Bcl-xL Inhibitor ABT-737 Research Articles

Synergistic cytotoxicity of perifosine and ABT-737 to colon cancer cells.

An acidic environment and hypoxia within the tumour are hallmarks of cancer that contribute to cell resistance to therapy. Deregulation of the PI3K/Akt pathway is common in colon cancer. Numerous Akt-targeted therapies are being developed, the activity of Akt-inhibitors is, however, strongly pH-dependent. Combination therapy thus represents an opportunity to increase their efficacy. In this study, the cytotoxicity of the Akt inhibitor perifosine and the Bcl-2/Bcl-xL inhibitor ABT-737 was tested in colon cancer HT-29 and HCT-116 cells cultured in monolayer or in the form of spheroids. The efficacy of single drugs and their combination was analysed in different tumour-specific environments including acidosis and hypoxia using a series of viability assays. Changes in protein content and distribution were determined by immunoblotting and a "peeling analysis" of immunohistochemical signals. While the cytotoxicity of single agents was influenced by the tumour-specific microenvironment, perifosine and ABT-737 in combination synergistically induced apoptosis in cells cultured in both 2D and 3D independently on pH and oxygen level. Thus, the combined therapy of perifosine and ABT-737 could be considered as a potential treatment strategy for colon cancer.

Abstract P1-13-02: Autophagy inhibition and senolytics to improve the response to fulvestrant + palbociclib in ER positive breast cancer

Abstract While anti-estrogens or aromatase inhibitors in combination with cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors are the current standard of care for estrogen receptor-positive (ER+) Her-2 negative metastatic breast cancer, relapse after remission continues to be a serious limitation to the effectiveness of these therapies. Although these combination therapies prolong progression-free survival compared to endocrine therapy alone, the growth-arrested state of residual tumor cells is clearly transient. Tumor cells that escape what might be considered a dormant or quiescent state and regain proliferative capacity often acquire resistance to further therapies. Our studies are based upon the observation that breast tumor cells arrested by Fulvestrant + Palbociclib enter into a state of autophagy/senescence from which a subpopulation ultimately escapes, potentially contributing to disease recurrence. ER+ MCF-7 breast tumor cells that were induced into autophagy/senescence by Fulvestrant + Palbociclib in vitro were exposed to the autophagy inhibitors, chloroquine and bafilomycin, prior to as well as after growth arrest. However, autophagy inhibition only moderately enhanced the response to therapy and, at best, delayed proliferative recovery. These findings were confirmed by genetic inhibition of autophagy. Despite clear entry into a state of senescence, the Bcl-xl inhibitor and senolytic ABT-263 (navitoclax) failed to promote tumor cell death; this is likely a consequence of high levels of survivin in the MCF-7 breast tumor cell line. However, the BET inhibitor, ARV-825, eliminated a large fraction of the senescent cell population and significantly delayed proliferative recovery. Cell death did not appear to be a consequence of increased apoptosis. Additionally, analysis of three database sets demonstrated that higher breast cancer expression of BRD4, a primary target of ARV-825, correlated with lower recurrence-free survival compared to patients with cancers expressing lower levels of BRD4. These studies indicate that administration of BET inhibitors, which are currently being investigated in multiple clinical trials, may potentially improve standard of care therapy in metastatic ER+ breast cancer patients by prolonging progression-free survival. Citation Format: Ryan Michael Finnegan. Autophagy inhibition and senolytics to improve the response to fulvestrant + palbociclib in ER positive breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-13-02.

MDM2-BCL-XL PROTACs enable degradation of BCL-XL and stabilization of p53.

Inhibition or degradation of anti-apoptotic protein BCL-XL is a viable strategy for cancer treatment. Despite the recent development of PROTACs for degradation of BCL-XL, the E3 ligases are confined to the commonly used VHL and CRBN. Herein we report the development of MDM2-BCL-XL PROTACs using MDM2 as E3 ligase for degradation of BCL-XL. Three MDM2-BCL-XL PROTACs derived from MDM2 inhibitor Nutlin-3, which can also upregulate p53, and BCL-2/BCL-XL inhibitor ABT-263 with different linker length were designed, synthesized, and evaluated in vitro. We found BMM4 exhibited potent, selective degradation activity against BCL-XL and stabilized tumor suppressor p53 in U87, A549 and MV-4-11 cancer cell lines. Moreover, combination of BMM4 and BCL-2 inhibitor ABT-199 showed synergistic antiproliferative activity. The unique dual-functional PROTACs offers an alternative strategy for targeted protein degradation.

Photodynamic Therapy Combined with Bcl-2/Bcl-xL Inhibition Increases the Noxa/Mcl-1 Ratio Independent of Usp9X and Synergistically Enhances Apoptosis in Glioblastoma.

