BH3 Mimetic ABT-737 Research Articles

HA14-1, but not the BH3 mimetic ABT-737, causes Ca2+ dysregulation in platelets and human cell lines

HA14-1, but not the BH3 mimetic ABT-737, causes Ca2+ dysregulation in platelets and human cell lines

Selective triggering of platelet apoptosis, platelet activation or both

Anucleate platelets perform two fundamental processes, activation and apoptosis. We elaborated an approach for selective and concurrent stimulation of platelet apoptosis and/or activation, processes important in haemostasis and platelet clearance. Human platelets were treated with BH3 mimetic ABT-737, thrombin, calcium ionophore A23187 and matched diluents. Apoptosis was determined as mitochondrial inner membrane potential (ΔΨm) depolarization and activation as P-selectin exposure. At optimal treatment conditions (90-180min, 37°C), ABT-737 predominantly induced apoptosis, when 77-81% platelets undergo only ΔΨm depolarization. The ABT-737 impact on ΔΨm depolarization is strongly time- and temperature-dependent, and much higher at 37°C than at room temperature. In contrast, when platelets were treated with thrombin for 15-90min at either temperature, activation-only was predominantly (79-85%) induced, whereas A23187 triggers both apoptosis and activation (73-81%) when platelets were treated for 15-60min at 37°C or 15-90min at room temperature. These data demonstrate that, depending on the triggering stimulus, platelets predominantly undergo ΔΨm depolarization-only, P-selectin exposure-only, or both responses, indicating that platelet apoptosis and activation are different phenomena driven by different mechanisms. The described model provides a basis for studying differential pharmacological manipulation of platelet apoptosis and activation and their role in haemostasis, thrombosis and platelet clearance.

Functional inhibition of BCL2 is needed to increase the susceptibility to apoptosis to SMO inhibitors in diffuse large B-cell lymphoma of germinal center subtype

Previously, we have demonstrated that inhibition of Hedgehog pathway induces predominantly apoptosis in diffuse large B-cell lymphoma (DLBCL) cell lines of activated B-cell (ABC) type but predominantly cell cycle arrest in those of germinal center (GC). Here, we explored the possibility of overcoming the resistance to apoptosis to SMO inhibitors in five DLBCL cells of GC type using the combination of the SMO inhibitor HhAntag (Genentech Inc) with the BH3 mimetic ABT-737 (Abbott Laboratories). As controls we have used two DLBCL of ABC type (OCI-LY10 and OCI-LY3). Combinatorial treatments were performed with increasing concentrations of the HhAntag with low doses (equal or less than the IC20) of ABT-737. MTS assays were used to detect changes in cell viability and Annexin-V and PARP1 cleavage assays were used to detect apoptosis. Combining low doses of ABT-737 with increasing concentrations of HhAntag in GC DLBCL cell lines resulted in significantly increase of apoptosis in comparison to treatments with the SMO inhibitor alone. We concluded that in GC DLBCL cell lines, in contrast to those of ABC type, functional inhibition of BCL2 family members is usually needed to overcome the resistance to apoptosis to SMO inhibitors. These findings provide a rationale to explore the use of SMO and BCL2 inhibitors as adjuvant therapy for treatment of DLBCL of GC type.

Omi/HtrA2 and XIAP are components of platelet apoptosis signalling

Although platelets possess the hallmarks of apoptosis such as activation of caspases, cytochrome c release and depolarisation of the mitochondrial transmembrane potential (∆Ψm), their entire apoptotic-signalling pathway is not totally understood. Therefore we studied the expression of various apoptotic proteins and found that platelets contain the pro-apoptotic proteins Omi/HtrA2 and Smac/Diablo, as well as their target the X-linked inhibitor of apoptosis XIAP. Omi/HtrA2 and Smac/Diablo were released from mitochondria into the platelet cytosol together with cytochrome c after induction of apoptosis by the Ca2+ ionophore A23187 or the BH3 mimetic ABT-737, and to a lesser extent, after platelet stimulation with collagen and thrombin. Inhibition of Omi/HtrA2 led to decreased levels of activated caspase-3/7 and caspase-9, but did not abolish loss of ∆Ψm or prevent release of Omi/HtrA2 from mitochondria. These results indicate that platelets have a functional intrinsic apoptotic-signalling pathway including the pro-apoptotic protease Omi/HtrA2 and its target protein XIAP.

Upregulating Noxa by ER Stress, Celastrol Exerts Synergistic Anti-Cancer Activity in Combination with ABT-737 in Human Hepatocellular Carcinoma Cells

The human hepatocellular carcinoma (HCC) represents biologically aggressive and chemo-resistant cancers. Owing to the low affinity with the apoptotic factor Mcl-1, the BH3 mimetic drug ABT-737 failed to exert potent cancer-killing activities in variety of cancer models including HCC. The current study demonstrated that combining ABT-737 and Celastrol synergistically suppressed HCC cell proliferation, and induced apoptosis which was accompanied with the activation of caspase cascade and release of cytochrome c from mitochondria. Further study revealed that the enhanced Noxa caused by Celastrol was the key factor for the synergy, since small interfering RNA-mediated knockdown of Noxa expression in HCC cells resulted in decreased apoptosis and attenuated anti-proliferative effects of the combination. In addition, our study unraveled that, upon Celastrol exposure, the activation of endoplasmic reticulum (ER) stress, specifically, the eIF2α-ATF4 pathway played indispensable roles in the activation of Noxa, which was validated by the observation that depletion of ATF4 significantly abrogated the Noxa elevation by Celastrol. Our findings highlight a novel signaling pathway through which Celastrol increase Noxa expression, and suggest the potential use of ATF4-mediated regulation of Noxa as a promising strategy to improve the anti-cancer activities of ABT-737.

