ALBERT

Description: Abstract 3884 Signaling via the B-cell receptor (BCR) stimulates growth and survival of CLL leukemic cells and inhibits apoptosis by phosphorylating immunoreceptor tyrosine based activation motifs. This signaling subsequently activates PI3 Kinase/AKT, mTOR, ERK and other pathways. Activation of Akt in turn requires phosphorylation by mTOR kinase, which assembles in two complexes mTORC1 and mTORC2 and it is the mTORC2 complex that phosphorylates and activates Akt. This phosphorylation of Akt in CLL specimen's upregulates anti-apoptotic proteins such as Mcl-1, Bcl-xl and XIAP. We have identified that Rictor, a component of mTORC2 complex is over-expressed in CLL specimens as compared to normal peripheral mononuclear B cells. This over-expression was noted by real time PCR that showed 1.5 to 4 fold upregulation (n=12). Western blot analysis also showed Rictor overexpression in all the twelve CLL specimens tested. Rictor overexpression was also seen in Mantle cell lymphoma cell lines and to study its role in BCR signaling, stable Mantle cell lymphoma lines with SiRNA mediated Rictor knockdown were established. Rictor knockdown resulted in a significant decrease in Akt activation as phosphorylation (phospho S473) of Akt both in unstimulated cells and when the cells were stimulated with BCR crosslinking was decreased. To determine the effect of Rictor and mTORC2 inhibition on CLL specimens, we tested the activity of three compounds isolated via yeast two hybrid drug screen designed to identify molecules that inhibit Rictor/mTOR interaction. When tested on CLL specimens in the presence of BCR crosslinking, these mTORC2 inhibitor compounds inhibited the downstream phosphorylation of Akt S473. Functionally the inhibitors also induced apoptosis in CLL cells with 40–60% of CLL cells undergoing apoptosis (1.0mM, cells treated for 48 hours). In comparison, rapamycin (mTORC1 inhibitor) and ppp242 (mTORC1, 2 inhibitor) were comparatively less active in CLL specimens as they were less effective in the induction of apoptosis. Western blot analysis of mTORC2 inhibitor treated cells also showed PARP cleavage and an increase in the pro-apoptotic protein BAD. Our data indicates that Rictor overexpression in CLL specimens is required for Akt phosphorylation activation and downstream BCR signaling. Inhibition of this pathway by mTORC2 inhibitors in CLL will be an effective therapeutic strategy. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 926 The B-cell receptor (BCR) signaling is a hallmark of chronic lymphocytic leukemia cells and results in cell proliferation, survival and resistance to apoptosis. B cell receptor (BCR) signaling in CLL specimens activates the Ras/ERK/MAPK pathway and is critical for survival and growth of CLL cells. Rasgrf1 is a guanine exchange factor that accelerates the removal of inactive GDP from Ras so that active GTP can bind Ras that results in Ras activation. In an Affymetrix microarray expression analysis screen of CLL specimens (n=5) and peripheral blood normal B cells (n=2), Rasgrf1 was found to be over-expressed (ranked within top 100 overexpressed genes) in CLL. This finding was further confirmed by quantitative RT-PCR as a 5–300 fold overexpression in CLL specimens (n=20) was noted as compared to normal B cells. Western blot analysis data also showed over-expression of Rasgrf1 protein in CLL specimen lysates but interestingly the expressed protein is of a smaller size in CLL specimens (70-80 kDa) as compared to the Rasgrf1 in normal peripheral blood mononuclear cells (140 kDa) This truncated protein retains the important c-terminal GEF (guanine exchange factor) domain but lacks the regulatory domains and is known to function as a hyperactive GEF in other tumor model systems. Knockdown of Rasgrf1 in CLL specimens (n=4) resulted in inhibition of the BCR-Ras/ERK/MAPK pathway as determined by a decrease in ERK phosphorylation. Similar Rasgrf1 knockdown experiments with BCR crosslinking also showed identical results in additional CLL specimens. Transient SiRNA mediated Rasgrf1 knockdown also resulted in higher percentage of annexin positive or apoptotic CLL cells as compared to the scrambled SiRNA control transfection. Lysates from the Rasgrf1 knockdown cells showed an increase in PARP cleavage by western blot analysis as well indicating that continued expression of this protein was necessary for survival of the leukemic cells. To confirm that the Rasgrf1 knockdown resulted in a decrease in the GEF function, the active Ras (Ras-GTP) in the CLL specimens was evaluated by an ELISA assay. In two CLL specimens the assay confirmed that the active Ras in the CLL cells was decreased with Rasgrf1 knockdown. Overall the findings implicate a novel GEF protein, Rasgrf1 as an amplifier of BCR signaling in CLL cells. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1776 E-cadherin gene is aberrantly spliced in CLL cells. This results in a transcript that lacks exon 11 and is non-functional. As CLL specimens express higher proportion of these transcripts as compared to normal B cells, this aberrant splicing is a mechanism of E-cadherin inactivation that results in wnt pathway activation. To design ways to inhibit this aberrant splicing, we analyzed epigenetic histone modifications in CLL specimens as the histones are critically involved in gene transcription, exon definition and splicing. Our findings show that increasing histone acetylation by histone deacetylase inhibitors (HDACi) can increase the expression of correctly spliced transcript and at the same time reduce the aberrantly spliced transcript (exon 11 skipped) resulting in re-expression of this tumor suppressor gene. It is known that increase in histone acetylation upon HDACi exposure results in an open chromatin structure and an increase in transcription. Exposure of CLL specimens (n=10) to HDACi, MS275 (0.1-1μM for 24–48 hours) resulted in a 4–16 fold increase in E-cadherin