Myeloproliferative Neoplasms Disease Research Articles

LNK/SH2B3 Loss Exacerbates the Development of Myeloproliferative Neoplasms in CBL-deficient Mice.

Genetic variations of signaling modulator protein LNK (also called SH2B3) are associated with relatively mild myeloproliferative phenotypes in patients with myeloproliferative neoplasms (MPN). However, these variations can induce more severe MPN disease and even leukemic transformation when co-existing with other driver mutations. In addition to the most prevalent driver mutation JAK2V617F, LNK mutations have been clinically identified in patients harboring CBL inactivation mutations, but its significance remains unclear. Here, using a transgenic mouse model, we demonstrated that mice with the loss of both Lnk and Cbl exhibited severe splenomegaly, extramedullary hematopoiesis and exacerbated myeloproliferative characteristics. Moreover, a population of Mac1+ myeloid cells expressed c-Kit in aged mice. Mechanistically, we discovered that LNK could pull down multiple regulatory subunits of the proteosome. Further analysis confirmed a positive role of LNK in regulating proteasome activity, independent of its well-established function in signaling transduction. Thus, our work reveals a novel function of LNK in coordinating with the E3 ligase CBL to regulate myeloid malignancies.

IL-1β promotes MPN disease initiation by favoring early clonal expansion of JAK2-mutant hematopoietic stem cells

AbstractJAK2-V617F is the most frequent somatic mutation causing myeloproliferative neoplasm (MPN). JAK2-V617F can be found in healthy individuals with clonal hematopoiesis of indeterminate potential (CHIP) with a frequency much higher than the prevalence of MPNs. The factors controlling the conversion of JAK2-V617F CHIP to MPN are largely unknown. We hypothesized that interleukin-1β (IL-1β)–mediated inflammation can favor this progression. We established an experimental system using bone marrow (BM) transplantations from JAK2-V617F and GFP transgenic (VF;GFP) mice that were further crossed with IL-1β−/− or IL-1R1−/− mice. To study the role of IL-1β and its receptor on monoclonal evolution of MPN, we performed competitive BM transplantations at high dilutions with only 1 to 3 hematopoietic stem cells (HSCs) per recipient. Loss of IL-1β in JAK2-mutant HSCs reduced engraftment, restricted clonal expansion, lowered the total numbers of functional HSCs, and decreased the rate of conversion to MPN. Loss of IL-1R1 in the recipients also lowered the conversion to MPN but did not reduce the frequency of engraftment of JAK2-mutant HSCs. Wild-type (WT) recipients transplanted with VF;GFP BM that developed MPNs had elevated IL-1β levels and reduced frequencies of mesenchymal stromal cells (MSCs). Interestingly, frequencies of MSCs were also reduced in recipients that did not develop MPNs, had only marginally elevated IL-1β levels, and displayed low GFP-chimerism resembling CHIP. Anti–IL-1β antibody preserved high frequencies of MSCs in VF;GFP recipients and reduced the rate of engraftment and the conversion to MPN. Our results identify IL-1β as a potential therapeutic target for preventing the transition from JAK2-V617F CHIP to MPNs.

Research status and challenges of advanced myeloproliferative neoplasms

Classical myeloproliferative neoplasms (MPN), also known as Ph-MPN, includes polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Secondary myelofibrosis (sMF) and secondary acute myeloid leukemia (sAML) are important disease progressions of MPN. After MPN disease progression, hematopoietic stem cells undergo new clonal evolution, leading to drug resistance, poor treatment effect and poor survival of patients. In recent years, the exploration of the mechanism of disease progression and the precise diagnosis and treatment of MPN have attracted much attention. This article summarizes the research status of MPN disease progression, including the pathogenesis, risk stratification, and precision treatment, in order to provide reference for exploring new diagnosis and treatment methods of MPN disease progression.

Impact of Kidney Dysfunction on Overall Survival in Myeloproliferative Neoplasms: A Single-Center Retrospective Study

Introduction: Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell disorders that can lead to the deposition of fibrous tissue, and by a propensity toward extramedullary hematopoiesis. The progression of kidney function and frequency of chronic kidney disease (CKD) in patients with MPN is unknown, although CKD is linked to increased mortality. In our study, we aimed to investigate the relationship between kidney function as measured by glomerular filtration rate (eGFR) and MPN disease and risk of thrombosis. Moreover, we analyzing the risk factors for CKD, impact of different treatment modalities and effect of CKD on survival. Patients & Method: We haveretrospectively screened 762 patients between Jan 1989 to May 2019 with MPN at Princess Margaret Cancer Centre. Diagnosis, of Polycythemia (PV), essential thrombocytosis (ET), and myelofibrosis (MF; comprising PMF, post-ET-PMF, and post-PV-PMF) was required as defined by the WHO classification. Patients with other MPNs (CNL, CEL, HES, MDS/MPN, MPN-U, Mastocytosis accelerating phase MPN and blast phase) or missing serial creatinine measurement or their CKD attributed to other causes (diabetic nephropathy, High blood pressure, polycystic kidney disease obstructive uropathy, Glomerulonephritis before MPN diagnosis and other) were excluded, resulting in study sample of 232 patients. The total cohort was subdivided according to the calculated eGFR, (ml/min/1.73m2) into eGFR1 (≥90, n=154), eGFR2 (60-89, n=12), and eGFR3 (<60, n=66). eGFR was collected retrospectively, because of small number of patients, we combined eGFR2 and eGFR3 in one group. Overall Survival (OS) was calculated using the Kaplan-Meier and log-rank test was used to assess impact variables of interest. Cox proportional hazards model was used to assess for prognostic factors of OS as well as to assess the joint effect of potential prognostic factors. Results: Median age for the total cohort was 58 years (range;18-88.3), 54% were male. Median follow-up duration was 86 months (range:45.5-135). Diagnosis of MPN involved ET; n=21(9%)), PV; n=32 (14%) and MF; n=179 (77%). JAK2V617F status was documented in 219 patients; 67% were positive; of 106 patients analyzed for CARL 40.6% were positive. Cardiovascular risk factors were higher in PV and MF than in ET (59.4%, 57% &38%) respectively. A higher uric acid and LDH levels were found in PV and MF. A total of 19% had a history of thrombosis and rate was higher in ET (28.6%) than PV (21.9%) or MF (16.8%). Further patient's characteristics summarized in Table1. Kidney biopsy performed in 8 patients. The most prominent histological finding included focal segmental glomerulosclerosis (n=3), Ig A nephropathy (n=3), mesangial hypercellularity and sclerosis, extramedullary haematopoiesis (n=1) and lupus nephritis (n=1). The risk factors for CKD based on eGFR group are presented in (Table 2). MPN diagnosis is a significant risk factor for kidney function (p=0.0117). In MF a higher rate of eGFR 88.5% compared with PV and ET. JAK2V617 demonstrates a significant impact on abnormal eGFR (p=0.05);. IPSS and DIPSS score are a significant risk factor for kidney dysfunction (p=0.0014, and 0.0074 respectively). High uric acid levels and neutrophil counts were higher in high eGFR group (p=0.0014, and 0.0074 respectively). No association found between thrombosis and high eGFR. Hydroxyurea treatment does not have an impact on CKD, however, more patients received Ruxolitinb in abnormal eGFR (p=0.001). CKD has significant impact on OS. The 5 years OS for eGFR1 was 75% (95% CI 67%-81%), and for abnormal eGFR was 63.9% (95% CI 52%-73%), p=0004 (Fig 1). Conclusion: Higher incidence of kidney dysfunction associated in MF compared to PV and ET, there was no association between thrombosis and high eGFR as we excluded all other cause of CKD. MF with abnormal eGFR associated with high DIPSS score. MPN patients with kidney dysfunction significantly affects OS, which indicate close monitoring and prospective study is required.

