Proapoptotic Bcl-2 Family Member Bim Research Articles

Hexokinase 3 enhances myeloid cell survival via non-glycolytic functions

The family of hexokinases (HKs) catalyzes the first step of glycolysis, the ATP-dependent phosphorylation of glucose to glucose-6-phosphate. While HK1 and HK2 are ubiquitously expressed, the less well-studied HK3 is primarily expressed in hematopoietic cells and tissues and is highly upregulated during terminal differentiation of some acute myeloid leukemia (AML) cell line models. Here we show that expression of HK3 is predominantly originating from myeloid cells and that the upregulation of this glycolytic enzyme is not restricted to differentiation of leukemic cells but also occurs during ex vivo myeloid differentiation of healthy CD34+ hematopoietic stem and progenitor cells. Within the hematopoietic system, we show that HK3 is predominantly expressed in cells of myeloid origin. CRISPR/Cas9 mediated gene disruption revealed that loss of HK3 has no effect on glycolytic activity in AML cell lines while knocking out HK2 significantly reduced basal glycolysis and glycolytic capacity. Instead, loss of HK3 but not HK2 led to increased sensitivity to ATRA-induced cell death in AML cell lines. We found that HK3 knockout (HK3-null) AML cells showed an accumulation of reactive oxygen species (ROS) as well as DNA damage during ATRA-induced differentiation. RNA sequencing analysis confirmed pathway enrichment for programmed cell death, oxidative stress, and DNA damage response in HK3-null AML cells. These signatures were confirmed in ATAC sequencing, showing that loss of HK3 leads to changes in chromatin configuration and increases the accessibility of genes involved in apoptosis and stress response. Through isoform-specific pulldowns, we furthermore identified a direct interaction between HK3 and the proapoptotic BCL-2 family member BIM, which has previously been shown to shorten myeloid life span. Our findings provide evidence that HK3 is dispensable for glycolytic activity in AML cells while promoting cell survival, possibly through direct interaction with the BH3-only protein BIM during ATRA-induced neutrophil differentiation.

NFAT Factors Are Dispensable for the Development but Are Critical for the Maintenance of Foxp3+ Regulatory T Cells.

The transcription factors of the nuclear factor of activated T cell (NFAT) family play a crucial role in multiple aspects of T cell function. It has recently been reported that NFATs play an important role in the suppressive function of CD4+CD25+Foxp3+ regulatory T (Treg) cells. In this study, we have investigated the role of NFATs in the thymic development of Treg cells in mice. We show that NFAT factors are dispensable for the development of Foxp3+ Treg cells in the thymus but are critical for the maintenance of both the phenotype and survival of Treg cells in the thymus as well as in peripheral lymphoid organs. Specifically, the homeostasis of CD4+CD25+Foxp3+ but not the CD4+CD25−Foxp3+ fraction is severely perturbed when NFAT signaling is blocked, leading to a strongly reduced Treg population. We underscored this intriguing effect of NFAT on CD4+CD25+Foxp3+ Treg cells to the disruption of survival signals provided by interleukin 2 (IL-2). Accordingly, blocking Treg cell death by abolishing the activity of pro-apoptotic Bcl-2 family member Bim, compensated for the survival defects induced due to a lack of NFAT-IL-2-IL-2R signaling. Inhibition of NFAT activity led to a strong reduction in the number of Foxp3+ Treg cells; however, it did not influence the level of Foxp3 expression on an individual cell basis. In addition, we show a differential effect of IL-2 and IL-7 signaling on Foxp3+ Treg versus CD4+CD25− T cell development, again underlining the dispensability of NFAT signaling in the development, but not in the maintenance of Foxp3+ Treg cells.

Bim Expression Promotes the Clearance of Mononuclear Phagocytes during Choroidal Neovascularization, Mitigating Scar Formation in Mice.

Inflammation is increasingly recognized as an important modulator in the pathogenesis of neovascular age-related macular degeneration (nAMD). Although significant progress has been made in delineating the pathways that contribute to the recruitment of inflammatory cells and their contribution to nAMD, we know little about what drives the resolution of these inflammatory responses. Gaining a better understanding of how immune cells are cleared in the choroid will give a novel insight into how sustained inflammation could influence the pathogenesis of nAMD. The pro-apoptotic Bcl-2 family member Bim is a master regulator of immune cell homeostasis. In its absence, immune cell lifespan and numbers increase. Most therapeutic regimes that squelch inflammation do so by enhancing immune cell apoptosis through enhanced Bim expression and activity. To test the hypothesis that Bim expression tempers inflammation during the pathogenesis of nAMD, we used the mouse laser-induced choroidal neovascularization (CNV) model in which inflammation acts as a facilitator of CNV. Here, we showed minimal to no change in the recruitment of F4/80-, CD80-, CD11b-, and Iba1-positive myeloid-derived mononuclear phagocytes to the site of laser photocoagulation in the absence of Bim expression. However, the resolution of these cells from the choroid of Bim-deficient (Bim -/-) mice was significantly diminished following laser photocoagulation. With time, we noted increased scar formation, demonstrated by collagen I staining, in Bim -/- mice with no change in the resolution of neovascularization compared to wild-type littermates. We also noted that mice lacking Bim expression in mononuclear phagocytes (BimFlox/Flox; Lyz2-Cre (BimMP) mice) had delayed resolution of F4/80-, CD80-, CD11b-, and Iba1-positive cells, while those lacking Bim expression in endothelial cells (BimFlox/Flox; Cad5-Cre (BimEC) mice) had delayed resolution of only CD11b- and Iba1-positive cells. Both BimMP and BimEC mice demonstrated increased scar formation, albeit to differing degrees. Thus, our studies show that resolving inflammation plays an important role in moderating scar formation in nAMD, and it is impacted by Bim expression in both the endothelium and mononuclear phagocyte lineages.

