Bcl-2 Inhibitor ABT-737 Research Articles

Exploiting endogenous and therapy-induced apoptotic vulnerabilities in immunoglobulin light chain amyloidosis with BH3 mimetics

Immunoglobulin light chain (AL) amyloidosis is an incurable hematologic disorder typically characterized by the production of amyloidogenic light chains by clonal plasma cells. These light chains misfold and aggregate in healthy tissues as amyloid fibrils, leading to life-threatening multi-organ dysfunction. Here we show that the clonal plasma cells in AL amyloidosis are highly primed to undergo apoptosis and dependent on pro-survival proteins MCL-1 and BCL-2. Notably, this MCL-1 dependency is indirectly targeted by the proteasome inhibitor bortezomib, currently the standard of care for this disease and the related plasma cell disorder multiple myeloma, due to upregulation of pro-apoptotic Noxa and its inhibitory binding to MCL-1. BCL-2 inhibitors sensitize clonal plasma cells to multiple front-line therapies including bortezomib, dexamethasone and lenalidomide. Strikingly, in mice bearing AL amyloidosis cell line xenografts, single agent treatment with the BCL-2 inhibitor ABT-199 (venetoclax) produces deeper remissions than bortezomib and triples median survival. Mass spectrometry-based proteomic analysis reveals rewiring of signaling pathways regulating apoptosis, proliferation and mitochondrial metabolism between isogenic AL amyloidosis and multiple myeloma cells that divergently alter their sensitivity to therapies. These findings provide a roadmap for the use of BH3 mimetics to exploit endogenous and induced apoptotic vulnerabilities in AL amyloidosis.

Identification of Monobenzone as a Novel Potential Anti-Acute Myeloid Leukaemia Agent That Inhibits RNR and Suppresses Tumour Growth in Mouse Xenograft Model.

Simple SummaryThe clinical treatment of acute myeloid leukaemia is still dominated by chemotherapy. Clinically used anti-leukaemia drugs have shortcomings such as myelosuppression, toxicity and drug resistance. Therefore, the need to develop other chemotherapeutic drugs to meet more clinical needs is urgent. Ribonucleotide reductase (RNR) consists of a catalytic large subunit M1 (RRM1) and a regulatory small subunit M2 (RRM2), which provides dNTPs for DNA synthesis. The rapid proliferation of cancer cells requires large amounts of dNTPs. Therefore, the use of RNR inhibitors is a promising strategy for the clinical treatment of various malignancies. Monobenzone is an FDA-approved depigmenting agent for vitiligo patients. In this study, we demonstrate that monobenzone is a potent inhibitor of RNR enzyme activity by targeting RRM2 protein, and thus has significant anti-leukaemia efficacy in vitro and in vivo. This finding suggests that monobenzone has the potential to be optimized as a novel anti-AML therapeutic drug in the future. Acute myeloid leukaemia (AML) is one of the most common types of haematopoietic malignancy. Ribonucleotide reductase (RNR) is a key enzyme required for DNA synthesis and cell proliferation, and its small subunit RRM2 plays a key role for the enzymatic activity. We predicted monobenzone (MB) as a potential RRM2 target compound based on the crystal structure of RRM2. In vitro, MB inhibited recombinant RNR activity (IC50 = 0.25 μM). Microscale thermophoresis indicated that MB inhibited RNR activity by binding to RRM2. MB inhibited cell proliferation (MTT IC50 = 6–18 μM) and caused dose-dependent DNA synthesis inhibition, cell cycle arrest, and apoptosis in AML cells. The cell cycle arrest was reversed by the addition of deoxyribonucleoside triphosphates precursors, suggesting that RNR was the intracellular target of the compound. Moreover, MB overcame drug resistance to the common AML drugs cytarabine and doxorubicin, and treatment with the combination of MB and the Bcl-2 inhibitor ABT-737 exerted a synergistic inhibitory effect. Finally, the nude mice xenografts study indicated that MB administration produced a significant inhibitory effect on AML growth with relatively weak toxicity. Thus, we propose that MB has the potential as a novel anti-AML therapeutic agent in the future.

Abstract 356: Pre-clinical investigations utilizing Bcl2 inhibitors in glioblastoma

Abstract Glioblastoma (GBM) is a lethal malignant cancer of the central nervous system. The multimodal management approach does not extend survival beyond 2 years. Due to diverse variability in subpopulations of patients with glioma, treatment benefits are not uniform with apoptosis evasion being a key attribute of treatment resistance in GBM. Antiapoptotic protein, Bcl-2, Bcl-xL, and Mcl-1, are frequently upregulated in acquired chemo resistant cancer cells. Here in, we investigated the anti-proliferative effects of disrupting “Bcl-2-regulated pathways” in GBM. Of note, accessibility of FDA approved Bcl-2 inhibitors with known safety profile, makes it a biologically relevant target where intra- and inter-patient heterogeneity can be overcome successfully. Western Blot (WB) analysis was performed to compare the Bcl-2 family expression in human GBM cell lines (LN229, T98G, DI321, SA21). RT-PCR data was obtained for Bcl-2 family mRNA expression profile in present GBM cell lines. IC50s were determined for afore mentioned cell lines using MTS assay for Temozolomide (TMZ, alkylating agent), Bcl-2 inhibitor ABT199, Bcl-xL inhibitor A1331852, Mcl-1 inhibitor AZD5991, and BTK inhibitor Ibrutinib. Further, synergistic activity of TMZ, ABT199, A1331852, AZD5991 and Ibrutinib in several permutations. All experiments were performed with an n of 3/5. Our data suggests upregulation of antiapoptotic Bcl-2 family proteins in GBM cells including GSCs (DI321) and patient derived cells (SA21). There was significant decrease in viability of these cells when treated with ABT199 and A-1331852 (p< 0.0001) as compared to TMZ as a single agent. Other combinations ABT199+AZD5991 (p< 0.0001), AZD5991+A-1331852 (p< 0.0001) and TMZ+ABT (p<0.0002) also shows anti-tumor activity. Alternatively, we observed increased sensitivity in TMZ-resistant GBM cells against these drugs using MTS assay. GBM is an aggressive malignancy with 100% recurrence over time. Our preclinical data suggests synergism of Bcl-2 protein inhibitors in GBM cells, both TMZ-naïve and TMZ-resistant. Our ongoing research is investigating the biological relevance of Bcl-2 in conjugation with other tumor suppressor proteins, in vitro and in vivo. Citation Format: Srishti Kala, Leland Earp, Sonikpreet Aulakh. Pre-clinical investigations utilizing Bcl2 inhibitors in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 356.

