RESUMO
Analysis of the human hematopoietic progenitor compartment is being transformed by single-cell multimodal approaches. Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) enables coupled surface protein and transcriptome profiling, thereby revealing genomic programs underlying progenitor states. To perform CITE-seq systematically on primary human bone marrow cells, we used titrations with 266 CITE-seq antibodies (antibody-derived tags) and machine learning to optimize a panel of 132 antibodies. Multimodal analysis resolved >80 stem, progenitor, immune, stromal and transitional cells defined by distinctive surface markers and transcriptomes. This dataset enables flow cytometry solutions for in silico-predicted cell states and identifies dozens of cell surface markers consistently detected across donors spanning race and sex. Finally, aligning annotations from this atlas, we nominate normal marrow equivalents for acute myeloid leukemia stem cell populations that differ in clinical response. This atlas serves as an advanced digital resource for hematopoietic progenitor analyses in human health and disease.
Assuntos
Células-Tronco Hematopoéticas , Transcriptoma , Humanos , Medula Óssea , Perfilação da Expressão Gênica , Células da Medula ÓsseaRESUMO
This study provides a unique translational research opportunity to help both humans and dogs diagnosed with diseases that carry dismal prognoses in both species: histiocytic sarcoma (HS), hemangiosarcoma (HSA), and disseminated mastocytosis/mast cell tumor (MCT). Although exceedingly rare in humans, these so called "orphan diseases" are relatively more common in dogs. For these and other more commonplace cancers like lymphoma (Lym), dogs are an excellent translational model for human disease due to remarkably similar disease biology. In this study, assays were performed to assess the therapeutic potential of parthenolide (PTL), a known canonical nuclear factor kappa B (NF-κB) signaling inhibitor with additional mechanisms of antineoplastic activity, including alteration of cellular reduction-oxidation balance. Canine cell lines and primary cells are sensitive to PTL and undergo dose-dependent apoptosis after exposure to drug. PTL exposure also leads to glutathione depletion, reactive oxygen species generation, and NF-κB inhibition in canine cells. Standard-of-care therapeutics broadly synergize with PTL. In two canine HS cell lines, expression of NF-κB pathway signaling partners is downregulated with PTL therapy. Preliminary data suggest that PTL inhibits NF-κB activity of cells and extends survival time in a mouse model of disseminated canine HS. These data support further investigation of compounds that can antagonize canonical NF-κB pathway signaling in these cancers and pave the way for clinical trials of PTL in affected dogs. As dogs are an excellent natural disease model for these cancers, these data will ultimately improve our understanding of their human disease counterparts and hopefully improve care for both species. SIGNIFICANCE STATEMENT: Disseminated neoplasms in human and canine cancers are challenging to treat, and novel therapeutic approaches are needed to improve outcomes. Parthenolide is a promising treatment for histiocytic sarcoma, hemangiosarcoma, and mast cell neoplasia.
Assuntos
Hemangiossarcoma , Sarcoma Histiocítico , Sesquiterpenos , Camundongos , Humanos , Animais , Cães , NF-kappa B/metabolismo , Linhagem Celular Tumoral , Sarcoma Histiocítico/tratamento farmacológico , Hemangiossarcoma/tratamento farmacológico , Sesquiterpenos/farmacologia , Sesquiterpenos/uso terapêutico , ApoptoseRESUMO
Acute myeloid leukemia (AML) is characterized by the presence of leukemia stem cells (LSCs), and failure to fully eradicate this population contributes to disease persistence/relapse. Prior studies have characterized metabolic vulnerabilities of LSCs, which demonstrate preferential reliance on oxidative phosphorylation (OXPHOS) for energy metabolism and survival. In the present study, using both genetic and pharmacologic strategies in primary human AML specimens, we show that signal transducer and activator of transcription 3 (STAT3) mediates OXPHOS in LSCs. STAT3 regulates AML-specific expression of MYC, which in turn controls transcription of the neutral amino acid transporter gene SLC1A5. We show that genetic inhibition of MYC or SLC1A5 acts to phenocopy the impairment of OXPHOS observed with STAT3 inhibition, thereby establishing this axis as a regulatory mechanism linking STAT3 to energy metabolism. Inhibition of SLC1A5 reduces intracellular levels of glutamine, glutathione, and multiple tricarboxylic acid (TCA) cycle metabolites, leading to reduced TCA cycle activity and inhibition of OXPHOS. Based on these findings, we used a novel small molecule STAT3 inhibitor, which binds STAT3 and disrupts STAT3-DNA, to evaluate the biological role of STAT3. We show that STAT3 inhibition selectively leads to cell death in AML stem and progenitor cells derived from newly diagnosed patients and patients who have experienced relapse while sparing normal hematopoietic cells. Together, these findings establish a STAT3-mediated mechanism that controls energy metabolism and survival in primitive AML cells.
