Purpose Aggressive cutaneous squamous cell carcinoma (cSCC) is often a disfiguring and lethal disease. Very little is currently known about the mutations that drive aggressive cSCC. Experimental Design Whole exome sequencing was performed on 39 cases of aggressive cSCC to identify driver genes and novel therapeutic targets. Significantly mutated genes were identified with MutSig or complementary methods developed to specifically identify candidate tumor suppressors based upon their inactivating mutation bias. Results Despite the very high mutational background caused by UV exposure, 23 candidate drivers were identified including the well-known cancer-associated genes TP53, CDKN2A, NOTCH1, AJUBA, HRAS, CASP8, FAT1, and KMT2C (MLL3). Three novel candidate tumor suppressors with putative links to cancer or differentiation, NOTCH2, PARD3 and RASA1, were also identified as possible drivers in cSCC. KMT2C mutations were associated with poor outcome and increased bone invasion. Conclusions The mutational spectrum of cSCC is similar to that of head and neck squamous cell carcinoma and dominated by tumor suppressor genes. These results improve the foundation for understanding this disease and should aid in identifying and treating aggressive cSCC.

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During a clinical trial of the tyrosine kinase inhibitor dasatinib for advanced non–small cell lung cancer (NSCLC) one patient responded dramatically and remains cancer-free 4 years later. A comprehensive analysis of his tumor revealed a previously undescribed, kinase inactivating BRAF mutation (Y472CBRAF); no inactivating BRAF mutations were found in the non-responding tumors taken from other patients. Cells transfected with Y472CBRAF exhibited CRAF, MEK, and ERK activation – characteristics identical to signaling changes that occur with previously known kinase inactivating BRAF mutants. Dasatinib selectively induced senescence in NSCLC cells with inactivating BRAF mutations. Transfection of other NSCLC cells with these BRAF mutations also increased these cells’ dasatinib sensitivity, whereas transfection with an activating BRAF mutation led to their increased dasatinib resistance. The sensitivity induced by Y472CBRAF was reversed by the introduction of a BRAF mutation that impairs RAF dimerization. Dasatinib inhibited CRAF modestly, but concurrently induced RAF dimerization resulting in ERK activation in NSCLC cells with kinase inactivating BRAF mutations. The sensitivity of NSCLC with kinase impaired BRAF to dasatinib suggested synthetic lethality of BRAF and a dasatinib target. Inhibiting BRAF in NSCLC cells expressing wild-type BRAF likewise enhanced these cells’ dasatinib sensitivity. Thus, the patient’s BRAF mutation was likely responsible for his tumor’s marked response to dasatinib, suggesting that tumors bearing kinase impaired BRAF mutations may be exquisitely sensitive to dasatinib. Moreover, the potential synthetic lethality of combination therapy including dasatinib and BRAF inhibitors may lead to additional therapeutic options against cancers with wild-type BRAF.

Molecular annotated patient-derived xenograft (PDX) models are useful for the preclinical investigation of anticancer drugs and individualized anticancer therapy. We established 23 PDXs from 88 surgical specimens of lung cancer patients and determined gene mutations in these PDXs and their paired primary tumors by ultradeep exome sequencing on 202 cancer-related genes. The numbers of primary tumors with deleterious mutations in TP53, KRAS, PI3KCA, ALK, STK11, and EGFR were 43.5%, 21.7%, 17.4%, 17.4%, 13.0%, and 8.7%, respectively. Other genes with deleterious mutations in ≥3 (13.0%) primary tumors were MLL3, SETD2, ATM, ARID1A, CRIPAK, HGF, BAI3, EP300, KDR, PDGRRA and RUNX1. Of 315 mutations detected in the primary tumors, 293 (93%) were also detected in their corresponding PDXs, indicating that PDXs have the capacity to recapitulate the mutations in primary tumors. Nevertheless, a substantial number of mutations had higher allele frequencies in the PDXs than in the primary tumors, or were not detectable in the primary tumor, suggesting the possibility of tumor cell enrichment in PDXs or heterogeneity in the primary tumors. The molecularly annotated PDXs generated from this study could be useful for future translational studies.

The PI3K/AKT/mTOR pathway is frequently activated in head and neck squamous cell carcinoma (HNSCC), but pathway inhibition has variable efficacy. Identification of predictive biomarkers and mechanisms of resistance would allow selection of patients most likely to respond and novel therapeutic combinations. The purpose of this study was to extend recent discoveries regarding the PI3K/AKT/mTOR pathway in HNSCC by more broadly examining potential biomarkers of response, by examining pathway inhibitors with a diverse range of targets, and by defining mechanisms of resistance and potential combination therapies. We used reverse-phase protein arrays (RPPA) to simultaneously evaluate expression of 195 proteins; single-nucleotide polymorphism array to estimate gene copy number; and mass array to identify mutations. We examined altered signaling at baseline and after pathway inhibition. Likewise, we examined the activation of the PI3K/AKT/mTOR pathway in HNSCC tumors by RPPA. Cell lines with PIK3CA mutations were sensitive to pathway inhibitors, whereas amplification status did not predict sensitivity. While we identified a set of individual candidate biomarkers of response to pathway inhibitors, proteomic pathway scores did not correlate with amplification or mutation and did not predict response. Several receptor tyrosine kinases, including EGFR and ERK, were activated following PI3K inhibition in resistant cells; dual pathway inhibition of PI3K and EGFR or MEK demonstrated synergy. Combined MEK and PI3K inhibition was markedly synergistic in HRAS-mutant cell lines. Our findings indicate that clinical trials of single-agent PI3K/AKT/mTOR pathway inhibitors in selected populations and of PI3K-EGFR or PI3KMEK inhibitor combinations are warranted; we plan to conduct such trials.

