Cisplatin is a widely used anticancer drug with notable side effects including ototoxicity and nephrotoxicity. Macrophages, the major resident immune cells in the cochlea and kidney, are important drivers of both inflammatory and tissue repair responses. To investigate the roles of macrophages in cisplatin-induced toxicities, we used PLX3397, a U.S. Food and Drug Administration-approved inhibitor of the colony-stimulating factor 1 receptor, to eliminate tissue-resident macrophages. Mice treated with cisplatin alone had considerable hearing loss (ototoxicity) and kidney injury (nephrotoxicity). Macrophage ablation resulted in significantly reduced hearing loss and had greater outer hair cell survival. Macrophage ablation also protected against cisplatin-induced nephrotoxicity, as evidenced by markedly reduced tubular injury and fibrosis. Mechanistically, our data suggest that the protective effect of macrophage ablation against cisplatin-induced ototoxicity and nephrotoxicity is mediated by reduced platinum accumulation in both the inner ear and the kidney. Together, our data indicate that ablation of tissue-resident macrophages represents an important strategy for mitigating cisplatin-induced ototoxicity and nephrotoxicity.

The encoding of acoustic stimuli requires precise neuron timing. Auditory neurons in the cochlear nucleus (CN) and brainstem are well suited for accurate analysis of fast acoustic signals, given their physiological specializations of fast membrane time constants, fast axonal conduction, and reliable synaptic transmission. The medial olivocochlear (MOC) neurons that provide efferent inhibition of the cochlea reside in the ventral brainstem and participate in these fast neural circuits. However, their modulation of cochlear function occurs over time scales of a slower nature. This suggests the presence of mechanisms that reduce MOC inhibition of cochlear function. To determine how monaural excitatory and inhibitory synaptic inputs integrate to affect the timing of MOC neuron activity, we developed a novel in vitro slice preparation (“wedge-slice”). The wedge-slice maintains the ascending auditory nerve root, the entire CN and projecting axons, while preserving the ability to perform visually guided patch-clamp electrophysiology recordings from genetically identified MOC neurons. The “in vivo-like” timing of the wedge-slice demonstrates that the inhibitory pathway accelerates relative to the excitatory pathway when the ascending circuit is intact, and the CN portion of the inhibitory circuit is precise enough to compensate for reduced precision in later synapses. When combined with machine learning PSC analysis and computational modeling, we demonstrate a larger suppression of MOC neuron activity when the inhibition occurs with in vivo-like timing. This delay of MOC activity may ensure that the MOC system is only engaged by sustained background sounds, preventing a maladaptive hypersuppression of cochlear activity.

Characteristics Cell lines

Summary: Cancers often display immune escape, but the mechanisms are incompletely understood. Herein, we identify SMYD3 as a mediator of immune escape in human papilloma virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC), an aggressive disease with poor response to immunotherapy with pembrolizumab. SMYD3 depletion induces upregulation of multiple type I interferon (IFN) response and antigen presentation machinery genes in HNSCC cells. Mechanistically, SMYD3 binds to and regulates the transcription of UHRF1, encoding for a reader of H3K9me3, which binds to H3K9me3-enriched promoters of key immune-related genes, recruits DNMT1, and silences their expression. SMYD3 further maintains the repression of immune-related genes through intragenic deposition of H4K20me3. In vivo, Smyd3 depletion induces influx of CD8+ T cells and increases sensitivity to anti-programmed death 1 (PD-1) therapy. SMYD3 overexpression is associated with decreased CD8 T cell infiltration and poor response to neoadjuvant pembrolizumab. These data support combining SMYD3 depletion strategies with checkpoint blockade to overcome anti-PD-1 resistance in HPV-negative HNSCC.

