Myeloid leukemia of Down syndrome (ML-DS) is a tractable human model of acute myeloid leukemia. A preleukemia phase, transient abnormal myelopoiesis (TAM) and silent TAM, occurs in 28% of neonates ...with Down Syndrome (Roberts et al. Blood 2013). TAM is caused by trisomy 21 and acquired mutations in GATA1 that result in a N-terminal truncated protein, GATA1s, in hematopoietic stem and progenitor cells (HSPCs) of fetal origin. ML-DS evolves from TAM by acquisition of additional genetic lesions. The nature of these lesions and the mechanism of transformation are incompletely understood.
We performed exome sequencing and targeted resequencing of 141 ML-DS and 111 TAM patients to characterize the evolving mutational landscape from TAM to ML-DS. On average 1.6 acquired mutations were detected in ML-DS (in addition to GATA1 mutations), significantly more than in TAM (0.4 mutations per sample). Additional anticipated loss-of-function mutations acquired in ML-DS mainly affected cohesin components including CTCF (43% of patients), PRC2 components (13%), KANSL1 and other epigenetic regulators (14%). Conversely, anticipated gain-of-function mutations were most prevalent in signaling pathways, e.g. JAK kinases, MPL, KIT and RAS family members (40%). Importantly, we detected a novel recurrent hotspot mutation in 4% of patients (6/141 cases) in CSF2RB encoding the IL3-, IL5-, GM-CSF-receptor common beta chain. To test if the A455D/T variant in the CSF2RB transmembrane domain is a putative oncogenic driver, we ectopically expressed CSF2RBA455D in TF1 cells. Cells expressing CSF2RBA455D exhibited cytokine independent growth and STAT5 autonomous phosphorylation. In a CD34+-HSPC megakaryocytic differentiation assay, CSF2RBA455D blocked terminal megakaryocytic differentiation whilst increasing proliferation by 30-fold (P=0.046). Moreover, the median survival of NSG mice transplanted with CSF2RBA455DTF1 cells was shortened by 30 days compared to wild type TF1 cells (23 days compared to 53 days, P=0.0097).
To experimentally test the potential of loss-of-function mutations to transform TAM to ML-DS, we performed an in vivo murine isogenic transplantation screen using Gata1s expressing fetal hematopoietic cells from Cas9-knockin mice. We tested variants in 22 genes, recurrently detected in ML-DS, with a pool of prevalidated gRNAs. This resulted in short latency (n=18 mice; median survival 36 days) and high penetrance (100%) leukemia. Leukemia was not detected in mice infected with control gRNAs. Leukemias had a typical ML-DS megakaryoblastic phenotype (CD117+ and CD41a+). Amplicon sequencing revealed on average 2.9 gRNAs per leukemia and high representation (61% of all leukemias) of gRNAs directed to the tumor suppressor Trp53, which was alone sufficient to induce leukemia with 100% penetrance. When excluding the Trp53 gRNA from pools, leukemic cells from moribund mice contained gRNAs against negative regulators of the RAS and JAK-STAT signaling cascade, such as Nf1, Cbl and Sh2b3 (70% of the mice), Ezh2, Asxl1, Kdm6a,Bcor and other epigenetic modifiers (85%) or Ctcf (15%), closely resembling the mutational landscape of ML-DS. In contrast to ML-DS, gRNAs targeting cohesion components, such as Rad21 and Stag2, were not present in any of the leukemias.
In summary, we performed the largest genetic analysis of transforming events in ML-DS that cooperate with trisomy 21 and GATA1s and uncovered a previously undescribed activating mutation in CSR2B. We experimentally validated many of the loss-of-function mutations in a novel murine fetal leukemia assay for ML-DS. The field is now well-placed to study mechanisms of oncogenic cooperativity and identify novel therapeutic approaches for this leukemia.
Crispino:Scholar Rock: Research Funding; Forma Therapeutics: Research Funding.
