RESUMO
TP 53-mutant acute myeloid leukemia (AML) remains the ultimate therapeutic challenge. Epichaperomes, formed in malignant cells, consist of heat shock protein 90 (HSP90) and associated proteins that support the maturation, activity, and stability of oncogenic kinases and transcription factors including mutant p53. High-throughput drug screening identified HSP90 inhibitors as top hits in isogenic TP53-wild-type (WT) and -mutant AML cells. We detected epichaperomes in AML cells and stem/progenitor cells with TP53 mutations but not in healthy bone marrow (BM) cells. Hence, we investigated the therapeutic potential of specifically targeting epichaperomes with PU-H71 in TP53-mutant AML based on its preferred binding to HSP90 within epichaperomes. PU-H71 effectively suppressed cell intrinsic stress responses and killed AML cells, primarily by inducing apoptosis; targeted TP53-mutant stem/progenitor cells; and prolonged survival of TP53-mutant AML xenograft and patient-derived xenograft models, but it had minimal effects on healthy human BM CD34+ cells or on murine hematopoiesis. PU-H71 decreased MCL-1 and multiple signal proteins, increased proapoptotic Bcl-2-like protein 11 levels, and synergized with BCL-2 inhibitor venetoclax in TP53-mutant AML. Notably, PU-H71 effectively killed TP53-WT and -mutant cells in isogenic TP53-WT/TP53-R248W Molm13 cell mixtures, whereas MDM2 or BCL-2 inhibition only reduced TP53-WT but favored the outgrowth of TP53-mutant cells. Venetoclax enhanced the killing of both TP53-WT and -mutant cells by PU-H71 in a xenograft model. Our data suggest that epichaperome function is essential for TP53-mutant AML growth and survival and that its inhibition targets mutant AML and stem/progenitor cells, enhances venetoclax activity, and prevents the outgrowth of venetoclax-resistant TP53-mutant AML clones. These concepts warrant clinical evaluation.
Assuntos
Antineoplásicos , Leucemia Mieloide Aguda , Humanos , Animais , Camundongos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Apoptose , Células-Tronco/metabolismo , Linhagem Celular TumoralRESUMO
Acute myeloid leukemia (AML) is a heterogeneous group of aggressive hematological malignancies commonly associated with treatment resistance, high risk of relapse, and mitochondrial dysregulation. We identified six mitochondria-affecting compounds (PS compounds) that exhibit selective cytotoxicity against AML cells in vitro. Structure-activity relationship studies identified six analogs from two original scaffolds that had over an order of magnitude difference between LD50 in AML and healthy peripheral blood mononuclear cells. Mechanistically, all hit compounds reduced ATP and selectively impaired both basal and ATP-linked oxygen consumption in leukemic cells. Compounds derived from PS127 significantly upregulated production of reactive oxygen species (ROS) in AML cells and triggered ferroptotic, necroptotic, and/or apoptotic cell death in AML cell lines and refractory/relapsed AML primary samples. These compounds exhibited synergy with several anti-leukemia agents in AML, acute lymphoblastic leukemia (ALL), or chronic myelogenous leukemia (CML). Pilot in vivo efficacy studies indicate anti-leukemic efficacy in a MOLM14/GFP/LUC xenograft model, including extended survival in mice injected with leukemic cells pre-treated with PS127B or PS127E and in mice treated with PS127E at a dose of 5 mg/kg. These compounds are promising leads for development of future combinatorial therapeutic approaches for mitochondria-driven hematologic malignancies such as AML, ALL, and CML.
