ALBERT

Description: Chromosomal abnormalities of band 8p11 are associated with a distinct subtype of acute myeloid leukemia with French-American-British M4/5 morphology and prominent erythrophagocytosis by the blast cells. This subtype is usually associated with the t(8;16)(p11;p13), a translocation that has recently been shown to result in a fusion between the MOZ and CBP genes. We have cloned the inv(8)(p11q13), an abnormality associated with the same leukemia phenotype, and found a novel fusion between MOZ and the nuclear receptor transcriptional coactivatorTIF2/GRIP-1/NCoA-2. This gene has not previously been implicated in the pathogenesis of leukemia or other malignancies. MOZ-TIF2 retains the histone acetyltransferase homology domains of both proteins and also the CBP binding domain of TIF2. We speculate that the apparently identical leukemia cell phenotype observed in cases with the t(8;16) and the inv(8) arises by recruitment of CBP by MOZ-TIF2, resulting in modulation of the transcriptional activity of target genes by a mechanism involving abnormal histone acetylation.

Description: Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease with recognized variability in clinical outcome, genetic features, and cells of origin. To date, transcriptional profiling has been used to highlight similarities between DLBCL tumor cells and normal B-cell subtypes and associate genes and pathways with unfavorable outcome. Given the genetic heterogeneity in DLBCL, there are likely to be subsets of tumors with different pathogenetic mechanisms and possible treatment targets. To identify DLBCL subtypes that were sufficiently robust to be captured by multiple methods, we analyzed the profiles of 176 newly diagnosed DLBCLs using three different clustering algorithms (hierarchical clustering (HC), self-organizing maps (SOM), and probabilistic clustering (PC)), the top 5% of genes with the highest reproducibility across duplicate samples and largest variation across patient tumors, and a resampling-based method (consensus clustering) that automatically selects the most stable numbers of clusters with each algorithm. Three discrete subsets of DLBCLs -- “Oxidative Phosphorylation” (OxPhos), “B-cell Receptor/Proliferation” and “Host Response” (HR) -- were identified, characterized using gene set enrichment analysis and confirmed in an independent series of newly diagnosed DLBCLs with available array data. There was an association between cluster membership and examined genetic abnormalities in DLBCL. BCL2 translocations were more common in the OxPhos cluster whereas BCL6 translocations were more frequent in the BCR/proliferation cluster. Translocations of either type were uncommon in the HR cluster. Patients with HR DLBCLs were also significantly younger than those with OxPhos or BCR/proliferation tumors; HR patients also had a significantly higher incidence of splenic and BM involvement. The unique characteristics of HR tumors - fewer known genetic abnormalities and prominent host immune and inflammatory cell transcripts -prompted us to assess host immune cells in study tumors using morphologic and immunohistochemical approaches. HR DLBCLs contained significantly higher numbers of morphologically distinct CD2+/CD3+ tumor-infiltrating lymphocytes and interdigitating S100+/GILT+/CD1a-/CD123- dendritic cells. The HR cluster shared features of histologically defined T-cell/histiocyte-richBCL, including fewer known genetic abnormalities, younger age at presentation and frequent splenic and bone marrow involvement. The current study identifies tumor microenvironment and host inflammatory response as defining features in DLBCL, provides insights into the nature of the host immune response in a major DLBCL subtype and suggests rational treatment targets in newly identified tumor groups.

Description: Although diffuse large B-cell lymphoma (DLBCL) is potentially curable with current therapy, a significant number of DLBCL patients still die of their disease. In a broad-based screen for genes and pathways associated with poor DLBCL outcome, we previously identified a novel risk-related gene, termed BAL (B-aggressive lymphoma). Thereafter, we cloned and characterized a major BAL partner protein, BBAP (B-aggressive lymphoma and BAL binding partner), described the nuclear trafficking patterns of both proteins and demonstrated that BAL functions as a modulator of transcription. In the current study, we characterized BAL expression in normal tonsil and primary DLBCLs and defined BAL regulation and potential functions in both cell types. Immunohistochemical staining of normal tonsil revealed that BAL was expressed by small numbers of germinal center (GC) cells and interfollicular cells with the morphologic features of centroblasts (with the GC) and immunoblasts (within the interfollicular areas). In primary DLBCLs, BAL was expressed by the malignant B cells. To gain insights regarding the regulation of BAL and BBAP expression in DLBCLs, we analyzed a series of 176 primary tumor biopsies transcriptionally profiled an U133A/B Affymetrix microarrays. BAL/BBAP high-expressing DLBCLs had evidence of a brisk host immune response, including increased expression of T/NK-cell receptor and activation pathway components, complement cascade members, macrophage/dendritic cells markers and γIFN-induced transcripts, raising the possibility that BAL was induced by γIFN. Consistent with this hypothesis, γIFN treatment of DLBCL cell lines with low basal levels of BAL markedly increased BAL expression. In silico analysis revealed that the BAL and BBAP genes are located in 3q21 in head-to-head orientation and share the same CpG-related promoter. This shared promoter contains a conserved γIFN-responsive module composed of IRF and STAT binding elements. The BAL/BBAP bidirectional promoter was cloned into a pGL3 luciferase promoterless reporter vector and found to increase luciferase activity 〉 20-fold following γIFN stimulation. To gain further insights into regulation of BAL transcription, mutant versions of BAL promoter were generated and cloned. Luciferase assays demonstrated that the IRF binding site was necessary for γIFN-induced BAL transcription, whereas the STAT1 binding site had an accessory role. Taken together, these results demonstrate that BAL and BBAP genes are transcriptionally activated by γIFN in DLBCLs with features of a brisk host immune response including γIFN signaling. Preliminary studies suggest that BAL limits the efficacy of the observed host inflammatory response.

