Members of the RAS superfamily of small guanosine triphosphatases (GTPases) transition between GDP-bound, inactive and GTPbound, active states and thereby function as binary switches in the regulation of various cellular activities. Whereas HRAS, NRAS, and KRAS frequently acquire transforming missense mutations in human cancer, little is known of the oncogenic roles of other small GTPases, including Ras-related C3 botulinum toxin substrate (RAC) proteins. We show that the human sarcoma cell line HT1080 harbors both NRAS(Q61K) and RAC1(N92I) mutant proteins. Whereas both of these mutants were able to transform fibroblasts, knockdown experiments indicated that RAC1(N92I) may be the essential growth driver for this cell line. Screening for RAC1, RAC2, or RAC3 mutations in cell lines and public databases identified several missense mutations for RAC1 and RAC2, with some of the mutant proteins, including RAC1(P29S), RAC1(C157Y), RAC2(P29L), and RAC2(P29Q), being found to be activated and transforming. P29S, N92I, and C157Y mutants of RAC1 were shown to exist preferentially in the GTP-bound state as a result of a rapid transition from the GDP-bound state, rather than as a result of a reduced intrinsic GTPase activity. Activating mutations of RAC GTPases were thus found in a wide variety of human cancers at a low frequency; however, given their marked transforming ability, the mutant proteins are potential targets for the development of new therapeutic agents.oncogene | resequencing

Tumor molecular profiling is becoming a standard of care for patients with cancer, but the optimal platform for cancer sequencing remains undetermined. We established a comprehensive assay, the Todai OncoPanel ( TOP ), which consists of DNA and RNA hybridization capture‐based next‐generation sequencing panels. A novel method for target enrichment, named the junction capture method, was developed for the RNA panel to accurately and cost‐effectively detect 365 fusion genes as well as aberrantly spliced transcripts. The TOP RNA panel can also measure the expression profiles of an additional 109 genes. The TOP DNA panel was developed to detect single nucleotide variants and insertions/deletions for 464 genes, to calculate tumor mutation burden and microsatellite instability status, and to infer chromosomal copy number. Clinically relevant somatic mutations were identified in 32.2% (59/183) of patients by prospective TOP testing, signifying the clinical utility of TOP for providing personalized medicine to cancer patients.

Purpose: Recently, several comprehensive genomic analyses demonstrated NOTCH1 and NOTCH3 mutations in head and neck squamous cell carcinoma (HNSCC) in approximately 20% of cases. Similar to other types of cancers, these studies also indicate that the NOTCH pathway is closely related to HNSCC progression. However, the role of NOTCH4 in HNSCC is less well understood.Experimental Design: We analyzed NOTCH4 pathway and downstream gene expression in the TCGA data set. To explore the functional role of NOTCH4, we performed in vitro proliferation, cisplatin viability, apoptosis, and cell-cycle assays. We also compared the relationships among NOTCH4, HEY1, and epithelialmesenchymal transition (EMT)-related genes using the TCGA data set and in vitro assays.Results: HEY1 is specifically upregulated in HNSCC compared with normal tissues in the TCGA data set. NOTCH4 is more significantly related to HEY1 activation in HNSCC in comparison with other NOTCH receptors. NOTCH4 promotes cell proliferation, cisplatin resistance, inhibition of apoptosis, and cell-cycle dysregulation. Furthermore, NOTCH4 and HEY1 upregulation resulted in decreased E-cadherin expression and increased Vimentin, Fibronectin, TWIST1, and SOX2 expression. NOTCH4 and HEY1 expression was associated with an EMT phenotype as well as increased invasion and cell migration.Conclusions: In HNSCC, the NOTCH4-HEY1 pathway is specifically upregulated, induces proliferation and cisplatin resistance, and promotes EMT.

The dominant paradigm for HPV carcinogenesis includes integration into the host genome followed by expression of E6 and E7 (E6/E7). We explored an alternative carcinogenic pathway characterized by episomal E2, E4, and E5 (E2/E4/E5) expression. Half of HPV positive cervical and pharyngeal cancers comprised a subtype with increase in expression of E2/E4/E5, as well as association with lack of integration into the host genome. Models of the E2/E4/E5 carcinogenesis show p53 dependent enhanced proliferation in vitro, as well as increased susceptibility to induction of cancer in vivo. Whole genomic expression analysis of the E2/E4/E5 pharyngeal cancer subtype is defined by activation of the fibroblast growth factor receptor (FGFR) pathway and this subtype is susceptible to combination FGFR and mTOR inhibition, with implications for targeted therapy.

Although promoter-associated CpG islands have been established as targets of DNA methylation changes in cancer, previous studies suggest that epigenetic dysregulation outside the promoter region may be more closely associated with transcriptional changes. Here we examine DNA methylation, chromatin marks, and transcriptional alterations to define the relationship between transcriptional modulation and spatial changes in chromatin structure. Using human papillomavirus-related oropharyngeal carcinoma as a model, we show aberrant enrichment of repressive H3K9me3 at the transcriptional start site (TSS) with methylation-associated, tumor-specific gene silencing. Further analysis identifies a hypermethylated subtype which shows a functional convergence on MYC targets and association with CREBBP/EP300 mutation. The tumor-specific shift to transcriptional repression associated with DNA methylation at TSSs was confirmed in multiple tumor types. Our data may show a common underlying epigenetic dysregulation in cancer associated with broad enrichment of repressive chromatin marks and aberrant DNA hypermethylation at TSSs in combination with MYC network activation.

