RESUMO
MHC class I (MHC-I) molecules are critical for CD8+ T cell responses to viral infections and malignant cells, and tumors can downregulate MHC-I expression to promote immune evasion. In this study, using a genome-wide CRISPR screen on a human melanoma cell line, we identified the polycomb repressive complex 1 (PRC1) subunit PCGF1 and the deubiquitinating enzyme BAP1 as opposite regulators of MHC-I transcription. PCGF1 facilitates deposition of ubiquitin at H2AK119 at the MHC-I promoters to silence MHC-I, whereas BAP1 removes this modification to restore MHC-I expression. PCGF1 is widely expressed in tumors and its depletion increased MHC-I expression in multiple tumor lines, including MHC-Ilow tumors. In cells characterized by poor MHC-I expression, PRC1 and PRC2 act in parallel to impinge low transcription. However, PCGF1 depletion was sufficient to increase MHC-I expression and restore T cell-mediated killing of the tumor cells. Taken together, our data provide an additional layer of regulation of MHC-I expression in tumors: epigenetic silencing by PRC1 subunit PCGF1.
Assuntos
Histonas , Ubiquitina , Humanos , Histonas/metabolismo , Ubiquitina/metabolismo , Epigênese Genética , Complexo Repressor Polycomb 1/metabolismo , Linhagem Celular , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismoRESUMO
The efficacy of anthracycline-based chemotherapeutics, which include doxorubicin and its structural relatives daunorubicin and idarubicin, remains almost unmatched in oncology, despite a side effect profile including cumulative dose-dependent cardiotoxicity, therapy-related malignancies and infertility. Detoxifying anthracyclines while preserving their anti-neoplastic effects is arguably a major unmet need in modern oncology, as cardiovascular complications that limit anti-cancer treatment are a leading cause of morbidity and mortality among the 17 million cancer survivors in the U.S. In this study, we examined different clinically relevant anthracycline drugs for a series of features including mode of action (chromatin and DNA damage), bio-distribution, anti-tumor efficacy and cardiotoxicity in pre-clinical models and patients. The different anthracycline drugs have surprisingly individual efficacy and toxicity profiles. In particular, aclarubicin stands out in pre-clinical models and clinical studies, as it potently kills cancer cells, lacks cardiotoxicity, and can be safely administered even after the maximum cumulative dose of either doxorubicin or idarubicin has been reached. Retrospective analysis of aclarubicin used as second-line treatment for relapsed/refractory AML patients showed survival effects similar to its use in first line, leading to a notable 23% increase in 5-year overall survival compared to other intensive chemotherapies. Considering individual anthracyclines as distinct entities unveils new treatment options, such as the identification of aclarubicin, which significantly improves the survival outcomes of AML patients while mitigating the treatment-limiting side-effects. Building upon these findings, an international multicenter Phase III prospective study is prepared, to integrate aclarubicin into the treatment of relapsed/refractory AML patients.
Assuntos
Aclarubicina , Antraciclinas , Leucemia Mieloide Aguda , Animais , Feminino , Humanos , Masculino , Aclarubicina/farmacologia , Aclarubicina/uso terapêutico , Antraciclinas/uso terapêutico , Antineoplásicos/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/efeitos adversos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/mortalidade , Resultado do TratamentoRESUMO
The endolysosomal system fulfils a myriad of cellular functions predicated on regulated membrane identity progressions, collectively termed maturation. Mature or "late" endosomes are designated by small membrane-bound GTPases Rab7 and Arl8b, which can either operate independently or collaborate to form a joint compartment. Whether, and how, Rab7 and Arl8b resolve this hybrid identity compartment to regain functional autonomy is unknown. Here, we report that Arl8b employs its effector SKIP to instigate inactivation and removal of Rab7 from select membranes. We find that SKIP interacts with Rab7 and functions as its negative effector, delivering the cognate GAP, TBC1D15. Recruitment of TBC1D15 to SKIP occurs via the HOPS complex, whose assembly is facilitated by contacts between Rab7 and the KMI motif of SKIP. Consequently, SKIP mediates reinstatement of single identity Arl8b sub-compartment through an ordered Rab7-to-Arl8b handover, and, together with Rab7's positive effector RILP, enforces spatial, temporal and morphological compartmentalization of endolysosomal organelles.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Fatores de Ribosilação do ADP/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Compartimento Celular , Endossomos/metabolismo , Proteínas Ativadoras de GTPase/genética , Células HEK293 , Humanos , Lisossomos/metabolismo , Ligação Proteica , Transporte Proteico , Proteínas rab de Ligação ao GTP/genética , proteínas de unión al GTP Rab7RESUMO
Cancer immunotherapy has proven remarkably successful through instigation of systemic antitumor T cell responses. Despite this achievement, further advancements are needed to expand the scope of susceptible cancer types and overcome variation in treatment outcomes between patients. Small-molecule drugs targeting defined pathways and/or cells capable of immune modulation are expected to substantially improve efficacy of cancer immunotherapy. Small-molecule drugs possess unique properties compatible with systemic administration and amenable to both extracellular and intracellular targets. These compounds can modify molecular pathways to overcome immune tolerance and suppression towards effective antitumor responses. Here, we provide an overview of how such effects might be achieved by combining immunotherapy with conventional and/or new small-molecule chemotherapeutics.
