High Levels Of Bcl-xL Research Articles

Exploring the potential of BH3 mimetic therapy in squamous cell carcinoma of the head and neck

Squamous cell carcinoma of the head and neck (SCCHN) is the sixth most common cancer worldwide, with overall survival of less than 50%. Current therapeutic strategies involving a combination of surgery, radiation, and/or chemotherapy are associated with debilitating side effects, highlighting the need for more specific and efficacious therapies. Inhibitors of BCL-2 family proteins (BH3 mimetics) are under investigation or in clinical practice for several hematological malignancies and show promise in solid tumors. In order to explore the therapeutic potential of BH3 mimetics in the treatment of SCCHN, we assessed the expression levels of BCL-2, BCL-XL, and MCL-1 via Western blots and immunohistochemistry, in cell lines, primary cells derived from SCCHN patients and in tissue microarrays containing tumor tissue from a cohort of 191 SCCHN patients. All preclinical models exhibited moderate to high levels of BCL-XL and MCL-1, with little or no BCL-2. Although expression levels of BCL-XL and MCL-1 did not correlate with patient outcome, a combination of BH3 mimetics to target these proteins resulted in decreased clonogenic potential and enhanced apoptosis in all preclinical models, including tumor tissue resected from patients, as well as a reduction of tumor volume in a zebrafish xenograft model of SCCHN. Our results show that SCCHN is dependent on both BCL-XL and MCL-1 for apoptosis evasion and combination therapy targeting both proteins may offer significant therapeutic benefits in this disease.

DT2216, a BCL-XL Proteolysis Targeting Chimera (PROTAC), Is a Potent Anti T-Cell Lymphoma Agent That Does Not Induce Significant Thrombocytopenia

There is an urgent need for new therapeutic agents to treat patients with T-cell lymphoma (TCL). Multiple hematologic malignancies evade apoptosis through overexpression of anti-apoptotic proteins in the BCL-2 family, including BCL-2, BCL-XL, and MCL-1. We and others recently showed that a large fraction of cutaneous and peripheral TCL cell lines, patient-derived xenografts and primary patient samples depend on BCL-XL for survival (Koch et al, Blood. 2019; 133:566-575). These findings suggest that targeted inhibition of BCL-XL could offer therapeutic benefit for some TCL patients. Currently available small molecule BCL-XL inhibitors have failed during clinical development due to on-target and dose-limiting thrombocytopenia, as platelets depend on BCL-XL for survival. To overcome this toxicity, we developed DT2216, a novel proteolysis-targeting chimera (PROTAC) that targets BCL-XL to the Von Hippel Lindau (VHL) E3 ligase for proteasomal degradation. We selected the VHL ligase because platelets express very low levels of VHL, suggesting that they would be spared from the pro-apoptotic effects of DT2216. Here we examined the therapeutic potential of DT2216 against different TCL cell lines in vitro and in TCL xenograft mouse models. We first profiled the expression of different anti-apoptotic BCL-2 family proteins in multiple TCL cell lines (Fig. 1a) and tested their sensitivity to selective inhibitors for different BCL-2 family proteins as well as commonly-used chemotherapeutic agents (Table 1). The results showed that the TCL cells with higher levels of BCL-XL were more resistant to doxorubicin, etoposide and vincristine. DT2216 was more effective in reducing the viability of BCL-XL-dependent TCLs such as MyLa cells than ABT263 (a dual BCL-2/XL inhibitor). The EC50 value of DT2216 for MyLa TCL cells was less than 10 nM and DT2216 killed MyLa cells through induction of BCL-XL degradation and cellular apoptosis (Fig. 1b-f). Moreover, DT2216 was less toxic to human platelets than ABT263 in vitro with an EC50 > 3 μM. We further validated the effect and specificity of DT2216 in MJ cells, another BCL-XL-dependent TCL cell line, and demonstrated that its antitumor activity was dependent on proteasome activity. In vivo, when DT2216 was given to mice with MyLa TCL xenografts by i.p. injections at 10 mpk/q4d, it significantly inhibited tumor growth, whereas ABT263 at the same dose had no significant effect (Fig. 1g). More importantly, after MyLa xenografted mice failed to respond to ABT263 treatment, we subjected the mice to DT2216 (10 mpk/q4d), which induced rapid tumor regression and increased the survival of the mice without causing significant reduction of blood platelets (Fig. 1h). These effects were associated with a significant reduction in BCL-XL expression and activation of caspase cascade in tumor xenografts. However, some TCLs depend on not only BCL-XL but also BCL-2 or MCL-1 for evasion of apoptosis. Therefore, we next assessed the therapeutic potential of DT2216 in combination with a selective BCL-2 inhibitor using the TCL PDX DFTL-28776, which depends on both BCL-XL and BCL-2 for survival. We found that the combination of DT2216 with ABT199 (a selective BCL-2 inhibitor) could more effectively kill DFTL-28776 TCL cells than either agent alone in cell culture. The effect of the combination treatment on the growth of DFTL-28776 PDX in vivo is under examination. Collectively, our findings suggest that targeting BCL-XL using DT2216 can selectively kill BCL-XL-dependent TCL cells without causing significant platelet toxicity. Moreover, the combination of DT2216 with an inhibitor targeting other anti-apoptotic BCL-2 family proteins may have broad therapeutic utility against multiple TCL types and other cancers dependent on BCL-XL. Disclosures He: University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Khan:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer agents. Zhang:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer agents. Zheng:Dialectic Therapeutics: Equity Ownership, Other: Co-founders of Dialectic Therapeutics that develops Bcl-xl PROTACs as anti-cancer agents; University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Weinstock:Celgene: Research Funding. Zhou:Dialectic Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: co-founders of Dialectic Therapeutics that develops Bcl-xl PROTACs as anti-cancer agents; Unity Biotechnology: Equity Ownership, Other: Co-founder of Unity Biotechnology which develops small-molecule senolytic drugs; University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents.

