ALBERT

Description: Background: Myelofibrosis (MF) is a clonal neoplastic disease resulting in bone marrow fibrosis, splenomegaly, and debilitating constitutional symptoms. The Janus kinase (JAK) pathway is often dysregulated in MF, and agents targeting this pathway have demonstrated efficacy in this disease. Ruxolitinib (RUX), a potent JAK1/JAK2 inhibitor, demonstrated superiority in spleen volume reduction, symptom improvement, and survival compared with the control arm in the phase III COMFORT-I and COMFORT-II studies. Panobinostat (PAN), a potent pan-deacetylase inhibitor (pan-DACi), inhibits JAK signaling through disruption of the interaction of JAK2 with the protein chaperone heat shock protein 90. In phase I/II studies, PAN has shown splenomegaly reduction and improvement of bone marrow fibrosis. The combination of RUX and PAN demonstrated synergistic anti-MF activity in preclinical studies. These preliminary results led to the initiation of a phase Ib study evaluating the combination of RUX and PAN in patients (pts) with MF. The updated results from the expansion phase of this trial are presented here. Methods: Eligible pts had intermediate-1, -2, or high-risk primary MF, post-polycythemia vera MF, or post-essential thrombocythemia MF by International Prognostic Scoring System criteria, with palpable splenomegaly (≥ 5 cm below the costal margin). The primary objective was determination of the maximum tolerated dose (MTD) and/or recommended phase II dose (RPIID). Secondary objectives included safety, efficacy, and pharmacokinetics. Exploratory endpoints included assessment of improvement in bone marrow fibrosis and reduction of JAK2 V617F allele burden. The treatment schedule was RUX (5-15 mg) twice daily (bid) every day and PAN (10-25 mg) once daily 3 times per week (tiw; days 2, 4, and 6) every other week (qow) in a 28-day cycle. Following dose escalation and identification of the potential RPIID, additional pts were enrolled into the expansion phase and treated at this dose. Results: As of March 14, 2014, a total of 61 pts were enrolled (38 escalation phase and 23 expansion phase). The median duration of exposure to PAN and to RUX was 24.6 weeks and 24.0 weeks, respectively, for pts treated in the expansion phase. Three DLTs were observed in the escalation phase (grade 4 thrombocytopenia [n = 2], grade 3 nausea [n = 1]). No MTD was reached. The RPIID was confirmed to be RUX 15 mg bid and PAN 25 mg tiw qow in May 2014. Among the 34 pts treated at the RPIID, grade 3/4 adverse events (AEs) regardless of causality included anemia (32%), thrombocytopenia (24%), diarrhea (12%), asthenia (9%), and fatigue (9%). AEs led to discontinuation in 6% of pts treated at the RPIID. Two pts treated at the RPIID died due to causes unrelated to study treatment (1 due to myocardial infarction and 1 due to progression of myelofibrosis). Among the pts treated at the RPIID, 79% showed a 〉50% decrease in palpable spleen length, with 100% decrease (non-palpable spleen) being observed in 53% of pts. Additionally, 48% of pts treated at the RPIID in the expansion phase achieved ≥35% reduction in spleen volume (Figure). These results are similar to those observed for spleen volume response at 24 weeks among pts who received single-agent RUX on the phase III COMFORT-I (41.9%) and COMFORT-II (32%) studies. Conclusions: The combination of the JAK1/JAK2 inhibitor RUX and the pan-DACi PAN was well tolerated and resulted in high rates of reductions in splenomegaly in pts with intermediate- and high-risk MF. Although a relatively larger proportion of patients experienced spleen volume reductions at week 24 as compared to the COMFORT studies, the smaller sample size, shorter follow up times and potential differences in the patient populations preclude definitive comparisons. Similar to COMFORT-I and II trials, hematological AEs, specifically anemia and thrombocytopenia, were the most common AEs observed in pts treated with the combination therapy. Pts continue to be treated in the expansion phase at the RPIID. Updated safety, efficacy, and exploratory analyses on bone marrow fibrosis, JAK V617F allele burden, and biomarkers, including cytokines, will be presented. Figure Change in Spleen Volume in Expansion Phase Figure. Change in Spleen Volume in Expansion Phase Disclosures Kiladjian: Novartis: Honoraria, Research Funding, Speakers Bureau; Shire: Membership on an entity's Board of Directors or advisory committees; AOP Orphan: Honoraria, Research Funding. Heidel:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Vannucchi:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ribrag:Celgene: Consultancy; Pharmamar: Consultancy; Epizyme: Research Funding; Bayer: Consultancy, Research Funding; Servier: Consultancy, Honoraria, Research Funding. Conneally:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Kindler:Novartis: Consultancy. Acharyya:Novartis: Employment. Gopalakrishna:Novartis: Employment. Ide:Novartis: Employment, Equity Ownership. Loechner:Novartis: Employment. Mu:Novartis: Employment. Harrison:Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi: Consultancy, Honoraria; CTI: Consultancy, Honoraria; Gilead: Honoraria; SBio: Consultancy; Shire: Speakers Bureau.

