Combination Of ABT-737 Research Articles

Integrated stress response activation induced by usnic acid alleviates BCL-2 inhibitor ABT-199 resistance in acute myeloid leukemia

IntroductionABT-199 (venetoclax) is a BCL-2 suppressor with pronounced effects on acute myeloid leukemia (AML). However, its usefulness as a monotherapy or in combination with hypomethylating medicines like azacitidine is debatable due to acquired resistance. Usnic acid, a dibenzofuran extracted from lichen Usnea diffracta Vain, exhibits anticancer properties and may counteract multidrug resistance in leukemia cells. ObjectiveThis study investigated whether usnic acid at low-cytotoxicity level could enhance sensitivity of AML cells with acquired resistance to ABT-199 by targeting the integrated stress response pathways. MethodsTo investigate the combined effects on AML cells, we used a cell viability test, flow cytometry to quantify apoptosis, cell cycle analysis, and mitochondrial membrane potential measurement. RNA-seq and immunoblot were used to determine the potential mechanisms of ABT-199 + usnic acid combination. ResultsUsnic acid, at a low cytotoxicity level, successfully restored ABT-199 sensitivity in AML cell lines that had developed ABT-199 resistance and increased ABT-199′s antileukemic activity in a xenograft model. Mechanistically, the combination of usnic acid and ABT-199 cooperated to boost the expression of the integrated stress response (ISR)-associated genes ATF4, CHOP, and NOXA through the heme-regulated inhibitor kinase (HRI), while also promoting the degradation of the anti-apoptotic protein MCL-1. ISRIB, a compound that blocks the ISR, was able to reverse the growth suppression and cell death, the increase in expression of genes related with the ISR, and the inhibition of MCL-1 protein caused by combination therapy. Additionally, the downregulation of MCL-1 was linked to an increase in MCL-1 phosphorylation at serine 159 and subsequent destruction by the proteasome. ConclusionIn summary, usnic acid improves chemosensitivity to ABT-199 by triggering the integrated stress response, leading to decreased levels of MCL-1 protein, suggesting a potential treatment for AML cases resistant to Bcl-2 inhibitors.

The Effects of ABT-199 and Dihydroartemisinin Combination on Cell Growth and Apoptosis in Human U937 and KG-1 Cancer Cells.

Change in the balance of Bcl-2 family proteins is one of the main reasons for resistance of tumor cells to ABT-199. In this study, the effect of dihydroartemisinin on cell growth, apoptosis and sensitivity of the AML cells to ABT-199 was investigated. Cell proliferation and survival were assessed by trypan blue staining and MTT assay, respectively. Cell apoptosis was measured by Hoechst 33342 staining and caspase-3 activity assay. The expression levels of Bcl-2, Mcl-1 and Bax mRNA were tested by qRT-PCR. Our data showed that combination therapy significantly reduced the IC50 value and synergistically decreased the AML cell survival and growth compared with dihydroartemisinin or ABT-199 alone. Treatment with each of ABT-199 or dihydroartemisinin alone clearly enhanced the Bax mRNA expression and inhibited the expression of Mcl-1 and Bcl-2 mRNA. Inhibition of Mcl-1 mRNA by dihydroartemisinin was associated with enhancement of apoptosis induced by ABT-199 in AML cells. In conclusion, dihydroartemisinin not only triggers the intrinsic pathway of apoptosis, but also can increase the sensitivity of the AML cells to ABT-199 via suppression of Mcl-1 expression.

Pre-Clinical Evaluation of a New-Generation Orally Bioavailable Dual Bcl-2/Bcl-Xl Inhibitor LP-118 in Mantle Cell Lymphoma

