Anti-apoptotic Bcl-2 Family Proteins Research Articles

Human Serum Albumin and the p53-Derived Peptide Fusion Protein Promotes Cytotoxicity Irrespective of p53 Status in Cancer Cells.

Human serum albumin (HSA) fusion protein is a viable and effective approach to target and inhibit essential intracellular pathways. It has previously been shown that an HSA fusion protein containing a p53-reactivating peptide (rHSA-p53i) retains the binding activity to MDM2 and MDMX, resulting in p53 transcription-dependent apoptosis. Here, we demonstrate that rHSA-p53i is able to bind and neutralize anti-apoptotic Bcl-2 family proteins, Bcl-xL and Mcl-1. This interaction displaces pro-apoptotic Bak and subsequently leads to intrinsic apoptosis via mimicking a p53 transcription-independent pathway. Cytotoxicity induced by rHSA-p53i, via p53 transcription dependent and independent apoptotic pathways, is irrespective of the p53 status in MDA-MB-231, HeLa, and SJSA-1 cells possessing either mutant, deficient, or wild-type p53. The therapeutic potential is also confirmed by treating SJSA-1 and MDA-MB-231 xenograft mouse tumors with rHSA-p53i. These data reveal that rHSA-p53i interferes with at least four intracellular targets, making it a viable therapeutic protein for the treatment of a variety of cancers, as well as a carrier to deliver fatty acid-modified chemotherapeutics.

Precision Targeting of BFL-1/A1 and an ATM Co-dependency in Human Cancer

Cancer cells overexpress a diversity of anti-apoptotic BCL-2 family proteins, such as BCL-2, MCL-1, and BFL-1/A1, to enforce cellular immortality. Thus, intensive drug development efforts have focused on targeting this class of oncogenic proteins to overcome treatment resistance. Whereas a selective BCL-2 inhibitor has been FDA approved and several small molecule inhibitors of MCL-1 have recently entered phase I clinical testing, BFL-1/A1 remains undrugged. Here, we developed a series of stapled peptide design principles to engineer a functionally selective and cell-permeable BFL-1/A1 inhibitor that is specifically cytotoxic to BFL-1/A1-dependent human cancer cells. Because cancers harbor a diversity of resistance mechanisms and typically require multi-agent treatment, we further investigated BFL-1/A1 co-dependencies by mining a genome-scale CRISPR-Cas9 screen. We identified ataxia-telangiectasia-mutated (ATM) kinase as a BFL-1/A1 co-dependency in acute myeloid leukemia (AML), which informed the validation of BFL-1/A1 and ATM inhibitor co-treatment as a synergistic approach to subverting apoptotic resistance incancer.

Limonene and BEZ 235 induce apoptosis in COLO-320 and HCT-116 colon cancer cells

Deregulated apoptosis is the hall mark of many cancers, therefore every defect in apoptosis pathway could be a potential target for cancer treatment.The anticancer mechanism of limonene could be multifactorial. However, induction of apoptosis in cancer cells is proposed as the predominant mechanism in several of preclinical studies. Therefore, we determined to investigate the role of apoptosis in the anticancer activity of limonene and BEZ235 combination in COLO-320 and HCT-116 colon cancer cells. Cells after treatments were assessed for apoptosis by DAPI staining for fluorescent microscopic examination of apoptotic cells, estimation of caspases activities, Bcl-2 family proteins in addition to cell cycle analysis by flowcytometry. Results show that both drugs induced apoptosis as demonstrated by increased caspases activity, significant alterations in pro and anti-apoptotic proteins of Bcl-2 family in promoting apoptosis and cell cycle arrest at G1 phase. Over all, it is indicated that limonene and BEZ exerted anticancer activity is mediated through induction of apoptosis involving mitochondria mediated intrinsic death pathway in the selected CRC cells.

Discovery and development of substituted tyrosine derivatives as Bcl-2/Mcl-1 inhibitors

Anti-apoptotic Bcl-2 family proteins are vital for cancer cells to escape apoptosis, which make them attractive targets for cancer therapy. Recently, a lead compound 1 was found to modestly inhibit the binding of BH3 peptide to Bcl-2 protein with a Ki value of 5.2 µM. Based on this, a series of substituted tyrosine derivatives were developed and tested for their binding affinities to Bcl-2 protein. Results indicated that these compounds exhibited potent binding affinities to Bcl-2 and Mcl-1 protein but not to Bcl-XL protein. Promisingly, compound 6i inhibited the binding of BH3 peptide to Bcl-2 and Mcl-1 protein with a Ki value of 450 and 190 nM respectively, and showed obvious anti-proliferative activities against tested cancer cells.

Low cleaved caspase-7 levels indicate unfavourable outcome across all breast cancers.

