br Regulation of DHODH activity in cancer Regulation of

Regulation of DHODH activity in cancer Regulation of DHODH activity occurs primarily through activation of de novo pyrimidine biosynthesis via the CAD complex. When Sodium 4-Aminosalicylate synthesis are not preparing for growth, flux through the de novo pyrimidine pathway is slow and functions to generate RNA nucleotides primarily for protein synthesis (Coleman, Suttle, & Stark, 1977; Evans & Guy, 2004; Jones, 1980). In this state, flux is controlled through product feedback inhibition as high concentrations of uridine inhibit CAD. However, when cells prepare to divide, phosphorylation of CAD alters its affinity for uridine to overcome feedback inhibition (Carrey, Campbell, & Hardie, 1985; Sahay, Guy, Liu, & Evans, 1998). Selective phosphorylation of CAD at specific residues regulates flux through the de novo pyrimidine biosynthesis pathway (Huang & Graves, 2003). Flux through the pathway is increased when T456 of CAD is phosphorylated by mitogen-activated protein kinase (MAPK) or mechanistic target of rapamycin 1 complex (mTORC1) via S6 kinase (Ben-Sahra, Howell, Asara, & Manning, 2013; Robitaille et al., 2013; Sigoillot, Berkowski, Sigoillot, Kotsis, & Guy, 2003). After sufficient concentrations of nucleotides are reached, protein kinase A phosphorylates S1406 of CAD to down-regulate nucleotide biosynthesis (Kotsis et al., 2007). Overexpression of enzymes controlling CAD phosphorylation, such as MAPK or mTORC1, leads to increased flux through the pathway in cancer. For example, the breast cancer cell line MCF7 overexpressing MAPK kinase causes a 4-fold increase in the rate of de novo pyrimidine biosynthesis (Sigoillot, Sigoillot, & Guy, 2004). Modulation of flux through the pathway by mTORC1 has not been confirmed experimentally. In addition to phosphorylation, CAD localization also affects the rate of flux through the de novo pyrimidine pathway. Hindrance of CAD nuclear import has been found to decrease the rate of pyrimidine synthesis by 21% and decrease nucleotide concentrations by nearly 60% (Sigoillot et al., 2005). However, it is unclear how and why CAD localization affects de novo pyrimidine biosynthesis. Nonetheless, CAD\'s phosphorylation and localization play a significant role in regulation of DHODH activity. Beyond phosphorylation, cancer cells may alter pyrimidine biosynthesis through the activation of the proto-oncogenic transcription factor MYC. MYC is a master regulator of many different pathways and has significant influence on the expression of nucleotide metabolism genes. Previous studies have shown that overexpression of MYC significantly increased expression of nucleotide metabolism enzymes, including DHODH, which was validated as a direct MYC target gene (Liu et al., 2008). Additionally, shRNA knockdown of MYC decreased the expression of nucleotide metabolism genes and lowered the intracellular concentrations of nucleotides (Mannava et al., 2008). These results demonstrate MYC\'s control over DHODH expression. Surprisingly, inhibition of DHODH by brequinar or teriflunomide down-regulated MYC expression (Dorasamy, Choudhary, Nellore, Subramanya, & Wong, 2017). However, this may be context-dependent as leflunomide (the pro-drug of teriflunomide) demonstrates no effect on MYC expression, possibly due to insufficient conversion of leflunomide to teriflunomide (O\'Donnell et al., 2012), but this was not confirmed experimentally. Nonetheless, while these conflicting results complicate the understanding of MYC and DHODH\'s relationship, nucleotide biosynthesis is among the many pathways MYC activation influences to facilitate cellular proliferation. This is a unique relationship as other transcription factors, such as those in the E2F family, do not appear to increase expression of DHODH (Bester et al., 2011). It is possible that MYC and DHODH expression are linked through glutamine metabolism because MYC increases glutamine flux, which is the first substrate in de novo pyrimidine biosynthesis (Hsieh, Walton, Altman, Stine, & Dang, 2015).