Pro-survival Family Members Research Articles

Bcl-2 Up-Regulation Mediates Taxane Resistance Downstream of APC Loss.

Triple-negative breast cancer (TNBC) patients are treated with traditional chemotherapy, such as the taxane class of drugs. One such drug, paclitaxel (PTX), can be effective in treating TNBC; however, many tumors will develop drug resistance, which can lead to recurrence. In order to improve patient outcomes and survival, there lies a critical need to understand the mechanism behind drug resistance. Our lab made the novel observation that decreased expression of the Adenomatous Polyposis Coli (APC) tumor suppressor using shRNA caused PTX resistance in the human TNBC cell line MDA-MB-157. In cells lacking APC, induction of apoptosis by PTX was decreased, which was measured through cleaved caspase 3 and annexin/PI staining. The current study demonstrates that CRISPR-mediated APC knockout in two other TNBC lines, MDA-MB-231 and SUM159, leads to PTX resistance. In addition, the cellular consequences and molecular mechanisms behind APC-mediated PTX response have been investigated through analysis of the BCL-2 family of proteins. We found a significant increase in the tumor-initiating cell population and increased expression of the pro-survival family member Bcl-2, which is widely known for its oncogenic behavior. ABT-199 (Venetoclax), is a BH3 mimetic that specifically targets Bcl-2. ABT-199 has been used as a single or combination therapy in multiple hematologic malignancies and has shown promise in multiple subtypes of breast cancer. To address the hypothesis that APC-induced Bcl-2 increase is responsible for PTX resistance, we combined treatment of PTX and ABT-199. This combination treatment of CRISPR-mediated APC knockout MDA-MB-231 cells resulted in alterations in apoptosis, suggesting that Bcl-2 inhibition restores PTX sensitivity in APC knockout breast cancer cells. Our studies are the first to show that Bcl-2 functional inhibition restores PTX sensitivity in APC mutant breast cancer cells. These studies are critical to advance better treatment regimens in patients with TNBC.

Key residues in the VDAC2-BAK complex can be targeted to modulate apoptosis.

BAK and BAX execute intrinsic apoptosis by permeabilising the mitochondrial outer membrane. Their activity is regulated through interactions with pro-survival BCL-2 family proteins and with non-BCL-2 proteins including the mitochondrial channel protein VDAC2. VDAC2 is important for bringing both BAK and BAX to mitochondria where they execute their apoptotic function. Despite this important function in apoptosis, while interactions with pro-survival family members are well characterised and have culminated in the development of drugs that target these interfaces to induce cancer cell apoptosis, the interaction between BAK and VDAC2 remains largely undefined. Deep scanning mutagenesis coupled with cysteine linkage identified key residues in the interaction between BAK and VDAC2. Obstructive labelling of specific residues in the BH3 domain or hydrophobic groove of BAK disrupted this interaction. Conversely, mutating specific residues in a cytosol-exposed region of VDAC2 stabilised the interaction with BAK and inhibited BAK apoptotic activity. Thus, this VDAC2-BAK interaction site can potentially be targeted to either inhibit BAK-mediated apoptosis in scenarios where excessive apoptosis contributes to disease or to promote BAK-mediated apoptosis for cancer therapy.

MALT1 Protease Inhibition Overcomes BTK Inhibitor Resistance and Shows Synergistic Activity with Venetoclax in Models of B Cell Lymphoma and Leukemia

Background Specific B cell malignancies, including the aggressive non-germinal center B cell-like (GCB) subtype of diffuse large B cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL), are driven by constitutive activation of the transcription factor NF-kB. Chronic signaling resulting from mutations or antigen-induced activation of the B cell receptor (BCR) pathway drives NF-kB activity in these tumors resulting in sustained proliferation and survival pathways. Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) represents a key mediator of the BCR/NF-kB signal transduction pathway responsible for maintaining survival and driving proliferation of NF-kB-addicted B cell tumors by proteolytically cleaving NF-kB antagonists. Aims Pharmacological inhibition of MALT1 protease activity may provide an attractive treatment option for patients with these cancers. Further, as combination therapy is often required for the treatment of aggressive B cell malignancies, the identification of therapies that synergistically combine with MALT1 inhibitors could afford additional and promising treatment options. Methods A highly potent and orally bioavailable MALT1 protease inhibitor (ABBV-MALT1) was used to test the hypothesis that MALT1 inhibition will abrogate the proliferation of preclinical models of B cell malignancies in vitro and in vivo. Tumors treated with ABBV-MALT1 were subjected to transcriptomic and functional proteomic assays to elucidate molecular mechanisms of action and rational combination partners. Results ABBV-MALT1 potently inhibits MALT1 activity in vitro while showing high selectivity against a panel of proteases, kinases and other types of receptors and enzymes. Mechanistic studies reveal that ABBV-MALT1 effectively inhibits signal transduction of the BCR pathway and reduces NF-kB gene activation in non-GCB DLBCL cell lines resulting in cell cycle arrest and diminished viability. In vivo, oral administration of this compound demonstrates robust tumor growth inhibition in several models of B cell tumors, including non-GCB DLBCL models that are resistant to Bruton's tyrosine kinase (BTK) inhibitors. NF-kB target genes include the pro-survival family members BCL-XL and BCL2-A1, which aid in regulation of the intrinsic apoptosis pathway. As ABBV-MALT1-induced inhibition of the NF-kB pathway resulted in downregulation of these genes, we hypothesized that the associated tumor models would become increasingly dependent on the pro-survival family member BCL-2. To test this hypothesis, combination studies of ABBV-MALT1 and the selective BCL-2 inhibitor venetoclax were performed in both cell line and patient-derived xenograft models of DLBCL. Herein we show that concomitant administration of ABBV-MALT1 and venetoclax results in dramatic antitumor activity in all models tested in vivo. This efficacy also translates to primary patient CLL cells in vitro where the combination confers greater levels of apoptosis compared to either agent alone. Summary/Conclusion: ABBV-MALT1 demonstrates robust single agent anti-tumor activity in malignant B cell models that are resistant to BTK inhibitors. Moreover, combination of ABBV-MALT1 with the BCL-2 inhibitor venetoclax shows synergistic cell killing of B cell tumors in vitro and dramatic tumor regression in vivo. Together, these data indicate that MALT1 inhibition may overcome BTK inhibitor resistance and combine with venetoclax to effectively treat patients with DLBCL, CLL and other B cell malignancies. Disclosures All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.

