Pro-apoptotic Bcl-2 Family Member Research Articles

Inhibition of histone methyltransferase G9a attenuates liver cancer initiation by sensitizing DNA-damaged hepatocytes to p53-induced apoptosis

While the significance of acquired genetic abnormalities in the initiation of hepatocellular carcinoma (HCC) has been established, the role of epigenetic modification remains unknown. Here we identified the pivotal role of histone methyltransferase G9a in the DNA damage-triggered initiation of HCC. Using liver-specific G9a-deficient (G9aΔHep) mice, we revealed that loss of G9a significantly attenuated liver tumor initiation caused by diethylnitrosamine (DEN). In addition, pharmacological inhibition of G9a attenuated the DEN-induced initiation of HCC. After treatment with DEN, while the induction of γH2AX and p53 were comparable in the G9aΔHep and wild-type livers, more apoptotic hepatocytes were detected in the G9aΔHep liver. Transcriptome analysis identified Bcl-G, a pro-apoptotic Bcl-2 family member, to be markedly upregulated in the G9aΔHep liver. In human cultured hepatoma cells, a G9a inhibitor, UNC0638, upregulated BCL-G expression and enhanced the apoptotic response after treatment with hydrogen peroxide or irradiation, suggesting an essential role of the G9a-Bcl-G axis in DNA damage response in hepatocytes. The proposed mechanism was that DNA damage stimuli recruited G9a to the p53-responsive element of the Bcl-G gene, resulting in the impaired enrichment of p53 to the region and the attenuation of Bcl-G expression. G9a deletion allowed the recruitment of p53 and upregulated Bcl-G expression. These results demonstrate that G9a allows DNA-damaged hepatocytes to escape p53-induced apoptosis by silencing Bcl-G, which may contribute to the tumor initiation. Therefore, G9a inhibition can be a novel preventive strategy for HCC.

Discovery of Small Molecule Bak Activator for Lung Cancer Therapy.

Rationale: Bak is a major proapoptotic Bcl2 family member and a required molecule for apoptotic cell death. High levels of endogenous Bak were observed in both small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cell lines. Increased Bak expression was correlated with poor prognosis of NSCLC patients, suggesting that Bak protein is an attractive target for lung cancer therapy. The BH3 domain functions as death domain and is required for Bak to initiate apoptotic cell death. Thus, the BH3 domain is attractive target for discovery of Bak agonist.Methods: The BH3 death domain binding pocket (aa75-88) of Bak was chosen as a docking site for screening of small molecule Bak activators using the UCSF DOCK 6.1 program suite and the NCI chemical library (300,000 small molecules) database. The top 500 compounds determined to have the highest affinity for the BH3 domain were obtained from the NCI and tested for cytotoxicity for further screening. We identified a small molecule Bak activator BKA-073 as the lead compound. The binding affinity of BKA-073 with Bak protein was analyzed by isothermal titration calorimetry (ITC) assay. BKA-073-mediated Bak activation via oligomerization was analyzed by a cross-linking with Bis (maleimido) hexane (BMH). Sensitivity of BKA-073 to lung cancer cells in vitro was evaluated by dynamic BH3 profiling (DBP) and apoptotic cell death assay. The potency of BKA-073 alone or in combination with radiotherapy or Bcl2 inhibitor was evaluated in animal models.Results: We found that BKA-073 binds Bak at BH3 domain with high affinity and selectivity. BKA-073/Bak binding promotes Bak oligomerization and mitochondrial priming that activates its proapoptotic function. BKA-073 potently suppresses tumor growth without significant normal tissue toxicity in small cell lung cancer (SCLC) and NSCLC xenografts, patient-derived xenografts, and genetically engineered mouse models of mutant KRAS-driven cancer. Bak accumulates in radioresistant lung cancer cells and BKA-073 reverses radioresistance. Combination of BKA-073 with Bcl-2 inhibitor venetoclax exhibits strong synergy against lung cancer in vivo.Conclusions: Development of small molecule Bak activator may provide a new class of anticancer agents to treat lung cancer.

EXTH-50. ACTIVATION OF THE MITOCHONDRIAL CLPP PROTEASE IS SYNTHETICALLY LETHAL WITH HDAC1/2 INHIBITION IN GLIOBLASTOMA MODEL SYSTEMS

