ALBERT

Description: Key Points IFN-γ impairs maintenance of HSCs by directly reducing their proliferative capacity and impairing their restoration upon viral infection. IFN-γ induces SOCS1 expression in HSCs, which inhibits TPO-induced STAT5 phosphorylation, thereby deregulating key cell-cycle genes.

Description: The receptor tyrosine kinases FLT3 and KIT are highly expressed on the surface of leukemic blasts in most patients with acute myeloid leukemia. Although about one third of patients display activating mutations in FLT3 (and more rarely in KIT), the majority of patients have no mutations in FLT3 or KIT. Previously, we demonstrated that Cbl functions as the E3 ligase for both FLT3 and KIT, and that ligase-inactivating mutations of Cbl stabilize FLT3 and KIT on the cell surface by preventing endocytosis and degradation (Sargin et al, Blood 2007). Furthermore, we demonstrated that expression of E3-ligase deficient Cbl mutants led to the development of a myeloproliferative disease in a murine bone marrow transplantation model (Bandi et al, Blood 2009). However, Cbl mutations are rarely found in AML. Here, we investigated the role of the Cbl regulators suppressors of T-cell signaling 1 and 2(STS1 and STS2) in stabilizing wild-type FLT3 and KIT on the cell surface of hematopoietic stem and progenitor cells (HSPCs). STS1 is ubiquitously expressed, while STS2 expression is restricted to the hematopoietic tissue. STS1 and STS2 constitutively bind to Cbl, while their binding to FLT3 and KIT is dependent on ligand-activation by FL and SCF, respectively. Interestingly, STS1 (but not STS2) functions as a tyrosine phosphatase for both ligand-activated FLT3 and KIT. This required the PGM domain of STS1, as PGM point mutant of STS1 did not dephosphorylate FLT3 or KIT. In line with this, knockdown of STS1 using stably expressing shRNA constructs showed a significant hyperphosphorylation of FLT3 and KIT. By using STS1/STS2 single and double knockout mice, we analyzed the effects of STS1 and STS2 on hematopoietic stem and progenitor cells in vivo. We found that deficiency of STS1 causes an increase of both absolute number and frequency of LSK (lineage marker-, KIT+, Sca1+) cells, which contain HSPCs. This phenotype was even more pronounced in STS1 and STS2 double knockout (dKO) mice, and is mainly attributable to the short term hematopoietic stem cell (ST-HSC) and multipotent progenitor (MPP) cell population, as defined by both standard and SLAM markers. Colony assays using primary bone marrow cells revealed a significantly higher colony forming ability in STS1-KO and dKO cells compared to wild type (wt) cells, particularly after serial replating. A careful analysis of the cells derived from methylcellulose culture revealed an increased proportion of immature (Mac1- CD48+ CD16/32-) cells in STS1-KO and dKO cells. Competitive repopulation assays showed an advantage for dKO cells when compared to wt, suggesting that the LT-HSC compartment is also affected. Even more pronounced were the differences in CFU-S assays (colony forming units spleen), displaying significantly more colonies of dKO compared to wt donor cells, functionally demonstrating a significantly increased ST-HSC / MPP population in dKO donors. A detailed analysis of the downstream signaling events demonstrated that loss of STS1 specifically causes an activated PI3-Kinase / AKT pathway. In summary, our data demonstrates that STS1 functions as a phosphatase of FLT3 and KIT and, using genetic mouse models, indicates a critical role in the maintenance and proliferation of long-term and short-term hematopoietic stem cells. This may also affect sensitivity to kinase inhibitors. Disclosures: No relevant conflicts of interest to declare.

Description: Autophagy maintains hematopoietic stem cell integrity and prevents malignant transformation. In addition to bulk degradation, selective autophagy serves as an intracellular quality control mechanism and requires autophagy receptors, such as p62 (SQSTM1), to specifically bridge the ubiquitinated cargos into autophagosomes. Here, we investigated the function of p62 in acute myeloid leukemia (AML) in vitro and in murine in vivo models of AML. Loss of p62 impaired expansion and colony-forming ability of leukemia cells and prolonged latency of leukemia development in mice. High p62 expression was associated with poor prognosis in human AML. Using quantitative mass spectrometry, we identified enrichment of mitochondrial proteins upon immunoprecipitation of p62. Loss of p62 significantly delayed removal of dysfunctional mitochondria, increased mitochondrial superoxide levels, and impaired mitochondrial respiration. Moreover, we demonstrated that the autophagy-dependent function of p62 is essential for cell growth and effective mitochondrial degradation by mitophagy. Our results highlight the prominent role of selective autophagy in leukemia progression, and specifically, the importance of mitophagy to maintain mitochondrial integrity.