Abstract 6156: MALT1 protease inhibition overcomes BTK inhibitor resistance and shows synergistic activity with venetoclax in models of B cell lymphoma and leukemia

Abstract Introduction: Specific B cell malignancies, including CLL and the aggressive non-GCB subtype of DLBCL, are driven by constitutive activation of the B cell receptor (BCR) pathway and the transcription factor NF-κB. Pharmacological inhibition of MALT1 protease, a key mediator of the BCR/NF-κB signal transduction pathway, may therefore provide an attractive treatment option for patients with these cancers. Further, as combination therapy is often required for the treatment of aggressive B cell malignancies, the identification of therapies that synergistically combine with MALT1 inhibitors could afford additional and promising treatment options. Experimental Procedures: A highly potent and orally bioavailable MALT1 protease inhibitor (ABBV-MALT1) was used to test the hypothesis that MALT1 inhibition will abrogate the proliferation of preclinical models of B cell malignancies in vitro and in vivo. Tumors treated with ABBV-MALT1 were subjected to transcriptomic and functional proteomic assays to elucidate molecular mechanisms of action and rational combination partners. Results: Mechanistic studies reveal that ABBV-MALT1 effectively inhibits signal transduction of the BCR pathway and reduces NF-κB gene activation in non-GCB DLBCL cell lines resulting in cell cycle arrest and diminished viability. In vivo, oral administration of this compound demonstrates robust tumor growth inhibition in several models of B cell tumors, including non-GCB DLBCL models that are resistant to Bruton’s tyrosine kinase (BTK) inhibitors. NF-κB target genes include the pro-survival family members BCL-XL and BCL2-A1, which aid in regulation of the intrinsic apoptosis pathway. As ABBV-MALT1-induced inhibition of the NF-κB pathway resulted in downregulation of these genes, we hypothesized that the associated tumor models would become increasingly dependent on the pro-survival family member BCL-2. To test this hypothesis, combination studies of ABBV-MALT1 and the selective BCL-2 inhibitor venetoclax were performed in both cell line and patient-derived xenograft models of DLBCL. Herein we show that concomitant administration of ABBV-MALT1 and venetoclax results in dramatic antitumor activity in all models tested in vivo. This efficacy also translates to primary patient CLL cells in vitro where the combination confers greater levels of apoptosis compared to either agent alone. Conclusion: ABBV-MALT1 demonstrates robust single agent anti-tumor activity in malignant B cell models that are resistant to BTK inhibitors. Moreover, combination of ABBV-MALT1 with the BCL-2 inhibitor venetoclax shows synergistic cell killing of B cell tumors in vitro and dramatic tumor regression in vivo. Together, these data indicate that MALT1 inhibition may overcome BTK inhibitor resistance and combine with venetoclax to effectively treat patients with B cell malignancies. Citation Format: Joshua P. Plotnik, Raghuveer S. Mali, Velitchka Bontcheva, Colleen Dowell, Jun Chen, Adam E. Richardson, Ryan A. McClure, Paul Jung, Lan Pham, Andrew J. Souers, Jonathan A. Meulbroek, William N. Pappano. MALT1 protease inhibition overcomes BTK inhibitor resistance and shows synergistic activity with venetoclax in models of B cell lymphoma and leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6156.

Single-cell multiomics reveal the scale of multilayered adaptations enabling CLL relapse during venetoclax therapy

AbstractVenetoclax (VEN) inhibits the prosurvival protein BCL2 to induce apoptosis and is a standard therapy for chronic lymphocytic leukemia (CLL), delivering high complete remission rates and prolonged progression-free survival in relapsed CLL but with eventual loss of efficacy. A spectrum of subclonal genetic changes associated with VEN resistance has now been described. To fully understand clinical resistance to VEN, we combined single-cell short- and long-read RNA-sequencing to reveal the previously unappreciated scale of genetic and epigenetic changes underpinning acquired VEN resistance. These appear to be multilayered. One layer comprises changes in the BCL2 family of apoptosis regulators, especially the prosurvival family members. This includes previously described mutations in BCL2 and amplification of the MCL1 gene but is heterogeneous across and within individual patient leukemias. Changes in the proapoptotic genes are notably uncommon, except for single cases with subclonal losses of BAX or NOXA. Much more prominent was universal MCL1 gene upregulation. This was driven by an overlying layer of emergent NF-κB (nuclear factor kappa B) activation, which persisted in circulating cells during VEN therapy. We discovered that MCL1 could be a direct transcriptional target of NF-κB. Both the switch to alternative prosurvival factors and NF-κB activation largely dissipate following VEN discontinuation. Our studies reveal the extent of plasticity of CLL cells in their ability to evade VEN-induced apoptosis. Importantly, these findings pinpoint new approaches to circumvent VEN resistance and provide a specific biological justification for the strategy of VEN discontinuation once a maximal response is achieved rather than maintaining long-term selective pressure with the drug.

