Myelofibrosis Patient Samples Research Articles

Inflammation and bone marrow fibrosis: novel immunotherapeutic targets.

Myelofibrosis (MF) is primarily driven by constitutive activation of the Janus kinase/signal transducer of activators of transcription (JAK/STAT) pathway. While JAK inhibitors have shown to alleviate disease symptoms, their disease-modifying effects in MF are limited. The only curative treatment remains allogeneic stem cell transplantation, which can be applied to a minority of patients. As a result, there is a need to explore novel targets in MF to facilitate appropriate drug development and therapeutic pathways. Recent research has focused on identifying novel signals that contribute to the abnormal cross-talk between hematopoietic and stromal cells, which promotes MF and disease progression. Inflammation and immune dysregulation have emerged as key drivers of both the initiation and progression of MF. A growing number of actionable targets has been identified, including cytokines, transcription factors, signalling networks and cell surface-associated molecules. These targets exhibit dysfunctions in malignant and nonmalignant hematopoietic cells, but also in nonhematopoietic cells of the bone marrow. The study of these inflammation-related molecules, in preclinical models and MF patient's samples, is providing novel therapeutic targets. The identification of immunotherapeutic targets is expanding the therapeutic landscape of MF. This review provides a summary of the most recent advancements in the study of immunotherapeutic targets in MF.

CAMK2G is identified as a novel therapeutic target for myelofibrosis

AbstractAlthough JAK1/2 inhibition is effective in alleviating symptoms of myelofibrosis (MF), it does not result in the eradication of MF clones, which can lead to inhibitor-resistant clones emerging during the treatment. Here, we established induced pluripotent stem cells (iPSCs) derived from MF patient samples (MF-iPSCs) harboring JAK2 V617F, CALR type 1, or CALR type 2 mutations. We demonstrated that these cells faithfully recapitulate the drug sensitivity of the disease. These cells were used for chemical screening, and calcium/calmodulin-dependent protein kinase 2 (CAMK2) was identified as a promising therapeutic target. MF model cells and mice induced by MPL W515L, another type of mutation recurrently detected in MF patients, were used to elucidate the therapeutic potential of CAMK2 inhibition. CAMK2 inhibition was effective against JAK2 inhibitor-sensitive and JAK2 inhibitor-resistant cells. Further research revealed CAMK2 γ subtype was important in MF model cells induced by MPL W515L. We showed that CAMK2G hetero knockout in the primary bone marrow cells expressing MPL W515L decreased colony-forming capacity. CAMK2G inhibition with berbamine, a CAMK2G inhibitor, significantly prolonged survival and reduced disease phenotypes, such as splenomegaly and leukocytosis in a MF mouse model induced by MPL W515L. We investigated the molecular mechanisms underlying the therapeutic effect of CAMK2G inhibition and found that CAMK2G is activated by MPL signaling in MF model cells and is an effector in the MPL-JAK2 signaling pathway in these cells. These results indicate CAMK2G plays an important role in MF, and CAMK2G inhibition may be a novel therapeutic strategy that overcomes resistance to JAK1/2 inhibition.

A Novel Humanized Animal Model Reveals Clonal Architecture and Therapeutic Vulnerabilities in Myelofibrosis

