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Squamous cell carcinoma driven by human papillomavirus (HPV) is more sensitive to DNA-damaging therapies, such as radiation, than its HPV-negative counterpart. Here we show that p16, the clinically utilized surrogate for HPV positivity, renders cells more sensitive to radiation via a ubiquitin-dependent signaling pathway, linking high levels of this protein to increased activity of the transcription factor SP1, increased HUWE1 transcription and degradation of ubiquitin-specific protease 7 (USP7). Activation of this pathway in HPV-positive disease leads to an absence of TRIP12, decreased DNA damage repair, improved response to radiation and better clinical outcomes. Conversely, repression of this pathway in HPV-negative disease is druggable via USP7 inhibitors under clinical development, resulting in potentiation of radiation response. Our findings may lead to improved outcomes for patients with HPV-negative radioresistant tumors, while allowing decreased intensity of therapy for patients with HPV-positive tumors.

Human papillomavirus (HPV) drives the development of squamous cell carcinoma at several sites, including the oropharynx. Generally, the presence of HPV renders a tumor more sensitive to DNA-damaging therapies such as radiation; however, the mechanism behind this phenomenon is elusive. Previous studies have shown that p16, the clinically utilized surrogate for HPV tumor positivity, can render cells more sensitive to radiation. In the current manuscript, using a combination of immunoprecipitation mass spectrometry (IP/MS), in vivo and in vitro modulation and clinical tumor profiling, we identify a novel ubiquitin-dependent signaling pathway linking p16 to increased activity of the transcription factor SP1 leading to increased HUWE1 transcription and degradation of ubiquitin-specific protease 7 (USP7). This pathway is activated in HPV-positive tumor cells, leading to an absence of TRIP12, decreased DNA damage repair and increased mitotic death following radiation. As USP7 inhibitors are currently in clinical trials, this pathway provides a novel means by which radioresistant tumors may be targeted to increase response and improve outcome.

Despite radiation forming the curative backbone of over 50% of malignancies, there are no genomically-driven radiosensitizers for clinical use. Herein we perform in vivo shRNA screening to identify targets generally associated with radiation response as well as those exhibiting a genomic dependency. This identifies the histone acetyltransferases CREBBP/EP300 as a target for radiosensitization in combination with radiation in cognate mutant tumors. Further in vitro and in vivo studies confirm this phenomenon to be due to repression of homologous recombination following DNA damage and reproducible using chemical inhibition of histone acetyltransferase (HAT), but not bromodomain function. Selected mutations in CREBBP lead to a hyperacetylated state that increases CBP and BRCA1 acetylation, representing a gain of function targeted by HAT inhibition. Additionally, mutations in CREBBP/EP300 are associated with recurrence following radiation in squamous cell carcinoma cohorts. These findings provide both a mechanism of resistance and the potential for genomically-driven treatment., Mutations in histone acetyltransferases (HATs) CREBBP and EP300 are generally thought to lead to decreased function or absence of protein product. Here the authors describe a gain of function of several CREBBP mutations leading to baseline hyper-acetylation, increased homologous recombination and potential synergy between radiation and HAT inhibition in CREBBP/EP300 mutant tumors.

Despite radiation forming the curative backbone of over 50% of malignancies, there are no genomically-driven radiation sensitizers for clinical use. We performedin vivoshRNA screening to identify targets generally associated with radiation response as well as those exhibiting a genomic dependency. This identified the histone acetyltransferases CREBBP/EP300 as a target for radiosensitization in combination with radiation in cognate mutant tumors. Furtherin vitroandin vivostudies confirmed this phenomenon was due to repression of homologous recombination following DNA damage and can be reproduced using chemical inhibition of histone acetyltransferase (HAT), but not bromodomain function. Selected mutations in CREBBP lead to a hyperacetylated state that increases CBP and BRCA1 acetylation, representing a gain of function targets by HAT inhibition. Additionally, mutations in CREBBP/EP300 were associated with recurrence following radiation, in several squamous cell carcinoma cohorts. These findings represent both a novel mechanism of treatment resistance and the potential for genomically-driven treatment.

PURPOSE: Head and neck cancers (HNSCC) are routinely treated with radiotherapy; however normal tissue toxicity remains a concern. Therefore, it is important to validate treatment modalities combining molecularly targeted agents with radiotherapy to improve the therapeutic ratio. The aim of this study was to assess the ability of the PARP inhibitor niraparib (MK-4827) alone, or in combination with cell cycle checkpoint abrogating drugs targeting Chk1 (MK-8776) or Wee1 (MK-1775), to radiosensitize HNSCCs in the context of HPV status. MATERIALS AND METHODS: Accepted Manuscript PARP1, PARP2, Chk1 or Wee1 shRNA constructs were analyzed from an in vivo shRNA screen of HNSCC xenografts comparing radiosensitization differences between HPV(+) and HPV(−) tumors. Radiosensitization by niraparib alone or in combination with MK-8776 or MK-1775 was assessed by clonogenic survival in HPV(−) and HPV(+) cells; and the role of p16 in determining response was explored. Relative expressions of DNA repair genes were compared by PCR array in HPV(+) and HPV(−) cells, and following siRNA-mediated knockdown of TRIP12 in HPV(−) cells. RESULTS: In vivo shRNA screening showed a modest preferential radiosensitization by Wee1 and PARP2 in HPV(−) and Chk1 in HPV(+) tumor models. Niraparib alone enhanced the radiosensitivity of all HNSCC cell lines tested. However, HPV(−) cells were sensitized to a greater degree, as suggested by the shRNA screen. When combined with MK-8776 or MK-1775, radiosensitization was further enhanced in an HPV dependent manner with HPV(+) cells enhanced by MK-8776 and HPV(−) cells enhanced by MK-1775. A PCR array for DNA repair genes showed PARP and HR proteins BRCA1 and RAD51 were much lower in HPV(+) cells than in HPV(−). Similarly, directly knocking down p16-dependent TRIP12 decreased expression of these same genes. Overexpressing p16 decreased TRIP12 expression and increased radiosensitivity in HPV(−) HN5. However, while PARP inhibition led to significant radiosensitization in the control, it led to no further significant radiosensitization in p16 overexpressing cells. Forced p16 expression in HPV(−) HN5 increased accumulation in G1 and subG1 and limited progression to S phase, thus reducing effectiveness of PARP inhibition. CONCLUSIONS: Niraparib effectively radiosensitizes HNSCCs with a greater benefit seen in HPV(−). HPV status also plays a role in response to MK-8776 or MK-1775 when combined with niraparib due to differences in DNA repair mechanisms. This study suggests that using cell cycle abrogators in combination with PARP inhibitors may be a beneficial treatment option in HNSCC, but also emphasizes the importance of HPV status when considering effective treatment strategies.