Assuntos
Sistema ASC de Transporte de Aminoácidos/metabolismo , Leucemia Mieloide Aguda/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Sobrevivência Celular , Humanos , Células-Tronco Neoplásicas/citologia , Fosforilação Oxidativa , Células Tumorais CultivadasRESUMO
Venetoclax with azacitidine (ven/aza) is a lower-intensity therapeutic regimen that has been shown to improve outcomes in elderly patients with acute myeloid leukemia (AML). Measurable residual disease (MRD) using flow cytometry is a valuable tool for the prediction of relapse in AML using conventional therapies and ven/aza; however, the prognostic value for broadscale molecular MRD after ven/aza treatment is less clear. We aimed to determine the utility of retrospective assessment using multi-gene molecular MRD by droplet digital polymerase chain reaction (ddPCR). We found this approach correlates with outcomes in a cohort of patients receiving frontline ven/aza for AML. The predictive value of ddPCR MRD persisted when NPM1 mutations were removed from analysis, as well as after adjustment for the impact of stem cell transplant on outcomes. Late achievement of MRD negativity, including after SCT, was still associated with superior outcomes compared to persistently detectable MRD. We further explored the impact of ven/aza on the burden of different classes of mutations, and identified the persistence of splicing factor mutations, commonly associated with MDS, as a consistent finding after ven/aza treatment. These data add to our understanding of the effects of ven/aza on AML disease biology and provide details on molecular depth of remission that can guide prospective trials in the future.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica , Azacitidina , Compostos Bicíclicos Heterocíclicos com Pontes , Leucemia Mieloide Aguda , Mutação , Neoplasia Residual , Nucleofosmina , Sulfonamidas , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/diagnóstico , Neoplasia Residual/diagnóstico , Sulfonamidas/uso terapêutico , Sulfonamidas/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Idoso , Masculino , Feminino , Azacitidina/uso terapêutico , Azacitidina/administração & dosagem , Pessoa de Meia-Idade , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Reação em Cadeia da Polimerase/métodos , Prognóstico , Idoso de 80 Anos ou mais , Estudos Retrospectivos , Adulto , Resultado do TratamentoRESUMO
The treatment of blast phase chronic myeloid leukemia (bpCML) remains a challenge due at least in part to drug resistance of leukemia stem cells (LSCs). Recent clinical evidence suggests that the BCL-2 inhibitor venetoclax in combination with ABL-targeting tyrosine kinase inhibitors (TKIs) can eradicate bpCML LSCs. In this report, we employed preclinical models of bpCML to investigate the efficacy and underlying mechanism of LSC-targeting with venetoclax/TKI combinations. Transcriptional analysis of LSCs exposed to venetoclax and dasatinib revealed upregulation of genes involved in lysosomal biology, in particular lysosomal acid lipase A (LIPA), a regulator of free fatty acids. Metabolomic analysis confirmed increased levels of free fatty acids in response to venetoclax/dasatinib. Pre-treatment of leukemia cells with bafilomycin, a specific lysosome inhibitor, or genetic perturbation of LIPA, resulted in increased sensitivity of leukemia cells toward venetoclax/dasatinib, implicating LIPA in treatment resistance. Importantly, venetoclax/dasatinib treatment does not affect normal stem cell function, suggestive of a leukemia-specific response. These results demonstrate that venetoclax/dasatinib is an LSCselective regimen in bpCML and that disrupting LIPA and fatty acid transport enhances venetoclax/dasatinib response in targeting LSCs, providing a rationale for exploring lysosomal disruption as an adjunct therapeutic strategy to prolong disease remission.
