Abstract: 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.

Abstract: 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.

Abstract: Background: The NOTCH1 gene functions as either an oncogene or tumor suppressor in cancer depending upon the tumor type. Our group previously characterized the genomic alterations in head and neck squamous cell carcinoma (HNSCC), discovering that NOTCH1 is frequently altered with a pattern of inactivating mutations suggesting it is a tumor suppressor in this cancer type. However, recent work by others suggests NOTCH1 signaling plays a more complex role, possibly promoting a more aggressive phenotype or cancer stem cell-like properties in HNSCCs with wild type NOTCH1. Our present study aimed to systematically compare the phenotypic consequences of NOTCH1 signaling in HNSCC to better understand its function in cancer, and identify targets downstream of NOTCH1 signaling. Methods: Established HNSCC cell lines wild type for NOTCH1 (PJ34, FADU) or harboring an inactivating mutation (UMSCC22A) were engineered to express activated cleaved NOTCH1 (cl-NOTCH1) from a doxycycline-inducible promoter. In vitro cell growth was measured with clonogenic assays. Stem cell-like properties were measured by orosphere formation and anoikis resistance. Stem cell markers for HNSCC including Aldehyde dehydrogenase activity (ALDH), CD133, and CD44 expression were measured by flow cytometry. NOTCH1-regulated downstream gene expression changes were examined by RNA-seq and qRT-PCR. Results: Activation of NOTCH1 inhibited clonogenic growth of all three cell lines, regardless of original NOTCH1 gene status. Growth inhibition was frequently accompanied by spontaneous formation of spheroid-like structures, characteristic of stem cells. NOTCH1 activation in UMSCC22A and FADU cells promoted orosphere formation and anoikis resistance, conveying some stem cell-like properties. However, classical stem cell markers including ALDH activity, CD133, and CD44 expression were not affected by NOTCH1 activation. Furthermore, RNA-seq demonstrated that critical cancer-associated pathways, including proliferation, differentiation, and migration, were regulated by NOTCH1. NOTCH1 activation downregulated gene expression of ITGA3, ITGA4, ITGB1, ITGB6, and LAMC2, which are key adhesion molecules that human basal keratinocytes use for attachment to the basement membrane and maintenance of the stem cell compartment. Concomitantly, NOTCH1 activation increased the basal/superbasal marker SOX2, but also the early differentiation markers KRT4 and KRT13. SiRNA-mediated SOX2 silencing blocked NOTCH1-promoted anoikis resistance. Conclusion: NOTCH1 activation inhibits in vitro growth regardless of mutational status. We hypothesize that stem cell-like properties associated with NOTCH1 activation in HNSCC may be a consequence of pathways that recapitulate early differentiation, rather than true stem cell maintenance. Citation Format: Chenfei Huang, Shhyam Moorthy, Qiuli Li, Rami Saade, Jiping Wang, Xiayu Rao, Noriaki Tanaka, Jiexin Zhang, Lin Tang, Curtis R. Pickering, Patrick A. Zweidler-McKay, Abdullah A. Osman, Tong-Xin Xie, Eve Shinbrot, Liu Xi, David Wheeler, Adel K. El-Naggar, Jing Wang, Jeffrey N. Myers, Mitchell J. Frederick. NOTCH1 activation in head and neck squamous cell carcinoma leads to growth inhibition, changes in gene expression associated with early differentiation, and acquisition of stem cell-like properties [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5506.

Abstract: Purpose: Development of extranodal extension (ENE) has been associated with poor survival in patients with oral cavity squamous cell carcinoma (OSCC). Here, we sought to confirm the role of ENE as a poor prognostic factor, and identify genomic and epigenetic markers of ENE in order to develop a predictive model and improve treatment selection. Experimental Design: An institutional cohort (The University of Texas MD Anderson Cancer Center) was utilized to confirm the impact of ENE on clinical outcomes and evaluate the genomic signature of primary and ENE containing tissue. OSCC data from The Cancer Genome Atlas (TCGA) were analyzed for the presence of molecular events associated with nodal and ENE status. Results: ENE was associated with decreased overall and disease-free survival. Mutation of the TP53 gene was the most common event in ENE+ OSCC. The frequency of TP53 mutation in ENE+ tumors was higher compared with ENE− tumors and wild-type (WT) TP53 was highly represented in pN0 tumors. pN+ENE+ patients had the highest proportion of high-risk TP53 mutations. Both primary tumors (PT) and lymph nodes with ENE (LN) exhibited a high rate of TP53 mutations (58.8% and 58.8%, respectively) with no significant change in allele frequency between the two tissue sites. Conclusions: ENE is one of the most significant markers of OSCC OS and DFS. There is a shift toward a more aggressive biological phenotype associated with high-risk mutations of the TP53 gene. Prospective clinical trials are required to determine whether TP53 mutational status can be used for personalized treatment decisions. Clin Cancer Res; 24(7); 1727–33. ©2018 AACR.