Abstract Novel therapeutic targets are critical to unravel for recalcitrant malignancies, such as the most common primary brain tumor in adults, glioblastoma (GBM). Heterogeneity remains a hallmark of primary glial brain tumors and therefore targeting several pathways simultaneously is an appropriate approach. Here, we showed that pharmacological activation of the mitochondrial ClpP protease through utilization of the novel imipridone compounds (ONC206 and ONC212) in combination with global (Panobinostat) and selective (romidepsin) HDAC – inhibitors caused synergistic reduction of viability in established and patient-derived xenograft (PDX) cultures of human GBM. This effect occurred independent of TP53 status and was partially mediated by activation of a cell death with apoptotic features accompanied by activation of initiator and effector caspases as well as cleavage of PARP. Consistently, the combination treatment altered the expression of anti-apoptotic and pro-apoptotic Bcl-2 family members, resulting in down-regulation of Bcl-xL and Mcl-1. Knockdown experiments targeting Noxa, BIM, Bcl-xL and Mcl-1 confirmed a functional implication of these proteins in the reduction of cellular viability mediated by the combination treatment. Importantly, knockdown of the ClpP protease significantly rescued the reduction of cellular viability by ClpP activators and the combination treatment, respectively, suggesting critical involvement of this protein. Finally, using a PDX model, we demonstrated that the combination treatment of romidepsin and ONC206 reduced tumor growth more potently than single treatments or vehicle by enhanced reduction of cellular proliferation and pronounced induction of cell death in vivo. Collectively, these observations suggest that the efficacy of HDAC-inhibitors might be significantly enhanced through ClpP activators in model systems of human GBM.

Abstract PO-53: Combined EZH2 and BCL2 inhibitors as precision therapy for genetically defined DLBCL subtypes

Abstract Molecular alterations in the histone methyltransferase EZH2 and the antiapoptotic protein BCL2 frequently co-occur in diffuse large B-cell lymphoma (DLBCL). We hypothesized that EZH2 inhibition and BCL2 inhibition would be synergistic in DLBCL. To test this, we evaluated the EZH2 inhibitor tazemetostat and the BCL2 inhibitor venetoclax in DLBCL cells, 3D lymphoma organoids, and patient-derived xenografts (PDXs). We found that tazemetostat and venetoclax are synergistic in DLBCL cells that harbor both an EZH2 mutation and a BCL2/IGH translocation, as demonstrated by CI values <1 (CI: 0.034, 0.259 and 0.074 in SUDHL-6, WSU-DLCL2, and OCI-Ly1 respectively), but not in wild-type cells. Since cell lines in suspension do not reflect lymph node architecture, we developed a 3D lymphoma organoid culture system that consists of extracellular matrix, lymphoma cells, and stromal cells (Tian et al., Biomaterials 2015). We observed synergy between the two agents in two organoid model systems: 1) OCI-LY1; 2) PDX derived from a DLBCL with BCL2/IGH translocation and EZH2 mutation. To investigate mechanisms of synergy, we evaluated previously published RNA-seq profiles of DLBCL cell lines (n=26) treated with vehicle or tazemetostat to investigate changes in BCL2 family members (Brach et al., Mol Can Ther 2017). Tazemetostat-treated cells showed enhanced expression of proapoptotic BCL2 family members including BCL2L11 (p=0.012), BMF (p<0.001), and BCL2L14 (p=0.002), suggesting that these may be direct or indirect EZH2 target genes that are de-repressed upon EZH2 inhibition. To assess mitochondrial priming to apoptosis as a result of EZH2 inhibition, we performed BH3 profiling of DLBCL PDX organoids treated with vehicle vs. tazemetostat. Tazemetostat-treated cells had increased priming as evidenced by cytochrome c release in response to general apoptotic signaling peptides BIM and PUMA (p<0.0001) and to the BCL2 specific peptide BAD (p<0.0001), suggesting that pretreatment with tazemetostat increases mitochondrial sensitivity to BCL2 inhibition. We next evaluated combination therapy in vivo. In SUDHL-6 xenografts, the combination resulted in attenuation of tumor growth compared to either drug alone (combination vs. venetoclax p<0.0001, combination vs. tazemetostat p=0.0004) and improved overall survival. In DLBCL PDXs, combination therapy resulted in complete resolutions of tumors, which were durable over time and associated with improved overall survival. Strikingly, after 197 days of follow-up there was no detectable disease in any combination-treated animal. In summary, we demonstrate that combined BCL2 and EZH2 inhibition results in synergistic anti-lymphoma effects. We expect this combination to be especially effective as precision therapy for the newly identified cluster 3/EZB DLBCL subtype, which frequently harbors both EZH2 and BCL2 alterations. A clinical trial of this combination is currently in development. Citation Format: Hanna Scholze, Regan E. Stephenson, Raymond Reynolds, Shivem Shah, Rishi Puri, Matthew R. Teater, Herman van Besien, Destini Gibbs-Curtis, Hideki Ueno, Salma Parvin, Anthony G. Letai, Susan Mathew, Ankur Singh, Ethel Cesarman, Ari Melnick, Lisa Giulino-Roth. Combined EZH2 and BCL2 inhibitors as precision therapy for genetically defined DLBCL subtypes [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-53.

