Genome-wide open reading frame profiling identifies fibroblast growth factor signaling as a driver of PD-L1 expression in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinomas (HNSCC) are associated with significant treatment-related morbidity and poor disease-free and disease-specific survival, especially in the recurrent and metastatic (R/M HNSCC) setting. Inhibition of the programmed death-1/ligand-1 (PD-1/PD-L1) immune checkpoint is accepted as a first-line treatment strategy for R/M HNSCC and has expanded into the neoadjuvant, definitive, and adjuvant settings. To understand cellular signals modulating the PD-L1 in HNSCC, we profiled a HNSCC cell-line with a genome-wide open reading frame (ORF) library of 17,000 individual constructs (14,000 unique genes). We identified 335 ORFs enriched in PD-L1high cells and independently validated five of these ORFs (FGF6, IL17A, CD300C, KLR1C and NFKBIA) as drivers of PD-L1 upregulation. We showed that exogenous FGF ligand is sufficient to induce PD-L1 expression in multiple HNSCC cell lines and human immature dendritic cells. Accordingly, overexpression of FGFR1, FGFR3 or the FGFR3 S249C and D786N mutants common to HNSCC tumors also induced PD-L1 overexpression on tumor cells. Small molecule inhibition of FGF signaling abrogated PD-L1 upregulation in these models and also blocked "classical" IFNγ-regulated PD-L1 expression in a STAT1-independent manner. Finally, we found that FGF specifically upregulated a glycosylated form of PD-L1 in our study, and exogenous FGF led to concomitant upregulation of glycosyltransferases that may stabilize PD-L1 on the surface of HNSCC cells. Taken together, our study supports a potential role for FGF/FGFR pathway signaling as a mechanism driving immune escape and rationalizes further exploration of novel combination therapies to improve clinical responses to PD-1/PD-L1 axis inhibition in HNSCC.

ScRNA-seq defines dynamic T-cell subsets in longitudinal colon and peripheral blood samples in immune checkpoint inhibitor-induced colitis.

Immune checkpoint inhibitors (ICIs) are increasingly being used to manage multiple tumor types. Unfortunately, immune-related adverse events affect up to 60% of recipients, often leading to treatment discontinuation in settings where few alternative cancer therapies may be available. Checkpoint inhibitor induced colitis (ICI-colitis) is a common toxicity for which the underlying mechanisms are poorly defined. To better understand the changing colon-specific and peripheral immune environments over the course of progression and treatment of colitis, we collected blood and colon tissue from a patient with Merkel cell carcinoma who developed colitis on treatment with pembrolizumab. We performed single-cell RNA sequencing and T-cell receptor sequencing on samples collected before, during and after pembrolizumab and after various interventions to mitigate toxicity. We report T-cells populations defined by cytotoxicity, memory, and proliferation markers at various stages of colitis. We show preferential depletion of CD8+ T cells with biologic therapy and nominate both circulating and colon-resident T-cell subsets as potential drivers of inflammation and response to immune suppression. Our findings highlight the need for further exploration of the colon immune environment and rationalize future studies evaluating biologics for ICI-colitis, including in the context of ICI re-challenge.

Prognostic value of CD103+ tumor-infiltrating lymphocytes and programmed death ligand-1 (PD-L1) combined positive score in recurrent laryngeal squamous cell carcinoma.

