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As part of the Cancer Genome Atlas (TCGA) Network, our comprehensive genomic analysis of 279 head and neck squamous cell carcinomas (HNSCCs) found frequent chromosomal copy number variation (CNV) and mutations of potential biologic and therapeutic importance. This underscored an urgent need to identify cell line models that harbor genomic alterations representative of HNSCC. We performed whole exome-DNA and transcriptome RNA sequencing on 15 human papillomavirus HPV(−) and 11 HPV(+) HNSCC cell lines. HNSCC lines harbored chromosome gains (3q, 5p, 7p, 8q, 11q) and losses (3p, 5q, 8p, 9p, 18q), consistent with those found in HNSCC tumors by TCGA and previous karyotype studies. Integrative genome-wide analysis of CNV with gene expression uncovered over 1500 genes that display significant correlation between CNV and gene expression in both TCGA tumors and cell lines. Ingenuity Pathway Analysis revealed multiple genes that converge on key pathways and functions deregulated in HNSCC, including PI3K/AKT/mTOR, NF-κB, RAS/MAPK, TP53, death receptor signaling, inflammation, and differentiation. Intriguingly, 103 genes displaying significant amplification and increased expression were predominately located on chromosome 3q22-29. These genes encode components involved in the PI3K/AKT/mTOR, Hippo, TGF-beta and Wnt/beta-catenin pathways, cell cycle, translational and post-translational regulation, and mitochondrial biosynthesis. Fisher’s exact test and survival analysis showed significant co-occurrence and worse overall survival of 3q26.3 amplification and TP53 mutation in HNSCC patients from TCGA datasets; 3q26.3 encompasses 53 genes including PIK3CA, PLD1, ACTL6A and SOX2. HNSCC cell lines also harbor common mutations found in TCGA, such as TP53, FAT1 and NOTCH1, and novel and rare tumor suppressor genes, such as MYH9. Our findings suggest that these cell lines could serve as models for mechanistic studies and pharmacologic screening, and investigation of genomic and expression alterations as potential biomarkers for precision diagnosis and prognosis of HNSCC. (Supported by NIDCD/NIH intramural projects ZIA-DC-000073, ZIA-DC-000074. This study utilized the high-performance computational capabilities of the Biowulf Linux cluster at the National Institutes of Health, Bethesda, MD. (http://biowulf.nih.gov).) Citation Format: Hui Cheng, Xinping Yang, Han Si, Anthony Saleh, Jamie Coupar, Robert L. Ferris, Wendell G. Yarbrough, Mark E. Prince, Thomas E. Carey, Carter Van Waes, Zhong Chen. Chromosome 3q22-29 amplification is linked to increased expression of multiple genes in key pathways deregulated in head and neck cancer tumors and cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4374. doi:10.1158/1538-7445.AM2017-4374

FAT1 is a large transmembrane protein (502 kD), reportedly having dual role in different human cancers. Our lab has reported oncogenic role of FAT1 overexpression in glioblastoma multiforme (GBM, grade IV glioma) increasing migration-invasion as well as tumor inflammation and HIF1α activity. Morris et al. (2013), have reported that glioma patients with low FAT1-expressing tumors have significantly (p=0.037) longer survival (Rembrandt dataset), suggesting high FAT1 expression to have adverse effect on GBM patient survival. Since NFκB is a major transcription factor overexpressed in GBM and involve in regulating many oncognes at transcriptional level. There are no reports available on FAT1 upstream regulation. Therefore in this study we investigated the role of NFkB (RelA) in regulating FAT1 expression in GBM. In-silico analysis of FAT1 promoter predicted binding sites for several transcription factors including NFkB (RelA) having multiple binding motifs with high matrix score. We assessed the mRNA expression correlation of FAT1 and NFκB target gene (BCL2) in small cohort of GBM tumors (n=16). A significant positive correlation (0.647, p Citation Format: Chitrangda Srivastava, Khushboo Irshad, Parthaprasad Chattopadhyay, Chitra Sarkar, Ashish Suri, Subrata Sinha, Kunzang Chosdol. FAT1: A potential target of NFkB (RelA) in GBM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3534. doi:10.1158/1538-7445.AM2017-3534

Introduction: Population- and family-based studies suggest germline predisposition in up to 35% of colorectal cancer (CRC) cases. Known genetic factors, including three highly penetrant Mendelian cancer syndromes and approximately 45 low penetrant alleles, account for less than 15% of all CRC and only a small proportion of early onset CRC. Identifying the genes responsible for the “missing” CRC predisposition will inform prevention and management. Methods: Our discovery cohort were selected to enrich for likely heritable susceptibility and consisted of 127 CRC cases from 86 pedigrees selected from Canadian and Australian CRC Family Registries (CCFRs) that met the Familial Colorectal Cancer Type X definition (satisfy Amsterdam I Criteria and lack other features of known cancer syndromes). Germline DNA from multiple CRC–affected individuals per pedigree underwent whole exome sequencing. The two replication cohorts were composed of pedigrees with multiple generations affected by CRC, one an Australian cohort of 166 families from which multiple affected and unaffected members were genotyped for rare ( Outcomes: Using filter-based approaches in our discovery cohort, we identified rare, non-silent variants in FAT1 that co-segregated with CRC in Ontario (Canadian), Newfoundland (Canadian) and Australian pedigrees, including one stopgain variant that was found in all 4 affected of one Ontario pedigree and in 2 of 6 unaffected. Somatic loss of heterozygosity of FAT1 was seen in available FFPE tumors from 3 Ontario and 4 Australian pedigrees by either Sanger sequencing or microarray. Modified segregation analysis of genotyped multi-case families from the Australian CCFR demonstrated an overall increased risk for CRC in FAT1 carriers (HR 1.44, 95%CI 1.05-1.97), with greater hazard ratios (>2.5) for individual FAT1 variants identified in sufficient numbers of families. Deep sequencing in 496 Polish probands identified 4 additional rare truncating FAT1 variants. Using the ExAC database as controls, variant-level association analyses of the Polish cohort cases revealed two strongly predisposing FAT1 rare missense variants, namely c.T9440G (OR 7.53, 95%CI 4.1-12.8, Fisher's exact test, q < 0.05 by false discovery rates) and c.G3067A (OR 10.8, 95%CI 3.3-26.8, q < 0.05). Gene-level association testing was also significant for increased CRC risk when considering all rare, non-silent FAT1 variants (OR 2.2, 95%CI 1.8-2.6, q < 0.05). 30 FAT1 hemizygous and 28 wild type mice on a CD1 background, balanced for sex and weight, did not show differences in number or greatest size of aberrant crypts, adenoma or adenocarcinoma. Conclusions: We have shown that rare germline variants in FAT1 may predispose to CRC. Discovery of such causal genes and associated pathways will improve our understanding of early onset CRC for both familial and sporadic cases. Stratifying CRC risk by familial susceptibility genes should foster tailored, cost-effective primary and secondary prevention strategies. Citation Format: Ashton A. Connor, Jordan Lerner-Ellis, Mohammad R. Akbari, Cezary Cybulski, J Lubinski, Caroline Badouel, Helen McNeill, James G. Dowty, Mark Clendenning, Daniel D. Buchanan. Rare variants in the FAT1 gene may predispose to familial colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr A23.

