Head And Neck Squamous Cell Carcinoma Xenografts Research Articles

Two-color fluorescence-guided surgery for head and neck cancer resections.

Head and neck squamous cell carcinoma (HNSCC) has the sixth highest incidence worldwide, with cases annually. Surgery is the primary treatment option for HNSCC, during which surgeons balance two main goals: (1)complete cancer resection and (2)preservation of normal tissues to ensure post-surgical quality of life. Unfortunately, these goals are not synergistic, where complete cancer resection is often limited by efforts to preserve normal tissues, particularly nerves, and reduce life-altering comorbidities. Currently, no clinically validated technology exists to enhance intraoperative cancer and nerve recognition. Fluorescence-guided surgery (FGS) has successfully integrated into clinical medicine, providing surgeons with real-time visualization of important tissues and complex anatomy, where FGS imaging systems operate almost exclusively in the near-infrared (NIR, 650 to 900nm). Notably, this spectral range permits the detection of two NIR imaging channels for spectrally distinct detection. Herein, we evaluated the utility of spectrally distinct NIR nerve- and tumor-specific fluorophores for two-color FGS to guide HNSCC surgery. Using a human HNSCC xenograft murine model, we demonstrated that facial nerves and tumors could be readily differentiated using these nerve- and tumor-specific NIR fluorophores. The selected nerve-specific fluorophore showed no significant difference in nerve specificity and off-target tissue fluorescence in the presence of xenograft head and neck tumors. Co-administration of two NIR fluorophores demonstrated successful tissue-specific labeling of nerves and tumors in spectrally distinct NIR imaging channels. We demonstrate a comprehensive FGS tool for cancer resection and nerve sparing during HNSCC procedures for future clinical translation.

Histone-methyltransferase KMT2D deficiency impairs the Fanconi anemia/BRCA pathway upon glycolytic inhibition in squamous cell carcinoma

Histone lysine methyltransferase 2D (KMT2D) is the most frequently mutated epigenetic modifier in head and neck squamous cell carcinoma (HNSCC). However, the role of KMT2D in HNSCC tumorigenesis and whether its mutations confer any therapeutic vulnerabilities remain unknown. Here we show that KMT2D deficiency promotes HNSCC growth through increasing glycolysis. Additionally, KMT2D loss decreases the expression of Fanconi Anemia (FA)/BRCA pathway genes under glycolytic inhibition. Mechanistically, glycolytic inhibition facilitates the occupancy of KMT2D to the promoter/enhancer regions of FA genes. KMT2D loss reprograms the epigenomic landscapes of FA genes by transiting their promoter/enhancer states from active to inactive under glycolytic inhibition. Therefore, combining the glycolysis inhibitor 2-DG with DNA crosslinking agents or poly (ADP-ribose) polymerase (PARP) inhibitors preferentially inhibits tumor growth of KMT2D-deficient mouse HNSCC and patient-derived xenografts (PDXs) harboring KMT2D-inactivating mutations. These findings provide an epigenomic basis for developing targeted therapies for HNSCC patients with KMT2D-inactivating mutations.

A genome-wide CRISPR screen reveals that antagonism of glutamine metabolism sensitizes head and neck squamous cell carcinoma to ferroptotic cell death

Glutamine is a conditionally essential amino acid for the growth and survival of rapidly proliferating cancer cells. Many cancers are addicted to glutamine, and as a result, targeting glutamine metabolism has been explored clinically as a therapeutic approach. Glutamine-catalyzing enzymes are highly expressed in primary and metastatic head and neck squamous cell carcinoma (HNSCC). However, the nature of the glutamine-associated pathways in this aggressive cancer type has not been elucidated. Here, we explored the therapeutic potential of a broad glutamine antagonist, DRP-104 (sirpiglenastat), in HNSCC tumors and aimed at shedding light on glutamine-dependent pathways in this disease. We observed a potent antitumoral effect of sirpiglenastat in HPV- and HPV + HNSCC xenografts. We conducted a whole-genome CRISPR screen and metabolomics analyses to identify mechanisms of sensitivity and resistance to glutamine metabolism blockade. These approaches revealed that glutamine metabolism blockade results in the rapid buildup of polyunsaturated fatty acids (PUFAs) via autophagy nutrient-sensing pathways. Finally, our analysis demonstrated that GPX4 mediates the protection of HNSCC cells from accumulating toxic lipid peroxides; hence, glutamine blockade sensitizes HNSCC cells to ferroptosis cell death upon GPX4 inhibition. These findings demonstrate the therapeutic potential of sirpiglenastat in HNSCC and establish a novel link between glutamine metabolism and ferroptosis, which may be uniquely translated into targeted glutamine-ferroptosis combination therapies.

