RESUMO
Most human epithelial tumors harbor numerous alterations, making it difficult to predict which genes are required for tumor survival. To systematically identify cancer dependencies, we analyzed 501 genome-scale loss-of-function screens performed in diverse human cancer cell lines. We developed DEMETER, an analytical framework that segregates on- from off-target effects of RNAi. 769 genes were differentially required in subsets of these cell lines at a threshold of six SDs from the mean. We found predictive models for 426 dependencies (55%) by nonlinear regression modeling considering 66,646 molecular features. Many dependencies fall into a limited number of classes, and unexpectedly, in 82% of models, the top biomarkers were expression based. We demonstrated the basis behind one such predictive model linking hypermethylation of the UBB ubiquitin gene to a dependency on UBC. Together, these observations provide a foundation for a cancer dependency map that facilitates the prioritization of therapeutic targets.
Assuntos
Neoplasias/genética , Neoplasias/patologia , Linhagem Celular Tumoral , Humanos , Interferência de RNA , Software , Ubiquitina/genéticaRESUMO
Treatment results for patients with newly diagnosed FMS-like tyrosine kinase 3 (FLT3)-mutated (FLT3mut+) acute myeloid leukemia (AML) ineligible for intensive chemotherapy are disappointing. This multicenter, open-label, phase 3 trial randomized (2:1) untreated adults with FLT3mut+ AML ineligible for intensive induction chemotherapy to receive gilteritinib (120 mg/d orally) and azacitidine (GIL + AZA) or azacitidine (AZA) alone. The primary end point was overall survival (OS). At the interim analysis (August 26, 2020), a total of 123 patients were randomized to treatment (GIL + AZA, n = 74; AZA, n = 49). Subsequent AML therapy, including FLT3 inhibitors, was received by 20.3% (GIL + AZA) and 44.9% (AZA) of patients. Median OS was 9.82 (GIL + AZA) and 8.87 (AZA) months (hazard ratio, 0.916; 95% CI, 0.529-1.585; P = .753). The study was closed based on the protocol-specified boundary for futility. Median event-free survival was 0.03 month in both arms. Event-free survival defined by using composite complete remission (CRc) was 4.53 months for GIL + AZA and 0.03 month for AZA (hazard ratio, 0.686; 95% CI, 0.433-1.087; P = .156). CRc rates were 58.1% (GIL + AZA) and 26.5% (AZA) (difference, 31.4%; 95% CI, 13.1-49.7; P < .001). Adverse event (AE) rates were similar for GIL + AZA (100%) and AZA (95.7%); grade ≥3 AEs were 95.9% and 89.4%, respectively. Common AEs with GIL + AZA included pyrexia (47.9%) and diarrhea (38.4%). Gilteritinib steady-state trough concentrations did not differ between GIL + AZA and gilteritinib. GIL + AZA resulted in significantly higher CRc rates, although similar OS compared with AZA. Results support the safety/tolerability and clinical activity of upfront therapy with GIL + AZA in older/unfit patients with FLT3mut+ AML. This trial was registered at www.clinicaltrials.gov as #NCT02752035.
Assuntos
Azacitidina , Leucemia Mieloide Aguda , Adulto , Humanos , Idoso , Azacitidina/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/diagnóstico , Pirazinas/efeitos adversosRESUMO
PURPOSE: Allogeneic hematopoietic cell transplantation (HCT) improves outcomes for patients with AML harboring an internal tandem duplication mutation of FLT3 (FLT3-ITD) AML. These patients are routinely treated with a FLT3 inhibitor after HCT, but there is limited evidence to support this. Accordingly, we conducted a randomized trial of post-HCT maintenance with the FLT3 inhibitor gilteritinib (ClinicalTrials.gov identifier: NCT02997202) to determine if all such patients benefit or if detection of measurable residual disease (MRD) could identify those who might benefit. METHODS: Adults with FLT3-ITD AML in first remission underwent HCT and were randomly assigned to placebo or 120 mg once daily gilteritinib for 24 months after HCT. The primary end point was relapse-free survival (RFS). Secondary end points included overall survival (OS) and the effect of MRD pre- and post-HCT on RFS and OS. RESULTS: Three hundred fifty-six participants were randomly assigned post-HCT to receive gilteritinib or placebo. Although RFS was higher in the gilteritinib arm, the difference was not statistically significant (hazard ratio [HR], 0.679 [95% CI, 0.459 to 1.005]; two-sided P = .0518). However, 50.5% of participants had MRD detectable pre- or post-HCT, and, in a prespecified subgroup analysis, gilteritinib was beneficial in this population (HR, 0.515 [95% CI, 0.316 to 0.838]; P = .0065). Those without detectable MRD showed no benefit (HR, 1.213 [95% CI, 0.616 to 2.387]; P = .575). CONCLUSION: Although the overall improvement in RFS was not statistically significant, RFS was higher for participants with detectable FLT3-ITD MRD pre- or post-HCT who received gilteritinib treatment. To our knowledge, these data are among the first to support the effectiveness of MRD-based post-HCT therapy.
