Treatment Of HNSCC Research Articles

Targeted therapy in head neck tumours

Targeted therapy in head neck tumours

The Nedd8-activating enzyme inhibitor MLN4924 (TAK-924/Pevonedistat) induces apoptosis via c-Myc-Noxa axis in head and neck squamous cell carcinoma.

ObjectivesThe present study aimed to reveal expression status of the neddylation enzymes in HNSCC and to elucidate the anticancer efficacy and the underlying mechanisms of inhibiting neddylation pathway.Materials and methodsThe expression levels of neddylation enzymes were estimated by Western blotting in human HNSCC specimens and bioinformatics analysis of the cancer genome atlas (TCGA) database. Cell apoptosis was evaluated by Annexin V fluorescein isothiocyanate/propidium iodide (Annexin V‐FITC/PI) stain and fluorescence‐activated cell sorting (FACS). Small interfering RNA (siRNA) and the CRISPR‐Cas9 system were used to elucidate the underlying molecular mechanism of MLN4924‐induced HNSCC apoptosis.ResultsExpression levels of NAE1 and UBC12 were prominently higher in HNSCC tissues than that in normal tissues. Inactivation of the neddylation pathway significantly inhibited malignant phenotypes of HNSCC cells. Mechanistic studies revealed that MLN4924 induced the accumulation of CRL ligase substrate c‐Myc that transcriptionally activated pro‐apoptotic protein Noxa, which triggered apoptosis in HNSCC.ConclusionsThese findings determined the over‐expression levels of neddylation enzymes in HNSCC and revealed novel mechanisms underlying neddylation inhibition induced growth suppression in HNSCC cells, which provided preclinical evidence for further clinical evaluation of neddylation inhibitors (eg, MLN4924) for the treatment of HNSCC.

Patients\u2019 experience of recurrent/metastatic head and neck squamous cell carcinoma and their perspective on the EORTC QLQ-C30 and QLQ-H&N35 questionnaires: a qualitative study

BackgroundHead and neck squamous cell carcinoma (HNSCC) and its associated treatments may affect all aspects of patients’ health-related quality of life (HRQoL). Although the EORTC QLQ-H&N35 is regularly administered to patients with HNSCC, there is a paucity of studies re-assessing the conceptual relevance of this patient-reported outcome (PRO) measure from a patient perspective. Furthermore, the content validity of the EORTC QLQ-C30 has not been widely documented in patients with recurrent and/or metastatic HNSCC. The objectives of this study were to understand patients’ experiences of recurrent/metastatic HNSCC and its treatments, and to evaluate the conceptual relevance and acceptability of the EORTC QLQ-C30 and QLQ-H&N35 from a patient perspective for use in clinical trials.MethodsA literature review and clinician interviews were conducted to inform in-depth semi-structured telephone interviews with US patients who had received treatment for recurrent and/or metastatic HNSCC in the preceding 12 months. Interview transcripts were analysed thematically using ATLAS.ti v7; patient quotes were coded to identify concepts and themes to develop a conceptual model of HNSCC experience.ResultsFourteen patients were interviewed (71% male, aged 35–84 years). Patients reported few symptoms pre-diagnosis including neck lump/swelling (n = 7/14, 50%) and/or difficulty swallowing (n = 3/14, 21%). Treatments generally comprised surgery and chemotherapy and/or radiotherapy. A number of side effects from all treatments were reported. Numbness, difficulty speaking and pain were the most reported side effects of surgery (n = 4/8, 50%); weight loss and fatigue were the most reported side effects of chemotherapy and/or radiotherapy (n = 8/13, 61%). All side effects negatively impacted patients’ HRQoL. Patients generally found the QLQ-C30 and QLQ H&N35 content to be understandable and conceptually relevant; excessive mucous production and neuropathic symptoms were among the suggested additions.ConclusionsHNSCC and its diverse symptoms and treatments have a negative impact on many aspects of patients’ lives. A number of reported symptoms including difficulty speaking and swallowing, localised pain and fatigue may be important for treatment benefit evaluation in clinical trials from a patient perspective. The QLQ-C30 and QLQ-H&N35 are generally relevant and suitable for use in clinical trials. However, some items could be amended/added to ensure conceptual comprehensiveness of these measures.

A Review of Promising Natural Chemopreventive Agents for Head and Neck Cancer.

Head and neck squamous cell carcinoma (HNSCC) accounts for 300,000 deaths per year worldwide, and overall survival rates have shown little improvement over the past three decades. Current treatment methods including surgery, chemotherapy, and radiotherapy leave patients with secondary morbidities. Thus, treatment of HNSCC may benefit from exploration of natural compounds as chemopreventive agents. With excellent safety profiles, reduced toxicities, antioxidant properties, and general acceptance for use as dietary supplements, natural compounds are viewed as a desirable area of investigation for chemoprevention. Though most of the field is early in development, numerous studies display the potential utility of natural compounds against HNSCC. These compounds face additional challenges such as low bioavailability for systemic delivery, potential toxicities when consumed in pharmacologic doses, and acquired resistance. However, novel delivery vehicles and synthetic analogues have shown to overcome some of these challenges. This review covers 11 promising natural compounds in the chemoprevention of HNSCC including vitamin A, curcumin, isothiocyanate, green tea, luteolin, resveratrol, genistein, lycopene, bitter melon, withaferin A, and guggulsterone. The review discusses the therapeutic potential and associated challenges of these agents in the chemopreventive efforts against HNSCC. Cancer Prev Res; 11(8); 441-50. ©2018 AACR.

