Ashley E. Winkler

ERK1/2 regulation of CD44 modulates oral cancer aggressiveness

Carcinogen-induced oral cavity squamous cell carcinoma (OSCC) incurs significant morbidity and mortality and constitutes a global health challenge. To gain further insight into this disease, we generated cell line models from 7,12-dimethylbenz(a)anthracene-induced murine primary OSCC capable of tumor formation upon transplantation into immunocompetent wild-type mice. Whereas several cell lines grew rapidly and were capable of metastasis, some grew slowly and did not metastasize. Aggressively growing cell lines displayed ERK1/2 activation, which stimulated expression of CD44, a marker associated with epithelial to mesenchymal transition and putative cancer stem cells. MEK (MAP/ERK kinase) inhibition upstream of ERK1/2 decreased CD44 expression and promoter activity and reduced cell migration and invasion. Conversely, MEK1 activation enhanced CD44 expression and promoter activity, whereas CD44 attenuation reduced in vitro migration and in vivo tumor formation. Extending these findings to freshly resected human OSCC, we confirmed a strict relationship between ERK1/2 phosphorylation and CD44 expression. In summary, our findings identify CD44 as a critical target of ERK1/2 in promoting tumor aggressiveness and offer a preclinical proof-of-concept to target this pathway as a strategy to treat head and neck cancer.

A surprising cross-species conservation in the genomic landscape of mouse and human oral cancer identifies a transcriptional signature predicting metastatic disease

Purpose: Improved understanding of the molecular basis underlying oral squamous cell carcinoma (OSCC) aggressive growth has significant clinical implications. Herein, cross-species genomic comparison of carcinogen-induced murine and human OSCCs with indolent or metastatic growth yielded results with surprising translational relevance. Experimental Design: Murine OSCC cell lines were subjected to next-generation sequencing (NGS) to define their mutational landscape, to define novel candidate cancer genes, and to assess for parallels with known drivers in human OSCC. Expression arrays identified a mouse metastasis signature, and we assessed its representation in four independent human datasets comprising 324 patients using weighted voting and gene set enrichment analysis. Kaplan–Meier analysis and multivariate Cox proportional hazards modeling were used to stratify outcomes. A quantitative real-time PCR assay based on the mouse signature coupled to a machine-learning algorithm was developed and used to stratify an independent set of 31 patients with respect to metastatic lymphadenopathy. Results: NGS revealed conservation of human driver pathway mutations in mouse OSCC, including in Trp53, mitogen-activated protein kinase, phosphoinositide 3-kinase, NOTCH, JAK/STAT, and Fat1-4. Moreover, comparative analysis between The Cancer Genome Atlas and mouse samples defined AKAP9, MED12L, and MYH6 as novel putative cancer genes. Expression analysis identified a transcriptional signature predicting aggressiveness and clinical outcomes, which were validated in four independent human OSCC datasets. Finally, we harnessed the translational potential of this signature by creating a clinically feasible assay that stratified patients with OSCC with a 93.5% accuracy. Conclusions: These data demonstrate surprising cross-species genomic conservation that has translational relevance for human oral squamous cell cancer. Clin Cancer Res; 20(11); 2873–84. ©2014 AACR.

CXCR3 enhances a T cell dependent epidermal proliferative response and promotes skin tumorigenesis

The chemokine receptor CXCR3 has been proposed to play a critical role in host antitumor responses. In this study, we defined CXCR3-expressing immune cell infiltration in human skin squamous cell carcinomas and then used CXCR3-deficient mice to assess the contribution of CXCR3 to skin tumorigenesis. Our studies employed two established protocols for chemical skin carcinogenesis [methylcholanthrene (MCA) or 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) models]. CXCR3 deletion did not affect tumor development in the MCA model; however, CXCR3 was important in the DMBA/TPA model where gene deletion reduced the incidence of skin tumors. This decreased incidence of skin tumors did not reflect differences in epidermal development but rather was associated with reduced epidermal thickness and proliferation in CXCR3(-/-) mice, implicating the CXCR3 pathway in DMBA/TPA-induced epidermal inflammation and proliferation. Notably, CXCR3 expressed in CD4(+) and CD8(+) T cells was found to be important for enhanced epidermal proliferation. Specifically, CXCR3-deficient mice reconstituted with T cells isolated from wild-type mice treated with DMBA/TPA restored wild-type levels of epidermal proliferation in the mutant mice. Taken together, our findings establish that CXCR3 promotes epidermal tumorigenesis likely through a T-cell-dependent induction of keratinocyte proliferation.