Introduction Mantle cell lymphoma (MCL) is an aggressive form of non-Hopkins lymphoma with poor prognosis. The current frontline therapy of MCL is immunochemotherapy. For relapsed and refractory MCL, BTK inhibitor (BTKi) has become the standard of care, however, relapse is common after treatment with BTKi. Therefore, novel treatments with different mechanisms of action are needed for MCL patients. LP-118 is a new generation dual Bcl-2/Bcl-xL inhibitor with potent activity against wildtype and mutant Bcl-2 and moderate activity against Bcl-xL, and it is currently in Phase 1 clinical trials for multiple types of hematological cancers and solid tumors. In this study, we evaluated the preclinical activity of LP-118 in two MCL cell lines, REC-1 and Granta519, in vitro and in vivo. We also screened multiple combinations of LP-118 plus chemotherapeutic or targeted agents, in order to explore the optimal drug combinations for MCL treatment. Methods In vitro cell viability assay was performed with CellTiterGlo method in MCL cell lines REC-1 and Granta519 to examine cellular activity of LP-118. To determine the synergy of the combinations of LP-118 with standard of care agents, matrix combinations were performed with different concentrations of LP-118 and different drugs including vincristine, bendamustine, cytarabine, docetaxel, doxorubicin, cisplatin, and bortezomib. The data were analyzed using SynergyFinder (2.0), an online program for synergy scores. For in vivo experiments, Granta519 and REC-1-derived xenograft models were established and treated with different regimens: for Granta519 model, mice were treated with vehicle, BR (bendamustine, 25mg/kg, iv, day 1 and rituximab, 10mg/kg, iv, day 1), LP-118 (75mg/kg, po, qd, 28 days) in combination with BR. For REC-1 models, mice were treated with vehicle, LP-118 (50mg/kg, po, qd, 28 days) plus a BTK inhibitor LP-168 (25mg/kg, po, bid, 28 days), or ABT-199 (50mg/kg, po, qd, 28 days) plus ibrutinib (25mg/kg, po, qd,13 days, then bid, 28 days), and tumor growth/remission were examined to determine the in vivo anticancer activity. Results LP-118 is highly active in cell viability assays with Granta519 and REC-1 cells, with IC 50 of 0.6 nM and 1.96 nM, respectively. And compared to venetoclax, LP-118 is 8 to18 times more potent in REC-1 and Granta519 cells. In the matrix combination studies with REC-1 cells, combinations of LP-118 with SOC agents showed synergistic anticancer effects with all the tested drugs, including vincristine, bendamustine, cytarabine, docetaxel, doxorubicin, cisplatin, and bortezomib. Moreover, the combination of LP-118 with BR induced complete tumor regression of Grana519 xenografts in all mice (7/7) at day 45 with no significant body weight changes, whereas no complete tumor regression was observed in the BR group, indicating that combination of LP-118 with BR was more effective than the treatment with BR alone. Similarly, the combination of LP-118 with LP-168 (BTKi) also resulted in complete tumor regression in REC-1 xenograft models at day 40, while the combination of ABT-199 plus ibrutinib showed no significant tumor growth inhibition, suggesting that combination of LP-118 and LP-168, a new generation BTKi currently also in clinical trial, might be more effective against MCL than the combination of ABT-199 plus ibrutinib in clinic. Conclusion LP-118 showed encouraging anti-MCL activity as single agent and in combination with standard of care agents in vitro and in vivo, warranting further evaluation of these combinations in clinical trials.

Combination Therapies Targeting Apoptosis in Paediatric AML: Understanding the Molecular Mechanisms of AML Treatments Using Phosphoproteomics.

Paediatric acute myeloid leukaemia (AML) continues to present treatment challenges, as no "standard approach" exists to treat those young patients reliably and safely. Combination therapies could become a viable treatment option for treating young patients with AML, allowing multiple pathways to be targeted. Our in silico analysis of AML patients highlighted "cell death and survival" as an aberrant, potentially targetable pathway in paediatric AML patients. Therefore, we aimed to identify novel combination therapies to target apoptosis. Our apoptotic drug screening resulted in the identification of one potential "novel" drug pairing, comprising the Bcl-2 inhibitor ABT-737 combined with the CDK inhibitor Purvalanol-A, as well as one triple combination of ABT-737 + AKT inhibitor + SU9516, which showed significant synergism in a series of paediatric AML cell lines. Using a phosphoproteomic approach to understand the apoptotic mechanism involved, proteins related to apoptotic cell death and cell survival were represented, in agreement with further results showing differentially expressed apoptotic proteins and their phosphorylated forms among combination treatments compared to single-agent treated cells such upregulation of BAX and its phosphorylated form (Thr167), dephosphorylation of BAD (Ser 112), and downregulation of MCL-1 and its phosphorylated form (Ser159/Thr 163). Total levels of Bcl-2 were decreased but correlated with increased levels of phosphorylated Bcl-2, which was consistent with our phosphoproteomic analysis predictions. Bcl-2 phosphorylation was regulated by extracellular-signal-regulated kinase (ERK) but not PP2A phosphatase. Although the mechanism linking to Bcl-2 phosphorylation remains to be determined, our findings provide first-hand insights on potential novel combination treatments for AML.

