Bcl-2 Antagonist Research Articles

Mitochondrial Apoptotic Priming Is Associated with Clinical Response to the Bcl-2 Antagonist ABT-199 in Chronic Lymphocytic Leukemia

BACKGROUNDBH3 profiling is a functional assay that assesses the degree to which cells are primed to die via mitochondrial apoptosis. We previously used this technique to show that pre-treatment CLL cells from patients who responded to frontline chemoimmunotherapy had higher levels of apoptotic priming than cells from refractory patients (Davids et al., Blood, 2012). BH3 profiling also detects dependence on individual anti-apoptotic Bcl-2 family proteins. Recently, ABT-199, a selective, potent, small molecule antagonist of the anti-apoptotic protein Bcl-2, has been found to be highly active in patients with relapsed or refractory CLL, even those with high-risk markers such as del(17p) (Seymour et al., EHA, 2014). Unlike its Bcl-XL/Bcl-2-targeting predecessor navitoclax (ABT-263), ABT-199 spares platelets, which rely on Bcl-XL for survival. We hypothesized that baseline levels of apoptotic priming would be associated with the depth of initial clinical response to ABT-199 in CLL patients. METHODSPeripheral blood samples were obtained just prior to initial dosing on the phase I, first-in-human study of ABT-199 in relapsed/refractory CLL. Viability was determined by Annexin V–FITC and propidium iodine by FACS. BH3 profiling was performed by exposing gently permeabilized CLL cells to a panel of BH3-domain peptides, fixing the cells, and quantifying apoptotic priming by assessing cytochrome C release by FACS. Platelets from 3 healthy donors were also analyzed. Clinical response was assessed by comparing baseline values to first-re-staging, as follows: peripheral blood: % reduction in absolute lymphocyte count, lymph node: % reduction in the sum of the product of the diameters of the 6 target lesions, and bone marrow: % reduction in morphologic bone marrow intertrabecular space involvement. Linear regression was used to assess clinical responses as continuous variables, and non-parametric Mann-Whitney testing was used for intergroup comparisons. Two-tailed p values were used in all cases. RESULTSEx vivo treatment of platelets with navitoclax induced substantial apoptotic priming, whereas minimal baseline priming was unchanged after ex vivo treatment with ABT-199. Baseline blood samples from 14 relapsed/refractory CLL patients on the ABT-199 phase I study were analyzed for viability after ex vivo treatment with ABT-199 and for pre-treatment levels of apoptotic priming. With ex vivo treatment, ABT-199 induced dose-dependent apoptosis, with a median EC50 of 23 nM in CLL cells (n=14); however, the ABT-199 EC50 was not associated with clinical response in blood, lymph node, or bone marrow. In contrast, samples with higher levels of apoptotic priming as measured by BIM BH3 peptide had a significantly deeper response in the bone marrow (Figure 1, n=9, p=0.01) and blood (n=13, p = 0.05), with a trend toward better response in lymph node (n= 14, p = 0.19). Reflecting the observation in the clinic that ABT-199 has an equivalent response rate irrespective of del(17p) status, we found no difference in apoptotic priming in patients with (n=5) or without (n=9) del(17p). Interestingly, although patients who were fludarabine-refractory at study entry (n=8) were highly primed at baseline, they had lower levels of apoptotic priming (median 92% depolarization) than those who were not refractory (n=6) (median 98%) (p=0.04). To provide further validation of the on target effect of ABT-199 on Bcl-2 on CLL mitochondria, we also studied low concentration ABT-199 used like a BH3 peptide in the BH3 profiling assay. We found an excellent correlation between the amount of mitochondrial depolarization caused by ABT-199 and BAD BH3 peptide (Figure 2, n=13, p = 0.0001). CONCLUSIONSBH3 profiling provides valuable insights into the mechanism of action of the promising Bcl-2 antagonist ABT-199. In contrast to traditional EC50 assays, the pre-treatment level of apoptotic priming predicts initial clinical response to ABT-199 in CLL. An analysis of the association between apoptotic priming and progression free survival is ongoing. With additional validation, BH3 profiling may eventually guide optimal patient selection for Bcl-2 directed therapy, and potentially for other therapies in CLL. [Display omitted] DisclosuresDavids:Genentech: Consultancy; Infinity Pharmaceuticals: Consultancy. Brown:Sanofi, Onyx, Vertex, Novartis, Boehringer, GSK, Roche/Genentech, Emergent, Morphosys, Celgene, Janssen, Pharmacyclics, Gilead: Consultancy. Letai:Abbvie: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding.

