Pro-survival Bcl-2 Family Proteins Research Articles

Venetoclax-based Rational Combinations are Effective in Models of MYCN-amplified Neuroblastoma.

Venetoclax is a small molecule inhibitor of the prosurvival protein BCL-2 that has gained market approval in BCL-2-dependent hematologic cancers including chronic lymphocytic leukemia and acute myeloid leukemia. Neuroblastoma is a heterogenous pediatric cancer with a 5-year survival rate of less than 50% for high-risk patients, which includes nearly all cases with amplified MYCN We previously demonstrated that venetoclax is active in MYCN-amplified neuroblastoma but has limited single-agent activity in most models, presumably the result of other pro-survival BCL-2 family protein expression or insufficient prodeath protein mobilization. As the relative tolerability of venetoclax makes it amenable to combining with other therapies, we evaluated the sensitivity of MYCN-amplified neuroblastoma models to rational combinations of venetoclax with agents that have both mechanistic complementarity and active clinical programs. First, the MDM2 inhibitor NVP-CGM097 increases the prodeath BH3-only protein NOXA to sensitize p53-wild-type, MYCN-amplified neuroblastomas to venetoclax. Second, the MCL-1 inhibitor S63845 sensitizes MYCN-amplified neuroblastoma through neutralization of MCL-1, inducing synergistic cell killing when combined with venetoclax. Finally, the standard-of-care drug cocktail cyclophosphamide and topotecan reduces the apoptotic threshold of neuroblastoma, thus setting the stage for robust combination efficacy with venetoclax. In all cases, these rational combinations translated to in vivo tumor regressions in MYCN-amplified patient-derived xenograft models. Venetoclax is currently being evaluated in pediatric patients in the clinic, including neuroblastoma (NCT03236857). Although establishment of safety is still ongoing, the data disclosed herein indicate rational and clinically actionable combination strategies that could potentiate the activity of venetoclax in patients with amplified MYCN with neuroblastoma.

Targeting Bfl-1 via acute CDK9 inhibition overcomes intrinsic BH3-mimetic resistance in lymphomas

AbstractBH3 mimetics like venetoclax target prosurvival Bcl-2 family proteins and are important therapeutics in the treatment of hematological malignancies. We demonstrate that endogenous Bfl-1 expression can render preclinical lymphoma tumor models insensitive to Mcl-1 and Bcl-2 inhibitors. However, suppression of Bfl-1 alone was insufficient to fully induce apoptosis in Bfl-1–expressing lymphomas, highlighting the need for targeting additional prosurvival proteins in this context. Importantly, we demonstrated that cyclin-dependent kinase 9 (CDK9) inhibitors rapidly downregulate both Bfl-1 and Mcl-1, inducing apoptosis in BH3-mimetic–resistant lymphoma cell lines in vitro and driving in vivo tumor regressions in diffuse large B-cell lymphoma patient-derived xenograft models expressing Bfl-1. These data underscore the need to clinically develop CDK9 inhibitors, like AZD4573, for the treatment of lymphomas using Bfl-1 as a selection biomarker.

ANO1 Expression Orchestrates p27Kip1/MCL1-Mediated Signaling in Head and Neck Squamous Cell Carcinoma.

Simple SummaryOur aim was to elucidate the molecular mechanisms of how ANO1 contributes to oncogenic processes in squamous cell carcinoma of the head and neck (HNSCC). We explored transcriptional programs influenced by ANO1 knockdown in patient-derived UT-SCC cell lines with 11q13 amplification and ANO1 overexpression. ANO1 depletion led to downregulation of broad pro-survival BCL2 family protein members, including MCL1, and simultaneously induced upregulation of the cell cycle inhibitor p27Kip1 and its redistribution from the cytoplasm into the nucleus in the studied HNSCC cells. Gene set enrichment analysis highlighted pathways associated with perturbed cell cycle and apoptosis in the ANO1-depleted samples. Silencing of ANO1 and application of an ANO1-targeting small-molecule inhibitor led to ANO1 degradation and reduction of cell viability. These findings suggest that ANO1 has drug target potential that deserves further evaluation in preclinical in vivo models.Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of tumors that derive from the mucosal epithelium of the upper aerodigestive tract and present high mortality rate. Lack of efficient targeted-therapies and biomarkers towards patients’ stratification are caveats in the disease treatment. Anoctamin 1 (ANO1) gene is amplified in 30% of HNSCC cases. Evidence suggests involvement of ANO1 in proliferation, migration, and evasion of apoptosis; however, the exact mechanisms remain elusive. Aim of this study was to unravel the ANO1-dependent transcriptional programs and expand the existing knowledge of ANO1 contribution to oncogenesis and drug response in HNSCC. We cultured two HNSCC cell lines established from primary tumors harboring amplification and high expression of ANO1 in three-dimensional collagen. Differential expression analysis of ANO1-depleted HNSCC cells demonstrated downregulation of MCL1 and simultaneous upregulation of p27Kip1 expression. Suppressing ANO1 expression led to redistribution of p27Kip1 from the cytoplasm to the nucleus and associated with a cell cycle arrested phenotype. ANO1 silencing or pharmacological inhibition resulted in reduction of cell viability and ANO1 protein levels, as well as suppression of pro-survival BCL2 family proteins. Collectively, these data provide insights of ANO1 involvement in HNSCC carcinogenesis and support the rationale that ANO1 is an actionable drug target.

