PD-L1 is upregulated via BRD2 in head and neck squamous cell carcinoma models of acquired cetuximab resistance.

BACKGROUND: Tumor models resistant to EGFR tyrosine kinase inhibitors or cisplatin express higher levels of the immune checkpoint molecule PD-L1. We sought to determine whether PD-L1 expression is elevated in head and neck squamous cell carcinoma (HNSCC) models of acquired cetuximab resistance and whether the expression is regulated by bromodomain and extraterminal domain (BET) proteins. METHODS: Expression of PD-L1 was assessed in HNSCC cell line models of acquired cetuximab resistance. Proteolysis targeting chimera (PROTAC)- and RNAi-mediated targeting were used to assess the role of BET proteins. RESULTS: Cetuximab-resistant HNSCC cells expressed elevated PD-L1 compared to cetuximab-sensitive controls. Treatment with the BET inhibitor JQ1, the BET PROTAC MZ1, or RNAi-mediated knockdown of BRD2 decreased PD-L1 expression. Knockdown of BRD2 also reduced the elevated levels of PD-L1 seen in a model of acquired cisplatin resistance. CONCLUSIONS: PD-L1 is significantly elevated in HNSCC models of acquired cetuximab and cisplatin resistance where BRD2 is the primary regulator.

Gene targets of sulforaphane in head and neck squamous cell carcinoma.

Patients who have undergone curative­intent therapy for head and neck squamous cell carcinoma (HNSCC) exhibit a high rate of development of second primary tumors (SPTs), which are frequently lethal. A chemoprevention strategy that prevents SPTs would have a major impact on patient outcomes. Sulforaphane, a naturally­occurring compound derived from cruciferous vegetables exhibits chemopreventive activity against HNSCC in a preclinical model. The effects of sulforaphane are considered to be mediated, in large part, through increased protein expression of the transcription factor nuclear factor erythroid 2­related factor 2 (NRF2). Development of sulforaphane chemoprevention for HNSCC would benefit from the identification of robust biomarkers of sulforaphane activity in HNSCC cells and normal mucosal epithelial cells. The present study revealed that sulforaphane potently induces multiple oxidative stress­associated genes at the RNA and protein levels, in HNSCC cells and Het­1A cells, a non­tumorigenic mucosal epithelial cell line. In the present analysis, HMOX1 and HSPA1A were identified as the most highly upregulated genes following sulforaphane treatment, suggesting their potential value as biomarkers to guide clinical trials. Sulforaphane induction of HMOX1 and HSPA1A was validated in vivo in murine tissues. Furthermore, the impact of sulforaphane treatment of HNSCC cells on the expression levels of natural killer group 2D (NKG2D) and DNAX accessory molecule­1 (DNAM­1) ligands, which are activators of natural killer (NK) cells, was examined. NRF2­dependent upregulation of the NKG2D ligand MICA/B was observed. However, only one of the six HNSCC cell lines studied exhibited enhanced sensitivity to NK cell­mediated killing following sulforaphane treatment, suggesting that this may not be a general mechanism of sulforaphane chemopreventive activity in HNSCC. In summary, the present study identified robust biomarkers of sulforaphane activity in HNSCC and normal tissues, supporting their application in the development of sulforaphane chemoprevention approaches for HNSCC.

ATR inhibition sensitizes HPV- and HPV+ head and neck squamous cell carcinoma to cisplatin.

OBJECTIVES: Cisplatin is commonly used in the treatment of head and neck squamous cell carcinoma (HNSCC), and the repair of cisplatin-induced DNA damage involves activation of the DNA damage response protein ataxia telangiectasia and Rad3-related (ATR). Resistance to cisplatin therapy exacerbates adverse toxicities and is associated with poor outcomes. Since repair of cisplatin-induced DNA damage contributes to resistance, we hypothesized that inhibition of ATR using AZD6738, a well-tolerated and orally-bioavailable inhibitor, would enhance the sensitivity of HNSCC cells and tumors to cisplatin. MATERIALS AND METHODS: A panel of human papilloma virus-negative (HPV-) and HPV+ HNSCC cell lines were treated with cisplatin in the absence or presence of AZD6738, and effects on cell viability, colony formation, apoptosis signaling, and DNA damage were assessed. The impact of co-treatment with cisplatin plus AZD6738 on the growth of HPV- and HPV+ cell line- and patient-derived xenograft tumors was also examined. RESULTS: Inhibition of ATR with AZD6738 enhanced cisplatin-induced growth inhibition of HNSCC cell lines and tumors, in association with increased apoptosis signaling and DNA damage. Both HPV- and HPV+ models were sensitized to cisplatin by ATR inhibition. CONCLUSION: Inhibition of ATR promotes sensitization to cisplatin in preclinical in vitro and in vivo models of HPV- and HVP+ HNSCC, supporting clinical evaluation of this strategy in this disease.

BET Inhibition Overcomes Receptor Tyrosine Kinase-Mediated Cetuximab Resistance in HNSCC.

Cetuximab, the FDA-approved anti-EGFR antibody for head and neck squamous cell carcinoma (HNSCC), has displayed limited efficacy due to the emergence of intrinsic and acquired resistance. We and others have demonstrated that cetuximab resistance in HNSCC is driven by alternative receptor tyrosine kinases (RTK), including HER3, MET, and AXL. In an effort to overcome cetuximab resistance and circumvent toxicities associated with the administration of multiple RTK inhibitors, we sought to identify a common molecular target that regulates expression of multiple RTK. Bromodomain-containing protein-4 (BRD4) has been shown to regulate the transcription of various RTK in the context of resistance to PI3K and HER2 inhibition in breast cancer models. We hypothesized that, in HNSCC, targeting BRD4 could overcome cetuximab resistance by depleting alternative RTK expression. We generated independent models of cetuximab resistance in HNSCC cell lines and interrogated their RTK and BRD4 expression profiles. Cetuximab-resistant clones displayed increased expression and activation of several RTK, such as MET and AXL, as well as an increased percentage of BRD4-expressing cells. Both genetic and pharmacologic inhibition of BRD4 abrogated cell viability in models of acquired and intrinsic cetuximab resistance and was associated with a robust decrease in alternative RTK expression by cetuximab. Combined treatment with cetuximab and bromodomain inhibitor JQ1 significantly delayed acquired resistance and RTK upregulation in patient-derived xenograft models of HNSCC. These findings indicate that the combination of cetuximab and bromodomain inhibition may be a promising therapeutic strategy for patients with HNSCC.Significance: Inhibition of bromodomain protein BRD4 represents a potential therapeutic strategy to circumvent the toxicities and financial burden of targeting the multiple receptor tyrosine kinases that drive cetuximab resistance in HNSCC and NSCLC.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/15/4331/F1.large.jpg Cancer Res; 78(15); 4331-43. ©2018 AACR.