Functional Precision Medicine Identifies Novel Druggable Targets and Therapeutic Options in Head and Neck Cancer.

Purpose: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with high mortality and a lack of targeted therapies. To identify and prioritize druggable targets, we performed genome analysis together with genome-scale siRNA and oncology drug profiling using low-passage tumor cells derived from a patient with treatment-resistant HPV-negative HNSCC.Experimental Design: A tumor cell culture was established and subjected to whole-exome sequencing, RNA sequencing, comparative genome hybridization, and high-throughput phenotyping with a siRNA library covering the druggable genome and an oncology drug library. Secondary screens of candidate target genes were performed on the primary tumor cells and two nontumorigenic keratinocyte cell cultures for validation and to assess cancer specificity. siRNA screens of the kinome on two isogenic pairs of p53-mutated HNSCC cell lines were used to determine generalizability. Clinical utility was addressed by performing drug screens on two additional HNSCC cell cultures derived from patients enrolled in a clinical trial.Results: Many of the identified copy number aberrations and somatic mutations in the primary tumor were typical of HPV(-) HNSCC, but none pointed to obvious therapeutic choices. In contrast, siRNA profiling identified 391 candidate target genes, 35 of which were preferentially lethal to cancer cells, most of which were not genomically altered. Chemotherapies and targeted agents with strong tumor-specific activities corroborated the siRNA profiling results and included drugs that targeted the mitotic spindle, the proteasome, and G2-M kinases WEE1 and CHK1 We also show the feasibility of ex vivo drug profiling for patients enrolled in a clinical trial.Conclusions: High-throughput phenotyping with siRNA and drug libraries using patient-derived tumor cells prioritizes mutated driver genes and identifies novel drug targets not revealed by genomic profiling. Functional profiling is a promising adjunct to DNA sequencing for precision oncology. Clin Cancer Res; 24(12); 2828-43. ©2018 AACR.

Synergy between Prkdc and Trp53 regulates stem cell proliferation and GI-ARS after irradiation.

Ionizing radiation (IR) is one of the most widely used treatments for cancer. However, acute damage to the gastrointestinal tract or gastrointestinal acute radiation syndrome (GI-ARS) is a major dose-limiting side effect, and the mechanisms that underlie this remain unclear. Here we use mouse models to explore the relative roles of DNA repair, apoptosis, and cell cycle arrest in radiation response. IR induces DNA double strand breaks and DNA-PK mutant Prkdcscid/scid mice are sensitive to GI-ARS due to an inability to repair these breaks. IR also activates the tumor suppressor p53 to trigger apoptotic cell death within intestinal crypt cells and p53 deficient mice are resistant to apoptosis. To determine if DNA-PK and p53 interact to govern radiosensitivity, we compared the response of single and compound mutant mice to 8 Gy IR. Compound mutant Prkdcscid/scid/Trp53-/-mice died earliest due to severe GI-ARS. While both Prkdcscid/scid and Prkdcscid/scid/Trp53-/-mutant mice had higher levels of IR-induced DNA damage, particularly within the stem cell compartment of the intestinal crypt, in Prkdcscid/scid/Trp53-/-mice these damaged cells abnormally progressed through the cell cycle resulting in mitotic cell death. This led to a loss of Paneth cells and a failure to regenerate the differentiated epithelial cells required for intestinal function. IR-induced apoptosis did not correlate with radiosensitivity. Overall, these data reveal that DNA repair, mediated by DNA-PK, and cell cycle arrest, mediated by p53, cooperate to protect the stem cell niche after DNA damage, suggesting combination approaches to modulate both pathways may be beneficial to reduce GI-ARS. As many cancers harbor p53 mutations, this also suggests targeting DNA-PK may be effective to enhance sensitivity of p53 mutant tumors to radiation.

Induction of Liver Tumors in Mice with N-Ethyl-N-Nitrosourea or N-Nitrosodiethylamine.

Since the groundbreaking studies in the middle part of the last century showing liver cancer in rodents exposed to aromatic amines, the liver has been widely used as a model target organ of chemical carcinogenesis. This protocol describes a method for inducing liver tumors by injecting mice with the widely used alkylating agents N-ethyl-N-nitrosourea (ENU) and N-nitrosodiethylamine (DEN). ENU does not require metabolic activation and readily induces tumors in a number of tissues, including the lungs, stomach, and ovaries, as well as inducing lymphomas. Mice injected with DEN can also develop other tumors, including those of the gastrointestinal tract, skin, lungs, and lymphocytes, but because DEN is metabolized in the liver, it is most effective at inducing liver tumors.

Induction of Colon Cancer in Mice with 1,2-Dimethylhydrazine.

In this protocol, colon cancer is induced in mice through a series of injections with 1,2-dimethylhydrazine. Mice will develop primarily colon tumors starting at about 3 mo after the first injection. Tumors in the lung, uterus, and small intestine may also be seen, as well as lymphomas.

Induction of Lung Tumors in Mice with Urethane.

In this protocol, urethane (ethyl carbamate) is used to induce lung tumors in mice. The use of urethane as an experimental carcinogen is especially attractive as it is inexpensive, relatively safe to handle, stable, and water soluble, and the protocol involves simple intraperitoneal (i.p.) injections in young mice. Urethane typically induces bronchioalveolar adenomas and, to a lesser extent, adenocarcinomas that resemble the adenocarcinoma subtype of non-small cell lung carcinoma. On a sensitive genetic background such as A/J, mice develop multiple adenomas visible on the lung surface by 25 wk, followed by the appearance of adenocarcinomas by 40 wk. Less-sensitive strains such as B6/129 develop tumors with a longer latency.

ARF suppresses hepatic vascular neoplasia in a carcinogen-exposed murine model.

Hepatic haemangiosarcoma is a deadly malignancy whose aetiology remains poorly understood. Inactivation of the CDKN2A locus, which houses the ARF and p16(INK4a) tumour suppressor genes, is a common event in haemangiosarcoma patients, but the precise role of ARF in vascular tumourigenesis is unknown. To determine the extent to which ARF suppresses vascular neoplasia, we examined the incidence of hepatic vascular lesions in Arf-deficient mice exposed to the carcinogen urethane [intraperitoneal (i.p.), 1 mg/g]. Loss of Arf resulted in elevated morbidity and increased the incidence of both haemangiomas and incipient haemangiosarcomas. Suppression of vascular lesion development by ARF was heavily dependent on both Arf gene-dosage and the genetic strain of the mouse. Trp53-deficient mice also developed hepatic vascular lesions after exposure to urethane, suggesting that ARF signals through a p53-dependent pathway to inhibit the development of hepatic haemangiosarcoma. Our findings provide strong evidence that inactivation of Arf is a causative event in vascular neoplasia and suggest that the ARF pathway may be a novel molecular target for therapeutic intervention in haemangiosarcoma patients.