The deubiquitinase Usp27x as a novel regulator of cFLIPL protein expression and sensitizer to death-receptor-induced apoptosis

Death receptors are transmembrane proteins that can induce the activation of caspase-8 upon ligand binding, initiating apoptosis. Recent work has highlighted the great molecular complexity of death receptor signalling, in particular through ubiquitination/deubiquitination. We have earlier defined the deubiquitinase Ubiquitin-Specific Protease 27x (Usp27x) as an enzyme capable of stabilizing the pro-apoptotic Bcl-2 family member Bim. Here, we report that enhanced expression of Usp27x in human melanoma cells leads to the loss of cellular FLICE-like inhibitory protein (cFLIP) and sensitizes to Tumor necrosis factor receptor 1 (TNF-R1) or Toll-like receptor 3 (TLR3)-induced extrinsic apoptosis through enabling enhanced processing of caspase-8. The loss of cFLIPL upon overexpression of Usp27x was not due to reduced transcription, could be partially counteracted by blocking the ubiquitin proteasome system and was independent of the known cFLIPL destabilizing ubiquitin E3-ligases Itch and DTX1. Instead, Usp27x interacted with the E3-ligase TRIM28 and reduced ubiquitination of TRIM28. Reduction of cFLIPL protein levels by Usp27x-induction depended on TRIM28, which was also required for polyI:C-induced cell death. This work defines Usp27x as a novel regulator of cFLIPL protein expression and a deubiquitinase in fine tuning death receptor signalling pathways to execute apoptosis.

Single Agent and Synergistic Combinatorial Efficacy of First-in-Class Small Molecule Imipridone ONC201 in Hematological Malignancies In Vitro and In Vivo through Apoptosis Via the Integrated Stress Response Pathway

▪ONC201, founding member of the imipridone class of small molecules, is currently being evaluated in advancer cancer clinical trials. We explored single agent and combinatorial efficacy of ONC201 in preclinical models of hematological malignancies.ONC201 was evaluated in 20 human leukemia cell lines (Figure 1): 13 acute myeloid leukemia (AML), 5 acute lymphoblastic leukemia (ALL) and 2 chronic myelogenous leukemia (CML) cell lines. We observed a dose-dependent decrease in cell viability in AML, ALL and CML cells (GI50 1-8 µM). ONC201 also decreased cell viability in 2 primary chronic lymphocytic leukemia (CLL) patient samples with 48-72h treatment. Dose- and time-dependent induction of apoptosis (caspase-3 cleavage), inhibition of Akt and Foxo3a phosphorylation was observed in AML cells treated with ONC201 for 24-48 hr. Sub-G1 analysis confirmed that ONC201 induced apoptosis in ALL cells and a pan-caspase inhibitor reduced ONC201-mediated apoptosis. ONC201-mediated apoptosis in ALL cells involved PARP cleavage and caspase-9 activation. Anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xl were downregulated while the pro-apoptotic Bcl-2 family member Bim was upregulated in response to ONC201 treatment in ALL cells. ONC201 also downregulates the inhibitor of apoptosis (IAP) family proteins cIAP1 and cIAP2 in ALL cells, which are known to confer a poor clinical prognosis.Low micromolar concentrations of ONC201 also reduced multiple myeloma (MM) cell viability in a time- and dose-dependent manner. ONC201 reduced cell viability and induced apoptosis in dexamethasone-resistant MM.1R cells with efficacy comparable to dexamethasone-sensitive MM.1S cells (Figure 2). ONC201 caused activation of the integrated stress response (ISR) pathway, as observed with induction of ATF/CHOP in MM cells. XIAP is highly up-regulated in MM cells and sownregulation of IAP family proteins induces apoptosis. ONC201 treatment of MM cells resulted in a dose-dependent decrease in the anti-apoptotic proteins XIAP, cIAP1 and cIAP2.Next, ONC201 was evaluated in 33 human lymphoma cell lines: 3 anaplastic large cell lymphoma (ALCL), 7 Burkitt's lymphoma, 2 cutaneous T-cell lymphoma (CTCL), 8 diffuse large B-cell lymphoma (DLBCL), 1 MCL, 2 nodular sclerosis (NS) and 10 other lymphoma cell lines. ONC201 demonstrated low micromolar range efficacy (GI50 1-6 µM) across all lymphoma subtypes. Low micromolar concentrations of ONC201 reduced cell viability of MCL and DLBCL cell lines and primary cells in a time- and dose-dependent manner (Figure 3). ONC201 caused activation of the ISR pathway, as observed with induction of ATF4 in Pfeiffer DLBCL cells. In patient-derived MCL cells, ONC201 reduced cyclin D1 expression, a diagnostic feature of MCL.We then tested the efficacy of ONC201 in combination with approved therapies in hematological malignancies in vitro and in vivo. ONC201 demonstrated synergistic reduction in cell viability in combination with cytarabine confirmed by combination indices in AML cell lines CMK, THP-1, HL60 and ALL Kasumi-2 cells. ONC201 combined with cytarabine in a Burkitt's lymphoma xenograft model exhibited superior tumor growth inhibition compared to either agent alone (Figure 4). ONC201 demonstrated synergistic reduction in cell viability in combination with 5-azacytidine that was confirmed by combination indices in AML cell lines HL-60, MOLM14 and MV411. ONC201 synergistically reduced cell viability in combination with dexamethasone or ixazomib in RPMI-8226 MM cells. ONC201 in combination with bortezomib synergistically reduced cell viability in KARPAS 299 ALCL cells and RPMI-8226 MM cells and improved apoptosis induction in UPN2 MCL cells. ONC201 combined with bortezomib was evaluated in a Burkitt's lymphoma xenograft model. Histological evaluation of tumor xenografts revealed a reduced tumor cell density at 3-4 weeks and enhanced CHOP induction at 48 hours with combination treatment compared to either agent alone. The combination treatment did not result in body weight loss.These results serve as a rationale for ongoing ONC201 single agent trials in relapsed/refractory acute leukemia (NCT02392572), non-Hodgkin's lymphoma (NCT02420795), MM (NCT02609230) and combination trial with dexamethasone in MM (NCT02863991). Our findings provide pharmacodynamic biomarkers and identify synergistic combinatorial regimens that are well tolerated and can be explored in the clinic. [Display omitted] DisclosuresPrabhu:Oncoceutics: Employment, Equity Ownership. Grupp:Novartis Pharmaceuticals Corporation: Consultancy, Other: grant; Adaptimmune: Consultancy; University of Pennsylvania: Patents & Royalties; Jazz Pharmaceuticals: Consultancy. Oster:Oncoceutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Allen:Oncoceutics: Employment, Equity Ownership, Patents & Royalties. El-Deiry:Oncoceutics, Inc: Equity Ownership, Patents & Royalties.