Abstract 1972: Targeting the IL-6 signaling pathway overcomes cetuximab resistance in head and neck squamous cell carcinoma

Abstract Head and neck squamous cell carcinoma (HNSCC), the sixth most common cancer worldwide, has a five-year survival rate of 50%. The last few decades have seen only marginal improvements to the survival rate due to high rates of resistance to currently available treatments. The only oncogene-targeted agent that has been FDA approved for the treatment of HNSCC is cetuximab, a monoclonal antibody directed against the epidermal growth factor receptor (EGFR). The response rate for single-agent cetuximab is below 20%, and both intrinsic and acquired resistance to cetuximab remain major obstacles in the treatment of HNSCC. Identification and targeting of mediators that confer cetuximab resistance are needed to improve patient outcomes in HNSCC and in other cancers in which cetuximab is employed. To address this unmet need, we generated cetuximab-resistant variants of the human HNSCC cell lines PE/CA-PJ49 and FaDu. Because secretion of interleukin 6 (IL-6) has been proposed as a mechanism of cetuximab resistance in HNSCC, we assessed IL-6 levels in these models of acquired resistance. IL6 mRNA expression was increased in PE/CA-PJ49 and FaDu cells that have acquired resistance to cetuximab compared to the parental cells from which they were derived. In addition, cetuximab-resistant PE/CA-PJ49 variants exhibited increased levels of glycoprotein 130 (gp130), a molecule that oligomerizes with IL-6R alpha (IL-6Rα) to form the IL-6 receptor. Since IL-6 is known to promote tumor cell proliferation and survival, we hypothesized that cetuximab-resistant PE/CA-PJ49 and FaDu variants would be sensitive to inhibition of components of the IL-6 signaling pathway. To test this hypothesis, we treated PE/CA-PJ49 parental cells and cetuximab-resistant variants with cetuximab and/or inhibitors of the IL-6 pathway. Colony formation assays revealed that cetuximab-treated parental cells formed fewer colonies than vehicle-treated cells, while cetuximab treatment did not affect the ability of cetuximab-resistant PE/CA-PJ49 variants to form colonies. In contrast, siRNA-mediated knockdown of IL6 or IL6R (encoding IL-6Rα) markedly reduced the colony-forming ability of the cetuximab-resistant variants, but not parental cells. These results suggest that targeted inhibition of IL-6 signaling inhibits proliferation and/or survival in cetuximab-resistant PE/CA-PJ49 cells. Ongoing studies are testing our hypothesis using small-molecule and antibody-based inhibitors of the IL-6 pathway in our cell line models and in cetuximab-resistant patient-derived xenografts. By conducting these experiments, we aim to assess the therapeutic potential of targeting the IL-6 pathway in cetuximab-resistant HNSCC. Citation Format: Rachel A. O'Keefe, Neil E. Bhola, David S. Lee, Daniel E. Johnson, Jennifer R. Grandis. Targeting the IL-6 signaling pathway overcomes cetuximab resistance in head and neck squamous cell carcinoma [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 1972.

Abstract 3626: A genome scale screen to identify regulators of PD-L1 in HNSCC

Abstract The immune checkpoint inhibitor Pembrolizumab was recently approved for the treatment of metastatic and recurrent HNSCC, but only 18% of patients in the initial trial responded to therapy, and it remains difficult to predict patients likely to experience benefit. We expect a broader understanding of checkpoint regulation in HNSCC to lead to improved response rates and identification of potential responders. To this end, we sought to identify targetable genetic factors modulating PD-L1, a molecule that serves to dampen the anti-tumor immune response. We developed a high throughput screen employing a Genome-scale CRISPR Knock-Out (GeCKO) library in HNSCC cell lines to identify targetable genes and pathways that may regulate PD-L1 expression. In UM-SCC-49, stable knockout pools with representation of approximately 300 gRNAs for each of over 20,000 target genes were expanded and serially sorted to create stable sub-populations with enhanced or diminished PD-L1 expression. These sub-populations were sequenced to identify gRNAs that increased or reduced PD-L1 expression, indicating that their genetic targets may function to regulate PD-L1. We observed 70% library coverage (44,900 gRNAs) in the initial population and 15% and 11% coverage in the sub-populations selected for high and low PD-L1 expression, respectively. We prioritized for validation 61 gRNAs that were enriched at least 2 fold in High or Low PDL1 pool over the control pool. Future experiments focus on validation of these targets in UM-SCC-49 and other cell lines. As we expand this screen and validate hits across multiple genetic backgrounds, we expect to reveal signaling networks promoting immune evasion and potential targets for combination immunotherapeutic protocols. Citation Format: Jacqueline Mann, Megan Ludwig, Aditi Kulkarni, Judy Kafelghazal, Julia Eisenberg, Rebecca Hoesli, Alexey Nesvizhskii, Chad Brenner. A genome scale screen to identify regulators of PD-L1 in HNSCC [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 3626.