Purpose: This study examined the potential role of the nuclear deubiquitinating enzyme BRCA1-associated protein-1 (BAP1) in radioresistance in head and neck squamous cell cancer (HNSCC). Experimental Design: We overexpressed, knocked down, and rescued BAP1 expression in six HNSCC cell lines, three human papillomavirus (HPV)–negative and three HPV-positive, and examined the effects on radiosensitivity in vitro and in an HNSCC mouse xenograft model. Radiosensitivity was assessed by clonogenic cell survival and tumor growth delay assays; changes in protein expression were analyzed by immunofluorescence staining and Western blotting. We also analyzed The Cancer Genome Atlas HNSCC database to test for associations between BAP1 expression and outcome in patients. Results: Overexpression of BAP1 induced radioresistance in both cell lines and xenograft models; conversely, BAP1 knockdown led to increased ubiquitination of histone H2A, which has been implicated in DNA repair. We further found that BAP1 depletion suppressed the assembly of constitutive BRCA1 foci, which are associated with homologous recombination (HR), but had minimal effect on γ-H2AX foci and did not affect proteins associated with nonhomologous end joining, suggesting that BAP1 affects radiosensitivity in HNSCC by modifying HR. Finally, in patients with HNSCC, overexpression of BAP1 was associated with higher failure rates after radiotherapy. Conclusions: BAP1 can induce radioresistance in HNSCC cells, possibly via deubiquitination of H2Aub and modulation of HR, and was associated with poor outcomes in patients with HNSCC. BAP1 may be a potential therapeutic target in HNSCC. Clin Cancer Res; 24(3); 600–7. ©2017 AACR.

Purpose: The primary cause of death due to head and neck squamous cell carcinoma (HNSCC) is local treatment failure. The goal of this study was to examine this phenomenon using an unbiased approach. Experimental Design: We utilized human papilloma virus (HPV)-negative cell lines rendered radiation-resistant (RR) via repeated exposure to radiation, a panel of HPV-negative HNSCC cell lines and three cohorts of HPV-negative HNSCC tumors (n = 68, 97, and 114) from patients treated with radiotherapy and subjected to genomic, transcriptomic, and proteomic analysis. Results: RR cell lines exhibited upregulation of several proteins compared with controls, including increased activation of Axl and PI3 kinase signaling as well as increased expression of PD-L1. Additionally, inhibition of either Axl or PI3 kinase led to decreased PD-L1 expression. When clinical samples were subjected to RPPA and mRNA expression analysis, PD-L1 was correlated with both Axl and PI3K signaling as well as dramatically associated with local failure following radiotherapy. This finding was confirmed examining a third cohort using immunohistochemistry. Indeed, tumors with high expression of PD-L1 had failure rates following radiotherapy of 60%, 70%, and 50% compared with 20%, 25%, and 20% in the PD-L1–low expression group (P = 0.01, 1.9 × 10−3, and 9 × 10−4, respectively). This finding remained significant on multivariate analysis in all groups. Additionally, patients with PD-L1 low/CD8+ tumor-infiltrating lymphocytes high had no local failure or death due to disease (P = 5 × 10−4 and P = 4 × 10−4, respectively). Conclusions: Taken together, our data point to a targetable Axl–PI3 kinase–PD-L1 axis that is highly associated with radiation resistance. Clin Cancer Res; 23(11); 2713–22. ©2017 AACR.

BACKGROUND Human papillomavirus (HPV)-positive oropharyngeal carcinomas response better to X-ray therapy (XRT) than HPV-negative disease. Whether HPV status influences the sensitivity of head and neck cancer cells to proton therapy or the relative biological effectiveness (RBE) of protons versus XRT is unknown. METHODS Clonogenic survival was used to calculate the RBE; immunocytochemical analysis and neutral comet assay were used to evaluate unrepaired DNA double-strand breaks. RESULTS HPV-positive cells were more sensitive to protons and the unrepaired double-strand breaks were more numerous in HPV-positive cells than in HPV-negative cells (p 1.06). Cell line type and radiation fraction size influenced the RBE. CONCLUSION HPV-positive cells were more sensitive to protons than HPV-negative cells maybe through the effects of HPV on DNA damage and repair. The RBE for protons depends more on cell type and fraction size than on HPV status. © 2016 Wiley Periodicals, Inc. Head Neck 39: 708-715, 2017.