Description: Abstract 144 Background: Sorafenib is a multi-kinase inhibitor with activity against several oncogenic kinases, which may play a role in the pathogenesis of acute myeloid leukemia (AML). In-vitro data and results from non-randomized clinical trials suggest that sorafenib might be an effective drug for the treatment of AML. So far, no randomized-controlled data are available for treatment of newly diagnosed AML patients up to the age of 60 years. We present the first results from the randomized placebo-controlled SORAML trial of the Study Alliance Leukemia (SAL). Patients and Methods: Between March 2009 and October 2011, 276 patients from 25 centers were enrolled in the SORAML trial (NCT00893373). The main eligibility criteria were: newly diagnosed AML, age from 18 to 60 years and suitability for intensive therapy. The treatment plan for all patients included two cycles of induction with DA (daunorubicin 60 mg/m2 days 3–5 plus cytarabine 100 mg/m2 cont. inf. days 1–7), followed by three cycles of high-dose cytarabine consolidation (3 g/m2 b.i.d. days 1, 3, 5). Patients without response after DA I received second induction with HAM (cytarabine 3 g/m2 b.i.d. days 1–3 plus mitoxantrone 10 mg/m2 days 3–5). Allogeneic stem cell transplantation was scheduled for all intermediate-risk patients in first complete remission with a family donor and for all high-risk patients with a matched donor. At study inclusion, patients were randomized to receive either sorafenib (800 mg/day) or placebo as add-on to standard treatment. Block randomization at a ratio of 1:1 was performed within cytogenetic and molecular risk strata, allocation was concealed and treatment was double blinded. Study medication was given on days 10–19 of DA I+II or HAM, from day 8 of each consolidation until 3 days before the start of the next consolidation and as maintenance for 12 months after the end of consolidation. The primary endpoint of the trial is event-free survival (EFS) with an event being defined as either failure to achieve a complete remission (CR) after induction, relapse or death. Secondary endpoints were overall survival (OS), CR rate and incidence of adverse events (AE). We present the results of the planned interim analysis (intent to treat) after the occurrence of 50% of EFS events. The O'Brien/Fleming adjusted significance level was set at p=0.0052. Results: Out of 276 randomized patients, 264 were evaluable for EFS, 132 in each arm. Demographic and disease characteristics were equally distributed between the two arms; the FLT3-ITD incidence was 16%. The median cumulative dose of administered study medication was equal in both arms. The CR rates were 56% versus 60% in the placebo versus sorafenib arm (p=0.622). By the time of analysis, a total number of 100 events had occurred. After a median observation time of 18 months, the median EFS was 12.2 months in the placebo arm and was not reached in the sorafenib arm, corresponding to a 1-year EFS of 50% versus 64% (p=0.023). The median OS had not been reached in both arms, the 2-year OS was 66% versus 72% in placebo and sorafenib arms, respectively (p=0.367). The most common reported AEs CTC Grade ≥3 were infectious complications including fever and pneumonia, followed by bleeding events, cardiac and hepatic toxicity, hypertension, skin toxicity and headache. The risk for hepatic toxicity (relative risk 6.2, p=0.025) and bleeding events (relative risk 3.6, p=0.016) was significantly higher in the sorafenib arm while the incidence of all other AEs showed no significant differences. Conclusions: In younger AML patients, the addition of sorafenib to standard chemotherapy is feasible but associated with a higher risk of liver toxicity and bleeding events. Sorafenib treatment resulted in a marked EFS prolongation; this difference is not significant according to the adjusted significance level of this interim analysis. Results from the final analysis including post-hoc exploration of molecularly defined subgroups are necessary for drawing final conclusions on the efficacy of sorafenib. Disclosures: Off Label Use: sorafenib for the treatment of acute myeloid leukemia. Serve:Bayer: Research Funding. Ehninger:Bayer: Research Funding.

Description: Activating mutations in the receptor tyrosine kinase FLT3 are frequently found in acute myelogenous leukemia patients and confer poor clinical prognosis. It is unclear how leukemic blasts escape cytokine control that regulates normal hematopoiesis. We have recently demonstrated that FLT3-internal tandem duplication (ITD), when localized to the biosynthetic compartment, aberrantly activates STAT5. Here, we show that one of the target genes induced by STAT5 is suppressor of cytokine signaling (SOCS)1—a surprising finding for a known tumor suppressor. Although SOCS1 expression in murine bone marrow severely impaired cytokine-induced colony growth, it failed to inhibit FLT3-ITD–supported colony growth, indicating resistance of FLT3-ITD to SOCS1. In addition, SOCS1 coexpression did not affect FLT3-ITD–mediated signaling or proliferation. Importantly, SOCS1 coexpression inhibited interferon-α and interferon-γ signaling and protected FLT3-ITD hematopoietic cells from interferon-mediated growth inhibitory effects. In a murine bone marrow transplantation model, the coexpression of SOCS1 and FLT3-ITD significantly shortened the latency of a myeloproliferative disease compared with FLT3-ITD alone (P 〈 .01). Mechanistically, SOCS proteins shield FLT3-ITD from external cytokine control, thereby promoting leukemogenesis. The data demonstrate that SOCS1 acts as a conditional oncogene, providing novel molecular insights into cytokine resistance in oncogenic transformation. Restoring cytokine control may provide a new way of therapeutic intervention.

Description: Abstract 6 Background Simultaneous ATRA and chemotherapy (CHT) is the current gold standard for newly diagnosed APL resulting in ∼80% cure rates, while arsenic trioxide (ATO) is the treatment of choice for relapsed patients. ATO in variable combinations including ± ATRA ± CHT has also been tested as front-line therapy yielding encouraging results in several pilot studies as well as in two phase III studies conducted in China and the US. So far, no randomised studies have compared front-line CHT-free ATO+ATRA combination against the standard ATRA+CHT approach. Patients and Methods The phase III, randomised, prospective APL0406 trial was started in October 2007 by the Italian GIMEMA group and joined in November 2008 by the German SAL and AMLSG multicenter groups. Eligible patients were adults aged 18-15 d) grade ≥ 3 neutropenia and thrombocytopenia were significantly more frequent in patients in arm B as compared to those in arm A (P

Description: Background: Chemotherapy-free treatment with arsenic trioxide and all-trans retinoic acid (ATO/ATRA) of patients with acute promyelocytic leukemia (APL) with presenting white blood counts (WBC) 〈 10 G/l has been shown to be at least equivalent or even better with regard to survival and quality of life compared to standard treatment according to the AIDA scheme which includes idarubicin, mitoxantrone in combination with ATRA followed by a two-year maintenance therapy with 6-mercaptopurin, methotrexate and ATRA (Lo Coco F et al., N. Engl. J. Med. 2013;369(2):111-21; Efficace F et al J Clin Oncol. 2014 accepted). Aims: To evaluate costs in relation to benefits in a cost-effectiveness analysis comparing ATO/ATRA to standard treatment with AIDA in newly diagnosed APL with WBC

Description: Abstract 886 The diagnosis acute myeloid leukemia (AML) describes a heterogeneous group of myeloid stem cell disorders. Based on current concepts of disease development, the combination of at least two mutations is necessary for transformation, typically affecting transcription factors (e.g. RUNX1) blocking normal differentiation and growth promoting genes, e.g. receptor tyrosine kinases like FLT3. This model has been challenged by more recent results of genome-wide mutational analysis, which revealed a typical load of 8–12 mutations affecting several additional pathways, e.g. epigenetic regulation (DNMT3A, TET2 or IDH1). However, the sequence of acquisition and the individual impact of these mutations are largely unknown because these aspects are difficult to study. Here we describe a unique case of a donor cell leukemia giving unexpected insights into the development of AML in man. Case report and methods: In May 2004, a 51-year old male (P1) with an 8-year history of B-CLL received G-CSF mobilized peripheral blood stem cells after dose reduced conditioning from his HLA-identical sister, because he had relapsed after several lines of conventional therapy. He rapidly engrafted and showed complete donor chimerism (DC). In February 2012, he was admitted to the hospital with elevated WBC counts and circulating blasts. Bone marrow (BM) aspiration and morphology revealed an infiltration of the BM with 94% myeloid blasts (FAB M1). Cytogenetic and standard molecular assessment showed a normal female karyotype and NPM1 and FLT3-ITD mutations. STR-based analysis also revealed a persistent, 100% DC, thus the diagnosis of a donor cell AML was made, which developed almost 8 years after SCT. Interestingly, his sister, the donor (P2) had been also diagnosed with AML (FAB M2, cytogenetics: 47, XX,+8; NPM1 and FLT3-ITD neg.) only 3 months before her brother in Nov. 2011. Currently, P1 is in CR after re-SCT from an unrelated donor, whereas P2 relapsed and is scheduled for SCT after reinduction. Since DNA material of both individuals was available and due to this unique constellation, we performed next generation sequencing of whole exome enriched material using an Illumina HiSEQ 2000 platform after obtaining informed consent to compare both AMLs. Identified mutations were then confirmed using conventional Sanger sequencing and traced back by 454-based amplicon deep sequencing in a pre-SCT sample of the donor/P2 as well as several post SCT samples collected from P1 for the documentation of chimerism. Results: Comparison of the two AML-samples with a pre-SCT donor sample and a sample taken after 1.SCT as well as the HG19 and dbSNP135 releases revealed more than 100 unknown SNPs. Confirmation focused on cancer related changes or genes in critical pathways. In P1, in addition to the known NPM1 and FLT3-ITD mutations, we found somatic changes in CLCA1, PKHD1 and TET2, whereas in P2, we identified and confirmed somatic mutations in CDCA2, CBL, IDH1, NEK9 and PHF6. In addition, a typical DNMT3A R882C mutation was found in both leukemias. Interestingly, this mutation was also detectable by conventional Sanger sequencing in the pre-SCT sample of P2, but not in P2-germline DNA derived from buccal swaps. As shown in the figure, the 454-amplicon sequencing revealed a gradual increase of the TET2 (R1167G) mutational load over time in P1, and showed also that this mutation was present at low levels (4%) already in the pre-SCT sample of P2, but not in her final AML. FLT3-ITD and NPM1 mutations were detectable only in the AML-sample of P1, but not at any prior time points or in P2. Conclusions: These data indicate that mutations like the DNMT3A R882C can be present in normal appearing hematopoiesis at high levels years before the development of AML. The presence of the mutation in the absence of overt leukemia or MDS indicates that these mutations might not have a direct effect on the development of the disease, but favor the development of aberrant clones which then acquire additional changes in a latency phase. Other mutations (e.g. TET2) might give a small clonal advantage, but only the final acquisition of abnormalities like NPM1 and FLT3-ITD might transform this latency phase into a rapidly proliferating status, consistent with the “driver” status of these aberrations. The divergent mutational pattern found in the two AMLs emerging from the same DNMT3A-starting clone points to the high clonal diversity which might develop even within a single individual. Disclosures: Thiede: AgenDix GmbH: Employment, Equity Ownership.

