HPV-negative Head And Neck Squamous Cell Carcinoma Cell Research Articles

Inhibition of the chemokine receptors CXCR1 and CXCR2 synergizes with docetaxel for effective tumor control and remodeling of the immune microenvironment of HPV-negative head and neck cancer models

BackgroundRelapsed head and neck squamous cell carcinoma (HNSCC) unrelated to HPV infection carries a poor prognosis. Novel approaches are needed to improve the clinical outcome and prolong survival in this patient population which has poor long-term responses to immune checkpoint blockade. This study evaluated the chemokine receptors CXCR1 and CXCR2 as potential novel targets for the treatment of HPV-negative HNSCC.MethodsExpression of IL-8, CXCR1, and CXCR2 was investigated in HNSCC tissues and human cell line models. Inhibition of CXCR1/2 with the clinical stage, small molecule inhibitor, SX-682, was evaluated in vitro and in vivo using human xenografts and murine models of HNSCC, both as a monotherapy and in combination with the taxane chemotherapy, docetaxel.ResultsHigh levels of IL-8, CXCR1, and CXCR2 expression were observed in HPV-negative compared to HPV-positive HNSCC tumors or cell lines. Treatment of HPV-negative HNSCC cell lines in vitro with SX-682 sensitized the tumor cells to the cytotoxic activity of docetaxel. In vivo, treatment of HNSCC xenograft models with the combination of SX-682 plus docetaxel led to strong anti-tumor control resulting in tumor cures. This phenomenon was associated with an increase of microRNA-200c and a decreased expression of its target, tubulin beta-3, a protein involved in resistance to microtubule-targeting chemotherapies. In vivo treatment of a murine syngeneic model of HNSCC with SX-682 plus docetaxel led to potent anti-tumor efficacy through a simultaneous decrease in suppressive CXCR2+ polymorphonuclear, myeloid-derived suppressor cells and an increase in cytotoxic CD8+ T cells in the combination therapy treated tumors compared to controls.ConclusionsThis study reports, for the first time, mechanistic findings through which the combination of CXCR1/2 inhibition and docetaxel chemotherapy exhibits synergy in models of HPV-negative HNSCC. These findings provide rationale for the use of this novel combination approach to treat HPV-negative HNSCC patients and for future combination studies of CXCR1/2 inhibition, docetaxel, and immune-based therapies.

Inhibition of ATM or ATR in combination with hypo-fractionated radiotherapy leads to a different immunophenotype on transcript and protein level in HNSCC.

The treatment of head and neck tumors remains a challenge due to their reduced radiosensitivity. Small molecule kinase inhibitors (smKI) that inhibit the DNA damage response, may increase the radiosensitivity of tumor cells. However, little is known about how the immunophenotype of the tumor cells is modulated thereby. Therefore, we investigated whether the combination of ATM or ATR inhibitors with hypo-fractionated radiotherapy (RT) has a different impact on the expression of immune checkpoint markers (extrinsic), the release of cytokines or the transcriptome (intrinsic) of head and neck squamous cell carcinoma (HNSCC) cells. The toxic and immunogenic effects of the smKI AZD0156 (ATMi) and VE-822 (ATRi) in combination with a hypo-fractionated scheme of 2x5Gy RT on HPV-negative (HSC4, Cal-33) and HPV-positive (UM-SCC-47, UD-SCC-2) HNSCC cell lines were analyzed as follows: cell death (necrosis, apoptosis; detected by AnxV/PI), expression of immunostimulatory (ICOS-L, OX40-L, TNFSFR9, CD70) and immunosuppressive (PD-L1, PD-L2, HVEM) checkpoint marker using flow cytometry; the release of cytokines using multiplex ELISA and the gene expression of Cal-33 on mRNA level 48h post-RT. Cell death was mainly induced by the combination of RT with both inhibitors, but stronger with ATRi. Further, the immune phenotype of cancer cells, not dying from combination therapy itself, is altered predominantly by RT+ATRi in an immune-stimulatory manner by the up-regulation of ICOS-L. However, the analysis of secreted cytokines after treatment of HNSCC cell lines revealed an ambivalent influence of both inhibitors, as we observed the intensified secretion of IL-6 and IL-8 after RT+ATRi. These findings were confirmed by RNAseq analysis and further the stronger immune-suppressive character of RT+ATMi was enlightened. We detected the down-regulation of a central protein of cytoplasmatic sensing pathways of nucleic acids, RIG-1, and found one immune-suppressive target, EDIL3, strongly up-regulated by RT+ATMi. Independent of a restrictive toxicity, the combination of RT + either ATMi or ATRi leads to comprehensive and immune-modulating alterations in HNSCC. This includes pro-inflammatory signaling induced by RT + ATRi but also anti-inflammatory signals. These findings were confirmed by RNAseq analysis, which further highlighted the immune-suppressive nature of RT + ATMi.

