ALBERT

Description: Acute lymphoblastic leukemia (ALL) is the most frequent malignant disease in childhood. Advances in therapy, in particular stratification of patients to appropriate treatment according to risk factors improved long term survival attaining cure rates of up to 80 %. Despite the efforts achieved by the stratification strategies the majority of relapse patients are recruited from the low risk groups emphasizing the need for additional independent risk factors. In a recent study we transplanted primary leukemia samples obtained from pediatric patients with newly diagnosed B cell precursor ALL (BCP-ALL) into NOD/SCID mice. Time to leukemia (TTL) was analyzed for each patient sample transplanted from date of transplant to date of leukemia manifestation in the recipients. We demonstrated that patients whose leukemia cells engrafted rapidly leading to manifestation of the disease within 10 weeks (TTLshort) showed a clearly inferior relapse free survival in contrast to patient samples with prolonged in vivo growth (TTLlong). Interestingly, the same distinct difference in relapse free survival was observed in the low risk groups only. Multivariate analysis showed an almost 45- fold increased risk for relapse in TTLshort patients. In order to further characterize the biological properties of the leukemia cells in the two groups, gene expression profiles of samples with short versus long TTL in the xenograft model were analyzed using a human whole genome array (Affymetrix U133 Plus 2.0). Here, we used quantitative traits analysis (QTA) correlating gene expression values (relative expression) to the time from transplant to manifestation of leukemia in the NOD/SCID mice (TTL, in weeks). 14 different xenograft samples (TTLshort n= 7, mean TTL 8.14 weeks; TTLlong n= 7, mean TTL 19.71 weeks; T-test P = .03) isolated from leukemia bearing mice were investigated. All 14 patients included were stratified in low (standard or intermediate) risk groups. All patients were negative for TEL/AML1- fusion, BCR/ABL- fusion or MLL rearrangement. By QTA we identified 5 genes that were significantly correlated (Spearman correlation, for all 5 genes P 〈 .0001) to time from transplant to leukemia manifestation in the recipient mice (TTL). Analysis of the 14 xenograft samples using the 5 genes identified showed clustering of all but one sample in one group. The 5 genes were than explored for their power to predict relapse in an independent cohort of pediatric ALL patients. For these patients expression profiles were analyzed in leukemia samples obtained at diagnosis. All patients in this cohort were also stratified in low risk groups and negative for the presence of TEL/AML1-, BCR/ABL- or MLL- fusion transcripts. 8 of the 38 patients encountered relapse, 5 at early and 3 at late time points (〈 or 〉 24 months after diagnosis). Clustering according to the 5 genes identified by QTA lead to separation of the patients into two groups. Interestingly, all relapsed patients except one clustered in the group associated with the signature characteristic for TTLshort. Most importantly, all 5 patients with early relapse gathered in this cluster. Taken together, we used a novel approach directly correlating gene expression values to the continuous variable time to leukemia (TTL) which might be reflected more accurately by this strategy than comparing two groups. We applied this gene signature characteristic for TTLshort, thereby identifying poor overall survival in a set of independent pediatric ALL patients and found clustering of all early relapse patients in one group. This new independent risk factor might identify patients with high risk for early relapse avoiding xenografting in the mouse model.

Description: Approximately 25% of childhood acute lymphoblastic leukemias carry the ETV6/RUNX1 fusion gene. Despite their excellent initial treatment response, up to 20% of patients relapse. To gain insight into the relapse mechanisms, we analyzed single nucleotide polymorphism arrays for DNA copy number aberrations (CNAs) in 18 matched diagnosis and relapse leukemias. CNAs were more abundant at relapse than at diagnosis (mean 12.5 vs 7.5 per case; P = .01) with 5.3 shared on average. Their patterns revealed a direct clonal relationship with exclusively new aberrations at relapse in only 21.4%, whereas 78.6% shared a common ancestor and subsequently acquired distinct CNA. Moreover, we identified recurrent, mainly nonoverlapping deletions associated with glucocorticoid-mediated apoptosis targeting the Bcl2 modifying factor (BMF) (n = 3), glucocorticoid receptor NR3C1 (n = 4), and components of the mismatch repair pathways (n = 3). Fluorescence in situ hybridization screening of additional 24 relapsed and 72 nonrelapsed ETV6/RUNX1-positive cases demonstrated that BMF deletions were significantly more common in relapse cases (16.6% vs 2.8%; P = .02). Unlike BMF deletions, which were always already present at diagnosis, NR3C1 and mismatch repair aberrations prevailed at relapse. They were all associated with leukemias, which poorly responded to treatment. These findings implicate glucocorticoid-associated drug resistance in ETV6/RUNX1-positive relapse pathogenesis and therefore might help to guide future therapies.