TNFα is a key mediator of immune, chemotherapy and radiotherapy-induced cytotoxicity, but several cancers, including head and neck squamous cell carcinomas (HNSCC), display resistance to TNFα due to activation of the canonical NFκB pro-survival pathway. However, direct targeting of this pathway is associated with significant toxicity; thus, it is vital to identify novel mechanism(s) contributing to NFκB activation and TNFα resistance in cancer cells. Here, we demonstrate that the expression of proteasome-associated deubiquitinase USP14 is significantly increased in HNSCC and correlates with worse progression free survival in Human Papillomavirus (HPV)- HNSCC. Inhibition or depletion of USP14 inhibited the proliferation and survival of HNSCC cells. Further, USP14 inhibition reduced both basal and TNFα-inducible NFκB activity, NFκB-dependent gene expression and the nuclear translocation of the NFκB subunit RELA. Mechanistically, USP14 bound to both RELA and IκBα and reduced IκBα K48-ubiquitination leading to the degradation of IκBα, a critical inhibitor of the canonical NFκB pathway. Furthermore, we demonstrated that b-AP15, an inhibitor of USP14 and UCHL5, sensitized HNSCC cells to TNFα-mediated cell death, as well as radiation-induced cell death in vitro. Finally, b-AP15 delayed tumor growth and enhanced survival, both as a monotherapy and in combination with radiation, in HNSCC tumor xenograft models in vivo, which could be significantly attenuated by TNFα depletion. These data offer new insights into the activation of NFκB signaling in HNSCC and demonstrate that small molecule inhibitors targeting the ubiquitin pathway warrant further investigation as a novel therapeutic avenue to sensitize these cancers to TNFα- and radiation-induced cytotoxicity.

Co-occurence of TRAF3 and NF-kappaB pathway alterations

Supplementary Table from Inhibiting WEE1 and IKK-RELA Crosstalk Overcomes TNFα Resistance in Head and Neck Cancers

<p>Supplementary Methods, related references, and Figures: S1, 2 Effects TRAF3 on alternate pathway proteins; S2, Effects TRAF3 on alternate pathway proteins; S3, Effects of TNF/LTB on NF-kB reporter activity and TRAF3 siRNA on alternate pathway; S4, Effects TRAF3 on cisplatin and anti-apoptotic genes; S5, TRAF3 loss with passage; S6-8, TRAF3 effects on IRF expression; S9, TRAF3 effects on TP53, RB, p21; S10, TRAF3 effects on E6; S11, Diagram illustrating the genetic and cellular mechanisms linked to deficient TRAF3 in HPV+ HNSCC.</p>

Head and neck squamous cell carcinoma (HNSCC) remains a prevalent diagnosis with current treatment options that include radiotherapy and immune-mediated therapies, in which tumor necrosis factor-α (TNFα) is a key mediator of cytotoxicity. However, HNSCC and other cancers often display TNFα resistance due to activation of the canonical IKK–NFκB/RELA pathway, which is activated by, and induces expression of, cellular inhibitors of apoptosis proteins (cIAPs). Our previous studies have demonstrated that the IAP inhibitor birinapant sensitized HNSCC to TNFα-dependent cell death in vitro and radiotherapy in vivo. Furthermore, we recently demonstrated that the inhibition of the G2/M checkpoint kinase WEE1 also sensitized HNSCC cells to TNFα-dependent cell death, due to the inhibition of the pro-survival IKK-NFκB/RELA complex. Given these observations, we hypothesized that dual-antagonist therapy targeting both IAP and WEE1 proteins may have the potential to synergistically sensitize HNSCC to TNFα-dependent cell death. Using the IAP inhibitor birinapant and the WEE1 inhibitor AZD1775, we show that combination treatment reduced cell viability, proliferation and survival when compared with individual treatment. Furthermore, combination treatment enhanced the sensitivity of HNSCC cells to TNFα-induced cytotoxicity via the induction of apoptosis and DNA damage. Additionally, birinapant and AZD1775 combination treatment decreased cell proliferation and survival in combination with radiotherapy, a critical source of TNFα. These results support further investigation of IAP and WEE1 inhibitor combinations in preclinical and clinical studies in HNSCC.

