Search

Background This was a retrospective study of patients with human papillomavirus (HPV)-associated oropharyngeal cancer treated with concurrent systemic therapy and radiation. Methods Data were extracted through chart review, and statistical analyses included frequency tabulation, chi-square, and Kaplan–Meier tests. Results Three hundred thirty-nine patients were analyzed; 166 received neoadjuvant chemotherapy. One hundred thirty-six patients were treated with cisplatin, 123 with cetuximab, and 59 with carboplatin. The 2-, 3-, and 5-year actuarial overall survival rates were 92%, 88%, and 78%, respectively. There were no significant differences in survival or disease control when analyzed by systemic agent. Platin-treated patients had greater hematologic toxicity, and required more intravenous hydration. The incidence of confluent mucositis was highest among patients treated with cetuximab. Conclusion Platin and cetuximab seem to have similar efficacy when delivered concurrently with radiation in our retrospective population study. Although platin did cause greater hematologic toxicity, radiation-specific side effects seemed relatively comparable. © 2015 Wiley Periodicals, Inc. Head Neck, 2015

Purpose: Although the majority of patients with HPV+ oropharyngeal cancers have a favorable prognosis, there are some patients with tumors that are resistant to aggressive chemoradiotherapy with unusual patterns of locoregional and systemic recurrences. Therefore, more effective therapies are needed. In this study, we investigated the chemosensitizing efficacy of the selective Wee-1 kinase inhibitor, AZD-1775, in HPV+ head and neck squamous cell carcinoma (HNSCC). Experimental Design: Clonogenic survival assays and an orthotopic mouse model of HPV+ oral cancer were used to examine the in vitro and in vivo sensitivity of HPV+ HNSCC cell lines to AZD-1775 in combination with cisplatin, respectively. Cell-cycle analysis, DNA damage (γH2AX), homologous recombination (HR), and apoptosis were examined to dissect molecular mechanisms. Results: We found that AZD-1775 displays single-agent activity and enhances the response of HPV+ HNSCC cells to cisplatin both in vitro and in vivo. The sensitivity of the HPV+ HNSCC cells to AZD-1775 alone or in combination with cisplatin was associated with G2 checkpoint abrogation, persistent DNA damage, and apoptosis induction. This finding of AZD-1775 increasing the sensitivity of HPV+ HNSCC cells to cisplatin through apoptosis was not seen previously in the HPV− HNSCC cancer cells and is accompanied by a decreased expression of the antiapoptotic proteins, MCl-1and XIAP, which appear to be cleaved following AZD-1775 treatment. Conclusions: AZD-1775 selectively sensitizes HPV+ HNSCC cells and orthotopic oral xenografts to cisplatin through apoptosis and support the clinical investigation of AZD-1775 in combination with cisplatin particularly in patients with advanced and recurrent metastatic HPV+ HNSCC tumors. Clin Cancer Res; 21(21); 4831–44. ©2015 AACR.

Improved therapies are greatly needed for non-small cell lung cancer (NSCLC) that does not harbor targetable kinase mutations or translocations. We previously demonstrated that NSCLC cells that harbor kinase-inactivating BRAF mutations (KIBRAF) undergo senescence when treated with the multitargeted kinase inhibitor dasatinib. Similarly, treatment with dasatinib resulted in a profound and durable response in a patient with KIBRAF NSCLC. However, no canonical pathways explain dasatinib-induced senescence in KIBRAF NSCLC. To investigate the underlying mechanism, we used 2 approaches: gene expression and reverse phase protein arrays. Both approaches showed that DNA repair pathways were differentially modulated between KIBRAF NSCLC cells and those with wild-type (WT) BRAF. Consistent with these findings, dasatinib induced DNA damage and activated DNA repair pathways leading to senescence only in the KIBRAF cells. Moreover, dasatinib-induced senescence was dependent on Chk1 and p21, proteins known to mediate DNA damage-induced senescence. Dasatinib also led to a marked decrease in TAZ but not YAP protein levels. Overexpression of TAZ inhibited dasatinib-induced senescence. To investigate other vulnerabilities in KIBRAF NSCLC cells, we compared the sensitivity of these cells with that of WTBRAF NSCLC cells to 79 drugs and identified a pattern of sensitivity to EGFR and MEK inhibitors in the KIBRAF cells. Clinically approved EGFR and MEK inhibitors, which are better tolerated than dasatinib, could be used to treat KIBRAF NSCLC. Our novel finding that dasatinib induced DNA damage and subsequently activated DNA repair pathways leading to senescence in KIBRAF NSCLC cells represents a unique vulnerability with potential clinical applications.

Purpose: We determined the maximum tolerated dose (MTD), safety, pharmacokinetics, pharmacodynamics, and preliminary activity of OSI-906, a potent, oral, dual inhibitor of insulin-like growth factor-1 receptor (IGF1R) and insulin receptor (IR), in patients with advanced solid tumors. Experimental Design: This was a multicenter, open-label, dose escalation phase I study evaluating three intermittent dosing schedules of once-daily OSI-906 [schedule (S) 1, days 1–3 every 14 days; S2, days 1–5 every 14 days; S3, days 1–7 every 14 days]. A fed-fasting expansion cohort was included in the study. Results: Seventy-nine patients were enrolled: 62 in S1, 4 in S2, and 13 in S3. S2 was discontinued. Dose-limiting toxicity comprised grade 3–4 hyperglycemia, vomiting, fatigue, and prolonged QTc interval. The MTD and recommended phase II dose of OSI-906 was 600 mg for both S1 and S3 schedules. Other common adverse events were grade 1–2 nausea, vomiting, fatigue, and diarrhea. The pharmacokinetics of OSI-906 was dose linear, and the terminal half-life ranged between 2 and 6 hours. High-fat meals had a moderate effect on the pharmacokinetics of OSI-906. At the MTD, inhibition of IGF1R and IR was observed in peripheral blood mononuclear cells. An increase in plasma IGF1 concentrations, an indirect measure of IGF1R signaling inhibition, was seen at doses ≥ 450 mg. Two patients with adrenocortical carcinoma achieved partial responses. Conclusion: The MTD of 600 mg was well tolerated and associated with preliminary antitumor activity. These data support further evaluation of OSI-906 in solid tumors. Clin Cancer Res; 21(4); 693–700. ©2014 AACR. See related commentary by Yee, p. 667

Cutaneous squamous cell carcinoma (CSCC) is a very common malignancy in which most patients present with localized disease. Recurrent and metastatic disease is rare, and there is no standard therapy. These tumors frequently overexpress the epidermal growth factor receptor (EGFR). We conducted a phase 2 trial to determine the response rate to therapy with erlotinib, an EGFR tyrosine kinase inhibitor, in patients with locoregionally recurrent or metastatic CSCC that was not amenable to curative treatment (NCT01198028).Eligible patients had CSCC not amenable to curative intent therapy. Patients who had previously received anti-EGFR targeted therapy were excluded. All patients received oral therapy with erlotinib 150 mg daily. Response was assessed every 8 weeks, and treatment continued until progression, unacceptable toxicity, or withdrawal of consent. The primary endpoint was overall response rate according to RECIST 1.1 criteria.A total of 39 patients received treatment during the trial; 29 of these patients were evaluable for response. The overall response rate was 10% (3/29); all responses were partial responses. The disease control rate (partial response + stable disease) was 72% (21/29). The median progression-free survival was 4.7 months (95% confidence interval, 3.5-6.2 months); the median overall survival was 13 months (95% confidence interval, 8.4-20.5 months). No unexpected toxicities were seen.Erlotinib therapy was feasible for most patients with incurable CSCC and was associated with expected toxicities. However, only a modest response rate of 10% was observed. Further study of EGFR tyrosine kinase inhibitors in this patient population is not warranted. Cancer 2018;124:2169-73. © 2018 American Cancer Society.

There are few published studies to guide the treatment of carcinoma metastatic to the neck from an unknown primary (CUP). In this regard, the objective of the current study was to share the authors' current experience treating patients with CUP using intensity-modulated radiation therapy (IMRT), which principally targeted both sides of the neck, the nasopharynx, and the oropharynx.This was a retrospective study in which an institutional database search was conducted to identify patients with CUP who received IMRT. Data analysis included frequency tabulation, survival analysis, and multivariable analysis.Two-hundred sixty patients met inclusion criteria. The most common lymph node category was N2b (54%). IMRT volumes included the entire pharyngolaryngeal mucosa in 78 patients, the nasopharynx and oropharynx in 167 patients, and treatment limited to the involved neck in 11 patients. Eighty-four patients underwent neck dissections. The 5-year overall survival, regional control, and distant metastases-free survival rates were 84%, 91%, and 94%, respectively. Over 40% of patients had gastrostomy tubes during therapy, and 7% patients were diagnosed with chronic radiation-associated dysphagia. Higher lymph node burden was associated with worse disease-related outcomes, and in subgroup analysis, patients with human papillomavirus-associated disease had better outcomes. No therapeutic modality was statistically associated with either disease-related outcomes or toxicity.Comprehensive IMRT with treatment to both sides of the neck and to the oropharyngeal and nasopharyngeal mucosa results in high rates of disease control and survival. The investigators were unable to demonstrate that treatment intensification with chemotherapy or surgery added benefit or excessive toxicity. Cancer 2018;124:1415-27. © 2018 American Cancer Society.

