CDKS Blockade Enhances In Vivo Efficacy of EGFR Inhibition in Chordomas

Background: Chordomas are rare malignant bone tumors that arise in the cranial base, mobile spine, and sacrococcygeal region, affecting patients of all ages. Currently, there are no FDA-approved agents for chordoma patients; thus, there is a high-unmet need to develop effective treatments. The EGFR blockade showed significant but incompletely antitumor activity in chordoma, suggesting that inhibition of other growth promoting pathways may be required for enhanced efficacy. The purpose of this study was to evaluate the efficacy of cyclin-dependent kinases (CDKs 1/2/7/9)/JAK2/FLT3 inhibitor TG02 as a single agent or in combination with an EGFR inhibitor afatinib to identify novel therapies against chordoma.

Methods: Three validated human chordoma cell lines U-CH1, U-CH2, UMChor1 were used in this study. In vitro drug synergy was determined quantitatively by the combination-index (CI) method derived from the median-effect principle of Chou and Talalay. Cell Titer-Glo Luminescent Cell Viability Assay was used to measure cell viability, and growth inhibition was expressed as the percentage of surviving cells in drug-treated versus DMSO-treated control cells. To evaluate the anti-tumor effect of combination therapy, afatinib and TG02 were administered by oral gavage in U-CH1 cell line derived xenograft model as single and combined treatments. Tumor volume and body weight were measured weekly. Mice were euthanized if the tumors reached ethical limit of 2,000 mm³, and tumor tissue was collected for further analysis.

Results: In this study, single treatment with TG02 significantly reduced cell viability in a dose-dependent manner in all tested three cell lines, and co-treatment with TG02 and afatinib led to further significant reduction of cell viability compared to single treatments and in a synergistic fashion. In addition, co-treatment with TG02 and afatinib collectively reduced cell viability, inhibited colony formation and cell proliferation compared to single treatments. Cell apoptosis assay revealed that co-treatment with TG02 and afatinib increased the percentage of cells undergoing early stage apoptosis compared with single treatments, as indicated by a significant increase in percentage of AnnV+ /PI-cells exposed to co-treatment in all tested cells. In addition, cotreatment with TG02 and afatinib dramatically induced the translocation of apoptosis inducing factor (AIF) from the damaged mitochondria to the nucleus for DNA cleavage, leading to enhanced caspase-independent cell apoptosis. Notably, cotreatment with TG02 and afatinib significantly inhibited tumor growth in the chordoma xenograft model, compared with single treatments, indicating an additive effect on antitumor efficacy.

Conclusion: Our data demonstrated that TG02 enhanced in vivo efficacy of afatinib, and the insights gained from our study may provide a novel combination therapeutic strategy for chordoma patients.

Publication History

Article published online:
12 February 2021