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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.

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

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

Supplementary Table S1

Supplementary Materials and Methods

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.

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.

Single agent activity of B02 in HNSCC cell lines, Cell growth assay in HNSCC cells, Cell growth assay in HNSCC cells, and Synergy between B02 and AZD1775 is mediated in part through forced activation of CDK1 and inhibition of Chk1 associated with impaired Rad51-mediated homologous recombination repair in HNSCC cells.

Supplementary Materials and Methods

Live cell imaging of histone-H2B-RFP expressing PCI13-G245D cells in the presence of AZD1775.

Purpose: The cure rate for patients with advanced head and neck squamous cell carcinoma (HNSCC) remains poor due to resistance to standard therapy primarily consisting of chemoradiation. As mutation of TP53 in HNSCC occurs in 60% to 80% of non–HPV-associated cases and is in turn associated with resistance to these treatments, more effective therapies are needed. In this study, we evaluated the efficacy of a regimen combining vorinostat and AZD1775 in HNSCC cells with a variety of p53 mutations.Experimental Design: Clonogenic survival assays and an orthotopic mouse model of oral cancer were used to examine the in vitro and in vivo sensitivity of high-risk mutant p53 HNSCC cell lines to vorinostat in combination with AZD1775. Cell cycle, replication stress, homologous recombination (HR), live cell imaging, RNA sequencing, and apoptosis analyses were performed to dissect molecular mechanisms.Results: We found that vorinostat synergizes with AZD1775 in vitro to inhibit growth of HNSCC cells harboring high-risk mutp53. These drugs interact synergistically to induce DNA damage, replication stress associated with impaired Rad51-mediated HR through activation of CDK1, and inhibition of Chk1 phosphorylation, culminating in an early apoptotic cell death during the S-phase of the cell cycle. The combination of vorinostat and AZD1775 inhibits tumor growth and angiogenesis in vivo in an orthotopic mouse model of oral cancer and prolongs animal survival.Conclusions: Vorinostat synergizes with AZD1775 in HNSCC cells with mutant p53 in vitro and in vivo. A strategy combining HDAC and WEE1 inhibition deserves further clinical investigation in patients with advanced HNSCC. Clin Cancer Res; 23(21); 6541–54. ©2017 AACR.

GO enrichment analysis identified two significant pathways involving G1/S phase transition and cell cycle checkpoint as annotated in the RPPA heatmap.

Supplementary Materials and Methods and Figure Legends

Supplementary Figure S1. Synergistic effects of vorinostat and AZD1775 in vitro in established isogenic pair HNSCC cell lines with known TP53 mutational status. Supplementary Figure S2. Synergistic effects of vorinostat and AZD1775 in vitro in various established HNSCC cell lines with known TP53 mutational status. Supplementary Figure S3. Laser scanning confocal microscopy showing stress-induced membrane blebbing prior to apoptosis in selected populations of PCI13-G245D cells. Supplementary Figure S4. Combination of vorinostat and AZD1775 is associated with subsequent accumulation of cells in G2/M phase in HNSCC mutp53 cells. Supplementary Figure S5. Vorinostat interacts synergistically with AZD1775 in HNSCC to induce markers of DNA damge, replication stress and apoptosis. Supplementary Figure S6. Correlation between overall survival and expression levels of genes regulated by vorinostat alone or in combination with AZD1775 in HNSCC TCGA dataset. Supplementary Figure S7. Proteomic profiling of HNSCC mutant p53 cells treated with vorinostat and AZD1775 identifies proteins involved in cell cycle regulation. Supplementary Figure S8. Tumor volume growth curves for individual mice in each treatment group. Supplementary Figure S9. Immunostaining of p21, PCNA, RRM2 and survivin in tumor of HNSCC oral xenografts mice. Supplementary Figure S10. Combination of vorinostat and AZD1775 is well tolerated in an orthotopic mouse model of oral cancer. Supplementary Table S1. Combination index of vorinostat in combination with AZD1775 in various HNSCC cell lines.

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.

