Abstract
Cross-resistance to drugs remains an unsolved problem in cancer chemotherapy. This study elucidates a molecular mechanism of cross-resistance to diverse inhibitors of nicotinamide phosphoribosyltransferase (NAMPT) with anticancer activity. We generated a variant of the human colon cancer cell line HCT116, HCT116RFK866, which exhibited primary resistance to the potent NAMPT inhibitor FK866, and was approximately 1,000-fold less sensitive to the drug than the parental HCT116. HCT116RFK866 was found to be cross-resistant to diverse NAMPT inhibitors, including CHS-828, GNE-617, and STF-118804. Whole-exon sequencing revealed two point mutations (H191R and K342R) in NAMPT in HCT116RFK866, only one of which (K342R) was present in the parental HCT116. Importantly, the protein level, NAMPT enzyme activity, and intracellular NAD+ level were similar between HCT116RFK866 and HCT116. Hence, we investigated NAMPT-binding partners in both cell lines by focused proteomic analyses. The amount of NAMPT precipitated with anti-NAMPT monoclonal antibody was much higher in HCT116RFK866 than in the parental. Furthermore, in HCT116, but not in HCT116RFK866, NAMPT was revealed to interact with POTE ankyrin domain family member E and beta-actin. Thus, these results suggest that NAMPT usually interacts with the two partner proteins, and the H191R mutation may prevent the interactions, resulting in resistance to diverse NAMPT inhibitors.
Highlights
NAD+ is an essential redox cofactor in energy metabolism, including glycolysis, the TCA cycle, and oxidative phosphorylation [1, 2]
To elucidate the mechanisms underlying crossresistance to diverse nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, we generated a variant of the human colon cancer cell line HCT116 that was resistant to FK866, a potent inhibitor of NAMPT in vitro that has efficacy against various human tumors in in vivo xenograft models [11, 12]
As in previous experiments aimed at obtaining cells resistant to NAMPT inhibitors, our initial efforts focused on generating a NAMPT inhibitor–resistant HCT116 cell line with one point mutation in the NAMPT protein
Summary
NAD+ is an essential redox cofactor in energy metabolism, including glycolysis, the TCA cycle, and oxidative phosphorylation [1, 2]. It serves as a substrate for multiple enzymes, such as sirtuin and poly (ADP-ribose) polymerase, involved in cellular signaling processes [2], calcium homeostasis [3], gene regulation [4], genome integrity [5], and cell survival or death [6]. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of the salvage pathway, is considered an attractive target for the development of anticancer drugs [1, 8]. A number of new NAMPT inhibitors, including GNE617 [1, 17] and STF-118804 [18], are in the preclinical stages [8]
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