IL-21 and anti-CD40 restore Bcl-2 family protein imbalance in vitro in low-survival CD27+ B cells from CVID patients

Common variable immunodeficiency (CVID) is characterized by an abnormal B cell differentiation to memory and antibody-secreting B cells. The defective functionality of CVID patients’ B cells could be the consequence of alterations in apoptosis regulation. We studied the balance of Bcl-2 family anti-/pro-apoptotic proteins to identify molecular mechanisms that could underlie B cell survival defects in CVID. We used flow cytometry to investigate Bcl-2, Bcl-XL, Bax, and Bim expression in B cells ex vivo and after anti-CD40 or anti-BCR activation with or without IL-21, besides to spontaneous and stimulation-induced Caspase-3 activation and viable/apoptotic B cell subpopulations. We found increased basal levels of Bax and Bim in CVID B cells that correlated with low viability and high Caspase-3 activation only in CD27+ B cells, particularly in a subgroup of apoptosis-prone CVID (AP-CVID) patients with low peripheral B cell counts and high autoimmunity prevalence (mostly cytopenias). We detected a broad B cell defect in CVID regarding Bcl-2 and Bcl-XL induction, irrespective of the stimulus used. Therefore, peripheral CVID memory B cells are prompted to die from apoptosis due to a constitutive Bcl-2 family protein imbalance and defective protection from activation-induced apoptosis. Interestingly, anti-CD40 and IL-21 induced normal and even higher levels of Bcl-XL, respectively, in CD27+ B cells from AP-CVID, which was accompanied by cell viability increase. Thus low-survival memory B cells from AP-CVID can overcome their cell death regulation defects through pro-survival signals provided by T cells. In conclusion, we identify apoptosis regulation defects as disease-contributing factors in CVID. B cell counts and case history of cytopenias might be useful to predict positive responses to therapeutic approaches targeting T-dependent signaling pathways.

PO-027 Potential use of BH3 mimetics in the treatment of head and neck cancer

IntroductionHead and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. The limited success and significant toxicities associated with current therapies highlight the need for more efficacious treatment strategies. BH3 mimetics are an emerging class of drugs that target the anti-apoptotic BCL-2 family members, which are overexpressed proteins in many cancers, contributing to evasion of apoptosis and resistance to many therapeutic regimens. The BCL-2 specific inhibitor, venetoclax, is currently used to treat haematological malignancies, whereas specific inhibitors for BCL-XL and MCL-1 are entering clinical trials. We aim to determine the potential of BH3 mimetics in the treatment of HNSCC.Material and methodsHNSCC tissue microarrays (TMAs) were immunohistochemically stained for BCL-2, BCL-XL and MCL-1, protein levels assessed semi-quantitatively as low, moderate or high and correlated with patient survival. Cell lines and primary cells were treated with ABT-199 (BCL-2 specific), A1331852 (BCL-XL specific) and/or S63845 (MCL-1 specific), and apoptosis induction and replicative potential were assessed.Results and discussionsTMAs revealed that HNSCC tumours express moderate to high levels of BCL-XL and MCL-1, but low levels of BCL-2. Indeed treatment with a BCL-2 inhibitor had little effect on cell death in HNSCC cell lines; nor did treatment with A1331852 or S63845 as single agents. However, a combination of MCL-1 and BCL-XL inhibition induced extensive apoptotic cell death and a marked reduction in clonogenic cell growth, suggesting a dependence on these two anti-apoptotic proteins for tumour cell survival. A comparison of MCL-1 and BCL-XL expression levels against overall survival of HNSCC patients over a five year period post-surgery revealed that high MCL-1 levels were associated with low median survival. In contrast, the correlation between high BCL-XL expression and median survival was statistically not significant, although it may be clinically relevant.ConclusionBCL-2 is poorly expressed in HNSCC tumours, diminishing the potential of using venetoclax in HNSCC. In contrast, both BCL-XL and MCL-1 are highly expressed in HNSCC, and may be important prognostic indicators. Therefore targeting BCL-XL and MCL-1 together may be therapeutically beneficial, although the toxicity of this combination must be assessed before human trials can be considered.

