JAK Inhibitor Ruxolitinib Research Articles

Selective strong synergism of Ruxolitinib and second generation tyrosine kinase inhibitors to overcome bone marrow stroma related drug resistance in chronic myelogenous leukemia

The IC50 of TKIs is significantly increased when BCR-ABL+ K562 cell line is cultured in stroma conditioned media produced by BM mesenchymal cells. In particular, while the Imatinib IC50 in the stromal co-cultures was well above the in vivo through levels of the drug, the IC50s of second generation TKIs were still below their through levels. Moreover, we provide a formal comparison of the synergy between first and second generation TKIs with the JAK inhibitor Ruxolitinib to overcome BM stroma related TKI resistance. Taken together, our data provide a rationale for the therapeutic combination of TKIs and Ruxolitinib with the aim to eradicate primary BCR-ABL+ cells homed in BM niches.

Ruxolitinib Inhibits Dendritic Cell Function By Interfering With The MAP-Kinase/NF-Kb Signalling Pathways

The recently approved Jak-Inhibitor ruxolitinib improves clinical symptoms in patients with myelofibrosis and autoimmune diseases such as rheumatoid arthritis. It has been shown in myelofibrosis that the clinical benefit occurs independent of the Jak mutational status, indicating that not only diseased, but also healthy cells like immune cells might be affected by the treatment. In a previous study we investigated the effects of ruxolitinib on dendritic cells (DCs) as the most professional antigen presenting immune cells. We were able to show that ruxolitinib markedly impairs human and murine DC phenotype, DC activation and migration and the induction of allogeneic and antigen-specific T cell responses in vitro and in vivo. In regard to good clinical responses in myelofibrosis patients lacking the Jak-2-mutation, it seems rather unlikely that Jak-inhibition is the only responsible for the observed in vivo and in vitro effects. In the present study, we therefore aimed to investigate whether other signalling mechanisms might be additionally involved. To exclude significant effects on Jaks other than Jak-2, we first analyzed the more selective Jak-2 inhibitor TG101348 in our in vitro models. Here, we were able to show that both inhibitors impair DC phenotype and function to a similar extend. We next investigated the role of the MAP-kinase and the NF-kB signalling pathways. Therefore, human monocyte-derived immature and mature DCs were treated with different concentrations of ruxolitinib for distinct durations, followed by preparation of whole cell lysates and nuclear extracts to perform Western blot analyses. We found that ruxolitinib treatment resulted in a reduced phosphorylation and expression of the MAP-kinases ERK and p38, while the housekeeping gene GAPDH was not affected. This effect was independent of apoptosis induction as the expression of caspase 3 was constant. Moreover, the expression of the NF-kB family members RelA and cRel were markedly reduced.Taken together, we have shown that the Jak-inhibitior ruxolitinib significantly impairs DC phenotype and function resulting in a markedly reduced induction of allogeneic and antigen-specific T cell responses. To analyze potentially involved underlying signalling mechanisms, we were able to show that exposure of DCs to ruxolitinib leads to a distinct downregulation of the MAP-kinase ERK and p38 as well as a diminished expression of NF-kB family members downstream in the MAP-kinase signalling pathway. This may indicate that not only the Jak/Stat signalling pathway might be involved in the immunological effects of ruxolitinib in non-mutated cells, but also the MAP-kinase pathway downstream of Jak2. Our work might add a piece of information for a deeper understanding of the clinical efficacy of ruxolitinib in diseases characterized by an exaggerated inflammation, such as myelofibrosis or autoimmune disorders. Disclosures:Wolf:Novartis: Honoraria, Research Funding.

Myelofibrosis Survival Benefit Confirmed for Ruxolitinib

The Janus kinase 1 (JAK1) and JAK2 inhibitor ruxolitinib was shown in an early trial to reduce spleen size and improve symptoms in patients with

Tumoricidal effects of the JAK inhibitor Ruxolitinib (INC424) on hepatocellular carcinoma in vitro

