Bcl-2 Family Inhibitor ABT-737 Research Articles

Identification of senescent, TREM2-expressing microglia in aging and Alzheimer's disease model mouse brain.

Alzheimer's disease (AD) and dementia in general are age-related diseases with multiple contributing factors, including brain inflammation. Microglia, and specifically those expressing the AD risk gene TREM2, are considered important players in AD, but their exact contribution to pathology remains unclear. In this study, using high-throughput mass cytometry in the 5×FAD mouse model of amyloidosis, we identified senescent microglia that express high levels of TREM2 but also exhibit a distinct signature from TREM2-dependent disease-associated microglia (DAM). This senescent microglial protein signature was found in various mouse models that show cognitive decline, including aging, amyloidosis and tauopathy. TREM2-null mice had fewer microglia with a senescent signature. Treating 5×FAD mice with the senolytic BCL2 family inhibitor ABT-737 reduced senescent microglia, but not the DAM population, and this was accompanied by improved cognition and reduced brain inflammation. Our results suggest a dual and opposite involvement of TREM2 in microglial states, which must be considered when contemplating TREM2 as a therapeutic target in AD.

Depletion of preexisting B-cell lymphoma 2-expressing senescent cells before vaccination impacts antigen-specific antitumor immune responses in old mice.

The age-related decline in immunity reduces the effectiveness of vaccines in older adults. Immunosenescence is associated with chronic, low-grade inflammation, and the accumulation of senescent cells. The latter express Bcl-2 family members (providing resistance to cell death) and exhibit a pro-inflammatory, senescence-associated secretory phenotype (SASP). Preexisting senescent cells cause many aging-related disorders and therapeutic means of eliminating these cells have recently gained attention. The potential consequences of senescent cell removal on vaccine efficacy in older individuals are still ignored. We used the Bcl-2 family inhibitor ABT-263 to investigate the effects of pre-vaccination senolysis on immune responses in old mice. Two different ovalbumin (OVA)-containing vaccines (containing a saponin-based or a CpG oligodeoxynucleotide adjuvant) were tested. ABT-263 depleted senescent cells (apoptosis) and ablated the basal and lipopolysaccharide-induced production of SASP-related factors in old mice. Depletion of senescent cells prior to vaccination (prime/boost) had little effect on OVA-specific antibody and T-cell responses (slightly reduced and augmented, respectively). We then used a preclinical melanoma model to test the antitumor potential of senolysis before vaccination (prime with the vaccine and OVA boost by tumor cells). Surprisingly, ABT-263 treatment abrogated the vaccine's ability to protect against B16 melanoma growth in old animals, an effect associated with reduced antigen-specific T-cell responses. Some, but not all, of the effects were age-specific, which suggests that preexisting senescent cells were partly involved. Hence, depletion of senescent cells modifies immune responses to vaccines in some settings and caution should be taken when incorporating senolytics into vaccine-based cancer therapies.

Anti-apoptotic protein BCL-XL as a therapeutic vulnerability in gastric cancer.

New therapeutic targets are needed to improve the outcomes for gastric cancer (GC) patients with advanced disease. Evasion of programmed cell death (apoptosis) is a hallmark of cancer cells and direct induction of apoptosis by targeting the pro-survival BCL2 family proteins represents a promising therapeutic strategy for cancer treatment. Therefore, understanding the molecular mechanisms underpinning cancer cell survival could provide a molecular basis for potential therapeutic interventions. Here we explored the role of BCL2L1 and the encoded anti-apoptotic BCL-XL in GC. Using Droplet Digital PCR (ddPCR) technology to investigate the DNA amplification of BCL2L1 in GC samples and GC cell lines, the sensitivity of GC cell lines to selective BCL-XL inhibitors A1155463 and A1331852, pan-inhibitor ABT-263, and VHL-based PROTAC-BCL-XL was analyzed using (CellTiter-Glo) CTG assay in vitro. Western Blot (WB) was used to detect the protein expression of BCL2 family members in GC cell lines and the manner in which PROTAC-BCL-XL kills GC cells. Co-immunoprecipitation (Co-IP) was used to investigate the mechanism of A1331852 and ABT-263 kills GC cell lines. DDPCR, WB, and real-time PCR (RTPCR) were used to investigate the correlation between DNA, RNA, protein levels, and drug activity. The functional assay showed that a subset of GC cell lines relies on BCL-XL for survival. In gastric cancer cell lines, BCL-XL inhibitors A1155463 and A1331852 are more sensitive than the pan BCL2 family inhibitor ABT-263, indicating that ABT-263 is not an optimal inhibitor of BCL-XL. VHL-based PROTAC-BCL-XL DT2216 appears to be active in GC cells. DT2216 induces apoptosis of gastric cancer cells in a time- and dose-dependent manner through the proteasome pathway. Statistical analysis showed that the BCL-XL protein level predicts the response of GC cells to BCL-XL targeting therapy and BCL2L1 gene CNVs do not reliably predict BCL-XL expression. We identified BCL-XL as a promising therapeutic target in a subset of GC cases with high levels of BCL-XL protein expression. Functionally, we demonstrated that both selective BCL-XL inhibitors and VHL-based PROTAC BCL-XL can potently kill GC cells that are reliant on BCL-XL for survival. However, we found that BCL2L1 copy number variations (CNVs) cannot reliably predict BCL-XL expression, but the BCL-XL protein level serves as a useful biomarker for predicting the sensitivity of GC cells to BCL-XL-targeting compounds. Taken together, our study pinpointed BCL-XL as potential druggable target for specific subsets of GC.