Myelofibrosis (MF) is the deadliest subtype of myeloproliferative neoplasm (MPN) with a median survival of approximately 5 years. Ruxolitinib, a front line therapy for JAK2V617F mutant MPN, can alleviate symptoms of the disease, but does not eliminate the malignant clone and has minimal impact on BM fibrosis and overall survival. Current mouse models do not recapitulate the clinical heterogeneity, clonal genetic composition, or morphological features of MF. Most notably, these models do not generate robust reticulin fibrosis in the bone marrow, the most significant MF pathology. This lack of clinically relevant MF models presents a major barrier to deciphering the complex genetic drivers of the disease and developing effective therapies against it. We evaluated the ability of CD34+ hematopoietic stem and progenitor cells (HSPCs) from MF patients (that contain the MPN-disease initiating population) to give rise to MF in xenotransplanted NSGS mice. >5x104 FACS-sorted CD34+ HSPCs from the peripheral blood of MF patients with JAK2V617F (n=12), CALRindels (n=7) and MPLW515L (n=2) were transplanted into sublethally irradiated (200rads) NSGS mice via X-ray guided intra-tibial injection. We observed robust engraftment of patient-derived cells at 12 weeks post-transplant regardless of their genetic background or donor patient disease severity (Fig 1A). Post-transplant, BM analysis revealed robust expansion of phenotypically defined MF HSCs relative to cord blood CD34+ control recipients, suggesting a permissive niche for MF HSCs to undergo self-renewal. Remarkably, transplantation of CD34+ cells produced other hallmarks of MF in recipient animals such as splenomegaly, thrombocytosis and most importantly BM reticulin fibrosis in all recipients (Fig 1B). We assessed the clonal architecture of engrafted human cells compared to the primary disease in the donor patients through exome sequencing of CD34+ cells prior to transplantation and hCD45+ cells from MF xenografts. We found that the clonal and subclonal mutational landscape observed in CD34+ cells prior to transplantation was maintained in recipient mice (Fig 1C), suggesting that the PDX model accurately reflects the cellular composition of the primary disease. Intriguingly, in two of the xenografted patient samples, we identified additional mutations that were not detected in the primary patient samples using standard sequencing - namely TP53R248Q and EZH2Y663H respectively. Two years after we detected these mutations in PDXs, these MF patients transformed into sAML with acquisition of TP53R248Q and EZH2Y663H mutations. We performed droplet digital PCR and demonstrated that indeed rare pre-leukemic subclones containing these mutations were present at low levels (< 0.01% VAF) in chronic stage MF patients at least two years prior to sAML progression. These data also suggested that these rare subclones responsible for leukemic transformation expand significantly (>300 fold) under the selective pressure of transplantation in NSGS mice. Additional validation of these findings in a further six pre-sAML MF patient samples is currently ongoing. If successful, this model could be used to prospectively identify rising leukemic clones in chronic stage MF patients, which are below the level of detect of standard sequencing as a mechanism to stratify such patients for more aggressive treatments. While sequencing can identify ultra-rare variants, it cannot discern their functional potential for sAML transformation, which is the advantage of this approach. Finally, we harnessed this system for pre-clinical studies, initially focusing on inhibiting the JAK/STAT signaling pathway. Ruxolitinib treatment in MF PDXs produced remarkably similar phenotypes as observed in patients. We observed a small, but significant reduction in engraftment of MF cells in the BM and a sharp reduction in spleen size in Ruxolitinib-treated group compared to vehicle control. Ruxoltinib treatment however did not reduce the frequency of MF HSCs, the disease initiating population or lessen the degree of reticulin fibrosis. These data suggest that this system can be used as a reliable, clinically-relevant drug screening platform. Taken together, we offer the field a critical, previously missing biologically relevant screening system for validation of MPN drug targets identified in cell lines or genetic mouse models prior to moving forward into clinical trials. Disclosures Oh: Blueprint Medicines: Consultancy; Celgene/BMS: Consultancy; Constellation: Consultancy; CTI Biopharma: Consultancy; Disc Medicine: Consultancy; Gilead Sciences: Consultancy; Incyte Corporation: Consultancy; Kartos Therapeutics: Consultancy; Novartis: Consultancy; PharmaEssentia: Consultancy.

Cytokine production in myelofibrosis exhibits differential responsiveness to JAK-STAT, MAP kinase, and NFκB signaling.

The distinct clinical features of myelofibrosis (MF) have been attributed in part to dysregulated inflammatory cytokine production. Circulating cytokine levels are elevated in MF patients; a subset of which have been shown to be poor prognostic indicators. In this study, cytokine overproduction was examined in MF patient plasma and in MF blood cells ex vivo using mass cytometry. Plasma cytokines measured following treatment with ruxolitinib remained markedly abnormal, indicating that aberrant cytokine production persists despite therapeutic JAK2 inhibition. In MF patient samples, 14/15 cytokines measured by mass cytometry were found to be constitutively overproduced, with the principal cellular source for most cytokines being monocytes, implicating a non-cell-autonomous role for monocyte-derived cytokines impacting disease-propagating stem/progenitor cells in MF. The majority of cytokines elevated in MF exhibited ex vivo hypersensitivity to thrombopoietin (TPO), toll-like receptor (TLR) ligands, and/or tumor necrosis factor (TNF). A subset of this group (including TNF, IL-6, IL-8, IL-10) was minimally sensitive to ruxolitinib. All TPO/TLR/TNF-sensitive cytokines, however, were sensitive to pharmacologic inhibition of NFκB and/or MAP kinase signaling. These results indicate that NFκB and MAP kinase signaling maintain cytokine overproduction in MF, and that inhibition of these pathways may provide optimal control of inflammatory pathophysiology in MF.