OBJECTIVES: In an evolving era of immunotherapeutic options for persistent or recurrent laryngeal squamous cell carcinoma (LSCC), there is a need for improved biomarkers of treatment response and survival to inform optimal treatment selection and prognostication. Herein, our primary objective was to explore correlations between tumor infiltrating lymphocytes (TILs) and PD-L1 Combined Positive Score (CPS). Secondarily, we sought to explore their combined association with survival outcomes in patients with persistent or recurrent LSCC treated with salvage surgery. MATERIALS AND METHODS: This was a retrospective cohort study at a single academic medical center. Immunohistochemistry staining for TILs and PD-L1 was performed on a tissue microarray of persistent or recurrent LSCC pathologic specimens. Correlations between TIL subsets and PD-L1 CPS were examined using Pearson's correlation coefficient and survival outcomes were analyzed with the Kaplan-Meier method and log-rank tests. RESULTS: Only CD103+ TILs showed a statistically significant, weakly-positive correlation with PD-L1 CPS (r2 = 0.264, p < 0.015). No other TIL subsets correlated with PD-L1 CPS in our cohort. The most favorable survival outcomes were seen in patients with pathologic N0 tumors showing high CD103+ TILs and/or high PD-L1 CPS staining. CONCLUSION: Among patients with persistent or recurrent LSCC, CD103+ TILs only modestly correlated with PD-L1 CPS. A combined biomarker score incorporating CD103+ TILs and PD-L1 CPS greatly enhanced survival discrimination. This model may have additional utility in predicting the clinical benefit of immunotherapies in persistent or recurrent LSCC in the future.

Microbe-Mediated Activation of Toll-like Receptor 2 Drives PDL1 Expression in HNSCC.

As immunotherapies targeting the PDL1 checkpoint have become a mainstay of treatment for a subset of head and neck squamous cell carcinoma (HNSCC) patients, a detailed understanding of the mechanisms underlying PDL1-mediated immune evasion is needed. To elucidate factors regulating expression of PDL1 in HNSCC cells, a genome-wide CRISPR profiling approach was implemented to identify genes and pathways conferring altered PDL1 expression in an HNSCC cell line model. Our screen nominated several candidate PDL1 drivers, including Toll-like Receptor 2 (TLR2). Depletion of TLR2 blocks interferon-γ-induced PDL1 expression, and stimulation of TLR2 with either Staphylococcus aureus or a bacterial lipopeptide mimetic, Pam3CSK4, enhanced PDL1 expression in multiple models. The data herein demonstrate a role for TLR2 in modulating the expression of PDL1 in HNSCC models and suggest that microbiota may directly modulate immunosuppression in cancer cells. Our study represents a step toward disentangling the diverse pathways and stimuli regulating PDL1 expression in HNSCC and underscores a need for future work to characterize the complex microbiome in HNSCC patients treated with immunotherapy.

The molecular landscape of the University of Michigan laryngeal squamous cell carcinoma cell line panel.

BACKGROUND: Laryngeal squamous cell carcinomas (LSCCs) have a high risk of recurrence and poor prognosis. Patient-derived cancer cell lines remain important preclinical models for advancement of new therapeutic strategies, and comprehensive characterization of these models is vital in the precision medicine era. METHODS: We performed exome and transcriptome sequencing as well as copy number analysis of a panel of LSCC-derived cell lines that were established at the University of Michigan and are used in laboratories worldwide. RESULTS: We observed a complex array of alterations consistent with those reported in The Cancer Genome Atlas head and neck squamous cell carcinoma project, including aberrations in PIK3CA, EGFR, CDKN2A, TP53, and NOTCH family and FAT1 genes. A detailed analysis of FAT family genes and associated pathways showed disruptions to these genes in most cell lines. CONCLUSIONS: The molecular profiles we have generated indicate that as a whole, this panel recapitulates the molecular diversity observed in patients and will serve as useful guides in selecting cell lines for preclinical modeling.

Analysis of tumor-infiltrating CD103 resident memory T-cell content in recurrent laryngeal squamous cell carcinoma.

