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Deletion or loss of function mutations of the STK11/LKB1 tumor suppressor are associated with primary resistance to immunotherapy in KRAS-mutant lung adenocarcinoma (LUAD) and drive metabolic reprogramming of tumor cells. We observed that LKB1-deficient tumors were resistant to anti-angiogenic therapy in the hypoxic and nutrient-depleted or acidic tumor microenvironment (TME). We determined that MYC which is elevated in LKB1-deficient cells, regulated the expression of the lactate transporter, MCT4. Moreover, knockdown of MYC decreases glycolysis and cell proliferation. Therefore, we hypothesize that metabolic changes in LKB1-deficient tumor cells is at least partially driven by MYC upregulation, and that depletion of MYC or targeting of key enzymes or transporters downstream of MYC such as MCT4 may abrogate lactate reutilization and sensitize LKB1-deficient tumors to anti-angiogenic therapy. To explore the impact of MYC on lactate metabolism in LKB1-deficient NSCLC cells, we performed isotope tracing in KRAS mutant (K) and KRAS mutant LKB1 knockout (KL) syngeneic murine lung cancer cells. 13C3-lactate tracing revealed that isotopologues were significantly enriched in TCA components such as pyruvate, citrate and a-ketoglutarate in KL cells. Meanwhile, lactate-treated KL cells shows less reactive oxygen species (ROS), suggesting that LKB1-deficient tumor cells reutilize lactate as an energy source more efficiently than LKB1-intact cells. MCT4 KO significantly abrogated lactate incorporation into TCA cycle. Similarly, MYC knockdown or MCT4 KO decreased lactate-induced oxygen consumption, but increased ROS levels. In contrast, exhausted T cells were inefficient at lactate reutilization, and high levels of lactate increased ROS in T cells. Hence, LKB1-deficient tumors have a survival advantage over T cells in the lactate-rich TME. We queried scRNAseq data from K, KL and KL MCT4KO syngeneic models and similarly observed that KL tumor cells exhibited elevated hypoxia and angiogenesis gene expression signatures, which was reversed by MCT4 KO. However, ROS detoxification was decreased in T cells from KL tumors but increased in MCT4 KO tumors. Finally, we injected KL murine tumor cells into immunocompetent mice, and randomly treated them with vehicle or the VEGF blocking antibody, DC101. MYC knockdown or MCT4 KO sensitized KL tumors to VEGF inhibition, and significantly increased overall survival. Collectively, our data indicates that in LKB1-deficient tumors, upregulation of MYC promotes tumor cell metabolic reprogramming and that targeting MYC or MCT4 can inhibit lactate reutilization and enhance the efficacy of anti-angiogenic agents. These findings provide insight into the mechanisms driving the aggressive phenotype of KRAS-mutant LKB1-deficient tumors and identify a novel therapeutic strategy for targeting this patient population. Citation Format: Yu Qian, David Molkentine, Chendong Yang, Ana Galan Cobo, Irene Guijarro, Minghao Dang, Alissa Poteete, Peixin Jiang, Ferdinandos Skoulidis, Linghua Wang, Alexandre Reuben, John D. Minna, Ralph J. DeBerardinis, John V. Heymach. MYC mediates enhanced lactate reutilization and resistance to anti-angiogenesis therapy in preclinical models of LKB1-deficient NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 271.

Radiation and platinum-based chemotherapy form the backbone of therapy in HPV-negative head and neck squamous cell carcinoma (HNSCC). We have correlated focal adhesion kinase (FAK/PTK2) expression with radioresistance and worse outcome in these patients. However, the importance of FAK in driving radioresistance and its effects on chemoresistance in these patients remain unclear. We performed an in vivo shRNA screen using targetable libraries to address these questions and identified FAK as an excellent target for both radio- and chemosensitization. Because TP53 is mutated in over 80% of HPV-negative HNSCC, we hypothesized that mutant TP53 may facilitate FAK-mediated therapy resistance. FAK inhibitor increased sensitivity to radiation, increased DNA damage and repressed homologous recombination and non-homologous end joining repair in mutant, but not wild-type, TP53 HPV-negative HNSCC cell lines. Mutant TP53 cisplatin-resistant cell line had increased FAK phosphorylation compared to wild-type, and FAK inhibition partially reversed cisplatin resistance. To validate these findings, we utilized a HNSCC cohort to show that FAK copy number and gene expression were associated with worse disease-free survival in mutant TP53, but not wild-type TP53, HPV-negative HNSCC tumors. Thus, FAK may represent a targetable therapeutic sensitizer linked to a known genomic marker of resistance.