Combined EZH2 and Bcl-2 inhibitors as precision therapy for genetically defined DLBCL subtypes

AbstractMolecular alterations in the histone methyltransferase EZH2 and the antiapoptotic protein Bcl-2 frequently co-occur in diffuse large B-cell lymphoma (DLBCL). Because DLBCL tumors with these characteristics are likely dependent on both oncogenes, dual targeting of EZH2 and Bcl-2 is a rational therapeutic approach. We hypothesized that EZH2 and Bcl-2 inhibition would be synergistic in DLBCL. To test this, we evaluated the EZH2 inhibitor tazemetostat and the Bcl-2 inhibitor venetoclax in DLBCL cells, 3-dimensional lymphoma organoids, and patient-derived xenografts (PDXs). We found that tazemetostat and venetoclax are synergistic in DLBCL cells and 3-dimensional lymphoma organoids that harbor an EZH2 mutation and an IGH/BCL2 translocation but not in wild-type cells. Tazemetostat treatment results in upregulation of proapoptotic Bcl-2 family members and priming of mitochondria to BH3-mediated apoptosis, which may sensitize cells to venetoclax. The combination of tazemetostat and venetoclax was also synergistic in vivo. In DLBCL PDXs, short-course combination therapy resulted in complete remissions that were durable over time and associated with superior overall survival compared with either drug alone.

The Multifaceted Roles of the BCL-2 Family Member BOK

BCL-2–related ovarian killer (BOK) is—despite its identification over 20 years ago—an incompletely understood member of the BCL-2 family. BCL-2 family proteins are best known for their critical role in the regulation of mitochondrial outer membrane permeabilization during the intrinsic apoptotic pathway. Based on sequence and structural similarities to BAX and BAK, BOK is grouped with these “killers” within the effector subgroup of the family. However, the mechanism of how exactly BOK exerts apoptosis is not clear and controversially discussed. Furthermore, and in accordance with reports on several other BCL-2 family members, BOK seems to be involved in the regulation of a variety of other, “apoptosis-independent” cellular functions, including the unfolded protein response, cellular proliferation, metabolism, and autophagy. Of note, compared with other proapoptotic BCL-2 family members, BOK levels are often reduced in cancer by various means, and there is increasing evidence for BOK modulating tumorigenesis. In this review, we summarize and discuss apoptotic- and non–apoptotic-related functions of BOK, its regulation as well as its physiological and pathophysiological roles.

Abstract 6218: Discovery and preclinical evaluation of AMG 397, a potent, selective and orally bioavailable MCL1 inhibitor

Abstract MCL1 inhibitors are an emerging class of therapeutics targeting key protein-protein interactions between MCL1 and BH3 domain containing pro-apoptotic BCL-2 family members. Several MCL1 inhibitors have entered clinical trials including AMG 176, currently in Phase I clinical development for hematologic malignancies. However, all existing clinical stage MCL1 inhibitors are being administered intravenously. Here we describe the discovery and preclinical evaluation of AMG 397, the first orally administered MCL1 inhibitor in clinical development. The pursuit of an orally bioavailable MCL1 inhibitor with an acceptable human clinical dose required improvements to AMG 176 in both potency and pharmacokinetic properties. The chemistry effort to identify AMG 397 began with the observation that there were two conformations in AMG 176 bound to MCL1 with a difference of 2.8 kcal/mol in energy. A structure-guided approach in combination with ligand-based design to attempt to favor the more active conformation provided the advanced lead AM-3106 (Ki=100 pM) with significantly improved cell based potency (OPM2 viability IC50 = 19 nM) compared to AMG 176. Further refinements in potency and PK properties of AM-3106 culminated in the discovery of AMG 397. AMG 397 exhibited picomolar affinities for MCL1 (Ki=15 pM), selectively competing for binding to the BH3-binding groove of MCL1 with pro-apoptotic BCL-2 family members (e.g. BIM). In cells, AMG 397 potently disrupted the interaction between MCL1 and BIM. Treatment of OPM2 cells, an MCL1 dependent multiple myeloma cell line, induced clear increases in Caspase-3/7 activity within one hour. Subsequent effects on viability were observed across a treatment time course with maximal effects observed following 24 hours of continuous exposure (IC50=50 nM). Time course washout viability studies demonstrated that AMG 397 induced a committed step towards apoptosis and suggested that discontinuous dosing schedules would be sufficient to achieve antitumor effects in vivo. Cell lines from hematologic malignancies including AML, multiple myeloma and DLBCL exhibited greatest sensitivity to AMG 397 in a large tumor cell line profiling screen (n=300). In vivo, oral administration of AMG 397 demonstrated rapid and sustained dose dependent increases in activated BAK, cleaved Caspase-3 and cleaved PARP in established OPM2 xenografts. Discontinuous dosing schedules of once or twice weekly at 25 or 50 mg/kg of AMG 397 exhibited significant tumor regressions in mice bearing OPM2 xenografts, with 9 of 10 mice tumor free at the end of the study in both 50 mg/kg groups. AMG 397 was also tested in the MOLM-13 orthotopic model of AML where twice weekly administration at 10, 30 and 60 mg/kg achieved 47% tumor growth inhibition (TGI), 99% TGI and 75% regression respectively. Combination of AMG 397 at 10 mg/kg twice weekly and 50 mg/kg of venetoclax daily achieved 45% regression in the same model. Citation Format: Sean Caenepeel, Rex Karen, Brian Belmontes, Alla Verlinsky, Hong Tan, Yajing Yang, Xiaoyue Chen, Kexue Li, Jennifer Allen, Jan Wahlstrom, Jude Canon, Angela Coxon, Paul Hughes. Discovery and preclinical evaluation of AMG 397, a potent, selective and orally bioavailable MCL1 inhibitor [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6218.

