Sanjai Sharma

Gene transfer of CD40-ligand induces autologous immune recognition of chronic lymphocytic leukemia B cells.

CD40-CD40-ligand (CD154) interactions play a critical role in immune activation. Using replication defective adenovirus encoding mouse CD154 (Ad-CD154), we modified human chronic lymphocytic leukemia B cells to express a functional ligand for CD40. This not only induces expression of immune accessory molecules on the infected cell, but also allows it to trans-activate noninfected bystander leukemia B cells. Also, factors that impair the antigen-presenting capacity of leukemia B cells are downmodulated. Ad-CD154- infected leukemia cells are highly effective stimulators in mixed lymphocyte reactions and can induce generation of cytotoxic T lymphocytes specific for autologous nonmodified leukemia cells. As such, Ad-CD154 can induce a host antileukemia response that may have therapeutic potential.

Mechanism by Which Mammalian Target of Rapamycin Inhibitors Sensitize Multiple Myeloma Cells to Dexamethasone-Induced Apoptosis

Mammalian target of rapamycin (mTOR) inhibitors curtail cap-dependent translation. However, they can also induce post-translational modifications of proteins. We assessed both effects to understand the mechanism by which mTOR inhibitors like rapamycin sensitize multiple myeloma cells to dexamethasone-induced apoptosis. Sensitization was achieved in multiple myeloma cells irrespective of their PTEN or p53 status, enhanced by activation of AKT, and associated with stimulation of both intrinsic and extrinsic pathways of apoptosis. The sensitizing effect was not due to post-translational modifications of the RAFTK kinase, Jun kinase, p38 mitogen-activated protein kinase, or BAD. Sensitization was also not associated with a rapamycin-mediated increase in glucocorticoid receptor reporter expression. However, when cap-dependent translation was prevented by transfection with a mutant 4E-BP1 construct, which is resistant to mTOR-induced phosphorylation, cells responded to dexamethasone with enhanced apoptosis, mirroring the effect of coexposure to rapamycin. Thus, sensitization is mediated by inhibition of cap-dependent translation. A high-throughput screening for translational efficiency identified several antiapoptotic proteins whose translation was inhibited by rapamycin. Immunoblot assay confirmed rapamycin-induced down-regulated expressions of XIAP, CIAP1, HSP-27, and BAG-3, which may play a role in the sensitization to apoptosis. Studies in a xenograft model showed synergistic in vivo antimyeloma effects when dexamethasone was combined with the mTOR inhibitor CCI-779. Synergistic effects were associated with an enhanced multiple myeloma cell apoptosis in vivo. This study supports the strategy of combining dexamethasone with mTOR inhibitors in multiple myeloma and identifies a mechanism by which the synergistic effect is achieved.

Involvement of Hepcidin in the Anemia of Multiple Myeloma

Purpose: Hepcidin is a liver-produced peptide implicated in the anemia of inflammation. Because interleukin (IL)-6 is a potent inducer of hepcidin expression and its levels are elevated in multiple myeloma, we studied the role of hepcidin in the anemia of multiple myeloma. Experimental Design: Urinary hepcidin and serum levels of IL-6, ferritin, C-reactive protein, tumor necrosis factor-α, and IL-1β were studied in newly diagnosed myeloma patients. In vitro hepcidin induction assay was assessed by real-time PCR assay. Results: Pretreatment urinary hepcidin levels in 44 patients with stage III multiple myeloma were 3-fold greater than normal controls. In the subset of multiple myeloma patients without renal insufficiency (n = 27), a marked inverse correlation was seen between hemoglobin at diagnosis and urinary hepcidin level (P = 0.014) strongly supporting a causal relationship between up-regulated hepcidin expression and anemia. The urinary hepcidin also significantly (P < 0.05) correlated with serum ferritin and C-reactive protein, whereas its correlation with serum IL-6 levels was of borderline significance (P = 0.06). Sera from 14 multiple myeloma patients, with known elevated urinary hepcidin, significantly induced hepcidin mRNA in the Hep3B cells, whereas normal sera had no effect. For 10 patients, the ability of anti-IL-6 and anti-IL-6 receptor antibodies to prevent the serum-induced hepcidin RNA was tested. In 6 of these patients, hepcidin induction was abrogated by the anti-IL-6 antibodies, but in the other 4 patients, the neutralizing antibodies had no effect. Conclusions: These results indicate hepcidin is up-regulated in multiple myeloma patients by both IL-6-dependent and IL-6-independent mechanisms and may play a role in the anemia of multiple myeloma.

Interleukin-6 activates phosphoinositol-3' kinase in multiple myeloma tumor cells by signaling through RAS-dependent and, separately, through p85-dependent pathways.

