J. Jay Boniface, Vijay R. Baichwal, Daniel M. Cimbora, Lynn DeMie, Tracey C. Fleischer, Andrew D. Gassman, Siavash Ghaffari, Jeremy P. Green, Jeff S. Flick, Ryan T. Terry-Lorenzo, Damon I. Papac, Ashley Peterson, Rosann Robinson, Robert O. Carlson. Myrexis, Inc., Salt Lake City, UT
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 the magnitude of NAD production, which is a function of Nampt expression. MPC-9528 has the greatest effect on tumor cell lines with lower Nampt expression; therefore, a companion diagnostic based upon Nampt expression in primary tumor specimens could be used to select patients most likely to respond to MPC-9528 monotherapy in the clinic.
Abstract #2551"The Nampt inhibitor MPC-9528 synergizes with DNA damaging agents"
Ryan T. Terry-Lorenzo, Thomas B. Douce, Harry A. Austin, J. Jay Boniface, Daniel M. Cimbora, Tracey C. Fleischer, Robert O. Carlson. Myrexis, Inc., Salt Lake City, UT
Background: MPC-9528 reduces cellular NAD levels and causes cell death, by blocking the NAD salvage pathway through inhibition of nicotinamide phosphoribosyltransferase (Nampt). Many DNA damaging agents also reduce cellular NAD levels, by activating the NAD consuming enzyme poly(ADP-ribose) polymerase (Parp). We hypothesized that the combination of a Nampt inhibitor and a DNA damaging agent would synergize in killing cancer cells, due to a combined effect on NAD levels through two independent mechanisms.
Methods: Cellular NAD was measured using a coupled enzymatic assay. Drug combination experiments were performed in HCT116 colon carcinoma cells, using measurement of ATP levels as a cell viability endpoint. Synergy, antagonism, or additivity was assessed using the MacSynergy II program.
Results: In HCT116 cells, saturating doses of MPC-9528 induced depletion of NAD with a half-life of 5 hours and a decrease in ATP that was delayed approximately 14 hours relative to NAD. Lower, sublethal concentrations of MPC-9528 induced partial NAD depletion without a concomitant ATP loss. At these sublethal concentrations, MPC-9528 was found to synergize with the DNA alkylating agents temozolomide and streptozotocin, which are known to activate Parp. Additionally, MPC-9528 was found to synergize with two structurally different thymidylate synthase inhibitors, 5-fluorouracil (5-FU) and raltitrexed, neither of which have been reported to activate Parp. Individually, 5-FU and raltitrexed each caused NAD depletion in HCT116 cells, which was enhanced by combination with MPC-9528. Furthermore, both 5-FU- and raltitrexed-mediated NAD depletion and synergy with MPC-9528 were completely blocked by the Parp inhibitor olaparib.
Conclusions: Parp activation induced by the alkylating agents temozolomide and streptozotocin, or by the thymidylate synthase inhibitors 5-FU and raltitrexed, is the basis for tumoricidal synergy with the Nampt inhibitor MPC-9528. This synergy is a direct consequence of the NAD depletion resulting from Parp activation coupled with the inhibition of NAD synthesis due to Nampt inhibition. These results provide a basis for clinical combination of MPC-9528 with the agents studied here or with related agents that induce Parp activation.
Abstract #3526 "Coadministration of nicotinic acid with the Nampt inhibitor MPC-9528 enhances antitumor activity in Naprt deficient cancer cells in culture and in xenografts"
Tracey C. Fleischer, Vijay R. Baichwal, J. Jay Boniface, Daniel M. Cimbora, Lynn DeMie, Thomas B. Douce, Andrew D. Gassman, Damon I. Papac, Ashley Peterson, Rosann Robinson, Ryan T. Terry-Lorenzo, Robert O. Carlson. Myrexis, Inc., Salt Lake City, UT
Background: The tumoricidal small molecule MPC-9528 is a picomolar inhibitor of nicotinamide phosphoribosyltransferase (Nampt). Nampt catalyzes the first and rate-limiting step in NAD synthesis from nicotinamide. Nicotinic acid phosphoribosyltransferase (Naprt) catalyzes the first and rate-limiting step in an alternate pathway of NAD synthesis from nicotinic acid (NA). Cancer cells are particularly dependent on NAD and many cancer cell lines, but not most normal tissues, are deficient in Naprt activity. Therefore administration of NA could prevent MPC-9528-induced NAD depletion in normal tissues, but not in Naprt-deficient tumors, resulting in greater therapeutic index and efficacy.
Methods: Cell viability was determined based on ATP levels. Naprt protein expresson was quantified by western blot and qRT-PCR. NAD was acid-extracted from cells and quantified by a coupled reaction based on fluorescent resorufin. Xenograft studies were performed in nu/nu mice.