RESUMO
Venetoclax+azacitidine is the standard of care for newly-diagnosed patients with acute myeloid leukemia (AML) for whom intensive chemotherapy is inappropriate. Efforts to optimize this regimen are necessary. We designed a clinical trial to investigate two hypotheses: i) higher doses of venetoclax are tolerable and more effective, and ii) azacitidine can be discontinued after deep remissions. Forty-two newly diagnosed AML patients were enrolled in the investigator-initiated High Dose Discontinuation Azacitidine+Venetoclax (HiDDAV) Study (clinicaltrials gov. Identifier: NCT03466294). Patients received one to three "induction" cycles of venetoclax 600 mg daily with azacitidine. Responders received MRD-positive or MRDnegative "maintenance" arms: azacitidine with 400 mg venetoclax or 400 mg venetoclax alone, respectively. The toxicity profile of HiDDAV was similar to 400 mg venetoclax. The overall response rate was 66.7%; the duration of response (DOR), event-free survival (EFS) and overall survival were 12.9, 7.8 and 9.8 months, respectively. The MRD negativity rate was 64.3% by flow cytometry and 25.0% when also measured by droplet digital polymerase chain recation. MRD-negative patients by flow cytometry had improved DOR and EFS; more stringent measures of MRD negativity were not associated with improved OS, DOR or EFS. Using MRD to guide azacitidine discontinuation did not lead to improved DOR, EFS or OS compared to patients who discontinued azacitidine without MRD guidance. Within the context of this study design, venetoclax doses >400 mg with azacitidine were well tolerated but not associated with discernible clinical improvement, and MRD may not assist in recommendations to discontinue azacitidine. Other strategies to optimize, and for some patients, de-intensify, venetoclax+azacitidine regimens are needed.
Assuntos
Azacitidina , Leucemia Mieloide Aguda , Humanos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Compostos Bicíclicos Heterocíclicos com Pontes/efeitos adversos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/tratamento farmacológico , Neoplasia Residual/tratamento farmacológicoRESUMO
We have previously demonstrated that oxidative phosphorylation is required for the survival of human leukemia stem cells (LSCs) from patients with acute myeloid leukemia (AML). More recently, we demonstrated that LSCs in patients with de novo AML rely on amino acid metabolism to drive oxidative phosphorylation. Notably, although overall levels of amino acids contribute to LSC energy metabolism, our current findings suggest that cysteine may be of particular importance for LSC survival. We demonstrate that exogenous cysteine is metabolized exclusively to glutathione. Upon cysteine depletion, glutathione synthesis is impaired, leading to reduced glutathionylation of succinate dehydrogenase A (SDHA), a key component of electron transport chain complex (ETC) II. Loss of SDHA glutathionylation impairs ETC II activity, thereby inhibiting oxidative phosphorylation, reducing production of ATP, and leading to LSC death. Given the role of cysteine in driving LSC energy production, we tested cysteine depletion as a potential therapeutic strategy. Using a novel cysteine-degrading enzyme, we demonstrate selective eradication of LSCs, with no detectable effect on normal hematopoietic stem/progenitor cells. Together, these findings indicate that LSCs are aberrantly reliant on cysteine to sustain energy metabolism, and that targeting this axis may represent a useful therapeutic strategy.
