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Supplementary Figure S1. Crebbp deficiency results in accelerated germinal center derived B-cell lymphoma development in mice. Supplementary Figure S2. Ep300 deficiency results in accelerated germinal center derived B-cell lymphoma development in mice. Supplementary Figure S3. Western blots showing the knockdown efficiency of shRNAs against human CREBBP in human DLBCL MD901 cells and H3K27ac reduction in MD901 cells transduced with shRNAs against human CREBBP, and stacked bar plot showing the genome-wide distribution of CREBBP ChIPseq peaks in OCI-Ly7 cells. Supplementary Figure S4. GSEA enrichment plots showing correlation of genes with >25% loss of H3K27ac at enhancers in VavP-Bcl2/shCrebbp tumours with ranked expression change between B220+ lymphoma cells from VavP-Bcl2/shCrebbp tumours (n=3) and VavP-Bcl2/GFP tumours (n=3). NES, normalized enrichment score. Supplementary Figure S5. Venn diagram showing the overlap of H3K4me2+H3K4me3-H3K27ac+ (active enhancer) peaks between human tonsilar IgD+ Naïve B cells (NBs) and CD77+ germinal center B cells (GCBs). Supplementary Figure S6. Pathway analysis of down-regulated genes in top 500 differentially expressed genes between murine VavP-Bcl2/shCrebbp tumours and VavP-Bcl2/GFP tumours (left panel), or between CREBBP knockdown and scramble control human DLBLC MD901 cells (right panel). Supplementary Figure S7. Pathway analysis of genes with > 25% reduction of H3K27ac reads at promoters in murine VavP-Bcl2/shCrebbp tumors. Supplementary Figure S8. UCSC read-density tracks of normalized BCL6 (purple) and SMRT (orange) ChIP-seq reads in human tonsilar GCBs, H3K27ac ChIP-seq reads in human tonsilar NBCs (red) and GCBs (blue), and H3K27ac ChIP-seq in control scramble (Scr, green) and CREBBP KD (shCREBBP, turquoise) MD901 cells at the human CIITA and CD74 loci. BCL6 and SMRT peaks determined by MACS2 are indicated by grey bars under the read density track. Supplementary Figure S9. Stacked bar plots showing the frequencies of CREBBP somatic mutations in two cohorts of FL and one cohort of DLBCL primary patient samples. Supplementary Figure S10. The proportions of the Up (up-regulated) and Down (down-regulated) genes in the top 100, 500, and 1000 most differentially expressed genes of the respective cohorts profiled in Figure 3. Supplementary Figure S11. Representative flow cytometry analysis for FAS and GL7 of whole spleens from NS-DAD wild type (wt) or mutant (mut) animals. Supplementary Figure S12. Western blots showing the knockdown efficiency of shRNAs against human HDAC3 in DLBCL cell lines OCI-Ly7 and MD901 120 hr after induction of shRNA expression. The amount of HDAC3 was quantified by using ImageJ, and the ratio of shRNA knockdown samples and the control shLuc sample was indicated below the blots. Supplementary Figure S13. BCL6 and HDAC3 ChIP-qPCR at enhancers of CD74 or HLA-DOA (primer sequences please check Supplementary Table S12) in OCI-Ly1 cells transfected with either non-target siRNA (siNT) or BCL6 siRNA (siBCL6). Supplementary Figure S14. Quantification of HLA-DR measured by flow cytometry in shCREBBP or scramble transduced lymphoma cells MD901 and OCI-Ly18 in a second biological replicate experiment.

A list of CREBBP mutations in cohorts 1 and 2.

Cancer cells adapt their metabolic activities to support growth and proliferation. However, increased activity of metabolic enzymes is not usually considered an initiating event in the malignant process. Here, we investigate the possible role of the enzyme serine hydroxymethyltransferase-2 (SHMT2) in lymphoma initiation. SHMT2 localizes to the most frequent region of copy number gains at chromosome 12q14.1 in lymphoma. Elevated expression of SHMT2 cooperates with BCL2 in lymphoma development; loss or inhibition of SHMT2 impairs lymphoma cell survival. SHMT2 catalyzes the conversion of serine to glycine and produces an activated one-carbon unit that can be used to support S-adenosyl methionine synthesis. SHMT2 induces changes in DNA and histone methylation patterns leading to promoter silencing of previously uncharacterized mutational genes, such as SASH1 and PTPRM. Together, our findings reveal that amplification of SHMT2 in cooperation with BCL2 is sufficient in the initiation of lymphomagenesis through epigenetic tumor suppressor silencing. Parsa et al. report a mechanism of lymphoma initiation involving cooperation of BCL2 and increased activity of the metabolic enzyme SHMT2, which imparts changes in DNA and histone methylation.