Transcriptomic and epigenetic landscape of nimorazole-enhanced radiochemotherapy in head and neck cancer

BackgroundHypoxia remains a challenge for the therapeutic management of head and neck squamous cell carcinoma (HNSCC). The combination of radiotherapy with nimorazole has shown treatment benefit in HNSCC, but the precise underlying molecular mechanisms remain unclear. PurposeTo assess and to characterize the transcriptomic/epigenetic landscape of HNSCC tumor models showing differential therapeutic response to fractionated radiochemotherapy (RCTx) combined with nimorazole. Materials/methodsBulk RNA-sequencing and DNA methylation experiments were conducted using untreated and treated HNSCC xenografts after 10 fractions of RCTx with and without nimorazole. These tumor models (FaDu, SAS, Cal33, SAT and UT-SCC-45) previously showed a heterogeneous response to RCTx with nimorazole. The prognostic impact of candidate genes was assessed using clinical and gene expression data from HNSCC patients treated with primary RCTx within the DKTK-ROG. ResultsNimorazole responder and non-responder tumor models showed no differences in hypoxia gene signatures However, non-responder models showed upregulation of metabolic pathways. From that, a subset of 15 differentially expressed genes stratified HNSCC patients into low and high-risk groups with distinct outcome. ConclusionIn the present study, we found that nimorazole non-responder models were characterized by upregulation of genes involved in Retinol metabolism and xenobiotic metabolic process pathways, which might contribute to identify mechanisms of resistance to nitroimidazole compounds and potentially expand the repertoire of therapeutic options to treat HNSCC.

Methylation of histone H3 lysine 36 is a barrier for therapeutic interventions of head and neck squamous cell carcinoma.

Approximately 20% of head and neck squamous cell carcinomas (HNSCCs) exhibit reduced methylation on lysine 36 of histone H3 (H3K36me) due to mutations in histone methylase NSD1 or a lysine-to-methionine mutation in histone H3 (H3K36M). Whether such alterations of H3K36me can be exploited for therapeutic interventions is still unknown. Here, we show that HNSCC models expressing H3K36M can be divided into two groups: those that display aberrant accumulation of H3K27me3 and those that maintain steady levels of H3K27me3. The former group exhibits reduced proliferation, genome instability, and heightened sensitivity to genotoxic agents like PARP1/2 inhibitors. Conversely, H3K36M HNSCC models with constant H3K27me3 levels lack these characteristics unless H3K27me3 is elevated by DNA hypomethylating agents or inhibiting H3K27me3 demethylases KDM6A/B. Mechanistically, H3K36M reduces H3K36me by directly impeding the activities of the histone methyltransferase NSD3 and the histone demethylase LSD2. Notably, aberrant H3K27me3 levels induced by H3K36M expression are not a bona fide epigenetic mark because they require continuous expression of H3K36M to be inherited. Moreover, increased sensitivity to PARP1/2 inhibitors in H3K36M HNSCC models depends solely on elevated H3K27me3 levels and diminishing BRCA1- and FANCD2-dependent DNA repair. Finally, a PARP1/2 inhibitor alone reduces tumor burden in a H3K36M HNSCC xenograft model with elevated H3K27me3, whereas in a model with consistent H3K27me3, a combination of PARP1/2 inhibitors and agents that up-regulate H3K27me3 proves to be successful. These findings underscore the crucial balance between H3K36 and H3K27 methylation in maintaining genome instability, offering new therapeutic options for patients with H3K36me-deficient tumors.

The value of subcutaneous xenografts for individualised radiotherapy in HNSCC: Robust gene signature correlates with radiotherapy outcome in patients and xenografts

PurposeTo assess the robustness of prognostic biomarkers and molecular tumour subtypes developed for patients with head and neck squamous cell carcinoma (HNSCC) on cell-line derived HNSCC xenograft models, and to develop a novel biomarker signature by combining xenograft and patient datasets. Materials and methodsMice bearing xenografts (n = 59) of ten HNSCC cell lines and a retrospective, multicentre patient cohort (n = 242) of the German Cancer Consortium-Radiation Oncology Group (DKTK-ROG) were included. All patients received postoperative radiochemotherapy (PORT-C). Gene expression analysis was conducted using GeneChip Human Transcriptome Arrays. Xenografts were stratified based on their molecular subtypes and previously established gene classifiers. The dose to control 50 % of tumours (TCD50) was compared between these groups. Using differential gene expression analyses combining xenograft and patient data, a gene signature was developed to define risk groups for the primary endpoint loco-regional control (LRC). ResultsTumours of mesenchymal subtype were characterized by a higher TCD50 (xenografts, p < 0.001) and lower LRC (patients, p < 0.001) compared to the other subtypes. Similar to previously published patient data, hypoxia- and radioresistance-related gene signatures were associated with high TCD50 values. A 2-gene signature (FN1, SERPINE1) was developed that was prognostic for TCD50 (xenografts, p < 0.001) and for patient outcome in independent validation (LRC: p = 0.007). ConclusionGenetic prognosticators of outcome for patients after PORT-C and subcutaneous xenografts after primary clinically relevant irradiation show similarity. The identified robust 2-gene signature may help to guide patient stratification, after prospective validation. Thus, xenografts remain a valuable resource for translational research towards the development of individualized radiotherapy.