Human papillomavirus-associated head and neck squamous cell carcinoma cells lose viability during triggered myocyte lineage differentiation

Head and neck squamous cell carcinoma (HNSCC) is a highly malignant disease, and death rates have remained at approximately 50% for decades. New tumor-targeting strategies are desperately needed, and a previous report indicated the triggered differentiation of HPV-negative HNSCC cells to confer therapeutic benefits. Using patient-derived tumor cells, we created a similar HNSCC differentiation model of HPV+ tumor cells from two patients. We observed a loss of malignant characteristics in differentiating cell culture conditions, including irregularly enlarged cell morphology, cell cycle arrest with downregulation of Ki67, and reduced cell viability. RNA-Seq showed myocyte-like differentiation with upregulation of markers of myofibril assembly. Immunofluorescence staining of differentiated and undifferentiated primary HPV+ HNSCC cells confirmed an upregulation of these markers and the formation of parallel actin fibers reminiscent of myoblast-lineage cells. Moreover, immunofluorescence of HPV+ tumor tissue revealed areas of cells co-expressing the identified markers of myofibril assembly, HPV surrogate marker p16, and stress-associated basal keratinocyte marker KRT17, indicating that the observed myocyte-like in vitro differentiation occurs in human tissue. We are the first to report that carcinoma cells can undergo a triggered myocyte-like differentiation, and our study suggests that the targeted differentiation of HPV+ HNSCCs might be therapeutically valuable.

Inhibition of key DNA double strand break repair protein kinases enhances radiosensitivity of head and neck cancer cells to X-ray and proton irradiation

Ionising radiation (IR) is widely used in cancer treatment, including for head and neck squamous cell carcinoma (HNSCC), where it induces significant DNA damage leading ultimately to tumour cell death. Among these lesions, DNA double strand breaks (DSBs) are the most threatening lesion to cell survival. The two main repair mechanisms that detect and repair DSBs are non-homologous end joining (NHEJ) and homologous recombination (HR). Among these pathways, the protein kinases ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related (ATR) and the DNA dependent protein kinase catalytic subunit (DNA-Pkcs) play key roles in the sensing of the DSB and subsequent coordination of the downstream repair events. Consequently, targeting these kinases with potent and specific inhibitors is considered an approach to enhance the radiosensitivity of tumour cells. Here, we have investigated the impact of inhibition of ATM, ATR and DNA-Pkcs on the survival and growth of six radioresistant HPV-negative HNSCC cell lines in combination with either X-ray irradiation or proton beam therapy, and confirmed the mechanistic pathway leading to cell radiosensitisation. Using inhibitors targeting ATM (AZD1390), ATR (AZD6738) and DNA-Pkcs (AZD7648), we observed that this led to significantly decreased clonogenic survival of HNSCC cell lines following both X-ray and proton irradiation. Radiosensitisation of HNSCC cells grown as 3D spheroids was also observed, particularly following ATM and DNA-Pkcs inhibition. We confirmed that the inhibitors in combination with X-rays and protons led to DSB persistence, and increased micronuclei formation. Cumulatively, our data suggest that targeting DSB repair, particularly via ATM and DNA-Pkcs inhibition, can exacerbate the impact of ionising radiation in sensitising HNSCC cell models.

Autophagy is the main driver of radioresistance of HNSCC cells in mild hypoxia.

Hypoxia poses a significant challenge to the effectiveness of radiotherapy in head and neck squamous cell carcinoma (HNSCC) patients, and it is imperative to discover novel approaches to overcome this. In this study, we investigated the underlying mechanisms contributing to x-ray radioresistance in HPV-negative HNSCC cells under mild hypoxic conditions (1% oxygen) and explored the potential for autophagy modulation as a promising therapeutic strategy. Our findings show that HNSCC cells exposed to mild hypoxic conditions exhibit increased radioresistance, which is largely mediated by the hypoxia-inducible factor (HIF) pathway. We demonstrate that siRNA knockdown of HIF-1α and HIF-1β leads to increased radiosensitivity in HNSCC cells under hypoxia. Hypoxia-induced radioresistance was not attributed to differences in DNA double strand break repair kinetics, as these remain largely unchanged under normoxic and hypoxic conditions. Rather, we identify autophagy as a critical protective mechanism in HNSCC cells following irradiation under mild hypoxia conditions. Targeting key autophagy genes, such as BECLIN1 and BNIP3/3L, using siRNA sensitizes these cells to irradiation. Whilst autophagy's role in hypoxic radioresistance remains controversial, this study highlights the importance of autophagy modulation as a potential therapeutic approach to enhance the effectiveness of radiotherapy in HNSCC.

Radiobiology of Proton Therapy in Human Papillomavirus-Negative and Human Papillomavirus-Positive Head and Neck Cancer Cells.

