B) The biotin-conjugated MCL (biotin-MCL) and its inactive analogue S-MCL (biotin-S-MCL) were prepared as the positive- and negative- probe respectively. UKE1 and SET2 cell lysates were incubated with 20μM biotin-MCL or biotin-S-MCL for 1 hour at room temperature, then the mixtures were separately pulled down with streptavidin-coated agarose beads, and STAT3/STAT5 were detected with WB analysis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal