ALBERT

Description: Abstract 255 Introduction: Standard treatment of acute myeloid leukemia (AML) comprises one or two cycles of chemotherapy to induce complete remission (CR) followed by postremission treatment in order to prevent relapse of the disease (consolidation therapy). In 2003, we initiated a prospective multicenter randomized trial to investigate the impact of different consolidation strategies on long-term outcome in AML patients ≤ 60 years. Consolidation options comprised upfront allogeneic stem cell transplantation (allo SCT) in aplasia after induction therapy, autologous SCT, and three cycles of standard high-dose-cytarabine-based consolidation. For patients receiving high-dose cytarabine, the main study aim was to evaluate the benefit of adding additional mitoxantrone and amsacrine to cytarabine consolidation. Design: From 2003 to 2009, 1182 patients (median age, 48 years; range 16–60 years) with untreated AML were randomly assigned at diagnosis to different consolidation strategies after classical 7+3 induction. According to the risk-adapted treatment strategy of the trial, cytogenetically or molecular intermediate-risk (IR) and adverse-risk (AR) patients should receive an allo SCT as consolidation treatment if an HLA-identical-sibling donor (IR) or HLA-matched related or unrelated donor (AR) was available. IR and AR patients with no available donor should receive autologous SCT. All favorable risk patients and patients with no available donor were scheduled for high-dose cytarabine based consolidation. Half of the patients were randomized for high dose cytarabine based consolidation. Half of the patients were randomized for high dose cytarabine alone while the other half received high dose cytarabine with the addition of amsacrine and mitoxantrone. Standard chemotherapy consisted of three cycles with high dose cytarabine (2 × 3 g/m2, day 1,3,5) whereas combined consolidation contained two cycles of MAC (cytarabine 2 × 1g/m2, day 1–6, mitoxantrone 10 mg/m2, day 4–6) plus one cycle of MAMAC (cytarabine 2 × 1 g/m2, day 1–5, amsacrine 100 mg/m2, day 1–5). In order to evaluate the effect of the two cytarabine based consolidation strategies, we determined overall survival (OS) and event free survival (EFS) using the method of Kaplan Meyer. Survival distributions were compared using the log rank test. Results: 1182 patients were randomized for further intervention (Arm A+B: n=582, 49.3%; Arm C+D: n=600, 50.7 %). Median follow-up was 41.4 months (95%-CI 39.3–43.6). A total number of 375 patients received allogeneic (n=322) or autologous SCT (n=53) and 807 patients were consolidated with cytarabine. Of these patients, 407 were randomized for cytarabine alone and 400were randomized to receive cytarabine plus mitoxantrone and amsacrine (MAC/MAC/MAMAC). Complete remission rate (CR) after second induction therapy was 59.1% (n=698). Between the four arms, there were no significant differences of the CR rates. Five-year OS of patients receiving high dose cytarabine alone was 47.1% (95%-CI 42.0–52.2%), for patients receiving MAC/MAMAC as consolidation therapy it was 46.8% (95%-CI 42.3–51.3%; p = 0.610). Three-year event free survival (EFS) was also not significant with 30.5% (95%-CI 26.6–34.4%) for patients receiving high dose cytarabine alone and 35.6% (95%-CI 31.7–39.5%; p = 0.059) for patients receiving MAC/MAMAC. Conclusions: According to our data, the addition of mitoxantrone and amsacrine to high dose cytarabine consolidation confers no benefit for treatment outcome in younger AML patients. Disclosures: No relevant conflicts of interest to declare.

Description: Mantle cell lymphoma (MCL) cells are characterized by a discrepancy between high mRNA and low protein levels of the pro-apoptotic BH3-only protein NOXA. Modulation of NOXA protein expression has been described as a major mechanism of cell death induction in MCL cells. However, the efficiency of induction of apoptosis at lower concentrations is limited despite effective stabilization of NOXA. We therefore investigated whether the main binding partner of NOXA, the anti-apoptotic protein MCL-1 is co-regulated by these agents and whether dual targeting of NOXA and MCL-1 could be a promising strategy to enhance effectiveness of cell death induction in MCL cell lines. Screening a panel of compounds supposed to lead to NOXA stabilization in MCL, we identified the proteasome inhibitor Bortezomib, the fatty acid synthase inhibitor Orlistat and the ROS inducing agents Helenalin, a sesquesterpenone lactone from Arnica and the naphtoquinone derivative Menadione to kill MCL cells very effectively in a NOXA -dependent manner. Investigating the NOXA-/MCL-1-protein expression upon treatment with these agents, we could observe that at lower, sublethal concentrations of Bortezomib and Orlistat, NOXA protein was increased to a certain extent but also the anti-apoptotic MCL-1 was highly induced, counteracting induction of apoptosis. From this observation it has to be concluded that MCL-1 limits the apoptotic activity of NOXA stabilizing agents and dual targeting of MCL-1 and NOXA is required for optimal killing of MCL cells. In search for MCL-1 regulating agents we could identify the cdk-inhibitor Dinaciclib to be the most effective one. This compound rapidly downregulates Mcl-1 protein in a dose- and time-dependent manner presumably due to cdk 9-mediated inhibition of phosphorylation of the RNA-Polymerase II-subunit RPB1. To study the efficiency of dual targeting of NOXA/MCL-1 in killing of MCL cells we combined sublethal doses of Dinaciclib with NOXA stabilizing agents and observed a synergistic induction of apoptosis in MCL cells. Western Blot analysis showed that combination treatment decreased Mcl-1 and increased NOXA protein expression compared to single-agents. It could be shown that induction of cell death by treatment with the combined agents depends on NOXA as transfection of cells with NOXA-siRNA rescued cells from induction of apoptosis. Cell death upon treatment with Dinaciclib and ROS-inducing agents Helenalin and Menadione could furthermore be rescued by preincubation with the antioxidant GSH. Interestingly, combined treatment of cells with Orlistat and Dinaciclib killed most effectively when Dinaciclib was added for 8 hours after 16 hours of preincubation with the NOXA stabilizing agent. This observation contributes to the hypothesis that stabilization of NOXA protein leads to priming of MCL cells to induction of apoptosis by pharmacological downregulation of Mcl-1 with Dinaciclib. In summary, the NOXA-MCL-1 balance is critical for survival of MCL cells. Dual targeting of MCL-1 and NOXA efficiently kills MCL cells and appears to be of particular importance especially at lower concentrations of these compounds. These culture conditions most likely resemble the limited exposure after in vivo treatment. Therefore the combination of NOXA stabilizing agents with Dinaciclib appears to be a promising strategy to be tested in clinical trials. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 3150 Imatinib resistance is a major problem in treatment of Bcr-Abl positive leukemias, particularly in patients with ALL and advanced CML. One mechanism of this resistance is overexpression of Bcr-Abl. We investigated the effects of Imatinib deprivation in Bcr-Abl overexpressing Imatinib-resistant ALL cell lines. Removal of Imatinib from culture medium led to induction of a non-apoptotic cell death starting approximately 40 hours after Imatinib withdrawal. This cell death was preceded by a rapid and robust induction of Bcr-Abl autophosphorylation and concomitant overstimulation of PI3K-, MAPK-, and JAK/STAT signalling. Over-activation of Bcr-Abl downstream signalling was accompanied by a significant enhanced glucose and amino acid metabolism and an elevated intracellular protein and nucleic acid content. As a result of this enhanced metabolisms we observed a massive increase in cell size, multinucleation, cytoplasmic vacuolization, and reduced intracellular ATP level. The phase-lucent vacuoles did not incorporate the endosome/lysosome tracker, Lyso Tracker Red indicating that the vacuoles were formed by dilation of ER cisternae. To determine if Imatinib deprivation induces an ER stress response concurrent with vacuolization we investigated typical ER stress markers. Upon removal of Imatinib a massive induction of CHOP expression and eIF2 alpha phosphorylation, as well as alternative splicing of XPB could be detected. It has been demonstrated that severe ER stress promotes cell death either by induction of a BIM-dependent apoptitic process or by a TRAF2-RIP1 dependent pathway. Despite of a robust induction of BIM and posttranslational modification of RIP1, siRNA-mediated suppression of BIM and RIP1 had no effect on Imatinib deprivation-induced cell death whereas downmodulation of CHOP partially rescued those cells. Using different small molecule inhibitor libraries, we also identified inhibitors of glycogen synthase kinase-3 (GSK3) and p38-MAPK, as well as glucocorticoids as potent compounds which not only completely prevented metabolic stress and induction of cell death upon removal of Imatinib but also partially repressed induction of CHOP upon Imatinib withdrawal. Importantly, in the presence of glucocorticoids, we found a significant enhanced number of cell clones with Bcr-Abl overexpression in lympoid cell lines upon de novo transfection with Bcr-Abl. In conclusion, acute over-activation of the oncogene Bcr-Abl led to metabolic stress and ER stress followed by a delayed induction of a necrosis-like cell death. These cellular responses to Bcr-Abl-mediated oncogenic stress could be completely blocked by glucocorticoids. Therefore, treatment of glucocorticoid resistant ALL may generate selective pressure towards Bcr-Abl overexpession. Disclosures: No relevant conflicts of interest to declare.

Description: Imatinib is highly effective in inducing remissions in chronic phase CML. However, complete eradication of the malignant clone by Imatinib monotherapy is rare. This prompted us to explore the efficacy of combination of Imatinib with the DNA damaging agent Cisplatin and with Nutlin-3, a compound which induces p53 accumulation by interfering with its binding to Mdm2. We used a Bcr-Abl positive cell line characterized by the loss of Imatinib sensitivity in the presence of optimal growth factor concentrations. Whereas combined treatment of Imatinib with either Cisplatin or Nutlin-3 in low and nontoxic doses induced ∼15–20% cell death, simultaneous treatment of all three compounds was highly effective (∼50% cell death) even in the presence of optimal growth conditions. Importantly, comparable effects were also seen in CFU assays performed with primary CD34 positive cells from 5 CML patients. To examine the molecular mechanisms of these effects we analyzed p53 dependent cellular response to Cisplatin in our cell line model in the presence or absence of Imatinib and/or Nutlin-3. The active Bcr-Abl kinase caused superinduction of p53 protein after DNA damage. We found both an upregulated p53 accumulation and enhanced p21 and Mdm2 RNA and protein levels upon Cisplatin treatment. This phenomenon could be reversed both by siRNA-mediated inhibition of Bcr-Abl expression and by Imatinib-induced inhibition of Bcr-Abl kinase activity: despite of optimal growth conditions inhibition of Bcr-Abl caused a significant reduction of p53 expression and activation. In the presence of Nutlin-3, p53 expression was significantly upregulated upon Cisplatin treatment both with and without Imatinib. However, Nutlin-3 was not capable to restore the Imatinib-induced blockade of the transcriptional activity of p53 on mdm2. The reduced p53 activation observed in Bcr-Abl positive cells treated with Imatinib was paralleled by a shift from cell cycle arrest to cell death both in the presence and absence of Nutlin selectively in Bcr-Abl positive cells rendering them hypersensitive to Cisplatin. In summary, combined treatment of Imatinib with low concentrations of a DNA damaging agent and a p53 activator is highly effective in vitro. Such combined treatment may prove to be clinically relevant for complete eradication of the malignant clone in CML, provided that conditions are found where it does not affect adversely normal hematopoietic cells in vivo.

Description: The increasing impact of targeted cancer treatment demands strategies to identify and evaluate resistance mechanisms toward kinase inhibitors prior to their therapeutic application. Point mutations within the Bcr-Abl kinase domain constitute the major mechanism of resistance toward imatinib mesylate in Philadelphia-positive (Ph+) leukemia. Using Bcr-Abl-transformed Ba/F3 cells, we established a cell-based screening strategy for the prediction of specific kinase mutations that cause resistance toward kinase inhibitors. With imatinib at clinically relevant concentrations, we generated 368 resistant Ba/F3 sublines that were derived from resistant colonies. Thirty-two different single point mutations within the kinase domain of Bcr-Abl were identified in twenty-five per cent (liquid culture conditions) and seventy-two per cent (solid culture conditions) of these lines at known and novel positions. Using imatinib, the pattern and relative frequency of mutations reflected matters observed in patients with imatinib resistance. We then applied this screen to the pyrido-pyrimidine PD166326 (PD16), an investigational Abl kinase inihibitor. Compared to imatinib, we observed a five to seven times lower frequency of resistant colonies with equipotent concentrations of PD16. In addition, PD16 produced a distinct pattern of Bcr-Abl mutations. P-loop, A-loop and the known imatinib contact site T315 were affected with both inhibitors, whereas C-helix and SH2 contact sites were affected in imatinib resistant colonies exclusively. In contrast to imatinib, where kinase domain mutations were still widely distributed over the kinase domain even at at 4μM, mutations observed with PD16 at a concentration of 100nM narrowed to the exchange at position T315 to iseulicine. We did not detect mutations outside the kinase domain. Some resistant sublines displayed increased Bcr-Abl activity. Mutations that were derived from the screen were cloned and examined for the extent of cross-resistance to both inhibitors. The majority of mutations were effectively suppressed by PD16 at 50–500nM. In contrast, only few mutations were inhibited by imatinib at 5–10μM. However, exchanges at position F317 mediated resistance toward PD16, but were inhibited by standard concentrations of imatinib. Since this cell-based system produced results that are clinically significant, it may be used to predict resistance mutations in Bcr-Abl and other oncogenic kinases like cKit, EGFR, FIP1L1-PDGFRalpha or FLT3 towards clinically applicated and investigational drugs. Thus, this robust and simple screening strategy provides a rational basis for combinatorial and sequential treatment strategies.