Targeting anti-apoptotic pathways eliminates senescent melanocytes and leads to nevi regression

Human melanocytic nevi (moles) result from a brief period of clonal expansion of melanocytes. As a cellular defensive mechanism against oncogene-induced hyperplasia, nevus-resident melanocytes enter a senescent state of stable cell cycle arrest. Senescent melanocytes can persist for months in mice and years in humans with a risk to escape the senescent state and progress to melanoma. The mechanisms providing prolonged survival of senescent melanocytes remain poorly understood. Here, we show that senescent melanocytes in culture and in nevi express high level of the anti-apoptotic BCL-2 family member BCL-W but remain insensitive to the pan-BCL-2 inhibitor ABT-263. We demonstrate that resistance to ABT-263 is driven by mTOR-mediated enhanced translation of another anti-apoptotic member, MCL-1. Strikingly, the combination of ABT-263 and MCL-1 inhibitors results in synthetic lethality to senescent melanocytes, and its topical application sufficient to eliminate nevi in male mice. These data highlight the important role of redundant anti-apoptotic mechanisms for the survival advantage of senescent melanocytes, and the proof-of-concept for a non-invasive combination therapy for nevi removal.

Synergistic cytotoxicity of perifosine and ABT-737 to colon cancer cells.

An acidic environment and hypoxia within the tumour are hallmarks of cancer that contribute to cell resistance to therapy. Deregulation of the PI3K/Akt pathway is common in colon cancer. Numerous Akt-targeted therapies are being developed, the activity of Akt-inhibitors is, however, strongly pH-dependent. Combination therapy thus represents an opportunity to increase their efficacy. In this study, the cytotoxicity of the Akt inhibitor perifosine and the Bcl-2/Bcl-xL inhibitor ABT-737 was tested in colon cancer HT-29 and HCT-116 cells cultured in monolayer or in the form of spheroids. The efficacy of single drugs and their combination was analysed in different tumour-specific environments including acidosis and hypoxia using a series of viability assays. Changes in protein content and distribution were determined by immunoblotting and a "peeling analysis" of immunohistochemical signals. While the cytotoxicity of single agents was influenced by the tumour-specific microenvironment, perifosine and ABT-737 in combination synergistically induced apoptosis in cells cultured in both 2D and 3D independently on pH and oxygen level. Thus, the combined therapy of perifosine and ABT-737 could be considered as a potential treatment strategy for colon cancer.

Sensitization effect of kaempferol from persimmon leaves on HepG2 hepatoma cells with ABT-199 resistance and its molecular mechanisms.

ABT-199 (venetoclax) is the first-in-class selective B-cell lymphoma 2 (BCL2) inhibitor, which is known to be ineffective towards liver cancer cells. Here, we investigated the efficacy and the underlying molecular processes of the sensitization effect of kaempferol isolated from persimmon leaves (KPL) on the ABT-199-resistant HepG2 cells. The effects of various doses of KPL coupled with ABT-199 on the proliferation of HepG2 cells and on the H22 liver tumor-bearing mouse model were examined, as well as the underlying mechanisms. Our findings showed that ABT-199 alone, in contrast to KPL, had no significant impact on hepatoma cell growth, both in vitro and in vivo. Interestingly, the combination therapy showed significantly higher anti-hepatoma efficacy. Mechanistic studies revealed that combining KPL and ABT-199 may promote both early and late apoptosis, as well as decrease the mitochondrial membrane potential in HepG2 cells. Western blot analysis showed that combination of KPL and ABT-199 significantly reduced the expression of the anti-apoptotic proteins Bcl-2, Bcl-xL, and Mcl-1, raised the expression of Bax and cleaved caspase 3, and enhanced cytochrome C release and Bax translocation. Therefore, KPL combined with ABT-199 has a potential application prospect in the treatment of hepatocellular carcinoma.

IFN-γ enhances CLL cell resistance to ABT-199 by regulating MCL-1 and BCL-2 expression via the JAK-STAT3 signaling pathway

Although clinical outcomes of CLL have improved with the use of BCL-2 inhibitor, ABT-199, acquired resistance eventually occurs in many cases, which leads to CLL disease progression. Thus, understanding the mechanisms that mediate this relapse is important to design improved therapies. Herein, we report that cytokine IFN-γ, secreted by dysfunctional T cells, enhanced CLL cells resistance to ABT-199. IFN-γ stimulation significantly increased the expression of BCL-2, MCL-1 and BCL-xL. Blocking JAK1/2-STAT3 signaling pathway impaired the expression of these anti-apoptotic proteins after IFN-γ stimulation. The combination of ABT-199 with JAK1/2 inhibitor Ruxolitinib or STAT3 inhibitors Stattic and C188-9 increased malignant B cell death. In summary, we show that IFN-γ enhanced CLL cells resistance to ABT-199 at least in part by up-regulating BCL-2, MCL-1 and BCL-xL expression via JAK1/2-STAT3 pathway, and thus blocking this pathway with inhibitors increased ABT-199 efficiency to induce CLL cell apoptosis, suggesting a potential therapeutically relevant combination to overcome ABT-199 resistance.