Mcl-1 antagonism is a potential therapeutic strategy in a subset of solid cancers

Cancer cell survival is frequently dependent on the elevated levels of members of the Bcl-2 family of prosurvival proteins that bind to and inactivate BH3-domain pro-apoptotic cellular proteins. Small molecules that inhibit the protein–protein interactions between prosurvival and proapoptotic Bcl-2 family members (so-called “BH3 mimetics”) have a potential therapeutic value, as indicated by clinical findings obtained with ABT-263 (navitoclax), a Bcl-2/Bcl-xL antagonist, and more recently with GDC-0199/ABT-199, a more selective antagonist of Bcl-2. Here, we report study results of the functional role of the prosurvival protein Mcl-1 against a panel of solid cancer cell lines representative of different tumor types. We observed silencing of Mcl-1 expression by small interfering RNAs (siRNAs) significantly reduced viability and induced apoptosis in almost 30% of cell lines tested, including lung and breast adenocarcinoma, as well as glioblastoma derived lines. Most importantly, we provide a mechanistic basis for this sensitivity by showing antagonism of Mcl-1 function with specific BH3 peptides against isolated mitochondria induces Bak oligomerization and cytochrome c release, therefore demonstrating that mitochondria from Mcl-1-sensitive cells depend on Mcl-1 for their integrity and that antagonizing Mcl-1 function is sufficient to induce apoptosis. Thus, our results lend further support for considering Mcl-1 as a therapeutic target in a number of solid cancers and support the rationale for development of small molecule BH3-mimetics antagonists of this protein.

Recent advances in the pathogenesis and treatment of chronic lymphocytic leukemia.

Chronic lymphocytic leukaemia (CLL) is a common lymphoid malignancy characterized by the expansion and progressive accumulation of mature autoreactive B lymphocytes. The disease is clinically heterogeneous and incurable by standard chemotherapy. A major feature of the disease is the marked dependence of the leukaemic cells on various microenvironmental stimuli, which promote leukaemia cell growth, survival, and drug-resistance. Recently, considerable progress has been made in the understanding of the molecular mechanisms that drive CLL. The identification of recurrent genetic lesions using next generation sequencing technology has provided new data on the pathophysiology of the disease and has improved its prognostication. The recognition of the critical role of the B cell receptor (BCR) in driving the disease has resulted in the development of BCR pathway inhibitors that have the potential to completely transform CLL treatment in the near future. Other novel therapeutic agents, such as BCL2 antagonists and chimeric antigen receptor (CAR)-modified T-cells, are also showing great promise in clinical trials. In this review, we summarize some of these recent advances, with a particular focus on the BCR and corresponding pathway inhibitors.

Interaction between Na-K-ATPase and Bcl-2 proteins BclXL and Bak.

In silico analysis predicts interaction between Na-K-ATPase (NKA) and Bcl-2 protein canonical BH3- and BH1-like motifs, consistent with NKA inhibition by the benzo-phenanthridine alkaloid chelerythrine, a BH3 mimetic, in fetal human lens epithelial cells (FHLCs) (Lauf PK, Heiny J, Meller J, Lepera MA, Koikov L, Alter GM, Brown TL, Adragna NC. Cell Physiol Biochem 31: 257-276, 2013). This report establishes proof of concept: coimmunoprecipitation and immunocolocalization showed unequivocal and direct physical interaction between NKA and Bcl-2 proteins. Specifically, NKA antibodies (ABs) coimmunoprecipitated BclXL (B-cell lymphoma extra large) and BAK (Bcl-2 antagonist killer) proteins in FHLCs and A549 lung cancer cells. In contrast, both anti-Bcl-2 ABs failed to pull down NKA. Notably, the molecular mass of BAK1 proteins pulled down by NKA and BclXL ABs appeared to be some 4-kDa larger than found in input monomers. In silico analysis predicts these higher molecular mass BAK1 proteins as alternative splicing variants, encoding 42 amino acid (aa) larger proteins than the known 211-aa long canonical BAK1 protein. These BAK1 variants may constitute a pool separate from that forming mitochondrial pores by specifically interacting with NKA and BclXL proteins. We propose a NKA-Bcl-2 protein ternary complex supporting our hypothesis for a special sensor role of NKA in Bcl-2 protein control of cell survival and apoptosis.