BACKGROUND: Recurrent laryngeal squamous cell carcinomas (LSCCs) are associated with poor outcomes, without reliable biomarkers to identify patients who may benefit from adjuvant therapies. Given the emergence of tumor-infiltrating lymphocytes (TIL) as a biomarker in head and neck squamous cell carcinoma, we generated predictive models to understand the utility of CD4+, CD8+ and/or CD103+ TIL status in patients with advanced LSCC. METHODS: Tissue microarrays were constructed from salvage laryngectomy specimens of 183 patients with recurrent/persistent LSCC and independently stained for CD4+, CD8+, and CD103+ TIL content. Cox proportional hazards regression analysis was employed to assess combinations of CD4+, CD8+, and CD103+ TIL levels for prediction of overall survival (OS), disease-specific survival (DSS), and disease-free survival (DFS) in patients with recurrent/persistent LSCC. RESULTS: High tumor CD103+ TIL content was associated with significantly improved OS, DSS, and DFS and was a stronger predictor of survival in recurrent/persistent LSCC than either high CD8+ or CD4+ TIL content. On multivariate analysis, an "immune-rich" phenotype, in which tumors were enriched for both CD103+ and CD4+ TILs, conferred a survival benefit (OS hazard ratio: 0.28, p = 0.0014; DSS hazard ratio: 0.09, p = 0.0015; DFS hazard ratio: 0.18, p = 0.0018) in recurrent/persistent LSCC. CONCLUSIONS: An immune profile driven by CD103+ TIL content, alone and in combination with CD4+ TIL content, is a prognostic biomarker of survival in patients with recurrent/persistent LSCC. Predictive models described herein may thus prove valuable in prognostic stratification and lead to personalized treatment paradigms for this patient population.

The genomic landscape of UM-SCC oral cavity squamous cell carcinoma cell lines.

OBJECTIVES: We sought to describe the genetic complexity of 14 UM-SCC oral cavity cancer cell lines that have remained uncharacterized despite being used as model systems for decades. MATERIALS AND METHODS: We performed exome sequencing on 14 oral cavity UM-SCC cell lines and denote the mutational profile of each line. We used a SNP array to profile the multiple copy number variations of each cell line and use immunoblotting to compare alterations to protein expression of commonly amplified genes (EGFR, PIK3CA, etc.). RNA sequencing was performed to characterize the expression of genes with copy number alterations. RESULTS: The cell lines displayed a highly complex network of genetic aberrations that was consistent with alterations identified in the HNSCC TCGA project including PIK3CA amplification, CDKN2A deletion, as well as TP53 and CASP8 mutations, enabling genetic stratification of each cell line in the panel. Copy number FISH and spectral karyotyping analysis demonstrate that cell lines retain chromosomal heterogeneity. CONCLUSIONS: Collectively, we developed an important resource for future oral cavity HNSCC cell line studies and highlight the complexity of genomic aberrations in cell lines.

Fyn Regulates Binding Partners of Cyclic-AMP Dependent Protein Kinase A.

The cAMP-dependent protein kinase A (PKA) is a serine/threonine kinase involved in many fundamental cellular processes, including migration and proliferation. Recently, we found that the Src family kinase Fyn phosphorylates the catalytic subunit of PKA (PKA-C) at Y69, thereby increasing PKA kinase activity. We also showed that Fyn induced the phosphorylation of cellular proteins within the PKA preferred target motif. This led to the hypothesis that Fyn could affect proteins in complex with PKA. To test this, we employed a quantitative mass spectrometry approach to identify Fyn-dependent binding partners in complex with PKA-C. We found Fyn enhanced the binding of PKA-C to several cytoskeletal regulators that localize to the centrosome and Golgi apparatus. Three of these Fyn-induced PKA interactors, AKAP9, PDE4DIP, and CDK5RAP2, were validated biochemically and were shown to exist in complex with Fyn and PKA in a glioblastoma cell line. Intriguingly, the complexes formed between PKA-C and these known AKAPs were dependent upon Fyn catalytic activity and expression levels. In addition, we identified Fyn-regulated phosphorylation sites on proteins in complex with PKA-C. We also identified and biochemically validated a novel PKA-C interactor, LARP4, which complexed with PKA in the absence of Fyn. These results demonstrate the ability of Fyn to influence the docking of PKA to specific cellular scaffolds and suggest that Fyn may affect the downstream substrates targeted by PKA.

