Allogeneic hematopoietic stem cell transplantation (HSCT) is used effectively to treat a number of hematological malignancies. Its beneficial effects rely on donor-derived T cell–targeted leukemic cells, the so-called graft-versus-leukemia (GVL) effect. Induction of GVL is usually associated with concomitant development of graft-versus-host disease (GVHD), a major complication of allogeneic HSCT. The T cells that mediate GVL and GVHD are activated by alloantigen presented on host antigen-presenting cells of hematopoietic origin, and it is not well understood how alloantigen expression on non-hematopoietic cells affects GVL activity. Here we show, in mouse models of MHC-matched, minor histocompatibility antigen–mismatched bone marrow transplantation, that alloantigen expression on host epithelium drives donor T cells into apoptosis and dysfunction during GVHD, resulting in a loss of GVL activity. During GVHD, programmed death–1 (PD-1) and PD ligand–1 (PD-L1), molecules implicated in inducing T cell exhaustion, were upregulated on activated T cells and the target tissue, respectively, suggesting that the T cell defects driven by host epithelial alloantigen expression might be mediated by the PD-1/PD-L1 pathway. Consistent with this, blockade of PD-1/PD-L1 interactions partially restored T cell effector functions and improved GVL. These results elucidate a previously unrecognized significance of alloantigen expression on non-hematopoietic cells in GVL and suggest that separation of GVL from GVHD for more effective HSCT may be possible in human patients.
Shoji Asakura, Daigo Hashimoto, Shuichiro Takashima, Haruko Sugiyama, Yoshinobu Maeda, Koichi Akashi, Mitsune Tanimoto, Takanori Teshima
Somatic activating mutations in Notch1 contribute to the pathogenesis of T cell acute lymphoblastic lymphoma (T-ALL), but how activated Notch1 signaling exerts this oncogenic effect is not completely understood. Here we identify HIV-1 Rev–binding protein (Hrb), a component of the clathrin-mediated endocytosis machinery, as a critical mediator of Notch-induced T-ALL development in mice. Hrb was found to be a direct transcriptional target of Notch1, and Hrb loss reduced the incidence or delayed the onset of T-ALL in mouse models in which activated Notch1 signaling either contributes to or drives leukemogenesis. Consistent with this observation, Hrb supported survival and proliferation of hematopoietic and T cell precursor cells in vitro. We demonstrated that Hrb accelerated the uptake of transferrin, which was required for upregulation of the T cell protooncogene p21. Indeed, iron-deficient mice developed Notch1-induced T-ALL substantially more slowly than control mice, further supporting a critical role for iron uptake during leukemogenesis. Taken together, these results reveal that Hrb is a critical Notch target gene that mediates lymphoblast transformation and disease progression via its ability to satisfy the enhanced demands of transformed lymphoblasts for iron. Further, our data suggest that Hrb may be targeted to improve current treatment or design novel therapies for human T-ALL patients.
Shariq S. Khwaja, Hudan Liu, Caili Tong, Fang Jin, Warren S. Pear, Jan van Deursen, Richard J. Bram
Mice engineered to express c-Myc in B cells (Eμ-myc mice) develop lethal lymphomas in which the gene encoding the p53 tumor suppressor is frequently mutated. Whether the p53 homolog p73 also functions as a tumor suppressor in vivo remains controversial. Here we have shown that p73 loss does not substantially affect disease onset and mortality in Eμ-myc mice. However, it does alter the phenotype of the disease. Specifically, p73 loss decreased nodal disease and increased widespread extranodal dissemination. We further found that p53 acted as the dominant tumor suppressor during the onset of Eμ-myc–driven B cell lymphomagenesis, while p73 modulated tumor dissemination and extranodal growth. Immunophenotyping and expression profiling suggested that p73 loss allowed increased maturation of malignant B cells and deregulated genes involved in lymphocyte homing and dissemination of human lymphomas. Consistent with this, p73 expression was frequently downregulated in a large cohort of human mature aggressive B cell lymphomas, and both the incidence and degree of p73 downregulation in these tumors correlated with their extranodal dissemination status. These data indicate that p73 is a modifier of Myc-driven lymphomas in mice, favoring tumor dissemination, and suggest that p73 could be a biomarker for human B cell lymphoma dissemination, a notion that can now be tested in clinicopathologic correlation studies.
Alice Nemajerova, Oleksi Petrenko, Lorenz Trümper, Gustavo Palacios, Ute M. Moll
Antiapoptotic BCL2 family members have been implicated in the pathogenesis of acute myelogenous leukemia (AML), but the functional significance and relative importance of individual proteins (e.g., BCL2, BCL-XL, and myeloid cell leukemia 1 [MCL1]) remain poorly understood. Here, we examined the expression of BCL2, BCL-XL, and MCL1 in primary human hematopoietic subsets and leukemic blasts from AML patients and found that MCL1 transcripts were consistently expressed at high levels in all samples tested. Consistent with this, Mcl1 protein was also highly expressed in myeloid leukemic blasts in a mouse Myc-induced model of AML. We used this model to test the hypothesis that Mcl1 facilitates AML development by allowing myeloid progenitor cells to evade Myc-induced cell death. Indeed, activation of Myc for 7 days in vivo substantially increased myeloid lineage cell numbers, whereas hematopoietic stem, progenitor, and B-lineage cells were depleted. Furthermore, Mcl1 haploinsufficiency abrogated AML development. In addition, deletion of a single allele of Mcl1 from fully transformed AML cells substantially prolonged the survival of transplanted mice. Conversely, the rapid lethality of disease was restored by coexpression of Bcl2 and Myc in Mcl1-haploinsufficient cells. Together, these data demonstrate a critical and dose-dependent role for Mcl1 in AML pathogenesis in mice and suggest that MCL1 may be a promising therapeutic target in patients with de novo AML.