Molecular mechanisms underlying antitumor activity of camel whey protein against multiple myeloma cells

Treating drug-resistant cancer cells is a clinical challenge and it is also vital to screen for new cancer drugs. Multiple myeloma (MM) is a plasma cell clonal cancer that, despite many experimental therapeutics, remains incurable. In this study, two MM cell line lines U266 and RPMI 8226 were used to determine the impact of camel whey protein (CWP). The CWP IC50 was calculated by MTT examination, while the flow cytometry analysis was used to investigate the chemotaxis responses of MM cells in relation to CXCL12 and the pro-apoptotic effect of CHP. MM cells were treated with CWP and Western blot analysis was used to determine the underlying molecular mechanisms. Dose and time based on the impact of CWP on the cell viability of MM cells with IC50 of 50 μg/ml, without affecting the viability of normal healthy PBMCs. CWP reduced chemotaxis of MM cells significantly from the CXC chemokine ligand 12 (CXCL12). Using Western blot analysis, we found that CWP decreased the activation of AKT, mTOR, PLCβ3, NFαB and ERK, which was mechanistically mediated by CXCL12/CXCR4. In both U266 and RPMI 8226, CWP induced apoptosis by upregulating cytochrome C expression. In addition, CWP mediated the growth arrest of MM cells by robustly decreasing the expression of the anti-apoptotic Bcl-2 family members Bcl-2, Bcl-XL and Mcl-1. Conversely, the expression of pro-apoptotic Bcl-2 family members Bak, Bax and Bim was increased after treatment with CWP. Our data indicates CWP's therapeutic potential for MM 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.

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.

Abstract 318: Inhibition of FASN induces apoptosis and senescence in breast cancer

Abstract Fatty Acid Synthase (FASN), the key multienzyme in de novo lipogenesis, is upregulated and activated in numerous carcinomas, including breast cancer. Fatty acids play a fundamental role in cellular structure, energy production and storage and are intermediates in the biosynthesis of hormones and other biologic molecules. Therefore, FASN is a novel therapeutic target for treatment in cancer cells that have upregulated FASN protein. BT474 breast cancer cells, which overexpress FASN, were treated with a pharmacologic FASN inhibitor and resulted in apoptosis in a dose-dependent manner. Inhibition of FASN simultaneously upregulates the pro-apoptotic BCL2 family members Bim, Puma, and Noxa; additionally, FASN inhibition alters the NDAPH/NADP+ balance and induces reactive oxygen species (ROS) production. Most significantly, FASN inhibition induces sensitization of breast cancer cells to the BH3 mimetic navitoclax, a potential opportunity to use BH3 mimetics for the treatment of breast carcinomas. These results support further preclinical and potentially clinical development of a dual FASN and Bcl-2 treatment for breast cancer tumors that express FASN. In addition to apoptosis, there is an increase in cellular senescence in breast cancer cells overexpressing FASN when treated with FASN inhibitors. The increase in ROS, a known effect of FASN inhibition, is known to induce cellular stress, resulting in both apoptosis and senescence. Increased senescence was initially observed by x-gal staining of BT474 cells treated with FASN inhibitors via microscopy and FLOW. We also observed an increased expression of several pro-senescent genes both at the protein and mRNA levels, including p16/INK4a, p14ARF/p19ARF, p15, and p21. A hallmark of age-induced cellular senescence is the shortening of telomeres. However, telomere shortening was not observed, suggesting that senescence was a result of FASN inhibition and activation of senescence-induced genes. Therefore, inhibition of FASN appears to be an ideal target for therapeutic treatment of breast cancers that overexpress FASN through both apoptosis and senescence. Citation Format: Travis Vander Steen, Ruth Lupu. Inhibition of FASN induces apoptosis and senescence in breast 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 318.

Leukocyte Bim deficiency does not impact atherogenesis in ldlr\u2212/\u2212 mice, despite a pronounced induction of autoimmune inflammation

Proapoptotic Bcl-2 family member Bim is particularly relevant for deletion of autoreactive and activated T and B cells, implicating Bim in autoimmunity. As atherosclerosis is a chronic inflammatory process with features of autoimmune disease, we investigated the impact of hematopoietic Bim deficiency on plaque formation and parameters of plaque stability. Bim−/− or wild type bone marrow transplanted ldlr−/− mice were fed a Western type diet (WTD) for 5 or 10 weeks, after which they were immunophenotyped and atherosclerotic lesions were analyzed. Bim−/− transplanted mice displayed splenomegaly and overt lymphocytosis. CD4+ and CD8+ T cells were more activated (increased CD69 and CD71 expression, increased interferon gamma production). B cells were elevated by 147%, with a shift towards the pro-atherogenic IgG-producing B2 cell phenotype, resulting in a doubling of anti-oxLDL IgG1 antibody titers in serum of bim−/− mice. Bim−/− mice displayed massive intraplaque accumulation of Ig complexes and of lesional T cells, although this did not translate in changes in plaque size or stability features (apoptotic cell and macrophage content). The surprising lack in plaque phenotype despite the profound pro-atherogenic immune effects may be attributable to the sharp reduction of serum cholesterol levels in WTD fed bim−/− mice.

Silymarin and its active component silibinin act as novel therapeutic alternatives for salivary gland cancer by targeting the ERK1/2-Bim signaling cascade.

Approximately 20% of all salivary gland cancer patients who are treated with current treatment modalities will ultimately develop metastases. Its most common form, mucoepidermoid carcinoma (MEC) is a highly aggressive tumor with an overall 5-year survival rate of ~30%. Until now, several chemotherapeutic drugs have been tested for the treatment of salivary gland tumors, but the results have been disappointing and the drugs often cause unwanted side effects. Therefore, several recent studies have focused on the potential of alternative and/or complementary therapeutic options, including the use of silymarin. The effects of silymarin and its active component silibinin on salivary gland cancer-derived MC3 and HN22 cells and their underlying molecular mechanisms were examined using trypan blue exclusion, 4'-6-diamidino-2-phenylindole (DAPI) staining, Live/Dead, Annexin V/PI staining, mitochondrial membrane potential (ΔΨm) measurement, quantitative RT-PCR, soft agar colony formation and Western blotting analyses. We found that silymarin and silibinin dramatically increased the expression of the pro-apoptotic protein Bim in a concentration- and time-dependent manner and, concomitantly, induced apoptosis in MC3 and HN22 cells. We also found that ERK1/2 signaling inhibition successfully sensitized these cells to the apoptotic effects of silymarin and silibinin, which indicates that the ERK1/2 signaling pathway may act as an upstream regulator that modulates the silymarin/silibinin-induced Bim signaling pathway. Taken together, we conclude that ERK1/2 signaling pathway inhibition by silymarin and silibinin increases the expression of the pro-apoptotic Bcl-2 family member Bim which, subsequently, induces mitochondria-mediated apoptosis in salivary gland cancer-derived cells.

The Bcl-2 BH4 Antagonist Bda-366 Induces Apoptosis in CLL and DLBCL Cells through a Bcl-2 Independent Mechanism

Overexpression of the antiapoptotic protein Bcl-2 is considered primarily responsible for the increased apoptosis resistance and prolonged survival of the malignant B cells in chronic lymphocytic leukemia (CLL), follicular lymphoma and subsets of diffuse large B cell lymphoma (DLBCL). The importance of Bcl-2 overexpression is corroborated by the significant clinical activity of the recently approved Bcl-2 BH3 antagonist venetoclax. Venetoclax induces apoptosis by sequestering Bcl-2 from the pro-apoptotic Bcl-2 family members Bim, Bax and Bak, resulting in consequent Bax/Bak activation, mitochondrial outer membrane permeabilization and cytochrome c release. Although venetoclax is extremely toxic against CLL cells in vitro, resistance can arise in vivo because of overexpression of other anti-apoptotic Bcl-2 family members, such as Bcl-xL and Mcl-1.In the present study we investigated the activity of BDA-366, another small molecule Bcl-2 inhibitor that was recently reported to selectively induce apoptosis in Bcl-2-overexpressing lung cancer cells through a distinct mechanism (Han B et al, Cancer Cell. 2015;27:852-63). This drug was designed to selectively bind the BH4 domain of Bcl-2 and was reported to trigger apoptosis by inducing a conformational change in Bcl-2 that converts it into a pro-apoptotic protein. To investigate whether BDA-366 is also selectively toxic for CLL cells, we analyzed the viability of CLL (n=31) and normal peripheral blood mononuclear cells (PBMCs)(n=6) after 48 hours in culture with increasing concentrations of BDA-366. Annexin V/PI analysis showed that BDA-366 is significantly more toxic against CLL (LD50=1.11±0.46 mM) than normal PBMCs (LD50=2.03±0.31 mM, P<0.001). However, considerable variability was noted in the sensitivity of the different CLL samples, with approximatelly 50% of the cases demonstrating LD50 values in the nanomolar range and the remaining cases displaying sensitity similar to normal PBMC. To investigate the reasons for this variability, we correlated LD50 values with IGHV mutation status, ZAP-70 expression, cytogenetic abnormalities, and expression of various Bcl-2 family members, including Bcl-2, Bcl-xL, Mcl-1, BimEL, BimL, Bax, Bak and Hrk. The only positive finding from this analysis was the inverse correlation between LD50 and expression of BimL (n=23, r=-0.446, p=0.033), whereas no correlation was observed with Bcl-2 expression.Variable sensitivity to BDA-366 and absence of correlation with Bcl-2 expression was also observed in the analysis of a series of DLBCL and CLL cell lines. These cell lines showed LD50 values ranging from nanomolar (Pfeiffer, 0.19 µM; OCI-LY-18, 0.32 µM; OCI-LY-1, 0.33 µM; TOLEDO, 0.41 µM; OCI-Ly10, 0.50 µM; HT, 0.56 µM; Ri-1, 0.58 µM; and SU-DHL-6, 0.81 µM) to micromolar range (MEC1, 1.5 µM; HBL-1, 4.0 µM; Karpas, 4.34 µM; BJAB, 4.8 µM; and SU-DHL-4, 6.31 µM). Although most of the sensitive lines expressed Bcl-2, some had very low or even undetectable Bcl-2 expression (e.g., Pfeiffer and HT), suggesting that BDA-366 can induce apoptosis independently of Bcl-2. In support of this possibility, we observed that BDA-366 was equally effective against Bcl-2-negative HT cells and HT cells stably transfected with Bcl-2. In addition, transient overexpression of Bcl-2 in the DLBCL cell line BJAB and the CLL cell line MEC1 resulted in greater resistance to BDA-366, further suggesting that BDA-366 does not induce apoptosis by converting Bcl-2 into a proapoptotic protein.To determine whether mechanisms that induce resistance to venetoclax can also induce resistance to BDA-366, we investigated viability of BDA-366-treated CLL cells following stimulation with immobilized anti-IgM. Pre-treatment of CLL cells with immobilized anti-IgM resulted in increased resistance to both BDA-366 and venetoclax. Interestingly, the combination of BDA-366 and venetoclax was significantly more toxic than each agent alone (Figure 1), suggesting that combining these drugs could be a potential approach to overcome resistance induced by BCR signals.In conclusion, these data show that BDA-366 is selectively toxic at low nanomolar concentrations for a large proportion of primary CLL samples and DLBCL cell lines. The primary mechanism of action is still unclear, but the lack of a correlation with Bcl-2 expression suggests that it differs from the one initially proposed. [Display omitted] DisclosuresEfremov:Gilead: Honoraria.

Single Agent and Combinatorial Efficacy of First-in-Class Small Molecule ONC201 in Acute Leukemia and Multiple Myeloma

ONC201 is the founding member of the imipridone class of anti-cancer small molecules that possess a unique core chemical structure. ONC201 is currently being evaluated in several Phase I/II clinical trials for advanced cancers. In the current study, we evaluated the single agent and combinatorial efficacy of ONC201 in preclinical models of acute leukemia and multiple myeloma (MM).In acute leukemia, we evaluated ONC201 anti-cancer effects in acute myeloid leukemia (AML) (Kasumi-1, HL60) and acute lymphoblastic leukemia (ALL) (Reh, Jurkat and MOLT-4) cell lines. We observed a time- and dose-dependent decrease in cell viability for every cell line in the panel (EC50 1-5 µM). Vincristine-resistant cells HL60/VCR were also sensitive to single agent ONC201 with EC50 values on par with corresponding vincristine-sensitive parental cells. Dose- and time-dependent induction of apoptosis was noted in Western blot analysis of caspase-3 cleavage in AML cell lines treated with 2.5 µM or 5 µM of ONC201 for 48 hr. Western Blot analysis further demonstrated inhibition of Akt and Foxo3a phosphorylation in Kasumi-1 cells, in line with the previously reported late-stage signaling effects of ONC201 in solid tumor cells (Allen et al, 2013). Sub-G1 analysis indicated that ONC201 induces apoptosis in ALL cells and a pan-caspase inhibitor reduced ONC201-mediated apoptosis. Western blot analysis revealed ONC201-mediated apoptosis involves PARP cleavage and caspase-9 activation in ALL cells. Anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xl were downregulated while the pro-apoptotic Bcl-2 family member Bim is upregulated in response to ONC201 treatment in ALL cells. ONC201 also downregulates the inhibitor of apoptosis (IAP) family proteins cIAP1 and cIAP2 in ALL cells. We observed inhibition of Akt phosphorylation upon ONC201 treatment of ALL cells. Fresh AML patient cells were also found to be sensitive to ONC201 in cell viability and caspase 3/7 activity assays at 5µM.We observed that independent clones of cancer cells with acquired resistance to ONC201 were more sensitive to cytarabine compared to parental ONC201-sensitive cancer cells. In addition, ONC201 demonstrated synergistic reduction in cell viability in combination with cytarabine in AML cell lines. Determination of combination indices (CI) revealed synergy at several concentrations (CI 0.336-0.75 in CMK cells). Also, ONC201 combined additively with midostaurin in CMK cells and vincristine in HL60/VCR cells. Thus, ONC201 is a promising combinatorial partner for AML therapies based on these preclinical sensitization results.In accordance with ONC201-mediated activation of the integrated stress response that B cells are highly sensitive to (Kline et al and Ishizawa et al, 2016), MM was identified as one of the most ONC201-sensitive tumor types in the Genomics of Drug Sensitivity in Cancer collection of cell lines. Three human MM cell lines were used for validation (KMS18, MM.1S and RPMI-8226), which revealed a time- and dose-dependent decrease in cell viability (EC50 1-2.5 µM). Bortezomib-resistant cells MM.1S 33X were sensitive to ONC201 as a single agent with EC50 values comparable to bortezomib-sensitive parental cells. We observed an average of 10-fold induction of ONC201-mediated apoptosis using Sub-G1 analyses in MM cells at 5 µM, 48 hrs post-treatment. Rescue of ONC201-mediated apoptosis was demonstrated using the pan-caspase inhibitor (Z-VAD-FMK). In addition, Western blot analysis in MM cells indicated a dose-dependent decrease in the anti-apoptotic protein XIAP which is a key mediator of apoptosis inhibition and is reported to be highly up-regulated in MM cells. Furthermore, ONC201 demonstrated synergistic reduction in cell viability at various concentrations in combination with either ixazomib or dexamethasone, which are used in the clinical treatment of MM, in RPMI8226 cells (CI 0.228-0.75). Also, ONC201 combined additively with bortezomib in RPMI8226 and MM.1S 33X cells.In summary, these preclinical studies support the ongoing ONC201 single agent trials in acute leukemias and MM. Our findings suggest that ONC201 may be an important therapeutic option for patients with hematological malignancies who have developed resistance to approved therapies. Additionally, our results point to specific standard-of-care therapies that may be combined with ONC201 to exert durable responses without adding to the burden of toxicity. DisclosuresPrabhu:Oncoceutics: Employment. Tarapore:Oncoceutics: Employment, Equity Ownership. Oster:Oncoceutics: Employment, Equity Ownership. Allen:Oncoceutics: Employment, Equity Ownership. El-Deiry:Oncoceutics: Equity Ownership.