Assuntos
Cisteína/metabolismo , Complexo II de Transporte de Elétrons/antagonistas & inibidores , Leucemia Mieloide Aguda/metabolismo , Células-Tronco Neoplásicas/metabolismo , Trifosfato de Adenosina/metabolismo , Biomarcadores , Metabolismo Energético , Glutationa/metabolismo , Humanos , Oxirredução , Fosforilação Oxidativa , Espécies Reativas de Oxigênio/metabolismo , Succinato Desidrogenase/metabolismoRESUMO
Rheumatoid arthritis (RA) is a debilitating autoimmune disease characterized by chronic inflammation and progressive destruction of joint tissue. It is also characterized by aberrant blood phenotypes including anemia and suppressed lymphopoiesis that contribute to morbidity in RA patients. However, the impact of RA on hematopoietic stem cells (HSC) has not been fully elucidated. Using a collagen-induced mouse model of human RA, we identified systemic inflammation and myeloid overproduction associated with activation of a myeloid differentiation gene program in HSC. Surprisingly, despite ongoing inflammation, HSC from arthritic mice remain in a quiescent state associated with activation of a proliferation arrest gene program. Strikingly, we found that inflammatory cytokine blockade using the interleukin-1 receptor antagonist anakinra led to an attenuation of inflammatory arthritis and myeloid expansion in the bone marrow of arthritic mice. In addition, anakinra reduced expression of inflammation-driven myeloid lineage and proliferation arrest gene programs in HSC of arthritic mice. Altogether, our findings show that inflammatory cytokine blockade can contribute to normalization of hematopoiesis in the context of chronic autoimmune arthritis.
Assuntos
Artrite Experimental , Artrite Reumatoide , Doenças Autoimunes , Animais , Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Citocinas , Modelos Animais de Doenças , Humanos , CamundongosRESUMO
Drug resistance is a major hurdle in oncology. Responses of acute myeloid leukaemia (AML) patients to cytarabine (Ara-C)-based therapies are often short lived with a median overall survival of months. Therapies are under development to improve outcomes and include targeting the eukaryotic translation initiation factor (eIF4E) with its inhibitor ribavirin. In a Phase II clinical trial in poor prognosis AML, ribavirin monotherapy yielded promising responses including remissions; however, all patients relapsed. Here we identify a novel form of drug resistance to ribavirin and Ara-C. We observe that the sonic hedgehog transcription factor glioma-associated protein 1 (GLI1) and the UDP glucuronosyltransferase (UGT1A) family of enzymes are elevated in resistant cells. UGT1As add glucuronic acid to many drugs, modifying their activity in diverse tissues. GLI1 alone is sufficient to drive UGT1A-dependent glucuronidation of ribavirin and Ara-C, and thus drug resistance. Resistance is overcome by genetic or pharmacological inhibition of GLI1, revealing a potential strategy to overcome drug resistance in some patients.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Ácido Glucurônico/metabolismo , Glucuronosiltransferase/metabolismo , Proteínas Hedgehog/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Fatores de Transcrição/metabolismo , Linhagem Celular Tumoral , Citarabina/metabolismo , Citarabina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Deleção de Genes , Glucuronosiltransferase/biossíntese , Humanos , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/patologia , Ribavirina/metabolismo , Ribavirina/farmacologia , Transdução de Sinais , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Proteína GLI1 em Dedos de ZincoRESUMO
