Abstract 1060: Identification and validation of repurposed drugs for treating head and neck cancer
Authors: Bryant Jennifer L. , Batis Nikolaos , Kaul Baksho , Hartley Margaret , Ryan Gordon B. , Spruce Rachel J. , Mehanna Hisham 
Published: July 2017

Abstract
Background: Head and neck squamous cell carcinoma (HNSCC) prevalence is rising and new treatments are urgently required. Repurposing existing, clinically approved drugs to be used for cancer therapy is appealing due to the reduced time and cost required to achieve patient benefit.
Aim: The aim of this study was to identify repurposed drugs for use in HNSCC and to validate these drugs and their targets in vitro and in vivo.
Methods: A library of 100 BNF-approved drugs were screened against HNSCC cell lines using high-throughput AlamarBlue proliferation assays and the drug combination IHN56 and IHN29 was identified. To validate this finding, AlamarBlue assays were repeated using a range of concentrations either alone or in combination with cisplatin. In addition, the ability of IHN56 + IHN29 to reduce colony formation and promote apoptosis of HNSCC cell lines was quantified using clonogenic survival assays and Annexin V/PI FACS analysis. To determine the mechanisms of action of IHN56 + IHN29, CAL27 cells were exposed to drugs for 24 hours and the expression of STAT3, pSTAT3-Y705, pSTAT3-S727, EGFR, pEGFR, p21, p53, LC-3 and Ki67 were visualized by Western blot. Finally, in vivo mouse models were employed to confirm our in vitro findings. 5 x 105 CAL27 cells were injected into male NSG mice and allowed to reach 150 mm3. Mice then received IP injections of 310 mg/kg INH29 on days 3-14 after escalating doses on days 1-2, 40 mg/kg INH56 days 3-14, 2 mg/kg cisplatin days 3, 5 and 7, or vehicle control days 1-14. Weights and tumors were measured at least 3 times a week.
Results: The initial screen identified two promising drugs in combination; INH56 and INH29, originally used to treat helminthiasis and epilepsy. The IC50 of INH56 in CAL27 cells utilising the AlamarBlue assay was 1.47 μM and reduced to 0.66 µM in the presence of 0.9 mM INH29. Clonogenic assays demonstrated the ability of INH56 to reduce colony formation by 55% at 2 µM and by 85% when combined with 0.9 mM INH29. Furthermore, a dose-dependent increase in apoptosis was observed with INH56 treatment, which was markedly increased following the addition of INH29. Western blot analysis supports these observations through decreased Ki67 (proliferation), reduced STAT3 activation (oncogenic signaling), increased p21 (apoptosis) independent of p53, and increased LC3 (autophagy) protein levels. In vivo, CAL27 xenograft tumor growth was significantly impaired by IP administration of INH29 alone (P=0.002) and in combination with INH56 (P=0.002) after 11 days, without any obvious toxicity.
Conclusion: The combination of INH56 + INH29 demonstrates strong promise as a novel treatment for HNSCC. This study reveals that INH56 + INH29 alone and in combination with standard treatment (cisplatin and irradiation) reduces proliferation, impedes colony formation and increases apoptosis in vitro, and reduces tumor growth in vivo. We are progressing to a phase I/II trial in HNSCC patients starting in the new year.
Citation Format: Jennifer L. Bryant, Nikolaos Batis, Baksho Kaul, Margaret Hartley, Gordon B. Ryan, Rachel J. Spruce, Hisham Mehanna. Identification and validation of repurposed drugs for treating head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1060. doi:10.1158/1538-7445.AM2017-1060