Senolytic elimination of Cox2-expressing senescent cells inhibits the growth of premalignant pancreatic lesions

ObjectiveCellular senescence limits tumourigenesis by blocking the proliferation of premalignant cells. Additionally, however, senescent cells can exert paracrine effects influencing tumour growth. Senescent cells are present in premalignant pancreatic intraepithelial...

Senolytic compounds control a distinct fate of androgen receptor agonist- and antagonist-induced cellular senescent LNCaP prostate cancer cells

BackgroundThe benefit of inducing cellular senescence as a tumor suppressive strategy remains questionable due to the senescence-associated secretory phenotype. Hence, studies and development of senolytic compounds that induce cell death in senescent cells have recently emerged. Senescent cells are hypothesized to exhibit different upregulated pro-survival/anti-apoptotic networks depending on the senescent inducers. This might limit the effect of a particular senolytic compound that targets rather only a specific pathway. Interestingly, cellular senescence in prostate cancer (PCa) cells can be induced by either androgen receptor (AR) agonists at supraphysiological androgen level (SAL) used in bipolar androgen therapy or by AR antagonists. This challenges to define ligand-specific senolytic compounds.ResultsHere, we first induced cellular senescence by treating androgen-sensitive PCa LNCaP cells with either SAL or the AR antagonist Enzalutamide (ENZ). Subsequently, cells were incubated with the HSP90 inhibitor Ganetespib (GT), the Bcl-2 family inhibitor ABT263, or the Akt inhibitor MK2206 to analyze senolysis. GT and ABT263 are known senolytic compounds. We observed that GT exhibits senolytic activity specifically in SAL-pretreated PCa cells. Mechanistically, GT treatment results in reduction of AR, Akt, and phospho-S6 (p-S6) protein levels. Surprisingly, ABT263 lacks senolytic effect in both AR agonist- and antagonist-pretreated cells. ABT263 treatment does not affect AR, Akt, or S6 protein levels. Treatment with MK2206 does not reduce AR protein level and, as expected, potently inhibits Akt phosphorylation. However, ENZ-induced cellular senescent cells undergo apoptosis by MK2206, whereas SAL-treated cells are resistant. In line with this, we reveal that the pro-survival p-S6 level is higher in SAL-induced cellular senescent PCa cells compared to ENZ-treated cells. These data indicate a difference in the agonist- or antagonist-induced cellular senescence and suggest a novel role of MK2206 as a senolytic agent preferentially for AR antagonist-treated cells.ConclusionTaken together, our data suggest that both AR agonist and antagonist induce cellular senescence but differentially upregulate a pro-survival signaling which preferentially sensitize androgen-sensitive PCa LNCaP cells to a specific senolytic compound.

Blunting senescence boosts liver regeneration.

The mammalian liver possesses a unique capacity for regeneration. However, this regenerative potential declines with age due to unknown mechanisms. In this issue of Genes & Development, Ritschka and colleagues (pp. 489-494). compare liver regeneration upon partial hepatectomy in young and adult mice. Partial hepatectomy causes a transient increase in p21 in a subpopulation of hepatocytes that persists in adult mice. Remarkably, treatment with the BCL-2 family inhibitor ABT-737 blunts p21 expression, enhancing liver regeneration.