The IL-6-induced activation of the phosphatidylinositol-3′ kinase (PI3-K)/AKT cascade in multiple myeloma (MM) cells is critical for tumor cell proliferation and viability. Since the IL-6 receptor does not contain binding sites for the p85 regulatory portion of PI3-K, intermediate molecules must play a role. Coimmunoprecipitation studies in MM cell lines demonstrated the IL-6-induced formation of two independent PI3-K-containing complexes: one containing p21 RAS but not STAT-3 and a second containing STAT-3 but not RAS. Both complexes demonstrated IL-6-induced lipid kinase activity. IL-6 also generated kinase activity in a mutant p110 molecule that could not bind p85. Use of dominant-negative (DN) constructs confirmed the presence of two independent pathways of activation: a DN RAS prevented the IL-6-induced generation of lipid kinase activity in the mutant p110 molecule but had no effect on activity generated in the STAT-3-containing complex. In contrast, a DN p85 prevented the generation of kinase activity in the STAT-3-containing complex but had no effect on activity generated in the p110 molecule. Both DN constructs significantly prevented the IL-6-induced activation of AKT. MM cells expressing activating RAS mutations demonstrated enhanced IL-6-independent growth and constitutive PI3-K activity. These data indicate two potential independent pathways of PI3-K/AKT activation in MM cells: one mediated via signaling through RAS which is independent of p85 and a second mediated via p85 and due to a STAT-3-containing complex.

IL-6-Induced Stimulation of c-Myc Translation in Multiple Myeloma Cells Is Mediated by Myc Internal Ribosome Entry Site Function and the RNA-Binding Protein, hnRNP A1

Prior work indicates that c-myc translation is up-regulated in multiple myeloma cells. To test a role for interleukin (IL)-6 in myc translation, we studied the IL-6-responsive ANBL-6 and IL-6-autocrine U266 cell lines as well as primary patient samples. IL-6 increased c-myc translation, which was resistant to rapamycin, indicating a mechanism independent of mammalian target of rapamycin (mTOR) and cap-dependent translation. In contrast, the cytokine enhanced cap-independent translation via a stimulatory effect on the myc internal ribosome entry site (IRES). As known IRES-trans-activating factors (ITAF) were unaffected by IL-6, we used a yeast-three-hybrid screen to identify novel ITAFs and identified hnRNP A1 (A1) as a mediator of the IL-6 effect. A1 specifically interacted with the myc IRES in filter binding assays as well as EMSAs. Treatment of myeloma cells with IL-6 induced serine phosphorylation of A1 and increased its binding to the myc IRES in vivo in myeloma cells. Primary patient samples also showed binding between A1 and the IRES. RNA interference to knock down hnRNP A1 prevented an IL-6 increase in myc protein expression, myc IRES activity, and cell growth. These data point to hnRNP A1 as a critical regulator of c-myc translation and a potential therapeutic target in multiple myeloma.

Separable Mechanisms of Attachment and Cell Uptake during Retrovirus Infection

ABSTRACT In the absence of viral envelope gene expression, cells expressing human immunodeficiency virus type 1 (HIV-1) gag andpol, accessory HIV functions, and a vector genome RNA produce and secrete large amount of noninfectious virus-like particles (VLPs) into the conditioned medium. After partial purification, such HIV-1 VLPs can be made infectious in cell-free conditions in vitro by complex formation with lipofection reagents or with the G protein of vesicular stomatitis virus (VSV-G). The resulting in vitro-modified HIV-1 particles are able to infect nondividing cells. Infectivity of envelope-free HIV VLPs can also be induced by prior modification of target cells through exposure to partially purified VSV-G vesicles. Similarly, infection can be carried out by attachment of envelope-free noninfectious VLPs to unmodified cells followed by subsequent treatment of cells with VSV-G. We interpret these findings to indicate that interaction between a viral envelope and a cell surface receptor is not necessary for the initial virus binding to the cells but is required for subsequent cell entry and infection.

Aberrant splicing of the E-cadherin transcript is a novel mechanism of gene silencing in chronic lymphocytic leukemia cells

Premature termination codon (PTC) mutations are due to insertion or deletion of nucleotides causing a frameshift and premature termination codon in RNA. These transcripts are degraded by the nonsense-mediated decay pathway and have a very short half-life. We used a microarray technique to screen for genes that up-regulate their RNA signal upon nonsense-mediated decay pathway blockade in chronic lymphocytic leukemia (CLL) specimens and identified an E-cadherin transcript with PTC. Sequencing revealed an aberrant E-cadherin transcript lacking exon 11, resulting in a frameshift and PTC. The aberrant E-cadherin transcript was also identified in normal B cells, but occurred at a much lower level compared with CLL cells. In CLL specimens, E-cadherin expression was depressed more than 50% in 62% cases (relative to normal B cells). By real-time polymerase chain reaction analysis, the relative amounts of wild-type transcript inversely correlated with amounts of aberrant transcript (P = .018). Ectopic expression of E-cadherin in CLL specimens containing high amounts of aberrant transcript resulted in down-regulation of the wnt-beta-catenin pathway reporter, a pathway known to be up-regulated in CLL. Our data point to a novel mechanism of E-cadherin gene inactivation, with CLL cells displaying a higher proportion of aberrant nonfunctional transcripts and resulting up-regulation of the wnt-beta-catenin pathway.

