JAK2V617F-positive Cells Research Articles

Activating mutations in JAK2 and CALR differentially affect intracellular calcium flux in store operated calcium entry

BackgroundCalcium (Ca2+) signaling regulates various vital cellular functions, including integrin activation and cell migration. Store-operated calcium entry (SOCE) via calcium release-activated calcium (CRAC) channels represents a major pathway for Ca2+ influx from the extracellular space in multiple cell types. The impact of JAK2-V617F and CALR mutations which are disease initiating in myeloproliferative neoplasms (MPN) on SOCE, calcium flux from the endoplasmic reticulum (ER) to the cytosol, and related key signaling pathways in the presence or absence of erythropoietin (EPO) or thrombopoietin (TPO) is poorly understood. Thus, this study aimed to elucidate the effects of these mutations on the aforementioned calcium dynamics, in cellular models of MPN.MethodsIntracellular Ca2+ levels were measured over a time frame of 0–1080 s in Fura-2 AM labeled myeloid progenitor 32D cells expressing various mutations (JAK2-WT/EpoR, JAK2-V617F/EpoR; CALR-WT/MPL, CALR-ins5/MPL, and del52/MPL). Basal Ca2+ concentrations were assessed from 0–108 s. Subsequently, cells were stimulated with EPO/TPO in Ca2+-free Ringer solution, measuring Ca2+ levels from 109–594 s (store depletion). Then, 2 mM of Ca2+ buffer resembling physiological concentrations was added to induce SOCE, and Ca2+ levels were measured from 595–1080 s. Fura-2 AM emission ratios (F340/380) were used to quantify the integrated Ca2+ signal. Statistical significance was assessed by unpaired Student's t-test or Mann–Whitney-U-test, one-way or two-way ANOVA followed by Tukey's multiple comparison test.ResultsFollowing EPO stimulation, the area under the curve (AUC) representing SOCE significantly increased in 32D-JAK2-V617F cells compared to JAK2-WT cells. In TPO-stimulated CALR cells, we observed elevated Ca2+ levels during store depletion and SOCE in CALR-WT cells compared to CALR-ins5 and del52 cells. Notably, upon stimulation, key components of the Ca2+ signaling pathways, including PLCγ-1 and IP3R, were differentially affected in these cell lines. Hyper-activated PLCγ-1 and IP3R were observed in JAK2-V617F but not in CALR mutated cells. Inhibition of calcium regulatory mechanisms suppressed cellular growth and induced apoptosis in JAK2-V617F cells.ConclusionsThis report highlights the impact of JAK2 and CALR mutations on Ca2+ flux (store depletion and SOCE) in response to stimulation with EPO and TPO. The study shows that the JAK2-V617F mutation strongly alters the regulatory mechanism of EpoR/JAK2-dependent intracellular calcium balance, affecting baseline calcium levels, EPO-induced calcium entry, and PLCγ-1 signaling pathways. Our results reveal an important role of calcium flux in the homeostasis of JAK2-V617F positive cells.

Effects of Ruxolitinib on Immune Checkpoint Molecule Expression in JAK2 V617F-Positive Cells.

This study aimed to explore the clinical significance of ruxolitinib and its effects on the proliferation and apoptosis of human erythroleukemia (HEL) cells and the expression of immune checkpoint molecules programmed death-1 (PD-1), programmed death-ligand 1 (PD-L1), and regulatory T cells (Tregs) in HEL cells and JAK2 V617F-positive patients with myeloproliferative neoplasms (MPNs). JAK2 V617F-positive patients with MPNs admitted to the Baoding No. 1 Hospital from January 2016 to September 2023 were recruited, including 30 patients for the newly diagnosed group and 10 for the treatment group. Additionally, 15 healthy volunteers were selected as the control group. JAK2 V617F mutation was detected by using fluorescence quantitative PCR, and the expression levels of phosphorylated JAK2 (p-JAK2), PD-1, and PD-L1 in fresh bone marrow were examined by immunohistochemistry. HEL cells were treated with ruxolitinib at different concentrations (0, 50, 100, 250, 500, and 1,000 nmol/L). Cell viability was detected by CCK-8 assay. The mRNA expression levels of JAK2, PD-1, and PD-L1 were determined by using fluorescence quantitative PCR. The protein expression of p-JAK2 was detected by Western blot and those of PD-1 and PD-L1 were evaluated by flow cytometry. The expression of PD-1, PD-L1, and Tregs after the 48-hour co-culture of primary bone marrow cells and HEL cells were also analyzed by flow cytometry. In the newly diagnosed group, the bone marrow myeloid cells highly expressed p-JAK2, PD-1, and PD-L1. The Tregs expression in their peripheral blood increased and was significantly higher than those in the treatment and control groups (all p < 0.05). Ruxolitinib at different concentrations could inhibit the proliferation of HEL cells and was positively correlated with treatment time and dose. Additionally, ruxolitinib could reduce p-JAK2, PD-1, and PD-L1 expression in HEL cells and Tregs expression. Ruxolitinib reduces the expression of p-JAK2, PD-1, and PD-L1 in JAK2 V617F-positive cells by specifically inhibiting the JAK2 signaling pathway, thereby suppressing the progression of MPNs.

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

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

Antineoplastic effects of pharmacological inhibitors of aurora kinases in CSF3RT618I-driven cells

Myeloproliferative neoplasms (MPN) are consolidated as a relevant group of diseases derived from the malfunction of the hematopoiesis process and have as a particular attribute the increased proliferation of myeloid lineage. Among these, chronic neutrophilic leukemia (CNL) is distinguished, caused by the T618I mutation of the CSF3R gene, a trait that generates ligand-independent receptor activation and downstream JAK2/STAT signaling. Previous studies reported that mutations in BCR::ABL1 and JAK2V617F increased the expression of the aurora kinase A (AURKA) and B (AURKB) in Ba/F3 cells and their pharmacological inhibition displays antineoplastic effects in human BCR::ABL1 and JAK2V617F positive cells. Delimiting the current scenario, aspects related to the AURKA and AURKB as a potential target in CSF3RT618I-driven models is little known. In the present study, the cellular and molecular effects of pharmacological inhibitors of aurora kinases, such as aurora A inhibitor I, AZD1152-HQPA, and reversine, were evaluated in Ba/F3 expressing the CSF3RT618I mutation. AZD1152-HQPA and reversine demonstrated antineoplastic potential, causing a decrease in cell viability, clonogenicity, and proliferative capacity. At molecular levels, all inhibitors reduced histone H3 phosphorylation, aurora A inhibitor I and reversine reduced STAT5 phosphorylation, and AZD1152-HQPA and reversine induced PARP1 cleavage and γH2AX expression. Reversine more efficiently modulated genes associated with cell cycle and apoptosis compared to other drugs. In summary, our findings shed new insights into the use of AURKB inhibitors in the context of CNL.