Aberrant Activity of the Calcium Sensor STIM1 Underlies Congenital Platelet Disorders and Myeloproliferative Neoplasms

MB and VA equal contributors AB and STO co-corresponding authors This study was initiated following evaluation of a 32 year-old woman who presented with a history of thrombocytopenia identified in childhood who subsequently developed features of myelofibrosis (MF). A bone marrow biopsy demonstrated hypercellularity in conjunction with megakaryocyte hyperplasia and marked reticulin fibrosis. Molecular testing for JAK2, CALR, and MPL mutations was negative. Given the unusual association between congenital thrombocytopenia and MF in this patient, exome sequencing was performed, revealing a heterozygous R304W mutation in the coiled coil (CC) domain of STIM1. Activating mutations in the CC and EF hand domains of STIM1 have been associated with Stormorken syndrome, a rare congenital platelet disorder associated with abnormal store operated calcium entry (SOCE). We subsequently identified a second patient with MF associated with a STIM1 activating mutation. This individual was found to have severe thrombocytopenia at birth, and exome sequencing revealed a heterozygous S88G mutation in the EF hand of STIM1. A bone marrow biopsy obtained at 6 months of age revealed atypical megakaryocytes and grade 1-2 MF. A repeat biopsy at age 2 showed persistence of stable MF. The unusual finding of MF in these two patients suggested the possibility of altered calcium signaling as a shared mechanism driving congenital platelet disorders such as Stormorken syndrome and myeloproliferative neoplasms (MPNs) including MF. In support of this notion, we identified elevated STIM1 expression in MF vs normal megakaryocyte progenitors, as well as in platelets from patients with essential thrombocythemia (ET) vs healthy controls. Additionally, we found that STIM1 expression was significantly elevated in CD34+ hematopoietic stem/progenitor cells (HSPCs) from both ET and MF patients vs healthy controls. Notably, STIM1 expression was increased in both JAK2 and CALR-mutant MF patients. Collectively, these findings provide evidence of aberrant STIM1 expression in MPN patient cells. To determine the functional role of STIM1 in MPN disease development, colony assays and patient-derived xenograft (PDX) experiments were performed with MF patient CD34+ cells subjected to CRISPR ablation of STIM1. Strikingly discordant results were observed with JAK2 vs CALR-mutant patient samples. Abrogation of STIM1 in CALR-mutant CD34+ cells led to decreased colony formation, and NSGS mice engrafted in parallel with STIM1-targeted cells exhibited decreased human CD45+ cell engraftment in conjunction with prolonged survival. These findings suggest an important role for STIM1 in CALR-mutant MPN disease phenotypes. In contrast, targeting of STIM1 in JAK2-mutant CD34+ cells led to increased colony formation and exacerbated disease phenotypes in vivo as manifested by enhanced human CD45+ cell engraftment, worsened splenomegaly, and early lethality. Similar results were obtained in experiments utilizing pharmacologic inhibitors of SOCE activity. Taken together, these findings indicate that the consequences of aberrant STIM1 activity may be context-dependent relating to specific MPN driver mutations. To expand these observations, we identified a separate cohort of 9 family members in Italy with Stormorken syndrome and confirmed STIM1 EF hand mutations. In ex vivo megakaryocytic differentiation assays, cells from affected individuals exhibited a defect in proplatelet formation. These observations were corroborated by initial analyses of a newly generated Stim1 R304W conditional knock-in mouse which recapitulated the characteristic thrombocytopenia found in patients with Stormorken syndrome. In summary, this study represents the first demonstration of MF development in patients with Stormorken syndrome, thereby uncovering a previously unrecognized hallmark of altered calcium signaling via aberrant STIM1 activation underlying Stormorken syndrome and MPNs. Our findings suggest distinct mechanisms relating to the interaction between JAK2 vs CALR mutation and altered STIM1 activity. Further studies of these relationships may have important ramifications for potential therapeutic approaches targeting these pathways.

Loss of Complement Factor I (Cfi) a Negative Regulator of Complement Cascade Activity Exacerbates JAK2V617F-Dependent Phenotype

Background: The current evidence suggests that inflammatory phenotype contributes to the pathogenesis of myeloproliferative neoplasms (MPNs). While our understanding of complement cascade contributions to inflammation-mediated pathologies has expanded, role this cascade plays in etiology of myeloid malignancies has not been detailed. Previously, we uncovered an inactivating RNSV in complement factor I (CFI G119R), an inhibitor of the complement cascade, in 20% of PMF patients (n=10) using WGS, and in a separate patient cohort (n=10) we observed an increase in CH50 levels (n=3) and decrease in C3 levels (n=3) (Oakes et al., 2021, Blood, 138:1472).These findings prompted us to generate murine models allowing assessment of the Cfi loss-driven complement overactivity effects on hematopoiesis and development of MPNs. Methods: A conventional Cfi knockout (KO) mouse model was generated by targeting exon 2 of the Cfi gene using CRISPR/Cas9. Inactivation of one or two copies of Cfi was confirmed by genotyping and total loss (Cfi KO) or Cfi deficiency (Cfi HET) were confirmed in peripheral blood (PB) plasma. Complete blood counts (CBC) and the frequencies of granulocytes, B cells, plasmocytes, T cells, macrophages/monocytes, platelets, erythroid cells in the bone marrow were longitudinally assessed by flow cytometry for 40 weeks in Cfi WT, HET and KO mice. Cfi HET animals were crossed with JAK2V617F(fl/+);Mx1-cre(+) mice (obtained from crossing B6.Cg-Tg( Mx1- cre+ 1)Cgn/J; # 003556, JAX) and JAK2V617F expressing (B6N.129S6(SJL)- Jak2 tm1.2Ble/AmlyJ #031658, JAX) to generate Cfi(+/-)/JAK2V617F(fl/+);Tg(Mx1-cre(+/-) (CFI/JAK2) strain. Complete blood counts and survival were monitored for Cfi(+/+)/JAK2V617F(fl/+);Tg(Mx1-cre)(+/-) (CFI WT/JAK2 HET) Cfi(+/-)/JAK2V617F(fl/+);Tg(Mx1-cre)(+/-) (CFI HET/JAK2 HET) or Cfi(-/-)/JAK2V617F(fl/+); Tg(Mx1-cre)(+/-) (CFI KO/JAK2 HET) mice. Results:Cfi HET and KO mice were fertile and viable. Cohort of Cfi WT n=13, Het n=12, KO n=13 mice was subjected to analyses. Genotypes were confirmed via PCR and western blot. Flow cytometry demonstrated a copy loss dependent decrease in total T-cells (CD3+) and increase in mature resting B-cells (CD19+/B220+). CBC demonstrated a copy loss dependent decrease in both monocyte (p adj. KO vs. WT 0.002) and granulocyte percentage (p adj. KO vs. WT 0.003), and increase in lymphocyte percentage (p adj. KO vs. WT 0.001). In PB plasma we detected increased C3 activation in KO mice compared to WT and Het, demonstrated as decreased C3 alpha and presence of inactivated C3 due to increased proteolytical processing of C3 complex. Proteolytic cleavage of CFB compared to Cfi WT and Het was also noted in Cfi KO animals. A significant decrease in survival probability of CFI HET/JAK2 HET vs. CFI WT/JAK2 HET (p=0.03), CFI KO/JAK2 HET vs. CFI WT/JAK2 HET (p=0.003), and CFI HET/JAK2 HET vs. CFI KO/JAK2 HET (p=0.05) averaging 92 days for Cfi HET/JAK2 HET, 67 days for Cfi KO/JAK2 HET vs. 98 days for Cfi WT/JAK2 HET was noted (Fig. A). A granulocyte and monocyte percentage of CFI HET/JAK2 HET was increased as compared to CFI WT/JAK2 HET mice (Fig. B). Conclusions:An overactivation of complement cascade at the C3 level is noted in Cfi deficient animals, that is phenotypically linked to an increase in frequency of B cells. These findings may suggest involvement of Cfi directly, and/or in conjunction with generally overactive complement, in B-cell maturation and function. The absence of one copy of Cfi in JAK2V617F mice shows more severe MPN disease progression resulting in markedly decreased survival, linked to increased frequencies of monocytes and granulocytes and markedly decreased lymphocytes. In sum, these data indicate that increased complement activity may increase JAK2V617F-dependent phenotype severity. In depth mechanistic studies detailing observed phenotype are warranted.