The solid tumour microenvironment includes nerve fibres that arise from the peripheral nervous system(1,2). Recent work indicates that newly formed adrenergic nerve fibres promote tumour growth, but the origin of these nerves and the mechanism of their inception are unknown(1,3). Here, by comparing the transcriptomes of cancer-associated trigeminal sensory neurons with those of endogenous neurons in mouse models of oral cancer, we identified an adrenergic differentiation signature. We show that loss of TP53 leads to adrenergic transdifferentiation of tumour-associated sensory nerves through loss of the microRNA miR-34a. Tumour growth was inhibited by sensory denervation or pharmacological blockade of adrenergic receptors, but not by chemical sympathectomy of pre-existing adrenergic nerves. A retrospective analysis of samples from oral cancer revealed that p53 status was associated with nerve density, which was in turn associated with poor clinical outcomes. This crosstalk between cancer cells and neurons represents mechanism by which tumour-associated neurons are reprogrammed towards an adrenergic phenotype that can stimulate tumour progression, and is a potential target for anticancer therapy.

Purpose: TP53 mutations are highly prevalent in head and neck squamous cell carcinoma (HNSCC) and associated with increased resistance to conventional treatment primarily consisting of chemotherapy and radiation. Restoration of wild-type p53 function in TP53-mutant cancer cells represents an attractive therapeutic approach and has been explored in recent years. In this study, the efficacy of a putative p53 reactivator called COTI-2 was evaluated in HNSCC cell lines with different TP53 status. Experimental Design: Clonogenic survival assays and an orthotopic mouse model of oral cancer were used to examine in vitro and in vivo sensitivity of HNSCC cell lines with either wild-type, null, or mutant TP53 to COTI-2 alone, and in combination with cisplatin and/or radiation. Western blotting, cell cycle, live-cell imaging, RNA sequencing, reverse-phase protein array, chromatin immunoprecipitation, and apoptosis analyses were performed to dissect molecular mechanisms. Results: COTI-2 decreased clonogenic survival of HNSCC cells and potentiated response to cisplatin and/or radiation in vitro and in vivo irrespective of TP53 status. Mechanistically, COTI-2 normalized wild-type p53 target gene expression and restored DNA-binding properties to the p53-mutant protein in HNSCC. In addition, COTI-2 induced DNA damage and replication stress responses leading to apoptosis and/or senescence. Furthermore, COTI-2 lead to activation of AMPK and inhibition of the mTOR pathways in vitro in HNSCC cells. Conclusions: COTI-2 inhibits tumor growth in vitro and in vivo in HNSCC likely through p53-dependent and p53-independent mechanisms. Combination of COTI-2 with cisplatin or radiation may be highly relevant in treating patients with HNSCC harboring TP53 mutations.

Objectives Currently there are no standard biomarkers of head and neck squamous cell carcinoma (HNSCC) response to therapy. This is, due to a lack of adequate predictive tumor models. To this end, we established cancer organoid lines from individual patient’s tumors, and characterized their growth characteristics and response to different drug treatments with the objective of using these models for prediction of treatment response. Materials and Methods Forty-three patients’ samples were processed to establish organoids. To analyze the character of these organoids, immunohistochemistry, Western blotting, drug sensitivity assays, clonogenic survival assays, and animal experiments were performed. The HPV status and TP53 mutational status were also confirmed in these lines. Results HNSCC organoids were successfully established with success rate of 30.2%. Corresponding two-dimensional cell lines were established from HNSCC organoids at higher success rate (53.8%). These organoids showed similar histological features and stem cell, epithelial and mesenchymal marker expression to the original tumors, thus recapitulating many of the characteristics of the original tumor cells. The cisplatin and docetaxel IC50 were determined for HNSCC organoids and the corresponding 2D cell lines using drug sensitivity and clonogenic survival assays. Responses to drug treatment in vivo were found to be similar to the IC50 calculated from organoids by drug sensitivity assays in vitro. Conclusion We established novel in vitro HNSCC cancer organoid lines retaining many properties of the original tumors from they were derived. These organoids can predict in vivo drug sensitivity and may represent useful tools to develop precision treatments for HNSCC.