BCL2 levels are elevated in breast cancer where they are marker of good prognosis. BCL2 and active caspase levels correlate negatively; yet, active caspases indicate good outcome. Low BCL2 and low caspase-7 are highly prognostic of unfavourable outcome across all breast cancers. BCL2 levels indicate molecular subtype and tumour proliferation status in breast cancer.

Abstract A39: Estrogen-driven noncanonical WNT4 signaling is essential for proliferation and survival in lobular carcinoma cells

Abstract Invasive lobular carcinoma (ILC) is the second most common histologic subtype of breast cancer. Though >90% of ILC tumors are estrogen receptor (ER)-positive, ILC patients have poorer outcomes than others with ER-positive tumors, suggesting ER biology is unique in ILC cells. We previously identified the Wnt ligand WNT4 as an ILC-specific ER transcriptional target that is critical for both estrogen-induced growth and antiestrogen resistance in ILC cells. We hypothesize that characterizing the WNT4 signaling network in ILC cells will identify novel biomarkers and therapeutic targets for ILC patients. WNT4 signals in a paracrine manner in normal mammary gland and in breast cancer tissues, via secretion from luminal cells to nearby basal or basal-like cells. Inhibiting Wnt secretion with Porcupine-inhibitors (PORCNi) in these contexts blocks WNT4-driven phenotypes. However, we have observed that PORCNi have no effect on ILC cell proliferation. Further, PORCNi combined with siRNA knockdown of PORCN completely suppress secretion of overexpressed WNT3A but not of WNT4. These observations suggest that WNT4 engages its signaling cascade via an atypical mechanism in ILC cells, where WNT4 signals in an autocrine or intracellular manner or is secreted via PORCN-independent mechanisms. Understanding how WNT4 activates signaling is vital to developing appropriate therapeutic strategies, especially as PORCNi have been tested clinically for ILC patients. In both the normal mammary gland and in breast cancer cells, WNT4 engages the canonical Wnt pathway and activates β-catenin-mediated gene regulation. However, ILC cells and tumors lack β-catenin protein (related to loss of E-cadherin); WNT4 must be activating an alternative signaling cascade in ILC cells. To define downstream WNT4 signaling in ILC cells, we utilized reverse phase protein arrays (RPPA) and identified ER-mediated protein changes blocked by concurrent WNT4 knockdown. These experiments identified putative targets that link WNT4 signaling to control of proliferation and cell cycle as well as apoptosis and cell survival. In the former, coordinate suppression of p21 and induction of FOXM1 control G1/S and G2/M transitions, respectively, and WNT4 knockdown engages both checkpoints to halt cellular proliferation. In the latter, WNT4 activates distinct components of Akt/mTOR signaling in ILC versus IDC cells, as E2 treatment increases phosphorylation of mTOR/Rictor but not of Akt in ILC cells. Activation of mTOR correlates with post-transcriptional regulation of levels of the anti-apoptotic BCL2-family protein MCL-1; E2-mediated suppression of MCL-1 protein levels via mTOR may regulate cell survival and sensitivity to apoptotic stimuli. WNT4 mediates a critical ER-driven pathway in ILC cells, but the WNT4 signaling network has not been characterized to identify putative therapeutic approaches for ILC patients. Our data suggest that targeting FOXM1, mTOR, or BCL2-family proteins may modulate ER/WNT4 signaling and that ILC cells are unlikely to respond to PORCNi. Characterization of this unique signaling network will drive new predictive models and therapeutic approaches for ILC patients. Citation Format: Deviyani M. Rao, Rebecca Ferguson, Matthew J. Sikora. Estrogen-driven noncanonical WNT4 signaling is essential for proliferation and survival in lobular carcinoma cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A39.

ABT-199-mediated inhibition of Bcl-2 as a potential therapeutic strategy for nasopharyngeal carcinoma

BackgroundAberrant overexpression of Bcl-2 protein has been detected in 80% of nasopharyngeal carcinoma (NPC), and Bcl-2 family proteins are implicated in both NPC oncogenesis and chemotherapy resistance. Previous studies have shown that while treatment of NPC cells with Bcl-2 family inhibitors alone is rarely effective, concomitant treatment with a cytotoxic reagent such as cisplatin can increase efficacy through a synergistic effect. The aim of the current work was to determine how we might increase the efficacy of Bcl-2 family inhibitors in the absence of cytotoxic reagents, which are associated with negative side effect profiles. MethodsWe assessed cell proliferation in Bcl-2 high-expressing NPC cells by CCK-8 assay after treatment with the Bcl-2 inhibitor ABT-199 and/or the Mcl-1 inhibitor S63845. Apoptotic induction by ABT-199 was evaluated by Annexin V-FITC and PI double staining. We also evaluated Bcl-2 family protein expression (Bim, Mcl-1, Bcl-xL, Noxa) after treatment with ABT-199 by western blotting. Finally, xenografted Balb/c nude mice were used to test ABT-199 efficacy in vivo, H&E and immunohistochemistry assay were used to analyze tumor samples. ResultsABT-199 effectively induced NPC cell apoptosis in vitro and in the xenograft model. Following ABT-199 treatment in NPC cells, upregulation of Mcl-1 and Bcl-xL can lead to drug resistance, while concomitant Noxa overexpression partially neutralized the Mcl-1-caused resistance. Given that ABT-199 induces apoptosis in NPC cells through the Bcl-2/Noxa/Mcl-1 axis, treatment avenues further targeting this pathway should be promising. Indeed, the newly developed Mcl-1 inhibitor S63845 in combination with ABT-199 had a synergistic effect on NPC cell apoptosis. ConclusionBcl-2 inhibition in NPC cells with ABT-199 triggers apoptosis through the Bcl-2/Noxa/Mcl-1 axis, and dual inhibition of the anti-apoptotic Bcl-2 family proteins Bcl-2 and Mcl-1 provided a strong synergistic effect without the need for adjunctive cytotoxic agent treatment with cisplatin.