Photon-based radiotherapy (XRT) is one of the most frequently used treatment modalities for HPV-negative and HPV-positive locally advanced head and neck squamous cell carcinoma (HNSCC). However, locoregional recurrences and normal RT-associated toxicity remain major problems for these patients. Proton therapy (PT), with its dosimetric advantages, can present a solution to the normal toxicity problem. However, issues concerning physical delivery and the lack of insights into the underlying biology of PT hamper the full exploitation of PT. Here, we assessed the radiobiological processes involved in PT in HPV-negative and HPV-positive HNSCC cells. We show that PT and XRT activate the DNA damage-repair and stress response in both HPV-negative and HPV-positive cells to a similar extent. The activation of these major radiobiological mechanisms resulted in equal levels of clonogenic survival and mitotic cell death. Altogether, PT resulted in similar biological effectiveness when compared to XRT. These results emphasize the importance of dosimetric parameters when exploiting the potential of increased clinical effectiveness and reduced normal tissue toxicity in PT treatment.

CHD4 acts as a prognostic factor and drives radioresistance in HPV negative HNSCC

Despite great efforts in improving existing therapies, the outcome of patients with advanced radioresistant HPV-negative head and neck squamous cell carcinoma (HNSCC) remains poor. The chromatin remodeler Chromodomain helicase DNA binding protein 4 (CHD4) is involved in different DNA-repair mechanisms, but the role and potential in HNSCC has not been explored yet. In the present study, we evaluated the prognostic significance of CHD4 expression using in silico analysis of the pan-cancer dataset. Furthermore, we established a monoclonal HNSCC CHD4 knockdown cell clone utilizing the CRISPR/Cas9 system. Effects of lower CHD4 expression on radiosensitivity after increasing doses of ionizing radiation were characterized using clonogenic assays and cell numbers. The in silico analysis revealed that high CHD4 expression is associated with significant poorer overall survival of HPV-negative HNSCC patients. Additionally, the knockdown of CHD4 significantly increased the radiosensitivity of HNSCC cells. Therefore, CHD4 might be involved in promoting radioresistance in hard-to-treat HPV-negative HNSCC entities. We conclude that CHD4 could serve as a prognostic factor in HPV-negative HNSCC tumors and is a potential target protein overcoming radioresistance in HNSCC. Our results and the newly established cell clone laid the foundation to further characterize the underlying mechanisms and ultimately use CHD4 in HNSCC therapies.

Abstract 1729: SUV420H1 depletion reveals therapeutic potential in HPV-negative head and neck squamous cell carcinoma

Abstract Human Papilloma Virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) poses a significant clinical challenge with limited treatment options. SUV420H1, which encodes for a protein lysine methyltransferase (PMT) that methylates H4K20 (H4K20me3), is recurrently amplified in ~5% of HPV-negative HNSCC tumors and is the second most frequently amplified PMT in this disease, suggesting an oncogenic function in this subset of HPV-negative HNSCC patients. Gene set enrichment analysis using the TCGA database of HPV-negative HNSCC tumors revealed that SUV420H1 overexpressing tumors showed enrichment in the mitotic spindle related pathways, and repression of immune-related pathways. To assess whether SUV420H1 depletion affects the proliferation of HPV-negative HSNCC cells, CCK8 and colony formation assays were pursued after siRNA-mediated knockdown of SUV420H1 in five SUV420H1-overexpressing HPV-negative HNSCC cell lines and revealed a significant reduction in cell proliferation and colony forming capacity. SUV420H1 CRISPR knockout (KO) HPV-negative HNSCC human and mouse cell lines were generated. Proliferation and colony formation assays with the SUV420H1 KO cell lines are ongoing to validate the aforementioned phenotypes. To identify oncogenic mechanism of SUV420H1, genome-wide mapping of H4K20me3 after siRNA-mediated depletion or enzymatic inhibition of SUV420H1 (A-196) was pursued using CUT&RUN assays combined with RNA-seq in a SUV420H1 amplified cell line (SCC-151), and analysis is ongoing. Preliminary RNA-seq analysis revealed upregulation of a number of type I IFN response genes after siRNA-mediated knockdown of SUV420H1. In vivo mouse experiments using SUV420H1 KO mouse cell lines are planned in a syngeneic HPV-negative HNSCC flank mouse model (MOC1) with or without anti-PD-1 treatment with the goal to assess whether SUV420H1 depletion affects tumor growth and/or sensitizes cancer cells to anti-PD-1 therapy. These data provide preliminary support for the function of SUV420H1 as an oncogene in HPV-negative HNSCC. Citation Format: Arfa Moshiri, Sohyoung Kim, Marie Luff, Vassiliki Saloura. SUV420H1 depletion reveals therapeutic potential in HPV-negative head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1729.