Caspase-8 (CASP8) is one of the most frequently mutated genes in head and neck squamous carcinomas (HNSCCs), and CASP8 mutations are associated with poor survival. The distribution of these mutations in HNSCCs suggests that they are likely to be inactivating. Inhibition of CASP8 has been reported to sensitize cancer cells to necroptosis, a regulated cell death mechanism. Here, we show that knockdown of CASP8 renders HNSCCs susceptible to necroptosis by a second mitochondria-derived activator of caspase (SMAC) mimetic, birinapant, in combination with pan-caspase inhibitors Z-VAD-FMK or emricasan and radiation. In a syngeneic mouse model of oral cancer, birinapant, particularly when combined with radiation, delayed tumor growth and enhanced survival under CASP8 loss. Exploration of molecular underpinnings of necroptosis sensitivity confirmed that the level of functional receptor-interacting serine/threonine protein kinase 3 (RIP3) determines susceptibility to this mode of death. Although an in vitro screen revealed that low RIP3 levels rendered many HNSCC cell lines resistant to necroptosis, patient tumors maintained RIP3 expression and should therefore remain sensitive. Collectively, these results suggest that targeting the necroptosis pathway with SMAC mimetics, especially in combination with radiation, may be relevant therapeutically in HNSCC with compromised CASP8 status, provided that RIP3 function is maintained., Caspase-8 status sensitizes a murine model of head and neck squamous carcinoma to the combination of radiation and SMAC mimetic treatment through increased necroptosis.

Caspase-8 (CASP8) is one of the most frequently mutated genes in head and neck squamous carcinomas (HNSCC), and mutations ofCASP8are associated with poor overall survival. The distribution of these mutations in HNSCC suggests that they are likely to be inactivating. Inhibition ofCASP8has been reported to sensitize cancer cells to necroptosis, a unique cell death mechanism. Here, we evaluated howCASP8regulates necroptosis in HSNCC using cell line models and syngeneic mouse xenografts.In vitro, knockdown ofCASP8rendered HNSCCs susceptible to necroptosis induced by a second mitochondria-derived activator of caspase (SMAC) mimetic, Birinapant, when combined with pan-caspase inhibitors Z-VAD-FMK or Emricasan. Strikingly, inhibition ofCASP8function via knockdown or Emricasan treatment was associated with enhanced radiation killing by Birinapant through induction of necroptosis. In a syngeneic mouse model of oral cancer, Birinapant, particularly when combined with radiation delayed tumor growth and enhanced survival underCASP8loss. Exploration of molecular underpinnings of necroptosis sensitivity confirmed that the level of functional receptor-interacting serine/threonine-protein kinase-3 (RIP3), a key enzyme in the necroptosis pathway was crucial in determining susceptibility to this mode of death. Although anin vitroscreen revealed that many HNSCC cell lines were resistant to necroptosis due to low levels of RIP3, patient tumors maintain RIP3 expression and should therefore remain sensitive. Collectively, these results suggest that targeting the necroptosis pathway with SMAC mimetics, especially in combination with radiation, may be a relevant therapeutic approach in HNSCC with compromisedCASP8status, provided that RIP3 function is maintained.SignificanceCASP8status regulates necroptotic death in HNSCC and this pathway can be exploited therapeutically.

Despite recent advances in treatment modalities, patient survival for non-viral mediated oropharyngeal cancer patients continues to remain poor over the past years. Whole-exome sequencing studies reveal that TP53 is commonly mutated in human papillomavirus-negative HNSCC patients. Clinically, TP53 mutations are significantly associated with poor survival and tumor resistance to radiotherapy and chemotherapy in HNSCC patients, suggesting a potential utilization of the TP53 mutational status as a useful molecular prognostic marker to predict clinical response. Several approaches for restoration of wild-type p53 function in HNSCC tumors have been developed using gene transfer therapy via adenovirus and nanoparticles delivery. These approaches have shown relative success inducing cell-cycle arrest and apoptosis. Recently, small molecules that specifically reactivate mutant p53 and trigger mutant p53-dependent apoptosis have been developed and tested in clinical trials and show great promise. Targeting HNSCC cells harboring mutant p53 with ATR, Chk1/2 and Wee1 inhibitors alone or in combination with chemotherapy and/or radiation has become an intensive area of investigation. Numerous studies have revealed a novel function of p53 in regulation of cellular metabolism. A compelling evidence for a novel gain-of-function (GOF) of mutant p53 proteins in promoting metabolic changes has been recently demonstrated in HNSCC. We have demonstrated that TP53 mutations are associated with poor clinical outcome and increased radioresistance in HNSCC. We have also provided evidence that metformin acts as a radiosensitizer in vitro in TP53 mutant HNSCC by increasing expression of p21 and reactive oxygen species (ROS) levels, leading to induction of cellular senescence. Collectively, these novel strategies raise hopes for more effective therapies for head and neck cancers.