Abstract 3812: JAK2 and PTPN11 mutations as potential biomarkers for BCL-xL inhibition as monotherapy and in combination therapy for acute myeloid leukemia

Abstract Acute Myeloid Leukemia (AML) is a heterogeneous disease where many distinct functional mutations have been identified. While mutations in FLT3, NRAS, NPM1, and IDH genes have been well characterized in de novo AML, a subset of AML associated with leukemic transformation of chronic myeloproliferative neoplasms (MPNs) are enriched in mutated JAK2 and PTPN11. MPNs are a set of disorders characterized by the chronic and abnormal overproduction of blood cells which can ultimately progress to AML. Targeted therapy against mutated JAK2 and PTPN11 with small molecule inhibitors against the JAK-STAT and RAS-ERK pathways, respectively, are being actively investigated in AML. However, acquired resistance and non-durable responses are already being demonstrated in monotherapy treatment. Thus, AML that has arisen from leukemic transformation of MPNs remains an area of unmet medical need. This need is underscored by the fact that these patients suffer from poor outcomes and low response rates to standard chemotherapy. Together, these results indicate the need for rational combinations in this population. In this study, we sought to better define the dependencies of AML cells on BCL-2 family members by screening AML cell lines with selective small molecule inhibitors to the BCL-2 family members. The screen revealed that the majority of AML cell lines depend on BCL-2, or BCL-2 together with MCL1 for survival. Intriguingly, a subset of AML cell lines is exquisitely sensitive to BCL-xL inhibition. Treating these cell lines with BCL-xL-selective inhibitor or with navitoclax, a dual BCL2/BCL-xL inhibitor induces apoptosis. These BCL-xL inhibitor-sensitive cell lines express high levels of BCL-xL, but not BCL-2 and MCL1. Genomic analysis of BCL-xL inhibitor-sensitive AML cell lines revealed that this subset is also enriched in JAK2V16F and PTPN11 mutations. We further demonstrate positive combination effect between BCL-xL inhibitor or navitoclax with JAK inhibitor (ruxolitinb) and two MEK inhibitors (trametinib and AZD6244) in cell lines with JAK2V16F and PTPN11 mutations, respectively. Together, these results demonstrate the potential utility of a BCL-xL inhibition as a combination partner in JAK2 and PTPN11 mutated cancers. Importantly, JAK2 and PTPN11 mutations in addition to expression of BCL-xL, BCL-2, and MCL1 could also be further tested as potential biomarkers for the utility of BCL-xL inhibition based treatments for patients with these mutations. Citation Format: Richard J. Bellin, Valerie Hilgenberg, Relja Popovic, Tamar Uziel, Lloyd T. Lam. JAK2 and PTPN11 mutations as potential biomarkers for BCL-xL inhibition as monotherapy and in combination therapy for acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3812. doi:10.1158/1538-7445.AM2017-3812

Short-Term Grafting of Human Neural Stem Cells: Electrophysiological Properties and Motor Behavioral Amelioration in Experimental Parkinsons Disease.

Cell replacement therapy in Parkinsons disease (PD) still lacks a study addressing the acquisition of electrophysiological properties of human grafted neural stem cells and their relation with the emergence of behavioral recovery after transplantation in the short term. Here we study the electrophysiological and biochemical profiles of two ventral mesencephalic human neural stem cell (NSC) clonal lines (C30-Bcl-XL and C32-Bcl-XL) that express high levels of Bcl-XL to enhance their neurogenic capacity, after grafting in an in vitro parkinsonian model. Electrophysiological recordings show that the majority of the cells derived from the transplants are not mature at 6 weeks after grafting, but 6.7% of the studied cells showed mature electrophysiological profiles. Nevertheless, parallel in vivo behavioral studies showed a significant motor improvement at 7 weeks postgrafting in the animals receiving C30-Bcl-XL, the cell line producing the highest amount of TH+ cells. Present results show that, at this postgrafting time point, behavioral amelioration highly correlates with the spatial dispersion of the TH+ grafted cells in the caudate putamen. The spatial dispersion, along with a high number of dopaminergic-derived cells, is crucial for behavioral improvements. Our findings have implications for long-term standardization of stem cell-based approaches in Parkinsons disease.

MiR-377-dependent BCL-xL regulation drives chemotherapeutic resistance in B-cell lymphoid malignancies.

