ALBERT

Description: Abstract 1586 Background Mantle cell lymphoma (MCL) is a subtype of B-cell non-Hodgkin lymphoma (NHL) characterized by the t(11;14) translocation and concomitant over-expression of cyclin D1. MCL has a variable natural history; while some patients have prolonged survival similar to other indolent B-cell lymphomas, most follow an aggressive course with short survival. While the t(11;14) is pathognomonic of MCL, it is not necessary for disease pathogenesis, as a subset of MCL cases lack the translocation. Furthermore, in vivo models demonstrate that cyclin D1 over-expression alone is unable to bring about the disease, and that deregulation of additional cellular pathways is required for its pathogenesis. Assessment of microRNA (miR) expression in MCL may help determine mechanisms of gene deregulation and reveal pathways involved in disease pathogenesis. In this study we examined MCL in relation to both aggressive and indolent B-cell NHL to determine a miR signature that characterizes MCL. Design and Methods Total RNA from a training set of 36 B-cell NHL cases (19 indolent and 17 aggressive) and 32 MCL was applied to a high-throughput quantitative real-time PCR platform assessing the expression of 365 miRs [TaqMan Human MicroRNA Array v1.0 (Early Access) or TLDA]. miRs that were differentially expressed between MCL and aggressive NHL, and between MCL and indolent NHL were then validated using RNA from a second, independent, set of B-cell NHL cases (28 indolent and 28 aggressive) and 50 MCL cases. Validated miRs were determined and potential targets for each miR were examined. A map of targets common to the MCL miR signature was created, revealing important proteins involved in MCL pathogenesis. Results 66 miRs (11 over-expressed, 55 under-expressed) were differentially expressed between MCL and aggressive B-cell NHL and 8 miRs (7 over-expressed, 1 under-expressed) were differentially expressed between MCL and indolent B-cell NHL (false discovery rate = 0.2). 6 miRs from each group were chosen for validation in an independent set of MCL and NHL cases. Of these 12 miRs, 7 miRs validated (2 were under-expressed in MCL relative to aggressive B-cell NHL, and 5 were over-expressed in MCL relative to indolent B-cell NHL). Genes and pathways involved in disease pathogenesis are most likely targeted by multiple miRs. We thus determined a set of 123 genes predicted to be targets of this MCL miR signature, based on five miR target prediction databases from the mirDIP (microRNA data integration) portal. These genes were significantly enriched for focal adhesion and integrin signalling, proteasome-mediated degradation, and the PI3K signalling pathway. Conclusions Using the largest set of MCL cases evaluated to date, a 7-miR signature characteristic of MCL was discovered. The gene targets of these miRs are enriched for roles in proteasome-mediated protein degradation, consistent with the reported sensitivity of MCL to proteasome inhibitors. In addition, these miRs are predicted to be involved in regulation of PI3K/AKT signalling, confirming reports of the importance of this pathway in MCL pathogenesis. Enrichment of target genes involved in focal adhesion and integrin signalling indicate the importance of MCL-stromal interactions and motivates further study into the role of the tumor microenvironment in MCL pathogenesis. Disclosures: No relevant conflicts of interest to declare.

Description: We used the hCG-NuMA-RARα transgenic model of acute promyelocytic leukemia (APL) (Sukhai et al, 2004) to determine the downstream genetic targets of NuMA-RARα, one of five APL-associated fusion proteins (X-RARα). X-RARα retain the C-terminal domains of retinoic acid receptor (RAR) α, and are thought to interfere with retinoid signaling pathways and thus inhibit neutrophil differentiation. However, X-RARα have a wider range of DNA binding specificities compared to RARα, and may deregulate novel downstream targets that play critical roles in APL. Our group previously identified PPARγ signaling as being one such pathway deregulated by X-RARα (Kamel-Reid et al, 2003). We sought to identify additional, nonretinoid signaling, target genes of NuMA-RARα by using a combined experimental and in silico approach. Affymetrix oligonucleotide array analysis (mouse 430A arrays) was performed on RNA harvested from bone marrow cultures established from wild-type and transgenic mice. Genes deemed to be significantly deregulated and of biological relevance were validated by quantitative real-time RT-PCR. In our analysis, we identified 260 significantly over-expressed and 278 significantly under-expressed genes in transgenic bone marrow cultures, compared to wild-type. As NuMA-RARα is an aberrant transcriptional repressor, we focused on under-expressed genes: 251/278 were putative direct targets of NuMA-RARα. 82/251 were retinoid response genes; 169/251 were novel targets, external to retinoid signaling pathways. 150/251 had known function. Genes involved in regulation of apoptosis, cell cycle, signal transduction and transcription were over-represented within this dataset, in comparison to their frequency within the mouse genome. NuMA-RARα deregulated a number of myeloid transcription factors, including PU.1 (11.5-fold under-expression compared to wild-type), members of the C/EBP (3.5–5.0 fold under-expression) and GATA families (8.5–11.0-fold over-expression), as well as c-Myb and AML1. Binding sites for these transcription factors were overrepresented within the promoters of NuMA-RARα-deregulated genes. NuMA-RARα also deregulated a set of genes involved in cell cycle regulation and DNA damage response, including Gadd45α (1.9-fold under-expressed) and Dusp1 (3.8-fold under-expressed), and components of signal transduction pathways, including Jak2 (20.0-fold under-expressed). By mapping the chromosomal addresses of deregulated genes onto the murine genome, we determined that changes in chromosome copy number may not be responsible for deregulation. Finally, our comparison with other, previously published, microarray analyses indicated that NuMA-RARα shared some common target genes with other leukemia fusion genes, and that gene deregulation caused by NuMA-RARα could give rise to a molecular phenotype reminiscent of hematopoietic stem cells. Several genes identified as candidate downstream targets in these analyses were also previously identified as putative secondary events in APL mouse models. NuMA-RARα therefore has specific effects on the transcriptome, distinct from retinoid signaling, and from other leukemia fusion genes. Future functional studies are required to determine the cooperative relationship between these novel target genes and NuMA-RARα.

