RESUMO
Post-translational modifications of cellular substrates with ubiquitin and ubiquitin-like proteins (UBLs), including ubiquitin, SUMOs, and neural precursor cell-expressed developmentally downregulated protein 8, play a central role in regulating many aspects of cell biology. The UBL conjugation cascade is initiated by a family of ATP-dependent enzymes termed E1 activating enzymes and executed by the downstream E2-conjugating enzymes and E3 ligases. Despite their druggability and their key position at the apex of the cascade, pharmacologic modulation of E1s with potent and selective drugs has remained elusive until 2009. Among the eight E1 enzymes identified so far, those initiating ubiquitylation (UBA1), SUMOylation (SAE), and neddylation (NAE) are the most characterized and are implicated in various aspects of cancer biology. To date, over 40 inhibitors have been reported to target UBA1, SAE, and NAE, including the NAE inhibitor pevonedistat, evaluated in more than 30 clinical trials. In this Review, we discuss E1 enzymes, the rationale for their therapeutic targeting in cancer, and their different inhibitors, with emphasis on the pharmacologic properties of adenosine sulfamates and their unique mechanism of action, termed substrate-assisted inhibition. Moreover, we highlight other less-characterized E1s-UBA6, UBA7, UBA4, UBA5, and autophagy-related protein 7-and the opportunities for targeting these enzymes in cancer. SIGNIFICANCE STATEMENT: The clinical successes of proteasome inhibitors in cancer therapy and the emerging resistance to these agents have prompted the exploration of other signaling nodes in the ubiquitin-proteasome system including E1 enzymes. Therefore, it is crucial to understand the biology of different E1 enzymes, their roles in cancer, and how to translate this knowledge into novel therapeutic strategies with potential implications in cancer treatment.
Assuntos
Neoplasias , Enzimas Ativadoras de Ubiquitina , Humanos , Neoplasias/tratamento farmacológico , Processamento de Proteína Pós-Traducional , Ubiquitina/metabolismo , Enzimas Ativadoras de Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/metabolismo , Ubiquitinação , Ubiquitinas/metabolismoRESUMO
Mitochondrial DNA encodes 13 proteins that comprise components of the respiratory chain that maintain oxidative phosphorylation. The replication of mitochondrial DNA is performed by the sole mitochondrial DNA polymerase γ. As acute myeloid leukemia (AML) cells and stem cells have an increased reliance on oxidative phosphorylation, we sought to evaluate polymerase γ inhibitors in AML. The thymidine dideoxynucleoside analog, alovudine, is an inhibitor of polymerase γ. In AML cells, alovudine depleted mitochondrial DNA, reduced mitochondrial encoded proteins, decreased basal oxygen consumption, and decreased cell proliferation and viability. To evaluate the effects of polymerase γ inhibition with alovudine in vivo, mice were xenografted with OCI-AML2 cells and then treated with alovudine. Systemic administration of alovudine reduced leukemic growth without evidence of toxicity and decreased levels of mitochondrial DNA in the leukemic cells. We also showed that alovudine increased the monocytic differentiation of AML cells. Genetic knockdown and other chemical inhibitors of polymerase γ also promoted AML differentiation, but the effects on AML differentiation were independent of reductions in oxidative phosphorylation or respiratory chain proteins. Thus, we have identified a novel mechanism by which mitochondria regulate AML fate and differentiation independent of oxidative phosphorylation. Moreover, we highlight polymerase γ inhibitors, such as alovudine, as novel therapeutic agents for AML.