Human Papilloma Virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer type in the world and its outcomes are mostly unchanged for the past few decades, especially for HPV-negative HNSCC. The TCGA recently revealed a plethora of genetic alterations in chromatin modifiers in HPV-negative HNSCC, including protein lysine methyltransferases which methylate lysine residues on histone tails and regulate gene expression. One of these enzymes, SUV420H1, is known to trimethylate H4K20 and is recurrently amplified in approximately 6% of HPV-negative HNSCC tumors (TCGA). It has also been found to be significantly overexpressed in multiple HNSCC cell lines compared with normal keratinocytes. This study aims to investigate whether SUV420H1 has oncogenic activity and could be a novel therapeutic target in HPV-negative HNSCC. To evaluate the effect of SUV420H1 depletion on HPV-negative HNSCC cell lines, siRNA-mediated knockdown was performed in four SUV420H1 overexpressing HPV-negative HNSCC cell lines, and MTT assays showed significantly reduced cell viability by approximately 80-90% after 12 days of siRNA treatment. Colony formation assays, cell cycle analysis and apoptosis assays are ongoing, and these assays are planned in SUV420H1 CRISPR KO cell lines. Gene Set Enrichment Analysis in 434 HPV-negative HNSCC tumors (TCGA) showed significant enrichment of immune signatures, EMT, cell cycle and apoptosis pathways. Ongoing experiments aim to investigate the effects of SUV420H1 knockdown on the global histone methylome, and to identify direct downstream targets of SUV420H1 through combined genome-wide mapping of SUV420H1 using CUT&RUN assays and RNA-seq in HPV-negative HNSCC cell lines. Syngeneic and xenograft mouse models are also planned to evaluate the effect of SUV420H1 depletion on flank tumor growth in vivo. These studies may elucidate the oncogenic effects and mechanisms of SUV420H1, and may provide evidence to support SUV420H1 as a potential novel target in the treatment of HPV-negative HNSCC tumors with SUV420H1 amplification or overexpression. Citation Format: Arfa Moshiri, Hui Cheng, Sohyoung Kim, Vassiliki Saloura. SUV420H1 as a novel target in HPV-negative head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6284.

Head and neck squamous cell carcinoma (HNSCC) remains a lethal and prevalent diagnosis with limited treatment options for recurrent metastatic cases, particularly in patients with sporadic, human papillomavirus (HPV) negative disease. Recently, the Human Cancer Genome Project identified cell death and NF-κB signaling alterations in a subset of HPV- and HPV+ HNSCC. Co-amplification of Fas-associated death domain (FADD) and cellular inhibitor of apoptosis protein 1 (cIAP1) was identified in HPV- HNSCC, whereas Tumor Necrosis Factor receptor-associated factor 3 (TRAF3) deletion was linked with HPV+ HNSCC. Birinapant, a cIAP inhibitor with primary affinity for cIAP1, functions as a SMAC mimetic to modulate downstream TNF death signaling and promote apoptosis. Clinical trials with Birinapant have demonstrated tolerability and favorable pharmacokinetics but limited activity as a single agent. Our lab recently demonstrated a key interaction between TNF-NF-κB signaling and the G2/M checkpoint kinase WEE1, providing a possible rationale for combination treatment targeting these pathways. We hypothesize that dual-antagonist therapy has the potential to synergistically inhibit TNF-induced canonical NF- κB pro-survival signaling, while enhancing sensitization to TNF-caspase and G2/M mitotic cell death. To investigate this, in vitro studies of Birinapant in combination with Adavosertib, a potent WEE1 inhibitor, were performed. Birinapant and Adavosertib demonstrated drug synergism to varying degrees in all HPV- and HPV+ cell lines tested, both in the presence and absence of tumor necrosis factor alpha (TNF-α), according to the Chou-Talalay method. In the majority of cell lines, synergistic drug activity, as indicated by a low combination index, was positively correlated with percent inhibition. These results were confirmed by increased levels of apoptosis as demonstrated by flow cytometry and both early and sustained cell growth inhibition over time in impedance assays. Ongoing studies include additional characterization of the downstream effects of these agents on NF-κB pro-survival signaling and the cell cycle, along with evaluation in a preclinical murine xenograft model with combined radiotherapy. Citation Format: Tiffany Toni, Ethan Morgan, Ramya Viswanathan, Xinping Yang, Hui Cheng, Carter Van Waes. Combination treatment with cIAP and WEE1 inhibitors exhibits synergism in HPV-positive and HPV-negative head and neck squamous carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2997.