The BCL-2 family members NOXA and BIM mediate fluorizoline-induced apoptosis in multiple myeloma cells

Fluorizoline is a new synthetic molecule that induces apoptosis by selectively targeting prohibitins. In this study, we have assessed the pro-apoptotic effect of fluorizoline in 3 different multiple myeloma cell lines and 12 primary samples obtained from treatment-naïve multiple myeloma patients. Fluorizoline induced apoptosis in both multiple myeloma cell lines and primary samples at concentrations in the low micromolar range. All primary samples were sensitive to fluorizoline. Moreover, fluorizoline increased the mRNA and protein levels of the pro-apoptotic BCL-2 family member NOXA both in cell lines and primary samples analyzed. Finally, NOXA-depletion by CRISPR/Cas9 in cells that do not express BIM conferred resistance to fluorizoline-induced apoptosis in multiple myeloma cells. These results suggest that targeting prohibitins could be a new therapeutic strategy for myeloma multiple.

PRKCQ inhibition enhances chemosensitivity of triple-negative breast cancer by regulating Bim

BackgroundProtein kinase C theta, (PRKCQ/PKCθ) is a serine/threonine kinase that is highly expressed in a subset of triple-negative breast cancers (TNBC) and promotes their growth, anoikis resistance, epithelial-mesenchymal transition (EMT), and invasion. Here, we show that PRKCQ regulates the sensitivity of TNBC cells to apoptosis triggered by standard-of-care chemotherapy by regulating levels of pro-apoptotic Bim.MethodsTo determine the effects of PRKCQ expression on chemotherapy-induced apoptosis, shRNA and cDNA vectors were used to modulate the PRKCQ expression in MCF-10A breast epithelial cells or triple-negative breast cancer cells (MDA-MB231Luc, HCC1806). A novel PRKCQ small-molecule inhibitor, 17k, was used to inhibit kinase activity. Viability and apoptosis of cells treated with PRKCQ cDNA/shRNA/inhibitor +/-chemotherapy were measured. Expression levels of Bcl2 family members were assessed.ResultsEnhanced expression of PRKCQ is sufficient to suppress apoptosis triggered by paclitaxel or doxorubicin treatment. Downregulation of PRKCQ also enhanced the apoptosis of chemotherapy-treated TNBC cells. Regulation of chemotherapy sensitivity by PRKCQ mechanistically occurs via regulation of levels of Bim, a pro-apoptotic Bcl2 family member; suppression of Bim prevents the enhanced apoptosis observed with combined PRKCQ downregulation and chemotherapy treatment. Regulation of Bim and chemotherapy sensitivity is significantly dependent on PRKCQ kinase activity; overexpression of a catalytically inactive PRKCQ does not suppress Bim or chemotherapy-associated apoptosis. Furthermore, PRKCQ kinase inhibitor treatment suppressed growth, increased anoikis and Bim expression, and enhanced apoptosis of chemotherapy-treated TNBC cells, phenocopying the effects of PRKCQ downregulation.ConclusionsThese studies support PRKCQ inhibition as an attractive therapeutic strategy and complement to chemotherapy to inhibit the growth and survival of TNBC cells.

674 The effective killing of the difficult-to-treat melanomas with the combinations of MCL1 inhibitors S63845/MIK665 plus Navitoclax

Despite the advancement of melanoma care, a subset of melanoma patients do not respond or relapse with current targeted or immuno-therapies. Therefore, it is necessary to explore other biological processes can be therapeutic. The BCL-2 family of proteins contributes to a cancer cell’s resistance to treatment, and BH3 mimetics drugs that target BCL-2 are effective in hematological cancers. We used genetic knockdown and BH3 mimetics therapeutics to target BCL-2’s anti-apoptotic defenses, and identified MCL1 and BCLXL as crucial melanoma pro-survival members. We then examined the effects of combining BH3 mimetics that target MCL1 and BCLXL in vivo and in vitro. We prioritized clinical-trial-ready compounds, such as ABT263 (Navitoclax) and S63845/S64315 (MIK655). We used commercial cell lines, as well as cells derived from patients with difficult-to-treat melanomas. The melanoma panel included the subtypes acral, mucosal, superficial spreading, and nodular, as well as diverse mutations, such as wild type for BRAF, NRAS and NF1. In vitro, the combined inhibition of MCL1 and BCLXL resulted in greater killing than either single agent (p < 0.05) in multiple assays. Genetic knock-down and knock-out studies showed that the combination-induced cell death was not dependent on pro-apoptotic BCL2 family members BID, BIM, or NOXA. Moreover, in mouse xenograft model, the combination inhibited tumor growth of multiple melanomas (p <0.01), had tolerable toxicity, and reduced sphere forming capacity (p < 0.05) in a downstream in vitro assay. In summary, this study suggests that dual targeting of MCL1 and BCLXL can be an alternative treatment for melanoma patients who have exhausted current treatment options.