Simple SummaryGlioblastoma represents one of the most common malignant brain tumors in adults and is associated with a poor clinical outcome despite current therapeutic measures. Therefore, novel strategies for the treatment of this disease are urgently needed. In this work, we examined the antineoplastic effects of a combined treatment with photodynamic therapy and ABT-263 on different glioblastoma cells. Photodynamic therapy uses the selective uptake of a photosensitive molecule followed by activation by light of a specific wavelength to kill cancer cells. ABT-263 is a small molecule inhibitor that targets cancer cells by facilitating programmed cell death. This novel combinatorial therapeutic strategy synergistically killed glioblastoma cells. These results indicate that a combination of the two treatment modalities may be of benefit for the treatment of glioblastoma supporting further studies.The purpose of this study was to assess in vitro whether the biological effects of 5-aminolevulinic acid (5-ALA)-based photodynamic therapy are enhanced by inhibition of the anti-apoptotic Bcl-2 family proteins Bcl-2 and Bcl-xL in different glioblastoma models. Pre-clinical testing of a microcontroller-based device emitting light of 405 nm wavelength in combination with exposure to 5-ALA (PDT) and the Bcl-2/Bcl-xL inhibitor ABT-263 (navitoclax) was performed in human established and primary cultured glioblastoma cells as well as glioma stem-like cells. We applied cell count analyses to assess cellular proliferation and Annexin V/PI staining to examine pro-apoptotic effects. Western blot analyses and specific knockdown experiments using siRNA were used to examine molecular mechanisms of action. Bcl-2/Bcl-xL inhibition synergistically enhanced apoptosis in combination with PDT. This effect was caspase-dependent. On the molecular level, PDT caused an increased Noxa/Mcl-1 ratio, which was even more pronounced when combined with ABT-263 in a Usp9X-independent manner. Our data showed that Bcl-2/Bcl-xL inhibition increases the response of glioblastoma cells toward photodynamic therapy. This effect can be partly attributed to cytotoxicity and is likely related to a pro-apoptotic shift because of an increased Noxa/Mcl-1 ratio. The results of this study warrant further investigation.

CRISPR screens identify a novel combination treatment targeting BCL-XL and WNT signaling for KRAS/BRAF-mutated colorectal cancers.

Metastatic or recurrent colorectal cancer (CRC) patients require systemic chemotherapy, but the therapeutic options of targeted agents remain limited. CRC patients with KRAS or BRAF gene mutations exhibit a worse prognosis and are resistant to anti-EGFR treatment. Previous studies have shown that the expression of anti-apoptotic protein BCL-XL is increased in CRC patients with KRAS/BRAF mutations, suggesting BCL-XL as a therapeutic target for this subgroup. Here, we performed genome-wide CRISPR/Cas9 screens of cell lines with KRAS mutations to investigate the factors required for sensitivity to BCL-XL inhibitor ABT-263 using single-guide RNAs (sgRNAs) that induce loss-of-function mutations. In the presence of ABT-263, sgRNAs targeting negative regulators of WNT signaling (resulting in WNT activation) were enriched, whereas sgRNAs targeting positive regulators of WNT signaling (resulting in WNT inhibition) were depleted in ABT-263-resistant cells. The activation of WNT signaling was highly associated with an increased expression ratio of anti- to pro-apoptotic BCL-2 family genes in CRC samples. Genetic and pharmacologic inhibition of WNT signaling using β-catenin short hairpin RNA or TNIK inhibitor NCB-0846, respectively, augmented ABT-263-induced cell death in KRAS/BRAF-mutated cells. Inhibition of WNT signaling resulted in transcriptional repression of the anti-apoptotic BCL-2 family member, MCL1, via the functional inhibition of the β-catenin-containing complex at the MCL1 promoter. In addition, the combination of ABT-263 and NCB-0846 exhibited synergistic effects in in vivo patient-derived xenograft (PDX) models with KRAS mutations. Our data provide a novel targeted combination treatment strategy for the CRC patient subgroup with KRAS or BRAF mutations.

Mitochondrial Function Is Impaired in a Subset of Aged Haematopoietic Stem Cells in Response to Infection

Introduction The burden of age-related diseases is steadily increasing as our population ages. A better understanding of the physiological changes that occur with age may provide an insight into the processes that promote disease development which in turn may help to identify potential targets for the management of these diseases. Haematopoietic stem cells (HSCs) play a vital role in the bone marrow response to infection and previous work has shown that emergency granulopoiesis is supported by increased mitochondrial function in HSCs (1). HSC numbers have previously been shown to increase with age whilst mitochondrial function is known to decline with age. Here we investigate how mitochondrial health changes in ageing HSCs and how this impacts on HSC function and their ability to respond to stress. Methods C57Bl/6 mice were aged to 18-24 months and 8-12-week-old mice were used as controls. Both aged and young mice were sacrificed, and their BM was isolated. Flow cytometry was used to measure numbers of HSCs and mitochondrial content and function using MitoTracker Green and TMRM. Seahorse metabolic flux analysis was used to assess metabolic changes in young and aged HSC populations. Next young and aged C57Bl/6 mice were treated with lipopolycaccharide (LPS) and sacrificed after 16 hours. Their BM was isolated HSC populations were quantified and mitochondrial health was assessed using flow cytometry. Finally, ABT-263 was used to deplete senescent cells in the BM microenvironment. C57Bl/6 mice were treated with 100mg/kg of ABT-263 or vehicle control by oral gavage for 17 days, they were then treated with 0.4mg/kg of LPS and sacrificed after 12 hours. They BM was isolated and analysed by flow cytometry as described above. Results In aged mice HSC numbers were increase and this was associated with an increase in mitochondrial content but a decrease in mitochondrial function. Levels of oxidative phosphorylation (OXPHOS) or glycolysis were not significantly changed in aged HSCs in a steady state. In response to infection, however, in aged mice HSC mitochondrial content and function was not shown to increase following LPS treatment when compared to HSCs from young mice. Moreover, LPS significantly increased OXPHOS in young HSCs whilst aged HSCs were shown to favour glycolysis. Finally, aged HSCs were shown to have increased expression of the anti-apoptotic protein BCL-XL but not BCL-2. In vitro analysis of aged HSC showed the BCL-XL inhibitor ABT-263 but not the BCL-2 inhibitor ABT-199 induced HSC apoptosis. Finally, to determine the effect of targeting BCL-XL on HSCs in vivo we treated aged animals with ABT-263 daily for 17 days and then treated these animals with LPS. Results show that treating aged animals with ABT-263 allowed the HSC to use OXPHOS instead of glycolysis in response to stress. Conclusion Aged HSCs have altered mitochondrial content and function and this impairs their ability to respond to infection through changes in OXPHOS. Instead aged HSC have increased reliance on glycolysis in response to LPS treatment, which is less efficient, and this may impact on HSC function. Targeting BCL-XL with ABT-263 allows elimination of aged HSCs within the BM, reverses some of the age-related mitochondrial changes observed and improves the HSCP ability to respond to stress.

