Members Of Bcl-2 Protein Family Research Articles

Direct-Acting Small Molecule Activators of PP2A (SMAPs) in Myeloid Malignancies: Understanding Mechanisms of Cytotoxicity to Inform Rational Combinatorial Therapeutic Strategies

The protein phosphatase 2A (PP2A) functions as a tumor suppressor by counteracting oncogenic kinase signaling. Suppression of PP2A activity represents a critical process in the development and maintenance of many neoplasms, including myeloid malignancies; PP2A inhibition de-represses numerous proliferative and survival signaling pathways. However, complete loss of PP2A is lethal to cells. As such, indirect reactivation of PP2A has emerged as a promising therapeutic strategy for myeloid leukemias. Yet the broad applicability of such a strategy remains uncertain, as it relies on targeting endogenous PP2A inhibitors. In the present work we investigated the effects of direct acting small molecule activators of PP2A (SMAPs) (Sandokar et al. JCI 2017) on myeloid leukemia cells and uncovered a novel link between PP2A reactivation and NOXA-mediated apoptosis. This mechanism explains our observation of cooperativity between SMAPs and the BH3 mimetic ABT-199 (venetoclax). Additionally, we found that SMAPs elicit a protective stress response that can be overcome by inhibition ofprotein kinase R (PKR)-like endoplasmic reticulum kinase (PERK).Given the known role of PP2A inactivation in both acute and chronic myeloid leukemia (AML and CML), we first tested the activity of SMAPs against cell lines derived from both diseases. Across all cell lines tested (CML- K562, Meg-01; AML- MOLM-14, HL-60, OCI-AML3, THP-1) SMAPs caused a dose-dependent decrease in cell viability. It is known that reactivation of PP2A induces apoptosis in myeloid leukemia cells, though this area has yet to be thoroughly investigated. Therefore, we first sought to clarify the mode of cell death in response to PP2A reactivation. Both by biochemical and flow cytometric analyses, SMAPs showed activation of the intrinsic apoptotic cascade. Furthermore, SMAP-mediated cell death was blocked by co-treatment with the caspase inhibitor z-VAD-FMK (50 μM). We previously demonstrated the target specificity of SMAPs for PP2A with methods including expression of SV40 small T antigen and PP2A alpha subunit drug-binding mutants (Sandokar et al. JCI 2017). Here, we confirmed that pre-treatment with the PP2A inhibitor okadaic acid at a dose that retains selectivity for PP2A (10 nM) attenuated SMAP-induced cell death and cleavage of PARP and caspase-3.Next, we investigated the effectors of SMAP-mediated apoptosis. To this end, we performed Western blots to detect changes in abundance and/or phosphorylation of a panel of Bcl-2 family member proteins. Interestingly, a strong upregulation of the pro-apoptotic protein NOXA occurred consistently across AML and CML cell lines. Further RT-PCR studies showed this increase to occur at the transcript level. Knockdown studies confirmed the importance of NOXA for SMAP-mediated apoptosis. These data represent the first reported link between PP2A and NOXA regulation. Since SMAPs target the NOXA-MCL1 axis of the Bcl-2 family, we next tested their efficacy in combination with the Bcl-2-selective BH3 mimetic ABT-199. We found co-treatment with SMAPs and ABT-199 to exhibit enhanced cell death compared to either agent alone.Concurrent with NOXA elevation, SMAP treatment modulated protein expression and phosphorylation levels for several known pathways downstream of PP2A. Interestingly, we additionally observed increased phosphorylation of eukaryotic translation initiation factor 2 subunit alpha (eIF2α) in SMAP-treated cells which correlated with upregulation of mRNA and protein for the downstream transcription factor ATF4. Blockade of eIF2α phosphorylation with PERK inhibitor GSK2656157 (350 nM) prevented ATF4 elevation and enhanced SMAP-induced apoptosis. This result supports the idea that the integrated stress response (ISR) via PERK-eIF2α-ATF4 acts to counter apoptosis in the context of PP2A reactivation in myeloid leukemia cells. Thus, the efficacy of SMAPs and other PP2A-activating therapeutics may be enhanced by combination with both BH3 mimetics and inhibitors of the ISR. DisclosuresNarla:Mount Sinai School of Medicine: Patents & Royalties: patents on the small molecule PP2A activators (GN).

MAPK-ERK is a central pathway in T-cell acute lymphoblastic leukemia that drives steroid resistance.

(Patho-)physiological activation of the IL7-receptor (IL7R) signaling contributes to steroid resistance in pediatric T-cell acute lymphoblastic leukemia (T-ALL). Here, we show that activating IL7R pathway mutations and physiological IL7R signaling activate MAPK-ERK signaling, which provokes steroid resistance by phosphorylation of BIM. By mass spectrometry, we demonstrate that phosphorylated BIM is impaired in binding to BCL2, BCLXL and MCL1, shifting the apoptotic balance toward survival. Treatment with MEK inhibitors abolishes this inactivating phosphorylation of BIM and restores its interaction with anti-apoptotic BCL2-protein family members. Importantly, the MEK inhibitor selumetinib synergizes with steroids in both IL7-dependent and IL7-independent steroid resistant pediatric T-ALL PDX samples. Despite the anti-MAPK-ERK activity of ruxolitinib in IL7-induced signaling and JAK1 mutant cells, ruxolitinib only synergizes with steroid treatment in IL7-dependent steroid resistant PDX samples but not in IL7-independent steroid resistant PDX samples. Our study highlights the central role for MAPK-ERK signaling in steroid resistance in T-ALL patients, and demonstrates the broader application of MEK inhibitors over ruxolitinib to resensitize steroid-resistant T-ALL cells. These findings strongly support the enrollment of T-ALL patients in the current phase I/II SeluDex trial (NCT03705507) and contributes to the optimization and stratification of newly designed T-ALL treatment regimens.

BRD4 Inhibitors Enhance the Anti-Tumor Activity of Targeted Therapy in Chronic Lymphocytic Leukemia

Background: The development of drugs targeting BTK and BCL2 has dramatically improved the therapeutic landscape in chronic lymphocytic leukemia (CLL). However, resistance to these agents has been reported due to non-recurrent changes in oncogenic pathways and gene expression signatures. The bromodomain and extra terminal (BET) family proteins (BRD2, BRD3, BRD4 and BRDT) are epigenetic reader proteins that recognize acetylated lysine residues in histones. They play a critical role in mediating gene transcription and have been considered as highly promising targets in several diseases including cancer. Of these, the BRD4 protein is highly enriched in super enhancers and regulates transcription of relevant oncogenes such as MYC, CDK genes, cyclin-D1, BCL2 and MCL-1. BRD4 inhibitors (BRD4i) block the transcription of these key oncogenes through the displacement of BRDs and other epigenetic modifiers from chromatin. In this study, we investigated the preclinical activity of two BRD4i, AZD5153 and PLX51107, and their cooperative role with the BCL2 inhibitor venetoclax and BTK inhibitor ibrutinib in CLL. Methods: Primary tumor cells were isolated from 36 CLL patients. Peripheral blood mononuclear cells were isolated via histopaque-1077 (Sigma-Aldrich, St Louis MO) from peripheral blood apheresis samples from consenting adults under RPCCC protocol CIC-01-16. B-cells were then isolated from enriched lymphocytes by MACS separation using a human B-cell Isolation Kit II. CLL cells were seeded at 3 x106/mL in a 384 well tissue culture plates and exposed to different doses of AZD5153 (1-20uM) or PLX51107 (1-20uM) as a single agent or in combination with venetoclax (1-100nM) or ibrutinib (50-1000nM) for 48 and 72hrs (Selleck Chemicals, Houston TX). Cell viability was assessed using Cell Titer-glo (Promega, Madison WI) with all data being normalized to untreated controls. Synergy was calculated using Calcusyn (Cambridge, UK) software to determine Coefficient of Synergy (CI) values. In addition, CLL cells were plated in 6 well plates and exposed to AZD5153 or PLX51107 alone or in combination with either venetoclax or ibrutinib for 24 hrs. Induction of apoptosis and changes in B-cell receptor signaling, BCL2 and NF-kB pathway related proteins were evaluated by flow cytometry or Western blotting respectively. Results: The study included 33 treatment naïve and 3 relapsed/refractory CLL patients. Deletion 11q was detected in 6 patients and del 17p was seen in 1 patient. Both AZD5153 and PLX51107 displayed time and dose dependent induction of apoptosis of CLL cells. Activity was observed in both treatment naïve and relapsed/refractory samples. Moreover, activity was observed in del 17p or ATM deletion samples. Synergistic activity was observed between BRD4is and venetoclax. PLX51107 exhibited synergy with venetoclax in 83% of the samples tested. Similarly, AZD5153 exhibited synergistic effects when combined with venetoclax in 61% of the samples. To a lesser degree, BRD4is exhibited synergistic activity when combined with ibrutinib, (44% with PLX51107 and 39% with AZD5153). Induction of apoptosis was observed in CLL cells exposed to either BRD4i. Morevoer, an increase in the percentage of apoptosis was observed when BRD4i was combined with venetoclax. Western blotting showed a accumulation of p21, reduced levels of C-MYC, as well as modulation of BCL-2 family member proteins such as BCL2, MCL-1 following BRD4i ex vivo exposure. Conclusions: Our data indicate that BRD4 inhibition results in apoptosis of primary tumor cells isolated from patients with various subtypes of CLL. PLX51107 and AZD5153 exhibit synergistic effects when combined with venetoclax and to a lesser degree with ibrutinib. Modulation of BCL-2 family members following BRD4 inibition could play a role in the synergistic activity observed in our studies. A better understanding of the key signaling pathways involved in proliferation and survival of CLL cells impacted by BRD4i in combination with venetoclax and ibrutinib would provide a proof-of-concept rationale for studies validating BRD4i as epigenetic approach to target BCR signaling in CLL. Disclosures Hernandez-Ilizaliturri: Karyopharm: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Gilead: Consultancy; Pharmacyclics: Consultancy; Seattle Genetics: Consultancy; Astra Zeneca: Consultancy; Epyzome: Consultancy.

Mcl-1 and Bok transmembrane domains: Unexpected players in the modulation of apoptosis

The Bcl-2 protein family comprises both pro- and antiapoptotic members that control the permeabilization of the mitochondrial outer membrane, a crucial step in the modulation of apoptosis. Recent research has demonstrated that the carboxyl-terminal transmembrane domain (TMD) of some Bcl-2 protein family members can modulate apoptosis; however, the transmembrane interactome of the antiapoptotic protein Mcl-1 remains largely unexplored. Here, we demonstrate that the Mcl-1 TMD forms homooligomers in the mitochondrial membrane, competes with full-length Mcl-1 protein with regards to its antiapoptotic function, and induces cell death in a Bok-dependent manner. While the Bok TMD oligomers locate preferentially to the endoplasmic reticulum (ER), heterooligomerization between the TMDs of Mcl-1 and Bok predominantly takes place at the mitochondrial membrane. Strikingly, the coexpression of Mcl-1 and Bok TMDs produces an increase in ER mitochondrial-associated membranes, suggesting an active role of Mcl-1 in the induced mitochondrial targeting of Bok. Finally, the introduction of Mcl-1 TMD somatic mutations detected in cancer patients alters the TMD interaction pattern to provide the Mcl-1 protein with enhanced antiapoptotic activity, thereby highlighting the clinical relevance of Mcl-1 TMD interactions.

BNIP3 as a Regulator of Cisplatin-Induced Apoptosis.

BNIP3 is a member of Bcl-2 protein family involved in regulation of various forms of cell death. However, its role in these processes remains unclear and varies depending on the type of cancer cells and environmental factors (pH, O2 level, etc.). Here, the role of BNIP3 in apoptosis regulation in lung adenocarcinoma cells was investigated. The suppressed expression of BNIP3 caused inhibition of oxygen consumption and stimulated production of the mitochondrial reactive oxygen species, suggesting the role of BNIP3 in induction of mitochondrial dysfunction and its potential involvement in regulation of cell death. Indeed, cytochrome c release in the cells with BNIP3 knockout and knockdown was higher than in the wild-type (WT) upon apoptosis stimulation by cisplatin. Moreover, suppression of BNIP3 expression led to the increase in the caspase-3 activity and, as a consequence, accumulation of the apoptotic marker - p89 fragment of poly(ADP-ribose)-polymerase (PARP) - as compared to WT cells. Analysis of the SubG1 population by flow cytometry confirmed the elevated level of apoptosis in the BNIP3 knockout cells. Pretreatment with the antioxidant Trolox did not affect cell death, indicating that it was independent on reactive oxygen species. These data show that BNIP3 is involved in maintaining normal functioning of mitochondria and, as a result, can regulate the mitochondrial pathway of cell death.

Abstract CT133: Targeting BCL-2 and MCL-1 overcomes treatment resistance in relapsed and refractory non-Hodgkin lymphoma: Pre-clinical rationale and results from an open-label phase 1b study

Abstract Purpose: Treatment of patients with relapsed or refractory lymphoid neoplasms represents a significant clinical challenge, largely due to the genetic heterogeneity of these malignancies. Our study goal was to design a rational treatment strategy that simultaneously targets multiple disease drivers to provide safe, efficacious, and durable responses in patients with relapsed or refractory non-Hodgkin lymphoma (NHL). Experimental procedures and data summary: Venetoclax is a selective BCL-2 inhibitor that induces apoptosis and is efficacious in chronic lymphocytic leukemia; however, single-agent efficacy in other lymphoid neoplasms is limited. Using pharmacologic and genetic studies, we identified the pro-survival BCL-2 protein family member MCL-1 as a venetoclax resistance factor in NHL cell lines. We found that the monomethyl auristatin E (MMAE) payload of the antibody-drug conjugate polatuzumab vedotin promotes MCL-1 degradation via the ubiquitin/proteasome system, thus providing a strong rationale for combination with venetoclax. Mechanistically, polatuzumab vedotin combined with venetoclax promoted apoptotic NHL cell death. In NHL animal models, venetoclax, polatuzumab vedotin, and the anti-CD20 antibody obinutuzumab, that activates targeted antibody- and complement-dependent cell cytotoxicity, produced durable tumor regressions. In a phase 1b clinical trial, 33 patients with relapsed or refractory follicular lymphoma were evaluable for response over 6 dose levels with 19 complete responses and 6 partial responses (overall response rate 76%). All patients (8 of 8) treated with the recommended phase 2 regimen (venetoclax 800mg + polatuzumab vedotin 1.8mg/kg + obinutuzumab 1000mg) achieved complete responses at end of induction. The median duration of follow-up for all dose levels was 17.7 months. This triplet combination was well-tolerated by most patients and had an expected and acceptable safety profile. Conclusions: Our studies implicate MCL-1 as a key venetoclax resistance factor in NHL that can be overcome by polatuzumab vedotin, which promotes MCL-1 degradation. Pre-clinical NHL animal models and early clinical data confirmed that the combination of venetoclax, polatuzumab vedotin, and obinutuzumab is safe and promotes durable responses. Because MMAE and other anti-tubulin agents are likely not the only therapeutics that antagonize MCL-1, our study provides scientific rationale to pursue the broader strategy of identifying other therapeutics that similarly neutralize MCL-1 function. Such agents could be used in combination with venetoclax in other malignancies where MCL-1 is a venetoclax resistance factor. Our mechanism-based treatment regimen that directly targets oncogenic drivers in NHL patients may serve as a framework for treating other malignancies with complex etiologies. Citation Format: Dhara N. Amin, Rajat Bannerji, Raghuveer Singh Mali, Jason Oeh, Eva Lin, Anuradha Zindal, MaryAnn Go, Shang-Fan Yu, Maxwell Krem, Chris Arthur, Uwe Hahn, Anna M. Johnston, Vinit G. Karur, Nadia Khan, Paula Marlton, Tycel Phillips, Giuseppe Gritti, John F. Seymour, Monica Tani, Sam Yuen Yuen, Scott Martin, Matthew T. Chang, Christopher M. Rose, Victoria C. Pham, Elizabeth A. Lasater, Andrew G. Polson, YiMeng Chang, Jamie Hirata, Lisa Musick, Deepak Sampath, Christopher R. Flowers, Ingrid E. Wertz. Targeting BCL-2 and MCL-1 overcomes treatment resistance in relapsed and refractory non-Hodgkin lymphoma: Pre-clinical rationale and results from an open-label phase 1b study [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 CT133.

Cell Death Pathways in Lymphoid Malignancies.

This review highlights the importance of the Bcl-2 family members in lymphoma cell survival and discusses the approaches to modulate their function, directly or indirectly, to advance lymphoma therapeutics. The balance of cell death versus survival is ultimately leveraged at the mitochondria. Mitochondrial outer membrane permeabilization (MOMP) is the critical event that governs the release of pro-apoptotic molecules from the intermembrane mitochondrial space. MOMP is achieved through the coordinated actions of pro- and anti-apoptotic Bcl-2 family member proteins. Recognition of functional alterations among the Bcl-2 family member proteins led to identification of tractable targets to combat hematologic malignancies. A new class of drugs, termed BH3 mimetics, was introduced in the clinic. Venetoclax, a Bcl-2 inhibitor, received regulatory approvals in therapy of chronic lymphocytic leukemia and acute myeloid leukemia. Alternative pro-survival Bcl-2 family proteins, in particular Mcl-1, have been successfully targeted in preclinical studies using novel-specific BH3 mimetics. Finally, anti-apoptotic Bcl-2 family members may be targeted indirectly, via interference with the pro-survival signaling pathways, e.g., phosphoinotiside-3 kinase, B-cell receptor signaling, and NF-κB.

MAPK-ERK Pathway Activation: The Achilles' Heel of IL-7R‒Induced Steroid Resistance

INTRODUCTION. Mutations in the IL-7R signaling pathway (e.g.IL7Ra, JAK1/3, PTPN11, NF1, STAT5B, N/KRAS or AKT) are associated with steroid resistance and inferior relapse-free survival. These mutant molecules drive strong activation of the JAK-STAT, MAPK-ERK and/or PI3K-AKT signaling pathways. Also, activation of wild type RAS or AKT-reflecting physiological IL7-induced signaling-drives steroid resistance. These observations extend the findings in preB-ALL where RAS mutations were related to steroid resistance. Impaired transcription of pro-apoptotic BIM-an essential direct transcriptional target gene of the glucocorticoid receptor NR3C1 that mediates steroid-induced apoptosis-is observed in various steroid resistant PDX models. AIM. Given the central role of BIM in steroid-induced apoptosis, we investigated how mutations in IL7R or downstream signaling components would affect the function of BIM and other BCL2 protein family members (BCL2, BCLXL and MCL1) in steroid-exposed T-ALL. RESULTS. We generated doxycycline-inducible SUP-T1 and P12 Ichikawa cell line models that can overexpress specific IL-7R signaling mutants. Expression of mutant JAK1, NRAS or IL7Ra molecules results in robust activation of the MAPK-ERK pathway that confers steroid resistance. As a target of NR3C1, BIM transcription was not impaired in these steroid treated cell lines. Therefore, we investigated post-transcriptional mechanisms of steroid resistance. We observed strong phosphorylation of BIM downstream of MAPK-ERK activation under steroid treated and untreated conditions. Exposure to each of three MEK-inhibitors CI1040, Selumetinib or Trametinib abolished not only phospho-ERK but also phosphorylation of BIM. To study the consequences of BIM phosphorylation in relation to binding and inactivation of anti-apoptotic proteins, we immunoprecipitated BIM. Increased BIM levels following steroid exposure was related to increased binding to BCL2, BCLXL and MCL1 in steroid-sensitive control cells. In contrast, activation of mutant JAK1 and NRAS molecules resulted in phosphorylated BIM and reduced binding of BIM to these anti-apoptotic BCL2 family members. This indicates that preventing ERK-mediated phosphorylation of BIM enhances steroid responsiveness. T-ALL patients with activating IL-7R signaling mutations may therefore benefit from treatment combining steroids and MEK inhibitors. Indeed we observed strong synergy between MEK inhibitors and steroids in primary T-ALL patient samples. CONCLUSIONS AND FUTURE PERSPECTIVES. Treatment with MEK-inhibitors Selumetinib or Trametinib was shown effective in reversing steroid resistance, and therefore strongly synergize with steroid treatment. Our data highlights the importance of the dynamic interplay between BIM and anti-apoptotic BCL2 family members in MAPK-ERK-driven steroid resistance. Therefore, we are exploring whether MEK inhibitors (that prevent phosphorylation and inactivation of BIM) combined with BH3-mimetics (that block anti-apoptotic family members) will further enhance restoration of steroid responsiveness in IL-7R signaling mutant T-ALL patients. As physiological IL7-induced signaling is sufficient to raise cellular steroid resistance, these inhibitors could be applied for all IL-7R signaling-dependent T-ALL patients, also in the absence of IL-7R signaling mutations. Refractory/relapse T-ALL patients are now eligible for the current phase 1/2 MEK-inhibitor‒dexamethasone (SeluDex) trial. We are using mass-spectrometry to determine the exact phospho-sites in BIM that drives steroid resistance that can serve as a valuable biomarker. Disclosures No relevant conflicts of interest to declare.

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 >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 <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 <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 <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.

Concomitant targeting of Hedgehog signaling and MCL-1 synergistically induces cell death in Hedgehog-driven cancer cells.

In the present study, we show that concomitant inhibition of Hedgehog (HH) signaling by the glioma-associated oncogene homolog1 (GLI1)-targeting agent GANT61 and the antiapoptotic BCL-2 protein family member MCL-1 by A-1210477 synergistically induces cell death in HH-driven cancers, i.e. rhabdomyosarcoma (RMS) and medulloblastoma (MB) cells. Combined genetic and pharmacological inhibition emphasized that co-treatment of GANT61 and A-1210477 indeed relies on inhibition of GLI1 (by GANT61) and MCL-1 (by A-1210477). Mechanistic studies revealed that A-1210477 triggers the release of BIM from MCL-1 and its shuttling to BCL-xL and BCL-2. Indeed, BIM proved to be required for GANT61/A-1210477-induced cell death, as genetic silencing of BIM using siRNA significantly rescues cell death upon GANT61/A-1210477 co-treatment. Similarly, genetic silencing of NOXA results in a significant reduction of GANT61/A-1210477-mediated cell death. Also, overexpression of MCL-1 or BCL-2 significantly protects RMS cells from GANT61/A-1210477-triggered cell death. Addition of the pan-caspase inhibitor zVAD.fmk significantly decreases GANT61/A-1210477-stimulated cell demise, indicating apoptotic cell death. In conclusion, GANT61 and A-1210477 synergize to engage mitochondrial apoptosis. These findings provide the rationale for further evaluation of dual inhibition of HH signaling and MCL-1 in HH-driven cancers.

Discovery of β-carboline copper(II) complexes as Mcl-1 inhibitor and in vitro and in vivo activity in cancer models

Mcl-1 is an anti-apoptotic member of Bcl-2 family proteins. The development of inhibitors of Mcl-1 has been challenging. To develop metal-based Mcl-1inhibitors, twenty two copper(II) complexes 25–46 with 9-substituted β-carboline derivatives were reported. Complexes 38 and 39 showed higher cytotoxicity than the corresponding ligands or cisplatin. The most potent complex 39 presented higher selectivity to Mcl-1 than other Bcl-2 family proteins, and killed cancer cells via Bax/Bak mediated apoptosis. Complex 39 showed an excellent safety profile in mouse model, and significantly inhibited the tumor growth in NCI-H460 tumor bearing model, which is more potent than AZD5991 at the same dosage. Complex 39 prolonged the survival time of the tumor bearing mice. Complex 39 is the first metal-based Mcl-1 inhibitor acting as a potential anticancer agent.

Loss of Bcl-G, a Bcl-2 family member, augments the development of inflammation-associated colorectal cancer

Gastrointestinal epithelial cells provide a selective barrier that segregates the host immune system from luminal microorganisms, thereby contributing directly to the regulation of homeostasis. We have shown that from early embryonic development Bcl-G, a Bcl-2 protein family member with unknown function, was highly expressed in gastrointestinal epithelial cells. While Bcl-G was dispensable for normal growth and development in mice, the loss of Bcl-G resulted in accelerated progression of colitis-associated cancer. A label-free quantitative proteomics approach revealed that Bcl-G may contribute to the stability of a mucin network, which when disrupted, is linked to colon tumorigenesis. Consistent with this, we observed a significant reduction in Bcl-G expression in human colorectal tumors. Our study identifies an unappreciated role for Bcl-G in colon cancer.

The ReFRAME library as a comprehensive drug repurposing library to identify mammarenavirus inhibitors

Several mammarenaviruses, chiefly Lassa virus (LASV) in Western Africa and Junín virus (JUNV) in the Argentine Pampas, cause severe disease in humans and pose important public health problems in their endemic regions. Moreover, mounting evidence indicates that the worldwide-distributed mammarenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen of clinical significance. The lack of licensed mammarenavirus vaccines and partial efficacy of current anti-mammarenavirus therapy limited to an off-label use of the nucleoside analog ribavirin underscore an unmet need for novel therapeutics to combat human pathogenic mammarenavirus infections. This task can be facilitated by the implementation of “drug repurposing” strategies to reduce the time and resources required to advance identified antiviral drug candidates into the clinic. We screened a drug repurposing library of 11,968 compounds (Repurposing, Focused Rescue and Accelerated Medchem [ReFRAME]) and identified several potent inhibitors of LCMV multiplication that had also strong anti-viral activity against LASV and JUNV. Our findings indicate that enzymes of the rate-limiting steps of pyrimidine and purine biosynthesis, the pro-viral MCL1 apoptosis regulator, BCL2 family member protein and the mitochondrial electron transport complex III, play critical roles in the completion of the mammarenavirus life cycle, suggesting they represent potential druggable targets to counter human pathogenic mammarenavirus infections.

Abstract 2180: AMG 176 exhibits robust antitumor activity in combination with standard of care agents in models of acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is a heterogeneous malignancy involving the clonal expansion of abnormal myeloid progenitor cells. The anti-apoptotic BCL-2 protein family member MCL1 has been implicated in AML pathogenesis. AMG 176 is a potent and selective MCL1 inhibitor currently being evaluated in an AML Phase I clinical trial. Standard of care (SOC) treatment for AML involves “7+3” induction therapy with cytarabine and an anthracycline (i.e. doxorubicin). In patient’s ineligible for conventional induction therapy, the hypomethylating agent decitabine is a well-tolerated alternative. Here we investigate the activity of AMG 176 and AM-8621, a closely related analog of AMG 176 with similar potency and selectivity, in combination with SOC therapies in preclinical models of AML. We profiled a panel of AML cell lines (MOLM-13, MV-4-11, GDM-1 and EOL-1), testing AM-8621 in combination with cytarabine, doxorubicin and decitabine in 3-day viability assays. Evidence for synergistic activity was detected with each of the combinations across subsets of the cell lines. A time course of AM-8621 treatment revealed a rapid induction of caspase 3/7 activity, with significant increases observed within the first 2-4 hours of treatment while longer duration treatment (>8 hours) was required to activate caspase 3/7 with SOC agents. Combined treatment with AM-8621 and SOC agents exhibited significant improvements in caspase 3/7 activity beyond either single agent alone. To provide mechanistic insight into the ability of SOC agents to sensitize AML cell lines to AM-8621 treatment, signaling experiments were performed to characterize changes in BCL-2 protein family members following treatment with SOC agents. Clear changes in the levels of anti-apoptotic BCL-2 family members (MCL1, BCL-2 and BCL-XL), BH3-only domain containing proteins (BIM, NOXA, PUMA) and the executioner protein BAK were observed, providing insight into potential mechanisms of priming cells to MCL1 inhibition. Combinations of AMG 176 and each of the SOC therapies were also tested in a MOLM-13 orthotopic xenograft model of AML. Mice were treated with combinations of AMG 176 and cytarabine, decitabine or doxorubicin and compared against the single agent therapies. Significant reductions in tumor burden compared to single agent treatments were observed with the AMG 176 + doxorubicin (30% regression) and AMG 176 + decitabine (98% tumor growth inhibition) combinations. These data highlight the promise of combining AMG 176 with SOC therapies in the setting of AML. Citation Format: Sean Caenepeel, Brian Belmontes, Tao Osgood, Elaina Cajulis, Angela Coxon, Jude Canon, Paul E. Hughes. AMG 176 exhibits robust antitumor activity in combination with standard of care agents in models of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2180.

Abstract 2501: Combining BH3-mimetics to target both BCL-2 and MCL1 has potent activity in diffuse large B-cell lymphoma (DLBCL) pre-clinical models

Abstract Background: Upregulation of the anti-apoptotic protein Mcl-1 has been implicated in chemotherapy resistance and poor clinical outcomes in activated B-cell like (ABC)-DLBCL. We hypothesized that singular inhibition of Mcl-1 by AMG-176 and/or dual inhibition of MCL-1 and Bcl-2 by combining AMG-176 with venetoclax could be an effective strategy for inducing cell death in DLBCL pre-clinical models. Materials and Methods: Using a panel of DLBCL cell lines, we conducted pre-clinical targeting of Mcl-1 with AMG-176. Cells were exposed to AMG-176 as a single agent (0-20uM) over 24, 48, 72 hrs and IC50 concentrations were calculated for each cell line. Subsequently, DOHH-2, OCI-LY1, DHL4, DHL6, TMD8, and VAL cells were exposed to AMG-176 (0-20 uM) in combination with Doxorubicin (0-1uM), Cisplatin (0-10uM), Cytarabine (0-10uM), Vincristine (0-10uM), Ixazomib (0-100nM), Carfilzomib (0-12.5nM), and Venetoclax (0-10uM) for 72 hours. Cell viability was determined by Cell Titerglo. Coefficient of synergy was calculated using CalcuSyn. In addition, induction of apoptosis was evaluated by flow cytometry (Annexin V/PI staining) as well as changes in cell cycle (PI staining). Bcl-2 family member protein expression was determined by Western blotting. Primary tumor cells isolated from a patient with a composite lymphoma: angioimmunoblastic T-cell lymphoma (AITL) and EBV-driven DLBCL were exposed ex vivo to AMG-176 +/- venetoclax. By immune-phenotype, the primary tumor cells isolated and expanded were ABC-DLBCL. Results: In vitro, AMG-176 single agent exposure induced cell death in a dose- and time-dependent manner in all DLBCL cell lines tested. AMG-176 IC50 values varied from 0.03 to 11.42 uM for OCI-LY1 and ROS-50 respectively. Western blotting studies confirmed changes in Bcl-2 family members, specifically in Bok, Bcl-XL, Mcl-1 and Bcl-2 following in vitro exposure to AMG-176. The combination of AMG-176 with Doxorubicin, Cisplatin, Cytarabine, Vincristine, Ixazomib, Carfilzomib, or Venetoclax resulted in significant synergistic activity for each combination. The strongest coefficient of synergy was observed when AMG-176 was combined with venetoclax. Similar synergy results were noted in primary tumor cells isolated from the lymphoma patient. Annexin/PI staining with AMG-176 demonstrated caspase dependent apoptosis. No significant changes in cell cycle were observed with AMG-176 exposure. Conclusion: Our data suggests that AMG-176 exhibits strong synergistic activity with various chemotherapy agents and small molecule inhibitors especially venetoclax. In vivo experiments are planned. Combination therapy targeting Mcl-1 and Bcl-2 may provide an alternative therapeutic approach in DLBCL. Citation Format: Cory Mavis, Juan Gu, Matthew Barth, Sumera Khan, Pallawi Torka, Francisco Hernandez-Ilizaliturri. Combining BH3-mimetics to target both BCL-2 and MCL1 has potent activity in diffuse large B-cell lymphoma (DLBCL) pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2501.

The expanding role of venetoclax in chronic lymphocytic leukemia and small lymphocytic lymphoma.

The BCL-2 protein family members inhibit cellular apoptosis, and their overexpression represents a common survival adaption in cancer. Recently, a selective BCL-2 inhibitor ABT-199, venetoclax, has demonstrated remarkable activity in relapsed/refractory chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), both as a single agent and in combination with anti-CD20 immunotherapies, such as rituximab. In this article, we review the development and latest clinical data that have led to the expanded approval of venetoclax with rituximab in relapsed/refractory CLL/SLL. We also discuss ongoing and future clinical trials designed to evaluate the efficacy of venetoclax in previously untreated patients and to investigate venetoclax combinations with inhibitors of B-cell receptor signaling pathway. These studies hope to offer an expanded list of chemotherapy-free regimens for patients with CLL/SLL.

Logical complexity of Bcl-2 family proteins function in the intrinsic apoptosis

Apoptosis (type of programmed cell death) is an active process of cellular self-destruction in multicellular organisms. It is characterized by distinctive histomorphological, biochemical, and molecular features. Multiple cellular pathways trigger apoptosis, two of them are the best known: intrinsic and extrinsic. Multiple cellular signals and interactions can influence the course of apoptotic pathways. Bcl-2 family proteins play a key role in regulatory mechanisms of intrinsic apoptosis. Mitochondrial outer membrane permeabilization (MOMP) is an essential step for intrinsic apoptosis that is controlled by pro-apoptotic and anti-apoptotic members of Bcl-2 protein family. Pro-apoptotic effector proteins Bax and Bak represent the only Bcl-2 proteins inducing formation of MOMP, whose pores facilitate the subsequent releasing of several pro-apoptotic proteins from mitochondrial intermembrane space into cytosol. These proteins initiate a caspase cascade, resulting in rapid elimination of the doomed cells.

CLL Cells with 13q- Are Characterized By High BCL2 Levels and CLL Cells with Trisomy-12 By Increased Activation of STAT3 and NF-κB

CLL Cells with 13q- Are Characterized By High BCL2 Levels and CLL Cells with Trisomy-12 By Increased Activation of STAT3 and NF-κB

Targeting Mcl-1 and other Bcl-2 family member proteins in cancer therapy.

Regulation of both the extrinsic and the mitochondria-dependent intrinsic apoptotic pathways plays a key role in the development of the hematopoietic system, for sustaining cell survival during generation of various cell types, in eliminating cells with dual identities such as CD4/CD8 double-positive cells (Hettmann, Didonato, Karin, & Leiden, 1999; Ogasawara, Suda, & Nagata, 1995), for sustaining cells during the rapid clonal expansion phase (Schirmer, Vallejo, Weyand, & Gronzy, 1998), as well as eliminating cells during the contraction phase (Yajima et al., 2006). The anti-apoptotic protein Mcl-1 is necessary for sustaining hematopoietic stem cells (HPS) (Akashi et al., 2003; Akashi, Traver, Miyamoto, & Weissman, 2000). The anti-apoptotic factors Mcl-1, Bcl-2, and Bcl-xL were also found to be over-expressed in acute myeloid leukemia (AML) (Kaufmann et al., 2016) and acute lymphocytic leukemia (ALL) (Findley, Gu, Yeager, & Zhou, 1997), suggesting that dis-regulated apoptotic processes could be a factor in the instigation of leukemia and/or its relapse. Molecules targeting these proteins were used as single agents to treat leukemia. However, by using a set of recently developed specific molecule inhibitors targeting anti-apoptotic proteins, distinct roles are being discovered for these anti-apoptotic proteins during hematopoietic and tumor development. Furthermore, using these inhibitors in proper combinations can effectively induce apoptosis in various solid tumors, even though each agent on its own cannot induce apoptosis in them. These new findings suggest that inhibiting anti-apoptotic elements can induce apoptosis without external stimuli in most cells, but it comes with a risk that some combinations could also trigger apoptosis in healthy cells. One way to address the safety issue is by limiting exposure to all the agents to only cancer cells, thus making the combination safe and effective. In this article, we review this rapidly developing idea in cancer research.

Counting on Death - Quantitative aspects of Bcl-2 family regulation.

The Bcl-2 protein family members critically regulate mitochondrial outer membrane permeabilization (MOMP), a point-of-no-return in the intrinsic and extrinsic apoptosis pathways. The common view on qualitative interaction and activation patterns of the three subclasses, the BH3-only proteins, prosurvivals, and effectors, is static and currently being revolutionized by an emerging understanding of the complex dynamic equilibria that govern cellular fate. Recent experimental evidence on protein associations with the mitochondrial outer membrane, retrotranslocation to the cytosol, and differential binding affinities in aqueous and membranous environments instigate the development of a revised model of Bcl-2 family interplay. Likely, the dynamic processes and their respective timescales need to be taken into account to authentically understand and, by extension, to generate reliable predictions on cellular decision-making. Here, we review the quantitative aspects of Bcl-2 family-regulated MOMP. In particular, we discuss affinity binding constants of protein-protein associations and velocities of post-translational modifications, membrane (retro-) translocations, and effector oligomerization. Moreover, we provide insights into how these kinetic and network information enable systems biological approaches, further enhancing our understanding of the complex molecular mechanisms governing MOMP.