NOXA the BCL-2 Family Member behind the Scenes in Cancer Treatment

NOXA is a critical mediator of stress responses to anticancer drugs. This BH3-only protein sets the apoptotic threshold in cancer cells in response to chemotherapies by counteracting the prosurvival BCL-2 family protein MCL-1.

Role of Bcl-2 Family Proteins in Photodynamic Therapy Mediated Cell Survival and Regulation.

Photodynamic therapy (PDT) is a treatment modality that involves three components: combination of a photosensitizer, light and molecular oxygen that leads to localized formation of reactive oxygen species (ROS). The ROS generated from this promising therapeutic modality can be lethal to the cell and leads to consequential destruction of tumor cells. However, sometimes the ROS trigger a stress response survival mechanism that helps the cells to cope with PDT-induced damage, resulting in resistance to the treatment. One preferred mechanism of cell death induced by PDT is apoptosis, and B-cell lymphoma 2 (Bcl-2) family proteins have been described as a major determinant of life or death decision of the death pathways. Apoptosis is a cellular self-destruction mechanism to remove old cells through the biological event of tissue homeostasis. The Bcl-2 family proteins act as a critical mediator of a life–death decision of cells in maintaining tissue homeostasis. There are several reports that show cancer cells developing resistance due to the increased interaction of the pro-survival Bcl-2 family proteins. However, the key mechanisms leading to apoptosis evasion and drug resistance have not been adequately understood. Therefore, it is critical to understand the mechanisms of PDT resistance, as well as the Bcl-2 family proteins, to give more insight into the treatment outcomes. In this review, we describe the role of Bcl-2 gene family proteins’ interaction in response to disease progression and PDT-induced resistance mechanisms.

Regulation of Platelet Production and Life Span: Role of Bcl-xL and Potential Implications for Human Platelet Diseases.

Blood platelets have important roles in haemostasis, where they quickly stop bleeding in response to vascular damage. They have also recognised functions in thrombosis, immunity, antimicrobal defense, cancer growth and metastasis, tumour angiogenesis, lymphangiogenesis, inflammatory diseases, wound healing, liver regeneration and neurodegeneration. Their brief life span in circulation is strictly controlled by intrinsic apoptosis, where the prosurvival Bcl-2 family protein, Bcl-xL, has a major role. Blood platelets are produced by large polyploid precursor cells, megakaryocytes, residing mainly in the bone marrow. Together with Mcl-1, Bcl-xL regulates megakaryocyte survival. This review describes megakaryocyte maturation and survival, platelet production, platelet life span and diseases of abnormal platelet number with a focus on the role of Bcl-xL during these processes.