Description: Abstract 800 Mantle cell lymphoma (MCL) is a B-cell non-Hodgkin lymphoma (NHL) accounting for ~6% of all NHL. It is sensitive to combination chemotherapy, but remission durations are short without approaches such as stem cell transplantation (SCT). Most patients are incurable, but the clinical course is variable, with some patients succumbing quickly, while others survive 〉10 years. MicroRNAs (miRs) are small, non-coding RNAs that regulate gene expression by inhibiting mRNA translation. miRs are useful in the prognostic assessment of tumors, but work to date examining differences between MCL and normal lymphoid tissues, have only identified 2 miRs involved in MCL prognosis (Zhao JJ, Blood, 2010; Di Lisio L, Leukemia, 2010). We used a novel approach to identify a prognostic miR signature in MCL. We hypothesized that a miR signature defining aggressiveness can be obtained by comparing miR expression profiles of aggressive NHL with indolent NHL, and that this signature when applied to a set of MCL cases, may aid in MCL prognosis. Total RNA was extracted from 135 formalin-fixed paraffin-embedded samples obtained at primary diagnosis (Table 1). RNA from a training set of 19 indolent and 20 aggressive NHL cases was analyzed on a high-throughput quantitative real-time PCR (qRT-PCR) platform assessing the expression of 365 miRs and 3 endogenous controls (TaqMan Human MicroRNA Array v1.0: TLDA, ABI) using the DDCt method. A two-sample Wilcoxon Rank sum test corrected for false discovery rate was used to assess the significance of differential expression for each miR between aggressive and indolent NHL. The 14 most significantly differentially expressed miRs (p

Description: Acute promyelocytic leukemia (APL) is characterized by accumulation of abnormal promyelocytes in the bone marrow and peripheral blood, and sensitivity to treatment with all-trans retinoic acid. APL cases have a balanced chromosomal translocation involving retinoic acid receptor alpha (RARA) on chromosome 17. The resulting fusion proteins (X-RARA) are aberrant transcription factors and block ATRA-induced neutrophil differentiation. Loss of RARA signalling impairs granulopoiesis, but is not sufficient to cause a leukemic phenotype. We elucidated the identities of additional signalling pathways, which can potentially cooperate with X-RARA in APL, that are commonly modulated by multiple X-RARA. We used the U-937 hematopoetic cell line retrovirally transduced with NPM-RARA (Kamel-Reid et al, 2003), and NuMA-RARA, in addition to NB4 cells (expressing PML-RARA) to determine the common genes and pathways deregulated in APL. Gene expression analysis was carried out on RNA harvested in triplicate from control and X-RARA expressing cell lines, using the Affymetrix U133Plus2 array platform. Gene expression and pathways analysis of array data was carried out using a suite of analysis tools. Array data were validated in an independent sample set by real-time quantitative PCR. We observed a total of 311 genes deregulated at least 2-fold by NuMA-RARA (192 up-regulated, 119 down-regulated), 393 genes deregulated by NPM-RARA (292 up-regulated, 101 down-regulated), and 2056 genes deregulated by PML-RARA (1097 up-regulated, 959 down-regulated). A total of 65 genes, in 5 major interaction networks, were commonly deregulated by all three X-RARA (42/65 up-regulated, 23/65 down-regulated). The majority of these genes are involved in cellular signalling (14 genes, p-value 1.57E-07–7.77E-3), transcription (13 genes, p-value 5.68E-7–3.91E-3), cell proliferation (25 genes, p-value 9.73E-7–7.77E-3), apoptosis (26 genes, p-value 9.96E-7–7.71E-3), and cell movement (17 genes, p-value 1.43E-6–7.60E-3). Genes involved in the CEBPA interaction network (GFI1, TRIB2, ELA2), as well as other genes that we anticipated to be deregulated in APL including ID1, MMP9, and JUN were found through this analysis. NF-kB (p-value 2.10E-3), AHR (p-value 2.12E-3), IL-6 (p-value 5.40E-3), and G-protein coupled receptor (p-value 7.45E-3) signalling were among the top canonical pathways determined to be altered by X-RARA. Over-expression of a number of NF-kB downstream transcriptional targets, including VEGF, IL8, MMP9, cIAP2, and TNFAIP3, were also observed in multiple X-RARA expressing cell lines. In addition, in vitro results were compared to NuMA-RARA gene targets identified in primary bone marrow cultures derived from the hCG-NuMA-RARA transgenic mouse model (Sukhai et al, 2004). We observed that pathways involved in cell signalling, cell death, gene expression, proliferation, and cell cycle were significantly deregulated in both mouse and human datasets, indicating that these pathways may be important cooperating events in APL. Our data represent the first comparison of the genetic profiles of the variant fusion proteins NPM-RARA and NuMA-RARA in a haematopoietic cell system. Our studies are a significant step in identifying key targets that cooperate with X-RARA in the development of APL.