The descending auditory system modulates the ascending system at every level. The final descending, or efferent, stage comprises lateral olivocochlear and medial olivocochlear (MOC) neurons. MOC somata in the ventral brainstem project axons to the cochlea to synapse onto outer hair cells (OHC), inhibiting OHC-mediated cochlear amplification. MOC suppression of OHC function is implicated in cochlear gain control with changing sound intensity, detection of salient stimuli, attention and protection against acoustic trauma. Thus, sound excites MOC neurons to provide negative feedback of the cochlea. Sound also inhibits MOC neurons via medial nucleus of the trapezoid body (MNTB) neurons. However, MNTB-MOC synapses exhibit short-term depression, suggesting reduced MNTB-MOC inhibition during sustained stimuli. Further, due to high rates of both baseline and sound-evoked activity in MNTB neurons in vivo, MNTB-MOC synapses may be tonically depressed. To probe this, we characterized short-term plasticity of MNTB-MOC synapses in mouse brain slices. We mimicked in vivo-like temperature and extracellular calcium conditions, and in vivo-like activity patterns of fast synaptic activation rates, sustained activation and prior tonic activity. Synaptic depression was sensitive to extracellular calcium concentration and temperature. During rapid MNTB axon stimulation, postsynaptic currents in MOC neurons summated but with concurrent depression, resulting in smaller, sustained currents, suggesting tonic inhibition of MOC neurons during rapid circuit activity. Low levels of baseline MNTB activity did not significantly reduce responses to subsequent rapid activity that mimics sound stimulation, indicating that, in vivo, MNTB inhibition of MOC neurons persists despite tonic synaptic depression. Key points Inhibitory synapses from the medial nucleus of the trapezoid body (MNTB) onto medial olivocochlear (MOC) neurons exhibit short-term plasticity that is sensitive to calcium and temperature, with enhanced synaptic depression occurring at higher calcium concentrations and at room temperature. High rates of background synaptic activity that mimic the upper limits of spontaneous MNTB activity cause tonic synaptic depression of MNTB-MOC synapses that limits further synaptic inhibition. High rates of activity at MNTB-MOC synapses cause synaptic summation with concurrent depression to yield a response with an initial large amplitude that decays to a tonic inhibition.

Otolith organs of the inner ear are innervated by two parallel afferent projections to the brainstem and cerebellum. These innervations were proposed to segregate across the line of polarity reversal (LPR) within each otolith organ, which divides the organ into two regions of hair cells (HC) with opposite stereociliary orientation. The relationship and functional significance of these anatomical features are not known. Here, we show regional expression of Emx2 in otolith organs, which establishes LPR, mediates the neuronal segregation across LPR and constitutes the bidirectional sensitivity function. Conditional knockout (cKO) of Emx2 in HCs lacks LPR. Tmie cKO, in which mechanotransduction was abolished selectively in HCs within the Emx2 expression domain also lacks bidirectional sensitivity. Analyses of both mutants indicate that LPR is specifically required formice to swim comfortably and to traverse a balance beam efficiently, but LPR is not required formice to stay on a rotating rod.

AbstractTNFα is a key mediator of immune and radiotherapy-induced cytotoxicity, but many cancers, including head and neck squamous cell carcinomas (HNSCC), display TNF resistance due to activation of the canonical IKK–NF-κB/RELA pro-survival pathway. However, toxicities associated with direct targeting of the canonical pathway point to the need to identify mechanism(s) contributing to TNFα resistance and synthetic lethal targets to overcome such resistance in cancer cells. Here, RNAi screening for modulators of TNFα–NF-κB reporter activity and cell survival unexpectedly implicated the WEE1 and CDC2 G2–M checkpoint kinases. The IKKα/β-RELA and WEE1-CDC2 signaling pathways are activated by TNFα and form a complex in cell lines derived from both human papillomavirus (−) and (+) subtypes of HNSCC. WEE1 inhibitor AZD1775 reduced IKK/RELA phosphorylation and the expression of NF-κB–dependent pro-survival proteins Cyclin D1 and BCL2. Combination of TNFα and AZD1775 enhanced caspase-mediated apoptosis in vitro, and combination treatment with radiotherapy and AZD1775 potentiated inhibition of HNSCC tumor xenograft growth in vivo, which could be significantly attenuated by TNFα depletion. These data offer new insight into the interplay between NF-κB signaling and WEE1-mediated regulation of the G2–M cell-cycle checkpoint in HNSCC.Implications:Inhibiting WEE1 and IKK-RELA crosstalk could potentially enhance the effects of therapies mediated by TNFα with less systemic immune suppression and toxicity than observed with direct interruption of IKK-NF-κB/RELA signaling.