Combination of ruxolitinib with ABT-737 exhibits synergistic effects in cells carrying concurrent JAK2V617F and ASXL1 mutations.

The V617F mutation in Janus kinase 2 is considered one of the driver mutations leading to Philadelphia-negative myeloproliferative neoplasms (MPNs). Concurrent JAK2V617F and ASXL1 mutations accelerate the progression of myelofibrosis in patients with MPNs. Few therapies are currently available for patients with these two mutations. In our study, the combination of ruxolitinib with ABT-737 was evaluated in cells carrying JAK2V617F and ASXL1 double mutations. RNA sequencing indicated overactivated oxidative phosphorylation in JAK2V617F;Asxl1+/- cKit+ cells. The cell line model with JAK2V617F and ASXL1 double mutations (HEL-AKO cells) also exhibited dysregulated mitochondrial function with an increase in the reactive oxygen species levels and a decrease in the ATP levels. The colony growth inhibition rates of cells with JAK2V617F and ASXL1 double mutations were significantly lower than those of cells with only the JAK2V617F mutation. Combined treatment with ruxolitinib and ABT-737 promoted apoptosis and inhibited the proliferation of HEL-AKO cells. Cotreatment with the two drugs also inhibited the growth of bone marrow mononuclear cells isolated from patients with concurrent JAK2V617F and ASXL1 mutations. In conclusion, we provide preclinical evidence showing that the combination of ruxolitinib and ABT-737 is a promising therapeutic strategy for MPN patients with concurrent JAK2V617F and ASXL1 mutations.

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.

Abstract 1797: Bcl-2 up-regulation mediates taxane resistance downstream of APC loss

Abstract Patients with triple negative breast cancer (TNBC) are treated with traditional chemotherapy, such as paclitaxel (PTX). Despite the efficacy of taxanes, many tumors will recur due to drug resistance. Therefore, the need to understand the mechanism behind drug resistance is critical to improve patient outcome and survival. Our lab was the first to show that loss of the Adenomatous Polyposis Coli (APC) tumor suppressor caused resistance to PTX in the MDA-MB-157 human TNBC cell line. In the absence of APC, apoptosis induction was decreased, as measured through cleaved caspase 3 and annexin/PI staining. To understand the molecular mechanisms behind APC-mediated PTX response, we analyzed the BCL-2 family of proteins and found a robust increase of the pro-survival family member, Bcl-2. The BH3 mimetic, ABT-199 (Venetoclax), which specifically targets Bcl-2, has been used as a single or combination therapy in multiple hematologic malignancies. In addition, ABT-199 has shown promise in multiple subtypes of breast cancer. Therefore, we used ABT-199 in combination with PTX to address the hypothesis that APC-induced Bcl-2 increase is responsible for PTX resistance. Combination treatment in three CRISPR-mediated APC knockout TNBC cell lines (MDA-MB-157, MDA-MB-231, and SUM159) caused changes in cell proliferation and apoptosis. Combined, these data suggest restored sensitivity to PTX using ABT-199. Our studies are the first to show that Bcl-2 inhibition can restore PTX-sensitivity in APC mutant breast cancer cells. These studies are critical to advance better treatment regimens in patients with TNBC. Citation Format: Sara Maloney, Sarah Zabala, Grace Richmond, Jenifer R. Prosperi. Bcl-2 up-regulation mediates taxane resistance downstream of APC loss [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 1797.

Targeting PI3K/AKT/mTOR pathway to enhance the anti-leukemia efficacy of venetoclax

BackgroundThe treatment of acute myeloid leukemia (AML) is developing towards “targeted therapy”, which faces challenges such as low sensitivity and drug resistance. Therefore, targeted drugs need to be used in combination with other drugs to overcome clinical problems. ObjectiveAML cells and animal models were used to determine the synergistic anti-leukemic effect of Dactolisib (BEZ235) and Venetoclax (ABT199) and explore its mechanism. MethodsIn vitro experiments, we used cell counting kit-8 (CCK8), flow cytometry, real-time quantitative PCR (qPCR), and Western blot to detect the anti-leukemic effects of ABT199 and BEZ235. In vivo experiments, female nude mice were injected subcutaneously with THP-1 cells to form tumors, evaluate the combined effect of ABT199 and BEZ235 by indicators such as tumor size, tumor weight, Ki67 and cleaved-Caspase3 staining. The mice's body weight and HE staining were used to evaluate the liver injury and adverse drug reactions. ResultsThe combination of BEZ235 and ABT199 has a synergistic effect through promoting apoptosis and inhibiting proliferation. The BEZ235 increased the drug sensitivity of ABT199 by reducing the MCL-1 protein synthesis and promoted the degradation of MCL-1 protein, which is considered as the mechanism of reversing ABT199 resistance. Furthermore, the BEZ235 and ABT199 can synergistically enhance the inhibition of PI3K/AKT/mTOR pathway. ConclusionThe combination of BEZ235 and ABT199 exhibits a synergistic anti-tumor effect in AML by down-regulating MCL-1 protein.