Abstract 1810: PI-1840, a novel non-covalent and rapidly reversible proteasome inhibitor with anti-tumor activity

Abstract Non-covalent proteasome inhibitors that are less toxic and more effective against solid tumors than currently used proteasome inhibitors are desirable. Through HTS and hit-to-lead optimization efforts we have identified PI-1840, a potent and selective inhibitor for chymotrypsin-like (CT-L) (IC50 value = 27 ± 0.14 nM) over T-L and PGPH (IC50 values >100 µM) activities of the proteasome. Furthermore, PI-1840 is over 100-fold more selective for the constitutive proteasome over the immunoproteasome. Dialysis and mass spectrometry studies demonstrate that PI-1840 is a non-covalent and rapidly reversible CT-L inhibitor. PI-1840 inhibits CT-L activity, induces the accumulation of proteasome substrates p27, Bax and IκB-α, inhibits survival pathways and proliferation, and induces apoptosis in human cancer cells. The mdm2/p53 disruptor, nutlin, sensitizes cancer cells to PI-1840 in a p53-dependent manner. Furthermore, the pan Bcl-2 antagonist BH3-M6 also sensitizes cancer cells to PI-1840. Finally, in vivo, PI-1840 but not Bortezomib (clinically used proteasome inhibitor) suppresses the growth in nude mice of human breast xenografts. These results warrant further evaluation of non-covalent and rapidly reversible proteasome inhibitor as potential anticancer agents against solid tumors. Citation Format: Said M. Sebti, Aslamuzzaman Kazi, Sevil Ozcan, Awet G. Tecleab, Ying Sun, Harshani Lawrence. PI-1840, a novel non-covalent and rapidly reversible proteasome inhibitor with anti-tumor activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1810. doi:10.1158/1538-7445.AM2014-1810

Macrophage migration inhibitory factor limits activation-induced apoptosis of platelets via CXCR7-dependent Akt signaling.

Macrophage migration inhibitory factor (MIF) is released on platelet activation. Circulating MIF could potentially regulate platelets and thereby platelet-mediated inflammatory and regenerative mechanisms. However, the effect of MIF on platelets is unknown. The present study evaluated MIF in regulating platelet survival and thrombotic potential. MIF interacted with CXCR4-CXCR7 on platelets, defining CXCR7 as a hitherto unrecognized receptor for MIF on platelets. MIF internalized CXCR4, but unlike CXCL12 (SDF-1α), it did not phosphorylate Erk1/2 after CXCR4 ligation because of the lack of CD74 and failed in subsequent CXCR7 externalization. MIF did not alter the activation status of platelets. However, MIF rescued platelets from activation and BH3 mimetic ABT-737-induced apoptosis in vitro via CXCR7 and enhanced circulating platelet survival when administered in vivo. The antiapoptotic effect of MIF was absent in Cxcr7(-/-) murine embryonic cells but pronounced in CXCR7-transfected Madin-Darby canine kidney cells. This prosurvival effect was attributed to the MIF-CXCR7-initiated PI3K-Akt pathway. MIF induced CXCR7-Akt-dependent phosphorylation of BCL-2 antagonist of cell death (BAD) both in vitro and in vivo. Consequentially, MIF failed to rescue Akt(-/-) platelets from thrombin-induced apoptosis when challenged ex vivo, also in prolonging platelet survival and in inducing BAD phosphorylation among Akt(-/-) mice in vivo. MIF reduced thrombus formation under arterial flow conditions in vitro and retarded thrombotic occlusion after FeCl3-induced arterial injury in vivo, an effect mediated through CXCR7. MIF interaction with CXCR7 modulates platelet survival and thrombotic potential both in vitro and in vivo and thus could regulate thrombosis and inflammation.

Use of Spleen as an Internal Marker in Whole Body DWIBS Exam in Patients of Multiple Myeloma