Assuntos
Imunoterapia , Neoplasias , Bibliotecas de Moléculas Pequenas , Antineoplásicos/uso terapêutico , Humanos , Tolerância Imunológica , Neoplasias/terapia , Bibliotecas de Moléculas Pequenas/uso terapêutico , Linfócitos T/imunologiaRESUMO
The anthracycline doxorubicin (Doxo) and its analogs daunorubicin (Daun), epirubicin (Epi), and idarubicin (Ida) have been cornerstones of anticancer therapy for nearly five decades. However, their clinical application is limited by severe side effects, especially dose-dependent irreversible cardiotoxicity. Other detrimental side effects of anthracyclines include therapy-related malignancies and infertility. It is unclear whether these side effects are coupled to the chemotherapeutic efficacy. Doxo, Daun, Epi, and Ida execute two cellular activities: DNA damage, causing double-strand breaks (DSBs) following poisoning of topoisomerase II (Topo II), and chromatin damage, mediated through histone eviction at selected sites in the genome. Here we report that anthracycline-induced cardiotoxicity requires the combination of both cellular activities. Topo II poisons with either one of the activities fail to induce cardiotoxicity in mice and human cardiac microtissues, as observed for aclarubicin (Acla) and etoposide (Etop). Further, we show that Doxo can be detoxified by chemically separating these two activities. Anthracycline variants that induce chromatin damage without causing DSBs maintain similar anticancer potency in cell lines, mice, and human acute myeloid leukemia patients, implying that chromatin damage constitutes a major cytotoxic mechanism of anthracyclines. With these anthracyclines abstained from cardiotoxicity and therapy-related tumors, we thus uncoupled the side effects from anticancer efficacy. These results suggest that anthracycline variants acting primarily via chromatin damage may allow prolonged treatment of cancer patients and will improve the quality of life of cancer survivors.
Assuntos
Antineoplásicos/efeitos adversos , Cromatina/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Doxorrubicina/efeitos adversos , Animais , Linhagem Celular , Doxorrubicina/análogos & derivados , Doxorrubicina/síntese química , Doxorrubicina/metabolismo , Doxorrubicina/uso terapêutico , Cardiopatias/induzido quimicamente , Histonas , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , CamundongosRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal types of cancer, mainly due to its delayed diagnosis and lack of effective therapeutic options. Therefore, it is imperative to find novel treatment options for PDAC. Here, we tested a series of conventional chemotherapeutics together with anthracycline compounds as single agents or in combination, determining their effectivity against established commercial and patient-derived, low-passage PDAC cell lines. Proliferation and colony formation assays were performed to determine the anticancer activity of anthracyclines; aclarubicin and doxorubicin, on commercial and patient-derived, low-passage PDAC cell lines. In addition, the effect of standard-of-care drugs gemcitabine and individual components of FOLFIRINOX were also investigated. To evaluate which mechanisms of cell death were involved in drug response, cleavage of poly(ADP-ribose)polymerase was evaluated by western blot. Aclarubicin showed superior antitumor activity compared to other anthracyclines and standard of care drugs (gemcitabine and individual components of FOLFIRINOX) in a patient-derived, low-passage PDAC cell line and in commercial cell lines. Importantly, the combination of gemcitabine and aclarubicin showed a synergistic effect at a dose range where the single agents by themselves were ineffective. In parallel, evaluation of the antitumor activity of aclarubicin demonstrated an apoptotic effect in all PDAC cell lines. Aclarubicin is cytotoxic for commercial and patient-derived low-passage PDAC cell lines, at doses lower than peak serum concentrations for patient treatment. Our findings support a (re)consideration of aclarubicin as a backbone of new combination regimens for pancreatic cancer patients.