Background Curative treatment of the most aggressive solid tumors utilizes radiation therapy, either as a monotherapy or combined with surgery and/or chemotherapy. Previously, we linked the expression of PD-L1 with clinical radioresistance; however, the relationship between outcome following radiation and immune function is unclear. Methods We used two well-annotated cohorts to examine clinical outcomes. The discovery cohort included 94 patients diagnosed with Head and Neck squamous cell carcinoma (HNSCC) who were treated uniformly with surgery and adjuvant radiation. The validation cohort consisted of 97 patients with similar treatment. Immune infiltrates in tumors were derived from RNAseq gene expression in tumor tissues using xCell. The association between each immune cell type and clinical outcomes was tested using Cox proportional hazards models. Immune cell types significantly associated with overall survival (OS), distant metastasis (DM), or locoregional recurrence (LRR) in the discovery cohort were examined in the independent validation cohort. Genes that carry nonsynonymous somatic mutations or have mRNA expression that is associated with the abundance of CD4 + T helper 2 (Th2) cell infiltrate in tumors were also identified in the full TCGA HNSCC cohort as well as non-overlapping cohorts from the International Cancer Genome Consortium (ICGC) and the National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (CPTAC). Results In the discovery cohort, we identified intratumoral cell types significantly associated with OS (monocytes, CD4 + memory T cells & Th2 cells), DM (CD4 + memory T cells, sebocytes, and epithelial cells) or LRR (common lymphoid progenitor (CLP) cells, CD4 + memory T cells, Th2 cells, and immature dendritic cells (iDCs). However, only a positive association between high levels of Th2 cells and LRR was identified in the validation cohort. We also identified CREBBP/EP300 and CASP8 among the genes that carry somatic mutations significantly associated with the presence of Th2 cells. Additionally, pathway analysis of genes correlated with Th2 cells revealed potential repression of the antitumor immune response and activation of BRCA1-associated DNA damage repair in multiple cohorts. Conclusions Th2 infiltrate is enriched in HPV-negative HNSCC tumors and is associated with LRR in two independent cohorts of patients following surgery and radiation. Th2 infiltrate is associated with mutations in CASP8 and CREBBP/EP300 and pathways previously shown to impact the response to radiation. Further investigation is warranted to investigate the mechanisms underlying the role of Th2 cells in mediating radioresistance in HPV-negative HNSCC.

Background Human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) leads to the death of over 360,000 patients annually worldwide. Curative therapy for this disease commonly consists of surgery followed by radiation therapy. Previously, we linked the expression of PD-L1 with clinical radioresistance in HNSCC; however, the relationship between outcome following radiation and immune function is unclear. Methods We used two well-annotated cohorts to examine clinical outcomes. The discovery cohort included 94 patients diagnosed with HPV-negative HNSCC who were treated uniformly with surgery and adjuvant radiation. The validation cohort consisted of 97 patients with similar treatment. Immune infiltrates in tumors were derived from RNAseq gene expression in tumor tissues using xCell. The association between each immune cell type and clinical outcomes was tested using Cox proportional hazards models. Immune cell types significantly associated with overall survival (OS), distant metastasis (DM), or locoregional recurrence (LRR) in the discovery cohort were examined in the independent validation cohort. Genes that carry nonsynonymous somatic mutations or have mRNA expression that is associated with the abundance of CD4 + T helper 2 (Th2) cell infiltrate in tumors were also identified in the full TCGA HPV-negative HNSCC cohort as well as nonoverlapping cohorts from the International Cancer Genome Consortium (ICGC) and the National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (CPTAC). Results In the discovery cohort, we identified intratumoral cell types significantly associated with OS (monocytes, CD4 + memory T cells & Th2 cells), DM (CD4 + memory T cells, sebocytes and epithelial cells) or LRR (common lymphoid progenitor (CLP) cells, CD4 + memory T cells, Th2 cells, and immature dendritic cells (iDCs). However, only a positive association between high levels of Th2 cells and LRR was identified in the validation cohort. We also identified CREBBP/EP300 and CASP8 among the genes that carry somatic mutations significantly associated with the presence of Th2 cells. Additionally, pathway analysis of genes correlated with Th2 cells revealed potential repression of the antitumor immune response and activation of BRCA1-associated DNA damage repair in multiple cohorts. Conclusions Th2 infiltrate is enriched in HPV-negative HNSCC tumors and is associated with LRR in two independent cohorts of patients following surgery and radiation. Th2 infiltrate is associated with mutations in CASP8 and CREBBP/EP300 and pathways previously shown to impact the response to radiation. Further investigation is warranted to investigate the mechanisms underlying the role of Th2 cells in mediating radioresistance in HPV-negative HNSCC.