PURPOSE OF REVIEW: Venetoclax with backbone therapies have shown promising efficacy for newly diagnosed, previously untreated, older, unfit acute myeloid leukemia patients. This review discusses this data and potential reasons for the efficacy of these venetoclax-based combinations. RECENT FINDINGS: Venetoclax with hypomethylators and low-dose cytarabine have resulted in high response rates, promising response durations, and significant overall survival in relatively small, uncontrolled studies. There is emerging data that these responses are due to the effective targeting of leukemia stem cells through an alteration of the metabolic environment that is poorly tolerated by this population. SUMMARY: Venetoclax with a backbone therapy in older, untreated patients with acute myeloid leukemia has shown promising efficacy in preliminary clinical trials, and at least partially works through a novel mechanism that can target the leukemia stem cell population. Future investigations will help elucidate the mechanism and the contributions being made by each agent in the regimen.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Citarabina/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Sulfonamidas/farmacologia , Relação Dose-Resposta a Droga , Humanos , Leucemia Mieloide Aguda/patologia , Células-Tronco Neoplásicas/efeitos dos fármacosRESUMO
For more than 50 years, investigators have considered a malignant stem cell as the potential origin of and a key therapeutic target for acute myeloid leukemia (AML) and other forms of cancer.1-4 The nature and existence of tumor-initiating cells for leukemia and other malignancies have long been the subject of intense and rigorous study; indeed, the promise of the potential to eradicate such cells is clear. However, until recently, deficiencies in our understanding of the nature of these cell populations, coupled with a limited ability to therapeutically exploit their weaknesses, have been limiting factors in realizing the goal of targeting leukemic stem cells (LSCs). Exciting new insights into the fundamental underpinnings of LSCs are now being made in an era in which drug development pipelines offer the potential to specifically target pathways of significance. Therefore, the focus in this new era, characterized by the confluence of understanding LSCs and the ability to target them, is shifting from "if it can be done" to "how it will be done." Moving from a theoretical stage to this hopeful era of possibilities, new challenges expectedly arise, and our focus now must shift to determining the best strategy by which to target LSCs, with their well-documented heterogeneity and readily evident intra- and interpatient variability. The purpose of this review is therefore both to summarize the key scientific findings pertinent to AML LSC targeting and to consider methods of clinical evaluation that will be most effective for identifying successful LSC-directed therapies.
Assuntos
Leucemia Mieloide Aguda/patologia , Células-Tronco Neoplásicas/patologia , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Terapia de Alvo Molecular/métodos , Terapia de Alvo Molecular/tendências , Transdução de Sinais/efeitos dos fármacosRESUMO
Activating mutations in FMS-like tyrosine kinase 3 (FLT3) are common in acute myeloid leukemia (AML) and drive leukemic cell growth and survival. Although FLT3 inhibitors have shown considerable promise for the treatment of AML, they ultimately fail to achieve long-term remissions as monotherapy. To identify genetic targets that can sensitize AML cells to killing by FLT3 inhibitors, we performed a genome-wide RNA interference (RNAi)-based screen that identified ATM (ataxia telangiectasia mutated) as being synthetic lethal with FLT3 inhibitor therapy. We found that inactivating ATM or its downstream effector glucose 6-phosphate dehydrogenase (G6PD) sensitizes AML cells to FLT3 inhibitor induced apoptosis. Examination of the cellular metabolome showed that FLT3 inhibition by itself causes profound alterations in central carbon metabolism, resulting in impaired production of the antioxidant factor glutathione, which was further impaired by ATM or G6PD inactivation. Moreover, FLT3 inhibition elicited severe mitochondrial oxidative stress that is causative in apoptosis and is exacerbated by ATM/G6PD inhibition. The use of an agent that intensifies mitochondrial oxidative stress in combination with a FLT3 inhibitor augmented elimination of AML cells in vitro and in vivo, revealing a therapeutic strategy for the improved treatment of FLT3 mutated AML.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Leucêmica da Expressão Gênica , Glucosefosfato Desidrogenase/genética , Leucemia Mieloide Aguda/genética , Tirosina Quinase 3 Semelhante a fms/genética , Animais , Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Benzotiazóis/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quimioterapia Combinada , Feminino , Glucosefosfato Desidrogenase/antagonistas & inibidores , Glucosefosfato Desidrogenase/metabolismo , Humanos , Hidrazinas/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Camundongos Endogâmicos NOD , Pessoa de Meia-Idade , Oxirredução , Compostos de Fenilureia/farmacologia , Inibidores de Proteínas Quinases/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