Combination of the IGF-1 Receptor Inhibitor Picropodophylin and the BH3 Mimetic ABT-737 Has Synergistic Anti-Myeloma Activity

AbstractAbstract 4010Despite intensive research multiple myeloma (MM) is still an incurable disease. MM cells are strongly dependent on the BM micro-environment where growth factors are secreted. IGF-1 is one of the most important growth factors in MM and thus forms an attractive target for anti-cancer therapy. Previously, we demonstrated that picropodophylin (PPP), an IGF-1R kinase inhibitor, indeed has potent anti-MM effects both on human cells and in the 5T2MM and 5T33MM murine models. However, mice eventually relapsed and showed signs of morbidity. Therefore it would be an attractive approach to combine PPP with other cytotoxic drugs. ABT-737 is a BH3 mimetic that binds with high affinity to Bcl-xL, Bcl-2 and Bcl-w, but not Mcl-1. In MM, elevated expression of the Bcl-2 pro-survival family of proteins, especially Mcl-1 and to a lesser extent Bcl-2, has extensively been shown to cause resistance to drug induced apoptosis in MM cells. Consequently, ABT-737 was shown to have potent anti-MM activity but only on a subset of human cell lines. Only very recently, it was shown that the ABT-737 sensitivity appears to be determined both by the Bcl-2/Mcl-1 expression ratio and the interaction of these anti-apoptotic proteins with Bim. Interestingly, we demonstrated that IGF-1R inhibition reduces the expression of Mcl-1 and Bcl-xL and that IGF-1 down-regulates the expression of Bim. In addition, a protective effect of IL-6 and BMSC against ABT-737 has been reported. Together, all the above suggests that it would be beneficial to combine ABT-737 with agents that target growth factors, like PPP. Here, we investigated the potential synergistic anti-MM effects of PPP and ABT-737 and studied the underlying mechanisms using two human myeloma cell lines (OPM-2 and RPMI-8226) and the murine 5T33MM model. Both PPP and ABT-737 (kindly provided by Abbott Laboratories) alone were found to significantly decrease cell viability and induce apoptosis dose and time dependently as evidenced by a decrease in ATP levels and an increase in the number of AnnexV/7’AAD positive cells. However, in agreement with previous reported data, we observed differential sensitivity to ABT-737 between the cell lines used. Nevertheless, treatment with PPP/ABT-737 synergistically decreased cell viability and induced apoptosis in all cell lines. In addition, by western blot analysis we could observe increased cleavage of caspase- 3,- 9 and PARP. Mechanistically, PPP was found to circumvent the adverse effect of ABT-737 by blocking the ABT-737 induced Mcl-1 expression and increasing the expression of Noxa. Interestingly, while CD138+ 5T33MM cells were more sensitive to PPP and the CD138- cells more sensitive to ABT-737, treatment with PPP/ABT-737 targeted both MM cell subpopulations to an equal extent. Finally, we tested the combination of PPP and ABT-737 in the 5T33MM model in a prophylactic setting. Whereas, vehicle and ABT-737 treated mice exhibited progressive MM growth, PPP, and to a significant greater extent, PPP/ABT-737 reduced the tumor burden and prolonged overall survival (p≤0.001).In conclusion, PPP combined with ABT-737 appears to have synergistic anti-MM activity and might thus be a novel and promising therapy strategy for MM. Disclosures:No relevant conflicts of interest to declare.

Inhibition of the HDM-2 E3 Ligase Induces Types I and II Cell Death in Wild-Type and Mutant p53 Models of Multiple Myeloma, and Acts Synergistically in Both with ABT-737.

Abstract 2940 Background:The ubiquitin-proteasome pathway has been validated as a therapeutic target for multiple myeloma by the incorporation of proteasome inhibitors such as bortezomib and carfilzomib into our standards of care against this disease. A minority of patients achieve complete remission with single-agent therapy, however, and proteasome inhibitors may induce on- and off-target anti-apoptotic effects and clinical toxicities that limit their utility. Unlike proteasome inhibitors, which have a broad impact on intracellular proteolysis, E3 ligases are responsible for ubiquitination of only a small subset of proteins. Our hypothesis proposed that inhibition of the Human double minute (HDM)-2 E3 ligase, which in part is responsible for p53 ubiquitination, could be a more rational therapeutic target for myeloma, where p53 mutations are less common than is the case in solid tumors. Methods:We used Nutlin-3a and MI-63, which bind to the p53 binding pocket of HDM-2, as prototypic agents, and studied their activity against both wild-type (wt) and mutant (mut) p53 cell line models of multiple myeloma. These studies were supported by the M. D. Anderson Cancer Center SPORE in Multiple Myeloma. Results:Inhibition of HDM-2 in wt p53 (MM1.S, H929, MOLP-8) cells with Nutlin-3a or MI-63 reduced viability, with a median inhibitory concentration (IC50) in the 0.5–2 μM range. In wt cells, genomic and proteomic studies showed that MI-63 induced transcription of downstream gene targets including p21, PUMA and Bax, and promoted accumulation of p53, p21, p27, and Bax. This was accompanied by cell cycle arrest at G1, and induction of apoptosis as demonstrated by Annexin V staining and caspase activation. Interestingly, these agents also showed activity against mut p53 cells such as RPMI 8226 and U266, though with higher IC50’s of 30–40 μM. Knockdown of HDM-2 sensitized mut p53 myeloma cells to MI-63, suggesting that this agent was inducing cell death in an on-target manner. In p53 mutant cells, MI-63 induced both type I cell death and type II, autophagic cell death, as determined by conversion of microtubule-associated protein 1 light chain 3, and activation of transcription of autophagy (ATG) genes ATG3 and 5. Since autophagy can be a mechanism of cell survival or cell death, we tested the impact of the autophagy inhibitors chloroquine and 3-methyladenine, and found that they reduced the impact of MI-63 on viability. Moreover, knockdown of ATG5 helped rescue cells as well, supporting the possibility that autophagic activation was a mechanism of cell death in these myeloma models. Synergistic anti-myeloma effects were seen when MI-63 was combined with the BH3 mimetic ABT-737, with decreased viability and enhanced apoptosis, accompanied by increased Bax activation and caspase cleavage. Notably, ABT-737 also enhanced the effects of MI-63 on mut p53 myeloma models, where increased levels of both type I and II programmed cell death were seen. Conclusions:Together, our findings support the possibility that HDM-2 is a promising therapeutic target for multiple myeloma, and also provide a rationale for translation of HDM-2 inhibitors alone, and in combination with ABT-737, to the clinic. Disclosures:Leopold:Ascenta Therapeutics: Employment, Equity Ownership.

