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Purpose: Cisplatin (CDDP)-based chemotherapy is a first-line treatment for patients with advanced head and neck squamous cell carcinomas (HNSCC), despite a high rate of treatment failures, acquired resistance, and subsequent aggressive behavior. The purpose of this study was to study the mechanism of CDDP resistance and metastasis in HNSCC. We investigated the role of NRF2 pathway activation as a driven event for tumor progression and metastasis of HNSCC. Experimental Design: Human HNSCC cell lines that are highly resistant to CDDP were generated. Clonogenic survival assays and a mouse model of oral cancer were used to examine the impact of NRF2 activation in vitro and in vivo on CDDP sensitivity and development of metastasis. Western blotting, immunostaining, whole-exome sequencing, single-cell transcriptomic and epigenomic profiling platforms were performed to dissect clonal evolution and molecular mechanisms. Results: Implantation of CDDP-resistant HNSCC cells into the tongues of nude mice resulted in a very high rate of distant metastases. The CDDP-resistant cells had significantly higher expression of NRF2 pathway genes in the presence of newly acquired KEAP1 mutations, or via epigenomic activation of target genes. Knockdown of NRF2 or restoration of the wild-type KEAP1 genes resensitized resistant cells to CDDP and decreased distant metastasis (DM). Finally, treatment with inhibitor of glutaminase-1, a NRF2 target gene, alleviated CDDP resistance. Conclusions: CDDP resistance and development of DM are associated with dysregulated and epigenetically reprogrammed KEAP1–NRF2 signaling pathway. A strategy targeting KEAP1/NRF2 pathway or glutamine metabolism deserves further clinical investigation in patients with CDDP-resistant head and neck tumors.

Studies of the immune landscape led to breakthrough trials of programmed death-1 (PD-1) inhibitors for recurrent/metastatic head and neck squamous cell carcinoma therapy. This study investigated the timing, influence of somatic copy-number alterations (SCNAs), and clinical implications of PD-L1 and immune-cell patterns in oral precancer (OPC).The authors evaluated spatial CD3, CD3/8, and CD68 density (cells/mmEpithelial, but not CD68 immune-cell, PD-L1 expression was detected in 28% of OPCs, correlated with immune-cell infiltration, 9p21.3 loss of heterozygosity (LOH), and inferior oral cancer-free survival (OCFS), notably in OPCs with low CD3/8 cell density, dysplasia, and/or 9p21.3 LOH. High CD3/8 cell density in dysplastic lesions predicted better OCFS and eliminated the excess risk associated with prior oral cancer and dysplasia. PD-L1 and CD3/8 patterns revealed inferior OCFS in PD-L1 high intrinsic induction and dysplastic immune-cold subgroups.This report provides spatial insight into the immune landscape and drivers of OPCs, and a publicly available immunogenomic data set for future precancer interrogation. The data suggest that 9p21.3 LOH triggers an immune-hot inflammatory phenotype; whereas increased 9p deletion size encompassing CD274 at 9p24.1 may contribute to CD3/8 and PD-L1 depletion during invasive transition. The inferior OCFS in PD-L1-high, immune-cold OPCs support the development of T-cell recruitment strategies.

TP53 mutation is the most frequent genetic event in head and neck squamous cell carcinoma (HNSCC), found in more than 80% of patients with human papillomavirus–negative disease. As mutations in the TP53 gene are associated with worse outcomes in HNSCC, novel therapeutic approaches are needed for patients with TP53-mutated tumors. The National Cancer Institute sponsored a Clinical Trials Planning Meeting to address the issues of identifying and developing clinical trials for patients with TP53 mutations. Subcommittees, or breakout groups, were tasked with developing clinical studies in both the locally advanced and recurrent and/or metastatic (R/M) disease settings as well as considering signal-seeking trial designs. A fourth breakout group was focused on identifying and standardizing biomarker integration into trial design; this information was provided to the other breakout groups prior to the meeting to aid in study development. A total of 4 concepts were prioritized to move forward for further development and implementation. This article summarizes the proceedings of the Clinical Trials Planning Meeting with the goal of developing clinical trials for patients with TP53-mutant HNSCC that can be conducted within the National Clinical Trials Network.