Context: To determine mechanisms of IL-6 induced resistance to ABT-737 in multiple myeloma. Results: Bcl-2 family antagonists such as navitoclax (ABT-263) and ABT-199 are not predicted to be efficacious in multiple myeloma based on their inability to disrupt the interaction between Bim and Mcl-1, the predominant anti-apoptotic protein in both normal and malignant plasma cells. However, despite their dependence on Mcl-1, some human myeloma cell lines (HMCL) as well as freshly isolated myeloma patient cells are sensitive to ABT-737 in vitro. Knowing that myeloma normally requires the bone marrow niche for survival, we hypothesized that a stromal derived factor might mediate the resistance to ABT-737 in vivo and identified IL-6 as a key resistance factor. In order to understand the mechanisms of IL-6 mediated resistance to ABT-737, we have focused on the effect of IL-6 on the Bcl-2 family of proteins. In previous work from our lab, the ABT-737 sensitive HMCLs KMS18, MM.1s, and 8226 all showed increased binding of Bim to Bcl-2 and Bcl-xL compared to ABT-737 resistant lines. We therefore examined the effect of IL-6 on the distribution of Bim among Bcl-2, Bcl-xL, and Mcl-1. In KMS18, IL-6 stimulation increases Mcl-1 expression and Bim binding. We also examined Bim for IL-6 induced post-translational modifications that could alter its binding to Bcl-2 proteins. We observed Bim serine 69 phosphorylation within 5minutes of IL-6 stimulation in both KMS18 and MM.1s cells. Phosphorylation was reversible with 10mMof theMEK inhibitorU0126. Interestingly, the MEK inhibitor sensitized both KMS18 andMM.1s to ABT-737 and was able to partially overcome IL-6 induced resistance. Of note, the MEK inhibitor failed to prevent upregulation of Mcl-1 in KMS18, suggesting an alternative pathway mediates this effect. We are currently attempting to develop a flow cytometry based biomarker capable of identifying patients who might be sensitive or resistant to ABT-737.Conclusion: Targeting IL-6 or its downstream pathways may sensitize myeloma to Bcl-2 antagonists such as ABT-199 and navitoclax.

Transcriptional and Post-translational Regulation of the Bcl-2 Family by IL-6 Mediates Resistance to ABT-737 in Multiple Myeloma

While direct inhibition of Bcl-2 and/or Bcl-xL is an exciting new approach in the treatment of hematologic malignancies, current agents in clinical testing including navitoclax (ABT-263) and ABT-199 are not predicted to be efficacious in most cases of multiple myeloma. Navitoclax and the related molecule ABT-737 promote apoptosis by releasing the pro-apoptotic BH3 only protein Bim from Bcl-2 and Bcl-xL, but are incapable of disrupting the interaction between Bim and Mcl-1, the predominant anti-apoptotic protein in both normal and malignant plasma cells. However, despite their dependence on Mcl-1, some human myeloma cell lines (HMCL) as well as freshly isolated patient-derived myeloma cells are sensitive to ABT-737 in vitro. Knowing that myeloma normally requires the bone marrow niche for survival, we hypothesized that a stromal derived factor might mediate the resistance to ABT-737 in vivo and identified IL-6 as a key resistance factor. In our initial experiments, the HS-5 stromal cell line induced resistance to ABT-737 in the HMCL MM.1s, as did conditioned media from both HS-5 cells and patient derived bone marrow stromal cells. Blocking IL-6 with an IL-6 neutralizing antibody reversed the protective effect of conditioned media, while addition of 10 ng/ml IL-6 protected cells to the same degree as conditioned media. In order to understand the mechanisms of IL-6 mediated resistance to ABT-737, we have focused on the effect of IL-6 on the Bcl-2 family of proteins. In previous work from our lab, the ABT-737 sensitive HMCLs KMS18, MM.1s, and 8226 all showed increased binding of Bim to Bcl-2 and Bcl-xL compared to ABT-737 resistant lines. We therefore examined the effect of IL-6 on the distribution of Bim among Bcl-2, Bcl-xL, and Mcl-1. In KMS18, stimulation with 10 ng/ml IL-6 for 24 hours increases binding of Bim to Mcl-1. The increased binding correlates with a 2 fold increased Mcl-1 expression at both the RNA and protein level. The increased Mcl-1 expression in response to IL-6 may be limited to KMS18 as it was not observed in MM.1s, 8226, or KMS11. IL-6 also does not prevent ABT-737 from disrupting the interaction between Bim and Bcl-xL. We also examined Bim for IL-6 induced post-translational modifications that could alter its binding to Bcl-2 proteins. Bim is known to be phosphorylated on serine 69 by Erk in response to growth factor stimulation. We observed Bim serine 69 phosphorylation within 5 minutes of IL-6 stimulation in both KMS18 and MM.1s cells. Phosphorylation was reversible with 10 μM of the MEK inhibitor U0126. Although serine 69 phosphorylation has been reported to result in Bim degradation, we do not observe any change in Bim levels over the course of 24 hours. Interestingly, the MEK inhibitor sensitized both KMS18 and MM.1s to ABT-737 and was able to partially overcome IL-6 induced resistance. Inhibition of Akt with the PI3K inhibitor LY294002 had no effect on Bim serine 69 phosphorylation or IL-6 induced resistance. Of note, the MEK inhibitor failed to prevent upregulation of Mcl-1 in KMS18, suggesting an alternative pathway mediates this effect. We are currently studying the role of JAK signaling in Mcl-1 expression with the inhibitors AZD1480 and ruxolitinib, and are also extending our results to additional cell lines and patient samples. These results suggest that targeting IL-6 or its downstream pathways may sensitize myeloma to Bcl-2 antagonists such as ABT-199 and navitoclax. Disclosures: Kaufman: Onyx: Consultancy; Novartis: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Millennium Pharmaceuticals: Consultancy; Jansenn: Consultancy; Merck: Research Funding. Lonial: Millennium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; BMS: Consultancy; Sanofi: Consultancy; Onyx: Consultancy. Boise: Onyx Pharmaceuticals: Consultancy.