Focal adhesion kinase inhibition decreases cell viability and induces apoptosis of JAK2 V617F positive cells

Focal adhesion kinase inhibition decreases cell viability and induces apoptosis of JAK2 V617F positive cells

PTPN1 Deficiency Accelerates the Progression to Myelofibrosis in JAK2V617F-Driven Myeloproliferative Neoplasm

The deletion of chromosome 20q (del20q) is frequently associated with myeloid malignancies including myeloproliferative neoplasms (MPN), myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The del20q cytogenetic abnormality was observed most frequently in patients with myelofibrosis (MF). However, the key target genes within 20q involved in the pathogenesis of myelofibrosis has remained elusive. We previously reported that PTPN1 gene encoding protein tyrosine phosphatase non-receptor type 1 is a potential tumor suppressor within 20q in MPN/MF (Jobe et al. Leukemia 2017). The del20q involving PTPN1 deletion was more commonly found in MPN/MF in comparison to other myeloid neoplasms. We also observed significant co-occurrence of del20q involving PTPN1 deletion with JAK2V617F mutation in patients with MF. So, we hypothesize that PTPN1 deficiency may cooperate with JAK2V617F in the pathogenesis of MF. In this study, we investigated the effects of heterozygous and homozygous PTPN1 deletion in JAK2V617F-induced MPN using a conditional PTPN1 knockout allele and a JAK2V617F knock-in mouse model of MPN. We previously reported the generation of a conditional JAK2V617F knock-in mouse. Mice expressing heterozygous JAK2V617F (JAK2VF/+) exhibit a PV-like MPN characterized by increase in red blood cells (RBC), white blood cells (WBC), neutrophils and platelets in the peripheral blood. Heterozygous deletion of PTPN1 significantly increased WBC, neutrophil and platelet counts but reduced RBC and hemoglobin levels in JAK2VF/+ mice. Flow cytometric analysis showed that heterozygous deletion of PTPN1 significantly increased myeloid (Gr-1+Mac-1+) and megakaryocytic (CD41+CD61+) precursors in the BM and spleens of PTPN1-deficient JAK2VF/+ mice compared with JAK2VF/+ mice. Additionally, deletion of PTPN1 significantly increased hematopoietic stem cells and myeloid progenitors in the BM and spleens of PTPN1-deficient JAK2VF/+ mice compared to JAK2VF/+ mice. Spleen weight was significantly increased in JAK2VF/+ mice, while deletion of PTPN1 further enhanced splenomegaly in PTPN1-deficient JAK2VF/+ mice. Furthermore, deletion of PTPN1 significantly enhanced bone marrow fibrosis in JAK2VF/+mice. Homozygous deletion of PTPN1 resulted in more robust and accelerated development of lethal myelofibrosis in JAK2VF/+ mice. The cooperative effects of JAK2V617F and PTPN1 deficiency in myelofibrotic transformation is associated with increased production of proinflammatory cytokines including IL-1beta, IL-6 and TGF-beta. We also observed enhanced activation of STAT1, STAT3, ERK1/2 and NF-kB in PTPN1-deficient JAK2VF/+ mice bone marrow compared with JAK2VF/+ or WT mice bone marrow. CRISPR/Cas9-mediated deletion of PTPN1 also resulted in enhanced activation of these signaling molecules and increased proliferation in JAK2V617F-positive mouse and human hematopoietic cells. Furthermore, deletion of PTPN1 significantly reduced ruxolitinib sensitivity against JAK2V617F-positive hematopoietic cells, suggesting that del20q involving PTPN1 deletion may cause ruxolitinib resistance in MPN. Overall, this study demonstrates that PTPN1 is an important target in del20q-associated MPN, and deficiency of PTPN1 collaborates with JAK2V617F in the progression to myelofibrosis.

P989: DYNAMIC CHANGES IN THE HISTONE H3 LYSINE 27 TRIMETHYLATION EPIGENETIC LANDSCAPE FOLLOWING RUXOLITINIB ADMINISTRATION

Background: Dysregulation of normal epigenetic processes is increasingly recognised as a feature of myeloid malignancy generally and implicated in the pathogenesis of MPN. Epigenetic changes may directly result from the mutation of key epigenetic regulators including TET2, ASXL1 and EZH2 or indirectly from the activation of intracellular signalling cascades. We have previously observed that ruxolitinib, a JAK1/2 inhibitor, may alter the epigenetic landscape of histone modifications in MPN. CUT&RUN is a novel experimental approach providing high resolution profiling of epigenetic modifications and transcription factor binding. Aims: To comprehensively profile the H3K27me3 epigenetic landscape of JAK2 V617F positive cells resulting from JAK inhibition and determine the dynamic changes occurring between short term and persistent ruxolitinib therapy to identify potential genetic contributors to pathogenesis and options for future therapeutics. Methods:JAK2 V617F positive UKE1 cells were cultured in duplicate in the presence of 1000nM ruxolitinib or DMSO 0.1% vehicle control continuously for approximately five weeks. Samples were harvested at 37 days for CUT&RUN analysis using H3K27me3, EZH2 and IgG (negative control) antibodies. CUT&RUN performed using Epicypher CUTANA Kit according to manufacturer instructions. Libraries prepared using NEBNext® Ultra™ II DNA Library Prep kit with 50 base pair, paired end sequencing undertaken on Illumina NovaSeq 6000. Resulting FASTQ files were trimmed using Trim Galore! and aligned to reference genome hg38 using Bowtie2. Duplicate reads were removed using SAMtools rmdup and peaks called with MACS2 callpeak on the usegalaxy.eu platform. Differential peak analysis undertaken with Diffbind package in R. Samples were normalised to library size prior to differential analysis with DESeq2 methodology. Bigwig files generated using deepTools bamcoverage for visualisation. Results: UKE1 cells remain sensitive to ruxolitinib after a period of treatment with high dose ruxolitinib lasting over 5 weeks. CUT&RUN libraries were sequenced to a minimum depth of 3.6 million unique reads per sample. Initial comparisons between Day 37 high dose treated cells and vehicle control revealed 1004 differentially enriched H3K27me3 peaks. Of these, 545 (54.2%) peaks were increased in the ruxolitinib treated cells and 459 (45.7%) peaks were increased in the control cells. Analysis of the difference between day 37 and day 1 high dose ruxolitinib treated cells revealed 290 peaks differentially enriched. Of these peaks, 279 (96.2%) were increased and 11 (3.8%) were decreased at day 37. Analysis of the overlap between these results identified 33 peaks increased at D37 in comparison to DMSO control and day 1 treatment samples. Analysis of the differential binding patterns and direct visualisation of the results in a genome browser revealed particular genetic locations of interest including the promoter region of SPRY1, a potential regulator of erythropoiesis and JAK signalling, UGCG and BTBD11. Figure 1 shows an example. Differential H3K27me3 was also noted at regions associated with PLXNA1 and NRP2 genes which act as co-receptors for class III semaphorin signalling. Image:Summary/Conclusion: The dynamic changes in the H3K27me3 epigenetic landscape of JAK2 V617F positive cells following persistent high dose ruxolitinib administration has potential therapeutic implications and may sensitise cells to epigenetic therapies. Specific gene loci identified demonstrating differential H3K27me3 may offer some insight into JAK2 V617F mediated pathology and require further investigation.