Abstract 3194: Development of an in vitro drug profiling platform with primary Rhabdomyosarcoma cells for tailoring patient-specific treatments

Abstract The paradigm of cancer therapy currently shifts from broadly used cytotoxic drug cocktails to patient specific precision therapies. These novel therapies are normally directed towards tumor specific driver oncogenes, identified by genomic analysis of individual tumors. In case of tumors with a very low mutational burden and/or currently undruggable driver oncogenes this approach is less suitable. There, direct drug profiling of in vitro cultured tumor cells represents a promising alternative for identification of patient specific therapies. Conditions to maintain primary tumor cells in culture for this purpose however are largely unknown for most tumor entities. Here, we aimed to establish an in vitro drug profiling platform for Rhabdomyosarcoma (RMS), a childhood cancer with above described characteristics. Towards this aim, we isolated cells from 6 alveolar and 8 embryonal RMS patient derived xenografts and systematically tested 18 different culture conditions in order to find suitable parameters for in vitro propagation of primary RMS cells. This approach revealed that cells from most tumors survive and proliferate only in serum-free medium, whereas in serum containing medium tumor cells are progressively lost over time. Importantly, cells preserve the clonal composition and phenotypic characteristics of the primary PDX under these conditions, as assessed by genomic and copy number analysis. Drug profiles established with a library of 204 drugs revealed, beside patient-specific vulnerabilities, a yet unrecognized sensitivity of a subgroup of alveolar RMS towards AKT inhibitors. Furthermore, screening with the same drugs for resensitization of resistant relapse samples identified the BCL-2 family inhibitor ABT-263 as most potent resensitizer towards standard-of-care chemotherapy. Detailed molecular analysis revealed that this effect is based on blockade of the BCL-XL-MCL-1 axis. Overall, our proof of concept study highlights the feasibility of in vitro drug profiling of primary RMS cells for patient-specific treatment selection in a co-clinical setting. Citation Format: Marco Wachtel, Gabriele Manzella, Michaela Römmele, Luduo Zhang, Joelle Tchinda, Felix Niggli, Beat Schäfer. Development of an in vitro drug profiling platform with primary Rhabdomyosarcoma cells for tailoring patient-specific treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3194.

High-Throughput Functional Genetic and Compound Screens Identify Targets for Senescence Induction in Cancer

Senescence is a proliferation arrest that can result from a variety of stresses. Cancer cells can also undergo senescence, but the stresses that provoke cancer cells to undergo senescence are unclear. Here, we use both functional genetic and compound screens in cancer cells harboring a reporter that is activated during senescence to find targets that induce senescence. We show that suppression of the SWI/SNF component SMARCB1 induces senescence in melanoma through strong activation of the MAP kinase pathway. From the compound screen, we identified multiple aurora kinase inhibitors as potent inducers of senescence in RAS mutant lung cancer. Senescent melanoma and lung cancer cells acquire sensitivity to the BCL2 family inhibitor ABT263. We propose a one-two punch approach for the treatment of cancer in which a drug is first used to induce senescence in cancer cells and a second drug is then used to kill senescent cancer cells.

Intrinsic Resistance to 5-Fluorouracil in a Brain Metastatic Variant of Human Breast Cancer Cell Line, MDA-MB-231BR.

Although drug resistance is often observed in metastatic recurrence of breast cancer, little is known about the intrinsic drug resistance in such metastases. In the present study, we found, for the first time, that MDA-MB-231BR, a brain metastatic variant of a human breast cancer cell line, was refractory to treatment with 5-fluorouracil (5-FU) even without chronic drug exposure, compared to its parent cell line, MDA-MB-231, and a bone metastatic variant, MDA-MB-231SCP2. Both the mRNA and protein levels of COX-2 and BCL2A1 in MDA-MB-231BR were significantly higher than those in MDA-MB-231 or MDA-MB-231SCP2. Neither the COX-2 inhibitor celecoxib nor the NF-κB inhibitor BAY11-7082 could sensitize MDA-MB-231BR to 5-FU, indicating that COX-2 plays little, if any, role in the resistance of MDA-MB-231BR to 5-FU. Although BCL2-family inhibitor ABT-263 failed to sensitize MDA-MB-231BR to 5-FU at a dose at which ABT-263 is considered to bind to BCL2, BCL2-xL, and BCL2-w, but not to BCL2A1, ABT-263 did sensitize MDA-MB-231BR to 5-FU to a level comparable to that in MDA-MB-231 at a dose of 5 μM, at which ABT-263 may disrupt intracellular BCL2A1 protein interactions. More importantly, BCL2A1 siRNA sensitized MDA-MB-231BR to 5-FU, whereas the overexpression of BCL2A1 conferred 5-FU-resistance on MDA-MB-231. These results indicate that BCL2A1 is a key contributor to the intrinsic 5-FU-resistance in MDA-MB-231BR. It is interesting to note that the drug sensitivity of MDA-MB-231BR was distinct from that of MDA-MB-231SCP2 even though they have the same origin (MDA-MB-231). Further investigations pertinent to the present findings may provide valuable insight into the breast cancer brain metastasis.