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.

Purpose/Objective(s) Our group has previously demonstrated focal adhesion kinase (FAK) expression is associated with radioresistance and worse outcome in human papillomavirus negative (HPV-) head and neck squamous cell carcinoma (HNC). However, the relative importance of FAK in driving therapeutic resistance is unclear. FAK function can be inhibited by wild type (wt) p53, but p53 is mutated in over 80% of HPV(-) HNC. Based on these observations, we tested the hypothesis that mutant (mut) p53 permits or even drives FAK-mediated radioresistance. Materials/Methods Two cohorts of HPV(-) HNC tumors treated with radiation (RT), single institution (n = 81) and TCGA (n = 324), were stratified by TP53 mutational status (wt versus mut) and FAK copy number/mRNA expression. In vivo shRNA screening in 5 human HNSCC xenograft flank models was performed using libraries focused on known “druggable” proteins and DNA damage repair combined with RT or carboplatin. Correlation between FAK protein expression and sensitivity to cisplatin was evaluated in 20 TP53 mut HNC cell lines. The relationship between p53, FAK and radioresponse was examined in vitro using UMSCC (p53 null) HNC cells forced to express wt or mut TP53. FAK function was modulated in these cells using shRNA or chemical inhibition (defactinib) and response to radiation or carboplatin was evaluated via immunoblot for DNA damage repair proteins and clonogenic assay. FAK and p53 interactions were evaluated via immunoprecipitation. An in vivo immunodeficient flank model using HN31 cells expressing FAK shRNA was used to assess in vivo radioresponse. Results High levels of FAK copy number and gene expression were associated with worse disease-free survival in TP53 mut HPV(-) HNC, but not TP53 wt tumors. In vivo shRNA screening identified FAK as an excellent target for either radio- or chemosensitization. FAK protein expression was correlated with cisplatin IC50 (P = 0.0083, r = 0.572) in our HNC cell line panel. Inhibition of FAK via shRNA in the HN31(TP53 mut) cell line led to radiosensitization both in vitro and in a xenograft flank model. Defactinib treatment led to increased sensitivity to radiation or carboplatin in TP53 mut (HN31) but not TP53 wt (HN30) tumor cell lines. In UMSCC1 cells pretreated with defactinib, TP53 mut expression resulted in significant radiosensitization, but TP53 wt expression did not. To examine the physical interaction between p53 and FAK, different TP53 constructs were expressed in UMSCC1 cells. Interestingly, missense mutation does not impair the ability of p53 to bind to FAK. However, FAK phosphorylation was higher in cells expressing mut p53 compared to wt. Moreover, FAK inhibition combined with radiation led to higher levels of ɣ-H2AX in mut p53 expressing cells. Conclusion FAK is a potentially important target for therapeutic sensitization, primarily in TP53 mutated HNSCC.

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.

