Depletion of mutant IDH1 impairs chondrosarcoma growth by downregulating integrins.

Abstract

11031 Background: chondrosarcomas are malignant bone tumors that produce cartilaginous matrix. Mutations in isocitrate dehydrogenase enzymes (IDH1/2) were recently described in several cancers, including chondrosarcomas. These mutations lead to the inability of IDH to convert isocitrate into α-ketoglutarate (α-KG). Instead, α-KG is reduced into 2-hydroxyglutarate (D-2HG), an oncometabolite. We sought to determine the role of IDH1 mutation in the tumorigenesis of human chondrosarcomas by inactivating mutant IDH1 (mIDH1) using genetic approaches. Methods: we employed two human chondrosarcoma cell lines, JJ012 and HT1080 that carry endogenous IDH1 mutations. D-2HG levels were measured by tandem mass spectrometry. mIDH1 was knocked out via the CRISPR/Cas9 technique. Colony-forming ability and cell migration were evaluated using soft agar assay and Transwell assay, respectively. The effect of mIDH1 on chondrosarcoma growth was analyzed in murine xenograft models. RNA-seq from total RNA was performed to identify differentially expressed genes (DEGs) between the parental and knockout cells. The biological functions of DEGs were identified through Ingenuity pathway analysis. Results: we found that mIDH1 knockout almost completely depleted D-2HG production, dramatically inhibited colony formation by 35% - 60% in JJ012 ( p < 0.01) and by 40% - 80% in HT1080 ( p < 0.01), and significantly reduced the migratory cell numbers by around 40% in both cell lines ( p < 0.01). Moreover, we observed that loss of mIDH1 led to a marked attenuation of chondrosarcoma formation, whereby the mean volume of the tumors derived from mIDH1-knockout cells was approximately 30% of that of control tumors, in both cell lines (JJ012 and HT1080) ( p < 0.01). Interestingly, RNA-seq data showed a significant downregulation of integrin genes such as ITGA10, ITGA5 and ITGB5 in response to mIDH1 knockout (adjusted p < 0.001). These results were confirmed by qRT-PCR and Immunoblotting. Conclusions: our findings demonstrate that mutant IDH1 plays a vital role in chondrosarcoma tumor formation through aberrant activation of integrins.