Interaction between the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGF-1R) has been well established in many cancer types. We investigated the effects of cetuximab (EGFR antibody) and IMC-A12 (IGF-1R antibody) on the response of head and neck squamous cell carcinoma (HNSCC) to radiation therapy (RT). The effects of cetuximab and IMC-A12 on cell viability and radiosensitivity were determined by clonogenic cell survival assay. Formation of nuclear γ-H2AX and 53BP1 foci was monitored by immunofluorescence. Alterations in target signaling were analyzed by Western blots. In vivo tumor growth delay assay was performed to determine the efficacy of triple therapy with IMC-A12, cetuximab, and RT. In vitro data showed that cetuximab differentially affected the survival and the radiosensitivity of HNSCC cells. Cetuximab suppressed DNA repair that was evident by the prolonged presence of nuclear γ-H2AX and 53BP1 foci. IMC-A12 did not have any effect on the cell survival. However, it increased the radiosensitivity of one of the cell lines. EGFR inhibition increased IGF-1R expression levels and also the association between EGFR and IGF-1R. Addition of IMC-A12 to cetuximab did not increase the radiosensitivity of these cells. Tumor xenografts exhibited enhanced response to RT in the presence of either cetuximab or IMC-A12. Concurrent treatment regimen failed to further enhance the tumor response to cetuximab and/or RT. Taken together our data suggest that concomitant inhibition of both EGFR and IGF-1R pathways did not yield additional therapeutic benefit in overcoming resistance to RT.

Purpose: We investigated whether vandetanib, an inhibitor of the tyrosine kinase activities of vascular endothelial growth factor receptor-2 (VEGFR-2), epidermal growth factor receptor (EGFR), and rearranged during transfection (RET), could augment the antitumor activity of radiation with or without cisplatin in preclinical in vitro and in vivo models of human head and neck squamous cell carcinoma (HNSCC). Experimental Design: OSC-19 and HN5 HNSCC cells that were cisplatin and radioresistant were treated with vandetanib, cisplatin, and radiation alone or in combination in vitro and in vivo using an orthotopic nude mouse model. Treatment effects were assessed using clonogenic survival assay, tumor volume, bioluminescence imaging, tumor growth delay, survival, microvessel density, tumor and endothelial cell apoptosis, and EGFR and Akt phosphorylation data. Results: Vandetanib plus cisplatin radiosensitized HNSCC cells in vitro and in vivo. The combination treatment with vandetanib, cisplatin, and radiation was superior to the rest of treatments (including the double combinations) in antitumoral effects, prolonging survival, decreasing cervical lymph node metastases in vivo. It also increased both tumor and tumor-associated endothelial cell apoptosis and decreased microvessel density in vivo. An analysis of tumor growth delay data revealed that vandetanib plus cisplatin enhanced radioresponse in vivo. All vandetanib-containing treatments inhibited EGFR and Akt phosphorylation in vitro and in vivo. Conclusion: The addition of vandetanib to combination therapy with cisplatin and radiation was able to effectively overcome cisplatin and radioresistance in in vitro and in vivo models of HNSCC. Further study of this regimen in clinical trials may be warranted. Clin Cancer Res; 17(7); 1815–27. ©2011 AACR.

Purpose: Radiotherapy plays an integral role in the treatment of head and neck squamous cell carcinoma (HNSCC). Although proteins involved in DNA repair may predict HNSCC response to radiotherapy, none has been validated in this context. We examined whether differential expression of double-strand DNA break (DSB) repair proteins in HNSCC, the chief mediators of DNA repair following irradiation, predict for treatment outcomes. Experimental Design: Archival HNSCC tumor specimens (n = 89) were assembled onto a tissue microarray and stained with antibodies raised against 38 biomarkers. The biomarker set was enriched for proteins involved in DSB repair, in addition to established mechanistic markers of radioresistance. Staining was correlated with treatment response and survival alongside established clinical and pathologic covariates. Results were validated in an independent intramural cohort (n = 34). Results: Ku80, a key mediator of DSB repair, correlated most closely with clinical outcomes. Ku80 was overexpressed in half of all tumors, and its expression was independent of all other covariates examined. Ku80 overexpression was an independent predictor for both locoregional failure and mortality following radiotherapy (P < 0.01). The predictive power of Ku80 overexpression was confined largely to HPV-negative HNSCC, where it conferred a nine-fold greater risk of death at two years. Conclusions: Ku80 overexpression is a common feature of HNSCC, and is a candidate DNA repair-related biomarker for radiation treatment failure and death, particularly in patients with high-risk HPV-negative disease. It is a promising, mechanistically rational biomarker to select individual HPV-negative HNSCC patients for strategies to intensify treatment. Clin Cancer Res; 17(7); 2035–43. ©2011 AACR.

The p300-CBP1 coactivator family members (CREB-binding protein (CREBBP) and E1A binding protein p300 (EP300)) share significant homology and act as transcriptional co-activators for a large number of target genes. These genes are collectively mutated in approximately 14% of all head and neck squamous cell cancers carcinomas (HNSCC) and are targeted by multiple agents in clinical development. Because of this the p300-CBP1 coactivator family presents a promising target in HNSCC. Recently, our group performed in-vivo screening utilizing a barcoded shRNA library of targets of known anti-neoplastic agents. A total of 5 HNSCC cell lines of varying HPV status (HPV (-): Cal27, HN31, UMSCC22A; HPV (+): UMSCC47, UPCISCC152) were transduced with the shRNA library and allowed to form subcutaneous, flank tumors, which were then treated with IR (2 Gy/day) to achieve an approximate 20% reduction in tumor volume. The tumors were then harvested and barcodes were sequenced. Interestingly, this screen revealed that CREBBP or EP300 depletion led to dramatic sensitization to IR, but only in tumors harboring a mutation in either CREBBP or EP300, independent of HPV status. To investigate this phenomenon further, we utilized chemical inhibition of CREBBP as well as shRNA to CREBBP in multiple HNSCC cell lines of varying HPV and CREBBP mutational statuses. In those cell lines harboring a CREBBP mutation, we found significant in vitro sensitization to IR on clonogenic assay following CREBBP inhibition, however this phenomenon was not observed in CREBBP wild type cells. All cell lines tested were generally resistant to IR-induced apoptosis, as measured by TUNEL assay and PARP cleavage. However, IR combined with inhibition of CREBBP led to dramatically increased IR-associated apoptosis as well as increased γ-H2AX and decreased BRCA1 foci following radiation. We next examined CREBBP inhibition using two shRNA clones specific for CREBBP in an in vivo UMSCC47 xenograft model. In this model, inhibition of CREBBP alone or IR alone (2 Gy x 8 d) led to minimal tumor volume reduction (tumor growth delay (TGD) 6 and 2 days respectively). However, the combination of each shCREBBP clone and IR led to a TGD of 24 and 23 days (p Citation Format: Manish Kumar, Kathleen Bridges, David Molkentine, Liangpeng Yang, Aakash Sheth, Raymond Meyn, Mitchell Frederick, Jeffrey Myers, Curtis Pickering, Heath D. Skinner. In vivo shRNA screening identifies synthetic cytotoxicity in CREBBP/EP300 mutant head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 978.

