Abstract 577: Basal NAD levels and Nampt expression correlate with in vitro and in vivo sensitivity of tumor cell lines to the Nampt inhibitor MPC-9528

Background: MPC-9528 is a potent and selective inhibitor of the NAD biosynthetic enzyme nicotinamide phosphoribosyltransferase (Nampt). Inhibition of Nampt by MPC-9528 causes depletion of cellular NAD followed by a decrease in ATP and cell death. Cancer cells develop dependence on Nampt due to increased metabolic demands and the elevated activity of enzymes such as poly(ADP-ribose) polymerases (Parps) that consume NAD. MPC-9528 has shown anti-tumor activity, ranging from no response to complete regression in a variety of xenograft models. Materials and Methods: In vitro Nampt activity and cellular NAD levels were measured in coupled biochemical reactions. Cellular Parp activity was measured by immunofluorescent detection of poly(ADP-ribose) (PAR). Enzyme protein and mRNA levels were quantified by western blot and qRT-PCR, respectively. Mechanism of cell death was determined by Caspase 3/7 activity, Caspase 3 and Parp1 cleavage, and SytoxGreen staining. Cell viability was based on ATP levels. Xenografts were performed in nu/nu mice. Results: MPC-9528 inhibited Nampt activity in vitro with an average IC50 of 40 pM and suppressed cellular NAD levels and nuclear PAR levels, with potencies of 170 pM and 120 pM, respectively. In a screen of 93 cancer cell lines of diverse origin, MPC-9528 had a median TC50 of 2.8 nM with a range of 100 pM to 62 nM. Similar to cultured cells, a range of tumor responses was observed in six different xenograft models. In HCT116 colon carcinoma and HT1080 fibrosarcoma xenografts, oral administration of MPC-9528 at 75 mg/kg intermittently resulted in tumor regressions. In contrast, similar treatment of MIA PaCa-2 pancreatic cancer, N87 gastric carcinoma or HCC827 and NCI-H460 lung cancer xenografts led to partial tumor growth inhibition or no response. The effects in xenografts correlated with TC50 values for MPC-9528 for these cell lines in culture, which ranged from 260 pM to 24 nM. The TC50 values also correlated well with basal cellular NAD levels, IC50 values for MPC-9528-induced NAD depletion, and Nampt protein expression, but not with expression of three other enzymes involved in NAD metabolism – Naprt, Qprt or Parp1. The mechanism of cell death induced by MPC-9528 was cell type dependent and did not correlate with MPC-9528 potency in culture. Conclusions: NAD levels in cancer cell lines are primarily dependent upon the Nampt pathway. The differential sensitivity of tumor cells to the Nampt inhibitor MPC-9528 is likely due to