Pancreas-specific deletion of protein kinase D attenuates inflammation, necrosis, and severity of acute pancreatitis

BackgroundProtein kinase D (PKD) family, which includes PKD/PKD1, PKD2, and PKD3, has been increasingly implicated in the regulation of multiple cellular functions and human diseases. We recently reported that pharmacologic inhibition of PKD ameliorated the pathologic responses and severity of pancreatitis. However, to further investigate the importance of PKD family members in pancreatitis, it is necessary to explore the effects of pancreas-specific genetic inhibition of PKD isoform on pathology of pancreatitis. MethodsWe generated a mouse model (referred as PKD3Δpanc mice) with pancreas-specific deletion of PKD3, the predominant PKD isoform in mouse pancreatic acinar cells, by crossing Pkd3flox/flox mice with Pdx1-Cre transgenic mice which express Cre recombinase under the control of the mouse Pdx1 promoter. Pancreas-specific deletion of the PKD3 gene and PKD3 protein was confirmed by PCR and Western blot analysis. Experimental pancreatitis was induced in PKD3Δpanc and Pkd3flox/flox (control mice) littermates by intraperitoneal injections of cerulein or L-arginine. ResultsCompared to the control mice, PKD3Δpanc mice displayed significant attenuation in inflammation, necrosis, and severity of pancreatitis in both experimental models. PKD3Δpanc mice had markedly decreased NF-κB and trypsinogen activation, pancreatic mRNA expression of multiple inflammatory molecules, and the receptor-interacting protein kinase 1 (RIP1) activation in pancreatitis. PKD3Δpanc mice also had less pancreatic ATP depletion, increased pro-survival Bcl-2 family protein expression, and autophagy promotion. ConclusionWith PKD3Δpanc mouse model, we further demonstrated that PKD plays a critical role in pathobiological process of pancreatitis and PKD constitutes a novel therapeutic target to treat this disorder.

Regulation of Parkin-dependent mitophagy by Bcl-2-associated athanogene (BAG) family members.

Regulation of Parkin-dependent mitophagy by Bcl-2-associated athanogene (BAG) family members.

Discovery of a Copper-Based Mcl-1 Inhibitor as an Effective Antitumor Agent.

Myeloid cell leukemia 1 (Mcl-1), which belongs to the Bcl-2 family of prosurvival proteins, is a key regulator of cancer cell survival. To date, few drug-like Mcl-1 inhibitors have been reported. Herein, we report the preparation of 10 copper complexes with 9-substituted β-carboline ligands that act as metal-based Mcl-1 inhibitors. Complex 14 was identified as a potent and selective Mcl-1 inhibitor with strong in vitro antitumor activity. Mechanistic studies demonstrated that complex 14 disrupted Mcl-1-Bax/Bak heterodimerization and induced Bax/Bak-dependent apoptosis. In addition, complex 14 significantly (P < 0.001) inhibited tumor growth in vivo, induced tumor necrosis, and extended survival time in an NCI-H460 xenograft model. Furthermore, complex 14 showed no apparent toxicity in mice. Together, these findings indicate that complex 14 is a copper-based Mcl-1 inhibitor with high efficacy and low toxicity that could be developed for the treatment of Mcl-1-related cancers.

Abstract B20: Patient-derived organoids demonstrate synergistic effect of co-targeting Aurora kinase and prosurvival BCL2 family proteins

Abstract Many melanomas are resistant to standard-of-care immune checkpoint blockade and inhibitors of mutated BRAF. New effective therapies that work via immune and BRAF-independent mechanisms are urgently needed. We have previously demonstrated that an inhibitor of mitotic kinase Aurora A (AURKAi) can induce a distinct type of stable cell cycle known as senescence in melanoma tumors independent of their genetic background. Here we hypothesized that combination of AURKAi with a drug that selectively kills senescent cells, such as BCL2 inhibitor (BCLi) navitoclax, will be synthetically lethal to melanoma cells. Melanoma patient-derived organoids (PDOs) were used to test efficacy of AURKAi and BCLi combination treatment. To generate organoids, patient-derived tumor cells were cultured ex vivo in 3D matrix. Drug response was evaluated using fluorescent viability assay. Fifteen out of twenty PDOs were highly sensitive to AURKAi and BCLi combination treatment, exhibiting robust induction of cell death. No cell death was observed when either drug was added individually. NextGen DNA sequencing indicated that all sensitive PDOs retained wild-type TP53, while resistant tumors had mutations in this gene. Knockout or knockdown of TP53 abrogated AURKAi and BCLi-induced cell death in melanoma cells, suggesting that transcription factor p53 encoded by TP53 is a key mediator of drug response. Mechanistically, p53 was induced by AURKAi treatment independent of BCLi. However, single-agent AURKAi treatment resulted in activation of p53-mediated cell cycle arrest based on increased expression of CDK inhibitor p21 and decreased expression of proliferation markers. In contrast, addition of BCLi to AURKAi activated proapoptotic p53 target BAX and induced cleavage of PARP and caspases 3, 7, and 8. Treatment with pan-caspase inhibitor abrogated cell death, suggesting caspase-dependent apoptosis. Electron microscopy revealed morphologic signs of apoptosis in combination-treated cells, including cell blebbing, apoptotic bodies, and nuclear fragmentation. This suggests that BCLi shifts cell fate decision in AURKAi-treated cells towards apoptosis. In conclusion, combination treatment with AURKAi and BCl2i induced p53-dependent apoptosis. Preclinical evaluation of this drug combination in PDO model demonstrated robust efficacy against melanomas with wild-type p53. Since p53 mutations are relatively rare in melanoma, a combination of AURKAi and BCLi presents a promising therapeutic option for this deadly disease. Citation Format: Vijaya Bharti, Ashlyn Blevins, Vivian L. Weiss, Sheau C Chen, Roman Uzhachenko, Ann Richmond, Anna E. Vilgelm. Patient-derived organoids demonstrate synergistic effect of co-targeting Aurora kinase and prosurvival BCL2 family proteins [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B20.