Disruption of conserved polar interactions causes a sequential release of Bim mutants from the canonical binding groove of Mcl1

Mcl1 is an important anti-apoptotic member of the Bcl2 family proteins that are upregulated in several cancer malignancies. The canonical binding groove (CBG) located at the surface of Mcl1 exhibits a critical role in binding partners selectively via the BH3-domain of pro-apoptotic Bcl2 family members that trigger the downregulation of Mcl1 function. There are several crystal structures of point-mutated pro-apoptotic Bim peptides in complex with Mcl1. However, the mechanistic effects of such point-mutations towards peptide binding and complex stability still remain unexplored. Here, the effects of the reported point mutations in Bim peptides and their binding mechanisms to Mcl1 were computationally evaluated using atomistic-level steered molecular dynamics (SMD) simulations. A range of external-forces and constant-velocities were applied to the Bim peptides to uncover the mechanistic basis of peptide dissociation from the CBG of Mcl1. Although the peptides showed similarities in their dissociation pathways, the peak rupture forces varied significantly. According to simulations results, the disruption of the conserved polar contacts at the complex interface causes a sequential release of the peptides from the CBG of Mcl1. Overall, the results obtained from the current study may provide valuable insights for the development of novel anti-cancer peptide-inhibitors that can downregulate Mcl1's function.

Blockade of HSP70 by VER-155008 synergistically enhances bortezomib-induced cytotoxicity in multiple myeloma

Proteasome inhibitor bortezomib is one of the most effective drugs currently available for the treatment of multiple myeloma (MM). However, the intrinsic and acquired resistance to bortezomib can limit its effectiveness. The activation of heat shock response has been characterized as a potential resistance mechanism protecting MM cells from bortezomib-induced cell death. In this study, in response to bortezomib therapy, we discovered that HSP70 is one of the most substantially upregulated heat shock proteins. In order to further explore approaches to sensitizing bortezomib-based treatment for MM, we investigated whether targeting HSP70 using a specific inhibitor VER-155008 combined with bortezomib could overcome the acquired resistance in MM. We found that HSP70 inhibitor VER-155008 alone significantly decreased MM cell viability. Moreover, the combination of VER-155008 and bortezomib synergistically induced MM cell apoptosis markedly in vitro. Notably, the combined treatment was found to increase the cleavage of PARP, an early marker of chemotherapy-induced apoptosis. Importantly, the reduction of anti-apoptotic Bcl-2 family member Bcl-2, Bcl-xL, and Mcl-1 and the induction of pro-apoptotic Bcl-2 family member BH3-only protein NOXA and Bim were confirmed to be tightly associated with the synergism. Finally, the ER stress marker CHOP (CCAAT-enhancer binding protein homologous protein), which can cause transcriptional activation of genes involved in cell apoptosis, was markedly induced by both VER-155008 and bortezomib. Taken together, our finding of a strong synergistic interaction between VER-155008 and bortezomib may support for combination therapy in MM patients in the future.

Conserved pathways of apoptosis in leukemia: A short communication

The stem cell nature of leukemia and several other cancers has long been known now. Given that the leukemic blast cells (the large population of aberrantly differentiated blood cancer cells) arise from a rare and quiet population of stem cells called leukemic stem cells (LSCs) which have accumulated multiple mutations in multiple steps spread over the years, the particular hypothesis that cancer results from the loss-of-function mutation in apoptotic gene of the apoptotic pathway would mean that the LSCs and the leukemic blast cells do not undergo apoptosis at all. However, this is not the case; in reality, there are experimental and/or clinical studies showing that the LSCs and the leukemic blast cells do undergo apoptosis in a fully grown cancer, though its rate is less than the proliferation rate. I hypothesize in this article that this apoptosis by itself is significant and its importance cannot be ruled out, though it may not be significant when compared with the apoptosis of blood cells in a healthy person. This means that though leukemia arises through multiple mutations which may include mutations in apoptotic genes too, there are left some conserved pathways of apoptosis in fully grown cancer. A few of these possible conserved pathways of apoptosis in a fully developed leukemia, that I list here, include P53, pro-apoptotic Bcl-2 family members, microRNA, and short interfering RNA.