Assuntos
Antineoplásicos , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Aclarubicina/farmacologia , Aclarubicina/uso terapêutico , Antraciclinas/farmacologia , Antraciclinas/uso terapêutico , Antibióticos Antineoplásicos/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Citotoxinas/farmacologia , Citotoxinas/uso terapêutico , Humanos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias PancreáticasRESUMO
Anthracyclines are effective drugs in the treatment of various cancers, but their use comes with severe side effects. The archetypal anthracycline drug, doxorubicin, displays two molecular modes of action: DNA double-strand break formation (through topoisomerase IIα poisoning) and chromatin damage (via eviction of histones). These biological activities can be modulated and toxic side effects can be reduced by separating these two modes of action through alteration of the aminoglycoside moiety of doxorubicin. We herein report on the design, synthesis, and evaluation of a coherent set of configurational doxorubicin analogues featuring all possible stereoisomers of the 1,2-amino-alcohol characteristic for the doxorubicin 3-amino-2,3-dideoxyfucoside, each in nonsubstituted and N,N-dimethylated forms. The set of doxorubicin analogues was synthesized using appropriately protected 2,3,6-dideoxy-3-amino glycosyl donors, equipped with an alkynylbenzoate anomeric leaving group, and the doxorubicin aglycon acceptor. The majority of these glycosylations proceeded in a highly stereoselective manner to provide the desired axial α-linkage. We show that both stereochemistry of the 3-amine carbon and N-substitution state are critical for anthracycline cytotoxicity and generally improve cellular uptake. N,N-Dimethylepirubicin is identified as the most potent anthracycline that does not induce DNA damage while remaining cytotoxic.
Assuntos
Antraciclinas , Antineoplásicos , Antibióticos Antineoplásicos , DNA Topoisomerases Tipo II , DoxorrubicinaRESUMO
Proteasome inhibitors are established therapeutic agents for the treatment of hematological cancers, as are anthracyclines such as doxorubicin. We here present a new drug targeting approach that combines both drug classes into a single molecule. Doxorubicin was conjugated to an immunoproteasome-selective inhibitor via light-cleavable linkers, yielding peptide epoxyketone-doxorubicin prodrugs that remained selective and active toward immunoproteasomes. Upon cellular uptake and immunoproteasome inhibition, doxorubicin is released from the immunoproteasome inhibitor through photoirradiation. Multiple myeloma cells in this way take a double hit: immunoproteasome inhibition and doxorubicin-induced toxicity. Our strategy, which entails targeting of a cytotoxic agent, through a covalent enzyme inhibitor that is detrimental to tumor tissue in its own right, may find use in the search for improved anticancer drugs.
