Neutrophil extracellular traps promote invasion and metastasis via NLRP3-mediated oral squamous cell carcinoma pyroptosis inhibition.

Neutrophil extracellular traps (NETs) are reticular structures composed of neutrophil elastase (NE), cathepsin G (CG) and DNA-histone enzyme complexes. Accumulating evidence has revealed that NETs play important roles in tumor progression, metastasis, and thrombosis. However, our understanding of its clinical value and mechanism of action in oral squamous cell carcinoma (OSCC) is limited and has not yet been systematically described. Here, we aimed to investigate the clinical significance of NETs in OSCC and the mechanisms by which they affect its invasive and metastatic capacity. Our results demonstrated that high enrichment of NETs is associated with poor prognosis in OSCC, and mechanistic studies have shown that NE in NETs promotes invasion and metastasis via NLRP3-mediated inhibition of pyroptosis in OSCC. These findings may provide a new therapeutic approach for OSCC.

The efficacy and safety of dalpiciclib, a cyclin-dependent kinase 4/6 inhibitor, in patients with advanced head and neck mucosal melanoma harboring CDK4 amplification.

BACKGROUND: Mucosal melanoma (MM) is a rare but devastating subtype of melanoma. Our previous studies have demonstrated robust anti-tumor effects of cyclin-dependent kinase 4/6 (CDK 4/6) inhibitors in head and neck MM (HNMM) patient-derived xenograft models with CDK4 amplification. Herein, we aimed to investigate the efficacy and safety of dalpiciclib (SHR6390), a CDK4/6 inhibitor, in HNMM patients harboring CDK4 amplification. METHODS: The anti-tumor efficacy of dalpiciclib was assessed by HNMM patient-derived xenograft (PDX) models and patient-derived tumor cells (PDC) in vivo and in vitro. Immunohistochemical analyses and western blot were then performed to assess the markers of cell proliferation and CDK4/6 signaling pathway. For the clinical trial, advanced recurrent and/or metastatic HNMM patients with CDK4 amplification were treated with dalpiciclib 125 mg once daily for 21 consecutive days in 28-day cycles. The primary endpoint was disease control rate (DCR). Secondary endpoints included safety, objective response rate (ORR), progression-free survival (PFS), and overall survival (OS). RESULTS: Dalpiciclib profoundly suppressed growth of HNMM-PDX and PDC with CDK4 amplification, whereas it showed relatively weak suppression in those with CDK4 wild type compared with vehicle. And dalpiciclib resulted in a remarkable reduction in the expression levels of Ki-67 and phosphorylated Rb compared with control group. In the clinical trial, a total of 17 patients were enrolled, and 16 patients were evaluable. The ORR was 6.3%, and the DCR was 81.3%. The estimated median PFS was 9.9 months (95% CI, 4.8-NA), and the median OS was not reached. The rate of OS at 12 months and 24 months was 68.8% (95% CI, 0.494-0.957) and 51.6% (95% CI, 0.307-0.866), respectively. The most frequent adverse events were neutrophil count decrease, white blood cell count decrease, and fatigue. CONCLUSIONS: Dalpiciclib was well-tolerated and displayed a durable benefit for HNMM patients with CDK4 amplification in this study. Further studies on CDK4 inhibitors and its combination strategy for MM are worth further exploration. TRIAL REGISTRATION: ChiCTR2000031608.

Apatinib potentiates the therapeutic effect of anti-PD-1 in locally advanced head and neck cancers.

OBJECTIVES: Antiangiogenic inhibitors have been shown to synergize with immune checkpoint blockade, but the underlying mechanisms of the synergistic response are not fully understood. PATIENTS AND METHODS: We investigate the impact of VEGFR2 inhibition on tumor-infiltrating immune cells in vivo and the activity of the combination of apatinib and anti-PD-1 in synergistic mouse model of HNSCC. A patient with squamous cell carcinoma of the left tongue with cervical lymph node were received with combined induction treatment of camrelizumab and apatinib to validate the efficacy of neoadjuvant immunotherapy before surgery. RESULTS: We found that apatinib increased the infiltration of CD8+ T cells and decreased the population of Tregs in a preclinical syngeneic mouse model. The proportions of CD8+ PD1+ T cells were significantly increased in apatinib-treated tumors. The combined treatment of apatinib and anti-PD-1 demonstrated better therapeutic benefit than each treatment alone. The patient with squamous cell carcinoma of the left tongue with cervical lymph node achieved major pathologic response (MPR) after two cycles of combined induction treatment. CONCLUSION: Our study demonstrated that apatinib therapy synergized with an anti-PD-1 antibody in preclinical cancer models and in patient with advanced HNSCC. These results provide a new rationale for advancing this neoadjuvant immunotherapy in large scale of clinical trials of HNSCC.

Fibroblast growth factor receptors 1 and 4 combined with lymph node metastasis predicts poor prognosis in oral cancer.

OBJECTIVES: The fibroblast growth factor receptor (FGFR) members including FGFR1-4 have been identified as promising novel therapeutic targets and prognostic markers in multiple solid tumors. However, the predictive role of the expression of FGFR proteins in oral squamous cell carcinoma (OSCC) requires further exploration. MATERIALS AND METHODS: Immunohistochemical evaluation of FGFR1-4 was performed on 161 paired OSCC samples. The associations of FGFRs with clinicopathologic and prognostic parameters were analyzed. To further assess the contribution of FGFRs to OSCC proliferation, cell lines, and one PDX model was utilized to examine the anti-tumor effect of the pan-FGFR inhibitor AZD4547. RESULTS: All FGFR members were found to be overexpressed in OSCC tumors when compared to normal tissues, and their expression was significantly associated with poor overall survival and disease-free survival. Multivariate Cox regression analysis revealed high expression of FGFR1 (p = 0.014) and FGFR4 (p = 0.009) were independent prognostic factors and co-overexpression of FGFR1 and FGFR4 with lymph node metastasis increased HR for death (p = 0.02). The pan-FGFR inhibitor AZD4547 showed anti-tumor activity in cell lines and in a patient-derived xenograft of OSCC. CONCLUSIONS: This study highlights the co-overexpression of FGFR1 and FGFR4 as a significantly poor prognosis indicator in OSCC when combined with lymph node metastasis.

Pharmacogenomic landscape of head and neck squamous cell carcinoma informs precision oncology therapy.

Head and neck squamous cell carcinoma (HNSCC) is a common and frequently lethal cancer with few therapeutic options. In particular, there are few effective targeted therapies. Development of highly effective therapeutic strategies tailored to patients with HNSCC remains a pressing challenge. To address this, we present a pharmacogenomic study to facilitate precision treatments for patients with HNSCC. We established a large collection of 56 HNSCC patient-derived cells (PDCs), which recapitulated the molecular features of the original tumors. Pharmacological assessment of HNSCCs was conducted using a three-tiered high-throughput drug screening using 2248 compounds across these PDC models and an additional 18 immortalized cell lines. We integrated genomic, transcriptomic, and pharmacological analysis to predict biomarkers, gene-drug associations, and validated biomarkers. These results supported drug repurposing for multiple HNSCC subtypes, including the JAK2 inhibitor fedratinib, for low KRT18-expressing HNSCC cases, and the topoisomerase inhibitor mitoxantrone, for IL6R-activated HNSCC cases. Our results demonstrated concordance between susceptibility predictions from the PDCs and the matched patients' responses to standard clinical medication. Moreover, we identified and experimentally confirmed that high expression of ITGB1 elicited therapeutic resistance to docetaxel and high SOD1 expression conferred resistance to afatinib. We further validated ITGB1 as a predictive biomarker for the efficacy of docetaxel therapy in a phase 2 clinical trial. In summary, our study shows that this HNSCC cell resource, as well as the resulting pharmacogenomic profiles, is effective for biomarker discovery and for guiding precision oncology therapies in HNSCCs.

Candidate therapeutic agents in a newly established triple wild-type mucosal melanoma cell line.

BACKGROUND: Mucosal melanoma has characteristically distinct genetic features and typically poor prognosis. The lack of representative mucosal melanoma models, especially cell lines, has hindered translational research on this melanoma subtype. In this study, we aimed to establish and provide the biological properties, genomic features and the pharmacological profiles of a mucosal melanoma cell line that would contribute to the understanding and treatment optimization of molecularly-defined mucosal melanoma subtype. METHODS: The sample was collected from a 67-year-old mucosal melanoma patient and processed into pieces for the establishment of cell line and patient-derived xenograft (PDX) model. The proliferation and tumorigenic property of cancer cells from different passages were evaluated, and whole-genome sequencing (WGS) was performed on the original tumor, PDX, established cell line, and the matched blood to confirm the establishment and define the genomic features of this cell line. AmpliconArchitect was conducted to depict the architecture of amplified regions detected by WGS. High-throughput drug screening (HTDS) assay including a total of 103 therapeutic agents was implemented on the established cell line, and selected candidate agents were validated in the corresponding PDX model. RESULTS: A mucosal melanoma cell line, MM9H-1, was established which exhibited robust proliferation and tumorigenicity after more than 100 serial passages. Genomic analysis of MM9H-1, corresponding PDX, and the original tumor showed genetic fidelity across genomes, and MM9H-1 was defined as a triple wild-type (TWT) melanoma subtype lacking well-characterized "driver mutations". Instead, the amplification of several oncogenes, telomerase reverse transcriptase (TERT), v-Raf murine sarcoma viral oncogene homolog B1 (BRAF), melanocyte Inducing transcription factor (MITF) and INO80 complex ATPase subunit (INO80), via large-scale genomic rearrangement potentially contributed to oncogenesis of MM9H-1. Moreover, HTDS identified proteasome inhibitors, especially bortezomib, as promising therapeutic candidates for MM9H-1, which was verified in the corresponding PDX model in vivo. CONCLUSIONS: We established and characterized a new mucosal melanoma cell line, MM9H-1, and defined this cell line as a TWT melanoma subtype lacking well-characterized "driver mutations". The MM9H-1 cell line could be adopted as a unique model for the preclinical investigation of mucosal melanoma.

Palbociclib-based high-throughput combination drug screening identifies synergistic therapeutic options in HPV-negative head and neck squamous cell carcinoma.

BACKGROUND: Deregulation of cell-cycle pathway is ubiquitously observed in human papillomavirus negative (HPVneg) head and neck squamous cell carcinoma (HNSCC). Despite being an attractive target, CDK4/6 inhibition using palbociclib showed modest or conflicting results as monotherapy or in combination with platinum-based chemotherapy or cetuximab in HPVneg HNSCC. Thus, innovative agents to augment the efficacy of palbociclib in HPVneg HNSCC would be welcomed. METHODS: A collection of 162 FDA-approved and investigational agents was screened in combinatorial matrix format, and top combinations were validated in a broader panel of HPVneg HNSCC cell lines. Transcriptional profiling was conducted to explore the molecular mechanisms of drug synergy. Finally, the most potent palbociclib-based drug combination was evaluated and compared with palbociclib plus cetuximab or cisplatin in a panel of genetically diverse HPVneg HNSCC cell lines and patient-derived xenograft models. RESULTS: Palbociclib displayed limited efficacy in HPVneg HNSCC as monotherapy. The high-throughput combination drug screening provided a comprehensive palbociclib-based drug-drug interaction dataset, whereas significant synergistic effects were observed when palbociclib was combined with multiple agents, including inhibitors of the PI3K, EGFR, and MEK pathways. PI3K pathway inhibitors significantly reduced cell proliferation and induced cell-cycle arrest in HPVneg HNSCC cell lines when combined with palbociclib, and alpelisib (a PI3Kα inhibitor) was demonstrated to show the most potent synergy with particularly higher efficacy in HNSCCs bearing PIK3CA alterations. Notably, when compared with cisplatin and cetuximab, alpelisib exerted stronger synergism in a broader panel of cell lines. Mechanistically, RRM2-dependent epithelial mesenchymal transition (EMT) induced by palbociclib, was attenuated by alpelisib and cetuximab rather than cisplatin. Subsequently, PDX models with distinct genetic background further validated that palbociclib plus alpelisib had significant synergistic effects in models harboring PIK3CA amplification. CONCLUSIONS: This study provides insights into the systematic combinatory effect associated with CDK4/6 inhibition and supports further initiation of clinical trials using the palbociclib plus alpelisib combination in HPVneg HNSCC with PIK3CA alterations.

Repurposing Ponatinib as a Potent Agent against KIT Mutant Melanomas.

Rationale: Mutations in KIT, a major cancer driver gene, are now considered as important drug targets for the treatment of melanomas arising from mucosal and acral tissues and from chronically sun-damaged sites. At present, imatinib is the only targeted drug for KIT-mutation-bearing melanomas that is recommended by the National Comprehensive Cancer Network (NCCN) Clinical Practice guidelines. Patients with KIT mutations, however, are either insensitive or rapidly progress to imatinib insensitivity, which restricts its clinical use. Thus, effective inhibitors of KIT-mutation-bearing melanomas are urgently needed. Methods: A cohort of patient-derived tumor xenograft (PDX) models and corresponding PDX-derived cells (PDCs) from patients with melanomas harboring KIT mutations (KITV560D, KITK642E and KITD816V) were established, characterized, and then used to test the in vitro and, subsequently, in vivo inhibitory effects of a panel of known KIT inhibitors. Results: Ponatinib was more potent than imatinib against cells bearing KIT mutations. In vivo drug efficacy evaluation experiments showed that ponatinib treatment caused much stronger inhibition of KIT-mutation-bearing melanomas than did imatinib. Mechanistically, molecular dynamics (MD) simulations revealed a plausible atomic-level explanation for the observation that ponatinib has a higher affinity for the KITD816V mutant protein than does imatinib. Conclusions: Our study of KIT-mutation-and KITWT-bearing melanomas demonstrates that ponatinib is a far more potent inhibitor than is imatinib for KIT-mutation-bearing melanomas and thus underscores that ponatinib should be given priority consideration for the design of precision treatments for melanoma patients triaged to have KIT mutations. Moreover, our work provides a rationale for undertaking clinical trials to examine the repurposing of ponatinib, which is already approved for use in leukemia, for use in treating a large subset of melanoma patients.

Analysis of Mucosal Melanoma Whole-Genome Landscapes Reveals Clinically Relevant Genomic Aberrations.

PURPOSE: Unlike advances in the genomics-driven precision treatment of cutaneous melanomas, the current poor understanding of the molecular basis of mucosal melanomas (MM) has hindered such progress for MM patients. Thus, we sought to characterize the genomic landscape of MM to identify genomic alterations with prognostic and/or therapeutic implications. EXPERIMENTAL DESIGN: Whole-genome sequencing (WGS) was performed on 65 MM samples, including 63 paired tumor blood samples and 2 matched lymph node metastases, with a further droplet digital PCR-based validation study of an independent MM cohort (n = 80). Guided by these molecular insights, the FDA-approved CDK4/6 inhibitor palbociclib was tested in an MM patient-derived xenograft (PDX) trial. RESULTS: Besides the identification of well-recognized driver mutations of BRAF (3.1%), RAS family (6.2%), NF1 (7.8%), and KIT (23.1%) in MMs, our study also found that (i) mutations and amplifications in the transmembrane nucleoporin gene POM121 (30.8%) defined a patient subgroup with higher tumor proliferation rates; (ii) enrichment of structural variations between chromosomes 5 and 12 defined a patient subgroup with significantly worse clinical outcomes; (iii) over 50% of the MM patients harbored recurrent focal amplification of several oncogenes (CDK4, MDM2, and AGAP2) at 12q13-15, and this co-occurred significantly with amplification of TERT at 5p15, which was verified in the validation cohort; (iv) the PDX trial demonstrated robust antitumor effects of palbociclib in MMs harboring CDK4 amplification. CONCLUSIONS: Our largest-to-date cohort WGS analysis of MMs defines the genomic landscape of this deadly cancer at unprecedented resolution and identifies genomic aberrations that could facilitate the delivery of precision cancer treatments.See related commentary by Shoushtari, p. 3473.

Decreasing graphene synthesis temperature by catalytic metal engineering and thermal processing.

Chemical vapor deposition (CVD) from gaseous hydrocarbon sources has shown great promise for large-scale graphene growth, but the high growth temperature, typically 1050 °C, requires precise and expensive equipment and makes the direct deposition of graphene in electronic device manufacturing processes unfeasible due to the severe physical damage to substrates. Here we demonstrate a facile route to synthesize graphene by catalytic metal engineering and thermal processing. The engineered catalytic metal (copper) with carbon implantation could lower the synthetic temperature to 700 °C. And the resulting graphene shows few defects, uniform morphology and high carrier mobility, comparable to CVD graphene grown at 1050 °C. This technique could expand the applications of graphene in electronic and optoelectronic device manufacturing and is compatible with conventional microelectronics technology.