Assessment of the degree of synergy between cisplatin and MK-1775 in HN30 (wild-type TP53) and HN31 (mutant TP53) HNSCC cells respectively using the Chou and Talalay method.

Supplementary figure 3. Correlation of PI3K mutation, amplification, PI3K score, and mTOR score with sensitivity to GDC0941

In vivo RNA sequencing and pair-wise comparison between 4 distinct groups in HN30-P and HN30-R8 HNSCC tumors.

PDF file, 810KB.

Whole exome sequencing showing mutational status in the parental HN30-P versus HN30-R8 cisplatin resistant cell lines.

Synergy between B02 and AZD1775 is associated with impaired Rad51-mediated homologous recombination repair in HPV-positive HNSCC cells

Supplemental Figure 2. IR triggers a temporary, reversible perturbation in cellular reducing potential.

Dual phospho-H3 staining for mitotic cells and PI flow cytometric analysis (FACS).

Supplementary Table S1

PDF file - 746K, Forest plot of overall survival, disease-free survival, locoregional recurrence-free survival, and distant metastasis-free survival in OSCC patients treated with surgery and post-operative radiotherapy with and without TPF induction chemotherapy.

Supplementary Materials and Methods

PDF file - 132K, Differentially expressed genes between resistant/bevacizumab tumors compared to parental/bevacizumab tumors.

We have previously demonstrated that PD-1 blockade decreased the incidence of high-grade dysplasia in a carcinogen-induced murine model of oral squamous cell carcinoma (OSCC). It remains unknown, however, whether there are additional factors involved in escape from immune surveillance that could serve as additional targets for immunoprevention. We performed this study to further characterize the immune landscape of oral premalignant lesions (OPL) and determine the impact of targeting of the PD-1, CTLA-4, CD40, or OX40 pathways on the development of OPLs and oral carcinomas in the 4-nitroquinoline 1-oxide model. The immune pathways were targeted using mAbs or, in the case of the PD-1/PD-L1 pathway, using PD-L1–knockout (PD-L1ko) mice. After intervention, tongues and cervical lymph nodes were harvested and analyzed for malignant progression and modulation of the immune milieu, respectively. Targeting of CD40 with an agonist mAb was the most effective treatment to reduce transition of OPLs to OSCC; PD-1 alone or in combination with CTLA-4 inhibition, or PD-L1ko, also reduced progression of OPLs to OSCC, albeit to a lesser extent. Distinct patterns of immune system modulation were observed for the CD40 agonists compared with blockade of the PD-1/PD-L1 axis with or without CTLA-4 blockade; CD40 agonist generated a lasting expansion of experienced/memory cytotoxic T lymphocytes and M1 macrophages, whereas PD-1/CTLA-4 blockade resulted in a pronounced depletion of regulatory T cells among other changes. These data suggest that distinct approaches may be used for targeting different steps in the development of OSCC, and that CD40 agonists merit investigation as potential immunoprevention agents in this setting.Prevention Relevance:PD-1/PD-L1 pathway blockade, as well as activation of the CD40 pathway, were able to prevent OPL progression into invasive OSCC in a murine model. A distinct pattern of immune modulation was observed when either the CD40 or the PD-1/PD-L1 pathways were targeted.

Clustering of HNSCC cell lines by DDR genes.

Supplementary Tables and Methods

Supplementary figure 2. Representative FISH data

PDF file - 81K, Supplemental table S1: Survival comparison between the cN2 patients treated with and without TPF induction chemotherapy. Supplemental table S2: Univariate Cox model analysis. Supplemental table S3: Multivariate Cox model analysis with interaction analysis. Supplemental table S4: Correlation between cyclin D1 expression and response to TPF induction chemotherapy. Supplemental table S5: Survival comparison between the cN2 patients treated with and without TPF induction chemotherapy according to cyclin D1 expression.

Supplementary Figures PDF - file 648K, This file contains supplementary figures S1-S5. Figure S1 has additional copy number analysis. Figure S2 expands on the integration of gene expression and copy number analysis. Figures S3-S4 provide additional data on the function and mechanism of activated NOTCH1 expression in cell lines. Figure S5 shows inhibition of CASP8 promotes tumorigenesis

Restoration of KEAP1/NRF2 pathways increases cisplatin sensitivity in resistant HNSCC cells through ferroptosis.