RSK1 Is an Exploitable Dependency in Myeloid Malignancies

Myeloid malignancies harbor distinct molecular drivers but share convergence of oncogenic signaling pathways and propagation by ripe pro-inflammatory niches. To delineate these hallmarks across the spectrum of myeloid disease states, we established the largest comprehensive atlas of myeloproliferative neoplasms (MPN) and secondary acute myeloid leukemia (sAML) through RNA-sequencing and mass cytometry (CyTOF) of 370 primary samples encompassing CD34+ hematopoietic stem/progenitor cells and CD14+ monocytes, revealing aberrant of PI3K/AKT/mTOR signalling and NFκB-mediated hyper-inflammation. As a central mediator of these hyperactive signaling pathways, ribosomal S6 kinases (RSKs) represent potentially attractive novel therapeutic targets warranting further evaluation. Using both genetic approaches and a first-in-class oral RSK inhibitor, PMD-026, that is currently being evaluated in phase 1/1b clinical trials (NCT04115306) in metastatic and triple negative breast cancer, we found that RSK1 ( RPS6KA1) inhibition potently induced apoptosis and G2/M arrest in leukemia models in conjunction with suppression of PI3K/AKT/mTOR and NFκB signaling. Further NFκB cascade profiling following RSK1 perturbation revealed reduced phosphorylation of IKKα/β and p65/RELA and prevention of p65/RELA nuclear translocation. Using RNA-seq, ATAC-seq and H3K27ac CUT&Tag, we found that PMD-026 treatment altered chromatin accessibility and inhibited NFKB1, TNF, and cell cycle regulators CCNA1/2 and CDK1/2. In cytokine CyTOF, PMD-026 nearly abrogated all induction of inflammatory cytokines including TNF, IL-6, IL-8, CCL3, and CCL4 in primary MPN monocytes to levels substantially below basal conditions. We further extended our findings to de novo AML harboring FLT3 internal tandem duplication ( FLT3-ITD), where FLT3-ITD patients exhibited elevated RPS6KA1 expression and those who developed resistance to gilteritinib demonstrated progressively increased RPS6KA1 expression. FLT3-ITD cells exhibited preferentially sensitivity to RSK1 inhibition, where we uncovered bi-directional regulation. Through cycloheximide, MG-132, and ubiquitination assays, we found that RSK1 regulates FLT3-ITD activity and protein stability through deubiqutinase USP1, and where either RPS6KA1 or USP1 inhibition resulted in concomitant downregulation of FLT3 protein and cell lethality. Multivariate analysis of the TCGA and BeatAML cohorts (n= 568 patients) revealed high median USP1 or RPS6KA1 expression to be significantly associated with poor survival, thus defining a RSK1-USP1-FLT3 axis with prognostic relevance. Across in vivo models, PMD-026 ameliorated MPN disease features driven by MPL W515L including leukocytosis, splenomegaly, and bone marrow fibrosis, while prolonging survival, in conjunction with suppression of inflammatory cytokines including TNF, IL-6 and IL-1b. We then evaluated PMD-026 across five patient-derived xenograft (PDX) models spanning the spectrum of myeloid malignancies including myelofibrosis (MF), sAML, and chronic myelomonocytic leukemia (CMML) encompassing various driver and multiple high-risk mutations. Treatment in MF PDX mice decelerated disease progression, extended survival, and reduced splenomegaly, while in CMML/sAML PDXs, PMD-026 led to a 67 to 96 percent reduction in human CD45+ cells engraftment. Finally, PMD-026 treatment in both mice xenografted with MV4-11 cells and syngeneic Flt3ITDTet2KO mice significantly reduced leukemic cell engraftment and prolonged survival. These data indicate potent efficacy of RSK inhibition across diverse models of chronic and acute myeloid malignancies. Lastly, we investigated the impact of PMD-026 on hematological parameters from 41 breast cancer patients enrolled in the phase 1/1b clinical trial. Overall, there was no sustained suppressive effect on hematological parameters and there were no hematological PMD-026-related toxicities meeting criteria for dose modification during the trial period. These clinical data demonstrate that PMD-026 did not cause myelosuppression. Our findings uncover a novel therapeutic avenue for a conserved RSK1 dependency across chronic and acute myeloid neoplasms. The potent and consistent disease-ameliorating effects across numerous leukemia mouse models demonstrates promise for repurposing PMD-026 for the treatment of myeloid malignancies.

The Function and Mechanism Research of NLRP6 in Bone Marrow Microenvironment in Myeloproliferative Neoplasms

Since the important role of inflammation in myeloproliferative neoplasms (MPN) progression, inflammasome is an important regulator of inflammation involved in the development of tumor. Here, we aimed to investigate the function of NLRP6 in bone marrow (BM) microenvironment in MPN in the hope of getting better treatment for MPN. We first analyzed that NLRP6 was highly expressed in BM mononuclear cells from individuals newly diagnosed with ET, PV, PMF patients (with or without JAK2 V617Fmutation). Similar results were obtained in BM cells and BM stromal cells of MPL W515L transduced C57BL/6 mice (C57BL/6 MPN). The results were validated that NLRP6 of the BM microenvironment was involved in the development of MPN. We thus hypothesized that the absence of NLRP6 in BM microenvironment affects the development of MPN. NLRP6 -/- mice and C57BL/6 mice MPN models were established by the same method. The experiments demonstrated that overall survival of NLRP6 -/- MPN mice was significantly prolonged compared to C57BL/6 MPN mice. Loss of NLRP6 in BM microenvironment interfered with hematopoiesis of BM, but it had no effect on the extramedullary hematopoiesis (EMH) in spleen. Pathological analysis showed that the destruction of spleen structure in NLRP6 -/- MPN mice and the infiltration of atypical megakaryocytes were less than C57BL/6 MPN mice. The tumor load in spleen was significantly reduced and the structure of spleen was intact, the hematopoietic function of the BM returned to normal and the tumor load in the BM was significantly reduced at 5 weeks in NLRP6 -/- MPN mice after sequential transplantation. It suggested that knockout of NLRP6 in the BM microenvironment significantly attenuated the tumorigenicity of malignant HSCs, delayed or even inhibited the development of MPN disease. The expression of inflammatory cytokines, abnormal megakaryocytes and the phenomenon of aggregation and reticular fibers in BM microenvironment were significantly reduced in NLRP6 -/- MPN group compared with the control group. After sequential transplantation, the destruction of BM structure was alleviated, megakaryocytes aggregation and heteromorphic megakaryocytes in BM were reduced, and reticular fibers were significantly diminished. Furthermore, the cytokines promoting disease progression in BM microenvironment were significantly reduced in NLRP6 -/- MPN group, suggesting that knockout of NLRP6 can improve the BM microenvironment and alleviate the progression of myelofibrosis. Finally, to gain better insight into the role of NLRP6 inflammasome in MPN pathogenesis, we sought to dissect the changes with scRNA-seq and pathway identification based on available data and bioinformatic pathway analysis in NLRP6 -/- mice. The results of scRNA-seq analysis showed that C18 population of malignant HSCs of recipients in primary transplantation was added, C16, C13 and C9 cell populations disappeared after deletion of NLRP6 in BM microenvironment, while C5, C6, C7, C10 and C12 cell populations increased significantly. Regulon of various subpopulations of malignant HSCs changed significantly, mainly involving cell development and differentiation, immune response, inflammatory BM microenvironment, vascular endothelial homeostasis and tumor related genes. Our results indicate that knockout of NLRP6 in the BM microenvironment may affect the progression of MPN by altering some malignant HSCs subpopulations and regulons, participating in immune, inflammatory, tumor metabolism and other signaling pathways to regulate the functional characteristics of tumor stem cells of MPN.Our data here provide new evidence for NLRP6 as potential target in the treatment of MPN.