Recombinant immunotoxin induces tumor intrinsic STING signaling against head and neck squamous cell carcinoma

The innate immune stimulator of interferon genes (STING) pathway is known to activate type I interferons (IFN-I) and participate in generating antitumor immunity. We previously produced hDT806, a recombinant diphtheria immunotoxin, and demonstrated its efficacy against head and neck squamous cell carcinoma (HNSCC). However, it’s unknown whether the tumor-intrinsic STING plays a role in the anti-HNSCC effects of hDT806. In this study, we investigated the innate immune modulation of hDT806 on HNSCC. hDT806 significantly upregulated the level of STING and the ratio of p-TBK1/TBK1 in the HNSCC cells. Moreover, intratumoral hDT806 treatment increased the expression of STING-IFN-I signaling proteins including IFNA1, IFNB, CXCL10 and MX1, a marker of IFN-I receptor activity, in the HNSCC xenografts. Overexpression of STING mimicked the hDT806-induced upregulation of the STING-IFN-I signaling and induced apoptosis in the HNSCC cells. In the mouse xenograft models of HNSCC with STING overexpression, we observed a significant suppression of tumor growth and reduced tumor weight with increased apoptosis compared to their control xenograft counterparts without STING overexpression. Collectively, our data revealed that hDT806 may act as a stimulator of tumor-intrinsic STING-IFN-I signaling to inhibit tumor growth in HNSCC.

Prognostic biomarkers for the response to the radiosensitizer nimorazole combined with RCTx: a pre-clinical trial in HNSCC xenografts

BackgroundTumor hypoxia is associated with resistance to radiotherapy and chemotherapy. In head and neck squamous cell carcinoma (HNSCC), nimorazole, an oxygen mimic, combined with radiotherapy (RT) enabled to improve loco-regional control (LRC) in some patients with hypoxic tumors but it is unknown whether this holds also for radiochemotherapy (RCTx). Here, we investigated the impact of nimorazole combined with RCTx in HNSCC xenografts and explored molecular biomarkers for its targeted use.MethodsIrradiations were performed with 30 fractions in 6 weeks combined with weekly cisplatin. Nimorazole was applied before each fraction, beginning with the first or after ten fractions. Effect of RCTx with or without addition of nimorazole was quantified as permanent local control after irradiation. For histological evaluation and targeted gene expression analysis, tumors were excised untreated or after ten fractions. Using quantitative image analysis, micromilieu parameters were determined.ResultsNimorazole combined with RCTx significantly improved permanent local control in two tumor models, and showed a potential improvement in two additional models. In these four models, pimonidazole hypoxic volume (pHV) was significantly reduced after ten fractions of RCTx alone. Our results suggest that nimorazole combined with RCTx might improve TCR compared to RCTx alone if hypoxia is decreased during the course of RCTx but further experiments are warranted to verify this association. Differential gene expression analysis revealed 12 genes as potential for RCTx response. When evaluated in patients with HNSCC who were treated with primary RCTx, these genes were predictive for LRC.ConclusionsNimorazole combined with RCTx improved local tumor control in some but not in all HNSCC xenografts. We identified prognostic biomarkers with the potential for translation to patients with HNSCC.

Abstract 3628: RAC1 aberrations in head and neck cancer affect immune microenvironments

Abstract RAC1 is a Rho GTPase well-identified as an oncogene overexpressed in various cancer types. RAC1 amplification or gain account for ~40% of head and neck squamous cell carcinoma (HNSCC) while mutation of RAC1 occurs at ~3% according to TCGA-HNSCC cohort. HNSCC patients with RAC1 aberrations, including RAC1 mutations and amplification or gain, are associated with poor overall and disease-free survival in HNSCC. Significantly higher tumor mutational burden is observed in RAC1-mutated group (p=0.0336) or in RAC1-amplified/gain group (p=0.0109) vs. wildtype patients in TCGA-HNSCC cohort. As increases in TMB are often associated with immune cell infiltration in cancers, we hypothesized that RAC1 aberrations impact the tumor immune microenvironment (TIME) of HNSCC, potentially contributing to disease progression.PD-L1 (CD274) is an important immunosuppressive immune checkpoint molecule expressed in tumor cells. Here, we first reported that ectopic overexpression of RAC1 p.P29S and p.A159V mutations in a human HNSCC cell line (PECAPJ41 clone D2, ATCC, USA) could cause upregulation of PD-L1 at both mRNA (by RNA-seq) and protein levels (by Western blotting), first demonstrating a direct effect of RAC1 mutations on potential HNSCC TIME modulation via PD-L1. Furthermore, by tumor immune estimation resources (TIMER) analysis, we found that RAC1-mutated tumors have significantly higher level of neutrophils immune infiltration of 38.63% (p=0.027) compared to the wildtype tumors. From the CIBERSORTx analysis, M2 Macrophages was significantly increased by 40.42% in RAC1-mutated tumors vs wildtype in the TCGA-HNSCC cohort (p=0.031). We also validated this finding by immunofluorescent staining of PD-L1 and M2 Macrophages in immunocompetent mouse HNSCC xenografts models expressing RAC1 p.P29S and p.A159V mutations as compared to controls. Our in silico, in vitro and in vivo findings first uncover an important role of RAC1 aberrations in HNSCC TIME immunosuppression by regulating PD-L1 expression, neutrophil and M2 macrophages infiltrations. Clinical activities of PD-L1 inhibitors in RAC1-mutated HNSCC patients worth future investigations. Acknowledgement: This research is funded by the General Research Fund (Ref. No.:14168517) and the Research Impact Fund (Ref. No.: R4015-19) from the Research Grants Council in Hong Kong. VWYL is supported by Start-up Fund, Georgia Cancer Center, Medical College of Georgia at Augusta University. Citation Format: Helen Hoi Yin Chan, Yuchen Liu, Hoi Lam Ngan, Vivian Wai Yan Lui. RAC1 aberrations in head and neck cancer affect immune microenvironments. [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 3628.