Assuntos
Diferenciação Celular/efeitos dos fármacos , DNA Polimerase gama/antagonistas & inibidores , Didesoxinucleosídeos/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Mitocôndrias/patologia , Monócitos/patologia , Fosforilação Oxidativa/efeitos dos fármacos , Animais , Antivirais/farmacologia , Apoptose , Proliferação de Células , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Timidina/química , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ovarian cancer is the fifth leading cause of cancer-related mortalities in women. Epithelial ovarian cancer (EOC) represents approximately 90% of all ovarian malignancies. Most EOC patients are diagnosed at advanced stages and current chemotherapy regimens are ineffective against advanced EOC due to the development of chemoresistance. It is important to better understand the molecular mechanisms underlying acquired resistance to effectively manage this disease. In this study, we examined the expression of the Wnt/ß-catenin signaling components in the paired cisplatin-sensitive (A2780s) and cisplatin-resistant (A2780cp) EOC cell lines. Our results showed that several negative regulators of Wnt signaling are downregulated, whereas a few Wnt ligands and known Wnt/ß-catenin target genes are upregulated in A2780cp cells compared to A2780s cells, suggesting that Wnt/ß-catenin signaling is more active in A2780cp cells. Further analysis revealed nuclear localization of ß-catenin and higher ß-catenin transcriptional activity in A2780cp cells compared to A2780s cells. Finally, we demonstrated that chemical inhibition of ß-catenin transcriptional activity by its inhibitor CCT036477 sensitized A2780cp cells to carboplatin, supporting a role for ß-catenin in carboplatin resistance in A2780cp cells. In conclusion, our data suggest that increased Wnt/ß-catenin signaling activity contributes to carboplatin resistance in A2780cp cells.
Assuntos
Antineoplásicos/farmacologia , Carboplatina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , beta Catenina/genética , Sequência de Bases , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Éxons , Feminino , Humanos , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Ovarianas/metabolismo , Ovário/efeitos dos fármacos , Ovário/metabolismo , Ativação Transcricional/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/antagonistas & inibidores , beta Catenina/metabolismoRESUMO
OBJECTIVE: Resistance to platinum-based therapeutic agents represents a major hurdle in the treatment of epithelial ovarian cancer (EOC). There is an urgent need to better understand the underlying mechanisms. Here, we investigated the role of RUNX3 in carboplatin resistance in EOC cells. METHODS: Expression of RUNX3 was determined in human EOC cell line A2780s (cisplatin-sensitive) and A2780cp (cisplatin-resistant), human ovarian surface epithelium (OSE) and primary EOC cells. The effects of RUNX3 expression on sensitivity to carboplatin were determined in A2780s and A2780cp cells using neutral red uptake and clonogenic assays. Carboplatin-induced apoptosis was determined by measuring cleaved PARP using Western blotting. The expression of cellular inhibitor of apoptosis protein-2 (cIAP2) and its regulation by RUNX3 were assessed by quantitative RT-PCR and Western blotting. RESULTS: The expression of RUNX3 was elevated in A2780cp cells compared to A2780s cells and in EOC tissues from chemoresistant patients compared to those from chemosensitive patients. Overexpression of RUNX3 rendered A2780s cells more resistant to carboplatin, whereas inhibition of RUNX3 increased sensitivity to carboplatin in A2780cp cells. Inhibition of RUNX3 potentiated carboplatin-induced apoptosis in A2780cp cells as demonstrated by more pronounced PARP cleavage. Interestingly, the expression of cIAP2 was elevated in A2780cp cells compared to A2780s cells. Overexpression of RUNX3 increased cIAP2 expression in A2780s cells, whereas inhibition of RUNX3 decreased cIAP2 expression and potentiated carboplatin-induced decrease of cIAP2 in A2780cp cells. CONCLUSIONS: RUNX3 contributes to carboplatin resistance in EOC cells and may hold promise as a therapeutic target to treat EOC and/or a biomarker to predict chemoresistance.