Assuntos
Compostos de Anilina , Transplante de Células-Tronco Hematopoéticas , Leucemia Mieloide Aguda , Mutação , Pirazinas , Tirosina Quinase 3 Semelhante a fms , Humanos , Tirosina Quinase 3 Semelhante a fms/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/mortalidade , Masculino , Feminino , Pessoa de Meia-Idade , Pirazinas/uso terapêutico , Adulto , Compostos de Anilina/uso terapêutico , Idoso , Sequências de Repetição em Tandem , Adulto Jovem , Neoplasia Residual , Inibidores de Proteínas Quinases/uso terapêutico , Quimioterapia de Manutenção , Duplicação GênicaRESUMO
PURPOSE: Gilteritinib is a type 1 FLT3 inhibitor active as monotherapy for relapsed or refractory FLT3-mutated AML. We investigated the safety, tolerability, and efficacy of gilteritinib incorporated into intensive induction and consolidation chemotherapy, and as maintenance therapy for adult patients with newly diagnosed, non-favorable-risk AML. METHODS: In this phase IB study (2215-CL-0103; ClinicalTrials.gov identifier: NCT02236013), 103 participants were screened and 80 were allocated to treatment. The study was divided into four parts: dose escalation, dose expansion, investigation of alternate anthracycline and gilteritinib schedule, and continuous gilteritinib during consolidation. RESULTS: After dose escalation, 120 mg gilteritinib once daily was chosen for further study. There were 58 participants evaluable for response at this dose, 36 of whom harbored FLT3 mutations. For participants with FLT3-mutated AML, the composite complete response (CRc) rate was 89% (83% were conventional complete responses), all achieved after a single induction cycle. The median overall survival time was 46.1 months. Gilteritinib was well-tolerated in this context although the median time to count recovery during induction was approximately 40 days. Longer time-to-count recovery was associated with higher trough levels of gilteritinib, which, in turn, were associated with azole use. The recommended regimen is gilteritinib at a dose of 120 mg once daily from days 4 to 17 or 8 to 21 of a 7 + 3 induction with either idarubicin or daunorubicin and from day 1 continuously with high-dose cytarabine consolidation. Maintenance therapy with gilteritinib was well-tolerated. CONCLUSION: These results demonstrated the safety and tolerability of gilteritinib incorporated into an induction and consolidation chemotherapy regimen, and as single-agent maintenance therapy for patients with newly diagnosed FLT3-mutant AML. The data herein provide an important framework for the design of randomized trials comparing gilteritinib with other FLT3 inhibitors.
Assuntos
Quimioterapia de Consolidação , Leucemia Mieloide Aguda , Adulto , Humanos , Recidiva Local de Neoplasia/tratamento farmacológico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Idarubicina , Inibidores de Proteínas Quinases/uso terapêutico , Tirosina Quinase 3 Semelhante a fms/genética , MutaçãoRESUMO
Genetic and chemical perturbations impact diverse cellular phenotypes, including multiple indicators of cell health. These readouts reveal toxicity and antitumorigenic effects relevant to drug discovery and personalized medicine. We developed two customized microscopy assays, one using four targeted reagents and the other three targeted reagents, to collectively measure 70 specific cell health phenotypes including proliferation, apoptosis, reactive oxygen species, DNA damage, and cell cycle stage. We then tested an approach to predict multiple cell health phenotypes using Cell Painting, an inexpensive and scalable image-based morphology assay. In matched CRISPR perturbations of three cancer cell lines, we collected both Cell Painting and cell health data. We found that simple machine learning algorithms can predict many cell health readouts directly from Cell Painting images, at less than half the cost. We hypothesized that these models can be applied to accurately predict cell health assay outcomes for any future or existing Cell Painting dataset. For Cell Painting images from a set of 1500+ compound perturbations across multiple doses, we validated predictions by orthogonal assay readouts. We provide a web app to browse predictions: http://broad.io/cell-health-app. Our approach can be used to add cell health annotations to Cell Painting datasets.