Abstract 1797: Bcl-2 up-regulation mediates taxane resistance downstream of APC loss

Abstract Patients with triple negative breast cancer (TNBC) are treated with traditional chemotherapy, such as paclitaxel (PTX). Despite the efficacy of taxanes, many tumors will recur due to drug resistance. Therefore, the need to understand the mechanism behind drug resistance is critical to improve patient outcome and survival. Our lab was the first to show that loss of the Adenomatous Polyposis Coli (APC) tumor suppressor caused resistance to PTX in the MDA-MB-157 human TNBC cell line. In the absence of APC, apoptosis induction was decreased, as measured through cleaved caspase 3 and annexin/PI staining. To understand the molecular mechanisms behind APC-mediated PTX response, we analyzed the BCL-2 family of proteins and found a robust increase of the pro-survival family member, Bcl-2. The BH3 mimetic, ABT-199 (Venetoclax), which specifically targets Bcl-2, has been used as a single or combination therapy in multiple hematologic malignancies. In addition, ABT-199 has shown promise in multiple subtypes of breast cancer. Therefore, we used ABT-199 in combination with PTX to address the hypothesis that APC-induced Bcl-2 increase is responsible for PTX resistance. Combination treatment in three CRISPR-mediated APC knockout TNBC cell lines (MDA-MB-157, MDA-MB-231, and SUM159) caused changes in cell proliferation and apoptosis. Combined, these data suggest restored sensitivity to PTX using ABT-199. Our studies are the first to show that Bcl-2 inhibition can restore PTX-sensitivity in APC mutant breast cancer cells. These studies are critical to advance better treatment regimens in patients with TNBC. Citation Format: Sara Maloney, Sarah Zabala, Grace Richmond, Jenifer R. Prosperi. Bcl-2 up-regulation mediates taxane resistance downstream of APC loss [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1797.

The Effect of the Proteasome Inhibitor Ixazomib (MLN9708) and UPR Mediated Therapy in Multiple Myeloma Cells and Bone Marrow Niche

Background: The concept of disrupting proteostasis in order to weaken cancer cells and challenge their microenvironment has shown striking clinical success, especially by applying proteasome inhibitors (PIs) in multiple myeloma (MM). The proteasome inhibitor MLN9708 (ixazomib) demonstrated significant preclinical and clinical anti-myeloma activity combined with a favorable toxicity profile. Proteostasis is also maintained by different adaptive pathways, including heat shock response (HSR) and the unfolded protein response (UPR). MM cells are characterized by mis- and overproduction and thus accumulation of immunoglobulin (Ig) within the bone marrow, relying on the UPR system to overcome stress due to this accumulation of unfolded proteins. The UPR mediates its function through three pathways, namely IRE-1α, PERK and ATF6. The most conserved UPR pathway is mediated by IRE-1α which reduces the ER stress load by splicing the XBP1 mRNA through removal of a 26-nucleotide intron.Aim: The UPR represents a vulnerability of MM cells which we aimed to harness with a novel combination therapy of UPR inhibition and proteasome inhibition.Methods: We used two MM cell lines (KMS11 and RPMI-8226) to validate the cytotoxic effects of ixazomib together with the IRE-1α inhibitors A106 and STF-083010 by measuring cell viability and proliferation, assessing synergistic or additive effects with the Bliss formula and quantifying the apoptotic cell fraction. For gene expression assays, total RNA was isolated and relative quantification of target gene expression was calculated by the comparative threshold cycle method.Results: Our in vitro results demonstrate that the combination of ixazomib and IRE-1α inhibitors has a synergistic effect on reducing of viability and proliferation of multiple myeloma cells and induces apoptosis as a single agent and in combination with IRE-1α inhibitors (Viability of KMS11 with ixazomib treatment 13.5% ± 0.35 vs 7.3% ± 0.23 combination with STF-083010, RPMI-8226 with ixazomib treatment 33.4% ± 0.56 vs 15.7% ± 0.07 combination with STF-083010; apoptotic fraction of KMS11 with ixazomib treatment 67.1% vs 76.9% combination with STF-083010, RPMI-8226 with ixazomib treatment 31.7% vs 36.3% combination with STF-083010). Ixazomib in combination with STF-083010 increased proteolytic cleavage of PARP, a signature of apoptosis and downstream target of Caspase-3, increased G1 phase regulators (p21cip1, p27kip1) and up-regulated pro-apoptotic regulator protein, BAX. In addition, we found that combination treatment increased the expression of pro-apoptotic BH3-only family members, PUMA and NOXA and decreased the expression of Bcl2 pro-survival family members (Bcl2, Bcl-xl).Mesenchymal stromal cells (MSCs) mimic the positive effects of bone marrow niche on MM cells in vitro . Co-culture of MM cells with Telomerase-immortalized Human Mesenchymal Stem Cell (hMSC-TERT) resulted in support of survival and cell growth of the two MM cell lines, (KMS11-hMSC-TERT with ixazomib treatment 55.4% ± 2.3 vs 24.2% ± 1.0 combination with STF-083010, RPMI-8226 with ixazomib treatment 68.6% ± 0.14 vs 17.8% ± 0.42 combination with STF-083010). However, the combination therapy of ixazomib with IRE-1α inhibitors significantly reduced the viability of multiple myeloma cells even when protected by the bone marrow niche microenvironment, confirming the notion that this combination therapy abrogates microenvironmental support of myeloma cells.Quantitative PCR (qRT-PCR) revealed a relevant up-regulation of XBP1s mRNA levels by ixazomib monotherapy in the two MM cell lines whereas the combination of proteasome inhibition by ixazomib with IRE-1α inhibition significantly reduced the expression level of XBP1s (KMS11 with ixazomib 77.3% ± 3.4 vs 34.8% ± 2.7 combination with STF-083010, RPMI-8226 with ixazomib 22.2.0% ± 1.7 vs 11.9% ± 1.7 combination with STF-083010), again demonstrating the IRE-1α pathway of the UPR as an important target in the therapy of multiple myeloma. A second experimental set with the IRE-1α inhibitor A106 showed similar results, for which data are not explicitly mentioned here.Conclusion: Taken together, our findings demonstrate that targeting the UPR specifically via inhibition of IRE-1α in combination with ixazomib is a promising therapeutic strategy in multiple myeloma. DisclosuresSalimi:Takeda: Consultancy, Research Funding. Schroeder:Takeda: Consultancy, Research Funding. Vieri:Takeda: Consultancy, Research Funding. Brümmendorf:Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Takeda: Consultancy, Research Funding. Appelmann:Takeda: Consultancy, Research Funding. Kharabi:Takeda: Consultancy, Research Funding.