In Vivo Ablation of NFκB Cascade Effectors Alleviates Disease Burden in Myeloproliferative Neoplasms

ABAL and TK equal contributors Multilevel regulation of NFκB activation exists in response to microenvironmental cues. Aberrant hyperactivation of the NFκB cascade propagates oncogenic signaling and inflammation, which together augment disease burden in myeloproliferative neoplasms (MPNs). Here, we systematically ablated NFκB signaling effectors to identify core dependencies using a series of primary samples and syngeneic and patient-derived xenograft (PDX) mouse models. We first examined the impact of Rela abrogation in the Jak2 V617F knock-in mouse model that exemplifies polycythemia vera hallmarks and generated Jak2V617FRela flox/flox Ubc-Cre-ERT2 ( Jak2- Rela fl) mice. Evident suppression of leukocytosis was observed within a week of Rela excision and sustained until endpoint. Elevated hematocrit, monocytes, and CD45.2 myeloid cells were suppressed in both the peripheral blood (PB) and bone marrow (BM) and Jak2- Rela flmice had significant reduction of splenomegaly. We then adopted a second MPN model, where MPL W515L retroviral expression produces a phenotype resembling myelofibrosis. Consistent reduction of pathologic WBC counts, monocytes, and PB and BM CD45.2 myeloid cells were observed in MPL- Rela fl mice. Rela ablation also reduced hematocrit, platelet counts, and CD45.2 GFP+ cells. Importantly, we observed a significant attenuation of disease hallmarks with a decrease of spleen weight and dysmegakaryopoiesis, enhanced survival, and reduction of BM reticulin deposition. We next established PDX models where CD34+ cells from MF patients and healthy BM donors (NBM) were transduced with an sgRNA targeting RELA and engrafted into NSGS mice. Across two MPN PDXs, there was significant suppression of leukemic cells and improvement of disease burden, reduced spleen weights and prolonged survival, and subsequent BM histology revealing improved sclerosis, fibrosis, and dysmegakaryopoiesis. In contrast, NSGS mice engrafted with NBM CD34+ cells did not yield disease symptoms, nor did RELA targeting in healthy cells affect hematopoiesis or measured disease hallmarks. RNA-seq on CD34+ cells from four MPN patients following RELA abrogation revealed augmented and altered expression of genes that are 1) regulators of survival and proliferative pathways: MDM2, TP53INP1, and FAS 2) major ferroptosis mediator GPX4, and 3) cell cycle effectors: CDKN1A (p21), E2F4, and CHEK2. In further investigation of NFκB pathway effectors, we genetically targeted upstream effector Myd88 and transduced Kit+ cells from Myd88-null mice with MPL W515L. Consistent with Rela-knockout, Myd88-deficiency also diminished pathogenic WBC and monocyte count upsurgence, inhibited the accumulation of BM CD45.2 myeloid cells, and reduced spleen weights. The rapid lethality governed by mutant MPL was also delayed upon Myd88 loss, consistent with reduced pathology in spleen and BM sections. While Myd88 and Rela activate NFκB, miR-146a is repressive. We performed further knock-out of MIR146A using PDX models and transplanted MIR146A-KO CD34+ cells into NSGS mice and observed enhanced leukemic engraftment, increased spleen weights, earlier lethality, and evidence of BM sclerosis or fibrosis across both MF PDXs. Importantly, these disease parameters remained unchanged in a NBM PDX following MIR146A-KO. Lastly, in our WashU Cohort of RNA-sequencing performed on 90 CD34+ samples encompassing the spectrum of MPN and secondary acute myeloid leukemia (AML) with NBM controls, we observed significant upregulation of IRAK4 and IL1B in MF and sAML compared to NBM, and their significant expression correlation in MPN. We then pharmacologically inhibited IRAK4 using a small molecule, CA-4948, which is currently under evaluation in phase 1/2a trials of relapsed/refractory AML/myelodysplastic syndrome. In PDX experiments, CA-4948 treatment effectively reduced leukemic engraftment and spleen weights of two MF models without inducing toxicity in a NBM PDX. Through qRT-PCR and mass cytometry, we demonstrate that CA-4948 treatment dampened inflammatory cytokine production induced by IL-1β in primary MPN CD14+ monocytes. Overall, we demonstrate that targeting mediators including Rela, Myd88, and IRAK4 specifically alleviated MPN disease burden without toxicity to healthy tissue and further establish CA-4948 as a promising therapeutic avenue for the treatment of MPN.

Establishment of Human Bone Marrow-on-Chip As a Preclinical Model to Evaluate Drug-Induced Toxicities and Myelofibrosis Patient-Specific Pathophysiology

Hematopoiesis takes place in the bone marrow (BM) and is supported by a complex cellular and molecular network in the BM microenvironment. The inaccessibility of living human BM hampers the study of its pathophysiology under stress induced by drugs, radiation, or genetic mutations. Commonly used models of the human BM microenvironment rely on mouse models and 2D or 3D static tissue cultures. Although existing in vitro hematopoiesis models have led to advances in the field, they fail to recapitulate the biology of cellular interactions, cell extrinsic factors, and complex functions of living human BM hematopoiesis. This has also limited our understanding of human BM biology and the influence of BM niche cells in BM defects in diseases such as myeloproliferative neoplasms (MPNs) and leukemias. Consequently, there is an unmet need to develop a preclinical human BM hematopoiesis model that supports growth and differentiation of multilineage hematopoietic stem cells to enable evaluation of investigational compounds for myeloerythroid toxicity and efficacy. Microphysiologic systems (MPS), such as organ-on-chips, emulate the complex interplay between intrinsic and extrinsic cell factors in the BM microenvironment, the 3D tissue architecture, and are more clinically relevant compared with static co-culture approaches (PMID: 31988457; PMID: 37359774). Here we present the development of a human BM-on-chip MPS model to recapitulate both healthy and MPN disease and evaluate the alterations in the BM niche environment. We utilized a commercially available organ-on-chip microfluidic platform (Emulate, Boston, MA) to develop a living human BM with a functional hematopoietic niche in vitro by perfusion with culture medium. This fluidic-controlled modular chip comprises an osteogenic apical channel separated from a vascular basal channel by a semiporous membrane, which allows for media perfusion, cellular crosstalk, and intercellular signaling. The osteogenic apical channel is filled with a fibrin gel that allows the co-culture of all critical cells of the BM niche, including osteoblasts, endothelial cells, mesenchymal stem cells, hematopoietic stem and progenitor cells. The parallel basal channel is lined by human vascular endothelium and is perfused with culture medium optimized to allow survival and development of functional hematopoietic lineage niche cell populations. This unidirectional fluidic MPS model allows for collection of cell effluents for evaluation of cell-extrinsic factors such as cytokines, extracellular vesicles, and metabolites. The modular and accessible nature of this system provides an advantage to investigate the effects of the microenvironment and drug candidates individually on the osteogenic and vascular compartments. This human BM-on-chip has been tested with cells from several healthy and myelofibrosis donors. Utilizing imaging, flow cytometry, cytokine profiling, and transcriptomics, we demonstrate that the vascularized human BM-on-chip recapitulates the living human hematopoietic niche, with BM relevant cytokine levels, retains hematopoietic stem and progenitor cells in in vivo-like proportions for over 3 weeks in culture, allows for the differentiation of BM niche lineage cell populations similar to human BM aspirate, and captures human donor variabilities. We also show that BM-on-chips derived from MPN patients recapitulate the hematopoietic defects to understand the biology and disease response in myelofibrosis. Our robust experimental workflow and bioinformatics platform will be utilized to evaluate candidate compounds for BM myelotoxicities, drug mechanisms of action, and biomarkers in a multifactorial manner to support preclinical programs. This biomimetic platform offers a new approach for analysis of drug responses and toxicities in BM as well as for study of hematopoiesis and hematologic diseases such as myelofibrosis.