Inhibition of SHP2 Ameliorates JAK2V617F-Induced Myeloproliferative Neoplasms

The oncogenic JAK2V617F mutation has been found in a majority of patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Ruxolitinib, a JAK1/JAK2 inhibitor, has been approved for treatment of MF and advanced PV. Although ruxolitinib treatment can reduce splenomegaly and alleviate constitutional symptoms, it does not induce disease remission or cure bone marrow (BM) fibrosis in patients with MPN/MF. Thus, there is a critical need to identify new therapeutic target and develop novel targeted therapies for MPN/MF. We found that SHP2, a protein tyrosine phosphatase that positively regulates growth factor and cytokine signaling, is constitutively hyperphosphorylated in mouse and human MPN hematopoietic cells expressing JAK2V617F. We found that genetic ablation of SHP2 markedly inhibited the development of PV disease and BM fibrosis in Jak2V617F knock-in mouse models of MPN. So, we hypothesize that targeting of SHP2 alone or in combination with JAK2 inhibition might be useful for treatment of JAK2V617F-evoked MPN. In this study, we investigated the efficacy of SHP2 inhibition using an allosteric SHP2 inhibitor, SHP099, in hematopoietic cells and progenitors expressing JAK2V617F and a Jak2V617F knock-in mouse model of MPN.Treatment of SHP099 alone significantly reduced proliferation of murine BA/F3 cells expressing JAK2V617F. SHP099 treatment also significantly inhibited human JAK2V617F-positive UKE-1 cells. However, SHP099 only modestly affected proliferation of wild type JAK2-expressing BA/F3 cells at higher concentrations. SHP099 treatment also resulted in marked apoptosis in BA/F3-EpoR-JAK2V617F and UKE-1 cells but not in wild-type JAK2 expressing BA/F3-EpoR cells. We also found that SHP099 and ruxolitinib synergistically induced apoptosis in JAK2V617F-expressing hematopoietic cells. Furthermore, SHP099 significantly inhibited hematopoietic progenitor colony growth in MPN/MF patient CD34+ cells.We previously generated conditional Jak2V617F knock-in mice. Whereas expression of heterozygous Jak2V617F (Jak2 VF/+) induces a PV-like disease, homozygous Jak2V617F (Jak2 VF/VF) expression promotes rapid progression to myelofibrosis. We utilized heterozygous Jak2V617F mice to test the in vivo efficacy of SHP099 against PV. Treatment of SHP099 significantly reduced the increase in WBC, neutrophil and RBC counts and markedly reduced splenomegaly in heterozygous Jak2V617F (Jak2 VF/+) mice. SHP099 treatment also significantly reduced hematopoietic stem/progenitors (HSPC) and myeloid precursors (Gr-1 +/Mac1 +) in the BM and spleens of Jak2V617F mice. Erythropoietin (Epo)-independent erythroid colony formation in the BM and spleens, a hall-mark feature of PV disease, was observed in Jak2V617F mice. Treatment of SHP099 markedly inhibited the Epo-independent CFU-E colonies in the BM and spleens of heterozygous Jak2V617F mice.We also tested the efficacy of SHP099 alone or in combination with ruxolitinib against myelofibrosis using our homozygous Jak2V617F (Jak2 VF/VF) mice. SHP099 treatment alone or in combination with ruxolitinib significantly reduced WBC and neutrophil counts and markedly reduced splenomegaly in Jak2 VF/VF mice. Treatment of SHP099 or SHP099/ruxolitinib combination also significantly reduced the HSPC and myeloid precursor cells in the BM and spleens of Jak2 VF/VF mice. Histopathologic analyzes revealed extensive BM fibrosis in vehicle-treated Jak2 VF/VFmice. SHP099 treatment alone caused marked reduction of BM fibrosis in Jak2 VF/VF mice whereas combined treatment of SHP099 and ruxolitinib almost completely inhibited BM fibrosis in Jak2 VF/VF mice. Additionally, we performed competitive BM transplantation assays using Mx1Cre; Jak2 VF/VF GFP+ and WT mice BM cells followed by drug treatments. We observed that combined treatment of SHP099 and ruxolitinib significantly reduced the percentage of Jak2V617F mutant GFP+ LSK and myeloid cells in the BM of chimeric mice, suggesting that SHP099 and ruxolitinib combination preferentially inhibits oncogenic Jak2V617F mutant hematopoietic progenitors. Overall, our results suggest that SHP099 alone or in combination with ruxolitinib may have therapeutic potential against PV and MF and support the clinical investigation of SHP2 inhibitor in patients with PV/MF. DisclosuresNo relevant conflicts of interest to declare.

The Unfolded Protein Response Is a Major Driver of LCN2 Expression in BCR-ABL- and JAK2V617F-Positive MPN.

Simple SummaryLipocalin 2 (LCN2) is a proinflammatory mediator increased in the blood of patients with myeloproliferative neoplasms (MPN) and other hematologic malignancies, significantly contributing to MPN disease initiation and progression. Here, we investigated the underlying mechanisms of LCN2 overexpression in MPN. We found a strong correlation between BCR–ABL and JAK2V617F driver oncogenes and LCN2 expression. Furthermore, LCN2 expression is strongly induced by endoplasmic reticulum (ER) stress, independent of oncogenic kinase activity. We identified the IRE1–JNK–NF-κB–C/EBP axis as a major mediator of ER stress-induced LCN2 expression. Our findings provide novel insights into the regulation of LCN2 and present a basis for innovative, targeted treatment approaches in MPN.Lipocalin 2 (LCN2), a proinflammatory mediator, is involved in the pathogenesis of myeloproliferative neoplasms (MPN). Here, we investigated the molecular mechanisms of LCN2 overexpression in MPN. LCN2 mRNA expression was 20-fold upregulated in peripheral blood (PB) mononuclear cells of chronic myeloid leukemia (CML) and myelofibrosis (MF) patients vs. healthy controls. In addition, LCN2 serum levels were significantly increased in polycythemia vera (PV) and MF and positively correlated with JAK2V617F and mutated CALR allele burden and neutrophil counts. Mechanistically, we identified endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) as a main driver of LCN2 expression in BCR-ABL- and JAK2V617F-positive 32D cells. The UPR inducer thapsigargin increased LCN2 expression >100-fold, and this was not affected by kinase inhibition of BCR-ABL or JAK2V617F. Interestingly, inhibition of the UPR regulators inositol-requiring enzyme 1 (IRE1) and c-Jun N-terminal kinase (JNK) significantly reduced thapsigargin-induced LCN2 RNA and protein expression, and luciferase promoter assays identified nuclear factor kappa B (NF-κB) and CCAAT binding protein (C/EBP) as critical regulators of mLCN2 transcription. In conclusion, the IRE1–JNK-NF-κB–C/EBP axis is a major driver of LCN2 expression in MPN, and targeting UPR and LCN2 may represent a promising novel therapeutic approach in MPN.