Check point kinases (Chk) play a major role in facilitating DNA repair upon radiation exposure. We tested the potency of a novel inhibitor of Chk1 and Chk2, XL-844 (provided by Exelixis Inc., CA, USA), to radiosensitize human cancer cells grown in culture and investigated the underlying mechanisms. HT-29 cells (a human colon cancer line) were exposed to XL-844, radiation, or both, and assessed for clonogenic cell survival. Treatment-dependent effects on phosphorylated forms of Chk proteins were assessed by Western blots. Further mechanistic investigations in HT-29 cells included cell cycle analysis by flowcytometry and assessment of DNA repair kinetics by immuno-cytochemistry (ICC) for nuclear appearance of the phosphorylated form of histone 2AX protein (γ-H2AX) staining. Cells undergoing mitotic catastrophe were identified by irregular pattern of mitotic spindle markers α and γ-tubulin staining by ICC. XL-844 enhanced radiosensitivity in a dose and schedule-dependent manner and the enhancement factor was 1.42 at 0.5 survival fraction. Mechanistically XL-844 abrogated radiation-induced Chk2 phosphorylation, induced pan-nuclear γ-H2AX, and prolonged the presence of radiation-induced γ-H2AX foci, and promoted mitotic catastrophe. In conclusion, our data showed that inhibition of Chk2 activity by XL-844 enhanced cancer cell radiosensitivity that was associated with inhibition of DNA repair and induction of mitotic catastrophe.

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.

Introduction: The radiation doses used to treat unresectable lung cancer are often limited by the proximity of normal tissues. Overexpression of c-Met, a receptor tyrosine kinase, occurs in about half of non–small-cell lung cancers (NSCLCs) and has been associated with resistance to radiation therapy and poor patient survival. We hypothesized that inhibiting c-Met would increase the sensitivity of NSCLC cells to radiation, enhancing the therapeutic ratio, which may potentially translate into improved local control. Methods: We tested the radiosensitivity of two high-c-Met–expressing NSCLC lines, EBC-1 and H1993, and two low-c-Met–expressing lines, A549 and H460, with and without the small-molecule c-Met inhibitor MK-8033. Proliferation and protein expression were measured with clonogenic survival assays and Western blotting, respectively. γ-H2AX levels were evaluated by immunofluorescence staining. Results: MK-8033 radiosensitized the high-c-Met–expressing EBC-1 and H1993 cells but not the low-c-Met–expressing cell lines A549 and H460. However, irradiation of A549 and H460 cells increased the expression of c-Met protein at 30 minutes after the irradiation. Subsequent targeting of this up-regulated c-Met by using MK-8033 followed by a second radiation dose reduced the clonogenic survival of both A549 and H460 cells. MK-8033 reduced the levels of radiation-induced phosphorylated (activated) c-Met in A549 cells. Conclusions: These results suggest that inhibition of c-Met could be an effective strategy to radiosensitize NSCLC tumors with high basal c-Met expression or tumors that acquired resistance to radiation because of up-regulation of c-Met.

Background and purpose Although inhibition of epidermal growth factor receptor (EGFR) signaling during radiation led to improvement of tumor control and survival, novel strategies are needed to further improve the outcome of patients with locally advanced head and neck carcinoma. Because EGFR is known to interact with c-Src kinases, the present study investigated dasatinib (BMS-354825), an inhibitor of c-Src kinases, for its efficacy in enhancing radiosensitivity of human head and neck squamous cell carcinomas (HNSCC) in vitro and examined the underlying mechanisms for this effect. Materials and methods Six HNSCC lines were exposed to dasatinib, radiation, or both, and assessed for c-Src and EGFR expression, cell survival and colony forming ability. Among these cell lines, HN-5 and FaDu lines were analyzed for induction of apoptosis, cell cycle re-distribution and for nuclear localization of EGFR, γ-H2AX and 53BP1 proteins. Immuno-precipitation and Western blots were performed to analyze the levels and binding of proteins involved in cell survival, apoptosis and DNA repair pathways. Suppression of c-Src by siRNA and subsequent clonogenic assay was performed in HN-5 cells. Results All six HNSCC lines that were examined expressed high levels of c-Src. Two (HN-5 and MDA-183) expressed higher levels of EGFR than other lines. Dasatinib suppressed cell survival of all cell lines tested independent of c-Src or EGFR levels but enhanced the radiosensitivity of HN-5 and MDA-183. HN-5 and FaDu were analyzed further. Dasatinib suppressed phosphorylation of c-Src in both cell lines, but decreased repair of radiation-induced DNA damage in HN-5 cells only as evidenced by suppression of c-Abl and Nbs-1 activity, inhibition of the association between c-Src and EGFR or Her-2, prolongation of nuclear γ-H2AX and 53BP1 foci and inhibition of EGFR nuclear localization and its association with DNA-PKcs. Finally, partial suppression of c-Src resulted in a small increase in HN-5 cell radiosensitivity. Conclusions Our data demonstrate that dasatinib induces apoptosis and blocks DNA repair in EGFR-expressing HNSCC cells and improves radiotherapy outcome. These findings warrant further investigation using in vivo tumor models for potential translation into clinical testing.