Description: Mutations within the FMS-like tyrosine kinase 3 (FLT3) gene on chromosome 13q12 have been detected in up to 35% of acute myeloid leukemia (AML) patients and represent one of the most frequently identified genetic alterations in AML. Over the last years, FLT3 has emerged as a promising molecular target in therapy of AML. Here, we review results of clinical trials and of correlative laboratory studies using small molecule FLT3 tyrosine kinase inhibitors (TKIs) in AML patients. We also review mechanisms of primary and secondary drug resistance to FLT3-TKI, and from the data currently available we summarize lessons learned from FLT3-TKI monotherapy. Finally, for using FLT3 as a molecular target, we discuss novel strategies to overcome treatment failure and to improve FLT3 inhibitor therapy.

Description: Acute myeloid leukemia (AML) cannot be cured by chemotherapy in approximately 60% of cases. Several prognostic factors have been evaluated, such as cytogenetic changes or molecular mutations. Length mutations of the FLT3-gene (internal tandem duplications, FLT3-ITD) confer a significantly worse prognosis with an increased rate of relapsed and refractory disease upon chemotherapy. The high rate of induction failure and of relapse upon chemotherapy in FLT3-ITD positive patients raises the question whether dysregulation at the level of the apoptotic machinery promotes resistance of AML blasts. Myeloid cell leukemia-1 (Mcl-1) protein is an anti-apoptotic member of the Bcl-2 family and blocks cytochrome c-release from mitochondria by interacting with proapoptotic members of the BCL-2 protein family, e.g. BAX and BAK, thereby preventing their activation and mitochondrial outer membrane permeabilization (MOMP). By Western blotting, high levels of Mcl-1 protein expression could be demonstrated in 6/6 FLT3-ITD positive patient samples versus 2/6 in FLT3-wildtype patient samples. Upregulation of Mcl-1 at a RNA and protein level could also be demonstrated in FLT3-ITD positive cell lines using transfected murine 32D cells (32D-FLT3-ITD vs 32D-FLT3- wt) and human FLT3-ITD positive cell lines (MV4;11 (ITD positive) vs RS4;11 (ITD negative)). To functionally investigate the role of Mcl-1 overexpression in resistance to chemotherapy, 32D-FLT3-ITD cells were transfected with a murine Mcl-1-wildtype construct. 32D-FLT3-ITD positive cells stably expressing Mcl-1 and controls were tested for induction of apoptosis upon cytotoxic treatment using various apoptosis assays (TMRE, AnnexinV-Staining, DNA content analysis by FACS). Overexpression of Mcl- 1 in 32D-FLT3-ITD cells conferred a striking decrease in induction of apoptosis upon chemotherapy (daunorubicine/cytarabine) and tyrosine kinase inhibitor treatment in comparison to the empty vector control. To analyze the influence of Mcl-1 expression on drug resistance in primary blasts, we perfomed siRNA knockdown experiments on primary AML blasts; siRNA silencing of Mcl-1 expression in primary AML-blasts was shown to result in increased apoptosis rates of up to 25% upon growth factor starvation or treatment with cytotoxic drugs. Constitutively activated FLT3-receptor phosphorylates and activates downstream signaling nodes as AKT and ERK, which are known upstream modifiers of Mcl-1. Thus, we hypothesized that phosphorylation of Mcl-1 by these pathways may be involved in differential Mcl-1-expression in FLT3-ITD positive AML. To investigate the role of Mcl-1 phosphorylation on drug resistance, a wildtype MCL-1 construct was mutagenized at different phosphorylation sites (serine/threonine to alanine). Experiments analyzing the functional role of mutated Mcl-1 when stably expressed in the hematopoietic cell line 32D-FLT3-ITD are in progress and will be presented. In conclusion, we here present evidence that Mcl-1 is critically involved in mediating resistance in FLT3-ITD positive AML. Our findings provide a rationale to clinically investigate agents that inactivate Mcl-1 in FLT-ITD positive AML.