Description: Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy accounting for 80% of leukemias. The involvement of the central nervous system (CNS) by ALL is a major clinical problem and occurs in about 50% of the children without adequate treatment. The introduction of CNS-directed therapy consisting of intrathecal and high dose systemic chemotherapy and, occasionally, cranial irradiation, reduced relapse rate to less than 5% and has become a prerequisite for treating children with ALL. However, substantial neurotoxicity associated with this therapy is a major concern. Moreover the CNS is involved in up to a third from all relapses. To date very little is known about the pathogenesis of CNS leukemia. Our research was promoted by the previous observation that high mRNA expression of interleukin 15 (IL15) in leukemic blasts is associated with increased risk for CNS involvement (Cario et al JCO 2007;25:4813-20). As IL15 is a strong stimulant of Natural Killer (NK) cells, we hypothesized that the increased expression of IL15 may activate NK cells which, in turn, will control residual ALL cells in the peripheral blood but not in the relatively protected central nervous system. To investigate this hypothesis, we utilized two mouse models, a S49-derived T lymphoblastic leukemia syngeneic model and a novel human xenograft ALL model in immune-deficient mice. We found that constitutive expression of IL15 in mouse T lymphoblastic leukemia cells transplanted in neonatal Balb/c mice markedly slowed the development of systemic disease and caused CNS leukemia characterized by pronounced clinical CNS symptoms and subarachnoid infiltration of leukemia cells. This phenotype was accompanied by increase in activated natural killer (NK) cells (from 0.16% to 18.6% P

Description: Although improved treatment of pediatric Acute Lymphoblastic Leukemia (ALL) has become increasingly successful with cure rates of up to 90%, leukemia reoccurrence is clearly associated with inferior prognosis. Treatment failure and resistance in leukemias are considered to involve defects in apoptosis signaling, which forms the basis for apoptogenic treatment approaches such as the use of Bcl-2 inhibitors. Identification of alternative cell death programs like lysosomal membrane permeabilization-induced cell death may provide a novel strategy for the treatment of ALL. Previous studies showed that lysosomal activity, as well as their traffic properties are greatly altered during tumorigenesis. Intralysosomal hydrolases such as cathepsin B, L and D have been implicated in cancer progression and high expression levels are generally associated with poor prognosis. Lysosomotropic agents modulating lysosomal integrity may overcome cell death resistance and might therefore also improve the outcome of high risk or ALL relapse patients. In this study, we investigated the efficacy of the lysosomotropic compound B10, a derivative of Betulinic acid, as well as its combination with the Bcl-2 inhibitor ABT-263 in B-cell precursor- (BCP-) ALL. In BCP-ALL cell lines, B10 showed induction of cell death combined with increased DNA fragmentation. Interestingly, additional treatment with the pan-caspase inhibitor zVAD.fmk only partially rescued B10 triggered loss of cell viability, indicating that B10 is not exclusively inducing caspase-dependent apoptosis, but also induces an additional alternative cell death program. B10 permeabilized lysosomes as indicated by the significant decrease of LysotrackerRED positive populations detected by flow cytometry, and treatment with E-64d, a potent inhibitor of thiol proteases and cathepsins, reduced B10-induced cell death, thus emphasizing the cathepsin-dependent effect of B10. To exclude that B10-induced lysosomal permeabilization might represent a secondary effect, a panel of different substances was tested for their activity on lysosomal membrane integrity. While treatment with dexamethasone, chloroquine, vincristine and asparaginase also showed lysosomal permeabilization, co-treatment with E-64d did not reduce cell death indicating a secondary effect of these compounds on lysosomes. In addition to cell lines, we tested the effect of B10 on 15 primary leukemia samples isolated from ALL bearing mice of established patient-derived NOD/SCID/hu BCP-ALL xenografts. In a majority of these individual patient-derived leukemias, B10 induced cell death that could be inhibited by E-64d, thus demonstrating a lysosomal protease-dependent death program also in primary ALL. In addition to its classical role in triggering the intrinsic mitochondria dependent apoptosis pathway, the pro-apoptotic Bcl-2 family member Bax was previously described to be involved in the regulation of lysosomal membrane integrity, pointing to a possible synergistic effect of B10 and BH3-mimetics. Intriguingly, in cell fractionation assays we observed increased Bax recruitment to lysosomal membranes when B10 is present. Interestingly, co-treatment with the Bcl-2 inhibitor ABT-263, which displaces Bcl-2 from its inhibitory binding to Bax, led to increased lysosomal permeabilization, loss of mitochondrial membrane potential, and caspase activation indicating involvement of the intrinsic apoptosis pathway. The importance of lysosomal Bax recruitment for the activity of ABT-263 and its concomitant effect on lysosomal membrane permeabilization was further supported by Bax knockdown experiments, since induction of lysosomal disruption, release of cathepsins and their subsequent effect on cell death activation by B10 was reduced. Taken together, our findings suggest an important role of lysosomal membrane permeabilization-induced cell death for the activity of Bcl-2 inhibitors such as ABT-263. The combination of BH3 mimetics with lysosomotropic compounds may provide the basis for novel molecular directed treatment strategies. Disclosures No relevant conflicts of interest to declare.