Abstract TS3-1: Beyond B cells: Targeting BCL-2 pro-survival proteins in breast cancer

Abstract Resisting cell death is a hallmark of cancer. B-cell lymphoma 2 (BCL-2) is the prototypic member of a 'pro-survival' family that protects against cell death (apoptosis). Considerable progress has been made in deciphering its mechanism of action since it was first identified as the gene involved in the t(14;18) chromosomal translocation in follicular lymphoma about 30 years ago. BCL-2 and other pro-survival family members MCL-1 and BCL-XL have been shown to play a central role in the intrinsic apoptotic pathway: They keep the pro-apoptotic 'effector' proteins BAK and BAX in check and also neutralize a group of 'sensor' proteins that can be activated by stress signals (including cytotoxic and targeted therapies). These sensor proteins dock onto a surface hydrophobic groove on BCL-2 through their BH3 domain. BCL-2 proteins therefore play a vital role as guardians against apoptosis by inhibiting sensor and effector protein activity. Potent and specific 'BH3 mimetics' have recently been developed that mimic the BH3 domain of the sensor proteins. BH3 mimetics bind to and neutralize BCL-2 (and/or its pro-survival relatives) to help trigger apoptosis. Venetoclax (ABT-199/GDC-0199) is a potent and selective BCL-2 inhibitor. Its utility in targeting BCL-2 in hematological malignancies has recently become apparent, with remarkable clinical responses observed in heavily pre-treated patients with treatment refractory chronic lymphocytic leukemia, resulting in the FDA granting fast-track approval. Combination therapy with venetoclax is also showing activity in other hematological malignancies. BCL-2 is overexpressed in approximately 80% of ER-positive (ER+) primary breast cancer, while MCL-1 overexpression is a feature of TNBC. Our group has explored the role of the BCL-2 inhibitor venetoclax and MCL-1 inhibitor S63845 in pre-clinical models of breast cancer. Using patient-derived xenograft (PDX) models of ER+ breast cancer, venetoclax was found to synergize with tamoxifen to improve tumor response by increasing apoptosis. Similarly, the MCL-1 inhibitor S63845 augmented tumor response to docetaxel in PDX models of TNBC. These findings have led to a phase 1b dose escalation and expansion study, 'mBEP', combining venetoclax with tamoxifen in ER+ and BCL-2+ metastatic breast cancer. Findings from the dose escalation component have revealed that the combination is safe and well-tolerated and identified the recommended phase 2 dose. In the dose expansion phase, notable clinical activity has been observed. Since venetoclax therapy can be associated with 'on-target' lymphopenia, its effect on peripheral blood immune subsets was also explored. A role for combining venetoclax with 'modern day' endocrine therapies is not known. This question may in part be informed by a randomised phase II study of fulvestrant with or without venetoclax in women with ER+ metastatic breast cancer that has progressed on a CKD4/6 inhibitor (VERONICA, WO40181), recently commenced. In addition, preliminary pre-clinical modeling suggests that combining venetoclax with fulvestrant and a CDK4/6 inhibitor can augment tumor response. Since CDK4/6 inhibitors have recently been shown to modulate immune responses, establishing the immune effects of combination therapy could also be important. Citation Format: Lindeman G. Beyond B cells: Targeting BCL-2 pro-survival proteins in breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr TS3-1.