Epigallocatechin Gallate (EGCG), an Active Phenolic Compound of Green Tea, Inhibits Tumor Growth of Head and Neck Cancer Cells by Targeting DNA Hypermethylation

Head and neck cancers are among the deadliest cancers, ranked sixth globally in rates of high mortality and poor patient prognoses. The prevalence of head and neck squamous cell carcinoma (HNSCC) is associated with smoking and excessive alcohol consumption. Despite several advances in diagnostic and interventional methods, the morbidity of subjects with HNSCC has remained unchanged over the last 30 years. Epigenetic alterations, such as DNA hypermethylation, are commonly associated with several cancers, including HNSCC. Thus, epigenetic changes are considered promising therapeutic targets for chemoprevention. Here, we investigated the effect of EGCG on DNA hypermethylation and the growth of HNSCC. First, we assessed the expression levels of global DNA methylation in HNSCC cells (FaDu and SCC-1) and observed enhanced methylation levels compared with normal human bronchial epithelial cells (NHBE). Treatment of EGCG to HNSCC cells significantly inhibited global DNA hypermethylation by up to 70-80% after 6 days. Inhibition of DNA hypermethylation in HNSCC cells was confirmed by the conversion of 5-methylcytosine (5-mc) into 5-hydroxy methylcytosine (5hmC). DNA methyltransferases regulate DNA methylation. Next, we checked the effect of EGCG on the expression levels of DNA methyltransferases (DNMTs) and DNMT activity. Treatment of EGCG to HNSCC cells significantly reduced DNMT activity to 60% in SCC-1 and 80% in FaDu cells. The protein levels of DNMT3a and DNMT3b were downregulated in both cell lines after EGCG treatment. EGCG treatment to HNSCC cells reactivated tumor suppressors and caused decreased cell proliferation. Our in vivo study demonstrated that administration of EGCG (0.5%, w/w) as a supplement within an AIN76A diet resulted in inhibition of tumor growth in FaDu xenografts in nude mice (80%; p < 0.01) compared with non-EGCG-treated controls. The growth inhibitory effect of dietary EGCG on the HNSCC xenograft tumors was associated with the inhibition of DNMTs and reactivation of silenced tumor suppressors. Together, our study provides evidence that EGCG acts as a DNA demethylating agent and can reactivate epigenetically silenced tumor suppressors to inhibit the growth of HNSCC cells.

Evaluation of Human Mesenchymal Stromal Cells as Carriers for the Delivery of Oncolytic HAdV-5 to Head and Neck Squamous Cell Carcinomas.

Human multipotent mesenchymal stromal cells (hMSCs) are of significant therapeutic interest due to their ability to deliver oncolytic adenoviruses to tumors. This approach is also investigated for targeting head and neck squamous cell carcinomas (HNSCCs). HAdV-5-HexPos3, a recently reported capsid-modified vector based on human adenovirus type 5 (HAdV-5), showed strongly improved infection of both hMSCs and the HNSCC cell line UM-SCC-11B. Given that, we generated life cycle-unmodified and -modified replication-competent HAdV-5-HexPos3 vector variants and analyzed their replication within bone marrow- and adipose tissue-derived hMSCs. Efficient replication was detected for both life cycle-unmodified and -modified vectors. Moreover, we analyzed the migration of vector-carrying hMSCs toward different HNSCCs. Although migration of hMSCs to HNSCC cell lines was confirmed in vitro, no homing of hMSCs to HNSCC xenografts was observed in vivo in mice and in ovo in a chorioallantoic membrane model. Taken together, our data suggest that HAdV-5-HexPos3 is a potent candidate for hMSC-based oncolytic therapy of HNSCCs. However, it also emphasizes the importance of generating optimized in vivo models for the evaluation of hMSC as carrier cells.