Critical Interferon-Response Genes Are Induced By JAK2V617F but Repressed By BCR-ABL, and They Are Responsible for the Differential Effects of Interferon in PV Vs. CML

Interferon-alpha (IFNa) is successfully used as monotherapy in JAK2V617F positive myeloproliferative neoplasms (MPN) such as polycythemia vera (PV), but with only limited success in chronic myeloid leukemia (CML). Here, we investigated the mechanisms of IFNa in JAK2V617F vs. BCR-ABL positive cells.In 32D cells stably transduced with JAK2V617F, IFNa alone (100 U/mL) significantly decreased the viability to less than 50% of control but had no significant effect on 32D-BCR-ABL cell viability, as assessed by MTT assays. Gene expression microarray data of 32D-JAK2V617F cells revealed strong upregulation of a number of interferon-stimulated genes compared to empty vector-transduced (EV) cells, and RT-PCR validation confirmed significantly increased expression of Stat1, Stat2 and Irf9 and reversal of these effects by ruxolitinib treatment. Conversely, BCR-ABL led to a decrease of these three genes, and this was reversed by imatinib. Similar responses were seen in human HEL and K562 cells. Histone (H3K9) acetylation of Stat1, Stat2 and Irf9 promoters was significantly higher in 32D-JAK2V617F vs. 32-BCR-ABL cells, correlating with gene expression. IFNa treatment increased Stat1, Stat2 and Irf9 mRNA in both 32D-JAK2V617F and 32D-BCR-ABL cells, and this effect was reduced by ruxolitinib but potentiated by imatinib. In line with these results, the phosphorylation of STAT1 (P-STAT1) was induced by IFNa in all cell lines. In addition, Stat1, Stat2 and Irf9 mRNA was significantly downregulated in peripheral blood samples from CML but not PV patients, as compared to healthy controls (all subjects provided written informed consent, as approved by the local ethics committee). Likewise, Stat1 and Stat2 expression was significantly downregulated in SCLtTA/BCR-ABL but not JAK2V617F transgenic mice.We next analyzed whether ectopic expression of STAT1 or STAT2 or their dominant-negative Y/F mutants affected the IFNa sensitivity of 32D-JAK2V617F vs. 32D-BCR-ABL cells. While there was little apoptosis in empty-vector- or STAT1-overexpressing 32D-BCR-ABL cells, overexpression of the STAT1Y701F mutant in these cells showed a significantly increased basal level of apoptosis, which was further enhanced by IFNa (p<0.05). This was associated with IFNa-induced P-STAT1 but complete absence of P-STAT2. Conversely, EV- or STAT1-overexpressing 32D-JAK2V617F cells showed strong IFNa-induced apoptosis, while STAT1Y701F overexpression in these cells led to abrogation of IFNa-induced apoptosis. This was associated with decreased P-STAT1 but preserved P-STAT2. On the other hand, STAT2 overexpression decreased P-STAT1 and P-STAT2 and led to mild IFNa-induced apoptosis in 32D-BCR-ABL cells, but preserved the high P-STAT1 and P-STAT2 as well as potent IFNa responsiveness in 32D-JAK2V617F cells.In conclusion, JAK2V617F and BCR-ABL induce opposite effects on IFNa target genes in vitro and in vivo, suggesting that JAK2V617F but not BCR-ABL sensitizes cells to interferon. Moreover, while the major factor of IFNa-induced apoptosis in JAK2V617F positive cells is an increase of P-STAT1, the major determinant in BCR-ABL positive cells is a decrease of P-STAT2. Together, these results may possibly explain the differences of clinical responses to IFNa monotherapy in PV vs. CML. DisclosuresBrümmendorf:Takeda: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding. Koschmieder:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Roche: Other: Clinical Trial participation.

Higher Rates of Molecular Response to Peg-IFNa in JAK2V617F Vs. Calr Mutant MPN Patients Are Due to JAK1-Mediated STAT1 Activation and Autoregulation

Introduction: Pegylated interferon alpha (Peg-IFNa) treatment has been described to induce molecular remission in patients (pts) with myeloproliferative neoplasms (MPN), with partial molecular remission (PMR) being achieved in approximately 30-40% of pts. However, it is unclear whether pts with CALR mutations exhibit the same response as pts with JAK2V617F mutations. Also, the precise mechanisms accounting for the responses are still unknown.Methods: We report on hematological and molecular responses, according to ELN guidelines, in 52 MPN pts (JAK2V617F n=26 vs. CALR mutation n=26; ET n=25, prePMF n=8, PMF n=19), treated at Roskilde hospital with Peg-IFNa. The mechanism of IFNa response was assessed in stably transduced 32D murine cell lines and in a cohort of MPN patients within the Czech MPN registry MIND. All pts provided written informed consent, as approved by the local ethics committees. Cell proliferation was assessed by cell counting and/or MTT assay, signaling molecules were quantified using RT-PCR, flow cytometry and/or Western blotting.Results: Pts harboring JAK2V617F mutations were more likely to achieve PMR than those harboring CALR mutations (60% vs. 23%, p<0.05 by Chi square testing), while there was no significant difference in allelic burden before Peg-IFNa treatment. Accordingly, the median allelic burden decrease from baseline in the IFNa responding pts was 66% (range 15-99%) in JAK2V617F- vs. 29% (range 5-82%) in CALR-mutant pts. These differences were present independently of the fractions of ET, prePMF, or PMF pts in the two groups. There was no significant difference in the rates of hematological response. When examining primary cells from the Czech MPN cohort, cells from JAK2V617F positive pts showed higher expression of IFNa response-related genes such as Irf1 (p=0.0350) and Stat1 (trend at p=0.0874). Moreover, IFNa receptor expression was higher in JAK2V617F positive pts (p=0.0471) and the expression levels were associated with hemoglobin (p=0.0139) and hematocrit levels (p=0.0251). This was reflected in 32D cell lines, ectopically expressing both the MPL receptor and either JAK2V617F or CALR del 52. Here, the expression of Irf7, Irf-9, Ifi-27, and Stat2 was significantly higher in JAK2VF vs. CALR mutants (all p<0.001). In addition, JAK2V617F positive cells showed higher STAT1 phosphorylation and Stat1 mRNA expression levels (p=0.0499). In particular, STAT1 showed constitutive phosphorylation in JAK2V617F but not CALR mutant cells, indicating priming of these cells towards an IFNa response. Overexpression of STAT1 but not of a dominant-negative STAT1YF mutant was able to rescue cellular IFNa-induced growth arrest in CALR positive cells to the same level of JAK2V617F positive cells. In contrast, manipulation of STAT2 expression did not alter IFNa signaling. IFNa-induced growth arrest was counteracted by selective JAK1 inhibition (using itacitinib or filgotinib) but not selective JAK2 inhibition (using BMS-911543).Conclusions: MPN pts harboring JAK2V617F mutations show superior Peg-IFNa sensitivity against the malignant clone, as detected by molecular responses, compared to pts harboring CALR mutations. The higher IFNa sensitivity of JAK2V617F mutant cells was reflected in mutant-specific cell lines and is at least in part due to JAK1-mediated increases in STAT1 phosphorylation and STAT1 transcription. These results are of considerable importance for future clinical trials of Peg-IFNa as monotherapy or in combination with JAK inhibitors. DisclosuresBrümmendorf:Takeda: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Mayer:Eisai: Research Funding; Novartis: Research Funding. Knudsen:Novartis: Other: Payed for ASH2016 registration fee, travel expenses and accommodation. Hasselbalch:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; AOR Orphan: Membership on an entity's Board of Directors or advisory committees. Koschmieder:Roche: Other: Clinical Trial participation; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support.

