Amos Gaikwad

Cell-Specific Gene Expression in Langerhans Cell Histiocytosis Lesions Reveals a Distinct Profile Compared with Epidermal Langerhans Cells

Langerhans cell histiocytosis (LCH) is a rare disease characterized by heterogeneous lesions containing CD207+ Langerhans cells (LCs) and lymphocytes that can arise in almost any tissue and cause significant morbidity and mortality. After decades of research, the cause of LCH remains speculative. A prevailing model suggests that LCH arises from malignant transformation and metastasis of epidermal LCs. In this study, CD207+ cells and CD3+ T cells were isolated from LCH lesions to determine cell-specific gene expression. Compared with control epidermal CD207+ cells, the LCH CD207+ cells yielded 2113 differentially expressed genes (false discovery rate < 0.01). Surprisingly, the expression of many genes previously associated with LCH, including cell-cycle regulators, proinflammatory cytokines, and chemokines, were not significantly different from control LCs in our study. However, several novel genes whose products activate and recruit T cells to sites of inflammation, including SPP1 (osteopontin), were highly overexpressed in LCH CD207+ cells. Furthermore, several genes associated with immature myeloid dendritic cells were overexpressed in LCH CD207+ cells. Compared with the peripheral CD3+ cells from LCH patients, the LCH lesion CD3+ cells yielded only 162 differentially regulated genes (false discovery rate < 0.01), and the expression profile of the LCH lesion CD3+ cells was consistent with an activated regulatory T cell phenotype with increased expression of FOXP3, CTLA4, and SPP1. Results from this study support a model of LCH pathogenesis in which lesions do not arise from epidermal LCs but from accumulation of bone marrow-derived immature myeloid dendritic cells that recruit activated lymphocytes.

A plant triterpenoid, avicin D, induces autophagy by activation of AMP-activated protein kinase.

Avicins, a family of plant triterpene electrophiles, can trigger apoptosis-associated tumor cell death, and suppress chemical-induced carcinogenesis by its anti-inflammatory, anti-mutagenic, and antioxidant properties. Here, we show that tumor cells treated with benzyloxycarbonylvalyl-alanyl–aspartic acid (O-methyl)–fluoro-methylketone, an apoptosis inhibitor, and Bax−/−Bak−/− apoptosis-resistant cells can still undergo cell death in response to avicin D treatment. We demonstrate that this non-apoptotic cell death is mediated by autophagy, which can be suppressed by chloroquine, an autophagy inhibitor, and by specific knockdown of autophagy-related gene-5 (Atg5) and Atg7. Avicin D decreases cellular ATP levels, stimulates the activation of AMP-activated protein kinase (AMPK), and inhibits mammalian target of rapamycin (mTOR) and S6 kinase activity. Suppression of AMPK by compound C and dominant-negative AMPK decreases avicin D-induced autophagic cell death. Furthermore, avicin D-induced autophagic cell death can be abrogated by knockdown of tuberous sclerosis complex 2 (TSC2), a key mediator linking AMPK to mTOR inhibition, suggesting that AMPK activation is a crucial event targeted by avicin D. These findings indicate the therapeutic potential of avicins by triggering autophagic cell death.

The JAK kinase inhibitor CP-690,550 supresses the growth of human polycythemia vera cells carrying the JAK2V617F mutation

The somatic activating janus kinase 2 mutation (JAK2)V617F is detectable in most patients with polycythemia vera (PV). Here we report that CP‐690,550 exerts greater antiproliferative and pro‐apoptotic activity against cells harboring JAK2V617F compared with JAK2WT. CP‐690,550 treatment of murine factor‐dependent cell Patersen–erythropoietin receptor (FDCP‐EpoR) cells harboring human wild‐type or V617F JAK2 resulted in inhibition of cell proliferation with a 50% inhibitory concentration (IC50) of 2.1 µM and 0.25 µM, respectively. Moreover, CP‐690,550 induced a significant pro‐apoptotic effect on murine FDCP‐EpoR cells carrying JAK2V617F, whereas a lesser effect was observed for cells carrying wild‐type JAK2. This activity was coupled with inhibition of phosphorylation of the key JAK2V617F‐dependent downstream signaling effectors signal transducer and activator of transcription (STAT)3, STAT5, and v‐akt murine thymoma viral oncogene homolog (AKT). Furthermore, CP‐690,550 treatment of ex‐vivo‐expanded erythroid progenitors from JAK2V617F‐positive PV patients resulted in specific, antiproliferative (IC50 = 0.2 µM) and pro‐apoptotic activity. In contrast, expanded progenitors from healthy controls were less sensitive to CP‐690,550 in proliferation (IC50 > 1.0 µM), and apoptosis assays. The antiproliferative effect on expanded patient progenitors was paralleled by a decrease in JAK2V617F mutant allele frequency, particularly in a patient homozygous for JAK2V617F. Flow cytometric analysis of expanded PV progenitor cells treated with CP‐690,550 suggests a possible transition towards a pattern of erythroid differentiation resembling expanded cells from normal healthy controls. (Cancer Sci 2008; 99: 1265–1273)