Assuntos
Antineoplásicos/farmacologia , Carboplatina/farmacologia , Subunidade alfa 3 de Fator de Ligação ao Core/biossíntese , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Proteína 3 com Repetições IAP de Baculovírus , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Resistencia a Medicamentos Antineoplásicos , Técnicas de Silenciamento de Genes , Humanos , Proteínas Inibidoras de Apoptose/biossíntese , Proteínas Inibidoras de Apoptose/genética , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Ovarianas/genética , Ubiquitina-Proteína Ligases/biossíntese , Ubiquitina-Proteína Ligases/genéticaRESUMO
OBJECTIVES: KRAS mutations, particularly KRASG12C, are prevalent in non-small cell lung cancer (NSCLC). Immune checkpoint inhibitors (ICIs) have been a frontline treatment, but recently developed KRASG12C-selective inhibitors, such as sotorasib, present new therapeutic options. We conducted a multi-center retrospective cohort study to gain insights into real-world treatment patterns and outcomes in patients with KRASG12C-positive advanced NSCLC receiving systemic therapy post-ICI treatment. METHODS: From the CAnadian CAncers With Rare Molecular Alterations-Basket Real-world Observational Study (CARMA-BROS), a cohort of 102 patients with KRASG12C-positive advanced NSCLC across 9 Canadian centers diagnosed between 2015 and 2021 was analyzed. Clinico-demographic and treatment data were obtained from electronic health records. Survival outcomes were assessed using Kaplan-Meier curves and Cox proportional hazards models. RESULTS: The patients (median age 66 years; 58 % female; 99 % current/former tobacco exposure; 59 % PD-L1 ≥ 50 %), exhibited heterogeneous treatment patterns post-ICI. Most patients received ICIs as a first-line therapy, with varying subsequent lines including chemotherapy and targeted therapy. In patients receiving systemic therapy post-ICI, median overall survival was 12.6 months, and real-world progression-free survival was 4.7 months. KRASG12C-selective targeted therapy post-ICI (n = 20) showed longer real-world progression-free survival compared to single-agent chemotherapy (aHR = 0.39, p = 0.012). CONCLUSION: This study contributes valuable real-world data on KRASG12C-positive advanced NSCLC post-ICI treatment. The absence of a standard treatment sequencing post-ICI underscores the need for further investigation and consensus-building in the evolving landscape of KRASG12C-targeted therapies.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Inibidores de Checkpoint Imunológico , Neoplasias Pulmonares , Proteínas Proto-Oncogênicas p21(ras) , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Feminino , Masculino , Estudos Retrospectivos , Inibidores de Checkpoint Imunológico/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Idoso , Proteínas Proto-Oncogênicas p21(ras)/genética , Canadá/epidemiologia , Pessoa de Meia-Idade , Mutação , Idoso de 80 Anos ou mais , Resultado do Tratamento , AdultoRESUMO
Cellular thermal shift assay (CETSA) is based on the thermal stabilization of the protein target by a compound binding. Thus, CETSA can be used to measure a compound's cellular target engagement and permeability. HiBiT CETSA method is quantitative and has higher throughput compared to the traditional Western-based CETSA. Here, we describe the protocol for a HiBiT CETSA, which utilizes a HiBiT tag derived from the NanoLuciferase (NanoLuc) that upon complementation by LgBiT NanoLuc tag produces a bright signal enabling tracking of the effects of increasing temperature on the stability of a protein-of-interest in the presence/absence of various compounds. Exposure of a HiBiT-tagged protein to increasing temperatures induces protein denaturation and thus decreased LgBiT complementation and NanoLuc signal. As the stability of proteins at higher temperatures can be influenced by the compound binding, this method enables screening for target engagement in living or permeabilized cells.
Assuntos
Temperatura Alta , Proteínas , TemperaturaRESUMO
Close monitoring after diagnosis of patients with stage I-III non-small cell lung cancer (NSCLC) may result in fitter patients with lower disease burden at the time of metastatic recurrence or progression compared to patients diagnosed initially as stage IV (de novo). We compared the presentation, treatments, and outcomes of patients with KRASG12C-mutated NSCLC with de novo versus recurrent stage IV disease. Of 109 patients, 94% had a smoking history. When compared to patients with KRASG12C-mutated NSCLC who developed stage IV disease at recurrence (n = 38), de novo stage IV patients (n = 71) had worse ECOG performance status (p = 0.007), greater numbers of extra-thoracic metastatic sites (p = 0.001), and were less likely to receive 2nd/3rd line systemic therapy (p = 0.05, p = 0.002) or targeted therapy (p = 0.001). De novo metastatic patients had shorter overall survival than metastatic patients at recurrence (9.1 versus 24.2 months; adjusted-hazard-ratio=1.94 (95% CI: 1.14-3.28; p = 0.01)). There is a critical need for well-tolerated targeted therapies in the first-line setting for metastatic patients with de novo, high-burden, stage IV KRASG12C-mutated NSCLCs.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas p21(ras) , Prognóstico , Resultado do TratamentoRESUMO
Proteolysis targeting chimeras (PROTACs) are an emerging class of targeted protein degraders that coopt the intracellular degradation machinery to selectively deplete their respective targets. PROTACs act as bifunctional degraders that comprise ubiquitin E3 ligase- and target-binding moieties connected by chemical linkers with appropriate physicochemical properties. Through this bivalent structure, PROTACs induce the degradation of their targets via proximity-based pharmacology. Compared to conventional inhibitors, PROTACs exhibit superior pharmacologic properties in terms of efficacy, potency, selectivity, the durability of response, and efficacy against undruggable proteins. Over the last few years, the scientific community has witnessed significant endeavors to advance this field and expand the armamentarium of PROTACs. In this perspective, we highlight these advances with an emphasis on emerging PROTAC variants, PROTACtability and degradability of protein targets, expression-guided PROTACs, multivalent PROTACs, preclinical resistance, candidates evaluated in clinical trials, and prospects for the use of PROTACs as a therapeutic modality.