Hepatocellular carcinoma (HCC) is an aggressive tumour with limited treatment options. The Janus kinase/signal transducers and activators of transcription (JAK/STAT) signalling pathway plays a key role in promoting tumorigenesis in HCC. Recently a new JAK inhibitor Ruxolitinib (INC424) has been developed by Novartis Pharmaceuticals and it shows high affinity for JAK signalling with very low affinity for non-JAK targets. Clinical trials have demonstrated that Ruxolitinib has good therapeutic efficacy for the treatment of myelofibrosis and is currently FDA approved for the treatment of advanced stages of this disease. Our study tested the effects of Ruxolitinib on HCC tumorigenesis in vitro. Ruxolitinib effectively inhibited JAK/STAT signalling in HCC cells with a significant reduction in the expression of JAK downstream targets pSTAT1 and pSTAT3. Ruxolitinib also caused a marked reduction in the proliferation and colony formation of HCC cells. The antiproliferative effect of Ruxolitinib on HCC cells is unlikely due to off-target effects with no inhibition of key regulators of other cell proliferative pathways. To our knowledge this study is the first to report on the effect of Ruxolitinib on liver cancer cells.

The characteristics and treatment of patients with primary myelofibrosis at the University medical centre Ljubljana in the period 2008–2012

Background: Primary myelofibrosis (PMF) is a rare hematologic condition belonging to the group of classical myeloproliferative neoplasms. For the diagnosis of PMF, bone marrow histology examination and fibrosis evaluation is mandatory. No recurrent cytogenetic abnormality has been identified and JAK2 mutation is present only in approximately half of the patients. Treatment is limited mainly to alleviation of B symptoms, splenomegaly and anemia. Only allogeneic hematopoietic stem-cell transplant (HSCT) can offer a cure from the disease. A new drug, JAK2 inhibitor ruxolitinib, that has been available since recently, will change treatment options mainly for patients not eligible for HSCT. Methods: Hipokrat medical database was searched for patients diagnosed with PMF between 2008–2012. Data were collected retrospectively and analysed using basic statistics. Results: PMF was newly diagnosed in 28 patients (18/64.3 % men). Two (7.1 %) patients presented with systemic symptoms and ten (35.7 %) had enlarged spleen. Their median blood counts and lactate dehydrogenase activity (LDH) results were: leukocytes 11.25 x 109/l (3.00–72.00), hemoglobin 117 g/l (68–173), platelets 420 x 109/l (111–1477), LDH 7.45 μkat/l (3.00–20.71). In six (21.4 %) patients only bone marrow cytology examination was performed without histologic examination. Cytogenetics was performed in 11 (39.3 %) patients with three abnormalities detected: complex, +9 and del(13q14). JAK 2 mutation was identified in 12 (60.0 %) of 20 examined cases. Patients were treated with acetylsalicylic acid (n = 12/42.9 %), hydroxyurea (n = 12/42.9 %), blood transfusions or epoetins (n = 12), anagrelide (n = 4/14.3 %), methylprednisolone (n = 4/14.3 %), immunomodulatory drugs (n = 3/10.7 %), interferon (n = 2/7.1 %), spleen irradiation (n = 2/7.1 %) and venipunctures (n = 2/7.1 %). At the end of 2011, first eligible patients were treated with ruxolitinib. Conclusions: PMF is a rare disorder with various presentations and treatment suited specifically to patients’ complaints. The treatment is symptomatic with HSCT being the only curable option available. Ruxolitinib will play an importatnt role in PMF treatment in the future.

Targeting JAK1/2 and mTOR in murine xenograft models of Ph-like acute lymphoblastic leukemia

AbstractCRLF2 rearrangements, JAK1/2 point mutations, and JAK2 fusion genes have been identified in Philadelphia chromosome (Ph)–like acute lymphoblastic leukemia (ALL), a recently described subtype of pediatric high-risk B-precursor ALL (B-ALL) which exhibits a gene expression profile similar to Ph-positive ALL and has a poor prognosis. Hyperactive JAK/STAT and PI3K/mammalian target of rapamycin (mTOR) signaling is common in this high-risk subset. We, therefore, investigated the efficacy of the JAK inhibitor ruxolitinib and the mTOR inhibitor rapamycin in xenograft models of 8 pediatric B-ALL cases with and without CRLF2 and JAK genomic lesions. Ruxolitinib treatment yielded significantly lower peripheral blast counts compared with vehicle (P < .05) in 6 of 8 human leukemia xenografts and lower splenic blast counts (P < .05) in 8 of 8 samples. Enhanced responses to ruxolitinib were observed in samples harboring JAK-activating lesions and higher levels of STAT5 phosphorylation. Rapamycin controlled leukemia burden in all 8 B-ALL samples. Survival analysis of 2 representative B-ALL xenografts demonstrated prolonged survival with rapamycin treatment compared with vehicle (P < .01). These data demonstrate preclinical in vivo efficacy of ruxolitinib and rapamycin in this high-risk B-ALL subtype, for which novel treatments are urgently needed, and highlight the therapeutic potential of targeted kinase inhibition in Ph-like ALL.

