Mitchell J. Frederick

Exome Sequencing of Head and Neck Squamous Cell Carcinoma Reveals Inactivating Mutations in NOTCH1

The mutational profile of head and neck cancer is complex and may pose challenges to the development of targeted therapies. Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. To explore the genetic origins of this cancer, we used whole-exome sequencing and gene copy number analyses to study 32 primary tumors. Tumors from patients with a history of tobacco use had more mutations than did tumors from patients who did not use tobacco, and tumors that were negative for human papillomavirus (HPV) had more mutations than did HPV-positive tumors. Six of the genes that were mutated in multiple tumors were assessed in up to 88 additional HNSCCs. In addition to previously described mutations in TP53, CDKN2A, PIK3CA, and HRAS, we identified mutations in FBXW7 and NOTCH1. Nearly 40% of the 28 mutations identified in NOTCH1 were predicted to truncate the gene product, suggesting that NOTCH1 may function as a tumor suppressor gene rather than an oncogene in this tumor type.

Adenovirus-mediated p53 gene transfer in patients with advanced recurrent head and neck squamous cell carcinoma.

PURPOSE Standard therapies of head and neck squamous cell carcinoma (HNSCC) often cause profound morbidity and have not significantly improved survival over the last 30 years. Preclinical studies showed that adenoviral vector delivery of the wild-type p53 gene reduced tumor growth in mouse xenograft models. Our purpose was to ascertain the safety and therapeutic potential of adenoviral (Ad)-p53 in advanced HNSCC. PATIENTS AND METHODS Patients with incurable recurrent local or regionally metastatic HNSCC received multiple intratumoral injections of Ad-p53, either with or without tumor resection. Patients were monitored for adverse events and antiadenoviral antibodies, tumors were monitored for response and p53 expression, and body fluids were analyzed for Ad-p53. RESULTS Tumors of 33 patients were injected with doses of up to 1 x 10(11) plaque-forming units (pfu). No dose-limiting toxicity or serious adverse events were noted. p53 expression was detected in tumor biopsies despite antibody responses after Ad-p53 injections. Clinical efficacy could be evaluated in 17 patients with nonresectable tumors: two patients showed objective tumor regressions of greater than 50%, six patients showed stable disease for up to 3.5 months, and nine patients showed progressive disease. One resectable patient was considered a complete pathologic response. Ad-p53 was detected in blood and urine in a dose-dependent fashion, and in sputum. CONCLUSION Patients were safely injected intratumorally with Ad-p53. Objective antitumor activity was detected in several patients. The infectious Ad-p53 in body fluids was asymptomatic, and suggests that systemic or regional treatment may be tolerable. These results suggest the further investigation of Ad-p53 as a therapeutic agent for patients with HNSCC.

Integrative Genomic Characterization of Oral Squamous Cell Carcinoma Identifies Frequent Somatic Drivers

The survival of patients with oral squamous cell carcinoma (OSCC) has not changed significantly in several decades, leading clinicians and investigators to search for promising molecular targets. To this end, we conducted comprehensive genomic analysis of gene expression, copy number, methylation, and point mutations in OSCC. Integrated analysis revealed more somatic events than previously reported, identifying four major driver pathways (mitogenic signaling, Notch, cell cycle, and TP53) and two additional key genes (FAT1, CASP8). The Notch pathway was defective in 66% of patients, and in follow-up studies of mechanism, functional NOTCH1 signaling inhibited proliferation of OSCC cell lines. Frequent mutation of caspase-8 (CASP8) defines a new molecular subtype of OSCC with few copy number changes. Although genomic alterations are dominated by loss of tumor suppressor genes, 80% of patients harbored at least one genomic alteration in a targetable gene, suggesting that novel approaches to treatment may be possible for this debilitating subset of head and neck cancers.

BRAK/CXCL14 Is a Potent Inhibitor of Angiogenesis and a Chemotactic Factor for Immature Dendritic Cells

BRAK/CXCL14 is a CXC chemokine constitutively expressed at the mRNA level in certain normal tissues but absent from many established tumor cell lines and human cancers. Although multiple investigators cloned BRAK, little is known regarding the physiologic function of BRAK or the reason for decreased expression in cancer. To understand the possible significance associated with loss of BRAK mRNA in tumors, we examined the pattern of BRAK protein expression in normal and tumor specimens from patients with squamous cell carcinoma (SCC) of the tongue and used recombinant BRAK (rBRAK) to investigate potential biological functions. Using a peptide-specific antiserum, abundant expression of BRAK protein was found in suprabasal layers of normal tongue mucosa but consistently was absent in tongue SCC. Consistent with previous in situ mRNA studies, BRAK protein also was expressed strongly by stromal cells adjacent to tumors. In the rat corneal micropocket assay, BRAK was a potent inhibitor of in vivo angiogenesis stimulated by multiple angiogenic factors, including interleukin 8, basic fibroblast growth factor, and vascular endothelial growth factor. In vitro, rBRAK blocked endothelial cell chemotaxis at concentrations as low as 1 nmol/L, suggesting this was a major mechanism for angiogenesis inhibition. Although only low affinity receptors for BRAK could be found on endothelial cells, human immature monocyte-derived dendritic cells (iDCs) bound rBRAK with high affinity (i.e., Kd, ∼2 nmol/L). Furthermore, rBRAK was chemotactic for iDCs at concentrations ranging from 1 to 10 nmol/L. Our findings support a hypothesis that loss of BRAK expression from tumors may facilitate neovascularization and possibly contributes to immunologic escape.