Lncrna-Iκbα Promotes Erythroid Differentiation By Inhibiting NF-Κb Pathway As a Cerna Against Mir-24-3p in Polycythemia Vera

Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem/progenitor cell diseases characterized by the overproduction of one or more mature myeloid blood cell lineages, mainly including primary thrombocythemia (ET) and polycythemia vera (PV). The acquired kinase mutation Jak2V617F plays a central role in these disorders, but the mechanisms responsible for same genetic mutation leading to different clinical features are not fully understood. LncRNAs are critical regulators of diverse physiological and pathological cellular processes, including normal and malignant hematopoiesis. Although thousands of erythroid stage-specific lncRNAs have been identified, the biological functions of the vast majority of lncRNAs in PV remains to be elucidated. In an attempt to identify lncRNAs associated with the occurrence and development of PV progression, we conducted three rounds of screening sequentially. Firstly, RNA-Seq datasets were generated form ET, PV patients and normal peoples. In total, we identified 32259 lncRNAs, including 31841 annotated and 418 novel lncRNAs. Secondly, 446 differentially expressed lncRNAs (DELs) were identified between normal peoples and patients (ET and PV). Meanwhile, 53 DELs were identified between ET and PV. Lastly, 26 DELs involved in PV were identified by intersecting these two DELs datasets with each other. KEGG pathway analysis showed that these lncRNAs were mainly concentrated in the NF-κB signal pathway. Subsequently, expression of 9 lncRNAs were confirmed by real-time PCR. From these 9 lncRNAs, 2 relative high-expression and most differentially expressed lncRNAs (foldchange &amp;gt; 4) were subjected to loss-of-function analysis in JAK2V617F positive HEL cells. We focused on a novel lncRNA and named it lncRNA-IκBα, which inhibited hemin-induced erythroid differentiation in HEL cells. The expression of γ-globin and CD235a were significantly inhibited (~ reduced to 50%) in lncRNA-IκBα knockdown HEL cells compared to control cells. Likewise, flow cytometry revealed that the frequency of CD235+ cells and CD71+ cells was also lower in lncRNA-IκBα knockdown HEL cells than that in control cells (5% vs 29% , 1.64% vs 6.55%). Additionally, CCK-8 assays showed that the proliferation of lncRNA-IκBα knockdown cell was significantly slower than control cells . We assessed in vivo incorporation of Brdu to investigate the cell-cycle kinetics and found that 36% of lncRNA-IκBα knockdown cells and 44% of control cells were Brdu+, which indicated that knockdown of lncRNA-IκBα inhibit the proliferation. Analysis of lncRNA-miRNA-mRNA co-regulatory network showed that lncRNA-IκBα may target IκBα through competing binding with mir-24-3p. LncRNA-IκBα and IκBα have consistent expression patterns in MPN patients. In addition, lncRNA-IκBα has predicted binding site for mir-24-3p. Over-expression of lncRNA-IκBα in HEL cells can significantly up-regulate the expression of IκBα (~50%). Taken together, we suggested that lncRNA-IκBα regulates erythroid differentiation by regulating NF-κB signaling pathway through competitive binding endogenous miR-24-3p with IκBα. Furthermore, lncRNA-IκBα may affects the occurrence and development of PV by regulating erythroid differentiation. Disclosures No relevant conflicts of interest to declare.

Exploring redox vulnerabilities in JAK2V617F-positive cellular models

BackgroundIn Philadelphia chromosome-negative myeloproliferative neoplasm (MPN) models, reactive oxygen species (ROS) are elevated and have been implicated in genomic instability, JAK2/STAT signaling amplification, and disease progression. Although the potential effects of ROS on the MPN phenotype, the effects of ruxolitinib treatment on ROS regulation have been poorly explored. Herein, we have reported the impact of ruxolitinib on redox signaling transcriptional network, and the effects of diphenyleneiodonium (DPI), a pan NOX inhibitor, in JAK2V617F-driven cellular models. MethodRedox signaling-related genes were investigated in SET2 cells upon ruxolitinib treatment by RNA-seq (GEO accession GSE69827). SET2 and HEL cells, which represent JAK2V617F-positive MPN cellular models with distinct sensitivity to apoptosis induced by ruxolitinib, were used. Cell viability was evaluated by MTT, apoptosis by annexin V/PI and flow cytometry, and cell signaling by quantitative PCR and Western blot. Main resultsRuxolitinib impacted on a network composed of redox signaling-related genes, and DUOX1 and DUOX2 were identified as potential modulators of ruxolitinib response. In SET2 and HEL cells, DPI reduced cell viability and, at low doses, it significantly potentiated ruxolitinib-induced apoptosis. In the molecular scenario, DPI inhibited STAT3, STAT5 and S6 ribosomal protein phosphorylation and induced PARP1 cleavage in JAK2V617F-positive cells. DPI combined with ruxolitinib increased PARP1 cleavage in SET2 cells and potentiated ruxolitinib-reduced STAT3, STAT5 and S6 ribosomal protein in HEL cells. ConclusionOur study reveals a potential adaptation mechanism for resistance against ruxolitinib by transcriptionally reprogramming redox signaling in JAK2V617F cells and exposes redox vulnerabilities with therapeutic value in MPN cellular models.