Purpose:Cisplatin (CDDP)-based chemotherapy is a first-line treatment for patients with advanced head and neck squamous cell carcinomas (HNSCC), despite a high rate of treatment failures, acquired resistance, and subsequent aggressive behavior. The purpose of this study was to study the mechanism of CDDP resistance and metastasis in HNSCC. We investigated the role of NRF2 pathway activation as a driven event for tumor progression and metastasis of HNSCC.Experimental Design:Human HNSCC cell lines that are highly resistant to CDDP were generated. Clonogenic survival assays and a mouse model of oral cancer were used to examine the impact of NRF2 activation in vitro and in vivo on CDDP sensitivity and development of metastasis. Western blotting, immunostaining, whole-exome sequencing, single-cell transcriptomic and epigenomic profiling platforms were performed to dissect clonal evolution and molecular mechanisms.Results:Implantation of CDDP-resistant HNSCC cells into the tongues of nude mice resulted in a very high rate of distant metastases. The CDDP-resistant cells had significantly higher expression of NRF2 pathway genes in the presence of newly acquired KEAP1 mutations, or via epigenomic activation of target genes. Knockdown of NRF2 or restoration of the wild-type KEAP1 genes resensitized resistant cells to CDDP and decreased distant metastasis (DM). Finally, treatment with inhibitor of glutaminase-1, a NRF2 target gene, alleviated CDDP resistance.Conclusions:CDDP resistance and development of DM are associated with dysregulated and epigenetically reprogrammed KEAP1–NRF2 signaling pathway. A strategy targeting KEAP1/NRF2 pathway or glutamine metabolism deserves further clinical investigation in patients with CDDP-resistant head and neck tumors.

Tumor volume growth curves for individual mice in each treatment arm.

Supplemental figure legends - detailed text

Supplementary Tables -XLSX file 2328K, This file contains supplemental tables S1-S14. These tables describe the clinical, pathological and genomic information for this study. Also included are GISTIC peak information, expression and copy number correlations, additional expression and methylation data, sequencing coverage and mutation data for patients and cell lines, and expanded pathway and targeted event information

Induction chemotherapy is likely to be effective for biologically distinct subgroups of patients with cancer with biomarker detection. To investigate the prognostic and predictive values of cyclin D1 expression in patients with oral squamous cell carcinoma (OSCC) who were treated in a prospective, randomized, phase III trial evaluating standard treatment with surgery and postoperative radiotherapy preceded or not by induction docetaxel, cisplatin, and 5-fluorouracil (TPF), immunohistochemical staining for cyclin D1 was conducted in pretreatment biopsy specimens of 232 out of 256 clinical stage III/IVA OSCC patients randomized to the clinical trial. Cyclin D1 index was estimated as the proportion of tumor cells with cyclin D1 nuclear staining. A low cyclin D1 expression predicted significantly better overall survival (OS; P = 0.001), disease-free survival (P = 0.005), locoregional recurrence-free survival (P = 0.003), and distant metastasis-free survival (DMFS; P = 0.002) compared with high cyclin D1 expression. Cyclin D1 expression levels were not predictive of benefit from induction TPF in the population overall. However, patients with nodal stage cN2 whose tumors had high cyclin D1 expression treated with TPF had significantly greater OS (P = 0.025) and DMFS (P = 0.025) when compared with high cyclin D1 cN2 patients treated with surgery upfront. Patients with low cyclin D1 level or patients with cN0 or cN1 disease did not benefit from induction chemotherapy. This study indicates that cN2 OSCC patients with high cyclin D1 expression can benefit from the addition of TPF induction chemotherapy to standard treatment. Cyclin D1 expression could be used as a biomarker in further validation studies to select cN2 patients that could benefit from induction therapy. Mol Cancer Ther; 12(6); 1112–21. ©2013 AACR.

Combined inhibition or Rad51 and Wee1 enhances the DNA damage response in HPV-positive HNSCC cells

Determination of drug synergy by the combination-index and isobologram analyses using the CalcuSyn software.