Despite recent advances in treatment options for Human Papillomavirus (HPV)- head and neck squamous cell carcinoma (HNSCC), the overall survival (OS) rate for HNSCC is low, demonstrating the need for novel therapies for these cancers. Targeting the ubiquitin-proteasome system (UPS) has emerged as a potential target for the development of novel anti-cancer therapies. Bortezomib, a first generation proteasome inhibitor clinically approved for the treatment of Multiple Myeloma, has previously been demonstrated to induce tumor regression in a subset of HNSCC patients through the inhibition of canonical NFκB activity; however, the failure of Bortezomib to inhibit other pro-survival pathways such as MAPK and STAT3 signalling resulted in heterologous responses. A recently developed alternative to proteasome inhibitors is the small molecule b-AP15, which inhibits the proteasomal deubiquitinases USP14 and UCHL5, thus allowing more specificity and less toxicity than proteasome inhibitors. Here, we show b-AP15 inhibits proliferation and colony formation in multiple HPV- and HPV+ HNSCC cell lines, with minimal effects in normal oral keratinocytes. Furthermore, b-AP15 induces significant caspase-dependent apoptosis. Using siRNA targetting USP14 and UCHL5, only USP14 depletion significantly reduced cell proliferation and colony formation. Mechanistically, b-AP15 treatment reduces TNFα-induced NFκB activity and the expression of pro-survival proteins such as cIAP2 and TRAF2, promoting the formation of the TNFR complex II and sensitizing cells to TNFα-induced cell death. Finally, TCGA data demonstrates that USP14 is highly expressed in HNSCC when compared with normal tissue and is significantly correlated with worse overall survival. Together, we have identified that inhibition of proteasomal deubiquitinases inhibits the proliferation and survival of HNSCC cells and enhanced TNFα-induced cell death via the inhibition of NFκB activity. Our data suggest that combination therapies with b-AP15 could potentially offer a clinical benefit in HNSCC patients by promoting TNFα-induced cytotoxicity. Further studies will focus on the mechanism by which USP14 regulates NFκB signalling in HNSCC cells and the effect of b-AP15 activity in combination with radiation treatment in in vivo mouse xenograft models. Citation Format: Ethan L. Morgan, Tiffany Toni, Xinping Yang, Hui Cheng, Ramya Viswanathan, Zhong Chen, Carter Van Waes. Proteasomal deubiquitinases represent an attractive therapeutic target in head and neck squamous cell carcinomas (HNSCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2988.

Human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer type in the world and is associated with an overall poor prognosis. The protein methyltransferase SET and MYND domain-containing 3 (SMYD3), which trimethylates H3K4 and H4K20, can stimulate gene transcription and activate several oncogenic pathways, including epithelial-mesenchymal transition, and cell cycle related pathways. Additionally, its expression is associated with a poor prognosis in various cancer types, such as colon and ovarian cancer. This study aims to decipher whether SMYD3 has direct oncogenic effects and relevant mechanisms in HPV-negative HNSCC. Our preliminary results have demonstrated that SMYD3 is significantly overexpressed in HPV-negative HNSCC compared to normal and dysplastic epithelium. SMYD3 knockdown decreased cellular proliferation and clonal capacity in CFAs after siRNA-mediated SMYD3 knockdown in several HPV-negative HNSCC cell lines or CRISPR-mediated SMYD3 knockout. Cell cycle analysis of CRISPR SMYD3 KO cell lines showed a significant decrease in the S phase, supporting that cell cycle arrest may be a mechanism through which this decreased cellular proliferation occurs. Nuclear/cytoplasmic dissociation and western blotting of 6 HPV-negative HNSCC cell lines, and immunohistochemistry for SMYD3 in primary and recurrent/metastatic HPV-negative HNSCC tumor tissues have identified the expression of SMYD3 in both the cytoplasm and nucleus, with greater expression in the cytoplasm than the nucleus. Ongoing experiments aim to investigate the effects of SMYD3 knockdown on the global proteome, as well as its effects on histone post-translational modifications in HPV-negative HNSCC cell lines. Genome-wide mapping for SMYD3, H3K4me3 and H4K20me3 in HPV- negative HNSCC cells using CUT&RUN assays are ongoing with the goal to identify direct downstream gene targets regulated by human SMYD3. Finally, we plan to investigate if SMYD3 knockout decreases tumor growth in NSG xenografts. This study may provide the biological rational to target SMYD3 as a novel therapeutic avenue in HPV-negative HNSCC. Citation Format: Madhavi Murali, Daniel Tsai, Nupur Nigam, Kyunghee Burkitt, Sohyoung Kim, Hui Cheng, Vassiliki Saloura. Investigating the oncogenic effects and mechanisms of the SMYD3 methyltransferase in HPV-negative head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2959.