Inhibition of Anti-Apoptotic Bcl-2 Proteins in Preclinical and Clinical Studies: Current Overview in Cancer.

The dynamic interplay between pro-death and pro-survival Bcl-2 family proteins is responsible for a cell’s fate. Due to the recognized relevance of this family in cancer progression and response to therapy, different efforts have made in recent years in order to develop small molecules able to target anti-apoptotic proteins such as Bcl-2, Bcl-xL and Mcl-1. The limitations of the first Bcl-2 family targeted drugs, regarding on-target and off-target toxicities, have been overcome with the development of venetoclax (ABT-199), the first BH3 mimetic inhibitor approved by the FDA. The purpose of this review is to discuss the state-of-the-art in the development of drugs targeting Bcl-2 anti-apoptotic proteins and to highlight the potential of their application as single agents or in combination for improving anti-cancer therapy, focusing in particular on solid tumors.

Comparison of putative BH3 mimetics AT-101, HA14-1, sabutoclax and TW-37 with ABT-737 in platelets

Platelet lifespan is regulated by intrinsic apoptosis. Platelet apoptosis can be triggered by BH3 mimetics that inhibit the pro-survival Bcl-2 family protein, Bcl-xL. Here, we investigated several small molecules that are reported to act as BH3 mimetics and compared their effects to the well-established BH3 mimetic, ABT-737. Platelet phosphatidylserine (PS) exposure was determined by flow cytometry. Changes in cytosolic Ca2+ signaling were detected using Cal-520. Plasma membrane integrity was determined by calcein leakage. The roles of caspases and calpain in these processes were determined using Q-VD-OPh and calpeptin, respectively. As previously reported, ABT-737 triggered PS exposure in a caspase-dependent manner and calcein loss in a caspase and calpain-dependent manner. In contrast, AT-101 and sabutoclax triggered PS exposure independently of caspases. HA14-1 also triggered PS exposure in a caspase-independent but calpain-dependent manner. There were also significant differences in the pattern and protease-dependency of cytosolic Ca2+ signaling in response to these drugs compared to ABT-737. Since there are clear differences between the action of ABT-737 and the other putative BH3 mimetics investigated here, AT-101, HA14-1 and sabutoclax cannot be considered as acting as BH3 mimetics in platelets. Furthermore, the platelet death caused by these drugs is likely to be distinct from apoptosis.

Abstract BL1: Deciphering stem and progenitor cells to understand breast cancer

Abstract Breast cancer is a highly heterogeneous disease at the molecular and pathological levels. To understand heterogeneity and potential ‘cells of origin’ of breast cancer, it is important to dissect the normal mammary epithelial differentiation hierarchy as well as the cellular composition of breast tumors. Discrete populations of mouse and human mammary epithelial cells have been isolated on the basis of cell surface marker expression, yielding three primary populations including a stem cell-enriched population and two luminal populations. Their responsiveness to female steroid hormones was investigated, revealing the importance of paracrine regulation of ER/PR– stem/progenitor cells by ER/PR+ luminal ‘hormone sensor’ cells. RANKL was found to be a key paracrine effector of progesterone signaling, underscoring the central role that female hormones play in normal mammary gland homeostasis and cancer. Consistent with this observation, a RANK+ luminal progenitor cell was found to be the likely target cell that gives rise to breast cancer in BRCA1 mutation carriers. Pre-clinical studies further indicate that it may be possible to target this ‘culprit’ cell with the RANKL inhibitor denosumab. This finding is now being tested in a randomized phase III prevention study, ‘BRCA-P’. In further studies, a quiescent stem cell that remains hormone responsive has been identified. Lineage tracing, combined with high resolution 3D imaging (visualizing large regions of intact tissue), was carried out to track stem and progenitor cells in situ. These studies have revealed the presence of long-lived stem cells that replenish mammary epithelium, while unipotent progenitors appear to play an essential role in sustaining the independent lineages on a day-to-day basis. In order to generate a comprehensive single cell expression resource and explore molecular heterogeneity in the mouse mammary gland, single cell profiling at key developmental stages was performed. This has revealed novel intermediates and unexpected changes in the transcriptional landscape that could inform a role for distinct cell types during mammary oncogenesis. To gain insight into cancer heterogeneity and clonal dynamics at cellular resolution, 3D imaging was combined with lineage tracing to track specific cells during cancer development. Through the application of a novel pipeline, the epithelial-to-mesenchymal transition (EMT) was found to be a frequent event, thus highlighting the inherent plasticity of mammary tumors. These findings are currently being extended to human tumors to further understand inter- and intra-tumoral heterogeneity. In parallel, our group has established a bank of patient-derived xenograft (PDX) and patient derived organoid (PDO) models to facilitate the transfer of laboratory findings to the clinic. These serve as useful pre-clinical models to test promising agents. Initial efforts have focused on BH3 mimetics, a new class of drug that targets the BCL-2 family of pro-survival proteins. This work is leading to early phase clinical trials, currently focused on patients with metastatic ER+ breast cancer. Citation Format: JE Visvader, GJ Lindeman. Deciphering stem and progenitor cells to understand breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr BL1.

In silico prediction of Anti-apoptotic BCL-2 proteins Modulation by Afzelin in MDA-MB-231 Breast cancer cell

Triple-negative breast cancer (TNBC) has aggressive characteristics, and lower overall- and disease-free survival compared to other breast cancer subtypes. TNBC tends to be apoptotic resistant, which allegedly related to dysregulation of anti-apoptotic Bcl-2 family proteins. Afzelin is a chemical compound that has anti-cancer potentials. The purposes of the study were analysing the effect of afzelin on apoptosis of MDA-MB-231 in vitro, and the interaction between afzelin and anti-apoptotic Bcl-2 family proteins through in silico approach. Apoptosis induced by afzelin was analysed by fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection Kit with propidium iodide (PI) through flow cytometry, with subsequent ANOVA analysis. Identification of pro-survival Bcl-2 family proteins and its key amino acid residues was based on literature reviews, followed with protein structures mining from Protein Data Bank (PDB). Afzelin chemical structure was obtained from PubChem. Reverse docking performed by Autodock Vina. Afzelin significantly increased apoptosis on MDA-MB-231 in a dose-dependent manner. The interactions of afzelin and anti-apoptotic Bcl-2 family proteins were based on BH3-mimetic mode of action. Reverse docking in BH3-hydrophobic groove showed that afzelin interact with Bcl-XL, Bcl-B, and MCL1, in the order from the highest to lower binding energy. Afzelin and corresponding BH3-only proteins formed hydrogen bonds with the same amino acid residues when interacted with Bcl-XL, Bcl-B, and MCL1. The outcomes predicted that afzelin induced apoptosis in MDA-MB-231 breast cancer cells through BH3 mimetic effect, particularly on Bcl-XL.

Mcl-1 inhibition overcomes intrinsic and acquired regorafenib resistance in colorectal cancer.

