JAK2V617F Cells Research Articles

JAK2V617F-dependent down regulation of SHP-1 expression participates in the selection of myeloproliferative neoplasm cells in the presence of TGF-β.

Myeloproliferative neoplasms (MPNs) are characterized by an increased production of blood cells due to the acquisition of mutations such as JAK2V617F. TGF-β, whose secretion is increased in MPN patients, is known to negatively regulate haematopoietic stem cell (HSC) proliferation. Using an isogenic JAK2V617F or JAK2 wild-type UT-7 cell line we observed that JAK2V617F cells resist to TGF-β antiproliferative activity. Although TGF-β receptors and SMAD2/3 expressions are similar in both cell types, TGF-β-induced phosphorylation of SMAD2/3 is reduced in UT-7 JAK2V617F cells compared with JAK2 WT cells. We confirmed that JAK2V617F mutated cells are resistant to the antiproliferative effect of TGF-β in a competitive assay as we observed a positive selection of JAK2V617F cells when exposed to TGF-β. Using cell lines, CD34-positive cells from MPN patients and bone marrow cells from JAK2V617F knock-in mice we identified a down regulation of the SHP-1 phosphatase, which is required for the regulation of HSC quiescence by TGF-β. The transduction of SHP-1 cDNA (but not a phosphatase inactive cDNA) restores the antiproliferative effect of TGF-β in JAK2V617F mutated cells. Finally, SC-1, a known agonist of SHP-1, antagonized the selection of JAK2V617F mutated cells in the presence of TGF-β. In conclusion, we show a JAK2-dependent down regulation of SHP-1 in MPN patients' cells which is related to their resistance to the antiproliferative effect of TGF-β. This may participate in the clonal selection of cancer cells in MPNs.

Neutrophil-specific expression of JAK2-V617F or CALRmut induces distinct inflammatory profiles in myeloproliferative neoplasia

BackgroundNeutrophils play a crucial role in inflammation and in the increased thrombotic risk in myeloproliferative neoplasms (MPNs). We have investigated how neutrophil-specific expression of JAK2-V617F or CALRdel re-programs the functions of neutrophils.MethodsLy6G-Cre JAK2-V617F and Ly6G-Cre CALRdel mice were generated. MPN parameters as blood counts, splenomegaly and bone marrow histology were compared to wild-type mice. Megakaryocyte differentiation was investigated using lineage-negative bone marrow cells upon in vitro incubation with TPO/IL-1β. Cytokine concentrations in serum of mice were determined by Mouse Cytokine Array. IL-1α expression in various hematopoietic cell populations was determined by intracellular FACS analysis. RNA-seq to analyse gene expression of inflammatory cytokines was performed in isolated neutrophils from JAK2-V617F and CALR-mutated mice and patients. Bioenergetics of neutrophils were recorded on a Seahorse extracellular flux analyzer. Cell motility of neutrophils was monitored in vitro (time lapse microscopy), and in vivo (two-photon microscopy) upon creating an inflammatory environment. Cell adhesion to integrins, E-selectin and P-selection was investigated in-vitro. Statistical analysis was carried out using GraphPad Prism. Data are shown as mean ± SEM. Unpaired, two-tailed t-tests were applied.ResultsStrikingly, neutrophil-specific expression of JAK2-V617F, but not CALRdel, was sufficient to induce pro-inflammatory cytokines including IL-1 in serum of mice. RNA-seq analysis in neutrophils from JAK2-V617F mice and patients revealed a distinct inflammatory chemokine signature which was not expressed in CALR-mutant neutrophils. In addition, IL-1 response genes were significantly enriched in neutrophils of JAK2-V617F patients as compared to CALR-mutant patients. Thus, JAK2-V617F positive neutrophils, but not CALR-mutant neutrophils, are pathogenic drivers of inflammation in MPN. In line with this, expression of JAK2-V617F or CALRdel elicited a significant difference in the metabolic phenotype of neutrophils, suggesting a stronger inflammatory activity of JAK2-V617F cells. Furthermore, JAK2-V617F, but not CALRdel, induced a VLA4 integrin-mediated adhesive phenotype in neutrophils. This resulted in reduced neutrophil migration in vitro and in an inflamed vessel. This mechanism may contribute to the increased thrombotic risk of JAK2-V617F patients compared to CALR-mutant individuals.ConclusionsTaken together, our findings highlight genotype-specific differences in MPN-neutrophils that have implications for the differential pathophysiology of JAK2-V617F versus CALR-mutant disease.

Genomic and functional impact of Trp53 inactivation in JAK2V617F myeloproliferative neoplasms

Classical myeloproliferative neoplasms (MPNs) are characterized by the proliferation of myeloid cells and the risk of transformation into myelofibrosis or acute myeloid leukemia (AML) and TP53 mutations in MPN patients are linked to AML. However, JAK2V617F has been reported to impact the TP53 response to DNA damage, suggesting potential overlapping role of TP53 inactivation in MPN. We established a mouse model showing that JAK2V617F/Vav-Cre/Trp53−/− mice displayed a similar phenotype to JAK2V617F/Vav-Cre mice, but their proliferation was outcompeted in competitive grafts. RNA-Seq revealed that half of the genes affected by JAK2V617F were affected by p53-inactivation, including the interferon pathway. To validate this finding, mice were repopulated with a mixture of wild-type and JAK2V617F (or JAK2V617F/Vav-Cre/Trp53−/−) cells and treated with pegylated interferonα. JAK2V617F-reconstituted mice entered complete hematological remission, while JAK2V617F/Vav-Cre /Trp53−/−-reconstituted mice did not, confirming that p53 loss induced interferon-α resistance. KEGG and Gene Ontology analyses of common deregulated genes showed that these genes were mainly implicated in cytokine response, proliferation, and leukemia evolution, illustrating that in this mouse model, the development of MPN is not affected by TP53 inactivation. Taken together, our results show that many genetic modifications induced by JAK2V617F are influenced by TP53, the MPN phenotype may not be. Trp53 loss alone is insufficient to induce rapid leukemic transformation in steady-state hematopoiesis in JAK2V617F MPN, and Trp53 loss may contribute to interferon resistance in MPN.