Most cancers evolve over time as patients initially responsive to therapy acquire resistance to the same drugs at relapse. Cancer stem cells have been postulated to represent a therapy-refractory reservoir for relapse, but formal proof of this model is lacking. We prospectively characterized leukemia stem cell populations (LSCs) from a well-defined cohort of patients with acute myelogenous leukemia (AML) at diagnosis and relapse to assess the effect of the disease course on these critical populations. Leukemic samples were collected from patients with newly diagnosed AML before therapy and after relapse, and LSC frequency was assessed by limiting dilution analyses. LSC populations were identified using fluorescent-labeled cell sorting and transplantation into immunodeficient NOD/SCID/interleukin 2 receptor γ chain null mice. The surface antigen expression profiles of pretherapy and postrelapse LSCs were determined for published LSC markers. We demonstrate a 9- to 90-fold increase in LSC frequency between diagnosis and relapse. LSC activity at relapse was identified in populations of leukemic blasts that did not demonstrate this activity before treatment and relapse. In addition, we describe genetic instability and exceptional phenotypic changes that accompany the evolution of these new LSC populations. This study is the first to characterize the evolution of LSCs in vivo after chemotherapy, identifying a dramatic change in the physiology of primitive AML cells when the disease progresses. Taken together, these findings provide a new frame of reference by which to evaluate candidate AML therapies in which both disease control and the induction of more advanced forms of disease should be considered.
Assuntos
Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Células-Tronco Neoplásicas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores Tumorais/imunologia , Estudos de Coortes , Progressão da Doença , Feminino , Humanos , Imunofenotipagem , Leucemia Mieloide Aguda/imunologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Transplante de Neoplasias , Células-Tronco Neoplásicas/imunologia , Estudos Prospectivos , Recidiva , Adulto JovemRESUMO
Although multidrug approaches to cancer therapy are common, few strategies are based on rigorous scientific principles. Rather, drug combinations are largely dictated by empirical or clinical parameters. In the present study we developed a strategy for rational design of a regimen that selectively targets human acute myelogenous leukemia (AML) stem cells. As a starting point, we used parthenolide, an agent shown to target critical mechanisms of redox balance in primary AML cells. Next, using proteomic, genomic, and metabolomic methods, we determined that treatment with parthenolide leads to induction of compensatory mechanisms that include up-regulated NADPH production via the pentose phosphate pathway as well as activation of the Nrf2-mediated oxidative stress response pathway. Using this knowledge we identified 2-deoxyglucose and temsirolimus as agents that can be added to a parthenolide regimen as a means to inhibit such compensatory events and thereby further enhance eradication of AML cells. We demonstrate that the parthenolide, 2-deoxyglucose, temsirolimus (termed PDT) regimen is a potent means of targeting AML stem cells but has little to no effect on normal stem cells. Taken together our findings illustrate a comprehensive approach to designing combination anticancer drug regimens.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Desoxiglucose/farmacologia , Feminino , Humanos , Leucemia Mieloide Aguda/patologia , Masculino , NADP/biossíntese , Células-Tronco Neoplásicas/patologia , Sesquiterpenos/farmacologia , Sirolimo/análogos & derivados , Sirolimo/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