Tumor necrosis factor alpha (TNFalpha) is an immunomodulatory and proinflammatory cytokine implicated in neuroinflammation and neuronal damage in response to cerebral ischemia. Tumor necrosis ...factor-alpha converting enzyme (TACE or ADAM17) is a key sheddase that releases TNFalpha from its inactive cell-bound precursor. Using a selective small molecule inhibitor of TACE, DPH-067517, we tested the hypothesis that inhibition of TNFalpha formation might have a salutary effect in ischemic stroke induced by embolic occlusion of the middle cerebral artery (MCAO). DPH-067517 selectively inhibited TACE enzyme activity in vitro (K(i) = 2.8 nM), and effectively suppressed ischemia-induced increase in soluble TNFalpha in brain tissue after systemic administration. DPH-067517 (3 and 30 mg/kg, i.p. administered 15 min before MCAO) produced 43% (n = 8, p = 0.16) and 58% (n = 8, p < 0.05) reduction in infarct size and 36% (p < 0.05) and 23% (p < 0.05) reduction in neurological deficits, respectively. The salutary effect of DPH-067517 in ischemic brain injury was also observed when the first dose was administrated 60 min after the onset of ischemia. Inhibition of TACE had no effect on apoptosis measured by levels of active caspase-3 expression and DNA fragmentation. Our data suggest that inhibition of TACE might be a potential therapeutic strategy for neuroprotection after focal ischemic stroke.
The human cytokine, interferon-inducible protein-10 (IP-10), is a small glycoprotein secreted by activated monocytes, T cells, keratinocytes, astrocytes, and endothelial cells and is structurally ...related to the α subfamily of chemotactic cytokines called chemokines (Taub and Oppenheim, Cytokine 5:175, 1993). However, in contrast to other α chemokines that induce neutrophil migration, IP-10 has been shown to chemoattract monocytes and T lymphocytes in vitro, suggesting a role in T-cell–mediated immune responses. We therefore examined the effects of human IP-10 after in vivo administration. IP-10 induces significant mononuclear cell infiltration after subcutaneous injections in normal mice. In an effort to study the in vivo effects of IP-10 on human leukocyte migration, we then examined the ability of recombinant human IP-10 (rhlP-10) to induce human T-cell infiltration using a human/severe combined immune deficiency (SCID) mouse model. SCID mice received an intraperitoneal injection of human peripheral blood lymphocytes (108 cells), followed by a subcutaneous injection of rhlP-10 (1 μg/injection) in the hind flank for 4 hours or sequential injections for 3 days. The skin and underlying tissue from the rhlP-10 injection site were then biopsied and examined for the extent of mononuclear cell infiltration. rhlP-10 again induced significant mononuclear cell accumulation 72 hours after injection. Immunohistologic evaluation determined that a significant number of human CD3+ T cells were recruited in response to rhlP-10 injections. These results show that rhlP-10 is capable of inducing human T-cell migration in vivo and may play an important role in monocyte and lymphocyte recruitment into inflammatory sites.
This is a US government work. There are no restrictions on its use.
Janus kinase 3 (JAK3) is a cytoplasmic tyrosine kinase required for T cell development and activated by cytokines that utilize the interleukin-2 (IL-2) receptor common gamma chain (gamma(c)). Genetic ...inactivation of JAK3 is manifested as severe combined immunodeficiency disease (SCID) in humans and mice. These findings have suggested that JAK3 represents a pharmacological target to control certain lymphoid-derived diseases. Here we provide novel evidence that AG-490 potently inhibits the autokinase activity of JAK3 and tyrosine phosphorylation and DNA binding of signal transducer and activator of transcription 5a and 5b (STAT5a/b). Similar inhibitory effects were observed with other cytokines that use gamma(c). AG-490 also inhibited IL-2-mediated proliferative growth in human T cells with an IC50) = 25 microM that was partially recoverable. Moreover, we demonstrate that this inhibitor prevented tetanus toxoid antigen-specific T cell proliferation and expansion but failed to block activation of Zap70 or p56Lck after anti-CD3 stimulation of human T cells. Taken together, these findings suggest that AG-490 inhibits the JAK3-mediated Type II signaling pathway but not the T cell receptor-derived Type I pathway and possesses therapeutic potential for T cell-derived pathologies such as graft-versus-host disease, allergy, and autoimmune disorders.
