SUMMARY Hepatocyte Nuclear Factor (HNF)4α is a central regulator of gene expression in cell types that play a critical role in metabolic homeostasis, including hepatocytes, enterocytes, and pancreatic β-cells. Although fatty acids were found to occupy the HNF4α ligand-binding pocket and proposed to act as ligands, there is controversy about both the nature of HNF4α ligands as well as the physiological role of the binding. Here, we report the discovery of potent synthetic HNF4α antagonists through a high-throughput screen for effectors of the human insulin promoter. These molecules bound to HNF4α with high affinity and modulated the expression of known HNF4α target genes. Notably, they were found to be selectively cytotoxic to cancer cell lines in vitro and in vivo, although in vivo potency was limited by suboptimal pharmacokinetic properties. The discovery of bioactive modulators for HNF4α raises the possibility that diseases involving HNF4α, such as diabetes and cancer, might be amenable to pharmacologic intervention by modulation of HNF4α activity.

Fanconi anemia (FA) is a recessive genome instability syndrome characterized by heightened cellular sensitivity to DNA damage, aplastic anemia and cancer susceptibility. Leukemias and squamous cell carcinomas (SCC) are the most predominant FA associated cancers, with the latter exhibiting markedly early disease onset and aggressiveness. While studies of hematopoietic cells derived from FA patients have provided much insight into bone marrow deficiencies and leukemogenesis, molecular transforming events in FA deficient keratinocytes, which are the cell type of origin for SCC, are poorly understood. We describe here the growth and molecular properties of FANCA-deficient versus FANCA-corrected, HPV E6/E7 immortalized keratinocytes in monolayer and organotypic epithelial raft culture. In response to DNA damage, FANCA-deficient patient-derived keratinocyte cultures displayed a G2/M phase arrest, senescence and apoptosis. Organotypic raft cultures exhibited DNA repair associated defects with more 53BP1 foci and TUNEL positive cells over their corrected counterparts. Interestingly, together with reduced rates of DNA damage, FA correction resulted in a marked decrease in epithelial thickness and the presence of fewer cell layers. The observed FANCA mediated suppression of hyperplasia correlated with the detection of fewer cells transiting through the cell cycle in the absence of gross differentiation abnormalities or apoptotic differences. Importantly, the knockdown of either FANCA or FANCD2 in HPV positive keratinocytes was sufficient for increasing epithelial hyperplasia. Our findings support a new role for FA pathways in the maintenance of differentiation-dependent cell cycle exit, with the implication that FA deficiencies may contribute to the high risk of FA patients for developing HPV-associated SCC.

k High-risk human papillomaviruses (HPVs) deregulate epidermal differentiation and cause anogenital and head and neck squamous cell carcinomas (SCCs). The E7 gene is considered the predominant viral oncogene and drives proliferation and genome instability. While the implementation of routine screens has greatly reduced the incidence of cervical cancers which are almost exclusively HPV positive, the proportion of HPV-positive head and neck SCCs is on the rise. High levels of HPV oncogene expression and genome load are linked to disease progression, but genetic risk factors that regulate oncogene abundance and/or genome amplification remain poorly understood. Fanconi anemia (FA) is a genome instability syndrome characterized at least in part by extreme susceptibility to SCCs. FA results from mutations in one of 15 genes in the FA pathway, whose protein products assemble in the nucleus and play important roles in DNA damage repair. We report here that loss of FA pathway components FANCA and FANCD2 stimulates E7 protein accumulation in human keratinocytes and causes increased epithelial proliferation and basal cell layer expansion in the HPV-positive epidermis. Additionally, FANCD2 loss stimulates HPV genome amplification in differentiating cells, demonstrating that the intact FA pathway functions to restrict the HPV life cycle. These findings raise the possibility that FA genes suppress HPV infection and disease and suggest possible mechanism(s) for reported associations of HPV with an FA cohort in Brazil and for allelic variation of FA genes with HPV persistence in the general population.H uman papillomaviruses (HPVs) are double-stranded DNA viruses comprised of more than 100 subtypes, some of which, designated high risk, cause cervical and approximately one quarter of head and neck squamous cell carcinomas (HNSCCs) (1,20,21). HPV is associated primarily with HNSCCs that occur in the oropharynx, which includes the back of the tongue, walls of the throat, the soft palate, and the tonsils. Approximately 60% of oropharyngeal SCCs are caused by one of the high-risk types of HPV, and the vast majority of those are positive for HPV type 16 (HPV16) (10). Importantly, these HNSCCs are a distinct type distinguishable from HPV-negative tumors which have alcohol and tobacco use as the predominant risk factors (1).HPV is the causative agent of nearly all cervical cancers and is best studied in that disease. Although HPV infections are common, with up to 90% of women estimated to be infected in their lifetime, most infections are cleared by the immune system and do not progress to cancer. A small fraction of HPV infections, however, lead to low-grade and high-grade squamous intraepithelial lesions and cervical cancer. Persistent infection is the most significant risk factor for developing cervical cancer. While the exact progression of events is still not entirely understood, replication, persistence, integration of the viral genome, and increased expression of the viral E6 and E7 oncogenes are important steps in malignant pro...