Assuntos
Ubiquitina-Proteína Ligases , Humanos , Proteínas/metabolismo , Proteólise , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Histone lysine methylation is a post-translational modification that plays a key role in the epigenetic regulation of a broad spectrum of biological processes. Moreover, the dysregulation of histone lysine methyltransferases (KMTs) has been implicated in the pathogenesis of several diseases particularly cancer. Due to their pathobiological importance, KMTs have garnered immense attention over the last decade as attractive therapeutic targets. These endeavors have culminated in tens of chemical probes that have been used to interrogate many aspects of histone lysine methylation. Besides, over a dozen inhibitors have been advanced to clinical trials, including the EZH2 inhibitor tazemetostat approved for the treatment of follicular lymphoma and advanced epithelioid sarcoma. In this Review, we highlight the chemical biology and pharmacology of KMT inhibitors and targeted protein degraders focusing on the clinical development of EZH1/2, DOT1L, Menin-MLL, and WDR5-MLL inhibitors. We also briefly discuss the pharmacologic targeting of other KMTs.
Assuntos
Histonas , Neoplasias , Biologia , Epigênese Genética , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisina/metabolismo , Metilação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Processamento de Proteína Pós-TraducionalRESUMO
Pancreatic ductal adenocarcinoma is characterized by increased generation of reactive oxygen species that can cause lethal oxidative stress. Here, we evaluated the combined inhibition of the glutathione and thioredoxin antioxidant systems in preclinical models of pancreatic ductal adenocarcinoma, using buthionine sulfoximine (BSO) that targets glutathione synthesis, and auranofin that targets thioredoxin recycling. BSO potentiated the cytotoxicity of auranofin and induced lethal oxidative stress in primary pancreatic cancer cells. As assessed by the cellular thermal shift assay, auranofin engaged with thioredoxin reductase 1 in primary cells at concentrations known to induce cell death. Moreover, we used imaging mass cytometry to map the biodistribution of atomic gold in patient-derived xenografts treated with auranofin, and the drug was readily detectable throughout the epithelial and stromal compartments after treatment with a clinically relevant dose. In conclusion, combinatorial treatment with BSO and auranofin could serve as a potential therapeutic strategy in pancreatic ductal adenocarcinoma.
RESUMO
Anticancer drug response is determined by genetic and epigenetic mechanisms. To identify the epigenetic regulators of anticancer drug response, we conducted a chemical epigenetic screen using chemical probes that target different epigenetic modulators. In this screen, we tested 31 epigenetic probes in combination with 14 mechanistically diverse anticancer agents and identified 8 epigenetic probes that significantly potentiate the cytotoxicity of TAK-243, a first-in-class ubiquitin-activating enzyme (UBA1) inhibitor evaluated in several solid and hematologic malignancies. These probes are TP-472, GSK864, A-196, UNC1999, SGC-CBP30, and PFI-4 (and its related analogues GSK6853 and GSK5959), and they target BRD9/7, mutant IDH1, SUV420H1/2, EZH2/1, p300/CBP, and BRPF1B, respectively. In contrast to epigenetic probes, negative control compounds did not have a significant impact on TAK-243 cytotoxicity. Potentiation of TAK-243 cytotoxicity was associated with reduced ubiquitylation and induction of apoptosis. Mechanistically, these epigenetic probes exerted their potentiation by inhibiting the efflux transporter ATP-binding cassette subfamily G member 2 (ABCG2) without inducing significant changes in the ubiquitylation pathways or ABCG2 expression levels. As assessed by docking analysis, the identified probes could potentially interact with ABCG2. Based on these data, we have developed a cell-based assay that can quantitatively evaluate ABCG2 inhibition by drug candidates. In conclusion, our study identifies epigenetic probes that profoundly potentiate TAK-243 cytotoxicity through off-target ABCG2 inhibition. We also provide experimental evidence that several negative control compounds cannot exclude a subset of off-target effects of chemical probes. Finally, potentiation of TAK-243 cytotoxicity can serve as a quantitative measure of ABCG2-inhibitory activity.