Abstract CT170: A phase II, multicenter study to evaluate the efficacy and safety of autologous tumor infiltrating lymphocytes (LN-145) for the treatment of patients with recurrent and/or metastatic squamous cell carcinoma of the head and neck

Abstract Adoptive cell therapy (ACT) may be effective in treating immunogenic tumors with high mutational load, such as melanoma and virally-associated tumors, like cervical cancer, with several patients in studies performed at various institutions achieving durable, complete responses for years. Despite the heterogeneity of squamous cell carcinomas of the head and neck (HNSCC), most tumors are either virally-associated (e.g., HPV in oropharyngeal) or carry high mutational load (e.g., tobacco-related) providing an increased diversity of potential targets ideal for the polyclonal nature of ACT. Furthermore, outcomes for patients with recurrent and/or metastatic HNSCC remain poor. Therefore, a clear rationale exists for the potential application of ACT in patients with HNSCC. Clinical trial C-145-03 (NCT03083873) is a prospective phase II multicenter, open-label study evaluating the efficacy of a single autologous tumor infiltrating lymphocyte infusion (LN-145) followed by IL-2 after a non-myeloablative lymphodepletion (NMA-LD) regimen in patients with recurrent and/or metastatic HNSCC. Study-related therapy begins with resection of a tumor lesion that is then shipped to a central GMP manufacturing facility where TIL are extracted, expanded, packaged, and shipped for administration (LN-145). One week prior to LN-145 infusion, patients undergo NMA-LD consisting of cyclophosphamide (60 mg/kg) daily x 2 days followed by fludarabine (25 mg/m2) daily x 5 days. LN-145 is infused 24 hours after the last dose of fludarabine followed by up to 6 doses of IL-2 (600,000 IU/kg) every 8-12 hours. The primary efficacy endpoints are the objective response rate per RECIST v1.1 and the safety summarization of treatment-emergent adverse events (AEs) including serious AEs, AEs leading to discontinuation, and clinical laboratory tests. Secondary efficacy endpoints include CR, DOR, PFS, and OS. Patients must have been treated with at least one systemic chemotherapy or immunotherapy treatment for recurrent and/or metastatic HNSCC and, in addition to the tumor targeted for excision and TIL manufacture, must have an additional measurable lesion for assessment of response. Additional eligibility criteria include amongst others: adequate bone marrow, liver, pulmonary, cardiac, and renal function; ECOG performance status of 0 or 1. Systemic steroids greater than 10 mg/day prednisone equivalents are prohibited as are a history of serious immunotherapy-related adverse events. Citation Format: Rom Leidner, Ammar Sukari, Christine Chung, James Ohr, Missak Haigentz, Ezra Cohen, Robert Brown, Sam Suzuki, Igor Gorbatchevsky, Maria Fardis, Robert L. Ferris. A phase II, multicenter study to evaluate the efficacy and safety of autologous tumor infiltrating lymphocytes (LN-145) for the treatment of patients with recurrent and/or metastatic squamous cell carcinoma of the head and neck [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 CT170.

PO-490 Expression of cortactin and focal adhesion kinase in early stages of laryngeal tumorigenesis and clinical application for cancer risk-stratification

IntroductionCortactin (CTTN) and the Focal Adhesion Kinase (FAK) are two major candidate genes to respectively drive 11q13- and 8q24-associated aggressive behaviour in various cancers. Recent evidence uncovered their clinical relevance in early stages of tumorigenesis as promising biomarkers for cancer risk assessment.Material and methodsUsing a multicenter validation study CTTN and FAK expression was evaluated by immunohistochemistry in a cohort of 109 patients with laryngeal precancerous lesions, and correlated with clinicopathologic parameters and laryngeal cancer risk. The pathophysiological role of CTTN and FAK was further investigated using functional studies in cellular models.Results and discussionsIncreased CTTN and FAK expression was detected in 49 (41%) and 35 (32%) laryngeal dysplasias, respectively. Univariate Cox analysis showed that CTTN and FAK expression but not histological grading were significantly associated with both recurrence risk and laryngeal cancer risk. Patients carrying strong CTTN- or FAK-expressing lesions experienced the highest laryngeal cancer incidence (log-rank p<0.001). In multivariate stepwise analysis, FAK expression (HR=13.91, 95% CI 4.82–40.15; p<0.001) and alcohol consumption (HR=2.22, 95% CI 1.17–4.20; p=0.014) were significant independent predictors of laryngeal cancer development. Targeting FAK by either RNAi or pharmacological inhibitors effectively blocked cell growth, colony formation and invasion into 3D collagen matrices.ConclusionCTTN and FAK emerge as powerful predictors of laryngeal cancer risk beyond histological grading, thus encouraging their clinical application as complementary markers for risk-stratification. Furthermore, our findings unveil that pharmacological targeting of FAK could constitute a promising therapeutic strategy for HNSCC prevention and treatment.