MAPKAPK2-centric transcriptome profiling reveals its major role in governing molecular crosstalk of IGFBP2, MUC4, and PRKAR2B during HNSCC pathogenesis

Transcriptome analysis of head and neck squamous cell carcinoma (HNSCC) has been pivotal to comprehending the convoluted biology of HNSCC tumors. MAPKAPK2 or MK2 is a critical modulator of the mRNA turnover of crucial genes involved in HNSCC progression. However, MK2-centric transcriptome profiles of tumors are not well known. This study delves into HNSCC progression with MK2 at the nexus to delineate the biological relevance and intricate crosstalk of MK2 in the tumor milieu. We performed next-generation sequencing-based transcriptome profiling of HNSCC cells and xenograft tumors to ascertain mRNA expression profiles in MK2-wild type and MK2-knockdown conditions. The findings were validated using gene expression assays, immunohistochemistry, and transcript turnover studies. Here, we identified a pool of crucial MK2-regulated candidate genes by annotation and differential gene expression analyses. Regulatory network and pathway enrichment revealed their significance and involvement in the HNSCC pathogenesis. Additionally, 3'-UTR-based filtering recognized important MK2-regulated downstream target genes and validated them by nCounter gene expression assays. Finally, immunohistochemistry and transcript stability studies revealed the putative role of MK2 in regulating the transcript turnover of IGFBP2, MUC4, and PRKAR2B in HNSCC. Conclusively, MK2-regulated candidate genes were identified in this study, and their plausible involvement in HNSCC pathogenesis was elucidated. These genes possess investigative values as targets for diagnosis and therapeutic interventions for HNSCC.

Doublecortin-like kinase 1 is a therapeutic target in squamous cell carcinoma.

Doublecortin like kinase 1 (DCLK1) plays a crucial role in several cancers including colon and pancreatic adenocarcinomas. However, its role in squamous cell carcinoma (SCC) remains unknown. To this end, we examined DCLK1 expression in head and neck SCC(HNSCC) and anal SCC(ASCC). We found that DCLK1 is elevated in patient SCC tissue, which correlated with cancer progression and poorer overall survival. Furthermore, DCLK1 expression is significantly elevated in human papilloma virusnegative HNSCC, which are typically aggressive with poor responses to therapy. To understand the role of DCLK1 in tumorigenesis, we used specific shRNA to suppress DCLK1 expression. This significantly reduced tumor growth, spheroid formation, and migration of HNSCC cancer cells. To further the translational relevance of our studies, we sought to identify a selective DCLK1 inhibitor. Current attempts to target DCLK1 using pharmacologic approaches have relied on nonspecific suppression of DCLK1 kinase activity. Here, we demonstrate that DiFiD (3,5-bis [2,4-difluorobenzylidene]-4-piperidone)binds to DCLK1 with high selectivity. Moreover, DiFiD mediated suppression of DCLK1 led to G2/M arrest and apoptosis and significantly suppressed tumor growth of HNSCC xenografts and ASCC patient derived xenografts, supporting that DCLK1 is critical for SCC growth.

FUT6 inhibits the proliferation, migration, invasion, and EGF-induced EMT of head and neck squamous cell carcinoma (HNSCC) by regulating EGFR/ERK/STAT signaling pathway.

Glycosylation change is one of the landmark events of tumor occurrence and development, and tumor cells may be inhibited by regulating the aberrant expression of glycosyltransferases. Currently, fucosyltransferase VI (FUT6), which is involved in the synthesis of α-1, 3 fucosyl bond, has been detected to be closely associated with multiple tumors, but its function and mechanism in head and neck squamous cell carcinoma (HNSCC) still need further research. In this study, FUT6 knockdown and overexpression strategies were used to investigate the effects of FUT6 on cell proliferation, migration, and invasion, as well as the growth and metastasis of HNSCC in a xenografts mouse model. The protein expression levels of epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase (ERK), Signal Transducer and Activator of Transcription (STAT), protein kinase B (AKT), c-Myc, and epithelial-mesenchymal transition (EMT) markers were determined by western blot analysis. Our research found that the mRNA expression of FUT6 was lower in HNSCC tissues than in normal mucosal epithelial tissues. In Cal-27 and FaDu cells, FUT6 overexpression inhibited cell proliferation, migration and invasion, causing upregulation of ZO-1 and E-cadherin, downregulation of N-cadherin and Vimentin, and finally decreased the phosphorylation levels of EGFR, ERK, STAT, and c-Myc. In HSC-3 cells, knockdown of FUT6 promoted cell proliferation, migration and invasion, downregulating ZO-1 and E-cadherin, upregulating N-cadherin and Vimentin, and increased the phosphorylation levels of EGFR, ERK, STAT, and c-Myc. In the HNSCC xenografts mouse, FUT6 overexpression inhibited tumor growth and metastasis. In summary, FUT6 controls the proliferation, migration, invasion, and EGF-induced EMT of HNSCC by regulating EGFR/ERK/STAT signaling pathway, indicating its potential future therapeutic application for HNSCC.

Pretargeted PET Imaging with a TCO-Conjugated Anti-CD44v6 Chimeric mAb U36 and [89Zr]Zr-DFO-PEG5-Tz.