Abstract 5337: Tolinapant (ASTX660) enhances the anti-leukemic activity of Venetoclax and Dexamethasone in T cell acute lymphoblastic leukemia (T-ALL)

Abstract Background: Inhibitors of apoptosis proteins (IAPs) are overexpressed in ALL leading to resistance to apoptosis and chemo-resistance. Tolinapant, a non-peptidomimetic antagonist of the cellular and X-linked inhibitors of apoptosis (cIAP1/2 and XIAP) being evaluated in Phase 1/2 clinical trial [NCT02503423] showed single-agent activity in T-cell lymphoma. Objectives: Herein, we analyzed the preclinical activity of tolinapant in combination with ABT199 and Dexamethasone (DEX) in T-ALL (in vitro and ex-vivo patient derived xenografts (PDX). Results: Using a panel of 8 T-ALL cell lines we analyzed the single agent activity of tolinapant. Loucy and SUP-T11 were most sensitive (IC50 = 190-309 nM). CCRF-CEM and ALL-SIL showed moderate sensitivity (IC50 = 10-18 µM) while Jurkat, MOLT16, MOLT4 and PF382 were resistant. Western blotting (WB) showed decreased levels of cIAP1and cIAP2 with no change in XIAP in response to tolinapant as a single agent. We next investigated the efficacy of tolinapant with Bcl2 inhibitor ABT199. The combination of ABT199 and ASTX660 was synergistic in Loucy cell line with combination index (CI) of 0.14. Cell death was increased to 64±3% in combination v/s 27±0.9% in ABT199. WB showed decreased cIAP2 and increased levels of cleaved caspase 7 and cleaved caspase 9 in combination compared to single agents, suggesting increased apoptosis. Treatment of PDX-derived cells with tolinapant and ABT199 was more effective than monotherapy in inducing apoptosis in CD45+ bulk (46±0.7% to 63±6%, p<0.0001) and leukemia initiating cells (LICs, CD45+ CD7+ CD19- CD34+) (39±3% to 54±8%, p=0.003). Next, we checked the effect of combination of DEX with tolinapant in T-ALL cell lines. CCRF-CEM cell line is from a relapsed patient and is resistant to DEX. The combination was synergistic with CI of 0.26 and cell death of 50±4% compared to 20±3% by DEX. A triple combination of DEX, ABT199 and tolinapant increased apoptotic response to 82.9±1%, compared to DEX+ABT199 dual combination where apoptosis was induced to 52.4±2%. Strong synergy in terms of both cytoreduction as well as apoptosis induction was observed in SUP-T11 cells. Tolinapant sensitized SUP-T11 cells to DEX with ED50 value of 542 nM in combination compared to ~2 µM with DEX treatment. Simultaneous analysis of cell proliferation, stress response and DNA damage using single-cell proteomics analysis showed downregulation of proliferation (Ki-67), stress response (ATF4, LC3B) and increased levels of cleaved PARP, cleaved caspase 3 suggesting increased apoptosis in combination of tolinapant with DEX. Treatment of PDX-derived cells with tolinapant enhanced the cytotoxic effect of DEX in CD45+ bulk (58±2% to 71±0.1%, p<0.0001) and LICs (62±2% to 78±0.9%, p<0.0001). Conclusion: Tolinapant synergizes with the anti-leukemic activity of ABT199 and DEX, establishing a therapeutic rationale for IAP antagonist in T-ALL. Citation Format: Priyanka Sharma, Sujan Piya, Huaxian Ma, Natalia Baran, Muharrem Muftuoglu, Mahesh Basyal, Vivian Ruvolo, George Ward, Tomoko Smyth, Martin J. Sims, Michael Andreeff, Gautam Borthakur. Tolinapant (ASTX660) enhances the anti-leukemic activity of Venetoclax and Dexamethasone in T cell acute lymphoblastic leukemia (T-ALL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5337.

BCL2 inhibitor ABT-199 and BCL2L1 inhibitor WEHI-539 coordinately promote NOXA-mediated degradation of MCL1 in human leukemia cells

Human leukemia U937 cells that were continuously treated with hydroquinone (HQ) were transformed into U937/HQ cells with increased MCL1 and BCL2L1 expression. Compared with their parental cells, U937/HQ cells were less sensitive to ABT-263 (BCL2/BCL2L1 inhibitor)/ABT-199 (BCL2 inhibitor) cytotoxicity. The combination of WEHI-539 (BCL2L1 inhibitor) with either ABT-199 or ABT-263 showed synergistic cytotoxicity to U937 and U937/HQ cells. Therefore, we further investigated the cytotoxic mechanism induced by the combination of WEHI-539 and ABT-199. The combined treatment of WEHI-539 and ABT-199 induced NOX4/ROS/p38 MAPK axis-mediated autophagy, which in turn accelerated β-TrCP mRNA turnover. Downregulation of β-TrCP increased Sp1 expression, thereby promoting Sp1-mediated NOXA transcription, which in turn induced NOXA-dependent MCL1 degradation. Enforced expression of MCL1 alleviated the cytotoxicity of WEHI-539 plus ABT-199 to induce the loss of mitochondrial membrane potential and cell viability. WEHI-539 alone induced Sp1/NOXA axis-mediated MCL1 downregulation, while ABT-199 significantly decreased the dose of WEHI-539 by approximately 350- and 50-fold to induce MCL1 suppression in parental and HQ-selected cells, respectively. Furthermore, WEHI-539 sensitized ABT-199-resistant U937 cells to ABT-199 cytotoxicity by inducing NOXA-mediated degradation of MCL1. Collectively, the data in this study indicate that ABT-199 and WEHI-539 cooperatively induce NOXA-dependent MCL1 degradation, and the inhibition of MCL1 mainly explains their combined cytotoxicity in parental, HQ-selected, and ABT-199-resistant U937 cells.