Atypical APC/C-dependent degradation of Mcl-1 provides an apoptotic timer during mitotic arrest.

The initiation of apoptosis in response to the disruption of mitosis provides surveillance against chromosome instability. Here, we show that proteolytic destruction of the key regulator Mcl‐1 during an extended mitosis requires the anaphase‐promoting complex or cyclosome (APC/C) and is independent of another ubiquitin E3 ligase, SCFFbw7. Using live‐cell imaging, we show that the loss of Mcl‐1 during mitosis is dependent on a D box motif found in other APC/C substrates, while an isoleucine‐arginine (IR) C‐terminal tail regulates the manner in which Mcl‐1 engages with the APC/C, converting Mcl‐1 from a Cdc20‐dependent and checkpoint‐controlled substrate to one that is degraded independently of checkpoint strength. This mechanism ensures a relatively slow but steady rate of Mcl‐1 degradation during mitosis and avoids its catastrophic destruction when the mitotic checkpoint is satisfied, providing an apoptotic timer that can distinguish a prolonged mitotic delay from normal mitosis. Importantly, we also show that inhibition of Cdc20 promotes mitotic cell death more effectively than loss of APC/C activity through differential effects on Mcl‐1 degradation, providing an improved strategy to kill cancer cells.

Abstract 3522: Both mitochondrial function and composition of BCL2 family proteins determines sensitivity to Cisplatin in ovarian cancer cells and are promising targets to overcome Cisplatin resistance in ovarian cancer

Abstract Mitochondria are critical target structures of platinum drugs. To study whether mitochondrial activities of Cisplatin are critical for its cytotoxic function we studied mitochondrial mass, mitochondrial function and composition of pro- and anti-apoptotic BCL2 family proteins in three resistant and three sensitive high-grade serous carcinoma (HGSC) cell lines. Sensitive cell lines differed from resistant cells by higher mitochondrial mass (MM), higher levels of mitochondrial reactive oxygen species (mtROS) and higher basal oxygen consumption rate (OCR) levels as well as a higher ratio of pro- vs. anti-apoptotic BCL2 family proteins. Cell death upon Cisplatin in sensitive cells was dependent on induction of reactive oxygen species (ROS), loss of mitochondrial membrane potential (MMP), induction of BAX-BAK pores and caspases. Genetically alteration of the BCL2 family protein ratio by siRNA mediated knockdown of pro-apoptotic NOXA reduced acute response in sensitive cell lines, whereas knockdown of anti-apoptotic BCLw rendered resistant cells sensitive. In analogy, pharmacological inhibition of anti-apoptotic proteins by ABT737 also completely re-sensitized Cisplatin resistant cell lines. Cisplatin exposure increased MM, mtROS and OCR in both sensitive and insensitive HGSC cell lines. However, these parameters reached higher peak levels in cell lines sensitive to induction of cell death by Cisplatin. Knockdown of PGC-1α (the master regulator of mitochondrial biogenesis) partially rescues sensitive cells from apoptosis. In addition, pharmacological inhibition of ATP synthase by Oligomycin A blocks MMP and mtROS induction by Cisplatin and inhibits Cisplatin induced cell death. This supports the hypothesis that induction of mitochondria leading to increased release of mtROS contributes to cell death by Cisplatin. We finally tested whether other modulators of mitochondrial function restore sensitivity to Cisplatin in resistant cells. Whereas Complex-I and Complex-III inhibitors did not affect cytotoxicity of Cisplatin, the iron chelator VLX600 had significant synergistic activity with Cisplatin in resistant cancer cells. VLX600 inhibits oxygen consumption almost completely in combination with Cisplatin whereas both compounds had no significant effect on survival when used as monotherapy. In conclusion, cytotoxicity of Cisplatin in HGSC cell lines depends on efficient induction of MM and mtROS. Resistance can efficiently be targeted by modulating the mitochondrial function by VLX600. These observations support the view that mitochondria are attractive targets for increasing Cisplatin activity on cancer cells. Citation Format: Markus Kleih, Simon Heine, Kathrin Böpple, Meng Dong, Heiko van der Kuip, Walter E. Aulitzky. Both mitochondrial function and composition of BCL2 family proteins determines sensitivity to Cisplatin in ovarian cancer cells and are promising targets to overcome Cisplatin resistance in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3522.

