RESUMO
To identify therapeutic targets for glioblastoma (GBM), we performed genome-wide CRISPR-Cas9 knockout (KO) screens in patient-derived GBM stem-like cells (GSCs) and human neural stem/progenitors (NSCs), non-neoplastic stem cell controls, for genes required for their in vitro growth. Surprisingly, the vast majority GSC-lethal hits were found outside of molecular networks commonly altered in GBM and GSCs (e.g., oncogenic drivers). In vitro and in vivo validation of GSC-specific targets revealed several strong hits, including the wee1-like kinase, PKMYT1/Myt1. Mechanistic studies demonstrated that PKMYT1 acts redundantly with WEE1 to inhibit cyclin B-CDK1 activity via CDK1-Y15 phosphorylation and to promote timely completion of mitosis in NSCs. However, in GSCs, this redundancy is lost, most likely as a result of oncogenic signaling, causing GBM-specific lethality.
Assuntos
Sistemas CRISPR-Cas/genética , Proteínas de Ciclo Celular/genética , Genoma Humano , Proteínas de Membrana/genética , Células-Tronco Neoplásicas/metabolismo , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Proteína Quinase CDC2/antagonistas & inibidores , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Ciclina B/metabolismo , Receptores ErbB/metabolismo , Biblioteca Gênica , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Microscopia de Vídeo , Mitose , Células-Tronco Neoplásicas/citologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Pirimidinonas , Interferência de RNA , Imagem com Lapso de Tempo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
To identify key regulators of human brain tumor maintenance and initiation, we performed multiple genome-wide RNAi screens in patient-derived glioblastoma multiforme (GBM) stem cells (GSCs). These screens identified the plant homeodomain (PHD)-finger domain protein PHF5A as differentially required for GSC expansion, as compared with untransformed neural stem cells (NSCs) and fibroblasts. Given PHF5A's known involvement in facilitating interactions between the U2 snRNP complex and ATP-dependent helicases, we examined cancer-specific roles in RNA splicing. We found that in GSCs, but not untransformed controls, PHF5A facilitates recognition of exons with unusual C-rich 3' splice sites in thousands of essential genes. PHF5A knockdown in GSCs, but not untransformed NSCs, astrocytes, or fibroblasts, inhibited splicing of these genes, leading to cell cycle arrest and loss of viability. Notably, pharmacologic inhibition of U2 snRNP activity phenocopied PHF5A knockdown in GSCs and also in NSCs or fibroblasts overexpressing MYC. Furthermore, PHF5A inhibition compromised GSC tumor formation in vivo and inhibited growth of established GBM patient-derived xenograft tumors. Our results demonstrate a novel viability requirement for PHF5A to maintain proper exon recognition in brain tumor-initiating cells and may provide new inroads for novel anti-GBM therapeutic strategies.
Assuntos
Neoplasias Encefálicas/fisiopatologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Glioblastoma/fisiopatologia , Interferência de RNA , Animais , Neoplasias Encefálicas/genética , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Proliferação de Células , Sobrevivência Celular/genética , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Estudo de Associação Genômica Ampla , Glioblastoma/genética , Humanos , Camundongos , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Splicing de RNA , Proteínas de Ligação a RNA , Transativadores , Transplante HeterólogoRESUMO
UNLABELLED: To identify new candidate therapeutic targets for glioblastoma multiforme, we combined functional genetics and glioblastoma network modeling to identify kinases required for the growth of patient-derived brain tumor-initiating cells (BTIC) but that are dispensable to proliferating human neural stem cells (NSC). This approach yielded BUB1B/BUBR1, a critical mitotic spindle checkpoint player, as the top-scoring glioblastoma lethal kinase. Knockdown of BUB1B inhibited expansion of BTIC isolates, both in vitro and in vivo, without affecting proliferation of NSCs or astrocytes. Mechanistic studies revealed that BUB1B's GLE2p-binding sequence (GLEBS) domain activity is required to suppress lethal kinetochore-microtubule (KT-MT) attachment defects in glioblastoma isolates and genetically transformed cells with altered sister KT dynamics, which likely favor KT-MT instability. These results indicate that glioblastoma tumors have an added requirement for BUB1B to suppress lethal consequences of altered KT function and further suggest that sister KT measurements may predict cancer-specific sensitivity to BUB1B inhibition and perhaps other mitotic targets that affect KT-MT stability. SIGNIFICANCE: Currently, no effective therapies are available for glioblastoma, the most frequent and aggressive brain tumor. Our results suggest that targeting the GLEBS domain activity of BUB1B may provide a therapeutic window for glioblastoma, as the GLEBS domain is nonessential in untransformed cells. Moreover, the results further suggest that sister KT distances at metaphase may predict sensitivity to anticancer therapeutics targeting KT function.