Description: BACKGROUND: MF is a myeloproliferative neoplasm characterized by bone marrow (BM) fibrosis, splenomegaly, and debilitating constitutional symptoms. RUX is a potent JAK1/JAK2 inhibitor that has demonstrated superiority in spleen volume reduction, symptom improvement, and survival in the phase 3 COMFORT studies compared with placebo and best available therapy. PAN, a potent pan-deacetylase inhibitor, inhibits JAK signaling by disrupting the interaction between JAK2 and heat shock protein 90, a protein chaperone. PAN has demonstrated reductions in splenomegaly and improvement of BM fibrosis in phase 1/2 studies. The combination of RUX and PAN demonstrated synergistic activity in preclinical MF models. Thus, a phase 1b study evaluating RUX plus PAN in pts with MF was initiated. We present results from the expansion phase of this study confirming the recommended phase 2 dose (RP2D) of RUX plus PAN combination therapy and its tolerability in pts with MF. METHODS: Eligible pts had primary MF, post-polycythemia vera MF, or post-essential thrombocythemia MF classified as intermediate-1, -2, or high risk by International Prognostic Scoring System criteria and splenomegaly ≥ 5 cm by palpation. The primary objective was to determine the maximum tolerated dose and/or the RP2D of RUX-PAN combination therapy. Additional objectives included safety and efficacy. Exploratory endpoints included assessment of changes in BM fibrosis, JAK2 allele burden, and levels for 59 cytokines, with a focus on those known to be altered with RUX treatment. Pts received RUX 5-15 mg twice daily (bid) and PAN 10-25 mg 3 times weekly (tiw; days 2, 4, and 6) every other wk (qow) in a 28-day cycle. Following dose escalation and identification of the RP2D, additional pts were enrolled into the expansion phase and treated at this dose. Results: At data cutoff (17 December 2014), 61 pts received treatment (escalation phase, n = 38; expansion phase, n = 23. Three dose-limiting toxicities were observed in the escalation phase (grade 4 thrombocytopenia, n = 2; grade 3 nausea, n = 1). The RP2D was confirmed to be RUX 15 mg bid and PAN 25 mg tiw qow. Among the 34 pts treated at the RP2D, 65% remained on treatment, and 21% discontinued due to adverse events (AEs); 65% had ≥ 1 dose interruption/change. The median duration of exposure to PAN and to RUX in these pts was 67.1 and 68.7 wk, respectively. The most common grade 3/4 hematologic AEs among pts treated at the RP2D, regardless of causality, were anemia (32%) and thrombocytopenia (29%); grade 3/4 nonhematologic AEs included diarrhea (18%), asthenia (12%), and fatigue (9%). Three deaths (due to progression of underlying disease, myocardial infarction, and hypoxic cardiac arrest) occurred on or within 30 days of treatment and were assessed by the treating investigator as unrelated to study treatment. Most pts treated in the expansion phase had a reduction in spleen volume at wk 24 (87%; 20/23) and at wk 48 (74%; 17/23); 57% (13/23) and 39% (9/23) of pts achieved a ≥ 35% reduction from baseline in spleen volume at wk 24 and 48, respectively (Figure). Of 12 evaluable pts assessed for BM fibrosis grade by central review, 4 had improved fibrosis at wk 48, 6 had no change, and 2 worsened. Of the 17 pts in the expansion phase who were JAK2 V617F positive at baseline, 5 (29%) had a ≥ 20% decrease in allele burden by wk 48; most pts had a continuous decline in allele burden over time. Additionally, elevated levels of various markers of inflammation (IL-18, MMP-9, and MPO) normalized on treatment, whereas leptin levels increased, an effect that is associated with improvement in weight loss. CONCLUSIONS: The combination of RUX and PAN was well tolerated and resulted in reductions in splenomegaly over the longer period of follow-up. 57% and 39% of pts achieved a spleen response at wk 24 and 48, respectively. Although