Description: Background: Acute myeloid leukemia (AML) is the second most common form of leukemia and the most frequent cause of leukemia-related deaths in the United States. While complete response rates can be as high as 80% in patients undergoing initial induction chemotherapy, the majority of AML patients will relapse. Refractory or relapsed patients have a bleak prognosis. As there is currently no standard regimen for these patients, a great clinical need exists for new treatment options. Selinexor, an oral first-in-class Selective Inhibitor of Nuclear Export (SINE), inhibits XPO1 mediated nuclear export inducing cytotoxicity in cells with genomic damage. Preclinical data with Ara-C and selinexor significantly prolonged the survival of leukemic mice. A phase I clinical study demonstrated encouraging results in AML patients. The role of selinexor as mono-therapy or in combination is currently under investigation in phase II trials of AML. Aims: This phase II trial investigates the efficacy & tolerability of Ara-C and Idarubicin in combination with selinexor in patients with relapsed or refractory AML. Methods: Patients with relapsed/refractory AML were treated with Ara-C (100 mg/m2, continuous infusion, day 1-7), idarubicin (10 mg/m2, day 1, 3, 5) every 4 weeks. Selinexor was administered twice weekly orally starting on day 2 (40 mg/m2). A small cohort of patients received selinexor after registration and before first induction cycle for correlative studies. The primary endpoint was percentage of patients achieving a complete remission or complete remission without normalization of peripheral blood counts (CRi). Secondary endpoints were partial remission rate, percentage of patients undergoing subsequent allogeneic stem cell transplantion, early death rate, overall survival (OS), event-free survival and toxicity. Results: As of June-16-2015, 21 patients with AML have been enrolled at 3 sites in Germany (NCT02249091) after obtaining informed consent of which 20 had received ≥ 1 induction cycle and were evaluable for efficacy and toxicity. Median age was 60 (range 34-78) years. Seven patients had a complex and 6 a normal karyotype (unknown in 7 cases). On average, patients had received 3.5 (range 1-6) prior therapies. 2 patients had primary refractory disease and 11 patients an early relapse and 7 patients a late relapse. 7 patients had prior allogeneic transplantation. Overall response rate was 60% (25% of patients achieved CR, 25% of patients achieved CRi, 10% of patients achieved PR). Sixty percent of patients received or were planned for stem cell transplantion or donor lymphocyte infusion. The most frequent non-hematologic AES were vomiting, diarrhoea, nausea, fatigue, anorexia and neutropenic fever. One treatment-related death occurred. The patient developed subarachnoid haemorrhage during thrombocytopenia grade 4 and died. Summary / Conclusion: The prognosis of relapsed/refractory AML patients is remarkably poor. Our findings suggest that Ara-C and Idarubicin in combination with selinexor resulted in a remarkable response rate and is a promising regimen in this particularly unfavourable cohort of patients without unexpected toxicities enabling the majority of them to proceed to first or second allogeneic stem cell transplantation. Disclosures Fiedler: Teva, Amgen, Astellas: Other: Travel Grant; Pfizer, Amgen, Kolltan: Research Funding; Karyopharm: Research Funding. Off Label Use: Selinexor has been used in combination with AraC and idarubicin in relapsed/refractory AML patients.. Chromik:Karyopharm: Research Funding. Theile:GSO Hamburg: Employment. Kranich:GSO Global: Employment. Heuser:Karyopharm: Research Funding.

