Integrated Human and Murine Clinical Study Establishes Clinical Efficacy of Ruxolitinib in Chronic Myelomonocytic Leukemia.

PURPOSE: Chronic myelomonocytic leukemia (CMML) is a rare leukemia characterized by peripheral monocytosis with no disease-modifying therapies. CMML cells are uniquely hypersensitive to granulocyte-macrophage colony-stimulating factor (GM-CSF) and robustly engraft in immunocompromised mice that secrete human cytokines. To leverage these unique biological features, we conducted an integrated human and murine study evaluating ruxolitinib, a JAK1/2 inhibitor that potently downregulates intracellular GM-CSF signaling. PATIENTS AND METHODS: A total of 50 patients with WHO-defined CMML were enrolled in this open-label, multi-institution phase I/II clinical study, with a ruxolitinib dose of 20 mg twice daily studied in phase II. In parallel, 49 patient-derived xenografts (PDX) derived from 13 study participants were generated and randomized to receive ruxolitinib or vehicle control. RESULTS: The most common grade 3/4 treatment-related toxicities observed were anemia (10%) and thrombocytopenia (6%). The clinical overall response rate was 38% by Myelodysplastic Syndrome/Myeloproliferative Neoplasm (MDS/MPN) International Working Group (IWG) criteria and 43% of patients with baseline splenomegaly achieved a spleen response. Profiling of cytokine levels and somatic mutations at baseline failed to identify predictive biomarkers. PDX models derived from screening samples of study participants recapitulated responses seen in humans, particularly spleen responses, and corroborated ruxolitinib's clinical efficacy in a randomized murine study not feasible in human trials. CONCLUSIONS: Ruxolitinib demonstrated clinical efficacy and an acceptable adverse event profile in patients with CMML, identifying a potential novel therapeutic in this rare malignancy. Furthermore, this study demonstrates proof of concept that PDX modeling can recapitulate responses of patients treated on clinical trial and represents a novel correlative study that corroborates clinical efficacy seen in humans.See related commentary by Shastri and Adrianzen-Herrera, p. 6069.

Assessment of ASC specks as a putative biomarker of pyroptosis in myelodysplastic syndromes: an observational cohort study.