BACKGROUNDCisplatin is widely used to treat adult and pediatric cancers. It is the most ototoxic drug in clinical use, resulting in permanent hearing loss in approximately 50% of treated patients. There is a major need for therapies that prevent cisplatin-induced hearing loss. Studies in mice suggest that concurrent use of statins reduces cisplatin-induced hearing loss.METHODSWe examined hearing thresholds from 277 adults treated with cisplatin for head and neck cancer. Pretreatment and posttreatment audiograms were collected within 90 days of initiation and completion of cisplatin therapy. The primary outcome measure was a change in hearing as defined by the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE).RESULTSAmong patients on concurrent atorvastatin, 9.7% experienced a CTCAE grade 2 or higher cisplatin-induced hearing loss compared with 29.4% in nonstatin users (P < 0.0001). A mixed-effect model analysis showed that atorvastatin use was significantly associated with reduced cisplatin-induced hearing loss (P ≤ 0.01). An adjusted odds ratio (OR) analysis indicated that an atorvastatin user is 53% less likely to acquire a cisplatin-induced hearing loss than a nonstatin user (OR = 0.47; 95% CI, 0.30-0.78). Three-year survival rates were not different between atorvastatin users and nonstatin users (P > 0.05).CONCLUSIONSOur data indicate that atorvastatin use is associated with reduced incidence and severity of cisplatin-induced hearing loss in adults being treated for head and neck cancer.TRIAL REGISTRATIONClinicalTrials.gov identifier NCT03225157.FUNDINGFunding was provided by the Division of Intramural Research at the National Institute on Deafness and Other Communication Disorders (1 ZIA DC000079, ZIA DC000090).

Head and neck squamous cell carcinomas (HNSCC) induced by human papillomavirus (HPV) have increased recently in the US. However, the distinct alterations of molecules involved in the death pathways and drug effects targeting inhibitor of apoptosis proteins (IAPs) have not been extensively characterized in HPV(+) HNSCC cells. In this study, we observed the distinct genomic and expression alterations of nine genes involved in cell death in 55% HNSCC tissues, which were associated with HPV status, tumor staging, and anatomic locations. Expression of four genes was statistically correlated with copy number variation. A panel of HPV(+) HNSCC lines showed abundant TRAILR2 and IAP1 protein expression, but were not sensitive to IAP inhibitor birinapant alone, while combinatory treatment with TNFα or especially TRAIL enhanced this drug sensitivity. The death agonistic TRAILR2 antibody alone showed no cell inhibitory effects, whereas its combination with birinapant and/or TRAIL protein demonstrated additive or synergistic effects. We observed predominantly late apoptosis mode of cell death after combinatorial treatments, and pan-caspase (ZVAD) and caspase-8 (ZIETD) inhibitors attenuated treatment-induced cell death. Our genomic and expression data-driven study provides a framework for identifying relevant combinatorial therapies targeting death pathways in HPV(+) HNSCC and other squamous cancer types.