Comparative Analysis of Tumor- Infiltrating Lymphocytes in a Syngeneic Mouse Model of Oral Cancer

Objective To perform a comparative analysis of infiltrating immune cells in a newly developed C57BL/6 background syngeneic transplantable mouse oral cancer (MOC) model. Study Design/Setting Scientific study in an academic medical center. Methods Use of carcinogen-induced tumorigenesis, tissue culture, cell line transplantation, and flow cytometric analysis techniques. Results Previously, the authors established a series of cell line models that displayed dichotomous growth phenotypes when transplanted into immunocompetent mice. They now show that the indolent growth pattern of the MOC1-generated tumors is associated with increased baseline and inducible major histocompatibility complex class I expression and increased CD8+ T-cell infiltration into the tumor microenvironment. Conversely, the aggressive and metastatic pattern of MOC2-generated tumors has decreased basal and inducible class I expression and is associated with FOXP3+CD4+ regulatory T-cell infiltration. Delayed primary tumor growth after targeted monoclonal antibody therapy of these FOXP3+ regulatory cells further suggests that these immune cells contribute to the aggressive phenotype of MOC2. Conclusion These data validate that key infiltrating immune cells identified here parallel findings in human head and neck cancer, making this newly developed syngeneic model a critical platform for the continued dissection of tumor-host interactions in head and neck cancer.

Biomarker and Tumor Responses of Oral Cavity Squamous Cell Carcinoma to Trametinib: A Phase II Neoadjuvant Window of Opportunity Clinical Trial

Purpose: Ras/MEK/ERK pathway activation is common in oral cavity squamous cell carcinoma (OCSCC). We performed a neoadjuvant (preoperative) trial to determine the biomarker and tumor response of OCSCC to MEK inhibition with trametinib. Experimental Design: Patients with stage II–IV OCSCC received trametinib (2 mg/day, minimum 7 days) prior to surgery. Primary tumor specimens were obtained before and after trametinib to evaluate immunohistochemical staining for p-ERK1/2 and CD44, the primary endpoint. Secondary endpoints included changes in clinical tumor measurements and metabolic activity [maximum standardized uptake values (SUVmax) by F-18 fluorodeoxyglucose positron emission tomography/CT), and in tumor downstaging. Drug-related adverse events (AE) and surgical/wound complications were evaluated. Results: Of 20 enrolled patients, 17 (85%) completed the study. Three patients withdrew because of either trametinib-related (n = 2: nausea, duodenal perforation) or unrelated (n = 1: constipation) AEs. The most common AE was rash (9/20 patients, 45%). Seventeen patients underwent surgery. No unexpected surgical/wound complications occurred. Evaluable matched pre- and posttrametinib specimens were available in 15 (88%) of these patients. Reduction in p-ERK1/2 and CD44 expression occurred in 5 (33%) and 2 (13%) patients, respectively. Clinical tumor response by modified World Health Organization criteria was observed in 11 of 17 (65%) evaluable patients (median 46% decrease, range 14%–74%). Partial metabolic response (≥25% reduction in SUVmax) was observed in 6 of 13 (46%) evaluable patients (median 25% decrease, range 6%–52%). Clinical-to-pathologic tumor downstaging occurred in 9 of 17 (53%) evaluable patients. Conclusions: Trametinib resulted in significant reduction in Ras/MEK/ERK pathway activation and in clinical and metabolic tumor responses in patients with OCSCC. Clin Cancer Res; 23(9); 2186–94. ©2016 AACR.

Genomic analysis to define molecular basis of aggressiveness in a mouse model of oral cancer

To investigate the molecular basis underlying aggressive behavior in oral squamous cell carcinoma (OSCC), our laboratory developed a carcinogen-induced mouse oral cancer (MOC) cell line model that encompasses the growth and metastasis spectrum of its human counterpart. We performed next-generation sequencing (NGS) and gene expression microarray profiles to explore the genomic and transcriptional backgrounds of the differential MOC line phenotypes, as well as, the cross-species relevance of the model. Here we describe the comparative analysis of NGS (www.ncbi.nlm.nih.gov/biosample?LinkName=bioproject_biosample_all&from_uid=247825) and expression microarray (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE50041) data from the MOC lines and corresponding human data, as described in our recent publication [1].