BackgroundBCL-xL is an anti-apoptotic BCL-2 family protein that inhibits apoptosis and is overexpressed in many cancers. We have reported that acquired resistance to the BCL-2 inhibitor ABT-199 (venetoclax) is associated with increased BCL-xL expression. Yet, how BCL-xL mediates chemoresistance in hematopoietic malignancies is not clear. This finding may help in design of new strategies for therapeutic intervention to overcome acquired chemoresistance mediated by BCL-xL.ResultsWe now show that the increased BCL-xL expression was inversely correlated with that of miR-377 in ABT-199-resistant cells. This finding was also extended to a panel of B-cell lymphoid lines and primary chronic lymphocytic leukemia (CLL) cells. miR-377 suppressed BCL-xL expression by recognizing two binding sites in the BCL-xL 3’-UTR. Mutation of these two miR-377 consensus-binding sites completely abolished its regulatory effect. Expression of a miR-377 mimic downregulated BCL-xL protein expression and significantly increased apoptotic cell death. Expression of a miR-377 inhibitor restored BCL-xL protein expression and limited cell death caused by the hypomethylating agent 5-azacytidine. Thus, miR-377-dependent BCL-xL regulation drives acquired therapeutic resistance to ABT-199. We further show that CLL patients who received a diverse array of chemotherapy regimens also had significantly higher BCL-xL and lower miR377 expression, indicating that exposure to chemotherapy might trigger transcriptional silencing of miR-377, which results in high levels of BCL-xL. Importantly, CLL patients with high BCL-xL/low miR-377 expression had an advanced tumor stage. Moreover, the high BCL-xL expression correlated with short treatment-free survival in 76 CLL patients. miR-377 is located at 14q32 in the DLK1-DIO3 region, which encodes the largest tumor suppressor miRNA cluster in humans. Examination of five additional 14q32 miRNAs revealed that the majority were significantly down-regulated in most CLL patients as well as in ABT-199-resistant cell lines. Remarkably, four of these miRNAs had significantly decreased expression in chemotherapy-treated CLL patients as compared to those untreated. These findings indicate a reduced expression of multiple miRNAs that may reflect a global silencing of this miRNA cluster in therapy-resistant lymphoid cells.ConclusionsThese findings reveal a novel mechanism by which down-regulation of miR-377 increases BCL-xL expression, promoting chemotherapy resistance in B-cell lymphoid malignancies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0460-8) contains supplementary material, which is available to authorized users.

HDAC3 is required for thymocyte positive selection (HEM2P.236)

Abstract In order to generate functional peripheral T cells, proper gene regulation during T cell development is critical. Development involves both the activation and repression of genes as cells progress through each developmental checkpoint. Histone acetylation/deacetylation is one mechanism regulating gene expression. Acetylation by histone acetyl transferases (HATs) allows for gene expression and deacetylation by histone deacetylase (HDAC) enzymes promote gene silencing, and their role in regulating T cell development is not clear. In this study, our lab focused on HDAC3. We examined the role HDAC3 plays during T cell development by utilizing a CD2-Cre-mediated loss of HDAC3 (CD2icre HDAC3-cKO), which deletes early in thymocyte development. When HDAC3 is absent, there is a developmental block during positive selection, leading to the production of very few CD4 and CD8 SP thymocytes leading to a severe defect in peripheral T cell numbers. During positive selection, HDAC3-deficient DP thymocytes fail to down-regulate RORγt as they progress to the SP stage. Consistent with the failure to down-regulate RORγt, thymocytes post-positive selection express high levels of Bcl-xl and fail to express Bcl-2, which is required for their survival. Moreover, HDAC3-deficient thymocytes show increased apoptosis. These results suggest that HDAC3 is required for the down-regulation of RORγt during positive selection.

Elimination of quiescent/slow-proliferating cancer stem cells by Bcl-XL inhibition in non-small cell lung cancer.

Lung cancer is the most common cause of cancer-related mortality worldwide, urging the discovery of novel molecular targets and therapeutic strategies. Stem cells have been recently isolated from non-small cell lung cancer (NSCLC), thus allowing the investigation of molecular pathways specifically active in the tumorigenic population. We have found that Bcl-XL is constantly expressed by lung cancer stem cells (LCSCs) and has a prominent role in regulating LCSC survival. Whereas chemotherapeutic agents were scarcely effective against LCSC, the small molecule Bcl-2/Bcl-XL inhibitor ABT-737, but not the selective Bcl-2 inhibitor ABT-199, induced LCSC death at nanomolar concentrations. Differently from gemcitabine, which preferentially eliminated proliferating LCSC, ABT-737 had an increased cytotoxic activity in vitro towards quiescent/slow-proliferating LCSC, which expressed high levels of Bcl-XL. In vivo, ABT-737 as a single agent was able to inhibit the growth of LCSC-derived xenografts and to reduce cancer stem cell content in treated tumors. Altogether, these results indicate that quiescent/slow-proliferating LCSC strongly depend on Bcl-XL for their survival and indicate Bcl-XL inhibition as a potential therapeutic avenue in NSCLC.

Possible Mechanisms Of Resistance To The Novel BH3-Mimetic ABT-199 In In Vitro Lymph Node Models Of CLL – The Role Of Abl and Btk