Head and neck squamous cell carcinoma (HNSCC) represents a biologically complex disease process with a heterogeneous collection of tumors in which multiple genes and pathways are altered. With the advancements in molecular and genetic research techniques and bioinformatics, there has been an explosion of new discoveries regarding the molecular biology and genetic alterations underpinning the pathogenesis of HNSCC. In this chapter, we review the specific genetic alterations in HNSCC and their clinical implications and future applications. The most common mutations in head and neck cancer, including the TP53, NOTCH1, EGFR, HRAS, PIK3CA, and CDKN2A/p16 genes, are discussed as well as human papillomavirus as the primary etiologic agent in oropharyngeal SCC. The main topics of discussions are focused around three clinical applications of cancer genomics in head and neck cancers—diagnostic, prognostic, and therapeutic applications.

International audience; Facioscapulohumeralmuscular dystrophy (FSHD) is linked to copy-number reduction (N < 10) of the 4q D4Z4 subtelomeric array, in association with DUX4-permissive haplo-types. This main form is indicated as FSHD1. FSHD-like phenotypes may also appear in the absence of D4Z4 copy-number reduction. Variants of the SMCHD1 gene have been reported to associate with D4Z4 hypomethylation in DUX4-compatible hap-lotypes, thus defining FSHD2. Recently, mice carrying a muscle-specific knockout of the protocadherin gene Fat1 or its consti-tutive hypomorphic allele were shown to develop muscular and nonmuscular defects mimicking human FSHD. Here, we report FAT1 variants in a group of patients presenting with neuro-muscular symptoms reminiscent of FSHD. The patients do not carry D4Z4 copy-number reduction, 4q hypomethylation, or SM-CHD1 variants. However, abnormal splicing of the FAT1 transcript is predicted for all identified variants. To determine their pathogenicity, we elaborated a minigene approach coupled to an antisense oligonucleotide (AON) assay. In vitro, four out of five selected variants induced partial or complete alteration of splicing by creating new splice sites or modifying splicing regulators. AONs confirmed these effects. Altered transcripts may affect FAT1 protein interactions or stability. Altogether, our data suggest that defective FAT1 is associated with an FSHD-like phenotype. Hum Mutat 36:443–453, 2015. C 2015 Wiley Periodicals, Inc.

Esophageal squamous cell carcinoma (ESCC) is a world-wide prevalent cancer, which is particularly common in certain regions of Asia. Here we report the whole-exome or targeted deep sequencing of 139 paired ESCC cases, and analysis of somatic copy number variations (SCNV) of over 180 ESCCs. We identified novel significantly mutated genes such as FAT1, FAT2, ZNF750 and KMT2D, in addition to previously discovered ones (TP53, PIK3CA and NOTCH1). Further SCNV evaluation, immunohistochemistry and biological analysis suggested their functional relevance in ESCC. Notably, RTK-MAPK-PI3K pathways, cell cycle and epigenetic regulation are frequently dysregulated by multiple molecular mechanisms in this cancer. Moreover, our approaches uncovered many novel druggable candidates, and XPO1 was further explored as a therapeutic target because of its mutation and protein overexpression. Together, our integrated study unmasks a number of novel genetic lesions in ESCC and provides an important molecular foundation for understanding esophageal tumors and developing therapeutic targets.

In pancreatic cancer, there is a clear unmet need to identify new serum markers for either early diagnosis, therapeutic stratification or patient monitoring. Proteomic analysis of tumor cell secretomes is a promising approach to indicate proteins released from tumor cells \8\textit {in vitro}\). Ectodomain shedding of transmembrane proteins has previously been shown to contribute significant fractions the tumor cell secretomes and to generate valuable serum biomarkers. Here we introduce a soluble form of the giant cadherin Fat1 as a novel biomarker candidate. Fat1 expression and proteolytic processing was analyzed by mass spectrometry and Western blotting using pancreatic cancer cell lines as compared to human pancreatic ductal epithelial cells. RNA expression in cancer tissues was assessed by \(\textit {in silico}\) analysis of publically available microarray data. Involvement of ADAM10 (A Disintegrin and metalloproteinase domain-containing protein 10) in Fat1 ectodomain shedding was analyzed by chemical inhibition and knockdown experiments. A sandwich ELISA was developed to determine levels of soluble Fat1 in serum samples. In the present report we describe the release of high levels of the ectodomain of Fat1 cadherin into the secretomes of human pancreatic cancer cells \(\textit {in vitro}\), a process that is mediated by ADAM10. We confirm the full-length and processed heterodimeric form of Fat1 expressed on the plasma membrane and also show the p60 C-terminal transmembrane remnant fragment corresponding to the shed ectodomain. Fat1 and its sheddase ADAM10 are overexpressed in pancreatic adenocarcinomas and ectodomain shedding is also recapitulated \(\textit {in vivo}\) leading to increased Fat1 serum levels in some pancreatic cancer patients. We suggest that soluble Fat1 may find an application as a marker for patient monitoring complementing carbohydrate antigen 19-9 (CA19-9). In addition, detailed analysis of the diverse processed protein isoforms of the candidate tumor suppressor Fat1 can also contribute to our understanding of cell biology and tumor behavior.

Omega-3 long-chain polyunsaturated fatty acid (n-3 LC-PUFA), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are essential nutrients for human health. However, vertebrates, including humans, have lost the abilities to synthesize EPA and DHA de novo, majorly due to the genetic absence of delta-12 desaturase and omega-3 desaturase genes. Fishes, especially those naturally growing marine fish, are major dietary source of EPA and DHA. Because of the severe decline of marine fishery and the decrease in n-3 LC-PUFA content of farmed fishes, it is highly necessary to develop alternative sources of n-3 LC-PUFA. In the present study, we utilized transgenic technology to generate n-3 LC-PUFA-rich fish by using zebrafish as an animal model. Firstly, fat1 was proved to function efficiently in fish culture cells, which showed an effective conversion of n-6 PUFA to n-3 PUFA with the n-6/n-3 ratio that decreased from 7.7 to 1.1. Secondly, expression of fat1 in transgenic zebrafish increased the 20:5n-3 and 22:6n-3 contents to 1.8- and 2.4-fold, respectively. Third, co-expression of fat2, a fish codon-optimized delta-12 desaturase gene, and fat1 in fish culture cell significantly promoted n-3 PUFA synthesis with the decreased n-6/n-3 ratio from 7.7 to 0.7. Finally, co-expression of fat1 and fat2 in double transgenic zebrafish increased the 20:5n-3 and 22:6n-3 contents to 1.7- and 2.8-fold, respectively. Overall, we generated two types of transgenic zebrafish rich in endogenous n-3 LC-PUFA, fat1 transgenic zebrafish and fat1/fat2 double transgenic zebrafish. Our results demonstrate that application of transgenic technology of humanized fat1 and fat2 in farmed fishes can largely improve the n-3 LC-PUFA production.

Fludarabine refractoriness (FR) represents an unsolved clinical problem of chronic lymphocytic leukemia (CLL) management. Although next-generation sequencing studies have led to the identification of a number of genes frequently mutated in FR-CLL, a comprehensive evaluation of the FR-CLL genome has not been reported. Toward this end, we studied 10 FR-CLLs by combining whole-exome sequencing and copy number aberration (CNA) analysis, which showed an average of 16.3 somatic mutations and 4 CNAs per sample. Screening of recurrently mutated genes in 48 additional FR-CLLs revealed that ~70% of FR-CLLs carry ≥1 mutation in genes previously associated with CLL clinical course, including TP53 (27.5%), NOTCH1 (24.1%), SF3B1 (18.9%), and BIRC3 (15.5%). In addition, this analysis showed that 10.3% of FR-CLL cases display mutations of the FAT1 gene, which encodes for a cadherin-like protein that negatively regulates Wnt signaling, consistent with a tumor suppressor role. The frequency of FAT1-mutated cases was significantly higher in FR-CLL than in unselected CLLs at diagnosis (10.3% vs 1.1%, P = .004), suggesting a role in the development of a high-risk phenotype. These findings have general implications for the mechanisms leading to FR and point to Wnt signaling as a potential therapeutic target in FR-CLL.

