Effects of Oncometabolite L-2-hydroxyglutarate (L2HG) on differentiation of normal renal proximal tubule cells Mary Taub, Biochemistry, University at Buffalo, and Sunil Sudarshan, Urology, University of Alabama at Birmingham Previously, we reported that the level of oncometabolite L-2-hydroxyglutarate (L-2HG) is elevated in human renal cell carcinomas (RCCs). L-2HG inhibits the demethylation of histones, as well as genomic DNA, thereby regulating the epigenetic landscape of cells, and cellular differentiation. Thus, it was of interest to determine whether L-2HG alters the differentiation of renal proximal tubule (RPT) cells, the cells of origin of the RCC. Towards these ends, L-2HG dehydrogenase (L2HGDH) was knocked down (>80%) in primary cultures of normal rabbit RPT cells using either L2HGDH siRNA, or lentiviral L2HGDH shRNA, resulting in a significant increase in L2HG, as well as changes in the level of metabolic products of 2-oxoglutarate-dependent dioxygenases. The effect of these changes on the ability of primary RPT cells to form tubules in matrigel was examined. Normally, EGF stimulates tubulogenesis by in primary RPT cells in matrigel. However, tubulogenesis was significantly impaired in primary RPT cells with an L2HGDH was knockdown. This impairment was associated with reduced expression of mRNAs encoding for a number of differentiated RPT functions (including the Na+/Pi cotransporter NaPi2a (slc34a1), the Na+/glucose cotransporter SGLT2 (SLC5A2), the water transporter Aquaporin 1 (AQP1), as well as the Na,K-ATPase b1 subunit (atp1b1)). In addition, the expression of genes affecting tissue architecture and cell migration was altered, including CDH1 (E-cadherin), PLAU (Plasminogen Activator), and CBY1 (Chibby Homologue 1). This could be explained by the altered methylation of genomic DNA in primary cultures with an L2HGDH KD (as observed in the promoters of these genes during kidney development). Consistent with this hypothesis, a) the level of 5-Ho-MeC was decreased in cultures with an L2HGDH KD and b) the inhibition of the TET1 and TET2 DNA demethylases by Bobcat 339 prevented tubulogenesis. Moreover, the glutaminase inhibitor CB839 prevented the inhibitory effect of elevated L2HG on tubulogenesis (presumably by increasing 2-OG levels). Thus, our findings support a role of 2-OG dependent demethylases in renal differentiation that can be impaired by the accumulation of 2HG produced as a consequence of reduced L2HGDH. The implications of this in the development of RCCs is currently being studied. This work is funded by RO1 CA200653.