Purpose: The present study investigated the effect of AC480, a small molecule pan-HER tyrosine kinase inhibitor, on in vitro radiosensitivity and in vivo radioresponse of a human head and neck squamous cell carcinoma cell line. Methods: HN-5 cells were exposed to γ-radiation with and without AC480 and assayed for proliferation, clonogenic survival, apoptosis, cell cycle distribution, and DNA damage. The cells were analyzed by immunoprecipitation and western blotting for proteins involved in apoptosis, cell cycle regulation, and the EGFR pathway. The effect of AC480 on tumor radioresponse was assessed by tumor growth delay assay using HN5 tumor xenografts generated in nude mice. Results: At the molecular level, in HN-5 cells the agent inhibited the expression of pEGFR, pHER2, cyclins D and E, pRb, pAkt, pMAPK, pCDK1 and 2, CDK 6, and Ku70 proteins. The drug also induced accumulation of cells in the G1 cell cycle phase, inhibited cell growth, enhanced radiosensitivity, and prolonged the presence of γ-H2AX foci up to 24 h after radiation. AC480 did not increase the percentage of cells undergoing radiation-induced apoptosis. The drug given before and during irradiation improved the radioresponse of HN5 tumors in vivo. Conclusion: AC480 significantly enhanced the radiosensitivity of HN-5 cells, expressing both EGFR and Her2. The mechanisms involved in the enhancement included cell cycle redistribution and inhibition of DNA repair. Both in vitro and in vivo data from our study suggest that AC480 has potential to increase tumor response to radiotherapy.

Purpose: Integrins are implicated in resistance of solid tumors to radiation therapy, suggesting that their inhibition would enhance therapeutic efficacy. We investigated the efficacy of DI17E6, a therapeutic antibody targeting alpha v (αv) integrins, in enhancing the radiosensitivity of non-small cell lung carcinoma (NSCLC) and head & neck cancer (HNSCC) cells using in vitro and in vivo models, and compared to the previously reported effects of the cyclic peptide cilengitide (inhibits αvβ3 and αvβ5 integrins). Methods: NSCLC lines (A549 and H460) and HNSCC lines (FaDu and HN-5) were examined. Basal level of αv integrin was assessed by viable cell flow-cytometry and by Western blot. The effects of DI17E6 on cell viability were determined by MTS assay, and on cellular radiosensitivity by clonogenic cell survival assay after exposing the cells to γ-radiation with or without DI17E6. In vivo effect of DI17E6 on radioresponse of xenografts was evaluated by tumor growth delay assay. DI17E6 was given i.p.; as a single loading dose of 180 mg/kg followed by weekly doses of 10, 30 or 100 mg/kg given for 6 weeks; fractionated doses of local tumor irradiation was delivered using 137Cs γ-ray source (2 Gy daily for 7 days). When combined, the loading dose of DI17E6 was given either 4 h before or after the first radiation dose. Results: DI17E6 (0.01-10 µg/ml, 24 h) had a minimal effect on the cell viability. However, DI17E6 reduced the plating efficiency of A549, H460 and FaDu with no effect on HN-5 cells. When combined with radiation, DI17E6 enhanced the radiosensitivity of NSCLC cells but not the HNSCC cells. These data are consistent with previously observed effects of cilengitide in vitro. In vivo experiments with A549 xenografts showed that DI17E6 as a single agent had no antitumor activity, but in combination with radiation it suppressed the tumor growth in a schedule- and dose-dependent manner. These data were compared to the previously reported effect of cilengitide, which did not enhance the response of A549 xenografts to radiation therapy. DI17E6 has an extended pharmacokinetics (with a half-life of >7 days) and blocks more αv integrins than cilengitide. The results imply that when αvβ3 and αvβ5 integrins were inhibited by cilengitide either the duration of blockade was insufficient, or that other αv integrins may have provided tumor growth advantage. In vivo studies on other tumor types are underway. Conclusions: Inhibition of αv integrins by DI17E6 in combination with radiation enhanced radiosensitivity of NSCLC cell lines in vitro and in vivo enhanced A549 tumor xenograft response to radiation in mice. These results suggest that DI17E6 may improve the treatment outcome of patients with NSCLC when combined with radiotherapy in a clinical setting. Supported by EMD Serono Merck KGaA, Germany Citation Format: Amit A. Deorukhkar, David P. Molkentine, David R. Valdecanas, Kathryn A. Mason, Uma Raju. Preclinical evaluation: efficacy of DI17E6, a therapeutic antibody against alpha v integrins, compared to cilengitide in sensitizing human cancers to radiation therapy. [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 857. doi:10.1158/1538-7445.AM2014-857

Purpose/Objective(s): Interaction between the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGF-1R) has been well established inmany cancer types. Ligands of these receptors are capable of activating both pathways. We investigated the effects of cetuximab (EGFR antibody) and IMC-A12 (IGF-1R antibody) on the response of head and neck squamous cell carcinoma (HNSCC) to radiation therapy (IR). Materials/Methods: SixHNSCCmodels expressing different levels of EGFR and IGF-1R were studied. The effects of cetuximab and IMC-A12 on cell viability (MTS assay) and radiosensitivity (clonogenic survival assay) were determined. Formation of nuclear g-H2AX and 53BP1 foci was monitored by immunofluorescence to assess the IR-induced DNA damage and repair kinetics. Alterations in target signaling were analyzed by Western blots. In vivo tumor growth delay assay was performed to determine the efficacy of IMCA12 in enhancing tumor response to cetuximab and radiation using two HNSCC tumor xenograft models generated in mice. Results: In vitro studies in six HNSCC lines showed that exposure to cetuximab resulted in increased cell radiosensitivity in two cell lines. EGFR inhibition suppressed DNA repair that was evident by the prolonged presence of nuclear g-H2AX and 53BP1 foci. Western blot analyses showed that EGFR inhibition increased IGF-1R expression levels and also the association between EGFR and IGF-1R. When IMC-A12 was added concurrently to the treatment regimen along with cetuximab, a further increase in radiosensitivity was observed in one of the three cell lines tested. In vivo tumor growth delay data in two of the HNSCC tumor models showed that tumor xenografts exhibited enhanced tumor response to RT in the presence of either cetuximab or IMC-A12 depending on the tumor type. Addition of IMC-A12 to cetuximab and IR treatment regimen failed to further enhance the tumor response to cetuximab as well as to IR. Conclusions: Taken together our data suggest that concomitant inhibition of both EGFR and IGF-1R pathways using their respective antibodies did not yield additional therapeutic benefit in overcoming resistance to RT. Author Disclosure: U. Raju: None. D.P. Molkentine: None. D.R. Valdecanas: None. A.A. Deorukhkar: None. K.A. Mason: None. T.A. Buchholz: None. R.E. Meyn: None. K.K. Ang: None. H. Skinner: None.