MOTIVATION: Somatic variant calling typically requires paired tumor-normal tissue samples. Yet, paired normal tissues are not always available in clinical settings or for archival samples. RESULTS: We present SomVarIUS, a computational method for detecting somatic variants using high throughput sequencing data from unpaired tissue samples. We evaluate the performance of the method using genomic data from synthetic and real tumor samples. SomVarIUS identifies somatic variants in exome-seq data of â¼150 × coverage with at least 67.7% precision and 64.6% recall rates, when compared with paired-tissue somatic variant calls in real tumor samples. We demonstrate the utility of SomVarIUS by identifying somatic mutations in formalin-fixed samples, and tracking clonal dynamics of oncogenic mutations in targeted deep sequencing data from pre- and post-treatment leukemia samples. AVAILABILITY AND IMPLEMENTATION: SomVarIUS is written in Python 2.7 and available at http://www.sjdlab.org/resources/ CONTACT: subhajyoti.de@ucdenver.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Software , Exoma , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , NeoplasiasRESUMO
Aldehyde dehydrogenase 1A1 (ALDH1A1) activity is high in hematopoietic stem cells and functions in part to protect stem cells from reactive aldehydes and other toxic compounds. In contrast, we found that approximately 25% of all acute myeloid leukemias expressed low or undetectable levels of ALDH1A1 and that this ALDH1A1- subset of leukemias correlates with good prognosis cytogenetics. ALDH1A1- cell lines as well as primary leukemia cells were found to be sensitive to treatment with compounds that directly and indirectly generate toxic ALDH substrates including 4-hydroxynonenal and the clinically relevant compounds arsenic trioxide and 4-hydroperoxycyclophosphamide. In contrast, normal hematopoietic stem cells were relatively resistant to these compounds. Using a murine xenotransplant model to emulate a clinical treatment strategy, established ALDH1A1- leukemias were also sensitive to in vivo treatment with cyclophosphamide combined with arsenic trioxide. These results demonstrate that targeting ALDH1A1- leukemic cells with toxic ALDH1A1 substrates such as arsenic and cyclophosphamide may be a novel targeted therapeutic strategy for this subset of acute myeloid leukemias.
Assuntos
Aldeído Desidrogenase/deficiência , Quimioterapia Combinada/métodos , Leucemia Mieloide Aguda/tratamento farmacológico , Família Aldeído Desidrogenase 1 , Animais , Trióxido de Arsênio , Arsenicais/uso terapêutico , Células Cultivadas , Ciclofosfamida/uso terapêutico , Xenoenxertos , Humanos , Leucemia Mieloide Aguda/enzimologia , Camundongos , Terapia de Alvo Molecular , Óxidos/uso terapêutico , Retinal DesidrogenaseRESUMO
A series of succinamide derivatives of melampomagnolide B have been synthesized by coupling MMB monosuccinate (2) with various heterocyclic amines to afford compounds 3a-3l. MMB monosuccinate was also reacted with terminal diaminoalkanes to afford dimeric succinamido analogs of MMB (4a-4h). These succinamide analogs of MMB were evaluated for their anti-cancer activity against a panel of sixty human cancer cell lines. Analogs 3d-3i and dimers 4f-4g exhibited promising anti-cancer activity with GI50 values ranging from 0.28 to 33.5µM against most of the cell lines in the panel. The dimeric analogs 4f and 4g were identified as lead compounds with GI50 values in the nanomolar range (GI50=280-980nM) against several cell lines in the panel; i.e. leukemia cell lines CCRF-CEM, HL-60(TB), K-562, MOLT-4, RPMI-8226 and SR; and solid tumor cell lines NCI-H522 (non-small cell lung cancer), SW-620 and HCT-116 (colon cancer), LOX IMVI (melanoma), RXF 393 (renal cancer), and MCF7, BT-549 and MDA-MB-468 (breast cancer). Succinamide analogs 3a, 3c-3l and 4b-4h were also evaluated for their apoptotic activity against M9-ENL1 acute myelogenous leukemia cells; compounds 3h-3j and 4g were equipotent with parthenolide, exhibiting LC50 values in the range 4.1-8.1µM. Molecular docking studies indicate that these molecules interact covalently with the highly conserved Cys-46 residue of the N-terminal lobe (1-109) of human IKKß to inhibit the NFκB transcription factor complex, resulting in down-regulation of anti-apoptotic genes under NFκB control.