Targeting antioxidant enzymes enhances the therapeutic efficacy of the BCL-XL inhibitor ABT-263 in KRAS-mutant colorectal cancers

Cancer chemotherapeutic drugs exert cytotoxic effects by modulating intracellular reactive oxygen species (ROS) levels. However, whether ROS modulates the efficacy of targeted therapeutics remains poorly understood. Previously, we reported that upregulation of the anti-apoptotic protein, BCL-XL, by KRAS activating mutations was a potential target for KRAS-mutant colorectal cancer (CRC) treatment. Here, we demonstrated that the BCL-XL targeting agent, ABT-263, increased intracellular ROS levels and targeting antioxidant pathways augmented the therapeutic efficacy of this BH3 mimetic. ABT-263 induced expression of genes associated with ROS response and increased intracellular ROS levels by enhancing mitochondrial superoxide generation. The superoxide dismutase inhibitor, 2-methoxyestradiol (2-ME), exhibited synergism with ABT-263 in KRAS-mutant CRC cell lines. This synergistic effect was attributed to the inhibition of mTOR-dependent translation of the anti-apoptotic MCL-1 protein via caspase 3-mediated cleavage of AKT and S6K. In addition, combination treatment of ABT-263 and 2-ME demonstrated a synergistic effect in in vivo patient-derived xenografts harboring KRAS mutations. Our data suggest a novel role for ROS in BH3 mimetic-based targeted therapy and provide a novel strategy for treatment of CRC patients with KRAS mutations.

Abstract 1821: Reversal of PD-L1-mediated chemoresistance by MCL-1/BCL-2 antagonism in colon cancer cells

Abstract Background: PD-L1 is an immune checkpoint protein that may display intrinsic, non-immune functions in human cancer cells. Previously, we discovered that PD-L1 is regulated by BRAFV600E via YAP and c-Jun, and whose deletion can confer chemoresistance due to a decrease in the BH3-only BIM protein in colorectal cancer (CRC) cells. Here, we hypothesize that release of pro-apoptotic BIM from its sequestration by MCL-1 or BCL-2 can overcomes drug resistance mediated by depletion of PD-L1. Materials and Methods: Parental and PD-L1 knockout human (RKO) and murine (MC38) CRC cells with or without reconstituted PD-L1 were utilized. RKO cells with knockdown of MCL-1 or BCL-2 by shRNA were generated. Cells were treated with the MCL-1 selective inhibitor AZD5591 or the BCL-2/BCL-xL inhibitor ABT-263 alone or combined with the MEK inhibitor, cobimetinib. Apoptosis was measured by immunoblotting of cleaved caspase-3 and by Annexin V labeling using FACS. Clonogenic cell survival assay was performed. Protein/protein interaction was analyzed by immunoprecipitation assay. Results: BIM induction by cobimetinib was insufficient to overcome chemoresistance due to loss of PD-L1. Using a potent and selective inhibitor of MCL-1 (AZD-5591), we demonstrate that concurrent antagonism of MCL-1 and MEK can overcome apoptosis resistance in both human and murine PD-L1 knockout CRC cells. Specifically, inhibition of MCL-1 by AZD-5591 or gene knockdown combined with cobimetinib suppresses clonogenic cell survival and enhances apoptosis in cells with PD-L1 knockout, wild type or those with reconstituted PD-L1. Mechanistically, the interaction of BIM with MCL-1 is increased in PD-L1 knockout (versus wild-type cells) in the presence of cobimetinib, and enhanced binding of BIM to MCL-1 is associated with BIM phosphorylation at Thr116. Targeting MCL-1 using AZD-5591 or MCL-1 shRNA releases BIM from its sequestration by MCL-1 and results in enhanced apoptotic signaling induced by cobimetinib to an equivalent extent in wild type and PD-L1 knockout cells with or without reconstitution of PD-L1. Similarly, targeting BCL-2 by ABT-263 or by BCL-2 shRNA releases BIM from its binding to BCL-2, and enhances apoptosis induction by cobimetinib independent of PD-L1 status. Enhanced binding of BIM to BCL-2 was also associated with BIM phosphorylation at Thr116. Conclusion: Chemoresistance mediated by loss of PD-L1 can be reversed by the use of BH3 mimetic drugs combined with MEK inhibition in CRC cells to exploit their apoptotic dependency on BIM expression. Citation Format: Lei Sun, Arpad Patai, Bo Qin, Frank A. Sinicrope. Reversal of PD-L1-mediated chemoresistance by MCL-1/BCL-2 antagonism in colon cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1821.