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.

CREB/Sp1-mediated MCL1 expression and NFκB-mediated ABCB1 expression modulate the cytotoxicity of daunorubicin in chronic myeloid leukemia cells

Although some studies have hinted at the therapeutic potential of daunorubicin (DNR) in chronic myeloid leukemia (CML), the mechanism by which DNR induces CML cell death is unclear. Therefore, this study aimed to investigate DNR-induced cell death signaling pathways in CML cell lines K562 and KU812. DNR-triggered apoptosis in K562 cells was characterized by inhibition of MCL1 expression, while restoration of MCL1 expression protected K562 cells from DNR-mediated cytotoxicity. In addition, DNR induced NOX4-dependent ROS production, leading to the activation of p38 MAPK and inactivation of Akt and ERK. Activated p38 MAPK stimulated protein phosphatase 2A-dependent dephosphorylation of CREB. Since Akt-mediated activation of ERK reduced β-TrCP mRNA stability, the inactivation of Akt-ERK axis increased β-TrCP expression, which in turn promoted proteasomal degradation of Sp1. Inhibition of CREB phosphorylation and Sp1 expression simultaneously reduced MCL1 transcription and protein expression. DNR-induced MCL1 suppression was not reliant on its ability to induce DNA damage. In addition, DNR induced the expression of drug exporter ABCB1 in K562 cells through the p38 MAPK/NFκB-mediated pathway, while imatinib or ABT-199 inhibited the DNR-induced effect. The combination of imatinib or ABT-199 with DNR showed synergistic cytotoxicity in K562 cells by increasing intracellular DNR retention. Cumulatively, our data indicate that DNR induces MCL1 downregulation in K562 cells by promoting p38 MAPK-mediated dephosphorylation of CREB and inhibiting the Akt-ERK axis-mediated Sp1 protein stabilization. Furthermore, experimental evidence indicates that DNR-induced death of KU812 cells occurs through a similar pathway.

Novel Irreversible Menin Inhibitor, BMF-219, Shows Potent Single Agent Activity in Clinically Relevant DLBCL Cells

Novel Irreversible Menin Inhibitor, BMF-219, Shows Potent Single Agent Activity in Clinically Relevant DLBCL Cells

AXL/MERTK inhibitor ONO-7475 potently synergizes with venetoclax and overcomes venetoclax resistance to kill FLT3-ITD acute myeloid leukemia.

FMS-like Tyrosine Kinase 3 (FLT3) mutation is associated with poor survival in acute myeloid leukemia (AML). The specific Anexelekto/MER Tyrosine Kinase (AXL) inhibitor, ONO-7475, kills FLT3-mutant AML cells with targets including Extracellular-signal Regulated Kinase (ERK) and Myeloid Cell Leukemia 1 (MCL1). ERK and MCL1 are known resistance factors for Venetoclax (ABT-199), a popular drug for AML therapy, prompting the investigation of the efficacy of ONO-7475 in combination with ABT-199 in vitro and in vivo. ONO-7475 synergizes with ABT-199 to potently kill FLT3-mutant acute myeloid leukemia cell lines and primary cells. ONO-7475 is effective against ABT-199-resistant cells including cells that overexpress MCL1. Proteomic analyses revealed that ABT-199-resistant cells expressed elevated levels of pro-growth and anti-apoptotic proteins compared to parental cells, and that ONO-7475 reduced the expression of these proteins in both the parental and ABT-199-resistant cells. ONO-7475 treatment significantly extended survival as a single in vivo agent using acute myeloid leukemia cell lines and PDX models. Compared to ONO-7474 monotherapy, the combination of ONO-7475/ABT-199 was even more potent in reducing leukemic burden and prolonging the survival of mice in both model systems. These results suggest that the ONO-7475/ABT-199 combination may be effective for AML therapy.