The survival of patients with oral squamous cell carcinoma (OSCC) has not changed significantly in several decades, leading clinicians and investigators to search for promising molecular targets. To this end, we conducted comprehensive genomic analysis of gene expression, copy number, methylation, and point mutations in OSCC. Integrated analysis revealed more somatic events than previously reported, identifying four major driver pathways (mitogenic signaling, Notch, cell cycle, and TP53) and two additional key genes (FAT1, CASP8). The Notch pathway was defective in 66% of patients, and in follow-up studies of mechanism, functional NOTCH1 signaling inhibited proliferation of OSCC cell lines. Frequent mutation of caspase-8 (CASP8) defines a new molecular subtype of OSCC with few copy number changes. Although genomic alterations are dominated by loss of tumor suppressor genes, 80% of patients harbored at least one genomic alteration in a targetable gene, suggesting that novel approaches to treatment may be possible for this debilitating subset of head and neck cancers.Significance: This is the first integrated genomic analysis of OSCC. Only through integrated multiplatform analysis was it possible to identify four key pathways. We also discovered a new disease subtype associated with CASP8 and HRAS mutation. Finally, many candidate targetable events were found and provide hope for future genomically driven therapeutic strategies. Cancer Discov; 3(7); 770–81. ©2013 AACR.See related commentary by Iglesias-Bartolome et al., p. 722This article is highlighted in the In This Issue feature, p. 705

Ionizing radiation is the primary nonsurgical treatment modality for solid tumors. Its effectiveness is impacted by temporal constraints such as fractionation, hypoxia, and development of radioresistant clones. Biomarkers of acute radiation response are essential to developing more effective clinical algorithms. We hypothesized that acute perturbations in tumor lactate levels act as a surrogate marker of radiation response. In vitro experiments were carried out using validated human-derived cell lines from three histologies: anaplastic thyroid carcinoma (ATC), head and neck squamous cell carcinoma (HNSCC), and papillary thyroid carcinoma (PTC). Cellular metabolic activity was measured using standard biochemical assays. In vivo validation was performed using both an orthotopic and a flank derivative of a previously established ATC xenograft murine model. Irradiation of cells and tumors triggered a rapid, dose-dependent, transient decrease in lactate levels that was reversed by free radical scavengers. Acute lactate perturbations following irradiation could identify hypoxic conditions and correlated with hypoxia-induced radioresistance. Mutant TP53 cells and cells in which p53 activity was abrogated (shRNA) demonstrated a blunted lactate response to irradiation, consistent with a radioresistant phenotype. Lactate measurements therefore rapidly detected both induced (i.e., hypoxia) and intrinsic (i.e., mutTP53-driven) radioresistance. We conclude that lactate is a quantitative biomarker of acute genotoxic stress, with a temporal resolution that can inform clinical decision making. Combined with the spatial resolution of newly developed metabolic imaging platforms, this biomarker could lead to the development of truly individualized treatment strategies. Mol Cancer Ther; 14(12); 2901–8. ©2015 AACR.

Supplemental Figure 6. Loss of p53 function results in decreased radiosensitivity in HNSCC cell lines.

PDF file, 2343KB.

Supplementary figure 4. GDC0941 effect on cell cycle

PDF file - 3255K, Subgroup analysis of overall survival, disease-free survival, locoregional recurrence-free survival and distant metastasis free-survival in 232 OSCC patients with low and high cyclin D1 expression

DNA promoter methylation of tumor suppressor genes and global DNA hypomethylation are common features of head and neck cancers. Our goal was to identify early DNA methylation changes in oral premalignant lesions (OPL) that may serve as predictive markers of developing oral squamous cell carcinoma (OSCC). Using high-throughput DNA methylation profiles of 24 OPLs, we found that the top 86 genes differentially methylated between patients who did or did not develop OSCC were simultaneously hypermethylated, suggesting that a CpG island methylation phenotype may occur early during OSCC development. The vast majority of the 86 genes were nonmethylated in normal tissues and hypermethylated in OSCC versus normal mucosa. We used pyrosequencing in a validation cohort of 44 patients to evaluate the degree of methylation of AGTR1, FOXI2, and PENK promoters CpG sites that were included in the top 86 genes and of LINE1 repetitive element methylation, a surrogate of global DNA methylation. A methylation index was developed by averaging the percent methylation of AGTR1, FOXI2, and PENK promoters; patients with a high methylation index had a worse oral cancer–free survival (P = 0.0030). On the other hand, patients with low levels of LINE1 methylation had a significantly worse oral cancer–free survival (P = 0.0153). In conclusion, AGTR1, FOXI2, and PENK promoter methylation and LINE1 hypomethylation may be associated with an increased risk of OSCC development in patients with OPLs. Cancer Prev Res; 8(11); 1027–35. ©2015 AACR.