Abstract LB-171: Potential chemopreventative treatments in Fanconi anemia related HNSCC

Abstract Fanconi anemia (FA) is a DNA repair mechanism genetic disorder which leads to bone marrow failure and cancer. Risk of solid tumor malignancy in FA patients occurs decades earlier and at a rate several hundred-fold higher than the general population, necessitating regular cancer surveillance. Development of an adjunct therapy or preventative survival-enhancing therapy with a minimal side effect profile will benefit this population in which chemotherapy and radiation are particularly damaging. To this end, we established a cell line from a post bone marrow transplant FA patient with a T2N2bM0 oral SCC grown as an adherent monolayer culture. We tested pioglitazone, metformin, and N1-hexyl-N5-benzyl-biguanide (HBB), emerging chemopreventative therapies for head and neck squamous carcinoma, for growth inhibition in head and neck cancer cell lines, including our FA SCC cell line. Additional cell lines include UMSCC 11A (laryngeal squamous cell carcinoma), CA 9-22 (oral squamous cell carcinoma), Beas2B (SV40 immortalized normal bronchial epithelium), and MSK Leuk1 (oral leukoplakia cell line). All cell lines are HPV negative. MTT assays determined cell viability after 1, 2, and 3 days of treatment. Pioglitazone at all concentrations (5, 10, 20, 40 μM) decreases cell proliferation compared to solvent controls (P&amp;lt;0.0001). Metformin (1 μM - 1 mM) unexpectedly increased cell proliferation or was otherwise equal in growth with the controls, and at higher doses (10 mM - 100 mM) metformin decreased cell proliferation or resulted in cytotoxicity (P&amp;lt;0.0001). Pioglitazone (10 μM) and metformin (above 1 mM) resulted in larger, cell line dependent, decreases in cell proliferation than either agent alone. In the FA cell line, pioglitazone and HBB resulted in increased efficacy. HBB treatment resulted in decreases in proliferation and percent live cells at 20 μM equivalent to the effect of 10 to 100 mM metformin. Combining HBB with 10 μM pioglitazone decreased cell proliferation and percent live cells over pioglitazone alone. HBB also caused S phase blockade in a pilot FA cell cycle experiment. Pioglitazone combined with HBB appears to be a promising chemopreventative adjunct treatment for HNSCC in FA patients. Further studies are underway to evaluate the relative contribution of HBB to decreased cell proliferation versus apoptotic mechanisms. Recent studies of HBB in breast cancer indicate cytochrome P450 arachidonic acid epoxygenase activity as an HBB target and suggest further exploration of this target in head and neck squamous cell carcinoma. Citation Format: Kimberly A. Miller, Beverly R. Wuertz, Anna M. Haynes, David A. Potter, Frank G. Ondrey. Potential chemopreventative treatments in Fanconi anemia related HNSCC [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 LB-171.

2192 The association of preoperative functional capacity and outcomes for head and neck cancer patients undergoing definitive surgical treatment

OBJECTIVES/SPECIFIC AIMS: To study the role functional capacity plays in surgical outcomes for head and neck cancers. METHODS/STUDY POPULATION: In this single-institution cohort study, we combined preoperative anesthesia assessment information with oncology registry data for newly-diagnosed patients with squamous cell carcinoma of the oral cavity, pharynx, and larynx (HNSCC) treated with definitive surgery at Siteman Cancer Center from 2012 to 2016. Patient-reported exercise capacity was assessed as metabolic equivalents. Metabolic equivalents&lt;4 was defined as poor functional capacity. The primary outcome measure was overall survival (OS). Kaplan-Meir survival analysis was used to compare the survival of patients with poor functional capacity (PFC) and patients with normal functional capacity (NFC). Cox proportional hazard regression was used to explore the independent prognostic role of functional capacity on overall survival after controlling for other factors. RESULTS/ANTICIPATED RESULTS: A total of 671 patients underwent surgical treatment for HNSCC. The average age was 62 years (range: 19–94 years). Majority of the patients were male (n=481; 72%), White race (n=589; 88%), and smokers (n=528; 79%). Of 671 patients, 22% (n=146) had PFC. Two-year OS rate in PFC patients was 70% compared with 85% in NFC patients (15% difference; 95% CI: 7%–23%). Unadjusted Cox proportional hazard analysis showed that PFC patients had 2.2 times higher risk of death (95% CI: 1.5–3.2) than NFC patients. After adjustment for age at surgery, BMI, preoperative weight loss, comorbidity score, tumor site, and TNM stage the magnitude of the association between functional capacity and OS decreased (aHR=1.3; 95% CI: 0.88–1.98). DISCUSSION/SIGNIFICANCE OF IMPACT: Poor functional capacity is associated with decreased overall survival, but the magnitude of the association, while clinically meaningful, decreases after controlling for other important patient and tumor factors. Nevertheless, we believe preoperative functional capacity status is an important patient factor to consider when discussing prognosis and attempting risk stratification. We also believe that functional capacity may be associated with 30-day unplanned readmissions and 90-day complications and are currently performing chart review to ascertain this information.

HTERT gene knockdown enhances response to radio- and chemotherapy in head and neck cancer cell lines through a DNA damage pathway modification

The aim of the study was to analyze the effect of hTERT gene knockdown in HNSCC cells by using novel in vitro models of head and neck cancer (HNSCC), as well as improving its personalized therapy. To obtain the most efficient knockdown siRNA, shRNA-bearing lentiviral vectors were used. The efficiency of hTERT silencing was verified with qPCR, Western blot, and immunofluorescence staining. Subsequently, the type of cell death and DNA repair mechanism induction after hTERT knockdown was assessed with the same methods, followed by flow cytometry. The effect of a combined treatment with hTERT gene knockdown on Double-Strand Breaks levels was also evaluated by flow cytometry. Results showed that the designed siRNAs and shRNAs were effective in hTERT knockdown in HNSCC cells. Depending on a cell line, hTERT knockdown led to a cell cycle arrest either in phase G1 or phase S/G2. Induction of apoptosis after hTERT downregulation with siRNA was observed. Additionally, hTERT targeting with lentiviruses, followed by cytostatics administration, led to induction of apoptosis. Interestingly, an increase in Double-Strand Breaks accompanied by activation of the main DNA repair mechanism, NER, was also observed. Altogether, we conclude that hTERT knockdown significantly contributes to the efficacy of HNSCC treatment.