BH3 Profiling Predicts 5-Azacytidine Response in Malignant Myeloid Cells

AbstractAbstract 1432Previously we demonstrated that inhibition of anti-apoptotic BCL-2 family members sensitize leukemic cells to 5-Azacytidine (5-Aza), using siRNA and pharmacological inhibition with the BH3-mimetic ABT-737, both in vitro and ex vivo (Bogenberger, JM., et. al. ASH Annual Abstracts 2011;118:Abstract 3513). Crucially, several anti-apoptotic BCL-2 members (e.g. BCL-2 and BCL-XL) required concurrent inhibition for potent and universal sensitization to 5-Aza. Anti-apoptotic BCL-2 proteins block the execution of programmed cell death (apoptosis) by binding to and counteracting two types of pro-apoptotic BCL-2 family proteins: the “BH3-only” proteins, including both activators (BIM and BID) and sensitizers (e.g. NOXA, PUMA, HRK), and the multi-domain effector proteins (BAX and BAK). Cells dependent on anti-apoptotic BCL-2 family members for survival have been defined as “primed for death” (Certo, M., et. al. Cancer Cell 2006 May;9(5):351-65). Importantly, the priming status reflects a dependence on anti-apoptotic BCL-2 family proteins and can be characterized with the BH3 profiling functional assay. This assay measures induction of mitochondrial outer membrane permeabilization (MOMP) in response to treatment with peptides derived from BH3-only proteins (Ni Chonghaile, T., et. al. Science 2011 Nov 25;334(6059);1129-33). Thus, the unique BH3 profile associated with a specific malignant cell population, is a function of the anti-apoptotic BCL-2 family member/s contextual expression in that cell population. Based on our observations of potent 5-Aza sensitization in combination with anti-apoptotic BCL-2 protein family inhibition, we hypothesized that BH3-profiling would predict response to 5-Aza.To address the potential utility of BH3 profiling in predicting response to 5-Aza in myeloid malignancies, we assayed a broad panel of AML-derived cell lines (N=13) by BH3 profiling and correlated BH3 metrics with 5-Aza response. Identical cell line passages were assayed by BH3 profiling and in 5-Aza drug dose response assays. The cell panel comprised lines derived from AML FAB subtypes M7, M6, M5, M4, M2 and M0, as well as diverse cytogenetics such as t(11;21), t(9;11), t(4;11), t(6;11), del 5q, del 7, and a broad spectrum of mutations such as FLT3, N-RAS, CDKN2A, NPM1 and DNMT3A. The panel included a normal karyotype AML cell line (CG-SH) (Munker, R., et. al. Leuk Res 2009 Oct:33(10):1405-8) and a blastic cell line derived from a patient with MDS (MDS-L) (Tohyama, K., et. al. Br J Haematol 1994 Jun;87(2):235-42). Partition modeling using several BH3 metrics discriminates 5-Aza response with statistical significance (N=13, Mann-Whitney p<0.01) between more sensitive (EC50<2uM) and less sensitive (EC50>2uM) AML lines. For instance, the Puma BH3 peptide alone distinguishes more sensitive from less sensitive cell lines (N=13, Mann-Whitney p=0.0046). Several more complex parameters, such as “Puma+Hrk”, “Puma+Noxa,” and “Bim+Puma+Noxa+Hrk,” also significantly discriminate 5-Aza response (Mann-Whitney p=0.0011). Using continuous variable analysis, a Puma+Hrk metric exhibited the strongest correlation with 5-Aza response (R2=0.85, p<0.0001), while an individual Puma metric yielded the second strongest correlation (R2=0.70, p=0.0004). In conclusion, BH3 profiling discriminates 5-Aza response and confirms a central role for anti-apoptotic BCL-2 members in 5-Aza response. Furthermore, HRK priming indicates that BCL-XL is an important, although not the sole, anti-apoptotic component determining 5-Aza response in myeloid cells. These BH3 profiling results are in agreement with our functional 5-Aza RNAi and BCL-XL/BCL-2 inhibitor data in combination with 5-Aza. Based on these results, we are currently evaluating BH3 profiles from 5-Aza responding and non-responding AML and MDS patients to validate BH3 profiling-derived metrics in predicting clinical outcomes to 5-Aza. [Display omitted] [Display omitted] Disclosures:Pierceall:Eutropics Pharmaceuticals: Employment, Equity Ownership. Lena:Eutropics Pharmaceuticals: Employment. Mesa:Sanofi: Research Funding; Incyte: Research Funding; Lilly: Research Funding; NS Pharma: Research Funding; YM Bioscience: Research Funding. Cardone:Eutropics Pharmaceuticals: Employment, Equity Ownership.

The BCL-2-Specific BH3-Mimetic ABT-199 (GDC-0199) Is Active and Well-Tolerated in Patients with Relapsed Non-Hodgkin Lymphoma: Interim Results of a Phase I Study