Abstract 1734: Dissecting the bifaceted function of SMYD3 as a transcriptional activator and repressor in HPV-negative HNSCC

Abstract Background: Approximately 75% of Human-papilloma-virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) tumors carry genetic and expression alterations in the family of protein methyltransferases and demethylases, underscoring the importance of these enzymes in the pathogenesis of HNSCC. SMYD3 a lysine methyltransferase that is overexpressed in HPV-negative HNSCC. We recently reported that nuclear SMYD3 plays dual role in immune-related gene regulation through H3K4me3 or H4K20me3, leading to gene activation or silencing, respectively in HPV-negative HNSCC. While SMYD3 has been reported to write and bind to H3K4me3 activating target gene expression, its function in gene silencing is understudied. This study aims to determine the mechanism(s) through which SMYD3 functions both as a transcriptional activator and a repressor within the same cell context, specifically in HPV-negative HNSCC. Methods: immunoprecipitation (IP) of SMYD3 was performed from nuclear extracts isolated from HN-6 cells to identify candidate interacting partners of SMYD3. The nuclear immunoprecipitates were sent for MS analysis to identify candidate nuclear interacting partners of SMYD3. CUT&RUN assays for selected candidate interacting proteins and SMYD3 will be performed in control HN-6 cells versus SMYD3 KO cells to assess genomic co-occupancy with SMYD3. Further, CUT&RUN assays for repressive marks H4K20me3, H3K27me3 and H3K9me3 have been performed in HN-6 and SMYD3 KO cells to assess co-occupancy with SMYD3 as well as its effect of SMYD3 on the genome-wide mapping of these marks. Finally, CUT&RUN assays for SMYD3 followed by re-CUT&RUN of repressive histone marks are ongoing to identify co-occupied loci. Results: Preliminary analysis of our nuclear SMYD3 IP results has identified a chromatin modifier with known repressive function. Co-immunoprecipitation experiments to validate this interaction are planned. CUT&RUN assays for repressive histone marks in HN-6 versus SMYD3 KO cells have revealed impact on H4K20me3 and H3K27me3. Conclusions: Our preliminary results suggest that SMYD3 may exert its silencing function by regulating the genome-wide deposition of H4K20me3.Our nuclear SMYD3 IP analysis has identified candidate interacting partners that may assist SMYD3 in promoting the deposition of these repressive marks. CUT&RUN assays for SMYD3 followed by re-CUT&RUN for H4K20me3 and H3K27me3 bound chromatin may provide further insight about whether SMYD3 co-occupies specific genomic regions together with these repressive marks. This project is expected to provide mechanistic insights into the bifaceted functions of SMYD3 within the same cell context using HPV-negative HNSCC cell lines as an experimental model system.. Citation Format: Jawad Akhtar, Sohyoung Kim, Vassiliki Saloura. Dissecting the bifaceted function of SMYD3 as a transcriptional activator and repressor in HPV-negative HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1734.

Abstract 2884: Resistance to pan-FGFR inhibition is determined by the EMT phenotype associating with clinical outcome in HNSCC

Abstract Receptor tyrosine kinases (RTK) and integrins coalesce at focal adhesions. These cell membrane areas serve as huge signaling hubs critically contributing to central aspects of cancer cell survival and therapy resistance. Despite the fact that these receptors mutually interact, co-dependencies between these receptors and associated therapeutically exploitable vulnerabilities remain largely unclear in HPV-negative head and neck squamous cell carcinoma (HNSCC). Here, we evaluated the cytotoxic and radiochemosensitizing potential of targeting 10 RTK and β1 integrin in up to 20 different 3D matrix grown HNSCC cell models. RNA sequencing and protein-based biochemical assays were performed for molecular and pathway characterization. Bioinformatically identified transcriptomic signatures were applied to patient cohorts to show clinical relevance. Our observations demonstrate that fibroblast growth factor receptors (FGFR 1-4) present with the strongest cytotoxic and radiosensitizing potential, both as monotherapy and in combination with β1 integrin inhibition, surpassing the efficacy of the other investigated RTK. Pharmacological pan-FGFR inhibition elicited a variable response spectrum ranging from cytotoxicity/radiochemosensitization to resistance/radioprotection. Transcriptomic characterization revealed an association of these contrasting responses to FGFR inhibition with a mesenchymal-to-epithelial transition (MET) for sensitive cell models and a partial epithelial-to-mesenchymal transition (EMT) for resistant cell models. Accordingly, deactivation of EMT associated kinases like EGFR, PKC and PAK proved effective in diminishing the adaptive FGFR-driven resistance. Importantly, the translation of the adaptive resistance profiles to HNSCC patient cohorts showed its prognostic value and provided conclusive validation of the presence of EMT-related vulnerabilities that can be strategically used for therapeutic intervention. In conclusion, our study demonstrates that pan-FGFR inhibition induces both a highly beneficial radiochemosensitizing and a detrimental radioprotective effect in HNSCC cell models. Adaptive EMT-associated resistance appears to be of clinical importance, and we provide effective molecular approaches to exploit this therapeutically. Citation Format: Felix Broghammer, Mahesh Gouda, Irina Korovina, Cornelia Brunner, Robert P. Coppes, Olivier Gires, Michael Seifert, Nils Cordes. Resistance to pan-FGFR inhibition is determined by the EMT phenotype associating with clinical outcome in HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2884.