expression with a decrease in the aberrant transcript (undetectable levels in 6 CLL specimens). Similar results were obtained with other HDACi, SAHA and Valproic acid and the increase in expression was also confirmed by western blot analysis. Chromatin immunoprecipitation (ChIP) experiments with a pan-acetyl H3 and H4 antibody confirms that the E-cadherin promoter and exon 11 region of this gene are indeed hypoacetylated as compared to PBMC and the treatment with HDAC inhibitor increases histone acetylation at both the promoter and exon 11 region. We investigated the wnt pathway as lack of E-cadherin expression is a mechanism of wnt pathway activation in CLL cells. Re-expression of E-cadherin by HDACi led to inhibition of this pathway as two downstream effectors, LEF and cyclinD1 genes were downregulated by real time PCR analysis indicating that this strategy could be used to inhibit this pathway in a clinical setting as well. Apoptosis was also observed in CLL specimens treated with HDACi. To analyze the global changes induced by histone modifications and their reversal by HDACi, we analyzed a defined gene set PCR ChIP promoter array. CLL specimens were compared to PBMC and also analyzed for the change in histone patterns upon HDAC inhibitor treatment. From this array a number of interesting candidate genes that are also affected by histone modifications were identified including E-cadherin, BAX, a bcl2 inhibitor and CDKN2B, a gene that inhibits activation of CDK kinases were hypoacetylated in CLL specimens as compared PBMC, however underwent hyperacetylation with HDACi treatment. BCL2 and NFKB1 promoters were hyperacetylated or transcriptionally active in CLL specimens. Besides the histone modifications on the ChIP array, the change in expression of these genes upon HDACi treatment was also confirmed by real time PCR analysis. With the E-cadherin gene as a model of histone epigenetic changes in CLL specimens, the studies highlight a signature pattern of these modifications in CLL cells. It is expected that these epigenetic histone changes will influence gene expression levels, RNA isoforms and splice variants and will have a role in CLL biology. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4582 High throughput sequencing of cellular mRNA provides a comprehensive analysis of the transcriptome. Besides identifying differentially expressed genes in different cell types, it also provides information of mRNA isoforms and splicing alterations. We have analyzed two CLL specimens and a normal peripheral blood B cells mRNA by this approach and performed data analysis to identify differentially expressed and spliced genes. The result showed CLLs specimens express approximately 40% more transcripts compared to normal B cells. The FPKM data (fragment per kilobase of exon per million) revealed a higher transcript expression on chromosome 12 in CLL#1 indicating the presence of trisomy 12, which was confirmed by fluorescent in-situ hybridization assay. With a two-fold change in FPKM as a cutoff and a p value cutoff of 0.05 as compared to the normal B cell control, 415 genes and 174 genes in CLL#1 and 676 and 235 genes in CLL#2 were up and downregulated or differentially expressed. In these two CLL specimens, 45% to 75% of differentially expressed genes are common to both the CLL specimens indicating that genetically disparate CLL specimens have a high percentage of a core set of genes that are potentially important for CLL biology. Selected differentially expressed genes with increased expression (selectin P ligand, SELPLG, and adhesion molecule interacts with CXADR antigen 1, AMICA) and decreased (Fos, Jun, CD69 and Rhob) expression based on the FPKM from RNA-sequencing data were also analyzed in additional CLL specimens by real time PCR analysis. The expression data from RNA-seq closely matches the fold-change in expression as measured by RT-PCR analysis and confirms the validity of the RNA-seq analysis. Interestingly, Fos was identified as one of the most downregulated gene in CLL. Using the Cufflinks and Cuffdiff software, the splicing patterns of genes in CLL specimens and normal B cells were analyzed. Approximately, 1100 to 1250 genes in the two CLL specimens were significantly differentially spliced as compared to normal B cells. In this analysis as well, there is a core set of 800 common genes which are differentially spliced in the two CLL specimens. The RNA-sequencing analysis accurately identifies differentially expressed novel genes and splicing variations that will help us understand the biology of CLL. Disclosures: No relevant conflicts of interest to declare.

Description: Recent reports indicate that human herpesvirus (HHV)-6 reactivation occurs in 40–65% of patients undergoing hematopoietic stem cell transplantation (HSCT).But the complication after HSCT that predispose to HHV-6 viremia are not well characterized. The aim of our research is to study the potential relationship between HHV-6 activation and acute graft-versus-host disease (aGVHD) after hematopoietic cell transplantation (HSCT) in chinese patients. Peripheral blood samples were collected before and weekly after HSCT from 40 consecutive recipients who underwent HSCT between March 2005 and Joungry 2007 (2 autologous and 38 allogeneic transplants) .HHV-6 DNAemia was monitored by real-time PCR. The genotypes of HHV-6 were identified by Hind III restriction assay. Of the 40 patients, HHV-6 DNAemia were detected in only 1 patient (2.5%) before HSCT, and the viral load was 420 copies/ml.After HSCT there were 18(45%) patients detected HHV-6 DNA on a median of day 14.5 (range, 0– 23 days), and the median HHV-6 viral load of 4884.4±374.4 copies/ml (range, 282 – 43400 copies/ml). Respectively, HHV-6B was identified as the predominant variant.Grade I – IV aGHVD occurred in 18 (45%) on a median of day 20 (range, 8–40 days). The median onset time of HHV-6 DNAemia was significantly earlier than that of aGHVD (P