Establishment of a Primary Screening Assay for the DHX9 Helicase.

The RNA helicase DHX9 is an ATP-dependent DExH box helicase that can unwind DNA and RNA. Much evidence has implicated DHX9 at multiple levels of gene expression regulation ranging from genome stability and replication, to transcriptional control and translation regulation. Its association with the EWS-FLI1 fusion product, as well as the finding that its suppression can be synthetic lethal with the BCL-2 family inhibitor ABT-737 indicates a potential role in tumor maintenance. Hence, to identify small molecules that could interfere with its activity, we developed a homogenous RNA-dependent ATPase assay. We show that aurintricarboxylic acid, a promiscuous protein-nucleic acid inhibitor prevents DHX9-mediated hydrolysis demonstrating that the assay is also capable of detecting compounds that impinge on DHX9:RNA association.

Abstract B37: Targeting Myc-reprogrammed glutamine metabolism for treatment of neuroblastoma

Abstract Oncogenic Myc is known to alter cellular metabolism resulting in glutamine addiction, and the MYCN-amplified subclass of neuroblastoma is associated with extremely poor prognosis. Successful identification of therapeutic agents that are effective in neuroblastoma, particularly in tumors with MYCN amplification, could significantly increase patient survival. To explore the effect of inhibiting glutamine metabolism in neuroblastoma, we used the glutamine antagonist DON (6-diazo-5-oxo-norleucine). DON is an irreversible competitive inhibitor which alkylates enzymes that utilize glutamine. Although first studied in the 1950s as a possible cancer chemotherapeutic, DON was associated with severe nausea which limited its use in patients. DON remains a potent inhibitor, and a useful tool to study the effect of modulating glutamine metabolism in vivo. We tested the DON sensitivity of six human neuroblastoma cell lines (Kelly, IMR-32, SK-N-BE2, SK-N-AS, SK-N-FI, and SH-SY5Y) compared to immortalized BJ fibroblast control cells. DON effectively inhibited all neuroblastoma cell lines tested with minimal effect on BJ cells. Furthermore, MycN overexpression in SK-N-AS cells, a cell line which is not MYCN-amplified and expresses low levels of cMyc, was sufficient to make cells significantly more sensitive to DON. Next, we tested the effect of DON treatment on subcutaneous xenografts. For these experiments we used the cell lines SK-N-AS (not MYCN-amplified), SK-N-BE2 (MYCN-amplified with moderate MycN levels), and IMR-32 (MYCN-amplified with high MycN levels). Mice were treated twice per week by intraperitoneal injection with DON (50 or 100mg/kg) or water control. Treatment with 50mg/kg DON significantly inhibited tumor growth of all three cell lines, suggesting neuroblastoma tumors may be broadly sensitive to glutamine inhibitors. IMR-32 tumors were the most sensitive to DON treatment, leading to a 6-fold reduction in tumor size after 2.5 weeks of treatment. Tumor BrdU incorporation decreased following DON treatment, and IMR-32 tumors showed increased levels of cleaved caspase 3, a marker of apoptosis. At the higher 100mg/kg dose, DON treatment led to a more substantial decrease in BrdU incorporation, and at this dose BE2 tumors also showed significantly increased levels of cleaved caspase 3, which were not detected in AS tumors. These results suggest that MYCN-amplified tumors may be particularly sensitive to glutamine inhibition. To examine the mechanism of DON-induced cell death, we treated cells with the pan-caspase inhibitor QVD. QVD treatment for 48 or 72hr significantly rescued both MYCN-amplified cell lines (Kelly, IMR-32) and cells that express high levels of cMyc (SY5Y), while having minimal effect in the remaining neuroblastoma cell lines with low Myc levels. These results confirm that DON induces cell death partially by activating apoptosis in high Myc expressing cell lines. Next, we used shRNA to show that in Kelly cells DON-induced apoptosis occurs by signaling through Bax, but not Bak. Lastly, we tested DON in combination with pro-apoptotic compounds in vitro. We found that DON treatment combined with the Bcl-2 family inhibitor ABT-263 had synergistic effects on cell death in the MYCN-amplified cell lines Kelly and BE2. Our results suggest that combined targeting of glutamine metabolism while reducing the threshold for tumor cell apoptosis may be beneficial for neuroblastoma treatment. Citation Format: Rachelle R. Olsen, Michelle N. Mary-Sinclair, Zhirong Yin, Kevin W. Freeman. Targeting Myc-reprogrammed glutamine metabolism for treatment of neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B37.