Efficacy of ALK5 inhibition in myelofibrosis

Myelofibrosis (MF) is a bone marrow disorder characterized by clonal myeloproliferation, aberrant cytokine production, extramedullary hematopoiesis, and bone marrow fibrosis. Although somatic mutations in JAK2, MPL, and CALR have been identified in the pathogenesis of these diseases, inhibitors of the Jak2 pathway have not demonstrated efficacy in ameliorating MF in patients. TGF-β family members are profibrotic cytokines and we observed significant TGF-β1 isoform overexpression in a large cohort of primary MF patient samples. Significant overexpression of TGF-β1 was also observed in murine clonal MPLW515L megakaryocytic cells. TGF-β1 stimulated the deposition of excessive collagen by mesenchymal stromal cells (MSCs) by activating the TGF-β receptor I kinase (ALK5)/Smad3 pathway. MSCs derived from MPLW515L mice demonstrated sustained overproduction of both collagen I and collagen III, effects that were abrogated by ALK5 inhibition in vitro and in vivo. Importantly, use of galunisertib, a clinically active ALK5 inhibitor, significantly improved MF in both MPLW515L and JAK2V617F mouse models. These data demonstrate the role of malignant hematopoietic stem cell (HSC)/TGF-β/MSC axis in the pathogenesis of MF, and provide a preclinical rationale for ALK5 blockade as a therapeutic strategy in MF.

Preliminary Results from a Phase I Dose Escalation Trial of Ruxolitinib and the PI3Kδ Inhibitor TGR-1202 in Myelofibrosis

Background: Therapies for myelofibrosis (MF) are limited and most are palliative. The JAK1/2 inhibitor ruxolitinib reduces spleen size and MF-related symptoms and improves survival, but can be limited by dose-dependent anemia and thrombocytopenia. Moreover, nearly half of ruxolitinib responders relapse within 5 years. PI3Kd is highly expressed in MF patient samples, independent of ruxolitinib pre-exposure. In JAK2-mutated cell lines, inhibition of PI3K/AKT signaling reduced proliferation and clonogenic potential. The once daily, next generation PI3Kd inhibitor TGR-1202 inhibited PI3K/AKT signaling and led to apoptosis in leukemia and lymphoma cell lines and was well-tolerated in clinical studies, with a toxicity profile distinct from that of ruxolitinib and other PI3Kd inhibitors. We hypothesized that adding TGR-1202 to ruxolitinib could resensitize or augment the response of MF patients with lost or suboptimal response to single-agent ruxolitinib.Objective: To assess safety of TGR-1202 in combination with ruxolitinib in MF patientsSecondary Objectives: Hematologic response, symptom assessmentMethods: MF patients who had sub-optimal responses to ruxolitinib continued their highest tolerated dose of ruxolitinib without change for ≥ 8 weeks, and were assigned to escalating doses of TGR-1202 in a standard 3+3 algorithm. Adverse events (AEs) were graded by NCI-CTCAE v4.03. Efficacy was assessed according to IWG-MRT consensus response criteria. Symptoms were assessed by the MPN symptom assessment form total symptom score (TSS). All patients received Pneumocystis pneumonia prophylaxis after cycle 1.Results: Eleven MF patients were enrolled and received 400 mg (n=3), 800 mg (n=6), or 600 mg TGR-1202 (n=2) daily. Nine were evaluable for response. Median age was 66y, 73% were male. All had ECOG PS 0-1. Five patients had mutations in JAK2, 4 in CALR, and 3 in MPL; these were mutually exclusive with exception of 1 patient with CALR and MPL mutations (Table 1). Median number of cycles of TGR-1202 + ruxolitinib treatment was 5 (1-13). Grade 2 anemia was the most common AE (Table 2). Two patients had asymptomatic Grade 3 elevations in amylase and lipase that persisted after drug was held, meeting criteria for dose limiting toxicities (DLTs) in 2 separate cohorts (TGR-1202 800mg+ruxolitinib 15mg BID and TGR-1202 800mg+ruxolitinib 10mg BID). Both patients had peak plasma TGR-1202 concentrations 1.5-2x higher than the other patients receiving 800mg TGR-1202, although steady-state levels were equivalent. The maximum tolerated dose (MTD) of TGR-1202 in combination with ruxolitinib was not established. Two patients went off-study due to AEs, and 3 due to progressive disease. One of 9 evaluable patients achieved complete remission and 7 had stable disease. Seven of the 9 evaluable patients had improvement in hematologic parameters and 8 had reduced MF symptoms with a median 33% decrease in TSS (Fig. 1).Conclusions: TGR-1202 + ruxolitinib was well-tolerated. Pharmacokinetic data were consistent with single-agent TGR-1202 (unpublished data), indicating that ruxolitinib does not alter absorption or metabolism of TGR-1202. Grade 3 elevations in amylase and lipase were considered DLTs, per protocol. Although the clinical significance of these asymptomatic laboratory findings is not clear, the protocol was amended to further assay these labs and to exclude concomitant medications with the potential to increase amylase/lipase. Importantly, no grade ≥3 hepatotoxicity, colitis, or thrombocytopenia was seen and no MTD was found. Although only one patient achieved CR, 89% demonstrated clinical benefit with the addition of TGR-1202 to ruxolitinib, supporting further exploration of this combination. [Display omitted] DisclosuresStrickland:Alexion Pharmaceuticals: Consultancy; Ambit: Consultancy; Baxalta: Consultancy; Boehringer Ingelheim: Consultancy, Research Funding; CTI Biopharma: Consultancy; Daiichi Sankyo: Consultancy; Sunesis Pharmaceuticals: Consultancy, Research Funding; Abbvie: Research Funding; Astellas Pharma: Research Funding; Celator: Research Funding; Cyclacel: Research Funding; GlaxoSmithKline: Research Funding; Karyopharm Therapeutica: Research Funding; Sanofi: Research Funding. Miskin:TG Therapeutics, Inc: Employment, Equity Ownership. Cavers:TG Therapeutics: Employment, Equity Ownership. Sportelli:TG Therapeutics, Inc.: Employment, Equity Ownership. Michaelis:Pfizer: Equity Ownership; Cellgene Corporation: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte Corporation: Consultancy, Honoraria. Mesa:CTI: Research Funding; Promedior: Research Funding; Celgene: Research Funding; Gilead: Research Funding; Incyte: Research Funding; Galena: Consultancy; Ariad: Consultancy; Novartis: Consultancy. Savona:Amgen Inc.: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Research Funding; Ariad: Membership on an entity's Board of Directors or advisory committees; Takeda: Research Funding; Sunesis: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees.