PDF file - 2826K, Overall survival (A), disease-free survival (B), locoregional recurrence-free survival (C), and distant metastasis-free survival (D) in 232 OSCC patients with low and high cyclin D1 expression.

Despite advances in surgery, chemotherapy, and radiation, there are limited treatment options for advanced head and neck squamous cell carcinoma (HNSCC) and survival remains very poor. Therefore, effective therapies are desperately needed. Recently, selective exploitation of DNA damage and replication stress responses has become a novel approach for cancer treatment. Wee1 kinase and Rad51 recombinase are two proteins involved in regulating replication stress and homologous recombination repair in cancer cells. In this study, we investigated the combined effect of Rad51 inhibitor (B02) and Wee1 inhibitor (AZD1775) in vitro and in vivo in various HNSCC cell lines. Clonogenic survival assays demonstrated that B02 synergized with AZD1775 in vitro in all HNSCC cell lines tested. The synergy between these drugs was associated with forced CDK1 activation and reduced Chk1 phosphorylation leading to induction of excessive DNA damage and replication stress, culminating in aberrant mitosis and apoptosis. Our results showed that elevated Rad51 mRNA expression correlated with worse survival in HNSCC patients with HPV-positive tumors. The combination of B02 and AZD1775 significantly inhibited tumor growth in vivo in mice bearing HPV-positive HNSCC tumors as compared to HPV-negative HNSCC. This differential sensitivity appears to be linked to HPV-positive tumors having more in vivo endogenous replication stress owing to transformation by E6 and E7 oncogenes. Furthermore, addition of B02 radiosensitized the HPV-negative HNSCC tumors in vitro and in vivo. In conclusion, our data implicate that a novel rational combination with Rad51 and Wee1 inhibitors holds promise as synthetic lethal therapy, particularly in high-risk HPV-positive HNSCC.

Anaplastic thyroid carcinoma (ATC) accounts for more than 50% of thyroid cancer mortality and is generally refractory to conventional treatment. On the basis of recent studies, we hypothesized that ATC metabolism can be targeted to improve response to chemoradiotherapy. Eight established and authenticated ATC cell lines were sequenced at 140 sites contained within 26 commonly mutated genes to identify novel potential therapeutic targets. Cellular proliferation, energy, and reducing potential stores were measured under conditions of specific nutrient deprivation. Tumor metabolism was evaluated using hyperpolarized 13C MRI in a murine orthotopic xenograft model of ATC. Sensitivity to chemotherapeutic agents and radiation (XRT) was assayed using cytotoxicity assays. We identified mutations in BRAF, NRAS, and KIT but failed to identify generalized novel targets for therapeutic intervention. ATC cell lines exhibited a mesenchymal phenotype and generalized dependence on glucose for energy, reducing potential and survival. Glycolytic inhibition using 2-deoxyglucose (2-DG) sensitized ATC cells to conventional chemotherapy and external beam radiation. In vivo, 2-DG induced a transient, but significant reduction in ATC metabolic activity. Generalized dependence of ATC cells on glucose catabolism makes them susceptible to the sensitizing effects of 2-DG for radiation therapy and chemotherapy. Under in vivo conditions, 2-DG can inhibit ATC metabolism. However, the modest magnitude and transient nature of this effect suggest the need for antimetabolic agents with more favorable pharmacodynamics to achieve therapeutic effects. Mol Cancer Ther; 11(6); 1373–80. ©2012 AACR.