Prevalence and clinical correlates of JAK2 mutations in Down syndrome acute lymphoblastic leukaemia

Recurrent, prognostically significant chromosomal abnormalities occur in approximately 75% of paediatric acute lymphoblastic leukaemia (ALL), but only infrequently in children with Down syndrome (DS) and ALL. Recently, novel somatic activating mutations in the gene Janus kinase 2 (JAK2) were reported in 18% of DS ALL. Here we report identification and clinical correlates of JAK2 mutations in an independent cohort. JAK2 activating mutations occurred in 10/53 DS ALL cases (18·9%). Mutations were overrepresented in males (P < 0·03), occurred once in association with high hyperdiploidy and were not significantly correlated with age, initial white blood count, or event‐free survival. Our results confirm the significance of JAK–STAT pathway activation in DS ALL.

Lower Induction of p53 and Decreased Apoptosis in NQO1-Null Mice Lead to Increased Sensitivity to Chemical-Induced Skin Carcinogenesis

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a cytosolic protein that catalyzes metabolic detoxification of quinones and protects cells against redox cycling and oxidative stress. NQO1-null mice deficient in NQO1 protein showed increased sensitivity to 7,12-dimethylbenz(a)anthracene- and benzo(a)pyrene-induced skin carcinogenesis. In the present studies, we show that benzo(a)pyrene metabolite benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide and not benzo(a)pyrene quinones contributed to increased benzo(a) pyrene-induced skin tumors in NQO1-null mice. An analysis of untreated skin revealed an altered intracellular redox state due to accumulation of NADH and reduced levels of NAD/NADH in NQO1-null mice as compared with wild-type mice. Treatment with benzo(a)pyrene failed to significantly increase p53 and apoptosis in the skin of NQO1-null mice when compared with wild-type mice. These results led to the conclusion that altered intracellular redox state along with lack of induction of p53 and decreased apoptosis plays a significant role in increased sensitivity of NQO1-null mice to benzo(a)pyrene-induced skin cancer.

Triterpenoid Electrophiles (Avicins) Suppress Heat Shock Protein-70 and X-Linked Inhibitor of Apoptosis Proteins in Malignant Cells by Activation of Ubiquitin Machinery: Implications for Proapoptotic Activity

Avicins are plant-derived triterpenoid stress metabolites that have both proapoptotic and cytoprotective properties. Avicins induce apoptosis in Jurkat T leukemia cells by targeting mitochondria and release of cytochrome c that occurs in a p53-independent manner. However, postmitochondrial antiapoptotic barriers, such as increased expression of heat shock proteins (Hsp) and X-linked inhibitor of apoptosis proteins (XIAP), frequently exist in cancer cells and often account for resistance to chemotherapy and a poor prognosis. In this article, we show the role of avicins in the activation of stress-regulated ubiquitination and degradation of Hsp70 and XIAP. This is the first report showing the regulation of Hsp70 via the ubiquitin/proteasome pathway. We also show the induction of E3α ubiquitin ligase in avicin-treated Jurkat T leukemia cells, and its involvement in the degradation of XIAP. Avicin-mediated suppression of Hsp70 and XIAP was further confirmed in other leukemic/lymphoma cell lines and freshly isolated peripheral blood lymphocytes from Sezary syndrome patients. No change in the Hsp70 and XIAP proteins was observed in peripheral blood lymphocytes from normal donors. We propose that the ability of avicins to induce ubiquitination and regulate the degradation of Hsp70 and XIAP in leukemia cells could have important implications in the treatment of drug-resistant neoplasia and inflammatory disorders.

Presence and induction of the enzyme NAD(P)H: Quinone oxidoreductase 1 in the central nervous system

NAD(P)H:quinone oxidoreductase 1 (NQO1) catalyzes a reductive detoxification that is thought to protect cells against the adverse effects of quinones and related compounds. NQO1 activity is present in all tissues. Absence of the enzyme produces abnormalities in the redox state and seizures, suggesting an important role of the protein in the central nervous system. Immunohistochemical analysis showed that the protein was found throughout the brain of the adult rat and mouse, with complete absence of the protein in brains from NQO1‐/‐ mice. NQO1 was not seen in any neuronal population, but was localized to Bergmann glial in the cerebellum and a subset of the oligodendrocytes throughout the brain. Prolonged seizures induced in adult rats with kainic acid resulted in an increase in activity of the enzyme throughout the brain, most prominently in the cerebellum, but immunoreactivity did not appear in neurons. Comparison of the axons in the corpus callosum from a wild‐type mouse to a knockout mouse showed that myelin is produced in the absence of NQO1, but there appears to be more small‐diameter axons in the knockout animal. These results suggest that NQO1 has a role in myelination in the central nervous system or in the insulating/wrapping function of glial cells. J. Comp. Neurol. 471:289–297, 2004.