Abstract 1877: Selective inhibitor of nuclear export (SINE) compound, eltanexor (KPT-8602), synergizes with venetoclax (ABT-199) to eliminate leukemia cells and extend survival in an in vivo model of acute myeloid leukemia

Abstract SINE compounds (KPT-330/selinexor and KPT-8602/eltanexor) are a novel class of small molecules with therapeutic potential across a broad range of cancers. SINE compounds target exportin 1 (XPO1), which is an essential nucleo-cytoplasmic transport protein, responsible for the nuclear export of >200 cargo proteins including many tumor-suppressor proteins (TSPs) and oncogenes, such as p53, IκB, BCL-2, eIF4E and MYC. XPO1 is overexpressed in numerous solid and hematologic malignancies, often correlating with poor prognosis. An important family of oncoproteins regulated by SINE compounds are the pro-survival BCL-2 family proteins, BCL-2 and MCL-1, as both are transported from the nucleus by forming a complex with the XPO1 cargo protein, elF4E. BCL-2 and MCL-1 are dysregulated and linked to maintenance and survival in numerous cancers. Early studies with venetoclax (VEN/ABT-199), a potent, selective inhibitor of BCL-2, have revealed response rates from 50-80% across a variety of hematologic malignancies. However, resistance often arises via upregulation of other BCL-2 family antiapoptotic proteins, most commonly MCL-1. Thus, we hypothesized that co-treatment of VEN and SINE compounds would show enhanced cell killing in AML due to the ability of SINE to further block the prosurvival signals of both BCL-2 and MCL-1. Co-treatment with eltanexor and VEN demonstrated synergistic loss of viability in multiple hematologic malignancy cell lines, particularly MV-4-11 cells. Further in vitro analysis showed this enhanced cell death was due to an increase in apoptosis that led to a loss of clonogenicity in methylcellulose assays. We used in vivo studies to test the effect of this combination in a systemic AML xenograft model. For this, MV-4-11 cells were transplanted into NSGS mice and allowed to engraft before treating the mice with vehicle, eltanexor, VEN, or a combination of the two compounds. When the vehicle mice became moribund at 4 weeks after transplant, all treatment groups were sacrificed to compare leukemic burden in the hematopoietic tissues. Flow cytometric analysis revealed a drastic elimination of leukemic cells in the bone marrow, spleen, and peripheral blood of the combination-treated mice compared to vehicle and single-treatment groups. Immunohistochemical analysis of the spleen and bone marrow confirmed this finding, and also revealed the reduction in leukemic cells was due, in part, to an induction of apoptosis (an increase in cleaved caspase 3 and decrease in survivin) in splenic tissue sections, in agreement with the in vitro results. Finally, the dramatic clearance of leukemia cells in the mouse caused by eltanexor and VEN treatment led to prolonged survival. Our results demonstrate the potential of this combination regimen in the treatment of AML. Citation Format: Melissa A. Fischer, Pia M. Arrate, Hua Chang, Agnieszka E. Gorska, Londa S. Fuller, Haley E. Ramsey, Trinayan Kashyap, Christian Argueta, William Senapedis, Erkan Baloglu, Yosef Landesman, Sharon Shacham, Michael R. Savona. Selective inhibitor of nuclear export (SINE) compound, eltanexor (KPT-8602), synergizes with venetoclax (ABT-199) to eliminate leukemia cells and extend survival in an in vivo model of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1877.

PO-072 ω3-Polyunsaturated fatty acids inhibit cell growth and invasion of human hypopharyngeal carcinoma cells