Although treatment paradigms have not changed significantly, radiotherapy, surgery, and imaging techniques have improved, leading us to investigate oncologic and survival outcomes for oral cavity squamous cell cancer (OCSCC) patients treated with surgery followed by postoperative IMRT.Records of patients with pathological diagnosis of OCSCC treated between 2000 and 2012 were retrospectively reviewed. Patients' demographic, disease, and treatment criteria were extracted. Kaplan-Meier method was used to calculate survival curves.Two hundred eighty-nine patients were analyzed. Median follow-up was 35months. Two hundred sixty-eight had neck dissections (93%), of which 66% had nodal involvement, and 51% of those positive dissections had extracapsular extension. Forty patients received induction chemotherapy and 107 received concurrent chemotherapy. Median dose to high risk clinical target volume was 60Gy/30 fractions. The 5-year locoregional control and overall survival rates were 76% and 57%, respectively. Tumors with1.5cm depth of invasion had significantly higher risk of local failure compared with ≤1.5cm (p0.001). In multivariate analysis, positive and no neck dissection (p=0.01), positive lymphovascular invasion (p=0.006) and1.5cm depth of invasion (p=0.003) were independent predictors of poorer survival.Disease outcomes were consistent with historical data and did not appear compromised by the use of IMRT.

BACKGROUND. EGFR and Src family kinases are upregulated in head and neck squamous cell carcinoma (HNSCC). EGFR interacts with Src to activate STAT3 signaling, and dual EGFR-Src targeting is synergistic in HNSCC preclinical models. pSrc overexpression predicted resistance to the EGFR inhibitor, erlotinib, in a prior window trial. We conducted a 4-arm window trial to identify biomarkers associated with response to EGFR and/or Src inhibition. METHODS. Patients with operable stage II-IVa HNSCC were randomized to 7-21 days of neoadjuvant erlotinib, the Src inhibitor dasatinib, the combination of both, or placebo. Paired tumor specimens were collected before and after treatment. Pharmacodynamic expression of EGFR and Src pathway components was evaluated by IHC of tissue microarrays and reverse-phase protein array of tissue lysates. Candidate biomarkers were assessed for correlation with change in tumor size. RESULTS. From April 2009 to December 2012, 58 patients were randomized and 55 were treated. There was a significant decrease in tumor size in both erlotinib arms (P = 0.0014); however, no effect was seen with dasatinib alone (P = 0.24). High baseline pMAPK expression was associated with response to erlotinib (P = 0.03). High baseline pSTAT3 was associated with resistance to dasatinib (P = 0.099). CONCLUSIONS. Brief exposure to erlotinib significantly decreased tumor size in operable HNSCC, with no additive effect from dasatinib. Baseline pMAPK expression warrants further study as a response biomarker for anti-EGFR therapy. Basal expression of pSTAT3 may be independent of Src, explain therapeutic resistance, and preclude development of dasatinib in biomarker-unselected cohorts. TRIAL REGISTRATION. NCT00779389. FUNDING. National Cancer Institute, American Cancer Society, Pennsylvania Department of Health, V Foundation for Cancer Research, Bristol-Myers Squibb, and Astellas Pharma.

The genomic alterations identified in head and neck squamous cell carcinoma (HNSCC) tumors have not resulted in any changes in clinical care, making the development of biomarker-driven targeted therapy for HNSCC a major translational gap in knowledge. To fill this gap, we used 59 molecularly characterized HNSCC cell lines and found that mutations of AJUBA, SMAD4 and RAS predicted sensitivity and resistance to treatment with inhibitors of polo-like kinase 1 (PLK1), checkpoint kinases 1 and 2, and WEE1. Inhibition or knockdown of PLK1 led to cell-cycle arrest at the G2/M transition and apoptosis in sensitive cell lines and decreased tumor growth in an orthotopic AJUBA-mutant HNSCC mouse model. AJUBA protein expression was undetectable in most AJUBA-mutant HNSCC cell lines, and total PLK1 and Bora protein expression were decreased. Exogenous expression of wild-type AJUBA in an AJUBA-mutant cell line partially rescued the phenotype of PLK1 inhibitor-induced apoptosis and decreased PLK1 substrate inhibition, suggesting a threshold effect in which higher drug doses are required to affect PLK1 substrate inhibition. PLK1 inhibition was an effective therapy for HNSCC in vitro and in vivo. However, biomarkers to guide such therapy are lacking. We identified AJUBA, SMAD4 and RAS mutations as potential candidate biomarkers of response of HNSCC to treatment with these mitotic inhibitors.

Introduction Dysregulation of cell cycle progression has an established link to neoplasia and cancer progression. Components of the cyclin D-CDK4/6-INK4-Rb pathway are frequently altered in squamous cell carcinomas (SCCs) by diverse mechanisms, including viral oncogene-induced degradation, mutation, deletion, and amplification. Activation of the CDK4/6 pathway may predict response to CDK4/6 inhibitors and provide clinical biomarkers. Recently, the CDK4/6 inhibitor palbociclib showed clinical efficacy in combination with cetuximab in HNSCC patients. Areas covered This review focuses on the current research on the use of CDK4/6 inhibitors, comprising preclinical animal studies through phase II clinical trials across all SCCs. Expert opinion CDK4/6 inhibitors have a proven clinical benefit in breast cancer, but data on SCCs are sparse. Although frequent dysregulation of the cyclin D-CDK4/6-INK4-Rb pathway in SCCs suggests that targeting CDK4/6 may hold promise for improved clinical outcomes, single-agent activity has been modest in preclinical studies and absent in clinical studies. Combinations with immunotherapy or inhibitors of the PI3 K/mTOR or EGFR pathway may be effective. Given that SCCs caused by human papillomavirus have high levels of p16 and low levels of Rb, the CDK4/6 inhibitors are predicted to be ineffective in these cancers.

Background Spindle epithelial tumor with thymus-like differentiation (SETTLE) is a rare cancer of thymic origin with little information available for treating clinicians. Methods We performed a comprehensive literature search and updates were requested from all authors. We report one recent case. Results We present the most comprehensive review of the clinical literature for SETTLE. We identified 41 published cases. SETTLE usually presents as an asymptomatic neck mass. Most patients successfully underwent surgical resection of the primary tumor. The mean latency to develop metastasis was 10 years. SETTLE is uniformly responsive to radiotherapy and combination chemotherapy in the rare cases where response data are available. Conclusion Although SETTLE is initially indolent, it has the capacity to metastasize late in its course. The malignant potential of this tumor might be underestimated because of the lack of long-term follow-up. In cases in which complete surgical resection is not possible, radiotherapy or combination chemotherapy is effective. © 2014 Wiley Periodicals, Inc. Head Neck 37: 746–754, 2015

Importance A major goal of primary transoral robotic surgery (TORS) for oropharyngeal cancer is to optimize swallowing outcomes by personalized treatment based on pathologic staging. However, swallowing outcomes after TORS are uncertain, as are the outcomes compared with nonsurgical options. Objectives To estimate rates of acute dysphagia and recovery after TORS and to compare swallowing outcomes by primary treatment modality (TORS or radiotherapy). Design, Setting, and Participants This case series study was a secondary analysis of prospective registry data from 257 patients enrolled from March 1, 2015, to February 28, 2018, at a single academic institution who, according to theAJCC Staging Manual, 7th edition TNM classification, had low- to intermediate-risk human papillomavirus–related oropharyngeal squamous cell carcinoma possibly resectable by TORS. Exposure Patients were stratified by primary treatment (75 underwent TORS and 182 received radiotherapy). Main Outcomes and Measures Modified barium swallow (MBS) studies graded per Dynamic Imaging Grade of Swallowing Toxicity (DIGEST) and the MD Anderson Symptom Inventory–Head and Neck Module (MDASI-HN) questionnaires were administered at standard intervals. Prevalence and severity of dysphagia were estimated per DIGEST before and after TORS and 3 to 6 months after treatment. Moderate-severe dysphagia (DIGEST grade ≥2) was assessed using logistic regression and compared by primary treatment group. The MDASI swallowing symptom severity item scores during and after radiotherapy were compared using generalized estimating equations by treatment status at the start of radiotherapy, after induction, and after TORS. Results A total of 257 patients (mean [SD] age, 59.54 [9.07] years; 222 [86.4%] male) were included in the study. Dysphagia severity (per DIGEST) was significantly worse after TORS (r = −0.63; 95% CI, −0.78 to −0.44): 17 patients (22.7%; 95% CI, 13.8%-33.8%) had moderate-severe (DIGEST grade ≥2) acute post-TORS dysphagia significantly associated with primary tumor volume (odds ratio, 1.43; 95% CI, 1.11-1.84). DIGEST improved by 3 to 6 months but remained worse than that at baseline; at 3 to 6 months, the number of patients with DIGEST grade 2 or higher dysphagia was 5 (6.7%; 95% CI, 2.2%-14.9%) after primary TORS and 29 (15.9%; 95% CI, 10.9%-22.1%) after radiotherapy. At the start of radiotherapy, MDASI swallowing symptom severity item scores were significantly worse in the post-TORS group compared with postinduction (mean [SD] change, 2.6 [1.1]) and treatment-naive (mean [SD] change, 1.7 [0.3]) patients. This result inverted at radiotherapy end, and all groups converged at 3 to 6 months. Conclusions and Relevance Subacute swallowing outcomes were similar regardless of primary treatment modality among patients with low- to intermediate-risk oropharyngeal squamous cell carcinoma.

Human papilloma virus (HPV) is an oncogenic driver for a subset of head and neck squamous cell carcinomas (HNSCC), primarily from the oropharyngeal tissue subsite (OPSCC). These tumors are increasing in incidence and have recently surpassed cervical cancer as the most common HPV-driven malignancy in the United States. Fortunately, these tumors generally respond well to radiation-based therapy (XRT), and long-term (5 yr) survival is around 85%. However, the XRT treatment can generate significant morbidity, including problems with speech and swallowing. There is a clinical effort to reduce the treatment-related morbidity without compromising survival outcomes, through de-escalation treatment protocols. However, there is a subset of HPV+ OPSCC patients who do not respond to the current therapies and should not be given less intense treatment. This has generated the need to stratify patients based on their risk of recurrence or death, but currently no molecular biomarkers are available for risk assessment in OPSCC. By analyzing genomic data from The Cancer Genome Atlas (TCGA) we have identified a gene expression signature associated with expression of HPV genes. This signature identified 2 groups within the HPV+ tumors that demonstrate different levels of HPV function. One group seems to have reduced HPV function and present with intermediate phenotypes between HPV+ and HPV- tumors. Importantly, this signature is also highly prognostic in HPV+ OPSCC (p Citation Format: Frederico O. Gleber-Netto, Meng Gao, Xiayu Rao, Christopher P. Vellano, Joseph R. Marszalek, Jing Wang, Faye M. Johnson, Curtis R. Pickering. Variations in HPV function are associated with patient outcome and identify new candidate therapeutic approaches [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 4942.

