Enhanced Degradation of Dihydrofolate Reductase through Inhibition of NAD Kinase by Nicotinamide Analogs

Abstract

Dihydrofolate reductase (DHFR), because of its essential role in DNA synthesis, has been targeted for the treatment of a wide variety of human diseases, including cancer, autoimmune diseases, and infectious diseases. Methotrexate (MTX), a tight binding inhibitor of DHFR, is one of the most widely used drugs in cancer treatment and is especially effective in the treatment of acute lymphocytic leukemia, non-Hodgkin's lymphoma, and osteosarcoma. Limitations to its use in cancer include natural resistance and acquired resistance due to decreased cellular uptake and decreased retention due to impaired polyglutamylate formation and toxicity at higher doses. Here, we describe a novel mechanism to induce DHFR degradation through cofactor depletion in neoplastic cells by inhibition of NAD kinase, the only enzyme responsible for generating NADP, which is rapidly converted to NADPH by dehydrogenases/reductases. We identified an inhibitor of NAD kinase, thionicotinamide adenine dinucleotide phosphate (NADPS), which led to accelerated degradation of DHFR and to inhibition of cancer cell growth. Of importance, combination treatment of NADPS with MTX displayed significant synergy in a metastatic colon cancer cell line and was effective in a MTX-transport resistant leukemic cell line. We suggest that NAD kinase is a valid target for further inhibitor development for cancer treatment.

Suppression of Cytosolic NADPH Pool by Thionicotinamide Increases Oxidative Stress and Synergizes with Chemotherapy

“…1C). This experiment, together with our previous study showing that NADK inhibition lowered NADPH levels (Hsieh et al, 2013), established NADK as a valid target for drug development. Thionicotinamide is the active moiety of two previously identified NADK inhibitors, NADS and NADPS (Fig.…”

“…1A). Treatment of C85 cancer cells with thionicotinamide resulted in an identical loss of dihydrofolate reductase levels, a G1/S block (Hsieh et al, 2013), and similar toxicity profiles as NADS and NADPS; that is, thionicotinamide is a prodrug and is converted intracellularly to NADPS (Fig. 1, B and C).…”

“…Therefore, thionicotinamide, by conversion to NADPS, acts not only as an inhibitor of NADK but also as an inhibitor for G6PD, thus both activities may contribute to its anticancer effects. As previously noted, the level of nicotinamide in the medium used for culturing cells has a large effect on the toxicity of NADS and NADPS (Hsieh et al, 2013). To investigate whether nicotinamide levels affect thionicotinamide toxicity, we performed a colony-formation assay, varying the levels of nicotinamide (0, 8.2, and 32.8 mM) to assess the effect on cells treated with thionicotinamide or cells with knocked down NADK (Fig.…”

“…The inhibition of NAD 1 kinase (NADK) in cancer cells may represent a novel treatment strategy (Hsieh et al, 2013). Cytosolic NADK is an enzyme responsible for generating NADP, which is then rapidly converted to NADPH by reductases.…”

Extramedullary relapse of multiple myeloma defined as the highest risk group based on deregulated gene expression data

“…Inhibition of NAD kinase enhances the degradation of the dihydrofolate reductase (DHFR), which plays a key role in DNA synthesis and for many diseases, including cancer; it represents a therapeutic target 23 . Degradation of DHFR is then associated with inhibition of tumor cell growth.…”