Patients with human papillomavirus associated (HPV+) head and neck cancer (HNC) demonstrate significantly improved survival outcome compared to those with HPV− negative (HPV−) tumors. Published data examining this difference offers conflicting results to date. We systematically investigated the radiation sensitivity of all available validated HPV+ HNC cell lines and a series of HPV− HNC cell lines using in vitro and in vivo techniques. HPV+ HNCs exhibited greater intrinsic radiation sensitivity (average SF2 HPV− 0.59 vs. HPV+ 0.22, p<0.0001), corresponding with a prolonged G2/M cell cycle arrest and increased apoptosis following radiation exposure (percent change 0% vs. 85%, p=0.002). A genome-wide microarray was used to compare gene-expression 24 hours following radiation between HPV+ and HPV− cell lines. Multiple genes in TP53 pathway were upregulated in HPV+ cells (Z score 4.90), including a 4.6 fold increase in TP53 (p<0.0001). Using immortalized human tonsillar epithelial cells, increased radiation sensitivity was seen in cell expressing HPV-16 E6 despite the effect of E6 to degrade p53. This suggested that low levels of normally functioning p53 in HPV+ HNC cells could be activated by radiation, leading to cell death. Consistent with this, more complete knockdown of TP53 by siRNA resulted in radiation resistance. These results provide clear evidence, and a supporting mechanism, for increased radiation sensitivity in HPV+ HNC relative to HPV− HNC. This issue is under active investigation in a series of clinical trials attempting to de-escalate radiation (and chemotherapy) in selected patients with HPV+ HNC in light of their favorable overall survival outcome.

Best disease (BD) is an inherited degenerative disease of the human macula that results in progressive and irreversible central vision loss. It is caused by mutations in the retinal pigment epithelium (RPE) gene BESTROPHIN1 (BEST1), which, through mechanism(s) that remain unclear, lead to the accumulation of subretinal fluid and autofluorescent waste products from shed photoreceptor outer segments (POSs). We employed human iPS cell (hiPSC) technology to generate RPE from BD patients and unaffected siblings in order to examine the cellular and molecular processes underlying this disease. Consistent with the clinical phenotype of BD, RPE from mutant hiPSCs displayed disrupted fluid flux and increased accrual of autofluorescent material after long-term POS feeding when compared with hiPSC-RPE from unaffected siblings. On a molecular level, RHODOPSIN degradation after POS feeding was delayed in BD hiPSC-RPE relative to unaffected sibling hiPSC-RPE, directly implicating impaired POS handling in the pathophysiology of the disease. In addition, stimulated calcium responses differed between BD and normal sibling hiPSC-RPE, as did oxidative stress levels after chronic POS feeding. Subcellular localization, fractionation and co-immunoprecipitation experiments in hiPSC-RPE and human prenatal RPE further linked BEST1 to the regulation and release of endoplasmic reticulum calcium stores. Since calcium signaling and oxidative stress are critical regulators of fluid flow and protein degradation, these findings likely contribute to the clinical picture of BD. In a larger context, this report demonstrates the potential to use patient-specific hiPSCs to model and study maculopathies, an important class of blinding disorders in humans.

Spliceosome mutations are common in MDS and AML, yet the oncogenic changes due to these mutations have not been identified. A global analysis of exon usage in AML samples revealed distinct molecular subsets containing alternative spliced isoforms of inflammatory and immune genes. IRAK4 was the dominant alternatively spliced isoform in MDS/AML and is characterized by a longer isoform that retains exon 4, encoding a protein, IRAK4-Long (L) that assembles with the Myddosome, results in maximal activation of NF-κB, and is essential for leukemic cell function. Expression of IRAK4-L is mediated by mutant U2AF1 and is associated with oncogenic signaling in MDS/AML. Inhibition of IRAK4-L abrogates leukemic growth, particularly in AML cells with higher expression of the IRAK4-L isoform. Collectively, mutations in U2AF1 induce expression of therapeutically targetable “active” IRAK4 isoforms and provide a genetic link to activation of chronic innate immune signaling in MDS and AML.