Description: Over the past decades, the use of optimized multi-agent chemotherapy protocols and improved risk stratification have led to superior patient outcome. However, avoiding therapy-related toxicity, decreasing the relapse rate of currently almost 20 % and improving the outcome of relapsed patients remain challenging problems. Aberrant activity of pathways involved in the regulation of survival and cell death contribute to development of leukemia and therapy failure. Anti-apoptotic BCL-2 family proteins are key regulators of apoptosis providing a promising target for novel directed therapies. The BH3 mimetic ABT-199 binds to BCL-2, counteracts its anti-death function and leads to apoptosis induction by direct release of pro-death BCL-2 family proteins such as BAX. BH3 mimetics are currently used pre-clinically and in first clinical trials. However the intrinsic or acquired resistance indicates the need for predictive markers and for effective combination treatment strategies. In this study, we addressed the activity of ABT-199 in B cell precursor (BCP) ALL. The effects of ABT-199 as single agent and in combination with chemotherapeutic drugs used in remission-induction therapy of pediatric ALL were analyzed using BCP-ALL cell lines (n=5) and a set of patient-derived primograft samples (n=9) established by transplantation of primary leukemia cells obtained from pediatric BCP-ALL patients at diagnosis onto immunodeficient NOD/SCID mice. Since ABT-199 does not bind to all antiapoptotic BCL-2 family members, sparing MCL-1, we included the cyclin-dependent kinase (CDK) inhibitor dinaciclib, which also exerts MCL-1 inhibitory effects. Cell viability was determined by flowcytometry according to fw/sc criteria and half maximal inhibitory concentrations (IC50) were estimated upon exposure to single agents and drug combinations. Combination indices (CI) indicating synergistic or additive effects were estimated according to Chou-Talalay. BCL-2 and MCL-1 protein levels were investigated by western blot analysis. Despite sensitivity in most of the BCP-ALL cell lines with IC50 values in the nanomolar range (mean IC50, 212 nM), one cell line, Nalm-6, showed ABT-199 resistance with an IC50 〉10 µM. Interestingly, low protein levels of BCL-2 and increased MCL-1 expression were identified in this insensitive line, in contrast to high level BCL-2 and low MCL-1 expression in ABT-199 sensitive leukemias, indicating a potential marker to identify sensitive and resistant ALL. A pronounced synergism of ABT-199 in combination with chemotherapeutic agents was found with vincristine (CI 0.31 and 0.40, cell lines RS4;11 and Nalm-6) and even strong synergism with asparaginase and dexamethasone in ABT-199 monotherapy resistant Nalm-6 cells (CI 0.002, 0.14). Strikingly, the combination of the MCL-1 inhibitor dinaciclib with ABT-199 was highly effective and revealed a strong synergism for both compounds (CI 0.03). When we addressed the effects of ABT-199 on primograft ALL samples isolated from mice with full-blown leukemia, a pattern of high sensitivity (IC50 from 16 to 156 nM) similar to the cell lines was observed. In contrast, peripheral blood (PB) samples from healthy control donors were resistant to ex vivo exposure with ABT-199 (n=3, IC50 values 〉 1 µM). However, one primograft sample derived from a high risk patient showed ABT-199 insensitivity with an IC50 value of 〉 1 µM similar to healthy PBMCs. This primograft sample was also characterized by low BCL-2/high MCL-1 protein expression similar to Nalm-6. Most importantly, this resistant leukemia was also rendered ABT-199 sensitive by co-treatment with dinaciclib (CI 0.04). Taken together, we found efficacy of ABT-199 in the majority BCP-ALL cell lines and patient-derived primograft ALL samples and ABT-199 synergized with conventional chemotherapeutic agents. ABT-199 resistant leukemias characterized by low BCL-2/high MCL-1 expression were resensitized by addition of dinaciclib leading to strong synergism. These data indicate effective cell death sensitization by ABT-199 and potential strategies to overcome ABT-199 resistance in BCP-ALL. Disclosures No relevant conflicts of interest to declare.