Combined IL6 and CCR2 blockade potentiates antitumor activity of NK cells in HPV-negative head and neck cancer

BackgroundWhile T cell-activating immunotherapies against recurrent head and neck squamous cell carcinoma (HNSCC) have shown impressive results in clinical trials, they are often ineffective in the majority of patients. NK cells are potential targets for immunotherapeutic intervention; however, the setback in monalizumab-based therapy in HNSCC highlights the need for an alternative treatment to enhance their antitumor activity.MethodsSingle-cell RNA sequencing (scRNA-seq) and TCGA HNSCC datasets were used to identify key molecular alterations in NK cells. Representative HPV-positive ( +) and HPV-negative ( −) HNSCC cell lines and orthotopic mouse models were used to validate the bioinformatic findings. Changes in immune cells were examined by flow cytometry and immunofluorescence.ResultsThrough integration of scRNA-seq data with TCGA data, we found that the impact of IL6/IL6R and CCL2/CCR2 signaling pathways on evasion of immune attack by NK cells is more pronounced in the HPV − HNSCC cohort compared to the HPV + HNSCC cohort. In orthotopic mouse models, blocking IL6 with a neutralizing antibody suppressed HPV − but not HPV + tumors, which was accompanied by increased tumor infiltration and proliferation of CD161+ NK cells. Notably, combining the CCR2 chemokine receptor antagonist RS504393 with IL6 blockade resulted in a more pronounced antitumor effect that was associated with more activated intratumoral NK cells in HPV − HNSCC compared to either agent alone.ConclusionsThese findings demonstrate that dual blockade of IL6 and CCR2 pathways effectively enhances the antitumor activity of NK cells in HPV-negative HNSCC, providing a novel strategy for treating this type of cancer.

Resistance of HNSCC cell models to pan-FGFR inhibition depends on the EMT phenotype associating with clinical outcome

BackgroundFocal adhesion signaling involving receptor tyrosine kinases (RTK) and integrins co-controls cancer cell survival and therapy resistance. However, co-dependencies between these receptors and therapeutically exploitable vulnerabilities remain largely elusive in HPV-negative head and neck squamous cell carcinoma (HNSCC).MethodsThe cytotoxic and radiochemosensitizing potential of targeting 10 RTK and β1 integrin was determined in up to 20 3D matrix-grown HNSCC cell models followed by drug screening and patient-derived organoid validation. RNA sequencing and protein-based biochemical assays were performed for molecular characterization. Bioinformatically identified transcriptomic signatures were applied to patient cohorts.ResultsFibroblast growth factor receptor (FGFR 1–4) targeting exhibited the strongest cytotoxic and radiosensitizing effects as monotherapy and combined with β1 integrin inhibition, exceeding the efficacy of the other RTK studied. Pharmacological pan-FGFR inhibition elicited responses ranging from cytotoxicity/radiochemosensitization to resistance/radiation protection. RNA sequence analysis revealed a mesenchymal-to-epithelial transition (MET) in sensitive cell models, whereas resistant cell models exhibited a partial epithelial-to-mesenchymal transition (EMT). Accordingly, inhibition of EMT-associated kinases such as EGFR caused reduced adaptive resistance and enhanced (radio)sensitization to FGFR inhibition cell model- and organoid-dependently. Transferring the EMT-associated transcriptomic profiles to HNSCC patient cohorts not only demonstrated their prognostic value but also provided a conclusive validation of the presence of EGFR-related vulnerabilities that can be strategically exploited for therapeutic interventions.ConclusionsThis study demonstrates that pan-FGFR inhibition elicits a beneficial radiochemosensitizing and a detrimental radioprotective potential in HNSCC cell models. Adaptive EMT-associated resistance appears to be of clinical importance, and we provide effective molecular approaches to exploit this therapeutically.

Preclinical Evaluation of the ATR Inhibitor BAY 1895344 as a Radiosensitizer for Head and Neck Squamous Cell Carcinoma

Despite aggressive multimodal treatment that typically includes definitive or adjuvant radiation therapy (RT), locoregional recurrence rates approach 50% for patients with locally advanced human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC). Thus, more effective therapeutics are needed to improve patient outcomes. We evaluated the radiosensitizing effects of ataxia telangiectasia and RAD3-related (ATR) inhibitor (ATRi) BAY 1895344 in preclinical models of HNSCC. Murine and human HPV-negative HNSCC cells (MOC2, MOC1, JHU-012) were treated with vehicle or ATRi with or without 4 Gy. Checkpoint kinase 1 phosphorylation and DNA damage (γH2AX) were evaluated by Western blot, and ATRi half-maximal inhibitory concentration was determined by MTT assay for HNSCC cells and immortalized murine oral keratinocytes. In vitro radiosensitization was tested by clonogenic assay. Cell cycle distribution and mitotic catastrophe were evaluated by flow cytometry. Mitotic aberrations were quantified by fluorescent microscopy. Tumor growth delay and survival were assessed in mice bearing MOC2 or JHU-012 transplant tumors treated with vehicle, ATRi, RT (10 Gy × 1 or 8 Gy × 3), or combined ATRi + RT. ATRi caused dose-dependent reduction in checkpoint kinase 1 phosphorylation at 1 hour post-RT (4 Gy) and dose-dependent increase in γH2AX at 18 hours post-RT. Addition of RT to ATRi led to decreased BAY 1895344 half-maximal inhibitory concentration in HNSCC cell lines but not in normal tissue surrogate immortalized murine oral keratinocytes. Clonogenic assays demonstrated radiosensitization in the HNSCC cell lines. ATRi abrogated the RT-induced G2/M checkpoint, leading to mitosis with unrepaired DNA damage and increased mitotic aberrations (multinucleated cells, micronuclei, nuclear buds, nucleoplasmic bridges). ATRi and RT significantly delayed tumor growth in MOC2 and JHU-012 in vivo models, with improved overall survival in the MOC2 model. These findings demonstrated that BAY 1895344 increased in vitro and in vivo radiosensitivity in HPV-negative HNSCC preclinical models, suggesting therapeutic potential warranting evaluation in clinical trials for patients with locally advanced or recurrent HPV-negative HNSCC.