Proportion of CD4 and CD8 tumor infiltrating lymphocytes predicts survival in persistent/recurrent laryngeal squamous cell carcinoma.

Tumor infiltrating lymphocytes (TILs) have been shown to be an important prognostic factor in patients with previously untreated head and neck cancer. After organ preservation therapy for laryngeal cancer and subsequent persistence/recurrence, the prognostic value of TILs is unknown. Our goal was to determine if TILs have value as a prognostic biomarker in patients with surgically salvageable persistent/recurrent laryngeal squamous cell carcinoma. Levels of TILs were quantified on tissue microarrays from 183 patients undergoing salvage total laryngectomy for persistent/recurrent laryngeal cancer after radiation or chemoradiation between 1997 and 2014. Demographic and clinical data were abstracted. Immunohistology evaluation included CD4, CD8, PDL-1, p16, CD31, Vimentin, EGFR, and p53. Elevated levels of either CD8 or CD4 positive TILs were associated with improved disease specific survival (CD8: HR 0.46, 95% CI 0.24-0.88, CD4: HR 0.43; 95% CI 0.21-0.89) and disease free survival (CD8: HR 0.53, 95% CI 0.29-0.94, CD4: HR 0.52; 95% CI 0.27-0.99). Levels of CD8 (HR 0.74; 95% CI 0.47-1.17) or CD4 (HR 0.66; 95% CI 0.40-1.08) TILs were not significantly associated with overall survival. In bivariate analysis, patients with elevated CD4 and/or CD8 TILs had significantly improved disease specific survival (HR 0.42; 95% CI 0.21-0.83) and disease free survival (HR 0.45; 95% CI 0.24-0.84) compared to patients with low levels of CD4 and CD8. PDL-1, p16, CD31, Vimentin, EGFR, and p53 were not significant prognostic factors. On multivariate analysis, elevated CD8 TILs were associated with improved disease specific survival (HR 0.35; 95% CI 0.14-0.88, p = .02) and disease free survival (HR 0.41; 95% CI 0.17-0.96, p = .04). CD8, and possibly CD4, positive TILs are associated with favorable disease free and disease specific survival for recurrent/persistent laryngeal cancer.

Identification of Targetable ERBB2 Aberrations in Head and Neck Squamous Cell Carcinoma.

IMPORTANCE: ERBB2 (formerly HER2) is an important drug target in breast cancer, where anti-ERBB2 therapy has been shown to lead to improvements in disease recurrence and overall survival. ERBB2 status in head and neck squamous cell carcinoma (HNSCC) has not been well studied. Identification of ERBB2-positive tumors and characterization of response to ERBB2 therapy could lead to targeted treatment options in HNSCC. OBJECTIVE: To identify ERBB2 aberrations in HNSCCs and investigate the potential for ERBB2-targeted therapy in HNSCCs. DESIGN, SETTING, AND PARTICIPANTS: A retrospective case series of patients with laryngeal (42 tumor specimens) and oral cavity (94 tumor specimens) SCC enrolled in the University of Michigan Head and Neck Specialized Program of Research Excellence was conducted. Publicly available sequencing data (The Cancer Genome Atlas), as well as data from other studies, were reviewed to identify additional mutations and overexpression in ERBB2 in HNSCC. Established HNSCC cell lines were used for follow-up in vitro analysis. The study was conducted from October 1, 2014, to August 30, 2015. INTERVENTIONS: With the use of targeted, amplicon-based sequencing with the Oncomine Cancer Panel, the copy number and mutation status of commonly altered genes in HNSCCs were assessed. Immunohistochemical staining was performed on tissue microarrays of HNSCCs to assess the expression of ERBB2. Western blotting for HNSCC cell line ERBB2 expression and cell survival assays after treatment with ERBB2 inhibitors were performed. MAIN OUTCOMES AND MEASURES: The prevalence of ERBB2 genetic aberrations and ERBB2 overexpression in laryngeal and oral cavity SCCs, prevalence of ERBB2 aberrations in HNSCC in The Cancer Genome Atlas, ERBB2 protein expression in HNSCC cell lines, and response of HNSCC cell lines to targeted ERBB2 inhibitors. RESULTS: Of the 42 laryngeal SCC samples screened by targeted sequencing, 4 (10%) were positive for ERBB2 amplification. Two of these samples showed ERBB2 overexpression on immunohistochemistry. Two of the 94 oral cavity SCC samples (2%) were positive for ERBB2 on immunohistochemistry. Analysis of 288 patients from publicly available HNSCC sequencing data revealed 9 amplifications (3%) in ERBB2. Protein expression was variable across HNSCC cell lines, and a subset of these cell lines showed responsiveness to anti-ERBB2 therapy. CONCLUSIONS AND RELEVANCE: ERBB2 aberrations were identified in a subset of HNSCCs. These tumors may be responsive to targeted therapy against ERBB2. Screening for ERBB2 aberrations and applying targeted therapy in ERBB2-positive patients may be useful in personalized therapy trials, particularly in patients who are refractory to current treatment paradigms.