The cadherin gene FAT1, located on chromosome 4q34-35 (ref. 1) within a region frequently deleted in human cancers,2 encodes a large protein with 34 extracellular cadherin repeats.3 In solid tumors, aberrant expression of FAT1 was found to be associated with disease progression.4 Although the gene was originally cloned from a human T-cell acute lymphoblastic leukemia (T-ALL) cell line,4 FAT1 just recently gained interest owing to its altered gene expression levels and the detection of somatic mutations identified by next-generation sequencing (NGS) in acute leukemia.2, 5, 6, 7 FAT1 was shown to be aberrantly expressed in pediatric patients with acute leukemia, whereas hematopoietic progenitors from healthy donors lacked FAT1 expression.5, 8 In addition, a recent report correlated high FAT1 expression with a higher probability of relapse in a small cohort of pediatric patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) based on an in silico analysis comprising two BCP-ALL data sets including 32 and 27 patients.5

Novel target discovery is warranted to improve treatment in adult T-cell acute lymphoblastic leukemia (T-ALL) patients. We provide a comprehensive study on mutations to enhance the understanding of therapeutic targets and studied 81 adult T-ALL patients. NOTCH1 exhibitedthe highest mutation rate (53%). Mutation frequencies of FBXW7 (10%), WT1 (10%), JAK3 (12%), PHF6 (11%), and BCL11B (10%) were in line with previous reports. We identified recurrent alterations in transcription factors DNM2, and RELN, the WNT pathway associated cadherin FAT1, and in epigenetic regulators (MLL2, EZH2). Interestingly, we discovered novel recurrent mutations in the DNA repair complex member HERC1, in NOTCH2, and in the splicing factor ZRSR2. A frequently affected pathway was the JAK/STAT pathway (18%) and a significant proportion of T-ALL patients harboured mutations in epigenetic regulators (33%), both predominantly found in the unfavourable subgroup of early T-ALL. Importantly, adult T-ALL patients not only showed a highly heterogeneous mutational spectrum, but also variable subclonal allele frequencies implicated in therapy resistance and evolution of relapse. In conclusion, we provide novel insights in genetic alterations of signalling pathways (e.g. druggable by γ-secretase inhibitors, JAK inhibitors or EZH2 inhibitors), present in over 80% of all adult T-ALL patients, that could guide novel therapeutic approaches.

The extrinsic apoptosis pathway is initiated by binding of death ligands to death receptors resulting in the formation of the death-inducing signaling complex (DISC). Activation of procaspase-8 within the DISC and its release from the signaling complex is required for processing executor caspases and commiting cell death. Here, we report that the atypical cadherin FAT1 interacts with caspase-8 preventing the association of caspase-8 with the DISC. We identified FAT1 in a genome-wide siRNA screen for synthetic lethal interactions with death receptor-mediated apoptosis. Knockdown of FAT1 sensitized established and patient-derived glioblastoma cell lines for apoptosis transduced by cell death ligands. Depletion of FAT1 resulted in enhanced procaspase-8 recruitment to the DISC and increased formation of caspase-8 containing secondary signaling complexes. In addition, FAT1 knockout cell lines generated by CRISPR/Cas9-mediated genome engineering were more susceptible for death receptor-mediated apoptosis. Our findings provide evidence for a mechanism to control caspase-8-dependent cell death by the atypical cadherin FAT1. These results contribute towards the understanding of effector caspase regulation in physiological conditions.

Introduction: Despite mapping of the mutational landscape in patients (pts) with chronic lymphocytic leukemia (CLL), there remains limited information regarding the functional or clinical impact of less common, recurrently mutated genes. Over the past several years, the influence of high-risk mutations has been reported. With the advent of readily available next generation sequencing (NGS), physicians receive a large amount of genomic data, many times without information regarding the clinical impact of less validated, mutations. Using results from a commercially available NGS platform, we identified a relatively high prevalence of mutations in the gene, FAT1, in treatment naïve (TN) CLL pts. FAT1 encodes a protein in the cadherin superfamily, important in regulating WNT signaling, with a role in tumor suppression. Aberrant mutated FAT1 expression has been associated with acute lymphoblastic leukemias with an intermediate clinical course. Loss of protein, through chromosomal deletions has been implicated in many solid tumors and associated with tumor progression. The majority of mutations arise in the cadherin repeats, previously shown to diminish antagonism of β-catenin, allowing its nuclear localization (Morris Nat Genetics 2013). In CLL, FAT1 mutations have been demonstrated to enrich in fludarabine refractory pts (10.3%) but with a relatively low prevalence in untreated pts (1.1%) (Messina, Blood 2014). Given a high prevalence of FAT1 mutations in our database and evidence suggesting mutated FAT1 contributes to tumor evolution, we investigated the clinical impact of clonal FAT1 mutations in pts with CLL. Methods: Patients were identified for inclusion if mutational analysis was performed prior to, or within 12 months of treatment start date. Only pts treated initially with a signal transduction inhibitor (STI) were included. Treated pts were either on protocol or received their STI as standard of care. All pts underwent whole exome profiling with a lymphoid specific NGS panel, including 75 genes (Genoptix Inc.). Analysis was performed on peripheral blood or bone marrow aspirates. The primary outcome was time to first treatment (TTFT). The log-rank test was used to compare Kaplan-Meir curves. Double-sided P values Results: In total we analyzed 172 pts, 118 (69%) remain on observation, 54 (31%), have been treated at data cutoff. Nineteen (11%) pts were found to be FAT1 mutated (mFAT1) and 153 (89%) were FAT1 wild type (wtFAT1). The median time from diagnosis to mutational profiling for the cohort was 35 months (range 0-301). We identified 21 total mutations, 17 unique (82% cadherin domain, 18% EGF like region). Recurrent mutations were identified, each occurring in 2 pts (p.A636T, p.P1614L, p.R1257Q, p.R2041H). One pt had an indel and 1 pt had an inframe deletion, the remaining were missense. We found no significant differences between groups in regards to age, IGVH mutation status or co-occurrence of high-risk mutations in NOTCH1, SF3B1, TP53 or ATM. Only 17p deletions occurred significantly more in mFAT1 pts (24%) vs. wtFAT1 (7%), p=0.04, other cytogenetic abnormalities were balanced. mFAT1 pts had significantly shorter TTFT, 50 versus 143 months respectively, p=0.02. We investigated if mFAT1 status could stratify IGVH mutated pts but found no differences in TTFT. Eleven mFAT1 pts have received treatment (82% ibrutinib, 18% acalabrutinib), 8 remain on observation. All mFAT1 pts have responded. Conclusions: Given the increasing use of rapidly available NGS testing, physicians receive genomic information with limited data regarding clinical impact. We identified a higher prevalence of FAT1 mutations in TN pts, than that previously reported. FAT1 mutations associated with deletion 17p but not other high-risk genetic mutations. Recurrent FAT1 mutations were identified and commonly found in the cadherin domain. mFAT1 pts have a significantly shorter TTFT than wtFAT1 pts. When treated with novel agents, there was no difference in response rates. These findings suggest FAT1 mutations in TN pts may be more common than previously reported and identify an intermediate risk for progression. Additional studies investigating the influence of recurrent FAT1 mutations and association with 17p deletions in TN pts are warranted. Disclosures Allan: Pharmacyclics: Speakers Bureau.

