ABSTRACT
The BCL2 inhibitor venetoclax promotes apoptosis in blood cancer cells and is approved for treatment of chronic lymphocytic leukemia and acute myeloid leukemia. However, multiple myeloma cells are frequently more dependent on MCL-1 for survival, conferring resistance to venetoclax. Here we report that mevalonate pathway inhibition with statins can overcome resistance to venetoclax in multiple myeloma cell lines and primary cells. In addition, statins sensitize to apoptosis induced by MCL-1 inhibitor, S63845. In retrospective analysis of venetoclax clinical studies in multiple myeloma, background statin use was associated with a significantly enhanced rate of stringent complete response and absence of progressive disease. Statins sensitize multiple myeloma cells to venetoclax by upregulating two proapoptotic proteins: PUMA via a p53-independent mechanism and NOXA via the integrated stress response. These findings provide rationale for prospective testing of statins with venetoclax regimens in multiple myeloma. SIGNIFICANCE: BH3 mimetics including venetoclax hold promise for treatment of multiple myeloma but rational combinations are needed to broaden efficacy. This study presents mechanistic and clinical data to support addition of pitavastatin to venetoclax regimens in myeloma. The results open a new avenue for repurposing statins in blood cancer.
Subject(s)
Antineoplastic Agents , Hematologic Neoplasms , Hydroxymethylglutaryl-CoA Reductase Inhibitors , Multiple Myeloma , Humans , Myeloid Cell Leukemia Sequence 1 Protein , Multiple Myeloma/drug therapy , Proto-Oncogene Proteins c-bcl-2/genetics , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Retrospective Studies , Prospective Studies , Antineoplastic Agents/pharmacology , Hematologic Neoplasms/drug therapyABSTRACT
Myelodysplastic syndromes (MDS) are heterogeneous neoplastic disorders of hematopoietic stem cells (HSCs). The current standard of care for patients with MDS is hypomethylating agent (HMA)-based therapy; however, almost 50% of MDS patients fail HMA therapy and progress to acute myeloid leukemia, facing a dismal prognosis due to lack of approved second-line treatment options. As cancer stem cells are the seeds of disease progression, we investigated the biological properties of the MDS HSCs that drive disease evolution, seeking to uncover vulnerabilities that could be therapeutically exploited. Through integrative molecular profiling of HSCs and progenitor cells in large patient cohorts, we found that MDS HSCs in two distinct differentiation states are maintained throughout the clinical course of the disease, and expand at progression, depending on recurrent activation of the anti-apoptotic regulator BCL-2 or nuclear factor-kappa B-mediated survival pathways. Pharmacologically inhibiting these pathways depleted MDS HSCs and reduced tumor burden in experimental systems. Further, patients with MDS who progressed after failure to frontline HMA therapy and whose HSCs upregulated BCL-2 achieved improved clinical responses to venetoclax-based therapy in the clinical setting. Overall, our study uncovers that HSC architectures in MDS are potential predictive biomarkers to guide second-line treatments after HMA failure. These findings warrant further investigation of HSC-specific survival pathways to identify new therapeutic targets of clinical potential in MDS.
Subject(s)
Bridged Bicyclo Compounds, Heterocyclic , Myelodysplastic Syndromes , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Hematopoietic Stem Cells/pathology , Humans , Myelodysplastic Syndromes/drug therapy , Myelodysplastic Syndromes/genetics , Myelodysplastic Syndromes/pathology , Proto-Oncogene Proteins c-bcl-2/genetics , SulfonamidesABSTRACT
Despite high initial response rates, acute myeloid leukemia (AML) treated with the BCL-2-selective inhibitor venetoclax (VEN) alone or in combinations commonly acquires resistance. We performed gene/protein expression, metabolomic and methylation analyses of isogenic AML cell lines sensitive or resistant to VEN, and identified the activation of RAS/MAPK pathway, leading to increased stability and higher levels of MCL-1 protein, as a major acquired mechanism of VEN resistance. MCL-1 sustained survival and maintained mitochondrial respiration in VEN-RE cells, which had impaired electron transport chain (ETC) complex II activity, and MCL-1 silencing or pharmacologic inhibition restored VEN sensitivity. In support of the importance of RAS/MAPK activation, we found by single-cell DNA sequencing rapid clonal selection of RAS-mutated clones in AML patients treated with VEN-containing regimens. In summary, these findings establish RAS/MAPK/MCL-1 and mitochondrial fitness as key survival mechanisms of VEN-RE AML and provide the rationale for combinatorial strategies effectively targeting these pathways.