PI3Kδ Inhibition Elicits Anti-Leukemic Effects through Bim-Dependent Apoptosis

Introduction: Phosphatidylinositol-3-kinase δ (PI3Kδ)-signaling provides key maintenance, proliferation, and survival cues during both normal and malignant B-lymphocyte development. Consequently, isoform-selective PI3Kδ inhibitors (PI3Kδi) have generated huge interest as a potential treatment for lymphoid malignancies. In particular, PI3Kδi demonstrate impressive clinical efficacy in combination with anti-CD20 monoclonal antibodies (mAbs) for relapsed chronic lymphocytic leukemia (CLL). However, these combinations function primarily to delay disease progression, but are not curative. With the ever-rising number of new targeted therapeutics, the challenge is to identify combinations that will ultimately deliver curative regimes. In order to guide these selections, a detailed mechanistic understanding is required. To date, only limited data are available regarding the exact in vivo therapeutic mechanism of PI3Kδi. Currently, studies identify immunomodulation, inhibition of BCR-, chemokine/cytokine-signaling, and induction of apoptosis as putative therapeutic mechanisms. Here we characterize the molecular mechanisms responsible for PI3Kδi-induced cytotoxicity and determine the relative contribution toward in vivo therapeutic responses utilizing the Eµ-TCL1-Tg mouse model of CLL alongside human CLL samples.Methods: The molecular mechanisms of PI3Kδi alone or in combination with anti-CD20 mAbs were assessed using the Eµ-TCL1-Tg mouse model, an in vivo model system of CLL. To inhibit PI3Kδ, the δ isoform-selective inhibitor GS-9820 was chosen, as it is highly structurally related to idelalisib, and critically demonstrates improved pharmacokinetic properties in the mouse in comparison to idelalisib. In vitro GS-9820 IC50 are as follows: PI3Kδ 27 nM; PI3Kα 83,424 nM; PI3Kβ 14,899 nM; and PI3Kγ 15,606 nM. Assays to measure its effects on BCR-mediated kinase activation, chemokine signaling/chemotaxis, and cytokine- and cell-mediated support were performed. GS-9820 was administered in vivo at 10 mg/kg per os BID (formulated in 0.5% methylcellulose, 0.05% tween-80), once leukemias were detected, and maintained throughout the treatment period (GS-9820 Cmax 3114 nM, Ctrough 48.6 nM).Results: GS-9820 induced substantive in vitro cell death and disruptedBCR-mediated kinase activation, chemokine signaling/chemotaxis, and inhibited both cytokine- and cell-mediated support in murine (Eµ-TCL1) and human CLL cells. In vivo administration of GS-9820 imparted significant therapeutic responses in Eµ-TCL1-bearing animals, reducing leukemic burden by 75% and splenic tumor deposits by 66% 4 weeks posttreatment. GS-9820 appeared well tolerated in recipient animals with no obvious toxicity apparent (e.g. weight loss or behavioral symptoms). When in combination with anti-CD20 mAbs, GS-9820 extended leukemia depletion by several weeks. GS-9820 enhanced overall survival by 66% in comparison with vehicle control-recipient animals and enhanced the survival benefit of anti-CD20 mAb therapy. These therapeutic responses were associated with a 2-fold increase in expression of the pro-apoptotic BH3-only Bcl-2 family member Bim and a 3-fold increase in the extent of Bim/Bcl-2 interaction. Accordingly, Bim-/- Eµ-TCL1-Tg leukaemias exhibited profound resistance to PI3Kδi-induced cytotoxicity, were refractory to PI3Kδi in vivo, and failed to display combination efficacy with anti-CD20 mAbs. These findings informed the rational design of a GS-9820 + ABT199 (Venetoclax) complementary drug combination strategy. Combinations of GS-9820 and ABT199 were well tolerated with an absence of weight loss or altered behavioral symptoms. The GS-9820 + ABT199 combination effectively halted leukemia progression in vivo with increased efficacy compared to monotherapy regimes, resulting in a 90% reduction in leukemic burden at the end of the treatment period.Conclusions: Bim-dependent apoptosis represents the key in vivo effector mechanism for PI3Kδi in the Eµ-TCL1-Tg mouse model, both alone and in combination with anti-CD20 mAbs. As such, combinations of PI3Kδ and Bcl-2 inhibitors may represent an efficacious drug combination strategy. DisclosuresTannheimer:Gilead Sciences: Employment. Packham:Karus Therapeutics: Other: Share Holder & Founder; Aquinox Pharmaceuticals: Research Funding. Cragg:Baxalta: Consultancy; Roche: Consultancy, Research Funding; Bioinvent International: Consultancy, Research Funding; Gilead Sciences: Research Funding; GSK: Research Funding.