Assuntos
Células/citologia , Previsões/métodos , Processamento de Imagem Assistida por Computador/métodos , Algoritmos , Bioensaio , Linhagem Celular , Humanos , Aprendizado de Máquina , Microscopia , FenótipoRESUMO
There are few options for prophylaxis after exposure to Bacillus anthracis, especially in children and women of childbearing potential. Faropenem is a beta-lactam in the penem subclass that is being developed as an oral prodrug, faropenem medoxomil, for the treatment of respiratory tract infections. Faropenem was shown to have in vitro activity against B. anthracis strains that variably express the bla1 beta-lactamase (MIC range, Assuntos
Antraz/tratamento farmacológico
, Antraz/prevenção & controle
, Antibacterianos/farmacocinética
, Bacillus anthracis/efeitos dos fármacos
, beta-Lactamas/farmacocinética
, Animais
, Antraz/mortalidade
, Antibacterianos/sangue
, Proteínas Sanguíneas/metabolismo
, Modelos Animais de Doenças
, Relação Dose-Resposta a Droga
, Feminino
, Exposição por Inalação
, Camundongos
, Camundongos Endogâmicos BALB C
, Modelos Biológicos
, beta-Lactamases/metabolismo
, beta-Lactamas/sangue
RESUMO
We hypothesized that candidate dependencies for which there are small molecules that are either approved or in advanced development for a nononcology indication may represent potential therapeutic targets. To test this hypothesis, we performed genome-scale loss-of-function screens in hundreds of cancer cell lines. We found that knockout of EGLN1, which encodes prolyl hydroxylase domain-containing protein 2 (PHD2), reduced the proliferation of a subset of clear cell ovarian cancer cell lines in vitro. EGLN1-dependent cells exhibited sensitivity to the pan-EGLN inhibitor FG-4592. The response to FG-4592 was reversed by deletion of HIF1A, demonstrating that EGLN1 dependency was related to negative regulation of HIF1A. We also found that ovarian clear cell tumors susceptible to both genetic and pharmacologic inhibition of EGLN1 required intact HIF1A. Collectively, these observations identify EGLN1 as a cancer target with therapeutic potential. SIGNIFICANCE: These findings reveal a differential dependency of clear cell ovarian cancers on EGLN1, thus identifying EGLN1 as a potential therapeutic target in clear cell ovarian cancer patients.
Assuntos
Estudo de Associação Genômica Ampla , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Neoplasias Ovarianas/genética , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Ovarianas/patologia , Interferência de RNARESUMO
CRISPR-Cas9 provides the means to perform genome editing and facilitates loss-of-function screens. However, we and others demonstrated that expression of the Cas9 endonuclease induces a gene-independent response that correlates with the number of target sequences in the genome. An alternative approach to suppressing gene expression is to block transcription using a catalytically inactive Cas9 (dCas9). Here we directly compare genome editing by CRISPR-Cas9 (cutting, CRISPRc) and gene suppression using KRAB-dCas9 (CRISPRi) in loss-of-function screens to identify cell essential genes. CRISPRc identified 98% of previously defined cell essential genes. After optimizing library construction by analysing transcriptional start sites (TSS), CRISRPi identified 92% of core cell essential genes and did not show a bias to regions involved in copy number alterations. However, bidirectional promoters scored as false positives in CRISRPi. We conclude that CRISPRc and CRISPRi have different off-target effects and combining these approaches provides complementary information in loss-of-function genetic screens.
Assuntos
Sistemas CRISPR-Cas , Deleção de Genes , Genes Essenciais , Genoma Humano , Células A549 , Catálise , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , DNA/análise , Variações do Número de Cópias de DNA , Endonucleases/metabolismo , Reações Falso-Positivas , Edição de Genes , Expressão Gênica , Biblioteca Gênica , Células HT29 , Humanos , FenótipoRESUMO
PURPOSE: This study characterized the pharmacokinetics, mass balance, routes and extent of elimination, metabolites, and safety of a single oral dose of (14)C-linsitinib, an IGF-1R/IR inhibitor, in patients with advanced solid tumors. The tolerability of linsitinib after multiple-dose administration was assessed in those patients who wished to continue treatment beyond the single (14)C-linsitinib dose. METHODS: Five patients received a single oral dose of 150 mg (14)C-linsitinib, followed by collection of blood, plasma, urine, and feces for 10 days. The collected material was analyzed for total radioactivity, linsitinib, and metabolites. The safety of 150 mg of unlabeled linsitinib administered twice daily until disease progression was also assessed. RESULTS: The median time to reach the maximum plasma concentration of linsitinib was 3.0 h, median maximum plasma concentration was 1789 ng/mL, median terminal half-life was 2.4 h, and median apparent oral clearance was 12.45 L/h. After a single dose of (14)C-linsitinib, 5.44 and 76.4 % of mean total radioactivity administered were recovered in urine and feces, respectively. Eighteen linsitinib metabolites (M1-M18) were detected in plasma, urine, and feces samples, and their structures were elucidated. The main metabolic reactions of linsitinib in humans were oxidation and sulfate conjugation. Linsitinib was well tolerated after a single dose of (14)C-linsitinib, and fatigue was the most frequent adverse event following multiple doses of unlabeled linsitinib. CONCLUSIONS: (14)C-linsitinib is rapidly absorbed and extensively metabolized. Linsitinib excretion via bile into feces is the predominant elimination route from plasma with minor renal elimination.
