Forced Expression of the “IY” Mutant Inosine Monophosphate Dehydrogenase II Results in Physiologically Significant Resistance to Mycophenolic Acid In Vitro.

Abstract Nonmyeloablative conditioning regimens followed by allogeneic hematopoietic cell transplantation (HSCT) rely on pre and post transplant immunosuppression to overcome host-versus-graft reactions and control graft-versus-host-disease (GVHD). Patients with less than 50% of donor T cells by day 14 after transplant have a higher risk of graft rejection and relapse than those with more than 50% donor chimerism (Blood 2004; 104(8):2254). However, with increasing donor T-cell chimerism the risk of acute GVHD increases. Immunosuppression may inhibit the beneficial graft-versus-infection and graft-versus-leukemia effects mediated by mature donor T cells. Genetically modifying T cells with a drug resistance gene is one way to confer protection from the effects of the immunosuppressive agents, retain donor T cell function, and increase donor T cell chimerism by day 14. Mycophenolate mofetil (MMF) has been used in the post transplant setting to decrease the severity of GVHD and reduce the risk of host vs. graft reaction leading to graft failure. The active metabolite of MMF, mycophenolic acid (MPA), inhibits the enzyme inosine monophosphate dehydrogenase (IMPDH). The type II isoform of IMPDH is the rate-limiting enzyme in de novo guanosine synthesis and is preferentially expressed in activated T and B cells which lack the salvage pathway for guanosine synthesis. A number of investigators have identified IMPDH II mutants that have altered MPA binding capacity and normal guanosine synthetic activity ex vivo (BBA 2002; 1594:27, JBC. 1997; 272:961, BBA. 1994; 1217:156). We have generated six mutants of IMPDH II based on in vitro data and x-ray crystallography of the MPA binding site and transduced them into a human T cell line (Jurkat), a canine large granular lymphocyte cell line (CLGL-90) and a fibroblast cell line (NIH3T3) using oncoretroviral transduction. The mutants include: S276A (Amut), S276F (Fmut), T333I + S351Y (IYmut), L30F + Q277R (FRmut), Q277R + A462T (RTmut), and S276Y (Ymut). We generated bicistronic (IRES containing) retroviral vectors that expressed either HA-tagged wild type human IMPDH II (WT) or an HA-tagged IMPDH mutant and EGFP. We show that forced expression of WT will increase the MPA IC50 3 fold (IC50=3uM) compared to non-transduced cells. Forced expression of only the IY mutant in all cell lines tested was able to transfer resistance to MPA in excess of WT (IC50>50uM). This is clinically relevant since MPA concentrations measured after transplant in patients on MMF are around 8uM (Blood. 2005; 106(13):4381). To show that resistance was related to the expression of the mutant enzyme we correlated GFP by FACS with HA-IMPDH II expression by western blot analysis, and correlated this with resistance to MPA in vitro. This was true regardless of selection in MPA or transduction and cell sorting for >90% GFP positive cells. Analysis of IMPDH activity in lysates of transduced cells is currently underway. This is the first report where forced expression only of the IY mutant provides a clear enhanced resistance to MPA in vitro compared to cells over expressing wild type IMPDH II. Currently, we are generating bicistronic maloney based oncoretroviral vectors which carry a suicide gene (CD34-TK) and the IMPDH-HA IY mutant which will allow us to pharmacologically modulate the level of donor T cells after allogeneic stem cell transplantation.