Weina Chen

Differential regulation and properties of MAPKs.

Mitogen-activated protein kinases (MAPKs) regulate diverse cellular programs including embryogenesis, proliferation, differentiation and apoptosis based on cues derived from the cell surface and the metabolic state and environment of the cell. In mammals, there are more than a dozen MAPK genes. The best known are the extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK(1–3)) and p38(α, β, γ and δ) families. ERK3, ERK5 and ERK7 are other MAPKs that have distinct regulation and functions. MAPK cascades consist of a core of three protein kinases. Despite the apparently simple architecture of this pathway, these enzymes are capable of responding to a bewildering number of stimuli to produce exquisitely specific cellular outcomes. These responses depend on the kinetics of their activation and inactivation, the subcellular localization of the kinases, the complexes in which they act, and the availability of substrates. Fine-tuning of cascade activity can occur through modulatory inputs to cascade component from the primary kinases to the scaffolding accessory proteins. Here, we describe some of the properties of the three major MAPK pathways and discuss how these properties govern pathway regulation and activity.

Ischemic Preconditioning Activates Phosphatidylinositol-3- Kinase Upstream of Protein Kinase C

The present study is designed to test whether phosphatidylinositol 3-kinase (PI3-kinase) has a role in the signaling pathway in ischemic preconditioning (PC) and whether it is proximal or distal to protein kinase C (PKC). Before 20 minutes of global ischemia, Langendorff-perfused rat hearts were perfused for 20 minutes (control); preconditioned with 4 cycles of 5-minute ischemia and 5-minute reflow (PC); treated with either wortmannin (WM) or LY 294002 (LY), each of which is a PI3-kinase inhibitor, for 5 minutes before and throughout PC; treated with 1,2-dioctanoyl-sn-glycerol (DOG), an activator of PKC for 10 minutes (DOG); treated identically to the DOG group except with WM added 10 minutes before and during perfusion with DOG; or treated with either WM or LY for 25 minutes. Recovery of left ventricular developed pressure (LVDP; percentage of initial preischemic LVDP), measured after 30 minutes of reflow, was improved by PC (72±2% versus 36±4% in control;P <0.001), and this was blocked by WM and LY (41±4% and 43±5%, respectively;P <0.05 compared with PC). DOG addition improved postischemic LVDP (67±6%;P <0.001 compared with control), but in contrast to its effect on PC, WM did not completely eliminate the protective effect of DOG (52±4%;P >0.05 compared with DOG;P <0.05 compared with control). PC induced phosphorylation of protein kinase B and translocation of PKC&egr;, and it increased NO production, and these effects were blocked by WM, which suggests a role for PI3-kinase in PC upstream of PKC and NO.

MYC/BCL2 protein coexpression contributes to the inferior survival of activated B-cell subtype of diffuse large B-cell lymphoma and demonstrates high-risk gene expression signatures: a report from The International DLBCL Rituximab-CHOP Consortium Program

Diffuse large B-cell lymphoma (DLBCL) is stratified into prognostically favorable germinal center B-cell (GCB)-like and unfavorable activated B-cell (ABC)-like subtypes based on gene expression signatures. In this study, we analyzed 893 de novo DLBCL patients treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone). We show that MYC/BCL2 protein coexpression occurred significantly more commonly in the ABC subtype. Patients with the ABC or GCB subtype of DLBCL had similar prognoses with MYC/BCL2 coexpression and without MYC/BCL2 coexpression. Consistent with the notion that the prognostic difference between the 2 subtypes is attributable to MYC/BCL2 coexpression, there is no difference in gene expression signatures between the 2 subtypes in the absence of MYC/BCL2 coexpression. DLBCL with MYC/BCL2 coexpression demonstrated a signature of marked downregulation of genes encoding extracellular matrix proteins, those involving matrix deposition/remodeling and cell adhesion, and upregulation of proliferation-associated genes. We conclude that MYC/BCL2 coexpression in DLBCL is associated with an aggressive clinical course, is more common in the ABC subtype, and contributes to the overall inferior prognosis of patients with ABC-DLBCL. In conclusion, the data suggest that MYC/BCL2 coexpression, rather than cell-of-origin classification, is a better predictor of prognosis in patients with DLBCL treated with R-CHOP.