Ruxolitinib, an oral JAK1 and JAK2 inhibitor, in myelofibrosis

Introduction: Myelofibrosis (MF) is a debilitating hematologic malignancy characterized by progressive splenomegaly, burdensome symptoms, cytopenias and shortened survival. Chronic alterations in Janus-associated kinase-signal transducer and activator of transcription (JAK-STAT) signaling have been identified in the pathogenesis of MF, making this pathway a target for drug development. Ruxolitinib is the first JAK1 and JAK2 inhibitor to be approved by the US Food and Drug Administration.Areas covered: This review describes the characteristics of MF, the current therapeutic options and need for effective therapies, the contribution of aberrant JAK-STAT signaling to various disease-specific manifestations and the pharmacodynamics, pharmacokinetics, efficacy and tolerability of ruxolitinib. Articles describing MF disease burden and results of ruxolitinib pre-clinical and clinical trials were identified and summarized.Expert opinion: Conventional MF treatments alleviate some MF symptoms but have limited efficacy, do not modify the natural history of the disease and are not approved for MF. The JAK1 and JAK2 inhibitor ruxolitinib has shown promising results in pre-clinical and clinical trials. In Phase III trials, ruxolitinib was shown to reduce splenomegaly and improve MF-related symptoms. Recent evidence also suggests that ruxolitinib may improve survival. The most common adverse events were anemia and thrombocytopenia, which were managed with dose adjustments (or red blood cell transfusions for anemia).

Ruxolitinib for myelofibrosis

Myelofibrosis (MF), a myeloproliferative neoplasm, is a disease associated with a significant burden of symptoms, shortened survival and an array of standard treatment regimens which have historically lacked impact and efficacy. The discovery in 2005 of the highly prevalent JAK2V617F-activating tyrosine kinase mutation, strongly associated with myeloproliferative neoplasms, led to the rapid development of a new class of drugs, JAK inhibitors, for the treatment of MF. These drugs have produced a profound effect upon splenomegaly, proliferative blood counts and constitutional symptoms, which are characteristic of MF, and have given hope to both patients and physicians who treat this debilitating disease. This article reviews the current evidence for the use of the JAK inhibitor ruxolitinib, which has completed Phase III trials and with which there is the most extensive clinical experience, as well as assessing other JAK inhibitors in clinical development.

Biology and clinical management of myeloproliferative neoplasms and development of the JAK inhibitor ruxolitinib.

Myeloproliferative neoplasms (MPN) are debilitating stem cell-derived clonal myeloid malignancies. Conventional treatments for the BCR-ABL1-negative MPN including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) have, so far, been unsatisfactory. Following the discovery of dysregulated JAK-STAT signaling in patients with MPN, many efforts have been directed toward the development of molecularly targeted therapies, including inhibitors of JAK1 and JAK2. Ruxolitinib (previously known as INCB018424; Incyte Corporation, Wilmington, Delaware, USA) is a rationally designed potent oral JAK1 and JAK2 inhibitor that has undergone clinical trials in patients with PV, ET, and PMF. Ruxolitinib was approved on November 16, 2011 by the United States Food and Drug Administration for the treatment of intermediate or high-risk myelofibrosis (MF), including patients with PMF, post-PV MF, and post-ET MF. In randomized phase III studies, ruxolitinib treatment resulted in significant and durable reductions in splenomegaly and improvements in disease-related symptoms in patients with MF compared with placebo or best available therapy. The most common adverse events were anemia and thrombocytopenia, which were manageable and rarely led to discontinuation. This review addresses the cellular and molecular biology, and the clinical management of MPN.

An open-label, multicenter, expanded access study assessing the safety and efficacy of oral ruxolitinib administered to patients with primary myelofibrosis (PMF), post-polycythemia myelofibrosis (PPV MF) or post-essential thrombocythemia myelofibrosis (PET-MF).