Lead optimization of novel quinolone chalcone compounds by a structure\u2013activity relationship (SAR) study to increase efficacy and metabolic stability

Many agents targeting the colchicine binding site in tubulin have been developed as potential anticancer agents. However, none has successfully made it to the clinic, due mainly to dose limiting toxicities and the emergence of multi-drug resistance. Chalcones targeting tubulin have been proposed as a safe and effective alternative. We have shown previously that quinolone chalcones target tubulin and maintain potent anti-proliferative activity vis-à-vis colchicine, while also having high tolerability and low toxicity in mouse models of cancer and refractivity to multi-drug resistance mechanisms. To identify the most effective anticancer chalcone compound, we synthesized 17 quinolone–chalcone derivatives based on our previously published CTR-17 and CTR-20, and then carried out a structure–activity relationship study. We identified two compounds, CTR-21 [((E)-8-Methoxy-3-(3-(2-methoxyphenyl)-3-oxoprop-1-enyl) quinolin-2(1H)-one)] and CTR-32 [((E)-3-(3-(2-ethoxyphenyl)-3-oxoprop-1-enyl) quinolin-2(1H)-one)] as potential leads, which contain independent moieties that play a significant role in their enhanced activities. At the nM range, CTR-21 and CTR-32 effectively kill a panel of different cancer cells originated from a variety of different tissues including breast and skin. Both compounds also effectively kill multi-drug resistant cancer cells. Most importantly, CTR-21 and CTR-32 show a high degree of selectivity against cancer cells. In silico, both of them dock near the colchicine-binding site with similar energies. Whereas both CTR-21 and CTR-32 effectively prevents tubulin polymerization, leading to the cell cycle arrest at G2/M, CTR-21 has more favorable metabolic properties. Perhaps not surprisingly, the combination of CTR-21 and ABT-737, a Bcl-2 inhibitor, showed synergistic effect in killing cancer cells, since we previously found the “parental” CTR-20 also exhibited synergism. Taken together, CTR-21 can potentially be a highly effective and relatively safe anticancer drug.

Abstract 1398: Impact of proteasome inhibitor specificity and efficacy on apoptosis induction by combination with ABT-199

Abstract Metastatic Soft-Tissue Sarcomas (STS) are a rare and highly heterogeneous group of mesenchymal malignancies that carry a poor prognosis. Therapy is hampered by a limited number of effective treatments, and the almost non-existing long-term survival rate illustrates the need of effective targeted treatment. As a potential approach, we investigated combination of the clinically approved BH3-mimetic drug ABT-199 with different proteasome inhibitors (PIs): Bortezomib, Carfilzomib and Ixazomib, each with proven efficacy, e.g., in multiple myeloma. ABT-199 selectively inhibits the anti-apoptotic protein Bcl-2 while Bortezomib (BOZ) and Ixazomib (IXZ) bind reversible to the β1 and β5 subunits of the 20S proteasome and Carfilzomib (CFZ) irreversibly blocks the subunits β2 and β5. We investigated whether the combination of ABT-199 with CFZ or IXZ shows synergistic activity as recently published for BOZ. SW982 sarcoma cells were cultured with PIs alone or in combination with ABT-199. Cell death was detected by flow cytometric analysis of mitochondrial membrane potential (TMRM) and exposure of phosphatidyl serine (Annexin V). To elucidate potential differences due to specificity of PIs, we analyzed expression of BCL-2 family proteins by Western Blot and performed analogue experiments in knock-out (BAXKO, BAKKO, BOKKO) cell lines. In combination, ABT-199&BOZ or ABT-199&CFZ showed comparable synergistic cell death induction in SW982 while ABT-199&IXZ less efficiently induced cell death. Engineered SW982 knock-out cell lines suggests specific relevance of BOK in IXZ induced apoptosis, whereas all tested PIs crucially depend on BAX for apoptosis induction in combination with ABT-199. Also, as shown for ABT-199&BOZ, both CFZ and IXZ, alone and in combination with ABT-199, efficiently induced expression of NOXA. Strikingly, and in line with augmented cell death induction, ABT-199&CFZ resulted in highest expression of NOXA as compared to BOZ and IXZ. ABT-199&PIs synergistically induce apoptotic cell death in SW982 and corresponding knock-out cell lines with CFZ showing exacerbated expression of NOXA. ABT-199&CFZ also induced strongest stabilization of p53, a transcriptional key regulator of NOXA. We suggest that simultaneous inhibition of anti-apoptotic BCL-2 by ABT-199 and the enhanced expression of NOXA double-hit on the BCL-2 and the MCL-1 signaling axis force the cells over the threshold of apoptosis. Enhanced efficacy of CFZ is dually caused by reduced degradation of NOXA and enhanced stabilization of p53, which in turn transactivates expression of NOXA. In conclusion, PI specificity and efficacy modulate apoptosis induction by combination with ABT-199. Future efforts, e.g. knock-out of relevant BH3-only proteins, will elucidate the observed variable efficacy of these and other PIs for prospective translation in clinical applications. Citation Format: Sandra Weller, Benjamin Schaefer, Tobias Beigl, Kathrin Böpple, Walter E. Aulitzky, Hans-Georg Kopp, Frank Essmann. Impact of proteasome inhibitor specificity and efficacy on apoptosis induction by combination with ABT-199 [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 1398.