Description: MicroRNAs (miR) are non-coding RNAs that regulate gene expression by pairing to 3UTRs of target genes inducing translational repression or mRNA cleavage. New evidence suggests that the latter mechanism markedly contributes to miRNA effects. Hence, global gene expression analyses may help elucidate the functional role of miRNA by recognizing pathways modified by their abnormal expression and identifying direct targets. MiR155, the product of the non coding gene BIC, is overexpressed in lymphomas and its role in tumorigenesis is supported by the development of B-cell malignancies in miR155 transgenic mice. However, the functional consequences of miR155 overexpression in tumor development remain unclear. To address this issue, we developed a semi-quantitative RT-PCR assay that specifically amplifies either the nuclear unspliced BIC mRNA (target of the RNase III Drosha) or the spliced BIC mRNA. We found a marked correlation between the expression levels of these two mRNAs, which in turn agreed with the levels of mature miR155 detected in northern blots. Of the 22 DLBCL cell lines studied, only 5 (DHL6, Ly3, Ly10, Farage, RCK-8) expressed significantly high levels of BIC and miR155. To isolate the effects of miR155 in DLBCL we genetically modified its expression and performed global transcription analysis on microarray. In brief, we cloned the BIC transcript in a MSCV-GFP bicistronic retrovirus and confirmed in transduced HeLa cells that the mature miR155 was expressed when this transcript was driven by an LTR promoter. Next, we used two DLBCL cell lines with low levels of miR155 (Ly8 and Ly19) to generate unique populations expressing miR155 or MSCV alone. RNA was isolated from GFP-sorted cells, hybridized to the Affymetrix U133Plus2.0 chip and the data analyzed with dChip. Remarkably, and in agreement with the role of miRNAs, supervised analysis (fold diff 〉1.7) revealed a vast predominance (〉90%) of downregulated genes when comparing miR155 expressing cells to MSCV only. These gene groups included predicted miR155 targets and were significantly enriched for molecules involved in the immune response (p

Description: Two distinct leukemia syndromes are associated with abnormalities of chromosome band 8p11. First, a myeloproliferative disorder with features characteristic of both chronic myeloid leukemia and non-Hodgkin's lymphoma and second, an acute myeloid leukemia (AML) with French-American-British (FAB) M4/5 morphology and prominent erythrophagocytosis. The two syndromes are exemplified by a t(8; 13)(p11; q12) and a t(8; 16)(p11; p13), respectively, but cytogenetic variants of both have been described. Recently, the t(8; 16) has been cloned and shown to fuse the MOZ gene at 8p11 to the CBP gene at 16p13. We have used fluorescence in situ hybridization (FISH), Southern blotting, and reverse transcriptase-polymerase chain reaction (RT-PCR) to refine the 8p11 breakpoint in three cases with t(8; 13)(p11; q12) and in a single case of AML-M5 with a clinical picture apparently identical to that found in patients with a t(8; 16), but characterized by an inv(8)(p11q13). FISH analysis was performed with several 8p11 CEPH yeast artificial chromosome (YAC) clones. YAC 782H11 was centromeric to the one case with t(8; 13) tested, but was telomeric to the inv(8). YAC 847B12 was telomeric to both the t(8; 13) and the inv(8), whereas YAC 829D12 was centromeric to the t(8; 13), but split by the inv(8). Southern blotting and PCR of YAC 829D12 showed that it contained the MOZ gene. A 900-bp MOZ fragment encompassing the published t(8; 16) breakpoint was amplified by PCR from normal peripheral blood leukocyte cDNA and used to probe Southern blots of patient DNA. A rearrangement was detected in the case with inv(8), but not in any of the three cases with t(8; 13). Southern blotting with a CBP probe and RT-PCR with MOZ and CBP primers suggested that the inv(8) does not result in a cryptic MOZ-CBP fusion. It is likely, therefore, that MOZ is fused to a novel gene at 8q13 in this case. We conclude that the t(8; 13) breakpoint is flanked by YACs 782H11 and 847B12 and is at least 1 Mb telomeric to MOZ. MOZ is involved, however, in a new variant of the t(8; 16).