JAK2V617F Activates IL-10R Signalling Potentially through Transactivation of JAK1

Classical myeloproliferative neoplasms (MPNs) arise from somatic mutations in haematopoietic stem cells (HSCs) that lead to unregulated proliferation of mature myeloid cells. The most common mutation associated with MPNs is JAK2 V617F, this causes constitutive JAK-STAT signalling leading to excessive production of mature myeloid cells. In humans, for MPN to arise, a JAK2 V617F HSC must have a selective advantage over WT HSC. However, in mouse MPN models Jak2 V617F HSCs do not display an overt selective advantage. This suggests that unique selective pressures may be present among those predisposed to acquire MPN that allow for the emergence of Jak2 V617F clones. We have identified defects in the IL-10R signalling pathway among MPN patients, with evidence that this defect is an intrinsic not acquired abnormality. In mice, we found that the blockade of IL-10R extends the cycling of WT but not Jak2 V617FHSC in response to inflammatory stimuli and hastens proliferation-induced WT HSC exhaustion. We hypothesized that IL-10R blockade may create an environment that affords Jak2 V617F HSC a selective advantage, indeed we found that IL-10R blocking antibody allowed Jak2 V617F cells to outcompete WT cells in mouse transplant experiments. From these mouse data, we hypothesized that Jak2 V617Fenhances IL-10R signalling. To test this hypothesis, we created Ba/F3 cell lines co-expressing IL-10Rα and Jak2 V617F. We found that Jak2 V617F enhances growth at limiting concentrations of IL-10. We also found that cytokine-independent IL-10Rα Jak2 V617F clones (but never IL-10Rα Jak2 WTor IL-10Rα cells) emerge at day 8-10 in culture after withdrawal of cytokines. We performed a limiting dilution assay to pinpoint the proportion of IL-10R Jak2 V617F cells capable of surviving IL-3 withdrawal. One in 126 IL-10Rα Jak2 V617F cells has transforming ability, whereas 1 in 1.4 MPL Jak2 V617F has transforming ability. Together, these data support our hypothesis that Jak2 V617F enhances IL-10R signalling and can activate IL-10Rα, however with weaker transformation potential compared to Jak2V617F's activation of MPL signalling. JAK2 canonically does not participate in the IL-10R pathway. It has been previously shown that JAK1 can transactivate JAK2 when JAK2 V617F cells are under the stress of persistent JAK2 inhibitor exposure, and this is the purported mechanism of JAK inhibitor resistance among MPN patients. We hypothesized that JAK2 V617F can transactivate JAK1, leading to the activation of IL-10R. The same transfected BaF3 cells aforementioned were serum starved for 4 hours and stimulated with mIL-10 for 15 minutes to induce signalling. By way of Western Blot analysis, cytokine-independent Jak2 V617F IL-10Rα Ba/F3 cells showed constitutive activation of phosphorylated JAK1 and JAK2 as well as STAT3 and STAT5, consistent with transactivation. Studies to demonstrate direct interactions between JAK2 V617F and JAK1 are currently ongoing.

Expansion and Blunted Inflammatory Responses of Jak2 V617F Cells in the Setting of Electronic Cigarette Aerosol Inhalation

Clonal hematopoiesis of intermediate potential (CHIP) increases the risk for hematological malignancies and cardiovascular disease with Jak2 being a commonly mutated gene in CHIP. Age is the strongest determinant of CHIP but environmental factors also play a role since only a fraction of the individuals with CHIP eventually present with overt clonal hematopoiesis. The popularity of electronic (E-) cigarette (E-cig) usage has increased but their health effects are not well-established. E-cigarettes have been associated with inflammation and oxidative stress and these can provide a selective pressure for the outgrowth of CHIP mutant cells. Here, we determined the impact of E-cig aerosol inhalation on the survival and/or expansion of Jak2 mutant hematopoietic cells by exposing bone marrow transplanted mice to E-cig aerosols. Competitive bone marrow transplant assays were established to determine expansion of Jak2V617F mutant cells to E-cigarette aerosols. Lethally irradiated recipient mice in the CD45.1/2 background were transplanted with equal numbers of whole bone marrow cells from wild type (WT) (CD45.1) and Jak2V617F(CD45.2) mice (WT:Jak2V617F), or WT (CD45.1) and WT (CD45.2) (WT:WT) as transplant controls. Eight weeks post-transplant, mice were exposed to E-cigarette aerosols using a nose-only inhalation exposure system for 2 hours/day, 4 days/week for 3 months. Control mice were exposed to room air using the nose-only inhalation approach. At regular intervals, peripheral blood was drawn to determine the percentage of WT and Jak2V617F cells by flow cytometry. At euthanasia, the percentage of WT and mutant cells in the bone marrow (BM) and spleen was determined. In competitive bone marrow transplants in a 1:1 ratio, this mouse model of Jak2V617F does not gain a selective advantage in relation to WT cells. Instead, we observe declining levels of Jak2V617F cells in the peripheral blood over time. The time point at which the Jak2V617F cells were at their minimum was recorded as time point 0 (T0). Mice were exposed for 12 weeks and then allowed to rest, to determine how E-cig exposure promotes Jak2V617F expansion, until termination of the experiment (Tend). Mice exposed to E-cig aerosols demonstrated significant expansion of Jak2V617F cells at the end of the experiment compared to air exposed controls (Wilcoxon paired test, p<0.05). Jak2V617F Lin-, c-Kit+, Sca-1+ (LKS) cells in the bone marrow were also slightly increased in the E-cig group compared to the air group. To assess how inflammation is altered in the setting of prior E-cig aerosol inhalation, bone marrow derived macrophages (BMDMs) from exposed mice were stimulated with lipopolysaccharide (LPS) and secretion of the pro-inflammatory cytokines, TNFα and IL-6, were quantified by ELISA. WT:WT BMDMs from the E-cig cohort produced more TNFα and IL-6 than the WT:WT BMDMs from the air cohort. However, interestingly E-cig exposed WT:Jak2V617F BMDMs produced significantly less TNFα and IL-6 than the E-cig exposed WT:WT BMDMs. This indicates that prior E-cig aerosol inhalation can sensitize WT cells to inflammatory stimuli while Jak2V617F mutant cells are more resistant to LPS mediated inflammation post E-cig exposure. This is particularly relevant in inflammation-induced hematopoietic stem cell exhaustion since a diminishing pool of WT HSCs provides selection pressure for the outgrowth of mutant HSCs. Environmental factors such as combustible cigarette smoke and E-cigarette products can play a role in the clinical consequences of CHIP. We observed that in vivo exposure to E-cigarette aerosols supports the survival and expansion of Jak2V617F cells in bone marrow transplanted mice. Our data also indicates that E-cig exposure sensitizes WT cells to inflammation more than Jak2V617F mutant cells. Future work is aimed at determining E-cig exposure mediated inflammatory and apoptotic effects on WT and mutant HSCs and subsets of mature hematopoietic cells.