Description: In a up-front randomized study, 939 adult patients up to the age of 60 years received a double induction therapy. One course of MAV (mitoxantrone 10 mg/m2 days 4–8, cytarabine 100 mg/m2 continuous infusion days 1–8, etoposide 100 m g/m2 days 4–8) was followed by one cycle of MAMAC (cytarabine 1 g/m2, every 12h days 1–5; amsacrine 100 mg/m2 days 1–5). Patients with intermediate risk cytogenetic (IRCG) and a HLA matched sibling received an allogeneic transplantation, those with poor risk cytogenetic (PRCG) were intended to be transplanted from a sibling or unrelated donor. All AML patients without an available donor received the randomly assigned first postremission therapy (PRT) mitoxantrone combined with intermediate-dose cytarabine (I-MAC; total dose 12 g/m2) or high-dose cytarabine (H-MAC; total dose 36 g/m2). As second PRT, patients with t(8;21) received an additional cycle of chemotherapy. An autologous transplantation was scheduled for IRCG and PRCG without an allogeneic donor. The CR rate was 88% for patients with t(8;21), with IRCG 71%, and 50% with PRCG. The 5-year-survival was 21% (95% CI: 16–27%) in the PRCG, 40% (95% CI: 36–45%) in the IRCG and 74% (95% CI: 60–88%) in the t(8;21) group. No difference was observed between the I-MAC and the H-MAC group. In a multivariate analysis, a significant (p

Description: Abstract 229 Background: The optimal timing of hematopoietic cell transplantation (HCT) in AML and its use in risk groups defined by genetic markers or early blast clearance is still under debate. We addressed this question in the AML 2003 study, a large multicenter, randomized, open-label study within the German SAL group. All patients received one cycle of induction therapy (IT) with a 3+7 regimen combining daunorubicin and continuous infusion cytarabine. Upfront cytogenetic and molecular characterization, HLA typing, related donor search and a preliminary search for an unrelated donor were performed for all patients. The subsequent transplant strategy was tailored to the biological risk and donor availability. Patients and Methods: Patients aged 18–60 years were randomly assigned upfront 1:1 to either of two transplant strategies: In the control arm allogeneic HCT was scheduled in first complete remission for patients with intermediate risk cytogenetics and an HLA-identical sibling and for patients with a complex karyotype (CK) and a related or unrelated HLA-compatible unrelated donor. In the experimental arm the indication for allogeneic HCT was extended to patients with an FLT3-ITD allelic ratio 〉0.8 (mutant/wild type), 〉10% marrow blasts on day 15 after IT1 and patients with adverse karyotypes, including: −7, −5, del(5q), inv(3q), t(3;3), t(6;9), t(6;11), t(11;19)(q23;p13.1). Furthermore, allogeneic HCT was scheduled at an early time-point, i.e. in aplasia after the first or the second cycle of IT in the experimental arm. Patients without an HLA-compatible donor assigned to the transplant strategy were scheduled for high-dose busulfan and cyclophosphamide followed by autologous SCT. Here, we present the final analysis of this study according to the intent-to-treat principle. Results: Between December, 1, 2003 and November, 26, 2009 1179 patients were randomized between the experimental (N=598) and the control intervention (N=581). The median age was 48 years (range, 18 to 60 years). The distribution of age, ECOG performance status, type of AML, cytogenetic risk groups, NPM1- and FLT3-ITD-mutations, LDH, white blood cell and platelet count was not statistically different between the two treatment arms. However, more males and patients with higher marrow blast counts at diagnosis were randomized to the experimental treatment arm. The median observation time for all patients was 52 months. The hazard ratio of the treatment effect (experimental versus control) was 0.92 (95% CI, 0.75 to 1.14; p=0.45) for primary endpoint overall survival and 0.85 (95% CI, 0.71 to 1.02; p=0.08) for the secondary endpoint