AbstractAbstract 304 Background:BCL-2 is highly expressed in indolent non-Hodgkin lymphomas (NHL), mantle cell lymphoma (MCL) and other selected aggressive lymphomas, and is a promising target for therapeutic intervention. The first-generation BCL-2 inhibitor navitoclax showed some activity in indolent lymphoma, but its co-inhibition of BCL-xL resulted in dose-limiting thrombocytopenia, precluding the full exploration of the potential of BCL-2 inhibition with this drug in NHL. ABT-199 is an orally bioavailable, second-generation BH3-mimetic that inhibits BCL-2 (Ki<0.10 nM), but has 500-fold less activity for BCL-xL (Ki=48 nM). ABT-199 demonstrated antitumor activity against a variety of human cell lines and xenograft models that include B cell NHL, follicular lymphomas (FL), diffuse large B-cell Lymphoma (DLBCL) and MCL. Methods:This is a phase-I dose-escalation trial using a modified Fibonacci design in patients with relapsed/refractory NHL. The primary objectives of this study are to determine the safety, pharmacokinetics (PK) and maximum tolerated dose (MTD) of ABT-199; to recommend a phase-2 dose; and to assess efficacy and biomarkers in patients with relapsed/refractory NHL. Adult patients requiring therapy, with ECOG performance status £1, and adequate marrow function received ABT-199 on Week 1 Day −7 (W1D-7), followed by continuous once-daily dosing from W1D1, until progressive disease (PD) or unacceptable toxicity. Due to concerns of potential tumor lysis, a strategy of commencing with a 2 to 3 week lead-in period with step-wise increases to the target cohort dose is being evaluated. In the first four cohorts, the starting dose increased from 50 to 200 mg (50, 100, 200, and 200 mg, respectively), with target cohort doses of 200 mg [n=3], 300 mg [n=3], 400 mg [n=4], and 600 mg [n=7]. Evaluations include: adverse events (AE; NCI-CTCAE-V4) and tumor response (IWG 2007 criteria). Results:To date, 17 patients (median age, 71 [35–85]) have been treated with ABT-199. Median prior therapies were 3 (range, 1–7) and 6 patients had bulky adenopathy (>5cm). Most common AEs (experienced by >2 patients) were nausea (41%), diarrhea (24%), dyspepsia (24%), fatigue (24%), extremity pain (24%); and anemia, constipation, upper respiratory tract infection and cough (18% each). Grade 3 or 4 AEs occurring in >1 patient were anemia (18%) and neutropenia (12%). Treatment-related thrombocytopenia has not been reported and no dose-limiting toxicities (DLTs) have been identified to date. After a single dose administration with a high-fat meal, ABT-199 reached Cmax at approximately 7 hrs with a terminal half-life of about 15 hrs. Food increased ABT-199 exposure by approximately 3-fold. With a median follow-up of 2.8 months (range, 1.2 to 10.8), 14 patients remain on study and 3 have discontinued due to PD. In patients who have completed at least a W6 assessment, reductions of >50% in target lesions have been observed in 8/15 patients (53%); 6/6 patients with MCL, 1/2 patients with WM and 1/2 patients with DLBCL. Additionally, 5 FL patients have been evaluated (3 with rituxan-refractory disease) with a median time on study of 6.4 months (range, 3.5 to 10.8). 4/5 FL patients had nodal disease reductions ranging from 18% to 40%. Conclusions:ABT-199 shows single agent anti-tumor activity in patients with NHL; particularly in MCL. Activity is also observed in DLBCL and WM. To date, no DLTs have been identified and tumor lysis syndrome related to ABT-199 has not been reported. Dose escalation is continuing to identify the optimal dosing regimen and MTD of ABT-199 in NHL. Updated results will be presented. Disclosures:Roberts:Abbott: Research Funding; Genentech: Research Funding. Anderson:Genentech: Research Funding; Abbott: Research Funding; Walter and Eliza Hall Institute of Medical Research: Employment, receives commercial income related to ABT-199, receives commercial income related to ABT-199 Other. Kahl:Genentech: Consultancy, Research Funding; Abbott: Research Funding. Darden:Abbott: Employment, owner of Abbott stock Other. Nolan:Abbott: Employment, own Abbott stock Other. Gressick:Abbott: Employment, stock owner Other. Yang:Abbott: Employment, own Abbott stock Other. Chyla:Abbott: Employment, Stock owner Other. Busman:Abbott: Employment, Stock owner Other. Graham:Abbott: Employment, Stock owner Other. Cerri:Abbott: Employment, Stock owner Other. Enschede:Abbott: Employment, own Abbott stocks Other. Humerickhouse:Abbott: Employment, own Abbott stocks Other. Seymour:Roche: Advisory board member, Advisory board member Other, Consultancy; Genentech: Advisory board member, Advisory board member Other, Consultancy.

The BCL-2-Specific BH3-Mimetic ABT-199 (GDC-0199) Is Active and Well-Tolerated in Patients with Relapsed/Refractory Chronic Lymphocytic Leukemia: Interim Results of a Phase I First-in-Human Study