HSP27: A Therapeutic Target in Myelofibrosis

▪Myelofibrosis (MF) is the most aggressive myeloproliferative neoplasms (MPN) with the highest degree of morbidity and mortality, including progressive bone marrow fibrosis resulting into bone marrow failure. JAK2 kinase inhibitors have been successfully used for a few years in MPN and more particularly for MF treatment. Despite their beneficial effects on spleen size and symptoms, JAK2 inhibitors induce low molecular and survival responses underscoring the urgent need for other therapeutic approaches. Recently, heat shock protein 90 (HSP90) - known to stabilize JAK2 - has been reported as a promising therapeutic target in MPN. However HSP90 inhibitors show toxicity and induce the expression of stress-inducible proteins like HSP70 and, most likely HSP27 as previously shown in other cancers. In addition, we and others have shown that HSP27, was strongly expressed in patients with idiopathic pulmonary, lung or kidney tubulointerstitial fibrosis, underlying a relevant role of HSP27 in fibrotic processes. Taking into account both the beneficial effects of HSP inhibitors in leukemia and in MPN, and the possible implication of HSP27 in fibrosis, we have evaluated in this work, the status of HSP27 in MF patient's samples and assess the effectiveness of an HSP27 oligonucleotide inhibitor called OGX-427.In this study, we first assessed the extracellular and intracellular level of HSPs from MF patients by ELISA, flow cytometry and by immunohistochemistry. We observed for the first time a specific increase in both intracellular and extracellular HSP27 in CD34+ circulating hematopoietic progenitor cells (n=9-16; P=0.0097) and in the sera of patients (n=24-27; P<0.0001) with MF compared with healthy donors, respectively. Moreover, we identified the presence of HSP27 in the bone marrow’s MF patients.We then investigated the in vivo impact of OGX-427, a specific inhibitor of HSP27, or an oligonucleotide control in a murine TPO-induced MF mouse model. The use of OGX-427 limited the progression of the disease in our MF mouse model (n=9). In particular, OGX-427 was associated with a marked reduction in both the spleen weight and size. Also, we noted a decrease of megakaryocyte hyperplasia in the bone marrow accompanied by a visible restoration of spleen structure and lymphoid white pulp territories with OGX-427.Taking altogether, our results support a key role of HSP27 in the pathophysiology in MF and highlight the potential therapeutic benefit of HSP27 inhibitors in this disorder. DisclosuresNo relevant conflicts of interest to declare.