Description: Acute lymphoblastic leukemia (ALL) is the most frequent malignant disorder in children and adolescents. Despite successful treatment, relapse of the disease remains a major problem and is associated with poor prognosis. This emphasizes the need for novel treatment strategies to be applied in addition to established chemotherapy regimens without increasing general toxicity. Previously, we described a strong association of leukemia cell engraftment of primary patient B cell precursor (BCP) ALL samples transplanted in a NOD/SCID/huALL mouse model and patient outcome. Rapid onset of leukemia related morbidity (time to leukemia, TTLshort) is indicative for patient relapse and characterized by a specific gene expression profile. Among the top differentially regulated genes, the gene coding for CD70 was identified to be significantly up-regulated in TTLshort/high risk ALL. CD70 is a member of the tumor necrosis factor (TNF) family expressed on activated B- and T-lymphocytes and dendritic cells. Binding of CD70 to its receptor CD27 is involved in regulation of T- and B-cells including priming and generation of memory and plasma cells. CD70 has been described to be constitutively expressed on different cancers including hematological malignancies. However, expression and targeting of CD70 in B- cell precursor lymphoblastic leukemia has so far not been investigated. In this study, we addressed expression of CD70 in patient-derived primograft leukemia samples and primary patient specimens obtained at diagnosis from pediatric patients. Furthermore, we evaluated CD70 as a therapeutic target for directed immunotherapy in vitro and in our BCP-ALL xenograft system in vivo. Flow cytometric analyses of CD70 surface expression in all together 19 patient-derived xenograft samples (TTLshort n= 7, TTLlong n=12) revealed a higher expression of CD70 on ALL cells with a TTLshort/early relapse phenotype compared to TTLlongsamples. We also investigated expression of the CD70 receptor CD27 and found no significant difference in surface expression between both TTL subgroups. Moreover, we investigated the transcript expression levels of 198 BCP-ALL specimens obtained at diagnosis. Interestingly, we found a heterogenous expression of CD70 with no association to cytogenetic subgroups, minimal residual disease (MRD) risk classes or patient outcome. Importantly, a significant higher CD70 expression was found in leukemia samples compared to healthy bone marrow controls indicating a general over-expression in BCP-ALL. CD27 however, did not show different transcript expression including healthy bone marrow controls. To take advantage of increased CD70 expression in BCP-ALL, we addressed CD70 as therapeutic target for immunotherapy. Co-culture in vitro experiments of primograft ALL cells with NK cells in the presence of specific anti-CD70 antibodies revealed five-fold increased antibody-dependent cell-mediated cytotoxicity (ADCC) as compared to the respective isotype control. To evaluate the efficacy of CD70 directed immunotherapy, we assessed leukemia development in NOD/SCID mice upon transplantation of primograft ALL with high CD70 expression either incubated with anti-CD70 antibody or the respective isotype control. Most importantly, a marked reduction of leukemia load in peripheral blood, bone marrow and spleens of the animals was detected in anti-CD70 treated cases. This indicates, that CD70 provides an immunotherapeutic target on ALL cells inducing ADCC by NK cells present in NOD/SCID mice. Most interestingly, this effect could be abrogated both by NK-cell depletion (pre-treatment with anti-mouse CD122 antibodies) in the recipient animals and by using NK-cell deprived NSG mice as recipients, confirming that decreased in vivo leukemia growth upon anti-CD70 treatment is mediated by NK-cell induced cytotoxicity of anti-CD70 bearing CD70 positive ALL cells. Taken together, we identified significantly up-regulated CD70 expression in BCP-ALL with varying expression among molecular and prognostic subgroups. BCP-ALL samples with high surface expression of CD70, as detected by flowcytometry, can be targeted by directed immunotherapy with anti-CD70 antibodies leading to efficient NK-cell dependent lysis of leukemia cells in vitro and decreased growth in an in vivo BCP-ALL model. Thus, CD70 provides a novel target for directed immunotherapy of BCP-ALL. Disclosures No relevant conflicts of interest to declare.