Abstract 44: Synergistic antitumor activity of olaparib combined with AZD1775 in head and neck squamous cell carcinoma

Abstract Purpose: Although the treatment of locally advanced head and neck cancer (HNSCC) has evolved in recent years, the cure rate for patients with aggressive tumors and high-risk of failure remains poor due to resistance to standard chemoradiotherapy. Since TP53, the gene that encodes the protein p53, is by far the most commonly mutated gene found in HNSCC, and is in turn associated with treatment failure and poor survival outcomes. Although HPV+ HNSCC has a good prognosis, a group of HPV+ patients' tumors remain refractory to conventional treatments and unusual patterns of recurrence and metastasis are emerging. Therefore, more effective therapies are urgently needed. Our recent work has demonstrated that increased DNA damage response associated induction of replication stress and impaired Rad51-mediated homologous recombination (HR) sensitizes HNSCC tumor cells to WEE1 kinase inhibition with AZD1775. While the use of single molecularly targeted agents has demonstrated limited therapeutic benefits, there is growing interest in targeting multiple pathways or multiple steps within a single pathway to achieve even more effective cancer treatments. In this context, combinations of agents that target the DNA damage and HR responses are an exciting new area of investigation. Given the synthetic lethality of PARP inhibitor, olaparib in BRCA1/2-mutant or HR repair-defective cancers, combining PARP inhibitors with agents that inhibit HR is another interesting area of intensive investigation. Therefore, we hypothesize that simultaneous targeting of PARP and WEE1 will decrease the clonogenic survival in vitro and inhibit tumor growth and prolong animal survival in treated mice in an in vivo orthotopic nude mouse model of oral cancer. Experimental Design: Clonogenic survival assays and an orthotopic mouse model of oral cancer were used to examine the in vitro and in vivo sensitivity of high-risk mutant p53 and HPV+ HNSCC cell lines to olaparib in combination with AZD1775 respectively. Cell cycle analysis, DNA damage (γH2AX), HR, live cell imaging, and apoptosis were performed to dissect molecular mechanisms. Results: We found that olaparib synergized with AZD1775 in vitro to inhibit growth of HNSCC cells irrespective of p53 status or HPV positivity (median CI = 0.07-0.19). Mechanistically, these drugs interact synergistically to induce DNA damage, replication stress, and impaired Rad51-mediated HR through activation of CDK1 and increased Chk1 phosphorylation preceded by enhanced activity of ATR, culminating in aberrant mitosis associated with apoptotic cell death. Assessment of olaparib and AZD1775 combination therapy in vivo in an orthotopic mouse model of oral cancer is currently underway. Conclusions: Olaparib synergizes with AZD1775 in HNSCC cells in vitro through apoptosis. Our data provide an early-preclinical evidence for the rational combination of WEE1 and PARP inhibitors in the treatment of advanced HNSCC. Citation Format: Hideaki Takahashi, Antje Lindemann, Ameeta A. Patel, Weiwei Liu, Noriaki Tanaka, Lin Tang, Mei Zhao, Walter N. Hittelman, Jeffrey N. Myers, Abdullah A. Osman. Synergistic antitumor activity of olaparib combined with AZD1775 in head and neck squamous cell carcinoma [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 44.

Abstract IA08: Personalizing HNSCC treatment based on TP53 mutational status

Abstract Head and neck squamous cell carcinoma (HNSCC) is a leading cause of cancer deaths worldwide, and resistance to standard treatments with radiation/chemotherapy accounts for the majority of HNSCC deaths. Mutation of TP53 occurs in 60-85% of non-HPV-associated HNSCC cases, and some mutant p53s display oncogenic properties, termed “gain-of-function (GOF),” which are independent of wild-type (WT) p53 functions. These GOF p53 mutant proteins can enhance cell transformation, increase tumor formation, and confer cellular resistance to chemo- and/or radio-therapy. Therefore, we are interested in the development of innovative therapeutic approaches to overcome this resistance. Pre-clinical studies both in vitro and in vivo have shown the promise of some novel targeted agents including Chk-1, Wee-1, and PARP inhibitors used alone or in combination with HDAC inhibition. In addition, COTI-2 is a novel drug with striking single-agent and radio-/chemo-sensitizing activity in pre-clinical models of HNSCC bearing TP53 mutations. Our preliminary data demonstrate that COTI-2 markedly decreases cell growth in vitro and induces p21, noxa, and puma expression in HNSCC cells. COTI-2 also negatively modulates the PI3K/AKT/mTOR survival pathway, which is constitutively activated in many types of cancer including HNSCC. In vitro studies in our laboratory have shown that COTI-2 has a nanomolar (IC50: 1.3-13.2 nm) activity in nine human HNSCC cell lines. Additionally, GOF mutant p53-bearing HNSCC cells are significantly delayed in outgrowth in an orthotopic nude mouse model of tongue cancer when treated with COTI-2. Current clinical studies are evaluating the safety and efficacy of the combination of COTI-2 alone and in combination with cisplatin in phase I/II studies of patients with refractory HNSCC and whether response is associated with TP53 mutational status of the tumor determined from sequencing of circulating cell free DNA. If this combination is found to safely provide sensitization of HNSCC to cisplatin-based chemotherapy in the majority of human patients who have TP 53 mutant tumors, this should make a major impact on the survival of individuals suffering from HNSCC, and potentially other p53 driven epithelial malignancies. Citation Format: Jeffrey Myers. Personalizing HNSCC treatment based on TP53 mutational 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 IA08.