Oral Cavity Squamous Cell Carcinoma Xenografts Retain Complex Genotypes and Intertumor Molecular Heterogeneity

SUMMARY Herein, we report an oral cavity squamous cell carcinoma (OCSCC) patient-derived xenograft (PDX) platform, with genomic annotation useful for co-clinical trial and mechanistic studies. Genomic analysis included whole-exome sequencing (WES) and transcriptome sequencing (RNA-seq) on 16 tumors and matched PDXs and additional whole-genome sequencing (WGS) on 9 of these pairs as a representative subset of a larger OCSCC PDX repository (n = 63). In 12 models with high purity, more than 90% of variants detected in the tumor were retained in the matched PDX. The genomic landscape across these PDXs reflected OCSCC molecular heterogeneity, including previously described basal, mesenchymal, and classical molecular subtypes. To demonstrate the integration of PDXs into a clinical trial framework, we show that pharmacological intervention in PDXs parallels clinical response and extends patient data. Together, these data describe a repository of OCSCC-specific PDXs and illustrate conservation of primary tumor genotypes, intratumoral heterogeneity, and co-clinical trial application.

Abstract PR01: Genomic and functional correlates from a phase II clinical trial of trametinib in surgically resectable oral cavity squamous cell carcinoma

Background: Neoadjuvant trametinib treatment of patients with locally advanced oral cavity squamous cell carcinoma (OSCC) demonstrates metabolic- and biomarker-based responses (Uppaluri et al., Clin. Can. Res, 2016). Patient response, defined as both metabolic response and decreased ERK phosphorylation in trametinib-treated tumors in comparison to baseline biopsies, was seen in 4/20 patients. We hypothesized that genomic and transcriptomic analyses of the pre- and post-treatment samples would provide insight into biomarkers of response for stratification of patients in future clinical trials. Methods: Tumor biopsies were collected at baseline and post-treatment at surgical resection. To generate patient-derived xenografts (PDX), biopsies from both baseline and post-treatment were implanted into immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Whole exome and RNA sequencing (WES, RNAseq) were performed on baseline specimens and RNA-Seq was also performed on matched post-treatment tumor samples. Successful PDX models were also interrogated with whole exome and RNA-Seq. For PDX therapy, tumor bearing NSG mice were treated with trametinib (GSK1120212) via daily oral gavage (3 mg/kg/dose) or vehicle control and monitored for tumor growth. Results:WES—The mutational landscape of our cohort largely reflected the constellation of mutations present in the TCGA OSCC samples. Mutations in TP53 occurred in 15/20 (75%) of patients, CDKN2A in 8/20 (40%), NOTCH1 in 6/20 (30%), FAT1 in 5/20 (25%) and CASP8 in 5/20 (25%) of patients. Although gain of function mutations in RAS are associated with activation of the RAS/RAF/MEK/ERK pathway, no patients in our trial had RAS, RAF or ERK mutations. RNA-Seq—We identified crosstalk between the MEK/RAF/ERK pathway and the Hippo pathway. Responder patients had significantly higher YAP1 expression at baseline than the non-responders (p=0.0141). Furthermore, treatment with trametinib induced substantial reduction in Hippo pathway associated genes. Matched RNA-Seq analysis of two of three responder patients showed decreased expression of YAP1 and its downstream targets FOXM1 and BIRC5, known mediators of cell survival and metastasis. PDX—We successfully established PDXs in 22 of 39 tumor samples. Comparison of the WES in a subset of early passage PDXs to their matched primary tumors (n=9) demonstrated conservation with the primary tumor mutational content. To evaluate if treatment of the PDX model would reflect in vivo responsiveness of the primary tumor we treated the baseline biopsy PDX of a responder with daily trametinib or vehicle control (n=8 each). Consistent with findings in the patient, all PDXs treated with trametinib had significant decrease in tumor size after 14 days of daily treatment whereas the control treated tumors grew progressively (average tumor volume 52 mm3 and 714 mm3, respectively. p=0.013). As the 14-day short window phase did not allow full tumor responses to mature in patients, we modeled continuous treatment beyond 14 days in the PDX to evaluate for clinical response. Whereas 8/8 vehicle treated PDXs displayed progressive growth, 7/8 trametinib-treated PDXs were dramatically attenuated until eventual escape and outgrowth between days 40-60. Conclusion: In this genomic and functional analysis of samples from a trametinib window trial in OSCC, we identified YAP1 overexpression to be associated with clinical and biomarker response. Functional inhibition of this pathway with trametinib was identified in 2/3 patients with reductions in YAP1, FOXM1 and BIRC5 message. Finally, we demonstrated successful prospective establishment of PDX models that prove useful for confirming patient responses and for pathway specific molecular dissection. This abstract is also being presented as Poster 65. Citation Format: Paul A. Zolkind, Katie M. Campbell, Tianxiang Lin, Zach Skidmore, Ashley Winkler, Erica Barnell, Tusar Giri, Douglas R. Adkins, Malachi Griffith, Gavin P. Dunn, Obi L. Griffith, Ravindra Uppaluri. Genomic and functional correlates from a phase II clinical trial of trametinib in surgically resectable oral cavity 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 PR01.

