SummaryEmerging evidence indicates that the tumour microenvironment (TME) regulates the behaviour of chronic lymphocytic leukaemia (CLL). However, the precise mechanism and molecules involved in this process remain unknown. Gene expression profiles of CLL cells from lymph node (LN), bone marrow (BM) and peripheral blood (PB) indicate overexpression of a tolerogenic signature in CLL cells in lymph nodes (LN-CLL). Based on their role in B cell biology, the progression of CLL, or immune regulation, a few genes of this 83-gene signature were selected for further analyses. We observed a significant correlation between the clinical outcomes and the expression of CAV1 (P = 0·041), FGFR1 isoform 8 (P = 0·032), PTPN6 (P = 0·031) and ZWINT (P < 0·001). CAV1, a molecule involved in the regulation of tumour progression in other cancers, was seven-fold higher in LN-CLL cells compared to BM-and PB-CLL cells. Knockdown of CAV1 expression in CLL cells resulted in significantly decreased migration (P = 0·016) and proliferation (P = 0·04). When CAV1 was knocked down in B and T cell lines, we observed an inability to form immune synapses. Furthermore, CAV1 knockdown in CLL cells impaired their ability to form immune synapses with autologous T lymphocytes and allogeneic, healthy T cells. Subsequent analyses of microarray data showed differential expression of cytoskeletal genes, specifically those involved in actin polymerization. Therefore, we report a novel role for CAV1 in tumour-induced immunosuppression during the progression of CLL.
Smoking is an established risk factor for pancreatic cancer (PC), but late diagnosis limits the evaluation of its mechanistic role in the progression of PC. We used a well-established genetically engineered mouse model (LSL-K-rasG12D) of PC to elucidate the role of smoking during initiation and development of pancreatic intraepithelial neoplasia (PanIN). The 10-week-old floxed mice (K-rasG12D; Pdx-1cre) and their control unfloxed (LSL-K-rasG12D) littermates were exposed to cigarette smoke (total suspended particles: 150 mg/m3) for 20 weeks. Smoke exposure significantly accelerated the development of PanIN lesions in the floxed mice, which correlated with tenfold increase in the expression of cytokeratin19. The systemic accumulation of myeloid-derived suppressor cells (MDSCs) decreased significantly in floxed mice compared with unfloxed controls (P<0.01) after the smoke exposure with the concurrent increase in the macrophage (P<0.05) and dendritic cell (DCs) (P<0.01) population. Further, smoking-induced inflammation (IFN-γ, CXCL2; P<0.05) was accompanied by enhanced activation of pancreatic stellate cells and elevated levels of serum retinoic acid-binding protein 4, indicating increased bioavailability of retinoic acid which contributes to differentiation of MDSCs to tumor-associated macrophages (TAMs) and DCs. TAMs predominantly contribute to the increased expression of heparin-binding epidermal growth factor-like growth factor (EGFR ligand) in pre-neoplastic lesions in smoke-exposed floxed mice that facilitate acinar-to-ductal metaplasia (ADM). Further, smoke exposure also resulted in partial suppression of the immune system early during PC progression. Overall, the present study provides a novel mechanism of smoking-induced increase in ADM in the presence of constitutively active K-ras mutation.
1243 Poster Board I-265 Chronic lymphocytic leukemia (CLL) is the most common adult leukemia and is clinically very heterogeneous. Following diagnosis, some patients do not require treatment for several years whereas others have a more aggressive disease thus requiring immediate treatment. Understanding the molecular basis of clinical heterogeneity in CLL will enhance our ability to treat this presently incurable disease effectively. CLL cells in the patient body proliferate/survive for a long time resulting in their accumulation in bone marrow (BM), lymph nodes (LN), and blood (PB). However, CLL cells do not survive for a long time once they are removed from the body, suggesting that an in vivo microenvironment provides essential proliferation/survival signals to CLL cells. Therefore, to elucidate the precise role of microenvironments on the CLL cell proliferation/survival and migration, in this study, we have analyzed CLL cells from PB (n=20), BM (n=18), and LN (n=15) from patients for their gene expression profiles using microarray. Differentially expressed genes and their associated cellular pathways were identified using significant analysis of microarray (SAM) and gene set enrichment analyses (GSEA). Among the six pathways/gene expression signatures identified (BCR-, BAFF/April-, NFκB-, PI3K/Akt, cytokine-, and tolerogenic) the most significant pathways in CLL biology are the BCR-, NFκB-, PI3K/Akt pathways and tolerogenic signature associated genes with immune dysregulation particularly with CLL cells from LN. We have already reported the differential expression of CLL cell proliferation and survival related genes belonging to BCR and NFαB pathways (Mittal et al, 2008 Blood-ASH Annual Meeting Abstract 546, page 112). In this report we have focused on differentially expressed genes associated with the tolerogenic signature and PI3K/Akt pathway. Among the eighty-three differentially expressed genes in the tolerogenic signature, based on their known role in immune regulation and/or level of significant expression comparing CLL cells from PB or BM, a few selected genes were further studied to understand their possible role in clinical heterogeneity of CLL. These genes are: CAV1, CD47, CCNB2, IL2Rαa, FOXP3, ZWINT, TGFβR1, IL22, IL10Rαa, INDO, APC, and STAT1. There was a significant increase in the expression of CD47, IL-10Rαa, CAV1, APC, CCNB2, and STAT1 in the LN cells from CLL patients; whereas the expression of IL-22R was decreased in the LN cells. These genes have been shown to be associated with immunosuppression indicating a lack of immune response against CLL in the lymph node. In addition, MAPK pathway associated genes are known to increase the survival/proliferation of tumor cells, including CLL cells. Specifically, genes associated with PI3K/Akt pathway, a part of MAPK pathways are also overexpressed in CLL cells from LN that includes AKT, 4E-BP1, PSMC4 and PDK1 genes indicating the importance of PI3K/Akt pathway in proliferation/survival of CLL cells in LN microenvironment. Based on these results, we hypothesize that differentially expressed genes belonging to the tolerogenic signature in LN of CLL patients down regulate the immune response against CLL, thus leading to enhanced disease progression whereas, overexpressed proliferation/survival-related genes belong to PI3K/Akt pathway promote proliferation/survival of CLL cells. (This work was supported by the CLL Foundation, Houston, TX and National Institutes of Health, Bethesda, MD, INBRE Grant # P20 RR016469). Disclosures No relevant conflicts of interest to declare.