Subject(s)
Bridged Bicyclo Compounds, Heterocyclic , Leukemia, Myeloid, Acute , MAP Kinase Signaling System , Myeloid Cell Leukemia Sequence 1 Protein , Proto-Oncogene Proteins c-bcl-2 , Sulfonamides , ras Proteins , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Line, Tumor , Drug Resistance, Neoplasm , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , MAP Kinase Signaling System/drug effects , Myeloid Cell Leukemia Sequence 1 Protein/genetics , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Sulfonamides/pharmacologyABSTRACT
Therapeutic outcomes achieved in head and neck squamous cell carcinoma (HNSCC) patients by concurrent cisplatin-based chemoradiotherapy initially reflect both tumor regression and tumor stasis. However, local and distant metastasis and disease relapse are common in HNSCC patients. In the current work, we demonstrate that cisplatin treatment induces senescence in both p53 wild-type HN30 and p53 mutant HN12 head and neck cancer models. We also show that tumor cells can escape from senescence both in vitro and in vivo. We further establish the effectiveness of the senolytic, ABT-263 (Navitoclax), in elimination of senescent tumor cells after cisplatin treatment. Navitoclax increased apoptosis by 3.3-fold (P ≤ 0.05) at day 7 compared with monotherapy by cisplatin. Additionally, we show that ABT-263 interferes with the interaction between B-cell lymphoma-x large (BCL-XL) and BAX, anti- and pro-apoptotic proteins, respectively, followed by BAX activation, suggesting that ABT-263-induced apoptotic cell death is mediated through BAX. Our in vivo studies also confirm senescence induction in tumor cells by cisplatin, and the promotion of apoptosis coupled with a significant delay of tumor growth after sequential treatment with ABT-263. Sequential treatment with cisplatin followed by ABT-263 extended the humane endpoint to â¼130 days compared with cisplatin alone, where mice survived â¼75 days. These results support the premise that senolytic agents could be used to eliminate residual senescent tumor cells after chemotherapy and thereby potentially delay disease recurrence in head and neck cancer patients. SIGNIFICANCE STATEMENT: Disease recurrence is the most common cause of death in head and neck cancer patients. B-cell lymphoma-x large inhibitors such as ABT-263 (Navitoclax) have the capacity to be used in combination with cisplatin in head and neck cancer patients to eliminate senescent cells and possibly prevent disease relapse.
Subject(s)
Aniline Compounds/administration & dosage , Antineoplastic Agents/administration & dosage , Cellular Senescence/drug effects , Cisplatin/administration & dosage , Head and Neck Neoplasms/drug therapy , Sulfonamides/administration & dosage , Tumor Suppressor Protein p53/genetics , bcl-2-Associated X Protein/genetics , Aniline Compounds/pharmacology , Animals , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Cisplatin/pharmacology , Drug Synergism , Female , Gene Expression Regulation, Neoplastic/drug effects , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/metabolism , Humans , Mice , Mutation , Sulfonamides/pharmacology , bcl-2-Associated X Protein/metabolismSubject(s)
Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Sulfonamides/therapeutic use , Antineoplastic Agents/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Line, Tumor , Drug Resistance, Neoplasm , Gene Expression Regulation, Leukemic/drug effects , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Mutation/drug effects , Sulfonamides/pharmacologySubject(s)
Aminopyridines/pharmacology , Antineoplastic Agents/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Enzyme Inhibitors/pharmacology , Isocitrate Dehydrogenase/genetics , Leukemia, Myeloid, Acute/drug therapy , Sulfonamides/pharmacology , Triazines/pharmacology , Aminopyridines/therapeutic use , Animals , Antineoplastic Agents/therapeutic use , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Cell Line, Tumor , Enzyme Inhibitors/therapeutic use , Humans , Isocitrate Dehydrogenase/antagonists & inhibitors , Leukemia, Myeloid, Acute/genetics , Mutation/drug effects , Sulfonamides/therapeutic use , Triazines/therapeutic useABSTRACT
