RESUMEN
To establish novel and effective treatment combinations for chronic myelomonocytic leukemia (CMML) preclinically, we hypothesized that supplementation of CMML cells with the human oncogene Meningioma 1 (MN1) promotes expansion and serial transplantability in mice, while maintaining the functional dependencies of these cells on their original genetic profile. Using lentiviral expression of MN1 for oncogenic supplementation and transplanting transduced primary mononuclear CMML cells into immunocompromised mice, we established three serially transplantable CMML-PDX models with disease-related gene mutations that recapitulate the disease in vivo. Ectopic MN1 expression was confirmed to enhance the proliferation of CMML cells, which otherwise did not engraft upon secondary transplantation. Furthermore, MN1-supplemented CMML cells were serially transplantable into recipient mice up to 5 generations. This robust engraftment enabled an in vivo RNA interference screening targeting CMML-related mutated genes including NRAS, confirming that their functional relevance is preserved in the presence of MN1. The novel combination treatment with azacitidine and the MEK-inhibitor trametinib additively inhibited ERK-phosphorylation and thus depleted the signal from mutated NRAS. The combination treatment significantly prolonged survival of CMML mice compared to single-agent treatment. Thus, we identified the combination of azacitidine and trametinib as an effective treatment in NRAS-mutated CMML and propose its clinical development.
Asunto(s)
Antineoplásicos/farmacología , Evaluación Preclínica de Medicamentos , Leucemia Mielomonocítica Crónica/tratamiento farmacológico , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Azacitidina/farmacología , Evolución Clonal , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos/métodos , Evaluación Preclínica de Medicamentos/normas , Sinergismo Farmacológico , Femenino , GTP Fosfohidrolasas/genética , Humanos , Leucemia Mielomonocítica Crónica/genética , Leucemia Mielomonocítica Crónica/mortalidad , Leucemia Mielomonocítica Crónica/patología , Proteínas de la Membrana/genética , Ratones , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridonas/farmacología , Piridonas/uso terapéutico , Pirimidinonas/farmacología , Pirimidinonas/uso terapéutico , ARN Interferente Pequeño/genética , Receptor Notch1/genética , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Short hairpin RNA (shRNA) screens are powerful tools to probe genetic dependencies in loss-of-function studies, such as the identification of therapeutic targets in cancer research. Lentivirally delivered shRNAs embedded in endogenous microRNA contexts (shRNAmiRs) mediate efficient long-term suppression of target genes suitable for numerous experimental contexts and clinical applications. Here, an easy-to-use laboratory protocol is described, covering the design and pooled assembly of focused shRNAmiR libraries into an optimized, Tet-inducible all-in-one lentiviral vector, packaging of viral particles, followed by retrieval and quantification of hairpin sequences after cellular DNA-recovery. Starting from a gene list to the identification of hits, the protocol enables shRNA screens within 6 weeks.
Asunto(s)
Biblioteca de Genes , Vectores Genéticos/genética , Lentivirus/genética , MicroARNs/genética , ARN Interferente Pequeño/genética , Tetraciclina , Línea Celular , HumanosRESUMEN
Genome editing with the CRISPR-Cas9 system has enabled unprecedented efficacy for reverse genetics and gene correction approaches. While off-target effects have been successfully tackled, the effort to eliminate variability in sgRNA efficacies-which affect experimental sensitivity-is in its infancy. To address this issue, studies have analyzed the molecular features of highly active sgRNAs, but independent cross-validation is lacking. Utilizing fluorescent reporter knock-out assays with verification at selected endogenous loci, we experimentally quantified the target efficacies of 430 sgRNAs. Based on this dataset we tested the predictive value of five recently-established prediction algorithms. Our analysis revealed a moderate correlation (r = 0.04 to r = 0.20) between the predicted and measured activity of the sgRNAs, and modest concordance between the different algorithms. We uncovered a strong PAM-distal GC-content-dependent activity, which enabled the exclusion of inactive sgRNAs. By deriving nine additional predictive features we generated a linear model-based discrete system for the efficient selection (r = 0.4) of effective sgRNAs (CRISPRater). We proved our algorithms' efficacy on small and large external datasets, and provide a versatile combined on- and off-target sgRNA scanning platform. Altogether, our study highlights current issues and efforts in sgRNA efficacy prediction, and provides an easily-applicable discrete system for selecting efficient sgRNAs.
Asunto(s)
Algoritmos , Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas , Edición Génica/métodos , Marcación de Gen/métodos , ARN Guía de Kinetoplastida/genética , Composición de Base , Secuencia de Bases , Proteína 9 Asociada a CRISPR/metabolismo , Línea Celular Tumoral , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Células HEK293 , Humanos , Leucocitos/citología , Leucocitos/metabolismo , Conformación de Ácido Nucleico , ARN Guía de Kinetoplastida/química , ARN Guía de Kinetoplastida/metabolismoRESUMEN
RNA interference (RNAi) and CRISPR-Cas9-based screening systems have emerged as powerful and complementary tools to unravel genetic dependencies through systematic gain- and loss-of-function studies. In recent years, a series of technical advances helped to enhance the performance of virally delivered RNAi. For instance, the incorporation of short hairpin RNAs (shRNAs) into endogenous microRNA contexts (shRNAmiRs) allows the use of Tet-regulated promoters for synchronous onset of gene knockdown and precise interrogation of gene dosage effects. However, remaining challenges include lack of efficient cloning strategies, inconsistent knockdown potencies and leaky expression. Here, we present a simple, one-step cloning approach for rapid and efficient cloning of miR-30 shRNAmiR libraries. We combined a human miR-30 backbone retaining native flanking sequences with an optimized all-in-one lentiviral vector system for conditional RNAi to generate a versatile toolbox characterized by higher doxycycline sensitivity, reduced leakiness and enhanced titer. Furthermore, refinement of existing shRNA design rules resulted in substantially improved prediction of powerful shRNAs. Our approach was validated by accurate quantification of the knockdown potency of over 250 single shRNAmiRs. To facilitate access and use by the scientific community, an online tool was developed for the automated design of refined shRNA-coding oligonucleotides ready for cloning into our system.