Inhibition of Bruton tyrosine kinase (BTK) is a breakthrough therapy for certain B cell lymphomas and B cell chronic lymphatic leukemia. Covalent BTK inhibitors (e.g., ibrutinib) bind to cysteine C481, and mutations of this residue confer clinical resistance. This has led to the development of noncovalent BTK inhibitors that do not require binding to cysteine C481. These new compounds are now entering clinical trials. In a systematic BTK mutagenesis screen, we identify residues that are critical for the activity of noncovalent inhibitors. These include a gatekeeper residue (T474) and mutations in the kinase domain. Strikingly, co-occurrence of gatekeeper and kinase domain lesions (L512M, E513G, F517L, L547P) in cis results in a 10- to 15-fold gain of BTK kinase activity and de novo transforming potential in vitro and in vivo. Computational BTK structure analyses reveal how these lesions disrupt an intramolecular mechanism that attenuates BTK activation. Our findings anticipate clinical resistance mechanisms to a new class of noncovalent BTK inhibitors and reveal intramolecular mechanisms that constrain BTK's transforming potential.

Significance BCL2 selective inhibitors are promising agents currently under clinical investigation for treatment of BCL2-dependent cancers. However, the clinical activity of BCL2 inhibitors in patients with diffuse large B cell lymphoma (DLBCL) has been disappointing. In this study, we identified PMAIP1/NOXA gene amplification as a marker of sensitivity to BCL2 inhibitors in DLBCL. Cells lacking NOXA amplification were less sensitive to BCL2 inhibitors due to codependency on MCL1 and BCL2 proteins. We show that pharmacologic induction of NOXA by the HDAC inhibitor panobinostat primes DLBCL to BCL2 inhibitor-induced cell death by disrupting the codependency on BCL2 and MCL1, mimicking the biologic effects of NOXA genetic amplification. Our data provide a mechanistic rationale for combining HDAC inhibitors with BCL2 inhibitors in DLBCL., Although diffuse large B cell lymphoma (DLBCL) cells widely express the BCL2 protein, they rarely respond to treatment with BCL2-selective inhibitors. Here we show that DLBCL cells harboring PMAIP1/NOXA gene amplification were highly sensitive to BCL2 small-molecule inhibitors. In these cells, BCL2 inhibition induced cell death by activating caspase 9, which was further amplified by caspase-dependent cleavage and depletion of MCL1. In DLBCL cells lacking NOXA amplification, BCL2 inhibition was associated with an increase in MCL1 protein abundance in a BIM-dependent manner, causing a decreased antilymphoma efficacy. In these cells, dual inhibition of MCL1 and BCL2 was required for enhanced killing. Pharmacologic induction of NOXA, using the histone deacetylase inhibitor panobinostat, decreased MCL1 protein abundance and increased lymphoma cell vulnerability to BCL2 inhibitors in vitro and in vivo. Our data provide a mechanistic rationale for combination strategies to disrupt lymphoma cell codependency on BCL2 and MCL1 proteins in DLBCL.

Somatic mutations in CREBBP occur frequently in B-cell lymphoma. Here, we show that loss of CREBBP facilitates the development of germinal center (GC)–derived lymphomas in mice. In both human and murine lymphomas, CREBBP loss-of-function resulted in focal depletion of enhancer H3K27 acetylation and aberrant transcriptional silencing of genes that regulate B-cell signaling and immune responses, including class II MHC. Mechanistically, CREBBP-regulated enhancers are counter-regulated by the BCL6 transcriptional repressor in a complex with SMRT and HDAC3, which we found to bind extensively to MHC class II loci. HDAC3 loss-of-function rescued repression of these enhancers and corresponding genes, including MHC class II, and more profoundly suppressed CREBBP-mutant lymphomas in vitro and in vivo. Hence, CREBBP loss-of-function contributes to lymphomagenesis by enabling unopposed suppression of enhancers by BCL6/SMRT/HDAC3 complexes, suggesting HDAC3-targeted therapy as a precision approach for CREBBP-mutant lymphomas. Significance: Our findings establish the tumor suppressor function of CREBBP in GC lymphomas in which CREBBP mutations disable acetylation and result in unopposed deacetylation by BCL6/SMRT/HDAC3 complexes at enhancers of B-cell signaling and immune response genes. Hence, inhibition of HDAC3 can restore the enhancer histone acetylation and may serve as a targeted therapy for CREBBP-mutant lymphomas. Cancer Discov; 7(1); 38–53. ©2016 AACR. See related commentary by Höpken, p. 14. This article is highlighted in the In This Issue feature, p. 1

Grainy head (Grh) is a conserved transcription factor (TF) controlling epithelial differentiation and regeneration. To elucidate Grh functions, we identified embryonic Grh targets by ChIP-seq and gene expression analysis. We show that Grh controls hundreds of target genes. Repression or activation correlates with the distance of Grh binding sites to the transcription start sites of its targets. Analysis of 54 Grh-responsive enhancers during development and upon wounding suggests cooperation with distinct TFs in different contexts. In the airways, Grh repressed genes encode key TFs involved in branching and cell differentiation. Reduction of the POU-domain TF, Vvl, (ventral veins lacking) largely ameliorates the airway morphogenesis defects of grh mutants. Vvl and Grh proteins additionally interact with each other and regulate a set of common enhancers during epithelial morphogenesis. We conclude that Grh and Vvl participate in a regulatory network controlling epithelial maturation.