BCL2 antagonist of cell death (BAD) gene sequence and functional analysis of phosphorylation sites in ovarian cancer

BCL2 antagonist of cell death (BAD) gene sequence and functional analysis of phosphorylation sites in ovarian cancer

The BCL2 antagonist of cell death (BAD) signaling pathway and phospho-BAD protein levels are associated with triple-negative breast cancer clinical development and clinical outcome

The BCL2 antagonist of cell death (BAD) signaling pathway and phospho-BAD protein levels are associated with triple-negative breast cancer clinical development and clinical outcome

ABT-199 (GDC-0199) combined with rituximab (R) in patients (pts) with relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL): Interim results of a phase 1b study.

7013 Background: ABT-199 is a selective, orally bioavailable Bcl-2 antagonist that induces rapid apoptosis of CLL cells and > 80% response rate as monotherapy in pts with R/R CLL. R synergizes with...

Targeting BCL2 for the Treatment of Lymphoid Malignancies

The failure of apoptosis (programmed cell death) underpins the development of many tumors and often renders them resistant to cytotoxic therapies. In hematologic malignancies, this impairment of apoptosis is often caused by overexpression of the pro-survival protein BCL2. Because abnormally high levels of BCL2 sustain these tumors, there has been much interest in targeting BCL2 as a novel approach to treating various hematologic malignancies. One such approach is the development of BH3 mimetic compounds, small molecules that mimic the action of the BH3-only proteins, natural antagonists of BCL2 and its pro-survival relatives. These compounds act by restoring the ability of a cell to undergo apoptotic cell death. Some of them have shown very encouraging results in early-phase clinical trials that are currently underway, particularly in patients with chronic lymphocytic leukemia and some non-Hodgkin lymphomas, diseases marked by BCL2 overexpression. In this review, we discuss the rationale behind targeting BCL2, highlight the recent findings from clinical trials, and pinpoint the next steps in the clinical development of this interesting and promising class of targeted agents, particularly for the treatment of lymphoid malignancies.

Investigation of PKCδII and BAD protein complex formation (613.7)

Protein kinase C δ (PKCδ) has been shown to have dual functions both as a regulator of apoptosis and cellular survival due to alternative splicing. One splice variant, PKCδII, is integral in promoting cell survivability. The pro‐apoptotic protein, BCL‐2 antagonist of cell death (BAD), will complex with and inhibit the function of pro‐survival proteins. Upon phosphorylation at Ser‐136, BAD disassociates from these complexes, conferring survival. It has been found that the overexpression of PKCδII coincides with increased BAD phosphorylation. Although the exact mechanism is unknown, we believe PKCδII enhances this phosphorylation via direct interactions with BAD. We aim to elucidate the amino acid residues of PKCδII that bind BAD. We propose the interactions that complex PKCδII with BAD using information gained from sequence analyses and computational simulations. The amphipathic groove (Lys‐49, Val‐176, Leu‐220) of 14‐3‐3 has previously been demonstrated to interact with BAD. Upon alignment of the 14‐3‐3 and PKCδII amino acid sequences, it was noted that the residues that comprise the amphipathic groove may be conserved and also facilitate binding of PKCδII with BAD. The PKCδII residues Gln‐376, Val‐547, and Val‐586 may enable the formation of this complex. A homology model of PKCδII and BAD has aided in visualizing this interaction. Subsequent site‐directed mutagenesis will further solidify our understanding of this protein complex. These proposed interactions provide evidence to suggest that BAD complexes directly with PKCδII in a conserved amphipathic groove, thereby leading to increased survivability.Grant Funding Source: The CREST Program is supported by NSF‐DUE #1022793 and #1323414.