Assuntos
Imidazóis/farmacocinética , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacocinética , Pirazinas/farmacocinética , Idoso , Feminino , Humanos , Imidazóis/efeitos adversos , Masculino , Redes e Vias Metabólicas , Pessoa de Meia-Idade , Pirazinas/efeitos adversosRESUMO
UNLABELLED: The CRISPR/Cas9 system enables genome editing and somatic cell genetic screens in mammalian cells. We performed genome-scale loss-of-function screens in 33 cancer cell lines to identify genes essential for proliferation/survival and found a strong correlation between increased gene copy number and decreased cell viability after genome editing. Within regions of copy-number gain, CRISPR/Cas9 targeting of both expressed and unexpressed genes, as well as intergenic loci, led to significantly decreased cell proliferation through induction of a G2 cell-cycle arrest. By examining single-guide RNAs that map to multiple genomic sites, we found that this cell response to CRISPR/Cas9 editing correlated strongly with the number of target loci. These observations indicate that genome targeting by CRISPR/Cas9 elicits a gene-independent antiproliferative cell response. This effect has important practical implications for the interpretation of CRISPR/Cas9 screening data and confounds the use of this technology for the identification of essential genes in amplified regions. SIGNIFICANCE: We found that the number of CRISPR/Cas9-induced DNA breaks dictates a gene-independent antiproliferative response in cells. These observations have practical implications for using CRISPR/Cas9 to interrogate cancer gene function and illustrate that cancer cells are highly sensitive to site-specific DNA damage, which may provide a path to novel therapeutic strategies. Cancer Discov; 6(8); 914-29. ©2016 AACR.See related commentary by Sheel and Xue, p. 824See related article by Munoz et al., p. 900This article is highlighted in the In This Issue feature, p. 803.
Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Dosagem de Genes , Marcação de Genes , Genômica , Linhagem Celular Tumoral , Clivagem do DNA , Variações do Número de Cópias de DNA , Dano ao DNA , Pontos de Checagem da Fase G2 do Ciclo Celular , Amplificação de Genes , Edição de Genes , Expressão Gênica , Técnicas de Inativação de Genes , Marcação de Genes/métodos , Genes Essenciais , Genômica/métodos , Ensaios de Triagem em Larga Escala , Humanos , RNA Guia de CinetoplastídeosRESUMO
EGF receptor (EGFR) inhibitors are used in the therapy of lung and pancreatic cancers and effectively prevent cancers in multiple animal models. Although daily dosing with erlotinib is effective, weekly dosing may reduce toxicity and have advantages, particularly for prevention. We tested alternative dosing regimens for preventive/therapeutic efficacy in a rat mammary cancer model. For prevention, erlotinib was administered by gavage beginning 5 days after methylnitrosourea (MNU). For therapy and biomarker studies, rats with palpable mammary cancers were treated for six weeks or for six days, respectively. Experiment A, erlotinib (6 mg/kg body weight/day, intragastric): daily (7 times/week); one day on/one day off; and two days on/two days off. All regimens decreased tumor incidence, increased tumor latency, and decreased cancer multiplicity versus controls (P < 0.01). However, intermittent dosing was less effective than daily dosing (P < 0.05). Experiment B, erlotinib (6 mg/kg body weight/day) daily or two days on/two days off or one time per week at 42 mg/kg body weight. All regimens reduced cancer incidence and multiplicity versus controls (P < 0.01). Interestingly, daily and weekly dosing were equally effective (P > 0.5). Experiment C, erlotinib administered at 42 or 21 mg/kg body weight 1 time per week, decreased tumor incidence and multiplicity (P < 0.01). Erlotinib had a serum half-life of ≤ 8 hours and weekly treatment yielded effective serum levels for ≤ 48 hours. Daily or weekly treatment of cancer bearing rats reduced mammary tumor size 25% to 35%, whereas control cancers increased >250%. Levels of phosphorylated extracellular signal-regulated kinase (ERK) were strongly decreased in rats treated daily/weekly with erlotinib. Thus, altering the dose of erlotinib retained most of its preventive and therapeutic efficacy, and based on prior clinical studies, is likely to reduce its toxicity.