AbstractAbstract 3923 Background:BCL-2 is highly expressed in indolent non-Hodgkin lymphomas (NHL), mantle cell lymphoma (MCL) and other selected aggressive lymphomas, and is a promising target for therapeutic intervention. The first-generation BCL-2 inhibitor navitoclax showed some activity in indolent lymphoma, but its co-inhibition of BCL-xL resulted in dose-limiting thrombocytopenia, precluding the full exploration of the potential of BCL-2 inhibition with this drug in NHL. ABT-199 is an orally bioavailable, second-generation BH3-mimetic that inhibits BCL-2 (Ki<0.10 nM), but has 500-fold less activity for BCL-xL (Ki=48 nM). ABT-199 demonstrated antitumor activity against a variety of human cell lines and xenograft models that include B cell NHL, follicular lymphomas (FL), diffuse large B-cell Lymphoma (DLBCL) and MCL. Methods:This is a phase-I dose-escalation trial using a modified Fibonacci design in patients with relapsed/refractory NHL. The primary objectives of this study are to determine the safety, pharmacokinetics (PK) and maximum tolerated dose (MTD) of ABT-199; to recommend a phase-2 dose; and to assess efficacy and biomarkers in patients with relapsed/refractory NHL. Adult patients requiring therapy, with ECOG performance status £1, and adequate marrow function received ABT-199 on Week 1 Day −7 (W1D-7), followed by continuous once-daily dosing from W1D1, until progressive disease (PD) or unacceptable toxicity. Due to concerns of potential tumor lysis, a strategy of commencing with a 2 to 3 week lead-in period with step-wise increases to the target cohort dose is being evaluated. In the first four cohorts, the starting dose increased from 50 to 200 mg (50, 100, 200, and 200 mg, respectively), with target cohort doses of 200 mg [n=3], 300 mg [n=3], 400 mg [n=4], and 600 mg [n=7]. Evaluations include: adverse events (AE; NCI-CTCAE-V4) and tumor response (IWG 2007 criteria). Results:To date, 17 patients (median age, 71 [35–85]) have been treated with ABT-199. Median prior therapies were 3 (range, 1–7) and 6 patients had bulky adenopathy (>5cm). Most common AEs (experienced by >2 patients) were nausea (41%), diarrhea (24%), dyspepsia (24%), fatigue (24%), extremity pain (24%); and anemia, constipation, upper respiratory tract infection and cough (18% each). Grade 3 or 4 AEs occurring in >1 patient were anemia (18%) and neutropenia (12%). Treatment-related thrombocytopenia has not been reported and no dose-limiting toxicities (DLTs) have been identified to date. After a single dose administration with a high-fat meal, ABT-199 reached Cmax at approximately 7 hrs with a terminal half-life of about 15 hrs. Food increased ABT-199 exposure by approximately 3-fold. With a median follow-up of 2.8 months (range, 1.2 to 10.8), 14 patients remain on study and 3 have discontinued due to PD. In patients who have completed at least a W6 assessment, reductions of >50% in target lesions have been observed in 8/15 patients (53%); 6/6 patients with MCL, 1/2 patients with WM and 1/2 patients with DLBCL. Additionally, 5 FL patients have been evaluated (3 with rituxan-refractory disease) with a median time on study of 6.4 months (range, 3.5 to 10.8). 4/5 FL patients had nodal disease reductions ranging from 18% to 40%. Conclusions:ABT-199 shows single agent anti-tumor activity in patients with NHL; particularly in MCL. Activity is also observed in DLBCL and WM. To date, no DLTs have been identified and tumor lysis syndrome related to ABT-199 has not been reported. Dose escalation is continuing to identify the optimal dosing regimen and MTD of ABT-199 in NHL. Updated results will be presented. Disclosures:Seymour:Roche: Advisory Board member Other, Consultancy; Genentech: Advisory Board member, Advisory Board member Other, Consultancy. Anderson:Abbott: Research Funding; Genentech: Research Funding; Walter and Eliza Hall Institute of Medical Research: Employment, receives commercial income related to ABT-199, receives commercial income related to ABT-199 Other. Kipps:Abbott: Consultancy, Research Funding. Wierda:Abbott: Research Funding; Genentech: Consultancy, Research Funding; GlaxoSmithKline: Consultancy, Research Funding; AmGen: Research Funding; Merck: Consultancy; Celgene: Consultancy; Pharmacyclics: Consultancy; Genzyme: Consultancy. Kahl:Abbott: Research Funding; Genentech: Consultancy, Research Funding. Miller:Abbott: Research Funding; Genentech: Research Funding. Darden:Abbott: Employment, owner of Abbott stock Other. Nolan:Abbott: Employment, own Abbott stock Other. Gressick:Abbott: Employment, stock owner Other. Xiong:Abbott: Employment, own Abbott stock Other. Huang:Genentech: Research Funding; Abbott: Research Funding; Walter and Eliza Hall Institiute of Medical Research: Employment, receives commercial income related to ABT-199, receives commercial income related to ABT-199 Other. Chyla:Abbott: Employment, Stock owner Other. Busman:Abbott: Employment, Stock owner Other. Graham:Abbott: Employment, Stock owner Other. Cerri:Abbott: Employment, Stock owner Other. Enschede:Abbott: Employment, own Abbott stocks Other. Humerickhouse:Abbott: Employment, own Abbott stocks Other. Roberts:Abbott: Research Funding; Genentech: Research Funding; Walter and Eliza Hall Institute of Medical Research: Employment, Receives commercial income related to ABT-199, Receives commercial income related to ABT-199 Other.

Inhibition of Autophagy by Chloroquine Sensitises Lymphoma Cells to ABT-737-Induced Apoptosis

AbstractAbstract 1625Diffuse large B-cell lymphoma (DLBCL) is characterised by overexpression of the anti-apoptotic protein Bcl-2. It has been recently observed that Bcl-2 also inhibits autophagy by binding and sequestering Beclin-1, an essential autophagy protein, but it is unclear whether Bcl-2 inhibits both apoptosis and autophagy in DLBCL cells. We aimed to determine the dual role of Bcl-2 in both apoptosis and autophagy in Bcl-2 positive cell lines (Su-DHL4 and CRL) and Bcl-2 negative cell lines (Su-DHL8 and Su-DHL10) using the BH3 mimetic compound ABT-737.The sensitivity of Bcl-2 positive and Bcl-2 negative cell lines to ABT-737-mediated mitochondrial depolarization (ΔΨmLOW) and cell death (DAPI positive) was assessed by flow cytometry. Treatment of the Bcl-2 positive cell lines Su-DHL4 and CRL with ABT-737 significantly increased (p<0.01) the percentage of both ΔΨmLOW cells, indicating mitochondrial damage as well as DAPI positive cells indicating cell death. Treatment with ABT-737 increased Bax activation and PARP cleavage in Bcl-2 positive cells, indicating that as expected, ABT-737-induced cell death is via apoptosis. ABT-737-induced cell death was not detected in Bcl-2 negative cell lines Su-DHL8 and Su-DHL10, demonstrating that, as expected, the sensitivity of DLBCL cell lines to ABT-737-induced apoptosis is Bcl-2 dependent.Treatment of Bcl-2 positive cells with ABT-737 also resulted in a decreased cellular co-localisation of Bcl-2 and Beclin-1 as detected by immunofluorescent staining. Degradation of p62 and LC3-II, selective substrates of autophagy, was detected by Western blotting in Bcl-2 positive but not in Bcl-2 negative cell lines after treatment with ABT-737 for 15 hours. LC3-I is a diffuse cytoplasmic protein which upon activation of autophagy becomes cleaved and lipidated to LC3-II which becomes punctate within cells. Punctuate LC3-II is a widely used marker of active autophagy. ABT-737-induced autophagosome formation was determined at an earlier time point (3 hours after ABT-737 treatment) using immune-fluorescent microscopy. ABT-737 induced increased numbers of larger punctate LC3-II in Bcl-2 positive Su-DHL4 and CRL cell lines but not in Bcl-2 negative cells, indicating that inhibition of Bcl-2 induces autophagy in Bcl-2 positive cells.We then determined whether autophagy affects ABT-737-induced apoptosis by blocking autophagy using an autophagy inhibitor chloroquine (CQ). Co-treatment with ABT-737 and CQ resulted in an increase in the percentage of ΔΨmLOW cells, DAPI positive cells and PARP cleavage compared to cells treated with ABT-737 alone in Bcl-2 positive cell lines. Combined, these results indicate that inhibition of autophagy by chloroquine further sensitises Bcl-2 positive cells to ABT-737-induced apoptosis.In summary, our results indicate that Bcl-2 inhibits autophagy in lymphoma cells by sequestering Beclin-1. Disruption of this interaction by ABT-737 induces autophagy which in turn inhibits apoptosis. Inhibition of autophagy results in increased sensitivity of Bcl-2 positive cells to ABT-737-induced apoptosis, suggesting a role for autophagy inhibitors in lymphoma treatment. Disclosures:No relevant conflicts of interest to declare.