Head and neck squamous cell carcinoma (HNSCC) is primarily driven by the loss of tumor suppressor genes. Although approaches to target driver oncogenes have been clinically successful, targeting tumor suppressors has been challenging. To address this translational gap, we previously demonstrated that HNSCC cell lines with loss of function (LOF) NOTCH1 mutations (NOTCH1MUT n=14) were more sensitive to 6 PI3K pathway inhibitors (Omipalisib, Alpelisib, Bimiralisib, Dactolisib, Copanlisib, Apitolisib) than NOTCH1WT cells (n=45). In contrast to PIK3CAMUT cell lines, NOTCH1MUT lines underwent significant apoptosis after PI3K/mTOR pathway inhibition. NOTCH1MUT lines also showed significantly reduced clonogenic growth and significant tumor growth inhibition in both oral orthotopic and subcutaneous xenograft models. We employed CRISPR-Cas9 gene editing technology to knock out NOTCH1 gene in two NOTCH1WT lines, PJ34 and UMSCC49. After Omipalisib treatment, NOTCH1-KO lines showed decreased cell viability compared to parental lines. The NOTCH1 knock out lines showed significantly increased apoptosis after Omipalisib treatment compared to parental lines (PJ34 KO: 1.62-fld, P Citation Format: Vaishnavi Sambandam, Li Shen, Shaohua Peng, Tuhina Mazumdar, Curtis Pickering, Jeffrey Myers, Jing Wang, Mitchell Frederick, Faye Johnson. PDK1 drives susceptibility of NOTCH1 mutant head and neck squamous carcinoma to PI3K/mTOR pathway inhibition [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 3913.

6008 Background: Anti-PD-1/PD-L1 are active in metastatic oropharynx squamous cell carcinoma (OPC). Durvalumab (durva) and tremelimumab (tremi) target respectively PD-L1 and CTLA-4, which in combination may be synergistic. Here we report the safety and interim results of durva vs. durva+tremi prior to surgery in a window of opportunity trial in OPC. Methods: Pts were randomized 1:1 to durva 1500 mg or durva 1500 mg + tremi 75 mg IV Q4W x 2 cycles. The primary objective was to quantify pre- and post-treatment differences in CD8+ tumor infiltrating lymphocytes for the two arms. Secondary objectives included safety, toxicity, ORR by RECIST, fraction of patients undergoing surgery at 8 wks, and percentage viable tumor cells in the surgical specimen. Serial pre- and post-treatment blood and tumor specimens were collected for ongoing correlative analyses. Results: 28 pts enrolled: median age 64y, 27 (96%) male, 19 (68%) newly diagnosed, most (63%) at stage IVA (AJCC 7th Ed), 9 (32%) had locoregional recurrence, 24 (86%) p16 positive, and 22 (79%) had ≤ 10 PPY smoking history. Median follow-up was 7.6 months. The most common AEs were fatigue (36%), leukopenia/lymphopenia (25%), transaminitis (25%), and rash (21%). Grade 3 AEs occurred in 4 (14%) pts: 2 elevated lipase, 1 diarrhea, and 1 hepatitis, all were manageable. There were no grade >3 AEs. ORR was 43%: 50% had SD (including 29% tumor shrinkage in 1 pt). Treatment effect in the surgical specimen was observed in 19 (79%) of 24 evaluable pts; 2 pts had major pathologic response (≤ 10% viable tumor) at the primary site. Efficacy was equivalent in both arms. The 2 pts with PD and 1 pt with SD were switched to chemotherapy after durva +/- tremi before resection; interestingly, each achieved a pCR in the primary. Most pts (57%) didn’t receive radiotherapy after surgery. There was a statistically significant association between ORR and treatment effect (p=0.014). The median percentage of viable tumor in the primary was 37.5% in pts with PR, and 82.5% in SD (p=0.003). Conclusions: Durva +/- tremi prior to surgery was well tolerated in OPC pts. Activity is encouraging with treatment effect seen in 79% of pts. The primary endpoint and complete efficacy data will be presented. Clinical trial information: NCT03144778.

IntroductionRNA-dependent protein kinase (PKR) is an independent prognostic variable in patients with non–small-cell lung cancer (NSCLC). In the current study, we investigated the correlation between PKR and 25 other biomarkers for NSCLC, identified the markers that could further improve the prognostic significance of PKR and elucidated the mechanisms of interaction between these markers and PKR.MethodsTissue microarray samples obtained from 218 patients with lung cancer were stained with an anti-PKR antibody and antibodies against 25 biomarkers. Immunohistochemical expression was scored and used for Kaplan–Meier survival analysis. The interaction between PKR and EphA2 in NSCLC cell lines was examined.ResultsWe found that PKR was associated with EphA2 and that the prognostic information regarding NSCLC provided by the combination of PKR and EphA2 (P/E) was significantly more accurate than that provided by either marker alone. The 5-year overall survival rate in patients with PKRlow/EphA2high (20%) was significantly lower than that of patients with PKRhigh/EphA2low (74%), patients with PKRhigh/EphA2high (55%), and patients with PKRlow/EphA2low (55%) (p < 0.0001). We also found that the PKR:EphA2 (P/E) ratio was significantly associated with prognosis (p < 0.0001). Univariate and multivariate Cox analyses revealed that this P/E combination or ratio was an independent predictor of overall survival. In addition, induction of PKR expression reduced EphA2 protein expression levels in NSCLC cell lines.ConclusionsPKR/EphA2 is a significant predictor of prognosis for NSCLC. PKR/EphA2 may be a promising approach to improving screening efficiency and predicting prognosis in patients with NSCLC.

Background The first generation CDK2/7/9 inhibitor seliciclib (CYC202) causes multipolar anaphase and apoptosis in lung cancer cells with supernumerary centrosomes (known as anaphase catastrophe). We investigated a new and potent CDK2/9 inhibitor, CCT68127 (Cyclacel). Methods CCT68127 was studied in lung cancer cells (three murine and five human) and control murine pulmonary epithelial and human immortalized bronchial epithelial cells. Robotic CCT68127 cell-based proliferation screens were used. Cells undergoing multipolar anaphase and inhibited centrosome clustering were scored. Reverse phase protein arrays (RPPAs) assessed CCT68127 effects on signaling pathways. The function of PEA15, a growth regulator highlighted by RPPAs, was analyzed. Syngeneic murine lung cancer xenografts (n = 4/group) determined CCT68127 effects on tumorigenicity and circulating tumor cell levels. All statistical tests were two-sided. Results CCT68127 inhibited growth up to 88.5% (SD = 6.4%, P < .003) at 1 μM, induced apoptosis up to 42.6% (SD = 5.5%, P < .001) at 2 μM, and caused G1 or G2/M arrest in lung cancer cells with minimal effects on control cells (growth inhibition at 1 μM: 10.6%, SD = 3.6%, P = .32; apoptosis at 2 μM: 8.2%, SD = 1.0%, P = .22). A robotic screen found that lung cancer cells with KRAS mutation were particularly sensitive to CCT68127 ( P = .02 for IC 50 ). CCT68127 inhibited supernumerary centrosome clustering and caused anaphase catastrophe by 14.1% (SD = 3.6%, P < .009 at 1 μM). CCT68127 reduced PEA15 phosphorylation by 70% (SD = 3.0%, P = .003). The gain of PEA15 expression antagonized and its loss enhanced CCT68127-mediated growth inhibition. CCT68127 reduced lung cancer growth in vivo ( P < .001) and circulating tumor cells ( P = .004). Findings were confirmed with another CDK2/9 inhibitor, CYC065. Conclusions Next-generation CDK2/9 inhibition elicits marked antineoplastic effects in lung cancer via anaphase catastrophe and reduced PEA15 phosphorylation.

We focused on a cohort of radiation naïve patients who had recurrent oral cavity cancer (recurrent OCC) to assess their outcomes with salvage multimodal therapy.A retrospective single institutional study was performed of patients with recurrent OCC. Disease recurrence and survival outcomes were assessed.Seventy-eight patients were analyzed. All patients had salvage surgery and intensity-modulated radiotherapy (IMRT) and 74% had chemotherapy. Five-year overall survival, recurrence-free survival, and locoregional control rates were 59%, 60%, and 74%, respectively.Outcomes of radiation naïve patients with recurrent OCC are fair, and seem similar with patients with locally advanced nonrecurrent OCC treated with multimodal therapy. © 2016 Wiley Periodicals, Inc. Head Neck 39: 633-638, 2017.

