The Application of Inosine 5′-Monophosphate Dehydrogenase Activity Determination in Peripheral Blood Mononuclear Cells for Monitoring Mycophenolate Mofetil Therapy in Children with Nephrotic Syndrome

In pediatric nephrotic syndrome, recommended mycophenolic acid (MPA) pharmacokinetics are higher than those for transplant recipients. In MPA therapeutic monitoring, inosine-5′-monophosphate dehydrogenase (IMPDH) activity may be useful. We modified the method established for renal transplant recipients and determined IMPDH activity in peripheral blood mononuclear cells (PBMCs) from healthy volunteers and children (4–16 years) with nephrotic syndrome treated with mycophenolate mofetil (MMF). From children, four blood samples were collected, and MPA concentrations were also determined. IMPDH activity was calculated using xanthosine monophosphate (XMP) normalized with adenosine monophosphate (AMP), both determined with the HPLC-UV method. The modified method was accurate, precise, and linear for AMP and XMP within 0.50–50.0 μmoL/L. Mean IMPDH activity in volunteers was 45.97 ± 6.24 µmoL·s−1·moL−1 AMP, whereas for children, the values were variable and amounted to 39.23 ± 27.40 µmoL·s−1·moL−1 AMP and 17.97 ± 15.24 µmoL·s−1·moL−1 AMP before the next MMF dose and 1 h afterward, respectively. The modified method may be applied to IMPDH activity determination in children with nephrotic syndrome treated with MMF. IMPDH activity should be determined after one thawing of PBMCs due to the change in AMP and XMP concentrations after subsequent thawing. For children, the lowest IMPDH activity was observed concomitantly with the highest MPA concentration.

Serum Metabolomics Uncovers a New Therapeutic Target in Diffuse Large B Cell Lymphoma (DLBCL)

Abstract 1648 DLBCL is a molecularly heterogeneous disease usually treated with chemoimmunotherapy ultimately curing ∼65% of pts. In order to improve therapy for these pts, the identification of broadly relevant therapeutic targets is critical. One such target is HSP90. Tumor cells are enriched for a fraction of HSP90 found in higher-order multi-chaperone complexes. Tumor-enriched HSP90 (teHSP90) displays higher affinity for HSP90 inhibitors than normal tissues, which contain latent, uncomplexed HSP90. Many client proteins are depleted upon exposure to teHSP90 inhibitors. PU-H71 is a highly teHSP90 selective inhibitor with an excellent toxicity profile currently being tested for DLBCL in clinical trials. Combination therapies involving teHSP90 inhibitors may synergize with drugs targeting client proteins of teHSP90 by more powerfully inhibit survival pathways. Cell replication involves substantial metabolic demands and PU-H71-induced degradation of enzymatic client proteins may affect critical metabolic pathways. We hypothesized that by identifying PU-H71-induced metabolic changes, metabolomics could point to potential new targets for combinatorial therapeutic intervention. We therefore analyzed the global metabolic consequence of teHSP90 inhibition in DLBCL by PU-H71. The metabolome was analyzed in the serum of LY7 DLBCL xenografted mice treated with 75 mg/m2 of PU-H71 for 24 h (n=5) or vehicle (n=5) by HPLC/MS. Bioinformatic analysis revealed significant changes in 122 metabolites in PUH-71- vs. vehicle-treated mice; including significantly lower levels of xanthine, hypoxanthine, adenosine, xanthosine monophosphate (XMP), depletion of the guanine nucleoside pool, together with higher levels of inosine and inosine monophosphate (IMP). These metabolic changes pointed towards possible PU-H71 mediated inhibition of inosine monophosphate dehydrogenase (IMPDH). IMPDH catalyzes the NAD-dependent oxidation of IMP to XMP, which is the committed step in de novo guanosine nucleotide biosynthesis. This reaction is particularly important to lymphocytes, which depend on IMPDH activity to generate the guanosine nucleotide levels needed to initiate a proliferative response to antigen. Increased IMPDH activity has also been observed in leukemia and lymphoma, mostly as consequence of up-regulation of the IMPDH2 isoform. In order to determine whether IMPDH1/2 stability depends on teHSP90, we treated a panel of 6 DLBCL cell lines (including LY7) with the mean GI50 of PU-H71 (1 μM) for up to 24 h and checked for IMPDH1/2 abundance. We found a time-dependent decrease in IMPDH2 protein levels. Similar results were obtained with the chemically unrelated HSP90 inhibitor 17-DMAG. To confirm that IMPDH2 binds to teHSP90, we took advantage of an affinity-based PUH-71 pull-down method we recently developed. In this assay PU-H71-beads preferentially bind to teHSP90 complexes pecipitating cancer-related client proteins. By using this assay, we determined that IMPDH2 was indeed a teHSP90 client in DLBCL cells. We also found that PU-H71 (and 17-DMAG) inhibited the activity of IMPDH in DLBCL cells by shortening its half-live (IMPDH t1/2from 2 h to 45 min). To determine whether the combination of teHSP90 inhibitors will synergize with IMPDH inhibitors in killing DLBCL, we treated a panel of 10 DLBCL cell lines with the combination of PU-H71 and two IMPDH inhibitors in clinical use, mycophenolic acid (MPA, an uncompetitive inhibitor) and ribavirin (RIB, a competitive inhibitor). We found that most cell lines showed synergistic killing effect when treated with the combination of drugs compared to each drug alone (determined by isobologram method). This prompted us to test the combination in vivo. SCID mice were xenografted with LY7 and SUDHL6 and once tumor developed, they were treated with vehicle, PU-H71, mycophenolate mofetil (MMF, the pro-drug of MPA), RIB and the combination of PU-H71 and MMF or RIB. We found that mice treated with the combination of drugs exhibited greater effect that each drug alone (p=0.002 for SU-DHL6 and p>0.001 for LY7 for PU-H71+MMF, and p=0.01 for SU-DHL6 and p=0.02 for LY7 for PU-H71+RIB, all T-test day 10). There were no toxic effects. In sum, our work uses serum metabolomics to provide new insights into the pharmacological targets of a particular HSP90 inhibitor, and unveiled a critical survival pathway in DLBCL that was harnessed to develop a rationally combined targeted therapy. Disclosures: No relevant conflicts of interest to declare.

The Association of the Polymorphisms of IMPDH1 Gene and Acute Graft-Versus-Host Disease After Related and Unrelated Hematopoietic Stem Cell Transplantation

Abstract 2377 Background: Mycophenolate mofetil (MMF) has been widely used in the prophylaxis and treatment of graft-versus-host disease (GVHD) in allogeneic hematopoietic stem cell transplantation (allo-HSCT). Inosine monophosphate dehydrogenase (IMPDH) is the target of mycophenolic acid (MPA), the active metabolite of MMF. IMPDH is the key enzyme in the de-novo synthesis of nucleotides and induces the rate-limiting step in this synthesis. There are two isoforms of IMPDH, IMPDH1 is constructively expressed in all cell types, whereas IMPDH2 is only expressed in particular cell types. The proliferation of lymphocytes depends on the synthesis of nucleotides by IMPDH, whereas other types of cells have a salvage pathway for the synthesis of nucleotides. This makes MPA a drug that specifically inhibits the proliferation of the lymphocytes. Interindividual variability in IMPDH activity has been observed in healthy volunteers as well as transplant patients. The considerable variability in baseline IMPDH activity and MPA response may logically be under the control of genetic variation within the IMPDH gene or in gene expression. Analysis of genetic variants could provide the explantation for the variability of IMPDH activity and MMF response in transplant patients. The single nucleotide polymorphism (SNP) of IMPDH1 gene has recently reported to be relevant to acute rejection in renal transplant patients receiving MMF. There are no data about the impact of the polymorphisms of IMPDH1 gene on the outcome of allo-HSCT. The objective of this study was to investigate IMPDH1 genetic variants in allo-HSCT patients and to retrospectively look for the association of these polymorphisms with aGVHD. Methods: The entire study population consisted of 240 consecutive pairs of transplant recipients and their donors who were transplanted from 2001 to 2009 in our Center, including 138 pairs of recipients and their unrelated donors and 102 pairs of recipients and their HLA-identical sibling donors. Both in the unrelated and sibling transplantation cohorts, the patients received the same GVHD prophylaxis consisting of cyclosporin A, a short-term methotrexate and MMF. Genomic DNA was extracted from peripheral blood samples obtained from recipients and donors before transplantation. Four SNPs of IVS7 +125 G>A (rs2278293), IVS8-106 G>A (rs2278294), Exon15 1572 G>A (rs2228075) and 5` flanking region C>T (rs714510) in IMPDH1 gene were analyzed by Multiplex SnaPshot. Results: (1) The IMPDH1 IVS8 -106 G/G genotypes in recipients were significantly associated with a higher incidence of aGVHD than recipients with other genotypes either in the unrelated transplantation cohort or in the sibling transplantation cohort (in the unrelated cohort: 83.3% vs 63.9%, P=0.048; in the sibling cohort: 47.6% vs17.3%, P=0.008). Furthermore, in the unrelated transplantation cohort, the IMPDH1 IVS8 -106 G/G genotypes in recipients were also associated with a higher incidence of grades II-IV aGVHD (63.3% vs 38.0%, P=0.021). However donor IMPDH1 IVS8 -106 genotype had no significant influence on the incidence of aGVHD. (2) In the combined cohort, multivariate analysis confirmed that recipients with the IVS8 -106 G/G genotype were significantly associated with higher risk of developing aGVHD (RR=2.018, 95%CI: 1.354–3.009, P=0.001). Other three variables associated with the risk of aGVHD were myeloablative conditioning (RR=3.309, 95%CI: 1.538–7.121, P=0.002), donor female and recipient male (RR=1.679, 95%CI: 1.139–2.475, P=0.009), and unrelated donor (RR=4.633, 95%CI: 2.934–7.315, P<0.001). (3) The genotypes of IVS7 +125, Exon15 1572 and 5` flanking region were not found to be associated with the risk of aGVHD. Conclusions: These results, which is the first report of IMPDH1 gene polymorphic features of Chinese population with the risk of aGVHD, suggest an interaction of the recipient IMPDH1 IVS8 -106 genotypes on the risk of aGVHD. These results are helpful for predicting allo-HSCT outcome, monitoring MMF therapy on an individual patient basis. Disclosures: No relevant conflicts of interest to declare.