TPS6640 Background: Myelofibrosis (MF) can be primary in origin or can progress from polycythemia vera (PPV-MF) or essential thrombocythemia (PET-MF). MF is characterized by cytopenias, reactive bone marrow fibrosis, splenomegaly, and constitutional symptoms including weight loss, fever, and night sweats. MF is associated with dysregulation of the Janus kinase pathway, irrespective of JAK2V617F mutation status. Recently, ruxolitinib (INCB018424) was approved for the treatment of MF on the basis of results from 2 phase 3 trials (COMFORT-I and -II). These trials demonstrated marked reductions in splenomegaly and symptom burden, and improvements in health-related quality of life (QoL) measures. Methods: JAK Inhibitor rUxolitinib in Myelofibrosis Patients (JUMP) is a global, phase 3b expanded access trial (NCT01493414) designed to assess the safety and efficacy of ruxolitinib in adult pts with PMF, PPV-MF or PET-MF who are treatment naïve, and are intolerant of, or had progressed on any prior therapy. Inclusion criteria: peripheral blast count of &lt;10%, adequate liver and renal function and intermediate-2 or high risk MF according to IPSS criteria or intermediate-1 risk MF with palpable spleen length ≥ 5 cm. The primary endpoint is to assess the safety of ruxolitinib. Additional endpoints include the proportion of pts with a &gt; 50% reduction in palpable spleen length, % change in white blood cell and platelet counts from baseline (BL), and change in packed red blood cell transfusion requirements. Changes from BL in QoL scores will be assessed using validated ECOG PS, FACT-Lymphoma, and FACIT instruments. Pts with a BL platelet count &gt; 200,000/μL will receive oral ruxolitinib 20 mg BID; pts with a BL platelet count of 100,000/μL to 200,000/μL will receive 15 mg BID; dose modifications are permitted for safety and efficacy. Global enrollment is open and currently includes 277 pts. This is planned to be the largest study ever conducted in pts with MF. [Table: see text]

In vivo monitoring of JAK/STAT and PI3K/mTOR signal transduction inhibition in pediatric CRLF2-rearranged acute lymphoblastic leukemia (ALL).

9506 Background: Therapy intensification for children with B-precursor ALL with high-risk genetic lesions has improved relapse-free survival. CRLF2 rearrangements and JAK2 and IL7RA mutations occur in 10-15% of adult and pediatric ALL patients, most of whom relapse. We and others identified aberrant kinase signatures and perturbed JAK/STAT and PI3K/mTOR signal transduction via in vitro studies of CRLF2-rearranged (CRLF2r) ALLs, suggesting the therapeutic relevance of signal transduction inhibitors (STIs). Our creation of CRLF2r ALL xenograft models has enabled rapid preclinical testing of STIs and measurement of in vivo target inhibition. We hypothesized that inhibition of JAK/STAT and PI3K/mTOR phosphosignaling correlates with therapeutic responses in these models. Methods: NOD/SCID/γc-null (NSG) mice well-engrafted with pediatric ALL samples were treated with the JAK inhibitor ruxolitinib, the mTOR inhibitor sirolimus, or vehicle for 72 hours (for signaling response) or 4 weeks (for therapeutic response). Splenocytes were briefly stimulated ex vivo with thymic stromal lymphopoietin (ligand for CRLF2) and stained with human-specific surface and intracellular phosphoantibodies for multi-parameter phosphoflow cytometry analysis. Results: Ruxolitinib-induced inhibition of phospho (p)-JAK2 and pSTAT5 was most pronounced in non-CRLF2r ALLs with novel JAK2-activating BCR-JAK2 and IL7RA/LNK mutations. Sirolimus potently inhibited pS6 and other PI3K/mTOR pathway phosphoproteins in the CRLF2r r ALLs. PSTAT5 and pS6 inhibition correlated with longer-term ruxolitinib- and sirolimus-induced decreases in ALL cell burden, demonstrating therapeutic responses to STIs. Conclusions: Ruxolitinib inhibited JAK/STAT phosphosignaling and markedly decreased leukemic burden in the JAK2-activating BCR-JAK2 and IL7RA/LNK mutant ALL xenografts. Sirolimus potently inhibited PI3K/mTOR (as well as some JAK/STAT) phosphosignaling and had greater therapeutic efficacy than ruxolitinib in the CRLF2r ALLs. The safety of ruxolitinib and of temsirolimus with cytotoxic chemotherapy are currently being established in Children’s Oncology Group Phase I trials.