Favorable outcomes for human papillomavirus-associated oropharyngeal cancer have led to interest in identifying a subgroup of patients with the lowest risk of disease recurrence after therapy. De-intensification of therapy for this group may result in survival outcomes that are similar to those associated with current therapy but with less toxicity. To advance this effort, this study analyzed the outcomes of oropharyngeal cancer patients treated with or without systemic therapy.This was a retrospective study of patients with oropharyngeal cancer treated between 1985 and 2012. The criteria for inclusion were ≤10 pack-years of cigarette smoking and stage III/IVA cancer limited to T1-3, N1-N2b, and T3N0 disease. A survival analysis was performed with the primary endpoint of progression-free survival (PFS).The cohort included 857 patients. Systemic therapy was given to 439 patients (51%). The median survival was 80 months. The 2-year PFS rate was 91%. When the analysis was limited to 324 patients irradiated without systemic therapy, the 2- and 5-year PFS rates were 90% and 85%, respectively. Furthermore, for these 324 patients, the 5-year PFS rates for T1, T2, and T3 disease were 90%, 83%, and 70%, respectively. The 5-year PFS rate for patients treated with systemic therapy for T3 disease was 77% (P = .07).According to the low-risk definition currently established in cooperative trials, the patients had a 2-year PFS rate approximating 90%. When patients who were treated with radiation alone were evaluated, no compromise was observed in this high rate of PFS, which is higher than the 2-year PFS thresholds used in current cooperative trials. Cancer 2016;122:1702-7. © 2016 American Cancer Society.

Purpose: The inhibition of c-Src results in a striking reduction in cancer cell invasion, but the effect on cell survival is modest. Defining mechanisms that limit apoptosis following c-Src inhibition could result in an ideal therapeutic approach that both inhibits invasion and leads to apoptosis. In this regard, we discovered a novel feedback loop that results in STAT3 reactivation following sustained c-Src inhibition. Here we define the mechanism underlying this feedback loop and examine the effect of inhibiting it in vivo. Experimental Design: We measured levels and activity of pathway components using PCR, Western blotting, and kinase assays following their manipulation using both molecular and pharmacologic approaches. We used a heterotransplant animal model in which human oral squamous cancer is maintained exclusively in vivo. Results: Following c-Src inhibition, STAT5 is durably inhibited. The inhibition of STAT5A, but not STAT5B, subsequently reduces the expression of suppressors of cytokine signaling 2 (SOCS2). SOCS2 inhibits Janus kinase 2 (Jak2) activity and Jak2–STAT3 binding. SOCS2 expression is necessary for STAT3 inhibition by c-Src inhibitors. Overexpression of SOCS2 is adequate to prevent STAT3 reactivation and to enhance the cytotoxic effects of c-Src inhibition. Likewise, the combination of Jak and c-Src inhibitors led to significantly more apoptosis than either agent alone in vivo. Conclusions: To our knowledge, ours is the first study that fully defines the mechanism underlying this feedback loop, in which sustained c-Src inhibition leads to diminished SOCS2 expression via sustained inhibition of STAT5A, allowing activation of Jak2 and STAT3, Jak2–STAT3 binding, and survival signals. Clin Cancer Res; 18(1); 127–39. ©2011 AACR.

The nonreceptor protein tyrosine kinase Src plays a crucial role in the signal transduction pathways involved in cell division, motility, adhesion, and survival in both normal and cancer cells. Although the Src family kinases (SFKs) are activated in various types of cancers, the exact mechanisms through which they contribute to the progression of individual tumors remain to be defined. The activation of Src in human cancers may occur through a variety of mechanisms that include domain interaction and structural remodeling in response to various activators or upstream kinases and phosphatastes. Because of Src's prominent roles in invasion and tumor progression, epithelial-to-mesenchymal transition, angiogenesis, and the development of metastasis, Src is a promising target for cancer therapy. Several small molecule inhibitors of Src are currently being investigated in clinical trials. In this article, we will summarize the mechanisms regulating Src kinase activity in normal and cancer cells and discuss the status of Src inhibitor development against various types of cancers.

Purpose: c-Src inhibition in cancer cells leads to an abrogation of invasion but a variable effect on apoptosis. The pathways downstream of c-Src promoting survival are not well characterized. Because cancer therapy that both decreases invasion and induces significant apoptosis would be ideal, we sought to characterize the mechanisms of resistance to c-Src inhibition. Experimental Design: c-Src was inhibited in a panel of oral cancer cell lines and subsequent survival and signaling measured. The interactions between c-Src and c-Met were evaluated using immunoprecitation and an in vitro kinase assay. Cytotoxicity was measured and the Chou–Talalay combination index calculated. An orthotopic model of oral cancer was used to assess the effects of c-Met and c-Src inhibitors. Results: Inhibition of c-Src resulted in c-Met inhibition in sensitive cells lines, but not in resistant cell lines. Isolated c-Met was a c-Src substrate in both sensitive and resistant cells, but there was no interaction of c-Src and c-Met in intact resistant cells. To examine the biological consequences of this mechanism, we demonstrated synergistic cytotoxicity, enhanced apoptosis, and decreased tumor size with the combination of c-Src and c-Met inhibitors. Conclusions: Sustained c-Met activation can mediate resistance to c-Src inhibition. These data suggest that the differences between c-Met and c-Src signaling in sensitive and resistant cells are due to distinct factors promoting or inhibiting interactions, respectively, rather than to intrinsic structural changes in c-Src or c-Met. The synergistic cytotoxic effects of c-Src and c-Met inhibition may be important for the treatment of head and neck cancers. Clin Cancer Res; 17(3); 514–24. ©2010 AACR.

BACKGROUND: Treatment options for patients with advanced head and neck squamous cell carcinoma (HNSCC) are scarce. This phase 2 study was conducted to evaluate the safety, tolerability, pharmacokinetics, and efficacy of dasatinib in this setting. METHODS: Patients with recurrent and/or metastatic HNSCC after platinum-based therapy were treated with dasatinib either orally or via percutaneous feeding gastrostomy (PFG). Primary endpoints were 12-week progression-free survival (PFS) and objective response rate with a 2-stage design and early withdrawal if the 12-week PFS rate was ≤20% and no patients had an objective response (OR). Forty-nine serum cytokines and angiogenic factors (CAFs) were analyzed from treated patients. RESULTS: Of the 15 patients enrolled, 12 were evaluable for response, and all patients were evaluable for toxicity. No OR was observed and 2 patients (16.7%) had stable disease (SD) at 8 weeks. The median treatment duration was 59 days, the median time to disease progression was 3.9 weeks, and the median survival was 26 weeks. One patient required a dose reduction, 3 patients required dose interruptions, and 4 patients were hospitalized for toxicity. Dasatinib inhibited c-Src both when administered orally and via PFG. Greater mean drug exposure, decreased half-life, and greater maximum concentration were observed in patients receiving dasatinib via PFG. Eleven baseline CAFs were associated with treatment outcome and 1 CAF, macrophage migration inhibitory factor, was found to be differentially modulated in correlation with SD versus disease progression. CONCLUSIONS: Single-agent dasatinib failed to demonstrate significant activity in patients with advanced HNSCC, despite c-Src inhibition. The toxicity profile was consistent with that reported in other solid tumors, and the drug can be given via PFG tube. Cancer 2011. © 2010 American Cancer Society.

Genomic alterations in the PI3K/mTOR pathway occur in 54% of HNSCC patients. To identify novel biomarkers of response to PI3K/mTOR pathway inhibitors in HNSCC, we tested the efficacy of 7 PI3K/mTOR pathway inhibitors in 59 HNSCC cell lines and determined the association between drug sensitivity and genomic alterations. We identified that NOTCH1mut lines were significantly more sensitive to PI3K/mTOR pathway inhibitors than NOTCHWT lines: GSK2126458 (12/14 NOTCH1Mut lines), BYL719 (6/14), PQR309 (12/14), BKM120 (14/16), BEZ235 (12/16), BAY806942 (13/14) and GDC0980 (5/14 lines). In contrast to PIK3CAmut cell lines that experienced cell cycle arrest, after PI3K/mTOR pathway inhibition, NOTCH1mut lines underwent significant apoptosis in addition to G1/S cell cycle arrest. NOTCH1mut lines also showed reduced clonogenic growth in vitro and tumor growth inhibition in vivo in both oral orthotopic and subcutaneous xenograft mouse models. NOTCH1 knock out (KO) by CRISPR-Cas9 system in a NOTCH1WT line (PJ34) rendered it more sensitive to PI3K/mTOR inhibition. After PI3K/mTOR inhibition, PJ34-NOTCH1 KO showed significant reduction in clonogenic growth (1.57-fold; P As no canonical pathways account for the underlying mechanism of sensitivity, we measured the level of 301 proteins by reverse phase protein array (RPPA) in 3 NOTCH1mut and 3 NOTCH1WT lines after GSK2126458 treatment. Several proteins related to cell cycle were differentially regulated in NOTCH1mutcells compared to wild type lines. Notably, both mRNA and protein levels of Aurora B were significantly decreased in NOTCH1mutcells but not in NOTCHwt cells following PI3K/mTOR inhibition. Aurora B is an important cell cycle regulator and deregulation of Aurora kinases leads to defective chromosomal segregation and mitotic catastrophe in numerous cancers. Aurora kinase inhibitors as single agent are highly effective in a panel of NOTCHwt cell lines as demonstrated by decreased colony formation ability and proliferation as well as G2/M arrest and apoptosis. Inhibition of Aurora kinases in combination with PI3K inhibitors displayed synergy (Combination Index This work is significant because inactivating NOTCH1 mutations, which occur in 18% of HNSCC patients and SCCs of the lung, esophagus, and other sites, may serve as a biomarker for response. Our present work may uncover potential combination therapies for HNSCC. Citation Format: Vaishnavi Sambandam, Li Shen, Pan Tong, Shaohua Peng, Tuhina Mazumdar, Ratnakar Singh, Curtis R. Pickering, Jeffrey N. Myers, Jing Wang, Mitchell Frederick, Faye M. Johnson. PI3K/mTOR pathway inhibition induces Aurora B mediated cell death in NOTCH1 mutant head and neck squamous (HNSCC) cells [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 2977.