BH3 mimetics are increasingly used as anti-cancer therapeutics either alone or in conjunction with other chemotherapies. However, mounting evidence has also demonstrated that BH3 mimetics modulate varied amounts of apoptotic signaling in healthy immune populations. In order to maximize their clinical potential, it will be essential to understand how BH3 mimetics affect discrete immune populations and to determine how BH3 mimetic pressure causes immune system adaptation. Here we focus on the BCL-2 specific inhibitor venetoclax (ABT-199) and its effects following short-term and long-term BCL-2 blockade on T cell subsets. Seven day "short-term" ex vivo and in vivo BCL-2 inhibition led to divergent cell death sensitivity patterns in CD8+ T cells, CD4+ T cells, and Tregs resulting in shifting of global T cell populations towards a more memory T cell state with increased expression of BCL-2, BCL-XL, and MCL-1. However, twenty-eight day "long-term" BCL-2 blockade following T cell-depleted bone marrow transplantation did not lead to changes in the global T cell landscape. Despite the lack of changes in T cell proportions, animals treated with venetoclax developed CD8+ and CD4+ T cells with high levels of BCL-2 and were more resistant to apoptotic stimuli following expansion post-transplant. Further, we demonstrate through RNA profiling that T cells adapt while under BCL-2 blockade post-transplant and develop a more activated genotype. Taken together, these data emphasize the importance of evaluating how BH3 mimetics affect the immune system in different treatment modalities and disease contexts and suggest that venetoclax should be further explored as an immunomodulatory compound.
Subject(s)
Antineoplastic Agents/therapeutic use , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Cell Death/drug effects , Memory T Cells/drug effects , Sulfonamides/therapeutic use , Animals , Antineoplastic Agents/pharmacology , Apoptosis , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Line, Tumor , Humans , Mice , Sulfonamides/pharmacologyABSTRACT
The covalent inhibitor of Bruton's tyrosine kinase ibrutinib and the specific Bcl-2 inhibitor venetoclax are both highly efficacious single-agent drugs in the treatment of chronic lymphocytic leukemia (CLL). Based on their complementary modes of action, ibrutinib and venetoclax are hypothesized to act in a synergistic fashion. Currently, it is unclear whether combined treatment is indeed superior to continuous single-agent treatment and what mechanisms underlie the resistance to combination treatment. In addition, the effects of such treatment on the skewed T-cell compartment characteristic of CLL are as yet unknown. In the murine Eµ-TCL1 adoptive transfer model resembling aggressive CLL, we found that combined treatment resulted in the deepest responses, with the longest duration related to a combination of decreased proliferation and increased induction of apoptosis. In addition, alterations in T-cell subsets were most prominent after combination treatment, with increased naive cells and reduced effector memory cells. Remarkably, effects of single agents but also combination treatment were eventually interrupted by relapse, and we found downregulation of BIM expression as a plausible cause of acquired drug resistance. Nevertheless, in this murine model, the combination of venetoclax and ibrutinib has increased efficacy over single agents, accompanied by a restoration of the T-cell compartment.
Subject(s)
Leukemia, Lymphocytic, Chronic, B-Cell , Adenine/analogs & derivatives , Animals , Bridged Bicyclo Compounds, Heterocyclic , Disease Models, Animal , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Mice , Piperidines , Proto-Oncogene Proteins , Pyrazoles , Pyrimidines , SulfonamidesABSTRACT
Cyclin-dependent kinase 9 (CDK9) is a serine/threonine kinase involved in the regulation of transcription elongation. An inhibition of CDK9 downregulates a number of short-lived proteins responsible for tumor maintenance and survival, including the antiapoptotic BCL-2 family member MCL-1. As pan-CDK inhibitors under development have faced dosing and toxicity challenges in the clinical setting, we generated selective CDK9 inhibitors that could be amenable to an oral administration. Here, we report the lead optimization of a series of azaindole-based inhibitors. To overcome early challenges with promiscuity and cardiovascular toxicity, carboxylates were introduced into the pharmacophore en route to compounds such as 14 and 16. These CDK9 inhibitors demonstrated a reduced toxicity, adequate pharmacokinetic properties, and a robust in vivo efficacy in mice upon oral dosing.