Assuntos
Neoplasias Hematológicas , Leucemia Mielogênica Crônica BCR-ABL Positiva , Leucemia Mieloide Aguda , Trifosfato de Adenosina/metabolismo , Animais , Neoplasias Hematológicas/metabolismo , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucemia Mieloide Aguda/patologia , Leucócitos Mononucleares/patologia , Camundongos , Mitocôndrias/metabolismoRESUMO
Acute myeloid leukemias (AML) are a group of aggressive hematologic malignancies resulting from acquired genetic mutations in hematopoietic stem cells that affect patients of all ages. Despite decades of research, standard chemotherapy still remains ineffective for some AML subtypes and is often inappropriate for older patients or those with comorbidities. Recently, a number of studies have identified unique mitochondrial alterations that lead to metabolic vulnerabilities in AML cells that may present viable treatment targets. These include mtDNA, dependency on oxidative phosphorylation, mitochondrial metabolism, and pro-survival signaling, as well as reactive oxygen species generation and mitochondrial dynamics. Moreover, some mitochondria-targeting chemotherapeutics and their combinations with other compounds have been FDA-approved for AML treatment. Here, we review recent studies that illuminate the effects of drugs and synergistic drug combinations that target diverse biomolecules and metabolic pathways related to mitochondria and their promise in experimental studies, clinical trials, and existing chemotherapeutic regimens.
RESUMO
The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes more complex, the goal of targeting mitochondria using various compounds that inflict mitochondrial damage (so-called mitocans) is becoming quite popular. Unfortunately, many existing cytotoxicity assays, such as those based on tetrazolium salts or resazurin require functional mitochondrial enzymes for their performance. The damage inflicted by compounds that target mitochondria often compromises the accuracy of these assays. Here, we describe a modified protocol based on differential staining with two fluorescent dyes, one of which is cell-permeant (Hoechst 33342) and the other of which is not (propidium iodide). The difference in staining allows living and dead cells to be discriminated. The assay is amenable to automated microscopy and image analysis, which increases throughput and reduces bias. This also allows the assay to be used in high-throughput fashion using 96-well plates, making it a viable option for drug discovery efforts, particularly when the drugs in question have some level of mitotoxicity. Importantly, results obtained by Hoechst/PI staining assay show increased consistency, both with trypan blue exclusion results and between biological replicates when the assay is compared to other methods.
Assuntos
Bioensaio/métodos , Núcleo Celular/metabolismo , Mitocôndrias/metabolismo , Automação , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Mitocôndrias/efeitos dos fármacos , Reprodutibilidade dos Testes , Rotenona/farmacologiaRESUMO
Acute myeloid leukemia (AML) is an aggressive group of cancers with high mortality rates and significant relapse risks. Current treatments are insufficient, and new therapies are needed. Recent discoveries suggest that AML may be particularly sensitive to chemotherapeutics that target mitochondria. To further investigate this sensitivity, six compounds that target mitochondria [IACS-010759, rotenone, cytarabine, etoposide, ABT-199 (venetoclax), and carbonyl cyanide m-chlorophenylhydrazone] were each paired with six compounds with other activities, including tyrosine kinase inhibitors (midostaurin and dasatinib), glycolytic inhibitors (2-deoxy-D-glucose, 3-bromopyruvate, and lonidamine), and the microtubule destabilizer vinorelbine. The 36 resulting drug combinations were tested for synergistic cytotoxicity against MOLM-13 and OCI-AML2 AML cell lines. Four combinations (IACS-010759 with vinorelbine, rotenone with 2-deoxy-D-glucose, carbonyl cyanide m-chlorophenylhydrazone with dasatinib, and venetoclax with lonidamine) showed synergistic cytotoxicity in both AML cell lines and were selective for tumor cells, as survival of healthy PBMCs was dramatically higher. Among these drug pairs, IACS-010759/vinorelbine decreased ATP level and impaired mitochondrial respiration and coupling efficiency most profoundly. Some of these four treatments were also effective in K-562, KU812 (chronic myelogenous leukemia) and CCRF-CEM, MOLT-4 (acute lymphoblastic leukemia) cells, suggesting that these treatments may have value in treating other forms of leukemia. Finally, two of the four combinations retained high synergy and strong selectivity in primary AML cells from patient samples, supporting the potential of these treatments for patients.