The new BH3-mimetic ABT-199 antagonizes Bcl-2 and avoids the thrombocytopenia associated with clinical application of its predecessor ABT-263 (navitoclax). Chronic lymphocytic leukemia (CLL) cells are highly sensitive to ABT-199 and the first clinical results show clear reductions in peripheral and bone marrow CLL cells and in lymph node size. In the lymph node, CLL cells receive pro-survival signals that upregulate Bcl-XL, Mcl-1 and Bfl-11. These Bcl-2 family members are not targeted by ABT-199, which poses the potential risk of remaining clones with residual viability. Here, we aimed to define the signals that determine sensitivity for ABT-199 and ABT-737 in an in vitro lymph node model of CLL.We applied CD40 and cytokine stimulation in combination with kinase inhibitors that are known to change microenviroenmental signals and increase drug resistance in CLL. Stimulation via CD40 plus IL-4 or IL-21 differentially affected the expression of Mcl-1, Bcl-XL, Bfl-1 and Noxa and this correlated with strong alterations in sensitivity to ABT-737 and ABT-199 (see table 1 for LC 50 values). As reported before2, in vitro CD40 stimulation reduced sensitivity to ABT-737 by 100-fold, and this was further decreased by IL-4. Strikingly, CD40+IL-4 stimulation in primary CLL cells resulted in full resistance to 10 μM ABT-199, probably due to very high levels of Bcl-XL.Table 1The LC50 of ABT-737 or ABT-199 for CLL cells stimulated with CD40L and IL-21 or IL-4 (averaged values n=8)StimulationLC50 (μM)ABT-737ABT-1993T3 (control)0.0050.0013T40L0.781> 103T40L + IL-210.1950.2103T40L + IL-46.772> 103T40L + IL-21 + IL-40.4269.121We next sought ways to circumvent resistance against ABT-199 induced in our in vitro model. We showed previously that the broad spectrum kinase inhibitor dasatinib prevented CD40-mediated resistance to various drugs, including ABT-7373. We therefore first characterized the targets of dasatinib in primary CLL by solid-phase pull-down, mass-spectrometry and competition binding. Abl and Btk were identified as dominant and specific interactors of dasatinib. Importantly, resistance for BH3-mimetics could be overcome by dasatinib (see table 2) and the Abl inhibitor imatinib, but not by the more selective Btk inhibitor ibrutinib. Conversely, BCR- and chemokine-controlled adhesion could be abolished by dasatinib and ibrutinib, but not by imatinib. Thus, Abl and Btk function in two key pro-survival arms; chemoresistance and localization in the protective environment.Table 2The LC50 of ABT-737 or ABT-199 for CLL cells stimulated with CD40L in combination with Dasatinib (averaged values n=4)StimulationLC50 (μM)ABT-737ABT-1993T30.0050.0013T40L0.781> 103T40L + 100 nM Dasatinib0.0810.0663T40L + 1000 nM Dasatinib0.0370.020The observed resistance to ABT-199 induced in our in vitro a co-culture system designed to simulate the CLL microenvironment does not reflect the observations from clinical trials in patients. Nevertheless, long-term clinical application of ABT-199 in CLL might select for resistant clones at protective niches. Our data suggest that this may be overcome by combination treatment with kinase inhibitors that either directly abrogate anti-apoptotic signals or cause egress from lymph node sites and prevent the resistance mechanism from coming into play.1. Smit LA, Hallaert DY, Spijker R et al. Differential Noxa/Mcl-1 balance in peripheral versus lymph node chronic lymphocitic leukemia cells correlates with survival capacity. Blood 2007;109:1660-1668.2. Vogler M, Butterworth M, Majid A et al. Concurrent up-regulation of BCL-XL and BCL2A1 induces approximately 1000-fold resistance to ABT-737 in chronic lymphocytic leukemia. Blood 2009;113:4403-4413.3. Hallaert DY, Jaspers A, van Noesel CJ et al. c-Abl kinase inhibitors overcome CD40-mediated drug resistance in CLL; Implications for therapeutic targeting of chemoresistant niches. Blood 2008;112:5141-5149. Disclosures:No relevant conflicts of interest to declare.

Bcl-XL overexpression prevents B cell receptor driven autophagy in IgM+ lymphoma (P3026)

Abstract We previously showed that B cell receptor (BCR) induced apoptosis in lymphoma required suppression of PI-3K [Carey GB, Scott DW (2001) J. Immunol, 166: 1618-]. PI-3K stimulates mTOR, a suppressor of autophagy and mitophagy. In addition, Bcl-XL blocks BCR-driven apoptosis in lymphoma, although the underlying mechanism is unclear. In contrast, a recent study demonstrated that Bcl2 overexpression blocked BCR-driven apoptosis, but not the induction of autophagy [Watanabe et al BBRC (2008) 19;374(2):274-]. Therefore the present study examined autophagic and mitophagic responses following BCR crosslinking on parental WEHI-231 lymphoma cells or on a derivative expressing high levels of Bcl-XL (WEHI-231/Bcl-XL). The results confirmed dramatic mitochondrial dysfunction followed by apoptosis in WEHI-231 compared to WEHI-231/Bcl-XL. BCR-crosslinking resulted in rapid and massive induction of the autophagic response in parental WEHI-231 compared to WEHI-231/Bcl-XL. Furthermore, treatment with STF-62247, a small molecule inducer of autophagy, resulted in apoptosis in WEHI-231 but not WEHI-231/Bcl-XL cells. These data suggest that Bcl-XL prevents BCR-driven apoptosis in lymphoma via blocking autophagic and mitophagic responses. Hence, these studies demonstrate specific roles of BclXL and bcl2 in BCR-driven death responses. Lastly, combinations of small molecule autophagy inducers and Bcl-XL antagonists may prove to be effective in addressing lymphoma.