Determining which HPV+ oropharyngeal squamous cell carcinoma (OPSCC) patients will respond to therapy is of utmost important for implementation of treatment de-escalation to reduce morbidity or testing of novel therapeutic approaches. Due to the lack of reliable indicators of treatment response, we explored the gene expression profile of OPSCC in order to better understand the biology of these tumors and identify novel biomarkers. In this study, we investigated human genes associated with HPV transcription in 80 OPSCC samples from The Cancer Genome Atlas (TCGA). This exploratory analysis provided a list of 582 human genes associated with HPV biology. Hierarchical clustering analysis using the 582 human genes was used to separate HPV+ OPSCC in two groups with significantly distinct survival (log-rank test p < 0.001) and differential expression of HPV genes. A refinement of this analysis generated a prognostic gene expression signature for HPV+ OPSCC containing 41 human genes. These results were validated in two independent cohorts of HPV+ OPSCC (n=83 and n=47) and one independent cohort of cervical cancer (n=83). In all three validation cohorts our 41 gene expression signature was able to identify a group of HPV+ OPSCC patients with worse survival. Further refinement and validation of the signature is ongoing. In order to understand the biology related to poor outcome in HPV+ OPSCC, a whole transcriptome analysis between the two initial HPV+ OPSCC TCGA groups was performed. Pathway analysis identified cell metabolism, cell stress, and DNA damage related genes were highly associated with poor outcome. Similar patterns of gene expression were found in vitro in a panel of 10 HPV+ cell lines, and markers of the poor outcome were found to be associated with reduced radiation sensitivity in vitro. Our study has identified prognostic biomarkers in HPV+ OPSCC with potential clinical importance. These biomarkers may be useful for selection of low risk patients for treatment de-escalation or selection of high risk patients for novel therapeutic approaches. These biomarkers are also functionally linked to radiation sensitivity and may include new therapeutic targets. Citation Format: Frederico O. Gleber-Netto, Xiayu Rao, Kelly Erikson, Keiko Akagi, Faye M. Johnson, Jing Wang, Joseph Califano, Maura L. Gillison, Jeffrey N. Myers, Curtis R. Pickering. Risk stratification and biomarker discovery in HPV-positive oropharynx squamous cell carcinoma determined by HPV and human gene expression profile associations [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 4621.

Purpose. To address the unmet need for biomarker-driven, effective, targeted therapy for human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC) and cervical epithelial squamous cell carcinoma (CESC), we conducted a high-throughput drug screen (HTDS) using 1122 compounds in all readily available HPV-positive HNSCC and CESC cell lines and an equal number of matched HPV-negative lines. Methods. Cells were incubated in drug concentrations ranging from 0.01 μM to 3.16 μM for 72 h, fixed and stained with DAPI, and counted. Of the 1122 analyzable compounds, 865 unique drugs were tested because of overlap. All drugs were assigned to one of 36 classes based on their primary targets. Drug concentrations resulting in a 50% reduction in cell proliferation (GI50) and the area under the dose response curve were calculated. Two biological replicates were performed for all cell lines on separate days and at least 1 week apart. Results. The HTDS was conducted using 24 cell lines. We identified 493 highly effective compounds, which we defined as those with GI50 values less than 0.5 μM in 2 or more of the cell lines screened. The most effective drug classes were inhibitors of polo-like kinase, proteasomes, histone deacetylase, and Aurora kinases. Of the 19 Aurora kinase inhibitors tested, 18 were highly effective. We confirmed the efficacy of 3 Aurora kinase inhibitors using colony formation assays in 15 cell lines. Treatment with a dual Aurora A/B inhibitor, danusertib, led to G2M arrest and apoptosis in all 6 tested cell lines. Additionally, danusertib treatment decreased tumor size compared to controls in patient-derived xenograft mouse models of HNSCC. To identify biomarkers predicting response to Aurora kinase inhibitors, we tested for associations between mutations in the cell lines and sensitivity to the Aurora kinase inhibitors using whole exome mutation data for the 50 most common driver mutations in HNSCC. To validate our findings in an independent dataset, we queried the Genomics of Drug Sensitivity in Cancer database. In both data sets, cancer cell lines with KMT2D (MLL2) mutations were more sensitive to Aurora kinase inhibitors than cells without mutations. KMT2D mutations are inactivating; experiments to knock down KMT2D in wild-type cell lines and assess sensitivity to Aurora kinase inhibitors are ongoing. Conclusions. We identified Aurora kinase inhibitors as effective and understudied drugs in HNSCC and CESC. These drugs cause apoptosis and cell cycle arrest in vitro and decrease tumor size in vivo. This is the first published study to demonstrate that mutations in KMT2D (MLL2), which are common in many cancers (16% HNSCC, 12% CESC), correlate with drug sensitivity in 2 independent data sets. Citation Format: Tuhina Mazumdar, Nene N. Kalu, Shaohua Peng, Pan Tong, Li Shen, Jing Wang, Jeffrey N. Myers, Curtis R. Pickering, David Brunell, Clifford C. Stephan, Faye M. Johnson. Pharmacogenomic screen identifies KMT2D mutations as a biomarker of sensitivity to Aurora kinase inhibition in head and neck and cervical 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 4646.

Introduction: Plk1 is a serine-threonine protein kinase that is overexpressed in cancer cells, and plays a major role in regulating tumor growth. Plk1 inhibitors are well tolerated, but only a few unselected patients with non-small cell lung cancer (NSCLC) respond to single-agent therapy. Our lab discovered that mesenchymal NSCLC cell lines are more sensitive to Plk1 inhibitors than epithelial cell lines in vitro and in vivo. However, mechanisms of resistance to Plk1 inhibitors have not been elucidated and this unknown is a major gap in knowledge. Experimental procedure: To study the mechanisms of Plk1 inhibitor-induced apoptosis we used 3 pairs of isogenic epithelial NSCLC cell lines induced to a mesenchymal phenotype with TGF-β. These isogenic pairs were treated with the Plk1 inhibitor volasertib for 24 h and levels of 301 proteins and phosphoproteins were simultaneously measured using reverse phase protein array (RPPA). Volasertib acquired resistance (VAR) cell lines were generated by exposing cells to increasing doses of volasertib. Results: The induction of a mesenchymal phenotype using TGF-β increased Plk1 inhibition-induced apoptosis in all 3 cell lines. To further elucidate mechanisms of resistance, we compared protein expression in these isogenic cell lines, 24 h after Plk1 inhibition. There were 33 proteins differentially regulated following Plk1 inhibition in parental vs TGF-β induced isogenic cells (p-value < 0.05). Notably, phosphorylated c-Met (Y1234/1235), FAK (Y397) and Src (Y416) were consistently inhibited following Plk1 inhibition in the mesenchymal lines. These changes were confirmed by Western blotting. Total c-Met, FAK and Src protein levels were not affected, implicating a post-translational changes. Likewise, VAR cell lines exhibited an epithelial phenotype and c-Met phosphorylation was persistent even after Plk1 inhibition. Simultaneous c-Met and Plk1 inhibition or silencing increased apoptosis in NSCLC cell lines tested compared to single agent inhibition or silencing. Combination of Plk1 and c-Met inhibitors decreased tumor volume and increased mouse survival in vivo in patient derived and cell line xenograft models. Similarly VAR cells also showed more apoptosis when treated with combination of Plk1 and c-Met inhibitors. Levels of the c-Met ligand HGF were unchanged after Plk1 inhibition and further mechanistic studies are on-going. Conclusion: NSCLC cell lines have diverse sensitivities to Plk1 inhibition, which is consistent with the results of clinical trials of Plk1 inhibitors in solid tumors. This study reveals a novel mechanism of non-canonical c-Met activation in resistant epithelial NSCLC after Plk1 inhibition. We demonstrate a profound effect of combination Plk1 and c-Met inhibition in vivo in multiple mouse models that could be a novel therapy for NSCLC patients. Citation Format: Ratnakar Singh, Pavitra Viswanath, Shaohua Peng, Vaishnavi Sambandam, Li Shen, Lerong Li, Jing Wang, Bingliang Fang, Faye M. Johnson. Noncanonical c-Met activation mediates de novo and acquired resistance to polo-like kinase 1 inhibitor-induced apoptosis in non-small cell lung 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 895.

Src tyrosine kinases regulate multiple genetic and signaling pathways involved in the proliferation, survival, angiogenesis, invasion, and migration of various types of cancer cells They are frequently expressed and activated in many cancer types, including lung cancer. Several Src inhibitors, including dasatinib, saracatinib, bosutinib, and KX2-391, are currently being investigated in clinical trials. Preliminary results of the use of single-agent Src inhibitors in unselected patients with lung cancer show that these inhibitors have a favorable safety profile and anticancer activity. Their combination with cytotoxic chemotherapy, other targeted therapy, and radiation therapy is currently being explored. In this review, we summarize the rationale for and the current status of Src inhibitor development and discuss future directions based on emerging preclinical data.

Although the role of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway has been most extensively studied in hematopoietic cells and hematologic malignancies, it is also activated in epithelial tumors, including those originating in the lungs and head and neck. The canonical pathway involves the activation of JAK following ligand binding to cytokine receptors. The activated JAKs then phosphorylate STAT proteins, leading to their dimerization and translocation into the nucleus. In the nucleus, STATs act as transcription factors with pleiotropic downstream effects. STATs can be activated independently of JAKs, most notably by c-Src kinases. In cancer cells, STAT3 and STAT5 activation leads to the increased expression of downstream target genes, leading to increased cell proliferation, cell survival, angiogenesis, and immune system evasion. STAT3 and STAT5 are expressed and activated in head and neck squamous cell carcinoma (HNSCC) where they contribute to cell survival and proliferation. In HNSCC, STATs can be activated by a number of signal transduction pathways, including the epidermal growth factor receptor (EGFR), alpha7 nicotinic receptor, interleukin (IL) receptor, and erythropoietin receptor pathways. Activated STATs are also expressed in lung cancer, but the biological effects of JAK/STAT inhibition in this cancer are variable. In lung cancer, STAT3 can be activated by multiple pathways, including EGFR. Several approaches have been used to inhibit STAT3 in the hopes of developing an antitumor agent. Although several STAT3-specific agents are promising, none are in clinical development, mostly because of drug delivery and stability issues. In contrast, several JAK inhibitors are in clinical development. These orally available, ATP-competitive, small-molecule kinase inhibitors are being tested in myeloproliferative disorders. Future studies will determine whether JAK inhibitors are useful in the treatment of HNSCC or lung cancer.