no formal comparison can be made due to the small sample size of this study, combination therapy led to a higher proportion of pts achieving a spleen response vs ruxolitinib alone in the COMFORT studies. Reductions in JAK2 V617F allele burden and improvements in BM fibrosis were noted in some pts. Anemia, thrombocytopenia, and diarrhea were the most common AEs; AE rates were consistent with those observed with RUX and PAN monotherapies. Overall, the combination of RUX and PAN was associated with substantial treatment benefits in pts with MF and warrants further investigation through larger studies. Disclosures Harrison: Shire: Speakers Bureau; Sanofi: Honoraria, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; CTI Biopharma: Consultancy, Honoraria, Speakers Bureau; Gilead: Honoraria. Off Label Use: Ruxolitinib is a kinase inhibitor indicated for treatment of patients with intermediate or high-risk myelofibrosis, including primary myelofibrosis, post-polycythemia vera myelofibrosis and post-essential thrombocythemia myelofibrosis. Panobinostat is a histone deacetylase inhibitor indicated for the treatment of patients with multiple myeloma who have received at least 2 prior regimens . Kiladjian:Novartis: Consultancy; Incyte Corporation: Consultancy; Novartis: Other: Travel grant; Research Funding paid to institution (Hôpital Saint-Louis et Université Paris Diderot). Heidel:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Vannucchi:Novartis: Other: Research Funding paid to institution (University of Florence), Research Funding; Shire: Speakers Bureau; Baxalta: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Passamonti:Novartis: Consultancy, Honoraria, Speakers Bureau. Conneally:Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Acharyya:Novartis Pharmaceuticals Corporation: Employment. Gopalakrishna:Novartis Pharma AG: Employment. Ide:Novartis: Employment, Equity Ownership. Liu:Novartis: Employment. Mu:Novartis: Employment. Ribrag:Esai: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmamar: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Description: Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative disorder of early childhood with often fatal outcome. Despite many attempts to develop alternative treatment options allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative modality. In the past our group has linked the prognosis of JMML to differential DNA methylation patterns (Olk-Batz, Blood 2011;117:4871-80 and Poetsch, Epigenetics 2014;9:1252-60), suggesting a key role of epigenetic modifications in JMML pathophysiology. To overcome the lack of suitable preclinical JMML research models we have developed an ex vivo JMML xenotransplantation system using neonatal Rag2-/- gamma-c-/- mice. Transplantation of 1x106 primary JMML cells resulted in stable xenologous engraftment and reproduced a characteristic JMML phenotype including myelomonocytic expansion; infiltration of spleen, liver and, notably, lung; splenomegaly; and reduced survival (median 26 weeks). Persistent human engraftment and leukemic organ infiltration was confirmed by both flow cytometry and immunohistology. Ras pathway mutations present in xenotransplanted patient samples were invariably confirmed in engrafted tissues. In addition, the model sustained serial transplantations and can therefore be used to amplify scarce patient material. We first tested if DNA methylation patterns in JMML cells were stable even after xenologous engraftment because such stability would be a prerequisite if the model were to be used for preclinical investigation of DNA methyltransferase inhibitors. JMML cells before xenotransplantation and those retrieved from the bone marrow of engrafted mice were profiled for global CpG methylation using Illumina 450K arrays. DNA methylation patterns in JMML were patient-specific and surprisingly robust