Effect of Integrin α 4β 1 -VCAM-1 Interactions in JAK2V617F Myeloproliferative Neoplasm Pro-Thrombotic Interactions

Introduction: The JAK2 V617Fmutation, which occurs in up to 95% of polycythemia vera (PV) myeloproliferative neoplasm (MPN) patients, increases thrombosis risk 6-fold. A recent study in PV mice demonstrated that blocking JAK2 V617F+ leukocyte-mediated β1/β2 integrin interactions with anti-VCAM-1 antibodies decreases thrombosis. Interestingly, hydroxyurea (HU), a common PV treatment, reduces leukocyte and red cell α4β1 integrin expression. Overall, JAK2 V617F+ mouse models show inconsistent thrombosis and bleeding phenotype, hindering pre-clinical study reproducibility; therefore, we have developed an in vitro endothelialized microfluidic model to assess MPN-flow behavior. We hypothesized that in our microfluidic model, disrupting α4β1 integrin expression on JAK2 V617F+ leukocytes would reduce pro-adhesive interactions. We tested this hypothesis by assessing JAK2 V617F+ MPN blood flow in untreated and hydroxyurea treated subjects and through addition of a monoclonal antibody against α4β1 integrin, natalizumab. Methods: Primary human umbilical vein endothelial cells (HUVECs) were exposed to venous shear (15 dynes/cm 2) in microfluidics for a 4-day culture period. On day 4, endothelialized microfluidics were treated +/- 10 ng/mL TNFα for 4 hours. Blood samples were obtained from consented JAK2 V617F MPN individuals aged >18 years with ET, PV, or MF as diagnosed by the WHO 2016 Criteria identified in the M Health Fairview Masonic Cancer Center Clinics. Healthy age, race, and sex-matched blood samples served as controls. Clinical data on patients was collected and stored in REDCAP. Platelets and leukocytes were visualized using Calcein AM. Samples were treated +/- 0.25 μg/mL natalizumab for 30 minutes, then perfused through the microfluidic under a microscope to observe blood-endothelial interactions in real-time. Post-experiment, videos were analyzed to quantify velocity and adhesion of platelets and leukocytes for each treatment condition. Results: When perfused over TNF-α activated endothelium, both controls ( JAK2 negative, n=11) and JAK2 V617F MPN (n=20) had a significant reduction in cell velocity. Furthermore, compared to controls, JAK2 V617F MPN individuals exhibited significantly lower cell velocities. In multi-variate analysis, reduction in cell velocity was independent of white blood cell, hemoglobin, hematocrit or platelet counts. Hydroxyurea is a standard therapy in JAK2 V617F MPN and is known to reduce α4β1 expression. Therefore, we performed subgroup analysis in our JAK2 V617F MPN cohort based on treatment. When perfused over TNF-α activated endothelium, individuals with JAK2 V617F MPN (n=9) not on therapy (newly diagnosed) or on phlebotomy/aspirin only had a significant reduction in cell velocity. In the same conditions, HU-treated JAK2 V617F MPN patients (n=9) had no change in cell velocity. There was no difference in complete blood counts between these groups. In a separate subgroup analysis, compared to untreated JAK2 V617F MPN patients (n=9), interferon-treated JAK2 V617F MPN patients (n=5) had a greater reduction in cell velocity. Last, we specifically targeted α4β1 integrin by adding natalizumab to either control (n=7) or untreated JAK2 V617F MPN (n=3) samples. On inactivated endothelium, treatment of either control or JAK2 V617F MPN blood with natalizumab did not significantly change cell velocity. On a TNF-α activated endothelium, treatment of control (n=7) and JAK2 V617F MPN blood (n=3) with natalizumab led to a recovery in cell velocity. Conclusion: Our data suggests that the interaction between integrin α4β1 on JAK2 V617F cell surface and VCAM-1 on activated endothelium is important for initiation of pro-thrombotic interactions. JAK2 V617F MPN individuals treated with HU exhibit decreased adhesion to activated endothelium compared to control; JAK2 V617F MPN individuals treated with interferon exhibit increased adhesion. In the microfluidic system, based on current findings, blockage of integrin α4β1-VCAM-1 interactions using natalizumab is a potential therapeutic strategy. Further mechanistic studies evaluating interferon and cell populations changed by therapies as they pertain to integrin α4β1 are underway.

DUSP1 Specific Protac Selectively Targets the JAK2 V617F Expressing Cells

Constitutive JAK-STAT signaling resulting from mutations in JAK2 V617F and certain growth-factor receptors has provided a strong rationale for targeting JAK2 kinase activity in myeloproliferative neoplasms (MPNs). Although several JAK2 inhibitors have been approved for MPN treatment, their clinical efficacy is limited to alleviating the constitutional symptoms associated with inflammatory cytokine signaling. These inhibitors are not cytotoxic and only exert a cytostatic response, failing to eradicate the malignant clone, induce molecular remission, or prevent progression to acute myeloid leukemia (AML). The inability of JAK2 TKIs to fully suppress the inflammatory cytokine overproduction, a key feature of MPN development and transition to AML, contributes to clonal persistence and TKI refractoriness. Recently, we reported that the cytokine signaling, in the context of JAK2 V617F signaling induces the expression of DUSP1, which enhances P53 degradation that leads to TKI refractoriness. Enhanced DUSP1 expression blocks P53 phosphorylation at Serine 15 (S15 phosphorylation prevents its interaction with MDM2) by inhibiting the P38 kinase activity. Consequently, reduced phosphorylation under JAK2 V617F signaling enhances P53 turnover. Deletion of DUSP1 enhances P38 kinase activity, leading to greater P53 stabilization and expression, which selectively induce cell death in JAK2 V617F expressing MPN cells while sparing normal cells. However, chemical inhibition of DUSP1 using a small molecule inhibitor (BCI) was ineffective in achieving synthetic lethality to JAK2 V617F cells, mainly due to pERK1/2 rebound caused by the off-target inhibition of DUSP6. Reintroduction of DUSP6 and BCI treatment restored JAK2 V617F clonal selectivity. This study provided the first mechanistic basis of TKI refractoriness in JAK2 targeted MPNs and suggested that selectively targeting DUSP1 could offer a stable or curative response in JAK2 V617F -driven MPNs. To address this, we developed a DUSP1-protac, a novel and highly potent DUSP1 selective PROTAC. Treatment of BaF3 cells expressing JAK2 V617F with DUSP1-protac resulted in dose- and time-dependent degradation of DUSP1, accompanied by increased P38 activity and P53 stabilization. The maximal level of DUSP1 degradation (Dmax) achieved was 95% with a DC50 of 70 nM/L within 24 hours. Pre-treatment with the proteasome inhibitor MG132, but not the lysosomal inhibitor chloroquine, reversed DUSP1 degradation, confirming that degradation occurs via the 26S-proteasome. As expected, DUSP1-protac induced cytotoxicity specifically in JAK2 V617F expressing cells, with a cellular IC50 of 50 nM. No toxicity was observed in BaF3 cells or those expressing MPL or CALR variants at concentrations up to 1000 nM, confirming its selectivity. In conclusion, our data suggest that DUSP1-protac is an exceptionally potent first-in-class DUSP1 selective PROTAC. It exerts a cytotoxic response specifically in JAK2 V617F expressing cells by enhancing P53 expression. Cellular uptake experiments and efficacy studies in primary cells are currently underway and will be presented in subsequent reports. The development of DUSP1-protac represents a promising approach for targeting JAK2 V617F -driven MPNs, offering potential benefits in achieving a stable or curative therapeutic response.