FDA Approval of the JAK Inhibitor Ruxolitinib and the Management of Patients with Myelofibrosis

FDA Approval of the JAK Inhibitor Ruxolitinib and the Management of Patients with Myelofibrosis

Treatment with Ruxolitinib (INCB018424) Induced Changes of Microrna Expression in Granulocytes of Patients with Polycythemia Vera and Essential Thrombocythemia,

Abstract 3852Protocol #18424-256 is a Phase 2 study of the JAK1 and JAK2 inhibitor ruxolitinib (INCB01842) in patients with advanced polycythemia vera (PV) and essential thrombocythemia (ET) refractory to hydroxyurea; preliminary results have been reported (Verstovsek S et al, ASH meeting, 2010, Abstract 313). As part of this protocol we analyzed the microRNA (miRNA) expression profile in gradient purified granulocytes (GN) in 5 PV and 11 ET patients at both baseline (Ba) and at week 4 (W4) after ruxolitinib initiation. Total RNA was purified using RNeasy kit and the purity/integrity assessed by Agilent 6000 Nano LabChips. The RNA (500ng) was labeled with the FlashTag® Biotin HSR kit and hybridized to Affymetrix Gene Chip miRNA Array 1.0. Image files were generated with Affymetrix Expression Console package and managed with Partek GS. Differentially expressed miRNAs were obtained and comparing using a paired t-test with a p-value cutoff of <0.01. Twenty microRNAs out of more than 46,000 probe included in the chip were significantly modified after treatment; of these, 12 (miR-708, -493, -367, -508-5p, -548m, -509-5p, -150, -146a, -31, -1275, -342, -32) were increased and 8 decreased (miR-1182, 142-3p, -374a, -101, 7f, 499-5p, -187, -548). Integrated analysis indicated that most target genes were involved in regulatory pathways potentially associated with MPN pathogenesis, including the TGF-β, MAPK, mTOR, JAK-STAT, VEGF and Notch, as well as chemokine and T-cell and B-cell receptor signaling pathways. miR-150, the levels of which increased with ruxolitinib treatment, has a negative effect on erythropoiesis by regulating the megakaryocyte-erythroid progenitor fate through antagonism to the transcription factor MYB; reduced miR-150 levels were reported in PMF granulocytes and PV reticulocytes, and its expression appeared to be inversely correlated with JAK2V617F burden in PV. miR146a, which was up-regulated post-ruxolitinib, has been reported to inhibit megakaryocytopoiesis by targeting CXCR4 mRNA. Of note, its over-expression also resulted in inhibition of Interleukin(IL)-1β-mediated release of pro-inflammatory cytokines, including IL-8, and suppression of the expression of NF-kB targets such as IL-1β, IL-6, IL-8. miR-31 and miR-342, which both increased whit ruxolitinib, were found to be down-regulated in PMF granulocytes and their levels inversely correlated with JAK2 V617F burden in PV. miR-32, up-regulated during ruxolitinib treatment, is believed to be involved in early erythroid commitment. Finally, miR-101 which is normally up-regulated during megakaryocytopoiesis as well as a negative regulator of EZH2 expression, significantly decreased with treatment. We hypothesize that these miRNAs might represent targets of the activated JAK/STAT pathway and potentially contribute to disease pathogenesis. To this end, we analyzed changes in miRNA expression profile in the SET-2 cell line, a heterozygous JAK2V617F mutated cell line derived from a patient with ET, at 3 and 6 hr of treatment with ruxolitinib (1.5 mM). Using paired t-statistics we identified significant changes vs baseline in 39 and 43 miRNAs tested in the SET2 cell line samples at 3h and 6h, respectively. Of these, 22 were up-regulated and 17 down-regulated at 3h; 8 up-regulated and 35 down-regulated at 6h of treatment. Four of the significantly modified miRNAs, namely miR-32, miR-31, miR-101, and miR-1275, showed changes consistent with these detected in granulocytes of patients treated with ruxolitinib, suggesting they might represent direct targets of this agent.However, the exact mechanisms of changes in miRNA expression following JAK inhibitor treatment remain to be defined.In summary, this study has documented for the first time in vivo changes of miRNA expression in granulocytes of patients receiving ruxolitinib, that could mechanistically contribute to the clinical efficacy of the drug. Disclosures:Vaddi:Incyte corporation: Employment. Burn:Incyte corporation: Employment. Contel:Incyte corporation: Employment. Verstovsek:Incyte Corporation: Research Funding. Vannucchi:Incyte corporation: Research Funding.