The recent advances in the production of engineered antibodies have facilitated the development and application of tailored, target-specific antibodies. Positron emission tomography (PET) of these antibody-based drug candidates can help to better understand their in vivo behavior. In this study, we report an in vivo proof-of-concept pretargeted immuno-PET study where we compare a pretargeting vs targeted approach using a new 89Zr-labeled tetrazine as a bio-orthogonal ligand in an inverse electron demand Diels–Alder (IEDDA) in vivo click reaction. A CD44v6-selective chimeric monoclonal U36 was selected as the targeting antibody because it has potential in immuno-PET imaging of head-and-neck squamous cell carcinoma (HNSCC). Zirconium-89 (t1/2 = 78.41 h) was selected as the radionuclide of choice to be able to make a head-to-head comparison of the pretargeted and targeted approaches. [89Zr]Zr-DFO-PEG5-Tz ([89Zr]Zr-3) was synthesized and used in pretargeted PET imaging of HNSCC xenografts (VU-SCC-OE) at 24 and 48 h after administration of a trans-cyclooctene (TCO)-functionalized U36. The pretargeted approach resulted in lower absolute tumor uptake than the targeted approach (1.5 ± 0.2 vs 17.1 ± 3.0% ID/g at 72 h p.i. U36) but with comparable tumor-to-non-target tissue ratios and significantly lower absorbed doses. In conclusion, anti-CD44v6 monoclonal antibody U36 was successfully used for 89Zr-immuno-PET imaging of HNSCC xenograft tumors using both a targeted and pretargeted approach. The results not only support the utility of the pretargeted approach in immuno-PET imaging but also demonstrate the challenges in achieving optimal in vivo IEDDA reaction efficiencies in relation to antibody pharmacokinetics.

Focal Adhesion Kinase Drives Resistance to Therapy in HPV-Negative Head and Neck Squamous Cell Carcinoma in a p53-Dependent Manner

<h3>Purpose/Objective(s)</h3> Focal adhesion kinase (FAK) expression is associated with radioresistance and worse outcome in human papillomavirus negative [HPV (-)] head and neck squamous cell carcinoma (HNSCC). However, the relative importance of FAK in driving therapeutic resistance is unclear. FAK function can be inhibited by wild type (wt) p53, but TP53 is mutated in over 80% of HPV (-) HNSCC. Based on these observations, we tested the hypothesis that mutant (mut) TP53 permits or potentially drives FAK-mediated therapy resistance. <h3>Materials/Methods</h3> <i>In vivo</i> shRNA screening in 3 human HNSCC xenograft flank models was performed using libraries focused on known "druggable" proteins combined with RT or carboplatin. An <i>in vivo</i> immunodeficient flank model using HN31 (mut TP53) cells expressing FAK shRNA was used to assess <i>in vivo</i> radioresponse. The relationship between p53, FAK, and radioresponse was examined <i>in vitro</i> using HNSCC cells: UMSCC (p53 null) with wt or mut TP53 expressed, HN30 (wt TP53), and HN31 (mut TP53). FAK function was modulated in these cells using siRNA, shRNA, or chemical inhibition (defactinib); response to radiation (RT) or carboplatin was evaluated via immunoblot for DNA damage repair proteins, survival clonogenic assay, MTT assay, DCFDA reactive oxygen species (ROS) assay, and fluorescence-based homologous recombination (HR) and non-homologous end joining (NHEJ) reporter assays. FAK and p53 interactions were evaluated via immunoprecipitation. Two cohorts of HPV (-) HNSCC tumors treated with RT, single institution (n=81) and TCGA (n=324), were stratified by TP53 mutational status and FAK copy number/mRNA expression. <h3>Results</h3> <i>In vivo</i> shRNA screening identified FAK as an excellent target for both radio- and chemosensitization. Inhibition of FAK via shRNA in the HN31 (mut TP53) cell line led to radiosensitization in a xenograft flank model. Defactinib treatment led to increased sensitivity to RT or carboplatin in mut TP53 (HN31) but not wt TP53 (HN30) tumor cell lines. In UMSCC1 cells treated with defactinib, mut TP53 expression resulted in significant radiosensitization, but wt TP53 expression did not. Both wt p53 and mut p53 retained the ability to bind FAK. Basal FAK phosphorylation was higher in cells expressing mut p53 compared to wt p53. FAK inhibition combined with radiation led to higher levels of ɣ-H2AX and ROS and decreased HR and NHEJ repair in mut TP53 expressing cells but had minimal effect in wt TP53 cells. High levels of FAK copy number and gene expression were associated with worse disease-free survival in mut TP53 HPV (-) HNSCC, but not wt TP53 tumors in both patient cohorts. <h3>Conclusion</h3> FAK is a potentially important target for therapeutic sensitization, primarily in mutated TP53 HPV (-) HNSCC.

Abstract P185: Preliminary antitumor activity of MCLA-158, an IgG1 bispecific antibody targeting EGFR and LGR5, in advanced head and neck squamous cell carcinoma

Abstract In the expansion part of an ongoing phase 1 study, MCLA-158 is being investigated at the recommended phase 2 dose (RP2D) in patients (pts) with advanced solid tumors, including head and neck squamous cell carcinoma (HNSCC). Epidermal growth factor receptor (EGFR) and WNT signaling are known oncogenic and mitogenic drivers in several cancers, including HNSCC. MCLA-158 is a human common light chain IgG1 bispecific antibody with enhanced antibody-dependent cellular cytotoxicity (ADCC) activity. It targets EGFR and the leucine-rich, repeat-containing, G-protein coupled receptor 5 (LGR5), a transmembrane receptor associated with tumor initiating cells, particularly cancer stem cells. Potent antitumor activity was seen with MCLA-158 in pt-derived HNSCC xenograft models. The RP2D of MCLA-158 was determined to be 1500 mg every 2 weeks (q2w), with 4-week cycles, during the dose escalation part of the study, based on safety, PK and receptor occupancy prediction. A maximum tolerated dose was not reached. The primary objective of the expansion part is to characterize the safety and tolerability of single-agent MCLA-158 and confirm the RP2D. Secondary objectives include assessment of antitumor activity (investigator-assessed overall response rate [ORR] per RECIST 1.1 and duration of response). Key eligibility criteria include prior exposure to standard therapy, ECOG performance status (PS) 0-1, measurable disease (RECIST 1.1), and availability of a baseline tumor biopsy. At the interim data cutoff date of 15 June 2021, 7 pts with advanced recurrent/metastatic HNSCC were enrolled and treated in the expansion phase. Median age was 63 years (range 50-74), ECOG PS 0/1: 2/5. Primary tumor locations were oropharynx (2 pts), hypopharynx (1 pts), larynx (3 pts), and unknown primary (1 pt). All pts had a histology of squamous cell carcinoma. Prior treatment included platinum-based chemotherapy in all pts, and anti-PD-1/PD-L1 in 6 pts. No pts received prior cetuximab. A median of 3 treatment cycles (range 1-8) were administered to the 7 pts, 4 of whom were continuing with therapy at the cutoff. Of the 5 pts who had a postbaseline assessment, 2 had confirmed partial responses (5+ and 8 cycles initiated), and 2 pts had stable disease (reduction in the sum of target lesions of 7% and 17%; 4+ and 5 cycles initiated, respectively). Scheduled first postbaseline tumor assessments for 2 pts occurred after the data cutoff date. Among 26 pts who were treated at the RP2D in the dose escalation and expansion cohorts, the most frequent adverse events regardless of causality (all grades/grade 3) were infusion-related reactions (73%/8%), rash (39%/0%), asthenia (35%/4%), decreased appetite (27%/4%), nausea (27%/4%), and acneiform dermatitis (23%/4%). There were no treatment-related grade 4 or 5 adverse events. In conclusion, MCLA-158 shows promising signs of antitumor activity in pretreated HNSCC, and a well-tolerated and favorable safety profile. Citation Format: Antoine Hollebecque, Irene Brana, Lara Iglesias, Caroline Even, Kato Shumei, Marc Díez García, Mateo Bover, Patricia Martin-Romano, Rocio Garcia-Carbonero, Guillen Argilés, Josep Tabernero, Rajan Khanna, Viktoriya Stalbovskaya, Jeroen Lammerts van Bueren, Kees Bol, Mohamed Bekradda, Andrew Joe, Ernesto Wasserman, Ezra E.W. Cohen. Preliminary antitumor activity of MCLA-158, an IgG1 bispecific antibody targeting EGFR and LGR5, in advanced head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P185.

Molecular Response to Combined Molecular- and External Radiotherapy in Head and Neck Squamous Cell Carcinoma (HNSCC).

Simple SummaryOur previous preclinical trial in a head and neck squamous cell carcinoma (HNSCC) xenograft model showed a high potential for the improvement of curative treatment outcome upon the combination treatment of a radiolabeled (Yttrium-90) anti-EGFR antibody (Cetuximab) and external radiotherapy. We aim to elucidate the molecular response of HNSCC tumors upon this combination. Here, we show that the combination treatment leads to an increasing number and complexity of DNA double strand breaks. The upregulation of p21cip1/waf1 expression and cleaved caspase-3 suggest a blockage of cell cycle transition and an induction of programmed cell death. Collectively, a complex interplay between molecular mechanisms involved in cell death induction, cell cycle arrest, and DNA double strand break repair accounts for the beneficial potential using Yttrium-90-Cetuximab in combination with external radiotherapy. Combination treatment of molecular targeted and external radiotherapy is a promising strategy and was shown to improve local tumor control in a HNSCC xenograft model. To enhance the therapeutic value of this approach, this study investigated the underlying molecular response. Subcutaneous HNSCC FaDuDD xenografts were treated with single or combination therapy (X-ray: 0, 2, 4 Gy; anti-EGFR antibody (Cetuximab) (un-)labeled with Yttrium-90 (90Y)). Tumors were excised 24 h post respective treatment. Residual DNA double strand breaks (DSB), mRNA expression of DNA damage response related genes, immunoblotting, tumor histology, and immunohistological staining were analyzed. An increase in number and complexity of residual DNA DSB was observed in FaDuDD tumors exposed to the combination treatment of external irradiation and 90Y-Cetuximab relative to controls. The increase was observed in a low oxygenated area, suggesting the expansion of DNA DSB damages. Upregulation of genes encoding p21cip1/waf1 (CDKN1A) and GADD45α (GADD45A) was determined in the combination treatment group, and immunoblotting as well as immunohistochemistry confirmed the upregulation of p21cip1/waf1. The increase in residual γH2AX foci leads to the blockage of cell cycle transition and subsequently to cell death, which could be observed in the upregulation of p21cip1/waf1 expression and an elevated number of cleaved caspase-3 positive cells. Overall, a complex interplay between DNA damage repair and programmed cell death accounts for the potential benefit of the combination therapy using 90Y-Cetuximab and external radiotherapy.