Purpose: Emerging data show crosstalk among receptor tyrosine kinases (RTK), e.g., epidermal growth factor receptor (EGFR) and insulin-like growth factor receptor (IGF-1R). Higher IGF-1R expression was associated with resistance to EGFR inhibitors and ionizing radiation (IR). We investigated the effects of cetuximab (EGFR antibody) and A12 (IGF-1R antibody) on the response of head and neck squamous cell carcinoma (HNSCC) to IR, escape mechanisms from EGFR blockade leading to radioresistance, and strategies to overcome it. Methods: Three HNSCC models (HN-5, FaDu, and UMSCC-1) expressing different levels of EGFR and IGF-1R were studied. The effects of cetuximab and A12 (from Imclone) on cell viability (MTS assay) and radiosensitivity (clonogenic survival assay) were determined. Various scheduling of drugs were examined. Alterations in target signaling were analyzed by Western blots. Results: Cetuximab inhibited viability of cells (14 to 53%) in a cell type dependent manner. The highest EGFR expressing HN-5 was the most responsive to cetuximab. A12 had no effect on cell viability but elicited a moderate radiosensitization of all three cell lines (enhancement factors, EFs, ranged from 1.11 to 1.67). Cetuximab, given 6 h before till 66 h after IR, significantly enhanced the radiosensitivity of HN-5 (EF, 2.55) and FaDu (EF, 1.46), but no effect on UMSCC-1. A 48 h exposure to cetuximab before IR, however, increased radioresistance of HN-5 and FaDu without affecting UMSCC-1. Continuing with cetuximab for 72 h after IR resulted in a net radiation sensitization in HN-5, partially reversed radioresistance in FaDu, and enhanced the radiosensitivity of UMSCC-1. Western blots revealed that cetuximab (48 h) upregulated IGF-1R in FaDu (2.28 fold) and UMSCC-1 (1.26 fold) but, not in HN-5; however, EGFR was upregulated in HN-5. IGF-1R inhibition had no effect on HN-5, reverted the radiation response partially in FaDu, and increased the net radiosensitivity of UMSCC-1. Compared to EGFR inhibition alone, co-targeting of both pathways did not yield a better effect on HN-5, reverted radiosensitivity to the baseline level in FaDu, but further increased UMSCC-1 radiosensitivity. Conclusion: This study showed that tumors responded to prolonged EGFR inhibition by upregulating other pro-survival RTK signaling, e.g. IGF-1R. In HN-5, EGFR was a main pro-survival signaling as EGFR inhibition yielded consistent sensitization that was not enhanced by IGF-1R blockade. UMSCC-1 seemed to rely on EGFR and IGF-1R signaling as dual inhibition yielded better sensitization than single pathway blockade. In FaDu, IGF-1R could become a major pro-survival signaling as it was associated with increased resistance that was only reverted by co-targeting IGF-1R. Taken together our data suggest that therapeutic benefit would result from combining cetuximab and A12 with IR in some HNSCC. Supported by P0-1 CA-06294 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2488. doi:10.1158/1538-7445.AM2011-2488

Purpose:, Targeting signaling pathways that are implicated in cancer cell resistance to therapy is being investigated to improve outcome. XL228 is a small molecule inhibitor of IGF-1R, cSrc, BCR/Abl and aurora kinases, which are frequently overexpressed or hyperactive in many cancer types including head and neck squamous cell carcinomas (HNSCCs) and non small cell lung cancers (NSCLCs). This study investigated the potential of XL228 in enhancing cancer cell sensitivity to radiation. Materials and Methods: Three HNSCC (HN-5, FaDu and UMSCC-1) and three NSCLC lines (H460, A549 and H1299) were used for this in vitro study. Cell viability assay was used to assess the single agent activity of 5-100 nM of XL228 for 1-3 d. In addition, the effects of XL228 (10 or 100 nM) on apoptosis (TUNEL assay), and cell cycle redistribution (flow cytometry), migration and invasion were evaluated. Clonogenic cell survival assayed (in HN-5, FaDu, A549 and H460) after exposing the cells to graded doses of γ-radiation, with or without XL228 (10, 50 or 100 nM) was used to quantify the effect on radiosensitivity. Results: XL228, at 5-100 nM, reduced survival of all cell lines tested by 10-70% in a dose and time dependent manner and inhibited migration and invasion of two tumors with high propensity to metastasize, FaDu and H460. At 50 and 100 nM XL228 abolished the ability of H460, A549 and FaDu cells to form colony. At 10 nM XL228 significantly increased the radiosensitivity of H460, A549 and FaDu cells by enhancement factors (EF, at the survival fraction of 0.5) of 1.52, 1.31 and 1.67 respectively. But, sensitization of HN-5 cells occurred only at 100 nM (EF = 2.27). Flow cytometry analysis showed that 10 nM of XL228 (that enhanced radiosensitivity of three cell lines) had no effect on the cell cycle distribution and it did not induce apoptosis tested at 24 h after exposure to XL228. However, in HN-5 cells 100 nM of XL228 induced accumulation of cells at the radiation sensitive G2/M phase of the cell cycle and induced apoptosis in 32% of cells. Mechanistic studies including inhibition of DNA repair processes are ongoing. Conclusions: This study demonstrated that XL228 as a single agent reduced viability, inhibited proliferation, migration and invasion of cells. In combination with radiation, XL228 enhanced HNSCC and NSCLC radiosensitivity. The underlying processes leading to enhanced radiosensitivity may be cell type specific. These data warrant in vivo investigations aiming at taking best regimens to the clinic testing. (XL228 was provided by Exelixis, Inc. South San Francisco, CA. Supported by P0-1 CA-06294) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2487. doi:10.1158/1538-7445.AM2011-2487