Polygenic Germline Risk of Common Haematological Traits Drives Clonal Selection on JAK2V617F and Development of Myeloproliferative Neoplasms

Polygenic germline loci are a major contributor to population variation in common blood cell traits, but little is known about how such predisposition influences clonal selection and cancer. Myeloproliferative neoplasms (MPN) are rare chronic haematological cancers characterised by the abnormal overproduction of mature blood cells leading to elevated blood cells parameters, and are driven by somatic mutations, e.g. JAK2V617F. JAK2 mutations result in a spectrum of phenotypes from asymptomatic clonal haematopoiesis (CH) to distinct MPN subtypes characterised by increased red blood cells (polycythaemia vera; PV) and platelets (essential thrombocythaemia; ET) respectively. Compared to MPN, mutant JAK2 is much more common in the population, the vast majority of whom do not meet, or go on to develop, disease defining characteristics of MPN. Little is understood about why only a minority of individuals with mutated JAK2 develop more severe haematological manifestations that fulfil the clinical criteria of MPN and the factors that influence blood count heterogeneity in MPN. We assessed how polygenic germline scores (PGS), based on associated genetic variants, for 29 haematological traits drive JAK2V617F positivity in UK Biobank (UKBB) (n=540 mutation carriers, 161,994 non-carriers). Accounting for the recognised predisposition towards MPN driven by the JAK2 46/1 haplotype and other known germline variants associated with MPN, we found significant positive associations with JAK2V617Fclones for the PGSs of plateletcrit (PGS PCT) and monocyte count (PGS MONO) ( P =9.510 -4 and 0.0036 respectively with FDR<0.05 for small clones [VAF <0.1, n=397]; 0.033 and 0.0022 with FDR adjusted P = 0.31 and 0.064 for large clones [VAF 0.1 or greater, n=143]). Empowered by genome-wide association study data associated with JAK2V617F positivity in the full UKBB data (n = 1,125 mutation carriers, 338,919 non-carriers), we identified strong evidence that genetically predicted monocytes (odds ratio [OR] 1.31, 95% confidence interval [CI]=1.15-1.49, P=4.610 -5) and plateletcrit (OR 1.52, CI=1.29-1.78; P=3.010 -7) are causal risk factors for JAK2V617F clonal expansion. We genotyped genome-wide SNPs in 761 MPN patients whom had detailed somatic mutation data, and built PGSs for blood cell traits in patients and 30,305 healthy controls. Germline predisposition to several higher red-cell traits increased the odds of PV (OR 1.22, CI=1.01-1.47 to 1.57, CI=1.34-1.86]), while high PGSs for two platelet traits predisposed to ET (OR 1.56, CI=1.44-1.70; OR 1.72, CI=1.44-2.02). Results were validated in MPN and healthy controls from UKBB. Importantly, extreme PGS for blood cell traits associated with an MPN diagnosis in the absence of somatic driver mutations in both patients and UKBB, potentially mimicking the haematological phenotype of clonal disease. We confirmed that the heritability of MPN subtype via polygenic predisposition to normal blood traits was independent of known germline MPN and CH risk loci. In summary, by analysing two large MPN disease cohorts and UKBB, we provide new insights into the interaction between germline polygenic variation involved in basic haematopoiesis and clonal selection on somatic driver mutations in blood. We demonstrate that genetically predicted haematological traits can causally influence clonal selection on mutant JAK2, and the polygenic predisposition of blood cell traits influences the phenotype of subsequent blood cancer. Our results highlight an independent and causal new component of the overall susceptibility to clonal disease and provide a novel framework for considering an individual's genetic background in the context of their clinical presentation.

JAK2-V617F Promotes up-Regulation of Pro-Inflammatory Cytokines Including IL-1 upon Adhesion-Induced Activation of β1/β2 Integrins

Introduction In JAK2-V617F (JAK2-VF) mutated cells, over-activation of β1/β2 integrin inside-out signaling has been described (Edelmann-Stephan et al., 2017, JCI). Integrins play a fundamental role in cell interaction and cell invasion during inflammatory processes. In patients suffering from Myeloproliferative Neoplasms (MPNs), highly elevated pro-inflammatory cytokine levels including IL-1β play an important role in pathophysiology. Importantly, Rai et al. (2023, BioRχ) have shown that IL-1β promotes initiation of MPN. In this study, we investigated a potential link between integrin over-activation and elevated pro-inflammatory cytokines including IL-1α and IL-1β in JAK2-VF induced disease. Methods We employed (1) murine myeloid progenitor 32D cells co-transfected with EPO-R and JAK2-WT or JAK2-VF, (2) granulocytes and hematopoietic progenitor cell populations from total bone marrow of Vav1-Cre x JAK2 +/+ and Vav1-Cre x JAK2 VF/+ mice (described by Mullally et al. 2010), and (3) human granulocytes isolated from PB of JAK2-VF positive patients and healthy volunteers. Cells were allowed to adhere on immobilized VCAM-1/ICAM-1 or on BSA (control) and subsequently subjected to further analysis including (1) qPCR analysis and ELISA of a panel of inflammatory cytokines (IL-1β, IL-1α, TNF-α, IL-6, CXCL10), (2) Western-blotting monitoring integrin outside-in signaling pathways, (3) FLICA assays assessing adhesion-induced active caspase-1, (4) RNAseq of mouse and human granulocytes (3 JAK2-VF patients, 2 controls). Results In the 32D cell model, adhesion on mVCAM-1/mICAM-1 strongly induced mRNA expression of IL-1α, IL1-β, IL-6, TNF-α and CXCL10. Interestingly, cytokine RNA induction was significantly higher in 32D JAK2-VF cells as compared to the 32D JAK2-WT cells (Figure 1). By applying a panel of integrin signaling inhibitors we could show an essential involvement of pivotal signaling nodes such as PI3K, Syk, FAK and NFκB, respectively. Next, cytokine proteins were investigated by ELISA. Adhesion on mVCAM-1/mICAM-1 increased intracellular IL-1α and IL-1β cytokine levels. Again, this effect was strongly amplified by the JAK2-VF mutation (Figure 2). Importantly, both cytokines were found in their mature form, indicating intact protein processing. RNAseq of bone marrow (BM) derived mouse granulocytes (including immature pro-and pre-neutrophils) also showed significant upregulation of a number of pro-inflammatory genes including Nlrp3, Il1a, Il1b, Tnf, Cxcl2 and Cxcl10 upon adhesion on mVCAM-1/mICAM-1. However, no significant differences between WT and VF cells were observed. In contrast, RNAseq of VCAM-1/ICAM-1 stimulated human JAK2-VF positive PB granulocytes (which represent terminally differentiated cells) revealed no cytokine up-regulation. Based on this finding, we hypothesize that the adhesion-induced cytokine phenotype is restricted to more immature cell populations as observed in the 32D cell model. Therefore, we are currently investigating (by qPCR and intracellular IL-1 immunophenotyping) hematopoietic progenitor cell populations from Vav1-Cre x JAK2 +/+ and Vav1-Cre x JAK2 VF/+ mice. Since active IL-1β requires processing by the inflammasome, FLICA assays for active caspase-1 were performed using mouse hematopoietic progenitor cells. Importantly, CD41+ cells and megakaryocyte progenitors (MKP) showed an increased FLICA signal upon adhesion on mVCAM-1/mICAM-1. This indicates that caspase 1 is activated upon adhesion in distinct progenitor cell populations. Finally, to support our hypothesis that JAK2-VF augments induction of pro-inflammatory cytokines upon adhesion to VCAM-1/ICAM-1, Vav1-Cre x JAK2 VF/+ mice (n=8) were treated in vivo with anti-β1/β2-integrin antibodies for 1 week. As expected we found a strong reduction of IL-1α concentration in serum of antibody treated mice (528.7 ± 82.2 pg/ml) as compared to PBS-treated control mice (1302.3 ± 370.4 pg/ml, p = 0.0541) Conclusion Here, we show a functional link between elevated cytokine levels and integrin activation in JAK2-VF induced disease. Adhesion to VCAM-1/ICAM-1 promoted the expression of a number of pro-inflammatory cytokines which are known to play important roles in initiation, clinical symptoms and prognosis of MPNs. Further studies aimed at precisely defining the primary cell populations involved in the mouse MPN disease models as well as in humans are currently ongoing.