Rapalogs induce non-apoptotic, autophagy-dependent cell death in HPV-negative TP53 mutant head and neck squamous cell carcinoma.

TP53 is the most frequently mutated gene in head and neck squamous cell carcinoma (HNSCC). Patients with HPV-negative TP53 mutant HNSCC have the worst prognosis, necessitating additional agents for treatment. Sincemutant p53 causes sustained activation of the PI3K/AKT/mTOR signaling pathway, we investigated the effect of rapalogs RAD001 and CCI-779 on HPV-negative mutTP53 HNSCC cell lines and xenografts. Rapalogs significantly reduced cell viability and colony formation. Interestingly, rapalogs-induced autophagy with no effect on apoptosis. Pretreatment with autophagy inhibitors, 3-methyladenine (3-MA) and ULK-101 rescued the cell viability by inhibiting rapalog-induced autophagy, suggesting that both RAD001 and CCI-779 induce non-apoptotic autophagy-dependent cell death (ADCD). Moreover, rapalogs upregulated the levels of ULK1 and pULK1 S555 with concomitant downregulation of the mTORC1 pathway. However, pretreatment of cells with rapalogs prevented the ULK-101-mediated inhibition of ULK1 to sustained autophagy, suggesting that rapalogs induce ADCD through the activation of ULK1. To further translate our in vitro studies, we investigated the effect of RAD001 in HPV-negative mutTP53 (HN31 and FaDu) tumor cell xenograft model in nude mice. Mice treated with RAD001 exhibited a significant tumor volume reduction without induction of apoptosis, and with a concomitant increase in autophagy. Further, treatment with RAD001 was associated with a considerable increase in pULK1 S555 and ULK1 levels through the inhibition of mTORC1. 3-MA reversed the effect of RAD001 on FaDu tumor growth suggesting that RAD001 promotes ACDC in HPV-negative mutTP53 xenograft. This is the first report demonstrating that rapalogs promote non-apoptotic ADCD in HPV-negative mutTP53 HNSCC via the ULK1 pathway. Further studies are required to establish the promising role of rapalogs in preventing the regrowth of HPV-negative mutTP53 HNSCC.

Dietary Melatonin and Omega-3 Fatty Acids Induce Human Cancer Xenograft Regression In Vivo in Rats by Suppressing Linoleic Acid Uptake and Metabolism.

Melatonin, the circadian nighttime neurohormone, and eicosapentaenoic acid (EPA) and docosahexaenoic acids (DHA), which are omega-3 fatty acids (FA) found in high concentrations in fish oil (FO) and plants, abrogate the oncogenic effects of linoleic acid (LA), an omega-6 FA, on the growth of rodent tumors and human breast, prostate, and head and neck squamous cell carcinoma (HNSCC) xenografts in vivo. Here we determined and compared the long-term effects of these inhibitory agents on tumor regression and LA uptake and metabolism to the mitogenic agent 13-[S]-hydroxyoctadecadienoic acid (13-[S]-HODE) in human prostate cancer 3 (PC3) and FaDu HNSCC xenografts in tumor-bearing male nude rats. Rats in this study were split into 3 groups and fed one of 2 diets: one diet containing 5% corn oil (CO, high LA), 5% CO oil and melatonin (2 μg/mL) or an alternative diet 5% FO (low LA). Rats whose diet contained melatonin had a faster rate of regression of PC3 prostate cancer xenografts than those receiving the FO diet, while both in the melatonin and FO groups induced the same rate of regression of HNSCC xenografts. The results also demonstrated that dietary intake of melatonin or FO significantly inhibited tumor LA uptake, cAMP content, 13-[S]-HODE formation, [³H]-thymidine incorporation into tumor DNA, and tumor DNA content. Therefore, long-term ingestion of either melatonin or FO can induce regression of PC3 prostate and HNSCC xenografts via a mechanism involving the suppression of LA uptake and metabolism by the tumor cells.