DNA intercalators based on (1,10-phenanthrolin-2-yl)isoxazolidin-5-yl core with better growth inhibition and selectivity than cisplatin upon head and neck squamous cells carcinoma

((3RS,5SR)- and ((3RS,5RS)-2-(2-methoxybenzyl)-3-(1,10-phenanthrolin-2-yl)isoxazolidin-5-yl)methanol have been synthesized, according to 1,3-dipolar cycloaddition methodology, as DNA intercalating agents and evaluated for their anticancer activity against human cervical carcinoma HeLa and head and neck squamous cells carcinoma cell lines. The synthesized compounds exhibited good cytotoxic activity with IC50 better than cisplatin, used as the main and effective treatment for HNSCC, and a 24.3–72.0-fold selectivity respect to the 184B5 non-cancerous immortalized breast epithelial cell lines. Unwinding assay, circular dichroism data, and Uv-vis melting experiments confirmed that these compounds act as DNA intercalators with a binding constant in the order of 104 M−1. Docking studies showed that both compounds can interact as intercalating agent with both poly-d(AT)2 and poly-d(GC)2, preferring an entrance by the minor groove of the poly-d(AT)2.

A chemical genetics approach to identify therapeutic vulnerabilities in Gefitinib resistant, EGFR T790M

Background: The first generation EGFR inhibitor, Gefitinib, has been approved for the treatment of non-small cell lung carcinoma (NSCLC) with activating EGFR mutations. However resistance to Gefitinib quickly develops and is mainly driven by the EGFR T790M mutation in the kinase domain. On the other hand, while EGFR was found to be overexpressed in the majority of HNSCC, clinical trials on the use of Gefitinib for treatment of HNSCC has been disappointing thus far. This is mainly due to the lack of activating mutations in the EGFR kinase domain of HNSCC patients, as well as treatment resistance. In this study, we sought to identify alternative therapeutic vulnerabilities in Gefitinib resistant HNSCC that are negative for EGFR T790M mutation. Methods: We developed a pair of isogenic Gefitinib sensitive (TM17S) and resistant (TM17R) cell lines through stepwise chronic treatment of the TM17S cell line with Gefitinib. The TM17S and TM17R cell lines were characterized for their mutational profile using POLARIS Xplora cancer panel and through gene expression studies. High-throughput chemical screen with two annotated compound libraries was carried out to identify alternative therapeutic vulnerabilities in the TM17R cell line. Results: While the TM17R cell line was derived to be resistant to Gefitinib, a first generation EGFR tyrosine kinase inhibitor (TKI), TM17R cells were found to be resistant to all 3 generations of EGFR TKIs. Comparison of the mutational profiles of TM17S and TM17R cells did not reveal obvious mutational drivers of resistance. Strikingly the EGFR T790M mutation was not detected in both TM17S and TM17R cells. We also observed a down regulation of EGFR gene and protein expression. All these could account for the resistance of TM17R cells to all 3 generations of EGFR TKIs, and suggests an alternative mechanism of resistance. Results from the high-throughput chemical screen revealed an alternative therapeutic vulnerability of TM17R cells to Aurora kinase inhibitors. This therapeutic vulnerability was also observed in 2 other Gefitinib sensitive and resistant pairs of HNSCC cell lines. Conclusions: In EGFR T790M negative cohorts, a possible mechanism of resistance to EGFR TKIs would involve the downregulation of EGFR. While this could lead to resistance to all 3 generations of EGFR TKIs, resistant cells developed sensitivity to Aurora kinase inhibitors instead. Comparison of the gene expression profiles of these cell lines with similar therapeutic vulnerabilities could potentially identify biomarkers of Gefitinib resistance in HNSCC. Legal entity responsible for the study: NA Funding: The Agency for Science, Technology and Research (A*STAR) of Singapore Disclosure: All authors have declared no conflicts of interest.

IN VIVO AND IN VITRO EFFECTS OF CURCUMIN ON HEAD AND NECK CARCINOMA: A SYSTEMATIC REVIEW

Background Head and neck squamous cell carcinoma (HNSCC) contributes globally to a great number of deaths and morbidity, in spite of new therapeutic strategies. There is a great need of new drugs that are significantly effective and less deleterious to the patients' general health. In this sense, phytotherapy is a tendency, with results pointing to its use as a chemo-preventive and adjuvant therapy. Therefore, the objective of this systematic review was to investigate the effects of curcumin on proliferation and survival of HNSCC. Materials and methods The search was conducted on six databases: Cochrane, LILACS, EMBASE, MEDLINE, PubMed, and Web of Science. In vitro and in vivo studies that evaluated the effects of curcumin on cell viability, tumor growth, cell cycle and/or cell death pattern in HNSCC cell lines or animal models were selected. Results Of the 525 initially gathered studies, 30 met the inclusion criteria. These studies demonstrated that curcumin induces cytotoxicity, apoptosis (via intrinsic pathway), and cell cycle arrest in G2/M phase in HSNCC cell lines. It also reduces tumor measurements in animal models. These events were mostly studied through MTT assay, flow cytometry, and cell cycle- and apoptosis-related proteins expression. Conclusion This systematic review demonstrated that curcumin is effective on HNSCC cell proliferation and survival, reinforcing the currently available evidence that curcumin could be an adjuvant drug in HNSCC treatment.