Abstract 1946: Synergistic cell death induction in breast cancer cell lines by combining ABT-199 and cisplatin irrespective of estrogen receptor and p53 expression

Abstract 50 Years after the generation of the most studied human breast cancer cell line, MCF-7, many research results fundamentally changed patient outcomes for the better. The mortality of breast cancer in women has decreased significantly, especially in the last 30 years. But the total number of deaths is rising again in the last ten years. Considering that breast cancer is the most common cancer in women and the second most common cancer overall, with incidences of 24,2% and 11,6%, respectively, total numbers of new cases are in the millions every year. First line drugs for the treatment of breast cancer often target microtubules (e.g. Vinblastine and Paclitaxel) or induce DNA damage (e.g. Carboplatin and Cisplatin (CisPt)). In addition to these classic therapeutic drugs new compounds that specifically target cancer-driving molecular alterations have been developed. Among these is the group of BH3-mimetics. The efficacy of several BH3-mimetics is investigated in numerous clinical trials while the BCL-2 specific inhibitor ABT-199 (Venetoclax) is already approved for clinical application in chronic lymphatic leukemia (CLL), acute myeloid leukemia (AML) and small lymphocytic lymphoma (SLL). BH3-mimetics target the apoptosis machinery by interacting with anti-apoptotic Bcl-2 proteins, mimicking the function of pro-apoptotic BH3-only proteins. In turn, the pro-apoptotic potential of pore-forming BCL-2 effectors BAX, BAK and BOK is released inducing mitochondrial outer membrane permeabilization and cell death. Recently we reported synergistic cell death induction by ABT-199 in combination with the proteasome inhibitor Bortezomib (BOZ) in soft tissue sarcoma (STS) cells. ABT-199 and BOZ induce accumulation of BOK and the BH3-only protein NOXA (Muenchow et al. 2020). Here, we combined ABT-199, CisPt and Nutlin-3 in breast cancer cell lines that differ in expression of the estrogen receptor (ER) and p53 activity. Similar to the results in STS cells synergistic induction of apoptotic cell death was observed by FACS analysis of the mitochondrial membrane potential (TMRM) and exposure of phosphatidyl serine (Annexin-V). Western blot analysis revealed that ABT-199 induced accumulation of NOXA and MCL-1, irrespective of the ER status or p53 mutation. Simultaneous treatment with ABT-199 and CisPt or Nutlin-3 further increased expression of NOXA and MCL-1. Interestingly, qRT-PCR revealed enhanced expression of NOXA and MCL-1 mRNA in ABT-199 treated cells indicating transcriptional regulation. However, transcriptional induction of NOXA was also detected independent of p53 activity. Thus, combined therapeutic approaches using CisPt and ABT-199 should be effective irrespective of ER and p53 expression. Nevertheless, in p53 proficient tumors Nutlins might increase therapeutic efficacy. Citation Format: Benjamin Schaefer, Sandra Weller, Tobias Beigl, Kathrin Boepple, Reiner Hoppe, Klaus Schulze-Osthoff, Hans-Georg Kopp, Walter E. Aulitzky, Frank Essmann. Synergistic cell death induction in breast cancer cell lines by combining ABT-199 and cisplatin irrespective of estrogen receptor and p53 expression [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 1946.

BCL-2 Inhibition with ABT-199 Shows Efficacy in AML Cell Lines with Synergistic Effects upon Addition of the MEK Inhibitor GDC-0973 and Prolongs Survival in an NAS/BCL-2-Mediated Mouse Model of AML Post-MDS