Fedratinib Overcomes Ruxolitinib Resistance through Inhibition of the Interferon Signaling Pathway

Background: Myelofibrosis (MF) is a clonal myeloproliferative neoplasm characterized by hyperactive JAK-STAT signaling, bone marrow fibrosis, extramedullary hematopoiesis and splenomegaly. Ruxolitinib (RUX) and Fedratinib are both JAK inhibitors used for the treatment of patients with intermediate or higher risk MF. While RUX is the first line treatment for patients with MF, about 50% of the patients discontinue treatment due to lack of efficacy or intolerance (Harrison et al., Ann. Hematol. 2020) after 3 years. Fedratinib is used as a second line therapy in MF based on efficacy from the JAKARTA2 study. The recently concluded FREEDOM and FREEDOM2 studies further support this. In this work, using in vitro approaches, we have explored the mechanistic basis of fedratinib's ability to overcome RUX resistance. Pharmacologically, Fedratinib binds with high affinity to the substrate-binding site and with low affinity to the ATP-binding site of JAK2(Kesarwani etal., Sci. Rep. 2015) while RUX binds only to the ATP-binding site. Previous studies on RUX resistance have shown acquired mutations driving resistance to RUX but not to Fedratinib. Here, we use RUX sensitive and resistant BaF3 (expressing human JAK2 WT or V617F) cell lines to examine the mechanism of resistance to RUX and the mechanism of efficacy of Fedratinib in RUX resistance. Methods: BaF3 cell lines overexpressing JAK2 WT or JAK2 activating mutation V617F were incrementally exposed to RUX over 3 weeks (up to IC 90) to induce resistance. Cell proliferation and phosphorylation of STAT5 in Fedratinib and RUX-treated cells were measured using celltiter glo and immunoblotting, respectively. RNAseq and ATAC-seq analyses were performed in triplicates by treating the relevant cell lines with DMSO, RUX or Fedratinib at two timepoints (6 and 15 hours). Pathway analysis was performed on RNAseq data using Metacore®. Results and Discussion: Ruxolitinib resistant BaF3 JAK2V617F cells showed minimal inhibition of proliferation (<10%) when treated with RUX but maintained sensitivity towards Fedratinib (IC 50 650nM vs 1552nM in RUX sensitive and resistant cells respectively) in 24-hour proliferation assays. Biochemical analysis revealed that Fedratinib inhibited JAK-STAT signaling in the RUX resistant cell line while RUX was unable to do so as measured by p-STAT5. Similar results were observed in BaF3-JAK2-WT cells as well. To further understand the mechanism of RUX-resistance and differential activity of RUX and Fedratinib, we performed RNAseq analysis on our RUX sensitive and resistant models before and after RUX and Fedratinib treatment. Myelofibrosis is associated with aberrant cytokine production and pro-inflammatory signaling that are mediated through activation of TNF-α and NF-κB signaling (Dunbar et al., Blood 2023). Consistent with this, RNAseq analysis of BaF3 JAK2 V617F cells treated with RUX or Fedratinib showed effective inhibition of these pathways by both agents as early as 6 hours. In the RUX resistant cells, while RUX did not have any significant pathway level changes, the top enriched pathways upon treatment with Fedratinib were predominantly interferon signaling pathways which includes JAK-STAT signaling suggesting that Fedratinib's efficacy in MF is not impacted by RUX resistance. To understand the mechanism of resistance, comparison of gene expression data from RUX senstive and RUX resistant cells without any drug treatment revealed minimal gene expression/pathway level differences suggesting that the mechanism of resistance could be driven by genetic (Downes et al. NPJ Precis Oncol, 2021) or epigenetic changes(Dunbar et al., Blood 2023). Ongoing analyses includes whole genome sequencing and ATAC-seq to investigate potential mutations and chromatin accessibility changes during resistance acquisition to RUX and how Fedratinib may overcome these. Conclusion: Our stable RUX resistant preclinical model demonstrates that Fedratinib is effective in overcoming RUX resistance by continuing to target key pathways in MPN biology including interferon signaling. Resistance to RUX on the other hand seems to be driven through potential genetic or epigenetic changes based on the minimal changes observed in gene expression profiles. Our ongoing analyses will reveal these potential genetic and epigenetic modifications associated with RUX resistance and explain the differential activity of RUX versus Fedratinib.

Use of Combination Therapies Including the XPO1 Inhibitor Selinexor in Is a Potential Effective Therapeutic Strategy to Treat Myelofibrosis Patients