Purpose: Head and neck squamous cell carcinoma (HNSCC) is commonly treated with radiotherapy, and local failure after treatment remains the major cause of disease-related mortality. To date, human papillomavirus (HPV) is the only known clinically validated, targetable biomarkers of response to radiation in HNSCC. Experimental Design: We performed proteomic and transcriptomic analysis of targetable biomarkers of radioresistance in HPV-negative HNSCC cell lines in vitro, and tested whether pharmacologic blockade of candidate biomarkers sensitized cells to radiotherapy. Candidate biomarkers were then investigated in several independent cohorts of patients with HNSCC. Results: Increased expression of several targets was associated with radioresistance, including FGFR, ERK1, EGFR, and focal adhesion kinase (FAK), also known as PTK2. Chemical inhibition of PTK2/FAK, but not FGFR, led to significant radiosensitization with increased G2–M arrest and potentiated DNA damage. PTK2/FAK overexpression was associated with gene amplification in HPV-negative HNSCC cell lines and clinical tumors. In two independent cohorts of patients with locally advanced HPV-negative HNSCC, PTK2/FAK amplification was highly associated with poorer disease-free survival (DFS; P = 0.012 and 0.034). PTK2/FAK mRNA expression was also associated with worse DFS (P = 0.03). Moreover, both PTK2/FAK mRNA (P = 0.021) and copy number (P = 0.063) were associated with DFS in the Head and Neck Cancer subgroup of The Cancer Genome Atlas. Conclusions: Proteomic analysis identified PTK2/FAK overexpression is a biomarker of radioresistance in locally advanced HNSCC, and PTK2/FAK inhibition radiosensitized HNSCC cells. Combinations of PTK2/FAK inhibition with radiotherapy merit further evaluation as a therapeutic strategy for improving local control in HPV-negative HNSCC. Clin Cancer Res; 22(18); 4643–50. ©2016 AACR.

Devices that combine magnetic resonance imaging with linear accelerators (MRL) represent a novel tool for MR-guided radiotherapy. However, whether magnetic fields (MFs) generated by these devices affect the radiosensitivity of tumors is unknown. We investigated the influence of a 1.5-T MF on cell viability and radioresponse of human solid tumors. Human head/neck cancer and lung cancer cells were exposed to single or fractionated 6-MV X-ray radiation; effects of the MF on cell viability were determined by cell plating efficiency and on radioresponsiveness by clonogenic cell survival. Doses needed to reduce the fraction of surviving cells to 37% of the initial value (D0s) were calculated for multiple exposures to MF and radiation. Results were analyzed using Student's t-tests. Cell viability was no different after single or multiple exposures to MRL than after exposure to a conventional linear accelerator (Linac, without MR-generated MF) in 12 of 15 experiments (all P > 0.05). Single or multiple exposures to MF had no influence on cell radioresponse (all P > 0.05). Cells treated up to four times with an MRL or a Linac further showed no changes in D0s with MF versus without MF (all P > 0.05). In conclusion, MF within the MRL does not seem to affect in vitro tumor radioresponsiveness as compared with a conventional Linac. Bioelectromagnetics. 37:471-480, 2016. © 2016 Wiley Periodicals, Inc.

Background Photon (X-ray) radiotherapy (XRT) kills cells via DNA damage, however, how proton radiotherapy (PRT) causes cell death in head and neck squamous cell carcinoma (HNSCC) is unclear. We investigated mechanisms of HNSCC cell death after XRT versus PRT. Methods We assessed type of death in 2 human papillomavirus (HPV)-positive and two HPV-negative cell lines: necrosis and apoptosis (Annexin-V fluorescein isothiocyanate [FITC]); senescence (β-galactosidase); and mitotic catastrophe (γ-tubulin and diamidino-phenylindole [DAPI]). Results The XRT-induced or PRT-induced cellular senescence and mitotic catastrophe in all cell lines studied suggested that PRT caused cell death to a greater extent than XRT. After PRT, mitotic catastrophe peaked in HPV-negative and HPV-positive cells at 48 and 72 hours, respectively. No obvious differences were noted in the extent of cell necrosis or apoptosis after XRT versus PRT. Conclusion Under the conditions and in the cell lines reported here, mitotic catastrophe and senescence were the major types of cell death induced by XRT and PRT, and PRT may be more effective.

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.