Identification of Genotype-Specific Therapeutic Vulnerabilities By Comparative Dynamic BH3 Profiling Analysis of Human and Murine CLL

Identification of Genotype-Specific Therapeutic Vulnerabilities By Comparative Dynamic BH3 Profiling Analysis of Human and Murine CLL

MCL-1 Inhibition By the Selective MCL-1 Inhibitor AMG-176 Induces in Vitro Activity Against Burkitt Lymphoma Cell Lines and Synergistically Enhances the Cytotoxic Effect of Chemotherapy and BH3 Mimetics

Background: Although &gt;90% of Burkitt Lymphoma (BL) patients will be cured by intensive multi-agent immunochemotherapy, relapsed and refractory BL patients have poor prognoses demonstrating the need to identify novel therapies. Myeloid cell leukemia-1 (MCL-1) is a pro-survival protein within the BCL-2 family that has been investigated as a therapeutic target in several leukemia and lymphoma pre-clinical models and clinical studies. Analysis of pediatric BL tumors has implicated MCL-1 in BL pathogenesis (Giulino-Roth et al., Blood 2012) and targeting MCL-1 may be effective in MYC-driven lymphomas (Kelly et al., Genes Dev 2014). High MCL-1 expression has also been associated with therapy resistance and can confer resistance to BH3 mimetic agents (Carrington et al., Cell Death Differ 2017). AMG176, a selective MCL-1 inhibitor, is an effective promoter of apoptosis and is currently under investigation in clinical trials for relapsed and refractory multiple myeloma, lymphoma, and acute myeloid leukemia. Hypothesis: We hypothesized that MCL-1 inhibition with AMG176 will promote apoptosis and increase sensitivity of BL therapy-sensitive (TSCL) and therapy-resistant (TRCL) cell lines to other chemotherapy drugs. Methods: A panel of TSCLs (Raji, Ramos and Daudi) and one TRCL (Raji 4RH) BL cell lines were exposed to increasing concentrations of AMG176 as a single agent and in combination with Doxorubicin or Venetoclax at different time points. Cell viability was analyzed using PrestoBlue. Synergy in drug combination exposures was determined by Calcusyn software and reported as a combination index (CI) value with values increasingly &lt;1 indicating increasing synergistic activity. Apoptosis induction was analyzed by flow cytometry for Annexin V (AV) and Sytox-7AAD and by western blotting for PARP. Effects of MCL-1 inhibition on the expression of other pro- and anti-apoptotic BCL-2 family member proteins was assessed by western blotting. AMG176 mechanism(s) of action was evaluated by co-immunoprecipitation followed by western blotting for pro-apoptotic BCL-2 family members known to be bound by MCL-1. Results: Single agent AMG176 exposure of BL cell lines exhibited a dose and time-dependent decrease in viable cells. 48hr IC50 values of Ramos (0.3097uM) and Raji (1.652uM) showed an increased sensitivity to AMG176 as compared to Daudi (9.616uM) and Raji 4RH (12.45uM). Exposure of cells to AMG176 also resulted in altered expression levels of pro-survival and pro-apoptotic proteins of the BCL-2 family proteins. Co-immunoprecipitation performed after 48hrs of AMG176 exposure exhibited a decrease in MCL-1:BIM and MCL-1:BAK complexes in Raji and Ramos cells promoting a pro-apoptotic environment. After 48hr exposure to AMG176, induction of apoptosis was observed in a dose-dependent manner in all cell lines as evidenced by increased PARP cleavage on western blot and AV-Sytox positivity on flow cytometry. To investigate the ability of MCL-1 inhibition to enhance the activity of cytotoxic chemotherapy, BL cells were exposed to AMG176 in combination with Doxorubicin. Daudi and Raji exhibited strong synergy with peak synergistic activity observed at 72hrs (Daudi CI values &lt;0.1 at all AGM176 concentrations with 0.1-0.2uM Doxorubicin; Raji CI ranged from 0.233-0.295 at multiple AMG176 concentrations with &lt;0.4uM Doxorubicin) while a minimal effect was observed in Ramos and Raji 4RH. As high MCL-1 is associated with resistance to Venetoclax by sequestering free BIM following Venetoclax exposure, the combination of AMG176 and Venetoclax was investigated and also showed significant synergy in BL cells. A minimal effect was observed in Daudi and Raji 4RH in Venetoclax combination but Ramos and Raji, the more AMG176 sensitive cell lines, showed peak synergy at 24hrs at high doses of Venetoclax (Ramos CI values ranged from 0.3-0.478; Raji CI ranged from 0.511 to 0.67). Conclusion: MCL-1 inhibition with AMG176 demonstrates dose- and time-dependent anti-lymphoma activity impairing in vitro proliferation and inducing apoptosis in BL cells. AMG176 exhibits synergistic activity in combination with cytotoxic chemotherapy in both therapy-sensitive and therapy-resistant BL cell lines and synergistically enhances response to the BH3 mimetic agent Venetoclax. These findings highlight the potential of MCL-1 inhibition as a therapeutic option in BL. This work was supported by T35 NIH training grant (T35AI089693). Disclosures No relevant conflicts of interest to declare.