FAK Drives Resistance to Therapy in HPV-Negative Head and Neck Cancer in a p53-Dependent Manner.

Radiation and platinum-based chemotherapy form the backbone of therapy in human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC). We have correlated focal adhesion kinase (FAK/PTK2) expression with radioresistance and worse outcomes in these patients. However, the importance of FAK in driving radioresistance and its effects on chemoresistance in these patients remains unclear. We performed an in vivo shRNA screen using targetable libraries to identify novel therapeutic sensitizers for radiation and chemotherapy. We identified FAK as an excellent target for both radio- and chemosensitization. Because TP53 is mutated in over 80% of HPV-negative HNSCC, we hypothesized that mutant TP53 may facilitate FAK-mediated therapy resistance. FAK inhibitor increased sensitivity to radiation, increased DNA damage, and repressed homologous recombination and nonhomologous end joining repair in mutant, but not wild-type, TP53 HPV-negative HNSCC cell lines. The mutant TP53 cisplatin-resistant cell line had increased FAK phosphorylation compared with wild-type, and FAK inhibition partially reversed cisplatin resistance. To validate these findings, we utilized an HNSCC cohort to show that FAK copy number and gene expression were associated with worse disease-free survival in mutant TP53, but not wild-type TP53, HPV-negative HNSCC tumors. FAK may represent a targetable therapeutic sensitizer linked to a known genomic marker of resistance.

In vitro antineoplastic effects of MK0752 in HPV-positive head and neck squamous cell carcinoma

PurposeGamma-secretase inhibitor MK0752 has shown a high therapeutic potential in different solid malignant tumors. Up to now, its antineoplastic effects were not investigated in head and neck squamous cell carcinoma (HNSCC) and particularly in human-papillomavirus (HPV)-positive tumors.MethodsWe conducted cytotoxic, migration, and clonogenic assays in two HPV-negative HNSCC cell lines (Cal27 and FaDu) and one HPV-positive cell line (SCC154). Furthermore, in order to assess the pro-apoptotic effects of MK0752, a Caspase 3/7 Glo assay was performed.ResultsOur experiments revealed antineoplastic effects of MK0752 in all three cell lines. Strong cytotoxic and antimigratory potential was shown in all cell lines, with strongest effects observed in the HPV-positive cell line. Meanwhile, anticlonogenic effects were only shown in Cal27 and SCC154. Most importantly, MK0752 induced apoptosis solely in HPV-positive SCC154.ConclusionsOur novel findings indicate a therapeutic potential of MK0752 in HPV-positive HNSCC. Indeed, further investigation is needed for validation of our results and for the assessment of the mechanistic background.

Palbociclib + cetuximab versus cetuximab in patients with CDKN2A-altered, anti-PD-1 resistant, HPV-negative head and neck squamous cell carcinoma (HNSCC): A phase 3 trial.