Structure-Guided Rescaffolding of Selective Antagonists of BCL-XL.

Because of the promise of BCL-2 antagonists in combating chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphoma (NHL), interest in additional selective antagonists of antiapoptotic proteins has grown. Beginning with a series of selective, potent BCL-XL antagonists containing an undesirable hydrazone functionality, in silico design and X-ray crystallography were utilized to develop alternative scaffolds that retained the selectivity and potency of the starting compounds.

MiR-125b inhibitor enhance the chemosensitivity of glioblastoma stem cells to temozolomide by targeting Bak1

Temozolomide (TMZ) is a promising chemotherapeutic agent for treating glioblastomas. However, resistance develops quickly with a high frequency. Glioblastoma stem cells (GSCs) causing resistance to drug therapy were considered to be one of key factors. The mechanisms underlying GSCs resistance to TMZ are not fully understood. MicroRNAs (miRNAs) have emerged to play important roles in tumorigenesis and drug resistance. Previous study showed that miR-125b was necessary for GSCs fission and for making stem cells insensitive to chemotherapy. Thus, exploring the functions and mechanisms of miR-125b action on TMZ-treated GSCs would be valuable. In this study, we found that miR-125b was up-regulated in TMZ-resistant cells, inhibition of which caused a marked increase of TMZ-induced cytotoxicity and apoptosis and a subsequent decrease in the resistance to TMZ in GSCs. Moreover, we demonstrated that the pro-apoptotic Bcl-2 antagonist killer 1 (Bak1) was a direct target of miR-125b. Down-regulation of Bak1 inhibited TMZ-induced apoptosis and led to an increased resistance to TMZ. Restoring Bak1 expression recovered TMZ sensitivity on GSCs. Taken together; our data strongly support an important role for miR-125b on conferring TMZ resistance through targeting Bak1 expression.

Selective BCL-2 Inhibition by ABT-199 Causes On-Target Cell Death in Acute Myeloid Leukemia

B-cell leukemia/lymphoma 2 (BCL-2) prevents commitment to programmed cell death at the mitochondrion. It remains a challenge to identify those tumors that are best treated by inhibition of BCL-2. Here, we demonstrate that acute myeloid leukemia (AML) cell lines, primary patient samples, and murine primary xenografts are very sensitive to treatment with the selective BCL-2 antagonist ABT-199. In primary patient cells, the median IC50 was approximately 10 nmol/L, and cell death occurred within 2 hours. Our ex vivo sensitivity results compare favorably with those observed for chronic lymphocytic leukemia, a disease for which ABT-199 has demonstrated consistent activity in clinical trials. Moreover, mitochondrial studies using BH3 profiling demonstrate activity at the mitochondrion that correlates well with cytotoxicity, supporting an on-target mitochondrial mechanism of action. Our protein and BH3 profiling studies provide promising tools that can be tested as predictive biomarkers in any clinical trial of ABT-199 in AML.

The use of a neutral peptide aptamer scaffold to anchor BH3 peptides constitutes a viable approach to studying their function

The B-cell CLL/lymphoma-2 (Bcl-2) family of proteins are important regulators of the intrinsic pathway of apoptosis, and their interactions, driven by Bcl-2 homology (BH) domains, are of great interest in cancer research. Particularly, the BH3 domain is of clinical relevance, as it promotes apoptosis through activation of Bcl-2-associated x protein (Bax) and Bcl-2 antagonist killer (Bak), as well as by antagonising the anti-apoptotic Bcl-2 family members. Although investigated extensively in vitro, the study of the BH3 domain alone inside cells is more problematic because of diminished secondary structure of the unconstrained peptide and a lack of stability. In this study, we report the successful use of a novel peptide aptamer scaffold – Stefin A quadruple mutant – to anchor and present the BH3 domains from Bcl-2-interacting mediator of cell death (Bim), p53 upregulated modulator of apoptosis (Puma), Bcl-2-associated death promoter (Bad) and Noxa, and demonstrate its usefulness in the study of the BH3 domains in vivo. When expressed intracellularly, anchored BH3 peptides exhibit much the same binding specificities previously established in vitro, however, we find that, at endogenous expression levels, Bcl-2 does not bind to any of the anchored BH3 domains tested. Nonetheless, when expressed inside cells the anchored PUMA and Bim BH3 α-helices powerfully induce cell death in the absence of efficient targeting to the mitochondrial membrane, whereas the Noxa helix requires a membrane insertion domain in order to kill Mcl-1-dependent myeloma cells. Finally, the binding of the Bim BH3 peptide to Bax was the only interaction with a pro-apoptotic effector protein observed in this study.

