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    • Extracellular uridine can be transported across the cell

    2020-02-07

    Extracellular uridine can be transported across the cell membrane to enter the nucleotide salvage pathway. A colon cancer cell line supplemented with uridine was capable of growing normally in the presence of 1 μM brequinar (Peters et al., 1992). The supplemented uridine may be transported into the cell by the solute carrier 28 family (concentrative nucleoside transporters, CNT1–3), solute carrier 29 family (equilibrative nucleoside transporter, ENT1–4), or select members of the solute carrier 35 family (SLC35) (Fig. 12) (Song, 2013; Young, Yao, Baldwin, Cass, & Baldwin, 2013). Once inside the cell, uridine can be converted to UMP by uridine kinase, bypassing pyrimidine depletion induced by DHODH inhibition (Greenberg, Schumm, & Webb, 1977). Thus, an extensive network of salvage enzymes and transporters can generate the required nucleotides from extracellular sources of nucleobases/nucleosides to sustain cell growth. However, it is unclear why combinations of DHODH inhibitors, such as brequinar with DPM, have been unsuccessful. A potential reason why this did not produce significant efficacy is that this combination may be cytostatic, with cancer AP-III-a4 synthesis regaining the ability to grow when the drugs are removed. We predict that the combination of DHODH inhibition with cytotoxic chemotherapy or radiation is likely to be cytotoxic rather than cytostatic and may improve efficacy. DPM halts the flux of nucleobases/nucleosides across the cell membrane by targeting the ENT isoforms (Young et al., 2013). While other nucleoside transporters are present, the ENT family is thought to be the predominant source of uridine flux across the membrane as most ENT isoforms catalyze facilitative diffusion and have a higher turnover rate in comparison to CNTs (ENT1: 200 uridine molecules/s; CNT1: 10 uridine molecules/s) (Smith et al., 2004; Young et al., 2013). Despite the slower rate of transport, it is possible that the sodium- or proton-coupled CNTs sustain the required intracellular nucleotide concentrations (Young et al., 2013). The role of nucleoside transporters from the SLC35 family is unknown. Select members of the large family transport UDP analogues across membranes. For example, the SLC35 family member UGT catalyzes the transport of UDP-galactose and UDP-N-acetylgalactosamine across the membrane, but requires UMP as an antiport exchange substrate (Song, 2013). It is unclear if these transporters would be active in cells with low UMP concentrations. For successful combination therapy, inhibitors of both CNT and ENT administered with DHODH inhibitors might be necessary.
    Future of DHODH-targeted therapy We believe the future of DHODH-targeted therapy in cancer lies in multi-drug combination treatments to produce in vivo synergy. Despite the setbacks of brequinar in clinical trials, DHODH remains a viable anticancer target. The pyrimidine depletion induced by DHODH inhibition may sensitize cells to better outcomes with current chemotherapy options. In fact, several studies have previously implicated DHODH inhibition as key to overcoming chemotherapy resistance (Brown et al., 2017; He et al., 2014; Shukla et al., 2017). DHODH inhibition sensitizes cancer cells to conventional chemotherapy and overcomes resistance mechanisms by targeting metabolic dependencies. For example, leflunomide is key to overcoming chemotherapy resistance in triple-negative breast cancer cell lines (Brown et al., 2017). Triple-negative breast cancer cell lines exposed to genotoxic agents increased flux through the de novo pyrimidine pathway, resulting in increased nucleotide concentrations to facilitate DNA repair and decreasing their sensitivity to genotoxic agents. Pretreatment with leflunomide induced pyrimidine depletion in triple-negative breast cancer cells and overcame doxorubicin resistance (Brown et al., 2017). Additionally, as previously described, brequinar increased cell sensitivity to TRAIL therapy (He et al., 2014). Beyond doxorubicin and TRAIL, DHODH inhibitors have been shown to sensitize cells to DNA substrate mimics. When leflunomide and gemcitabine were used in combination, the effect was more significant than with single-agent dosing. Similar to combinations of brequinar and 5-FU, leflunomide-induced pyrimidine depletion may have led to a higher incorporation of gemcitabine (Shukla et al., 2017). Similar combinations, such as teriflunomide with 5-azacytidine, were evaluated in resistant cell lines in 5-azacytidine-resistant leukemic cells (Imanishi et al., 2017), and leflunomide with fludarabine in fludarabine-resistant chronic lymphocytic leukemia cells (Dietrich et al., 2012). It is unclear if these combinations would have a similar effect clinically as brequinar and 5-FU. However, preclinical data suggests that DHODH-induced pyrimidine depletion may be used to overcome certain acquired resistance mechanisms.