in functional regions over several months of engraftment time (on average, 0.29% of 30877 promoters and 0.25 % of 30725 intragenic regions were called as "differentially methylated" between source and xenograft; 0.2 β-value change cutoff). These findings confirm the suitability of the xenograft model to investigate JMML epigenetics and, more importantly, indicate that patient-specific epigenetic profiles originate in leukemia-initiating stem cells, reinforcing a fundamental role of these alterations in JMML biology. Our group recently published a retrospective case series demonstrating unprecedented clinical efficacy of the DNA methyltransferase inhibitor 5-azacytidine (5AC) to induce partial or complete remissions in JMML before allogeneic HSCT (Cseh, Blood 2015;125:2311-3). To further investigate the drug on the preclinical level we administered 5AC to Rag2-/- gamma-c-/- mice xenografted with primary JMML cells. After a leukemia establishment phase the mice were divided into treatment or mock groups and treated with 5AC (3mg/kg body weight i.p., N=6) or saline (N=6) for 2 cycles (1 dose daily for 5 days; 9 days of recovery). This regimen was tolerated well by the animals. We found that 5AC reduced JMML infiltration in all organs analyzed, with most pronounced effects in spleen (human CD45+ fraction of all CD45+ cells, 0.24% +/- 0.04% vs 39.78% +/- 10.72%; p

Description: Erythropoiesis is a multi-step process in which the development of red blood cells occurs through expansion and differentiation of hematopoietic stem cells (HSCs) into more committed progenitors and finally into erythrocytes. Erythropoietin (Epo) is strictly required for erythropoiesis as it promotes survival and late maturation. In vivo and in vitro studies have pointed out the major role of erythropoietin receptor (EpoR) signalling through JAK2 tyrosine-kinase and STAT5a/b as a central regulator of erythropoiesis. STAT5a/b is essential in regulating early erythroblast survival, however, with regard to differentiation of erythroid progenitors current data are not definitive in establishing a critical, non-redundant role. Phospholipase C gamma 1 (PLCγ1) is known to act as key mediator of calcium-signalling that can substitute for PI3K/AKT in oncogenic models. Interestingly, genetic deletion of murine PLCγ1 in embryonic development using a conventional knockout mouse model resulted in lethality at E9.0 due to generalized growth failure and there was absence of erythrogenesis and vasculogenesis. Here, we revisited the role of Plcγ1 and investigated its function in signalling, differentiation and transcriptomic/epigenetic regulation of erythropoiesis: Upon Epo stimulation, we were able to demonstrate that Plcγ1 is a downstream target of EpoR/Jak2 signalling in lymphoid (Ba/F3) and myeloid (32D) progenitor cell lines (both transfected with EpoR and Jak2-WT) and in a erythroid progenitor (I/11) cell line. In order to specifically assess its role in erythroid development downstream of the EpoR-Jak2 axis, we focused on the murine pro-erythroblast cell line I/11 which is able to differentiate upon dexamethasone-/stem cell factor-withdrawal combined with erythropoietin stimulation. Interestingly, knockdown of Plcγ1 led to a dramatic delay (scr CD44high 21% vs. Plcγ1 shRNA CD44high 64%, p=0.02) in erythroid differentiation and accumulation of immature erythroid progenitors as assessed by flow cytometry technology. Knockdown of Plcγ1 did alter neither proliferation of cells nor the cell cycle distribution and activation of other EpoR downstream molecules as Stat5, Mek and Akt was not impaired. In addition, we analysed the colony-forming potential of Plcγ1-deficient I/11 and fetal liver cells (FLC) compared to controls. Colony formation was dramatically impaired in both - I/11 (scr 138 vs. Plcγ1 shRNA 32, p=0.03) and primary FLC (scr 107 vs. Plcγ1 shRNA 28, p