Abstract: 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.

Abstract: Checkpoint kinase inhibitors (CHKi) exhibit striking single-agent activity in certain tumors, but the mechanisms accounting for hypersensitivity are poorly understood. We screened a panel of 49 established human head and neck squamous cell carcinoma (HNSCC) cell lines and report that nearly 20% are hypersensitive to CHKi monotherapy. Hypersensitive cells underwent early S-phase arrest at drug doses sufficient to inhibit greater than 90% of CHK1 activity. Reduced rate of DNA replication fork progression and chromosomal shattering were also observed, suggesting replication stress as a root causative factor in CHKi hypersensitivity. To explore genomic underpinnings of CHKi hypersensitivity, comparative genomic analysis was performed between hypersensitive cells and cells categorized as least sensitive because they showed drug IC50 value greater than the cell panel median and lacked early S-phase arrest. Novel association between CDKN2A/p16 copy number loss, CDK2 activation, replication stress, and hypersensitivity of HNSCC cells to CHKi monotherapy was found. Restoring p16 in cell lines harboring CDKN2A/p16 genomic deletions alleviated CDK2 activation and replication stress, attenuating CHKi hypersensitivity. Taken together, our results suggest a biomarker-driven strategy for selecting HNSCC patients who may benefit the most from CHKi therapy. Significance: These results suggest a biomarker-driven strategy for selecting HNSCC patients who may benefit the most from therapy with CHK inhibitors. Cancer Res; 78(3); 781–97. ©2017 AACR.

Abstract: Determining which HPV+ oropharyngeal squamous cell carcinoma (OPSCC) patients will respond to therapy is of utmost important for implementation of treatment de-escalation to reduce morbidity or testing of novel therapeutic approaches. Due to the lack of reliable indicators of treatment response, we explored the gene expression profile of OPSCC in order to better understand the biology of these tumors and identify novel biomarkers. In this study, we investigated human genes associated with HPV transcription in 80 OPSCC samples from The Cancer Genome Atlas (TCGA). This exploratory analysis provided a list of 582 human genes associated with HPV biology. Hierarchical clustering analysis using the 582 human genes was used to separate HPV+ OPSCC in two groups with significantly distinct survival (log-rank test p & lt; 0.001) and differential expression of HPV genes. A refinement of this analysis generated a prognostic gene expression signature for HPV+ OPSCC containing 41 human genes. These results were validated in two independent cohorts of HPV+ OPSCC (n=83 and n=47) and one independent cohort of cervical cancer (n=83). In all three validation cohorts our 41 gene expression signature was able to identify a group of HPV+ OPSCC patients with worse survival. Further refinement and validation of the signature is ongoing. In order to understand the biology related to poor outcome in HPV+ OPSCC, a whole transcriptome analysis between the two initial HPV+ OPSCC TCGA groups was performed. Pathway analysis identified cell metabolism, cell stress, and DNA damage related genes were highly associated with poor outcome. Similar patterns of gene expression were found in vitro in a panel of 10 HPV+ cell lines, and markers of the poor outcome were found to be associated with reduced radiation sensitivity in vitro. Our study has identified prognostic biomarkers in HPV+ OPSCC with potential clinical importance. These biomarkers may be useful for selection of low risk patients for treatment de-escalation or selection of high risk patients for novel therapeutic approaches. These biomarkers are also functionally linked to radiation sensitivity and may include new therapeutic targets. Citation Format: Frederico O. Gleber-Netto, Xiayu Rao, Kelly Erikson, Keiko Akagi, Faye M. Johnson, Jing Wang, Joseph Califano, Maura L. Gillison, Jeffrey N. Myers, Curtis R. Pickering. Risk stratification and biomarker discovery in HPV-positive oropharynx squamous cell carcinoma determined by HPV and human gene expression profile associations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4621.