Intrinsic and acquired resistance to targeted therapies is a significant clinical problem in cancer. We previously showed that resistance to regorafenib, a multi-kinase inhibitor for treating colorectal cancer (CRC) patients, can be caused by mutations in the tumor suppressor FBW7, which block degradation of the pro-survival Bcl-2 family protein Mcl-1. We tested if Mcl-1 inhibition can be used to develop a precision combination therapy for overcoming regorafenib resistance.Methods: Small-molecule Mcl-1 inhibitors were tested on CRC cells with knock-in (KI) of a non-degradable Mcl-1. Effects of Mcl-1 inhibitors on regorafenib sensitivity were determined in FBW7-mutant and -wild-type (WT) CRC cells and tumors, and in those with acquired regorafenib resistance due to enriched FBW7 mutations. Furthermore, translational potential was explored by establishing and analyzing FBW7-mutant and -WT patient-derived organoid (PDO) and xenograft (PDX) tumor models.Results: We found that highly potent and specific Mcl-1 inhibitors such as S63845 overcame regorafenib resistance by restoring apoptosis in multiple regorafenib-resistant CRC models. Mcl-1 inhibition re-sensitized CRC tumors with intrinsic and acquired regorafenib resistance in vitro and in vivo, including those with FBW7 mutations. Importantly, Mcl-1 inhibition also sensitized FBW7-mutant PDO and PDX models to regorafenib. In contrast, Mcl-1 inhibition had no effect in FBW7-WT CRCs.Conclusions: Our results demonstrate that Mcl-1 inhibitors can overcome intrinsic and acquired regorafenib resistance in CRCs by restoring apoptotic response. FBW7 mutations might be a potential biomarker predicting for response to the regorafenib/Mcl-1 inhibitor combination.

Cell Death Pathways in Lymphoid Malignancies.

This review highlights the importance of the Bcl-2 family members in lymphoma cell survival and discusses the approaches to modulate their function, directly or indirectly, to advance lymphoma therapeutics. The balance of cell death versus survival is ultimately leveraged at the mitochondria. Mitochondrial outer membrane permeabilization (MOMP) is the critical event that governs the release of pro-apoptotic molecules from the intermembrane mitochondrial space. MOMP is achieved through the coordinated actions of pro- and anti-apoptotic Bcl-2 family member proteins. Recognition of functional alterations among the Bcl-2 family member proteins led to identification of tractable targets to combat hematologic malignancies. A new class of drugs, termed BH3 mimetics, was introduced in the clinic. Venetoclax, a Bcl-2 inhibitor, received regulatory approvals in therapy of chronic lymphocytic leukemia and acute myeloid leukemia. Alternative pro-survival Bcl-2 family proteins, in particular Mcl-1, have been successfully targeted in preclinical studies using novel-specific BH3 mimetics. Finally, anti-apoptotic Bcl-2 family members may be targeted indirectly, via interference with the pro-survival signaling pathways, e.g., phosphoinotiside-3 kinase, B-cell receptor signaling, and NF-κB.

