In kidney transplantation acute allograft rejection is the most common cause of late allograft loss. Changes in indoleamine 2,3 dioxygenase (IDO) activity, which catabolizes the degradation of tryptophan to kynurenine, may predict rejection. However, exogenous IDO is immunosuppressive in rodent kidney transplantation. Thus, the increase in IDO activity observed in acute allograft rejection is insufficient to prevent rejection. To address this question, we assessed the regulation of IDO and its role in acute rejection in a porcine model of kidney transplant. In tissue samples from rejecting kidney allografts, we showed a 13-fold increase in IDO gene transcription and 20-fold increase in IDO enzyme activity when compared with autotransplanted kidneys. Allografts also demonstrated an over fourfold increase in tissue interferon (IFN)-γ, with marked increases in tumor necrosis factor (TNF)-α, TNF-β and interleukin 1β. Gene transcription and protein levels of kynurenine 3-monooxygenase (KMO) were decreased. KMO generates the immunosuppressive kynurenine, 3-hydroxykynurenine. The results of these studies demonstrate a clear association between rejection and increased allograft IDO expression, likely driven in part by IFN-γ and facilitated by other cytokines of the allogeneic response. Moreover, the loss of downstream enzymatic activity in the IDO metabolic pathway may suggest novel mechanisms for the perpetuation of rejection.

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Background Stem Cell leukemia/lymphoma syndrome (SCLL) presents as a myeloproliferative disease which can progress to acute myeloid leukemia and is associated with the coincident development of B-cell and T-cell lymphomas. SCLL is driven by the constitutive activation of fibroblast growth factor receptor-1 (FGFR1) as a result of chromosome translocations with poor outcome. Mouse models have been developed which faithfully recapitulate the human disease and have been used to characterize the molecular genetic events that are associated with development and progression of the disease. Methods CRISPR/Cas9 approaches were used to generate SCLL cells null for Interleukin receptor associated kinase 1 (IRAK1) and interferon gamma (IFNG) which were introduced into syngeneic hosts through tail vein injection. Development of the disease and changes in immune cell composition and activity were monitored using flow cytometry. Bead-based immunoassays were used to compare the cytokine and chemokine profiles of control and knock out (KO) cells. Antibody mediated, targeted depletion of T cell and MDSCs were performed to evaluate their role in antitumor immune responses. Results In SCLL, FGFR1 activation silences miR-146b-5p through DNMT1-mediated promoter methylation, which derepresses the downstream target IRAK1. IRAK1 KO SCLL cells were xenografted into immunocompetent syngeneic mice where the typical rapid progression of disease was lost and the mice remained disease free. IRAK1 in this system has no effect on cell cycle progression or apoptosis and robust growth of the KO cells in immunodeficient mice suggested an effect on immune surveillance. Depletion of T-cells in immunocompetent mice restored leukemogenesis of the KO cells, and tumor killing assays confirmed the role of T cells in tumor clearance. Analysis of the immune cell profile in mice transplanted with the IRAK1 expressing mock control (MC) cells shows that there is an increase in levels of myeloid-derived suppressor cells (MDSCs) with a concomitant decrease in CD4+/CD8+ T-cell levels. MDSC suppression assays and depletion experiments showed that these MDSCs were responsible for suppression of the T cell mediated leukemia cell elimination. Immuno-profiling of a panel of secreted cytokines and chemokines showed that activation of IFN-γ is specifically impaired in the KO cells. In vitro and in vivo expression assays and engraftment with interferon gamma receptor-1 (IFNGR1) null mice and IFNG KO SCLL cells, showed the leukemia cells produced IFN-γ directly participating in the induction of MDSCs to establish immune evasion. Inhibition of IRAK1 using pacritinib suppresses leukemogenesis with impaired induction of MDSCs and attenuated suppression of CD4+/CD8+ T-cells. Conclusions IRAK1 orchestrates a previously unknown FGFR1-directed immune escape mechanism in SCLL, through induction of MDSCs via regulation of IFN-γ signaling from leukemia cells, and targeting IRAK1 may provide a means of suppressing tumor growth in this syndrome by restoring immune surveillance.

