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PDF file - 39K, Summary of Combination Matrices for Navitoclax and G-963 in Pancreatic Cancer Cell Lines. The data reflect the average of three independent runs performed in quadruplicate

PDF file - 680K, Overall Survival and Bcl-xL H Score.

To examine the potential of combining Bcl-2 family inhibitors with chemotherapy in ovarian cancer, we evaluated a panel of 27 ovarian cancer cell lines for response to the combination of navitoclax (formerly ABT-263) and paclitaxel or gemcitabine. The majority of cell lines exhibited a greater than additive response to either combination, as determined by the Bliss independence model, and more than 50% of the ovarian cell lines exhibited strong synergy for the navitoclax/paclitaxel combination. To identify biomarkers for tumors likely to respond to this combination, we evaluated the protein levels of intrinsic apoptosis pathway components. Bcl-xL seems necessary, but not sufficient, for navitoclax/paclitaxel synergy in vitro, suggesting that exclusion of patients whose tumors have low or undetectable Bcl-xL would enrich for patients responsive to the combination. We evaluated Bcl-xL levels in ovarian cancer tumor tissue from 40 patients (20 taxane responsive and 20 with poor response to taxane) and found that patients with high Bcl-xL were less sensitive to taxane treatment (10 of 12) Bcl-xL positive patients, P = 0.014). These data support the use of navitoclax in combination with taxane-based therapy in ovarian cancer patients with high levels of Bcl-xL. Mol Cancer Ther; 11(4); 1026–35. ©2012 AACR.

BCL2, BCL2L1 and MCL1 mRNA in CD138 primary MM patient samples and sensitivity to veneteclax ex vivo

PDF file - 575K, ROC for Bcl-xL H Score.

Legends for Tables S1 to S3 and Figures S1 to S5

Caspase-dependent degradation of MCL-1 in NCI-H929 HMCL following treatment with bortezomib treatment

BCL-2 and BCL-XL IHC correlates with qPCR and immunobot analysis of HMCLs

BCL-2 family proteins dictate survival of human multiple myeloma cells, making them attractive drug targets. Indeed, multiple myeloma cells are sensitive to antagonists that selectively target prosurvival proteins such as BCL-2/BCL-XL (ABT-737 and ABT-263/navitoclax) or BCL-2 only (ABT-199/GDC-0199/venetoclax). Resistance to these three drugs is mediated by expression of MCL-1. However, given the selectivity profile of venetoclax it is unclear whether coexpression of BCL-XL also affects antitumor responses to venetoclax in multiple myeloma. In multiple myeloma cell lines (n = 21), BCL-2 is expressed but sensitivity to venetoclax correlated with high BCL-2 and low BCL-XL or MCL-1 expression. Multiple myeloma cells that coexpress BCL-2 and BCL-XL were resistant to venetoclax but sensitive to a BCL-XL–selective inhibitor (A-1155463). Multiple myeloma xenograft models that coexpressed BCL-XL or MCL-1 with BCL-2 were also resistant to venetoclax. Resistance to venetoclax was mitigated by cotreatment with bortezomib in xenografts that coexpressed BCL-2 and MCL-1 due to upregulation of NOXA, a proapoptotic factor that neutralizes MCL-1. In contrast, xenografts that expressed BCL-XL, MCL-1, and BCL-2 were more sensitive to the combination of bortezomib with a BCL-XL selective inhibitor (A-1331852) but not with venetoclax cotreatment when compared with monotherapies. IHC of multiple myeloma patient bone marrow biopsies and aspirates (n = 95) revealed high levels of BCL-2 and BCL-XL in 62% and 43% of evaluable samples, respectively, while 34% were characterized as BCL-2High/BCL-XLLow. In addition to MCL-1, our data suggest that BCL-XL may also be a potential resistance factor to venetoclax monotherapy and in combination with bortezomib. Mol Cancer Ther; 15(5); 1132–44. ©2016 AACR.