BACKGROUND: The recently developed the Src and Abelson (Abl) kinase inhibitor dasatinib has antitumor effects in epithelial and mesenchymal tumors. Preclinical data have indicated that dasatinib is metabolized primarily through cytochrome P450 3A4 (CYP3A4) and may cause QT prolongation. In light of its improved tolerability, the authors were interested in the safety of a once-daily dasatinib regimen. METHODS: The authors conducted a phase 1 trial of dasatinib in 29 patients with advanced solid tumors. Segment 1 of the trial was short term and sequential and was designed to determine whether the coadministration of the potent CYP3A4 inhibitor ketoconazole had an effect on the pharmacokinetics of dasatinib. Segment 2 was designed to evaluate the safety of dasatinib as dosing was increased. QT intervals were monitored closely in both segments. Efficacy was assessed in Segment 2 using both positron emission tomography and computed tomography. RESULTS: Hematologic toxicities were markedly less than those observed in patients with leukemia, whereas nonhematologic toxicities were similar. The authors determined that the maximum recommended dose was 180 mg once daily based on the incidence of pleural effusion. Coadministration of ketoconazole led to a marked increase in dasatinib exposure, which was correlated with an increase in corrected QT (QTc) values of approximately 6 msec. No adverse cardiac events were observed. CONCLUSIONS: The dose-limiting toxic effect for dasatinib was pleural effusion. The pharmacokinetic and cardiac studies indicated that coadministration of dasatinib with potent CYP3A4 inhibitors or agents that prolong the QTc interval should be avoided if possible. Close monitoring for toxicity and dose reduction should be considered if the coadministration of such agents cannot be avoided. Cancer 2010. © 2010 American Cancer Society.

Overexpression of the receptor tyrosine kinase EphA2 occurs in non–small cell lung cancer (NSCLC) and a number of other human cancers. This overexpression correlates with a poor prognosis, smoking, and the presence of Kirsten rat sarcoma (K-Ras) mutations in NSCLC. In other cancers, EphA2 has been implicated in migration and metastasis. To determine if EphA2 can promote NSCLC progression, we examined the relationship of EphA2 with proliferation and migration in cell lines and with metastases in patient tumors. We also examined potential mechanisms involving AKT, Src, focal adhesion kinase, Rho guanosine triphosphatases (GTPase), and extracellular signal–regulated kinase (ERK)-1/2. Knockdown of EphA2 in NSCLC cell lines decreased proliferation (colony size) by 20% to 70% in four of five cell lines (P < 0. 04) and cell migration by 7% to 75% in five of six cell lines (P < 0. 03). ERK1/2 activation correlated with effects on proliferation, and inhibition of ERK1/2 activation also suppressed proliferation. In accordance with the in vitro data, high tumor expression of EphA2 was an independent prognostic factor in time to recurrence (P = 0.057) and time to metastases (P = 0.046) of NSCLC patients. We also examined EphA2 expression in the putative premalignant lung lesion, atypical adenomatous hyperplasia, and the noninvasive bronchioloalveolar component of adenocarcinoma because K-Ras mutations occur in atypical adenomatous hyperplasia and are common in lung adenocarcinomas. Both preinvasive lesion types expressed EphA2, showing its expression in the early pathogenesis of lung adenocarcinoma. Our data suggest that EphA2 may be a promising target for treating and preventing NSCLC.

While African Americans in the United States are disproportionately affected by HIV, they are less likely to take antiretroviral therapies. Different first-line antiretroviral therapies are associated with short-term and long-term adverse event (AE) risks. We estimated the willingness of antiretroviral-naïve, HIV-positive African Americans to accept risks of acute AEs with known outcomes and long-term AEs with uncertain outcomes in exchange for virologic suppression. We estimated the relative importance of short-term and long-term AE risks. Two hundred thirty-five subjects were recruited through eight clinics in the United States. One hundred fifty-eight subjects met study inclusion criteria. One hundred fifty-three subjects completed a series of choice-format conjoint trade-off tasks. In each task, subjects were asked to choose between two hypothetical treatments with varying levels of virologic failure, risks of hypersensitivity reaction, decreases in bone mineral density (BMD), and renal impairment, and outcome uncertainty associated with the risks of decreased BMD and renal impairment. Attributes were expressed as probabilities of occurrence. We calculated the relative importance of each AE and the level of risk subjects would accept to reduce the risk of virologic failure. Subjects indicated that short-term AEs with relatively certain outcomes are preferred to long-term AEs with uncertain outcomes. Subjects were strongly averse to the risk of decreased BMD that could not be treated successfully or when the outcome was uncertain and to the risk of renal impairment that could not be treated successfully. Subjects were willing to accept increased risks of AEs in exchange for lower risk of virologic failure.

BACKGROUND Epithelial–mesenchymal transformations (EMT) are critical for the invasion, progression, and metastasis of epithelial carcinogenesis. The role of EMT in head and neck squamous carcinoma (HNSC) tumorigenesis remains unexplored. In the current study, the expressions of several factors associated with the induction of EMT in HNSC cell lines and tumor specimens were investigated to define their functional and pathologic role in HNSC. METHODS Eleven HNSC cell lines and 50 primary tumor tissue specimens formed the materials of this study. Western blot analysis as well as immunohistochemical, and functional techniques were used to assess the status of activated Src (p-Src), E-cadherin, and vimentin in both cell lines and tumor tissues and the results were correlated with patients' clinicopathologic parameters. RESULTS The results demonstrated the inverse expression of p-Src and E-cadherin in the majority of cell lines and in primary tumor tissues compared with normal squamous mucosa. Elevated levels of p-Src were accompanied by down-regulation of E-cadherin and the expression of vimentin in epithelial tumor cells. In vitro inhibition of Src led to E-cadherin reexpression and increased cell contact in squamous carcinoma cell lines. Immunophenotypic analysis of these markers in primary tumor tissues demonstrated a significant correlation between increased p-Src, decreased E-cadherin, and vimentin expression and aggressive tumor features including penetrating invasive fronts, high-grade sarcomatoid transformation, and lymph node metastasis. CONCLUSIONS The results of the current study indicate that Src and E-cadherin may play an important role in EMT, invasion, and aggressive clinicopathologic features of HNSC. These proteins may be targeted for the therapeutic intervention of patients with HNSC. Cancer 2008. © 2008 American Cancer Society.

The c-Met receptor tyrosine kinase has been implicated in cellular transformation induced by mutant Ras, a commonly activated proto-oncogene in non-small cell lung cancer (NSCLC). However, the role of c-Met has not been defined in K-ras-mutant NSCLC, a disease for which no effective targeted therapeutic options currently exist. To acquire a greater understanding of its role, we used genetic and pharmacologic approaches to inhibit c-Met in mice and cultured cells. In KrasLA1 mice, which develop premalignant lung lesions that progress to multifocal lung adenocarcinomas owing to somatic mutations in K-ras, c-Met was expressed in multiple cell types within premalignant lung lesions, and high concentrations of HGF were detected in bronchoalveolar lavage samples. Short-term treatment with PHA-665752, a c-Met inhibitor, decreased the numbers of premalignant lung lesions and induced apoptosis in tumor cells and vascular endothelial cells within lesions. In cell culture, PHA-665752 induced apoptosis of a lung adenocarcinoma cell line derived from KrasLA1 mice (LKR-13) and a murine lung endothelial cell line (MEC). c-Met depletion by siRNA transfection induced apoptosis of MECs but not LKR-13 cells. Collectively, these findings suggest that apoptosis was an on-target effect of PHA-665752 in MECs but not in LKR-13 cells. We conclude that PHA-665752 inhibited lung tumorigenesis in KrasLA1 mice and may provide a novel therapeutic approach to the prevention of K-ras-mutant NSCLC. [Mol Cancer Ther 2008;7(4):952–60]

Malignant pleural mesothelioma (MPM) is a deadly disease with few systemic treatment options. One potential therapeutic target, the non–receptor tyrosine kinase c-Src, causes changes in proliferation, motility, invasion, survival, and angiogenesis in cancer cells and may be a valid therapeutic target in MPM. To test this hypothesis, we determined the effects of c-Src inhibition in MPM cell lines and examined c-Src expression and activation in tissue samples. We analyzed four MPM cell lines and found that all expressed total and activated c-Src. Three of the four cell lines were sensitive by in vitro cytotoxicity assays to the c-Src inhibitor dasatinib, which led to cell cycle arrest and increased apoptosis. Dasatinib also inhibited migration and invasion independent of the cytotoxic effects, and led to the rapid and durable inhibition of c-Src and its downstream pathways. We used immunohistochemical analysis to determine the levels of c-Src expression and activation in 46 archived MPM tumor specimens. The Src protein was highly expressed in tumor cells, but expression did not correlate with survival. However, expression of activated Src (p-Src Y419) on the tumor cell membrane was higher in patients with advanced-stage disease; the presence of metastasis correlated with higher membrane (P = 0.03) and cytoplasmic (P = 0.04) expression of p-Src Y419. Lower levels of membrane expression of inactive c-Src (p-Src Y530) correlated with advanced N stage (P = 0.02). Activated c-Src may play a role in survival, metastasis, and invasion of MPM, and targeting c-Src may be an important therapeutic strategy. [Mol Cancer Ther 2007;6(7):1962–72]

The role of Src-family kinases (SFKs) in non-small cell lung cancer (NSCLC) has not been fully defined. Here we addressed this question by examining SFK phosphorylation in NSCLC biopsy samples and using genetic and pharmacological approaches to inhibit SFK expression and activity in cultured NSCLC cells. Immunohistochemical analysis of NSCLC biopsy samples using a Tyr416 phosphorylation-specific, pan-SFK antibody revealed staining in 123 (33%) of 370 tumors. Because c-Src is known to be both an upstream activator and downstream mediator of epidermal growth factor receptor (EGFR), we next investigated SFK phosphorylation in a panel of NSCLC cell lines, including ones that depend on EGFR for survival. The EGFR-dependent NSCLC cell lines HCC827 and H3255 had increased phosphorylation of SFKs, and treatment of these cells with an SFK inhibitor (PP1 or SKI-606) induced apoptosis. PP1 decreased phosphorylation of EGFR, ErbB2, and ErbB3 and strikingly enhanced apoptosis by gefitinib, an EGFR inhibitor. HCC827 cells transfected with c-Src short hairpin RNA exhibited diminished phosphorylation of EGFR and ErbB2 and decreased sensitivity to apoptosis by PP1 or gefitinib. We conclude that SFKs are activated in NSCLC biopsy samples, promote the survival of EGFR-dependent NSCLC cells, and should be investigated as therapeutic targets in NSCLC patients.