Dual Trade of Bcl-2 and Bcl-xL in Islet Physiology

Apoptosis is now recognized as a predominant mechanism by which β cells are destroyed in both type 1 and type 2 diabetes (1). Pro- and antiapoptotic members of the Bcl-2 family are central regulators involved in β-cell fate decision between life and death in response to physiological insults. The intricate interplay and balance between members of this family regulate apoptosis by controlling mitochondrial cell death signaling, the so-called intrinsic pathway (2). The Bcl-2 family is divided into three subgroups as follows: 1 ) the antiapoptotic Bcl-2–like proteins (Bcl-2, Bcl-xL, Bcl-W, Mcl-1, and A1/Bfl1) that possess four independent and highly conserved Bcl-2 homology (BH) domains (BH-1 to -4); 2 ) the proapoptotic Bax-like proteins (Bax, Bak, and Bok/Mtd), which also harbor the four BH domains; and 3 ) proapoptotic members typified by a single BH-3 domain (Bid, Bim/Bod, Bad, Bmf, Bik/Nbk, Blk, NoxA, Puma/Bbc3, and Hrk/DP5). Members of this last subgroup initiate the apoptotic-signaling cascade by interacting with antiapoptotic members (3). In addition, intracellular calcium activity ([Ca2+]i) is an important second messenger that initiates the apoptotic program (4) as well as being essential in metabolism secretion coupling that drives the Ca2+ sensor of the exocytotic machinery (5). Given the importance of Bcl-2 and Bcl-xL as prosurvival sentinels of the mitochondria, combined with the indispensable function of this organelle in β-cell metabolism secretion coupling, these proteins have been the targets of numerous studies in which expression levels were manipulated with the goal of improving islet viability. The study by Zhou et al. (6) in 2000 was the first to provide proof of concept that overexpression of Bcl-xL in β cells blunted stressed-induced apoptosis. Unexpectedly, one of the transgenic lines expressing high levels of Bcl-xL also displayed severe hyperglycemia. Glucose intolerance in these animals was linked to …

Abstract 4955: TIMP-1 inhibits apoptosis in lung adenocarcinoma cells via Bcl-2 interaction

Abstract Tissue inhibitors of matrix metalloproteinases have multifunctional properties, orchestrating diverse biological activities. Previous studies revealed a novel function of TIMP-1 as an inhibitor of apoptosis in mammalian cells. However, the mechanisms by which TIMP-1 exerts its anti-apoptotic effect are not completely understood. The antiapoptotic effect has been correlated with higher levels of Bcl-Xl. Our earlier studies in a CNS model of metastasis, using HB-1, a TIMP-1 overexpressing lung adenocarcinoma cell line (H2009), resulted in more aggressive tumor kinetics and increased vasculature. The present study was undertaken to elucidate the role of TIMP-1 in apoptosis in our model. Utilizing an apoptosis -specific gene array, we found that TIMP-1 overexpression results in 3-fold increased expression of Bcl-2. H2009 and the TIMP1 over-expressing clone - HB1 were treated with staurosporine to induce apoptosis, followed by analysis for poly ADP-ribose polymerase (PARP) cleavage. HB1 showed marked reduction in PARP cleavage in comparison to the controls. Treatment of HB1 cells with ABT737, an inhibitor of Bcl-2, restored apoptosis resulting in PARP cleavage in this clone. To further confirm this mechanism, we induced transient inhibition of Bcl-2 with siRNA, which also restored PARP cleavage in the HB-1 clone. Thus both chemical and biological inhibition resulted in increased apoptosis. Additionally, immunoprecipitation of Bcl-2 from cell lysate co-immunoprecipitated TIMP-1 suggesting a direct interaction between TIMP-1 and Bcl-2. These investigations identify a relationship between TIMP-1 and Bcl-2 in apoptosis inhibition, contributing to aggressive tumor kinetics, utilizing non-MMP dependent mechanisms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4955. doi:1538-7445.AM2012-4955

Navitoclax (ABT-263) Reduces Bcl-xL–Mediated Chemoresistance in Ovarian Cancer Models