Assuntos
Antibióticos Antineoplásicos/uso terapêutico , Doxorrubicina/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/radioterapia , Óptica e Fotônica/métodos , Inibidores de Proteassoma/uso terapêutico , Antibióticos Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Humanos , Modelos Moleculares , Inibidores de Proteassoma/farmacologiaRESUMO
Anthracyclines comprise one of the most effective anticancer drug classes. Doxorubicin, daunorubicin, epirubicin, and idarubicin have been in clinical use for decades, but their application remains complicated by treatment-related toxicities and drug resistance. We previously demonstrated that the combination of DNA damage and histone eviction exerted by doxorubicin drives its associated adverse effects. However, whether the same properties dictate drug resistance is unclear. In the present study, we evaluate a library of 40 anthracyclines on their cytotoxicity, intracellular uptake, and subcellular localization in K562 wildtype versus ABCB1-transporter-overexpressing, doxorubicin-resistant cells. We identify several highly potent cytotoxic anthracyclines. Among these, N,N-dimethyl-idarubicin and anthracycline (composed of the idarubicin aglycon and the aclarubicin trisaccharide) stand out, due to their histone eviction-mediated cytotoxicity toward doxorubicin-resistant cells. Our findings thus uncover understudied anthracycline variants warranting further investigation in the quest for safer and more effective anticancer agents that circumvent cellular export by ABCB1.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP , Doxorrubicina , Resistencia a Medicamentos Antineoplásicos , Idarubicina , Humanos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Doxorrubicina/farmacologia , Doxorrubicina/química , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Idarubicina/farmacologia , Idarubicina/química , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Células K562 , Relação Estrutura-AtividadeRESUMO
Cardiotoxicity remains a major cause of drug withdrawal, partially due to lacking predictability of animal models. Additionally, risk of cardiotoxicity following treatment of cancer patients is treatment limiting. It is unclear which patients will develop heart failure following therapy. Human pluripotent stem cell (hPSC)-derived cardiomyocytes present an unlimited cell source and may offer individualized solutions to this problem. We developed a platform to predict molecular and functional aspects of cardiotoxicity. Our platform can discriminate between the different cardiotoxic mechanisms of existing and novel anthracyclines Doxorubicin, Aclarubicin, and Amrubicin. Doxorubicin and Aclarubicin unlike Amrubicin substantially affected the transcriptome, mitochondrial membrane integrity, contractile force and transcription factor availability. Cardiomyocytes recovered fully within two or three weeks, corresponding to the intermittent clinical treatment regimen. Our system permits the study of hPSC-cardiomyocyte recovery and the effects of accumulated dose after multiple dosing, allowing individualized cardiotoxicity evaluation, which effects millions of cancer patients treated annually.
RESUMO
The nuclear envelope (NE) is central to the architecture of eukaryotic cells, both as a physical barrier separating the nucleus from the cytoplasm and as gatekeeper of selective transport between them. However, in open mitosis, the NE fragments to allow for spindle formation and segregation of chromosomes, resulting in intermixing of nuclear and cytoplasmic soluble fractions. Recent studies have shed new light on the mechanisms driving reinstatement of soluble proteome homeostasis following NE reformation in daughter cells. Here, we provide an overview of how mitotic cells confront this challenge to ensure continuity of basic cellular functions across generations and elaborate on the implications for the proteasome - a macromolecular machine that functions in both cytoplasmic and nuclear compartments.
Assuntos
Núcleo Celular , Proteostase , Humanos , Núcleo Celular/metabolismo , Mitose , Citoplasma/metabolismo , Membrana Nuclear/genéticaRESUMO
The anthracycline anti-cancer drugs are intensely used in the clinic to treat a wide variety of cancers. They generate DNA double strand breaks, but recently the induction of chromatin damage was introduced as another major determinant of anti-cancer activity. The combination of these two events results in their reported side effects. While our knowledge on the structure-activity relationship of anthracyclines has improved, many structural variations remain poorly explored. Therefore, we here report on the preparation of a diverse set of anthracyclines with variations within the sugar moiety, amine alkylation pattern, saccharide chain and aglycone. We assessed the cytotoxicity in vitro in relevant human cancer cell lines, and the capacity to induce DNA- and chromatin damage. This coherent set of data allowed us to deduce a few guidelines on anthracycline design, as well as discover novel, highly potent anthracyclines that may be better tolerated by patients.
Assuntos
Antraciclinas , Neoplasias , Humanos , Antraciclinas/farmacologia , Antraciclinas/química , Doxorrubicina/farmacologia , Antibióticos Antineoplásicos/química , Inibidores da Topoisomerase II , Cromatina , DNA/metabolismo , Neoplasias/tratamento farmacológicoRESUMO
Glioblastoma (GBM) patients have one of the highest risks of venous thromboembolism (VTE), which is even further increased upon treatment with chemotherapy. Tissue factor (TF) is the initiator of the extrinsic coagulation pathway and expressed by GBM cells. In this study, we aimed to examine the effect of routinely used chemotherapeutic agents Temozolomide (TMZ) and Lomustine (LOM) on TF procoagulant activity and expression in GBM cells in vitro. Three human GBM cell lines (U-251, U-87, U-118) were exposed to 100 µM TMZ or 30 µM LOM for 72 h. TF procoagulant activity was assessed via an FXa generation assay and TF gene and protein expression through qPCR and Western blotting. The externalization of phosphatidylserine (PS) was studied using Annexin V flow cytometry. Treatment with TMZ and LOM resulted in increased procoagulant activity in all cell lines. Furthermore, both agents induced procoagulant activity in the supernatant and tumor-cell-secreted extracellular vesicles. In line, TF gene and protein expression were increased upon TMZ and LOM treatment. Additionally, PS externalization and induction of inflammatory-associated genes were observed. Overall, the chemotherapeutic modalities TMZ and LOM induced procoagulant activity and increased TF gene and protein expression in all GBM cell lines tested, which may contribute to the increased VTE risk observed in GBM patients undergoing chemotherapy.