Human papilloma virus (HPV) is associated with multiple types of squamous cell carcinoma, including that of the head and neck (HNSCC). The presence of HPV leads to increased expression of p16, to the extent that p16 is used as a surrogate marker of HPV positivity. HPV/p16 expression is associated with improved response to genotoxic therapy and improved outcome; however, the mechanism of this phenomenon is unclear. Previously, we showed that HPV/p16 expression repressed the DNA damage repair protein TRIP12 and led to radiosensitivity. In the current project we identify USP7 and HUWE1 as key links in this signaling pathway. Ubiquitin-specific-processing protease 7 (USP7) is a deubiquitinating enzyme that has previously been shown to bind to TRIP12, which we confirmed in HPV (-) HNSCC cell lines. We found that forced p16 overexpression in a panel of SCC cell lines led to decreased levels of USP7 and TRIP12 protein, while inhibition of p16 in HPV (+) HNSCC cell lines led to their increase. Forced p16 expression did not affect USP7 mRNA expression, however did lead to increased K48-linked ubiquitinylation and degradation of USP7. Chemical or shRNA mediated inhibition of USP7 in p16/HPV (-) cells led to decreased 53BP1 and BRCA1 foci and increased cell death following radiation. Expression of USP7 shRNA in a HPV (-), radioresistant cell derived xenograft (HN5) lead to a tumor growth delay (TGD) of 10 days following radiation (4 Gy x 5d), compared to a TGD of 1.4 days in control tumors (p Citation Format: David Molkentine, Kathleen Bridges, Aakash Sheth, David Valdecanas, Curtis Pickering, Heath D. Skinner. p16 modulates a novel ubiquitin signaling cascade which regulates radioresponse and offers clinically relevant therapeutic targets in squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2919.

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.

Proton radiotherapy (PRT) has shown to be less toxic in the treatment of head and neck cancers (HNSCC) than photon radiotherapy (XRT). XRT causes multiple types of cancer cell death by inducing DNA double-strand breaks. The DNA damage induced by PRT is more complex and has more severe biological consequences versus XRT. The present study was undertaken to uncover the cell death caused by PRT versus XRT in human papillomavirus positive (HPV+, frequently p53 wild-type, p16 positive; p53, p16 are key factors for cell death) and HPV-negative (HPV-, frequently p53 mutant, p16 negative) HNSCC cells. HNSCC cell lines HN5, SqCC/Y1 (HPV-) and UMSCC-47, UPCI-SCC-154 (HPV+) were used. Single doses of 4 Gy irradiation induced cell apoptosis, necrosis, mitotic catastrophe, and senescence were determined. Clinical 200-MeV proton beams (18 cm × 18 cm field) or 6-MV X-ray beams (25 cm × 25 cm field) were used. Cells were positioned in the centers of the irradiation fields (in the middle of the spread-out Bragg peak for PRT). At 4 hours (h), 24h and 48h after PRT or XRT, cell necrosis and apoptosis were determined by Annexin VFITC-conjugated staining. XRT and PRT caused increased cell necrosis in the HPV– SqCC/Y1 (moderate at 24h, significant at 48h [p all < 0.05]) and HN5 (moderate at 24h, 48h) cells, at slightly higher rates in PRT versus XRT treated cells; and in the HPV+ UPCI-SCC-154 (moderate at 4h) and UMSCC-47 (moderate at 48h) cells, with no difference between PRT versus XRT treatment groups. XRT and PRT induced a small percentage of cell apoptosis (range: 1.1 ± 0.5 to 7.6 ± 2.9). Cell apoptosis was only moderately increased in the HPV+ UMSCC-47 (at 4h, 24h, 48h) and HPV– SqCC/Y1 (at 24h, 48h) cells, with slightly more apoptosis seen in the XRT versus the PRT group. Cell senescence was assessed using a well-established senescence-associated biomarker (SA-β-gal). XRT and PRT induced significantly increased cellular senescence at both 4 and 6 days in all four cell lines (p all < 0.01), with PRT treatment leading to a significantly higher percentage of senescence in cells than XRT (p all < 0.01). Mitotic catastrophe, an important cell death mechanism by which solid tumors respond to clinical radiotherapy, was determined using co-staining for cytoplasm with γ- tubulin (with Texas red) and for nucleus with Dapi (blue). XRT and PRT both led to a significantly increased number of cells undergoing mitotic catastrophe at 4h, 24h, 48h and 72h (p all < 0.05) in all four cell lines, with significantly higher rates seen in PRT treated versus XRT treated (p all < 0.05). Moreover, the peak of cell mitotic catastrophe induced by PRT occurred at 48h in the HPV– cells, versus at 72h in the HPV+ cells; while no obvious peak was observed in the XRT treated group. In conclusion, mitotic catastrophe and senescence are the major types of cell death induced by both XRT and PRT, in which PRT inducing higher levels of both types of cell death as compared to XRT. Citation Format: Li Wang, Shichao Han, Jinming Zhu, Xiaochun Wang, Yuting Li, Zeming Wang, Eric Lin, Xiaofang Wang, David P. Molkentine, Pierre Blanchard, Yining Yang, Ruiping Zhang, Narayan Sahoo, Michael Gillin, Xiaorong Ronald Zhu, Xiaodong Zhang, Jeffrey N. Myers, Steven J. Frank. Proton versus photon irradiation induced cell death in head and neck cancer cells with different human papillomavirus status [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 69.