Clinical Burden and Progression of Myelofibrosis in a Controlled Study Population of Placebo-Treated Patients (COMFORT-I)

Abstract 5146 Background:Myelofibrosis (MF) is characterized by splenomegaly, debilitating symptoms, hematologic abnormalities, and shortened survival. COMFORT-I is a Phase III randomized, double-blind, placebo-controlled study of the JAK1 and JAK2 inhibitor ruxolitinib in patients (pts) with MF. The placebo arm of this ongoing study provides a unique population in which to assess the clinical burden of MF in a controlled setting. Methods:151 pts with intermediate-2 or high-risk primary-MF (PMF), post-polycythemia vera-MF (PPV-MF), or post-essential thombocythemia-MF (PET-MF) who were not candidates for other therapies were randomized to receive placebo in COMFORT-I after a 28-day washout of previous MF therapies. Assessments included MF symptoms measured daily using an electronic diary (modified Myelofibrosis Symptom Assessment Form [MFSAF] v2.0), spleen volume measured every 12 wks by blinded abdominal MRI or CT scans, and laboratory measures at least monthly through wk 24. The Patient-Reported Outcomes Measurement System (PROMISE) Fatigue and Patient Global Impression of Change (PGIC) questionnaires were administered at each visit. Pts with missing data (baseline or post-baseline assessment measured), or pts who withdrew or crossed over to active treatment before a study visit were not included in analyses. Results:151 pts received 1 or more doses of placebo (median exposure 204 days). The study included 65% high-risk and 35% intermediate-2 risk pts, with a median of 2.5 years since diagnosis. 55% had PMF, 31% PPV-MF, and 14% PET-MF. Regardless of prior MF therapy (57% were on hydroxyurea), there was continual worsening of symptoms over time as assessed by the MFSAF Total Symptom Score (TSS, comprised of scores for night sweats, itching, abdominal pain and discomfort, early satiety and bone/muscle pain) (see Figure).At wk 24, mean/median spleen volume increased by +8.1%/+8.5%, and both intermediate-2 and high-risk pts demonstrated progressive disease over the course of the study. Mean/median PROMIS Fatigue score worsened by 9.1%/6.5%. Mean/median score for the PGIC (a 7-point scale ranging from 1 = very much improved to 7 = very much worse, with 4 = no change) was 4.2/4.0 at wk 24; 19.6% of pts reported feeling much worse or very much worse. Mean/median total body weight changed from 72.0 kg/72.2 kg to 70.1 kg/69.7 kg at wk 24. Hematology assessments (median, baseline vs wk 24) were as follows: hemoglobin, 105 vs 106 g/L; platelets, 233 vs 219 x109/L; neutrophils, 14.8 vs 16.0 x109/L; white blood cell count, 18.4 vs 20.1 x109/L. Two of 151 pts had new-onset (new or worsening) grade 3 or grade 4 thrombocytopenia, and 23/151 pts had new-onset grade 3 or grade 4 anemia. 16 pts met protocol-specified criteria for unblinding and crossover to active treatment before wk 24, which was a 25% increase in MRI-based spleen volume either with or without increased pain or weight loss; thus, mean and median values reported at wk 24 may represent an underestimate of the degree of worsening in disease parameters. Conclusion:The signs and symptoms of MF continued to worsen over a 6-month time frame in this placebo population. The data illustrate the progressive nature of MF and support the need for effective therapeutic interventions. Moreover, treatment benefit must be benchmarked against continual evolution of the disease in the absence of treatment. Early recognition of the debilitating symptoms of MF and intervention with effective therapy to prevent continual worsening of the disease should be strongly considered. [Display omitted] Disclosures:Verstovsek:Incyte: Research Funding. Mesa:Incyte: Research Funding; Lilly: Research Funding; SBio: Research Funding; Astra Zeneca: Research Funding; NS Pharma: Research Funding; Celgene: Research Funding. Gotlib:Incyte: Consultancy, Research Funding. Levy:Incyte: Employment, Equity Ownership. Gupta:Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Catalano:Incyte: Honoraria; Novartis: Honoraria. Deininger:BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Genzyme: Research Funding. Miller:Incyte: Research Funding; Novartis: Honoraria, Research Funding, Speakers Bureau. Winton:Incyte: Consultancy. Arcasoy:Incyte: Research Funding. Lyons:Amgen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Incyte: Research Funding; Telik: Research Funding; Alexion: Consultancy, Honoraria; Novartis: Research Funding. Vaddi:Incyte: Employment. Erickson-Viitanen:Incyte: Employment. Sun:Incyte: Employment. Sandor:Incyte: Employment. Kantarjian:Incyte: Research Funding; Novartis: Consultancy, Research Funding.