ATR-101, a Selective and Potent Inhibitor of Acyl-CoA Acyltransferase 1, Induces Apoptosis in H295R Adrenocortical Cells and in the Adrenal Cortex of Dogs.

ATR-101 is a novel, oral drug candidate currently in development for the treatment of adrenocortical cancer. ATR-101 is a selective and potent inhibitor of acyl-coenzyme A:cholesterol O-acyltransferase 1 (ACAT1), an enzyme located in the endoplasmic reticulum (ER) membrane that catalyzes esterification of intracellular free cholesterol (FC). We aimed to identify mechanisms by which ATR-101 induces adrenocortical cell death. In H295R human adrenocortical carcinoma cells, ATR-101 decreases the formation of cholesteryl esters and increases FC levels, demonstrating potent inhibition of ACAT1 activity. Caspase-3/7 levels and terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate nick end labeled-positive cells are increased by ATR-101 treatment, indicating activation of apoptosis. Exogenous cholesterol markedly potentiates the activity of ATR-101, suggesting that excess FC that cannot be adequately esterified increases caspase-3/7 activation and subsequent cell death. Inhibition of calcium release from the ER or the subsequent uptake of calcium by mitochondria reverses apoptosis induced by ATR-101. ATR-101 also activates multiple components of the unfolded protein response, an indicator of ER stress. Targeted knockdown of ACAT1 in an adrenocortical cell line mimicked the effects of ATR-101, suggesting that ACAT1 mediates the cytotoxic effects of ATR-101. Finally, in vivo treatment of dogs with ATR-101 decreased adrenocortical steroid production and induced cellular apoptosis that was restricted to the adrenal cortex. Together, these studies demonstrate that inhibition of ACAT1 by ATR-101 increases FC, resulting in dysregulation of ER calcium stores that result in ER stress, the unfolded protein response, and ultimately apoptosis.

Novel autophosphorylation sites of Src family kinases regulate kinase activity and SH2 domain-binding capacity.

Src family tyrosine kinases (SFKs) are critical players in normal and aberrant biological processes. While phosphorylation importantly regulates SFKs at two known tyrosines, large-scale phosphoproteomics have revealed four additional tyrosines commonly phosphorylated in SFKs. We found these novel tyrosines to be autophosphorylation sites. Mimicking phosphorylation at the C-terminal site to the activation loop decreased Fyn activity. Phosphomimetics and direct phosphorylation at the three SH2 domain sites increased Fyn activity while reducing phosphotyrosine-dependent interactions. While 68% of human SH2 domains exhibit conservation of at least one of these tyrosines, few have been found phosphorylated except when found in cis to a kinase domain.