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.

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.

To assess radiosensitzing potential of huachansu (HCS) and delineate the underlying mechanisms.Lung cancer cell lines were exposed to HCS, radiation or both and subjected to survival assays, Western blots, apoptosis assay and immunocytochemical analysis.HCS suppressed the viability of all three lung lines tested and enhanced radiosensitivity of H460 and A549 (wild-type p53) only with no effect on H1299 (p53 null) cells. HCS prolonged the presence of radiation-induced γH2AX foci and increased radiation-induced apoptosis. Western blots showed that HCS increased cleaved caspase-3 and cleaved poly-(ADP-ribose) polymerase (PARP) levels, as well as reducing BCL-2 and p53 protein levels in H460 cells.HCS-enhanced radiosensitivity of human lung cancer lines appeared to be p53-dependent. Inhibition of DNA repair and increase in radiation-induced apoptosis may have served as underlying mechanisms. These data suggest that HCS may have potential to improve the efficacy of radiotherapy.

The IGF1/IGF-1R signaling pathway has emerged as a potential determinant of radiation resistance in human cancer cell lines. Therefore we investigated the potency of monoclonal anti-IGF-1R antibody, A12, to enhance radiation response in upper respiratory tract cancers.Cell lines were assessed for IGF-1R expression and IGF1-dependent response to A12 or radiation using viability and clonogenic cancer cell survival assays. In vivo response of tumor xenografts to 10 or 20 Gy and A12 (0.25-2 mg × 3) was assessed using growth delay assays. Combined treatment effects were also analyzed by immunohistochemical assays for tumor cell proliferation, apoptosis, necrosis, and vascular endothelial growth factor expression at Days 1 and 6 after start of treatment.A12 enhanced the radiosensitivity of HN5 and FaDu head-and-neck carcinomas in vitro (p0.05) and amplified the radioresponse of FaDu xenografts in a dose-dependent manner, with enhancement factors ranging from 1.2 to 1.8 (p0.01). Immunohistochemical analysis of FaDu xenografts demonstrated that A12 inhibited tumor cell proliferation (p0.05) and vascular endothelial growth factor expression. When A12 was combined with radiation, this resulted in apoptosis induction that persisted until 6 days from the start of treatment and in increased necrosis at Day 1 (p0.01, respectively). Combined treatment with A12 and radiation resulted in additive or subadditive growth delay in H460 or A549 xenografts, respectively.The results of this study strengthen the evidence for investigating how anti-IGF-1R strategies can be integrated into radiation and radiation-cetuximab regimen in the treatment of cancer of the upper aerodigestive tract cancers.

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.

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

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.

Introduction: Triple negative breast cancer (TNBC) is known to be relatively resistant to radiation therapy resulting in a comparatively higher rates of locoregional recurrence compared with non-TNBC patients. The mechanisms behind this relative radio resistance remain unknown. The goal of this study is to investigate the role of three miRNAs namely, miR-34a, miR-21, and miR-210, which are implicated in DNA damage-repair process, in the response of TNBC cell lines to radiation therapy. Methods: Seven TNBC breast cancer cell lines were used for the study. The levels of miR-34a, miR-21 and miR-210 were evaluated by taqman- based quantitative PCR assay. These cells were exposed to 2 Gy radiation dose and colonogenic survival was assessed after about 2 weeks. Survival fraction after 2 Gy (SF2), which is the ratio of number of cell colonies formed after 2 Gy radiation vs. the number of cell colonies with out radiation, was determined. The SF2 factor is a reliable index to measure radio sensitivity, greater the SF2 factor more radio resistant the cells are. Results: The SF2 value of the TNBC cells varied from 0.31 to 0.74 with MDA-MB-468, HCC-70 being the most radio sensitive cell lines and MDA-MB-436, BT-20 being the most radio resistant cell lines. MDA-MB- 231, BT-549 and Hs 578T were found to be moderately radio resistant breast cancer lines. The oncomiR -21 expression levels in these 7 TNBC cell lines varied from 0.85 to 4.18 fold with a very poor correlation with SF2 (r2=0.08). The oncomiR 210 levels varied from 0.49 to 9.19 fold and also did not exhibit any correlation with radio resistance in TNBC cells (r2= 0.141). Whereas the tumor suppressor miR, miR-34a expression showed an inverse correlation with radio resistance (r2=0.8) in TNBC cells. The radio resistant cell line (e.g., MDA-MB-436, SF2 = 0.74) expressed significantly low levels of miR-34a (0.016 fold) relative to miR -34a levels (set as 1) in HCC 70, which was found to be a radiosensitive cell line with SF2 of 0.309. Conclusions: Our preliminary data indicates that miR 34a may play a role in the response of TNBC cells to radiotherapy. Citation Format: Anjana Bhardwaj, Nivetha Ganesan, David Molkentine, Uma Raju, Isabelle Bedrosian. miR-34a as a radio sensitizer of triple negative breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4160. doi:10.1158/1538-7445.AM2013-4160

Purpose: Targeting signaling pathways that are implicated in cancer cell resistance to therapy are being investigated to improve therapy outcome. One of the receptor tyrosine kinases, c-Met (Met) is frequently overexpressed in many cancer types including non small cell lung cancers (NSCLCs) and head and neck squamous cell carcinomas (HNSCCs) and is associated with resistance to radiation. EMD1214063 is a small molecule inhibitor that suppresses the kinase activity of Met. The current study investigated the potential of EMD1214063 (provided by EMD Serono Merck Inc) in enhancing cancer cell sensitivity to radiation. Materials and Methods: Three NSCLC lines (A549, H460 and H1993) and three HNSCC lines (FaDu, HN-5 and UMSCC-1) with different levels of Met protein were used for the study. The effects of EMD1214063 on Met expression, cell viability, migration and cell radiosensitivity were assessed. Various scheduling effect on the magnitude of increased cell radiosensitivity was evaluated. 53BP1 foci formation was assayed for DNA repair kinetics. In vivo tumor growth delay assays on two NSCLCs and two HNSCCs generated in mice were performed to test the efficacy of EMD1214063 on tumor growth and tumor response to radiation. Results: NSCLC lines expressed higher levels of Met than HNSCC lines. EMD1214063 reduced survival of NSCLC lines markedly and had a minimal effect on HNSCC lines in vitro. Western blots showed three major Met protein bands with molecular weights of about 145 kDa, 60 kDa and 45 kDa. Radiation increased the expression levels of the 145 kDa and 60 kDa fragments of Met in a dose dependent manner. EMD1214063 suppressed the radiation-induced expression of Met and prolonged the presence of 53BP1 foci. It potently enhanced the radiosensitivity of A549, H1993, HN-5 and FaDu but not that of H460 and UMSCC-1. The enhancement factors at the survival fraction of 0.5 ranged from 1.11 to 2.24, with H1993 being the most responsive cell line. In vivo data showed lack of single agent activity of EMD1214063 on the growth of tumor xenografts tested (A549, H460 and FaDu). However, it showed a schedule dependent effect in enhancing A549 tumor xenograft response to radiation: a marked enhancement was observed only when EMD1214063 was given concurrently with fractionated doses of radiation. In vivo studies on other tumor types are ongoing. Conclusions: This study demonstrated that EMD1214063 reduced viability and migration of NSCLC cells and it selectively enhanced radiosensitivity of cells that express higher levels of basal or induced Met and the underlying mechanism may be inhibition of DNA repair. In vivo study data showed EMD1214063 induced a schedule dependent enhancement of tumor response to radiation in a NSCLC line. These data warrant further in vivo investigations aiming at taking best regimens to the clinic testing. This study is being supported by EMD Serono Merck via an independent medical grant. Citation Format: Uma Raju, David P. Molkentine, Jessica M. Molkentine, David N. Valdecanas, K Kian Ang. Targeting Met signaling to overcome human cancer cell resistance to radiation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4435. doi:10.1158/1538-7445.AM2013-4435

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.