Littérature scientifique sur le sujet « Bcatenin pathway »

Abstract Abstract 2958 Poster Board II-934 The interferon consensus sequence binding protein (ICSBP) is an interferon regulatory transcription factor (also referred to as IRF8). Similar to other IRF proteins, ICSBP regulates transcription of genes involved in the inflammatory response. However, ICSBP also functions as a myeloid leukemia tumor suppressor. Specifically, decreased ICSBP expression in myeloid progenitor cells results in cytokine hypersensitivity and resistance to apoptosis in response to Fas-activation or IL3 withdrawal. Consistent with function as a tumor suppressor, decreased ICSBP-expression is found in the bone marrow of human subjects with chronic myeloid leukemia (CML). Expression of ICSBP increases during remission and decreasing ICSBP expression is associated with progression to CML blast crisis (BC). In murine models, ICSBP-deficiency induces a myeloproliferative disorder (MPD) which resembles CML. And, ICSBP overexpression blocks MPD in mice transplanted with Bcr/abl expressing bone marrow. Increased Bcatenin activity is also associated with CML-BC. However, the impact of ICSBP on Bcatenin expression or activity has not been previously investigated. In these studies, we hypothesized that ICSBP-deficiency in CML-BC increases Bcatenin-expression. In support of this hypothesis, we found that Bcatenin protein and activity were increased in myeloid cell lines with ICSBP-knock-down and decreased in cells with ICSBP-overexpression. Bcatenin protein and activity were also increased in primary myeloid progenitors from ICSBP-/- mice in comparison to wild type or ICSBP+/- mice, in an ICSBP-dose dependent manner. Expression of Bcr/abl decreased ICSBP-expression in myeloid cells and increased Bcatenin protein and activity. Also supporting our hypothesis, the effect of Bcr/abl on Bcatenin was abolished by re-expression of ICSBP in Bcr/abl expressing myeloid cell lines or primary murine myeloid progenitor cells. In each of these situations, the increase in Bcatenin protein was not due to increased expression of Bcatenin mRNA. These results suggest ICSBP influences expression of target genes involved in Bcatenin protein stability. Therefore, these studies identify a pathway by which Bcr/abl activity impairs ICSBP expression in immature myeloid cells, thereby increasing stability of Bcatenin protein and Bcatenin activity. Poor prognosis and BC are associated with Fas resistance and increased Bcatenin activity in CML leukemia stem cells (LSC). Bcr/abl activity is also associated with decreased expression of ICSBP. Our prior studies implicated ICSBP-deficiency in Fas-resistance in CML via the ICSBP target gene PTPN13. Our current studies implicate ICSBP-deficiency in increased Bcatenin expression in CML. These results suggest that decreased ICSBP expression drives multiple aspects of the poor prognosis LSC phenotype in CML. Disclosures: No relevant conflicts of interest to declare.

e22046 Background: APC mutations are believed to constitutively activate the wnt pathway in colorectal tumors. Our goal was to comprehensively characterize Wnt signaling components in a set of APC-associated FAP tumors both at the DNA and RNA levels. Methods: Sixty adenomas from FAP cases harboring pathogenic APC mutations were included (10 adenomas and 1 normal mucosa per case). Somatic APC and KRAS alterations, β-catenin immunostaining and qRT-PCR of APC, MYC, AXIN2 and SFRP1 were analyzed. aCGH was also assessed in 26 FAP adenomas and 24 additional paired normal-adenoma-carcinoma samples. Results: A second APC alteration was present in 30 (50%) of adenomas (26 LOH and 4 point mutations). LOH was only occasionally associated with loss of genetic material. In 3 of 6 cases APC mRNA was overexpressed in macroscopically normal mucosa. In these cases diminished APC levels mRNA were observed in 11 of 30 adenomas analyzed.. In the remaining 3 cases APC mRNA was already underexpressed in normal mucosa with only 3 of 30 adenomas showing further underexpression. In FAP normal mucosae MYC was overexpressed (logratio range: 3.37–5.66). All adenomas showed further MYC (logratio range: 1.78–2.92) and AXIN2 overexpression (logratio range: 1.62–4.65), corregulating with APC mRNA levels (r=0.31 for MYC; r=0.59 for AXIN2). β-catenin nuclear immunostaining was detected in 80% of adenomas correlating with MYC mRNA levels. SFRP1 was underexpressed in all tumors (logratio range= -3.06–27). While copy number changes were rare in adenomas (median number :2.5; range 0–5). DNA changes were more often detected in areas containing wnt genes when FAP and sporadic tumors were jointly analyzed (p=0.02). Conclusions: Wnt pathway is altered, at the DNA and/or RNA level, in all APC mutant FAP tumors. MYC overexpression is a universal event that correlates with APC and AXIN2 expression levels as well as bcatenin nuclear accumulation. No significant financial relationships to disclose.