Children with Down syndrome (DS) with acute megakaryocytic leukemia (AMkL) have very high survival rates compared with non-DS AMkL patients. Somatic mutations identified in the X-linked transcription ...factor gene, GATA1, in essentially all DS AMkL cases result in the synthesis of a shorter (40 kDa) protein (GATA1s) with altered transactivation activity and may lead to altered expression of GATA1 target genes. Using the Affymetrix U133A microarray chip, we identified 551 differentially expressed genes between DS and non-DS AMkL samples. Transcripts for the bone marrow stromal-cell antigen 2 (BST2) gene, encoding a transmembrane glycoprotein potentially involved in interactions between leukemia cells and bone marrow stromal cells, were 7.3-fold higher (validated by real-time polymerase chain reaction) in the non-DS compared with the DS group. Additional studies confirmed GATA1 protein binding and transactivation of the BST2 promoter; however, stimulation of BST2 promoter activity by GATA1s was substantially reduced compared with the full-length GATA1. CMK sublines, transfected with the BST2 cDNA and incubated with HS-5 bone marrow stromal cells, exhibited up to 1.7-fold reduced cytosine arabinoside (ara-C)-induced apoptosis, compared with mock-transfected cells. Our results demonstrate that genes that account for differences in survival between DS and non-DS AMkL cases may be identified by microarray analysis and that differential gene expression may reflect relative transactivation capacities of the GATA1s and full-length GATA1 proteins.
We investigated the relationship between parents' empathic responses prior to their children undergoing cancer treatment procedures and children's pain/distress during the procedures. We ...hypothesized: (1) parents' empathic distress would be positively associated with children's pain/distress, (2) parents' empathic concern would be negatively associated with children's pain/distress; and (3) parents' enduring dispositions and social support would be associated with their empathic responses. Parents completed: (1) measures of dispositions and perceived social support several weeks before their children underwent the procedures, and (2) state measures of empathic distress and empathic concern just before the procedures. Empathic distress was positively associated with children's pain; empathic concern was negatively associated with children's pain/distress. Predictions about dispositions and social support were also substantially confirmed.
CD40-mediated interactions play an important role in the response to infections, transplantation, and cancer by affecting the development, activation, proliferation and differentiation of a variety ...of immune cells. In the current study we examined the role of CD40-mediated interactions in immune responses to bladder, pancreatic and breast carcinomas as well as melanoma cell lines using soluble human CD40L (rhCD40L) or anti-CD40 mAb in vitro. CD40 expression was readily detected in a large proportion of the cell lines and was augmented but not induced de novo by treatment with IFNγ. Treatment of CD40-positive cell lines with rhCD40L or anti-CD40mAb enhanced cell surface expression of ICAM-1 and FAS and stimulated the production of IL-6, IL-8, GROα, GM-CSF and TNFα but not IL-4, IL-10, TGFβ, MCP-1, RANTES, MIP-1β, or IP-10. In addition, incubation of CD40+ tumour cell lines with immobilised rhCD40L or anti-CD40 mAb in vitro resulted in significant inhibition of proliferation and a corresponding decrease in viability. This CD40-mediated inhibition of cell growth was due, at least in part, to alterations in cell cycle and the induction of apoptosis. Transfection of CD40-negative tumour cell lines with the cDNA for CD40 conferred responsiveness to rhCD40L and anti-CD40 antibody. Finally, the presence of CD40 on the surface of carcinoma lines was found to be an important factor in the generation of tumour-specific T cell responses.
PDF
