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Unresectable pancreatic cancer is almost universally lethal because chemotherapy and radiation cannot completely stop the growth of the cancer. The major problem with using radiation to approximate surgery in unresectable disease is that the radiation dose required to ablate pancreatic cancer exceeds the tolerance of the nearby duodenum. WR-2721, also known as amifostine, is a well-known radioprotector, but has significant clinical toxicities when given systemically. WR-2721 is a prodrug and is converted to its active metabolite, WR-1065, by alkaline phosphatases in normal tissues. The small intestine is highly enriched in these activating enzymes, and thus we reasoned that oral administration of WR-2721 just before radiation would result in localized production of the radioprotective WR-1065 in the small intestine, providing protective benefits without the significant systemic side effects. Here, we show that oral WR-2721 is as effective as intraperitoneal WR-2721 in promoting survival of intestinal crypt clonogens after morbid irradiation. Furthermore, oral WR-2721 confers full radioprotection and survival after lethal upper abdominal irradiation of 12.5 Gy × 5 fractions (total of 62.5 Gy, EQD2 = 140.6 Gy). This radioprotection enables ablative radiation therapy in a mouse model of pancreatic cancer and nearly triples the median survival compared to controls. We find that the efficacy of oral WR-2721 stems from its selective accumulation in the intestine, but not in tumors or other normal tissues, as determined by in vivo mass spectrometry analysis. Thus, we demonstrate that oral WR-2721 is a well-tolerated, and quantitatively selective, radioprotector of the intestinal tract that is capable of enabling clinically relevant ablative doses of radiation to the upper abdomen without unacceptable gastrointestinal toxicity.

Unresectable pancreatic cancer is almost universally lethal because chemotherapy and radiation cannot completely stop the growth of the cancer. The major problem with using radiation to approximate surgery in unresectable disease is that the radiation dose required to ablate pancreatic cancer exceeds the tolerance of the nearby duodenum. WR-2721, also known as amifostine, is a well-known radioprotector, but has significant clinical toxicities when given systemically. WR-2721 is a prodrug and is converted to its active metabolite, WR-1065, by alkaline phosphatases in normal tissues. The small intestine is highly enriched in these activating enzymes, and thus we reasoned that oral administration of WR-2721 just before radiation would result in localized production of the radioprotective WR-1065 in the small intestine, providing protective benefits without the significant systemic side effects. Here, we show that oral WR-2721 is as effective as intraperitoneal WR-2721 in promoting survival of intestinal crypt clonogens after morbid irradiation. Furthermore, oral WR-2721 confers full radioprotection and survival after lethal upper abdominal irradiation of 12.5Gy x 5 fractions (total of 62.5Gy, D2EQ=140.6Gy), which would likely ablate pancreatic cancer. We find that the efficacy of oral WR-2721 stems from its selective accumulation in the intestine, but not in tumors or other normal tissues, as determined by in vivo mass spectrometry analysis. Thus, we demonstrate that oral WR-2721 is a well-tolerated, and quantitatively selective, radioprotector of the intestinal tract that is capable of enabling clinically relevant ablative doses of radiation to the upper abdomen without unacceptable gastrointestinal toxicity.

Patients with cancer often experience a high symptom burden prior to the start of treatment. As disease and treatment-related neurotoxicities appear to be additive, targeting disease related symptoms may attenuate overall symptom burden for cancer patients and improve the tolerability of treatment. It has been hypothesized that disease-related symptoms are a consequence of tumor-induced inflammation. We tested this hypothesis using a syngeneic heterotopic murine model of human papilloma virus (HPV)-related head and neck cancer. This model has the advantage of being mildly aggressive and not causing cachexia and weight loss. We previously showed that this tumor leads to increased IL-6, IL-1β, and TNF-α expression in the liver and increased IL-1β expression in the brain. The current study confirmed these features and demonstrated that the tumor itself exhibits high inflammatory cytokine expression (e.g., IL-6, IL-1β, and TNF-α) compared to healthy tissue. While there is a clear relationship between cytokine levels and behavioral deficits in this model, the behavioral changes are surprisingly mild. Therefore, we sought to confirm the relationship between behavior and inflammation by amplifying the effect using a low dose of lipopolysaccharide (LPS, 0.1 mg/kg). In tumor-bearing mice LPS induced deficits in nest building, tail suspension, and locomotor activity approximately 24h after LPS. However, these mice did not display an exacerbation of LPS-induced weight loss, anorexia, or anhedonia. Further, while heightened serum IL-6 was observed there was minimal priming of liver or brain cytokine expression. Next we sought to inhibit tumor-induced burrowing deficits by reducing inflammation using minocycline. Minocycline (~50 mg/kg/day in drinking water) was able to attenuate tumor-induced inflammation and burrowing deficits. These data provide evidence in favor of an inflammatory-like mechanism for the behavioral alterations associated with tumor growth in a syngeneic murine model of HPV-related head and neck cancer. However, the inflammatory state and behavioral changes induced by this tumor clearly differ from other forms of inflammation-induced sickness behavior.