BACKGROUND: NLRP3 inflammasome-directed pyroptotic cell death drives ineffective haemopoiesis in myelodysplastic syndromes. During inflammasome assembly, the apoptosis-associated speck-like protein containing a CARD (PYCARD, commonly known as ASC) adaptor protein polymerises into large, filamentous clusters termed ASC specks that are released upon cytolysis. Specks are resistant to proteolytic degradation because of their prion-like structure, and therefore might serve as a biomarker for pyroptotic cell death in myelodysplastic syndromes. METHODS: This observational cohort study was done at the H Lee Moffitt Cancer Center (Tampa, FL, USA). Patients with myelodysplastic syndromes, healthy controls, and patients with non-myelodysplastic syndrome haematological cancers or type 2 diabetes were recruited. We used confocal and electron microscopy to visualise, and flow cytometry to quantify, ASC specks in peripheral blood and bone marrow plasma samples. Speck percentages were compared by t test or ANOVA, correlations were assessed by Spearman's rank correlation coefficient, and biomarker efficiency was assessed by receiver operating characteristics and area under the curve (AUC) analysis. FINDINGS: Between Jan 1, 2005, and Jan 12, 2017, we obtained samples from 177 patients with myelodysplastic syndromes and 29 healthy controls for the discovery cohort, and 113 patients with myelodysplastic syndromes and 31 healthy controls for the validation cohort. We also obtained samples from 22 patients with del(5q) myelodysplastic syndromes, 230 patients with non-myelodysplastic syndrome haematological cancers and 23 patients with type 2 diabetes. After adjustment for glucose concentration, the log10-transformed mean percentage of peripheral blood plasma-derived ASC specks was significantly higher in the 177 patients with myelodysplastic syndromes versus the 29 age-matched, healthy donors (-0·41 [SD 0·49] vs -0·67 [0·59], p=0·034). The percentages of ASC specks in samples from patients with myelodysplastic syndromes were significantly greater than those in samples from individuals with every other haematological cancer studied (all p<0·05) except myelofibrosis (p=0·19). The findings were confirmed in the independent validation cohort (p<0·0001). Peripheral blood plasma danger-associated molecular pattern protein S100-A8 and protein S100-A9 concentrations from 144 patients with myelodysplastic syndromes from the discovery cohort directly correlated with ASC speck percentage (r=0·4, p<0·0001 for S100-A8 and r=0·2, p=0·017 for S100-A9). Patients with at least two somatic gene mutations had a significantly greater mean percentage of peripheral blood plasma ASC specks than patients with one or no mutation (-0·22 [SD 0·63] vs -0·53 [0·44], p=0·008). The percentage of plasma ASC specks was a robust marker for pyroptosis in myelodysplastic syndromes (AUC=0·888), in which a cutoff of 0·80 maximised sensitivity at 0·84 (95% CI 0·65-0·91) and specificity at 0·87 (0·58-0·97). INTERPRETATION: Our results underscore the pathobiological relevance of ASC specks and suggest that ASC specks are a sensitive and specific candidate plasma biomarker that provides an index of medullary pyroptotic cell death and ineffective haemopoiesis in patients with myelodysplastic syndromes. FUNDING: T32 Training Grant (NIH/NCI 5T32 CA115308-08), Edward P Evans Foundation, The Taub Foundation Grants Program, the Flow Cytometry, Analytic Microscopy, and Tissue Core Facilities at the H Lee Moffitt Cancer Center and Research Institute, a National Cancer Institute-designated Comprehensive Cancer Center (P30-CA076292).

Lenalidomide induces lipid raft assembly to enhance erythropoietin receptor signaling in myelodysplastic syndrome progenitors.

Anemia remains the principal management challenge for patients with lower risk Myelodysplastic Syndromes (MDS). Despite appropriate cytokine production and cellular receptor display, erythropoietin receptor (EpoR) signaling is impaired. We reported that EpoR signaling is dependent upon receptor localization within lipid raft microdomains, and that disruption of raft integrity abolishes signaling capacity. Here, we show that MDS erythroid progenitors display markedly diminished raft assembly and smaller raft aggregates compared to normal controls (p = 0.005, raft number; p = 0.023, raft size). Because lenalidomide triggers raft coalescence in T-lymphocytes promoting immune synapse formation, we assessed effects of lenalidomide on raft assembly in MDS erythroid precursors and UT7 cells. Lenalidomide treatment rapidly induced lipid raft formation accompanied by EpoR recruitment into raft fractions together with STAT5, JAK2, and Lyn kinase. The JAK2 phosphatase, CD45, a key negative regulator of EpoR signaling, was displaced from raft fractions. Lenalidomide treatment prior to Epo stimulation enhanced both JAK2 and STAT5 phosphorylation in UT7 and primary MDS erythroid progenitors, accompanied by increased STAT5 DNA binding in UT7 cells, and increased erythroid colony forming capacity in both UT7 and primary cells. Raft induction was associated with F-actin polymerization, which was blocked by Rho kinase inhibition. These data indicate that deficient raft integrity impairs EpoR signaling, and provides a novel strategy to enhance EpoR signal fidelity in non-del(5q) MDS.

JAK2-V617F-mediated signalling is dependent on lipid rafts and statins inhibit JAK2-V617F-dependent cell growth.