A Redox-Based Mechanism for Cardioprotection Induced by Ischemic Preconditioning in Perfused Rat Heart

Recent studies have suggested that mild redox alterations can regulate cell function. Therefore, we tested the hypothesis that alteration in the thiol redox state might be responsible for the cardioprotective effects conferred by ischemic preconditioning in the perfused rat heart. We find that preconditioning with four 5-minute periods of ischemia, each separated by 5 minutes of reflow, is associated with a significant loss of glutathione (3.98 +/- 0.32 mumol/g dry wt, n = 8) compared with no preconditioning (6.38 +/- 0.24 mumol/g dry wt, n = 14). We further find that the addition of N-acetylcysteine (NAC, a glutathione precursor and antioxidant) during the preconditioning protocol not only blocks the loss of glutathione (5.60 +/- 0.31 mumol/g dry wt, n = 9) but also blocks the protective effects of preconditioning. It is observed that after 20 minutes of ischemia followed by 20 minutes of reflow, untreated hearts recover 38 +/- 7% (n = 5) of their initial preischemic contractile function, whereas preconditioned hearts recover 91 +/- 11% (n = 7). Hearts preconditioned in the presence of NAC recover 24 +/- 3% (n = 7) of their preischemic function. Similarly, the addition of NAC reverses the protective effect of preconditioning on creatine kinase release. On reflow after 60 minutes of ischemia, creatine kinase release from control hearts was 271 +/- 20 IU.20 min-1.g dry wt-1 (n = 5), whereas preconditioned hearts release only 170 +/- 26 IU.20 min-1.g dry wt-1 (n = 6), and hearts preconditioned in the presence of NAC release 361 +/- 30 IU.20 min-1.g dry wt-1 (n = 5). We also find that hearts preconditioned in the presence of NAC have less attenuation of the decline in pHi than hearts preconditioned in the absence of drug. Thus, a redox-sensitive mechanism may be involved in the protection afforded by ischemic preconditioning.

IDH mutations in acute myeloid leukemia

Acute myeloid leukemia is a heterogeneous group of diseases. Mutations of the isocitrate dehydrogenase (IDH) genes represent a novel class of point mutations in acute myeloid leukemia. These mutations prevent oxidative decarboxylation of isocitrate to α-ketoglutarate and confer novel enzymatic activity, facilitating the reduction of α-ketoglutarate to d-2-hydroxyglutarate, a putative oncometabolite. IDH1/IDH2 mutations are heterozygous, and their combined frequency is approximately 17% in unselected acute myeloid leukemia cases, 27% in cytogenetically normal acute myeloid leukemia cases, and up to 67% in acute myeloid leukemia cases with cuplike nuclei. These mutations are largely mutually exclusive. Despite many similarities of IDH1 and IDH2 mutations, it is possible that they represent distinct molecular or clinical subgroups of acute myeloid leukemia. All known mutations involve arginine (R), in codon 132 of IDH1 or codon 140 or 172 of IDH2. IDH1(R132) and IDH2(R140) mutations are frequently accompanied by normal cytogenetics and NPM1 mutation, whereas IDH2(R172) is frequently the only mutation detected in acute myeloid leukemia. There is increasing evidence that the prognostic impact of IDH1/2 mutations varies according to the specific mutation and also depends on the context of concurrent mutations of other genes. IDH1(R132) mutation may predict poor outcome in a subset of patients with molecular low-risk acute myeloid leukemia, whereas IDH2(R172) mutations confer a poor prognosis in patients with acute myeloid leukemia. Expression of IDH1/2 mutants induces an increase in global DNA hypermethylation and inhibits TET2-induced cytosine 5-hydroxymethylation, DNA demethylation. These data suggest that IDH1/2 mutations constitute a distinct mutational class in acute myeloid leukemia, which affects the epigenetic state, an important consideration for the development of therapeutic agents.

MEK Inhibition Enhances ABT-737-Induced Leukemia Cell Apoptosis via Prevention of ERK activated MCL-1 induction and Modulation of MCL-1/BIM Complex

Recently, strategies for acute myeloid leukemia (AML) therapy have been developed that target anti-apoptotic BCL2 family members using BH3-mimetic drugs such as ABT-737. Though effective against BCL2 and BCL-XL, ABT-737 poorly inhibits MCL-1. Here we report that, unexpectedly, ABT-737 induces activation of the extracellular receptor activated kinase and induction of MCL-1 in AML cells. MEK inhibitors such as PD0325901 and CI-1040 have been used successfully to suppress MCL-1. We report that PD0325901 blocked ABT-737-induced MCL-1 expression, and when combined with ABT-737 resulted in potent synergistic killing of AML-derived cell lines, primary AML blast and CD34+38-123+ progenitor/stem cells. Finally, we tested the combination of ABT-737 and CI-1040 in a murine xenograft model using MOLM-13 human leukemia cells.Whereas control mice and CI-1040-treated mice exhibited progressive leukemia growth, ABT-737, and to a significantly greater extent, ABT-737+CI-1040 exerted major anti-leukemia activity. Collectively, results demonstrated unexpected anti-apoptotic interaction between the BCL2 family-targeted BH3-mimetic ABT-737 and mitogen-activated protein kinase signaling in AML cells: the BH3 mimetic is not only restrained in its activity by MCL-1, but also induces its expression. However, concomitant inhibition by BH3 mimetics and MEK inhibitors could abrogate this effect and may be developed into a novel and effective therapeutic strategy for patients with AML.