Assuntos
Amidas/química , Sesquiterpenos/química , Succinatos/química , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Sítios de Ligação , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Proteínas I-kappa B/antagonistas & inibidores , Proteínas I-kappa B/metabolismo , Simulação de Acoplamento Molecular , Estrutura Terciária de Proteína , Sesquiterpenos/síntese química , Sesquiterpenos/farmacologiaRESUMO
A series of carbamate derivatives of the antileukemic sesquiterpene melampomagnolide B (MMB) has been synthesized utilizing a 1,2,4-triazole carbamate conjugate of MMB as an intermediate synthon. Five imidazole- and benzimidazole-carbamate analogs of MMB (8a-8e) were prepared and evaluated for anti-leukemic activity against cultured M9 ENL1 AML cells. All the analogs exhibited improved anti-leukemic activity (EC50=0.90-3.93µM) when compared to parthenolide and the parent sesquiterpene, MMB (EC50=7.0µM and 15.5µM, respectively). The imidazole carbamate analog, 8a (EC50=0.9µM), was 16 times more potent than MMB. The comparative bioavailabilities of 8a and MMB were determined in BALB/c mice following oral dosing of these compounds. It has been demonstrated that the absolute plasma bioavailabilities of MMB and 8a were 6.7±0.8%, and 45.5±2%, respectively. These results indicate that, compared to MMB, the PK parameters for 8a display significantly improved bioavailability and exposure after oral administration. Analog 8a is considered to be a potential clinical candidate for treatment of acute myelogenous leukemia.
Assuntos
Antineoplásicos/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Sesquiterpenos/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Sesquiterpenos/síntese química , Sesquiterpenos/química , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Leukemia stem cells (LSCs) account for the development of drug resistance and increased recurrence rate in acute myeloid leukemia (AML) patients. Targeted drug delivery to leukemia stem cells remains a major challenge in AML chemotherapy. Overexpressed interleukin-3 receptor alpha chain, CD123, on the surface of leukemia stem cells was reported to be a potential target in AML treatment. Here, we designed and developed an antibody drug conjugate (CD123-CPT) by integrating anti-CD123 antibody with a chemotherapeutic agent, Camptothecin (CPT), via a disulfide linker. The linker is biodegradable in the presence of Glutathione (GSH, an endogenous component in cells), which leads to release of CPT. Anti-CD123 antibody conjugates showed significant higher cellular uptake in CD123-overexpressed tumor cells. More importantly, CD123-CPT demonstrated potent inhibitory effects on CD123-overexpressed tumor cells. Consequently, these results provide a promising targeted chemotherapeutical strategy for AML treatment.
Assuntos
Anticorpos Monoclonais/química , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Camptotecina/química , Camptotecina/farmacologia , Desenho de Fármacos , Indóis/química , Subunidade alfa de Receptor de Interleucina-3/imunologia , Anticorpos Monoclonais/imunologia , Antineoplásicos/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Parthenolide is a naturally occurring terpene with promising anticancer properties, particularly in the context of acute myeloid leukemia (AML). Optimization of this natural product has been challenged by limited opportunities for the late-stage functionalization of this molecule without affecting the pharmacologically important α-methylene-γ-lactone moiety. Here, we report the further development and application of a chemoenzymatic strategy to afford a series of new analogs of parthenolide functionalized at the aliphatic positions C9 and C14. Several of these compounds were determined to be able to kill leukemia cells and patient-derived primary AML specimens with improved activity compared to parthenolide, exhibiting LC50 values in the low micromolar range. These studies demonstrate that different O-H functionalization chemistries can be applied to elaborate the parthenolide scaffold and that modifications at the C9 or C14 position can effectively enhance the antileukemic properties of this natural product. The C9-functionalized analogs 22a and 25b were identified as the most interesting compounds in terms of antileukemic potency and selectivity toward AML versus healthy blood cells.