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 laser-capture 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 of PD-1 ligands and their induction by JAK2, defining the PD-1 pathway and JAK2 as complementary rational therapeutic targets.

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 antitumor immune responses and foster tumor cell survival. PD-1 blockade has shown promise in multiple malignancies but should be directed toward 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 Epstein–Barr virus (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, more than 70% of EBV⁺ posttransplant 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. Clin Cancer Res; 18(6); 1611–8. ©2012 AACR.

The role of B-cell receptor (BCR)–mediated survival signals in diffuse large B-cell lymphoma (DLBCL) remains undefined. Ligand-induced BCR signaling induces receptor oligomerization, Igα/β immunoreceptor tyrosine-based activation motif (ITAM) phosphorylation, and activation of the spleen tyrosine kinase (SYK), which initiates downstream events and amplifies the initial BCR signal. BCRs also transmit low-level tonic survival signals in the absence of receptor engagement. Herein, we assess the role of SYK-dependent tonic BCR survival signals in DLBCL cell lines and primary tumors and evaluate the efficacy of an ATP-competitive inhibitor of SYK, R406, in vitro. R406 induced apoptosis of the majority of examined DLBCL cell lines. In R406-sensitive DLBCL cell lines, R406 specifically inhibited both tonic- and ligand-induced BCR signaling (autophosphorylation of SYK525/526 and SYK-dependent phosphorylation of the B-cell linker protein [BLNK]). The majority of examined primary DLBCLs also exhibited tonic- and ligand-induced BCR signaling; in these primary tumors, BCR signaling was also inhibited by R406. Of note, BCR-dependent and R406-sensitive DLBCL cell lines were independently identified as “BCR-type” tumors by transcriptional profiling. Therefore, SYK-dependent tonic BCR signaling is an important and potentially targetable survival pathway in some, but not all, DLBCLs. In addition, R406-sensitive DLBCLs can be identified by their transcriptional profiles.

Classical Hodgkin lymphomas (cHLs) contain small numbers of neoplastic Reed–Sternberg (RS) cells within an extensive inflammatory infiltrate that includes abundant T helper (Th)-2 and T regulatory (T _reg ) cells. The skewed nature of the T cell infiltrate and the lack of an effective host antitumor immune response suggest that RS cells use potent mechanisms to evade immune attack. In a screen for T cell-inhibitory molecules in cHL, we found that RS cells selectively overexpressed the immunoregulatory glycan-binding protein, galectin-1 (Gal1), through an AP1-dependent enhancer. In cocultures of activated T cells and Hodgkin cell lines, RNAi-mediated blockade of RS cell Gal1 increased T cell viability and restored the Th1/Th2 balance. In contrast, Gal1 treatment of activated T cells favored the secretion of Th2 cytokines and the expansion of CD4 ⁺ CD25 ^high FOXP3 ⁺ T _reg cells. These data directly implicate RS cell Gal1 in the development and maintenance of an immunosuppressive Th2/T _reg -skewed microenvironment in cHL and provide the molecular basis for selective Gal1 expression in RS cells. Thus, Gal1 represents a potential therapeutic target for restoring immune surveillance in cHL.