Effect of HA14‐1 on Apoptosis‐Regulating Proteins in HeLa Cells

Overexpression of Bcl-2 has been recognized in various malignancies. Recently, HA14-1, a Bcl-2 antagonist, has been identified for its anti-apoptotic effect. However, mode of action of HA14-1 still remains to be elucidated. In this study, we examined HA14-1 binding efficiency with receptor proteins through molecular docking. Cell viability using HeLa cells was evaluated through MTT assay after exposure to different concentration of HA14-1. Moreover, after HA14-1 exposure, expressions of tumor suppressor protein (p53), BH3-only protein (Puma) and apoptosis-associated proteins were analyzed by Western blotting. From the results, it was found that HA14-1 occupied all three domains; BH1, BH2, and BH3 within the hydrophobic pocket of Bcl-2. However, HA14-1 occupied only BH1 and BH3 of Bcl-xl, conversely, no such stable bond was observed for Bax and Bak. ARG107 and TYR101 were the amino acids involved in the binding of HA14-1 to Bcl-2 and Bcl-xl, respectively. Additionally, decrease in Bcl-2 and Bcl-xl expression along with increase in p53 and Puma expression after exposure to HA14-1 was observed. The results suggested p53 pathway to be the probable mechanism of action for the induction of apoptosis in HeLa cell by downregulating the effect of anti-apoptotic proteins suggesting that HA14-1 may provide therapeutic potential for the treatment of human cervical cancer.

Predictive Simulation Based Design and Validation Of Repurposed Novel Therapeutics With Multi-Target Mechanisms For Multiple Myeloma

IntroductionDevelopment of resistance to single agent therapy is a significant clinical obstacle in the treatment of multiple myeloma (MM). Genetic mutations and the bone marrow micro-environment are major determinants of MM resistance mechanisms. Given the complexity of MM, the need for combinatorial therapeutic regimens targeting multiple biological mechanisms of action is pressing. Repurposing has the advantage of using drugs with known clinical history. MethodologyWe used a predictive simulation-based approach that models MM disease physiology in plasma cells by integrating and aggregating signaling and metabolic networks across all disease phenotypes. We tested the efficacy of over 50 repurposed molecularly targeted agents both individually and in combination across simulation avatars of the MM cell lines OPM2 and U266. OPM2 harbors mutations in KRAS, CDKN2A/2C, PTEN, RASSF1A and P53, whereas U266's mutational components include BRAF, CDKN2A, P53, P73, RASSF1A and RB1. These cell lines were used as models because they possess mutations in genes classically known to be involved in myeloma. The predicted activity of novel combinations of existing drug agents was validated in vitro using standard molecular assays. MTT and flow cytometry were used to assess cellular proliferation. Western blotting was used to monitor the combinatorial effects on apoptotic and cellular signaling pathways. Synergy was analyzed using isobologram plots and the Bliss independence model. ResultsThrough simulation modeling, we identified two novel therapeutic regimens for MM using repurposed drugs: (1) AT101 (Bcl2 antagonist) and tesaglitazar (PPAR α/γ agonist) and (2) Ursolic acid (UA, inhibitor of NFκβ) and SP600125 (pan-JNK inhibitor). Simulation predictions showed that combining the IC30 concentrations with respect to viability of AT101 and tesaglitazar reduced proliferation by 40% and viability by 50%. Similarly simulation predictions showed that the combination of the IC30 concentrations of UA and SP600125 reduced proliferation by 50% and viability by 40%.Corroborating our predictive simulation assays, 10 µM tesaglitazar and 2 µM AT101 caused minimal growth inhibition as single agents in OPM2 and U266 MM cell lines. Growth inhibition in these cell lines is synergistically enhanced when the drugs are used in combination, reducing cellular viability by 88% and 77% in OPM2 and U266 cells, respectively. Similarly, proliferation was reduced by 34% with 7.5 μM UA and 25% with 10 μM SP600125 in OPM2 cells. When used in combination, cellular proliferation was synergistically reduced by 64%. In addition, isobologram analysis predicted synergy of lowered doses of the drugs in combination. Both combinations synergistically inhibited proliferation and induced apoptosis as evidenced by an increase in the percentage sub-G1 phase cells and cleavage of caspase 3 and poly ADP ribose polymerase (PARP). ConclusionsThese results highlight and validate the use of our predictive simulation approach to design therapeutic regimens with novel biological mechanisms using drugs with known chemistries. This allows for design of personalized treatments for patients using their tumor genomic signature beyond the “one-gene, one-drug” paradigm. The reuse of existing drugs with clinical data facilitates a rapid translational path into clinic and avoids the uncertainties associated with new chemistry. The corroboration of these results with patient derived cell lines will be pursued and discussed. Disclosures:No relevant conflicts of interest to declare.