The solid tumor microenvironment includes nerve fibers that arise from the peripheral nervous system. Recent work indicates that newly formed adrenergic nerve fibers promote tumor growth, but the origin of these nerves and the mechanism of their inception are unknown. Here, by comparing the transcriptomes of cancer-associated trigeminal sensory neurons with those of endogenous neurons in multiple mouse models of oral cancer, we identified an adrenergic differentiation signature. We show that loss of TP53 leads to adrenergic transdifferentiation of tumor-associated sensory nerves through loss of the microRNA miR-34a. Tumor cells that expressed wild-type p53 protein released vesicles containing microRNAs that were taken up by neighboring neurons. MiR-34a blocks neuronal proliferation, and the neurons were maintained in their current state. By contrast, tumors that had a mutant versions of the gene encoding p53 released vesicles that lacked miR-34a. In this case, neurons increased in number in the vicinity of the tumor, and these cells were reprogrammed as adrenergic neurons that express the molecule noradrenaline. These neurons had more axonal branches than did those near tumors that expressed wild-type p53. Interactions between adrenergic neurons and the tumor aided cancer growth. Tumor growth was inhibited by sensory denervation or pharmacological blockade of adrenergic receptors, but not by chemical sympathectomy of pre-existing adrenergic nerves; and when mice received a transplant of p53-deficient tumor cells, treatment with a drug (carvedilol) that blocks adrenergic signaling pathways slowed tumor growth. A retrospective analysis of tumor samples from patients with oral cancer revealed that p53 status was associated with nerve density, which was in turn associated with poor clinical outcomes. Neural regulation represents an emerging targetable pathway for the treatment of cancer. The peripheral adrenergic nervous system has previously been shown to regulate cancer tumorigenesis. In contrast to previous findings using a prostate cancer mouse model, in our oral cancer mouse model, ablation of the sympathetic nervous system before tumor inoculation neither abrogated the development of adrenergic neo-nerves nor inhibited tumor growth. Our present study reveals that the emergence of adrenergic neonerves in the tumor microenvironment accompanies the initial phase of oral cavity squamous cell carcinoma (OCSCC) development in a transgenic OCSCC mouse models. We have identified crosstalk between the peripheral nervous system and head and neck tumors and described a phenotypic switch, induced by cancer cells, in which sensory nerves differentiate into adrenergic neo-neurons. This crosstalk between cancer cells and neurons represents mechanism by which tumor-associated neurons are reprogrammed towards an adrenergic phenotype that can stimulate tumor progression, and is a potential target for anticancer therapy. Our findings show that in p53-deficient tumors, an miRNA-based mechanism mediates neuronal responses to environmental cues and determines the fate of cancer-associated neurons. We have shown that axonal sprouting and autonomic reprogramming of existing nerves occur as a result of exosomal miRNA shuttling from cancer cells to neurons. These miRNAs orchestrate gene expression via combined dominantly negative (for example, miR-34a) and positive (for example, miR-21 and miR-324) effects, activating transcriptional programs that establish neuronal identity. In our mouse model of OCSCC, surgical ablation of sensory nerves prevented the development of these adrenergic neo-nerves; and as tumors evolve, neo-neural networks develop in and around the tumor stroma, providing signals that coordinate cancer progression. Our results thus show that the peripheral sensory nerves may be reprogrammed during the development of cancer in a manner similar to that of neural progenitors that initiate adrenergic neurogenesis during tumor formation. These results are consistent with recent preclinical data suggesting that sympathetic fibers accumulate in the normal vicinity of solid tumor tissues and infiltrate into the stroma. Furthermore, clinical data show that cancer patients treated with β-blockade have improved survival, supporting the role of adrenergic nerve activity in cancer progression. Although further studies will be required to dissect the molecular events that link tumor-associated neuritogenesis to cancer progression, our data raise the tantalizing possibility that drugs that target both axonal growth and the adrenergic nervous system could be useful for the treatment of cancer. Moreover, our discovery that the absence of functional p53 influences the formation of neighboring neurons might have relevance for interpreting reports showing that fluctuations in the levels of wild-type p53 are observed in nerve regeneration. Thus, these findings might have repercussions that reach beyond the field of cancer research to regenerative medicine. Citation Format: Moran Amit, Hideaki Takahashi, Mihnea-Paul Dragomir, Simone Anfossi, Antje Lindemann, Frederico Netto-Glebber, Samantha Tam, Abdullah Osman, Erik Kuntsen, Curtis R. Pickering, Mei Zhao, Xiayu Rao, Jing Wang, Deborah A. Silverman, Carlos Caulin, Assaf Zinger, Ennio Tasciotti, Patrick Dougherty, Adel El-Naggar, George A. Calin, Jeffrey N. Myers. Cancer takes a nerve: Loss of p53 drives neuron reprogramming in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr NG07.