Autocrine TNF-α Signaling in Hematopoietic Stem Cells Promotes Myeloproliferative Disease Progression through Activation of TNFR2

The JAK2V617F mutation is a unifying feature in the majority of patients with myeloproliferative neoplasms (MPNs). The presence of JAK2V617 in a hematopoietic stem (HSC) or progenitor cell confers a proliferative advantage over native JAK2 counterparts. Several inflammatory cytokines are elevated in MPN patients and in murine models of JAK2V617F-driven MPN. In particular, expression of tumor necrosis factor alpha (TNF-α) is increased by constitutive JAK2 kinase activity and imparts a competitive advantage on JAK2V617Fexpressing cells.To identify cell types responsible for increased levels of TNF-α we performed intracellular cytokine staining in leukocytes from myelofibrosis (MF) patients and normal controls. Using a panel of markers to define cellular subsets we found expression of TNF-α was uniformly low in unstimulated cells. However, when cells were treated with lipopolysaccharide (LPS), TNF-α expression was 16-fold higher in HSCs of MF patients compared to normal controls (p<0.005), while expression in mature populations was not different. In a murine model of JAK2V617F driven MPN, where JAK2V617F cells are identified by GFP, TNF-α expression was 3-fold higher in JAK2V617F-expressing HSCs (GFP+) compared to GFP-controls (p<0.005), irrespective of LPS stimulation.To evaluate anti-TNF therapy in MPNs, we treated JAK2V617F mice with etanercept, a soluble TNF receptor fusion protein that binds and inactivates TNF-α. Etanercept did not significantly restrain JAK2V617F-associated WBC or hematocrit increases over a 10-week period, despite suppression of TNF-α activity in plasma. As TNF-α may activate pro-apoptotic (predominantly through TNF receptor 1, TNFR1) and pro-survival pathways (predominantly through TNF receptor 2, TNFR2), we reasoned that global blockade of TNF-α may not shift the balance in favor of normal hematopoiesis. To investigate this we sorted primitive (Lin-, cKit+) cells from mice with JAK2V617F-induced MPN by TNF receptor expression. TNFR2+ cells showed significantly increased colony formation compared to TNFR1+ cells, demonstrating that TNFR2 expression is associated with increased clonogenic potential. Additionally we treated these primitive cells with specific antibodies blocking TNFR1 or TNFR2. Colony assays performed after 3 days in liquid culture with TNFR-blocking antibodies confirmed that interrupting TNFR2-mediated survival signaling resulted in decreased colony formation (61% reduction) while blocking TNFR1 increased colony formation (5% increase). In addition we tested CD34+ cells from MF patient samples with TNFR-blocking antibodies. As seen with the murine MPN cells, colony formation was decreased with the TNFR2 blocking antibody (30% reduction) and increased with the TNFR1 blocking antibody (48% increase) consistent with a differential activation of survival signals by TNF-α in MPN cells. These studies were performed without addition of exogenous TNF-α, which confirms the ability and requirement for these primitive JAK2V617Fcells to utilize TNF-α production to actively support survival.Our data suggest that TNF-α generated by primitive MPN cells promotes their survival through activation of TNFR2, while TNFR1 activation is suppressive. If selective inhibition of TNFR2 shifts the equilibrium toward normal hematopoietic cells, this would support the use of TNFR2 blockade to treat MF and other myeloproliferative neoplasms. Genotyping of human MF and murine MPN colonies cultured with TNF receptor blocking antibodies and experiments in normal human CD34+ cells are ongoing and will be presented. DisclosuresDeininger:BMS, Novartis, Celgene, Genzyme, Gilead: Research Funding; BMA, ARIAD, Novartis, Incyte, Pfizer: Advisory Board, Advisory Board Other; BMS, ARIAD, Novartis, Incyte, Pfizer: Consultancy.