NT157 has antineoplastic effects and inhibits IRS1/2 and STAT3/5 in JAK2V617F-positive myeloproliferative neoplasm cells

Recent data indicate that IGF1R/IRS signaling is a potential therapeutic target in BCR-ABL1-negative myeloproliferative neoplasms (MPN); in this pathway, IRS2 is involved in the malignant transformation induced by JAK2V617F, and upregulation of IGF1R signaling induces the MPN phenotype. NT157, a synthetic compound designed as an IGF1R-IRS1/2 inhibitor, has been shown to induce antineoplastic effects in solid tumors. Herein, we aimed to characterize the molecular and cellular effects of NT157 in JAK2V617F-positive MPN cell lines (HEL and SET2) and primary patient hematopoietic cells. In JAK2V617F cell lines, NT157 decreased cell viability, clonogenicity, and cell proliferation, resulting in increases in apoptosis and cell cycle arrest in the G2/M phase (p < 0.05). NT157 treatment inhibited IRS1/2, JAK2/STAT, and NFκB signaling, and it activated the AP-1 complex, downregulated four oncogenes (CCND1, MYB, WT1, and NFKB1), and upregulated three apoptotic-related genes (CDKN1A, FOS, and JUN) (p < 0.05). NT157 induced genotoxic stress in a JAK2/STAT-independent manner. NT157 inhibited erythropoietin-independent colony formation in cells from polycythemia vera patients (p < 0.05). These findings further elucidate the mechanism of NT157 action in a MPN context and suggest that targeting IRS1/2 proteins may represent a promising therapeutic strategy for MPN.

Hypoxia-inducible factor 1 (HIF-1) is a new therapeutic target in JAK2V617F-positive myeloproliferative neoplasms.

Classical Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) are a heterogeneous group of hematopoietic malignancies including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The JAK2V617F mutation plays a central role in these disorders and can be found in 90% of PV and ~50-60% of ET and PMF. Hypoxia-inducible factor 1 (HIF-1) is a master transcriptional regulator of the response to decreased oxygen levels. We demonstrate the impact of pharmacological inhibition and shRNA-mediated knockdown (KD) of HIF-1α in JAK2V617F-positive cells. Inhibition of HIF-1 binding to hypoxia response elements (HREs) with echinomycin, verified by ChIP, impaired growth and survival by inducing apoptosis and cell cycle arrest in Jak2V617F-positive 32D cells, but not Jak2WT controls. Echinomycin selectively abrogated clonogenic growth of JAK2V617F cells and decreased growth, survival, and colony formation of bone marrow and peripheral blood mononuclear cells and iPS cell-derived progenitor cells from JAK2V617F-positive patients, while cells from healthy donors were unaffected. We identified HIF-1 target genes involved in the Warburg effect as a possible underlying mechanism, with increased expression of Pdk1, Glut1, and others. That was underlined by transcriptome analysis of primary patient samples. Collectively, our data show that HIF-1 is a new potential therapeutic target in JAK2V617F-positive MPN.

RMC-4550, an Allosteric Inhibitor of SHP2, Displays Therapeutic Efficacy in Pre-Clinical Models of Myeloproliferative Neoplasms

Philadelphia chromosome negative myeloproliferative neoplasms (MPNs) are JAK2-driven disorders resulting from mutations in JAK2, MPL, or CALR. Ruxolitinib, the only FDA-approved JAK2 inhibitor for MPNs, alleviates patient symptomology and improves quality of life, but has little effect on reducing mutant allele burden. This persistent survival of MPN cells in the face of ruxolitinib, as well as other JAK2 inhibitors that have been clinically tested, is a major clinical bottleneck to the development of an effective targeted therapy for MPN patients. Identifying new therapeutic targets which play critical roles in MPN cells and/or in JAK2 inhibitor persistence may lead to improved MPN therapies. SHP2 is an oncogenic tyrosine phosphatase that is an effector of growth factor and cytokine receptor signaling. SHP2 plays a critical role in the activation of the RAS-ERK pathway and regulates JAK-STAT signaling via numerous phosphatase-dependent mechanisms. Activating mutations of SHP2(PTPN11) have been identified in leukemia, including 8% of MPN patients whose disease progressed to acute myeloid leukemia (AML). In addition, SHP2 has been shown to mediate adaptive resistance to targeted therapies in several cancers. Given the role of SHP2 in cytokine and JAK-STAT signaling, we envisaged a potential role of SHP2 in MPN cell growth and/or survival and ruxolitinib persistence. Treatment of JAK2-V617F-driven MPN model cell lines (UKE1, SET2, and BaF3-JAK2-V617F) with ruxolitinib blocked constitutive tyrosine phosphorylation of SHP2, including phosphorylation of Y542, a marker for activated SHP2. This phosphorylation, however, was restored in ruxolitinib persistent cells. Combination treatment of the allosteric SHP2 inhibitor RMC-4550 (Revolution Medicines) with ruxolitinib prevented the development of ruxolitinib persistent cells and pre-established persistent cells remained sensitive to SHP2 inhibition. RMC-4550 treatment led to significantly reduced levels of pERK consistent with the role of SHP2 in RAS signaling. Interestingly, pERK levels in persistent cells were more sensitive to SHP2 inhibition compared to drug naïve cells suggesting pERK was more dependent on SHP2 in ruxolitinib persistent cells. SHP2 inhibitor treatment increased pSTAT5(Y694) in drug naïve cells but this increase was not observed in similarly treated persistent cells. Furthermore, while ruxolitinib inhibited pERK levels in UKE1 and SET2 cells, pERK levels recovered within 24 hrs of treatment. SHP2 inhibition prevented the recovery of pERK in the presence of ruxolitinib. Collectively, these data suggest that signaling pathways in MPN cells treated with ruxolitinib can become rewired, gaining greater dependence on SHP2, concomitant with sustained pERK and cell survival/growth. Interestingly, we identified a known activating SHP2 mutation (F71L) in UKE1 cells obtained from two independent sources - consistent with the presence of PTPN11 mutations in post-MPN AML. The persistent survival of UKE1 cells in ruxolitinib was antagonized by CRISPR-mediated reduction of SHP2 expression, providing further evidence that SHP2 contributes to ruxolitinib persistence. To assess the effects of a SHP2 inhibitor on MPN progression in vivo, we employed the MPLW515Lbone marrow transplant mouse model of MPN. Initial assessment of therapeutic treatment of mice with an established MPN phenotype indicated that once daily treatment of RMC-4550 (10 or 30 mg/kg) antagonized the MPN phenotype. Complete blood counts indicated a significant reduction in white blood cells, monocytes, and neutrophils compared to vehicle treated mice, while flow cytometry analysis indicated RMC-4550 diminished CD11b+ cell numbers to near that observed in mice transplanted with MPLWT-transduced bone marrow. RMC-4550 improved the overall health of diseased mice, as indicated by increased weight, and significantly reduced organomegaly of the spleen and liver compared to vehicle treated MPN mice. Finally, erythropoietin independent erythroid colony formation of JAK2V617F-positive MPN patient cells was suppressed following SHP2 inhibition, which synergized or enhanced the inhibition induced by ruxolitinib in this assay. In summary, our results suggest that SHP2 inhibition may represent a potential MPN therapy in both ruxolitinib naïve and resistant patients and is an attractive therapeutic target for future clinical investigation. Disclosures Epling-Burnette: Incyte Corporation: Research Funding; Forma Therapeutics: Research Funding; Celgene Corporation: Patents &amp; Royalties, Research Funding. Reuther:Incyte Corporation: Research Funding.