event-free survival. Patients in both arms had an excellent long-term overall survival of 50% (95% CI, 46% to 54%) at 5 years after enrollment in the experimental arm and 47% (95% CI, 42% to 51%) in the control arm (see Figure). Across all risk groups the rate of early allogeneic HCT performed per protocol was 22% in the experimental arm and 8% in the control arm. Among patients with high risk cytogenetics this rate was 48% and 17%, respectively. These numbers point at the difficulty to deliver allogeneic HCT within two months after diagnosis of AML in the context of a multicenter trial. However, since HLA-typing and preliminary donor search was done for all patients and allogeneic HCT was standard for relapsed or refractory AML in both arms, the rates of allogeneic HCT as first post remission therapy or after induction failure/relapse were substantially higher than in previous studies and almost equal in the experimental and control arm (63% versus 56%). Conclusions: Although a survival benefit by early allogeneic HCT could not be demonstrated in this large randomized study in newly diagnosed AML, the study treatment resulted in excellent overall survival rates in both arms. Relatively small differences between the per-protocol transplantation rates and crossover effects may partially explain why a benefit of early allogeneic HCT could not be demonstrated. Early awareness of donor availability and the option of allogeneic HCT in patients with primary induction failure or AML relapse resulted in a high overall rate of allogeneic transplantation. This may have contributed to the excellent overall survival in both arms. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1656 Mantle cell lymphoma (MCL) is an aggressive type of B-cell lymphoma characterized by a t(11;14)(q13;q32) chromosomal translocation resulting in a constitutive over-expression of cyclin D1. Cyclin D1 is a multifunctional protein not only regulating cell cycle progression but also affecting other cellular functions including glucose and lipid metabolism. In this study, we investigated the effects of the fatty acid synthase (FASN) inhibitors orlistat and C75 on viability of different MCL cell lines including JEKO-1, MINO, Granta-519, JVM-2, and Rec-1. In all cell lines inhibition of FASN alone induced apoptosis. In contrast, normal peripheral blood lymphocytes were resistant to these compounds. This proapoptotic effect was dependent on cyclin D1 as silencing of cyclin D1 partially rescued cells from induction of cell death following FASN inhibition. Inhibition of FASN led to a strong induction of the proapoptotic protein NOXA (PMAIP) in all MCL cell lines investigated independent of the p53 status. Pre-treatment of cells with NOXA siRNA significantly reduced induction of cell death demonstrating the predominant role of this proapoptotic protein for the observed effects. We then analyzed the combined effects of inhibition of FASN with a panel of compounds with potential clinical relevance. Combination of FASN inhibitors with the BH3 mimetic ABT737, the proteasome inhibitor Bortezomib, and the glycolysis inhibitor 2-deoxy-D-glucose (2-DG) led to an almost complete loss of viability. These combinatory effects were selective for MCL cells as the same treatments had almost no effects on cell viability of primary PBMCs or fibroblasts from healthy donors. Bortezomib, 2-DG, and ABT737 enhanced the proapoptotic effect of FASN inhibitors by further disturbing the balance of pro- and antiapoptotic Bcl-2 family proteins: FASN inhibitor mediated induction of NOXA was enhanced by Bortezomib whereas 2-DG significantly reduced the NOXA antagonist Mcl-1. In summary, our results suggest that FASN inhibitors exert significant antitumoral activity especially in combination with Bcl-2 family modulators in different MCL cell lines and may therefore represent an attractive model for the design of new therapeutic approaches for this entity. Disclosures: No relevant conflicts of interest to declare.