Exon 11 Skipping of E-Cadherin RNA Downregulates Its Expression in Head and Neck Cancer Cells

E-cadherin is an important tumor suppressor gene whose expression is lost when cells acquire a metastatic phenotype. We analyzed the role of E-cadherin missplicing as a mechanism of its downregulation by analyzing a misspliced E-cadherin transcript that lacks exon 11 of this gene. This results in a frameshift and a premature termination codon that targets this transcript for degradation. Tumor tissues, including breast (20%, n = 9), prostate (30%, n = 9) and head and neck (75%, n = 8) cancer, express the exon 11-skipped transcripts (vs. nonmalignant controls) and its levels inversely correlate with E-cadherin expression. This is a novel mechanism of E-cadherin downregulation by missplicing in tumor cells, which is observed in highly prevalent human tumors. In the head and neck cancer model, nontumorigenic keratinocytes express exon 11–skipped splice product two- to sixfold lower than the head and neck tumor cell lines. Mechanistic studies reveal that SFRS2 (SC35), a splicing factor, as one of the regulators that increases missplicing and downregulates E-cadherin expression. Furthermore, this splicing factor was found to be overexpressed in 5 of 7 head and neck cell lines and primary head and neck tumors. Also, methylation of E-cadherin gene acts as a regulator of this aberrant splicing process. In 2 head and neck cell lines, wild-type transcript expression increased 16- to 25-folds, whereas the percentage of exon 11-skipped transcripts in both the cell lines decreased five- to 30-folds when cells were treated with a hypomethylating agent, azacytidine. Our findings reveal that promoter methylation and an upregulated splicing factor (SFRS2) are involved in the E-cadherin missplicing in tumors. Mol Cancer Ther; 10(9); 1751–9. ©2011 AACR.

E-cadherin gene re-expression in chronic lymphocytic leukemia cells by HDAC inhibitors

BackgroundThe tumor suppressor gene E-cadherin gene is frequently silenced in chronic lymphocytic leukemia (CLL) cells and results in wnt-pathway activation. We analyzed the role of histone epigenetic modifications in E-cadherin gene silencing.MethodsCLL specimens were treated with histone deacetylase inhibitor (HDACi) MS-275 and analyzed for E-cadherin expression with western blot and RT-PCR analysis. The downstream effects of HDACi treated leukemic cells were studied by analyzing the effect on wnt-pathway signaling. HDACi induced alterations in E-cadherin splicing were investigated by transcript specific real time PCR analysis.ResultsTreatment of CLL specimens with histone deacetylase inhibitors (HDACi) treatment resulted in an increase of the E-cadherin RNA transcript (5 to 119 fold increase, n=10) in eight out of ten CLL specimens indicating that this gene is down regulated by histone hypoacetylation in a majority of CLL specimens. The E-cadherin re-expression in CLL specimens was noted by western blot analysis as well. Besides epigenetic silencing another mechanism of E-cadherin inactivation is aberrant exon 11 splicing resulting in an alternatively spliced transcript that lacks exon 11 and is degraded by the non-sense mediated decay (NMD) pathway. Our chromatin immunoprecipitation experiments show that HDACi increased the acetylation of histones H3 and H4 in the E-cadherin promoter region. This also affected the E-cadherin exon 11 splicing pattern as HDACi treated CLL specimens preferentially expressed the correctly spliced transcript and not the exon 11 skipped aberrant transcript. The re-expressed E- cadherin binds to β-catenin with inhibition of the active wnt-beta-catenin pathway in these cells. This resulted in a down regulation of two wnt target genes, LEF and cyclinD1 and the wnt pathway reporter.ConclusionThe E-cadherin gene is epigenetically modified and hypoacetylated in CLL leukemic cells. Treatment of CLL specimens with HDACi MS-275 activates transcription from this silent gene with expression of more correctly spliced E-cadherin transcripts as compared to the aberrant exon11 skipped transcripts that in turn inhibits the wnt signaling pathway. The data highlights the role of epigenetic modifications in altering gene splicing patterns.

Amplification of B cell receptor–Erk signaling by Rasgrf-1 overexpression in chronic lymphocytic leukemia

Abstract Rasgrf-1 is a guanine exchange factor (GEF) that catalyzes the exchange of GDP for GTP. In a RNA microarray analysis of chronic lymphocytic leukemia (CLL) specimens (n = 5), this gene was found to be overexpressed in CLL as compared to normal peripheral blood mononuclear cell (PBMC) CD19 + B cells (n = 3). CLL specimens (n = 29) expressed Rasgrf-1 RNA at levels 5–300-fold higher as compared to normal B cells. CLL specimens expressed a 75 kDa isoform that was smaller than the expected full-length protein (140 kDa) and the truncated variant had higher GEF activity. Knockdown of Rasgrf-1 in CLL specimens inhibited active GTP-bound Ras and the Ras/Erk/mitogen-activated protein kinase (MAPK) pathway. Rasgrf-1 was phosphorylated and activated by B cell receptor (BCR) signaling that increased its GEF function, and this phosphorylation was blocked by Src and Brutons tyrosine kinase (BTK) inhibitors. Rasgrf-1 is a novel GEF protein that has a role in BCR signaling and its overexpression further activates the Ras/Erk/MAPK pathway in CLL specimens.