Assessment of intestinal status in MPLW515L mutant myeloproliferative neoplasms mice model

The MPLW515L mutation is a prevalent genetic mutation in patients with myeloproliferative neoplasms (MPN), and utilizing this mutation in mice model can provide important insights into the disease. However, the relationship between intestinal homeostasis and MPN mice model remains elusive. In this study, we utilized a retroviral vector to transfect hematopoietic stem cells with the MPLW515L mutation, creating mutated MPN mice model to investigate their intestinal status. Our results revealed that the MPLW515L in MPN mice model aggravated inflammation in the intestines, decreased the levels of tight junction proteins and receptors for bacteria metabolites. Additionally, there was increased activation of the caspase1/IL-1β signaling pathway and a significant reduction in phos-p38 levels in the intestinal tissue in MPN mice. The MPLW515L mutation also led to up-expression of anti-microbial genes in the intestinal tract. Though the mutation had no impact on the alpha diversity and dominant bacterial taxa, it did influence the rare bacterial taxa/sub-communities and consequently impacted intestinal homeostasis. Our findings demonstrate the significance of MPLW515L mice model for studying MPN disease and highlight the mutation's influence on intestinal homeostasis, including inflammation, activation of the IL-1β signaling pathway, and the composition of gut microbial communities.

Optimization of cardiovascular risk factor management in patients with BCR::ABL1 negative chronic myeloproliferative neoplasms, current knowledge, and perspectives.

The exact prognostic role of cardiovascular (CV) risk factors in patients with BCR::ABL1 negative chronic myeloproliferative neoplasms (MPNs) remains unknown as it is often masked by other MPN-related features that bear strong prognostic impact on thrombotic risk. Therefore, current MPN treatment is not primarily guided by presence of CV risk factors. Treatment of CV risk factors in MPN patients usually mirrors that from the general population, despite the fact that CV risk factors in MPNs have their own specificities. Moreover, the optimal target levels for different metabolic deflections in MPNs (i.e., low-density lipoprotein, serum uric acid, or glycated hemoglobin levels) have not been defined. In the current review, we separately discuss the most important aspects of every individual CV risk factor (arterial hypertension, hyperlipidemia, chronic kidney disease, smoking, diabetes mellitus, hyperuricemia, and obesity and cachexia) in MPNs, summarize recent advances in the field, and propose future directions and research areas which may be needed to appropriately manage CV risk factors in MPNs.

Myeloproliferative neoplasms - blurring the lines between cancer and chronic inflammatory disorder.

Myeloproliferative Neoplasm (MPN) is a group of chronic blood cancers that arise from a hematopoietic stem cell (HSC) clone with somatic mutations causing constitutive activation of myeloid cytokine receptor signaling. In addition to elevated blood cell counts, MPN typically presents with increased inflammatory signaling and inflammation symptoms. Therefore, while being a clonally derived neoplasm, MPN has much in common with chronic non-cancerous inflammatory conditions, such as rheumatoid arthritis, lupus, and many more. MPN and chronic inflammatory disease (CID) share similar chronicity, symptoms, dependency on the immune system, environmental triggers, and treatments. Overall, we will highlight the similarities between an MPN and CID. We highlight that while MPN is classified as a cancer, its behavior is more aligned to that of a chronic inflammatory disease. We propose that MPN should inhabit a fluid/spectrum between auto-inflammatory disease and cancer.

Burden of Symptoms and Symptom Experience of Filipino Patients with Myeloproliferative Neoplasm: A Qualitative Phenomenological Approach.

Myeloproliferative neoplasms (MPN) are a heterogeneous group of disorders characterized by the cellular proliferation of one or more hematologic cell lines. Patients with MPN who are Philadelphia-negative such as those with Polycythemia Vera (PV), Essential Thrombocytosis (ET), or Myelofibrosis (MF) experience a cluster of symptoms related to the disease activity which can affect their quality of life. This study aimed to explore the symptoms and symptom experience as well as lived experience of Filipino patients with MPN using a qualitative phenomenological approach to get a deeper understanding of the disease symptomatology. Twenty-three patients with myeloproliferative neoplasms were purposively selected according to: 1) type of MPN (PV, ET, MF) 2) status of MPN disease (newly diagnosed vs. chronic) 3) age (≤50 years old; >50 years) and 4) sex (male vs. female). The investigators conducted key informant interviews using a semi-structured interview guide. Interview scripts and narratives were transcribed and analyzed using categorical aggregation and thematic analysis. Twenty patients proceeded with the interview (8 PV, 6 ET, 6 MF). The meta-themes identified were 1) symptom experience and 2) disease perception. Three sub-themes under symptom experience were a) heterogenous and complex symptomatology; b) dynamic nature of symptoms; c) living and coping with symptoms. Three sub-themes under disease perception were a) struggle with the concept of the disease; b) anxiety and uncertainty; c) acceptance and hope. The most common symptoms experienced by the patients were fatigue, bone pain, and abdominal discomfort. Vascular symptoms specifically headache, numbness, and problems in concentration were commonly reported by patients with PV. Fever and weight loss were the least common. Sources of anxiety/uncertainty include the unpredictability of symptom occurrence and blood counts, the burden of taking maintenance medications, the financial burden of living with MPN, and the unpredictability of disease and complications. Patients with MPN had heterogenous, co-occurring, and dynamic symptoms which affected their overall productivity both at home and at work. Patients with MF had the most symptom burden while patients with ET had the least. This study provided valuable insights on disease perceptions, sources of anxiety, and coping mechanisms of patients with MPN. A deeper understanding of the symptom experience and disease perceptions of the patients will enhance the physician-patient interaction especially when discussing management options.

