Loss of CREBBP and KMT2D cooperate to accelerate lymphomagenesis and shape the lymphoma immune microenvironment.

Despite regulating overlapping gene enhancers and pathways, CREBBP and KMT2D mutations recurrently co-occur in germinal center (GC) B cell-derived lymphomas, suggesting potential oncogenic cooperation. Herein, we report that combined haploinsufficiency of Crebbp and Kmt2d induces a more severe mouse lymphoma phenotype (vs either allele alone) and unexpectedly confers an immune evasive microenvironment manifesting as CD8+ T-cell exhaustion and reduced infiltration. This is linked to profound repression of immune synapse genes that mediate crosstalk with T-cells, resulting in aberrant GC B cell fate decisions. From the epigenetic perspective, we observe interaction and mutually dependent binding and function of CREBBP and KMT2D on chromatin. Their combined deficiency preferentially impairs activation of immune synapse-responsive super-enhancers, pointing to a particular dependency for both co-activators at these specialized regulatory elements. Together, our data provide an example where chromatin modifier mutations cooperatively shape and induce an immune-evasive microenvironment to facilitate lymphomagenesis.

Cooperative super-enhancer inactivation caused by heterozygous loss of CREBBP and KMT2D skews B cell fate decisions and yields T cell-depleted lymphomas.

Mutations affecting enhancer chromatin regulators CREBBP and KMT2D are highly co-occurrent in germinal center (GC)-derived lymphomas and other tumors, even though regulating similar pathways. Herein, we report that combined haploinsufficiency of Crebbp and Kmt2d (C+K) indeed accelerated lymphomagenesis. C+K haploinsufficiency induced GC hyperplasia by altering cell fate decisions, skewing B cells away from memory and plasma cell differentiation. C+K deficiency particularly impaired enhancer activation for immune synapse genes involved in exiting the GC reaction. This effect was especially severe at super-enhancers for immunoregulatory and differentiation genes. Mechanistically, CREBBP and KMT2D formed a complex, were highly co-localized on chromatin, and were required for each-other's stable recruitment to enhancers. Notably, C+K lymphomas in mice and humans manifested significantly reduced CD8 + T-cell abundance. Hence, deficiency of C+K cooperatively induced an immune evasive phenotype due at least in part to failure to activate key immune synapse super-enhancers, associated with altered immune cell fate decisions. SIGNIFICANCE: Although CREBBP and KMT2D have similar enhancer regulatory functions, they are paradoxically co-mutated in lymphomas. We show that their combined loss causes specific disruption of super-enhancers driving immune synapse genes. Importantly, this leads to reduction of CD8 cells in lymphomas, linking super-enhancer function to immune surveillance, with implications for immunotherapy resistance.

Unique Immune Cell Coactivators Specify Locus Control Region Function and Cell Stage.

Locus control region (LCR) functions define cellular identity and have critical roles in diseases such as cancer, although the hierarchy of structural components and associated factors that drive functionality are incompletely understood. Here we show that OCA-B, a B cell-specific coactivator essential for germinal center (GC) formation, forms a ternary complex with the lymphoid-enriched OCT2 and GC-specific MEF2B transcription factors and that this complex occupies and activates an LCR that regulates the BCL6 proto-oncogene and is uniquely required by normal and malignant GC B cells. Mechanistically, through OCA-B-MED1 interactions, this complex is required for Mediator association with the BCL6 promoter. Densely tiled CRISPRi screening indicates that only LCR segments heavily bound by this ternary complex are essential for its function. Our results demonstrate how an intimately linked complex of lineage- and stage-specific factors converges on specific and highly essential enhancer elements to drive the function of a cell-type-defining LCR.

Selective Inhibition of HDAC3 Targets Synthetic Vulnerabilities and Activates Immune Surveillance in Lymphoma.