Associations Between Improvements in Myelofibrosis (MF) Symptoms and Quality of Life Measures with Splenomegaly Reduction in COMFORT-I: A Randomized, Double-Blind, Phase III Trial of the JAK1 and JAK2 Inhibitor Ruxolitinib Versus Placebo in Patients with MF,

Abstract 3842▪▪This icon denotes a clinically relevant abstract Background:Dysregulated JAK-STAT signaling is a key feature in MF, which is characterized by splenomegaly, debilitating symptoms, poor quality of life (QoL), cytopenias, and shortened survival. Ruxolitinib is a selective JAK1 and JAK2 inhibitor with clinical activity in MF. COMFORT-I is a Phase III, double-blinded, placebo-controlled study of ruxolitinib in patients with MF. At wk 24, 41.9% of patients receiving ruxolitinib reached the protocol-defined threshold of a ≥35% reduction in spleen volume (SV) compared to 0.7% in the placebo group (p<0.0001). The objective of this analysis was to evaluate the relationship between SV reduction achieved with ruxolitinib treatment and changes in symptoms and QoL. Methods:309 patients were randomized to receive ruxolitinib (n=155) or placebo (n=154). Initial dosing was based on baseline platelet counts: 15 mg BID for counts of 100–200 x109/L and 20 mg BID for >200 x109/L. SV was measured by blinded imaging (MRI or CT scan) every 12 wks. Via electronic diary, patients reported MF symptoms daily using the modified Myelofibrosis Symptom Assessment Form (MFSAF) v2.0. The Total Symptom Score (TSS) from the MFSAF represents a combined score for the individual symptoms of abdominal discomfort, pain under ribs on left side, early satiety, itching, night sweats, and bone/muscle pain. The European Organization for Research and Treatment of Cancer Quality-of-Life 30 (EORTC QLQ-C30) and Patient-Reported Outcomes Measurement System (PROMISE) Fatigue questionnaires were administered at each visit; Patient Global Impression of Change (PGIC) was administered at every visit beginning at wk 4. The PGIC rates a patient’s overall sense of whether a treatment has been beneficial or not on a 7-point scale, ranging from 1 = very much improved to 7 = very much worse, with 4 = no change. Results:Baseline QoL scores reflected debilitating disease, and were similar to scores for similar-aged patients with other cancers (Scott NW, Fayers PM, Aaronson NK, et al. EORTC QLQ-C30 Reference Values Manual, July 2008). The impact on patients was particularly evident on the PROMIS Fatigue scale and QLC-C30 Global Health, Physical Functioning, Role Functioning, and Social Functioning subscales.Baseline Symptom and QoL Measures*Parameter MeasuredRuxolitinib (mean)Placebo (mean)Maximum Score PossibleMFSAF - TSS18.016.560 (worst possible)PROMIS Fatigue43.443.2100 (worst possible)EORTC QLQ C-30 SubscalesEORTC Global Health52.654.5100 (best possible)Physical Functioning69.567.6100 (best possible)Role Functioning64.665.1100 (best possible)Emotional Functioning73.776.3100 (best possible)Cognitive Functioning81.179.3100 (best possible)Social Functioning68.065.9100 (best possible)*Baseline measures for patients with both baseline and wk 24 data.Associations between changes in symptoms and QoL with the objectively measured changes in SV were evaluated. Patients receiving ruxolitinib were grouped according to SV reduction at 24 wks of treatment: ≥35% reduction in SV (n=65), 10-<35% (n=51), and <10% (n=23). Symptom and QoL responses were evaluated among the various subgroups (of the SV reduction percentage strata) and comparisons were made across these and the total placebo group (n=106). Shown in the Figure are mean changes at wk 24 relative to baseline for TSS, QoL indicators, and PGIC scores. Conclusions:Changes in SV as low as 10% in ruxolitinib-treated patients were associated with improvements on EORTC QLQ-C30 Global Health subscale, PROMISE Fatigue scale, and the MFSAF v2.0 diary TSS. Moreover, commensurate improvements in these outcomes were observed as the SV reduction increased. Importantly, the simple PGIC questionnaire allowed patients to characterize a meaningful change in their condition that was consistent with more comprehensive measures of MF symptoms and quality of life, reflecting a doctor-patient assessment easily applied to clinical practice. [Display omitted] Disclosures:Mesa:Incyte: Research Funding; Lilly: Research Funding; SBio: Research Funding; Astra Zeneca: Research Funding; NS Pharma: Research Funding; Celgene: Research Funding. Gotlib:Incyte: Consultancy, Research Funding. Gupta:Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Catalano:Incyte: Honoraria; Novartis: Honoraria. Deininger:BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Genzyme: Research Funding. Shields:Incyte: Consultancy. Miller:Incyte: Research Funding; Novartis: Honoraria, Research Funding, Speakers Bureau. Winton:Incyte: Consultancy. Hare:Incyte: Employment. Erickson-Viitanen:Incyte: Employment. Sun:Incyte: Employment. Sandor:Incyte: Employment. Levy:Incyte: Employment, Equity Ownership. Kantarjian:Incyte: Research Funding; Novartis: Consultancy, Research Funding. Verstovsek:Incyte: Research Funding.