JAK1/2 inhibitor therapy reduces the degree of splenomegaly and improves systemic symptoms of myelofibrosis (MF) patients, but does not delay disease progression and modestly prolongs overall survival. HDM2 antagonists (nutlins) have been reported to selectively eliminate MF CD34 + cells by activating the p53 pathway. Nutlins alone target malignant cells with wild type (WT) p53 and in clinical trials, HDM2 antagonists have led to reductions in spleen size, improvement of systemic symptoms and reduction in MPN driver mutation allelic burdens (Lu et al, Blood.2012; Mascarenhas et al, Blood. 2019). Although most patients with MF have WT TP53, mutations are present in a limited number of such patients and are associated with poor outcomes. We have searched for combinations of drugs to serve as a more effective and tolerable MF stem cell depleting therapy. Exportin 1 (XPO1) mediates the nuclear export of various proteins and RNAs (Azmi, et al. Nat Rev Clin Oncol. 2021) including tumour-suppressor proteins and growth regulators, such as RB1, p53, p63, p73, p21, cyclin B1 or D1 (Azmi, 2021). XPO1 is frequently overexpressed and/or mutated in human cancers and functions as an oncogenic driver. Suppression of XPO1-mediated nuclear export has been shown to be a potential therapeutic strategy to treat MF patients (Yan, et al. Clin Cancer Res. 2019). Selinexor (SEL) is an investigational, oral XPO1 inhibitor that may inhibit multiple pathways relevant in MF including JAK/STAT, ERK, and AKT (Tantravahi, ASH 2021). In the current study, we tested the effects of SEL on apoptosis, and cell cycle arrest in JAK2V617F cells with either TP53WT or TP53mut. We also evaluated the ability of SEL alone to eliminate MF CD34 + cells and in combination with other candidate drugs such as HDM201 (HDM2 antagonist), ruxolitinib (RUX), pan-BET inhibitor (JQ1) or a Bcl-2/Bcl-xL inhibitor (navitoclax). Treatment with SEL alone induced apoptosis of both TP53WT (UKE-1) and TP53mut (SET-2) MPN CD34 + cell lines in a dose-dependent fashion. The transcript levels of TP53 as well as its downstream genes, such as p21, PUMA, NOXA (intrinsic apoptotic pathway) and DR5 and Trail (extrinsic apoptotic pathway) were increased by 2-3 fold after SEL treatment of TP53WT and TP53mut cells. Importantly, SEL also increased TP63 and TP73 by ~2 fold but decreased the transcript levels of the anti-apoptotic Bcl-xL and c-Myc (by 20% and 50%), TP63 and TP73 are TP53 family members which promote p53 downstream events independently of WT p53. In addition, treatment with SEL for 2 days increased the proportion of cells in sub-G0/G1 phase from 16% (control) to 60% in UKE-1 cells and from 15% (control) to 53% in SET-2 cells. SEL and HDM201 alone induced apoptosis of primary MF but not healthy donor (HD) CD34 + cells in a dose dependent fashion. The combination of SEL with suboptimal doses of either HDM201, RUX, JQ1 or navitoclax showed additive pro-apoptotic effects on MF CD34 + cells. Colony formation assays showed that SEL, HDM201 and navitoclax alone decreased CFU-GM colony numbers generated by MF CD34 + cells in a dose-dependent fashion, yet had no effects on HD CD34 + cells, while RUX reduced both MF and ND colony formation. Combination treatment with SEL and 50nM of HDM201 or RUX led to a greater degree of reduction of MF CFU-GM colony numbers, but only SEL+HDM201 did not affect HD CFU-GM colony numbers (Figure 1). Furthermore, JAK2 genotyping showed that treatment with SEL, Rux and HDM201 alone decreased the absolute number of JAK2V167F+ colonies. Furthermore, SEL+HDM201 and SEL+RUX decreased the number of JAK2V617F+ colonies to greater extent than either drug alone, but SEL+HDM201 treatment led to the persistence of a greater numbers of JAK2WT colonies than SEL+RUX (Table 1). SEL+navitoclax did not further decrease JAK2V167F+ colonies than SEL alone (data not showed). These data suggested that SEL induces apoptosis and inhibits MPN CD34 + cell proliferation by affecting both p53-dependent and -independent pathways. SEL alone induces apoptosis and inhibits MF progenitor cells but not HD cells. The effects of SEL combined with either RUX or an HDM2 antagonist more effectively depleted mutated MF CD34+ cells than either drug alone. The combination of SEL+HDM201 spared the reservoir of WT progenitors, and reduced JAK2 mutated progenitor cells to a great degree suggesting that this combination might be associated with less hematological toxicity and greater efficacy.

The telomerase inhibitor imetelstat differentially targets JAK2V617F versus CALR mutant myeloproliferative neoplasm cells and inhibits JAK-STAT signaling.

Imetelstat shows activity in patients with myeloproliferative neoplasms, including primary myelofibrosis (PMF) and essential thrombocythemia. Here, we describe a case of prolonged disease stabilization by imetelstat treatment of a high-risk PMF patient enrolled into the clinical study MYF2001. We confirmed continuous shortening of telomere length (TL) by imetelstat treatment but observed emergence and expansion of a KRAST58I mutated clone during the patient's clinical course. In order to investigate the molecular mechanisms involved in the imetelstat treatment response, we generated induced pluripotent stem cells (iPSC) from this patient. TL of iPSC-derived hematopoietic stem and progenitor cells, which was increased after reprogramming, was reduced upon imetelstat treatment for 14 days. However, while imetelstat reduced clonogenic growth of the patient's primary CD34+ cells, clonogenic growth of iPSC-derived CD34+ cells was not affected, suggesting that TL was not critically short in these cells. Also, the propensity of iPSC differentiation toward megakaryocytes and granulocytes was not altered. Using human TF-1MPL and murine 32DMPL cell lines stably expressing JAK2V617F or CALRdel52, imetelstat-induced reduction of viability was significantly more pronounced in CALRdel52 than in JAK2V617F cells. This was associated with an immediate downregulation of JAK2 phosphorylation and downstream signaling as well as a reduction of hTERT and STAT3 mRNA expression. Hence, our data demonstrate that imetelstat reduces TL and targets JAK/STAT signaling, particularly in CALR-mutated cells. Although the exact patient subpopulation who will benefit most from imetelstat needs to be defined, our data propose that CALR-mutated clones are highly vulnerable.

Targeting PP2A-dependent autophagy enhances sensitivity to ruxolitinib in JAK2V617F myeloproliferative neoplasms

The Janus kinase 2 (JAK2)-driven myeloproliferative neoplasms (MPNs) are chronic malignancies associated with high-risk complications and suboptimal responses to JAK inhibitors such as ruxolitinib. A better understanding of cellular changes induced by ruxolitinib is required to develop new combinatory therapies to improve treatment efficacy. Here, we demonstrate that ruxolitinib induced autophagy in JAK2V617F cell lines and primary MPN patient cells through the activation of protein phosphatase 2A (PP2A). Inhibition of autophagy or PP2A activity along with ruxolitinib treatment reduced proliferation and increased the death of JAK2V617F cells. Accordingly, proliferation and clonogenic potential of JAK2V617F-driven primary MPN patient cells, but not of normal hematopoietic cells, were markedly impaired by ruxolitinib treatment with autophagy or PP2A inhibitor. Finally, preventing ruxolitinib-induced autophagy with a novel potent autophagy inhibitor Lys05 improved leukemia burden reduction and significantly prolonged the mice’s overall survival compared with ruxolitinib alone. This study demonstrates that PP2A-dependent autophagy mediated by JAK2 activity inhibition contributes to resistance to ruxolitinib. Altogether, our data support that targeting autophagy or its identified regulator PP2A could enhance sensitivity to ruxolitinib of JAK2V617F MPN cells and improve MPN patient care.

MAPK-negative feedback regulation confers dependence to JAK2V617F signaling.

Despite significant advances in developing selective JAK2 inhibitors, JAK2 kinase inhibitor (TKI) therapy is ineffective in suppressing the disease. Reactivation of compensatory MEK-ERK and PI3K survival pathways sustained by inflammatory cytokine signaling causes treatment failure. Concomitant inhibition of MAPK pathway and JAK2 signaling showed improved in vivo efficacy compared to JAK2 inhibition alone but lacked clonal selectivity. We hypothesized that cytokine signaling in JAK2V617F induced MPNs increases theapoptotic threshold that causes TKI persistence or refractoriness. Here, we show that JAK2V617F and cytokine signaling converge to induce MAPK negative regulator, DUSP1. Enhanced DUSP1 expression blocks p38 mediated p53 stabilization. Deletion of Dusp1 increases p53 levels in the context of JAK2V617F signaling that causes synthetic lethality to Jak2V617F expressing cells. However, inhibition of Dusp1 by a small molecule inhibitor (BCI) failed to impart Jak2V617F clonal selectivity due to pErk1/2 rebound caused by off-target inhibition of Dusp6. Ectopic expression of Dusp6 and BCI treatment restored clonal selectively and eradicated the Jak2V617F cells. Our study shows that inflammatory cytokines and JAK2V617F signaling converge to induceDUSP1, which downregulates p53 and establishes a higher apoptotic threshold. These data suggest that selectively targeting DUSP1 may provide a curative response in JAK2V617F-driven MPN.