Abstract 4108: Targeting IL-6 signaling overcomes cetuximab resistance in head and neck squamous cell carcinoma

Abstract Head and neck squamous cell carcinoma (HNSCC), the sixth most common cancer worldwide, has a five-year survival rate of only 50%. The first targeted agent FDA approved for treatment of HNSCC was cetuximab, a monoclonal antibody targeting the epidermal growth factor receptor (EGFR). Despite EGFR overexpression in up to 90% of HNSCC tumors and ample evidence supporting EGFR as a therapeutic target in HNSCC, the response rate for single-agent cetuximab is below 20%, and resistance to cetuximab-containing therapy remains a major obstacle in the effective treatment of HNSCC. Identification and targeting of mediators of cetuximab resistance is needed to improve patient outcomes. Secretion of the cytokine interleukin 6 (IL-6) has been proposed as a mechanism of resistance to cetuximab in HNSCC, and inhibition of signal transducer and activator of transcription 3 (STAT3), a downstream mediator of IL-6 signaling, has been shown to overcome cetuximab resistance in preclinical HNSCC models. Thus, we hypothesize that IL-6 signaling mediates resistance to cetuximab and that co-treatment with agents targeting the IL-6 pathway will enhance the therapeutic efficacy of cetuximab in HNSCC. We have generated cetuximab-resistant variants of the cetuximab-sensitive HNSCC cell lines Cal33 and PE/CA-PJ49. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis indicated that IL6 expression is increased 3.5- to 8-fold in cetuximab-resistant variants compared to the parental cells from which they were derived. In addition, a colony formation assay revealed that, as expected, cetuximab-treated parental cells formed fewer colonies than vehicle-treated cells, while cetuximab treatment had no effect on the ability of cetuximab-resistant PE/CA-PJ49 variants to form colonies. In contrast, treatment with tocilizumab, an IL-6 receptor-neutralizing monoclonal antibody, markedly reduced the colony-forming ability of the cetuximab-resistant variants, but not parental cells. These results suggest that targeted inhibition of IL-6 signaling may effectively inhibit proliferation in cetuximab-refractory HNSCC cells. Citation Format: Rachel A. O'Keefe, Neil Bhola, Toni M. Brand, Yan Zeng, Daniel E. Johnson, Jennifer R. Grandis. Targeting IL-6 signaling overcomes cetuximab resistance in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4108. doi:10.1158/1538-7445.AM2017-4108

Abstract 1667: Identifying intrinsic regulators of PD-L1 expression in cancer cells: A genome-scale CRISPR knock-out approach

Abstract Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most common cancer in the United States and affects 600,000 people each year worldwide. With a five-year survival rate of only 50% and a recent rise in HPV-associated HNSCCs, improved treatment protocols are urgently needed. Evidence of immunosuppression is often reported in HNSCC, making immunotherapy an attractive strategy for the management of this disease. The immune checkpoint inhibitor Pembrolizumab was recently approved for the treatment of metastatic and recurrent HNSCC, but only 18% of initial participants in a trial of Pembrolizumab responded, and it remains difficult to predict patients likely to experience benefit. To increase the number of patients that respond to immune checkpoint inhibition, we sought to identify targetable genetic factors modulating PD-L1, a molecule that serves to dampen the anti-tumor immune response. We show that several HNSCC models exhibit mild (2-4 fold) induction of PD-L1 expression following treatment with interferon-γ (adaptive expression), and recent reports suggest that common oncogenic pathways, including the EGFR and PI3K pathways, may provide a novel strategy to regulate PD-L1 expression. To this end, we developed a high throughput screen to identify targetable pathways that may be used to regulate PD-L1 expression in patients with specific genetic lesions. We have employed a Genome-scale CRISPR Knock-Out (GeCKO) screening technique in HNSCC cell lines and selected for genetic knockouts exhibiting altered PD-L1 expression. Stable knockout pools with representation of approximately 300 gRNAs per target gene (&amp;gt;20,000 target genes in the library) were expanded and serially sorted to create stable sub-populations with enhanced PD-L1 expression. These sub-populations were sequenced to identify gRNAs whose knockout causes a change in PD-L1 expression (e.g. genes that repress or enhance PD-L1 expression). We expect that these large-scale screens, when performed over multiple HNSCC cell lines with diverse genetic lesions, will identify patterns of targetable regulators that may ultimately be manipulated in combination with PD-1/PD-L1 inhibitors. Citation Format: Jacqueline E. Mann, Megan L. Ludwig, Rebecca C. Hoesli, Aditi Kulkarni, Simmy Patel, Judy Kafelghazal, Alexey Nesvizhskii, J Chad Brenner. Identifying intrinsic regulators of PD-L1 expression in cancer cells: A genome-scale CRISPR knock-out approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1667. doi:10.1158/1538-7445.AM2017-1667