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

Chemical biology: probes and therapeutics

In the Chemical Biology Minisymposium, cochaired by Alice Ting and Lisa Belmont, the first three talks focused on the development of novel probes for cell biology, while the last three presentations highlighted inhibitors of proteins previously believed to be “undruggable.” Daniel Hochbaum (Cohen lab, Harvard University) described an optical probe for imaging single action potentials using the fluorescence of a rhodopsin protein, Archaerhodopsin 3 (Arch), expressed in cultured rat hippocampal neurons. This voltage indicator exhibited a 10-fold improvement in sensitivity and speed over existing protein-based voltage indicators, with a twofold increase in brightness between −150 mV and +150 mV and a submillisecond response time. Arch detected single electrically triggered action potentials with a signal-to-noise ratio > 10. The mutant ArchD95N lacked endogenous proton pumping and showed 50% greater sensitivity than wild-type. Although it had a slower response (41 ms), ArchD95N resolved individual action potentials. Alice Ting (MIT) presented a novel approach to determining the proteomic composition of subcellular compartments by targeting a promiscuous biotin-conjugating enzyme to subcellular regions. The labeled proteins were enriched and identified by mass spectrometry. This approach was used to determine the proteomes of mitochondria and the endoplasmic reticulum of live mammalian cells without using subcellular fractionation. Katie White, a graduate student in Ting's lab, then described improvements to in vivo fluorophore labeling using mutants of lipoic acid ligase (LplA). She engineered LplA to accept a blue coumarin fluorophore and then used yeast display evolution to evolve LplA into a probe ligase with high activity in the secretory pathway. The LplA variants allowed imaging of intra- or intercellular protein–protein contacts. To expand the scope of bioluminescence imaging, Stephen Miller (University of Massachusetts Medical School) developed new aminoluciferin substrates for firefly luciferase that emit light at longer wavelengths than d-luciferin. Although these substrates were initially limited by product inhibition, this could be ameliorated by mutation of luciferase. Moreover, mutant luciferases were identified that displayed selectivity for these synthetic substrates over d-luciferin. This system has two advantages: it is potentially better suited for in vivo imaging, because tissue is more transparent to light at longer wavelengths, and orthogonal luciferase–luciferin pairs could allow multiplexed bioluminescence imaging. An NMR-based fragment screen for inhibitors of the Ras oncoprotein presented by Guowei Fang (Genentech) identified 25 compounds that inhibit Ras with two distinct mechanisms of action. One class of compounds binds to a small pocket between Switch I and Switch II that expands to accommodate the inhibitor. These compounds competitively inhibit nucleotide exchange by blocking the interaction of RasGDP with its nucleotide exchange factor, SOS. The second class of compounds binds in a pocket created by the interface of the Ras–SOS complex and acts by accelerating nucleotide release. Corey Nislow (University of Toronto) highlighted the power of yeast genetics by screening cell-active, structurally diverse compounds against 1100 heterozygous strains (haploid for essential genes) and 5000 homozygous deletion strains. He identified 55 novel drug targets, including Sec14 and septin. Sec14 coordinates lipid biosynthesis with signaling pathways and was thought to be undruggable. He then showed how chemical genetic profiling revealed that elesclomol, a compound demonstrating efficacy in metastatic melanoma, inhibits the electron transport chain. These approaches will continue to bear fruit as additional targets are confirmed and similar technology is applied to Candida albicans and other organisms. Lisa Belmont described a model for synergy between paclitaxel and navitoclax, a Bcl-2/Bcl-xL inhibitor, in which cells in mitotic arrest slowly degrade Mcl-1, while navitoclax causes acute inhibition of Bcl-xL. Across 50 cancer cell lines, cells with high levels of Bcl-xL relative to Mcl-1 exhibited lowered paclitaxel response and higher paclitaxel/navitoclax synergy. The cell line synergy translated to xenograft studies, and analysis of ovarian cancer tissue from paclitaxel-treated patients demonstrated that high Bcl-xL predicted poor response to paclitaxel. Taken together, the data suggest the paclitaxel–navitoclax combination might be effective in cancers expressing high levels of Bcl-xL.

Leflunomide Induces Apoptosis in Fludarabine-Resistant and Clinically Refractory CLL Cells

Environmental conditions in lymph node proliferation centers protect chronic lymphocytic leukemia (CLL) cells from apoptotic triggers. This situation can be mimicked by in vitro stimulation with CD40 ligand (CD40L) and interleukin 4 (IL-4). Our study investigates the impact of the drug leflunomide to overcome apoptosis resistance of CLL cells. CLL cells were stimulated with CD40L and IL-4 and treated with fludarabine and the leflunomide metabolite A771726. Resistance to fludarabine-mediated apoptosis was induced by CD40 activation alone stimulating high levels of BCL-XL and MCL1 protein expression. Apoptosis resistance was further enhanced by a complementary Janus-activated kinase (JAK)/STAT signal induced by IL-4. In contrast, CLL proliferation required both a CD40 and a JAK/STAT signal and could be completely blocked by pan-JAK inhibition. Leflunomide (A771726) antagonized CD40L/IL-4-induced proliferation at very low concentrations (3 μg/mL) reported to inhibit dihydroorotate dehydrogenase. At a concentration of 10 μg/mL, A771726 additionally attenuated STAT3/6 phosphorylation, whereas apoptosis of CD40L/IL-4-activated ("resistant") CLL cells was achieved with higher concentrations (IC(50): 80 μg/mL). Apoptosis was also effectively induced by A771726 in clinically refractory CLL cells with and without a defective p53 pathway. Induction of apoptosis involved inhibition of NF-κB activity and loss of BCL-XL and MCL1 expression. In combination with fludarabine, A771726 synergistically induced apoptosis (IC(50): 56 μg/mL). We thus show that A771726 overcomes CD40L/IL-4-mediated resistance to fludarabine in CLL cells of untreated as well as clinically refractory CLL cells. We present a possible novel therapeutic principle for attacking chemoresistant CLL cells.