By reducing global mRNA translation in several ways, 2-deoxyglucose lowers MCL-1 protein and sensitizes hemopoietic tumor cells to BH3 mimetic ABT737

Drugs targeting various pro-survival BCL-2 family members (‘‘BH3 mimetics’’) have efficacy in hemopoietic malignancies, but the non-targeted pro-survival family members can promote resistance. Pertinently, the sensitivity of some tumor cell lines to BH3 mimetic ABT737, which targets BCL-2, BCL-XL, and BCL-W but not MCL-1, is enhanced by 2-deoxyglucose (2DG). We found that 2DG augmented apoptosis induced by ABT737 in 3 of 8 human hemopoietic tumor cell lines, most strongly in pre-B acute lymphocytic leukemia cell line NALM-6, the focus of our mechanistic studies. Although 2DG can lower MCL-1 translation, how it does so is incompletely understood, in part because 2DG inhibits both glycolysis and protein glycosylation in the endoplasmic reticulum (ER). Its glycolysis inhibition lowered ATP and, through the AMPK/mTORC1 pathway, markedly reduced global protein synthesis, as did an ER integrated stress response. A dual reporter assay revealed that 2DG impeded not only cap-dependent translation but also elongation or cap-independent translation. MCL-1 protein fell markedly, whereas 12 other BCL-2 family members were unaffected. We ascribe the MCL-1 drop to the global fall in translation, exacerbated for mRNAs with a structured 5′ untranslated region (5′UTR) containing potential regulatory motifs like those in MCL-1 mRNA and the short half-life of MCL-1 protein. Pertinently, 2DG downregulated two other short-lived oncoproteins, MYC and MDM2. Thus, our results support MCL-1 as a critical 2DG target, but also reveal multiple effects on global translation that may well also affect its promotion of apoptosis.

Assessment of Dynamic BCL-2 Protein Shuttling Between Outer Mitochondrial Membrane and Cytosol.

BCL-2 proteins control stress-dependent commitment to the programmed cell death apoptosis. In nonapoptotic cells the proapoptotic BCL-2 proteins BAX and BAK but also prosurvival family members, like BCL-xL or MCL-1, translocate to the outer mitochondrial membrane (OMM) and retrotranslocate from the mitochondria back into the cytosol. The resulting equilibrium produces a broad range of localization pattern observed for BAX and BAK in human cells and shows correlation between relative BAX and BAK localizations and cellular predisposition to apoptosis. The retrotranslocation of BCL-2 proteins from the OMM can be measured using fluorescence-labeled protein in intact cells or endogenous protein from isolated heavy membrane fractions.

Hepatocyte growth factor renders BRAF mutant human melanoma cell lines resistant to PLX4032 by downregulating the pro-apoptotic BH3-only proteins PUMA and BIM.

A large proportion of melanomas harbour the activating BRAFV600E mutation that renders these cells dependent on MAPK signalling for their survival. Although the highly specific and clinically approved BRAFV600E kinase inhibitor, PLX4032, induces apoptosis of melanoma cells bearing this mutation, the underlying molecular mechanisms are not fully understood. Here, we reveal that PLX4032-induced apoptosis depends on the induction of the pro-apoptotic BH3-only protein PUMA with a minor contribution of its relative BIM. Apoptosis could be significantly augmented when PLX4032 was combined with an inhibitor of the pro-survival protein BCL-XL, whereas neutralization of the pro-survival family member BCL-2 caused no additional cell death. Although the initial response to PLX4032 in melanoma patients is very potent, resistance to the drug eventually develops and relapse occurs. Several factors can cause melanoma cells to develop resistance to PLX4032; one of them is the activation of the receptor tyrosine kinase cMET on melanoma cells by its ligand, hepatocyte growth factor (HGF), provided by the tumour microenvironment or the cancer cells themselves. We found that HGF mediates resistance of cMET-expressing BRAF mutant melanoma cells to PLX4032-induced apoptosis through downregulation of PUMA and BIM rather than by increasing the expression of pro-survival BCL-2-like proteins. These results suggest that resistance to PLX4032 may be overcome by specifically increasing the levels of PUMA and BIM in melanoma cells through alternative signalling cascades or by blocking pro-survival BCL-2 family members with suitable BH3 mimetic compounds.

Eliminating Legionella by inhibiting BCL-XL to induce macrophage apoptosis.

Human pathogenic Legionella replicate in alveolar macrophages and cause a potentially lethal form of pneumonia known as Legionnaires' disease(1). Here, we have identified a host-directed therapeutic approach to eliminate intracellular Legionella infections. We demonstrate that the genetic deletion, or pharmacological inhibition, of the host cell pro-survival protein BCL-XL induces intrinsic apoptosis of macrophages infected with virulent Legionella strains, thereby abrogating Legionella replication. BCL-XL is essential for the survival of Legionella-infected macrophages due to bacterial inhibition of host-cell protein synthesis, resulting in reduced levels of the short-lived, related BCL-2 pro-survival family member, MCL-1. Consequently, a single dose of a BCL-XL-targeted BH3-mimetic therapy, or myeloid cell-restricted deletion of BCL-XL, limits Legionella replication and prevents lethal lung infections in mice. These results indicate that repurposing BH3-mimetic compounds, originally developed to induce cancer cell apoptosis, may have efficacy in treating Legionnaires' and other diseases caused by intracellular microbes.