Abstract 95: Targeting BRD4 overcomes cetuximab resistance in HNSCC

Abstract Background: Nearly 600,000 people are diagnosed with head and neck cancer worldwide and 60% succumb to the disease within 5 years. The epidermal growth factor receptor (EGFR) is a major driver of HNSCC and in 2006 the EGFR monoclonal antibody cetuximab was FDA-approved for HNSCC treatment. However, cetuximab has not conferred significant long-term benefit compared to chemoradiation in patients with poorly differentiated recurrent-metastatic HNSCC. We and others have demonstrated that cetuximab treatment activates alternative receptor tyrosine kinases (RTKs) including Fibroblast Growth Factor (FGF) receptors, Met and Axl in HNSCC. To circumvent administration of multiple RTK inhibitors in combination with cetuximab, we sought to identify a common molecular target that regulates the expression of these RTKs. Bromodomain-containing protein-4 (BRD4) has been shown to regulate the transcription of RTKs in breast cancer models. Hypothesis: We hypothesized that targeting BRD4 will overcome cetuximab resistance by depleting the expression of alternative RTKs in HNSCC. We further hypothesize that genetic and pharmacological targeting of BRD4 will synergize with cetuximab. Results: Using a phospho-RTK array, 72-hour cetuximab treatment increased or sustained phosphorylated levels of EGFR, HER2, HER3, MET, and AXL in several HNSCC cell lines. Treatment with the BRD4 inhibitor JQ1 abrogated both phosphorylated and total RTK abundance in the presence of cetuximab. Cetuximab and JQ1 robustly decreased phosphorylated Src and induced the senescence marker p21. Phenotypically, cetuximab and JQ1 significantly decreased survival and increased apoptosis in 6 HNSCC cell line models, while the normal oral keratinocyte cell line NOKsi had approximately 10-fold higher IC50s for the BRD4 inhibitors, JQ1 and I-BET762, relative to HNSCC cell lines. Importantly, two HNSCC models of acquired cetuximab resistance exhibited robust sensitivity to pharmacological (JQ1, IBET-762) and genetic (RNAi) BRD4 targeting strategies. Moreover, exogneous overexpression of different RTKs (HER3, ALK, and ROR1) resulted in cetuximab resistance that was reversed upon BRD4 targeting (RNAi and JQ1). Combination of cetuximab and JQ1 in co-culture experiments with T cells decreased the CD4+/CD25+ Treg population and PD-L1 expression on HNSCC cell lines. Further, preliminary findings indicate that JQ1 treatment prevents outgrowth of cetuximab-treated HNSCC patient-derived xenograft models. Conclusion: Our findings indicate that targeting BRD4 decreases the activation and expression of multiple RTKs that mediate resistance to the FDA-approved EGFR inhibitor cetuximab. Furthermore, BRD4 abrogates expression of immunosuppressive markers, making it a promising tumor intrinsic and extrinsic therapeutic strategy for HNSCC. Citation Format: Toni Brand, Yan Zeng, Brandon Leonard, Rachel O' Keefe, Hua Li, Daniel Johnson, Jennifer Grandis, Neil E. Bhola. Targeting BRD4 overcomes cetuximab resistance in HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 95. doi:10.1158/1538-7445.AM2017-95

EGFR/CD16A tetravalent bispecific antibody AFM24 to engage NK-cells to kill EGFR expressing tumor cells and safety results in cynomolgus monkey studies.

e14001 Background: The epidermal growth factor receptor is a target for the treatment of solid tumors, like CRC, HNSCC and NSCLC. Therapeutic success of EGFR-targeting agents depends on mutations defining resistance to these agents, thus an effective EGFR-targeting therapy overcoming resistance would offer an attractive new treatment option. Natural killer (NK-) cells play a central role in the innate immune system and can destroy cancer cells. NK-cell-engaging bispecific antibodies may offer a safe and effective therapy to target EGFR-expressing tumor cells irrespective of their mutational status. Methods: AFM24 was generated using proprietary human anti-EGFR and anti-CD16A variable domains and characterized for binding, stability, manufacturability, efficacy and safety in biophysical and functional assays in vitro and in vivo. Assays for binding to target/effector cells and cytotoxicity were performed in the presence of physiologically relevant IgG levels using EGFR+ tumor cell lines with and without RAS. The safety profile of AFM24 was investigated in vitro for off-target activity and in vivo in cynomolgus monkeys. Results: AFM24 showed excellent biophysical properties and picomolar EC50 values in cytotoxicity assays. It was more potent than cetuximab, or an antibody with cetuximab-derived anti-EGFR variable domain. Importantly, AFM24 was less prone to IgG interference and eliminated cell lines harboring negative predictive biomarkers in in vitro cytotoxicity assays, even in the presence of relevant serum IgG levels. AFM24 demonstrated in vivo efficacy in a humanized hu-NOG mouse model. Furthermore, AFM24 exhibited a favorable safety profile in cynomolgus monkeys. It was well tolerated up to 94 mg/kg in a single dose, maximum tolerated dose study (MTD) when administered as a 2 hour infusion. Conclusions: Our data suggest that AFM24 is a highly potent and potentially safer drug candidate suitable for the treatment of EGFR-expressing cancers with the potential to overcome resistance to other EGFR-targeting agents and to avoid toxic side effects seen with other anti-EGFR therapeutics.