Assuntos
Antineoplásicos , Resistencia a Medicamentos Antineoplásicos , Enzimas Ativadoras de Ubiquitina , Antineoplásicos/farmacologia , Antineoplásicos/química , Epigênese Genética , Trifosfato de AdenosinaRESUMO
Protein methyltransferase 5 (PRMT5) symmetrically dimethylates arginine residues leading to regulation of transcription and splicing programs. Although PRMT5 has emerged as an attractive oncology target, the molecular determinants of PRMT5 dependency in cancer remain incompletely understood. Our transcriptomic analysis identified PRMT5 regulation of the activating transcription factor 4 (ATF4) pathway in acute myelogenous leukemia (AML). PRMT5 inhibition resulted in the expression of unstable, intron-retaining ATF4 mRNA that is detained in the nucleus. Concurrently, the decrease in the spliced cytoplasmic transcript of ATF4 led to lower levels of ATF4 protein and downregulation of ATF4 target genes. Upon loss of functional PRMT5, cells with low ATF4 displayed increased oxidative stress, growth arrest, and cellular senescence. Interestingly, leukemia cells with EVI1 oncogene overexpression demonstrated dependence on PRMT5 function. EVI1 and ATF4 regulated gene signatures were inversely correlated. We show that EVI1-high AML cells have reduced ATF4 levels, elevated baseline reactive oxygen species and increased sensitivity to PRMT5 inhibition. Thus, EVI1-high cells demonstrate dependence on PRMT5 function and regulation of oxidative stress response. Overall, our findings identify the PRMT5-ATF4 axis to be safeguarding the cellular redox balance that is especially important in high oxidative stress states, such as those that occur with EVI1 overexpression.
Assuntos
Leucemia Mieloide Aguda , Proteína-Arginina N-Metiltransferases , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Oxirredução , Estresse Oxidativo , Proteínas Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismoRESUMO
PURPOSE: Small cell lung cancer (SCLC) is an aggressive disease with an overall 5-year survival rate of less than 10%. Treatment for SCLC with cisplatin/etoposide chemotherapy (C/E) ± radiotherapy has changed modestly over several decades. The ubiquitin-proteasome system is an underexplored therapeutic target for SCLC. We preclinically evaluated TAK-243, a first-in-class small molecule E1 inhibitor against UBA1. EXPERIMENTAL DESIGN: We assessed TAK-243 in 26 SCLC cell-lines as monotherapy and combined with C/E, the PARP-inhibitor, olaparib, and with radiation using cell viability assays. We interrogated TAK-243 response with gene expression to identify candidate biomarkers. We evaluated TAK-243 alone and in combination with olaparib or radiotherapy with SCLC patient-derived xenografts (PDX). RESULTS: Most SCLC cell lines were sensitive to TAK-243 monotherapy (EC50 median 15.8 nmol/L; range 10.2 nmol/L-367.3 nmol/L). TAK-243 sensitivity was associated with gene-sets involving the cell cycle, DNA and chromatin organization, and DNA damage repair, while resistance associated with cellular respiration, translation, and neurodevelopment. These associations were also observed in SCLC PDXs. TAK-243 synergized with C/E and olaparib in vitro across sensitive and resistant SCLC cell lines. Considerable TAK-243-olaparib synergy was observed in an SCLC PDX resistant to both drugs individually. TAK-243 radiosensitization was also observed in an SCLC PDX. CONCLUSIONS: TAK-243 displays efficacy in SCLC preclinical models. Enrichment of gene sets is associated with TAK-243 sensitivity and resistance. TAK-243 exhibits synergy when combined with genotoxic therapies in cell lines and PDXs. TAK-243 is a potential therapeutic strategy to improve SCLC patient outcomes, both as a single agent and in combination with existing therapies.