Subject(s)
MTOR Inhibitors , Multiple Myeloma , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Cell Line, Tumor , Humans , Ikaros Transcription Factor , Multiple Myeloma/drug therapy , Multiple Myeloma/genetics , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Sulfonamides , TOR Serine-Threonine Kinases , Up-RegulationABSTRACT
BCL-XL, an antiapoptotic member of the BCL-2 family of proteins, drives tumor survival and maintenance and thus represents a key target for cancer treatment. Herein we report the rational design of a novel series of selective BCL-XL inhibitors exemplified by A-1293102. This molecule contains structural elements of selective BCL-XL inhibitor A-1155463 and the dual BCL-XL/BCL-2 inhibitors ABT-737 and navitoclax, while representing a distinct pharmacophore as assessed by an objective cheminformatic evaluation. A-1293102 exhibited picomolar binding affinity to BCL-XL and both efficiently and selectively killed BCL-XL-dependent tumor cells. X-ray crystallographic analysis demonstrated a key hydrogen bonding network in the P2 binding pocket of BCL-XL, while the bent-back moiety achieved efficient occupancy of the P4 pocket in a manner similar to that of navitoclax. A-1293102 represents one of the few distinct structural series of selective BCL-XL inhibitors, and thus serves as a useful tool for biological studies as well as a lead compound for further optimization.
ABSTRACT
Synovial sarcoma (SS) is frequently diagnosed in teenagers and young adults and continues to be treated with polychemotherapy with variable success. The SS18-SSX gene fusion is pathognomonic for the disease, and high expression of the anti-apoptotic BCL-2 pathologically supports the diagnosis. As the oncogenic SS18-SSX fusion gene itself is not druggable, BCL-2 inhibitor-based therapies are an appealing therapeutic opportunity. Venetoclax, an FDA-approved BCL-2 inhibitor that is revolutionizing care in some BCL-2-expressing hematological cancers, affords an intriguing therapeutic possibility to treat SS. In addition, there are now dozens of venetoclax-based combination therapies in clinical trials in hematological cancers, attributing to the limited toxicity of venetoclax. However, preclinical studies of venetoclax in SS have demonstrated an unexpected ineffectiveness. In this study, we analyzed the response of SS to venetoclax and the underlying BCL-2 family biology in an effort to understand venetoclax treatment failure and find a therapeutic strategy to sensitize SS to venetoclax. We found remarkably depressed levels of the endogenous MCL-1 inhibitor, NOXA, in SS compared to other sarcomas. Expressing NOXA led to sensitization to venetoclax, as did the addition of the MCL-1 BH3 mimetic, S63845. Importantly, the venetoclax/S63845 combination induced tumor regressions in SS patient-derived xenograft (PDX) models. As a very close analog of S63845 (S64315) is now in clinical trials with venetoclax in AML (NCT03672695), the combination of MCL-1 BH3 mimetics and venetoclax should be considered for SS patients as a new therapy.
ABSTRACT
Since gaining approval for the treatment of chronic lymphocytic leukemia (CLL), the BCL-2 inhibitor venetoclax has transformed the treatment of this and other blood-related cancers. Reflecting the large and hydrophobic BH3-binding groove within BCL-2, venetoclax has significantly higher molecular weight and lipophilicity than most orally administered drugs, along with negligible water solubility. Although a technology-enabled formulation successfully achieves oral absorption in humans, venetoclax tablets have limited drug loading and therefore can present a substantial pill burden for patients in high-dose indications. We therefore generated a phosphate prodrug (3, ABBV-167) that confers significantly increased water solubility to venetoclax and, upon oral administration to healthy volunteers either as a solution or high drug-load immediate release tablet, extensively converts to the parent drug. Additionally, ABBV-167 demonstrated a lower food effect with respect to venetoclax tablets. These data indicate that beyond-rule-of-5 molecules can be successfully delivered to humans via a solubility-enhancing prodrug moiety to afford robust exposures of the parent drug following oral dosing.