Platelet Production Occurs Independently of Both the Intrinsic and Extrinsic Apoptosis Pathways

AbstractAbstract 389The survival of megakaryocytes and platelets is regulated by the intrinsic apoptosis pathway. Both cell types express Bak and Bax, the essential mediators of intrinsic apoptosis, which must be kept in check for cellular viability to be maintained. In platelets, Bak and Bax are restrained by the pro-survival protein Bcl-xL. Mutations that reduce the pro-survival activity of Bcl-xL cause dose-dependent cell-intrinsic reductions in circulating platelet life span in mice. Accordingly, pharmacological blockade of Bcl-xL with the BH3 mimetic drugs ABT-737 or ABT-263 (Navitoclax) triggers platelet death and thrombocytopenia in mice, dogs and humans. In mice, loss of Bak and Bax almost doubles platelet life span, and renders platelets refractory to the effects of ABT-737. In megakaryocytes, we and others have recently demonstrated that Bcl-xLand its pro-survival relative Mcl-1 are essential for restraint of the intrinsic apoptosis pathway. Their loss triggers Bak/Bax-mediated death. Conversely, ablation of Bak and Bax can protect megakaryocytes from acute apoptotic insults, such as treatment with carboplatin. Combined with the fact that platelet production is normal in the absence of Bak and Bax, these studies have brought into question the long-standing theory that megakaryocytes deliberately undergo apoptosis in order to shed platelets. However, whilst it is clear that the intrinsic apoptosis pathway is not required for thrombopoiesis, the role of the extrinsic pathway—the other major route to apoptotic cell death—has not been established.In the current study we examined the functionality of, and physiological requirement for, the extrinsic apoptosis pathway in megakaryocytes and platelets. The extrinsic pathway is triggered when members of the tumor necrosis factor (TNF) superfamily such as Fas ligand (FasL) bind to cell surface death receptors (e.g. Fas). This induces receptor multimerization, recruitment of death domain adaptor proteins (e.g. FADD) and subsequent activation of Caspase-8, which is the essential mediator of extrinsic pathway-mediated cell death. We found that both megakaryocytes and platelets express critical components of the pathway, including FADD, Caspase-8 and Bid. Megakaryocytes, but not platelets, also expressed the death receptor Fas. Mature fetal liver-derived megakaryocytes treated with soluble FasL exhibited activation of Caspase-8 and the effector Caspases-3/7. This was accompanied by mitochondrial damage and a failure of pro-platelet formation.To establish the requirement for the extrinsic pathway in megakaryocyte development and platelet production, we conditionally deleted Caspase-8 from the megakaryocyte lineage. Platelet counts and platelet life span in Casp8Pf4Δ/Pf4Δ mice were indistinguishable from those of wild-type littermates. Megakaryocyte numbers, morphology, ploidy and in vitro pro-platelet formation capacity were also normal. Caspase-8-deficient megakaryocytes were resistant to FasL treatment. Casp8Pf4Δ/Pf4Δ animals responded to experimentally-induced thrombocytopenia in a manner similar to wild-type. Collectively, these data indicate that the extrinsic apoptosis pathway is dispensable for the generation and survival of platelets. To examine any potential redundancy between the extrinsic and intrinsic apoptosis pathways, we generated Bak−/−BaxPf4Δ/Pf4ΔCasp8Pf4Δ/Pf4Δ triple knockout mice. The ability of these animals to produce platelets, both at steady state and under conditions of thrombopoietic stress, was unperturbed. Megakaryocyte numbers and morphology were normal. Thus, platelet shedding by megakaryocytes does not require the intrinsic or extrinsic apoptosis pathways. Together with recent work demonstrating that the apoptotic initiator caspase, Caspase-9, is also dispensable for platelet production, we conclude that platelet biogenesis is not an apoptotic process. Disclosures:No relevant conflicts of interest to declare.

Preclinical Evaluation of Oncolytic Reovirus in Targeted Therapeutics for High-Risk Pediatric Leukemia