Aberrant JAK2 signalling plays an important role in the aetiology of myeloproliferative neoplasms (MPNs). JAK2 inhibitors, however, do not readily eliminate neoplastic MPN cells and thus do not induce patient remission. Further understanding JAK2 signalling in MPNs may uncover novel avenues for therapeutic intervention. Recent work has suggested a potential role for cellular cholesterol in the activation of JAK2 by the erythropoietin receptor and in the development of an MPN-like disorder in mice. Our study demonstrates for the first time that the MPN-associated JAK2-V617F kinase localizes to lipid rafts and that JAK2-V617F-dependent signalling is inhibited by lipid raft disrupting agents, which target membrane cholesterol, a critical component of rafts. We also show for the first time that statins, 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors, widely used to treat hypercholesterolaemia, induce apoptosis and inhibit JAK2-V617F-dependent cell growth. These cells are more sensitive to statin treatment than non-JAK2-V617F-dependent cells. Importantly, statin treatment inhibited erythropoietin-independent erythroid colony formation of primary cells from MPN patients, but had no effect on erythroid colony formation from healthy individuals. Our study is the first to demonstrate that JAK2-V617F signalling is dependent on lipid rafts and that statins may be effective in a potential therapeutic approach for MPNs.

Iron overload in myelodysplastic syndromes: diagnosis and management.

Myelodysplastic syndrome (MDS) is composed of a diverse spectrum of hematopoietic stem cell malignancies characterized by ineffective blood cell production. Many MDS patients are dependent on red blood cell (RBC) transfusions for symptomatic management of refractory anemia. Iron overload ensues when the iron acquired from transfused RBCs exceeds body storage capacity, thereby raising the risk for end organ damage. This is of greatest concern in patients with lower-risk MDS whose expected survival is measured in years. Transfusion dependence is associated with shorter survival and an increased risk for progression to acute myeloid leukemia (AML) in transfusion-dependent patients. Application of recent advances in the treatment of MDS can reduce or eliminate the need for transfusions, thus minimizing the risk of iron overload. Case control studies, prospective surveys, and phase II studies indicate that iron chelation therapy reduces iron load as measured by changes in serum ferritin and may prolong overall survival. Iron chelation strategies include oral agents such as deferasirox (Exjade, Novartis Pharmaceuticals Corp, East Hanover, NJ), deferiprone (Ferriprox, Apotex Europe BV, Leiden, the Netherlands) and, for those patients who are intolerant of or for whom oral therapy is ineffective, parenteral administration of deferoxamine (Desferal, Novartis). This review presents the data related to iron overload in MDS, including its prevalence, diagnosis, clinical impact, and management.

Identification of a novel inhibitor of JAK2 tyrosine kinase by structure-based virtual screening.

Janus kinase 2 (JAK2) plays a crucial role in the pathomechanism of myeloproliferative disorders and hematologic malignancies. A somatic mutation of JAK2 (Val617Phe) was previously shown to occur in 98% of patients with polycythemia vera and 50% of patients with essential thrombocythemia and primary myelofibrosis. Thus, effective JAK2 kinase inhibitors may be of significant therapeutic importance. Here, we applied a structure-based virtual screen to identify novel JAK2 inhibitors. One JAK2 inhibitor in particular, G6, demonstrated remarkable potency as well as specificity, which makes it as a potential lead candidate against diseases related to elevated JAK2 tyrosine kinase activity.

Immunomodulatory therapy for myelodysplastic syndromes.

Thalidomide and lenalidomide belong to the proprietary group of immunomodulatory drugs (IMiDs) that display broad biologic and pharmacologic properties. Encouraging results of clinical studies that evaluated the efficacy of thalidomide in patients with myelodysplastic syndromes (MDSs) led to the investigation of its structural analogue, lenalidomide, in patients with lower-risk MDS. The cumulative results of studies that tested lenalidomide in patients with interstitial deletion of chromosome 5q, ie, del(5q), showed a high frequency of both erythroid and cytogenetic responses (approximately 75% of patients), which led to US Food and Drug Administration approval of this agent for this cytogenetically defined MDS subset. A multicenter phase III study (MDS-002) that investigated the frequency of transfusion response in lower-risk non-del(5q) MDS patients showed that lenalidomide had significant erythropoietic activity, albeit less robust in lower-risk MDS without del(5q). These studies established lenalidomide as an active erythropoietic-remitting agent with novel cytogenetic-remitting activity in lower-risk MDS patients who would not otherwise benefit from therapy with erythropoietic growth factors. The National Comprehensive Cancer Network Clinical Practice Guidelines recently added lenalidomide to the therapeutic algorithm for MDS as front-line therapy for lower-risk MDS patients with del(5q) and transfusion-dependent anemia.