Despite the use of multimodality therapy using cisplatin to treat patients with advanced stage squamous cell carcinoma of the head and neck (HNSCC), there is an unacceptably high rate of treatment failure. TP53 is the most commonly mutated gene in HNSCC, and the impact of p53 mutation on response to cisplatin treatment is poorly understood. Here, we show unambiguously that wild-type TP53 (wtp53) is associated with sensitivity of HNSCC cells to cisplatin treatment, whereas mutation or loss of TP53 is associated with cisplatin resistance. We also show that senescence is the major cellular response to cisplatin in wtp53 HNSCC cells and that cisplatin resistance in p53-null or -mutant TP53 cells is due to their lack of senescence. Given the dependence on checkpoint kinase (Chk)1/2 kinases to mediate the DNA damage response in p53-deficient cells, there is potential to exploit this to therapeutic advantage through targeted inhibition of the Chk1/2 kinases. Treatment of p53-deficient HNSCC cells with the Chk inhibitor AZD7762 sensitizes them to cisplatin through induction of mitotic cell death. This is the first report showing the ability of a Chk kinase inhibitor to sensitize TP53-deficient HNSCC to cisplatin in a synthetic lethal manner, which has significance given the frequency of TP53 mutations in this disease and because cisplatin has become part of standard therapy for aggressive HNSCC tumors. These preclinical data provide evidence that a personalized approach to the treatment of HNSCC based on Chk inhibition in p53-mutant tumors may be feasible. Mol Cancer Ther; 12(9); 1860–73. ©2013 AACR.

Percentage of CD4+CD44hi

PDF file - 5 MB, Apoptosis is not the main mode of cell death in wtp53 HNSCC cells.

The Wee-1 inhibitor, MK-1775 Synergizes with cisplatin in vitro in established HNSCC cell lines with known TP53 mutational status; Dual phospho-H3 staining and PI flow cytometric analysis (FACS); The synergy between cisplatin and MK-1775 in HNSCC is not mediated by apoptosis.

NRF2 targeted suppression or restoration of wild type KEAP1 decreases distant metastasis in cisplatin resistant HNSCC cells.

Supplementary Figure 1: Flow-chart describing the selection of 86 candidate genes. Supplementary Figure 2: Promoter methylation of 62/86 candidate genes in head and neck cancer, normal mucosa and blood. Supplementary Figure 3: Promoter methylation of 13/86 candidate genes in normal tissues. Supplementary Fig. 4: Hierarchical clustering of paired normal, dysplastic lesions and carcinomas (in situ or invasive) collected from 10 patients (3), using methylation profiles of 60/86 of our candidate genes. Supplementary Figure 5: (A) Hierarchical cluster analysis of oral squamous cell carcinoma and oral normal mucosa using candidate genes expression profiles. Supplementary Figure 6: Correlation of the percentage methylation of AGTR1.

The PI3K/AKT/mTOR pathway is frequently activated in head and neck squamous cell carcinoma (HNSCC), but pathway inhibition has variable efficacy. Identification of predictive biomarkers and mechanisms of resistance would allow selection of patients most likely to respond and novel therapeutic combinations. The purpose of this study was to extend recent discoveries regarding the PI3K/AKT/mTOR pathway in HNSCC by more broadly examining potential biomarkers of response, by examining pathway inhibitors with a diverse range of targets, and by defining mechanisms of resistance and potential combination therapies. We used reverse-phase protein arrays (RPPA) to simultaneously evaluate expression of 195 proteins; SNP array to estimate gene copy number; and mass array to identify mutations. We examined altered signaling at baseline and after pathway inhibition. Likewise, we examined the activation of the PI3K/AKT/mTOR pathway in HNSCC tumors by RPPA. Cell lines with PIK3CA mutations were sensitive to pathway inhibitors, whereas amplification status did not predict sensitivity. While we identified a set of individual candidate biomarkers of response to pathway inhibitors, proteomic pathway scores did not correlate with amplification or mutation and did not predict response. Several receptor tyrosine kinases, including EGFR and ERK, were activated following PI3K inhibition in resistant cells; dual pathway inhibition of PI3K and EGFR or MEK demonstrated synergy. Combined MEK and PI3K inhibition was markedly synergistic in HRAS-mutant cell lines. Our findings indicate that clinical trials of single-agent PI3K/AKT/mTOR pathway inhibitors in selected populations and of PI3K/EGFR or PI3K/MEK inhibitor combinations are warranted; we plan to conduct such trials. Mol Cancer Ther; 13(11); 2738–50. ©2014 AACR.

Supplementary Table S4A. The KNC genes enriched in cluster 1 in HNSCC cells; Supplementary Table S4B. Gene activity score of cell surface markers in cluster 1 in HNSCC cells.