Treatments for recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) have limited efficacy. One potential therapeutic target for HNSCC is the RAS/RAF/MEK/ERK cascade, which is one of the major signaling pathways for HNSCC cell survival. In HNSCC, RAS can be activated either by HRAS mutation or by upstream signaling. The ABL inhibitor nilotinib acts as a weak RAF inhibitor that induces RAF dimerization and subsequent activation of MEK/ERK in other cancer cell lines with activated RAS, leading to an unexpected dependence on MEK/ERK for cell survival. We hypothesized that nilotinib and the MEK inhibitor MEK162 would be synergistic in HNSCC cell lines owing to the frequent activation of RAS. We treated HNSCC cell lines with nilotinib and performed immunoblotting and cell-viability experiments. We used an orthotopic mouse model to assess synergistic effects in vivo. Nilotinib induced significant BRAF-CRAF heterodimerization and ERK activation irrespective of RAS mutation status. In cell-viability assays, nilotinib synergized with MEK162. MEK162 alone induced G1 arrest that was minimally enhanced by nilotinib. In the mouse model, treatment with MEK162 alone or combined with nilotinib led to tumor growth inhibition. In HNSCC, nilotinib-induced RAF dimerization is independent of RAS mutation status, but this dimerization does not lead to MEK dependence for cell survival in all HNSCC cell lines. MEK inhibition alone leads to decreased proliferation both in vitro and in vivo. Although nilotinib has some synergistic effects with MEK162, other agents may be more effective against HNSCC when combined with MEK162.

This was a retrospective study of patients with human papillomavirus (HPV)-associated oropharyngeal cancer treated with concurrent systemic therapy and radiation.Data were extracted through chart review, and statistical analyses included frequency tabulation, chi-square, and Kaplan-Meier tests.Three hundred thirty-nine patients were analyzed; 166 received neoadjuvant chemotherapy. One hundred thirty-six patients were treated with cisplatin, 123 with cetuximab, and 59 with carboplatin. The 2-, 3-, and 5-year actuarial overall survival rates were 92%, 88%, and 78%, respectively. There were no significant differences in survival or disease control when analyzed by systemic agent. Platin-treated patients had greater hematologic toxicity, and required more intravenous hydration. The incidence of confluent mucositis was highest among patients treated with cetuximab.Platin and cetuximab seem to have similar efficacy when delivered concurrently with radiation in our retrospective population study. Although platin did cause greater hematologic toxicity, radiation-specific side effects seemed relatively comparable. © 2015 Wiley Periodicals, Inc. Head Neck 38: E1554-E1561, 2016.

Purpose: Epithelial tumors, including non–small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC), present clinical challenges. One potential target for systemic therapy is Src family nonreceptor tyrosine kinases, which are overexpressed in these tumors and induce pleiotropic effects, including increased proliferation, enhanced survival, stimulation of angiogenesis, and changes in motility. Dasatinib (BMS-354825), an ATP-competitive, small molecule tyrosine kinase inhibitor, suppresses the activity of these kinases at subnanomolar concentrations. Therefore, we tested the antitumor effects of this inhibitor in vitro to determine whether in vivo analyses were warranted.Experimental Design: The antitumor effects of dasatinib on HNSCC and NSCLC cells were evaluated using assays to measure cell cycle progression, apoptosis, migration, and invasion. Western blotting was used to monitor its effects on cell signaling.Results: Dasatinib inhibited migration and invasion in all cell lines and induced cell cycle arrest (blocking the G1-S transition) and apoptosis in some lines. The effects on migration and invasion correlated with the inhibition of Src and downstream mediators of adhesion [e.g., focal adhesion kinase (FAK), p130, and paxillin], and the cell cycle effects and apoptosis correlated with the induction of p27 and the dephosphorylation of Rb. Dasatinib also induced morphologic changes that were consistent with an upstream role for Src in regulating focal adhesion complexes.Conclusions: This study showed that Src inhibition in HNSCC and NSCLC has antitumor effects in vitro. This suggests that dasatinib would have therapeutic activity against these tumors. Clinical studies in these tumor types are warranted.

The combination of IP (irinotecan/cisplatin) has been shown to confer a survival benefit compared with EP (etoposide/cisplatin) in patients with extensive-stage small-cell lung cancer (SCLC). Based on this and potential synergy from sequential inhibition of topoisomerases I and II, we conducted a phase I study to assess the feasibility of weekly therapy alternating IP and EP. The doses of EP were fixed (etoposide 60 mg/m2 on days 1-3 and cisplatin 20 mg/m2 on day 1). The dose of irinotecan was escalated in serial cohorts at 3 dose levels: 80, 90, and 100 mg/m2 on day 1. Granulocyte colony-stimulating factor was given on days 2-5 and days 4-7 after IP and EP, respectively. Patients with limited-stage SCLC received chemoradiation during weeks 4-6 with etoposide 120 mg/m2 on days 1-3, cisplatin 60 mg/m2 on day 1, and thoracic radiation 1.5 Gy twice daily in 30 fractions. Patients received 12 weeks of therapy. To evaluate dose escalation in subsequent cohorts, dose-limiting toxicity (DLT) was initially assessed during weeks 1-3 of treatment. Characteristics of the 18 patients accrued are as follows: performance status 0/1, n = 9; female sex, n = 9; extended-stage SCLC, n = 16; and median age, 53 years. Four patients treated at irinotecan dose level 1 (80 mg/m2), 6 patients at dose level 2 (90 mg/m2), and 6 patients at dose level 3 (100 mg/m2) did not experience DLT in weeks 1-4 and completed therapy without major incident. The only 2 patients to experience DLT during weeks 1-4 were treated at dose level 2. Both were hospitalized during week 4 and subsequently died. However, patients had already been accrued at dose level 3 and tolerated therapy well. Therefore, the trial design was modified to assess DLT during weeks 1-4, and additional patients were cautiously added to the dose level 2 and 3 cohorts. Analysis of summary toxicity data resulted in a recommendation that dose level 3 be used in phase II based on the probability of DLT of 16% (95% CI, 3%-29%). Responses in 16 evaluable patients include complete response in 1 patient, partial response in 14 patients, and minor response in 1 patient. With the exception of the 2 deaths, the therapy was well tolerated and active. Phase II evaluation of the regimen in patients with extensive-stage SCLC is ongoing.

Background: Genomic alterations in the PI3K/mTOR pathway occur in 54% of HNSCC patients. However, clinical trials of PI3K/mTOR pathway inhibitors had limited success even in those tumors with pathway alterations, including PIK3CA mutations. To target genomic alterations in HNSCC, we tested the efficacy of 7 PI3K/mTOR pathway inhibitors in 59 HNSCC cell lines and determined the association between drug sensitivity and molecular characteristics in order to identify biomarkers of response. Methods: We systematically analyzed the association between drug sensitivity and genomic alterations in 59 HNSCC lines. Results: NOTCH1mut lines are significantly sensitive to PI3K/mTOR pathway inhibitors: GSK2126458 (13/16), BYL719 (6/16), PQR309 (13/16), BKM120 (14/16), BEZ235 (12/16), BAY806942 (14/16) and GDC0980 (13/16 lines). In contrast to PIK3CAmut cell lines, all 7 NOTCH1mut lines tested underwent apoptosis (14.3 fld; P Conclusion: In contrast to PIK3CAmut cells, NOTCH1mut HNSCC cells underwent apoptosis after PI3K/mTOR pathway inhibition in vitro and decreased tumor size in vivo. The ectopic activation of NOTCH1 rescued NOTCH1mut HNSCC cells from PI3K/mTOR inhibitor-mediated apoptosis. The underlying mechanism may involve differential effects on tumor metabolism and ROS production. This work is significant because inactivating NOTCH1 mutations, which occur in 18% of HNSCC patients and SCCs of the lung, esophagus, and other sites, may serve as a biomarker for response. Our future work may uncover previously unknown crosstalk between the PI3K/mTOR and NOTCH pathways in SCCs. Citation Format: Vaishnavi Sambandam, Li Shen, Pan Tong, Tuhina Mazumdar, Curtis Pickering, Jeffrey N. Myers, Jing Wang, Mitchell Frederick, Faye M. Johnson. Identification of NOTCH1 inactivating mutation as a therapeutic vulnerability to PI3K/mTOR pathway inhibition in head and neck squamous cell carcinoma (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 2992. doi:10.1158/1538-7445.AM2017-2992