CD123-Engager T Cells as a Novel Immunotherapeutic for Acute Myeloid Leukemia

Immunotherapy with CD123-specific T-cell engager proteins or with T cells expressing CD123-specific chimeric antigen receptors is actively being pursued for acute myeloid leukemia. T cells secreting bispecific engager molecules (ENG-T cells) may present a promising alternative to these approaches. To evaluate therapeutic potential, we generated T cells to secrete CD123/CD3-bispecific engager molecules. CD123-ENG T cells recognized primary acute myeloid leukemia (AML) cells and cell lines in an antigen-dependent manner as judged by cytokine production and/or tumor killing, and redirected bystander T cells to AML cells. Infusion of CD123-ENG T cells resulted in regression of AML in xenograft models conferring a significant survival advantage of treated mice in comparison to mice that received control T cells. At high effector to target ratios, CD123-ENG T cells recognized normal hematopoietic stem and progenitor cells (HSPCs) with preferential recognition of HSPCs from cord blood compared to bone marrow. We therefore introduced the CD20 suicide gene that can be targeted in vivo with rituximab into CD123-ENG T cells. The expression of CD20 did not diminish the anti-AML activity of CD123-ENG T cells, but allowed for rituximab-mediated ENG-T cell elimination. Thus, ENG-T cells coexpressing CD20 suicide and CD123 engager molecules may present a promising immunotherapeutic approach for AML.

Calnexin from Pisum sativum: cloning of the cDNA and characterization of the encoded protein.

A full-length cDNA of 1951 bp encoding a calnexin (CNX) protein was cloned from a Pisum sativum expression library. The open reading frame (ORF) within this cDNA encodes a 551-amino acid protein with a calculated molecular mass of 62.47 kDa that exhibits extensive homology with the CNX proteins from soybean (80%), Arabidopsis thaliana (70%), maize (70%), and dog (39%). The characteristic CNX signature motifs, KPEDWDE and GXW, generally found in molecular chaperones, are present in pea CNX (PsCNX), along with putative sites for Ca2+ binding and phosphorylation. In PsCNX, a signal sequence and a single transmembrane domain are also present at the N- and C-terminal ends, respectively. The PsCNX protein is expressed constitutively at the RNA level in vegetative and flowering tissues, as was evident from Northern analysis. Expression of PsCNX was light independent. In vitro translation of PsCNX cDNA yielded a 75-kDa precursor, which, in the presence of canine microsomal membranes, was cotranslationally processed into a 72.5-kDa product and was imported and localized to the endoplasmic reticulum. Trypsin treatment of the in vitro translated PsCNX in the presence of canine microsomes generated a further processed 67-kDa intraluminal form. The results with PsCNX also showed that the plant protein is a phosphoprotein containing phosphoserine residues, as evidenced by immunoprecipitation of PsCNX with anti-phosphoserine antibody. The PsCNX protein was also phosphorylated by endogenous kinases of pea microsomes.

Interleukin-6 levels predict event-free survival in pediatric AML and suggest a mechanism of chemotherapy resistance

The tumor microenvironment can protect cancer cells from conventional anticancer therapies. Thus, targeting these protective mechanisms could eradicate therapy-resistant cancer cells and improve outcomes. Interleukin-6 (IL-6) provides extrinsic protection for several solid tumors and multiple myeloma. In pediatric acute myeloid leukemia (AML), IL-6-induced STAT3 signaling frequently becomes stronger at relapse, and increases in IL-6-induced STAT3 activity are associated with inferior survival after relapse. These findings suggested that the IL-6-induced STAT3 pathway may promote chemotherapy resistance and disease progression. Thus, we investigated the dysregulation of IL-6 levels in the bone marrow niche in pediatric patients with AML and the association between IL-6 levels and outcome. We measured levels of over 40 cytokines and growth factors in plasma from diagnostic bone marrow aspirates of 45 pediatric AML patients and 7 healthy sibling controls. Of the measured cytokines, only IL-6 levels were associated with event-free survival. Importantly, the effect of elevated IL-6 was most striking among children classified as having a low risk of relapse. In these patients, 5-year event-free survival was 82.5% ± 11% for patients with low IL-6 levels at diagnosis (n = 14) compared with 17.3% ± 11% for patients with elevated IL-6 (n = 13, log-rank P = .0003). In vitro, exogenous IL-6 reduced mitoxantrone-induced apoptosis in cell lines and primary pediatric AML samples. These results suggest that IL-6 levels at diagnosis could be used to help identify children at high risk of relapse, particularly those who are otherwise classified as low risk by current algorithms. Moreover, the IL-6 pathway could represent a target for overcoming environment-mediated chemotherapy resistance.