IntroductionT-cell acute lymphoblastic leukaemia (T-ALL) is an aggressive hematologic malignancy resulting from the transformation of T-cell progenitors. Early T-cell progenitor ALL (ETP-ALL) subgroup accounts for 5% to 10% of T-ALL cases and associated with a poor prognosis and a very high risk for relapse. T-ALL leukemias develop a dependence on anti-apoptotic BCL-2 family proteins that is linked to the maturation stage of the blasts. Typical T-ALL is mostly dependent on BCL-XL, whereas ETP-ALL is more dependent on BCL-2. In recent years, there has been the development of a series of specific BH3 mimetics (ABT-199, ABT-263, WEHI 539) that can inhibit the function of anti-apoptotic BCL-2 proteins and induce apoptosis. T-ALL patients have blasts disseminated in the blood, bone marrow and spleen. Each niche may provide distinct extrinsic signals that could offer a survival advantage to leukemic cells.Material and methodsWe aim to assess in both ETP-ALL (Loucy cell line) and typical T-ALL (CEM-CCRF cell line) if distinct microenvironments affect the BCL-2 family proteins expression and the sensitivity to BH3 mimetics. To study the anti-apoptotic BCL-2 dependencies we will use the cutting-edge technology mitochondrial BH3 profiling. In brief, BH3 profiling is a functional assay that measures the response of mitochondria to exposure of known concentrations of synthetic BH3 peptides by measuring loss of mitochondrial membrane potential or cytochrome c release.Results and discussionsWe found that ETP-ALL cell line was BCL-2 dependent and sensitive to ABT-199 (BCL-2 specific BH3 mimetic). While the Typical T-ALL cell line was BCL-XL dependent and sensitive to both ABT-263 (BCL-2,BCL-XL and BCL-w) and WEHI 539 (BCL-XL) BH3 mimetics. Interestingly, when the ETP–ALL cell line Loucy was co-cultured with the human splenic fibroblast (HSF) cell line we found a reduction in BCL-2 dependency, as assessed by BH3 profiling, and a reduced sensitivity to ABT-199 (BCL-2 specific BH3 mimetic). Remarkably, we found the inverse in the typical T-ALL cell line co-cultured with the HSF. We found an increase in BCL-2 dependence and an increase in sensitivity to ABT-199.ConclusionCurrently, we are trying to identify the signalling pathways which are activated to cause this switch in BCL-2 family dependence in the different subtypes of T-ALL. In addition, we are assessing the BCL-2 family dependence of a xenograft cell line isolated from the blood and spleen of animals to determine if this switiching of BCL-2 dependence occurs in vivo.

PO-068 Determining how distinct microenvironments alter BCL-2 family dependence in T-cell acute lymphoblastic leukaemia

IntroductionT-cell acute lymphoblastic leukaemia (T-ALL) is an aggressive hematologic malignancy resulting from the transformation of T-cell progenitors. Early T-cell progenitor ALL (ETP-ALL) subgroup accounts for 5% to 10% of T-ALL cases and associated with a poor prognosis and a very high risk for relapse. T-ALL leukemias develop a dependence on anti-apoptotic BCL-2 family proteins that is linked to the maturation stage of the blasts. Typical T-ALL is mostly dependent on BCL-XL, whereas ETP-ALL is more dependent on BCL-2. In recent years, there has been the development of a series of specific BH3 mimetics (ABT-199, ABT-263, WEHI 539) that can inhibit the function of anti-apoptotic BCL-2 proteins and induce apoptosis. T-ALL patients have blasts disseminated in the blood, bone marrow and spleen. Each niche may provide distinct extrinsic signals that could offer a survival advantage to leukemic cells.Material and methodsWe aim to assess in both ETP-ALL (Loucy cell line) and typical T-ALL (CEM-CCRF cell line) if distinct microenvironments affect the BCL-2 family proteins expression and the sensitivity to BH3 mimetics. To study the anti-apoptotic BCL-2 dependencies we will use the cutting-edge technology mitochondrial BH3 profiling. In brief, BH3 profiling is a functional assay that measures the response of mitochondria to exposure of known concentrations of synthetic BH3 peptides by measuring loss of mitochondrial membrane potential or cytochrome c release.Results and discussionsWe found that ETP-ALL cell line was BCL-2 dependent and sensitive to ABT-199 (BCL-2 specific BH3 mimetic). While the Typical T-ALL cell line was BCL-XL dependent and sensitive to both ABT-263 (BCL-2,BCL-XL and BCL-w) and WEHI 539 (BCL-XL) BH3 mimetics. Interestingly, when the ETP–ALL cell line Loucy was co-cultured with the human splenic fibroblast (HSF) cell line we found a reduction in BCL-2 dependency, as assessed by BH3 profiling, and a reduced sensitivity to ABT-199 (BCL-2 specific BH3 mimetic). Remarkably, we found the inverse in the typical T-ALL cell line co-cultured with the HSF. We found an increase in BCL-2 dependence and an increase in sensitivity to ABT-199.ConclusionCurrently, we are trying to identify the signalling pathways which are activated to cause this switch in BCL-2 family dependence in the different subtypes of T-ALL. In addition, we are assessing the BCL-2 family dependence of a xenograft cell line isolated from the blood and spleen of animals to determine if this switiching of BCL-2 dependence occurs in vivo.