Measurement of minimal residual disease (MRD) maintains an important role in the clinical management of acute lymphoblastic leukemia (ALL). Recently, we identified Fat1 cadherin as a unique and independent prognostic factor for relapse-free and overall survival in pediatric pre-B-ALL. Here, we analyzed Fat1 mRNA for its potential as a novel marker of MRD in cases of pre-B- and T-ALL. Analyses of microarray data from 125 matched diagnosis/relapse samples across three independent datasets indicate that Fat1 mRNA is detectable in an average of 31.3% of diagnosed pre-B-ALL, of which 67.5% of cases remained positive at relapse. Furthermore, some 20% of cases with undetectable levels of Fat1 mRNA at diagnosis became positive upon relapse. T-ALL cases were 83.3% positive for Fat1 expression at diagnosis with 77.7% remaining positive at relapse. Towards proof of concept, we developed a quantitative polymerase chain reaction assay and demonstrate detection of Fat1 mRNA in leukemic cells mixed with normal peripheral blood cells at a sensitivity of 1 in 10 000 to 100 000 cells. Fat1 may therefore provide a new marker of MRD for patients with ALL lacking known genomic aberrations or within a multiplex approach to MRD detection.

Asperger syndrome is one of the Autism Spectrum Disorders (ASD's), characterized by significant difficulties in social interaction and nonverbal communication, alongside restricted and repetitive patterns of behavior and interests. ASD's have a strong and complex genetic basis that cannot be distinguished by the clinical presentation. A South African family with an affected father and three affected sons all with characteristics of Asperger syndrome including repetitive routine physical gestures and Sensory Processing Disorder was studied for the possible identification of the responsible genetic factor(s). Due to the significant proof of heritability and the extreme heterogeneity of Asperger syndrome, next generation sequencing was performed on all members of this family to extrapolate monoallelic variations in the affected father that have been inherited by all three of the affected sons probably through an autosomal dominant pattern of inheritance. These variations, validated by Sanger sequencing, were analyzed, prioritizing significant changes in the encoded protein. Variants that segregate with the affected individuals include one deletion in C17orf80, an unidentified protein expressed in the brain, (c.1745_1748delGTAA), three missense mutations that change highly conserved amino acids in PARK2, which codes for a component of a multiprotein E3 ubiquitin ligase complex that targets proteins for degradation also known to cause juvenile Parkinson disease (c.110 C>T, p.Pro37Leu), FAT1, member of a large cadherin family required for cell-cell association and actin organization, (c.2563 C>A p.Gly855Arg), and OR4C6, an olfactory receptor protein coding gene, (c.293 A>C p.Gln98Pro) and one nonsense mutation introducing a premature stop codon in HYAL4, a hyaluronidase that intracellularly degrades hyaluronan, one of the major glycosaminoglycans in the extracellular matrix (c.628 C>T p.Arg210STOP). The direct link of these previously reported variants to Asperger syndrome is yet unknown, however some of these genes such as PARK2, HYAL4 and FAT1 have previously been reported to be in involved in brain function and development, indicating a possible role in the onset of Asperger syndrome.

Background: Lung squamous cell carcinoma (SqCC) arises in the epithelial layer of the bronchial airways and is often preceded by the development of premalignant lesions. However, not all premalignant lesions will progress to lung SqCC and many of these lesions will regress without therapeutic intervention. Understanding the molecular events that contribute to progression of premalignant lesions in the airway will allow us to identify biomarkers for early detection and develop therapeutic strategies for early intervention. Methods: Bronchial brushings and biopsies were obtained from high-risk smokers undergoing lung cancer screening by auto-fluorescence bronchoscopy and CT at the Roswell Park Cancer Institute. For each subject (n = 30), both premalignant lesions (PMLs) and the cytologically normal mainstem bronchus were sampled repeatedly over time (n = 288 samples). DNA and RNA were isolated from a total of 197 bronchial biopsies of PML (average of 5 per subject) and 91 bronchial brushings. DNA was also isolated from the blood to serve as a matched normal. Exome capture was performed using the Agilent SureSelect Human All Exon+UTR 70MB kit and sequenced to a mean depth of coverage of 75x (n = 85 samples from 22 subjects). RNA libraries were prepared with Illumina TruSeq (mRNA-Seq: n = 288 samples from 30 subjects and miRNA-Seq: n = 183 samples from 26 subjects). Results: We identified gene and miRNA expression changes associated with histological grade as well as progressive/stable disease. The Hippo pathway, Wnt signaling, p53 signaling, and immune-related pathways are modulated with histological grade and disease progression. Genes associated with histological grade in the cytologically normal airway and in the biopsies were significantly concordantly enriched (FDR3/Mb) were taken from adjacent sites over two time points in the same individual with a history of lung squamous cell carcinoma. These lesions had a significantly overlapping set of mutations (p = 2.2 × 10−17) indicating a common evolutionary ancestor, and contained mutations in CREBBP and FAT1, suggesting they are at increased risk for progressing to frank malignancy. Conclusions: We performed genomic profiling of PMLs in the airways of high-risk smokers. The gene expression and somatic alterations that were observed in known cancer genes may be among the earliest events in cancer development. Citation Format: Joshua D. Campbell, Catalina Perdomo, Sarah Mazzilli, Yaron Geshalter, Samjot S. Dhillon, Gang Liu, Sherry Zhang, Hangqio Lin, Jessica Vick, Christopher Moy, Evan Johnson, Matthew Meyerson, Suso Platero, Marc Lenburg, Mary Reid, Avrum Spira, Jennifer Beane. Genomic characterization of premalignant lung squamous cell carcinoma lesions. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 895.

Review on FAT1 (FAT tumor suppressor homolog 1 (Drosophila)), with data on DNA, on the protein encoded, and where the gene is implicated.

Improved survival of patients with acute lymphoblastic leukemia (ALL) has emerged from identifying new prognostic markers; however, 20% of children still suffer recurrence. Previously, the altered expression of Fat1 cadherin has been implicated in a number of solid tumors. In this report, in vitro analysis shows that Fat1 protein is expressed by a range of leukemia cell lines, but not by normal peripheral blood (PB) and bone marrow (BM) cells from healthy donors. In silico analysis of expression of array data from clinical leukemias found significant levels of Fat1 transcript in 11% of acute myeloid leukemia, 29% and 63% of ALL of B and T lineages, respectively, and little or no transcript present in normal PB or BM. Furthermore, in two independent studies of matched diagnosis-relapse of precursor B-cell (preB) ALL pediatric samples (n=32 and n=27), the level of Fat1 mRNA expression was prognostic at the time of diagnosis. High Fat1 mRNA expression was predictive of shorter relapse-free and overall survival, independent of other traditional prognostic markers, including white blood cell count, sex and age. The data presented demonstrate that Fat1 expression in preB-ALL has a role in the emergence of relapse and could provide a suitable therapeutic target in high-risk preB-ALL.