The genomic alterations identified in head and neck squamous cell carcinoma (HNSCC) tumors have not resulted in any changes in clinical care, making the development of biomarker-driven targeted therapy for HNSCC a major translational gap in knowledge. To fill this gap, we used 59 molecularly characterized HNSCC cell lines and found that mutations of AJUBA,SMAD4 and RAS predicted sensitivity and resistance to treatment with inhibitors of polo-like kinase 1 (PLK1), checkpoint kinases 1 and 2, and WEE1. Inhibition or knockdown of PLK1 led to cell-cycle arrest at the G2/M transition and apoptosis in sensitive cell lines and decreased tumor growth in an orthotopic AJUBA-mutant HNSCC mouse model. AJUBA protein expression was undetectable in most AJUBA-mutant HNSCC cell lines, and total PLK1 protein expression was increased in cell lines wild-type for AJUBA. Exogenous expression of wild-type AJUBA in an AJUBA-mutant cell line partially rescued the phenotype of PLK1 inhibitor-induced apoptosis and decreased PLK1 substrate inhibition, suggesting a threshold effect in which higher drug doses are required to affect PLK1 substrate inhibition. PLK1 inhibition was an effective therapy for HNSCC in vitro and in vivo. However, biomarkers to guide such therapy are lacking. We identified AJUBA, SMAD4 and RAS mutations as potential candidate biomarkers of response of HNSCC to treatment with these mitotic inhibitors. Citation Format: Ming Zhang, Singh Ratnakar, Shaohua Peng, Mazumdar Tuhina, Shen Li, Pan Tong, Curtis Pickering, Jeffrey N. Myers, Jing Wang, Faye M. Johnson. Mutations of the lim protein ajuba mediate sensitivity of head and neck squamous cell carcinoma to treatment with cell cycle inhibitors [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 3816. doi:10.1158/1538-7445.AM2017-3816

Resistance to therapy, including conventional chemotherapy, targeted therapy and immunotherapy, continues to plague cancer treatment. Moreover, mechanisms governing resistance are poorly characterized leading to a dearth of rational combinatorial and sequential treatment strategies. While drug response data is abundant across myriad tumor types and drug classes, there exists no high-throughput method to probe such data with a query as simple as “If tumors are resistant to drug X, to what drug(s) are they sensitive?”- a seemingly trivial problem beset by immense data sets and imprecise definitions of sensitivity and resistance. Here, we present DISARM, a novel approach designed specifically to screen for drugs that are active in spite of resistance to a reference drug. DISARM selects candidates based on the proportion of samples that are resistant to a reference drug but sensitive to a candidate drug with simultaneous consideration to relatively lower IC50 values for candidate drugs and higher IC50 values for reference drugs. As candidates may work in only a subset of resistant models and precise delineation between sensitivity and resistance may vary between experimental settings, DISARM permits flexibility in dichotomizing drug data and uses grid search to optimize specifications. To illustrate, we analyzed publically available cell line data (IC50 data) from several cancer types for which platinum-based therapy is a standard of care, identifying multiple drugs that demonstrate activity in cisplatin-resistant models across tumor types such as the BCL-2 inhibitor obatoclax in small cell lung cancer, lung adenocarcinoma, gastric adenocarcinoma and bladder cancer, and the farnesyltransferase inhibitor tipifarnib in small cell lung cancer, bladder cancer, esophageal cancer, colon adenocarcinoma and head and neck squamous cell carcinoma. Frequently, multiple drugs from the same class were selected by DISARM for a single tumor type and, in these cases, we found statistically significant similarity between sensitive cell lines suggesting a subset of cisplatin-resistant cell lines that are repeatedly sensitive to a drug class. While translating preclinical observations into approved clinical use is often thwarted by an inability to identify predictive biomarkers, DISARM also allows us to select cell lines that are especially sensitive to candidate drugs or drug classes on which to perform biomarker analysis. To demonstrate this approach, we chose drugs with activity in multiple cancer types and compared mRNA and protein expression data to highlight potentially novel common and tumor-specific biomarkers for concomitant candidate drug sensitivity and cisplatin resistance. Thus, DISARM offers a simple yet effective approach for both drug and biomarker discovery within a specified clinical niche. Citation Format: Carl M. Gay, Pan Tong, Robert J. Cardnell, Xiao Su, Nene N. Kalu, Upasana Banerjee, Rasha O. Bara, Faye M. Johnson, John V. Heymach, Jing Wang, Lauren A. Byers. Differential sensitivity analysis for resistant malignancies (DISARM), a novel approach for drug screen analysis, identifies common candidate drugs across platinum-resistant cancer types [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 1560. doi:10.1158/1538-7445.AM2017-1560

Background: Inhibiting polo-like kinase 1 PLK1 may be an effective treatment for non-small cell lung cancer (NSCLC). PLK1 is a key regulator of mitosis and DNA damage checkpoints. PLK1 inhibitors are well tolerated, but only a few unselected patients with NSCLC respond to single-agent therapy. However, predictive biomarkers have not been used to select patients who are likely to experience a response to PLK1 inhibitors, and the mechanisms of resistance to PLK1 inhibitors have not been elucidated, making these unknowns a major gap in knowledge. To address this gap, we compared basal gene and protein expression in 63 NSCLC cell lines and discovered that mesenchymal NSCLC cell lines were more sensitive to PLK1 inhibitors than epithelial cell lines in vitro and in vivo. The induction of apoptosis in some NSCLC cell lines at very low drug concentrations and the need to find better therapy for mesenchymal NSCLC motivated us to further study PLK1 inhibition. Methods: To identify the pathways involved in PLK1 inhibitor-induced apoptosis, we used 3 pairs of isogenic NSCLC cell lines in which we had induced a mesenchymal phenotype using TGF-β. These isogenic lines were treated with the PLK1 inhibitor (volasertib) for 24 hours and levels of 301 proteins and phosphoproteins were simultaneously measured before and after treatment with volasertib using reverse phase protein array (RPPA). Results: The induction of a mesenchymal phenotype using TGF-β increased PLK1 inhibition-induced DNA damage and apoptosis in 3 NSCLC cell lines. To further elucidate mechanisms of resistance to PLK1 inhibition, we compared gene and protein expression in these isogenic cell lines, before and after PLK1 inhibition. There were 35, 12 and 43 proteins differentially regulated following PLK1 inhibition in epithelial vs. mesenchymal lines in HCC366, H1975, and HCC4006 cell lines, respectively at False Discovery rate 0.1. Phosphorylated FAK (Y397) and c-Met (Y1234/1235) were consistently inhibited following PLK1 inhibition in the mesenchymal lines but activated in the epithelial lines. These changes were confirmed by Western blotting. Total FAK and c-Met protein and mRNA levels were not affected, demonstrating post-translational changes. The inhibition of c-Met using EMD 1214063 led to FAK inhibition but FAK inhibition did not affect c-Met activation. The combination of c-Met inhibitor and volasertib increases sensitivity in NSCLC cell lines tested. The combinations led to more apoptosis than the single-agent inhibitors. Conclusions: NSCLC cell lines have diverse sensitivities to PLK1 inhibition, which is consistent with the results of clinical trials of PLK1 inhibitors in solid tumors, but no studies to date explain these diverse responses to PLK1 inhibition. We have identified c-Met activation as a previously unknown pathway of resistance to PLK1 inhibition in epithelial NSCLC. Citation Format: Ratnakar Singh, Li Shen, Pan Tong, Jing Wang, Faye M. Johnson. c-Met activation mediates resistance to polo-like kinase 1 inhibitor-induced apoptosis in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4095. doi:10.1158/1538-7445.AM2017-4095

6017 Background: In a single-arm, phase 2 study, we previously demonstrated that in pts with R/M HNSCC, cisplatin, docetaxel and E improved progression-free survival (PFS) compared to historical data (Kim et al., ASCO 2006). Herein, we evaluated this regimen in a single center, randomized, phase 2 trial. Methods: Pts with R/M HNSCC, with a performance status (PS) 0-2, were randomized (1:1) to receive up to 6 cycles of first-line chemotherapy with cisplatin 75 mg/m2 (or carboplatin AUC 6) and docetaxel 75 mg/m2 i.v. on day 1 every 21 days, plus placebo (P) vs. E 150 mg p.o. daily, followed by maintenance P or E until disease progression. The primary endpoint was PFS. With 120 pts, the study had 80% power to detect an improvement in median PFS from 3.0 to 4.9 months with a two-sided type I error rate of 0.1. Results: From 05/2010 to 07/2015, 120 pts were randomized to the P (N = 60) or E (N = 60) groups. All pts but one initiated treatment and were eligible for evaluation of the primary endpoint – 92 males; median age 62 years; 52 oropharynx, 40 oral cavity, 19 larynx, 8 hypopharynx cancer pts; 86 current/former smokers; 43 with recurrence within 6 months of completion of local treatment; 27 with prior exposure to EGFR inhibitors. Median PFS was 4.4 vs. 6.1 months for the P and E groups, respectively (hazard ratio [HR] 0.63, 95% confidence interval [CI] 0.42-0.95 months, p = 0.026). Response rates were 44% vs. 56% for P vs. E (p = 0.21). Median overall survival (OS) for P- and E-treated pts was 13.7 vs. 17.0 months (HR = 0.67, 95% CI 0.43-1.04, p = 0.07). Benefits from E on PFS and OS were more pronounced in pts with oropharyngeal tumors (p≤0.05 for interaction). In the E group, first-cycle rash grade 2-4 (34% pts) was associated with longer OS (HR = 0.40, p = 0.02). E-treated pts experienced a higher incidence of grade 3-4 adverse events (33.9 vs. 53.3%), including diarrhea (3 vs.17%), dehydration (5 vs. 15%), nausea (5 vs. 14%), rash (0 vs. 12%). Conclusions: This study met its primary endpoint. Addition of E to first-line platinum/docetaxel improved PFS and OS. This regimen may warrant further evaluation in randomized, phase 3 trials. Clinical trial information: NCT01064479.