Grainy head (Grh) is a conserved transcription factor (TF) controlling epithelial differentiation and regeneration. To elucidate Grh functions we identified embryonic Grh targets by ChIP-seq and gene expression analysis. We show that Grh controls hundreds of target genes. Repression or activation correlates with the distance of Grh-binding sites to the transcription start sites of its targets. Analysis of 54 Grh-responsive enhancers during development and upon wounding suggests cooperation with distinct TFs in different contexts. In the airways, Grh-repressed genes encode key TFs involved in branching and cell differentiation. Reduction of the POU domain TF Ventral veins lacking (Vvl) largely ameliorates the airway morphogenesis defects of grh mutants. Vvl and Grh proteins additionally interact with each other and regulate a set of common enhancers during epithelial morphogenesis. We conclude that Grh and Vvl participate in a regulatory network controlling epithelial maturation.

Epidermal injury initiates a cascade of inflammation, epithelial remodelling and integument repair at wound sites. The regeneration of the extracellular barrier and damaged tissue repair rely on the precise orchestration of epithelial responses triggered by the injury. Grainy head (Grh) transcription factors induce gene expression to crosslink the extracellular barrier in wounded flies and mice. However, the activation mechanisms and functions of Grh factors in re-epithelialization remain unknown. Here we identify stitcher (stit), a new Grh target in Drosophila melanogaster. stit encodes a Ret-family receptor tyrosine kinase required for efficient epidermal wound healing. Live imaging analysis reveals that Stit promotes actin cable assembly during wound re-epithelialization. Stit activation also induces extracellular signal-regulated kinase (ERK) phosphorylation along with the Grh-dependent expression of stit and barrier repair genes at the wound sites. The transcriptional stimulation of stit on injury triggers a positive feedback loop increasing the magnitude of epithelial responses. Thus, Stit activation upon wounding coordinates cytoskeletal rearrangements and the level of Grh-mediated transcriptional wound responses.

SummaryThe function of tubular epithelial organs like the kidney and lung is critically dependent on the length and diameter of their constituting branches. Genetic analysis of tube size control during Drosophila tracheal development has revealed that epithelial septate junction (SJ) components and the dynamic chitinous luminal matrix coordinate tube growth. However, the underlying molecular mechanisms controlling tube expansion so far remained elusive. Here, we present the analysis of two luminal chitin binding proteins with predicted polysaccharide deacetylase activities (ChLDs). ChLDs are required to assemble the cable-like extracellular matrix (ECM) and restrict tracheal tube elongation. Overexpression of native, but not of mutated, ChLD versions also interferes with the structural integrity of the intraluminal ECM and causes aberrant tube elongation. Whereas ChLD mutants have normal SJ structure and function, the luminal deposition of the ChLD requires intact cellular SJs. This identifies a new molecular function for SJs in the apical secretion of ChLD and positions ChLD downstream of the SJs in tube length control. The deposition of the chitin luminal matrix first promotes and coordinates radial tube expansion. We propose that the subsequent structural modification of chitin by chitin binding deacetylases selectively instructs the termination of tube elongation to the underlying epithelium.

The Drosophila Ret-like receptor, Stit, upholds signaling from the protein complex TORC1 during wing epithelial development, promoting growth under normal conditions and protecting tissues from an anabolic to catabolic switch in response to starvation., Body size in Drosophila larvae, like in other animals, is controlled by nutrition. Nutrient restriction leads to catabolic responses in the majority of tissues, but the Drosophila mitotic imaginal discs continue growing. The nature of these differential control mechanisms that spare distinct tissues from starvation are poorly understood. Here, we reveal that the Ret-like receptor tyrosine kinase (RTK), Stitcher (Stit), is required for cell growth and proliferation through the PI3K-I/TORC1 pathway in the Drosophila wing disc. Both Stit and insulin receptor (InR) signaling activate PI3K-I and drive cellular proliferation and tissue growth. However, whereas optimal growth requires signaling from both InR and Stit, catabolic changes manifested by autophagy only occur when both signaling pathways are compromised. The combined activities of Stit and InR in ectodermal epithelial tissues provide an RTK-mediated, two-tiered reaction threshold to varying nutritional conditions that promote epithelial organ growth even at low levels of InR signaling., Author Summary Growth of organs, or anabolism, is tightly controlled by nutritional and hormonal cues such as insulin-like peptides that also suppress autophagy through their receptors and downstream growth pathway. Starvation conditions induce growth arrest and catabolism (involving autophagy) in some tissues while sparing the growth of other prioritized organs. The mechanism behind this tissue-specific regulation of growth versus catabolism is largely unknown. In this study, we show that Stitcher, a Drosophila Ret-oncogene-like growth factor receptor, controls epithelial tissue growth. Stitcher, working in parallel with the Insulin receptor, endows epithelial organs, such as imaginal wing discs, with resistance to low nutrient and insulin conditions by suppressing autophagy and, at the same time, promotes cell division and growth in these tissues. Thus, Stitcher and the Insulin receptor work together to allow a two-threshold response to starvation in epithelial tissues. In cancer, this pathway is almost invariably constitutively stimulated, and so we postulate that oncogenic mutations of Ret promote tumor growth partly by counteracting the tumor suppressive effects of autophagy.