Purpose: Integrins are implicated in resistance of solid tumors to therapies, including radiation therapy, suggesting that their inhibition would enhance efficacy of tumor therapy. Because cilengitide, a cyclic Arg-Gly-Asp (RGD)-derived peptide, inhibits αVβ3 and αVβ5 integrins, we investigated the efficacy of cilengitide in enhancing in vitro cancer cell radiosensitivity and in vivo radioresponse of cancer xenografts. Methods: Three non-small cell lung carcinoma lines (NSCLCs) (H460, A549 and H1299) and five head and neck squamous cell carcinoma lines (HNSCCs) (FaDu, SCC-15, SCC-25, HN-5 and UMSCC-1) cell lines were used for in vitro experiments. Of these, H460 and FaDu were used for in vivo testing when grown as xenografts in nude mice. The effects of cilengitide (1 – 50 µg/ml, 24 h) on in vitro cell viability was determined by MTS assay and on cellular radiosensitivity by clonogenic cell survival assay after exposing the cells to graded single doses of γ-radiation with or without cilengitide (given 1 h before and continued for 23 h after irradiation). In vivo effect of cilengitide on radioresponse of xenografts was assessed by tumor growth delay. When tumor xenografts reached 7 mm in diameter cilengitide treatment (30 or 60 mg/d/5days) was initiated followed by a single dose of 15 Gy local radiation (using 137Cs γ-ray source), when the tumors reached 8 mm. Results: Cilengitide (5µg/ml, 24 h) reduced in vitro viability of 7 out of 8 cell lines tested, which ranged between 71.4 ± 2.2% (SCC-15) and 27.8 ± 4.2% (H1299). In general, NSCLCs were more sensitive to cilengitide than HNSCCs. When combined with radiation, cilengitide significantly enhanced the radiosensitivity of all 3 NSCLC, by enhancement factors of 1.35 for H460, 1.56 for A549 and 1.3 for H1299. In contrast, cilengitide exerted only an additive effect on radiosensitivity of HNSCC lines. In vivo experiments showed that cilengitide as a single agent had no antitumor activity, but in combination with radiation it significantly enhanced response of both H460 (NSCLC) and FaDu (HNSCC) tumor xenografts. The enhancement factors were 1.7 for H460 and 2.0 for FaDu. Conclusion: The results showed that cilengitide reduced in vitro cell viability of lung and head and neck cancer cells but had no effect on tumor growth in vivo. When combined with radiation, the drug enhanced radiosensitivity of lung but not head and neck carcinoma cells. In contrast, cilengitide was highly effective in enhancing radiation response of both lung (H460) and head and neck (FaDu) tumor xenografts. These results suggest that cilengitide has potential to improve the treatment outcome of patients with NSCLC and HNSCC when combined with radiotherapy. Mechanistic studies addressing cilengitide-radiation interactions both in vitro and in vivo are underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2509. doi:10.1158/1538-7445.AM2011-2509

Huachansu (HCS), an extract from dried toad (Bufo gargarizans or B. melanostictus) skin, contains biologically active substances including steroidal cardiac glycosides and indole alkaloids. It possesses antitumor activity and is well tolerated by cancer patients. The present study examined whether HCS enhances radiosensitivity of cancer cells. Three human lung cancer cell lines, H460 and A549 (p53 wild type), and H1299 (p53 null) were used. They exhibited dose-dependent cytostatic and/or cytotoxic responses to 24 h treatment with HCS, with an IC50 of approximately 20 mg/ml assayed by cell viability. IC50 dose was used in subsequent experiments in combination with IR. Treatment endpoint was clonogenic cell survival determined 12-14 days after irradiation with 2-6 Gy, with or without HCS. Cells were treated with HCS for 24 h before IR or was added immediately post exposure for 24 h. A 24 h pretreatment, but not post IR treatment, strongly enhanced radiosensitivity of H460 and A549 cells, with enhancement factors of 1.91 and 1.94, respectively, at 50% survival. Neither HCS treatment scheme affected radiosensitivity of H1299 cells. HCS pretreatment of H460 and A549 cells significantly prolonged the presence of radiation-induced double-strand breaks detected on the basis of γH2AX foci at 30 min, 4, 16 or 24h after 2 Gy IR. This suggests that inhibition of DNA repair may be an underlying mechanism of HCS-induced enhanced radiosensitivity. In addition, H460 and to lesser degree A549 cells treated with HCS were more susceptible to radiation-induced apoptosis. For example, the percentage of apoptotic cells (TUNEL) in H460 exposed both to HCS and 4 Gy (measured 24 h after IR) was 43.4 ± 1.1%, a value significantly higher than additive values of 5.2 ± 1.2% (HCS treatment alone), 10.5 ± 2.7% (IR alone). The percentage of apoptotic cells in untreated control was 4.2 ± 0.9%. This finding was supported molecularly by increased expression of cleaved caspase-3 and decreased expression of Bcl-2 analyzed by Western blot. Thus, increased susceptibility of cells to radiation-induced apoptosis is another mechanism underlying HCS-induced enhancement of cell radiosensitivity. Additional experiments showed that treatment of H460 and A549 cells with HCS induced no significant changes in cell cycle distribution to affect radiosensitivity. In conclusion, our findings demonstrated that HCS strongly enhanced radiosensitivity of human lung cancer cell lines and that the effect may be related to p53 status since only the p53 wt cell lines responded. Major underlying mechanisms of HCS-induced enhanced radiosensitivity include inhibition of DNA damage repair and increase in radiation-induced apoptosis. These data suggest that HCS may have potential to improve the efficacy of radiotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5674.