The CDK6 Inhibitor Palbociclib in Combination with Ruxolitinib Remarkably Improves Myelofibrosis in Murine Models

Myelofibrosis (MF) is the deadliest form of myeloproliferative neoplasms (MPN), characterized by deposition of fibrous tissues in the bone marrow, abnormal megakaryopoiesis, ineffective erythropoiesis and extramedullary hematopoiesis. The median survival for patients with MF is ~5 years. The JAK2V617F mutation, which results in constitutive activation of the JAK2 tyrosine kinase, has been found in 50-60% patients with MF. Additional mutations in the genes encoding thrombopoietin receptor (MPL) and calreticulin (CALR) have been found at a lower frequency than JAK2V617F in MF. The JAK1/JAK2 inhibitor ruxolitinib has been approved for treatment of MF. Although ruxolitinib can reduce splenomegaly and constitutional symptoms, it does not cure or prevent fibrosis in patients with MPN/MF. Additionally, ruxolitinib treatment does not improve survival in MF patients. So, there is a critical need to identify new therapeutic targets and develop novel therapies for MF. We have found that expression of CDK6 is significantly elevated in mouse and human MPN hematopoietic cells/progenitors. We also observed that knockdown of CDK6 significantly inhibited proliferation of BA/F3-EpoR-JAK2V617F and HEL cells expressing JAK2V617F but not wild-type JAK2 expressing BA/F3-EpoR cells, suggesting that CDK6 is a bona fide target in MPN. In this study, we investigated the efficacy of CDK4/6 inhibitor Palbociclib (PD-0332991) in hematopoietic cells expressing JAK2V617F and MPLW515L and murine models of MF. Since Palbociclib is an FDA-approved drug for treatment of breast cancer, information on the dose, pharmacokinetics and toxicity of Palbociclib in human is already available. Thus, it would be easier to move this drug into clinic and repurpose it for treatment of MPN/MF. We observed that Palbociclib treatment (0.25-0.5 uM) significantly reduced the proliferation of murine BA/F3 cells expressing JAK2V617F or MPLW515L. Palbociclib treatment also significantly inhibited human JAK2V617F-positive HEL, SET-2 and UKE-1 cells. However, Palbociclibonly modestly affected proliferation of wild type JAK2-expressing BA/F3 cells at higher concentrations. Palbociclib treatment also resulted in marked apoptosis in BA/F3-EpoR cells expressing JAK2V617F. Furthermore, Palbociclib and ruxolitinib synergistically induced apoptosis in JAK2V617F-expressing hematopoietic cells. Additionally, Palbociclib significantly inhibited hematopoietic progenitor colony formation in MPN/MF CD34+ cells. We previously generated conditional JAK2V617F knock-in mice. Whereas expression of heterozygous JAK2V617F induces a PV-like disease, homozygous JAK2V617F expression promotes rapid progression to myelofibrosis. We have utilized the homozygous JAK2V617F mice to test the in vivo efficacy of Palbociclib alone and in combination with ruxolitinib against MF. Treatment of Palbociclib alone significantly reduced the WBC and neutrophil counts and decreased the spleen size in homozygous JAK2V617F mice. Combined treatment of Palbociclib and ruxolitinib almost completely normalized the WBC and neutrophil counts and the spleen size in homozygous JAK2V617F mice. Histopathologic analysis revealed marked reduction in fibrosis in the bone marrow of homozygous JAK2V617F mice treated with Palbociclib or Palbociclib and ruxolitinib combination. We also have tested the in vivo efficacy of Palbociclib and Palbociclib/ruxolitinib combination in MPL mutant (MPLW515L) mouse model of MF. We observed similar decrease in WBC and neutrophil counts and reduction in splenomegaly in MPL mutant mice upon treatment of Palbociclib alone or in combination with ruxolitinib. Combined treatment of Palbociclib and ruxolitinib remarkably reduced fibrosis in the bone marrow of MPL mutant mice. Overall, our results suggest that palbociclib in combination with ruxolitinib may have therapeutic potential against MF and support the clinical investigation of palbociclib and ruxolitinib combination in patients with MF. Disclosures No relevant conflicts of interest to declare.

Differential roles of STAT1 and STAT2 in the sensitivity of JAK2V617F- vs. BCR-ABL-positive cells to interferon alpha

BackgroundInterferon alpha (IFNa) monotherapy is recommended as the standard therapy in polycythemia vera (PV) but not in chronic myeloid leukemia (CML). Here, we investigated the mechanisms of IFNa efficacy in JAK2V617F- vs. BCR-ABL-positive cells.MethodsGene expression microarrays and RT-qPCR of PV vs. CML patient PBMCs and CD34+ cells and of the murine cell line 32D expressing JAK2V617F or BCR-ABL were used to analyze and compare interferon-stimulated gene (ISG) expression. Furthermore, using CRISPR/Cas9n technology, targeted disruption of STAT1 or STAT2, respectively, was performed in 32D-BCR-ABL and 32D-JAK2V617F cells to evaluate the role of these transcription factors for IFNa efficacy. The knockout cell lines were reconstituted with STAT1, STAT2, STAT1Y701F, or STAT2Y689F to analyze the importance of wild-type and phosphomutant STATs for the IFNa response. ChIP-seq and ChIP were performed to correlate histone marks with ISG expression.ResultsMicroarray analysis and RT-qPCR revealed significant upregulation of ISGs in 32D-JAK2V617F but downregulation in 32D-BCR-ABL cells, and these effects were reversed by tyrosine kinase inhibitor (TKI) treatment. Similar expression patterns were confirmed in human cell lines, primary PV and CML patient PBMCs and CD34+ cells, demonstrating that these effects are operational in patients. IFNa treatment increased Stat1, Stat2, and Irf9 mRNA as well as pY-STAT1 in all cell lines; however, viability was specifically decreased in 32D-JAK2V617F. STAT1 or STAT2 knockout and reconstitution with wild-type or phospho-deficient STAT mutants demonstrated the necessity of STAT2 for IFNa-induced STAT1 phosphorylation in BCR-ABL- but not in JAK2V617F-expressing cells. STAT1 was essential for IFNa activity in both BCR-ABL- and JAK2V617F-positive cells. Furthermore, ChIP experiments demonstrate higher repressive and lower active chromatin marks at the promoters of ISGs in BCR-ABL-expressing cells.ConclusionsJAK2V617F but not BCR-ABL sensitizes MPN cells to interferon, and this effect was dependent on STAT1. Moreover, STAT2 is a survival factor in BCR-ABL- and JAK2V617F-positive cells but an IFNa-sensitizing factor solely in 32D-JAK2V617F cells by upregulation of STAT1 expression.