1367 Previously, we have shown that gene expression profiles (GEP) of CLL cells from lymph nodes (LN), bone marrow (BM), and peripheral blood (PB) are significantly different from each other. Among the major pathways associated with differential gene expression, a “tolerogenic signature” involved in host immune tolerance is significant in regulating CLL progression. The genes associated with the tolerogenic signature are significantly differentially expressed in patient LN-CLL compared to BM-CLL and PB-CLL, suggesting that LN-CLL cells induce this immune tolerance. From 83 differentially expressed genes identified by GEP that are associated with immune dysregulation, we selected eleven genes (CAV1, PTPN6, PKCb, ZWINT, IL2Ra, CBLC, CDC42, ZNF175, ZNF264, IL10, and HLA-G) for validation studies to determine whether these genes are also dysregulated in the Emu-TCL1 mouse model of CLL. The results demonstrate a trend of upregulation of these genes as determined by qRT-PCR in the LN-tumor microenvironment. To further evaluate the kinetics of selected gene expression during tumor progression, we determined the expression levels of Cav1, Ptpn6, and Pkcb at 12, 24, and 36 weeks of CLL development in the Em-TCL1 mouse model. We found that the expression of all three genes increased as a function of age, indicating a correlation of gene expression with disease progression. In addition, as CLL progressed in these mice there was a marked decrease in CD4+ and CD8+ T cells. The murine data were further validated using CLL cells from the same patients with indolent versus aggressive disease indicating a similar trend in expression as CLL progressed (n=4). Furthermore, patient data analyzed by Kaplan Meier analyses of the expression levels of the selected genes indicated a significant association between down-regulation of PTPN6 (p=0.031) and up-regulation of ZWINT (p<0.001) with clinical outcome as determined by a shorter time to treatment (p<0.05). Functional analysis by knockdown of CAV1 and PKCb in primary patient CLL cells determined by MTT assay showed a decrease in proliferation following knockdown of these genes (p<0.005). Protein-interaction modeling revealed regulation of CAV1 and PTPN6 by one another. Additionally, the PTPN6 protein regulates B cell receptor (BCR) signaling and subsequently the BCR regulates PKCb. Therefore, these data from both mice and humans with CLL, argue that an aggressive disease phenotype is paralleled by expression of genes associated with immune suppression. In particular, evidence presented here suggests, dysregulation of CAV1, PTPN6, ZWINT, and PKCb expression promotes CLL progression. Disclosures: No relevant conflicts of interest to declare.
The intent of this study was to characterize the effect OTK18 upregulation in monocytic cells had on neuronal survival. The human monocytic cell line, U937, was differentiated into macrophages or left as an undifferentiated monocyte. These cells were transfected with a plasmid containing the enhanced green fluorescent protein and OTK18 (pEGFP-OTK18) or an empty control vector (pEGFP-N3). The supernatants from the transfected U937 cells were used to culture rat neuronal cells (PC12). A live/dead assay was performed to determine the effect of culturing on cell survival. The protein levels of the neurotoxin, tumor necrosis factor alpha (TNF-alpha), and the neurotrophin, neurotrophin three (NT3), were determined by enzyme linked immunosorbent assay. The results of the live/dead assay showed differential cell survival between conditions with pEGFP-OTK18 when compared to the control empty vector. Quantitative real-time polymerase chain reaction assays demonstrated that OTK18 had an increased expression level when compared to the control. Lastly, NT3 protein levels were upregulated in treated cells with increased OTK18 expression, suggesting that OTK18 may play a role in neurotrophin production and consequently support neuronal survival.
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