Abstract IA19: Targeting the BCL-2 family in breast cancer

Abstract The steps taken to evade cell death are a recognized hallmark of cancer. Intense efforts over the last three decades have provided important insights into the molecular mechanisms that govern programmed cell death and how tumors recalibrate key survival pathways to sustain their growth. The pro-survival protein BCL-2 is overexpressed in approximately 75% of breast cancer, where it is has emerged as an important prognostic marker. Indeed, BCL-2 expression is a key component of the Oncotype DX assay. BCL-2 overexpression is even more prominent in luminal tumors (~85%), reflecting its estrogen-responsiveness. Other key pro-survival family members, MCL-1 and BCL-XL, are also commonly expressed across breast cancer subtypes. This group of pro-survival guardian proteins play a critical role in keeping pro-apoptotic effector proteins (such as BAX and BAK) in check. The effector proteins are essential for commitment to apoptosis, which occurs following mitochondrial cytochrome c release and caspase activation, resulting in cell death. BCL-2 guardian proteins also neutralize a group of sensor proteins (such as BIM). The sensor proteins are triggered by distinct cytotoxic stimuli (such as chemotherapy) to activate the effectors BAX and BAK. The sensor proteins are termed BH3 only proteins, as they lack the BCL-2 Homology (BH) domains BH1, BH2 and BH4 found in the two other subgroups. While BCL-2 is an important prognostic marker in breast cancer, its precise role as a predictive marker of tumor response has yet to be properly clarified. Nevertheless, there is increasing evidence that augmented levels of pro-survival BCL-2 proteins can prime tumors for death induced by conventional chemotherapy or endocrine therapy. This is due to the high occupation of pro-survival BCL-2 proteins by pro-apoptotic BH3 proteins, which can be activated by therapy, thereby committing tumor cells to apoptosis. Small molecule inhibitors termed BH3 mimetics that bind and neutralize BCL-2 pro-survival proteins have recently been described. These are showing considerable promise in early phase studies of lymphoid malignancies. We recently explored the feasibility of targeting luminal B tumors in combination therapy comprising endocrine therapy (tamoxifen) and a BH3 mimetic (ABT-737 or ABT-199) using patient derived xenograft (PDX) models of primary breast cancer. Tumor response and overall survival were significantly improved by combination therapy, when compared to tamoxifen alone. Moreover, synergy between BH3 mimetics and PI3K/mTOR inhibitors could be exploited by concomitant targeting of both survival pathways, a strategy that appeared both safe and effective. Since the potent and selective BCL-2 inhibitor ABT-199 was effective in these models (despite abundant BCL-XL expression), BCL-2 may be a crucial target for some luminal tumors, where we speculate it could provide a suitable companion biomarker for patient selection. This work has formed the basis for a Phase 1b study of BCL-2 inhibition with ABT-199 (venetoclax) in combination with tamoxifen in metastatic ER-positive breast cancer (ISRCTN98335443). The prospects for the development of BCL-XL and MCL-1 inhibitors will also be discussed. Citation Format: Geoffrey J. Lindeman, Delphine Merino, François Vaillant, James R. Whittle, Sheau Wen Lok, Kylie Shackleton, Jane E. Visvader. Targeting the BCL-2 family in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr IA19.

Targeting Cancer Stem Cell Survival in Plasma Cell Leukemia with a Pan-BCL2 Inhibitor

INTRODUCTIONRepresenting ten percent of all hematologic malignancies, multiple myeloma (MM) is typified by clonal plasma cell proliferation in the bone marrow (BM) and may progress to a therapy-resistant stage characterized by circulating malignant plasma cells, which is termed plasma cell leukemia, (PCL). Notably, copy number amplification involving the myeloid cell leukemia (MCL)-1 locus, and translocations at the BCL2 locus have been described in high-risk MM. Moreover, BCL2 family members, including both MCL1 and BCL2, are highly expressed in relapsed MM. Finally, downregulation of BCL2 family members was reported to increase sensitivity to lenalidomide, a standard treatment for MM, suggesting that BCL2-targeted treatments may obviate therapeutic resistance.Sabutoclax, a potent small molecule inhibitor of 6 anti-apoptotic BCL2 family proteins, was shown to inhibit cancer stem cell (CSC) survival in chronic myeloid leukemia (CML) (Goff et al Cell Stem Cell 2013). Thus, we investigated BCL2 pro-survival splice isoform expression in therapy-resistant MM and PCL and evaluated whether Sabutoclax reduces malignant plasma cell burden in humanized primagraft assays.METHODS AND RESULTSBCL2 pro survival expression in primary myeloma samplesMononuclear cells from MM or PCL samples were isolated by Ficoll-Paque density gradient separation and collected for RNA extraction or FACS Aria purified into CD138+ or CD34-/CD138- cancer stem cells (CSC) subsets. To evaluate BCL2 pro-survival family member expression, splice isoform-specific quantitative PCR was performed to measure expression of pro-survival long isoforms compared with short splice isoforms, which are pro-apoptotic. Interestingly, MM and PCL patients displayed higher levels of BCL2-L, MCL1-L, BCLX-L and BLF1-L compared to normal controls. Moreover, prolonged lenalidomide exposure increased BCL2-L and MCL1-L expression in the myeloma cell line H929, compared to untreated cells.Sabutoclax Treatment of a Novel Humanized Plasma Cell Leukemia Primagraft ModelMononuclear cells from three primary PCL patient samples were stably transduced with a GFP-luciferase lentiviral vector and transplanted intrahepatically in newborn RAG2γ-/- c-/- mice. Engraftment was monitored by peripheral blood free light chain ELISA assays. Flow cytometric analyses revealed robust engraftment of PCL cells in bone marrow, spleen, liver and peripheral blood. Once transplanted mice displayed significant tumor burden above background free light chain levels, animals were randomized by ELISA values in vehicle versus Sabutoclax groups. Sabutoclax was selected because this pan-BCL2 targeted compound, unlike related BCL2 inhibitors such as ABT-199, also inhibits MCL1. Sabutoclax (10mg/kg) was administered intravenously twice weekly for two to four doses. Sabutoclax treated PCL mice showed reduced human plasma cell burden in bone marrow and spleen tissues compared to vehicle controls.CONCLUSIONExpression of pro-survival BCL2 splice isoforms, including BCL2-L and MCL1-L, portends PCL engraftment in immunocompromised mice. Treatment of human PCL engrafted mice with Sabutoclax reduces malignant plasma cell survival in hematopoietic tissues. Thus, selective targeting of pro-survival isoform expressing CSC with a pan-BCL2 inhibitor may abolish BCL2 and MCL1-dependent therapeutic resistance in MM and PCL. DisclosuresJamieson:GlaxoSmithKline: Research Funding; Johnson & Johnson: Research Funding.