Abstract 299: A susceptible-quiescent model can describe biphasic cell-kill by MCL1 inhibitor AZD5991 in vitro

Abstract AZD5991 is a potent and selective inhibitor of Mcl-1, now in phase 1 clinical development. Mcl-1 is an anti-apoptotic Bcl2-family protein that is up-regulated by cancer cells in order to avoid apoptosis. Disruption of binding of Mcl-1 to pro-apoptotic BH3-only proteins such as Bax and Bak results in loss in cellular viability of several cell-line models of multiple myeloma (MM). As a function of time, many cell lines display a biphasic response, with an initial period of rapid cell kill, followed by a period of slower kill. Such biphasic kill curves are not uncommonly seen in bacteria. As a function of concentration, cell lines display a range of efficacy (extent of cell kill) and potency (concentration of half-maximal cell kill). Here we apply a model originally developed for bacterial cell-kill to parameterize the in vitro cell kill over time and across several orders of magnitude in concentration. Cells from 10 MM cell lines were grown in culture, then plated at uniform density in microtiter plates where they were incubated at concentrations of AZD5991 ranging from 0.3nM to 30µM (with DMSO control), each plate for a duration of 0.5h to 72h. After the desired incubation period, Cell-Titer Glo® was used to assess cellular viability by luminescence. Plates were run in duplicate. A model of cell survival was implemented in MATLAB, in which cells can be in one of two states: a proliferating state ‘S' in which they are susceptible to a saturable concentration-dependent drug-induced cell death, or a quiescent state ‘Q' in which they don't proliferate and have only a constitutive death rate independent of drug concentration. Transition between the two states is possible in either direction. By fitting the model to the data across time and over a wide range of concentrations, we can succinctly and precisely describe the rate of growth and drug-induced cell death, as well as transition between the susceptible and quiescent states. To our knowledge, this represents the first application of such a model to a BH3 mimetic such as AZD5991. We find that the cell lines show much greater variation in efficacy (i.e., maximum inducible cell kill rate) than in potency, suggesting that the main difference between these lines lies in their dependence on Mcl-1 for survival. Citation Format: Francis D. Gibbons, Matthew Belmonte, Paul Secrist, Alwin Schuller. A susceptible-quiescent model can describe biphasic cell-kill by MCL1 inhibitor AZD5991 in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 299.

Abstract 294: BCL-2 family compensation regulates T cell homeostasis and reveals a BIM:BCL-W axis in T-ALL

Abstract BIM is the master BH3-only BCL-2 family regulator of lymphocyte survival. To understand how long-term loss of BIM affects apoptotic resistance in T and B cells we examined peripheral blood counts in mice with T or B cell-specific deletion of Bim. In contrast to CD19CRE Bimfl/fl animals, LCKCRE Bimfl/fl mice had pronounced early lymphocytosis followed by lymphocyte count normalization as they aged. To explore BCL-2 family specific mechanism(s) leading to this normalization we compared cell death sensitivity and BCL-2 expression patterns of young versus old LCKCRE Bimfl/fl T cells. Young (6-12 weeks) and old (>40 weeks) LCKCRE Bimfl/fl mice had consistently abnormal thymocyte development hallmarked by a decrease in CD4+8+ double positive (DP) cells and accumulation of double negative (DN) and single positive (SP) cells as previously described in global Bim knockout mice. Thymocytes maintained cell death resistance to a number of apoptotic stimuli throughout all ages, reflecting a consistent loss of BIM. In contrast to thymocytes, mature splenic-derived LCKCRE Bimfl/fl T cells from young mice were equally sensitive to apoptotic stimuli as WT controls, indicating BCL-2 family compensation. Expression profiling in these cells revealed upregulation of other BH3-only proteins, including Noxa = Puma << Bmf, suggesting that these proteins functionally compensated for the loss of BIM. To determine if this was a T cell-intrinsic process and if the presence of other BH3-only proteins with pan-BCL-2 family binding affinities were required for lymphocyte number and apoptotic normalization, we analyzed peripheral blood from mice globally deleted in Bim, Bid, and Puma (TKO). TKO, like LCKCRE Bimfl/fl mice, developed profound early lymphocytosis followed by normalization as they aged. A subset of TKO mice developed T-ALL hallmarked by upregulation of Bmf and Bcl-w, an under characterized anti-apoptotic BCL-2 family protein, suggesting that BCL-W allowed for malignant T cell survival despite the presence of BMF. To determine if such a relationship exists in human disease, expression profiling of pediatric T-ALL patient samples revealed a significant negative correlation between BIM and BCL-W expression in developmentally immature and mature T-ALL. Our findings suggest a model in which the loss of BIM in T cells can be compensated for through upregulation of BMF, a BH3-only protein mechanistically similar to BIM. Loss of other ‘direct activating' BH3-only proteins increases the chances of malignant cell escape despite BMF upregulation, particularly when BCL-W is overexpressed. This work provides a developmental framework between BIM and BMF in T cells and for a BIM:BCL-W axis in T-ALL. Overexpression of BCL-W in BIMlow-expressing T-ALL may be an additional target responsible for early immature T-ALL therapeutic resistance. Citation Format: Lindsey M. Ludwig, Lauren E. Roach, Samuel G. Katz, Jill K. Fisher, Melissa Burns, Matthew R. Schnorenberg, Riyue Bao, Makda Zewde, Yusuke Nakamura, Alejandro Gutierrez, Loren D. Walensky, James L. LaBelle. BCL-2 family compensation regulates T cell homeostasis and reveals a BIM:BCL-W axis in T-ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 294.