TPS6103 Background: Cell cycle deregulation is ubiquitous in HNSCC. In HPV-negative HNSCC, the most common genomic cell-cycling alteration involves CDKN2A, which results in loss of p16 expression, hyper-activation of CDK4/6-cyclin D1, unrestrained cell cycling and tumor progression. The modest antitumor activity of cetuximab in HPV-negative HNSCC may be due to unregulated cell-cycling events downstream of EGFR. In cell-line, xenograft, and PDX models of HPV-negative HNSCC, selective CDK4/6 inhibition arrested cell cycling and inhibited tumor growth. CDKN2A alterations were predictive of efficacy. Palbociclib, a selective CDK4/6 inhibitor, in combination with an EGFR inhibitor synergistically reduced the viability of HPV-negative HNSCC cell lines. An exploratory analysis of a double-blind, randomized, phase 2 trial (PALATINUS) showed that overall survival (OS) was numerically superior in patients with CDKN2A altered, HPV-negative recurrent/metastatic (RM) HNSCC treated with palbociclib + cetuximab vs those treated with placebo + cetuximab (median 9.7 vs 4.6 months, HR 0.38); however, OS was similar between the two treatment arms in patients without CDKN2A-altered disease. Also, the TAPUR trial showed that palbociclib decreased target lesions in 25% of patients with CDKN2A-altered HN cancers. These data provide justification for a randomized trial of palbociclib + cetuximab vs cetuximab in patients with CDKN2A-altered, HPV-negative RM-HNSCC. Enrollment was limited to patients with anti-PD-1 resistant disease, since current treatments fail to improve OS in these patients. Methods: The primary aim of this open-label, phase 3 trial is to determine the OS of patients with CDKN2A-altered (mutation or deletion), anti-PD-1 resistant, HPV-negative HNSCC treated with palbociclib + cetuximab (Arm 1) vs cetuximab (Arm 2). Eligibility also requires 1-3 lines of prior therapy and no cetuximab for RM disease. Patients are randomized (2:1) to Arms 1 or 2, stratified by prior platinum or cetuximab (if given with curative-intent therapy). Patients receive 28-day cycles of palbociclib 125 mg/d orally on days 1-21 + cetuximab 400 mg/m2 IV cycle 1 day 1, then 250 mg/m2 weekly (Arm 1) or cetuximab (Arm 2). Tumor response is assessed using RECIST 1.1. A two-stage sequential design, including one interim and a final analysis, was planned. We hypothesized that the median OS of Arm 1 will be 9.7 months and of Arm 2 4.6 months. O'Brien-Fleming method was used to calculate the sample size at each stage and derive the boundaries of two-sided hypothesis with early stopping to reject or accept H0 (HR = 1). Assuming 22 months enrollment and 12 months follow-up, 66 patients (Arm 1: 44; Arm 2: 22) will be enrolled in the first stage. If the study continues, an additional 15 patients will be enrolled (total Arm 1: 54; Arm 2: 27) which achieves 80% power at a two-sided α 0.05. Clinical trial information: NCT04966481 .

Abstract 4756: NSD1/2 histone methyltransferases regulate cell growth in HPV-negative head and neck squamous cell carcinoma (HNSCC)

Abstract Members of the NSD protein family (NSD1, NSD2, and NSD3) are histone methyltransferases (HMTs) that catalyze lysine 36 dimethylation (K36me2) at histone H3. H3K36 modifications play an important role in regulating the function and structure of chromatin, affecting transcription, replication, and repair. Abnormal H3K36 methylation is often detected during tumor development and progression. Inactivating NSD1 mutations are frequent in head and neck squamous cell carcinoma (HNSCC). They commonly occur in HPV-negative oropharyngeal (OP) and laryngeal (LC) carcinomas, and define a prognostic subtype in LC, associated with significantly improved overall and progression-free survival. Notably, the SCC4 cell line, carrying a damaging mutation in the NSD1 gene demonstrated reduced dimethylation level of H3K36 compared to NSD1 wild-type HNSCC counterparts. To explore the biological impact of the NSD1/NSD2 loss of function in HNSCC, we established cell lines with doxycycline-inducible shRNA knockdown of NSD1 and NSD2 in the set of HNSCC cell lines originating from different sites (JHU011, JHU022, Cal27, and FaDu cell lines). The depletion of NSD1 and NSD2 led to reduction of K36me2, significant decrease in cell growth as measured by cell titer blue (CTB) and clonogenic assays. NSD1/NSD2 depletion in these HNSCC cells also caused a significant increase in apoptosis. Gene Set Enrichment Analysis (GSEA) of RNA-seq for NSD1 wt versus knockdown cells indicates that NSD1 knockdown reduced expression of E2F target genes. Among the E2F transcription factor family, E2F2 gene expression was significantly decreased in all NSD1 knockdown cell lines. NSD1 knockdown also activated gene pathways related to autophagy and response to starvation. NSD1 knockdown reduced the levels of autophagy initiation gene ULK1 at both mRNA and protein levels. We also probed for protein signaling in HNSCC cells following NSD1 depletion using a reverse protein phase array (RPPA) approach, and validated Phosphatidylinositol-5-Phosphate 4-Kinase Type 2 Beta (PIP4K2B), but not other members of this family (PIP4K2A and PIP4K2C), as NSD1-regulated. PIP4K2B was regulated at the mRNA level by NSD1 as well. The CHIP-qPCR assay demonstrated the loss of H3K36me2 at the promoter of the PIP4K2B gene in NSD1 knockdown cells, suggesting direct regulation by NSD1. Moreover, PIP4K2B siRNA depletion has also led to a significant decrease in HNSCC proliferation, which suggests that the NSD1 may regulate proliferative activity through PIP4K2B. Taken together, while this data supports the suggestion that NSD histone methyltransferases have multiple downstream targets, the underlying mechanism remain to be investigated in more detail. Further, NSD proteins are attractive targets for drug development for improving treatment strategies for HNSCC. Citation Format: Iuliia Topchu, Igor Bychkov, Petr Makhov, Evgeny Izumchenko, John Karanicolas, Jindan Yu, Jochen Lorch, Yanis Boumber. NSD1/2 histone methyltransferases regulate cell growth in HPV-negative head and neck squamous cell carcinoma (HNSCC). [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 4756.