Description: In normal and malignant lymphocytes the inhibitory cyclic-AMP (cAMP) signals are mainly controlled by the phosphodiesterase 4B (PDE4B). We previously showed that in diffuse large B-cell lymphoma (DLBCL) cAMP induces apoptosis via down regulation of PI3K/AKT activity independently of the effectors PKA and EPAC. These data implied the existence of a pathway linking cAMP to PI3K. However, the molecules that mediate this interaction are unknown. In B-cells, phosphorylation of PI3K’s regulatory subunit by SYK potently up-regulates its activity which is enhanced by stabilization of the catalytic domain to cell membrane via binding to RAS. Hence, we first asked if cAMP could decrease RAS activity and if that contributed to PI3K inhibition. To address this issue, we reconstituted PDE4B expression (wild-type gene [WT] or a phosphodiesterase inactive [PI] mutant) in PDE4B-null DLBCL cells. Subsequently, the intra-cellular levels of cAMP were increased with Forskolin (10μM) and the levels of activated RAS determined in pull-down assays with the Raf1- Ras Binding Domain-GST fusion protein. In cells lacking functional PDE4B (PI), high levels of cAMP resulted in a marked reduction of the RAS activity whereas it had no effects in the PDE4B-WT cells. After showing that cAMP decreases RAS activity in mature B-cells, we sought to link RAS inhibition to PI3K downregulation. Thus, we created cells expressing the dominant negative RASN17 mutant in a PDE4B-null background; the inhibitory role of these N17 mutants was confirmed in RAS activation assays. We reasoned that if RAS is necessary for cAMP-mediated inhibition of PI3K, its absence (N17 mutant) may block this event. To test this hypothesis we compared the effects of cAMP in PDE4B-WT, -PI or RASN17 cells on the phosphorylation of AKT (S473), a precise surrogate for PI3K activity in this model, and on downstream targets of mTOR, S6 ribosomal protein (S6R - S235/6) and 4EBP1 (Thr37/46). Whereas at baseline there was no significant difference in the phospho levels of these proteins, cAMP completely inhibited their phosphorylation in the PI cells but had a limited or no effect on RASN17 and WT cells, respectively. In agreement with these data, in proliferation assays RASN17 expression rendered PDE4B-null cells ∼ 50% more resistant (p=.001) to cAMP than their RAS intact counterpart. Taken together, these data indicate that RAS transduce most, but probably not all, of the cAMP effects towards PI3K/AKT. Therefore, we next investigated if modulation of the tyrosine kinase SYK could also be involved in the cAMP inhibition of PI3K. Using PDEB-null cells, we found that cAMP decreased the phosphorylation of residues essential for SYK function (Y525/26) to the same extent as the SYK inhibitor Piceatannol. As SYK is a candidate for targeted inhibition in a variety of disorders, this newly found interplay with cAMP/PDE4B may have clinical implications. For this reason, we created a PDE4B loss of function model (stable RNAi) to test the benefits of combining SYK and PDE4B inhibition in DLBCL. In these assays, Piceatannol (5–10μM) was more efficient in inhibiting the proliferation of PDE4B-RNAi cells than their control counterpart (60% vs. 30% inhibition, p=.004). These data suggest that in the absence of PDE4B cAMP signals lower the tonic activity of SYK and increase the effects of SYK inhibitors. Herein, we placed RAS and SYK in the pathway linking cAMP to PI3K and found evidence that combining PDE4 and SYK inhibitors may be beneficial in inflammatory, auto-immune and malignant conditions.

Description: Cyclic AMP (cAMP) potentiates glucocorticoid (GC) induced apoptosis in lymphoid cells but the mechanisms associated with these effects are unclear. We previously showed that cAMP inhibits PI3K/AKT activity and that in diffuse large B-cell lymphomas (DLBCL) overexpression of the phosphodiesterase 4B (PDE4B) blocks these effects. Recently, high AKT/mTOR activity was implicated in the GC resistance found in acute lymphoid leukemia (ALL) and rapamycin shown to modulate these effects. Therefore, we hypothesized that cAMP enhances GC responses by down-modulating the AKT/mTOR pathway and that PDE4B, by blunting these inhibitory effects, is at the center of GC resistance (and rapamycin activity) in DLBCL. If this notion is accurate, PDE4B expression may impinge on the same genes and pathways that control GC response in malignant lymphocytes. To test this idea, we used gene set enrichment analysis (GSEA) and found a marked association between gene sets of DLBCLs defined by high or low PDE4B expression and ALLs classified by their resistance or sensitivity to GC-induced apoptosis(p and FDR =.014). To further our investigation, we created PDE4B gain or loss of function models in DLBCL cell lines. We reconstituted PDE4B expression (wild-type [WT] or phosphodiesterase inactive [PI] mutant) in the PDE4B-null DHL6 cell line and constitutively expressed a PDE4B-specific RNAi, which decreased this gene expression 14-fold, in the PDE4B-high cell line Ly3 (Ly3Ri). The functional consequences of these changes were confirmed by measuring the intra-cellular levels of cAMP and further validated in cell proliferation assays. DHL6-PDE4B-WT cells became ∼ 50% more resistant to the inhibitory effects of cAMP (forskolin - 10μM) than their PI counterpart (p