Abstract Introduction: Aggressive histologic subtypes of lymphoma such as mantle cell (MCL) and activated B cell (ABC) are considered incurable and affected patients often have a short median survival despite multimodal therapy. It is well established that altered expression of oncogenes and epigenetic dysregulation of tumor suppressor and regulatory genes promote cellular transformation of normal B cells into malignant lymphoma. Hypermethylation of histone proteins (H3R8 and H4R3) by the protein arginine methyltransferase 5 (PRMT5) enzyme has been documented in multiple cancer types and has been shown to promote tumor cell growth and survival. Importantly, PRMT5 over expression does not occur in normal B cells (resting or activated) and is only detected in malignant lymphoma cells. We have previously shown that PRMT5 regulates the Polycomb-repressive complex 2 (PRC2) complex including EZH2, a core histone-lysine N methyl transferase. EZH2 has tumor suppressor functions and has been shown to regulate WNT antagonist’s gene expression. WNT/β-CATENIN signaling pathway has been associated with increased cell proliferation and survival in various forms of cancers including lymphoma. Until recently, the role of PRMT5 in controlling WNT/β-CATENIN signaling has been unclear. We hypothesized that PRMT5, through its ability to repress EZH2 expression, would control WNT/β-CATENIN signaling and orchestrate downstream pathways that are relevant to lymphomagenesis. Methods: PRMT5 inhibition of patient-derived lymphoma cell lines, primary lymphoma tumor cells and mouse primary Eμ-BRD2 transgenic lymphoma cells by infecting with sh-PRMT5 lentivirus (or sh-GFP control) or a selective small molecule PRMT5 inhibitor (tool compound CMP5). Gene expression was monitored by immunoblotting and reverse transcription (RT) real time PCR. Recruitment of target proteins to promoter regions was examined by ChIP assays. To evaluate PRMT5 and WNT antagonist expression in NHL patient samples, primary tumor samples were collected from 4 patients with MCL. Cellular growth and apoptosis was assessed by proliferation assay and FACS analysis. Results: PRMT5 supports WNT/β-CATENIN activity by direct transcriptional repression of AXIN2 and WIF1 via a PRMT5-EZH2 repressor complex. PRMT5 indirectly supports EZH2 expression via inactivation of the RB-E2F pathway. AXIN2 and WIF1 are two proteins that negatively regulate WNT/bCATENIN. Additionally, PRMT5 inhibition with shRNA or CMP5 leads to repression of the WNT/β-CATENIN signaling pathway by allowing de-repression of AXIN2 and WIF1, leading to decreased nuclear phospho-b-CATENIN and decreased transcription of the target genes CYCLIN D1, c-MYC and SURVIVIN. Reduced nuclear localization of phospho-β-catenin (S675) led to differential enhanced recruitment of co-repressors LSD and HDAC2 (and loss of epigenetic marks H3K4Me3 and H3K9Me3) and loss of activating epigenetic marks H3K9Ac and H3K14Ac on CYCLIN D1, c-MYC and SURVIVIN promoters. We also found that PRMT5 regulates target gene repression in primary blastic variant MCL patient samples and mouse primary lymphoma tumor cells. Significance: Our observations show that PRMT5 is an important epigenetic regulator that governs the expression of its own target genes, the PRC2 program as well as regulating the WNT/β-CATENIN-driven pro-growth and survival genes c-MYC, CYCLIND D1 and SURVIVIN. These results, together with the prevalence of PRMT5 and EZH2 over expression and activation of WNT targets in multiple lymphoma histologic subtypes, suggests that inhibiting PRMT5 is likely to result in removal of repressive histone arginine and lysine marks and promote restoration of normal growth and survival checkpoints in malignant lymphomas. Disclosures No relevant conflicts of interest to declare.