Subject(s)
Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Prodrugs/therapeutic use , Sulfonamides/therapeutic use , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Line, Tumor , Cross-Over Studies , Female , Healthy Volunteers , Humans , Prodrugs/pharmacology , Sulfonamides/pharmacologyABSTRACT
The antiapoptotic protein BCL2 plays critical roles in regulating lymphocyte development and immune responses, and has also been implicated in tumorigenesis and tumor survival. However, it is unknown whether BCL2 is critical for antitumor immune responses. We evaluated whether venetoclax, a selective small-molecule inhibitor of BCL2, would influence the antitumor activity of immune checkpoint inhibitors (ICI). We demonstrate in mouse syngeneic tumor models that venetoclax can augment the antitumor efficacy of ICIs accompanied by the increase of PD-1+ T effector memory cells. Venetoclax did not impair human T-cell function in response to antigen stimuli in vitro and did not antagonize T-cell activation induced by anti-PD-1. Furthermore, we demonstrate that the antiapoptotic family member BCL-XL provides a survival advantage in effector T cells following inhibition of BCL2. Taken together, these data provide evidence that venetoclax should be further explored in combination with ICIs for cancer therapy. SIGNIFICANCE: The antiapoptotic oncoprotein BCL2 plays critical roles in tumorigenesis, tumor survival, lymphocyte development, and immune system regulation. Here we demonstrate that venetoclax, the first FDA/European Medicines Agency-approved BCL2 inhibitor, unexpectedly can be combined preclinically with immune checkpoint inhibitors to enhance anticancer immunotherapy, warranting clinical evaluation of these combinations.This article is highlighted in the In This Issue feature, p. 1.
Subject(s)
Immune Checkpoint Inhibitors , T-Lymphocytes , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Sulfonamides/pharmacologyABSTRACT
BACKGROUND: Targeted therapies for triple-negative breast cancer (TNBC) are limited; however, the epidermal growth factor receptor (EGFR) represents a potential target, as the majority of TNBC express EGFR. The purpose of these studies was to evaluate the effectiveness of two EGFR-targeted antibody-drug conjugates (ADC: ABT-414; ABBV-321) in combination with navitoclax, an antagonist of the anti-apoptotic BCL-2 and BCL-XL proteins, in order to assess the translational relevance of these combinations for TNBC. METHODS: The pre-clinical efficacy of combined treatments was evaluated in multiple patient-derived xenograft (PDX) models of TNBC. Microscopy-based dynamic BH3 profiling (DBP) was used to assess mitochondrial apoptotic signaling induced by navitoclax and/or ADC treatments, and the expression of EGFR and BCL-2/XL was analyzed in 46 triple-negative patient tumors. RESULTS: Treatment with navitoclax plus ABT-414 caused a significant reduction in tumor growth in five of seven PDXs and significant tumor regression in the highest EGFR-expressing PDX. Navitoclax plus ABBV-321, an EGFR-targeted ADC that displays more effective wild-type EGFR-targeting, elicited more significant tumor growth inhibition and regressions in the two highest EGFR-expressing models evaluated. The level of mitochondrial apoptotic signaling induced by single or combined drug treatments, as measured by DBP, correlated with the treatment responses observed in vivo. Lastly, the majority of triple-negative patient tumors were found to express EGFR and co-express BCL-XL and/or BCL-2. CONCLUSIONS: The dramatic tumor regressions achieved using combined agents in pre-clinical TNBC models underscore the abilities of BCL-2/XL antagonists to enhance the effectiveness of EGFR-targeted ADCs and highlight the clinical potential for usage of such targeted ADCs to alleviate toxicities associated with combinations of BCL-2/XL inhibitors and systemic chemotherapies.