Successful Engraftment of Murine JAK2V617F MPN-like Disease into Unconditioned Mice

Background: JAK2V617F (JAK2VF) is the leading driver mutation present in hematopoietic cells of classical myeloproliferative neoplasms (MPN). The contribution of the non-hematopoietic compartment to MPN pathology remains obscure, although alterations and mutations in non-hematopoietic bone marrow (BM) cells (nonHC) can induce MPN-like diseases (reviewed in PMID32780848). Strikingly, the MPN-inducing JAK2VF mutation may be detected decades before MPN diagnosis, suggesting that the nonHC may act to support the expansion of the MPN clone. To examine this role of the nonHC within the BM during MPN disease, a model relying on low-level engraftment into an unaltered BM microenvironment is needed. Current transplantation models require myeloablative conditioning (irradiation) to ensure cell engraftment, which permanently alters the BM microenvironment (PMID24607941). We have developed a novel murine system that mimics JAK2VF+ MPN without the use of myeloablative conditioning, allowing the role of the BM microenvironment in the natural progression of MPN pathology to be studied. Methods: Floxed-JAK2VF (JAX#031658) were crossed with Mx1-Cre (JAX#003556) mice to produce a poly I:C-inducible MPN-like disease model (CD45.2). Five-week-old donor animals, designated JAK2MUT (Mx1-Cre(+);JAK2VF(+/-)) or JAK2CTRL (Mx1-Cre(-);JAK2VF(+/-)), were injected with poly I:C to activate the JAK2VF mutation. Five weeks later, BM was isolated and transplanted via single tail vein injection into age- and sex-matched C57BL/6 Ptprca (JAX#002014, CD45.1) recipient animals, at a concentration of 1.5x107 per animal. 10 mice (5 female, 5 male) were used as BM transplant (BMT) recipients of either JAK2MUT or JAK2CTRL BM. In addition, 10 total non-injected sex- and age-matched hosts were kept as a naïve (non-injected) group. Body weight (BW) of all 20 BMT recipients was monitored as an indicator of overall health. Blood from the submandibular vein was collected from all recipients every 4-6 weeks for complete blood cell counts (CBC) and flow cytometry analysis to determine alterations within the hematopoietic system and percentage of donor (CD45.2) chimerism within CD45.1 hosts. Results: At transplantation, JAK2MUT donors displayed advanced MPN-like disease (hematocrit (HCT) >70%, red blood cells (RBC) >1.9x107/µl, platelets (PLT) >1.1x106/µl). Over the course of the study, only male recipients receiving JAK2MUT donor cells showed a significantly different trend in weight change at 6 weeks post-transplantation with an average of 0.18% less BW gained. Hosts receiving JAK2CTRL cells displayed low engraftment efficiency <1% after 6 weeks post BMT, whereas hosts receiving JAK2MUT cells displayed ~4% observable engraftment within peripheral blood (Fig. A). Additionally, hosts receiving JAKMUT cells displayed reddening of extremities indictive of erythromelalgia, characteristic of JAK2VF+ MPN (Fig. A). When compared to donors, JAKMUT transplanted hosts displayed CBC abnormalities, characteristic of polycythemia (HCT >49%, RBC >25% naïve, unaltered levels of PLT) (PMID27956542). 2 of 10 recipients of JAK2MUT BMT died as the result of highly penetrant MPN 4-6 weeks after the transplant. Conclusion: While many ways to transplant hematopoietic cells within unconditioned animals have been described, we have successfully transplanted JAK2VF BM into the non-conditioned recipients and showed presence of diagnosable disease in animals displaying ~5% donor chimerism. Our model will offer advantages over existing methods for studying early MPN disease progression within an unconditioned murine BM. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Transcriptional and Metabolic Profiling Reveals Alpha-Ketoglutarate As a Novel Mediator of Aberrant Platelet Activity and Inflammation in Myeloproliferative Neoplasms

Prior studies by our group and others have demonstrated that platelets from patients with myeloproliferative neoplasms (MPNs) exhibit a hyperreactive phenotype. However, a complete understanding of platelet alterations in MPNs remains lacking, and the mechanisms by which platelets contribute to MPN-related thrombosis, as well as other MPN disease features, are incompletely understood. In this study, utilizing multiomic approaches to interrogate platelet phenotypes in patient samples, in conjunction with relevant MPN animal models (Figure 1A), we aim to investigate mechanisms of dysregulated platelet activity in MPNs, and explore how these findings may be leveraged to uncover novel therapeutic strategies. We initially studied platelet activation in peripheral blood from patients with essential thromobocythemia (ET) and compared to age-, sex-matched healthy controls. We found significantly increased P-selectin exposure in conjunction with increased platelet-leukocyte aggregates indicating activation of platelets from ET patients. To investigate the transcriptional signature of PBMCs and platelets at the single cell level, we performed single cell RNA-seq (scRNA-seq) in PBMCs from ET patients and healthy controls. Monocytes were increased in ET patients and displayed the highest inflammation index, implicating monocytes as the primary source of inflammation in ET. GSEA analysis revealed enrichment of platelet activation and oxidative phosphorylation (OXPHOS) genes in platelets from ET patients. Liquid chromatography-mass spectrometry (LC-MS) metabolomics analysis showed distinct metabolic phenotypes consisting of elevated tricarboxylic acid (TCA) cycle components, ATP generation and lower alpha-ketoglutarate (α-KG), in platelets from ET patients, all consistent with increased OXPHOS. α-KG is a key TCA cycle intermediate, which inhibits ATP generation via the suppression of ATP synthase. Extracellular flux analysis by Seahorse analysis confirmed bioenergetic alterations in MPN patient platelets. Enhanced mitochondrial respiration at both baseline and after ex vivo stimulation with the platelet agonist thrombin receptor activator peptide (TRAP6) was also observed from MPN patient platelets. Notably, α-KG supplementation drastically reduced oxygen consumption and ATP generation in platelets from MPN patients. We further investigated the effects of α-KG on MPN platelet activation. Ex vivo incubation of platelets from both MPN patients and Jak2 V617F knock-in mice with α-KG significantly reduced platelet surface P-selectin and integrin a2bb3 activation. Additionally, α-KG inhibited the spreading and adhesion of platelets from Jak2 V617F knock-in mice to fibrinogen-coated surfaces. Platelet phosphoblots demonstrated significant downregulation of p-STAT3, p-AKT and p-ERK after treatment with α-KG, suggesting these signaling pathways may be responsible for the inhibitory effects of α-KG on platelet activation. Thus, α-KG inhibited platelet activities in both human and mouse MPN samples. To test the therapeutic impact of α-KG on MPN disease features, we treated Jak2 V617F knock-in mice with α-KG for 6 weeks. Oral α-KG supplementation decreased splenomegaly and reduced elevated platelets and hematocrit. Additionally, monocytes were significantly decreased as early as 2 weeks after α-KG treatment in Jak2 V617F knock-in mice. In ex vivo studies with MPN patient CD34+ cells, α-KG treatment for 10 days led to a decrease in CD41+ CD61+ cells, suggesting decreased megakaryocyte commitment. We further observed that α-KG incubation significantly decreased the secretion of proinflammatory cytokines from sorted CD14+ human monocytes. Mass cytometry analysis of whole blood from MPN patients demonstrated inhibition of MAPK pathway signaling after α-KG treatment. Taken together, these results suggest that α-KG supplementation may exert therapeutic effects through both direct inhibition of MPN platelet activity and via quenching of monocyte hyper-inflammation (Figure 1B). In summary, these studies reveal a previously unrecognized metabolic disorder in platelets from MPN patients and highlight a prominent role for α-KG in aberrant MPN platelet activity and monocyte-driven inflammation. These findings have potential relevance for novel therapeutic approaches for MPN patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Jak2V617F Reversible Activation Shows an Essential Requirement for Jak2V617F in Myeloproliferative Neoplasms (MPNs)