GLS in Philadelphia-negative and JAK2 V617F-driven myeloproliferative neoplasms (MPNs).

e15092 Background: Philadelphia-negative myeloproliferative neoplasms (MPNs) are clonal hematopoietic malignancies. Nearly all MPNs are driven by somatic mutations in either: JAK2, CALR, or MPL. Although all three MPN driver mutations lead to constitutive activation of JAK/STAT signaling, JAK inhibitors are not curative and fail to alter disease progression and display unwanted side effects. Allogeneic stem cell transplantation remains the only curative therapy for MPNs, but is associated with substantial morbidity and mortality. Recently, glutaminolysis has been shown to play a critical role in cancer cell metabolism. During glutaminolysis, glutamine is metabolized through in a two-step reaction, the first of which involves the enzyme glutaminase (GLS) catalyzing the hydrolysis of glutamine to glutamate. Glutamate can then fuel for energy production into the TCA cycle, among several other possible metabolic fates. As many cancers have proven to be dependent on this pathway, targeting GLS has become an attractive therapeutic avenue. Because glutaminolysis has been understudied in MPNs, we sought to determine whether this pathway represents a novel, targetable vulnerability in MPNs. Methods: We evaluated the mRNA levels of GLS in peripheral blood mononuclear cells from 30 MPN patients and 5 healthy donors. We tested by qPCR for GLS and the protein levels of GLS by western blot in TF-1 cells, a human myeloid cell line, stably overexpressing either JAK2, MPL, or CALR mutated proteins. The latter also under treatment with the JAK inhibitor ruxolinitinib. We also evaluated the GLS enzyme activity through the use of a fluorimetric assay. Lastly, we tested the sensitivity of MPN cells to GLS inhibition with a GLS inhibitor, CB-839, which is currently in advanced phase clinical trials for other cancers including Myelodysplastic Syndromes. Results: GLS mRNA expression was increased in all MPN patients regardless of their driver mutation, where expression in JAK2V617F patients was higher in MF versus ET patients (p < 0.001). GLS protein expression and activity were increased in TF-1 cells expressing JAK2, MPL, and CALR mutations. We also found that GLS mRNA and protein expression was up-regulated in a JAK/STAT-dependent manner. Interestingly, despite increased expression of GLS across all MPN driver mutations, only JAK2 V617F cells demonstrated significant sensitivity to GLS inhibition with CB-839 in vitro and with preliminary data in vivo. We found that combination treatment with JAK inhibitor ruxolitinib further inhibited cell viability. Conclusions: GLS up-regulation is a common feature of all MPNs, and is JAK/STAT dependent. JAK2 V617F expressing cells show significant sensitivity to GLS inhibition. Combination treatment with ruxolitinib further enhances this effect. Treatment with CB-839 may thus represent a novel therapeutic avenue for JAK2 V617F+ MPNs.

Targeting RSK1-Mediated Dependency and Inflammation in Myeloid Malignancies

Myeloproliferative neoplasms (MPNs) are a group of hematological malignancies driven by hyperactive JAK-STAT signaling. Disease progression and potential transformation to secondary acute myeloid leukemia (sAML) are a consequence of the complex interplay between oncogenic drivers, distinct cellular populations, and temporal interactions, and most recently, a fourth pillar encompassing the pro-inflammatory cytokine milieu driven in part by NFκB pathway signaling. As such, current therapeutic approaches primarily targeting JAK2 demonstrate symptom control but are not curative, thus necessitating advances into novel approaches that overcome these combinatorial risk factors. We previously demonstrated that RPS6KA1 (RSK1) is overexpressed in AML, critical for cell survival, and associated with worse patient survival outcomes. Although RSK1 remains a promising therapeutic target, current pre-clinical inhibitors demonstrate poor pharmacokinetic profiles, thus limiting further translational development. Here, we perform mechanistic studies to characterize and exploit this RSK1 dependency in MPN/sAML. We further target RSK1 in vitro, ex vivo, and in vivo with PMD-026, a first-in-class oral RSK1-4 inhibitor currently undergoing Phase 1/1b clinical trials in patients with metastatic breast cancer. We first performed RNA-sequencing on CD34+ cells from 45 MPN patients and 11 healthy donors. Gene set enrichment analysis revealed augmented TNF/NFκB, JAK-STAT and PI3K/AKT/mTOR signaling pathways in myelofibrosis (MF) patients with elevated RPS6KA1 expression. RNA-seq of CD34+ cells from an additional 34 sAML patients showed similar enhancement of driver pathways. Following, in JAK2-V617F cell lines, PMD-026 suppressed oncogenic RSK1, mTOR, and S6 signaling and induced apoptosis and cell cycle arrest. Ex vivo, PMD-026 preferentially inhibited colony formation of MPN CD34+ cells compared to those from healthy donors, while RPS6KA1 knockdown also inhibited colony formation from diseased but not healthy donors. Moreover, PMD-026 potently alleviated leukocytosis and disease burden in conjunction with prolonged survival in the MPL W515L retroviral MF mouse model, both alone and in combination with ruxolitinib (Fig. A). Similar elimination of leukemic cells was observed in patient-derived xenograft NSGS mice that were transplanted with CD34+ cells from an sAML patient after RPS6KA1 perturbation. These results indicate potency and specificity of RSK1 inhibition in targeting MPN/sAML. Moreover, we observed elevated expression of RPS6KA1 in patients with acute myelomonocytic (M4) and monocytic (M5) leukemia, compared to other AML FAB subtypes, in addition to correlations between RPS6KA1 and CD14 expression across our MPN, TCGA LAML and BeatAML cohorts. We thus performed further RNA-seq on CD34- CD14+ monocytes from 54 MPN patients and 14 healthy donors (Fig. B). Differential gene and pathway analyses revealed consistent upregulation of JAK-STAT, inflammatory cytokine, and TNF/NFκB pathways, and enrichment of key effectors including TNF, IL6, CCL4, and CCL5 in patient monocytes. Following, we utilized high-resolution, multi-omic approaches in single cell RNA-seq and mass cytometry and observed monocyte-driven enrichment of inflammatory and NFκB signatures with concurrent elevation of RPS6KA1 expression in monocytes concordant with disease progression in serial samples at MF post-PV compared to antecedent PV of an MPN patient. Lastly, we investigated whether RSK1 inhibition could also suppress disease progression in part through modulating this monocyte-mediated inflammatory niche. Acute PMD-026 treatment of MPN patient CD14+ monocytes resulted in suppression of TNF, IL6, CXCL8, CCL4 and CCL5 cytokine mRNAs, while simultaneously reducing NFKB1 and NFKB2 expression. At the protein level, PMD-026 also inhibited NFκB signaling by reversal of TNF-induced IκB⍺ degradation and p65/RelA S536 phosphorylation in JAK2 mutant cell lines. Thus, RSK1 may play a critical role in augmenting the pro-inflammatory microenvironment through potent effector monocytes. Our findings are first in demonstrating pre-clinical efficacy of a clinical grade RSK1 inhibitor, PMD-026, for the treatment of myeloid malignancies and highlight RSK1 inhibition to be two-pronged, by suppressing oncogenic signaling and inhibiting the CD14+ monocyte-driven pro-inflammatory milieu. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Efficacy of WWQ-131, a highly selective JAK2 inhibitor, in mouse models of myeloproliferative neoplasms

