The RAS pathway is among the most frequently activated signaling nodes in cancer. However, the mechanisms that alter RAS activity in human pathologies are not entirely understood. The most prevalent post-translational modification within the GTPase core domain of NRAS and KRAS is ubiquitination at lysine 128 (K128), which is significantly decreased in cancer samples compared to normal tissue. Here, we found that K128 ubiquitination creates an additional binding interface for RAS GTPase-activating proteins (GAPs), NF1 and RASA1, thus increasing RAS binding to GAP proteins and promoting GAP-mediated GTP hydrolysis. Stimulation of cultured cancer cells with growth factors or cytokines transiently induces K128 ubiquitination and restricts the extent of wild-type RAS activation in a GAP-dependent manner. In KRAS mutant cells, K128 ubiquitination limits tumor growth by restricting RAL/ TBK1 signaling and negatively regulating the autocrine circuit induced by mutant KRAS. Reduction of K128 ubiquitination activates both wild-type and mutant RAS signaling and elicits a senescence-associated secretory phenotype, promoting RAS-driven pancreatic tumorigenesis.

• MeSH
• GTP-fosfohydrolasy metabolismus   genetika   MeSH
• lidé MeSH
• lysin metabolismus   MeSH
• membránové proteiny metabolismus   genetika   MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• neurofibromin 1 MeSH
• protein aktivující GTPasu p120 metabolismus   genetika   MeSH
• protein-serin-threoninkinasy metabolismus   genetika   MeSH
• protoonkogenní proteiny p21(ras) * metabolismus   genetika   MeSH
• Ras proteiny metabolismus   genetika   MeSH
• signální transdukce MeSH
• ubikvitinace * MeSH
• vazba proteinů * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

With the rapid advancement of sequencing technologies, next generation sequencing (NGS) analysis has been widely applied in cancer genomics research. More recently, NGS has been adopted in clinical oncology to advance personalized medicine. Clinical applications of precision oncology require accurate tests that can distinguish tumor-specific mutations from artifacts introduced during NGS processes or data analysis. Therefore, there is an urgent need to develop best practices in cancer mutation detection using NGS and the need for standard reference data sets for systematically measuring accuracy and reproducibility across platforms and methods. Within the SEQC2 consortium context, we established paired tumor-normal reference samples and generated whole-genome (WGS) and whole-exome sequencing (WES) data using sixteen library protocols, seven sequencing platforms at six different centers. We systematically interrogated somatic mutations in the reference samples to identify factors affecting detection reproducibility and accuracy in cancer genomes. These large cross-platform/site WGS and WES datasets using well-characterized reference samples will represent a powerful resource for benchmarking NGS technologies, bioinformatics pipelines, and for the cancer genomics studies.

• MeSH
• benchmarking MeSH
• genom lidský * MeSH
• genomika MeSH
• individualizovaná medicína MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory genetika   MeSH
• sekvenování celého genomu * MeSH
• sekvenování exomu * MeSH
• výpočetní biologie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• dataset MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

The lack of samples for generating standardized DNA datasets for setting up a sequencing pipeline or benchmarking the performance of different algorithms limits the implementation and uptake of cancer genomics. Here, we describe reference call sets obtained from paired tumor-normal genomic DNA (gDNA) samples derived from a breast cancer cell line-which is highly heterogeneous, with an aneuploid genome, and enriched in somatic alterations-and a matched lymphoblastoid cell line. We partially validated both somatic mutations and germline variants in these call sets via whole-exome sequencing (WES) with different sequencing platforms and targeted sequencing with >2,000-fold coverage, spanning 82% of genomic regions with high confidence. Although the gDNA reference samples are not representative of primary cancer cells from a clinical sample, when setting up a sequencing pipeline, they not only minimize potential biases from technologies, assays and informatics but also provide a unique resource for benchmarking 'tumor-only' or 'matched tumor-normal' analyses.

• MeSH
• benchmarking * MeSH
• datové soubory jako téma MeSH
• lidé MeSH
• mutace MeSH
• mutační analýza DNA normy   MeSH
• nádorové buněčné linie MeSH
• nádory prsu genetika   MeSH
• referenční standardy MeSH
• reprodukovatelnost výsledků MeSH
• sekvenování celého genomu normy   MeSH
• vysoce účinné nukleotidové sekvenování normy   MeSH
• zárodečné buňky MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Clinical applications of precision oncology require accurate tests that can distinguish true cancer-specific mutations from errors introduced at each step of next-generation sequencing (NGS). To date, no bulk sequencing study has addressed the effects of cross-site reproducibility, nor the biological, technical and computational factors that influence variant identification. Here we report a systematic interrogation of somatic mutations in paired tumor-normal cell lines to identify factors affecting detection reproducibility and accuracy at six different centers. Using whole-genome sequencing (WGS) and whole-exome sequencing (WES), we evaluated the reproducibility of different sample types with varying input amount and tumor purity, and multiple library construction protocols, followed by processing with nine bioinformatics pipelines. We found that read coverage and callers affected both WGS and WES reproducibility, but WES performance was influenced by insert fragment size, genomic copy content and the global imbalance score (GIV; G > T/C > A). Finally, taking into account library preparation protocol, tumor content, read coverage and bioinformatics processes concomitantly, we recommend actionable practices to improve the reproducibility and accuracy of NGS experiments for cancer mutation detection.

• MeSH
• benchmarking * MeSH
• buněčné linie MeSH
• lidé MeSH
• mutace MeSH
• nádorové buněčné linie MeSH
• nádory genetika   patologie   MeSH
• reprodukovatelnost výsledků MeSH
• sekvenční analýza DNA normy   MeSH
• sekvenování celého genomu normy   MeSH
• sekvenování exomu normy   MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH