We report that Heat shock protein 90 (Hsp90) inhibitors selectively kill Diffuse Large B-cell Lymphomas (DLBCL) that are biologically dependent on the Bcl6 transcriptional repressor. Endogenous Hsp90 was found to interact with Bcl6 in DLBCL cells and could stabilize both Bcl6 mRNA and protein. Hsp90 formed a complex with Bcl6 at its target promoters and Hsp90 inhibitors de-repressed Bcl6 target genes. A stable mutant of Bcl6 rescued DLBCL cells from Hsp90 inhibitor induced apoptosis. Bcl6 and Hsp90 were almost invariantly co-expressed in the nuclei of primary DLBCL cells, suggesting that their interaction is relevant in this disease. We examined the pharmacokinetics, toxicity and efficacy of PU-H71, a recently developed purine derived Hsp90 inhibitor. PU-H71 preferentially accumulated in lymphomas compared to normal tissues and selectively suppressed Bcl6-dependent DLBCLs in vivo, inducing reactivation of key Bcl6 target genes and apoptosis. PU-H71 also induced cell death in primary human DLBCL specimens.

IntroductionOn T-cell dependent activation, resting/naive B cells (NBCs) can be induced to migrate into lymphoid follicles and form germinal centers (GCs). 1,2 GC B cells subsequently undergo massive clonal expansion and mutagenesis mediated by activation-induced cytosine deaminase (AICDA). 2 Tolerance of simultaneous proliferation and genomic instability is a hallmark of the GC B-cell phenotype and is required for development of B-cell clones able to generate high-affinity antibodies. 1,2 AICDA not only induces mutations within the immunoglobulin loci but also localizes to many other sites of the genome including promoters and coding sequences of actively transcribed genes enriched in RGYW DNA motifs. [3][4][5][6] AICDA-induced mutations can thus occur at many sites throughout the genome in normal GCs. 3,6 In accordance with these observations, AICDA has been demonstrated to play a critical role in lymphomagenesis. 7 While genetic diversity of B-cell clones within GCs is important for the emergence of cells encoding high-affinity immunoglobulins, it also provides opportunities for the emergence of malignant clones. In fact a majority of B-cell neoplasms originate from cells that have transited the GC reaction. 1 Induction of the GC phenotype requires that NBCs undergo major changes in gene expression patterning, the basis of which are not fully understood. These shifts are mediated in part by transcription factors such as BCL6 and BACH2 [8][9][10] and histone modifying enzymes such as EZH2. 11 However, differential methylation of CpG dinucleotides is also known to control tissue specific gene expression. 12,13 CpG methylation is mediated by a family of DNA methyltransferase enzymes (DNMTs). 14 Of these, DNMT1 primarily mediates maintenance methylation, because of its preference for hemimethylated DNA 15 ; while DNMT3A and 3B primarily mediate de novo DNA methylation. Differential methylation occurs at the earliest stages of lymphopoiesis 16 and Dnmt1 hypomorphic mice accordingly display skewed hematopoietic differentiation toward the myeloid lineage, 17 but the role of DNMT1 in mature B cells has not been studied in a detailed manner.Both aberrant DNA hypermethylation and hypomethylation have been shown to occur in lymphomas derived from GC B-cells such as diffuse large B-cell lymphomas (DLBCL). 18,19 Furthermore, DLBCLs with GCB (Germinal Center B-cell like) versus ABC (Activated B cell-like) gene expression signatures display distinct DNA methylation profiles, 18 suggesting that cytosine methylation may contribute to the distinct phenotypes of these tumors. Very little is known regarding mechanisms of DNA demethylation, but reports have suggested that cytosine deamination mediated by AICDA followed by base excision The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. For personal use onl...