Hyperactivation of the Janus kinase 2 (JAK2) signaling pathway leads to myeloproliferative neoplasms (MPNs) and targeting JAK2 can be used as an effective strategy for the treatment of MPNs. Here, our study indicated that WWQ-131 was a highly selective JAK2 inhibitor (IC50 =2.36 nM), with 182-fold and 171-fold more selective to JAK1 and JAK3, respectively. In JAK2V617F-dependent cell lines, WWQ-131 efficaciously inhibited cell proliferation, induced cell cycle arrest at the G2/M phase and apoptosis, and blocked the aberrant activation of JAK2 signaling pathway. In a mouse Ba/F3_JAK2V617F driven disease model, WWQ-131 effectively suppressed STAT5 phosphorylation in spleen and liver, and inhibited Ba/F3_JAK2V617F cells spreading and proliferation in vivo. In addition, WWQ-131 suppressed rhEPO-induced extramedullary erythropoiesis and polycythemia in mice, as well as hematocrits and spleen sizes, especially had no effect on white blood cell count. Furthermore, WWQ-131 (75 mg/kg) exhibited stronger therapeutic effects than fedratinib (120 mg/kg) in these two MPN models. Taken together, this study suggests that WWQ-131 will be a promising candidate for the treatment of MPNs.

Inhibition of Osteoblast Differentiation by JAK2V617F Megakaryocytes Derived From Male Mice With Primary Myelofibrosis.

Past studies described interactions between normal megakaryocytes, the platelet precursors, and bone cell precursors in the bone marrow. This relationship has also been studied in context of various mutations associated with increased number of megakaryocytes. The current study is the first to examine the effects of megakaryocytes from transgenic mice carrying the most common mutation that causes primary myelofibrosis (PMF) in humans (JAK2V617F) on bone cell differentiation. Organ level assessments of mice using micro-computed tomography showed decreased bone volume in JAK2V617F males, compared to matching controls. Tissue level histology revealed increased deposition of osteoid (bone matrix prior mineralization) in these mutated mice, suggesting an effect on osteoblast differentiation. Mechanistic studies using a megakaryocyte-osteoblast co-culture system, showed that both wild type or JAK2V617F megakaryocytes derived from male mice inhibited osteoblast differentiation, but JAK2V617F cells exerted a more significant inhibitory effect. A mouse mRNA osteogenesis array showed increased expression of Noggin, Chordin, Alpha-2-HS-glycoprotein, Collagen type IV alpha 1 and Collagen type XIV alpha 1 (mostly known to inhibit bone differentiation), and decreased expression of alkaline phosphatase, Vascular cell adhesion molecule 1, Sclerostin, Distal-less homeobox 5 and Collagen type III alpha 1 (associated with osteogenesis) in JAK2V617F megakaryocytes, compared to controls. This suggested that the mutation re-programs megakaryocytes to express a cluster of genes, which together could orchestrate greater suppression of osteogenesis in male mice. These findings provide mechanistic insight into the effect of JAK2V617F mutation on bone, encouraging future examination of patients with this or other PMF-inducing mutations.

S193: GENOMIC AND FUNCTIONAL IMPACT OF TP53 INACTIVATION IN JAK2V617F MYELOPROLIFERATIVE NEOPLASMS: A TRANSGENIC MOUSE MODEL APPROACH.

Background: MPN are characterized by increased proliferation of myeloid cells and a risk of transformation to AML. MPN are due to the acquisition of mutations (JAK2, CALR, MPL) leading to JAK2 pathway activation but cooperating mutations involved in MPN evolution have been described. Mutations of TP53 in JAK2V617F patients are linked to AML transformation, illustrated by development of leukemias after retroviral overexpression of JAK2V617F in TP53 inactivated (KO) cells transplanted in irradiated animals, thus a possible cooperation between TP53 inactivation and JAK2 signaling in transformation process has been postulated. However, JAK2V617F signaling has also been reported as able to reduce p53 expression per sesuggesting a redundant role of p53 inactivation in JAK2V617F cells. Aims: Determine the role of TP53 in JAK2V617F induced genetic and phenotypic modifications. Methods: To better understand the role of TP53 in MPN phenotype and evolution in steady state hematopoiesis, we developed a transgenic model of vav-cre induced JAK2V617F expression in TP53 knock-out (KO) animals and analyzed phenotype, survival, genetic modifications using stem cell compartments RNA-Seq and response to therapy of this triple transgenic mice. Results: JAK2V617F vav-inductible-Tg/TP53 KO mice were generated to recombine JAK2V617F at an endogenous level in TP53 inactivated hematopoietic cells. During follow-up, JAK2V617F/TP53KO mice developed the same phenotype than JAK2V617F transgenic mice without leukemia. In competitive repopulations with grafts with different ratio of JAK2V617F, JAK2V617F/TP53KO and wild type cells, we observed that JAK2V617F/TP53KO cells outcompete JAK2V617F cells. In order to better define TP53-dependent and independent pathways linked to these phenotypes, LT-HSC, ST-HSC, MPP, CMP, MEP and GMP from normal, JAK2V617F and JAK2V617F/TP53 KO mice were cell sorted and RNA-Seq analysis was performed. Principal component analysis demonstrated that JAK2V617F/TP53KO cells were closer to normal cells than JAK2V617F cells whatever the (stem) cell compartment, illustrating that JAK2V617F-induced modifications are largely p53 dependent. Further analysis demonstrated that approximatively half of JAK2V617F deregulated genes in the different compartments are TP53 dependent including the IFN pathways. In order to validate this finding, mice repopulated with a mix of WT and JAK2V617F (either p53 KO or wild-type) cells were treated for 8 weeks with recombinant murine pegylated IFN-a. JAK2V617F reconstituted animals entered in complete hematological remission while JAK2VF/TP53KO reconstituted animals did not, illustrating that loss of TP53 in this context induced resistance to IFN-a. On the other hand, since MPN develop in the same way in JAK2V617F-only and in JAK2V617F/TP53KO mouse, this suggests that JAK2V617F-specific pathways also found differentially expressed in JAK2V617F/TP53KO are linked to the MPN phenotype. KEGG and GO analysis demonstrated that these genes were mainly implicated in cytokine response, cell proliferation, differentiation, and leukemia evolution illustrating that the development of MPN and its possible risk of transformation in this mouse model is largely TP53 independent. Summary/Conclusion: Taken together, our results show that a large part of genetic modifications induced by JAK2V617F mutation are p53 dependent but MPN phenotype is not, TP53 loss is insufficient to induce quick leukemic transformation in steady-state hematopoiesis in Jak2 V617F MPN despite it increases LT-HSC cell proliferation and finally that TP53 loss could be involved in IFN resistance in MPN.