Evolving applications of lenalidomide in the management of anemia in myelodysplastic syndromes.

Lenalidomide has emerged as an effective therapeutic alternative for the management of anemia in lower-risk myelodysplastic syndromes (MDS). Compelling results from phase I and phase II clinical studies prompted the US Food and Drug Administration to approve lenalidomide for the treatment of transfusion-dependent MDS patients with interstitial deletion of chromosome 5q [del(5q)]. Subsequently, the National Comprehensive Cancer Network (NCCN) has incorporated lenalidomide into their current treatment algorithm for the treatment of lower-risk del(5q) patients. This discussion examines the current NCCN guidelines for the treatment of these patients, including the management of anemia in lower-risk MDS, and discusses the potential future therapeutic applications of lenalidomide in this disease.

Vascular endothelial growth factor receptor-1 and receptor-2 initiate a phosphatidylinositide 3-kinase-dependent clonogenic response in acute myeloid leukemia cells.

OBJECTIVE: Vascular endothelial growth factor (VEGF) interacts with two high-affinity receptor tyrosine kinases (RTK) on vascular endothelium to initiate complementary but disparate biologic responses. We previously reported that acute myeloid leukemia (AML) cells express VEGF and one or both VEGF-A receptors, Flt-1 (VEGFR-1) and KDR (VEGFR-2). To evaluate receptor-selective trophic response to VEGF-A in AML cells, we investigated receptor-specific ligand activation responsible for VEGF-initiated clonogenic response. MATERIALS AND METHODS: Using KG1 (VEGFR-1+/VEGFR-2+) and HL60 (VEGFR-1+) cells with differential VEGF receptor display, we investigated ligand-induced clonogenic response and receptor-initiated signaling after stimulation with VEGF-A, the VEGFR-1 selective ligand placental growth factor (PlGF), or receptor-specific antibody agonists. RESULTS: Recombinant human (rhu)-VEGF increased S-phase fraction and stimulated colony formation in both KG1 and HL60 cells. Ligation of VEGFR-1 or VEGFR-2 with receptor-specific antibody agonists triggered equivalent and concentration-dependent stimulation of colony recovery in KG1 cells, whereas clonogenic response in HL60 cells was restricted to VEGFR-1 activation by antibody or PlGF. In serum-deprived KG1 and HL60 cells, rhu-VEGF stimulated rapid and sustained phosphorylation of Akt/PKB that was inhibited by the phosphatidyl inositol 3-kinase (PI3-K) kinase inhibitor wortmannin. Preincubation with wortmannin inhibited VEGF-induced colony formation in a concentration-dependent fashion. rhu-VEGF-induced clonogenic response and Akt phosphorylation was abolished by the VEGF-RTK inhibitor SU-5416 at concentrations greater than 10 microM, whereas MEK inhibition by PD98059 (1 and 10 microM) was ineffective. In vivo suppression of Akt phosphorylation was confirmed in myeloblast lysates from three patients with advanced myeloid malignancies treated with SU5416. CONCLUSION: These data indicate that VEGF interaction with either VEGFR-1 or VEGFR-2 initiates a clonogenic response in AML cells that is PI3-kinase dependent. RTK inhibitors with broad specificity for angiogenic receptors represent novel therapeutics that merit further clinical investigation in AML.