JAK inhibitors (JAKi) improve symptoms and outcomes in MPNs; however, mutant allele burden does not substantively change with chronic therapy. Alternative MAPK signaling and/or cooperating mutations are thought to contribute to MPN cell persistence; however, we hypothesized current JAKi fail to effectively abrogate mutant JAK2 signaling. To that end, we developed a conditionally inducible mouse model allowing for sequential activation, then deletion, of Jak2VF from its endogenous locus using a Dre-rox/Cre-lox (Jak2RL) dual recombinase system (Figure 1A). Dre mRNA electroporation of lineage-negative UbcCreER-Jak2RL bone marrow (BM) was used to induce Jak2VFex vivo, followed by transplant into lethally-irradiated recipient mice in competition with Cd45.1 BM. Transplanted mice developed a highly penetrant MPN characterized by leukocytosis, polycythemia, hepatosplenomegaly, and BM megakaryocytic hyperplasia. Tamoxifen administration 12 weeks post-transplant to delete Jak2VF resulted in abrupt normalization of blood cell parameters, spleen weights, and serum pro-inflammatory cytokines, with a marked prolongation in overall survival (OS) compared to MPN control mice. Reduced peripheral blood (PB) and BM Cd45.2 mutant fraction was also observed, including within hematopoietic stem/progenitor cell (HSPC) compartments and to a greater degree than ruxolitinib (RUX) therapy. Notably, treatment with CHZ868, a type II JAKi, showed improvements in regard to hematocrit and spleen weight reduction on par with deletion, as well as significant reductions in BM HSPC mutant fractions, albeit to a lesser degree than with Jak2VF reversion (Figure 1B). Transplant of Jak2VF deleted BM failed to form phenotypic disease in secondary recipient mice consistent with loss of disease-propagating MPN stem cells. RNA-Seq on sorted LSKs following Jak2VF deletion showed loss of STAT5, MAPK, and MTORC1 target gene expression, as well as negative enrichment in TGFb and TNFa/NFkB pathways consistent with reduced pro-inflammatory and proliferative output. The alterations in MAPK signaling were supported by Ybx1 RT-PCR of BM cKit+ cells and BM pERK IHC, both of which were increased with RUX but potently suppressed with deletion suggesting improved JAK2 targeting can overcome MAPK-mediated persistence. The initial proximity of Jak2RLlox sites grants Cre-mediated recombination prior to Dre-mediated Jak2VF knock-in allowing for assessment of Jak2VF oncogenic dependency alone or in the context of co-occurring mutation. Given the known effects of Tet2 loss on enhanced MPN HSC fitness, we evaluated Tet2 loss on sustaining MPN with Jak2VF reversion. UbcCreER-Jak2RL/Tet2f/f mice were pulsed with TAM to knock-out Tet2 followed by sacrifice and Dre Jak2RL knock-in 6 weeks later. Mice transplanted with UbcCreER-Jak2VF/Tet2-/- cells exhibited enhanced leukocytosis, spleen weights and PB/BM chimerism compared to Jak2VF mice; however, Jak2VF reversion resulted in similar reductions in MPN disease parameters, including Cd45.2 fraction, suggesting Tet2loss does not dramatically alter dependence on mutant JAK/STAT signaling. Notably, while Jak2VF/Tet2-/- mutant cells displayed increased serial replating capacity, this was abrogated with Jak2VF reversion suggesting despite the enhanced fitness of Tet2-/- HSCs, Jak2VF creates a unique dependency that renders double-mutant cells susceptible to eradication with Jak2VF reversion. In sum, Jak2VF deletion abrogates MPN features, extends OS, and reduces mutant cell fraction with depletion of disease-sustaining MPN HSCs, either alone or in the setting of cooperating Tet2 loss. These data suggest JAK2VF mutant-selective inhibition offers greater therapeutic potential than current JAKi and a potential curative option for the treatment of MPN. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

High JAK2V617F allele burden is associated with an increased burden of coronary artery calcification in MPNs

Abstract Background Patients with the haematological cancers Philadelphia-negative Myeloproliferative Neoplasms (MPNs), have an increased risk of coronary artery and aortic valve calcification, and an increased risk of myocardial infarctions. The most common acquired mutation in MPNs, the JAK2V617F mutation, has been linked to increased risk of thrombosis. Aims To exam the association between the JAK2V617F mutation, its allele burden, and coronary artery and aortic valve calcification. Methods One hundred and sixty one patients with MPN disease from one specialized haematological outpatient clinic where included. Information on demographics, smoking, alcohol habits and co-morbidities were registered. Blood samples were drawn for determination of the JAK2V617 mutation and allele burden. Patients were examined by cardiac computer tomography in order to determine their coronary artery calcium score (CACS) and aortic valve calcification (AVC) score. The association between the JAK2V617F mutation, its allele burden and coronary artery and aortic calcification was investigated with univariate and multivariate logistic regression analysis, adjusting for age, sex, ischemic heart disease (IHD), stroke, smoking, obesity, hypertension, hypercholesterolemia, diabetes mellitus, and family history of IHD or stroke in the multivariate analysis. Results Of the 161 patients (52% male, mean age 65.5±10.5 years), 137 (85%) were JAK2V617F positives, and the JAK2V617F allele burden was quantified in 120. The median JAK2V617F allele burden was 12% (IQR range 6–33%). There were 42 (26%) patients with a CACS &amp;gt;400, and 93 (58%) patients AVC. Among the JAK2V617F positive patients, 38 (24%) had a CACS &amp;gt;400 and 81 (59%) had AVC. In the 32 patients with a JAK2V617F allele burden&amp;gt;33%, 19 (59%) had a CACS &amp;gt;400 and 26 (81%) had AVC. In the univariate logistic regression analysis the presence of JAK2V617F mutation was not associated with a CACS &amp;gt;400 (Odds Ratio (OR) 1.92, 95% confidence interval (CI) 0.62–5.98, p=0.26). Similar result was found for the analysis on AVC (OR 1.45, 95% CI 0.61–3.45, p=0.41). In contrast, a JAK2V617F allele burden &amp;gt;33% was significantly associated with a CACS &amp;gt;400 (OR 5.31, 95% CI 2.23–12.66, p=0.0002), and similarly with AVC (OR 4.14, 95% CI 1.55–11.05, p=0.0045). In the multivariate adjusted analysis, a JAK2V617F allele burden &amp;gt;33% was significantly associated with a CACS &amp;gt;400 (OR 2.08, 95% CI 0.43–10.10, p=0.36), but not with AVC (OR 0.90, 95% CI 0.27–3.03, p=0.86). In the adjusted analysis the association between a JAK2V617F allele burden &amp;gt;33% and CACS &amp;gt;400 was significant (OR3.86, 95% CI 1.14–13.09, p=0.031), but the analysis on AVC was not (OR 1.59, 95% CI 0.42–6.04, p=0.50). Conclusion There is a significant association between a JAK2V617F allele burden &amp;gt;33% and the burden of coronary calcification in MPNs, measured as a CACS &amp;gt;400. The association remains significant after adjustment for cardiovascular risk factors. Funding Acknowledgement Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): Region Sjællands Sundhedsvidenskabelige ForskningsfondTømrermester Jørgen Holm og hustru Elisa F. Hansens Mindelegat