Targeting Bcl-2 Proteins in Myeloma

Escape from apoptosis is a hallmark of malignant cells that constitute an important mechanism associated to therapeutic resistance. Hematologic malignant cells are described as primed for apoptosis, a state in which pro-survival BCL2 family proteins sequester elevated levels of pro-apoptotic BH3-only proteins or even pre-activated BAX/BAK to ensure survival. Then, mimicking BH3-only proteins might preferentially turn on apoptosis in malignant cells. The understanding of BCL2 biology combined to drug discovery efforts have succeeded to the development of BH3-mimetics that bind with high affinity to a specific anti-apoptotic protein, promoting the release of pro-apoptotic members, leading to BAX and/or BAK activation and apoptosis. Venetoclax (ABT-199) is the first-in-class oral BH3-mimetic targeting selectively BCL2 and approved by the FDA for the treatment of CLL patients with 17p deletion. More recently, BH3-mimetics targeting MCL1 have been also described and are currently undergoing early phase clinical evaluation. Multiple myeloma (MM) displays a molecular heterogeneity, which includes hyperdiploid patients and patients with an Ig translocation with different chromosomes (4, 11, 6 or 16) leading to an over-expression of MMSET, CCND1, CCND3 or MAF genes, respectively. Analysis of apoptotic machinery diversity supports the notion that MM heterogeneity is extended to the BCL2 family content. The combined profile of BCL2, BCLXL and MCL1 mRNA expression in the main MM molecular subgroups was sufficient to discriminate each of them, suggesting that the composition of anti-apoptotic proteins should be taken into account for targeted therapies. MCL1 is over-expressed in MM and its strongest expression was found in the MMSET and MAF subgroups associated with an adverse prognosis. In contrast, the CCND1 subgroup expresses high levels of BCL2 and low level of MCL1 and BCLXL. In accordance to the heterogeneity of BCL2 member expression, different approaches such as MCL1 gene editing, BH3 profiling and in vitro sensitivity to specific BH3-mimetic demonstrate that myeloma cells are dependent on BCL2, MCL1 or BCLXL or co-dependent on two or three anti-apoptotic proteins. A plasticity of myeloma cell dependency was also reported according to disease status. Preclinical evaluation of venetoclax reveals that the sensitivity to venetoclax was mainly found in the CCND1 subgroup but not exclusively. The ratio of BCL2/BCLXL appears as a predictor of venetoclax sensitivity. A phase 1 clinical trial of venetoclax in relapsed/refractory myeloma patients demonstrate its efficacy mainly in t(11;14) patients with 40% of overall response. These results validate that targeting BCL2 as a very promising strategy for some patients, nevertheless stratification of patients based on t(11 ;14) and/or BCL2/BCLXL ratio is not sufficient to predict venetoclax response. Recent findings suggest that ex vivo pretreatment sensitivity could predict the clinical response to venetoclax. The reliability of the different approaches used to determine BCL2 dependency will be further discussed. A better comprehension of how to increase BCL2 priming is one of the most interesting perspectives to find the best combination regimens with venetoclax. Indeed, dexamethasone increases the BH3-only Bim protein and shifts it's binding towards BCL2. Phase 1 study of the combination of dexamethasone/venetoclax confirms the interest of this combination for t(11;14) patients. Finally, the possibility to overcome the resistance to venetoclax by targeting directly or indirectly the BCXL and MCL1 resistant factors could be of particular relevance. In conclusion, how basic research should work hand-in hand with clinical practice to design pertinent protocol to evaluate BCL2 targeted therapy will be examined. Disclosures No relevant conflicts of interest to declare.

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

EBV BCL-2 homologue BHRF1 drives chemoresistance and lymphomagenesis by inhibiting multiple cellular pro-apoptotic proteins.

Epstein-Barr virus (EBV), which is ubiquitous in the adult population, is causally associated with human malignancies. Like many infectious agents, EBV has evolved strategies to block host cell death, including through expression of viral homologues of cellular BCL-2 pro-survival proteins (vBCL-2s), such as BHRF1. Small molecule inhibitors of the cellular pro-survival BCL-2 family proteins, termed 'BH3-mimetics', have entered clinical trials for blood cancers with the BCL-2 inhibitor venetoclax already approved for treatment of therapy refractory chronic lymphocytic leukaemia and acute myeloid leukaemia in the elderly. The generation of BH3-mimetics that could specifically target vBCL-2 proteins may be an attractive therapeutic option for virus-associated cancers, since these drugs would be expected to only kill virally infected cells with only minimal side effects on normal healthy tissues. To achieve this, a better understanding of the contribution of vBCL-2 proteins to tumorigenesis and insights into their biochemical functions is needed. In the context of Burkittlymphoma (BL), BHRF1 expression conferred strong resistance to diverse apoptotic stimuli. Furthermore, BHRF1 expression in mouse haematopoietic stem and progenitor cells accelerated MYC-induced lymphoma development in a model of BL. BHRF1 interacts with the cellular pro-apoptotic BCL-2 proteins, BIM, BID, PUMA and BAK, but its capability to inhibit apoptosis could not be mapped solely to one of these interactions, suggesting plasticity is a key feature of BHRF1. Site-directed mutagenesis revealed a site in BHRF1 that was critical for its interaction with PUMA and blocking DNA-damage-induced apoptosis, identifying a potentially therapeutically targetable vulnerability in BHRF1.

Multiple myeloma with amplification of chromosome 1q is highly sensitive to MCL-1 targeting

Pro-survival BCL-2 family proteins are potent inhibitors of apoptosis and are often overexpressed in lymphoid malignancies. In multiple myeloma (MM), BCL-2 inhibitor Venetoclax induces cell death, particularly in presence of t(11;14) and high BCL-2 expression. The most prominent BCL-2 family member in MM is MCL-1, whose expression contributes to survival of malignant plasma cells and overexpression correlates with poor prognosis. Amplification of 1q21, the chromosome region that contains the MCL1 locus, occurs in approximately 40% of newly diagnosed MM, and 70% at relapse.