Abstract Human papilloma virus positive (HPV+) head and neck squamous cell carcinomas (HNSCC) exhibit a better prognosis and response to therapies than HPV(-) cancers. Analysis of HNSCC TCGA dataset provides evidence for distinct alterations in expression of components of the NF-κB and cell death pathways in HPV(+/-) HNSCC. Previously, we have found that birinapant, a novel SMAC mimetic that inhibits inhibitor of apoptosis proteins (IAPs), sensitizes a subset of HPV(-) HNSCC cell lines to death agonists like TNF-α and TRAIL. In our current study, we have observed that birinapant also sensitizes several HPV(+) cell lines to TNF-α and TRAIL in vitro. The IC50 for birinapant was under 50nM with TRAIL and TNF-α for the HPV (+) UPCI-SCC-90 and UM-SCC-47 cell lines. As TRAIL is known as a selective cancer cell death inducer, we explored its potential for enhancing death signaling in HPV(+) HNSCC cells via the effects of an agonistic polyclonal TRAILR2 antibody. Flow cytometry analysis confirmed the presence of TRAILR2 expression on the cell surface of the two HPV(+) cell lines. Treatment of cells with the antibody or TRAIL alone showed little or no inhibitory effect on growth of either cell lines. However, treatment with birinapant plus antibody, and especially the triple combination of birinapant, TRAIL, and antibody showed additive or synergistic effects to inhibit cell proliferation in a dose dependent manner. The Fixed Apoptotic Necrotic test using anti-phosphatidylserine and Zombie fixable dye showed that late apoptosis was the predominant mode of cell death for all combination groups for both cell lines at 48 hours post treatment. The proportion of cells undergoing apoptosis/necrosis was highest in the triple combination group in both cell lines, followed by birinapant and TRAIL combination for UM-SCC-47, or birinapant and antibody combination for UPCI-SCC-90, which was consistent with the relative percentage of sub-G0 DNA determined by flow cytometry analysis using propidium iodide staining. Enhanced apoptosis/necrosis with the triple combination was observed earlier than with single or dual treatment (12 versus 24 hours post treatment). Specific cell death mechanism was analyzed using pan-caspase (ZVAD), caspase-8 (ZIETD), and RIPK1 (necrostatin) inhibitors. In both UPCI-SCC-90 and UM-SCC-47 cells, caspase inhibition (ZVAD and ZIETD) completely reversed the effects of the double or triple combination treatments, whereas necrostatin did not. This suggests that the TRAILR2 agonist antibody mediated cell death in combination with birinapant is predominantly caspase-8 dependent. These results indicate that the TNF-α, TRAIL, and TRAILR2 agonist antibody sensitized birinapant anti-tumor activity, and triple combination exhibited synergistic effects. Supported by NIDCD projects ZIA-DC-000016, -73, -74, and the Medical Research Scholars Program. Citation Format: Yi An, Jun W. Jeon, Lillian Sun, Adeeb Derakhshan, Jianhong Chen, Hui Cheng, Xinping Yang, Christopher Silvin, Carter Van Waes, Zhong Chen. Birinapant enhances death agonist antibody against TRAILR2 anti-tumor activity in HPV-positive head and neck squamous cell carcinomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5175.

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide with five-year overall survival rate of about 50%. Analysis of The Cancer Genome Atlas (TCGA) dataset showed that one or more molecules linked to the tumor necrosis factor receptor (TNF)-NF-κB/REL signal pathway are genetically altered in most HNSCC, and experimental studies show this pathway drives expression of pro-proliferative, inflammatory, angiogenic, and therapeutic resistance genes. Such prevalence and effect across diverse genes suggest that the TNF-NF-κB/REL pathway interacts with molecules that are involved in multiple pathways, but their identity and mechanistic relationship to increased NF-κB signaling have not been studied in detail. Towards this hypothesis, we established a stable NF-κB β−lactamase reporter cell line and performed a genome-wide RNAi screening to identify key regulators of NF-κB oncogenic signaling and proliferation. As expected, the screening and validation data confirmed most components of the TNF receptor complex and downstream NF-κB pathway genes, such as IKKs, contribute to TNFα induced NF-κB activation and cell viability. We further identified and validated a novel sets of genes, which unexpectedly co-modulate NF-κB activity, including those related to the G2M cell cycle checkpoint, such as WEE1, PLK1, AURKA, TTK, CDC2, CDC7 and CALM2, as well as components related to kinetochore such as TPR, NDC80, and NUF2. After confirming sustained knockdown of selected mRNAs and proteins using siRNA by 48-72-hours post transfection, their effects on UM-SCC 1 were evaluated by real-time cell impedance assay. Cell index was significantly decreased by NDC80, NUF2, PLK1, or AURKA knockdown as compared to control siRNA knockdown. In addition, TNF-α treatment further decreased cellular density after NUF2, TPR, PLK1, and AURKA knockdown. The targeted drugs such as volasertib (PLK1), alisertib (AURKA), and adavosertib (WEE1) decreased cell density in a dose-dependent manner, while adavosertib and volasertib also attenuated NF-κB signaling. Thus, we demonstrate a novel link between NF-κB signaling with G2M kinases and kinetochore-related molecules to co-modulate tumor cell growth and survival. Integrated genomics and mechanistic studies are currently underway to understand how G2M and kinetochore-related molecules modulate NF-κB activation and their potential for cancer therapeutics. Supported by NIDCD projects ZIA-DC-000016, -73, -74, and the Medical Research Scholars Program (JWJ). Citation Format: Jun W. Jeon, Anthony D. Saleh, Shaleeka Cornelius, Sophie Carlson, Scott Martin, Pinar Ormanoglu, Hui Cheng, Xinping Yang, Chris Silvin, Zhong Chen, Carter Van Waes. Linkage of NF-κB pathway and mitosis-related molecules using integrated RNAi screening and transcriptomic analysis of head and neck squamous cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 605.