Expression profiling has shown 2 main and clinically distinct subtypes of diffuse large B-cell lymphomas (DLBCLs): germinal-center B cell-like (GCB) and activated B cell-like (ABC) DLBCLs. Further work has shown that these subtypes are partially characterized by distinct genetic alterations and different survival. Here, we show with the use of an assay that measures DNA methylation levels of 50 000 CpG motifs distributed among more than 14 000 promoters that these 2 DLBCL subtypes are also characterized by distinct epigenetic profiles. DNA methylation and gene expression profiling were performed on a cohort of 69 patients with DLBCL. After assigning ABC or GCB labels with a Bayesian expression classifier trained on an independent dataset, a supervised analysis identified 311 differentially methylated probe sets (263 unique genes) between ABC and GCB DLBCLs. Integrated analysis of methylation and gene expression showed a core tumor necrosis factor-␣ signaling pathway as the principal differentially perturbed gene network. Sixteen genes overlapped between the core ABC/GCB methylation and expression signatures and encoded important proteins such as IKZF1. This reduced gene set was an accurate predictor of ABC and GCB subtypes. Collectively, the data suggest that epigenetic patterning contributes to the ABC and GCB DLBCL phenotypes and could serve as useful biomarker. IntroductionDiffuse large B-cell lymphoma (DLBCL) is the most common B-cell malignancy and is highly heterogeneous from both clinical and molecular standpoints. 1 Gene expression profiling of primary DLBCL cases identified biologically distinct subtypes of DL-BCL. 2,3 One such approach classified DLBCL into germinal center B cell-like (GCB) and activated B cell-like (ABC) DLBCL subtypes, based on similarities of the respective gene signatures to normal germinal center B cells and activated peripheral B cells, respectively. 2 This subclassification is clinically significant and predicts overall and progression-free survival in patients treated with cyclophosphamide, hydroxydaunorubicin hydrochloride, vincristine, and prednisone and rituximab with cyclophosphamide, hydroxydaunorubicin hydrochloride, vincristine, and prednisone (R-CHOP). 2,4,5 Several years after the discovery of these subtypes, the mechanisms controlling gene expression in ABC and GCB DLBCLs are still only partially understood. For example, ABC DLBCLs feature aberrant activity of nuclear factor-B (NF-B) signaling, in part because of mutations in upstream components of this pathway. 6,7 Chromosomal translocations, including 3q27 or t(14;18), often do not correlate with the affected protein expression and do not alone define lymphoma phenotypes. [8][9][10] Lenz et al 11 used genomewide copy number analysis to describe 30 recurrent but relatively infrequent chromosomal aberrations with DLBCL subtype-specific frequencies. ABC DLBCLs were characterized by deletion of SPIB, deletion of INK4a/ARF, and trisomy 3. GCB DLBCLs showed preferential deletion of PTEN and amplification of the locus enc...

Diffuse large B-cell lymphoma (DLBCL) is a biologically and clinically heterogeneous disease with marked genomic instability and variable response to conventional R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone) chemotherapy. More clinically aggressive cases of DLBCLs have high level of circulating interleukin 10 (IL10) cytokine and evidence of activated intracellular STAT3 (signal transducer and activator of transcription 3) signaling. We investigated the role of IL10 and its surface receptor in supporting the neoplastic phenotype of DLBCLs. We determined that IL10RA gene is amplified in 21% and IL10RB gene in 10% of primary DLBCLs. Gene expression of IL10, IL10RA and IL10RB was markedly elevated in DLBCLs. We hypothesized that DLBCLs depend for their proliferation and survival on IL10-STAT3 signaling and that blocking the IL10 receptor (IL10R) would induce cell death. We used anti-IL10R blocking antibody, which resulted in a dose-dependent cell death in all tested activated B-cell-like subtype of DLBCL cell lines and primary DLBCLs. Response of germinal center B-cell-like subtype of DLBCL cell lines to anti-IL10R antibody varied from sensitive to resistant. Cells underwent cell cycle arrest, followed by induction of apoptosis. Cell death depended on inhibition of STAT3 and, to a lesser extent, STAT1 signaling. Anti-IL10R treatment resulted in interruption of IL10-IL10R autostimulatory loop. We thus propose that IL10R is a novel therapeutic target in DLBCLs.

Research with dopamine D1 receptor antagonists or neuronal inactivating agents suggests dissociable regulation of cocaine-seeking behavior by the rostral and caudal basolateral amygdala. In the present study, discrete infusions of the D1 receptor agonist SKF 81297 (0.0–0.8 μg/side) were compared to the D1 receptor antagonist SCH 23390 (0.0–2.0 μg/side) to demonstrate directly the importance of D1 receptor mechanisms within the rostral and caudal basolateral amygdala for their functional heterogeneity in regulating cocaine-seeking behavior. Under a second-order schedule, cocaine-seeking behavior was studied during maintenance (cocaine and cocaine cues present) and reinstatement (only cocaine cues present). Food-maintained responding examined specificity of maximal behaviorally effective doses of SKF 81297 and SCH 23390. Results demonstrated that the D1 agonist (0.4 or 0.8 μg) increased and D1 antagonist (1.0 μg) decreased cocaine-seeking behavior during maintenance when infused into caudal but not rostral basolateral amygdala. Cocaine intake was not affected by the agonist and was decreased by the antagonist. During reinstatement, the D1 agonist (0.4 μg) increased and D1 antagonist (1.0 μg) decreased cocaine-seeking behavior when infused into rostral but not caudal basolateral amygdala. In tests for behavioral specificity, the above effective doses of SKF 81297 and SCH 23390 used in self-administration experiments did not alter food-maintained responding. However, the 2.0 μg dose of SCH 23390 suppressed drug- and food-maintained responding after infusion into both subregions. Collectively, findings indicate dissociable sensitivity to D1 receptor ligands within the caudal and rostral basolateral amygdala for altering cocaine-seeking behavior under different conditions modeling phases of addiction.