CREBBP mutations are highly recurrent in B-cell lymphomas and either inactivate its histone acetyltransferase (HAT) domain or truncate the protein. Herein, we show that these two classes of mutations yield different degrees of disruption of the epigenome, with HAT mutations being more severe and associated with inferior clinical outcome. Genes perturbed by CREBBP mutation are direct targets of the BCL6-HDAC3 onco-repressor complex. Accordingly, we show that HDAC3-selective inhibitors reverse CREBBP-mutant aberrant epigenetic programming, resulting in: (i) growth inhibition of lymphoma cells through induction of BCL6 target genes such as CDKN1A and (ii) restoration of immune surveillance due to induction of BCL6-repressed IFN pathway and antigen-presenting genes. By reactivating these genes, exposure to HDAC3 inhibitors restored the ability of tumor-infiltrating lymphocytes to kill DLBCL cells in an MHC class I and II-dependent manner, and synergized with PD-L1 blockade in a syngeneic model in vivo. Hence, HDAC3 inhibition represents a novel mechanism-based immune epigenetic therapy for CREBBP-mutant lymphomas. SIGNIFICANCE: We have leveraged the molecular characterization of different types of CREBBP mutations to define a rational approach for targeting these mutations through selective inhibition of HDAC3. This represents an attractive therapeutic avenue for targeting synthetic vulnerabilities in CREBBP-mutant cells in tandem with promoting antitumor immunity.This article is highlighted in the In This Issue feature, p. 327.

The serine hydroxymethyltransferase-2 (SHMT2) initiates lymphoma development through epigenetic tumor suppressor silencing.

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.

CREBBP Inactivation Promotes the Development of HDAC3-Dependent Lymphomas.

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. 14This article is highlighted in the In This Issue feature, p. 1.

Inducible deletion of the Blimp-1 gene in adult epidermis causes granulocyte-dominated chronic skin inflammation in mice.

B lymphocyte-induced maturation protein-1 (Blimp-1) is a transcriptional repressor important for the differentiation and function of several types of immune cells. Because skin serves as a physical barrier and acts as an immune sentinel, we investigated whether Blimp-1 is involved in epidermal immune function. We show that Blimp-1 expression is reduced in skin lesions of some human eczema samples and in stimulated primary keratinocytes. Epidermal-specific deletion of PR domain containing 1, with ZNF domain (Prdm1), the gene encoding Blimp-1, in adult mice caused spontaneously inflamed skin characterized by massive dermal infiltration of neutrophils/macrophages and development of chronic inflammation associated with higher levels of cytokines/chemokines, including granulocyte colony-stimulating factor (G-CSF), and enhanced myelopoiesis in bone marrow. Deletion of Prdm1 in the epidermis of adult mice also led to stronger inflammatory reactions in a tape-stripping test and in a disease model of contact dermatitis. The elevated G-CSF produced by keratinocytes after deletion of Prdm1 in vitro was mediated by the transcriptional activation of FBJ osteosarcoma oncogene (Fos) and fos-like antigen 1 (Fosl1). Systemic increases in G-CSF contributed to the inflammatory responses, because deletion of the G-CSF gene [colony stimulating factor 3, (Csf3)] prevented neutrophilia and partially ameliorated the inflamed skin in Prdm1-deficient mice. Our findings indicate a previously unreported function for Blimp-1 in restraining steady-state epidermal barrier immunity.

Induction of apoptosis in plasma cells by B lymphocyte-induced maturation protein-1 knockdown.

B lymphocyte-induced maturation protein-1 (Blimp-1) is a transcriptional repressor that plays an important role during plasmacytic differentiation and is expressed in normal and transformed plasma cells. We here investigated the importance of continuous Blimp-1 expression. We found that knockdown of Blimp-1 expression by lentiviral vector-delivered short hairpin RNA causes apoptosis in multiple myeloma cell lines and plasmacytoma cells, indicating that continued expression of Blimp-1 is required for cell survival. We examined the mechanism underlying Blimp-1 knockdown-mediated apoptosis and found that the Blimp-1 knockdown neither reversed the phenotypic markers of plasma cells nor caused cell cycle arrest. Instead, our results show that knockdown of Blimp-1 induced the proapoptotic protein Bim, reduced the antiapoptotic protein Mcl-1, and activated caspase-9 and caspase-3. We further link apoptosis in transformed plasma cells mediated by proteasome inhibitors, the effective therapeutic agent for multiple myeloma patients, with reduced expression of Blimp-1. Lastly, we show that Blimp-1-dependent cell survival may act downstream of IFN regulatory factor 4 (IRF4) because IRF4 knockdown leads to down-regulation of Blimp-1 and apoptosis in multiple myeloma cells and plasmacytoma cells. Together, our data suggest that Blimp-1 ensures the survival of transformed plasma cells.