Description: Background: Sorafenib is a multi-kinase inhibitor with activity against several oncogenic kinases that may play a role in the pathogenesis of acute myeloid leukemia (AML). In-vitro data and results from non-randomized clinical trials suggest that sorafenib might be an effective drug for the treatment of AML. We present the results of the randomized placebo-controlled SORAML trial testing sorafenib versus placebo as add-on to standard induction and consolidation treatment in AML patients ≤60 years. Patients and Methods: Between March 2009 and October 2011, 276 patients from 25 centers were enrolled in the SORAML trial (NCT00893373). The main eligibility criteria were newly diagnosed AML, age from 18 to 60 years and suitability for intensive therapy. The treatment plan for all patients included two cycles of induction with DA (daunorubicin 60 mg/m2 days 3-5 plus cytarabine 100 mg/m2 cont. inf. days 1-7), followed by three cycles of high-dose cytarabine consolidation (3 g/m2 b.i.d. days 1, 3, 5). Patients without response after DA I received second induction with HAM (cytarabine 3 g/m2 b.i.d. days 1-3 plus mitoxantrone 10 mg/m2 days 3-5). Allogeneic stem cell transplantation was scheduled for all intermediate-risk patients in first complete remission with a sibling donor and for all high-risk patients with a matched related or unrelated donor. At study inclusion, patients were randomized to receive either sorafenib (800 mg/day) or placebo as add-on to standard treatment in a double blinded fashion. Block randomization at a ratio of 1:1 was performed within cytogenetic and molecular risk strata, allocation was concealed and treatment was double blinded. Study medication was given on days 10-19 of DA I+II or HAM, from day 8 of each consolidation until 3 days before the start of the next consolidation and as maintenance for 12 months after the end of consolidation. The primary endpoint of the trial was event-free survival (EFS) with an event being defined as either failure to achieve a complete remission (CR) after induction, relapse or death. Secondary endpoints were relapse-free survival (RFS), overall survival (OS), CR rate and incidence of adverse events (AE). We present the results of the final analysis of the primary endpoint EFS (intent to treat) after the occurrence of 134 events. Results: Out of 276 enrolled patients, 267 received study treatment, 134 in the sorafenib arm and 133 in the placebo arm. Demographic and disease characteristics were equally distributed between the two arms; the incidence of FLT3-ITD was 17%. The median cumulative dose of administered study medication was similar in both arms. The CR rates were 59% versus 60% in the placebo versus sorafenib arm (p=0.764). After a median observation time of 36 months, the median EFS was 9.2 months in the placebo arm and 20.5 months in the sorafenib arm, corresponding to a 3-year EFS of 22% versus 40% (p=0.013). Median RFS after standard treatment plus placebo was 23 months and not yet reached after sorafenib treatment, corresponding to a 3-year RFS of 38% and 56%, respectively (p=0.017). The median OS had not been reached in either arm; the 3-year OS was 56% with placebo versus 63% with sorafenib (p=0.382). In 46 FLT3-ITD positive patients, no difference in EFS, but a trend for prolonged RFS and OS in favor of sorafenib was observed. The most common reported AEs Grade ≥3 were fever (40%), infections (22%) and bleeding events (2%). The risk for fever, bleeding events and hand-foot syndrome was significantly higher in the sorafenib arm while the incidence of all other AEs showed no significant differences. Conclusions: In younger AML patients, the addition of sorafenib to standard chemotherapy in a sequential manner is feasible and associated with antileukemic efficacy. We observed a higher incidence of infections and bleeding events under sorafenib. Whereas OS in both treatment arms was similar, sorafenib treatment resulted in a significantly prolonged EFS and RFS. Figure 1: Event-free survival Figure 1:. Event-free survival Disclosures Off Label Use: sorafenib for treatment of aml. Serve:Bayer HealthCare: Research Funding. Ehninger:Bayer HealthCare: Research Funding.