Zhifu Xiang, Hui Luo, Jacqueline E. Payton, Jennifer Cain, Timothy J. Ley, Joseph T. Opferman, Michael H. Tomasson
Hematopoietic stem and progenitor cell (HSPC) expansion is regulated by intrinsic signaling pathways activated by cytokines. The intracellular kinase JAK2 plays an essential role in cytokine signaling, and activating mutations in JAK2 are found in a number of hematologic malignancies. We previously demonstrated that lymphocyte adaptor protein (Lnk, also known as Sh2b3) binds JAK2 and attenuates its activity, thereby limiting HSPC expansion. Here we show that loss of Lnk accelerates and exacerbates oncogenic JAK2-induced myeloproliferative diseases (MPDs) in mice. Specifically, Lnk deficiency enhanced cytokine-independent JAK/STAT signaling and augmented the ability of oncogenic JAK2 to expand myeloid progenitors in vitro and in vivo. An activated form of JAK2, unable to bind Lnk, caused greater myeloid expansion than activated JAK2 alone and accelerated myelofibrosis, indicating that Lnk directly inhibits oncogenic JAK2 in constraining MPD development. In addition, Lnk deficiency cooperated with the BCR/ABL oncogene, the product of which does not directly interact with or depend on JAK2 or Lnk, in chronic myeloid leukemia (CML) development, suggesting that Lnk also acts through endogenous pathways to constrain HSPCs. Consistent with this idea, aged Lnk–/– mice spontaneously developed a CML-like MPD. Taken together, our data establish Lnk as a bona fide suppressor of MPD in mice and raise the possibility that Lnk dysfunction contributes to the development of hematologic malignancies in humans.
Alexey Bersenev, Chao Wu, Joanna Balcerek, Jiang Jing, Mondira Kundu, Gerd A. Blobel, Kudakwashe R. Chikwava, Wei Tong
CD4+CD25+Foxp3+ Tregs have an indispensable role in the maintenance of tolerance after allogeneic HSC transplantation (HSCT). Patients with chronic graft-versus-host disease (GVHD) have fewer circulating Tregs, but the mechanisms that lead to this deficiency of Tregs after HSCT are not known. Here, we analyzed reconstitution of Tregs and conventional CD4+ T cells (Tcons) in patients who underwent allogeneic HSCT after myeloablative conditioning. Following transplant, thymic generation of naive Tregs was markedly impaired, and reconstituting Tregs had a predominantly activated/memory phenotype. In response to CD4+ lymphopenia after HSCT, Tregs underwent higher levels of proliferation than Tcons, but Tregs undergoing homeostatic proliferation also showed increased susceptibility to Fas-mediated apoptosis. Prospective monitoring of CD4+ T cell subsets revealed that Tregs rapidly expanded and achieved normal levels by 9 months after HSCT, but Treg levels subsequently declined in patients with prolonged CD4+ lymphopenia. This resulted in a relative deficiency of Tregs, which was associated with a high incidence of extensive chronic GVHD. These studies indicate that CD4+ lymphopenia is a critical factor in Treg homeostasis and that prolonged imbalance of Treg homeostasis after HSCT can result in loss of tolerance and significant clinical disease manifestations.
Ken-ichi Matsuoka, Haesook T. Kim, Sean McDonough, Gregory Bascug, Ben Warshauer, John Koreth, Corey Cutler, Vincent T. Ho, Edwin P. Alyea, Joseph H. Antin, Robert J. Soiffer, Jerome Ritz
HDL has anti-atherogenic properties, and plasma levels of HDL cholesterol correlate inversely with risk of coronary artery disease. HDL reportedly functions as a cofactor to the anticoagulant activated protein C (APC) in the degradation of factor Va (FVa). The aim of the present study was to elucidate the mechanism by which HDL functions as cofactor to APC. Consistent with a previous report, HDL isolated from human plasma by ultracentrifugation was found to stimulate APC-mediated degradation of FVa. However, further purification of HDL by gel filtration revealed that the stimulating activity was not a property of HDL. Instead, the stimulating activity eluted completely separately from HDL in the high-molecular-weight void volume fractions. The active portion of these fractions stimulated FVa degradation by APC and supported the assembly of factor Xa and FVa into a functional prothrombinase complex. Both the procoagulant and anticoagulant activities were blocked by addition of annexin V, suggesting that the active portion was negatively charged phospholipid membranes. These results demonstrate that HDL does not stimulate the APC/protein S effect and that the activity previously reported to be a property of HDL is instead caused by contaminating negatively charged phospholipid membranes.