RESUMO
The anthracycline drug doxorubicin is among the most used-and useful-chemotherapeutics. While doxorubicin is highly effective in the treatment of various hematopoietic malignancies and solid tumours, its application is limited by severe adverse effects, including irreversible cardiotoxicity, therapy-related malignancies and gonadotoxicity. This continues to motivate investigation into the mechanisms of anthracycline activities and toxicities, with the aim to overcome the latter without sacrificing the former. It has long been appreciated that doxorubicin causes DNA double-strand breaks due to poisoning topoisomerase II. More recently, it became clear that doxorubicin also leads to chromatin damage achieved through eviction of histones from select sites in the genome. Evaluation of these activities in various anthracycline analogues has revealed that chromatin damage makes a major contribution to the efficacy of anthracycline drugs. Furthermore, the DNA-damaging effect conspires with chromatin damage to cause a number of adverse effects. Structure-activity relationships within the anthracycline family offer opportunities for chemical separation of these activities towards development of effective analogues with limited adverse effects. In this review, we elaborate on our current understanding of the different activities of doxorubicin and their contributions to drug efficacy and side effects. We then offer our perspective on how the activities of this old anticancer drug can be amended in new ways to benefit cancer patients, by providing effective treatment with improved quality of life.
Assuntos
Antineoplásicos/toxicidade , Doxorrubicina/toxicidade , Aclarubicina/toxicidade , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , HumanosRESUMO
Anthracycline anticancer drugs doxorubicin and aclarubicin have been used in the clinic for several decades to treat various cancers. Although closely related structures, their molecular mode of action diverges, which is reflected in their biological activity profile. For a better understanding of the structure-function relationship of these drugs, we synthesized ten doxorubicin/aclarubicin hybrids varying in three distinct features: aglycon, glycan, and amine substitution pattern. We continued to evaluate their capacity to induce DNA breaks, histone eviction, and relocated topoisomerase IIα in living cells. Furthermore, we assessed their cytotoxicity in various human tumor cell lines. Our findings underscore that histone eviction alone, rather than DNA breaks, contributes strongly to the overall cytotoxicity of anthracyclines, and structures containing N,N-dimethylamine at the reducing sugar prove that are more cytotoxic than their nonmethylated counterparts. This structural information will support further development of novel anthracycline variants with improved anticancer activity.
Assuntos
Aclarubicina/química , Antineoplásicos/química , DNA Topoisomerases Tipo II/metabolismo , Doxorrubicina/química , Polissacarídeos/química , Antraciclinas/química , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Histonas/metabolismo , Humanos , Relação Estrutura-AtividadeRESUMO
The topoisomerase II poisons doxorubicin and etoposide constitute longstanding cornerstones of chemotherapy. Despite their extensive clinical use, many patients do not respond to these drugs. Using a genome-wide gene knockout approach, we identified Keap1, the SWI/SNF complex, and C9orf82 (CAAP1) as independent factors capable of driving drug resistance through diverse molecular mechanisms, all converging on the DNA double-strand break (DSB) and repair pathway. Loss of Keap1 or the SWI/SNF complex inhibits generation of DSB by attenuating expression and activity of topoisomerase IIα, respectively, whereas deletion of C9orf82 augments subsequent DSB repair. Their corresponding genes, frequently mutated or deleted in human tumors, may impact drug sensitivity, as exemplified by triple-negative breast cancer patients with diminished SWI/SNF core member expression who exhibit reduced responsiveness to chemotherapy regimens containing doxorubicin. Collectively, our work identifies genes that may predict the response of cancer patients to the broadly used topoisomerase II poisons and defines alternative pathways that could be therapeutically exploited in treatment-resistant patients.