413 Next-generation sequencing identifies the mechanism of tumourigenesis caused by loss of SMARCB1 in malignant rhabdoid tumours

significant antitumor activity in preclinical models of childhood cancers that may be prioritized for clinical testing. The PPTP has characterized a panel of 23 cell lines and 57 xenograft models in the primary testing panel representing most of the more common pediatric solid malignancies and acute lymphoblastic leukemia subtypes (ALL). Additional cell line-derived and PDX models are available for expanded testing should an agent show activity against specific cancer types in the primary screen. Molecular characterization includes expression profiles, SNP analysis, and exome sequencing for all models. Agents tested include small molecule drugs, antibodies, antibody-conjugates, and replication competent viruses. The preclinical models accurately identified known clinically active drugs (vincristine, cyclophosphamide, cisplatin, topotecan), with an overall response rate of 40% (70/175 tests). In contrast, the overall response rate for 21 signaling inhibitors was ~2%, despite clear pharmacodynamic evidence showing target inhibition in several cases. For example, the MEK inhibitor selumetinib (AZD6244) was active against only a BRAF(V600E) mutated glioma xenograft among 45 models tested. Inhibitors of the IGF-1 receptor-TOR pathway induced regressions in 9 of 321 models tested (2.8%), with activity largely limited to sarcoma. Agents showing high activity at exposures in mice not achieved in patients at comparable schedules included the Aurora A kinase inhibitor alisertib, the Polo-like kinase inhibitor BI-6727, and the pre-pro-drug PR-104. At relevant patient exposures PR104 demonstrated activity only in T cell ALL models that have high AKR1C3 activity. The tubulin binding agent eribulin demonstrated broad activity against solid tumors and ALL models at dose levels relevant to clinical exposure, including 4 of 5 Ewing sarcoma models in which vincristine had low activity. Genomic explanations for ‘exceptional responders’ were identified for the MEK inhibitor selumetinib (see above) and for the PARP inhibitor BMN 673 (PALB2 biallelic mutations). Agents demonstrating high activity against leukemia models included the CD19-targeted antibody drug conjugate SAR-3419, the MDM2 inhibitor RG7112 and the Bcl-2 family inhibitor ABT-263. Agents (or combinations) that the PPTP identified as active that have advanced to clinical testing in children include: selumetinib in low-grade glioma (NCT01089101); alisertib in ALL and neuroblastoma (NCT01154816); NTX-010 (NCT01048892); temsirolimus + cyclophosphamide + vinorelbine for rhabdomyosarcoma (NCT01222715); BMN 673 + temozolomide (NCT02116777), and eribulin (NCT02082626). Supported by NO1-CM-42216 from the NCI.

Abstract 5116: Inhibition of PI3K/AKT signaling by NVP-BKM120 promotes ABT-737-induced toxicity in established and primary cultured glioblastoma cells

Abstract Identification of therapeutic strategies that might enhance the efficacy of Bcl-2 inhibitor ABT-737 is of great interest in many cancers including glioma. Our recent study suggested that Akt is a crucial mediator of apoptosis sensitivity in response to ABT-737 in glioma cell lines. Inhibitors of PI3-K/Akt are currently being assessed clinically in patients with glioma, as PI3K/Akt inhibition would be expected to have many pro-apoptotic effects, we hypothesized that there may be unique synergy between PI3-K inhibitors and BH3-mimetics. We assessed the combination of the PI3K inhibitor BKM120 and the Bcl-2 family inhibitor ABT-737 in established and primary cultured glioma cells. We found that the combined treatment with these agents led to a significant activation of caspase-8, 3, PARP and cell death irrespective of PTEN status. The enhanced lethality observed with the combination also appears dependent upon the loss of mitochondrial membrane potential, release of cytochrome c, smac/DIABLO and apoptosis-inducing factor to the cytosol. Further study revealed that the upregulation of Noxa, truncation of Bid, activation of Bax and Bak caused by these inhibitors was the key factor for the synergy. In addition, we demonstrated the release of proapoptotic proteins Bim and Bak from Mcl-1. We found defects in chromosome segregation leading to multinuclear cells and loss of colony forming ability, suggesting the potential use of BKM120 as a promising agent to improve the anti-cancer activities of ABT-737. Citation Format: Esther P. Jane, Daniel R. Premkumar, Alejandro Morales, Kimberly A. Foster, Ian Pollack. Inhibition of PI3K/AKT signaling by NVP-BKM120 promotes ABT-737-induced toxicity in established and primary cultured glioblastoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5116. doi:10.1158/1538-7445.AM2014-5116