The Grainy head (Grh) family of transcription factors is characterized by a unique DNA-binding domain that binds to a conserved consensus sequence. Nematodes and flies have a single grh gene, whereas mice and humans have evolved three genes encoding Grainy head-like (Grhl) factors. We review the biological function of Grh in different animals and the mechanisms modulating its activity. grh and grhl genes play a remarkably conserved role in epithelial organ development and extracellular barrier repair after tissue damage. Recent studies in flies and vertebrates suggest that Grh factors may be primary determinants of cell adhesion and epithelial tissue formation. Grh proteins can dimerize and act as activators or repressors in different developmental contexts. In flies, tissue-specific, alternative splicing generates different Grh isoforms with different DNA-binding specificities and functions. Grh activity is also modulated by receptor tyrosine kinases: it is phosphorylated by extracellular signal regulated kinase, and this phosphorylation is selectively required for epidermal barrier repair. Two mechanisms have been proposed to explain the repressive function of Grh on target gene transcription. First, Grh can target the Polycomb silencing complex to specific response elements. Second, it can directly compete for DNA binding with transcriptional activators. Understanding the molecular mechanisms of gene regulation by Grh factors is likely to elucidate phylogenetically conserved mechanisms of epithelial cell morphogenesis and regeneration upon tissue damage.

The Grainy head (Grh) family of transcription factors is characterized by a unique DNA-binding domain that binds to a conserved consensus sequence. Nematodes and flies have a single grh gene, whereas mice and humans have evolved three genes encoding Grainy head-like (Grhl) factors. We review the biological function of Grh in different animals and the mechanisms modulating its activity. grh and grhl genes play a remarkably conserved role in epithelial organ development and extracellular barrier repair after tissue damage. Recent studies in flies and vertebrates suggest that Grh factors may be primary determinants of cell adhesion and epithelial tissue formation. Grh proteins can dimerize and act as activators or repressors in different developmental contexts. In flies, tissue-specific, alternative splicing generates different Grh isoforms with different DNA-binding specificities and functions. Grh activity is also modulated by receptor tyrosine kinases: it is phosphorylated by extracellular signal regulated kinase, and this phosphorylation is selectively required for epidermal barrier repair. Two mechanisms have been proposed to explain the repressive function of Grh on target gene transcription. First, Grh can target the Polycomb silencing complex to specific response elements. Second, it can directly compete for DNA binding with transcriptional activators. Understanding the molecular mechanisms of gene regulation by Grh factors is likely to elucidate phylogenetically conserved mechanisms of epithelial cell morphogenesis and regeneration upon tissue damage.

The zinc finger proteins containing the Kruppel-associated box domain (KRAB-ZFPs) are the single largest class of transcription factors in human genome. Many of the KRAB-ZFPs are involved in cardiac development or cardiovascular diseases. Here, we have identified a novel human KRAB zinc finger gene, named ZNF328, from the human fetal heart cDNA library. The complete sequence of ZNF328 cDNA contains a 2376-bp open reading frame (ORF) and encodes a 792 amino acid protein with an N-terminal KRAB domain and classical zinc finger C2H2 motifs in the C-terminus. Northern blot analysis indicates that the protein is expressed in most of the examined human adult and embryonic tissues. ZNF328 is a transcription suppressor when fused to Gal-4 DNA-binding domain and cotransfected with VP-16. Overexpression of ZNF328 in COS-7 cells inhibits the transcriptional activities of SRE and AP-1. Deletion analysis with a series of truncated fusion proteins indicates that the KRAB motif is a basal repression domain when cotransfected with VP-16. Similar results were obtained when the truncated fusion proteins were assayed for the transcriptional activities of SRE and AP-1. These results suggest that ZNF328 protein may act as a transcriptional repressor in mitogen-activated protein kinase (MAPK) signaling pathway to mediate cellular functions.