Identifying the piece of DNA culprit of a genetic disease has not been as straightforward as expected with the evolution of sequencing capabilities. This difficulty is exemplified by facioscapulohumeral dystrophy (FSHD) study. Unexpectedly, contributions in completing this picture recently came from mice models carrying a muscle specific knock-out of the protocadherin gene Fat1 or its constitutive hypomorphic allele. Both have been shown to develop muscular and non-muscular defects mimicking human FSHD. In humans, this disease is linked to copy number reduction ( n

Spatial organization of cells and their appendages is controlled by the planar cell polarity pathway, a signaling cascade initiated by the protocadherin Fat in Drosophila . Vertebrates express 4 Fat molecules, Fat1–4. We found that depletion of Fat1 caused cyst formation in the zebrafish pronephros. Knockdown of the PDZ domain containing the adaptor protein Scribble intensified the cyst-promoting phenotype of Fat1 depletion, suggesting that Fat1 and Scribble act in overlapping signaling cascades during zebrafish pronephros development. Supporting the genetic interaction with Fat1, Scribble recognized the PDZ-binding site of Fat1. Depletion of Yes-associated protein 1 (YAP1), a transcriptional co-activator inhibited by Hippo signaling, ameliorated the cyst formation in Fat1-deficient zebrafish, whereas Scribble inhibited the YAP1-induced cyst formation. Thus, reduced Hippo signaling and subsequent YAP1 disinhibition seem to play a role in the development of pronephric cysts after depletion of Fat1 or Scribble. We hypothesize that Hippo signaling is required for normal pronephros development in zebrafish and that Scribble is a candidate link between Fat and the Hippo signaling cascade in vertebrates.

Recessive lethal mutations in the fat locus of Drosophila cause hyperplastic, tumor-like overgrowth of larval imaginal discs, defects in differentiation and morphogenesis, and death during the pupal stage. Clones of mutant cells induced by mitotic recombination demonstrate that the overgrowth phenotype is cell autonomous. Here we show that the fat locus encodes a novel member of the cadherin gene superfamily: an enormous transmembrane protein of over 5000 amino acids with a putative signal sequence, 34 tandem cadherin domains, four EGF-like repeats, a transmembrane domain, and a novel cytoplasmic domain. Two recessive lethal alleles contain alterations in the fat coding sequence, and the dominant fat allele, Gull, contains an insertion of a transposable element in the 33rd cadherin domain. Thus, this novel member of the cadherin gene superfamily functions as a tumor suppressor gene and is required for correct morphogenesis.

Purpose: Large-scale cancer genome analyses have uncovered the genetic landscape of common cancers. However, rate tumor types in adolescent, young adult (AYA) have not been well-characterized genetically. We performed a pilot study of patient-specific cancer genome analysis to know whether we can analyze individual cancer genome without the existing large-scale genomics data of same tumor types in advanced and rare AYA tumors. Methods: The patients were prospectively enrolled from a medical oncology clinic in a university hospital. Tissue samples of the study patients were acquired from the fresh tissue, formalin-fixed paraffin-embedded (FFPE) tissue and cancer cells from pleural effusion as well. Cancer genome data was acquired using massive parallel sequencing technique from seven AYA patients with advanced solid tumors. WES and WTS data were generated from 6 tumors with matched normal and 4 tumors, respectively. Somatic alterations of cancer genome were classified with 2 distinct categories with heuristic ways and level 1 to 3 depends on differences of genetic alterations based on clinical and biological relevance, and mutation context. Results: Each different tumor types of 7 study patients were as follows; atypical prostate cancer (AYA01, 30 years old), olfactory neuroblastoma (AYA02, 30 years old), tongue cancer (AYA04, 33 years old), urachal carcinoma (AYA06, 32 years old), germ cell tumor (AYA07, 21 years old), lung cancer (AYA09, 34 years old) and liposarcoma (AYA10, 33 years old). Patients with GCT and tongue cancer had higher mutation rate based on WES data; 476 and 97 non-synonymous somatic nucleotide variations respectively. However, there were only 13∼16 non-synonymous somatic nucleotide variations and 7∼18 indel somatic variations in patients with other four cancers. We identified one level-1 (strong) oncogenic alterations and eight level-1 tumor suppressor alterations as well as 19 level-2 (moderate) and level-3 (modest) alterations were found from 5 WES data (we excluded 1 hypermutated germ cell tumor sample). Some level-1/2 alterations were considered as targetable by developed or developing drugs. Each tumor was characterized by tumor-unique manner. AYA01 was characterized with concurrent tumor suppressor alterations of RasGAP family genes (NF1 and RASA2), AYA02 with chromosome-level copy number alteration and CDKN2C stopgain mutation, AYA04 with concurrent oncogenic (AMER3) and tumor suppressor (FAT1, LGR6, MSX1) alterations of Wnt signaling, AYA06 with KRAS mutation and AYA09 with a fusion of EML4-ALK. Conclusion: Our study showed that we can characterize rare AYA tumors with WES or WTS data without pre-existing large-scale genomics data of same tumor types, although there are much hurdles in identifying major driver and/or druggable genetic alterations. Citation Format: Soojin Cha, Jeongeun Lee, Jong-Yeon Shin, Ji-Yeon Kim, Jong-Il Kim, Se-Hoon Lee. Patient-specific genomic profiling for advanced cancers in young adults. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 75. doi:10.1158/1538-7445.AM2015-75

Esophageal squamous cell carcinoma (ESCC) is one of the most common and most aggressive cancers. The epidemiological features of ESCC are extremely complex, with remarkable geographic differentiation among world's populations. While rural Anyang in the Henan Province of China is a well-known high-incidence area, the causal factors in this population remain elusive. We performed exome sequencing of 81 tumor-normal pairs, identified TP53, PIK3CA and NOTCH1 as significantly mutated genes, and observed highly recurrent aberrations in several other genes previously reported for ESCC (ZNF750, MLL2, FAT1, FAT2, and FAT3). Our catalog of ∼7,000 single-nucleotide mutations revealed two main signatures: C>T transitions due to spontaneous deamination of 5-methyl-cytosine, and C>T and C>G mutations at TpCpN attributed to the APOBEC family of cytidine deaminases. Per-sample loadings of these two signatures are uncorrelated with the patient's smoking and drinking status. Since APOBEC activities are associated with exogenous viruses, the prominence of this signature suggests a role of HPV in ESCC etiology, consistent with our previous studies that detected HPV DNA in tumor samples and anti-HPV-E7 antibody in patient's blood. To characterize intratumoral heterogeneity we applied our newly developed method, CHAT, to summarize the clonal frequencies of copy number alternations and single nucleotide mutations in each tumor. Many tumors show a multi-modal distribution of the clonal frequencies, suggesting extensive within-tumor diversity. To better understand the patterns of growth, migration and metastatic potential among different cells within a tumor we performing exome sequencing to compare multiple samples in 10 ESCC patients. For each, we analyzed 4-6 sectors of the tumor, 2-4 samples of adjacent normal tissue, and 1-2 nearby lymph nodes. The spatial heterogeneity of molecular lesions within each tumor is expected to uncover major genes and pathways affected in each patient, as well as the temporal progression of tumorigenic events that may have driven the initiation and outgrowth of ESCC. By integrating the mutation signatures, introtumoral clonal heterogeneity, and clinical outcomes, we aim to gain a better understanding of the molecular bases and evolutionary path of this lethal disease. Citation Format: Qingxuan Song, Mengfei Liu, Jian Bai, Amir Abliz, Wenqing Yuan, Zhen Liu, Jingjing Liu, Changhong Zeng, Hong Cai, Yang Ke, Jun Li. Mutation signature and intratumor heterogeneity of esophageal squamous cell carcinoma in a Chinese cohort. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4797. doi:10.1158/1538-7445.AM2015-4797

The cadherin superfamily protein Fat1 is known to interact with the EVH1 domain of mammalian Ena/VASP. Here we demonstrate that: (i) the scaffolding proteins Homer-3 and Homer-1 also interact with the EVH1 binding site of hFat1 in vitro, and (ii) binding of Homer-3 and Mena to hFat1 is mutually competitive. Endogenous Fat1 binds to immobilised Homer-3 and endogenous Homer-3 binds to immobilised Fat1. Both, endogenous and over-expressed Fat1 exhibit co-localisation with Homer-3 in cellular protrusions and at the plasma membrane of HeLa cells. As Homer proteins and Fat1 have been both linked to psychic disorders, their interaction may be of patho-physiological importance.