Abstract 5510: ABT-737 synergizes with Vorinostat to induce apoptosis, mediated by Noxa upregulation, Bax activation and mitochondrial dysfunction in PTEN-intact malignant human glioma cell lines

Abstract We have previously observed that glioma cells are differentially sensitive to ABT-737 and, when used as a single-agent, this drug failed to induce apopotsis. Identification of therapeutic strategies to enhance the efficacy of the Bcl-2 inhibitor ABT-737 in human glioma is of interest. Histone deacetylation inhibitors (HDACi) are currently being assessed clinically in patients with glioma, as regulation of epigenetic abnormalities is expected to produce pro-apoptotic effects. We hypothesized that cotreatment of glioma with a BH3-mimetic and HDACi may synergistically produce cell death. We assessed the combination of the Bcl-2 family inhibitor ABT-737 and the HDACi vorinostat in established and primary cultured glioma cells. We found that combined treatment with these agents led to significant cellular death when compared to either drug as a single agent and demonstrated activation of the caspase cascade. The enhanced apoptosis observed with the combination also appears dependent upon the loss of mitochondrial membrane potential, release of cytochrome c, smac/DIABLO and apoptosis-inducing factor to the cytosol. We observed that the upregulation of Noxa, truncation of Bid, and the activation of Bax caused by this combination were important factors for synergistic cell death. We demonstrated that Noxa functioned to occupy Mcl-1 in cells exposed to the combination, yet, contrary to studies in other malignancies, this combination in glioma occurred without the release of proapoptotic proteins Bim and Bak from Mcl-1. We showed this combination to be less effective in PTEN-deficient glioma cells. Both genetic manipulation of PTEN status and pharmacologic inhibition of PI3 kinase sensitized PTEN-deleted glioma cells to the combination. This study demonstrates that antagonizing apoptosis-resistance pathways, such as the Bcl-2 family, and regulating epigenetic aberrancies, such as histone regulation, may induce cell death. Moreover, we reiterate that therapies to target the PI3-kinase/Akt/mTor pathway may be additionally necessary in cancers lacking PTEN functionality. Citation Format: Kimberly A. Foster, Daniel R. Premkumar, Esther P. Jane, Alejandro Morales, Ian F. Pollack. ABT-737 synergizes with Vorinostat to induce apoptosis, mediated by Noxa upregulation, Bax activation and mitochondrial dysfunction in PTEN-intact malignant human glioma cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5510. doi:10.1158/1538-7445.AM2014-5510

Inhibition of phosphatidylinositol 3-kinase/AKT signaling by NVP-BKM120 promotes ABT-737-induced toxicity in a caspase-dependent manner through mitochondrial dysfunction and DNA damage response in established and primary cultured glioblastoma cells.

Identification of therapeutic strategies that might enhance the efficacy of B-cell lymphoma-2 (Bcl-2) inhibitor ABT-737 [N-{4-[4-(4-chloro-biphenyl-2-ylmethyl)-piperazin-1-yl]-benzoyl}-4-(3-dimethylamino-1-phenylsulfanylmethyl-propylamino)-3-nitro-benzenesulfonamide] is of great interest in many cancers, including glioma. Our recent study suggested that Akt is a crucial mediator of apoptosis sensitivity in response to ABT-737 in glioma cell lines. Inhibitors of phosphatidylinositol 3-kinase (PI3K)/Akt are currently being assessed clinically in patients with glioma. Because PI3K/Akt inhibition would be expected to have many proapoptotic effects, we hypothesized that there may be unique synergy between PI3K inhibitors and Bcl-2 homology 3 mimetics. Toward this end, we assessed the combination of the PI3K/Akt inhibitor NVP-BKM120 [5-(2,6-dimorpholinopyrimidin-4-yl)-4-(trifluoromethyl)pyridin-2-amine] and the Bcl-2 family inhibitor ABT-737 in established and primary cultured glioma cells. We found that the combined treatment with these agents led to a significant activation of caspase-8 and -3, PARP, and cell death, irrespective of PTEN status. The enhanced lethality observed with this combination also appears dependent on the loss of mitochondrial membrane potential and release of cytochrome c, smac/DIABLO, and apoptosis-inducing factor to the cytosol. Further study revealed that the upregulation of Noxa, truncation of Bid, and activation of Bax and Bak caused by these inhibitors were the key factors for the synergy. In addition, we demonstrated the release of proapoptotic proteins Bim and Bak from Mcl-1. We found defects in chromosome segregation leading to multinuclear cells and loss of colony-forming ability, suggesting the potential use of NVP-BKM120 as a promising agent to improve the anticancer activities of ABT-737.