PDF file - 751K, Bliss Scores of A549 and H647 Across Dose Matrix. Percent inhibition relative to DMSO control was measured using CellTiterGlo. Bliss scores were calculated as described in the materials and methods

Although mitogen-activated protein (MAP)–extracellular signal-regulated kinase (ERK) kinase (MEK) inhibition is predicted to cause cell death by stabilization of the proapoptotic BH3-only protein BIM, the induction of apoptosis is often modest. To determine if addition of a Bcl-2 family inhibitor could increase the efficacy of a MEK inhibitor, we evaluated a panel of 53 non–small cell lung cancer and pancreatic cancer cell lines with the combination of navitoclax (ABT-263), a Bcl-2/Bcl-xL (BCL2/BCL2L1) antagonist, and a novel MAP kinase (MEK) inhibitor, G-963. The combination is synergistic in the majority of lines, with an enrichment of cell lines harboring KRAS mutations in the high synergy group. Cells exposed to G-963 arrest in G1 and a small fraction undergo apoptosis. The addition of navitoclax to G-963 does not alter the kinetics of cell-cycle arrest, but greatly increases the percentage of cells that undergo apoptosis. The G-963/navitoclax combination was more effective than either single agent in the KRAS mutant H2122 xenograft model; BIM stabilization and PARP cleavage were observed in tumors, consistent with the mechanism of action observed in cell culture. Addition of the phosphatidylinositol 3-kinase (PI3K, PIK3CA) inhibitor GDC-0941 to this treatment combination increases cell killing compared with double- or single-agent treatment. Taken together, these data suggest the efficacy of agents that target the MAPK and PI3K pathways can be improved by combination with a Bcl-2 family inhibitor. Mol Cancer Ther; 12(6); 853–64. ©2013 AACR.

PDF file - 69K, H Score and Clinical Features.

PDF file - 44K

PDF file - 115K, Synthetic route for G-963

Correlation of mRNA expression of BCL2 family members and sensitivity to venetoclax or navitoclax in HMCLs

Conceptual workflow for establishing BCL-2 family IHC cut-offs for analysis of MM patient tumor samples

PDF file - 558K, Biochemical Selectivity of G-963. Percent inhibition of various kinases treated with 1uM G-963. Assays were performed at Invitrogen using their SelectScreenR Kinase profiling service

qPCR primer sequences for Apopanel of BCL2 family members

PDF file - 581K, Correlative Biomarker Analysis. A) Western blots illustrating baseline levels of total Bad and phospho-S-112 Bad. Correlative analysis of Bliss sums and baseline levels of B) phospho-S-112 Bad (Spearman ρ = -0.46; P = 0.02)

Purpose: To explore the potential of navitoclax in combination with taxane-based chemotherapy in the treatment of non–small cell lung cancer (NSCLC) by defining mechanism of synergy and identifying correlative biomarkers.Experimental Design: We treated a panel of NSCLC lines with a dose matrix of paclitaxel and navitoclax (formerly ABT-263), an inhibitor of Bcl-2, Bcl-xL, and Bcl-w (1), and evaluated synergy. We next used time-lapse microscopy to explore mechanism of synergy. Finally, we developed an immunohistochemical assay and assessed prevalence of Bcl-xL in NSCLC tumor tissues.Results: All cell lines exhibit greater than additive response to the combination of navitoclax and a taxane. These results were extended to mouse xenograft tumor models, in which the combination is more efficacious than either single-agent docetaxel or navitoclax. Addition of navitoclax to paclitaxel decreases the time from mitotic entry to cell death and changes cell fate from mitotic slippage to death during mitotic arrest. The relative levels of Bcl-xL and Mcl-1 correlate with the extent of synergy, suggesting that cancers with elevated levels of Bcl-xL will be relatively resistant to taxane-based therapy but could benefit from the addition of navitoclax to taxane treatment. Finally, a significant percentage of NSCLC patient samples exhibit relatively high Bcl-xL levels.Conclusions: The addition of navitoclax to taxane-based chemotherapy in NSCLC has the potential to increase efficacy, particularly in patients whose tumors express high levels of Bcl-xL. Clin Cancer Res; 17(6); 1394–404. ©2011 AACR.