Introduction: Neovascularization in response to ischemia depends on inflammation, angiogenesis and reactive oxygen species (ROS). Copper (Cu) is implicated in inflammation and angiogenesis. We reported that cytosolic Cu chaperone Atox1 activates secretory Cu enzymes lysyl oxidase (LOX), while nuclear Atox1 functions as a Cu-dependent transcription factor to promote ROS/NFkB-dependent inflammation in endothelial cells (ECs). However, mechanism of Atox1 nuclear translocation as well as role of endothelial Atox1 in inflammatory angiogenesis in vivo remain unknown. SUMOylation and its deSUMOylation by SENPs regulates transcription factor function. Silica analysis identified a conserved putative SUMOylation motif at Lys(K3) of Atox1. Results: Atox1 expression was dramatically increased in angiogenic ECs in mice hindlimb ischemia model. EC-specific Atox1-deficient mice significantly reduced angiogenesis (CD31+, 67%) and Mac+ inflammatory cells in ischemic tissues. In cultured ECs, inflammatory cytokine TNFα or hypoxia promoted Atox1 nuclear translocation and Atox1 SUMOylation (3.6-fold), which were inhibited by antioxidant NAC or overexpression of “SUMO-dead” Atox1K3R. Mechanistically, TNFα induced Cys603 oxidation/inactivation of SENP1 in cytosol, which in turn increased Atox1 SUMOylation and nuclear translocation. Functionally, siAtox1or Atox1K3R inhibited TNFα-induced inflammatory/angiogenic genes VCAM/ICAM, IL-15 and RANTES. In nucleus with reduced state, ChIP assay using SUMO-Atox1 revealed that Atox1 deSUMOylation by nuclear SENP1 increases Atox1 transcriptional activity for inflammatory genes. In parallel, Atox1K3R which maintains Cu chaperone function inhibited TNFα-induced EC permeability by activating LOX. In vivo, Atox1 SUMOylation was increased after hindlimb ischemia while CRISPR/Cas9-generated SUMO-dead Atox1K3R knock-in mice showed impaired angiogenesis in hindlimb ischemia model. Conclusion: Atox1 SUMOylation via oxidative/inactivation of SENP1 in cytosol promotes: 1) its translocation to nucleus where deSUMOylated Atox1 can function as Cu-dependent transcription factor to drive inflammatory angiogenesis and 2) EC barrier dysfunction in inflamed/hypoxic ECs after ischemic injury.

The Philadelphia (Ph) 9;22 chromosome translocation has two common isoforms that are preferentially associated with distinct subtypes of leukemia. The p210 variant is the hallmark of CML whereas p190 is frequently associated with B-ALL. The only sequence difference between the two isoforms is the guanidine exchange factor (GEF) domain. This GEF is reported to activate RHO family GTPases in response to diverse extracellular stimuli. It is not clear whether and how RHOA contributes to the p210 CML progression. Here we show that knockout of RHOA in the K562 and KU812, p210-expressing cell lines leads to suppression of leukemogenesis in animal models in vivo. RNA-Seq analysis of the mock control and null cells demonstrates a distinct change in the gene expression profile as a result of RHOA deletion, with significant down regulation of genes involved in cell activation and cell adhesion. Cellular analysis revealed that RHOA knockout leads to impaired cell adhesion and migration, and most importantly, the homing ability of leukemia cells to the bone marrow, which may be responsible for the attenuated leukemia progression. We also identified IGFBP2 as an important downstream target of RHOA. Further mechanistic investigation shows RHOA activation leads to relocation of the serum response factor (SFR) into the nucleus, where it directly activates IGFBP2. Knockout of IGFBP2 in CML cells suppresses cell adhesion/invasion, as well as leukemogenesis in vivo. This elevated IGFBP2 expression is confirmed in primary CML samples. Thus, we demonstrate one mechanism whereby RHOA-SRF-IGFBP2 signaling axis contributes to development of leukemia in cells expressing the p210 BCRABL1 fusion kinase.