Wnt signaling is one of the key oncogenic pathways in multiple cancers, and targeting this pathway is an attractive therapeutic approach. However, therapeutic success has been limited because of the lack of therapeutic agents for targets in the Wnt pathway and the lack of a defined patient population that would be sensitive to a Wnt inhibitor. We developed a screen for small molecules that block Wnt secretion. This effort led to the discovery of LGK974, a potent and specific small-molecule Porcupine (PORCN) inhibitor. PORCN is a membrane-bound O-acyltransferase that is required for and dedicated to palmitoylation of Wnt ligands, a necessary step in the processing of Wnt ligand secretion. We show that LGK974 potently inhibits Wnt signaling in vitro and in vivo, including reduction of the Wnt-dependent LRP6 phosphorylation and the expression of Wnt target genes, such as AXIN2.LGK974 is potent and efficacious in multiple tumor models at well-tolerated doses in vivo, including murine and rat mechanistic breast cancer models driven by MMTV-Wnt1 and a human head and neck squamous cell carcinoma model (HN30). We also show that head and neck cancer cell lines with loss-of-function mutations in the Notch signaling pathway have a high response rate to LGK974. Together, these findings provide both a strategy and tools for targeting Wntdriven cancers through the inhibition of PORCN.Wnt inhibition | β-catenin | HNSCC

Background We established multiple UM-SCC (University of Michigan Squamous Cell Carcinoma) cell lines. With time, these have been distributed to other labs all over the world. Recent scientific discussions have noted the need to confirm the origin and identity of cell lines in grant proposals and journal articles. We genotyped the UM-SCC cell lines in our collection to confirm their unique identity. Design Early passage UM-SCC cell lines were genotyped and photographed. Results Thus far, 73 unique head and neck UM-SCC cell lines (from 65 donors including 21 lines from 17 females) were genotyped. In 7 cases separate cell lines were established from the same donor. Conclusions These results will be posted on the U of M Head and Neck SPORE Tissue Core website for other investigators to confirm that the UM-SCC cells used in their laboratories have the correct features. Publications using UM-SCC cell lines should confirm the genotype.

Importance Human papillomaviruses are now recognized as an etiologic factor in a growing subset of head and neck cancers and have critical prognostic importance that affects therapeutic decision making. There is no universally accepted gold standard for high-risk HPV (hrHPV) assessment in formalin-fixed, paraffin-embedded (FFPE) tissue specimens, nor is there a clear understanding of the frequency or role of hrHPV in sites other than oropharynx. Objective To determine the optimal assessment of hrHPV in FFPE head and neck tumors. Design Assessment of hrHPV by p16 immunohistochemical staining, in situ hybridization (ISH), and PCR-MassArray (PCR-MA), with L1 PGMY-PCR (PGMY-PCR) and sequencing to resolve method discordance, was applied to 338 FFPE oropharyngeal, nasopharyngeal, and oral cavity tumors. Relative sensitivity and specificity were compared to develop a standard optimal test protocol. Setting Large Midwestern referral center. Participants Tissue specimens from 338 head and neck cancer patients treated during the period 2001-2011 in the departments of Otolaryngology, Radiation Oncology and Medical Oncology. Patients with oropharyngeal and oral cancer were consented for IRB approved study through the Head and Neck SPORE. Tissue blocks from nasopharyngeal cancer patients were retrieved from pathology archives under IRB approval for existing tissue and data. Intervention Patients received standard therapy. Main outcomes and measurements Optimal hrHPV identification, detection, and activity in head and neck cancers. Results Using combined PCR-MA with PGMY-PCR and sequencing for conclusive results, we found PCR-MA to have 99.5% sensitivity and 100% specificity, p16 to have 94.2% sensitivity and 85.5% specificity, and ISH to have 82.9% sensitivity and 81% specificity. Among HPV-positive tumors, HPV16 was most frequently detected, but 10 non-HPV16 types accounted for 6-50% of tumors, depending on site. Overall, 86% of oropharynx, 50% of nasopharynx and 26% of oral cavity tumors were positive for hrHPV. Conclusions and relevance PCR-MA has a low DNA (5ng) requirement effective for testing small tissue samples, high throughput, rapid identification of HPV types, with high sensitivity and specificity. PCR-MA together with p16INK4a provided accurate assessment of HPV presence, type, and activity, and was determined to be the best approach for HPV testing in FFPE head and neck tumors.

BACKGROUND Few human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) cell lines exist. We established UM-SCC-104, a new HPV(+) HNSCC cell linefrom a recurrent oral cavity tumor, and characterized it for the presence of cancer stem cells (CSC). METHODS Tumor cells were tested for biomarker expression by immunohistology and the presence of HPV was assessed by several methods. RESULTS UM-SCC-104 has a unique genotype, contains HPV-16 and expresses E6/E7. Inoculation of (Aldehyde Dehydrogenase) ALDH(+) and ALDH(−) cells in an immunocompromised mouse resulted in tumor growth from the ALDH(+) cells after 6 weeks that recapitulated the histology of the primary, while ALDH(−) cells did not produce tumors. CONCLUSIONS UM-SCC-104, a new HPV-16, CSC-containing HNSCC cell line will aid in studying recurrent HPV(+) tumors. The aggressive nature of this tumor is consistent with high uniform expression of EGFR and a functionally significant proportion of ALDH(+) CSC.

Enhancements in behavior that accompany repeated, intermittent administration of abused drugs (sensitization) endure long after drug administration has ceased. Such persistence reflects changes in intracellular signaling cascades and associated gene transcription factors in brain regions that are engaged by abused drugs. This process is not characterized for the most potent psychomotor stimulant, methamphetamine. Using motor behavior as an index of brain state in rats, we verified that five once-daily injections of 2.5 mg/kg methamphetamine induced behavioral sensitization that was demonstrated (expressed) 3 and 14 days later. Using immunoblot procedures, limbic brain regions implicated in behavioral sensitization were assayed for extracellular signal-regulated kinase and its phosphorylated form (pERK/ERK, a signal transduction kinase), cAMP response element binding protein and its phosphorylated form (pCREB/CREB, a constitutively expressed transcriptional regulator), and ⌬FosB (a long-lasting transcription factor). pERK, ERK, and CREB levels were not changed for any region assayed. In the ventral tegmental area, pCREB and ⌬FosB also were not changed. pCREB (activated CREB) was elevated in the frontal cortex at 3 days withdrawal, but not at 14 days. pCREB levels were decreased at 14 days withdrawal in the nucleus accumbens and ventral pallidum. Accumbal and pallidal levels of ⌬FosB were increased at 3 days withdrawal, and this increase persisted to 14 days in the pallidum. Thus, only the ventral pallidum showed changes in molecular processes that consistently correlated with motor sensitization, revealing that this region may be associated with this enduring behavioral phenotype initiated by methamphetamine. The present findings expand our understanding of the neuroanatomical and molecular substrates that may play a role in the persistence of druginduced sensitization.