Abstract 3571 Purpose:Leukemia is the most common malignancy in children. Improved treatment strategies in recent decades have yielded substantially enhanced outcomes for children with leukemia, reaching survival rates >80%. However, there remain significant issues with current treatment. Certain subgroups of patients who are resistant to or relapse from current treatments have a dismal prognosis. Furthermore, there are significant late effects of intensive treatments, including secondary cancers, neurocognitive defects, cardiotoxicity, obesity and infertility. For these reasons, novel treatment strategies are urgently needed for high-risk leukemia in children.Reovirus type 3 Dearing is a wild-type double-stranded RNA virus that has shown great promise as a selective oncolytic agent by its ability to replicate in transformed cells but not in normal cells. Although a number of early phase clinical studies have been completed in patients with advanced, refractory solid tumors in adults, systematic evaluation of this agent in the treatment of refractory pediatric leukemia has not been reported. As an initial step towards developing an oncolytics based treatment approach, we report preclinical data with respect to the activity, target validation, target modulation and drug combinability of reovirus in childhood leukemia cells. Experimental Design:A panel of pediatric leukemia cell lines representing high-risk molecular features such as Bcr-Abl, MLL rearranged and mixed lineage was used (n =6). Expression of JAM-A, the cell surface receptor for reovirus, was assessed by flow cytometry. The Ras Activation Assay Kit (EMD Millipore) was used to assess activity of the RAS protein. Western Blots were used to assess the activation (phosphorylation) of the signaling partners downstream of RAS. Cells treated with reovirus, chemotherapy drugs, or both for distinct treatment schedules were assessed for cell viability by the CellTiter-Glo© Luminescent Cell Viability Assay (Promega), and cell death by apoptosis was confirmed by cleavage of PARP. Productive viral infection was assessed by measuring reoviral protein synthesis by Western Blots, and reoviral replication was assessed by virus plaque titration assay. Drug synergies were calculated according to the method of Chou and Talalay. Results:Target validation assays showed the expression of JAM-A, which facilitates effective viral entry into malignant cells, in five of six cell lines. These cell lines also demonstrated differential activation of RAS and downstream kinases, suggesting targeted susceptibility of these cells to reovirus oncolysis. To further test this, we infected cells with reovirus for 1–4 days and assessed cytopathic effects. Using phase contrast microscopy, we observed the virus treated cell lines to demonstrate morphological changes characteristic of cell death following infection. Cell viability assays were used to quantify this effect, and the mechanism of cell death was determined to be apoptotic as evidenced by caspase-dependent cleavage of PARP. Reovirus-induced cell death was correlated with viral protein production and replication. Next, we screened for the ability of reovirus to induce synergistic activity in a panel of conventional and novel targeted therapeutic agents. Our studies showed that, in contrast to the current antileukemic agents, the Bcl-2 inhibitor BH3 mimetic ABT-737 was able to significantly synergize with reovirus in all cell lines tested. Conclusions:In our in vitro studies, oncolytic reovirus as a single agent showed potent oncolytic activity against all pediatric leukemia cell lines tested that express the receptor for reovirus, regardless of the status of the RAS signaling pathway. Further, we found reovirus-induced oncolysis can be enhanced by combination with Bcl-2 inhibition but was unaltered or antagonized by the other drugs indicating a key relationship between the two pathways. As such, our data for the first time, show that pediatric leukemia cells carry the potential to be targeted by reovirus induced oncolysis and the identification of drug synergy and the biomarkers of target modulation provide the basis for further studies to develop this novel therapeutic approach for clinical studies in the near future. Disclosures:No relevant conflicts of interest to declare.

The BH3-mimetic ABT-737 sensitizes human melanoma cells to apoptosis induced by selective BRAF inhibitors but does not reverse acquired resistance

Although the introduction of selective v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) inhibitors has been a major advance in treatment of metastatic melanoma, approximately 50% of patients have limited responses including stabilization of disease or no response at all. This study aims to identify a novel means of overcoming resistance of melanoma to killing by BRAF inhibitors. We examined the influence of the BH3-mimetic ABT-737 on induction of apoptosis by the selective BRAF inhibitor PLX4720 in melanoma cells with or without BRAF V600E mutation. Included were cell lines established from four patients before and during treatment with selective BRAF inhibitors and 3D spheroids derived from these cell lines. Cell lines with no or low sensitivity to PLX4720 underwent synergistic increases and increased rates of apoptosis when combined with ABT-737. This degree of synergism was not seen in cell lines without BRAF V600E mutations. Apoptosis was mediated through the mitochondrial pathway and was due in part to upregulation of Bim as shown by inhibition of apoptosis following small interfering RNA knockdown of Bim. Similar effects were seen in cell lines established from patients prior to treatment but not in lines from patients clinically resistant to the selective BRAF inhibitors and in 3D spheroids derived from these cell lines. These results suggest that combination of selective BRAF inhibitors with ABT-737 or the related orally available compound ABT-263 may increase the degree and rate of responses in previously untreated patients with V600E melanoma but not in those with acquired resistance to these agents.

P01-24 The combination of the IGF-1 receptor inhibitor picropodophylin (PPP) and the BH3 mimetic ABT-737 has synergistic anti-myeloma activity

P01-24 The combination of the IGF-1 receptor inhibitor picropodophylin (PPP) and the BH3 mimetic ABT-737 has synergistic anti-myeloma activity

BCL2 Suppresses PARP1 Function and Nonapoptotic Cell Death

BCL2 suppresses apoptosis by binding the BH3 domain of proapoptotic factors and thereby regulating outer mitochondrial membrane permeabilization. Many tumor types, including B-cell lymphomas and chronic lymphocytic leukemia, are dependent on BCL2 for survival but become resistant to apoptosis after treatment. Here, we identified a direct interaction between the antiapoptotic protein BCL2 and the enzyme PARP1, which suppresses PARP1 enzymatic activity and inhibits PARP1-dependent DNA repair in diffuse large B-cell lymphoma cells. The BH3 mimetic ABT-737 displaced PARP1 from BCL2 in a dose-dependent manner, reestablishing PARP1 activity and DNA repair and promoting nonapoptotic cell death. This form of cell death was unaffected by resistance to single-agent ABT-737 that results from upregulation of antiapoptotic BCL2 family members. On the basis of the ability of BCL2 to suppress PARP1 function, we hypothesized that ectopic BCL2 expression would kill PARP inhibitor-sensitive cells. Strikingly, BCL2 expression reduced the survival of PARP inhibitor-sensitive breast cancer and lung cancer cells by 90% to 100%, and these effects were reversed by ABT-737. Taken together, our findings show that a novel interaction between BCL2 and PARP1 blocks PARP1 enzymatic activity and suppresses PARP1-dependent repair. Targeted disruption of the BCL2-PARP1 interaction therefore may represent a potential therapeutic approach for BCL2-expressing tumors resistant to apoptosis.