Lipase-triggered drug release from BCL2 inhibitor ABT-199-loaded nanoparticles to elevate anti-leukemic activity through enhanced drug targeting on the mitochondrial membrane

Selective BCL2 inhibitor ABT-199 has been approved to treat hematological malignancies including acute myeloid leukemia (AML). However, acquired drug resistance and severe side effects occur after extended treatment limiting the clinical usage of ABT-199. Here, we successfully encapsulated pure ABT-199 in amphiphilic mPEG-b-PTMC169 block copolymer, forming mPEG-b-PTMC169@ABT-199 nanoparticles (abbreviated as PEG-ABT-199), which presented better aqueous dispersion and higher efficiency of loading and encapsulation than pure ABT-199. We then compared the anti-leukemic ability of pure ABT-199 and PEG-ABT-199 in vitro and in vivo. PEG-ABT-199 had a lower IC50 value compared with pure ABT-199 in MV4-11 and MOLM-13 cell lines. In addition, PEG-ABT-199 significantly induced apoptosis and decreased colony number than pure ABT-199. Most importantly, PEG-ABT-199 markedly reduced leukemic burden, inhibited the infiltration of leukemic blasts in the spleen, and extended the overall survival (OS) in MLL-AF9-transduced murine AML compared with free ABT-199. Meanwhile, the blank PEG169 NP was non-toxic to normal hematopoiesis in vitro and in vivo, suggesting that PEG169 NP is a safe carrier. Mechanistically, PEG-ABT-199 enhanced mitochondria-targeted delivery of ABT-199 to trigger the collapse of mitochondrial membrane potential (MMP), the release of cytochrome c (cyt-c), and mitochondria-based apoptosis. In conclusion, our results suggest that PEG-ABT-199 has more vital anti-leukemic ability than pure ABT-199. PEG-ABT-199 has potential application in clinical trials to alleviate side effects and improve anti-leukemia ability. Statement of significanceATB-199, an orally selective inhibitor for BCL2 protein, presents marked activity in relapsed or refractory AML, T-ALL, and CLL patients. However, ABT-199 resistance severely limits the further clinical usage because of off-target effects, non-specific toxicities, and low delivery of drugs. To reduce the side-effects and improve the solubility and bioavailability, ABT-199 was encapsulated into the amphiphilic mPEG-b-PTMC block copolymer by co-assembly method to obtain mPEG-b-PTMC@ABT-199 nanoparticles (PEG-ABT-199). PEG-ABT-199 has several advantages compared with pure ABT-199. 1.PEG-ABT-199 presents better aqueous dispersion and higher efficiencies of loading and encapsulation than pure ABT-199. 2. PEG-ABT-199 substantially enhances the anti-leukemic ability in vitro and in vivo compared with pure ABT-199. 3. PEG-ABT-199 has little effects on normal cells. 4. PEG-ABT-199 can reduce treatment cost.

Bisphenol A impairs macrophages through inhibiting autophagy via AMPK/mTOR signaling pathway and inducing apoptosis

Bisphenol A (BPA) is a widespread endocrine disruptor that induces the impairment of immune cells, but the mechanism remains unknown. Macrophages are one of the most important immune cells in innate and adaptive immunity. In this study, we aimed to probe the effects of BPA on the damage of RAW264.7 cells and its mechanisms of action, especially focusing on the relationship between autophagy and apoptosis. Cells were pretreated with 10 mg/L LPS, or added autophagy activator RAPA, autophagy inhibitor 3-MA or Bcl-2 inhibitor ABT-737, then treated with BPA (0, 10, 100 and 200 μmol/L) for 12 h. Results have shown that BPA decreased the cell viability and disrupted secretory function by promoting pro-inflammatory cytokines TNF-α and IL-6 and reducing anti-inflammatory cytokines IL-10 TGF-β, as well as phagocytic ability. Moreover, autophagy was inhibited by BPA through decreasing p-AMPK/AMPK and increasing p-mTOR/mTOR, and further down-regulating autophagy proteins ATG6, LC3II/I ratio, and up-regulating autophagy flux protein p62. Additionally, BPA significantly increased Bax/Bcl-2 ratio, Caspase-3 expression and apoptosis rate. We found that RAPA ameliorated the cell viability, Bax/Bcl-2 ratio, and macrophage function damage induced by BPA. Intriguingly, ABT-737 might promote ATG6 expression. In summary, our study demonstrated that the effects of BPA on macrophages seemed to be mediated by inhibiting AMPK/mTOR-dependent autophagy and inducing apoptosis via endogenous mitochondrial pathway. Both Bcl-2 and ATG6 were involved in the regulation of apoptosis and autophagy by BPA. These findings provide a broader perspective for understanding the interaction between autophagy and apoptosis in BPA-induced immune cell injury.