Ruxolitinib in Combination with Nilotinib and Prednisolone, a New Synergistic Approach to Treat Myelofibrosis

Background: Finding the best therapy option to treat myelofibrosis (MF) represents a huge challenge. Ruxolitinib (R) is a potent JAK1/2 inhibitor that has demonstrated improved survival and symptomatology in MF patients. There are a lot researches looking for the best combination of JAK inhibitor to improve its efficacy. In a previous work, we screened different drugs to evaluate if they were synergistic with ruxolitinib, we found that ruxolitinib was synergistic with nilotinib (N) and Prednisolone (P) as well as other drugs including bortezomib and HSP-90 inhibitors.Aim: To study the synergistic behavior and mechanism of actions of ruxolitinib in combination with nilotinib and prednisolone both in patient samples and cell lines.Methods: We have studied 20 secondary or primary MF patients and cell line: BA/F3 transfected with mutated JAK2 V617F (BA/F3 JAK2V617F).Classical assay was performed for patient samples: mononuclear cells isolated from peripheral blood were cultured during 2 weeks in Methocult TM GF_H4535 with 20 ng/ml IL-3, and 50 ng/ml SCF, in presence of increasing concentrations of R, N or P or their combinations.Also, mononuclear cells were cultured as described above, during 2 weeks but without drugs. Then, cells were washed with PBS and cultured 72 hours in RPMI 10% FBS and plated at 15,000 per well in 96-well plates with increasing concentrations of drugs.After both assay, cells were labeled with Annexin V and CD13 and analyzed in the Exvitech platform, an automated multiparametric flow cytometry platform.For cell lines, these were cultured in RPMI 10% FBS in the presence of increasing concentration of drugs. After 48 h of incubation, we performed a wst-8 assay to evaluate cell viability.To analyzed effect of treatment on survival and proliferation signaling pathways, cell lines were treated for 30 min with R 0.032 µM, N 1.6 µM and P 0.8 µM or combinations and total lysates were collected. To perform western blot, we use phospho-STAT5 (Tyr 964) and STAT5 after stripping as primary antibody and β-actin as load control.Graphpad Prism or XLFit was used to analyze dose-response curves. Synergism will be evaluated by the Median Effect methods described by T-C Chou and P. Talalay.Results:In patient samples when performed the classical assay, we found synergistic interaction in all combination in most of the patients (Table 1). Briefly, all combinations showed synergetic behavior (C<1) and triple combination were strongly synergetic in all patient samples: CI = 0.213 and 0.348. [Display omitted] However, when pathologic cells were amplified in a methylcellulose culture and then analyzed, we found a strong potentiation, representing an important dose reduction of R in the presence of N (Dose-Reduction Index (DRI) = 6.10, (n = 4) and a DRI = 110.17 when R is combined with P (n =2). There was not a synergistic interaction between R with N or P, because R was not efficient enough in this model.Interestingly, R had more effect in the classical assay, always less than 0.500 µM, than in the amplification assay even on less sensitive patient samples (Table 2). R seems inhibit growth and development of hematopoietic stem cells but not differentiated cells. N had a wide range of action in classical assay, but in amplification assay the effect was homogeneous, around 9 µM. P had similar heterogeneous effect on both assays from high nM to low µM. [Display omitted] The 3 drugs were more potent in cell line BA/F3V617F than in patients: IC50 R = 15 nM; IC50 N = 3.35 µM; IC50 P = 4 nM. All drug combination tested were synergistic (CI<1). We choose [R] = 0.032 µM, [N] = 1.6 µM and [P] = 0.8 µM and their combinations to analyzed the signaling pathways by western blot because of their strong synergistic behavior: CI RN = 0.144; CI RP = 0.178; CI RNP = 0.223. We studied the effect of the different treatment in the phosphorylation on STAT5 after 30 min of incubation with drugs. We observed a decreased of phosphorylated form in R treatment and combination with N and P but not in N or P alone. The maximal effect was found when RNP combination was employed.Conclusions: we have showed that ruxolitinib have a synergistic effect with nilotinib and prednisone in MF patient samples. This effect is in part mediated through a greatest inhibition of STAT5 pathway. This preclinical study warranties a clinical trial with this combination in patients with MF. DisclosuresHernandez-Campo:Vivia Biotech: Employment. Gorrochategui:Vivia Biotech: Employment. Primo:Vivia Biotech: Employment. Ballesteros:Vivia Biotech: Employment, Equity Ownership. Martinez-Lopez:Vivia Biotech: Honoraria.