Classical Hodgkin lymphoma (cHL) and mediastinal large B-cell lymphoma (MLBCL) are lymphoid malignancies with certain shared clinical, histologic, and molecular features. Primary cHLs and MLBCLs include variable numbers of malignant cells within an inflammatory infiltrate, suggesting that these tumors escape immune surveillance. Herein, we integrate high-resolution copy number data with transcriptional profiles and identify the immunoregulatory genes, PD-L1 and PD-L2, as key targets at the 9p24.1 amplification peak in HL and MLBCL cell lines. We extend these findings to lasercapture microdissected primary Hodgkin Reed-Sternberg cells and primary MLBCLs and find that programmed cell death-1 (PD-1) ligand/9p24.1 amplification is restricted to nodular sclerosing HL, the cHL subtype most closely related to MLBCL. Using quantitative immunohistochemical methods, we document the association between 9p24.1 copy number and PD-1 ligand expression in primary tumors. In cHL and MLBCL, the extended 9p24.1 amplification region also included the Janus kinase 2 (JAK2) locus. Of note, JAK2 amplification increased protein expression and activity, specifically induced PD-1 ligand transcription and enhanced sensitivity to JAK2 inhibition. Therefore, 9p24.1 amplification is a disease-specific structural alteration that increases both the gene dosage IntroductionClassical Hodgkin lymphoma (cHL) is a B-cell malignancy that occurs frequently in Western countries and commonly affects young adults. 1 These tumors are characterized by small numbers of neoplastic Reed-Sternberg (RS) cells within an extensive inflammatory/immune cell infiltrate. There are 4 subtypes of cHL, 2 of which comprise Ϸ 90% of cases: nodular sclerosing Hodgkin lymphoma (NSHL; 60% of cases) and mixed cellularity Hodgkin lymphoma (MCHL; 30% of cases). cHLs lack surface immunoglobulin expression and B-cell receptor-mediated signals and rely on alternative survival pathways, including aberrant nuclear factorB signaling. 1 In previous studies, we and others have defined shared molecular features of cHL and a specific subtype of diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma (MLBCL). 2,3 Like cHL, MLBCLs have a T-helper cell type 2 (Th2)-skewed cytokine profile, decreased expression of B-cell receptor signaling pathway components, and constitutive activation of nuclear factorB. 2 MLBCL also exhibits certain clinical and histologic similarities to cHL, particularly the NSHL subtype. 4,5 For example, both diseases are most common in young adults and often present as an anterior mediastinal or localized nodal mass. 2,4,5 In addition, both MLBCLs and NSHLs include bands of sclerotic tissue and immune/inflammatory cell infiltrates. 4,5 However, the inflammatory infiltrate is less prominent in MLBCLs, which have a more diffuse growth pattern. 4 Although cHLs have an extensive polymorphous inflammatory infiltrate, there is little evidence of an effective host antitumor immune response. In fact, recent studies indicate that Hodgkin RS cells pro...

Purpose Programmed cell death ligand 1 (PD-L1) is a molecule expressed on antigen-presenting cells that engages the PD-1 receptor on T cells and inhibits T-cell receptor signaling. The PD-1 axis can be exploited by tumor cells to dampen host anti-tumor immune responses and foster tumor cell survival. PD-1 blockade has shown promise in multiple malignancies but should be directed towards patients in whom it will be most effective. In recent studies, we found that the chromosome 9p24.1 amplification increased the gene dosage of PD-L1 and its induction by JAK2 in a subset of patients with classical Hodgkin lymphoma (cHL). However, cHLs with normal 9p24.1 copy numbers also expressed detectable PD-L1, prompting analyses of additional PD-L1 regulatory mechanisms. Experimental Design Herein, we utilized immunohistochemical, genomic and functional analyses to define alternative mechanisms of PD-L1 activation in cHL and additional EBV+ lymphoproliferative disorders. Results We identified an AP-1-responsive enhancer in the PD-L1 gene. In cHL Reed Sternberg cells, which exhibit constitutive AP-1 activation, the PD-L1 enhancer binds AP-1 components and increases PD-L1 promoter activity. In addition, we defined EBV infection as an alternative mechanism for PD-L1 induction in cHLs with diploid 9p24.1. PD-L1 was also expressed by EBV-transformed lymphoblastoid cell lines as a result of latent membrane protein 1-mediated, JAK/STAT-dependent promoter and AP-1-associated enhancer activity. In addition, over 70% of EBV+ post-transplant lymphoproliferative disorders expressed detectable PD-L1. Conclusions AP-1 signaling and EBV infection represent alternative mechanisms of PD-L1 induction and extend the spectrum of tumors in which to consider PD-1 blockade.