Assuntos
Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Complexo de Endopeptidases do Proteassoma , Pirazóis , Pirimidinas , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/metabolismo , Sulfetos , Sulfonamidas , Ubiquitina , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Drug discovery in the scope of cancer therapy has been focused on conventional agents that nonselectively induce DNA damage or selectively inhibit the activity of key oncogenic molecules without affecting their protein levels. An emerging therapeutic strategy that garnered attention in recent years is the induction of Targeted Protein Degradation (TPD) of cellular targets by hijacking the intracellular proteolysis machinery. This novel approach offers several advantages over conventional inhibitors and introduces a paradigm shift in several pharmacological aspects of drug therapy. While TPD has been found to be the major mode of action of clinically approved anticancer agents such as fulvestrant and thalidomide, recent years have witnessed systematic endeavors to expand the repertoire of proteins amenable to therapeutic ablation by TPD. Such endeavors have led to three major classes of agents that induce protein degradation, including molecular glues, Proteolysis Targeting Chimeras (PROTACs) and Hydrophobic Tag (HyT)-based degraders. Here, we briefly highlight agents in these classes and key advances made in the field with a focus on clinical translation in cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas , Neoplasias/tratamento farmacológico , Proteólise/efeitos dos fármacos , Animais , Antineoplásicos/uso terapêutico , Fulvestranto/farmacologia , Fulvestranto/uso terapêutico , Humanos , Terapia de Alvo Molecular , Neoplasias/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Talidomida/farmacologia , Talidomida/uso terapêutico , Ubiquitinação/efeitos dos fármacosRESUMO
TAK-243 is a first-in-class inhibitor of ubiquitin-like modifier activating enzyme 1 that catalyzes ubiquitin activation, the first step in the ubiquitylation cascade. Based on its preclinical efficacy and tolerability, TAK-243 has been advanced to phase I clinical trials in advanced malignancies. Nonetheless, the determinants of TAK-243 sensitivity remain largely unknown. Here, we conducted a genome-wide CRISPR/Cas9 knockout screen in acute myeloid leukemia (AML) cells in the presence of TAK-243 to identify genes essential for TAK-243 action. We identified BEN domain-containing protein 3 (BEND3), a transcriptional repressor and a regulator of chromatin organization, as the top gene whose knockout confers resistance to TAK-243 in vitro and in vivo. Knockout of BEND3 dampened TAK-243 effects on ubiquitylation, proteotoxic stress, and DNA damage response. BEND3 knockout upregulated the ATP-binding cassette efflux transporter breast cancer resistance protein (BCRP; ABCG2) and reduced the intracellular levelsof TAK-243. TAK-243 sensitivity correlated with BCRP expression in cancer cell lines of different origins. Moreover, chemical inhibition and genetic knockdown of BCRP sensitized intrinsically resistant high-BCRP cells to TAK-243. Thus, our data demonstrate that BEND3 regulates the expression of BCRP for which TAK-243 is a substrate. Moreover, BCRP expression could serve as a predictor of TAK-243 sensitivity.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Resistencia a Medicamentos Antineoplásicos , Inibidores Enzimáticos , Regulação Neoplásica da Expressão Gênica , Leucemia Mieloide Aguda , Proteínas de Neoplasias/metabolismo , Pirazóis , Pirimidinas , Proteínas Repressoras/metabolismo , Sulfetos , Sulfonamidas , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Genoma , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Masculino , Camundongos , Proteínas de Neoplasias/genética , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Proteínas Repressoras/genética , Sulfetos/farmacologia , Sulfetos/uso terapêutico , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêuticoRESUMO
Leukemic stem cells (LSCs) rely on oxidative metabolism and are differentially sensitive to targeting mitochondrial pathways, which spares normal hematopoietic cells. A subset of mitochondrial proteins is folded in the intermembrane space via the mitochondrial intermembrane assembly (MIA) pathway. We found increased mRNA expression of MIA pathway substrates in acute myeloid leukemia (AML) stem cells. Therefore, we evaluated the effects of inhibiting this pathway in AML. Genetic and chemical inhibition of ALR reduces AML growth and viability, disrupts LSC self-renewal, and induces their differentiation. ALR inhibition preferentially decreases its substrate COX17, a mitochondrial copper chaperone, and knockdown of COX17 phenocopies ALR loss. Inhibiting ALR and COX17 increases mitochondrial copper levels which in turn inhibit S-adenosylhomocysteine hydrolase (SAHH) and lower levels of S-adenosylmethionine (SAM), DNA methylation, and chromatin accessibility to lower LSC viability. These results provide insight into mechanisms through which mitochondrial copper controls epigenetic status and viability of LSCs.