Description: Acute lymphoblastic leukemia (ALL) is the most common malignancy in childhood. While improved multi-agent chemotherapy regimens with individualized risk stratification have led to increased survival rates of approximately 80 percent, 20 percent of patients respond poorly to therapy or relapse. Therefore, novel therapeutic avenues are urgently needed to improve treatment outcome, overcome resistance and reduce side effects. Failure to undergo cell death represents a key survival mechanism of cancer cells and results in drug resistance and clonal escape. Since inhibitor of apoptosis proteins (IAPs) are often overexpressed in malignant cells and their overexpression correlates with inferior survival rates, they provide an attractive molecular target for therapeutic intervention. Small molecule inhibitors have been developed that act as SMAC mimetics (SMs) to counteract the cell death inhibitory function of IAPs. SMs can activate and/or modulate cell death pathways, and are currently being evaluated in clinical trials. Their successful therapeutic implementation requires identification of patients who could benefit from a SM-based treatment regimen ideally before start of therapy. Here, we analyzed the intrinsic activity of two monovalent (AT406 and LCL161) and two bivalent (Birinapant or BV6) SMs on 29 unselected patient-derived pediatric precursor B-cell (BCP)-ALL samples and identified a subset of BCP-ALL primografts to be sensitive to SM treatment (n=8). When we compared gene expression of SM-sensitive (n=8) and SM-insensitive (n=6) patient-derived BCP-ALL samples, we identified a characteristic gene expression signature with 127 differentially regulated genes, amongst them upregulation of TNFRSF1A (TNFR1) in the SM-sensitive subset. In line with previous reports, we confirmed a critical role of the TNF/TNFR1-axis for SM-induced cell death in BCP-ALL by functional analysis. Expression of TNFRSF1A alone, however, did not correlate with sensitivity to SM-induced cell death indicating that TNFR1 is not the only factor regulating cell fate decisions in response to SM treatment. To identify potential biomarker genes for prediction of patient response to SM monotherapy in BCP-ALL, we compared differentially regulated genes of SM responders and non-responders from our cohort with data from a published cohort. Interestingly, we found 4 genes to overlap between these two cohorts. Of these 4 genes TSPAN7, FAM69C, and TNFRSF1A were upregulated whereas MTX2 was downregulated in SM-sensitive samples. The signature identified may reflect a particular TNF network. Analysis of expression levels of these 4 genes in BCP-ALL cell lines (Nalm6, Reh, UoCB6 and RS4;11) revealed that Reh cells, sensitive to SM-induced cell death, exhibited the biomarker profile of primograft sensitivity, i.e. upregulation of TSPAN7, FAM69C, TNFRSF1A and downregulation of MTX2. Nalm6 cells resembled the expression pattern of SM-insensitive samples with a downregulation of TSPAN7, FAM69C, TNFRSF1A and an upregulation of MTX2 and were resistant to SM-induced cell death. RS4;11 and UoCB6 cells showed no pattern. Based on these findings we hypothesized that the respective expression patterns of TSPAN7, FAM69C, TNFRSF1A and MTX2 could predict sensitivity to SMs. An extended screen of additional primary BCP-ALL samples for their expression levels of TSPAN7, FAM69C, TNFRSF1A and MTX2 and response to SMs substantiated this hypothesis. In summary, the subset of primary BCP-ALL samples with sensitivity to SMs is characterized by a gene signature with MTX2 low and TSPAN7, FAM69C and TNFRSF1A high. By using this expression profile, sensitivity to SMs in BCP-ALL could be identified in cell lines and additional primografts. Based on these results, we suggest the identified gene expression pattern as a biomarker for selecting patients to be treated by SM monotherapy in clinical trials. Disclosures No relevant conflicts of interest to declare.

Description: Deregulation of cell death pathways is a hallmark of many cancers and contributes to leukemogenesis and treatment failure in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Different pro- and anti-apoptotic molecules control apoptosis signaling. While pro-apoptotic BCL-2 homology domain 3 (BH3) proteins induce cellular death, apoptosis induction is counter-regulated by anti-apoptotic molecules like B-cell lymphoma 2 (BCL-2). Therefore, inhibition of anti-apoptotic molecules has been developed as therapeutic strategy. Venetoclax (VEN) selectively binds to BCL-2, which leads to the release of pro-apoptotic molecules such as BIM resulting in apoptosis induction. In BCP-ALL, we and others showed preclinical activity of VEN and first clinical trials have started to evaluate VEN in ALL. Despite high activity, resistance can be acquired over time and acquisition of BCL2 mutations has been reported in CLL patients. In this study, we modeled VEN resistance in BCP-ALL and investigated underlying mechanisms in order to identify potential strategies to overcome VEN insensitivity. Starting from the BCP-ALL cell line RS4;11, five parallel VEN insensitive lines were generated by exposure to increasing concentrations of VEN over time (49 passages, over 8 months of continuous treatment). Simultaneously, five control lines were exposed to corresponding concentrations of solvent (DMSO). Measuring half maximal effective concentrations (EC50) over time showed increasing EC50 values from 4 nM to 26.2 µM in all VEN treated lines, reflecting acquired resistance in our model. Importantly, no mutations of the BCL2 gene were identified by sequencing at high coverage (Illumina AmpliSeq, 650- to 5951-fold), excluding acquisition of BCL2 mutations as mechanism of resistance. Next, we assessed expression levels of the mitochondrial apoptosis regulators BCL-2, MCL-1 and BCL-XL by western blot analysis. Comparing VEN insensitive to control cell lines, no differences in expression of the target molecule BCL-2 and similar levels of BCL-XL were observed. Most interestingly, all five VEN insensitive lines showed significant up-regulation of MCL-1 compared to all control lines. We next investigated the dependence of apoptosis signaling on different BCL-2 family members by exposing ALL cells to synthetic BH3-only peptides, which specifically bind to different regulators of mitochondrial apoptosis signaling (BH3-profiling), followed by analysis of apoptosis induction. Interestingly, dependence on BCL-2 was clearly reduced in all VEN insensitive lines indicated by almost lost mitochondrial priming. On the other hand, VEN insensitive ALL cells showed increased dependence on MCL-1. In order to analyze whether our modeled VEN resistance can be overcome by targeting MCL-1, we investigated sensitivity of these cells to the MCL-1 inhibitor S63845. High EC50 values for S63845 were found in all VEN insensitive and corresponding control lines, indicating low anti-MCL-1 activity. Importantly, the combination of S63845 with VEN synergistically induced cell death, showing that acquired VEN insensitivity in BCP-ALL can be overcome by co-targeting BCL-2 and MCL-1. To get insight into the molecular mechanisms underlying the synergism, we treated VEN insensitive ALL cells with VEN, S63845 or both and investigated binding of BIM to either BCL-2 or MCL-1 by immunoprecipitation. In the presence of VEN, clearly lower co-precipitation of BIM with BCL-2 but increased co-precipitation with MCL-1 was observed, indicating that pro-apoptotic BIM displaced from BCL-2 by VEN is sequestered by MCL-1, thereby counter-regulating VEN activity. Inversely, the MCL-1 inhibitor S63845 reduced binding of BIM to MCL-1 but increased BIM binding to BCL-2. Most interestingly, upon combination of both inhibitors, lower BIM binding to both BCL-2 and MCL-1 was found. This indicates that co-targeting MCL-1 can block the sequestration of pro-apoptotic BIM from BCL-2 to MCL-1, overcoming VEN resistance. Taken together, we show that acquired VEN resistance in BCP-ALL is characterized by up-regulated expression of counter-regulatory MCL-1 and can be overcome by simultaneous BCL-2 and MCL-1 inhibition, which prevents sequestration of BIM by MCL-1 after release from BCL-2. Disclosures Tausch: Roche: Consultancy, Honoraria, Speakers Bureau; AbbVie: Consultancy, Honoraria, Other: travel support, Speakers Bureau. Stilgenbauer:Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pharmacyclics: Other: Travel support; GSK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Hoffmann La-Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau.