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

Background Focal 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). Methods The 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. Results Fibroblast 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. Conclusions This 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. Supplementary Information The online version contains supplementary material available at 10.1186/s12943-024-01954-8.

Percentage values indicate the frequency of the alterations in all patients and were used for row ordering; column order represents mutual exclusivity across genes.B OncoPrint diagram of the mutational profile of the tested cell models.The 20 most frequently mutated genes of HPVnegative HNSCC patients (TCGA) were selected.Complete exome mutational information for the selected genes is presented in Table S2.C Mutational data of selected RTK target genes in the indicated cell models.All are classified as missense mutations; annotation includes ClinVar information and COSMIC occurrence in "Upper Aerodigestive tract" (v94).Complete exome mutational information for the selected target genes is presented in Table S2.D Log2-ratio heatmap of specific exome copy number profiles from SAS, FaDu, UT-SCC 5 and UT-SCC 15 cell models.Log2-ratios quantify putative DNA copy number changes in a specific cell model

Fig. S1
Fig. S1 Mutational data and protein expression of selected targets from the RNAi screen in the indicated cell models.A OncoPrint diagram of the 20 most frequently mutated genes of the HPVnegative HNSCC TCGA cohort (n = 415).Putative driver mutations were annotated by cBioPortal.
relative to the average counts across all cell models.Low log2-ratios (clearly < 0) indicate potential deletions affecting specific exomes, values around zero (grey) indicate exomes with unchanged copy numbers, and high log2-ratios (clearly < 0) indicate potential duplications affection specific exomes of a gene.E Summarizing diagram indicating significant cytotoxic effects of single RTK knockdowns at non-irradiated and 6 Gy X-ray irradiated conditions (IR) across the four cell models (derived from Fig. 1D-E and Fig. S2).A greater mean ER under IR (ER (IR) > ER (non-IR)), indicated by black dots, represents radiosensitization.F Western blot analysis of basal FGFR and β1 integrin expression from whole cell lysates of indicated 3D lrECM cell models.β-actin served as loading control.Representative blots from three independent experiments are shown, supplemented with corresponding long-exposure blots for low intensity markers.

Fig. S3
Fig. S3 Radiochemosensitizing potential of FGFR and β1 integrin targeting in various HNSCC cell models.A Cell viability of 3D lrECM HNSCC models upon indicated treatment combinations.DMSO/IgG were used as control (n = 3; mean; two-way ANOVA; Dunnett's multiple comparison

Fig. S4
Fig. S4 Characterization of most sensitive and resistant cell models towards AIIB2/FGFRi/CDDP/irradiation reveals adverse effects.A Cell viability of indicated 3D lrECM grown HNSCC models upon indicted treatment combinations.DMSO/IgG were used as control (n = 3; mean; two-way ANOVA; Dunnett's multiple comparison test to corresponding controls; ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05).B Cell viability of shown 3D lrECM grown HNSCC models upon indicated treatment combinations plus a single dose of 6 Gy X-rays.DMSO/IgG were used as control (n = 3; mean; two-way ANOVA; Dunnett's multiple comparison test to corresponding

Fig. S5
Fig. S5 Differential expressed genes of top FGFRi sensitive and resistant cell models delineate their intrinsic and induced response.A Principal component analysis of transcriptomic datasets

Fig. S6
Fig. S6 Basal and treatment-induced changes of EMT markers pinpoint the importance of this cancer hallmark.A Deviating bar plot of MsigDB-hallmark gene set enrichment analysis (GSEA) between UT-SCC 33 and UM-SCC 10a cell models at basal/untreated conditions.Normalized enrichment scores (NES) and their corresponding significance (adjusted p-value ≤ 0.05) are depicted.B Normalized NES summary graph of multiple GSEA of indicated gene sets (TableS3) for basal/untreated UT-SCC 33 vs. UM-SCC 10a cell model comparison.Results are presented by NES in the color of the cell model in which the respective signature is enriched.Significance levels (adjusted p-value ≤ 0.05) are indicated by triangle size.C GSEA enrichment plots for a proliferation gene signature (TableS3) for the indicated treatment comparisons of the cell model UM-SCC 10a.

Fig. S7
Fig. S7 Upregulated or strongly expressed kinases present promising candidates for specific pharmacological inhibitors to overcome FGFRi-induced resistance.A Normalized enrichment

Fig. S8
Fig. S8 Drug screen cell viability data with selected kinase inhibitors.Graph illustrating cell viability of non-irradiated (left panel) or 6 Gy X-ray irradiated (right panel) 3D lrECM UM-SCC 10a cell cultures treated as indicated.Bars display mean cell viability (n = 3; two-way ANOVA; Tukey multiple comparison test to corresponding non-irradiated (left panel) or irradiated (right panel)

Fig. S9
Fig. S9 Selected kinases deactivate FGFRi-induced resistance in UM-SCC 10a cells.A Effects of indicated kinase inhibitors on cell viability in UM-SCC 10a cells.Bars and the bottom annotation

Fig. S10
Fig. S10 Selected kinase inhibitors enhance FGFR targeting efficacy in sensitive and resistant HNSCC cell models.A Normalized cell viability of indicated non-irradiated cell models upon treatment with selected kinase inhibitors alone (left bar) or in combination with FGFRi (right bar).Applied kinase inhibitor concentrations are indicated.Bars represent mean cell viability (n = 3; two-way ANOVA; Tukey multiple comparison test; ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05).'S' indicates synergy calculated by the Bliss independence model.B Normalized cell viability of indicated 6 Gy X-ray irradiated cell models upon kinase inhibitor monotherapy (left bar) or in combination with FGFRi (right bar).Applied kinase inhibitor concentrations are indicated in Fig. S10A.Bars represent mean cell viability (n = 3; two-way ANOVA; Tukey multiple comparison test; ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05).'S' indicates synergy calculated by the Bliss independence model.

Fig. S11
Fig. S11 HNSCC organoids validate combinatory effectiveness of selected kinase inhibitors with FGFRi.A Mean cell viability (± standard deviation) of HNSCC organoids to a concentration range of indicated kinase inhibitors.Per data point, three technical replicates were averaged and normalized to the corresponding DMSO controls.Non-linear regression was used for IC20

Fig. S13
Fig. S13 Identified signature genes are expressed in both the HNSCC TCGA cohort and single HNSCC cells.A Gene expression analysis of the 20 OS and PFS signature genes in HPVnegative HNSCC patients of the TCGA cohort.Data of primary tumor (n = 415) and normal tissue (n = 44) were compared using unpaired t test (***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05).B OS and PFS gene signature expression in single HNSCC cells (n = 1891) from 10 HNSCC patients (GSE103322).Circle size represents the percentage of cells with recorded expression of the respective gene per tumor; circle color code indicates the average expression values.

Fig. S14
Fig. S14 PROGENy pathway inference delineates the most responsive EGFR/MAPK pathway genes upon FGFRi and irradiation.Gene-level PROGENy data for EGFR (upper panel) and