Supplementary Figures S1-S6; Supplementary Table S1.

Supplementary Figure 1 from Microphthalmia-Associated Transcription Factor Is a Critical Transcriptional Regulator of Melanoma Inhibitor of Apoptosis in Melanomas

Supplementary Figure 2 from Microphthalmia-Associated Transcription Factor Is a Critical Transcriptional Regulator of Melanoma Inhibitor of Apoptosis in Melanomas

Supplementary Figure 3 from Microphthalmia-Associated Transcription Factor Is a Critical Transcriptional Regulator of Melanoma Inhibitor of Apoptosis in Melanomas

Melanoma inhibitor of apoptosis (ML-IAP) is a potent inhibitor of apoptosis, which is highly expressed in melanomas and likely contributes to their resistance to chemotherapeutic treatments. Herein, we show that the lineage survival oncogene microphthalmia-associated transcription factor (MITF) is a critical regulator of ML-IAP transcription in melanoma cells. The ML-IAP promoter contains two MITF consensus sites, and analysis of MITF and ML-IAP mRNA levels revealed a high correlation in melanoma tumor samples and cell lines. In reporter assays, MITF promoted a strong stimulation of transcriptional activity from the ML-IAP promoter, and MITF bound the endogenous ML-IAP promoter in melanoma cells by chromatin immunoprecipitation and electrophoretic mobility shift assay. Strikingly, small interfering RNA (siRNA)–mediated knockdown of MITF in melanoma cells led to a dramatic decrease in ML-IAP mRNA and protein levels, establishing that ML-IAP expression in melanoma cells is MITF dependent. Additionally, cyclic AMP–mediated induction of MITF expression in melanocytes resulted in increased ML-IAP expression, suggesting that melanocytes can express ML-IAP when MITF levels are heightened. Disruption of MITF by siRNA led to a decrease in melanoma cell viability, which could be rescued by ectopic expression of ML-IAP. Collectively, these findings implicate MITF as a major transcriptional regulator of ML-IAP expression in melanomas, and suggest that ML-IAP contributes to the prosurvival activity of MITF in melanoma progression. [Cancer Res 2008;68(9):3124–32]

Purpose: To explore the potential of navitoclax in combination with taxane-based chemotherapy in the treatment of non–small cell lung cancer (NSCLC) by defining mechanism of synergy and identifying correlative biomarkers. Experimental Design: We treated a panel of NSCLC lines with a dose matrix of paclitaxel and navitoclax (formerly ABT-263), an inhibitor of Bcl-2, Bcl-xL, and Bcl-w (1), and evaluated synergy. We next used time-lapse microscopy to explore mechanism of synergy. Finally, we developed an immunohistochemical assay and assessed prevalence of Bcl-xL in NSCLC tumor tissues. Results: All cell lines exhibit greater than additive response to the combination of navitoclax and a taxane. These results were extended to mouse xenograft tumor models, in which the combination is more efficacious than either single-agent docetaxel or navitoclax. Addition of navitoclax to paclitaxel decreases the time from mitotic entry to cell death and changes cell fate from mitotic slippage to death during mitotic arrest. The relative levels of Bcl-xL and Mcl-1 correlate with the extent of synergy, suggesting that cancers with elevated levels of Bcl-xL will be relatively resistant to taxane-based therapy but could benefit from the addition of navitoclax to taxane treatment. Finally, a significant percentage of NSCLC patient samples exhibit relatively high Bcl-xL levels. Conclusions: The addition of navitoclax to taxane-based chemotherapy in NSCLC has the potential to increase efficacy, particularly in patients whose tumors express high levels of Bcl-xL. Clin Cancer Res; 17(6); 1394–404. ©2011 AACR.