Objective: Patients with human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) have better responses to radiotherapy and higher overall survival rates than do patients with HPV-negative HNSCC, but the mechanisms underlying this phenomenon are unknown. P16 is used as a surrogate marker for HPV infection. Our goal was to examine the role of p16 in HPV-related favorable treatment outcomes and to investigate the mechanisms by which p16 may regulate radiosensitivity. Methods: HNSCC cells and xenografts were used. P16-overexpressing (HPV/p16-negative HN5 and UMSCC-1) and p16 shRNA knockdown (HPV/p16-positive UMSCC-47 and UCPI-SCC154) cells, TRIP12 shRNA or siRNA knockdown (HPV/p16-negative HN5 and Fadu) cells were generated. The effects of p16 or TRIP12 on HNSCC cell radiosensitivity were determined by clonogenic cell survival. The effects of p16 on tumor xenografts (HPV/p16-negative HN5 and HPV/p16-positive UMSCC-47) radioresponse were evaluated by tumor growth delay assays. DNA double-strand breaks (DSBs) were assessed by immunofluorescence analysis of 53BP1 foci; DSB levels were determined by neutral comet assay; western blotting was used to evaluate protein changes; changes in protein half-life were tested with a cycloheximide assay; gene expression was examined by real-time polymerase chain reaction (PCR); and data from The Cancer Genome Atlas HNSCC project were analyzed. Results: P16 expression led to downregulation of TRIP12 protein both in vitro and in vivo via decreasing TRIP12 protein's half life, which in turn led to increased RNF168 levels and subsequently repressed DNA damage repair, represented by increased 53BP1 foci and neutral comet moment tails at 24 hours after irradiation. As a result, p16 expression enhanced radioresponsiveness both in vitro and in vivo. Inhibition of TRIP12 expression led to radiosensitization through repressed DNA damage repair, represented by decreased BRCA1 foci at 1 and 5 hours after irradiation; and increased neutral comet moment tails at 24 hours after irradiation. Furthermore, overexpression of TRIP12 was associated with poor survival in patients with HPV-positive HNSCC. Conclusions: The findings of our study reveal that p16 participates in radiosensitization through influencing DNA damage repair. P16 downregulates TRIP12 protein expression via post-translational regulation. Inhibition of TRIP12 sensitized HNSCC cells via prohibition of DNA damage repair. These findings support the rationale of blocking TRIP12 signaling to improve radiotherapy outcomes. Citation Format: Li Wang, Peijing Zhang, David Molkentine, Jessica Molkentine, Uma Raju, David Valdecanas, Ramesh Tailor, Howard Thames, Thomas Buchholz, Junjie Chen, Li Ma, Kathy Mason, Raymond Meyn, Heath D. Skinner. TRIP12 as a mediator of human papillomavirus/p16-related radiation enhancement effects. [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 1661.

Background: Head and neck squamous cell carcinoma (HNSCC) tumorigenesis induced by human papillomavirus (HPV) shows a greater clinical response to radiation therapy when compared to HPV negative patients. The underlying mechanism for this more favorable outcome is unknown, but the presumption is that DNA repair response must play a large role. P16INK4a is a known surrogate marker for HPV positivity; however the relationship between radiation sensitivity and P16INK4a has not been completely elucidated in this context. Ubiquitin specific protease 7 (USP7), also known as HAUSP, is a deubiquitylating enzyme that cleaves ubiquitin from substrates to stabilize target proteins. USP7 overexpression is a predictor of poor prognosis in lung carcinomas and correlates with disease progression and lower survival in gliomas. More recent studies have shown USP7 has a role in the stability of DNA damage response proteins such as RNF168, BRCA1 and Chk1; and USP7 knockdown has been shown to radio-sensitize breast cancer cells to radiation. We have investigated the possible role of USP7 in the radio-response of HNSCC cells in the context of HPV status. Methods: Four HPV(+) and four HPV(-) HNSCC were chosen for this study. The radio-response was determined by clonogenic survival assay. P16INK4a overexpression was generated using lentivirus. Western blot analysis was used for visualizing protein expression. The Cancer Genome Atlas was interrogated to examine the relationship between USP7 and clinical outcome. Results: Our data suggest P16INK4a negatively regulates USP7 and leads to the more radiosensitive phenotype associated with HPV(+)HNSCC. Radio-resistant HPV(-) HNSCC cell lines show higher levels of USP7 than HPV(+) lines and over-expression of P16INK4a in HPV(-) HN5 cells leads to down-regulation of USP7 and radio-sensitization. Conversely, P16INK4a siRNA knockdown in HPV(+) UMSCC-47 shows an increase of USP7 protein expression and radio-resistance. P22077, an inhibitor of USP7 activity, was also shown to radiosensitize a HPV(-) cell line. This shows proof of principle that inhibiting USP7 can be a viable approach to sensitizing HPV(-) HNSCC to radiation. USP7 overexpression was also associated with poorer overall survival in HNSCC. Conclusion: These results suggest that USP7 may be a marker of clinical radioresistance in HNSCC and that its inhibition has the potential to improve the treatment outcome of patients with HPV(-) HNSCC when combined with radiotherapy. Citation Format: David Molkentine, Li Wang, Kathleen Bridges, Kathy Mason, Raymond Meyn, Heath D. Skinner. P16INK4a over-expression sensitizes HPV(-) HNSCC to radiation through down-regulation of USP7. [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 1652.

Human papillomavirus (HPV) type 16 is a major cause of oropharyngeal carcinoma (OPC). P16INK4A has been suggested to be a reliable surrogate marker of HPV-associated OPC with 100% sensitivity and about 80% specificity. Increasing data showed tumor HPV positivity or p16 expression was strongly associated with significantly better prognosis in patients with OPC. As a tumor suppressor gene, p16 has biological functions including regulation of cell cycle progression at the G1/S boundary, angiogenesis, cell senescence, tumor invasion, cell spreading, apoptosis and anoikis. The present study was undertaken to assess the role of p16 in regulating tumor radioresponse and the underlying mechanisms in OPC cell lines. OPC cell lines HN-5 (HPV and p16 negative) and UMSCC-47 (HPV and p16 positive) were used. P16 overexpressing HN-5 cells and shRNA p16 knockdown UMSCC-47 cells were generated using lentivirus vectors. Treatment endpoint was clonogenic cell survival (CSA) determined 10-12 days (for HN5) or 17-20 days (for UMSCC-47) after exposing the cells to 2-10 Gy single doses of γ-radiation (IR). Compared with the control (scramble) cells, p16-overexpressing HN5 cells had significantly higher radiosensitivity (by a factor of 1.56 at 0.1 cell survival fraction); whereas, p16-knockingdown UMSCC-47 cells had less radiosensitivity (by a factor of 1.23 at 0.1 cell survival fraction). Overexpressing P16 in HN-5 cells significantly prolonged the presence of radiation-induced double-strand breaks detected on the basis of 53BP1 foci at 24h after 4 Gy IR. To directly gauge damaged DNA, an alkaline comet assay to detect both single- and double-strand DNA breaks was performed. P16 overexpressing HN-5 cells exhibited a 2.09-fold increase in the comet ‘tail moment’ 48 hours after IR. This finding was supported by increased expression of 53BP1 analyzed by Western blot. In conclusion, other than being a robust surrogate marker for tumor control and survival outcome, our findings demonstrated that p16 also functions as a potent radiation sensitizer. The major underlying mechanism of p16 regulating radiosensitivity is by inhibition of DNA damage repair. Citation Format: Li Wang, Peijing Zhang, David P Molkentine, Hailong Piao, Chunyan Chen, Jessica M Molkentine, Jinsong Zhang, David R Valdecanas, Heath Skinner, Thomas A Buchholz, Junjie Chen, Li Ma, Kathy A Mason, Kie-kian Ang, Raymond E Meyn. P16INK4A, a surrogate marker of HPV infection and prognosis for head and neck cancer, delays DNA damage repair and enhances radiation response. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4895. doi:10.1158/1538-7445.AM2014-4895

Docetaxel, a potent chemotherapeutic drug and a strong enhancer of tumor radioresponse, possesses immunomodulating properties. We previously reported that 40% of murine tumors responding to docetaxel by growth delay showed heavy infiltration with macrophages or lymphocytes. The present study explored the effect of whole body irradiation on antitumor action of docetaxel alone or docetaxel plus tumor irradiation. Mice bearing 8-mm MCa-K mammary carcinoma in the leg received 33 mg/kg docetaxel i.v., 5 to 65 Gy tumor irradiation, or both (radiation given 24 h after docetaxel). Docetaxel delayed tumor growth and enhanced the efficacy of radiation: it dramatically reduced TCD50 (radiation dose yielding 50% tumor cure) from the control value of 38.6 Gy to 11.8 Gy, for an enhancement factor of 3.27. In addition to enhancing tumor radioresponse, docetaxel decreased the lung metastatic rate in mice with local tumor control from 26% in mice receiving radiation alone to 11% in mice receiving docetaxel plus radiation. Docetaxel induced heavy infiltration of tumors with lymphocytes, determined 2-4 days after treatment: the percentage of lymphocytes increased from the control value of