Abstract 90: Inducing and targeting senescence in HPV-negative head and neck squamous cancer cells

Abstract Background: The loss of normal cell cycle regulation via disruption of CDKN2A in HPV-negative (-) head and neck squamous cell carcinoma (HNSCC) is universal. Targeting CDK4/6 with a specific inhibitor, such as palbociclib, is therefore a logical strategy in this disease. Previous work has demonstrated that CDK4/6 inhibition leads to cell cycle arrest and senescence in cancer cells, and specifically HPV(-) HNSCC. Recent studies have also shown that the BCL-xL inhibitor, ABT-263 (navitoclax), effectively targets senescent cells. This study aimed to evaluate the efficacy and mechanism of a combination approach utilizing palbociclib and ABT-263 in HNSCC. Methods: Three HPV (-) HNSCC cell lines (CAL27, HN31, PCI15B) were treated with palbociclib. Cresyl violet staining and annexin V apoptosis assays were used to assess cell viability in response to palbociclib, navitoclax, or the combination of the two drugs. Senescence following treatment with palbociclib was measured using β-galactosidase expression on western blot analysis, as well as via immunofluorescence staining assessed and quantified using confocal microscopy. Levels of BCL-xL were also measured using these methods. Results: Following treatment with palbociclib, all cell lines exhibited phenotypic evidence of senescence, and protein expression of β-galactosidase was significantly increased compared to untreated controls (mean fold change ± SD: CAL27 2.67±0.26; HN31 1.79±0.41; PCI15B 1.26±0.15). Protein expression of BCL-xL was concurrently increased under these conditions (mean fold change ± SD: CAL27 1.57±0.04; HN31 1.58±0.39; PCI15B 1.33±0.17). Immunofluorescent staining was also used to determine if cells exhibiting senescence specifically demonstrated higher BCL-xL expression. Co-expression of β-galactosidase and BCL-xL showed a strong positive correlation (CAL27 r = 0.94, p < 0.0001; HN31 r = 0.94, p < 0.0001; PCI15B r = 0.71, p < 0.0001). Treatment with the combination of palbociclib and navitoclax resulted in significantly decreased survival compared to each agent alone in all HNSCC cell lines tested (data summarized for the cell line panel: mean percent survival, navitoclax 76.40%; palbociclib 38.76%; combination 16.74%). Annexin V apoptosis assay also revealed that there was a significant increase in apoptosis observed in all cell lines tested comparing each agent to combination treatment (data summarized for the cell line panel: mean percent apoptotic cells, navitoclax 35.53%; palbociclib 29.95%; combination 60.98%). Apoptosis with combination of palbociclib and navitoclax approached or exceeded levels achieved with staurosporine treated controls. Conclusions: Palbociclib treatment leads to consistent induction of senescence in HPV (-) HNSCC. Adding a senolytic agent, such as the BCL-xL inhibitor ABT-263 (navitoclax) appears to be an effective strategy which holds promise in this disease. Citation Format: Nicholas Gadsden, Carlos Thomas, Daniel Li, Nitisha Shrivastava, Nicolas Schlecht, Michael Prystowsky, Jeffrey Segall, Thomas Ow. Inducing and targeting senescence in HPV-negative head and neck squamous cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 90.

NOXA upregulation by the prohibitin-binding compound fluorizoline is transcriptionally regulated by integrated stress response-induced ATF3 and ATF4.

Fluorizoline is a new synthetic molecule that induces p53-independent apoptosis, in several tumor cell lines and in primary leukemia cells, by selectively targeting prohibitins (PHBs). In this study, we describe how fluorizoline induces BCL-2 homology 3-only protein NOXA, without modulating the protein levels of anti-apoptotic B-cell lymphoma-2 (BCL-2) family members prior to caspase activation, as well as how it synergizes with the BCL-2 and BCL-XL inhibitor ABT-737 to induce apoptosis. Interestingly, fluorizolinetreatment triggers the activation of the integrated stress response (ISR) in HeLa and HAP1 cells, with increased eukaryotic translation initiation factor 2α phosphorylation, and induction of ATF3, ATF4, and CHOP. Moreover, PHB downregulation induces similar ISR activation and apoptosis as with fluorizoline treatment. In addition, we studied the essential role of the pro-apoptotic protein NOXA in fluorizoline-induced apoptosis and we describe its mechanism of induction in HeLa and HAP1 cells. Moreover, we identified ATF3 and ATF4 as the transcription factors that bind to NOXA promoter upon fluorizoline treatment. Furthermore, using ATF3 and ATF4 CRISPR HeLa and HAP1 cells, we confirmed that both factors mediate the induction of NOXA and apoptosis by fluorizoline. In conclusion, fluorizoline treatment triggers the activation of the ISR that results in the induction of ATF3 and ATF4, important regulators of NOXA transcription in fluorizoline-induced apoptosis.

Therapeutic Concentrations of Statins Hyperpolarize Mitochondria and Inhibit Cell Proliferation Without Promoting Cell Death in Hepatocarcinoma Cells

