RESUMO
Effective gene therapy for gain-of-function or dominant-negative disease mutations may require eliminating expression of the mutant copy together with wildtype replacement. We evaluated such a "knockdown-replace" strategy in a mouse model of DNM1 disease, a debilitating and intractable neurodevelopmental epilepsy. To challenge the approach robustly, we expressed a patient-based variant in GABAergic neurons - which resulted in growth delay and lethal seizures evident by postnatal week three - and delivered to newborn pups an AAV9-based vector encoding a ubiquitously-expressed, Dnm1-specific interfering RNA (RNAi) bivalently in tail-to-tail configuration with a neuron-specific, RNAi-resistant, codon-optimized Dnm1 cDNA. Pups receiving RNAi or cDNA alone fared no better than untreated, whereas the vast majority of mutants receiving modest doses survived with almost full growth recovery. Synaptic recordings of cortical neurons derived from treated pups revealed that significant alterations in transmission from inhibitory to excitatory neurons were rectified by bivalent vector application. To examine the mutant transcriptome and impact of treatment, we used RNAseq and functional annotation clustering. Mutants displayed abnormal expression of >1000 genes in highly significant and relevant functional clusters, clusters that were abrogated by treatment. Together these results suggest knockdown-replace as a potentially effective strategy for treating DNM1 and related genetic neurodevelopmental disease.
RESUMO
The systematic identification of genetic events driving cellular transformation and tumor progression in the absence of a highly recurrent oncogenic driver mutation is a challenge in cutaneous oncology. In cutaneous squamous cell carcinoma (cuSCC), the high UV-induced mutational burden poses a hurdle to achieve a complete molecular landscape of this disease. Here, we utilized the Sleeping Beauty transposon mutagenesis system to statistically define drivers of keratinocyte transformation and cuSCC progression in vivo in the absence of UV-IR, and identified both known tumor suppressor genes and novel oncogenic drivers of cuSCC. Functional analysis confirms an oncogenic role for the ZMIZ genes, and tumor suppressive roles for KMT2C, CREBBP and NCOA2, in the initiation or progression of human cuSCC. Taken together, our in vivo screen demonstrates an extremely heterogeneous genetic landscape of cuSCC initiation and progression, which can be harnessed to better understand skin oncogenic etiology and prioritize therapeutic candidates.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Transformação Celular Neoplásica/genética , Queratinócitos/patologia , Mutagênese Insercional/métodos , Análise de Sequência de DNA/métodos , Neoplasias Cutâneas/genética , Proteína de Ligação a CREB/genética , Carcinoma de Células Escamosas/patologia , Transformação Celular Neoplásica/patologia , Elementos de DNA Transponíveis , Proteínas de Ligação a DNA/genética , Progressão da Doença , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Coativador 2 de Receptor Nuclear/genética , Neoplasias Cutâneas/patologiaRESUMO
A central challenge in oncology is how to kill tumors containing heterogeneous cell populations defined by different combinations of mutated genes. Identifying these mutated genes and understanding how they cooperate requires single-cell analysis, but current single-cell analytic methods, such as PCR-based strategies or whole-exome sequencing, are biased, lack sequencing depth or are cost prohibitive. Transposon-based mutagenesis allows the identification of early cancer drivers, but current sequencing methods have limitations that prevent single-cell analysis. We report a liquid-phase, capture-based sequencing and bioinformatics pipeline, Sleeping Beauty (SB) capture hybridization sequencing (SBCapSeq), that facilitates sequencing of transposon insertion sites from single tumor cells in a SB mouse model of myeloid leukemia (ML). SBCapSeq analysis of just 26 cells from one tumor revealed the tumor's major clonal subpopulations, enabled detection of clonal insertion events not detected by other sequencing methods and led to the identification of dominant subclones, each containing a unique pair of interacting gene drivers along with three to six cooperating cancer genes with SB-driven expression changes.
Assuntos
DNA de Neoplasias/genética , Genes Neoplásicos/genética , Hibridização In Situ/métodos , Leucemia Mieloide/genética , Mutagênese Insercional/genética , Análise de Sequência de DNA/métodos , Algoritmos , Animais , Biomarcadores Tumorais/genética , Elementos de DNA Transponíveis , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Leucemia Mieloide/patologia , Masculino , Camundongos , Proteínas de Neoplasias/genética , Software , Transposases/genéticaRESUMO
Although nearly half of human melanomas harbor oncogenic BRAF(V600E) mutations, the genetic events that cooperate with these mutations to drive melanogenesis are still largely unknown. Here we show that Sleeping Beauty (SB) transposon-mediated mutagenesis drives melanoma progression in Braf(V600E) mutant mice and identify 1,232 recurrently mutated candidate cancer genes (CCGs) from 70 SB-driven melanomas. CCGs are enriched in Wnt, PI3K, MAPK and netrin signaling pathway components and are more highly connected to one another than predicted by chance, indicating that SB targets cooperative genetic networks in melanoma. Human orthologs of >500 CCGs are enriched for mutations in human melanoma or showed statistically significant clinical associations between RNA abundance and survival of patients with metastatic melanoma. We also functionally validate CEP350 as a new tumor-suppressor gene in human melanoma. SB mutagenesis has thus helped to catalog the cooperative molecular mechanisms driving BRAF(V600E) melanoma and discover new genes with potential clinical importance in human melanoma.