MTOR Inhibition Specifically Sensitizes Colorectal Cancers with KRAS or BRAF Mutations to BCL-2/BCL-XL Inhibition by Suppressing MCL-1

Colorectal cancers harboring KRAS or BRAF mutations are refractory to current targeted therapies. Using data from a high-throughput drug screen, we have developed a novel therapeutic strategy that targets the apoptotic machinery using the BCL-2 family inhibitor ABT-263 (navitoclax) in combination with a TORC1/2 inhibitor, AZD8055. This combination leads to efficient apoptosis specifically in KRAS- and BRAF-mutant but not wild-type (WT) colorectal cancer cells. This specific susceptibility results from TORC1/2 inhibition leading to suppression of MCL-1 expression in mutant, but not WT, colorectal cancers, leading to abrogation of BIM/MCL-1 complexes. This combination strategy leads to tumor regressions in both KRAS-mutant colorectal cancer xenograft and genetically engineered mouse models of colorectal cancer, but not in the corresponding KRAS-WT colorectal cancer models. These data suggest that the combination of BCL-2/BCL-XL inhibitors with TORC1/2 inhibitors constitutes a promising targeted therapy strategy to treat these recalcitrant cancers.

Abstract C263: mTOR inhibition specifically sensitizes colorectal cancers with KRAS or BRAF mutations to BCL-2/BCL-XL inhibition by suppressing MCL-1.

Abstract Colorectal cancers (CRCs) harboring KRAS or BRAF mutations are refractory to current targeted therapies. Using data from a high-throughput drug screen, we have developed a novel therapeutic strategy that combines targeting of the apoptotic machinery using the BCL-2 family inhibitor ABT-263 (navitoclax) in combination with a TORC1/2 inhibitor, AZD8055. This combination leads to efficient apoptosis specifically in KRAS mutant (MT) and BRAF MT but not wild-type (WT) CRC cells. This specific susceptibility results from TORC1/2 inhibition leading to suppression of MCL-1 expression in mutant, but not wild-type CRCs, leading to abrogation of BIM/MCL-1 complexes. This combination strategy leads to tumor regressions in both KRAS MT colorectal cancer xenograft and genetically-engineered mouse models of CRC, but not in the corresponding KRAS WT CRC models. These data suggest that the combination of BCL-2/XL inhibitors with TORC1/2 inhibitors constitutes a promising targeted therapy strategy to treat these recalcitrant cancers. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C263. Citation Format: Erin M. Coffee, Anthony C. Faber, Carlotta Costa, Anahita Dastur, Hiromichi Ebi, Aaron N. Hata, Alan T. Yeo, Elena J, Youngchul Song, Ah Ting Tam, Jessica L. Boisvert, Randy J. Milano, Jatin Roper, David P. Kodack, Rakesh K. Jain, Ryan B. Corcoran, Miguel N. Rivera, Sridhar Ramaswamy, Kenneth E. Hung, Cyril H. Benes, Jeffrey A. Engelman. mTOR inhibition specifically sensitizes colorectal cancers with KRAS or BRAF mutations to BCL-2/BCL-XL inhibition by suppressing MCL-1. [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 C263.