Background: Standard treatment for head and neck squamous cell carcinoma (HNSCC) includes platinum-based therapy given in combination with radiation. Although platinum has been a key drug of HNSCC treatment for decades, many patients develop resistance and the prognosis is unsatisfactory, and therefore development of novel treatment strategies is needed. Targeting ATR has been shown to be effective as a monotherapy in various type of cancers. Thus, inhibition of ATR could be a new approach when combined with DNA damaging agents to kill cancer cells. Here, we hypothesize that addition of ATR inhibitor AZD6738 can enhance the antitumor effects of cisplatin in HNSCC cells and result in better survival in preclinical models of HNSCC. Materials and Methods: Clonogenic survival assays, reverse phase protein array (RPPA), Western blotting, cell cycle analysis, and apoptosis assay were used to evaluate the in vitro sensitivity of HNSCC cells to AZD6738 and cisplatin and the molecular mechanisms associated with antitumor response. An orthotopic mouse model of oral cancer was used to explore in vivo efficacy of the combination treatment. Results: HNSCC cells treated with various concentrations of AZD6738 showed an IC50 value of 0.24-3.00 μmol/L and no differential sensitivity was observed among cells with different TP53 mutational status. Treatment with constant drug concentration ratios of AZD6738 and cisplatin (2:1) resulted in a combination index of Conclusions: Addition of AZD6738 synergizes with cisplatin in vitro and potentiates cisplatin response in vivo. Mechanistically, combination of cisplatin and AZD6738 induces DNA damage and replication stress, resulting in apoptotic cell death. Interestingly, proteins essential for the origin licensing as part of the DNA replication initiation machinery were decreased following combination treatment, offering a potential mechanistic insight and warranting further investigation. Citation Format: Antje Lindemann, Hideaki Takahashi, Ameeta A. Patel, Walter N. Hittelman, Abdullah A. Osman, Jeffrey N. Myers. Inhibition of ATR as a therapeutic strategy to overcome cisplatin resistance in HNSCC [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr PR08.

Oral squamous cell carcinoma (OSCC) is the most common type of oral cancer worldwide and in the United States. OSCC remains a major cause of morbidity and mortality in patients with head and neck cancers. Tobacco and alcohol consumption alone or with chewing betel nut are potential risk factors contributing to the high prevalence of OSCC. Multimodality therapies, including surgery, chemotherapy, biologic therapy, and radiotherapy, particularly intensity-modulated radiotherapy (IMRT), are the current treatments for OSCC patients. Despite recent advances in these treatment modalities, the overall survival remains poor over the past years. Recent data from whole-exome sequencing reveal that TP53 is commonly mutated in human papillomavirus–negative OSCC patients. Furthermore, these data stressed the importance of the TP53 gene in suppressing the development and progression of OSCC. Clinically, TP53 mutations are largely associated with poor survival and tumor resistance to radiotherapy and chemotherapy in OSCC patients, which makes the TP53 mutation status a potentially useful molecular marker prognostic and predictive of clinical response in these patients. Several forms of DNA damage have been shown to activate p53, including those generated by ionizing radiation and chemotherapy. The DNA damage stabilizes p53 in part via the DNA damage signaling pathway that involves sensor kinases, including ATM and ATR and effector kinases, such as Chk1/2 and Wee1, which leads to posttranscriptional regulation of a variety of genes involved in DNA repair, cell cycle control, apoptosis, and senescence. Here, we discuss the link of TP53 mutations with treatment outcome and survival in OSCC patients. We also provide evidence that small-molecule inhibitors of critical proteins that regulate DNA damage repair and replication stress during the cell cycle progression, as well as other molecules that restore wild-type p53 activity to mutant p53, can be exploited as novel therapeutic approaches for the treatment of OSCC patients bearing p53 mutant tumors.