2 TRANSLATIONAL RELEVANCEHPV negative HNSCC represents a distinct clinical entity from HPV positive HNSCC. Recently, alternative splicing has been shown to be closely related to tumor progression. However, the characterization of alternative splicing in HPV negative HNSCC is still largely undescribed. In this study, we identified 580 significant splicing events in HPV negative HNSCC and we identified a novel DOCK5 variant highly expressed in a separate primary tumor validation set using qRT-PCR. We demonstrated that the DOCK5 variant played an oncogenic role in HPV negative HNSCC, which could promote proliferation, migration and invasion in HPV negative HNSCC cell lines, and patients with higher expression of DOCK5 variant showed decreased overall survival. Identification of alternative splicing in HPV negative HNSCC allows for a promising avenue to identify novel therapeutic targets and expands the understanding of HNSCC carcinogenesis. KEY WORDSHead and neck squamous cell carcinoma, alternative splicing, HPV negative, DOCK5Research. Experimental Design: RNA sequencing data of 407 HPV negative HNSCC and 38 normal samples were obtained from The Cancer Genome Atlas (TCGA) and splice junctions were discovered using MapSplice.Outlier analysis was used to identify significant splicing junctions between HPV negative HNSCC and normal samples. To explore the functional role of the identified DOCK5 variant, we checked its expression with qRT-PCR in a separate primary tumor validation set and performed proliferation, migration and invasion assays.Results: 580 significant splicing events were identified in HPV negative HNSCC and the most common type of splicing events was an alternative start site (33.3%). The prevalence of a given individual ASE among the tumor cohort ranged from 9.8% and 64.4%. Within the 407 HPV negative HNSCC samples in TCGA, the number of significant ASEs differentially expressed in each tumor ranged from 17 to 290. We identified a novel candidate oncogenic DOCK5 variant confirmed using qRT-PCR in a separate primary tumor validation set. Loss-and gain-of-function experiments indicated that DOCK5 variant promoted proliferation, migration and invasion of HPV negative HNSCC cells, and patients with higher expression of DOCK5 variant showed decreased overall survival. Conclusion:Analysis of ASEs in HPV negative HNSCC identifies multiple alterations likely related to carcinogenesis, including an oncogenic DOCK5 variant.

The molecular characteristics of therapeutically-relevant targets and their clinicopathological implications in salivary duct carcinomas (SDCs) are poorly understood. We investigated the gene alterations and the immunoexpression of crucial oncogenic molecules in 151 SDCs. The mutation rates that were identified, in order of frequency, were as follows: TP53, 68%; PIK3CA, 18%; H-RAS, 16%; BRAF, 4%; and AKT1, 1.5%. PIK3CA/H-RAS/BRAF mutations were more common in de novo SDC than in SDC ex-pleomorphic adenoma. Furthermore, these mutations were mutually exclusive for HER2 overexpression/amplification. TP53 mutations were frequently detected in cases with the aberrant p53 expression, and TP53 missense and truncating mutations were associated with p53-extreme positivity and negativity, respectively. DISH analysis revealed no cases of EGFR amplification. The rates of PI3K, p-Akt, and p-mTOR positivity were 34%, 22%, and 66%, respectively; PTEN loss was observed in 47% of the cases. These expressions were correlated according to the signaling axis. Cases with PI3K negativity and PTEN loss appeared to show a lower expression of androgen receptor. In the multivariate analysis, patients with SDC harboring TP53 truncating mutations showed shorter progression-free survival. Conversely, p-Akt positivity was associated with a favorable outcome. This study might provide information that leads to advances in personized therapy for SDC.

Aberrant activation of the PI3K-mTOR signaling pathway occurs in >80% of head and neck squamous cell carcinomas (HNSCC), and overreliance on this signaling circuit may in turn represent a cancer-specific vulnerability that can be exploited therapeutically. mTOR inhibitors (mTORi) promote tumor regression in genetically defined and chemically induced HNSCC animal models, and encouraging results have been recently reported. However, the mTOR-regulated targets contributing to the clinical response have not yet been identified. Here, we focused on EIF4E-BP1 (4E-BP1), a direct target of mTOR that serves as key effector for protein synthesis. A systematic analysis of genomic alterations in the PIK3CA-mTOR pathway in HNSCC revealed that 4E-BP1 is rarely mutated, but at least one 4E-BP1 gene copy is lost in over 35% of the patients with HNSCC, correlating with decreased 4E-BP1 protein expression. 4E-BP1 gene copy number loss correlated with poor disease-free and overall survival. Aligned with a tumor-suppressive role, 4e-bp1/2 knockout mice formed larger and more lesions in models of HNSCC carcinogenesis. mTORi treatment or conditional expression of a mutant 4E-BP1 that cannot be phosphorylated by mTOR was sufficient to disrupt the translation-initiation complex and prevent tumor growth. Furthermore, CRISPR/Cas9-targeted 4E-BP1 HNSCC cells resulted in reduced sensitivity to mTORi in vitro and in vivo. Overall, these findings indicate that in HNSCC, mTOR persistently restrains 4E-BP1 via phosphorylation and that mTORi can restore the tumor-suppressive function of 4E-BP1. Our findings also support 4E-BP1 expression and phosphorylation status as a mechanistic biomarker of mTORi sensitivity in patients with HNSCC. Significance: These findings suggest that EIF4E-BP1 acts as a tumor suppressor in HNSCC and that 4E-BP1 dephosphorylation mediates the therapeutic response to mTORi, providing a mechanistic biomarker for future precision oncology trials.