Statins, widely used inhibitors of HMG-CoA reductase to decrease cholesterol, may also decrease the risk of developing liver, breast, and lung cancer. Concentrations of statins (10-100 µM) typically used in vitro are 1,000- to 10,000-fold higher than plasmatic therapeutic concentrations (10-100 nM). In preliminary work, we showed that simvastatin (SIM) and lovastatin at 10 µM increased mitochondrial membrane potential (ΔΨ) and decreased respiration and ATP production in HepG2, Huh7 and HCC4006 cells. We also showed that the bcl-XL inhibitor ABT-737 prevents mitochondrial hyperpolarization induced by the ATP synthase inhibitor, oligomycin. Here, we hypothesize that SIM at nanomolar concentrations increases ΔΨ contributing to decreased cell proliferation and that both mitochondrial hyperpolarization and inhibition of cell proliferation are influenced by bcl-XL. Confocal fluorescence microscopy of tetramethylrhodamine methyl ester assessed ΔΨ. Cholesterol levels were determined using Amplex Red after lipid extraction from whole HepG2 cells and isolated mitochondria. Respiration was assessed using a Seahorse XFe96 Analyzer and cell proliferation by direct counting of live/dead cells. SIM (20 nM to 100 nM) increased mitochondrial ΔΨ in a dose-dependent fashion in HepG2 cells at 24 h. SIM (100 nM for 24 h and 10 µM for 6 h) increased ΔΨ by ∼120 % and 160 % respectively. High ΔΨ promoted by SIM (100 nM and 10 µM) was sustained for at least 72 h. ABT-737 (1 µM) blocked mitochondrial hyperpolarization induced by SIM. SIM at 100 nM and 10 µM decreased cell proliferation by ∼49% and 82% respectively without promoting cell killing. The inhibition of cell proliferation was blocked by pretreatment with ABT-737. Therapeutic concentrations of statins decreased cell proliferation without promoting cell killing possibly by modulation of mitochondrial metabolism mediated by bcl-XL.

Targeting BCL-XL By Protac DT2216 Effectively Eliminates Leukemia Cells in T-ALL Pre-Clinical Models

Targeting BCL-XL By Protac DT2216 Effectively Eliminates Leukemia Cells in T-ALL Pre-Clinical Models

Anti-Apoptotic BCL-2 Family Members Confer Resistance to Calicheamicin-Based Antibody-Drug Conjugate Therapy of Acute Leukemia

BACKGROUND: With gemtuzumab ozogamicin (GO; targeting CD33) and inotuzumab ozogamicin (IO; targeting CD22), 2 antibody-drug conjugates delivering a toxic calicheamicin (CLM) derivative have recently been approved for the treatment of people with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), respectively. While effective in some, many patients do not benefit from these ADCs. It is unclear to what degree anti-apoptotic BCL-2 family members are involved in modulating efficacy of CLM-based ADCs, with limited studies coming to differing conclusions. Given the clinical availability of small molecule inhibitors for BCL-2 family proteins (BCLi), here we clarify the impact of BCL-2 family proteins on the anti-leukemic activity of CLM-ADCs. MATERIALS AND METHODS: Human AML and ALL cell lines were engineered to overexpress BCL-2, BCL-XL, and MCL-1 via lentiviral gene transfer. AML and ALL cell lines as well as AML patient samples were exposed to increasing concentrations of GO or IO with or without the BCL-2 inhibitor ABT-199 (venetoclax), the BCL-2/BCL-XL inhibitor ABT-263 (navitoclax), and the MCL-1 inhibitor AZD5991. Dead cells were enumerated by flow cytometry via 4',6-diamidino-2-phenylindole staining after 72 hours. For BH3 profiling of AML patient specimens, thawed AML patient specimen aliquots were exposed to JC-1 mitochondrial dye and BH3 peptides, and peptide-induced depolarization was then calculated as a percent relative to a CCCP positive control, yielding a priming score for each BH3 peptide. RESULTS: At a dose of 1000 pg/ml, GO killing of ML-1 (AML) cells decreased from 56±5% (mean±SEM) in parental cells to 32±7% (p<0.01) and 26±6% (p<0.01) in cells overexpressing BCL-2 and BCL-XL, respectively (all n=3). Similar results were seen in another AML cell line (HL-60). In REH (ALL) cells treated with IO, overexpression of BCL family members also reduced killing - at 500 pg/ml, 59±8% of cells were killed in contrast to 12±1% (p<0.01) of BCL-2-expressing and 11±1% (p<0.01) of BCL-XL-expressing cells, with similar results seen in another ALL cell line (RS4;11). Addition of ABT-199 or ABT-263 at 1 µM modestly increased GO-mediated killing of AML cell lines - for example, ML-1 cells treated with GO at 100 pg/ml, cytotoxicity increased from 41±6% to 57±7% (ABT-199, p<0.01) and 61±8% (ABT-263, p<0.01). The effect of BCLi was more pronounced on IO-mediated killing of ALL cell lines than on GO-mediated killing of AML lines. For example, killing of REH cells treated with IO at 25 pg/ml increased from 39±7% (without BCLi) to 72±8% (ABT-199 1 µM, p<0.01) and 87±9% (ABT-263 1 µM, p<0.01), with similar results seen in RS4;11 cells. BH3 peptide profiling of AML patient specimens treated with GO implicated MCL-1 as a potential additional modulator of AML response to GO. Consistent with this finding, overexpression of MCL-1 reduced leukemia cell death in HL-60 cells treated with GO (GO at 1000 pg/ml, 41±2 % vs. 26±1 %, p=0.01) and RS4;11 cells treated with IO (IO at 100 pg/ml, 76±2% vs. 27±6%, p<0.01). The MCL-1 inhibitor AZD5991 modestly increased the anti-leukemic efficacy of GO in ML-1 cells and AML patient specimens, but more dramatically enhanced IO killing of REH cells (IO at 10 pg/ml, 18±2% without AZD5991 vs. 70±2% with 0.1 µM AZD5991, p<0.01). The triplet combination of GO, ABT-199 and AZD5991 did not improve markedly on the ABT-199/AZD5991 combination in the absence of GO in cell lines or AML patient specimens, though the triplet combination of IO, ABT-199 and AZD5991 showed promising activity: in REH cells treated with 10 pg/ml IO, cytotoxicity was 18±2% without BCLi, 32±8% with ABT-199 0.1 µM, 19±2% with AZD5991 0.01 µM, and 56±14% with the triplet combination (p<0.01 for comparison of triplet combination with IO/BCLi doublet). CONCLUSIONS: Our studies establish an important role of anti-apoptotic BCL-2 family members as resistance factor for CLM-based ADC therapy of acute leukemia. These findings provide the rationale to explore the combination of small-molecule inhibitors of BCL-2 family members with CLM-ADCs as a combination strategy in the clinic to improve the efficacy of GO and, particularly, IO. These therapeutic strategies may incorporate the assessment of the relative contribution of specific BCL-2 family members to an individual cancer patient's disease. Disclosures Jean: Eutropics Pharmaceuticals: Employment. Cardone:Eutropics Pharmaceuticals: Employment, Equity Ownership. Walter:Seattle Genetics: Research Funding; Kite Pharma: Consultancy; Daiichi Sankyo: Consultancy; Jazz Pharmaceuticals: Consultancy; Agios: Consultancy; Amgen: Consultancy; Amphivena Therapeutics: Consultancy, Equity Ownership; Aptevo Therapeutics: Consultancy, Research Funding; Argenx BVBA: Consultancy; Astellas: Consultancy; BioLineRx: Consultancy; BiVictriX: Consultancy; Boehringer Ingelheim: Consultancy; Boston Biomedical: Consultancy; Covagen: Consultancy; New Link Genetics: Consultancy; Pfizer: Consultancy, Research Funding; Race Oncology: Consultancy.

Selective Targeting BET Family Bdii Bromodomain with Abbv-744 and BCL-2 with Venetoclax (ABT-199) Is Synergistic in Primary Acute Myeloid Leukemia Models

Despite advances in understanding of the biology of acute myeloid leukemia (AML), cure remains elusive for the majority of patients. ABT-199 (Venetoclax) is a small-molecule BH3 mimetic that selectively inhibits BCL-2 causing cell death. First generation BET inhibitor ABBV-075 and Venetoclax were recently shown to be synergistic in AML cell lines (Bui MH,Cancer Res 2017). ABBV-744 is a highly selective inhibitor for the BDII of BET family proteins, exhibiting greater than 300-fold more potent binding affinity to the BDII bromodomain of BRD4 relative to BDI (Warren Kati AACR 2018; Xiaoyu Lin AACR 2018). In this study, we evaluated the anti-leukemia efficacy of the concomitant BCL-2 blockade by venetoclax and of BDII inhibition with ABBV-744 in primary AML samples. Anti-leukemia activity of venetoclax and ABBV-744 was examined in 21 primary AML samples with diverse genomic alterations. The combination significantly enhanced cell death (57.0 ± 6.3%) compared to the single agent treatment (43.9 ± 5.7% in ABT-199 10 nM group, p<0.001 and 23.8 ± 2.9% in ABBV-744 20nM group, p<0.001, Fig.1A). ABBV-744 reduced viable cell numbers in the majority of AML cases (31.7 ± 5.2%) and the cell growth suppression was more profound in the combination group (77.2 ± 6.3 %, p<0.001, Fig.1B). In two AML primary samples tested, combination treatment of ABBV-744 and ABT-199 induced apoptosis with caspase-3 activation and PARP cleavage through regulation of the key proteins regulating survival and proliferation pathways (e.g. (BCL-2, BCL-XL, MCL-1, c-Myc)). To identify biomarkers of response to therapy, we performed the baseline transcriptome analysis of AML cells used for in vitro response assessment (n=25) by RNA-sequencing (RNA-seq), and correlated baseline gene expression levels with in vitro response to therapy. Based on response to venetoclax or combination, samples were divided into 3 groups: sensitive to venetoclax (n=10), samples with no response to single agent or combination ("low apoptosis", n=7) and samples resistant to venetoclax as a single agent but responsive to venetoclax/ ABBV-744 combination ("synergy", n=4). AML samples sensitive to venetoclax and venetoclax/ABBV-744 combination were characterized by high level of BCL2 and lower levels of MCL1 and BCL2L1 transcripts, consistent with known inability of venetoclax to inhibit MCL-1 and BCL2L1 (Fig.1C).The resistant samples additionally expressed higher levels of anti-apoptotic genes such as GADD45, BCL2L10, PMAIP1. AML cells that showed synergy between venetoclax/ABBV-744 expressed low levels of AR, IL1R1 genes and had high CCND1 expression. The gene expression analysis indicated that the genes differentially expressed in the synergy vs. low apoptosis samples overlap with the genes inhibited by dual BCL-2/BCL-XL inhibitor ABT-737. To test the efficacy of this regimen in vivo, we established a patient-derived xenograft (PDX) from an AML patient with FLT3-ITD, DNMT3A, EGFR, IDH1, NPM1, TET2 mutations in NSG mice. Upon engraftment, mice were randomized to receive vehicle; single agent venetoclax at 50 mg/kg; ABBV-744 at 9.4 mg/kg; or venetoclax plus ABBV-744 for 21 days. After 21 days of therapy, flow cytometry data demonstrated significantly reduced leukemia burden in venetoclax treated group (9.5% ± 1.7%) but not in ABBV-744 group (22.3% ± 5.8%) compared to controls (30.8% ± 3.9%), with lowest tumor burden in the combination group (5.0% ± 0.8%, p<0.01) (Fig. 1E). Combination of ABBV-744 and venetoclax treatment delayed AML progression and extended the survival compared to the untreated mice (median survival, 193 days vs 99 days, p<0.001) (Fig. 1D). No significant impact on mice' weight was noted, and no clinical signs of toxicity recorded over the course of therapy. In summary, combinatorial blockade of BDII bromodomain and of BCL-2 anti-apoptotic pathway facilitates apoptotic cell death, suppresses proliferation in the majority of primary AML cells and produces anti-AML activity in AML PDX models in vivo at tolerable doses of both agents. This combination is currently undergoing testing in a Phase I clinical trial in AML (NCT03360006). Disclosures Kuruvilla: The University of Texas M.D.Anderson Cancer Center: Employment. Lin:AbbVie: Employment. Uziel:AbbVie: Employment, Other: stock or other options. Lu:AbbVie: Employment. Zhang:AbbVie: Employment. Huang:AbbVie: Employment. Zhang:The University of Texas M.D.Anderson Cancer Center: Employment. Shen:AbbVie: Employment. Konopleva:Astra Zeneca: Research Funding; Ablynx: Research Funding; Eli Lilly: Research Funding; Kisoji: Consultancy, Honoraria; Ascentage: Research Funding; Agios: Research Funding; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Amgen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Genentech: Honoraria, Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Calithera: Research Funding; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Forty-Seven: Consultancy, Honoraria.