Telomerase Reverse Transcriptase Contains a BH3-Like Motif and Interacts with BCL-2 Family Members.

Upregulation of human telomerase reverse transcriptase (hTERT) expression is an important factor in the cellular survival and cancer. Although growing evidence suggests that hTERT inhibits cellular apoptosis by telomere-independent functions, the mechanisms involved are not fully understood. Here, we show that hTERT contains a BH3-like motif, a short peptide sequence found in BCL-2 family proteins, and interacts with anti-apoptotic BCL-2 family proteins MCL-1 and BCL-xL, suggesting a functional link between hTERT and the mitochondrial pathway of apoptosis. Additionally, we propose that hTERT can be categorized into the atypical BH3-only proteins that promote cellular survival, possibly due to the non-canonical interaction between hTERT and antiapoptotic proteins. Although the detailed mechanisms underlying the hTERT BH3-like motif functions and interactions between hTERT and BCL-2 family proteins have not been elucidated, this work proposes a possible connection between hTERT and BCL-2 family members and reconsiders the role of the BH3-like motif as an interaction motif.

Combination therapy with BH3 mimetic and hyperthermia tends to be more effective on anti-melanoma treatment

Malignant melanoma has shown increased incidence and high mortality rate in the last three decades. In this study, we investigated whether combination therapy with ch282-5 (a novel BH3 mimetic) and microwave hyperthermia could display synergistic antitumor effects against melanoma. Our results indicated that combination therapy reduced the viability and proliferation of melanoma cells. Through inhibiting the expression of anti-apoptotic proteins of Bcl-2 and IAP family and activating MAPK proteins, combined hyperthermia enhanced ch282-5-induced apoptosis. Combination therapy also synergistically disturbed the mTOR/p70S6k signaling pathway, which is important for cell survival and migration. Moreover, our results showed that combination therapy remarkably suppressed melanoma cell migration in vitro and significantly reduced experimental pulmonary metastasis in vivo. In conclusion, our results indicate that combination therapy with ch282-5 and hyperthermia has synergistic antitumor effects and provides a possible therapeutic strategy for advanced melanoma.

Synthetic lethality in CCNE1-amplified high grade serous ovarian cancer through combined inhibition of Polo-like kinase 1 and microtubule dynamics.

The taxanes are effective microtubule-stabilizing chemotherapy drugs that inhibit mitosis, induce apoptosis, and produce regression in a fraction of cancers that arise at many sites including the ovary. Novel therapeutic targets that augment taxane effects are needed to improve clinical chemotherapy response in CCNE1-amplified high grade serous ovarian cancer (HGSOC) cells. In this study, we conducted an siRNA-based kinome screen to identify modulators of mitotic progression in CCNE1-amplified HGSOC cells that may influence clinical paclitaxel response. PLK1 is overexpressed in many types of cancer, which correlates with poor prognosis. Here, we identified a novel synthetic lethal interaction of the clinical PLK1 inhibitor BI6727 and the microtubule-targeting drug paclitaxel in HGSOC cell lines with CCNE1-amplification and elucidated the underlying molecular mechanisms of this synergism. BI6727 synergistically induces apoptosis together with paclitaxel in different cell lines including a patient-derived primary ovarian cancer culture. Moreover, the inhibition of PLK1 reduced the paclitaxel-induced neurotoxicity in a neurite outgrowth assay. Mechanistically, the combinatorial treatment with BI6727/paclitaxel triggers mitotic arrest, which initiates mitochondrial apoptosis by inactivation of anti-apoptotic BCL-2 family proteins, followed by significant loss of the mitochondrial membrane potential and activation of caspase-dependent effector pathways. This conclusion is supported by data showing that BI6727/paclitaxel-co-treatment stabilizes FBW7, a component of SCF-type ubiquitin ligases that bind and regulate key modulators of cell division and growth including MCL-1 and Cyclin E. This identification of a novel synthetic lethality of PLK1 inhibitors and a microtubule-stabilizing drug has important implications for developing PLK1 inhibitor-based combination treatments in CCNE1-amplified HGSOC cells.

Epidermal growth factor receptor (EGFR) density may not be the only determinant for the efficacy of EGFR-targeted photoimmunotherapy in human head and neck cancer cell lines.