Despite the intense progress achieved in processing the magnetic particle imaging (MPI) technology, there is no suitable tracer that satisfies all MPI requirements. For this purpose, dextran-coated superparamagnetic iron oxide nanoparticles (SPIONs) were designed and synthesized as appropriate MPI tracer. To ensure a prospective biosafe application of these nanoparticles, we started to evaluate the biocompatibility, including cell toxicity and cellular uptake. The SPIONs internalized into mouse fibroblast cells were showing good labeling efficiencies and a cytosolic accumulation. Flow cytometry analysis demonstrated no difference of the cell viability of labeled cells in comparison with unlabeled control cells. But we observed decreased cell proliferation in response to increased SPION concentrations. In conclusion, SPIONs could reveal harmful properties and, therefore, further experiments should be considered.

6040Background: The p53 tumor suppressor is mutated in more than half of all cancers. COTI-2, an oral 3rd-generation thiosemicarbazone, restores the structure and function of mutant p53 proteins an...

Integrated genomic analysis of head and neck squamous cell carcinoma (HNSCC) has identified a heterogeneous spectrum of alterations dominated by tumor suppressor genes. An interesting new candidate driver gene is Caspase-8 (CASP8), which is mutated in 18% of oral cavity HPV-negative tumors. CASP8 is not obviously a part of any well-characterized driver pathway in HNSCC, and its role in this tumor type is unknown. In TCGA genomic data CAPS8 mutant tumors have a unique spectrum of alterations and expression changes, including a dramatic increase in markers of inflammation, which suggest they are a genomic subtype of HNSCC. CAPS8 is a key component of the extrinsic apoptotic pathway, but it also has a role in necrotic death, inflammation, and other pathways. We have characterized human and mouse CASP8 knock-out cell lines in vitro and in vivo in order to begin to understand the function of this gene in HNSCC. Loss of CASP8 function prevents extrinsic apoptosis and alters proliferation and tumorigenicity. It may also regulate the antitumor immune response. CASP8 is an exciting new candidate driver gene in HNSCC that may offer new opportunities for therapeutic intervention. Citation Format: Burak Uzunparmak, Antje Lindemann, Mitchell J. Frederick, Jeffrey N. Myers, Curtis R. Pickering. Caspase-8 is a novel candidate driver gene in head and neck 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 04.

Purpose: Although the treatment of locally advanced head and neck cancer (HNSCC) has evolved in recent years, the cure rate for patients with aggressive tumors and high-risk of failure remains poor due to resistance to standard chemoradiotherapy. Since TP53, the gene that encodes the protein p53, is by far the most commonly mutated gene found in HNSCC, and is in turn associated with treatment failure and poor survival outcomes. Although HPV+ HNSCC has a good prognosis, a group of HPV+ patients' tumors remain refractory to conventional treatments and unusual patterns of recurrence and metastasis are emerging. Therefore, more effective therapies are urgently needed. Our recent work has demonstrated that increased DNA damage response associated induction of replication stress and impaired Rad51-mediated homologous recombination (HR) sensitizes HNSCC tumor cells to WEE1 kinase inhibition with AZD1775. While the use of single molecularly targeted agents has demonstrated limited therapeutic benefits, there is growing interest in targeting multiple pathways or multiple steps within a single pathway to achieve even more effective cancer treatments. In this context, combinations of agents that target the DNA damage and HR responses are an exciting new area of investigation. Given the synthetic lethality of PARP inhibitor, olaparib in BRCA1/2-mutant or HR repair-defective cancers, combining PARP inhibitors with agents that inhibit HR is another interesting area of intensive investigation. Therefore, we hypothesize that simultaneous targeting of PARP and WEE1 will decrease the clonogenic survival in vitro and inhibit tumor growth and prolong animal survival in treated mice in an in vivo orthotopic nude mouse model of oral cancer. Experimental Design: Clonogenic survival assays and an orthotopic mouse model of oral cancer were used to examine the in vitro and in vivo sensitivity of high-risk mutant p53 and HPV+ HNSCC cell lines to olaparib in combination with AZD1775 respectively. Cell cycle analysis, DNA damage (γH2AX), HR, live cell imaging, and apoptosis were performed to dissect molecular mechanisms. Results: We found that olaparib synergized with AZD1775 in vitro to inhibit growth of HNSCC cells irrespective of p53 status or HPV positivity (median CI = 0.07-0.19). Mechanistically, these drugs interact synergistically to induce DNA damage, replication stress, and impaired Rad51-mediated HR through activation of CDK1 and increased Chk1 phosphorylation preceded by enhanced activity of ATR, culminating in aberrant mitosis associated with apoptotic cell death. Assessment of olaparib and AZD1775 combination therapy in vivo in an orthotopic mouse model of oral cancer is currently underway. Conclusions: Olaparib synergizes with AZD1775 in HNSCC cells in vitro through apoptosis. Our data provide an early-preclinical evidence for the rational combination of WEE1 and PARP inhibitors in the treatment of advanced HNSCC. Citation Format: Hideaki Takahashi, Antje Lindemann, Ameeta A. Patel, Weiwei Liu, Noriaki Tanaka, Lin Tang, Mei Zhao, Walter N. Hittelman, Jeffrey N. Myers, Abdullah A. Osman. Synergistic antitumor activity of olaparib combined with AZD1775 in head and neck 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 44.