Kinase domain mutations confer resistance to novel inhibitors targeting JAK2V617F in myeloproliferative neoplasms

The transforming JAK2V617F kinase is frequently associated with myeloproliferative neoplasms (MPNs) and thought to be instrumental for the overproduction of myeloid lineage cells. Several small molecule drugs targeting JAK2 are currently in clinical development for treatment in these diseases. We performed a high-throughput in vitro screen to identify point mutations in JAK2V617F that would be predicted to have potential clinical relevance and associated with drug resistance to the JAK2 inhibitor ruxolitinib (INCB018424). Seven libraries of mutagenized JAK2V617F cDNA were screened to specifically identify mutations in the predicted drug-binding region that would confer resistance to ruxolitinib, using a BaF3 cell-based assay. We identified 5 different non-synonymous point mutations that conferred drug resistance. Cells containing mutations had a 9 to 33-fold higher EC50 for ruxolitinib compared to native JAK2V617F. Our results further indicated that these mutations also conferred cross-resistance to all JAK2 kinase inhibitors tested, including AZD1480, TG101348, lestaurtinib (CEP-701) and CYT-387. Surprisingly, introduction of the ‘gatekeeper’ mutation (M929I) in JAK2V617F affected only ruxolitinib sensitivity (4-fold increase in EC50). These results suggest that JAK2 inhibitors currently in clinical trials may be prone to resistance as a result of point mutations and caution should be exercised when administering these drugs.

A simplified plate method for detecting gelatine-liquefying bacteria.

<h3>Abstract</h3> Unleashing anti-tumor T cell activity by checkpoint inhibition is effective in many cancer patients but clinical response rates remain limited. Myeloid derived suppressor cells erode antitumor lymphocyte numbers and function, and correlate with resistance to checkpoint inhibitors. By screening small molecule libraries, we identified JAK inhibitors’ ability to rescue T cell function. Despite its documented immune suppressive properties, the prototypical JAK inhibitor ruxolitinib enhanced the efficacy of immune checkpoint blockade in cancer. This effect correlated with loss of suppressive gene expression, and acquisition of immunostimulatory molecular markers and T cell stimulatory activity in myeloid cells. In preclinical models, ruxolitinib significantly improved the function and increased the total numbers of activated tumor-infiltrating NK and CD4 T cells compared to checkpoint blockade alone and the efficacy was conditional on granulocytic cells. In addition to myeloid reprogramming in the tumor, ruxolitinib blunts G-CSF signaling in the bone marrow to prevent expression of suppressive and chemotaxis genes in neutrophils. In a clinical trial of Hodgkin lymphoma patients resistant to checkpoint inhibitors, treatment with ruxolitinib significantly reduced neutrophil-to-lymphocyte ratios and levels of suppressive markers in myeloid cells but increased numbers of cytokine-producing T cells. These results support the therapeutic potential of JAK inhibition in combination with checkpoint inhibitors in cancer and highlight the potential of reshaped myeloid immunity to improve immunotherapy. One sentence summary: Ruxolitinib reshapes myeloid immunity to synergize with checkpoint inhibitors