Background and aims: BCL-2 activation plays a role in the progression of MDS to AML and BCL-2 inhibition may be a therapeutic target in such patients. ABT-199 is a small molecule which specifically inhibits BCL-2, approved for the treatment of CLL with TP53 mutation/17p deletion. Using the OCI-AML2 and MOLM13 AML cell lines, ABT-199 resistant cells have been derived to investigate the mechanism of drug resistance. The RAS/BCL-2 complex has been previously shown to co-localize in the mitochondria (Mito) in AML post-MDS mice and patients (Le Pogam Leuk Res 2013). To test the hypothesis that increased MCL-1 conferred ABT-199 resistance, OCI-MCL3 conditional lines in which MCL-1 could be switched on and off were studied for apoptosis and RAS/BCL-2 co-localization. In combination with our double transgenic mouse AML post-MDS preclinical model MRP8[NRASD12/BCL2] (Beurlet Blood 2013), we assessed the effect of ABT-199, and the MEK inhibitor GDC-0973.Methods: The OCI-AML2 or MOLM13 ABT-199 resistant cell lines were transfected with either a control GFP (CON) or MCL-1-GFP tetracycline (OFF) inducible constructs and after treating overnight with doxycycline (DOX) (100 ng/ml) were incubated with a) no treatment, b) ABT-199 (1 µM) (Abbvie), c) GDC-0973 (1 µM) (Genentech) or d) ABT-199+GDC-0973 and a reagent for apoptosis, annexin V conjugated with a cyanine fluorescent red dye. Viability and cell death were followed using the Incucyte® Zoom System (Essen Bioscience). For RAS/BCL-2 co-localization studies, cells were labeled using antibodies specific for RAS (Alexa 647), BCL-2 (TRITC), Mito (Tom-20- Alexa 488) and plasma membrane (PM) (Ezrin-Alexa 488). In the MRP8[hNRASD12/hBCL2] model mice were treated at 3 weeks of age (13 vehicle, 18 ABT-199 (75mg/kg), 4 GDC-0973 (10mg/kg) and 6 ABT-199+GDC-0973) 3 times weekly by oral gavage for 33 days and followed for survival.Results: Both OCI-AML2 ABT-199 sensitive and resistant cells had the RAS/BCL-2 complex localized primarily in the Mito. However, after ABT-199 treatment in the sensitive line the RAS/BCL-2 complex was still found in the Mito, whereas in the resistant line the complex was found in the PM. The OCI/GFP control (CON) cells showed resistance to ABT-199 with similar annexin V staining compared to untreated cells indicative of no change in apoptosis with similar viability in all conditions. The MCL-1 lines showed sensitivity to ABT-199 when MCL-1 was OFF (+DOX) and resistance when ON (-DOX) with an increased viability in the latter (Fig. 1A&B) and increased apoptosis in the former (Fig. 1C&D). The immunofluorescence profiles are summarized in Table 1, Fig. E&F. In the CON lines with no treatment, ABT-199 alone, ABT-199+GDC-0973 and the MCL-1 OFF cells (+ DOX), untreated and ABT-199 treated and the MCL-1 ON untreated cells the RAS/BCL-2 complex localized primarily in the Mito with a minority of PM localization. With GDC-0973 treatment of the CON and MCL-1 OFF lines or with the combined treatment of ABT-199+GDC-0973 of the MCL-1 OFF line and the MCL-1 ON treated with ABT-199 the complex was found equally in the Mito and PM. In the MCL-1 ON cells treated with GDC-0973 there was more complex in the PM than the Mito; with the combination of ABT-199+GDC-0973 no complex was detected, suggesting the clearance of the cells with the RAS/BCL-2 complex, which may explain the reduced viability and apoptosis profiles (Figure 1). The yellow staining cells in these panels were consistent with BCL-2 localizing in the PM and the Mito. The Mito localization of the RAS/BCL-2 complex indicates disease profile whereas PM localization resistance; regulation of MCL-1 appears to increase PM localization and increased resistance to ABT-199 or GDC-0973.Survival of mice treated with ABT-199 was significantly longer (median 30 days) than those of untreated or vehicle (median 10 days) treated mice (p<0.008), underscoring drug efficacy. GDC-0973-treated mice died after 3-8 administrations at the same time as the vehicle-treated mice. Five of 6 mice treated with the combination of ABT-199+GDC0973 all survived until the end of treatment (15 administrations for 33 days), suggesting that the combination of ABT-199 and GDC-0973 extends lifespan (survival>75 days).Conclusion:ABT-199 is effective in both in vitro AML lines and in vivo AML mice. The MEK inhibitor GDC-0973 synergises with ABT-199 in vitro and in vivo. RAS/BCL-2 co-localization may be a useful biomarker of response to treatments. [Display omitted] DisclosuresPadua:Abbvie: Research Funding; Genetech: Research Funding. Fenaux:Novartis: Honoraria, Research Funding; Astex: Honoraria, Research Funding; Astex: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Amgen: Honoraria, Research Funding.