Here, we provide gain-of-function, loss-of function, and molecular evidence supporting genetic interactions between metastasis associated protein 1 (MTA1) and Six3 and between Six3 and rhodopsin. We discovered that MTA1 physically interacts with the Six3 chromatin in a histone deacetylase-dependent manner, leading to transcriptional suppression of the Six3 gene. MTA1 is also a Six3-interacting corepressor that contributes to a self-negative regulation of Six3 transcription by Six3. In contrast, deletion of the MTA1 alleles in murine embryonic fibroblasts or its knockdown in rat retinal ganglion cells stimulates Six3 expression. MTA1 inactivation in the MTA1-null mice results in an elevated Six3 level and proliferation of the retina cells with no obvious abnormities in eye formation. However, unexpectedly, we discovered an enhanced recruitment of Six3 to the rhodopsin chromatin in retina from the MTA1-null mice; Six3's homeodomain interacts with specific DNA elements in the rhodopsin promoter to stimulate its transcription, resulting in increased rhodopsin expression. Further, in holoprosencephaly patients, Six3 protein with a naturally occurring deletion mutation in the helix 3 of the homeodomain does not bind to rhodopsin DNA or stimulate rhodopsin transcription, implying a potential defective rhodopsin pathway in the affected holoprosencephaly patients. Further Six3 cooperates with Crx or NRL in stimulating transcription from the rhodopsin-luc. These findings reveal a previously unrecognized role for the MTA1 as an upstream modifier of Six3 and indicate that Six3 is a direct stimulator of rhodopsin expression, thus revealing a putative role for the MTA1/ Six3/rhodopsin pathway in vertebrate eye.MTA1 ͉ transcription repressor ͉ histone deacetylase

Human cancer cell lines are the most frequently used preclinical models in the study of cancer biology and the development of therapeutics. Although anatomically diverse, human papillomavirus (HPV)-driven cancers have a common etiology and similar mutations that overlap with but are distinct from those found in HPV-negative cancers. Building on prior studies that have characterized subsets of head and neck squamous cell carcinoma (HNSCC) and cervical squamous cell carcinoma (CESC) cell lines separately, we performed genomic, viral gene expression, and viral integration analyses on 74 cell lines that include all readily-available HPV-positive (9 HNSCC, 8 CESC) and CESC (8 HPV-positive, 2 HPV-negative) cell lines and 55 HPV-negative HNSCC cell lines. We used over 700 human tumors for comparison. Mutation patterns in the cell lines were similar to those of human tumors. We confirmed HPV viral protein and mRNA expression in the HPV-positive cell lines. We found HPV types in three CESC cell lines that are distinct from those previously reported. We found that cell lines and tumors had similar patterns of viral gene expression; there were few sites of recurrent HPV integration. As seen in tumors, HPV integration did appear to alter host gene expression in cell lines. The HPV-positive cell lines had higher levels of p16 and lower levels of Rb protein expression than did the HPV-negative lines. Although the number of HPV-positive cell lines is limited, our results suggest that these cell lines represent suitable models for studying HNSCC and CESC, both of which are common and lethal.

BackgroundThe patient-derived xenograft (PDX) model is likely to reflect human tumor biology more accurately than cultured cell lines because human tumors are implanted directly into animals; maintained in an in vivo, three-dimensional environment; and never cultured on plastic. PDX models of head and neck squamous cell carcinoma (HNSCC) have been developed previously but were not well characterized at the molecular level. HNSCC is a deadly and disfiguring disease for which better systemic therapy is desperately needed. The development of new therapies and the understanding of HNSCC biology both depend upon clinically relevant animal models. We developed and characterized the patient-derived xenograft (PDX) model because it is likely to recapitulate human tumor biology.MethodsWe transplanted 30 primary tumors directly into mice. The histology and stromal components were analyzed by immunohistochemistry. Gene expression analysis was conducted on patient tumors and on PDXs and cell lines derived from one PDX and from independent, human tumors.ResultsFive of 30 (17%) transplanted tumors could be serially passaged. Engraftment was more frequent among HNSCC with poor differentiation and nodal disease. The tumors maintained the histologic characteristics of the parent tumor, although human stromal components were lost upon engraftment. The degree of difference in gene expression between the PDX and its parent tumor varied widely but was stable up to the tenth generation in one PDX. For genes whose expression differed between parent tumors and cell lines in culture, the PDX expression pattern was very similar to that of the parent tumor. There were also significant expression differences between the human tumors that subsequently grew in mice and those that did not, suggesting that this model enriches for cancers with distinct biological features. The PDX model was used successfully to test targeted drugs in vivo.ConclusionThe PDX model for HNSCC is feasible, recapitulates the histology of the original tumor, and generates stable gene expression patterns. Gene expression patterns and histology suggested that the PDX more closely recapitulated the parental tumor than did cells in culture. Thus, the PDX is a robust model in which to evaluate tumor biology and novel therapeutics.