Prophylactic treatment with the BH3 mimetic ABT-737 impedes Myc-driven lymphomagenesis in mice

As many oncogenic changes, such as Myc overexpression, promote apoptosis, the survival of emerging neoplastic clones may often initially depend upon endogenous levels of particular pro-survival members of the Bcl-2 protein family. Pertinently, we recently showed that in lymphoma-prone Eμ-myc transgenic mice, which overexpress Myc in all B-lymphoid cells, endogenous Bcl-x(L) is critical for the survival, as well as the expansion of preneoplastic B-lymphoid cells and the development of malignant disease. This discovery raised the possibility that pharmacological blockade of Bcl-x(L) might impede Myc-driven lymphoma development. Indeed, we report here that treatment of preleukaemic Eμ-myc transgenic mice with the Bcl-2 homology (BH)3 mimetic drug ABT-737, which inhibits Bcl-x(L), as well as Bcl-2 and Bcl-w, augmented apoptosis of preneoplastic B-lymphoid cells, reduced their numbers and greatly prolonged lymphoma-free survival. These findings reveal that BH3 mimetic drugs may provide a prophylactic strategy to prevent the development of certain tumours, particularly those driven by deregulated Myc expression. Moreover, such treatment may help in the management of patients with hereditary cancer syndromes and perhaps also in the prevention of tumour relapses.

Bcl-2, Bcl-xL, and Bcl-w are not equivalent targets of ABT-737 and navitoclax (ABT-263) in lymphoid and leukemic cells

AbstractThe BH3-mimetic ABT-737 and an orally bioavailable compound of the same class, navitoclax (ABT-263), have shown promising antitumor efficacy in preclinical and early clinical studies. Although both drugs avidly bind Bcl-2, Bcl-xL, and Bcl-w in vitro, we find that Bcl-2 is the critical target in vivo, suggesting that patients with tumors overexpressing Bcl-2 will probably benefit. In human non-Hodgkin lymphomas, high expression of Bcl-2 but not Bcl-xL predicted sensitivity to ABT-263. Moreover, we show that increasing Bcl-2 sensitized normal and transformed lymphoid cells to ABT-737 by elevating proapoptotic Bim. In striking contrast, increasing Bcl-xL or Bcl-w conferred robust resistance to ABT-737, despite also increasing Bim. Cell-based protein redistribution assays unexpectedly revealed that ABT-737 disrupts Bcl-2/Bim complexes more readily than Bcl-xL/Bim or Bcl-w/Bim complexes. These results have profound implications for how BH3-mimetics induce apoptosis and how the use of these compounds can be optimized for treating lymphoid malignancies.

Restoration of chemosensitivity for doxorubicin and cisplatin in chondrosarcoma in vitro: BCL-2 family members cause chemoresistance

Chondrosarcomas are malignant cartilage-forming tumors notorious for their resistance to conventional chemo- and radiotherapy. Postulated explanations describe the inaccessibility due to abundant hyaline cartilaginous matrix, presence of multidrug resistance (MDR) pumps, and expression of anti-apoptotic BCL-2 family members. We studied the sensitivity of chondrosarcoma cell lines (SW1353, CH2879, JJ012, OUMS27) and two primary cultures for doxorubicin and cisplatin. We examined the role of extracellular matrix using three-dimensional (3D) pellet models and MDR pump activity using fluorescence-activated cell sorter analysis. The role of BCL-2 family members was investigated using the BH3 mimetic ABT-737. Chondrosarcoma cells showed highest resistance to cisplatin. 3D cell pellets, morphologically strongly resembling chondrosarcoma in vivo, confirmed nuclear incorporation of doxorubicin. MDR pump activity was heterogeneous among cultures. Chondrosarcoma cells responded to ABT-737 and combination with doxorubicin led to complete loss of cell viability and apoptosis with cytochrome C release. Despite MDR pump activity and abundance of hyaline cartilaginous matrix, doxorubicin is able to accumulate in the cell nuclei. By repairing the apoptotic machinery, we were able to sensitize chondrosarcoma cells to doxorubicin and cisplatin, indicating an important role for BCL-2 family members in chemoresistance and a promising new treatment strategy for inoperable chondrosarcoma.

γ-Secretase inhibition promotes cell death, Noxa upregulation, and sensitization to BH3 mimetic ABT-737 in human breast cancer cells

IntroductionInappropriate Notch signaling, downstream of γ-secretase activity, is understood to have tumor-promoting function and to be associated with poor outcome in cancer, of the breast in particular. The molecular basis of antitumoral effects of its inhibitors, however, remains poorly characterized. Moreover, the effects of their combination with the pro-apoptotic pharmacologic inhibitor of Bcl-2/Bcl-xL, ABT-737, have never been evaluated. In this study, we thus specifically addressed the biologic consequences of targeting γ-secretase and Bcl-2/Bcl-xL, alone or simultaneously, in breast cancer cell lines as well as in a novel human breast cancer ex vivo assay.MethodsBy using in vitro 2D or 3D cultures of breast cancer cells plus a novel preclinical short-term ex vivo assay that correctly maintains human mammary tissue integrity and preserves tumor microenvironment, we tested the effects of the pharmacologic γ-secretase inhibitor GSIXII used as a single agent or in combination with ABT-737.ResultsWe show herein that the γ-secretase inhibitor, GSIXII, efficiently induces apoptosis in breast cancer cell lines by a process that relies on the induction of Noxa, a pro-apoptotic Bcl2-homology 3 domain (BH3)-only protein of the Bcl-2 family that functions as an inhibitor of antiapoptotic Mcl1. GSIXII also targets mammary cancer stem-like cells because it dramatically prevents in vitro mammosphere formation. Moreover, combining GSIXII treatment with ABT-737, a BH3-mimetic inhibitor of additional antiapoptotic proteins, such as Bcl-2 and Bcl-xL, leads to both a synergistic apoptotic response in breast cancer cells and to an inhibitory effect on mammosphere formation. These effects are also found when a Notch transcriptional inhibitor, SAHM1, is used. Finally, we evaluated individual human tumor responses to γ-secretase inhibition alone or in combination with ABT-737 in ex vivo assays. Analysis of a series of 30 consecutive tumors indicated that a majority of tumors are sensitive to apoptosis induction by GSIXII and that association of GSIXII with ABT-737 leads to an enhanced induction of apoptosis in tumor cells.ConclusionsWe thus provide evidence that γ-secretase, and downstream Notch signaling, are relevant targets in breast cancer. GSIXII, used as single agent or in combination with clinically relevant BH3-mimetics, is a promising innovative proapoptotic strategy to treat mammary tumors.