Identification of An Abnormal JAK2 WT CD34+ Cell Population Characterized by High Bcl-Xl Expression Levels Both in JAK2 Positive and Negative PV, ET and PMF Patients,

Abstract 3472 Background:The JAK2V617F gene mutation is a common feature of Essential Thrombocythemia (ET), Polycythemia Vera (PV) and Primary Myelofibrosis (PMF); however the disease can occur without the JAK2 mutation suggesting that ET, PV and PMF share a common molecular mechanisms which is still unknown. In addition, in patients with the JAK2V617F mutation a substantial proportion of progenitor cells do not bear the JAK2V617F mutation and it is still unclear whether this population is normal or not. Methods: 64 patients affected by MPNs and 12 healthy subjects were included in the study. Twenty three patients were affected by ET, 25 by PV and 22 by PMF. We analyzed the expression, of the anti-apoptotic gene Bcl-xL, both at mRNA and protein level by qRT-PCR and immunofluorescence assay with specific antibodies. Erythroblasts, CD34+ and CD34+CD38-cell populations were selected and analyzed. Moreover, we designed specific PNA (Peptide Nucleic Acid) for JAK2V617F and for Bcl-xL isoform to identify their presence/expression at single CD34+ cell level. Results: Bcl-xL protein and mRNA were found progressively over-expressed in erythroblasts, in CD34+ and in CD34+ CD38- cells from ET, PV and PMF patients without any differences between JAK2V617F and wild type (WT) patients. The median protein level in erythroblasts increased from 22 Relative Units (RU) in ET, to 68,5 in PV and to 85 in PMF. Similarly, in CD34+ Bcl-xL increased from 30 RU in ET, to 89 RU in PV, and to 135 RU in PMF. Finally, CD34+CD38-increased from 17.4 RU in ET, to 45.5 RU in PV, to 101.7 RU in PMF. In addition, analysis by PNA identified distinct progenitor cells population. In patients bearing the JAK2 mutation, three CD34+ cell sub-populations were found: (a) one JAK2 positive and with high levels of Bcl-xL; (b) one JAK2 negative and with high levels of Bcl-xL. Thus, the cell population showing high levels of Bcl-xL is always greater than the JAK2 positive one; (c) one double negative population. In patients without JAK2 mutation, two CD34+ cell populations are identified: (a) one characterized by high levels of Bcl-xL and (b) one double negative. The distribution of these cell subpopulations is different in the distinct MPN. Conclusions: We have identified a previously undisclosed CD34+ cell population which is characterized by high level of Bcl-xL expression and represents the majority of CD34+ cells both in wild type and mutated samples. Interestingly, in mutated samples, this population encompasses the JAK2 mutated one. These findings suggest that Bcl-xL plays an important role in MPN, although it remains undisclosed whether this activation is dependent from an upstream oncogenetic signal. Finally, with this technique we can now identify a putative residual normal CD34+ cells in MPN which may be useful for monitoring patients undergoing targeted therapy with inhibitors. Altogether, these findings disclose unfolded aspects of pathophysiology of MPN and may contribute to a better understanding of the pathogenesis of MPN. Disclosures:Barosi:Novartis: Membership on an entity's Board of Directors or advisory committees. Saglio:Novartis Pharmaceutical: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Speakers Bureau; Pfizer: Consultancy.

Bcl-xL Is a Dominant Antiapoptotic Protein that Inhibits Homoharringtonine-Induced Apoptosis in Leukemia Cells

Homoharringtonine (HHT) has been reported to be effective in a portion of patients with acute myeloid leukemia (AML) or chronic myeloid leukemia (CML). To investigate its mechanism of action, cell growth inhibition and cytotoxicity of HHT were investigated in three AML cell lines, HL-60, NB4, and U937, and in three CML cell lines, K562, KU812, and KCL22. AML cells were more sensitive than CML cells to HHT-induced cytotoxicity. Using HL-60 cells, it was revealed that HHT decreased the levels of myeloid cell leukemia 1 (Mcl-1), X-linked inhibitor of apoptosis protein (XIAP), survivin, and B-cell lymphoma 2 (Bcl-2)-homology domain 3 (BH3)-only proteins as well as the mitochondrial membrane potential. The levels of Bcl-2, Bcl-2-associated X protein (Bax), and Bcl-2 homologous antagonist/killer (Bak) proteins in HL-60 cells were not changed after HHT treatment. U937, K562, KU812, and KCL22 cells expressed B-cell lymphoma-extra large (Bcl-xL) and were less responsive to HHT-induced apoptosis than HL-60 cells. Silencing Mcl-1 or Bcl-xL, but not XIAP or survivin, enhanced HHT-induced apoptosis in U937 cells. The levels of HHT-induced apoptosis in K562, KCL22, and KU812 cells were inversely correlated with the levels of Bcl-xL but not those of Bcl-2 or Mcl-1. K562 cells expressing high levels of Bcl-xL but no Bcl-2 were less responsive to HHT-induced apoptosis than KCL22 cells that expressed lower levels of Bcl-xL and higher levels of Bcl-2 protein. In K562 cells, knockdown of Bcl-xL, but not of Mcl-1, enhanced HHT-induced apoptosis. Transfection of Bcl-xL into KCL22 cells attenuated HHT-induced apoptosis. These data suggest that Bcl-xL plays a more important role than Bcl-2 and Mcl-1 in protecting against HHT-induced apoptosis.

Navitoclax Enhances the Efficacy of Taxanes in Non–Small Cell Lung Cancer Models

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

Navitoclax enhances the activity of taxanes in non-small cell lung carcinoma (NSCLC) models

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