Pediatric multicellular tumor spheroid models illustrate a therapeutic potential by combining BH3 mimetics with Natural Killer (NK) cell-based immunotherapy

The induction of apoptosis is a direct way to eliminate tumor cells and improve cancer therapy. Apoptosis is tightly controlled by the balance of pro- and antiapoptotic Bcl-2 proteins. BH3 mimetics neutralize the antiapoptotic function of Bcl-2 proteins and are highly promising compounds inducing apoptosis in several cancer entities including pediatric malignancies. However, the clinical application of BH3 mimetics in solid tumors is impeded by the frequent resistance to single BH3 mimetics and the anticipated toxicity of high concentrations or combination treatments. One potential avenue to increase the potency of BH3 mimetics is the development of immune cell-based therapies to counteract the intrinsic apoptosis resistance of tumor cells and sensitize them to immune attack. Here, we describe spheroid cultures of pediatric cancer cells that can serve as models for drug testing. In these 3D models, we were able to demonstrate that activated allogeneic Natural Killer (NK) cells migrated into tumor spheroids and displayed cytotoxicity against a wide range of pediatric cancer spheroids, highlighting their potential as anti-tumor effector cells. Next, we investigated whether treatment of tumor spheroids with subtoxic concentrations of BH3 mimetics can increase the cytotoxicity of NK cells. Notably, the cytotoxic effects of NK cells were enhanced by the addition of BH3 mimetics. Treatment with either the Bcl-XL inhibitor A1331852 or the Mcl-1 inhibitor S63845 increased the cytotoxicity of NK cells and reduced spheroid size, while the Bcl-2 inhibitor ABT-199 had no effect on NK cell-mediated killing. Taken together, this is the first study to describe the combination of BH3 mimetics targeting Bcl-XL or Mcl-1 with NK cell-based immunotherapy, highlighting the potential of BH3 mimetics in immunotherapy.

MDM2-BCL-XL PROTACs enable degradation of BCL-XL and stabilization of p53.

Inhibition or degradation of anti-apoptotic protein BCL-XL is a viable strategy for cancer treatment. Despite the recent development of PROTACs for degradation of BCL-XL, the E3 ligases are confined to the commonly used VHL and CRBN. Herein we report the development of MDM2-BCL-XL PROTACs using MDM2 as E3 ligase for degradation of BCL-XL. Three MDM2-BCL-XL PROTACs derived from MDM2 inhibitor Nutlin-3, which can also upregulate p53, and BCL-2/BCL-XL inhibitor ABT-263 with different linker length were designed, synthesized, and evaluated in vitro. We found BMM4 exhibited potent, selective degradation activity against BCL-XL and stabilized tumor suppressor p53 in U87, A549 and MV-4-11 cancer cell lines. Moreover, combination of BMM4 and BCL-2 inhibitor ABT-199 showed synergistic antiproliferative activity. The unique dual-functional PROTACs offers an alternative strategy for targeted protein degradation.

ZCL-082, a boron-containing compound, induces apoptosis of non-Hodgkin's lymphoma via targeting p90 ribosomal S6 kinase 1/NF-κB signaling pathway

IntroductionDespite the rapid progress in the diagnosis and treatment, the prognosis of some types of non-Hodgkin's lymphoma (NHL), especially those with double-hit or double-expressor genotypes, remains poor. Novel targets and compounds are needed to improve the prognosis of NHL. MethodsWe investigated the effect of ZCL-082, a novel boron-containing compound with anti-proliferating activity against ovarian cancer cells, on NHL cells and human peripheral blood mononuclear cells by CCK-8 assay, Annexin V/PI double staining assay, RH123/PI double staining, Western blot, and immunohistochemistry. NF-κB pathway activity was analyzed using luciferase reporter gene assay and RT-PCR. The location of p65 was detected by immunofluorescence and nuclear/cytoplasmic fractionation assay. Immunoprecipitation and chromatin immunoprecipitation assays were used to detect the binding between p65 and p300. CETSA and molecular docking assay were carried out to test the interaction between ZCL-082 and p90 ribosomal S6 kinase 1 (RSK1). Kinase reaction was conducted to examine the inhibition of RSK1 kinase activity by ZCL-082. ResultsWe found that ZCL-082 can induce the apoptosis of various NHL cell lines in vitro and in vivo. ZCL-082 significantly inhibits TNFα- or LPS-induced NF-κB activation without disturbing TNFα-induced IκBα degradation or the nuclear translocation and DNA-binding ability of p65. However, ZCL-082 markedly suppresses the phosphorylation of p65 on Ser536 and the interaction between p65 and p300. The overexpression of the phosphomimetic mutant of p65 at Ser536 partially abrogates ZCL-082-induced cell death. We further found that ZCL-082 directly binds to and inhibits the activity of RSK1. RSK1 can phosphorylate RelA/p65 on Ser536 and its overexpression is associated with the poor prognosis of lymphoma. The overexpression of RSK1 partially rescues ZCL-082-induced cell death. Molecular docking studies show that ZCL-082 fits well with the N-terminal kinase domain of RSK1. Furthermore, the combination of ZCL-082 and BCL-2 inhibitor ABT-199 has a synergistic apoptosis-inducing effect against double-hit lymphoma cell line OCI-Ly10. DiscussionWe found that ZCL-082 is a highly promising anti-lymphoma compound that targets RSK1 and interferes with the RSK1/NF-κB signaling pathway. The combination of ZCL-082 with BCL-2 inhibitor may represent a novel strategy to improve the outcome of double-hit or double-expressor lymphoma.

Multimeric Anti-DR5 IgM Agonist Antibody IGM-8444 Is a Potent Inducer of Cancer Cell Apoptosis and Synergizes with Chemotherapy and BCL-2 Inhibitor ABT-199.

Death receptor 5 (DR5) is an attractive target for cancer therapy due to its broad upregulated expression in multiple cancers and ability to directly induce apoptosis. Though anti-DR5 IgG antibodies have been evaluated in clinical trials, limited efficacy has been attributed to insufficient receptor crosslinking. IGM-8444 is an engineered, multivalent agonistic IgM antibody with 10 binding sites to DR5 that induces cancer cell apoptosis through efficient DR5 multimerization. IGM-8444 bound to DR5 with high avidity and was substantially more potent than an IgG with the same binding domains. IGM-8444 induced cytotoxicity in a broad panel of solid and hematologic cancer cell lines but did not kill primary human hepatocytes in vitro, a potential toxicity of DR5 agonists. In multiple xenograft tumor models, IGM-8444 monotherapy inhibited tumor growth, with strong and sustained tumor regression observed in a gastric PDX model. When combined with chemotherapy or the BCL-2 inhibitor ABT-199, IGM-8444 exhibited synergistic in vitro tumor cytotoxicity and enhanced in vivo efficacy, without augmenting in vitro hepatotoxicity. These results support the clinical development of IGM-8444 in solid and hematologic malignancies as a monotherapy and in combination with chemotherapy or BCL-2 inhibition.