We identified 22 polymorphisms in the adipocyte fatty-acid binding protein (FABP4) gene, a strong positional candidate gene for the FAT1 locus in porcine chromosome 4. The most informative polymorphism, an insertion/deletion in intron 1, together with a single nucleotide polymorphism in intron 3, was genotyped in a cross between Iberian and Landrace pigs. After performing QTL, single marker, and haplotype analyses, we showed that there were at least 2 quantitative trait genes in the FAT1 region and that the FABP4 polymorphism was tightly associated to fatness. A comparison of allelic frequencies in a panel of pig breeds suggested that the Del2634C polymorphism was under indirect selection. We also showed that FABP4 is tightly associated to fatness but not growth. Furthermore, a haplotype analysis suggests that there is genetic heterogeneity at the FAT1 locus within the Landrace breed. ©2006 American Society of Animal Science. All rights reserved.

The domestication of the pig began about 9 000 years ago and many of the existing domestic breeds have been selected for phenotypic traits like lean meat and fast growth. Domestic pigs are phenotypically very different from the ancestral wild boar that has adapted to survive in their natural environment. Because of their divergence, crosses between domestic pigs and wild boars are suitable for constructing genetic maps and Quantitative trait locus (QTL) analyses. A cross between the Large White and the European wild boar was thus initiated in the late 1980s. A major QTL for fat deposition and growth, denoted FAT1, was found on chromosome 4. The aim of this thesis was to further characterise the FAT1 locus and to identify the causative gene(s) and mutation(s). We have identified new markers and constructed a high-resolution linkage and RH map of the FAT1 QTL interval. We also performed comparative mapping to the human genome and showed that the pig chromosome 4 is homologous to human chromosomes 1 and 8. The gene order is very well conserved between the two species. In parallel we have narrowed down the FAT1 QTL interval by repeated backcrossing to the domestic Large White breed for six generations. The QTL could be confirmed for fatness but not for growth. Furthermore, the data strongly suggested that there might be more than one gene underlying the FAT1 QTL. Depending on which hypothesis to consider, the one- or two-loci model, the FAT1 interval can be reduced to 3,3 or 20 centiMorgan (cM), respectively, based on the backcross experiments. In the last study we confirm the two-loci model with one locus primarily effecting abdominal fat and another locus primarily effecting subcutaneous fat. We have identified a missense mutation in the RXRG gene which is in strong association with the abdominal fat QTL and the mutation is a potential candidate for that locus. Brown adipose tissue (BAT) is a specific type of fat essential for non-shivering thermogenesis in mammals. Piglets appear to lack BAT and rely on shivering as the main mechanism for thermoregulation. Uncoupling protein 1 (UCP1) gene is exclusively expressed in BAT and its physiological role is to generate heat by uncoupling oxidative phosphorylation. We show that the UCP1 gene has been disrupted in the pig lineage about 20 years ago. The inactivation of UCP1 provides a genetic explanation for the poor thermoregulation in piglets.

Role of Fat1 in cell-cell contact formation of podocytes in puromycin aminonucleoside nephrosis and neonatal kidney. Background Fat1 is a member of the cadherin superfamily characterized by its 34 cadherin repeats in the extracellular domain. Fat1 was originally found as a component of the slit diaphragm of podocytes, but its function in podocytes remains obscure. To gain insight into its role in podocytes, we expanded our study of Fat1 expression to puromycin aminonucleoside (PAN) nephrosis, the neonatal kidney, and the primary podocyte culture, where slit diaphragms are absent or disappear. Methods Expression of Fat1 was examined in isolated glomeruli of PAN nephrosis by the ribonuclease protection assay and Western blot analysis and in the neonatal kidney by in situ hybridization. Fat1 localization in glomeruli and in the primary culture was confirmed by immunofluorescence or immunoelectron microscopy. Results In PAN nephrotic rats, glomerular expression of Fat1 increased rather than decreased at both transcript and protein levels in comparison with normal rats. Immunofluorescence microscopy revealed distinct staining for Fat1 along the glomerular capillary wall, where nephrin staining was weakened or disappeared. Immunoelectron microscopy demonstrated significant accumulation of immunogold particles for Fat1 at intercellular junctions newly formed between podocytes in the nephrosis. In the primary culture of podocytes, Fat1 was mainly localized at cell-cell contact sites and in tips of cellular processes. In the neonatal kidney, immature podocytes expressed Fat1 more intensely than mature podocytes as shown by in situ hybridization. Double-labeled immunostaining using anti-pan cadherin antibody revealed that Fat1 in podocytes colocalized with cadherin in immature glomeruli, indicating that junctional complexes of developing podocytes contain Fat1. Conclusion These findings suggest that Fat1 may be a fundamental component of intercellular junctions of podocytes, and may be involved in the initial step of cell contacts of podocytes.

In recent years, mutational surveys published by our group, The Cancer Genome Atlas, and others have revealed highly prevalent mutations and deletions targeting an intriguing family of genes encoding transmembrane proteins: the FAT family (FAT1, FAT2, FAT3 and FAT4). In particular, FAT1 and FAT4 appear to be the most frequently altered. Both genes harbor recurrent mutations and deletions in multiple types of human cancer, including glioblastoma (FAT1, 20%), colorectal (FAT1, 9%; FAT4, 20%), head and neck squamous cell (FAT1, 12%; FAT4, 10%), gastric (FAT4, 20%), serous ovarian (FAT1, 7%; FAT4, 3%) and pancreatic (FAT1, 4%; FAT4, 8%) cancers.1-7 These somatic alterations are intriguing, given the closely related Drosophila Fat gene, which has been identified as a tumor suppressor gene. In the fly, Fat acts upstream of the Hippo signaling pathway, which restrains cell growth; loss of Fat causes overgrowth of larval imaginal discs in flies. Fat is also a key developmental gene, controlling cell polarity along the apical-basal axis. The human FAT genes are all closely related, although recent phylogenetic analyses reveal that the closest human ortholog of Drosophila Fat is in fact FAT4, while FAT1, FAT2 and FAT3 are actually most closely related to the Drosophila gene Fatl (Fat-like).8 Nevertheless, all four human genes bear tight similarity to the Drosophila Fat. The FAT genes all encode large membrane proteins called protocadherins, which have around 30+ cadherin repeats, four or five EGF-like domains and laminin G-like domains. The FAT proteins are present in a wide range of species and are highly conserved. They are believed to represent the first form of cadherins and were accordingly named “protocadherins.” In certain cell types, FAT proteins can be concentrated at lamellipodia, filopodia and sites of cell-cell contact. Like the classical cadherins, the protocadherins often serve dual roles in cell adhesion and signaling. FAT4 is most closely related to Fat and mediates similar key developmental functions, such as planar cell polarity. It appears to have growth- and invasion-suppressive properties in several cancer cell lines, but the mechanisms remain obscure.7 In Drosophila, Fat is the apical member of the Hippo signaling pathway, which regulates organ growth and cell cycle progression in response to cell density. Although definitive data are still pending in the context of cancer, FAT4 appears to participate in mammalian Salvador/Warts/Hippo signaling during development. It is therefore quite possible that the frequent FAT4 alterations observed in cancer lead to pro-proliferation signals through a loss of proper regulation of the Hippo pathway. For FAT1, more complete genomic, functional and mechanistic evidence has recently emerged, revealing the molecular basis of its tumor-suppressive function. This gene is a target within a highly prevalent region of deletion at 4q35 observed across many types of human cancer. We have identified mutations in glioblastoma, colorectal and head and neck samples at rates of 7–20%.1 FAT1 appears to be among the most frequently mutated genes in squamous cell carcinoma of the head and neck.3 Both in vitro and in vivo, depletion of FAT1 leads to markedly accelerated cell growth and proliferation, while expression of FAT1 robustly suppresses tumor growth.1 These growth-suppressive effects are abrogated when mutations observed in tumors are present. Just as classical cadherin proteins can bind to β-catenin and regulate its transcriptional activity, FAT1 also binds β-catenin and limits its translocation to the cell nucleus. Mutations in FAT1’s intracytoplasmic domain result in a loss of this ability to regulate β-catenin. Therefore, loss of FAT1 in cells activates the Wnt signaling pathway, unleashing β-catenin-dependent transcriptional activity and upregulating pro-growth wnt transcriptional targets. Consistent with this, primary cancer samples with FAT1 alterations are defined by significant enhancement of Wnt signaling. The growth-suppressive functions of FAT1 are mediated by its intracytoplasmic, β-catenin binding domain, but the extracellular domain also mediates cell-cell adhesion, which may be a secondary mechanism by which FAT1 loss promotes tumor growth. The Wnt/β-catenin pathway has been causally linked to multiple types of cancer. For example, in colorectal cancer, the overwhelming majority of tumors are defined by alteration of the core genes, APC, CTNNB1 and AXIN1/2. However, in non-colorectal tumors, these genes are infrequently or never mutated. The precise causes of Wnt pathway activation in these types of cancer have not been elucidated. New data now implicates FAT1 mutation as a driver of Wnt activation in many of these tumors, and point to FAT1 as a potential molecular determinant for guiding use of new small-molecule inhibitors of Wnt signaling. Interestingly, FAT1 appears to play multiple, seemingly opposing, roles in development and cell growth. While the protein has a strong tumor-suppressive effect, it also binds to Ena/VASP, thereby promoting actin polymerization and cell motility. Indeed, in experimental systems, FAT1 promotes lamellipodial dynamics and cell migration and invasion. These different functions may be vestiges of FAT1’s role in development, where it may play a directional role in guiding organ development. For these reasons, it is conceivable that the function of FAT1 in tumorigenesis is multifaceted, such that the gene may not operate as a tumor suppressor via the same mechanisms in all cellular contexts. FAT2 and FAT3 have not been well characterized to date, although each gene has been observed to be undergo mutation in approximately 10% of colorectal and lung squamous cell cancers.5,6 These two genes have more similarity to FAT1 than FAT4,8 but it remains to be seen whether these protocadherins are also able to modulate Wnt signaling. As additional mutational data in diverse human cancers is reported, the precise implications of the recurrent alterations targeting these intriguing, large, ancient proteins will be more thoroughly illuminated. (Fig. 1) Figure 1. Model of FAT1 function. When FAT1 is present, β-catenin is held at the cell membrane. When FAT1 is inactivated by mutation or deleted in cancers, an excess of β-catenin is present in the cytoplasm. Some β-catenin ...

Fat cadherins constitute a subclass of the large cadherin family characterized by the presence of 34 cadherin motifs. To date, three mammalian Fat cadherins have been described; however, only limited information is known about the function of these molecules. In this paper, we describe the second fat cadherin in Drosophila, fat-like (ftl). We show that ftl is the true orthologue of vertebrate fat-like genes, whereas the previously characterized tumor suppressor cadherin, fat, is more distantly related as compared with ftl. Ftl is a large molecule of 4705 amino acids. It is expressed apically in luminal tissues such as trachea, salivary glands, proventriculus, and hindgut. Silencing of ftl results in the collapse of tracheal epithelia giving rise to breaks, deletions, and sac-like structures. Other tubular organs such as proventriculus, salivary glands, and hindgut are also malformed or missing. These data suggest that Ftl is required for morphogenesis and maintenance of tubular structures of ectodermal origin and underline its similarity in function to a reported lethal mouse knock-out of fat1 where glomerular epithelial processes collapse. Based on our results, we propose a model where Ftl acts as a spacer to keep tubular epithelia apart rather than the previously described adhesive properties of the cadherin superfamily.

While roles in adhesion and morphogenesis have been documented for classical cadherins, the nonclassical cadherins are much less well understood. Here we have examined the functions of the giant protocadherin FAT by generating a transgenic mouse lacking mFAT1. These mice exhibit perinatal lethality, most probably caused by loss of the renal glomerular slit junctions and fusion of glomerular epithelial cell processes (podocytes). In addition, some mFAT1(-/-) mice show defects in forebrain development (holoprosencephaly) and failure of eye development (anophthalmia). In contrast to Drosophila, where FAT acts as a tumor suppressor gene, we found no evidence for abnormalities of proliferation in two tissues (skin and central nervous system [CNS]) containing stem and precursor cell populations and in which FAT is expressed strongly. Our results confirm a necessary role for FAT1 in the modified adhesion junctions of the renal glomerular epithelial cell and reveal hitherto unsuspected roles for FAT1 in CNS development.

Facio Scapulo Humeral Dystrophy (FSHD) is a muscle disease that asymmetrically affects flat muscles. Non-muscle symptoms are deafness and retinopathies. Defects very evocative of FSHD have been observed in mouse model partially deficient for FAT1. In addition, genetic screening of neuromuscular disease patients enabled us to identify the FAT1 mutations in FSHD-like patients. The family of FAT protocadherins (FAT 1–4) is well known to be involved in the Wnt/PCP and in the Hippo pathway in Drosophila, while it plays key roles in homeostasis of mammalian tissues. FAT1 expression is localized in developing limb buds, somites and neural tube as well as in adult kidney. Based on these data and on our previous results, we postulated that FAT1 plays a role in generation and homeostasis of flat muscle tissues. To understand which proteins compose the transduction pathway led by FAT1 in mammals, we first looked at the stability and activation of YAP transcription factor in the Hippo pathway. Although preliminary, results confirmed that FAT1 overexpression affects YAP/TAZ phosphorylation, nuclear localization and transcriptional activity, which may stop cell proliferation and affect cell polarity. Indeed, localization of YAP/TAZ depends from elasticity of the cellular environment through signal transduction from the extracellular matrix (ECM). In mesenchymal stem cells, TAZ and YAP are cytoplasmic when cells are on flexible substrates while nuclear on rigid matrices. Therefore, we plan to test muscle cells reaction to different levels of matrix rigidity and to measure functional consequences of FAT1 alterations to these mechanical stresses. In perspective, our observations will help explain the molecular mechanism involved in the differentiation and tissue response to cellular environment. Moreover, FAT1 alteration may lead to modification of ECM stiffness perception during flat muscle fibers development, thus contributing to pathological mechanism of FSHD.