Human papillomavirus (HPV), a known etiology of a subset of head and neck squamous cell carcinomas (HNCs), causes numerous alterations in normal cellular functions. This article reviews the biology, detection and treatment of HPV-positive HNC. The role of HPV oncoproteins in tumor development, the natural history of HPV infection, and risk factors for and prevention of transmission of oral HPV are considered. Commonly used methods for detecting HPV infection including limitations of these methods are discussed to aid the practicing clinician in utilizing these tests in their clinical practice. Clinical characteristics of HPV-positive HNC including potential explanations for the improved outcomes seen in patients with HPV-positive HNC are assessed. Ongoing clinical trials specific for patients with HPV-positive HNC are described and areas in need of additional research are summarized. Until the results of ongoing trials are known, treatment of HPV-positive HNC should not differ in clinical practice from treatment of similar non-HPV related cancers.

Highly differentiated, pigmented hiPSC-RPE monolayers can undergo limited serial expansion while retaining key cytological and functional attributes. However, passaging hiPSC-RPE cultures beyond senescence leads to loss of such features. Our findings support limited, controlled passaging of patient-specific hiPSC-RPE to procure cells needed for in vitro disease modeling, drug screening, and cellular transplantation.

Purpose To develop a clinically relevant model system to study head and neck squamous cell carcinoma (HNSCC), we have established and characterized a direct-from-patient, tumorgraft model of Human Papillomavirus (HPV)-positive and HPV-negative cancers. Experimental Design Patients with newly diagnosed or recurrent HNSCC were consented for donation of tumor specimens. Surgically obtained tissue was implanted subcutaneously into immunodeficient mice. During subsequent passages, both formalin-fixed/paraffin embedded as well as flash frozen tissues were harvested. Tumors were analyzed for a variety of relevant tumor markers. Tumor growth rates and response to radiation, cisplatin, or cetuximab were assessed and early passage cell strains were developed for rapid testing of drug sensitivity. Results Tumorgrafts have been established in 22 of 26 patients to date. Significant diversity in tumorgraft tumor differentiation was observed with good agreement in degree of differentiation between patient tumor and tumorgraft (Kappa 0.72). Six tumorgrafts were HPV-positive on the basis of p16 staining. A strong inverse correlation between tumorgraft p16 and p53 or Rb was identified (Spearman correlations p=0.085 and p=0.002, respectively). Significant growth inhibition of representative tumorgrafts was demonstrated with cisplatin, cetuximab or radiation treatment delivered over a two-week period. Early passage cell strains showed high consistency in response to cancer therapy between tumorgraft and cell strain. Conclusions We have established a robust human tumorgraft model system for investigating HPV-positive and HPV-negative HNSCC. These tumorgrafts show strong correlation with the original tumor specimens and provide a powerful resource for investigating mechanisms of therapeutic response as well as preclinical testing.

No abstract

Degradation of photoreceptor outer segments (POS) by retinal pigment epithelium (RPE) is essential for vision, and studies have implicated altered POS processing in the pathogenesis of some retinal degenerative diseases. Consistent with this concept, a recently established hiPSC-RPE model of inherited macular degeneration, Best disease (BD), displayed reduced rates of POS breakdown. Herein we utilized this model to determine (i) if disturbances in protein degradation pathways are associated with delayed POS digestion and (ii) whether such defect(s) can be pharmacologically targeted. We found that BD hiPSC-RPE cultures possessed increased protein oxidation, decreased free-ubiquitin levels, and altered rates of exosome secretion, consistent with altered POS processing. Application of valproic acid (VPA) with or without rapamycin increased rates of POS degradation in our model, whereas application of bafilomycin-A1 decreased such rates. Importantly, the negative effect of bafilomycin-A1 could be fully reversed by VPA. The utility of hiPSC-RPE for VPA testing was further evident following examination of its efficacy and metabolism in a complementary canine disease model. Our findings suggest that disturbances in protein degradation pathways contribute to the POS processing defect observed in BD hiPSC-RPE, which can be manipulated pharmacologically. These results have therapeutic implications for BD and perhaps other maculopathies.