Cecilia Oslakovic, Eva Norstrøm, Björn Dahlbäck
Glutaredoxin 5 (GLRX5) deficiency has previously been identified as a cause of anemia in a zebrafish model and of sideroblastic anemia in a human patient. Here we report that GLRX5 is essential for iron-sulfur cluster biosynthesis and the maintenance of normal mitochondrial and cytosolic iron homeostasis in human cells. GLRX5, a mitochondrial protein that is highly expressed in erythroid cells, can homodimerize and assemble [2Fe-2S] in vitro. In GLRX5-deficient cells, [Fe-S] cluster biosynthesis was impaired, the iron-responsive element–binding (IRE-binding) activity of iron regulatory protein 1 (IRP1) was activated, and increased IRP2 levels, indicative of relative cytosolic iron depletion, were observed together with mitochondrial iron overload. Rescue of patient fibroblasts with the WT GLRX5 gene by transfection or viral transduction reversed a slow growth phenotype, reversed the mitochondrial iron overload, and increased aconitase activity. Decreased aminolevulinate δ, synthase 2 (ALAS2) levels attributable to IRP-mediated translational repression were observed in erythroid cells in which GLRX5 expression had been downregulated using siRNA along with marked reduction in ferrochelatase levels and increased ferroportin expression. Erythroblasts express both IRP-repressible ALAS2 and non-IRP–repressible ferroportin 1b. The unique combination of IRP targets likely accounts for the tissue-specific phenotype of human GLRX5 deficiency.
Hong Ye, Suh Young Jeong, Manik C. Ghosh, Gennadiy Kovtunovych, Laura Silvestri, Danilo Ortillo, Naoya Uchida, John Tisdale, Clara Camaschella, Tracey A. Rouault
Mutation of the von Hippel–Lindau (VHL) tumor suppressor protein at codon 200 (R200W) is associated with a disease known as Chuvash polycythemia. In addition to polycythemia, Chuvash patients have pulmonary hypertension and increased respiratory rates, although the pathophysiological basis of these symptoms is unclear. Here we sought to address this issue by studying mice homozygous for the R200W Vhl mutation (VhlR/R mice) as a model for Chuvash disease. These mice developed pulmonary hypertension independently of polycythemia and enhanced normoxic respiration similar to Chuvash patients, further validating VhlR/R mice as a model for Chuvash disease. Lungs from VhlR/R mice exhibited pulmonary vascular remodeling, hemorrhage, edema, and macrophage infiltration, and lungs from older mice also exhibited fibrosis. HIF-2α activity was increased in lungs from VhlR/R mice, and heterozygosity for Hif2a, but not Hif1a, genetically suppressed both the polycythemia and pulmonary hypertension in the VhlR/R mice. Furthermore, Hif2a heterozygosity resulted in partial protection against vascular remodeling, hemorrhage, and edema, but not inflammation, in VhlR/R lungs, suggesting a selective role for HIF-2α in the pulmonary pathology and thereby providing insight into the mechanisms underlying pulmonary hypertension. These findings strongly support a dependency of the Chuvash phenotype on HIF-2α and suggest potential treatments for Chuvash patients.
Michele M. Hickey, Theresa Richardson, Tao Wang, Matias Mosqueira, Evguenia Arguiri, Hongwei Yu, Qian-Chun Yu, Charalambos C. Solomides, Edward E. Morrisey, Tejvir S. Khurana, Melpo Christofidou-Solomidou, M. Celeste Simon
Mixed-lineage leukemia (MLL) is a proto-oncogene frequently involved in chromosomal translocations associated with acute leukemia. These chromosomal translocations commonly result in MLL fusion proteins that dysregulate transcription. Recent data suggest that the MYB proto-oncogene, which is an important regulator of hematopoietic cell development, has a role in leukemogenesis driven by the MLL-ENL fusion protein, but exactly how is unclear. Here we have demonstrated that c-Myb is recruited to the MLL histone methyl transferase complex by menin, a protein important for MLL-associated leukemic transformation, and that it contributes substantially to MLL-mediated methylation of histone H3 at lysine 4 (H3K4). Silencing MYB in human leukemic cell lines and primary patient material evoked a global decrease in H3K4 methylation, an unexpected decrease in HOXA9 and MEIS1 gene expression, and decreased MLL and menin occupancy in the HOXA9 gene locus. This decreased occupancy was associated with a diminished ability of an MLL-ENL fusion protein to transform normal mouse hematopoietic cells. Previous studies have shown that MYB expression is regulated by Hoxa9 and Meis1, indicating the existence of an autoregulatory feedback loop. The finding that c-Myb has the ability to direct epigenetic marks, along with its participation in an autoregulatory feedback loop with genes known to transform hematopoietic cells, lends mechanistic and translationally relevant insight into its role in MLL-associated leukemogenesis.
Shenghao Jin, Huiwu Zhao, Yan Yi, Yuji Nakata, Anna Kalota, Alan M. Gewirtz