Abstract 4951: Proteasome inhibitor carfilzomib induces apoptosis via downregulation of AKT pathway in HPV negative HNSCC

Abstract Background Head and neck squamous cell carcinoma (HNSCC) presents in heterogenous sites, varied etiology and risk factors, and has diverse genetic background which confer dissimilar response to treatments, including chemotherapy. Here we tested the hypothesis that investigation of HPV presence in a HNSCC cell line would identify pathway contributing to oncogenesis of other HNSCC. We sought to identify pathways that differentially affect HNSCC according to the genetic background, therefore identifying targets and suitable candidate for treatment. We used genetic and pharmacological approaches to inhibit AKT pathway in HNSCC cell lines. Materials & methods Chemical genetic library screening against 6 HNSCC cell lines were performed. The high throughput combinatorial small molecule screening for cell viability and apoptosis was carried out. Candidate agents were validated in both CAL-27 (HPV-negative) and UM-SCC-47 (HPV-positive) cell lines. Identified pathways and representative targeted agents were validated in vitro. Expression of related protein were confirmed by western blot assay. Results EGFR, PIK3CA, mTOR, CDK1, HSP90AB1, HDAC1, and PSMD1 pathways were identified to be susceptible pathways in HNSCC using high throughput chemical genetic library screening. In vitro validation of carfilzomib showed IC50 of 7.07nM in CAL-27 cells and IC50 of 26.3nM in UM-SCC-47 cells. Knockdown of PSMD1 transcripts by transfected siRNAs reduced CAL-27 cells viability. Carfilzomib regulates the AKT pathway by decreasing t-AKT, p-AKT, p-ERK, p-S6 proteins and increasing p21 in CAL-27. Conclusion Inhibition of AKT pathway treatment with proteasome inhibitor carfilzomib is effective in HPV-negative HNSCC cells. Taken together, our findings reveal carfilzomib induced the apoptotic pathway and suggest that carfilzomib, the representative of a new class of chemotherapeutic drug, may be utilized for overcoming cisplatin-resistance in human SCC. Citation Format: Woo-Jin Jeong, Jiyeong Kim, Hye-Yeon Lee, Zhiyong Wang, Walt Amornphimoltham, Silvio Gutkind. Proteasome inhibitor carfilzomib induces apoptosis via downregulation of AKT pathway in HPV negative HNSCC. [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 4951.

Abstract 6213: Disruption of XPF function by HPV promotes alternative end joining and sensitivity to cisplatin in HNSCC

Abstract Human papillomavirus (HPV) positive head and neck squamous cell carcinoma (HNSCC) usually show a tremendous better clinical outcome compared to their HPV-negative counterparts. Although the HPV associated survival advantage is partially explained by the enhanced cisplatin sensitivity in HNSCC (Clin Ca Res 2018, 24(23): 6001), the underlying mechanisms are still poorly elucidated. Cisplatin causes DNA interstrand crosslinks (ICLs) that requires Fanconi anemia (FA) pathway for DNA repair. Consistently, our study showed that HPV-positive HNSCC cells exhibited a FA defective cellular phenotype. In clonogenic cell survival assay, 1h treatment with 5μM cisplatin eliminated approximately 56.7% more HPV-positive HNSCC cells than HPV-negative ones. Furthermore, HPV-positive cells treated with cisplatin showed prolonged G2/M cell cycle arrest and more 53BP1 foci, indicating profound deficiency in repairing ICLs. Consistent with these findings, increased aberrant chromosome formation was observed in HPV-positive cells following Mitomycin C treatment. In order to reveal the mechanism, we further interrogated HPV-labeled HNSCC samples in TCGA database, and identified XPF, an endonuclease protein in FA pathway, was downregulated in HPV-positive HNSCC. Further analysis of cellular and clinical samples confirmed that both mRNA and protein expressions of XPF were significantly lower in HPV-positive HNSCC (P<0.001). To test the importance of XPF in HPV-induced cisplatin sensitivity, we performed a cell viability assay, and found that knock-down of XPF increased the effects of cisplatin on HPV-negative HNSCC by 39.54% (± 4.42%), while no significant change was observed in HPV-positive HNSCC (P>0.05). Notably, inhibition of XPF function by a small molecule inhibitor recapitulated the effect of HPV, resulted in enhanced error-prone DNA repair with alternative end-joining (alt-EJ). In a probe-based ddPCR assay, XPF inhibition increased 32.02% (± 5.80%, P<0.001) alt-EJ events in HPV-negative HNSCC cells, while little difference was detected in HPV-positive cells. Thus, we speculated that combined inhibition of XPF and alt-EJ may improve clinical outcome in the difficult-to-treat, HPV-negative HNSCC, which worth further research efforts. Citation Format: Qi Liu, Nan Zuo, Lin Ma, Lanlan Wei. Disruption of XPF function by HPV promotes alternative end joining and sensitivity to cisplatin in HNSCC. [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 6213.

Abstract 6284: SUV420H1 as a novel target in HPV-negative head and neck squamous cell carcinoma

Abstract 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.