Apoptosis is a cell suicide process with a major role in development and homeostasis in vertebrates and invertebrates. Inhibition of apoptosis enhances the survival of cancer cells and facilitates their escape from immune surveillance and cytotoxic therapies. Among the principal molecules contributing to this phenomenon are the inhibitor of apoptosis (IAP) proteins, a family of antiapoptotic regulators that block cell death in response to diverse stimuli through interactions with inducers and effectors of apoptosis. IAP proteins are expressed in the majority of human malignancies at elevated levels and play an active role in promoting tumor maintenance through the inhibition of cellular death and participation in signaling pathways associated with malignancies. Here, we discuss the role of IAP proteins in cancer and options for targeting IAP proteins for therapeutic intervention.

Inhibitor of apoptosis (IAP) proteins are critical regulators of cell death, preventing apoptosis in response to a variety of signals. Overexpression of IAP proteins has been demonstrated to confer protection against a variety of pro apoptotic stimuli, including chemotherapies, and is a marker for poor prognosis in a variety of solid tumors and hematologic malignancies. Antagonism of the IAP protein mediated inhibition of caspases is required for caspase dependent cell death, and can be achieved by the mitochondrial protein SMAC/DIABLO, which is released into the cytoplasm in response to pro apoptotic stimuli. Structure-based design was used to identify small molecule SMAC mimetics that potently antagonize IAP protein interactions. These SMAC mimetics induce apoptosis in select cancer cell lines and have proven efficacious at inhibiting tumor growth in mouse xenograft models of cancer. Remarkably, binding of small-molecule SMAC mimetics to cIAP1 or 2 results in induction of auto-ubiquitination activity and rapid proteasomal degradation of these proteins. The cIAP proteins (cIAP1 and 2) comprise several domains, including three baculovirus IAP repeat (BIR1-3) domains, a ubiquitin-binding (UBA) domain, a caspase-associated recruitment domain (CARD), and a RING E3 ligase domain. Binding of SMAC mimetics to the cIAP1-BIR3 domain results in homodimerization of cIAP1 through its RING domain and activation of its ubiquitin E3 ligase activity. To understand how this long-range communication takes place, we solved the crystal structure of a truncated form of cIAP1, comprising the BIR3 through RING domains (cIAP1-B3R) and found that cIAP1-B3R adopts a compact tertiary structure in which the RING domain is sequestered on three sides by the BIR3 domain, the UBA domain, and the CARD. SMAC mimetic binding triggers E3 ligase activity in cIAP1 by disrupting the BIR3:RING interface, thereby allowing RING dimerization. However, this view of cIAP1 as a ‘binary switch' does not fully explain the mechanism of activation, since the compact structure of the inactive state precludes binding of peptides and SMAC mimetics. We have subsequently used NMR spectroscopy to study micro- and millisecond motions of specific domain interfaces in cIAP1 and have use time-resolved small-angle X-ray scattering to observe the global conformational changes necessary for activation. Although motions within each interface of the ‘closed' monomer are insufficient to activate cIAP1, they enable associations with catalytic partners and activation factors. Together these results suggest that cIAP1 internal motions are tuned to minimize the time required for the rate-limiting conformational rearrangements, priming the protein for rapid activation while maintaining the integrity of the closed monomer. The internal dynamics of cIAP1 present an intriguing picture of a “spring-loaded” cell death trigger, with activation speed balanced precisely against the critical need for message integrity. Citation Format: Wayne J. Fairbrother. Mechanistic studies of IAP inhibitors. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr IA03.