Histone deacetylase inhibitors interrupt HSP90•RASGRP1 and HSP90•CRAF interactions to upregulate BIM and circumvent drug resistance in lymphoma cells.

Histone deacetylase (HDAC) inhibitors, which are approved for the treatment of cutaneous T cell lymphoma and multiple myeloma, are undergoing evaluation in other lymphoid neoplasms. How they kill susceptible cells is incompletely understood. Here we show that trichostatin A, romidepsin, and panobinostat induce apoptosis across a panel of malignant B cell lines, including lines that are intrinsically resistant to bortezomib, etoposide, cytarabine, and BH3 mimetics. Further analysis traces the pro-apoptotic effects of HDAC inhibitors to increased acetylation of the chaperone heat shock protein 90 (HSP90), causing release and degradation of the HSP90 client proteins RASGRP1 and CRAF, which in turn leads to downregulation of mitogen activated protein kinase pathway signaling and upregulation of the pro-apoptotic BCL2 family member BIM in vitro and in vivo. Importantly, these pro-apoptotic effects are mimicked by RASGRP1 siRNA or HSP90 inhibition and reversed by overexpression of constitutively active MEK1 or siRNA-mediated downregulation of BIM. Collectively, these observations not only identify a new HSP90 client protein, RASGRP1, but also delineate a complete signaling pathway from HSP90 acetylation through RASGRP1 and CRAF degradation to BIM upregulation that contributes to selective cytotoxicity of HDAC inhibitors in lymphoid malignancies.

Myt3 suppression sensitizes islet cells to high glucose-induced cell death via Bim induction.

Diabetes is a chronic disease that results from the body's inability to properly control circulating blood glucose levels. The loss of glucose homoeostasis can arise from a loss of β-cell mass because of immune-cell-mediated attack, as in type 1 diabetes, and/or from dysfunction of individual β-cells (in conjunction with target organ insulin resistance), as in type 2 diabetes. A better understanding of the transcriptional pathways regulating islet-cell survival is of great importance for the development of therapeutic strategies that target β-cells for diabetes. To this end, we previously identified the transcription factor Myt3 as a pro-survival factor in islets following acute suppression of Myt3 in vitro. To determine the effects of Myt3 suppression on islet-cell survival in vivo, we used an adenovirus to express an shRNA targeting Myt3 in syngeneic optimal and marginal mass islet transplants, and demonstrate that suppression of Myt3 impairs the function of marginal mass grafts. Analysis of grafts 5 weeks post-transplant revealed that grafts transduced with the shMyt3 adenovirus contained ~20% the number of transduced cells as grafts transduced with a control adenovirus. In fact, increased apoptosis and significant cell loss in the shMyt3-transduced grafts was evident after only 5 days, suggesting that Myt3 suppression sensitizes islet cells to stresses present in the early post-transplant period. Specifically, we find that Myt3 suppression sensitizes islet cells to high glucose-induced cell death via upregulation of the pro-apoptotic Bcl2 family member Bim. Taken together these data suggest that Myt3 may be an important link between glucotoxic and immune signalling pathways.

The natural compound forskolin synergizes with dexamethasone to induce cell death in myeloma cells via BIM.

We have previously demonstrated that activation of the cyclic adenosine monophosphate (cAMP) pathway kills multiple myeloma (MM) cells both in vitro and in vivo. In the present study we have investigated the potential of enhancing the killing of MM cell lines and primary MM cells by combining the cAMP-elevating compound forskolin with the commonly used MM therapeutic drugs melphalan, cyclophosphamide, doxorubicin, bortezomib and dexamethasone. We observed that forskolin potentiated the killing induced by all the tested agents as compared to treatment with the single agents alone. In particular, forskolin had a synergistic effect on the dexamethasone-responsive cell lines H929 and OM-2. By knocking down the proapoptotic BCL-2 family member BIM, we proved this protein to be involved in the synergistic induction of apoptosis by dexamethasone and forskolin. The ability of forskolin to maintain the killing of MM cells even at lower concentrations of the conventional agents suggests that forskolin may be used to diminish treatment-associated side effects. Our findings support a potential role of forskolin in combination with current conventional agents in the treatment of MM.

Abstract B09: Targeting the anti-apoptotic protein Mcl-1 with the mTOR inhibitor RAD001 sensitizes luminal breast cancers to the Bcl-2/Bcl-xL inhibitor ABT-263