Background: cSrc is often over-expressed in cancer cells including head and neck squamous cell carcinoma (HNSCC) and is implicated in promoting cell survival, proliferation, angiogenesis, cell adhesion and motility. Its role in tumor resistance to cytotoxic agents, such as radiation, is less clear. Present study investigated (1) the relationship between the level of activated cSrc expression and the degree of cell radioresistance and (2) the role of cSrc in the cellular response to radiation, using both in vitro and in vivo models. Materials and methods: Ten HNSCC lines (FaDu, HN-5, UMSCC-1, HN-30, MDA1386, UMSCC-17B, UMSCC-47, Sqccy-1, SN2 and PCI15B) were assessed for their sensitivity to ionizing radiation by clonogenic survival and for expression levels of active Src kinases using phospho-kinase antibody array kit (Proteome ProfilerTM, R&D Systems, Inc., Minneapolis, MN). cSrc expression levels were validated by Western blot. cSrc-siRNA was transfected by electroporation to suppress cSrc expression, after which the cells were assessed for radiosensitivity. We also tested the effect of dasatinib, an inhibitor of Src kinases, on in vitro cell radiosensitivity, and on the growth of tumor xenografts in nude mice. Results: HNSCC lines exhibited a wide range of both radiosensitivity and levels of phosphorylated Src family kinases. There was a highly positive correlation (r=0.883) between SF2 values (cell survival at 2 Gy) and the degree of basal phosphorylated cSrc kinase expression. As the latter increased the ability of cells to survive radiation, increased. Inhibition of cSrc expression by cSrc-siRNA in 2 cell lines tested (FaDu and UMSCC1) resulted in a significant increase in their radiosensitivity, suggesting a causal relationship between cSrc and cell radioresistance. Inhibition of cSrc by dasatinib also resulted in increase of radiosensitivity of 3 cell lines tested (FaDu, HN-5, UMSCC-1). Additionally, dasatinib enhanced in vivo tumor response to radiation as shown by a marked increase in radiation-induced growth delay of FaDu xenografts. Conclusions: Our data showed that (1) a strong correlation exists between the expression level of cSrc and cell radiosensitivity of HNSCCs, (2) inhibition of cSrc expression by siRNA can render tumor cells more radiosensitive, implicating a causal relationship between cSrc and radioresistance, and (3) dasatinib enhanced both the in vitro tumor cell radiosensitivity and in vivo tumor response to radiation. These results suggest that targeting cSrc signaling network may have potential to improve the efficacy of radiotherapy. Supported in part by Bristol-Myers Squibb Company Sponsored Agreement (PI: U. Raju), NIH PO-1 Grant CA-06294 (PI: KK. Ang) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 488.

Objective Despite advances in treatment, the survival of patients with Head and neck squamous cell carcinoma (HNSCC) has not significantly improved over the past several decades. While concurrent chemotherapy with cisplatin and radiation has become a standard of care for many patients with HNSCC, some HNSCCs are resistant and persist/recur after this type of treatment. To determine whether we could overcome this resistance, we evaluated whether vandetanib, an inhibitor of tyrosine kinase activities of vascular endothelial growth factor receptor-2 (VEGFR-2) and epidermal growth factor receptor (EGFR) could augment the anti-tumor activity of concurrent chemotherapy with cisplatin and radiation in pre-clinical in vitro and in vivo models of human HNSCC Method To determine the effects of cisplatin and irradiation on HNSCC cell proliferation in vitro, we first performed MTT assays with cisplatin and clonogenic survival assay with radiation using 18 HNSCC cell lines. We chose HN5 as a relatively cisplatin & irradiation-resistant cell line. Clonogenic survival assays with irradiaton + cisplatin on HN5 were also performed to see the interaction between cisplatin and irradiation. We used an orthotopic nude mouse model of HNSCC by injecting human HN5 lines into the tongue of the mouse. Nine days after cell inoculation, the animals were randomly divided into 8 groups to receive vehicle, cisplatin (1 mg/kg, intravenously, once a week for 2 weeks), vandetanib (20 mg/kg, daily oral gavage, for 2 weeks), vandetanib plus cisplatin, irradiation (5 Gy at day 10), cisplatin plus irradiation, vandetanib plus irradiation, or vandetanib plus cisplatin plus irradiation. We assessed in vivo effects of the treatment by tumor size. Results Cisplatin GI50 values spanned a 10-fold range from 1.031 to 10.58 μM and the surviving fraction at 2 Gy (SF2) values ranged from 0.22 to 0.8 with 18 HNSCC cell lines. The GI50 value of vandetanib was 9.084 μM and the SF2 value was 0.75 for HN5 which indicated this line was a relatively cisplatin and irradiation-resistant cell line. In the clonogenic survival assay with iradiation and cisplatin on HN5, cisplatin did not improve radiosensitivity. At day 35 after cell inoculation, the treatment with cisplatin alone, vandetanib alone, vandetanib plus cisplatin, irradiation alone, cisplatin plus irradiation, vandetanib plus irradiation, and vandetanib plus cisplatin plus irradiation reduced tumor size by 5.0% (P=0.6843), 41.9% (P=0.0370), 49.2% (P Conclusion These data indicate that the addition of vandetanib to combination therapy with cisplatin and radiation may be effective in overcoming cisplatin-radiation resistance HNSCC and has the potential as a novel therapeutic startegy for patients with advanced HNSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 369.