Transcriptional and Post-Translational Regulation Of The Bcl-2 Family By IL-6 Mediates Resistance To ABT-737 In Multiple Myeloma

While direct inhibition of Bcl-2 and/or Bcl-xL is an exciting new approach in the treatment of hematologic malignancies, current agents in clinical testing including navitoclax (ABT-263) and ABT-199 are not predicted to be efficacious in most cases of multiple myeloma. Navitoclax and the related molecule ABT-737 promote apoptosis by releasing the pro-apoptotic BH3 only protein Bim from Bcl-2 and Bcl-xL, but are incapable of disrupting the interaction between Bim and Mcl-1, the predominant anti-apoptotic protein in both normal and malignant plasma cells. However, despite their dependence on Mcl-1, some human myeloma cell lines (HMCL) as well as freshly isolated patient-derived myeloma cells are sensitive to ABT-737 in vitro. Knowing that myeloma normally requires the bone marrow niche for survival, we hypothesized that a stromal derived factor might mediate the resistance to ABT-737 in vivo and identified IL-6 as a key resistance factor. In our initial experiments, the HS-5 stromal cell line induced resistance to ABT-737 in the HMCL MM.1s, as did conditioned media from both HS-5 cells and patient derived bone marrow stromal cells. Blocking IL-6 with an IL-6 neutralizing antibody reversed the protective effect of conditioned media, while addition of 10 ng/ml IL-6 protected cells to the same degree as conditioned media. In order to understand the mechanisms of IL-6 mediated resistance to ABT-737, we have focused on the effect of IL-6 on the Bcl-2 family of proteins. In previous work from our lab, the ABT-737 sensitive HMCLs KMS18, MM.1s, and 8226 all showed increased binding of Bim to Bcl-2 and Bcl-xL compared to ABT-737 resistant lines. We therefore examined the effect of IL-6 on the distribution of Bim among Bcl-2, Bcl-xL, and Mcl-1. In KMS18, stimulation with 10 ng/ml IL-6 for 24 hours increases binding of Bim to Mcl-1. The increased binding correlates with a 2 fold increased Mcl-1 expression at both the RNA and protein level. The increased Mcl-1 expression in response to IL-6 may be limited to KMS18 as it was not observed in MM.1s, 8226, or KMS11. IL-6 also does not prevent ABT-737 from disrupting the interaction between Bim and Bcl-xL. We also examined Bim for IL-6 induced post-translational modifications that could alter its binding to Bcl-2 proteins. Bim is known to be phosphorylated on serine 69 by Erk in response to growth factor stimulation. We observed Bim serine 69 phosphorylation within 5 minutes of IL-6 stimulation in both KMS18 and MM.1s cells. Phosphorylation was reversible with 10 μM of the MEK inhibitor U0126. Although serine 69 phosphorylation has been reported to result in Bim degradation, we do not observe any change in Bim levels over the course of 24 hours. Interestingly, the MEK inhibitor sensitized both KMS18 and MM.1s to ABT-737 and was able to partially overcome IL-6 induced resistance. Inhibition of Akt with the PI3K inhibitor LY294002 had no effect on Bim serine 69 phosphorylation or IL-6 induced resistance. Of note, the MEK inhibitor failed to prevent upregulation of Mcl-1 in KMS18, suggesting an alternative pathway mediates this effect. We are currently studying the role of JAK signaling in Mcl-1 expression with the inhibitors AZD1480 and ruxolitinib, and are also extending our results to additional cell lines and patient samples. These results suggest that targeting IL-6 or its downstream pathways may sensitize myeloma to Bcl-2 antagonists such as ABT-199 and navitoclax. Disclosures:Kaufman:Onyx: Consultancy; Novartis: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Millennium Pharmaceuticals: Consultancy; Jansenn: Consultancy; Merck: Research Funding. Lonial:Millennium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; BMS: Consultancy; Sanofi: Consultancy; Onyx: Consultancy. Boise:Onyx Pharmaceuticals: Consultancy.