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SUMMARY B-cell receptor (BCR) signaling pathway components represent promising treatment targets in diffuse large B-cell lymphoma (DLBCL) and additional B-cell tumors. BCR signaling activates spleen tyrosine kinase (SYK) and downstream pathways including PI3K/AKT and NF-κB. In previous studies, chemical SYK blockade selectively decreased BCR signaling and induced apoptosis of BCR-dependent DLBCLs. Herein, we characterize distinct SYK/PI3K-dependent survival pathways in DLBCLs with high or low baseline NF-κB activity including selective repression of the pro-apoptotic HRK protein in NF-κB-low tumors. We also define SYK/PI3K-dependent cholesterol biosynthesis as a feed-forward mechanism of maintaining the integrity of BCRs in lipid rafts in DLBCLs with low or high NF-κB. In addition, SYK amplification and PTEN deletion are identified as selective genetic alterations in primary “BCR”-type DLBCLs.

Diffuse large B cell lymphomas (DLBCLs) often express BCL6, a transcriptional repressor required for the formation of normal germinal centers. In a subset of DLBCLs, BCL6 is deregulated by chromosomal translocations or aberrant somatic hypermutation; in other tumors, BCL6 expression may simply reflect germinal center lineage. DLBCLs dependent on BCL6-regulated pathways should exhibit differential regulation of BCL6 target genes. Genomic array ChIP-on-chip was used to identify the cohort of direct BCL6 target genes. This set of genes was enriched in modulators of transcription, chromatin structure, protein ubiquitylation, cell cycle, and DNA damage responses. In primary DLBCLs classified on the basis of gene expression profiles, these BCL6 target genes were clearly differentially regulated in ''BCR'' tumors, a subset of DLBCLs with increased BCL6 expression and more frequent BCL6 translocations. In a panel of DLBCL cell lines analyzed by expression arrays and classified according to their gene expression profiles, only BCR tumors were highly sensitive to the BCL6 peptide inhibitor, BPI. These studies identify a discrete subset of DLBCLs that are reliant on BCL6 signaling and uniquely sensitive to BCL6 inhibitors. More broadly, these data show how genome-wide identification of direct target genes can identify tumors dependent on oncogenic transcription factors and amenable to targeted therapeutics.targeted therapy ͉ transcriptional repression ͉ ChIP on ChIP ͉ integrative analysis ͉ gene expression profiling B CL6 is a BTB/POZ domain transcription repressor that is required for normal germinal center (GC) development and expressed by the majority of normal GC B cells and a subset of diffuse large B cell lymphomas (DLBCLs) (1, 2). BCL6 favors the survival and proliferation of GC B cells, which undergo somatic hypermutation of Ig variable regions and Ig class switch recombination; down-regulation of BCL6 is necessary for post-GC B cell maturation (3-5). Deregulation of BCL6, by chromosomal translocation or aberrant somatic hypermutation, is the most common genetic abnormality in DLBCL (6). Conclusive evidence for the oncogenic role of BCL6 comes from murine models in which constitutive BCL6 expression results in the development of a lymphoid malignancy resembling DLBCL (7,8). Although deregulated BCL6 clearly plays a pathogenetic role in a subset of human DLBCLs, other DLBCLs may simply express this transcriptional repressor because the lymphomas are derived from normal BCL6 ϩ GC B cells. Identification of BCL6-dependent tumors has important therapeutic implications because a recently described specific BCL6 peptide inhibitor (BPI) inhibits the growth of some but not all DLBCLs (9).To delineate functionally relevant DLBCL subsets, we and others have used gene expression signatures. In an earlier approach known as the cell of origin (COO) classification, subsets of DLBCLs were associated with specific types of normal B cells [GC B cells (GCB) or in vitro activated peripheral blood B cells (ABC)] or left unassigned if ...

BAL1 is a transcription modulator that is overexpressed in chemoresistant, diffuse large B-cell lymphomas (DLBCLs).

Summary Although the BBAP E3 ligase and its binding partner, BAL, are overexpressed in chemotherapy-resistant lymphomas, the role of these proteins in DNA damage responses remains undefined. Since BAL proteins modulate promoter-coupled transcription and contain structural motifs associated with chromatin remodeling and DNA repair, we reasoned that the BBAP E3 ligase might target nucleosomal proteins. Herein, we demonstrate that BBAP selectively monoubiquitylates histone H4 lysine 91 and protects cells exposed to DNA damaging agents. Disruption of BBAP-mediated monoubiquitylation of histone H4K91 is associated with the loss of chromatin-associated H4K20 methylase, mono- and dimethyl H4K20, and a delay in the kinetics of 53BP1 foci formation at sites of DNA damage. Since 53BP1 localizes to DNA damage sites, in part, via an interaction with dimethyl H4K20, these data directly implicate BBAP in the monoubiquitylation and additional post-translational modification of histone H4 and an associated DNA damage response.