PI3K inhibition potentiates Bcl‐2‐dependent apoptosis in renal carcinoma cells

Inhibitors of PI3-K/Akt are currently being assessed clinically in patients with advanced RCC. Identification of therapeutic strategies that might enhance the efficacy of PI3-K/Akt inhibitors is therefore of great interest. As PI3-K inhibition would be expected to have many pro-apoptotic effects, we hypothesized that there may be unique synergy between PI3-K inhibitors and BH3-mimetics. Towards this end, we assessed the combination of the PI3K inhibitor LY 294002 and the Bcl-2 family inhibitor ABT-737 in RCC cell lines. We found that the combinatorial treatment with these agents led to a significant increase in PARP cleavage and cell death in all RCC cell lines. The synergized cell death was correlated with decreased levels of Mcl-1 and XIAP, and increased levels in Bim, and appears critically dependent upon the activation of caspase 3 and 8. The enhanced lethality observed with the combination also appears dependent upon the regulation of XIAP, Mcl-1 and Bim levels. Our results suggest that the combination of PI3-K inhibitors with BH3-mimetics may be a viable therapeutic strategy in RCC.

Abstract LB-79: Synergistic anticancer effects of the 9.2.27PE immunotoxin and ABT-737 in melanoma involves silencing of the anti-apoptotic protein Mcl-1

Abstract In cancer, combinations of drugs targeting different cellular functions is well accepted to improve tumor control. We studied the effects of a Pseudomonas exotoxin A (PE)-based immunotoxin, the 9.2.27PE, and the Bcl-2 family inhibitor ABT-737 in a panel of melanoma cell lines. ABT-737 inhibits the Bcl-2, Bcl-xl and the Bcl-w, but not Mcl-1 and A1 of the Bcl-2 family. The 9.2.27PE + ABT-737 drug combination resulted in synergistic cytotoxicity in the melanoma cells independent of BRAF mutation status. The cell death observed was associated with apoptosis, as activation of caspase-3, inactivation of Poly (ADP-ribose) polymerase (PARP) and increased DNA fragmentation could be prevented by pre-treatment with caspase and cathepsin inhibitors. In addition, ABT-737 caused endoplasmic reticulum (ER) stress with increased GRP78, phosphorylated eIF2α protein levels and increased intracellular calcium levels. The increased intracellular calcium levels were enhanced by silencing of Mcl-1 by short hairpin RNA or by treatment with the 9.2.27PE immunotoxin. These results suggests that Mcl-1 has an inhibitory effect on calcium release from the ER, and that strongly decreased Mcl-1 protein levels after treatment with 9.2.27PE or shRNA may be responsible for the increased intracellular calcium levels in ABT-737 treated melanoma cells. Calcium release has been shown to take place through the inositol 1,4,5-triphosphate receptor in the ER membrane, and the Bcl-2 family proteins Bcl-2, Bcl-xl and Mcl-1 have been shown to interact with the inositol 1,4,5-triphosphate receptor, supporting our results.1 Notably, the combination of 9.2.27PE and ABT-737 caused growth delay in a human melanoma xenograft mice model. 1. Eckenrode EF et. Al., J Biol Chem. 2010 Apr 30;285(18):13678-84 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-79. doi:1538-7445.AM2012-LB-79

Abstract C14: Pim kinase inhibitors sensitize prostate cancer cells to apoptosis triggered by Bcl-2 family inhibitor ABT-737

Abstract Pim serine/threonine kinases contribute to prostate tumorigenesis and therapeutic resistance, yet Pim kinase inhibitors appear to have only limited effects on prostate cancer cell survival. Since overexpression of Bcl-2 family members are implicated in chemotherapeutic resistance in prostate cancer, we investigated the cooperative effects of Pim kinase inhibition with ABT-737, a small molecule antagonist of Bcl-2 family members. Strikingly, the addition of ABT-737 to Pim inhibitors triggered a robust apoptosis of prostate cancer cells in vitro and in vivo. Pim inhibitors decreased levels of the Bcl-2 family member Mcl-1, both by blocking 5′- cap dependent translation and decreasing protein half life. Additionally, Pim inhibition transcriptionally increased levels of the BH3 protein Noxa by activating the unfolded protein response (UPR), lead to eIF-2α phosphorylation and increased expression of CHOP. Increased levels of Noxa also inactivated the remaining levels of Mcl-1 protein activity. Notably, these specific protein changes were essential to the apoptotic process because ABT-737 did not inhibit Mcl-1 protein activity and Mcl-1 overexpression blocked the apoptotic activity of ABT-737. Our results therefore suggest that this combination treatment could be developed as a potential therapy for human prostate cancer where overexpression of Pim kinases and antiapoptotic Bcl-2 family members drives tumor cell resistance to current anticancer therapies. Citation Format: Jin H. Song, Andrew S. Kraft. Pim kinase inhibitors sensitize prostate cancer cells to apoptosis triggered by Bcl-2 family inhibitor ABT-737 [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C14.