Dual Akt and Bcl-2 inhibition induces cell-type specific modulation of apoptotic and autophagic signaling in castration resistant prostate cancer cell lines.

Cancer cell survival depends on the cross-regulation between apoptosis and autophagy which share common signaling pathways including PI3K/Akt/mTOR and Bcl-2. The aim of this study was to elucidate the modulation patterns between apoptosis and autophagy following dual inhibition by Akt inhibitor erufosine and Bcl-2 inhibitor ABT-737 in castration-resistant prostate cancer (CRPC) cell lines, PC-3 (Bax+) and DU-145 (Bax-). Cell cycle progression, apoptotic and autophagic signaling were examined by flow cytometry, multi-caspase assay, Hoechst staining, acridine orange staining of acidic vesicular organelles (AVOs), qRT-PCR and Western Blot. Dual inhibition increased G2/M arrest in PC-3 and DU-145, but not in the healthy prostate epithelium cells, PNT-1A. Only in PC-3, dual inhibition induced synergistic apoptotic and additive autophagic effects. In DU-145 and PNT-1A cells, ABT-737 did not display any remarkable effect on multicaspase activity and erufosine and ABT-737, neither alone nor in combination induced AVOs. By dual inhibition, AKT, BCL-2 and NF-κB gene expressions were downregulated in PC-3, both ATG-5 and BECLIN-1 gene expressions were upregulated in DU-145 but Beclin-1 protein expression was substantially reduced in both CRPC cells. Dual inhibition-induced synergistic multicaspase activation in PC-3 degrades and disrupts autophagic activity of Beclin-1, enhancing caspase-dependent apoptosis. However, in DU-145, following dual inhibition, rate of multicaspase induction and apoptosis are lower but autophagy is completely abolished despite markedly increased BECLIN-1 gene expression. In conclusion, antineoplastic drug combinations may display cell-type specific modulation of apoptotic and autophagic signaling and lack of protective autophagy may not necessarily indicate increased chemotherapeutic sensitivity in heterogenous tumor subpopulations.

Therapeutic Effects of the Bcl-2 Inhibitor on Bleomycin-induced Pulmonary Fibrosis in Mice.

Idiopathic pulmonary fibrosis (IPF) is a distressing lung disorder with poor prognosis and high mortality rates. Limited therapeutic options for IPF is a major clinical challenge. Well-known for its anti-apoptotic properties, B-cell lymphoma 2 (Bcl-2) plays a critical role in the pathology of malignancies and inflammatory diseases, including IPF. In this study, we aimed to investigate the therapeutic effect of a Bcl-2 homology domain 3 mimetic inhibitor, ABT-199, on bleomycin (BLM)-induced pulmonary fibrosis in mice, and explore possible underlying mechanism. The lung inflammation and fibrosis model was established by intratracheal instillation of a single dose of BLM. We observed elevated Bcl-2 in the alveolar macrophages and fibroblasts derived from BLM-instilled mice from day 7. Further, we obtained in vivo evidence that early therapeutic treatment with Bcl-2 inhibitor ABT-199 from day 3, and late treatment from day 10, both alleviated airway inflammation and lung fibrosis induced by BLM. Our data suggest that ABT-199 might be an effective antifibrotic agent that interferes with profibrogenic cells, which may be a promising therapy in the treatment of clinical IPF patients.

BCL-2 Inhibitor ABT-737 Effectively Targets Leukemia-Initiating Cells with Differential Regulation of Relevant Genes Leading to Extended Survival in a NRAS/BCL-2 Mouse Model of High Risk-Myelodysplastic Syndrome.

AbastractDuring transformation, myelodysplastic syndromes (MDS) are characterized by reducing apoptosis of bone marrow (BM) precursors. Mouse models of high risk (HR)-MDS and acute myelogenous leukemia (AML) post-MDS using mutant NRAS and overexpression of human BCL-2, known to be poor prognostic indicators of the human diseases, were created. We have reported the efficacy of the BCL-2 inhibitor, ABT-737, on the AML post-MDS model; here, we report that this BCL-2 inhibitor also significantly extended survival of the HR-MDS mouse model, with reductions of BM blasts and lineage negative/Sca1+/KIT+ (LSK) cells. Secondary transplants showed increased survival in treated compared to untreated mice. Unlike the AML model, BCL-2 expression and RAS activity decreased following treatment and the RAS:BCL-2 complex remained in the plasma membrane. Exon-specific gene expression profiling (GEP) of HR-MDS mice showed 1952 differentially regulated genes upon treatment, including genes important for the regulation of stem cells, differentiation, proliferation, oxidative phosphorylation, mitochondrial function, and apoptosis; relevant in human disease. Spliceosome genes, found to be abnormal in MDS patients and downregulated in our HR-MDS model, such as Rsrc1 and Wbp4, were upregulated by the treatment, as were genes involved in epigenetic regulation, such as DNMT3A and B, upregulated upon disease progression and downregulated upon treatment.

Inhibitors of Anti-apoptotic Bcl-2 Family Proteins Exhibit Potent and Broad-Spectrum Anti-mammarenavirus Activity via Cell Cycle Arrest at G0/G1 Phase.

ABSTRACTTargeting host factors is a promising strategy to develop broad-spectrum antiviral drugs. Drugs targeting anti-apoptotic Bcl-2 family proteins that were originally developed as tumor suppressors have been reported to inhibit multiplication of different types of viruses. However, the mechanisms whereby Bcl-2 inhibitors exert their antiviral activity remain poorly understood. In this study, we have investigated the mechanisms by which obatoclax (OLX) and ABT-737 Bcl-2 inhibitors exhibited a potent antiviral activity against the mammarenavirus lymphocytic choriomeningitis virus (LCMV). OLX and ABT-737 potent anti-LCMV activity was not associated with their proapoptotic properties but rather with their ability to induce cell arrest at the G0/G1 phase. OLX- and ABT-737–mediated inhibition of Bcl-2 correlated with reduced expression levels of thymidine kinase 1 (TK1), cyclin A2 (CCNA2), and cyclin B1 (CCNB1) cell cycle regulators. In addition, small interfering RNA (siRNA)–mediated knockdown of TK1, CCNA2, and CCNB1 resulted in reduced levels of LCMV multiplication. The antiviral activity exerted by Bcl-2 inhibitors correlated with reduced levels of viral RNA synthesis at early times of infection. Importantly, ABT-737 exhibited moderate efficacy in a mouse model of LCMV infection, and Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and severe acute respiratory syndrome coronavirus 2 (SARS–CoV-2). Our results suggest that Bcl-2 inhibitors, actively being explored as anticancer therapeutics, might be repositioned as broad-spectrum antivirals.IMPORTANCE Antiapoptotic Bcl-2 inhibitors have been shown to exert potent antiviral activities against various types of viruses via mechanisms that are currently poorly understood. This study has revealed that Bcl-2 inhibitors’ mediation of cell cycle arrest at the G0/G1 phase, rather than their proapoptotic activity, plays a critical role in blocking mammarenavirus multiplication in cultured cells. In addition, we show that Bcl-2 inhibitor ABT-737 exhibited moderate antimammarenavirus activity in vivo and that Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our results suggest that Bcl-2 inhibitors, actively being explored as anticancer therapeutics, might be repositioned as broad-spectrum antivirals.

A novel 3D culture model recapitulates primary FL B-cell features and promotes their survival

AbstractNon-Hodgkin B-cell lymphomas (B-NHL) mainly develop within lymph nodes as aggregates of tumor cells densely packed with their surrounding microenvironment, creating a tumor niche specific to each lymphoma subtypes. In vitro preclinical models mimicking biomechanical forces, cellular microenvironment, and 3D organization of B-cell lymphomas remain scarce, while all these parameters are key determinants of lymphomagenesis and drug resistance. Using a microfluidic method based on cell encapsulation inside permeable, elastic, and hollow alginate microspheres, we developed a new tunable 3D model incorporating lymphoma B cells, extracellular matrix (ECM), and/or tonsil stromal cells (TSC). Under 3D confinement, lymphoma B cells were able to form cohesive spheroids resulting from overexpression of ECM components. Moreover, lymphoma B cells and TSC dynamically formed self-organized 3D spheroids favoring tumor cell growth. 3D culture induced resistance to the classical chemotherapeutic agent doxorubicin, but not to the BCL2 inhibitor ABT-199, identifying this approach as a relevant in vitro model to assess the activity of therapeutic agents in B-NHL. RNA-sequence analysis highlighted the synergy of 3D, ECM, and TSC in upregulating similar pathways in malignant B cells in vitro than those overexpressed in primary lymphoma B cells in situ. Finally, our 3D model including ECM and TSC allowed long-term in vitro survival of primary follicular lymphoma B cells. In conclusion, we propose a new high-throughput 3D model mimicking lymphoma tumor niche and making it possible to study the dynamic relationship between lymphoma B cells and their microenvironment and to screen new anti-cancer drugs.

BAG1 down‐regulation increases chemo‐sensitivity of acute lymphoblastic leukaemia cells

BCL2‐associated athanogene‐1 (BAG1) is a multi‐functional protein that is found deregulated in several solid cancers and in paediatric acute myeloid leukaemia. The investigation of BAG1 isoforms expression and intracellular localization in B‐cell acute lymphoblastic leukaemia (B‐ALL) patient‐derived specimens revealed that BAG1 levels decrease during disease remission, compared to diagnosis, but drastically increase at relapse. In particular, at diagnosis both BAG1‐L and BAG1‐M isoforms are mainly nuclear, while during remission the localization pattern changes, having BAG1‐M almost exclusively in the cytosol indicating its potential cytoprotective role in B‐ALL. In addition, knockdown of BAG1/BAG3 induces cell apoptosis and G1‐phase cell cycle arrest and, more intriguingly, shapes cell response to chemotherapy. BAG1‐depleted cells show an increased sensitivity to the common chemotherapeutic agents, dexamethasone or daunorubicin, and to the BCL2 inhibitor ABT‐737. Moreover, the BAG1 inhibitor Thio‐2 induces a cytotoxic effect on RS4;11 cells both in vitro and in a zebrafish xenograft model and strongly synergizes with pan‐BCL inhibitors. Collectively, these data sustain BAG1 deregulation as a critical event in assuring survival advantage to B‐ALL cells.

Drug Screening Reveals That Ibrutinib (PCI-32765) Exhibits Synergy With BCL-2 and Proteasome Inhibitors In Models Of Mantle Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL)