Abstract CT070: Biomarker and clinical response of oral cavity squamous cell carcinoma to the MEK 1/2 inhibitor trametinib: A phase II neoadjuvant window of opportunity clinical trial

Purpose: Ras/MEK/ERK pathway activation is common in oral cavity squamous cell carcinoma (OCSCC). To determine biomarker and clinical tumor response of MEK inhibition in patients with OCSCC, we performed a neoadjuvant window of opportunity trial in which the MEK inhibitor trametinib was administered before surgery (NCT01553851). Patients and Methods: Patients with untreated Stage II-IV OCSCC were scheduled to receive trametinib 2 mg/day orally for 7-14 days prior to surgery (last dose 24 hours before surgery). Tumor specimens from the primary site obtained before and after trametinib underwent immunohistochemistry staining for p-ERK1/2 (a marker of Ras/MEK/ERK activation) and CD44 (a protein upregulated by ERK activation), which represented the primary endpoint. Secondary endpoints included comparison of changes in pre- and post-trametinib tumor measurement by clinical examination and in metabolic activity (SUVmax) by FDG-PET/CT (partial response: >25% reduction). Adverse events (AE) and surgical/wound complications were evaluated. Results: Of the 20 enrolled patients, 17 (85%) completed the study as planned. Three patients withdrew from the study due to AE, two (nausea; duodenal perforation) related to trametinib and one (constipation) related to narcotics. The most common drug-related AE was mild rash (9/20 patients, 45%). Nineteen patients (95%) underwent surgery and neck dissection with no unexpected surgical/wound complications. Fifteen patients (75%) were evaluable for the primary biomarker endpoint. 5 (25%) patients either had insufficient pre- or post-treatment biopsies or did not complete the trial. Reduction in p-ERK1/2 expression occurred in 7/15 evaluable patients (47%), whereas a reduction in CD44 occurred in 3/15 (20%). Reduction in tumor size (median 40%, range -74 to +17%) assessed by clinical examination was observed in 12/17 (71%) evaluable patients, and partial metabolic tumor response assessed by FDG-PET/CT was observed in 5/13 (38%) patients. Conclusions: Trametinib was safe to administer as a neoadjuvant treatment in patients with OCSCC and several patients displayed significant reduction in Ras/MEK/ERK pathway activation, and in clinical and metabolic tumor responses. Further exploration of trametinib response in OCSCC patients is warranted. Citation Format: Ravindra Uppaluri, Ashley Winkler, Tianxiang Lin, Jonathan Law, Bruce Haughey, Brian Nussenbaum, Randal Paniello, Jason Rich, Jason Diaz, Loren Michel, Tanya Wildes, Gavin Dunn, Dorina Kallogjeri, Paul Zolkind, Farrokh Dehdashti, Barry Siegel, Rebecca Chernock, James S. Lewis, Jay Piccirillo, Douglas Adkins. Biomarker and clinical response of oral cavity squamous cell carcinoma to the MEK 1/2 inhibitor trametinib: A phase II neoadjuvant window of opportunity clinical trial. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT070.

A Comparative Analysis of Immune Infiltrating Lymphocytes in a Murine Model of Oral Cancer

Objective: 1) Understand types of infiltrating immune cells that contribute to the tumor microenvironment. 2) Understand the relative difference in immune infiltrates between aggressive and indolent tumor phenotypes in a new syngeneic model of OSCC. Method: This research is a preclinical scientific study focusing on mouse derived oral cavity squamous cell carcinoma that was conducted over 6 months at Washington University in St Louis. This study analyzed tumor infiltrating immune cells by flow cytometry and identified individual infiltrating populations associated with either aggressive or indolent growth. Results: Indolent growing mouse oral cancer (MOC) lines were associated with increased expression of increased MHC class I expression and increased CD8+ T-cell infiltration into the tumor microenvironment. However, aggressive and metastatic MOC lines were associated with decreased class I expression and increased FOXP3+ CD4+ T-cell infiltration and targeting these regulatory T-cells delayed tumor growth in vivo. Conclusion: These data validate that key infiltrating immune cells identified here parallel findings in human head and neck cancer, making this newly developed syngeneic model a critical platform for the continued dissection of tumor-host interactions in head and neck cancer.