Obinutuzumab (GA101) is a novel glycoengineered type II, anti-CD20 monoclonal antibody induces a high level of direct cell death. As a result of glycoengineering, GA101 has increased affinity for FcγRIIIa on effector cells resulting in enhanced direct cell death and ADCC induction. GA101 is currently in pivotal clinical trials in chronic lymphocytic leukemia (CLL), indolent non-Hodgkins lymphoma (iNHL) and diffuse large B-cell lymphoma (DLBCL). ABT-199 (GDC-0199) is a novel, orally bioavailable, selective Bcl-2 inhibitor that induces robust apoptosis in preclinical models of hematological malignancies and is currently in clinical trials for CLL, NHL and multiple myeloma. Based on their complementary mechanisms of action involving increased apoptosis (GDC-0199) or direct cell death (GA101) the combination of anti-CD20 therapy with a Bcl 2 inhibitor has the potential for greater efficacy in treating B lymphoid malignancies. The combination of GA101 or rituximab with GDC-0199 was studied in vitro utilizing assays that measure direct cell death induction/apoptosis (AxV/Pi positivity) on WSU-DLCL2 NHL, SU-DHL4 DLBCL and Z138 mantle cell lymphoma cells by FACS and the impact of Bcl-2 inhibition on ADCC induction. In vivo efficacy of the combination of GA101 or rituximab and GDC-0199 was evaluated in SU-DHL4 and Z138 xenograft models. GA101 and rituximab enhanced cell death induction when combined with GDC-0199 in SU-DHL4, WSU-DLCL2 and Z138 cell lines. When combined at optimal doses of GA101 and GDC-0199 an additive effect of the two drugs was observed. GDC-0199 did not negatively impact the capability of GA101 or rituximab to induce NK-cell mediated ADCC. Combination of GDC 0199 with GA101 or rituximab in vivo induced greater than additive anti-tumor effects in the SU DHL4 and Z138 xenograft models resulting in tumor regressions and delay in tumor regrowth when compared to monotherapy. Importantly, continued single-agent treatment with GDC-0199 after combination with GA101 resulted in sustained in vivo efficacy in the SU-DHL4 xenograft model. Our data demonstrate that the combination of GA101 with GDC-0199 results in enhanced cell death and robust anti-tumor efficacy in xenograft models representing NHL sub-types that is comparable to combination therapy with rituximab. In addition, single-agent treatment with GDC-0199 following combination with GA101 sustains efficacy in vivo suggesting a potential benefit in continued maintenance therapy with GDC-0199. Collectively, our preclinical data strongly supports clinical investigation of GA101 and GDC-0199 combination therapy, which is currently being evaluated in a phase Ib clinical trial (clinical trial.gov identifier [NCT01685892][1]). Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A245. Citation Format: Deepak Sampath, Sylvia Herter, Ellen Ingalla, Frank Herting, Marina Bacac, Michelle Nannini, Wayne J. Fairbrother, Christian Klein. Combination of the glycoengineered Type II CD20 antibody obinutuzumab (GA101), and the novel Bcl-2 selective inhibitor, ABT-199 (GDC-0199), results in superior in vitro and in vivo anti-tumor activity in models of B-cell malignancies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A245. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01685892&atom=%2Fmolcanther%2F12%2F11_Supplement%2FA245.atom