Abstract In an effort to enhance therapeutic tumor cell killing in luminal breast cancers, we investigated the inhibition of the anti-apoptotic Bcl-2 family proteins Bcl-2, Bcl-xL and Mcl-1. Anti-apoptotic Bcl-2 proteins are inhibitors of the intrinsic apoptotic pathway that sequester and inhibit pro-apoptotic Bcl-2 family members. Sustained overexpression of anti-apoptotic Bcl-2 family members frequently occurs in cancers and supports tumor initiation, tumor progression, therapeutic resistance and poor patient survival. While only 2/324 and 1/324 luminal breast cancers curated by The Cancer Genome Atlas (TCGA) had gene amplification of BCL2 and BCL2L1 (encoding Bcl-xL) respectively, 21/324 (&amp;lt;8%) displayed MCL1 amplification. Wap-Myc transgenic mouse mammary tumors, which are highly enriched for the luminal A transcription signature, displayed profoundly increased Mcl-1 levels as compared to normal mammary tissue, suggesting a role for Mcl-1 in luminal breast cancer biology. Using a panel of four MCL1-amplified breast cancer cell lines, we found that two were marginally sensitive to treatment with ABT-263, a BH3-mimetic that targets Bcl-2 and Bcl-xL. Conversely, ectopic overexpression of Mcl-1 decreased sensitivity to ABT-263. ABT-263 induced minimal cell killing in MCF7 xenografts and had no effect on tumor growth. Similarly, ABT-263 did not affect tumor growth in luminal-like Wap-Myc mouse mammary tumors. Thus, luminal breast cancer cells expressing increased Mcl-1 are poorly responsive to Bcl-2/Bcl-xL inhibition. Upregulation of Mcl-1 expression and activity, an established ABT-263-resistance mechanism in leukemias and lymphomas, was observed in MCL1-amplified luminal breast cancer cell lines treated with ABT-263 in culture and in vivo. ABT-263 also increased the anti-apoptotic activity of Mcl-1 towards the pro-apoptotic Bcl-2 family member BIM, suggesting that Mcl-1 upregulation compensates for inhibition of Bcl-2 and Bcl-xL by ABT-263. There are currently no Mcl-1 specific inhibitors. However, Mcl-1 undergoes cap-dependent translation, and thus inhibition of cap-dependent translation has been proposed as a method to inhibit the protein expression of Mcl-1. Importantly, the mTOR pathway is required for efficient cap-dependent translation in many cancer cells. We found that the rapalogue RAD001/everolimus, an mTOR inhibitor, decreased Mcl-1 protein levels in MCL1-amplified luminal breast cancer cell lines. Furthermore, RAD001 blocked Mcl-1 protein upregulation in response to ABT-263, producing increased cell killing and decreased cell growth in all MCL1-amplified luminal breast cancer cell lines. Treatment of luminal Wap-Myc mammary tumors with RAD001 decreased tumor volume by 40%, while treatment with ABT-263 had no effect on tumor volume. These data suggest that Mcl-1 targeting may be superior to Bcl-2/Bcl-xL targeting in luminal breast cancers, particularly in luminal breast cancers with MCL1-amplification. Furthermore, we have identified the mTOR pathway as a potential therapeutic target for blocking Mcl-1 expression in luminal breast cancers. Citation Format: Michelle M. Williams, Linus Lee, Meghan M. Morrison, Andrew J. Williams, Violeta Sanchez, Donna Hicks, Rebecca Cook. Targeting the anti-apoptotic protein Mcl-1 with the mTOR inhibitor RAD001 sensitizes luminal breast cancers to the Bcl-2/Bcl-xL inhibitor ABT-263. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B09.

Low expression of pro-apoptotic Bcl-2 family proteins sets the apoptotic threshold in Waldenström macroglobulinemia.

Waldenström Macroglobulinemia (WM) is a proliferative disorder of IgM secreting, lymphoplasmacytoid cells that inhabit the lymph nodes and bone marrow. The disease carries a high prevalence of activating mutations in MyD88 (91%) and CXCR4 (28%). Because signaling through these pathways leads to Bcl-xL induction, we examined Bcl-2 family expression in WM patients and cell lines. Unlike other B-lymphocyte-derived malignancies, which become dependent on expression of anti-apoptotic proteins to counter expression of pro-apoptotic proteins, WM samples expressed both pro- and anti-apoptotic Bcl-2 proteins at low levels similar to their normal B-cell and plasma cell counterparts. Three WM cell lines expressed pro-apoptotic Bcl-2 family members Bim or Bax and Bak at low levels which determined their sensitivity to inducers of intrinsic apoptosis. In two cell lines, miR-155 upregulation, which is common in WM, was responsible for inhibition of FOXO3a and Bim expression. Both antagonizing miR-155 to induce Bim and proteasome inhibition increased the sensitivity to ABT-737 in these lines indicating a lowering of the apoptotic threshold. In this manner, treatments that increase pro-apoptotic protein expression increase the efficacy of agents treated in combination in addition to direct killing.

Unveiling a common mechanism of apoptosis in β-cells and neurons in Friedreich's ataxia.

Friedreich's ataxia (FRDA) is a neurodegenerative disorder associated with cardiomyopathy and diabetes. Effective therapies for FRDA are an urgent unmet need; there are currently no options to prevent or treat this orphan disease. FRDA is caused by reduced expression of the mitochondrial protein frataxin. We have previously demonstrated that pancreatic β-cell dysfunction and death cause diabetes in FRDA. This is secondary to mitochondrial dysfunction and apoptosis but the underlying molecular mechanisms are not known. Here we show that β-cell demise in frataxin deficiency is the consequence of oxidative stress-mediated activation of the intrinsic pathway of apoptosis. The pro-apoptotic Bcl-2 family members Bad, DP5 and Bim are the key mediators of frataxin deficiency-induced β-cell death. Importantly, the intrinsic pathway of apoptosis is also activated in FRDA patients' induced pluripotent stem cell-derived neurons. Interestingly, cAMP induction normalizes mitochondrial oxidative status and fully prevents activation of the intrinsic pathway of apoptosis in frataxin-deficient β-cells and neurons. This preclinical study suggests that incretin analogs hold potential to prevent/delay both diabetes and neurodegeneration in FRDA.