New cyclohexadepsipeptides of the enniatin type with potential anthelmintic properties were produced by two different strategies: 1. In vitro synthesis by use of the multienzyme enniatin synthetase, and 2. in vivo precursor feeding of enniatin producing strains Fusarium scirpi and Fusarium sambucinum. The compounds were analyzed by HPLC, various NMR measurements and mass spectrometry. The three N-methyl L-amino acid positions in the enniatin B molecule could be gradually replaced by other (N-methyl) L-amino acids, e.g. alanine, cysteine, threonine and serine. The latter two amino acids yield new enniatins with functional groups in the hydrophobic side chains. Similarly the three D-2-hydroxyisovalerate residues, present in all naturally occuring enniatins, could be substituted by D-2-hydroxybutyric acid and D-lactic acid. Despite its lower yield the in vitro synthesis has the advantage of a broader variety of products formed.

The precise roles that oxidants play in lifespan and aging are still unknown. Here, we report the discovery that chronologically aging yeast cells undergo a sudden redox collapse, which affects over 80% of identified thiol-containing proteins. We present evidence that this redox collapse is not triggered by an increase in endogenous oxidants as would have been postulated by the free radical theory of aging. Instead it appears to be instigated by a substantial drop in cellular NADPH, which normally provides the electron source for maintaining cellular redox homeostasis. This decrease in NADPH levels occurs very early during lifespan and sets into motion a cascade that is predicted to down-regulate most cellular processes. Caloric restriction, a near-universal lifespan extending measure, increases NADPH levels and delays each facet of the cascade. Our studies reveal a time line of events leading up to the system-wide oxidation of the proteome days before cell death.DOI:http://dx.doi.org/10.7554/eLife.00306.001.

The precise roles that oxidants play in lifespan and aging are still unknown. Here, we report the discovery that chronologically aging yeast cells undergo a sudden redox collapse, which affects over 80% of identified thiol-containing proteins. We present evidence that this redox collapse is not triggered by an increase in endogenous oxidants as would have been postulated by the free radical theory of aging. Instead it appears to be instigated by a substantial drop in cellular NADPH, which normally provides the electron source for maintaining cellular redox homeostasis. This decrease in NADPH levels occurs very early during lifespan and sets into motion a cascade that is predicted to down-regulate most cellular processes. Caloric restriction, a near-universal lifespan extending measure, increases NADPH levels and delays each facet of the cascade. Our studies reveal a time line of events leading up to the system-wide oxidation of the proteome days before cell death. DOI: http://dx.doi.org/10.7554/eLife.00306.001, eLife digest While most animals experience a physiological decline as they age, the underlying cause of this decline is not fully understood. According to the free radical theory of aging, chemicals known as reactive oxygen species build up in the body and then cause damage to various components within cells, including DNA and proteins. These species, which include hydrogen peroxide and peroxynitrite, can cause substantial oxidative damage. However, while there is definitely a relationship between aging and reactive oxygen species, it remains possible that oxidative damage is a byproduct of aging rather than the cause of it. In the past researchers have measured the carbonylation of proteins (that is, the oxidation of certain amino acids in proteins) as a proxy for damage caused by reactive oxygen species, but this method has a number of shortcomings. More recently, it has become possible to quantify the oxidation state of cysteine, an amino acid that contains sulfur, in proteins using a technique based on mass spectrometry. Building on previous work in which they used this technique to measure the oxidation state of 300 proteins in vivo in the yeast Saccharomyces cerevisiae, Brandes et al. have now determined how the oxidation state of these proteins changes over the lifespan of S. cerevisiae, which is a popular model system for analyzing aging in cells that are in a high metabolic state but are no longer dividing. This made it possible to identify protein targets that might—as a result of changes in their oxidation state caused by reactive oxygen species—contribute to the physiological alterations observed in aging organisms. It was also possible to establish a clear connection between the onset and extent of oxidative stress and lifespan. Brandes et al. discovered that several days before the yeast cells died, they underwent a sudden and global ‘redox collapse’ in which ∼80% of the 300 proteins being studied experienced an increase in their oxidation state (i.e., they lost electrons). This event was preceded by a large drop in the level of NADPH, a coenzyme that, by being a source of electrons, helps to counterbalance the removal of electrons by reactive oxygen species within cells. The drop in the concentration of NADPH occurred very early in the life cycle of the yeast, and set in motion a series of events that down-regulated most cellular processes. Intriguingly, these findings are consistent with the effect of caloric restriction, a condition that is known to extend the lifespan of animals. Caloric restriction increases cellular NADPH and delays the down-regulation of cellular processes. Brandes et al. propose that the underlying cause of aging is not the accumulation of reactive oxygen species: rather, these results suggest that aging is caused by a sudden and substantial decrease in available NADPH, which means that cells cannot maintain a stable oxidation state. If borne out by further work, these findings could have a significant impact on how we think about the aging process, and could require researchers to rethink how they study aging. DOI: http://dx.doi.org/10.7554/eLife.00306.002