The Transcriptome of Immunomodulator-Resistant Multiple Myeloma

Introduction: While the molecular target of immunomodulators such as pomalidomide (POM) and lenalidomide (LEN) has been identified, the mechanisms underlying therapeutic resistance remain incompletely understood. The uniformly emerging resistance to therapy over time in the absence of identifiable cereblon pathway mutations in the majority of patients raises questions about alternative mechanisms including aberrant gene expression. Methods: We performed gene expression profiling using an Affymetrix GeneChip Human Genome U133 Plus 2.0 microarray on CD138+ bone marrow cells from patients with relapsed / refractory (RRMM) and newly diagnosed (NDMM) multiple myeloma prior to initiating treatment. Patients were treated on two phase II clinical trial protocols (MC0789: POM ± dexamethasone in RRMM; MC0884: LEN ± dexamethasone in NDMM) between 2007 and 2012. We categorized patients based on their IMWG response as non-responders (SD) and responders (VGPR+). We selected 15 responders and 15 non-responders from MC0789 (n = 30) and compared overall survival, gene expression patterns, and involved cellular pathways between the two groups. We selected 5 responders and 5 non-responders from MC0884 (n = 10) for targeted validation of differentially expressed candidate genes. After data quality control and normalization of gene expression values, differential gene expression was estimated using limma. Statistical significance was adjusted for multiple testing in the discovery set using a false discovery rate-based approach for genome-wide experiments (q-value). We used Gene Ontology and PANTHER pathway analysis for functional annotation of differentially expressed genes. Overall survival estimates were calculated using the Kaplan-Meier method. Computation and visualization were performed in R. Results: Median age at treatment initiation on MC0789 was 65 years (40 - 82), 65% of the patients were male. Pomalidomide resistance was associated with an increase in mortality (median overall survival 1.6 versus 6.4 years, p = 0.009, Kaplan-Meier plot). There were 1076 differentially regulated genes between responders and non-responders (521 up- and 555 down-regulated, q &lt; 0.050 for all genes, volcano plot). Expression of CRBN was 1.5-fold down-regulated in non-responders (q = 0.005). Supervised hierarchical clustering of the top 500 differentially expressed genes demonstrated distinct patterns in pomalidomide-resistant disease (heatmap). Gene ontology analysis revealed protein synthesis as one of the most enriched biological processes (bar graph). Pathway analysis showed a 6-fold enrichment (FDR = 0.007) of the ubiquitin proteasome pathway in pomalidomide-resistant disease. Differentially expressed genes involved key protein degradation pathways, epigenetic modifiers, and transcription factors. Targeted validation in MC0884 revealed 13 common genes with at least 1.5-fold differential expression (5 up- and 8 down-regulated), 12 of which have previously been implicated in the regulation of apoptosis, tumor glucose metabolism, Rho and Wnt signaling, miRNA-driven resistance to chemotherapy, and ubiquitin-dependent protein degradation (Table and Sankey diagram). The most up-regulated gene in non-responders was MYRIP, a gene coding for a vesicle trafficking protein associated with platinum resistance and suppression of pro-apoptotic BCL-2 family members in solid malignancies. The most down-regulated gene was FRZB, a gene coding for a negative regulator of Wnt signaling, previously implicated in the progression of monoclonal gammopathy of undetermined significance to multiple myeloma. Conclusions: Overall survival of patients with pomalidomide-resistant RRMM remains poor. Pomalidomide resistance was associated with differential gene expression in several potentially targetable cellular pathways beyond the known drug target cereblon. Targeted validation of candidate genes revealed common cellular pathways in immunomodulator-resistant disease. Elucidating the exact molecular mechanisms underlying immunomodulator resistance is of considerable interest for biomarker development and the identification of novel therapeutic targets and warrants further exploration. Figure Disclosures Lacy: Celgene: Research Funding. Dispenzieri:Celgene: Research Funding; Alnylam: Research Funding; Intellia: Consultancy; Janssen: Consultancy; Pfizer: Research Funding; Akcea: Consultancy; Takeda: Research Funding. Kumar:Takeda: Research Funding; Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding.

The E3 ubiquitin ligase RNF40 suppresses apoptosis in colorectal cancer cells

BackgroundColorectal cancer (CRC) is the fourth leading cause of cancer-related deaths worldwide, and deciphering underlying molecular mechanism is essential. The loss of monoubiquitinated histone H2B (H2Bub1) was correlated with poor prognosis of CRC patients and, accordingly, H2Bub1 was suggested as a tumor-suppressive mark. Surprisingly, our previous work revealed that the H2B ubiquitin ligase RING finger protein 40 (RNF40) might exert tumor-promoting functions. Here, we investigated the effect of RNF40 loss on tumorigenic features of CRC cells and their survival in vitro.MethodsWe evaluated the effects of RNF40 depletion in several human CRC cell lines in vitro. To evaluate cell cycle progression, cells were stained with propidium iodide and analyzed by flow cytometry. In addition, to assess apoptosis rates, caspase 3/7 activity was assessed in a Celigo® S-based measurement and, additionally, an Annexin V assay was performed. Genomic occupancy of H2Bub1, H3K79me3, and H3K27ac was determined by chromatin immunoprecipitation. Transcriptome-wide effects of RNF40 loss were evaluated based on mRNA-seq results, qRT-PCR, and Western blot. To rescue apoptosis-related effects, cells were treated with Z-VAD-FMK.ResultsHuman CRC cell lines displayed decreased cell numbers in vitro after RNF40 depletion. While the differences in confluence were not mediated by changes in cell cycle progression, we discovered highly increased apoptosis rates after RNF40 knockdown due to elevated caspase 3/7 activity. This effect can be explained by reduced mRNA levels of anti-apoptotic and upregulation of pro-apoptotic BCL2 family members. Moreover, the direct occupancy of the RNF40-mediated H2B monoubiquitination was observed in the transcribed region of anti-apoptotic genes. Caspase inhibition by Z-VAD-FMK treatment rescued apoptosis in RNF40-depleted cells. However, knockdown cells still displayed decreased tumorigenic features despite the absence of apoptosis.ConclusionsOur findings reveal that RNF40 is essential for maintaining tumorigenic features of CRC cells in vitro by controlling the expression of genes encoding central apoptotic regulators.