P980: JAK2V617F-DEPENDENT DOWN REGULATION OF SHP1 EXPRESSION PARTICIPATES IN THE SELECTION OF MPN CELLS IN THE PRESENCE OF TGFΒ

Background: Myeloproliferative neoplasms (MPNs) are characterized by an increased production of blood cells, due to acquisition in HSC of mutations in the JAK2, CALR or MPL genes. The JAK2V617F mutation, leading to a constitutively active form of JAK2, is the most frequent mutation. During disease evolution the clonal selection of mutated cells is dependent on cell-intrinsic and extrinsic factors present in the niche microenvironment such as cytokines. TGFβ, whose local secretion is increased in MPNs, and more particularly in myelofibrosis is known to negatively regulate normal hematopoietic stem cells (HSC) proliferation. However, its mode of action is not clearly established. We hypothesized that JAK2V617F mutant HSC are resistant to TGFβ antiproliferative effect explaining the pathological stem cell invasion in myelofibrosis. Using multiomics approaches to study the intracellular signaling pathways in MPNs we recently identified the down regulation of SHP-1 expression in JAK2V617F cells compared to wild type cells. Of note, the phosphatase SHP-1 has recently been shown to be required for the TGFβ-induced quiescence of HSCs. Aims: We hypothesized a relationship between SHP-1 down regulation and the resistance of JAK2V617F mutated cells to the antiproliferative effect of TGFβ. The work presented herein aimed at verifying this hypothesis. Methods: We used the HEL erythroleukemia cell line which carries several copies of the JAK2V617F mutated gene. We also used an isogenic model obtained by transducing either the JAK2 wild type (JAK2wt) of JAK2V617F allele in the UT-7 megakaryoblastic cell line. Proliferation was measured using the CCK8. Protein expression was measured using western blot while gene expression was measured using RT-qPCR. Results: The expression of SHP-1 was reduced by 30% at the RNA level and 50% at the protein level in UT-7 cells transduced with JAK2V617F compared to JAK2wt -transduced cells. In bone marrow cells taken from JAK2V617F knock-in mice the protein level of SHP1 was reduced by 70% compared to wild-type mice. EPO treatment of UT-7 cells (which express the EPO-R) showed a down regulation of SHP-1 expression which was abrogated in the presence of the JAK2 inhibitor ruxolitinib. These results suggest that SHP-1 expression is modulated by JAK2 signaling leading to its down regulation in JAK2V617F cells. Although TGFβ receptor and SMAD2/3 expression was similar in both cell type, JAK2V617F UT-7 cells were less responsive to TGFβ signaling compared to JAK2wt cells (reduced phosphorylation of SMAD2/3). The proliferation of JAK2wt UT-7 cells was significantly reduced in the presence of TGFβ 10ng/mL while JAK2V617F UT-7 cells were not affected. To confirm the involvement of SHP-1 we transduced HEL cells with the cDNA coding for SHP-1 in order to restore higher levels of the protein in this JAK2V617F mutated cell line and observed a significant reduction in the proliferation when treated with TGFβ compared to cells transduced with the empty vector. Finally, we performed an in vitro competitive assay where JAK2V617F UT-7 cells transduced by an m-cherry fluorescent vector were mixed 50/50 with JAK2wt UT-7 and cultured with TGFβ. After 14 days of culture with TGFβ 10ng/mL we observed a significant positive selection of the JAK2 mutant cells over JAK2wt cells. Summary/Conclusion: In conclusion, we found a JAK2-dependent down-regulation of SHP1 expression which is related to the resistance of JAK2V617F cells to the antiproliferative effect of TGFβ explaining clonal selection of mutant stem cells in myelofibrosis

Differential cytotoxic activity of pharmacological inhibitors of IGF1R-related pathways in JAK2V617F driven cells

Myeloproliferative neoplasms (MPN) belong to a group of clonal diseases of hematopoietic stem cells characterized by aberrant proliferation of mature myeloid lineages. The constitutive activation of the JAK2/STAT signaling pathway is now well established to play a central role in MPN pathogenesis; however, accumulating evidence now indicates that the IGF1R-mediated signaling pathway contributes to the maintenance of the malignant phenotype. Studies using inhibitors of IGF1-mediated signaling have reported cytotoxic effects in cellular and murine models of MPN, but no consensus has been reached regarding the potency and efficacy of inhibitors of the IGF1R-related pathway in this context. In the present study, we compared the potency and efficacy of three inhibitors of IGF1R-related pathways in a JAK2V617F-driven cellular model. These inhibitors (NT157, OSI-906, and NVP-AEW54) present antineoplastic activity with similar efficacy in Ba/F3 JAK2V617F cells, with NT157 showing the greatest potency. Both the induction of apoptosis and reduction in cell proliferation were associated with the observed reduction in cell viability. Downregulation of JAK2/STAT signaling was an advantageous off-target effect of all three inhibitors. These preclinical studies reinforce the potential of the IGF1R-related pathway as a therapeutic target in MPN.

Transcriptional differences between JAK2-V617F and wild-type bone marrow cells in patients with myeloproliferative neoplasms

The JAK2-V617F mutation is the most common cause of myeloproliferative neoplasms. Although experiments have revealed that this gain-of-function mutation is associated with myeloid blood cell expansion and increased production of white cells, red cells, and platelets, the transcriptional consequences of the JAK2-V617F mutation in different cellular compartments of the bone marrow have not yet been fully elucidated. To study the direct effects of JAK2-V617F on bone marrow cells in patients with myeloproliferative neoplasms, we performed joint single-cell RNA sequencing and JAK2 genotyping on CD34+-enriched cells from eight patients with newly diagnosed essential thrombocythemia or polycythemia vera. We found that the JAK2-V617F mutation increases the expression of interferon-response genes (e.g., HLAs) and the leptin receptor in hematopoietic progenitor cells. Furthermore, we sequenced a population of CD34- bone marrow monocytes and found that the JAK2 mutation increased expression of intermediate monocyte genes and the fibrocyte-associated surface protein SLAMF7 in these cells.