Immunosuppression withdrawal from calcineurin inhibitors is only possible in ~20% of liver transplant recipients. However, mTOR inhibitors (sirolimus) appear to be more immunoregulatory and might promote a tolerant state for withdrawal. Our purpose was to determine if systemic (blood, marrow, allograft) signatures of immunoregulation are promoted by conversion from tacrolimus to sirolimus. We therefore performed the following serial assays before and after sirolimus conversion in liver transplant recipients to test for enhanced markers of immunoregulation: 1) Flow cytometry immunophenotyping of PBMC and bone marrow aspirates for regulatory T cells (Tregs: CD4+CD25+++FOXP3+) and regulatory dendritic cells (DCregs: ILT3+/4+); 2) liver biopsy immunohistochemical staining (FOXP3:CD3, CD4:CD8 ratios) and immunophenotyping of biopsy-derived Tregs after growth in culture; 3) effects of pre-vs. post-conversion sera on Treg generation in mixed lymphocyte reactions; 4) peripheral blood non-specific CD4 responses (Cylex® ImmuKnow); 5) peripheral blood gene transcripts and proteomic profiles. We successfully converted 20 non-immune, non-viremic recipients (age 57.2±8; 3.5±2.1 years post-LT) from tacrolimus to sirolimus for renal dysfunction. Our results demonstrated significant increases in Tregs in PBMC and marrow and DCregs in PBMC (p<0.01) following conversion. In biopsy immunohistochemistry, FOXP3:CD3 and CD4:CD8 ratios were significantly higher after conversion and a number of biopsy cultures developed new or higher FOXP3+ cell growth. Non-specific CD4 responses (Cylex® ImmuKnow) did not change. Both pre- and post-conversion sera inhibited mixed lymphocyte reactions, although only tacrolimus sera suppressed Treg generation. Finally, 289 novel genes and 22 proteins, several important in immunoregulatory pathways, were expressed after conversion. Conclusions Tacrolimus to sirolimus conversion increases systemic Tregs, DCregs and immunoregulatory proteogenomic signatures in liver transplant recipients and may therefore facilitate immunosuppression minimization or withdrawal.

NUP98 is a nucleoporin that plays complex roles in the nucleocytoplasmic trafficking of macromolecules. Rearrangements of the NUP98 gene in human leukemia result in the expression of numerous fusion oncoproteins whose effect on nucleocytoplasmic trafficking is poorly understood. The present study was undertaken to determine the effects of leukemogenic NUP98 fusion proteins on CRM1-mediated nuclear export. NUP98-HOXA9, a prototypic NUP98 fusion, inhibited the nuclear export of two known CRM1 substrates: mutated cytoplasmic nucleophosmin and HIV-1 Rev. In vitro binding assays revealed that NUP98-HOXA9 binds CRM1 through the FG repeat motif in a Ran-GTP-dependent manner similar to but stronger than the interaction between CRM1 and its export substrates. Two NUP98 fusions, NUP98-HOXA9 and NUP98-DDX10, whose fusion partners are structurally and functionally unrelated, interacted with endogenous CRM1 in myeloid cells as shown by co-immunoprecipitation. These leukemogenic NUP98 fusion proteins interacted with CRM1, Ran, and the nucleoporin NUP214 in a manner fundamentally different from that of wildtype NUP98. NUP98-HOXA9 and NUP98-DDX10 formed characteristic aggregates within the nuclei of a myeloid cell line and primary human CD34؉ cells and caused aberrant localization of CRM1 to these aggregates. These NUP98 fusions caused nuclear accumulation of two transcription factors, NFAT and NFB, that are regulated by CRM1-mediated export. The nuclear entrapment of NFAT and NFB correlated with enhanced transcription from promoters responsive to these transcription factors. Taken together, the results suggest a new mechanism by which NUP98 fusions dysregulate transcription and cause leukemia, namely, inhibition of CRM1-mediated nuclear export with aberrant nuclear retention of transcriptional regulators.

NUP98 gene rearrangements occur in acute myeloid leukemia and result in the expression of fusion proteins. One of the most frequent is NUP98-DDX10 that fuses a portion of NUP98 to a portion of DDX10, a putative DEAD-box RNA helicase. Here we show that NUP98-DDX10 dramatically increases proliferation and self-renewal of primary human CD34+ cells, and disrupts their erythroid and myeloid differentiation. It localizes to their nuclei and extensively deregulates gene expression. Comparison to another leukemogenic NUP98 fusion, NUP98-HOXA9, reveals a number of genes deregulated by both oncoproteins, including HOX genes, COX-2, MYCN, ANGPT1, REN, HEY1, SOX4, and others. These genes may account for the similar leukemogenic properties of NUP98 fusion oncogenes. The YIHRAGRTAR sequence in the DDX10 portion of NUP98-DDX10 represents a major motif shared by DEAD-box RNA helicases that is required for ATP binding, RNA-binding, and helicase functions. Mutating this motif diminished the in vitro transforming ability of NUP98-DDX10, indicating that it plays a role in leukemogenesis. These data demonstrate for the first time the in vitro transforming ability of NUP98-DDX10 and show that it is partially dependent on one of the consensus helicase motifs of DDX10. They also point to common pathways that may underlie leukemogenesis by different NUP98 fusions.

NUP98-HOXA9 is the prototype of a group of oncoproteins associated with acute myeloid leukemia. It consists of an N-terminal portion of NUP98 fused to the homeodomain of HOXA9 and is believed to act as an aberrant transcription factor that binds DNA through the homeodomain. Here we show that NUP98-HOXA9 can regulate transcription without binding to DNA. In order to determine the relative contributions of the NUP98 and HOXA9 portions to the transforming ability of NUP98-HOXA9, the effects of NUP98-HOXA9 on primary human CD34+ cells were dissected and compared to those of wild-type HOXA9. In contrast to previous findings in mouse cells, HOXA9 had only mild effects on the differentiation and proliferation of primary human hematopoietic cells. The ability of NUP98-HOXA9 to disrupt the differentiation of primary human CD34+ cells was found to depend primarily on the NUP98 portion, whereas induction of long-term proliferation required both the NUP98 moiety and an intact homeodomain. Using oligonucleotide microarrays in primary human CD34+ cells, a group of genes was identified whose dysregulation by NUP98-HOXA9 is attributable primarily to the NUP98 portion. These include RAP1A, HEY1, and PTGS2 (COX-2). Their functions may reflect the contribution of the NUP98 moiety of NUP98-HOXA9 to leukemic transformation. Taken together, these results suggest that the effects of NUP98-HOXA9 on gene transcription and cell transformation are mediated by at least two distinct mechanisms: one that involves promoter binding through the homeodomain with direct transcriptional activation, and another that depends predominantly on the NUP98 moiety and does not involve direct DNA binding.