Camel Whey Protein Disrupts the Cross-Talk Between PI3K and BCL-2 Signals and Mediates Apoptosis in Primary Acute Myeloid Leukemia Cells

In the present study, we investigated the impact of camel whey protein (CWP) on the survival of primary acute myeloid leukemia (AML) cells that were isolated from 20 patients diagnosed with AML. We found that CWP induced apoptosis in the primary AML cells without affecting the normal PBMCs that were isolated from healthy individuals, as determined by PI/annexin V double staining followed by flow-cytometry analysis. Furthermore, we demonstrated that these primary AML cells exhibited aberrant phosphorylation of AKT, mTOR and STAT3. Treatment of AML cells with CWP mediated significant reduction in the phosphorylation of AKT, mTOR and STAT3. Additionally, we demonstrated that blockade of PI3K/AKT signaling pathway by wortmannin (WM) impaired the expression of Bcl-2 and BclXL in the primary AML cells, suggesting an essential cross-talk between PI3K and Bcl-2 that maintains the survival of AML cells. In this context, treatment of AML cells with CWP disrupted the PI3K/Bcl-2 cross-talk; significantly downregulated the expression of anti-apoptotic Bcl-2 family members Bcl-2 and BclXL; markedly upregulated the expression of the pro-apoptotic Bcl-2 family members Bak and Bax; and subsequently sensitized tumor cells to growth arrest. Our data revealed the therapeutic potential of CWP and the underlying mechanisms against leukemia.

BIRC3 Expression Predicts CLL Progression and Defines Treatment Sensitivity via Enhanced NF-κB Nuclear Translocation.

Chronic lymphocytic leukemia (CLL) pathophysiology is characterized by a complex crosstalk of tumor cells with the microenvironment. In this regard, NF-κB signaling is considered as important signaling axis, with a variety of key molecules aberrantly expressed or genetically altered in patients with CLL. One of these molecules is BIRC3 (cIAP2), a central regulator of noncanonical NF-κB signaling that serves as pathway brake in the absence of microenvironmental signals. However, the contribution of BIRC3 expression to CLL progression and potential therapeutic implications is unknown.Experimental Design: We analyzed the role of BIRC3 mRNA expression in primary CLL samples in correlation to clinical datasets and used ex vivo assays to investigate functional consequences on the level of NF-κB signaling and downstream target gene regulation. For proof-of-principle experiments, we used genetically modified cell lines. We demonstrate that patients with CLL with low BIRC3 expression experience a more rapid disease progression, which coincides with an enhanced activation of canonical NF-κB target genes evidenced by an increased p65/Rel-B nuclear translocation ratio. As a consequence of enhanced canonical NF-κB target gene activation, both anti- and proapoptotic Bcl-2 family members were upregulated in BIRC3low primary CLL cells, which was associated with higher sensitivity to venetoclax treatment in vitro. Here we show the impact of BIRC3 expression in CLL disease progression in the absence of BIRC3 mutations and show altered canonical NF-κB target gene activation with therapeutic implications.

The interaction between Vav1 and EBNA1 promotes survival of Burkitt's lymphoma cells by down-regulating the expression of Bim

Vav1 is a guanine nucleotide exchange factor (GEF) predominantly expressed in hematopoietic cells, and functions in the development and antigen-stimulated response of lymphocytes. Burkitt's lymphoma (BL) is characterized as transformed B cell lymphoma, and is highly associated with Epstein-Barr virus (EBV). EBV nuclear antigen 1 (EBNA1) is the only viral protein expressed across all three types of latency and essential for the persistence of EBV genome. It is not clear yet how EBNA1 contributes to the growth advantage of latently infected cells such as in EBV+ lymphoma B cells. Here, we reported that Vav1 interacts with EBNA1 via its C-terminal SH3 domain. This interaction suppresses the expression of a pro-apoptotic Bcl-2 family member, Bim, resulting in the resistance of the BL cells to apoptotic inductions. Our data uncovered Vav1 as a novel target for EBNA1, and suggested a pro-survival role of Vav1 in the pathogenesis of EBV associated BLs.