The response of phyllodes tumor of the breast to anticancer therapy: An in vitro and ex vivo study.

Phyllodes tumors of the breast (PTB) are uncommon stromal-epithelial neoplasms, with the main recommended treatment being surgical removal. However, even with adequate resection, the risk of recurrence in the malignant form remains as high as 40%, and there is no recognized consensus on the most effective drugs for PTB. In the present study, an ex vivo model of malignant phyllodes and derived primary cell cultures were used to evaluate the effectiveness of a panel of different drugs, including the Bcl-2/Bcl-xL inhibitor ABT-263, salinomycin (SAL), doxorubicin (DOX), paclitaxel (TAX), vincristine (VCR), colchicine (COL) and cisplatin (CIS). ABT-263, SAL and DOX were highly effective towards phyllodes spindle cells when assessed in the ex vivo model, contributing to ~98% tumor cell death. Furthermore, ABT-263 was highly selective for tumor cells in this system, and exhibited little toxic effect on adjacent normal epithelial cells. Furthermore, consistent with findings in the ex vivo model, ABT-263 was significantly less toxic towards MCF 10A non-tumorigenic breast epithelial cells compared with SAL and DOX. A conditional reprogramming strategy was subsequently used, involving Rho kinase inhibition, to successfully generate primary phyllodes tumor cells that could be cultured for several passages. The primary cells were sensitive to DOX with an IC50 of 0.40±0.07 µM in a standard viability assay and the preliminary results were obtained indicating sensitivity to ABT-263 and SAL. The present study demonstrated the feasibility of using explants and primary cells for drug discovery, selectively targeting PTB cells.

Bcl-2/Bcl-xL inhibition predominantly synergistically enhances the anti-neoplastic activity of a low-dose CUSP9 repurposed drug regime against glioblastoma.

Drug repurposing represents a promising approach to safely accelerate the clinical application of therapeutics with anti-cancer activity. In this study, we examined whether inhibition of the anti-apoptotic Bcl-2 family proteins Bcl-2 and Bcl-xL enhances the biological effects of the repurposed CUSP9 regimen in an in vitro setting of glioblastoma. We applied 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assays to assess cellular proliferation. Annexin V/propidium iodide and tetramethylrhodamine, ethyl ester staining were used to examine apoptosis. Western blotting, RT-PCR, and specific knockdown experiments using siRNA were employed to examine molecular mechanisms of action. Bcl-2/Bcl-xL inhibition exerted synergistic anti-proliferative effects across established, primary cultured, and stem-like glioblastoma cells when combined with CUSP9 which had been reduced to only one tenth of its proposed original concentration (CUSP9-LD). The combination treatment also led to enhanced apoptosis with loss of mitochondrial membrane potential and activation of caspases. On the molecular level, CUSP9-LD counteracted ABT263-mediated up-regulation of Mcl-1. Silencing of Mcl-1 enhanced ABT263-mediated apoptosis which indicates that down-regulation of Mcl-1 is crucial for the induction of cell death by the combination treatment. These data suggest that Bcl-2/Bcl-xL inhibition enhances the susceptibility of glioblastoma cells towards CUSP9, allowing dramatic dose reduction and potentially decreased toxicity when applied clinically. A clinical trial involving the original CUSP doses (CUSP9v3) is currently ongoing in our institution (NCT02770378). The Bcl-2/Bcl-xL inhibitor ABT263 is in clinical trials and might represent a valuable adjunct to the original CUSP.

Microtubules play an essential role in the survival of primary acute lymphoblastic leukemia cells advancing through G1 phase

