Loss of synergistic transcriptional feedback loops drives diverse B-cell cancers.

BACKGROUND: The most common B-cell cancers, chronic lymphocytic leukemia/lymphoma (CLL), follicular and diffuse large B-cell (FL, DLBCL) lymphomas, have distinct clinical courses, yet overlapping "cell-of-origin". Dynamic changes to the epigenome are essential regulators of B-cell differentiation. Therefore, we reasoned that these distinct cancers may be driven by shared mechanisms of disruption in transcriptional circuitry. METHODS: We compared purified malignant B-cells from 52 patients with normal B-cell subsets (germinal center centrocytes and centroblasts, naïve and memory B-cells) from 36 donor tonsils using >325 high-resolution molecular profiling assays for histone modifications, open chromatin (ChIP-, FAIRE-seq), transcriptome (RNA-seq), transcription factor (TF) binding, and genome copy number (microarrays). FINDINGS: From the resulting data, we identified gains in active chromatin in enhancers/super-enhancers that likely promote unchecked B-cell receptor signaling, including one we validated near the immunoglobulin superfamily receptors FCMR and PIGR. More striking and pervasive was the profound loss of key B-cell identity TFs, tumor suppressors and their super-enhancers, including EBF1, OCT2(POU2F2), and RUNX3. Using a novel approach to identify transcriptional feedback, we showed that these core transcriptional circuitries are self-regulating. Their selective gain and loss form a complex, iterative, and interactive process that likely curbs B-cell maturation and spurs proliferation. INTERPRETATION: Our study is the first to map the transcriptional circuitry of the most common blood cancers. We demonstrate that a critical subset of B-cell TFs and their cognate enhancers form self-regulatory transcriptional feedback loops whose disruption is a shared mechanism underlying these diverse subtypes of B-cell lymphoma. FUNDING: National Institute of Health, Siteman Cancer Center, Barnes-Jewish Hospital Foundation, Doris Duke Foundation.

Enhancer sequence variants and transcription-factor deregulation synergize to construct pathogenic regulatory circuits in B-cell lymphoma.

Most B-cell lymphomas arise in the germinal center (GC), where humoral immune responses evolve from potentially oncogenic cycles of mutation, proliferation, and clonal selection. Although lymphoma gene expression diverges significantly from GC B cells, underlying mechanisms that alter the activities of corresponding regulatory elements (REs) remain elusive. Here we define the complete pathogenic circuitry of human follicular lymphoma (FL), which activates or decommissions REs from normal GC B cells and commandeers enhancers from other lineages. Moreover, independent sets of transcription factors, whose expression was deregulated in FL, targeted commandeered versus decommissioned REs. Our approach revealed two distinct subtypes of low-grade FL, whose pathogenic circuitries resembled GC B or activated B cells. FL-altered enhancers also were enriched for sequence variants, including somatic mutations, which disrupt transcription-factor binding and expression of circuit-linked genes. Thus, the pathogenic regulatory circuitry of FL reveals distinct genetic and epigenetic etiologies for GC B-cell transformation.

Overexpression of Runx2 directed by the matrix metalloproteinase-13 promoter containing the AP-1 and Runx/RD/Cbfa sites alters bone remodeling in vivo.

The activator protein-1 (AP-1) and runt domain binding (Runx/RD/Cbfa) sites and their respective binding proteins, c-Fos/c-Jun and Runx2 (Cbfa1), regulate the rat matrix metalloproteinase-13 (MMP-13) promoter in both parathyroid hormone (PTH)-treated and differentiating osteoblastic cells in culture. To determine the importance of these regulatory sites in the expression of MMP-13 in vivo, transgenic mice containing either wild-type (-456 or -148) or AP-1 and Runx/RD/Cbfa sites mutated (-148A3R3) MMP-13 promoters fused with the E. coli lacZ reporter were generated. The wild-type transgenic lines expressed higher levels of bacterial beta-galactosidase in bone, teeth, and skin compared to the mutant and non-transgenic lines. Next, we investigated if overexpression of Runx2 directed by the MMP-13 promoter regulated expression of bone specific genes in vivo, and whether this causes morphological changes in these animals. Real time RT-PCR experiments identified increased mRNA expression of bone forming genes and decreased MMP-13 in the tibiae of transgenic mice (14 days and 6 weeks old). Histomorphometric analyses of the proximal tibiae showed increased bone mineralization surface, mineral apposition rate, and bone formation rate in the transgenic mice which appears to be due to decreased osteoclast number. Since MMP-13 is likely to play a role in recruiting osteoclasts to the bone surface, decreased expression of MMP-13 may cause reduced osteoclast-mediated bone resorption, resulting in greater bone formation in transgenic mice. In summary, we show here that the 148 bp upstream of the MMP-13 transcriptional start site is sufficient and necessary for gene expression in bone, teeth, and skin in vivo and the AP-1 and Runx/RD/Cbfa sites are likely to regulate this. Overexpression of Runx2 by these regulatory elements appears to alter the balance between the bone formation-bone resorption processes in vivo.

Mechanisms of the regulation of EGF receptor gene expression by calcitriol and parathyroid hormone in UMR 106-01 cells.

BACKGROUND: We have previously demonstrated that parathyroid hormone (PTH) and calcitriol increase the expression of epidermal growth factor receptors (EGFR) in UMR 106-01 osteoblast-like cells. The effect of PTH is mediated by cAMP and it involves an increase in the level of EGFR mRNA. The present studies were designed to investigate the mechanisms involved in the regulation of EGFR expression by PTH and calcitriol. METHODS: To examine the mechanism of the effect of calcitriol on EGFR expression, confluent cultures of UMR 106-01 cells were exposed to calcitriol and levels of EGFR mRNA were determined by reverse transcription-polymerase chain reaction (RT-PCR). In order to study the effect of calcitriol on EGFR gene transcription, a candidate vitamin D-responsive element (VDRE) was identified in the EGFR gene promoter and complimentary 30-mer oligonucleotides spanning this region were tested for binding to recombinant VDR using EMSA. Transcriptional activity in response to calcitriol and PTH was tested in UMR 106-01 cells stably transfected with a luciferase reporter construct containing the full length EGFR gene promoter. The effect of calcitriol on EGFR mRNA stability was examined in transcriptionally arrested cells. RESULTS: Treatment with calcitriol resulted in a time and dose dependent increase in EGFR mRNA levels in confluent cultures of UMR 106-01 osteoblast-like cells. Using EMSA, we demonstrated that the putative human EGFR VDRE binds to recombinant VDR in a retinoid X receptor (RXR)-dependent manner; however, calcitriol failed to increase transcriptional activity from a luciferase reporter construct containing the full-length EGFR gene promoter in stably transfected UMR 106-01 cells. Therefore, EGFR mRNA degradation was examined in transcriptionally arrested cells and calcitriol was found to prolong the half life of EGFR mRNA. Treatment of the cultures with PTH resulted in a ninefold increase in luciferase activity after four hours of exposure, a finding that was reproduced by treatment with forskolin. CONCLUSIONS: These studies demonstrate that the calciotropic hormones PTH and calcitriol increase EGF receptor expression by different mechanisms. The former increases EGFR gene transcription whereas the latter increases EGFR mRNA stability.