Abstract Endothelial cells are relevant to normal hematopoiesis. Vascular niches within the bone marrow allow hematopoietic stem cell proliferation and differentiation. In vivo, the contemporary administration of endothelial progenitor cells in BALB/c mice fosters the homing of transplanted hematopoietic cells (Salter et al. Blood 2009; 113:2104). In vitro, the differentiation of CD34+ progenitors is facilitated by the presence of an underlying confluent layer of endothelial colony forming cells (ECFCs Ingram et al. Blood. 2004;104: 2752), even though this effect is highly dependent from the physical contact between hematopoietic and endothelial cells (Teofili et al, Blood , ASH Annual Meeting, 2012;120:1718). In Myelodysplastic Syndromes (MDS) the dysfunction of vascular niches might contribute to cause the ineffective hematopoiesis typical of the disease. Accordingly, we found that normal CD34+ cells cultured over ECFCs isolated from patients with low risk MDS exhibit a deep perturbation of the expression of lineage-specific genes, with higher expression of genes involved in the early differentiation (such as PU.1 and RUNX1) and lower expression of genes usually up-regulated during the late differentiation (such as MPO and GP1b) (Teofili et al, Blood, ASH Annual Meeting, 2012; 120:1718). In order to figure out the underlying pathogenesis, we compared the cytokine milieu in the media of MDS and normal ECFCs cultures (Bio-Plex pro-human cytokine 27-plex assay, (Bio-Rad). We found that in MDS cultures, significant higher amounts of IL13, IL1b, PDGFb and VEGF were secreted, whilst the levels of IL6, IL7, IL10. G-CSF, CXCL-10, CCL-2 and CCL-3 were significantly lower than in normal ECFC cultures (Teofili et al. Leukemia Research 2013; 37, Supp.1, S129-S130). Moreover, we evaluate through PCR arrays the gene expression profiles of normal and MDS ECFCs, focusing on genes involved in both endothelial cell biology and WNT signaling (QIAGEN, Milan, Italy). In comparison with normal ECFCs, MDS ECFCs overexpressed various adhesive molecules such as ICAM-1, L-selectin and V-CAM. This over-expression resulted in the more pronounced capacity of MDS ECFCs to adhere to hematopoietic cells in comparison with normal ECFCs, as demonstrated by the significant higher proportion of CFSE-labeled mononuclear cells trapped within the different adherent layer after 6 hours-incubation (40+11 % versus 14+4% in MDS and normal ECFC, respectively). Furthermore, at PCR arrays, we found that MDS ECFCs showed the down-regulation of several members of the canonical WNT pathway, including WNT3a, WNT5a and WNT5b. Basically, the WNT/bcatenin pathway is the main regulator of the microenvironment dependent hematopoietic stem cell fate, both in steady state and in regenerative processes. Therefore we investigated if the addition of WNT3a and WNT5a to MDS ECFC cultures affected their over-expression of adhesion molecules. To this purpose, we added to the culture medium 100 ng/ml of purified WNT3a and WNT5a (both purchased from R&D). We found that both WNT3a and WNT5a lowered the expression of several adhesion molecules in MDS ECFCs, including L-selectin, ICAM and VCAM, with WNT3a showing more pronounced and durable effects. On the whole our findings suggest that the ineffective hematopoiesis typical of MDS may be partly due to the abnormal entrapping of hematopoietic progenitors within the vascular niche, in the presence of a deeply perturbed surrounding cytokine milieu. Since our findings allege the defective WNT pathway as putative responsible defect, restoring the adequate WNT signaling could represent a potential therapeutic approach to MDS. The study was supported by the Grant AIRC 2012 n.11799 Disclosures: No relevant conflicts of interest to declare.