Subject(s)
Aniline Compounds/pharmacology , Antibodies, Monoclonal, Humanized/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Immunoconjugates/pharmacology , Sulfonamides/pharmacology , Triple Negative Breast Neoplasms/drug therapy , Aniline Compounds/therapeutic use , Animals , Antibodies, Monoclonal, Humanized/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Apoptosis/drug effects , Breast/pathology , Drug Resistance, Neoplasm/drug effects , ErbB Receptors/analysis , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/metabolism , Female , Humans , Immunoconjugates/therapeutic use , Mice , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/metabolism , Sulfonamides/therapeutic use , Triple Negative Breast Neoplasms/pathology , Xenograft Model Antitumor Assays , bcl-X Protein/antagonists & inhibitors , bcl-X Protein/metabolismABSTRACT
Herein we describe the discovery of A-1331852, a first-in-class orally active BCL-XL inhibitor that selectively and potently induces apoptosis in BCL-XL-dependent tumor cells. This molecule was generated by re-engineering our previously reported BCL-XL inhibitor A-1155463 using structure-based drug design. Key design elements included rigidification of the A-1155463 pharmacophore and introduction of sp3-rich moieties capable of generating highly productive interactions within the key P4 pocket of BCL-XL. A-1331852 has since been used as a critical tool molecule for further exploring BCL-2 family protein biology, while also representing an attractive entry into a drug discovery program.
ABSTRACT
Tumor cells undergo senescence in response to both conventional and targeted cancer therapies. The induction of senescence in response to cancer therapy can contribute to unfavorable patient outcomes, potentially including disease relapse. This possibiliy is supported by our findings that tumor cells induced into senescence by doxorubicin or etoposide can give rise to viable tumors in vivo. We further demonstrate sensitivity of these senescent tumor cells to the senolytic ABT-263 (navitoclax), therefore providing a "two-hit" approach to eliminate senescent tumor cells that persist after exposure to chemotherapy or radiation. The sequential combination of therapy-induced senescence and ABT-263 could shift the response to therapy toward apoptosis by interfering with the interaction between BCL-XL and BAX. The administration of ABT-263 after either etoposide or doxorubicin also resulted in marked, prolonged tumor suppression in tumor-bearing animals. These findings support the premise that senolytic therapy following conventional cancer therapy may improve therapeutic outcomes and delay disease recurrence.
Subject(s)
Aniline Compounds/pharmacology , Cellular Senescence , Sulfonamides/pharmacology , bcl-2-Associated X Protein/metabolism , bcl-X Protein/metabolism , Apoptosis/drug effects , Carcinogenesis/drug effects , Carcinogenesis/pathology , Cell Death/drug effects , Cell Line, Tumor , Cellular Senescence/drug effects , Doxorubicin/pharmacology , Etoposide/pharmacology , HEK293 Cells , Humans , Male , Models, Biological , Protein Binding/drug effects , Radiation , Topoisomerase Inhibitors/pharmacology , Tumor BurdenABSTRACT
PURPOSE: Patients with acute myeloid leukemia (AML) frequently do not respond to conventional therapies. Leukemic cell survival and treatment resistance have been attributed to the overexpression of B-cell lymphoma 2 (BCL-2) and aberrant DNA hypermethylation. In a phase Ib study in elderly patients with AML, combining the BCL-2 selective inhibitor venetoclax with hypomethylating agents 5-azacitidine (5-Aza) or decitabine resulted in 67% overall response rate; however, the underlying mechanism for this activity is unknown. EXPERIMENTAL DESIGN: We studied the consequences of combining two therapeutic agents, venetoclax and 5-Aza, in AML preclinical models and primary patient samples. We measured expression changes in the integrated stress response (ISR) and the BCL-2 family by Western blot and qPCR. Subsequently, we engineered PMAIP1 (NOXA)- and BBC3 (PUMA)-deficient AML cell lines using CRISPR-Cas9 methods to understand their respective roles in driving the venetoclax/5-Aza combinatorial activity. RESULTS: In this study, we demonstrate that venetoclax and 5-Aza act synergistically to kill AML cells in vitro and display combinatorial antitumor activity in vivo. We uncover a novel nonepigenetic mechanism for 5-Aza-induced apoptosis in AML cells through transcriptional induction of the proapoptotic BH3-only protein NOXA. This induction occurred within hours of treatment and was mediated by the ISR pathway. NOXA was detected in complex with antiapoptotic proteins, suggesting that 5-Aza may be "priming" the AML cells for venetoclax-induced apoptosis. PMAIP1 knockout confirmed its major role in driving venetoclax and 5-Aza synergy. CONCLUSIONS: These data provide a novel nonepigenetic mechanism of action for 5-Aza and its combinatorial activity with venetoclax through the ISR-mediated induction of PMAIP1.