A hallmark feature of cancer is the ability to evade cell death signals induced by stress response cues mediated, in part, by the the Bcl-2 family of pro-apoptotic and pro-survival proteins. ABT-199 is a potent BH3-only mimetic that selectively antagonizes the pro-survival function of Bcl-2. The work presented describes the efficacy of ABT-199 as a single agent and in combination with bortezomib in preclinical models of Multiple Myeloma (MM). Expression of Bcl-2 protein was detected in 95% of MM cell lines (n=21) evaluated and ABT-199 reduced cell viability in a sub-set of these lines (7/21) with EC50 values less than 0.5 μM. Expression of Bcl-xL and Mcl-1 at the mRNA and protein levels were also evaluated to determine predictors of drug sensitivity and resistance to ABT-199. MM lines that expressed higher levels of Bcl-2 relative to Bcl-xL and Mcl-1 were the most sensitive to ABT-199 treatment while those that were less sensitive expressed higher levels of Mcl-1 or Bcl-xL. Other predictors of sensitivity to ABT-199 included higher levels of Bcl-2/Bim complexes and t(11;14) status. Ex vivo, 75% of the MM bone marrow biopsies and aspirates (n=27) had high Bcl-2 levels whereas 50% had high Bcl-xL expression, demonstrating that a subset of patient samples (33%) have a favorable biomarker profile (Bcl-2-high/Bcl-xL-low) that may be predictive of ABT-199 activity. Efficacy of ABT-199 as a single agent and in combination with bortezomib was evaluated in vivo in MM xenograft models that expressed Bcl-2 (OPM-2, KMS-11, RPMI-8226, H929 and MM.1s). Bortezomib treatment alone at maximum tolerated doses resulted in tumor regressions or stasis in all xenograft models tested. ABT-199 at maximum tolerated doses was efficacious as a single agent in xenograft models that expressed higher protein levels of Bcl-2 but relatively lower levels of Bcl-xL. The combination of ABT-199 with bortezomib increased overall response rates, durability of anti-tumor activity and levels of apoptotic markers such as cleaved caspase 3 and PARP when compared to bortezomib alone even in MM xenograft models that also express relatively high levels of Mcl-1. Treatment with bortezomib in vivo increased levels of Noxa; a pro-apoptotic BH3-only protein that selectively antagonizes Mcl-1. Therefore, greater efficacy may be achieved when ABT-199 is combined with bortezomib due to inhibition of Bcl-2 by ABT-199 and neutralization of Mcl-1 by Noxa in tumors that express both pro-survival proteins. Our preclinical data supports the evaluation of ABT-199 as a single agent and in combination with bortezomib in MM patients in which relative expression of the Bcl-2 pro-survival proteins may serve as predictive biomarkers of drug activity. Citation Format: Deepak Sampath, Elizabeth Punnoose, Erwin Boghaert, Lisa Belmont, Franklin Peale, Nguyen Tan, Jun Chen, Walter Darbonne, Peng Yue, Jason Oeh, Leslie Lee, Wayne J. Fairbrother, Andrew J. Souers, Steven W. Elmore, Joel D. Leverson. Expression of Bcl-2 pro-survival family proteins predicts pharmacological responses to ABT-199, a novel and selective Bcl-2 antagonist, in multiple myeloma models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4607. doi:10.1158/1538-7445.AM2013-4607

The Inhibitor of Apoptosis (IAP) protein family members are widely expressed in many cancers, providing a mechanism to protect these cancer cells from apoptosis. The IAP proteins block activation of the canonical and non-canonical NF-kB pathway, the subsequent expression of TNFα and its initiation of the extrinsic apoptosis pathway. IAP proteins also directly bind and inhibit caspases activated during the induction of apoptosis. Using MDA-MB-231 cells as a model system to assess the effect of candidate IAP antagonists on markers of extrinsic and intrinsic pathway inhibition, we observe that the antagonism of the major IAP family members results in apoptosis in vitro and inhibits their tumor growth in nude mice in vivo. These effects are coincident with changes in the cellular levels of IAP family members such as c-IAP1, detected by Western analysis. These data are consistent with previously described autoubiquitination and subsequent proteasomal degradation of c-IAP1 following IAP antagonism. We have developed a high-throughput, quantifiable and sensitive assay to measure c-IAP1 levels in multiple cell lines, xenografts and in PBMCs using the MesoScale Discovery (MSD) Platform. The c-IAP1 MSD assay has a lower limit of quantification of 1.6 ng/ml of cell lysate, has precision of Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4409. doi:10.1158/1538-7445.AM2011-4409

Navitoclax (formerly ABT-263) is a small molecule that potently antagonizes the activity of Bcl-2, Bcl-xL, and Bcl-w. To determine the potential of this molecule in combination with taxane-based chemotherapy, we evaluated a panel of 33 human NSCLC cell lines with a dose matrix of paclitaxel and navitoclax. All cell lines exhibited a greater than additive response to the combination, suggesting a synergistic interaction. These results were extended to mouse xenograft tumor models, in which the combination was more efficacious than either single agent docetaxel or navitoclax. Live cell time-lapse microscopy was used to demonstrate that the addition of navitoclax to paclitaxel decreases the time from mitotic entry to cell death and changes cell fate from mitotic slippage to death during mitotic arrest. The relative levels of Bcl-xL and Mcl-1 correlate with the extent of synergy as measured by a Bliss independence model, suggesting that cancers with high levels of Bcl-xL will be relatively resistant to taxane-based therapy, but could benefit from addition of navitoclax to taxane treatment. Finally, a significant percentage of NSCLC patient samples exhibit high Bcl-xL levels, indicating the potential for broad utility of this combination in lung cancer.