A Combinatorial Strategy For Targeting Papillary Thyroid Carcinoma With SHP099 And Selumetinib

Background: Molecular targeted therapy has shown a good prospect of development and application for the advanced PTC management.Supported by the encouraging preclinical data,MEK inhibitor(MEKi)selumetinib has been approved for the treatment of advanced RAIR thyroid carcinomas(TC).In addition to the inhibition of growth of PTC harboring BRAFV600 E,selumetinib restores the expression of sodium iodide symporter(NIS)to reintroduce the RAI therapy to RAIR PTC,providing patients substantial clinical benefits.However,there are still two limitations for selumetinib in the process of its clinical application.Firstly and most importantly,the emergence of MEKi resistance is common.Despite initial promising results,a lasting response to MEKi is,however,infrequently seen.Furthermore,MEKi has less effect on thyroid cancer cells that lack the BRAFV600 E mutation.Therefore,it is worthwhile to explore combination strategies that can abolish the intrinsic resistance,and extend the application range.The combination of SHP2 inhibitor SHP099 and a MEK inhibitor has been proven to exhibit an excellent antitumor effect in various tumor types with different genetic backgrounds.However,the effect of this combination strategy for PTCs with distinct genetic alterations remains unclear.Furthermore,little is known on whether SHP099 can restore MEKi sensitivity in resistant PTC.Therefore,the aim of this study is to explore the therapeutic effect and mechanism of combination of SHP2 inhibitor shp099 and MEK inhibitor selumetinib on PTC with different genetic background,and to explore whether the combination strategy can eliminate the adaptive resistance of PTC cells to MEK inhibitor,which will become a promising and meaningful therapeutic strategy.Methods: 1.Western blot and RTKs assay were used to detect the phosphorylation level of ERK protein and SHP2 protein,and the activation state of RTKs in four PTC cell lines treated with MEK inhibitor selumetinib.Immunoblotting and real-time quantitative PCR were used to detect the effect of combination strategy in PTC cell lines,and whether the new SHP099 has an off-target effect.2.The clone formation,cell proliferation,cell cycle and the expression level of NIS of PTC cell lines was respectively detected by the monoclonal formation assay,CCK-8 assay,flow cytometry assay and Western blot.3.MEK inhibitor resistant cell lines were constructed.The clone formation,cell proliferation,cell cycle and expression level of NIS in resistant cell lines were respectively detected by monoclonal formation assay,CCK-8 assay,flow cytometry and Western blot.4.Xenografts model of PTC cell line were established for further confirmation.Results: 1.After treatment with MEKi,the p-ERK expression rapidly rebounded to the baseline or to an even higher level at 48 hours.The activation levels of several RTKs were upregulated and p-SHP2 expression significantly increased in all four cell lines treated with selumetinib.The activation of ERK in four PTC cell lines was significantly inhibited by the combination strategy.A similar trend was observed in two PTC cell lines treated with selumetinib after knockdown of SHP2,indicating that SHP099 was on-target.2.Compared with the single agent,the clone formation and growth of PTC cell lines were suppressed,the cell cycle was more significantly arrested in G1 phase,and NIS expression was increased by SHP099/selumetinib combination strategy.3.Compared with the single agent,the clone formation and cell proliferation of MEK-resistant PTC cell lines were significantly suppressed,the cell cycle was more significantly arrested in G1 phase,and the NIS expression was significantly increased by SHP099/ selumetinib combination strategy.4.The combination strategy can effectively inhibit growth in vivo.After long-term treatment with MEK inhibitors,xenografts were transplanted to other mice.The combination strategy could effectively inhibit the growth of transplanted mice.Conclusion: The combination of SHP099 and selumetinib abolished the RTK-mediated ERK rebound,resulting in growth inhibition.The combination of SHP099 and selumetinib may be a promising and meaningful therapeutic strategy against PTC.