BackgroundIbrutinib, a Bruton's Tyrosine Kinase inhibitor, is clinically effective in relapsed MCL and CLL, but responses are often partial and resistance eventually develops. To identify agents that synergize with ibrutinib, we performed an in vitro combinatorial drug screen and evaluated the most promising combinations in additional models. MethodsDrug screens were performed in JVM2 and Z138 MCL cell lines, combining ibrutinib with a diverse panel of secondary agents inhibiting targets within the B-cell receptor (BCR) pathway (SC514, PD325901, enzastaurin, R788, temsirolimus, dasatinib, idelalisib) and agents inhibiting targets outside the BCR pathway (ABT-199, bendamustine, carfilzomib, bortezomib, geldanamycin, panobinostat, PD0332991). Three doses (IC10, IC20, IC 30) for each drug were chosen and combined in a 3x3 format. Bliss Model of Additivity was used to quantitate synergistic cytotoxicity. Levels of cleaved PARP or Annexin V/PI staining were assessed for cell lines exposed to ibrutinib and synergistic agents, including bortezomib, carfilzomib, and ABT-199. The Ibrutinib + carfilzomib combination was further tested in MCL cell lines Rec1 and Jeko, in primary MCL and CLL patient (pt) samples, and in a MCL-SCID-hu mouse. Molecular analysis of the mechanistic interactions of the agents has been initiated using forward-phase antibody arrays (R and D Systems). ResultsInhibitors of targets within the BCR pathway were ineffective at potentiating the effects of ibrutinib (magnitude of synergy ranging -12%-9% in Z138, -25%-8% in JVM2). However, substantial synergistic cytotoxicity was seen with some agents that target outside the BCR pathway (magnitude of synergy ranging -5%-37% in Z138, -17%-48% in JVM2). Notably, we observed robust and reproducible synergistic cytotoxicity by combining ibrutinib with proteasome inhibitors carfilzomib and bortezomib as well as BCL-2 inhibitor ABT-199.The degree of synergyof ibrutinib + ABT-199 in MCL cell lines was durable across all 9 dosing combinations, (23-32% in Z-138, 10-26% in JVM-2). In CLL pt samples, ibrutinib + ABT-199 conferred modest synergy (10-20%) and near-complete cytotoxicity (61-70%). Flow cytometry for cleaved PARP demonstrated a high degree of apoptosis when combining ibrutinib with ABT-199 (94.5% in Z-138, 82.1% in Z-138).At optimal dosing, bortezomib conferred 19% synergy with ibrutinib in Z138 and 27% in JVM2. The best synergistic effect of carfilzomib and ibrutinib was 50% in Z138 and 65% in JVM2. The synergistic effect between carfilzomib and ibrutinib was maintained in Rec1 and Jeko MCL cell lines and in primary MCL pt samples. In CLL pt samples, ibrutinib + proteasome inhibition also conferred high degrees of total cytotoxicity (58-71% for carfilzomib, 42-62% for bortezomib). Flow cytometry for cleaved PARP or Annexin V/PI demonstrated increased apoptosis for ibrutinib +carfilzomib (92.3% in Z138, 61.2% in JVM2, 95% in Rec1, 90% in Jeko). The combination treatment increased the percent survival of MCL-SCID-hu mice in three-fold over single agent treatment in a 125-day trial.Preliminary analysis of proteomic changes induced by ibrutinib+ carfilzomib indicates synergistic alterations in cell cycle proteins and in apoptosis-regulating proteins, although the specific alterations differ between Z138 and JVM2 cell lines. DiscussionWhile agents within the BCR pathway did not display synergistic cytotoxicity with ibrutinib in our drug screen, several agents outside the BCR pathway did synergize with ibrutinib, notably ABT-199, bortezomib, and carfilzomib in two MCL cell lines. For all three drug combinations, this effect appeared to be mediated via apoptosis. The synergistic effect of ibrutinib + carfilzomib was further studied in 2 additional MCL cell lines, 3 MCL pt samples, and CLL pt samples. MCL-SCID-hu mice demonstrated three-fold increased survival when treated in combination over animals treated with either agent alone. The identification of unexpected synergistic cytotoxic interactions between inhibitors of apparently unrelated pathways is a benefit of our combinatorial screening approach, although the molecular mechanisms remain to be elucidated. The combination of ibrutinib with either proteasome or BCL-2 inhibition is proposed as promising novel treatments for MCL and CLL. A phase I/II clinical trial has been designed to test ibrutinib + carfilzomib in relapsed or refractory MCL and CLL. Disclosures:Williams:Celgene: Consultancy, Independent Data Safety Committee member Other, Research Funding; Genentech: Consultancy, Research Funding; Janssen: Research Funding; Millennium: Consultancy, Independent Data Safety Committee member, Independent Data Safety Committee member Other, Research Funding; Pharmacyclics: Consultancy, Research Funding; Onyx: Research Funding; Gilead: Research Funding. Wang:Onyx: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Millennium: Research Funding; Pharmacyclics: Research Funding; Janssen: Research Funding; Novartis: Research Funding.

Abstract 52: Mechanistic evaluation of anti-DR5 IgM antibody IGM-8444 with potent tumor cytotoxicity, without in vitro hepatotoxicity

Abstract Death receptor 5 (DR5) is a member of the tumor necrosis factor (TNF) receptor superfamily that activates the extrinsic apoptotic pathway when bound and multimerized by its ligand, TNF-related apoptosis inducing ligand (TRAIL). DR5 is broadly expressed on solid and hematologic cancers and has been targeted with both recombinant TRAIL and agonistic antibodies in the clinic. However, these therapeutics have been unsuccessful due to lack of efficacy or due to hepatotoxicity. We have developed IGM-8444, an engineered pentameric IgM with 10 binding sites specific for DR5, which is designed to multimerize DR5 to selectively and potently induce tumor cell apoptosis while sparing hepatocytes. Here, we describe the rationale behind the selection of IGM-8444 as our clinical candidate. A panel of agonistic DR5 antibodies were evaluated for DR5 binding affinity, epitope, and in vitro potency versus hepatotoxicity. Antibodies formatted as an IgM showed enhanced potency when compared to an IgG with the same binding domain. IGM-8444 binds an epitope on DR5 within cysteine-rich domain 1 (CRD1) that competes with TRAIL binding. While the binding affinities of the panel of anti-DR5 antibodies were comparable, IGM-8444 was selected from a subset of anti-DR5 IgM antibodies capable of potently killing tumor cells without exhibiting cytotoxicity of primary human hepatocytes in vitro. Further mechanistic studies examined the kinetics of apoptotic induction by IGM-8444 and other DR5 agonists. Interestingly, we noted that DR5 agonists with the fastest kinetics of tumor cell apoptotic induction also displayed the most hepatotoxicity in vitro. In spite of the kinetic differences, IGM-8444 has similar maximal cytotoxicity in vitro and comparable anti-tumor efficacy in xenograft mouse tumor models when compared with an IgM antibody targeting a different DR5 epitope. In cynomolgus monkeys, IGM-8444 showed no evidence of hepatotoxicity or other adverse events when dosed repeatedly up to 30 mg/kg, the highest dose tested. These preclinical properties of IGM-8444 provide an opportunity for enhanced tumor cytotoxicity without additional hepatotoxicity when combined with standard of care agents. Indeed, we have demonstrated enhanced anti-tumor efficacy by combining IGM-8444 with chemotherapies such as 5-FU and irinotecan in colorectal cancer models, as well as combining with Bcl-2 inhibitor ABT-199 in hematological malignancy models. In summary, we have evaluated the mechanism by which IGM-8444 agonizes DR5, which potently kills tumor cells without accompanying hepatotoxicity. IGM-8444 is currently being evaluated in a Phase 1 study as a single agent and in combination with chemotherapy-based regimens in patients with solid cancers and NHL (NCT04553692). Citation Format: Beatrice T. Wang, Tasnim Kothambawala, Kevin C. Hart, Xingjie Chen, Melanie Desbois, Susan E. Calhoun, Poonam Yakkundi, Rodnie A. Rosete, Yuan Cao, Katie Cha, Thomas J. Matthew, Ling Wang, Paul R. Hinton, Maya K. Leabman, Genevive Hernandez, Maya F. Kotturi, Eric W. Humke, Angus M. Sinclair, Bruce A. Keyt. Mechanistic evaluation of anti-DR5 IgM antibody IGM-8444 with potent tumor cytotoxicity, without in vitro hepatotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 52.