ABSTRACT We recently reported that primary acute lymphoblastic leukemia (ALL) cells are susceptible to the microtubule depolymerizing agent vincristine (VCR) in G1 phase. This finding prompted testing another G1 phase-active compound, palbociclib (PCB), a highly selective inhibitor of cyclin-dependent kinases 4/6 (CDK4/6), alone and in combination with VCR. PCB used alone caused G1 arrest in ALL cells with no effect on cell viability, and similar results were obtained for the retinoblastoma (RB)-proficient T98G glioblastoma cell line. In contrast, HeLa cells failed to arrest in the presence of PCB, consistent with their lack of dependence on the CDK4/6-RB pathway. When ALL cells were pretreated with PCB, they became refractory to death in G1 phase induced by VCR treatment, whereas HeLa cells retained VCR sensitivity after PCB pretreatment. Immunofluorescence microscopy showed that PCB did not disrupt the microtubule network nor prevent VCR from doing so. Furthermore, ALL cells pretreated with PCB retained susceptibility to the Bcl-2/Bcl-xL inhibitor ABT-263, indicating that downstream apoptotic signaling was unaffected. When released from PCB-enforced arrest, ALL cells reinitiated cycling and regained sensitivity to VCR. ALL cells treated with cycloheximide also arrested in G1 phase and became insensitive to VCR, independently reinforcing conclusions derived from PCB-imposed arrest. Thus, primary ALL cells advancing through G1 phase are strictly dependent on functional microtubules for survival whereas microtubules are dispensable for G1-arrested cells. These findings provide novel insight into interphase microtubule function and, from a therapy standpoint, strongly caution against combining microtubule targeting agents and CDK4/6 inhibitors for ALL.

Bcl-2 and Bcl-xL mediate resistance to receptor tyrosine kinase-targeted therapy in lung and gastric cancer.

Promising clinical efficacy has been observed with receptor tyrosine kinase inhibitors (TKIs) particularly in lung and gastric cancers with mutations or amplifications in the targeted receptor tyrosine kinases (RTKs). However, the efficacy and the duration of the response to these inhibitors are limited by the emergence of drug resistance. Here, we report treatment of RTK-dependent lung and gastric cancer cell lines with TKIs increased protein levels of Bcl-2 and Bcl-xL. The combination of the Bcl-2 and Bcl-xL inhibitor ABT-263 and TKIs was superior to TKIs alone in reducing cell viability and capacity of resistant colony formation. Furthermore, resistant cells established with exposure of RTK-dependent cells to increasing concentrations of TKIs also express higher levels of Bcl-2 or Bcl-xL compared with their parental cells. The combination of inhibitors of PI3K/AKT, MEK/ERK, and Bcl-2/Bcl-xL effectively reduced the viability of resistant cells and inhibited tumor size in a xenograft model derived from resistant cells by inducing apoptosis. Our results define a generalizable resistance mechanism to TKIs and rationalize inhibition of Bcl-2 and Bcl-xL as a strategy to augment responses and blunt acquired resistance to TKIs in lung and gastric cancer.

Dynein light chain 1 induces assembly of large Bim complexes on mitochondria that stabilize Mcl-1 and regulate apoptosis.

The Bcl-2 family protein Bim triggers mitochondrial apoptosis. Bim is expressed in nonapoptotic cells at the mitochondrial outer membrane, where it is activated by largely unknown mechanisms. We found that Bim is regulated by formation of large protein complexes containing dynein light chain 1 (DLC1). Bim rapidly inserted into cardiolipin-containing membranes in vitro and recruited DLC1 to the membrane. Bim binding to DLC1 induced the formation of large Bim complexes on lipid vesicles, on isolated mitochondria, and in intact cells. Native gel electrophoresis and gel filtration showed Bim-containing mitochondrial complexes of several hundred kilodaltons in all cells tested. Bim unable to form complexes was consistently more active than complexed Bim, which correlated with its substantially reduced binding to anti-apoptotic Bcl-2 proteins. At endogenous levels, Bim surprisingly bound only anti-apoptotic Mcl-1 but not Bcl-2 or Bcl-XL, recruiting only Mcl-1 into large complexes. Targeting of DLC1 by RNAi in human cell lines induced disassembly of Bim-Mcl-1 complexes and the proteasomal degradation of Mcl-1 and sensitized the cells to the Bcl-2/Bcl-XL inhibitor ABT-737. Regulation of apoptosis at mitochondria thus extends beyond the interaction of monomers of proapoptotic and anti-apoptotic Bcl-2 family members but involves more complex structures of proteins at the mitochondrial outer membrane, and targeting complexes may be a novel therapeutic strategy.

Bcl-2/Bcl-xL inhibitor ABT-737 sensitizes pancreatic ductal adenocarcinoma to paclitaxel-induced cell death.

Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignant disease that is resistant to various chemotherapeutic agents and commonly relapses. Efficient elimination of metastasized PDA is critical for a positive post-surgical treatment outcome. The present study analyzed the effect of the B-cell lymphoma-2 (Bcl-2)/B-cell lymphoma extra-large (Bcl-xL) inhibitor, ABT-737, on paclitaxel-induced PDA cell death. A total of 8 PDA cell lines were subjected to immunoblotting to compare the expression of Bcl-2/Bcl-xL and other factors associated with taxane resistance, including myeloid cell leukemia 1 and βIII-tubulin (TUBB3). The viability of PDA cells was analyzed following treatment with paclitaxel alone or a combination treatment with ABT-737 and paclitaxel. Treatment with the ABT-737/paclitaxel combination induced PDA cell death at a lower concentration of paclitaxel compared with paclitaxel alone. In addition, the viable cell population at the saturation point of paclitaxel was also decreased by co-treatment with ABT-737. ABT-737 lowered the half maximal inhibitory concentration (IC50) by >2-fold in PDA cells with high Bcl-2/Bcl-xL expression, but not in PDA cells with low Bcl-2/Bcl-xL expression and high TUBB3 expression. Knockdown of Bcl-xL lowered the IC50 of paclitaxel, but knockdown of TUBB3 did not. ABT-737 sensitized PDA to paclitaxel-induced cell death, and Bcl-xL expression was a key determinant of its sensitivity. ABT-737 is potential candidate for combination chemotherapy of PDA with high Bcl-xL expression levels.