The aim of this study was to investigate the effects of targeted photoimmunotherapy (PIT) in vitro on cell lines with various expression levels of epidermal growth factor receptor (EGFR) using an anti-EGFR targeted conjugate composed of Cetuximab and IR700DX, phthalocyanine dye. Relative EGFR density and cell binding assay was conducted in three human head & neck cancer cell lines (scc-U2, scc-U8, and OSC19) and one reference cell line A431. After incubation with the conjugate for 1 or 24 hours, cellular uptake and localization were investigated by confocal laser scanning microscopy and quantified by image analysis. Cell survival was determined using the MTS assay and alamarBlue assay after PIT with a 690 nm laser to a dose of 7 J.cm-2 (at 5 mW.cm-2 ). The mode of cell death was examined with flow cytometry using apoptosis/necrosis staining by Annexin V/propidium iodide, together with immunoblots of anti-apoptotic Bcl-2 family proteins Bcl-2 and Bcl-xL. A431 cells had the highest EGFR density followed by OSC19, and then scc-U2 and scc-U8. The conjugates were localized both on the surface and in the cytosol of the cells after 1- and 24-hour incubation. After 24-hour incubation the granular pattern was more pronounced and in a similar pattern of a lysosomal probe, suggesting that the uptake of conjugates by cells was via receptor-mediated endocytosis. The results obtained from the quantitative imaging analysis correlate with the level of EGFR expression. Targeted PIT killed scc-U8 and A431 cells efficiently; while scc-U2 and OSC19 were less sensitive to this treatment, despite having similar EGFR density, uptake and localization pattern. Scc-U2 cells showed less apoptotic cell dealth than in A431 after 24-hour targeted PIT. Immunoblots showed significantly higher expression of anti-apoptotic Bcl-2 and Bcl-xL proteins in scc-U2 cell lines compared to scc-U8. Our study suggests that the effectiveness of EGFR targeted PIT is not only dependent upon EGFR density. Intrinsic biological properties of tumor cell lines also play a role in determining the efficacy of targeted PIT. We have shown that in scc-U2 cells this difference may be caused by differences in the apoptopic pathway. Lasers Surg. Med. 50:513-522, 2018. © 2018 Wiley Periodicals, Inc.

Targeting MUC1-C suppresses BCL2A1 in triple-negative breast cancer

B-cell lymphoma 2-related protein A1 (BCL2A1) is a member of the BCL-2 family of anti-apoptotic proteins that confers resistance to treatment with anti-cancer drugs; however, there are presently no agents that target BCL2A1. The MUC1-C oncoprotein is aberrantly expressed in triple-negative breast cancer (TNBC) cells, induces the epithelial–mesenchymal transition (EMT) and promotes anti-cancer drug resistance. The present study demonstrates that targeting MUC1-C genetically and pharmacologically in TNBC cells results in the downregulation of BCL2A1 expression. The results show that MUC1-C activates the BCL2A1 gene by an NF-κB p65-mediated mechanism, linking this pathway with the induction of EMT. The MCL-1 anti-apoptotic protein is also of importance for the survival of TNBC cells and is an attractive target for drug development. We found that inhibiting MCL-1 with the highly specific MS1 peptide results in the activation of the MUC1-C→NF-κB→BCL2A1 pathway. In addition, selection of TNBC cells for resistance to ABT-737, which inhibits BCL-2, BCL-xL and BCL-W but not MCL-1 or BCL2A1, is associated with the upregulation of MUC1-C and BCL2A1 expression. Targeting MUC1-C in ABT-737-resistant TNBC cells suppresses BCL2A1 and induces death, which is of potential therapeutic importance. These findings indicate that MUC1-C is a target for the treatment of TNBCs unresponsive to agents that inhibit anti-apoptotic members of the BCL-2 family.

Loss of Wilms tumor 1 protein is a marker for apoptosis in response to replicative stress in leukemic cells

A remaining expression of the transcription factor Wilms tumor 1 (WT1) after cytotoxic chemotherapy indicates remaining leukemic clones in patients. We determined the regulation and relevance of WT1 in leukemic cells exposed to replicative stress and DNA damage. To induce these conditions, we used the clinically relevant chemotherapeutics hydroxyurea and doxorubicin. We additionally treated cells with the pro-apoptotic kinase inhibitor staurosporine. Our data show that these agents promote apoptosis to a variable extent in a panel of 12 leukemic cell lines and that caspases cleave WT1 during apoptosis. A chemical inhibition of caspases as well as an overexpression of mitochondrial, anti-apoptotic BCL2 family proteins significantly reduces the processing of WT1 and cell death in hydroxyurea-sensitive acute promyelocytic leukemia cells. Although the reduction of WT1 correlates with the pharmacological efficiency of chemotherapeutics in various leukemic cells, the elimination of WT1 by different strategies of RNA interference (RNAi) does not lead to changes in the cell cycle of chronic myeloid leukemia K562 cells. RNAi against WT1 does also not increase the extent of apoptosis and the accumulation of γH2AX in K562 cells exposed to hydroxyurea. Likewise, a targeted genetic depletion of WT1 in primary oviduct cells does not increase the levels of γH2AX. Our findings position WT1 as a downstream target of the apoptotic process that occurs in response to cytotoxic forms of replicative stress and DNA damage.