Targeting BCL-2 to enhance vulnerability to therapy in estrogen receptor-positive breast cancer.

The last three decades have seen significant progress in our understanding of the role of the pro-survival protein BCL-2 and its family members in apoptosis and cancer. BCL-2 and other pro-survival family members including Mcl-1 and BCL-XL have been shown to have a key role in keeping pro-apoptotic 'effector' proteins BAK and BAX in check. They also neutralize a group of 'sensor' proteins (such as BIM), which are triggered by cytotoxic stimuli such as chemotherapy. BCL-2 proteins therefore have a central role as guardians against apoptosis, helping cancer cells to evade cell death. More recently, an increasing number of BH3 mimetics, which bind and neutralize BCL-2 and/or its pro-survival relatives, have been developed. The utility of targeting BCL-2 in hematological malignancies has become evident in early-phase studies, with remarkable clinical responses seen in heavily pretreated patients. As BCL-2 is overexpressed in ~75% of breast cancer, there has been growing interest in determining whether this new class of drug could show similar promise in breast cancer. This review summarizes our current understanding of the role of BCL-2 and its family members in mammary gland development and breast cancer, recent progress in the development of new BH3 mimetics as well as their potential for targeting estrogen receptor-positive breast cancer.

Abstract 847: PIAS3 induction of apoptosis is p53-independent and has STAT3-independent mediators.

Abstract Protein inhibitor of activated STAT3 (PIAS3) is an endogenous inhibitor of STAT3 that negatively regulates STAT3 transcriptional activity and cell growth and demonstrates limited expression in the majority of human squamous cell carcinomas of the lung. In the present study we sought to determine if PIAS3 inhibits cell growth in non-small cell lung cancer (NSCLC) cell lines by induction of apoptosis and further determine the dependence of PIAS3 activity on p53 status by using both wild-type and p53-null cells. Our results demonstrate that over-expression of PIAS3 promotes caspase 3 activation and PARP cleavage. Furthermore, the expression of pro-survival family members BclXL and Bcl-2 is decreased. These effects were observed after both transient and regulated expression of exogenous PIAS3 and were independent of p53 status. PIAS3 inhibition of STAT3 luciferase activity was also p53 independent. Microarray experiments were performed to further investigate the STAT3-dependence of PIAS3-induced apoptosis by comparing its gene expression signature with that of STAT3 siRNA treated cells. IPA pathway analysis showed that a number of genes related to cell death were significantly increased by PIAS3. Furthermore, a subset of apoptotic genes, including CIDEC and DAPK2, were uniquely expressed only after PIAS3 expression and not after STAT3 siRNA. Thus, PIAS3 may represent a promising lung cancer therapeutic target because of its p53-independent efficacy as well as its potential to synergize with direct STAT3 inhibitors. Citation Format: Snehal Dabir, Yu Liu, Gary Wildey, Afshin Dowlati. PIAS3 induction of apoptosis is p53-independent and has STAT3-independent mediators. [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 847. doi:10.1158/1538-7445.AM2013-847

BCL2L1 (BCL-X) promotes survival of adult and developing retinal ganglion cells

The Bcl-2 family is responsible for regulating cell death pathways in neurons during development, after injury and in disease. The activation of the pro-death family member BAX is often the final step before cell death in neurons. Pro-survival family members such as BCL-X (BCL2L1) act to inhibit BAX activation. Overexpression studies have suggested that BCL-X could play an important physiological role in mediating neuronal viability. Loss-of-function studies performed in vivo have implicated BCL-X as a mediator of neuronal survival during the early stages of neurodevelopment. To assess whether BCL-X is needed to promote the survival of neurons in the central nervous system throughout life, Bcl-x was conditionally removed from the optic cup or throughout the adult mouse. During development BCL-X was required for the survival of differentiating retinal ganglion cells (RGCs) leading up to their normal window of developmental death. Despite its expression in adult RGCs, BCL-X was not required for maintaining RGC viability in adult retinas. However, the loss of BCL-X in adult RGCs did significantly increase the rate of death of RGCs after axonal injury. Thus, in developing and injured RGCs there appears to be an active cell survival program preventing neuronal death.

NMR Solution Structure of a Photoswitchable Apoptosis Activating Bak Peptide Bound to Bcl-xL

The Bcl-2 family of proteins includes the major regulators and effectors of the intrinsic apoptosis pathway. Cancers are frequently formed when activation of the apoptosis mechanism is compromised either by misregulated expression of prosurvival family members or, more frequently, by damage to the regulatory pathways that trigger intrinsic apoptosis. Short peptides derived from the pro-apoptotic members of the Bcl-2 family can activate mechanisms that ultimately lead to cell death. The recent development of photocontrolled peptides that are able to change their conformation and activity upon irradiation with an external light source has provided new tools to target cells for apoptosis induction with temporal and spatial control. Here, we report the first NMR solution structure of a photoswitchable peptide derived from the proapoptotic protein Bak in complex with the antiapoptotic protein Bcl-x(L). This structure provides insight into the molecular mechanism, by which the increased affinity of such photopeptides compared to their native forms is achieved, and offers a rationale for the large differences in the binding affinities between the helical and nonhelical states.