The Novel Imipridone ONC212 Induces Pronounced Anti-Leukemia Effects in Vitro and In Vivo and Is Highly Synergistic with the BCL-2 Inhibitor ABT-199

Imipridones constitute a novel class of molecules that includes the first-in-class anti-cancer compound ONC201. ONC201 has shown promising clinical activity against hematological and solid malignancies (Allen et al., Oncotarget, 2016). ONC212 is a second-generation imipridone with higher potency than ONC201. We first confirmed the overall in vitro anti-tumor effects of ONC212 using the collection of 1,088 human cancer cell lines available from the Genomic of Drug Sensitivity in Cancer Project. Ranking the sensitivity of these cancer cell lines to ONC212 by tumor type revealed that leukemias and lymphomas were especially sensitive. In fact, ONC212 exerted prominent apoptogenic effects in AML cell lines with > 20-fold potency compared to ONC201 (ED50s at 72 hr; 6.4 μM vs 258.7 nM, respectively for OCI-AML3 cells). Similar results were observed in primary AML cells, but there was no activity against normal bone marrow (BM) cells. We previously demonstrated that ONC201 induces apoptosis via an atypical integrated stress response (ISR, Ishizawa et al., Sci Signal, 2016). Similarly, ONC212 showed ISR activation accompanying its apoptogenic effects in AML cells which was agnostic to the mutation status of p53. However, because of the higher sensitivity of wild-type p53 cells to ONC212, we speculated that this effect could also reflect a p53-dependent apoptosis mechanism. Indeed, p53 knockdown reduced the ONC212-induced apoptosis in MV4;11 and MOLM13 cells, suggesting both, p53-dependent and -independent, mechanisms.We next investigated the effects of ONC212 in vivo in a systemic AML xenograft model using OCI-AML3 cells expressing luciferase. Biweekly oral administration of 50 mg/kg ONC212 markedly inhibited AML expansion and prolonged overall survival (p = 0.0003). Median survival increased from 43 d in controls to 49 d in the ONC212-treated group (+14%). For in vivo functional assessment of ONC212's anti-tumor effects against leukemia stem and progenitor cells, we utilized our patient-derived xenograft (PDX) mouse model. We treated primary PDX cells with ONC212 (250 nM, 36 hr) ex vivo, and then injected identical numbers of viable cells (treated or untreated) into recipient NSG mice. After one month, the percentages of human CD45+ cells in the peripheral blood, spleen, and BM were significantly decreased in the ONC212-treated group. The median survival in this group was remarkably different: 82 d compared to 36 d for controls (+128%, p<0.0001). These results indicated that ONC212 targets AML stem cells with potential to eradicate the disease.Because BCL-2 is generally considered protective against ISR-mediated apoptosis and ONC212 induced p53-mediated apoptosis as demonstrated above, we hypothesized that the BCL-2 inhibitor ABT-199 or the p53 activator nutlin-3a could further sensitize AML cells to ONC212. Indeed, the in vitro combination of ONC212 plus ABT-199 or nutlin-3a synergistically induced apoptosis in AML cells. Furthermore, the combination of ONC212 and ABT-199 showed highly significant synergistic anti-leukemia effects in vivo in a MOLM-13 xenograft mouse model. The combinatorial treatment prolonged overall median survival from 20 d for untreated controls to 21 d for each agent as monotherapy to 30 d for the combination.Since the G-protein-coupled receptor (GPCR) dopamine receptor D2 is the putative target of first-generation imipridone ONC201, we performed a PathHunter β-arrestin screening to determine if ONC212 also bound GPCRs. ONC212 did not engage DRD2 but instead specifically engaged the orphan GPCR GPR132, which is a putative tumor suppressor when activated. Interestingly, expression of GPR132 mRNA was highly correlated with sensitivity to ONC212. Furthermore, GPR132 overexpression induced cell death and ONC212 treatment induced GPR132 mRNA expression in AML cell lines, suggesting that GPR132 could be a potential therapeutic target in hematological malignancies.Taken together, ONC212, and its putative target GPR132, represent a promising novel anti-cancer strategy for AML therapy that warrants further development. DisclosuresPrabhu:Oncoceutics: Employment, Equity Ownership. Allen:Oncoceutics: Employment, Equity Ownership, Patents & Royalties. Oster:Oncoceutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Stogniew:Oncoceutics: Employment. Andreeff:Daiichi Sankyo: Consultancy.