Implication of Hypoxia-Inducible Factor-1 (HIF-1) As a New Therapeutic Target in JAK2V617F Positive Myeloproliferative Neoplasms (MPN)

Myeloproliferative neoplasms (MPN) are a heterogeneous group of malignancies including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). The JAK2V617F mutation can be found in 90% of PV and approximately 50% of ET and PMF patients. Hypoxia-inducible factors (HIFs) are master transcriptional regulators of the response to decreases in cellular oxygen levels. Unveiling the function of deregulated HIF-1 signaling in normal and malignant hematopoiesis was the aim of several recent publications, highlighting the importance of HIF-1 for the maintenance of leukemic stem cells (LSCs) in acute and chronic myeloid leukemia (AML/CML). In a JAK2V617F knock-in mouse model and in patients, JAK2V617F was shown to induce the accumulation of reactive oxygen species (ROS) in the hematopoietic stem cell compartment, leading to a stabilization of HIF-1α protein. Further, aberrant STAT5 and PI3K/AKT/mTOR signaling induced HIF-1α expression on the transcriptional and translational level. Ruxolitinib treatment inhibited growth and reduced the expression of HIF-1α and its target gene VEGF in the JAK2V617F human erythroleukemia cell line HEL. In several leukemic cell lines constitutive expression of HIF-1α was reported, even under normoxic conditions. However, it still remains unknown whether HIF-1α plays a role in JAK2V617F positive MPN. In this study, we investigated the role HIF-1α signaling in JAK2V617F positive MPN in vitro.We retrovirally transduced the murine bone marrow cell line 32D with JAK2V617F or JAK2WT. Western blot analysis revealed significant increases in HIF-1α protein levels in JAK2V617F positive cells compared to JAK2WT controls after cultivation in normoxic conditions and this effect was abrogated by treatment with the JAK1/JAK2 inhibitor ruxolitinib. Inhibition of HIF-1, binding to hypoxia response elements (HRE), by low doses of echinomycin (1 nM), significantly impaired proliferation and survival. Using an Annexin-V/7-AAD flow cytometry assay apoptosis was found to be selectively induced in JAK2V617F positive, but not JAK2WT cells after echinomycin treatment. Additionally, BrdU/7-AAD cell cycle analysis revealed that only JAK2V617F positive cells were significantly arrested in G0/1 phase. These findings were consistent with shRNA-mediated knockdown (KD) of HIF-1α in JAK2V617F transduced 32D cells in presence but not the absence of HIF-2 antagonist 2. Inhibition of HIF-2 was necessary due to a compensatory increase of HIF-2α protein levels, shown by Western Blot analysis, counteracting HIF-1α-KD mediated effects. We isolated PBMCs and BMMNCs from JAK2V617F positive patients or healthy controls using Ficoll density gradient centrifugation. Echinomycin significantly abrogated the colony formation ability alone and in combination with ruxolitinib. In vitro treatment with echinomycin significantly decreased cell number and viability of 8 JAK2V617F positive BMMNC samples (4 PV, 3 PMF, 1 preMF; p[1nM]=0.0169, p[5nM]=0.0009) and 7 PBMC samples (6 PV, 1 PMF; p[1nM]=0.0156, p[5nM]=0.0156) in a dose-dependent manner. In contrast, PBMCs from 6 healthy donors were unaffected by the treatment. The same effect was observed in heterozygous and homozygous iPS cell-derived progenitors from JAK2V617F positive PV patients, whereas JAK2WT cells were unaffected by the treatment. Collectively, our data indicate that targeting HIF-1 might represent a novel therapeutic approach in classical Philadelphia-chromosome-negative MPN. DisclosuresBrümmendorf:Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Janssen: Consultancy; Merck: Consultancy; Takeda: Consultancy.

The NF-KB Inhibitor DMAPT in Combination with Ruxolitinib Displays Efficacy in Jak2V617F Knock-in Mouse Model of Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPN) are a group of hematologic malignancies characterized by excessive proliferation of myeloid/erythroid lineage cells. The JAK2V617F mutation has been found in a majority of patients with MPN including polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF). Ruxolitinib, a JAK1/JAK2 inhibitor, has been approved for treatment of MF and PV. Although ruxolitinib treatment provides some benefits, it does not produce disease remission or reverse bone marrow (BM) fibrosis in patients with MPN/MF. This underscores the need to identify new therapeutic targets and develop novel therapies for treatment of MPN.In order to identify new therapeutic targets in MPN, we performed microarray gene expression analysis on sorted hematopoietic stem cells (HSC) from wild type and JAK2V617F mice. We found significant enrichment of the NF-κB signaling pathway in JAK2V617F HSC compared with wild type HSC. We also observed increased phosphorylation/activation of NF-κB (p65 RelA) in the BM of JAK2V617F mice. Hyper activation of NF-κB also has been observed in patients with MPN. Lentiviral shRNA-mediated knockdown of RelA significantly reduced the proliferation of murine Ba/F3-EpoR cells expressing JAK2V617F. RelA knockdown also significantly inhibited human JAK2V617F-positive HEL, SET-2 and UKE-1 leukemia cells. Furthermore, forced expression of a dominant negative IκBα mutant markedly reduced proliferation in Ba/F3-EpoR-JAK2V617F cells. These data suggest a role for the NF-κB signaling pathway in MPN pathogenesis.In this study, we investigated the efficacy of the NF-κB inhibitor, dimethylaminoparthenolide (DMAPT), in hematopoietic cells expressing JAK2V617F and in JAK2V617F knock-in mouse model of MPN. DMAPT is a water soluble, orally bioavailable NF-κB inhibitor that has shown promise against several solid tumors in pre-clinical models. Treatment of DMAPT (1-2.5 uM) significantly reduced the proliferation of Ba/F3-EpoR-JAK2V617Fcells. DMAPT (2.5-7.5 uM) treatment also significantly inhibited the human JAK2V617F-positive HEL, SET-2 and UKE-1 leukemia cells. Furthermore, DMAPT treatment significantly inhibited the hematopoietic progenitor colony outgrowth in JAK2V617F mice BM and MPN patient peripheral blood CD34+ cells.We previously generated conditional Jak2V617F knock-in mice. Whereas expression of heterozygous Jak2V617F induces a PV-like disease, homozygous Jak2V617F expression promotes rapid progression to myelofibrosis. We have utilized the homozygous Jak2V617F mice to test the in vivo efficacy of DMAPT alone and in combination with ruxolitinib against MPN. We observed that combined treatment of DMAPT and ruxolitinib significantly reduced the WBC and neutrophil counts and spleen size in Jak2V617F mice compared with vehicle or single drug treatment. Combined treatment of DMAPT and ruxolitinib also significantly reduced hematopoietic stem/progenitors and myeloid precursors in the BM and spleens of Jak2V617F mice. Furthermore, combined treatment of DMAPT and ruxolitinib significantly decreased the Jak2V617F mutant-expressing myeloid precursors in the BM and spleens of transplanted mice. Histopathologic analysis revealed significant reduction of fibrosis in the bone marrow of Jak2V617F mice treated with the combination of DMAPT and ruxolitinib. Overall, our results suggest that simultaneous inhibition of JAK2 and NF-κB pathways might be useful for treatment of MPN/MF. DisclosuresCrooks:University of Arkansas for Medical Sciences: Patents & Royalties: Inventor on the DMAPT patents.