Assuntos
Autorrenovação Celular , Leucemia Mieloide Aguda , Diferenciação Celular , Cobre , Humanos , Células-Tronco NeoplásicasRESUMO
Acute myeloid leukemia (AML) is an aggressive hematologic malignancy for which new therapeutic approaches are required. One such potential therapeutic strategy is to target the ubiquitin-like modifier-activating enzyme 1 (UBA1), the initiating enzyme in the ubiquitylation cascade in which proteins are tagged with ubiquitin moieties to regulate their degradation or function. Here, we evaluated TAK-243, a first-in-class UBA1 inhibitor, in preclinical models of AML. In AML cell lines and primary AML samples, TAK-243 induced cell death and inhibited clonogenic growth. In contrast, normal hematopoietic progenitor cells were more resistant. TAK-243 preferentially bound to UBA1 over the related E1 enzymes UBA2, UBA3, and UBA6 in intact AML cells. Inhibition of UBA1 with TAK-243 decreased levels of ubiquitylated proteins, increased markers of proteotoxic stress and DNA damage stress. In vivo, TAK-243 reduced leukemic burden and targeted leukemic stem cells without evidence of toxicity. Finally, we selected populations of AML cells resistant to TAK-243 and identified missense mutations in the adenylation domain of UBA1. Thus, our data demonstrate that TAK-243 targets AML cells and stem cells and support a clinical trial of TAK-243 in this patient population. Moreover, we provide insight into potential mechanisms of acquired resistance to UBA1 inhibitors.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Nucleosídeos/farmacologia , Sulfonamidas/farmacologia , Enzimas Ativadoras de Ubiquitina/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Proliferação de Células/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos SCID , Pirazóis , Pirimidinas , Sulfetos , Células Tumorais CultivadasRESUMO
The mitochondrial caseinolytic protease P (ClpP) plays a central role in mitochondrial protein quality control by degrading misfolded proteins. Using genetic and chemical approaches, we showed that hyperactivation of the protease selectively kills cancer cells, independently of p53 status, by selective degradation of its respiratory chain protein substrates and disrupts mitochondrial structure and function, while it does not affect non-malignant cells. We identified imipridones as potent activators of ClpP. Through biochemical studies and crystallography, we show that imipridones bind ClpP non-covalently and induce proteolysis by diverse structural changes. Imipridones are presently in clinical trials. Our findings suggest a general concept of inducing cancer cell lethality through activation of mitochondrial proteolysis.
Assuntos
Endopeptidase Clp/genética , Endopeptidase Clp/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/administração & dosagem , Leucemia Mieloide Aguda/tratamento farmacológico , Mitocôndrias/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Endopeptidase Clp/química , Feminino , Células HCT116 , Células HEK293 , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Imidazóis , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos , Modelos Moleculares , Mutação Puntual , Conformação Proteica/efeitos dos fármacos , Proteólise , Piridinas , Pirimidinas , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We evaluated outcomes of 100 patients with high risk AML treated with Ida-FLAG induction as first-line therapy. 72 achieved remission with one cycle; 19 did not. High risk cytogenetics and TP53 mutations were associated with failure to achieve remission. In those reaching remission, allogeneic bone marrow transplantation was associated with better relapse-free and overall survival. Those not achieving remission with induction therapy were extremely unlikely to reach remission with further therapy and had a dismal prognosis. Exploratory molecular analysis confirmed persistence of the dominant genetic mutations identified at diagnosis. Ex vivo chemosensitivity did not demonstrate significant differences between responders and non-responders. Thus, Ida-FLAG induction has a high chance of inducing remission in patients with high risk AML. Those achieving remission require allogeneic transplantation to achieve cure; those not achieving remission rarely respond to salvage chemotherapy and have a dismal outcome. Alternatives to conventional chemotherapy must be considered in this group.