Human immunodeficiency virus-associated plasmablastic lymphoma: Poor prognosis in the era of highly active antiretroviral therapy

Plasmablastic lymphoma (PBL) is a rare and aggressive B‐cell lymphoma strongly associated with human immunodeficiency virus (HIV) infection. The authors conducted a multi‐institutional, retrospective study to describe characteristics and determine prognostic factors in HIV‐associated PBL.

Plasmablastic lymphomas with MYC/IgH rearrangement: Report of three cases and review of the literature

We report detailed clinicopathologic features of 3 cases of plasmablastic lymphoma (PBL) with MYC/IgH rearrangement, representing one third of PBL cases diagnosed at our institution. This study brings the total number of reported cases in the literature to 6. All patients were HIV+ with very low CD4 counts at diagnosis. The involved locations were mediastinum, anus, and bone marrow. Tumors exhibited predominantly immunoblastic/plasmablastic morphologic features and had a plasma cell-like immunophenotype. Bright CD38 expression by flow cytometry had a tendency to be more common in these cases compared with PBL without MYC rearrangement. All cases were positive for Epstein-Barr virus-encoded RNA but lacked human herpesvirus-8 latent nuclear antigen. The 2 patients with follow-up died within 3 months. These findings show that PBL is often associated with MYC/IgH rearrangements and that this finding may portend an aggressive clinical course, suggesting that cytogenetic studies should be routinely applied in cases of PBL.

Ascorbate regulates haematopoietic stem cell function and leukaemogenesis

Stem-cell fate can be influenced by metabolite levels in culture, but it is not known whether physiological variations in metabolite levels in normal tissues regulate stem-cell function in vivo. Here we describe a metabolomics method for the analysis of rare cell populations isolated directly from tissues and use it to compare mouse haematopoietic stem cells (HSCs) to restricted haematopoietic progenitors. Each haematopoietic cell type had a distinct metabolic signature. Human and mouse HSCs had unusually high levels of ascorbate, which decreased with differentiation. Systemic ascorbate depletion in mice increased HSC frequency and function, in part by reducing the function of Tet2, a dioxygenase tumour suppressor. Ascorbate depletion cooperated with Flt3 internal tandem duplication (Flt3ITD) leukaemic mutations to accelerate leukaemogenesis, through cell-autonomous and possibly non-cell-autonomous mechanisms, in a manner that was reversed by dietary ascorbate. Ascorbate acted cell-autonomously to negatively regulate HSC function and myelopoiesis through Tet2-dependent and Tet2-independent mechanisms. Ascorbate therefore accumulates within HSCs to promote Tet activity in vivo, limiting HSC frequency and suppressing leukaemogenesis.

Activating FLT3 mutations are detectable in chronic and blast phases of chronic myeloproliferative disorders other than chronic myeloid leukemia

FLT3 gene mutations, either internal tandem duplication (ITD) or D835 point mutations, have been studied extensively in acute myeloid leukemia and myelodysplastic syndrome (MDS). Little is known about FLT3 mutations in chronic myeloproliferative diseases (CMPDs) or their relationship with V617F JAK2 mutations. We analyzed bone marrow samples from 142 patients with Philadelphia (Ph) chromosome- CMPD or CMPD/MDS and from 119 patients with Ph+ chronic myeloid leukemia (CML) using a multiplex polymerase chain reaction assay. FLT3 mutations, 11 ITD and 2 D835, were detected in 13 (9.2%) patients with CMPD or CMPD/MDS, 7 in blast phase and 6 in chronic phase. Analyses for JAK2 mutations in 11 of 13 cases were all negative. By contrast, no FLT3 mutations were detected in CML, including 108 chronic and 11 blast phase cases. FLT3 mutations occur in approximately 10% of CMPD and CMPD/MDS but are not observed in JAK2+ CMPD or in CML.