Despite progress in the therapeutic management of patients with squamous cell carcinoma of the head and neck (SCCHN), the mortality rate of patients presenting with advanced disease remains high. One approach to improve treatment efficacy is to add novel molecular targeted agents to the classical treatment regimens. Monoclonal antibodies targeting the epidermal growth factor receptor (EGFR) have shown clinical benefits in palliative and curative settings. However, only a minority of patients presenting with recurrent or metastatic (R/M) SCCHN have meaningful tumor regression with these agents and virtually all who do develop acquired tumor resistance after a few months of treatment. For these reasons, other inhibitors of EGFR or molecules that interfere with known molecular pathways activated in squamous cell carcinoma of the head and neck are of considerable interest, either as single agents or in combination with other treatment modalities.Recently, deep sequencing technology has allowed a better characterization of the implicated genes. Somatic mutations in TP53 (47–72%), NOTCH1 (14–19%), CDKN2A (9–22%), PIK3CA (6–21%), FBXW7 (5%), HRAS (4–8%), FAT1 (23%) and CASP8 (8%) have been reported. Beside these mutations, some genes or their related proteins have been found to be altered by other mechanims (amplification, deletion, epigenetic). Altogether, activating mutations in classical oncogenes seem relatively rare in SCCHN and most of the genetic alterations occur in tumor suppressor genes. These findings are important for the further development of novel therapies for SCCHN although developping new compounds to restore the activity of altered tumor suppressor genes is extremely challenging. In this review, we will discuss the different molecular therapeutic approaches explored in SCCHN. We will also briefly outline new trial designs that could be used to accelerate the investigation of emerging therapeutic agents in this disease. Disclosure: J. Machiels: Advisory booard: Boerhinger and Novartis (uncompensated).

Introduction FAT1 belongs to the FAT protocadherin family, a drosophila homologous gene involved in development processes. Recently, FAT1 gained large interest as it is mutated in various cancers. Besides the known function of cell-cell interaction and polarity, FAT1 loss of function mutations have been linked to dysregulation of the WNT pathway in solid tumors. In acute lymphoblastic leukemia (ALL), aberrantly high expression of FAT1 was claimed to be associated with inferior outcome in pediatric B-lineage ALL. Herein, we investigated the yet unknown frequency and relevance of FAT1 expression and mutation in a large, homogenously treated cohort of adult ALL patients. Patients and Methods We investigated FAT1 expression in diagnostic bone marrow (BM) samples of 112 T-ALL, 122 B-lineage ALL, and additional 63 early T-cell precursor (ETP) ALL patients by real time (RT)-PCR. Patients were enrolled in trials of the German Multicenter Study Group for Adult ALL (GMALL) and outcome was investigated for patients into GMALL trials 06/99 and 07/03. Using the T-cell line BE13 as reference, we defined patients with FAT1 expression higher than BE13 as FAT1pos (0.01-38.5) and patients with a lower expression as FAT1neg ( Results Normal hematopoietic cells including unselected BM cells, CD34+-progenitors, or CD3+ T-cells from healthy donors lacked FAT1 expression (30.000/µL at diagnosis compared to FAT1neg T-ALL patients (78% vs. 42%, p Conclusion This first comprehensive analysis on FAT1 in a large cohort of adult patients with ALL shows a high frequency of FAT1 expression. Higher FAT1 expression occurred in ALL patients with more mature immunophenotype linking FAT1 to cell-cell adhesion and polarity, thymic homing and interaction with the BM niche. This yet unreported high mutation rate of 12 % in adult T-ALL makes FAT1 to one of the most frequently mutated genes in T-ALL. The link of inactivating FAT1 mutations to aberrant activation of the WNT pathway, as reported in solid tumors, might allow the development of refined treatment options. In summary, these data make FAT1 a promising candidate for disease monitoring, risk stratification and development of targeted therapies. Disclosures: Krebs: Illumina: Honoraria. Greif:Illumina: Honoraria.

Generation of skeletal muscles with forms adapted to their function is essential for normal movement. Muscle shape is patterned by the coordinated polarity of collectively migrating myoblasts. Constitutive inactivation of the protocadherin gene Fat1 uncoupled individual myoblast polarity within chains, altering the shape of selective groups of muscles in the shoulder and face. These shape abnormalities were followed by early onset regionalised muscle defects in adult Fat1-deficient mice. Tissue-specific ablation of Fat1 driven by Pax3-cre reproduced muscle shape defects in limb but not face muscles, indicating a cell-autonomous contribution of Fat1 in migrating muscle precursors. Strikingly, the topography of muscle abnormalities caused by Fat1 loss-of-function resembles that of human patients with facioscapulohumeral dystrophy (FSHD). FAT1 lies near the critical locus involved in causing FSHD, and Fat1 mutant mice also show retinal vasculopathy, mimicking another symptom of FSHD, and showed abnormal inner ear patterning, predictive of deafness, reminiscent of another burden of FSHD. Muscle-specific reduction of FAT1 expression and promoter silencing was observed in foetal FSHD1 cases. CGH array-based studies identified deletion polymorphisms within a putative regulatory enhancer of FAT1, predictive of tissue-specific depletion of FAT1 expression, which preferentially segregate with FSHD. Our study identifies FAT1 as a critical determinant of muscle form, misregulation of which associates with FSHD., Author Summary Facioscapulohumeral muscular dystrophy (FSHD) is a hereditary human myopathy affecting groups of skeletal muscles in the face and shoulders. Despite recent advances on the molecular cascade initiated by its main genetic cause, with identification of DUX4 as the main pathogenic agent, how this leads to the specific clinical picture is still poorly understood. Here, we investigated the role of the FAT1 protocadherin gene, located near the FSHD locus, which was repressed by DUX4 in human muscle cells. Disruption of the mouse Fat1 gene causes muscular and non-muscular phenotypes highly reminiscent of FSHD symptoms. We show that Fat1 is required in migrating muscle precursors, and that the altered muscle shapes caused by Fat1 mutations are predictive of early onset defects in muscle integrity in adult mutants, with a topography matching the map of muscles affected in FSHD. In humans, we observed FAT1 lowering in muscle but not brain of foetal cases with canonical FSHD1, and identified deletions of conserved elements in the FAT1 locus predictive of changes in FAT1 expression, that were enriched among FSHD patients. Thus, deregulating Fat1 in FSHD-related tissues provides a unique means to mimic FSHD symptoms in mice and learn about pathogenesis of this complex disease.

WNT signalling is activated in some cancers by mutation of adenomatous polyposis coli (APC) or β-catenin (CTNNB1). However, the mechanism of WNT pathway activation in cancers that do not carry these alterations is unclear. Morris et al. found recurrent somatic inactivating mutations in FAT1, which encodes a cadherin-like protein, in several cancer types. Expression of wild-type FAT1 suppressed tumorigenesis, and cancer-associated FAT1 mutations enhanced tumour growth in mice. The authors showed that β-catenin binds FAT1, and that mutated FAT1 promotes the nuclear localization of β-catenin, leading to the expression of WNT–β-catenin target genes.