Abstract
GMX1778 and its prodrug GMX1777 represent a new class of cancer drugs that targets nicotinamide phosphoribosyltransferase (NAMPT) as a new strategy to interfere with biosynthesis of the key enzymatic cofactor NAD, which is critical for a number of cell functions, including DNA repair. Using a genome-wide synthetic lethal siRNA screen, we identified the folate pathway-related genes, deoxyuridine triphosphatase and dihydrofolate reductase, the silencing of which sensitized non-small cell lung carcinoma (NSCLC) cells to the cytotoxic effects of GMX. Pemetrexed is an inhibitor of dihydrofolate reductase currently used to treat patients with nonsquamous NSCLC. We found that combining pemetrexed with GMX1777 produced a synergistic therapeutic benefit in A549 and H1299 NSCLC cells in vitro and in a mouse A549 xenograft model of lung cancer. Pemetrexed is known to activate PARPs, thereby accelerating NAD consumption. Genetic or pharmacologic blockade of PARP activity inhibited this effect, impairing cell death by pemetrexed either alone or in combination with GMX1777. Conversely, inhibiting the base excision repair pathway accentuated NAD decline in response to GMX and the cytotoxicity of both agents either alone or in combination. These findings provide a mechanistic rationale for combining GMX1777 with pemetrexed as an effective new therapeutic strategy to treat nonsquamous NSCLC.
Highlights
Interest in nicotinamide phosphoribosyltransferase (NAMPT) as a potential anticancer target stems from its involvement as the major and rate-limiting step in the biosynthesis of NADþ [1, 2]
We demonstrate that GMX synergizes with PTX in vitro and in the A549 lung adenocarcinoma xenograft model
Protein was extracted from cells 48 hours after transfection and detected by Western blotting using antibodies to dihydrofolate reductase (DHFR) (EPR5285; Abcam), deoxyuridine triphosphatase (DUT) (AB137097; Abcam), PARP-1 (9542; Cell Signaling Technology), a-tubulin (DM1A; Sigma), g-H2AX (05-636; Millipore), or PAR proteins (MAB3192; Millipore)
Summary
Interest in nicotinamide phosphoribosyltransferase (NAMPT) as a potential anticancer target stems from its involvement as the major and rate-limiting step in the biosynthesis of NADþ [1, 2]. As a cellular response to DNA damage, PARP is activated and consumes its substrate NADþ for the poly-(ADP-ribosylation) (PAR) of proteins [3]. In this way, it contributes to increased NADþ demands to support the ongoing DNA repair mechanisms in cancer cells. NAMPT inhibitors, including GMX1778 (GMX) and FK866, have shown preclinical activity alone and in combination with several therapeutic DNA-damaging agents, but the mechanism of synergy has not been clearly elucidated [7,8,9,10]. We demonstrate that GMX synergizes with PTX in vitro and in the A549 lung adenocarcinoma xenograft model. The mechanism of synergy involves a PARP-1– mediated accelerated and sustained NADþ decline that results in enhanced cytotoxicity
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