Discovery of Marinopyrrole A (Maritoclax) as a Selective Mcl-1 Antagonist that Overcomes ABT-737 Resistance by Binding to and Targeting Mcl-1 for Proteasomal Degradation

The anti-apoptotic Bcl-2 family of proteins, including Bcl-2, Bcl-X(L) and Mcl-1, are well-validated drug targets for cancer treatment. Several small molecules have been designed to interfere with Bcl-2 and its fellow pro-survival family members. While ABT-737 and its orally active analog ABT-263 are the most potent and specific inhibitors to date that bind Bcl-2 and Bcl-X(L) with high affinity but have a much lower affinity for Mcl-1, they are not very effective as single agents in certain cancer types because of elevated levels of Mcl-1. Accordingly, compounds that specifically target Mcl-1 may overcome this resistance. In this study, we identified and characterized the natural product marinopyrrole A as a novel Mcl-1-specific inhibitor and named it maritoclax. We found that maritoclax binds to Mcl-1, but not Bcl-X(L), and is able to disrupt the interaction between Bim and Mcl-1. Moreover, maritoclax induces Mcl-1 degradation via the proteasome system, which is associated with the pro-apoptotic activity of maritoclax. Importantly, maritoclax selectively kills Mcl-1-dependent, but not Bcl-2- or Bcl-X(L)-dependent, leukemia cells and markedly enhances the efficacy of ABT-737 against hematologic malignancies, including K562, Raji, and multidrug-resistant HL60/VCR, by ∼60- to 2000-fold at 1-2 μM. Taken together, these results suggest that maritoclax represents a new class of Mcl-1 inhibitors, which antagonizes Mcl-1 and overcomes ABT-737 resistance by targeting Mcl-1 for degradation.

PD08-02: Targeting BCL-2 Expressing Breast Tumors with BH3-Mimetics – A New Class of Drugs in Breast Cancer?

Abstract Background: Impairment of apoptosis is a hallmark of cancer and can result in resistance to chemotherapy. Tumor resistance to apoptosis is frequently acquired through deregulated expression of BCL-2 family members or inactivation of the p53 tumor suppressor pathway. Over-expression of the pro-survival protein BCL-2 is common in breast cancer (where it is readily detected by immunostaining), and has been shown to be an important prognostic marker. A potential role for BCL-2 as a therapeutic target in breast cancer, however, has not been explored. Recently, small molecules termed ‘BH3-mimetics’ have been developed that mimic the action of pro-apoptotic BH3-only proteins. These bind and neutralize pro-survival proteins including BCL-2. Here we have derived a panel of primary breast tumor xenografts (that include basal-like breast tumors) to study the efficacy of the BH3-mimetic ABT-737 combined with docetaxel in targeting BCL-2-positive breast cancer. Methods and Results: We first studied the expression of BCL-2, pro-survival family members BCL-XL and MCL-1, and the pro-apoptotic protein BIM in tissue microarrays of 197 primary breast tumors, which were subtyped on the basis of ER, PR, HER2, CK5/6 and EGFR expression. BCL-2 was overexpressed in luminal (83.3%), HER2−positive (50.0%), basal-like (18.5%) and ‘marker-null’ (41.4%) breast cancers. BCL-2-positive tumors generally co-expressed BCL-XL (96.2%), MCL-1 (94.7%) and BIM (93.5%). To determine whether the BH3-mimetic ABT-737 (which neutralizes BCL-2, BCL-XL and BCL-W) was effective in targeting BCL-2-expressing breast tumors, we generated a panel of 28 primary breast tumor xenografts in immunocompromised NOG mice. Five xenograft lines (838T, 24T, 315T, 13T and 806T) were selected for further analysis. Four were basal-like, and one (315T) was a luminal B tumor, as determined by gene profiling. Mice bearing tumor xenografts were treated with ABT-737 (50 mg/kg i.p. d1-10), docetaxel (10 mg/kg i.p. d1) or a combination in q21d cycles. Tumor response and overall survival were significantly improved by combination therapy, but only for tumors that expressed elevated levels of BCL-2. Treatment with ABT-737 alone was ineffective, suggesting that ABT-737 sensitized tumors to docetaxel. Combination therapy was accompanied by a marked increase in apoptosis and dissociation of BIM from BCL-2, indicating that a perturbation of BIM complexes may contribute to the activation of the apoptotic cascade. Notably, ABT-737 appeared effective in targeting BCL-2-expressing basal-like tumor xenografts (838T and 24T) harboring p53 mutations. Discussion: Primary breast tumor xenograft models that recapitulate the phenotype of the primary tumor have been developed as useful ‘proof-of-principle', pre-clinical models. Here we provide the first in vivo evidence that BH3-mimetics can be used to sensitize primary BCL-2-expressing breast tumors to taxane chemotherapy. Our results suggest that elevated BCL-2 expression constitutes a predictive response marker in breast cancer. These findings provide a rationale for the development of clinical protocols using the oral analogue ABT-263 (navitoclax) as an adjunct to taxane chemotherapy in BCL-2-expressing basal-like and luminal breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD08-02.