In vivo expression of the novel CXC chemokine BRAK in normal and cancerous human tissue.

Using differential display, we cloned a gene with reduced expression in short-term explants of head and neck squamous cell carcinoma (HNSCC) tumors compared to cultured normal oral epithelial cells. The differentially expressed gene was identical to the recently cloned CXC chemokine BRAK, which is ubiquitously expressed in normal tissue extracts but is absent from many tumor cell lines in vitro. To define the cell populations expressing BRAK in vivo, in situ mRNA hybridization was performed on normal and cancerous tissues from six different histological sites. The predominant normal cell type constitutively expressing BRAK in vivo was squamous epithelium. Expression in tumors was heterogeneous, with the majority of HNSCCs and some cervical squamous cell carcinomas (SCCs) showing loss of BRAK mRNA. Although absent in unstimulated peripheral blood mononuclear cells, high levels of BRAK were consistently found in infiltrating inflammatory cells (with lymphocyte morphology) in nearly all cancers examined. Furthermore, BRAK expression was demonstrated in B cells and monocytes, after stimulation of peripheral blood mononuclear cells with lipopolysaccharide. This study demonstrates for the first time up-regulation of BRAK mRNA by inflammatory cells in the tumor microenvironment and lost expression from certain cancers in vivo. The data suggest that BRAK may have a role in host-tumor interactions.

Mutational Landscape of Aggressive Cutaneous Squamous Cell Carcinoma

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. Clin Cancer Res; 20(24); 6582–92. ©2014 AACR.

Chemokines in cancer.

Chemokines are small, chemotactic cytokines that direct migration of leukocytes, activate inflammatory responses and participate in many other pleiotropic functions, including regulation of tumour growth. Chemokines modulate tumour behaviour by three important mechanisms: regulation of tumour-associated angiogenesis, activation of a host tumour-specific immunological response, and direct stimulation of tumour cell proliferation in an autocrine fashion. All of these mechanisms are promising points of cancer intervention, and preclinical mouse models suggest that chemokine antagonists and agonists could become important in the development of new anticancer therapies.

Glucose, not Glutamine is the Dominant Energy Source Required for Proliferation and Survival of Head and Neck Squamous Carcinoma Cells

Tumor metabolism is an essential contributor to disease progression and response to treatment. An understanding of the metabolic phenotype of head and neck squamous cell carcinoma (HNSCC) will allow the development of appropriate antimetabolic strategies for this tumor type.

Intratumoral Myeloid Cells Regulate Responsiveness and Resistance to Antiangiogenic Therapy

Antiangiogenic therapy is commonly used in the clinic, but its beneficial effects are short-lived, leading to tumor relapse within months. Here, we found that the efficacy of angiogenic inhibitors targeting the VEGF/VEGFR pathway was dependent on induction of the angiostatic and immune-stimulatory chemokine CXCL14 in mouse models of pancreatic neuroendocrine and mammary tumors. In response, tumors reinitiated angiogenesis and immune suppression by activating PI3K signaling in all CD11b+ cells, rendering tumors nonresponsive to VEGF/VEGFR inhibition. Adaptive resistance was also associated with an increase in Gr1+CD11b+ cells, but targeting Gr1+ cells was not sufficient to further sensitize angiogenic blockade because tumor-associated macrophages (TAMs) would compensate for the lack of such cells and vice versa, leading to an oscillating pattern of distinct immune-cell populations. However, PI3K inhibition in CD11b+ myeloid cells generated an enduring angiostatic and immune-stimulatory environment in which antiangiogenic therapy remained efficient.

Chk1/2 Inhibition Overcomes the Cisplatin Resistance of Head and Neck Cancer Cells Secondary to the Loss of Functional p53

Despite the use of multimodality therapy using cisplatin to treat patients with advanced stage squamous cell carcinoma of the head and neck (HNSCC), there is an unacceptably high rate of treatment failure. TP53 is the most commonly mutated gene in HNSCC, and the impact of p53 mutation on response to cisplatin treatment is poorly understood. Here, we show unambiguously that wild-type TP53 (wtp53) is associated with sensitivity of HNSCC cells to cisplatin treatment, whereas mutation or loss of TP53 is associated with cisplatin resistance. We also show that senescence is the major cellular response to cisplatin in wtp53 HNSCC cells and that cisplatin resistance in p53-null or -mutant TP53 cells is due to their lack of senescence. Given the dependence on checkpoint kinase (Chk)1/2 kinases to mediate the DNA damage response in p53-deficient cells, there is potential to exploit this to therapeutic advantage through targeted inhibition of the Chk1/2 kinases. Treatment of p53-deficient HNSCC cells with the Chk inhibitor AZD7762 sensitizes them to cisplatin through induction of mitotic cell death. This is the first report showing the ability of a Chk kinase inhibitor to sensitize TP53-deficient HNSCC to cisplatin in a synthetic lethal manner, which has significance given the frequency of TP53 mutations in this disease and because cisplatin has become part of standard therapy for aggressive HNSCC tumors. These preclinical data provide evidence that a personalized approach to the treatment of HNSCC based on Chk inhibition in p53-mutant tumors may be feasible. Mol Cancer Ther; 12(9); 1860–73. ©2013 AACR.