Functional proteomics of patient derived head and neck squamous cell carcinoma cells reveal novel applications of trametinib

Abstract

In this study, we report a differential response of mitogen-activated protein kinase-kinase (MEK) inhibitor trametinib in 20 head and neck squamous cell carcinoma (HNSCC) patients' tumor-derived cell cultures. Relatively sensitive and resistant cases to trametinib were identified using high throughput metabolic assays and validated in extended dose response studies in vitro. High throughput metabolic assays exploring combination therapies with trametinib were subjected to synergy models and maximal synergistic dose analyses. These yielded several candidates, including axtinib, GDC-0032, GSK-690693, and SGX-523. The combination regimen of trametinib and AXL/MET/VEGFR inhibitor glesatinib showed initial efficacy both in vitro and in vivo (92% reduction in tumor volume). Sensitivity was validated in vivo in a patient-derived xenograft (PDX) model in which trametinib as a single agent effected reduction in tumor volume up to 72%. Reverse Phase Protein Arrays (RPPA) demonstrated differentially expressed proteins and phosphoproteins upon trametinib treatment. Furthermore, resistant cell lines showed a compensatory mechanism via increases in MAPK and non-MAPK pathway proteins that may represent targets for future combination regimens. Intrinsic-targeted options have potential to address paucity of medical treatment options for HNSCC cancer patients, enhance response to extrinsic targeted agents, and/or reduce morbidity as neoadjuvant to surgical treatments.

Keywords: Cancer; HNSCC; MAPK; PDX; Trametinib.

Figure 1.

Patient derived HNSCC cells were exposed to trametinib at a max concentration of 20 μM. (a) Differential AUCs of HNSCC cohort of 20 tumors. Highlighted selected cases: Sensitive cases (blue bars), resistant cases (red bars), HNSCC cells (black bars), and tumor matched normal tissues 10309 and 20004 (gray bars). Red line demarcates no drug effect. (b) At time of selection, sensitive cases that were <20% of the median IC50 values where selected for validation. Selected cases were validated in an extended dose response assay for 2 sensitive and 3 resistant cell lines.

Figure 2.

Patient-derived HNSCC tumor cells from 2 sensitive and 3 resistant lines were exposed to trametinib combination treatments in 7 × 7 drug matrices. Bliss beta values were generated for each cell line and drug combination. Bliss beta values >1 (blues) are considered to be synergistic, bliss beta values of 1 (white) are considered to be additive, and bliss beta values < 1 (yellow) are considered to be antagonistic.

Figure 3.

Patient-derived HNSCC tumor cells for patient 10004 were implanted and established at 350 mm³. Mice were treated with either trametinib (N = 6), trametinib + glesatinib (N = 6) combination treatment or vehicle (N = 8). Data represents the group mean ± SD. Change in mouse numbers displayed above data points for vehicle and trametinib groups as a fraction of the total cohort. Change in mouse number displayed below the graph for trametinib + glesatinib groups. P value shows range of statistical difference between vehicle and treatment groups.

Figure 4.

Sensitive and resistant cells were treated with trametinib for either 24 or 72 hours at 100 nM or their IC50 values, respectively. (a) Volcano plot shows log fold change of perturbed protein expression of pooled sensitive and resistant cell lines at both the 24-hour (gray) and 72-hour (yellow) time points. Proteins above the horizontal black line demonstrates statistically significant differential affinity with labeled proteins being statistically significant at both time points. (b) Venn diagrams demonstrate proteins with statistically significant increased (upper) and decreased (lower) affinity at the 72-hours after treatment with trametinib, stratified by trametinib-sensitive and trametinib-resistant.