2-Hidroksiglutarat Onkometabolitinin Kolon Kanseri Metabolizması Üzerindeki Etkisinin CRISPR/Cas9-Tabanlı Gen Terapisi ile Oluşturulan Hücre Modelleri ve In Vivo Modeller Üzerinde İncelenmesi

The normal concentration of D-2-hydroxyglutarate (D-2HG) is quite low but it is generated at a high level and role in colorectal carcinogenesis. This oncometabolite is generated by various cell-type-specific enzymes in addition to wild-type or mutant isocitrate dehydrogenase (IDH). This thesis aims to examine the D-2HG generation pathways and the impact of this oncometabolite on colon cancer metabolism. Firstly, the level of some intermediates in the TCA and Cori cycles, glutaminolysis, and glycolysis were determined in cell lines and xenograft models. The D-2HG has significantly been generated in both colon adenocarcinoma cell lines (Caco-2 and SW620), but it could not be detected in the colon epithelial cells (CCD-18Co). The Sanger results showed that there was no mutation in the IDH1/2 gene in SW620. Then, D-2HG generation was almost 2.5-fold decreased in wild-type IDH1 knockout cells (IDH1 KO) compared to the control, but still it generated. The mRNA level of the malate dehydrogenase (MDH2) enzyme which role in the L/D-2HG generation was considerably increased in IDH1 KO. After treatment with MDH and lactate dehydrogenase (LDH) enzyme inhibitors, the D-2HG could not be detected. Similar findings were obtained in the patient-derived xenograft (PDX) models. Finally, the R132H mutation was generated using the CRISPR/Cas9 tool to examine the effect of mutant IDH1 enzyme on D-2HG generation, and it was revealed that the D-2HG level did not change. In conclusion, the D-2HG generation pathway has been determined and a new therapeutic approach to colon cancer by inhibiting this oncometabolite generation is provided.