Results: In 44 out of 153 cancer cell lines surveyed, NA did not prevent MPC-9528-induced cell death, which correlated with low to undetectable levels of Naprt. MPC-9528-induced NAD depletion and cell death in HCT116 colon carcinoma cells were prevented by the addition of NA, consistent with high Naprt expression. A single dose of MPC-9528 at the maximum-tolerated dose (MTD) of 75 mg/kg caused tumor regression in HCT116 xenografts and NA coadministration completely blocked this effect. NA also completely blocked mortality in mice induced by 300 mg/kg MPC-9528, consistent with the finding that most mouse tissues have high Naprt expression. In Naprt-deficient MIA PaCa-2 xenografts, NA coadministration allowed tolerance of 200 mg/kg MPC-9528 with a substantially increased anti-tumor response relative to the MTD of 75 mg/kg MPC-9528 alone.
Conclusions: Low Naprt expression correlates with the lack of effect of NA on MPC-9528 tumoricidal activity. Because Naprt deficiency is prevalent in cancer cell lines and in primary tumor specimens, but not in normal tissues, NA coadministration with MPC-9528 should increase the tolerability and efficacy of MPC-9528 in patients with Naprt-deficient tumors. A companion diagnostic designed to measure Naprt expression or activity in tumors could be used to identify tumors that would most likely benefit from such combination therapy.
Abstract #4386 "Administration of nicotinic acid reduces or prevents adverse effects of MPC-9528, a potent and selective Nampt inhibitor"
Gary G. Mather, Valerie L. Belcher, Anna Costa, Lynn DeMie, Orvelin Roman, Lori Fotheringham, Chad Bradford. Myrexis, Inc., Salt Lake City, UT
Background: Inhibition of Nampt in cancer cell lines decreases NAD levels and induces cell death. We evaluate the potential toxicity of MPC-9528, a Nampt inhibitor, and investigate the effects of coadministration of nicotinic acid (NA), the substrate for an alternate pathway leading to NAD formation in normal tissues but lacking in many cancer cells.
Methods: SD rats 8/sex/group were administered MPC-9528 at doses of 0, 10, or 15 mg/kg/day, or 15 mg/kg/day + 200 mg/kg/day of NA. Three additional rats were used to evaluate recovery after 7 days without treatment. Pathology was assessed at scheduled necropsies and PK parameters were determined, organ weights recorded, and selected tissues examined microscopically. Subsequently, CD-1 mice were given single oral doses of MPC-9528 at levels known to be efficacious in xenografts or lethal (75 or 300 mg/kg, respectively). NAD levels and white cell counts were determined. Female rats (n=6) were administered MPC-9528 (15 mg/kg/day) with or without pretreatment with NA (200 mg/kg/day) for 9 days. NAD levels were quantified by LC/MS/MS.
Results: Cmax and AUC in females were approximately five times those in male rats. Two females died in the 10 mg/kg group and 8/11 females in the 15 mg/kg group. There were no deaths in male rats or in females dosed with MPC-9528 and NA. Leukocyte counts were reduced (36.4-77.0%) for all groups compared to controls. The reduction for rats treated concurrently with NA was less than groups treated with MPC-9528 alone. Leukocyte counts partially recovered 7 days off drug. There were no significant changes in clinical chemistry at any dose in male rats. AST and CPK were increased in a single female compared to controls. Albumin and total protein were reduced in females treated at 10 or 15 mg/kg/day. Thymus weights were reduced in all treated groups and spleen weights were reduced in females. These changes were not observed at the recovery sacrifice. Testis weight was reduced at both terminal and recovery in MPC-9528 treated males. Lymphoid depletion was noted on histopath. Clinical pathology, organ weight, and histopath changes were either reduced in severity or prevented by concurrent NA. NAD levels in mice given a single dose of 75 mg/kg were reduced >95% and white counts were reduced from a mean of 2.2 x 106/mL to 0.2 x 106/mL by Day 6. Lethality of a 300 mg/kg single dose in mice was completely prevented by coadministration of 1000 mg/kg NA. Similarly, NAD levels in rat blood decreased >50% by Day 6 in females administered MPC-9528 (15 mg/kg/day), however, with concurrent NA, NAD levels were reduced less than 30%.
Conclusion: Coadministration of NA prevented or reduced the severity of adverse effects associated with daily administration of higher doses of MPC-9528. These data suggest that coadministration of NA has the potential to increase the therapeutic margin and to abrogate adverse clinical events in future clinical studies of MPC-9528.
Abstract #LB-393 "The cancer metabolism inhibitor MPC-9528 induces tumor regression in xenograft models with multiple dosing schedules by causing rapid and sustained reduction in tumor NAD"
Vijay R. Baichwal, Adam J. Willardsen, Jeff W. Lockman, Brett J. Murphy, Ruth Gordillo, Tracey C. Fleischer, Chad L. Bradford, Damon I. Papac, Gary G. Mather, Robert O. Carlson. Myrexis, Inc., Salt Lake City, UT
--this is a late-breaking abstract and the full text abstract results are not yet available online