Head and neck cancer includes the squamous cell carcinomas of the oral cavity, pharynx and larynx and is one of the most common solid neoplasms worldwide. The malignancy is an important public health problem worldwide with more than 500000 new cases diagnosed each year. The change in survival over the last 20 years remains minimal and despite recent attention, the mortality rates are still high due to local tumor invasion and to a high predilection for the development of relapses and metastases. The cellular and molecular mechanisms responsible for tumor aggressiveness and its response to chemo- and radiation therapies remain mostly unknown.

Antje Lindemann,1 Kerstin Lüdtke-Buzug,2 Bianca M Fräderich,1 Ksenija Gräfe,2 Ralph Pries,1 Barbara Wollenberg11Department of Otorhinolaryngology, University Hospital of Schleswig-Holstein, Luebeck, Germany; 2Institute of Medical Engineering, University of Luebeck, Luebeck, GermanyBackground: As a tomographic imaging technology, magnetic particle imaging (MPI) allows high spatial resolution and sensitivity, and the possibility to create real-time images by determining the spatial distribution of magnetic particles. To ensure a prospective biosafe application of UL-D (University of Luebeck-Dextran coated superparamagnetic nanoparticles), we evaluated the biocompatibility of superparamagnetic iron oxide nanoparticles (SPIONs), their impact on biological properties, and their cellular uptake using head and neck squamous cancer cells (HNSCCs).Methods: SPIONs that met specific MPI requirements were synthesized as tracers. Labeling and uptake efficiency were analyzed by hematoxylin and eosin staining and magnetic particle spectrometry. Flow cytometry, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays, and real-time cell analyzer assays were used to investigate apoptosis, proliferation, and the cytokine response of SPION-labeled cells. The production of reactive oxygen species (ROS) was determined using a fluorescent dye. Experimental results were compared to the contrast agent Resovist®, a standard agent used in MPI.Results: UL-D nanoparticles and Resovist particles were taken up in vitro by HNSCCs via unspecific phagocytosis followed by cytosolic accumulation. To evaluate toxicity, flow cytometry analysis was performed; results showed that dose- and time-dependent administration of Resovist induced apoptosis whereas cell viability of UL-D-labeled cells was not altered. We observed decreased cell proliferation in response to increased SPION concentrations. An intracellular production of ROS could not be detected, suggesting that the particles did not cause oxidative stress. Tumor necrosis factor alpha (TNF-α) and interleukins IL-6, IL-8, and IL-1ß were measured to distinguish inflammatory responses. Only the primary tumor cell line labeled with >0.5 mM Resovist showed a significant increase in IL-1ß secretion.Conclusion: Our data suggest that UL-D SPIONs are a promising tracer material for use in innovative tumor cell analysis in MPI.Keywords: cell labeling, HNSCCs, biomedical imaging, iron oxide nanoparticles, cytotoxicity