Tumor-associated antigen PRAME exhibits dualistic functions that are targetable in diffuse large B cell lymphoma.

PRAME is a prominent member of the cancer testis antigen family of proteins, which triggers autologous T cell-mediated immune responses. Integrative genomic analysis in diffuse large B cell lymphoma (DLBCL) uncovered recurrent and highly focal deletions of 22q11.22, including the PRAME gene, which were associated with poor outcome. PRAME-deleted tumors showed cytotoxic T cell immune escape and were associated with cold tumor microenvironments. In addition, PRAME downmodulation was strongly associated with somatic EZH2 Y641 mutations in DLBCL. In turn, PRC2-regulated genes were repressed in isogenic PRAME-KO lymphoma cell lines, and PRAME was found to directly interact with EZH2 as a negative regulator. EZH2 inhibition with EPZ-6438 abrogated these extrinsic and intrinsic effects, leading to PRAME expression and microenvironment restoration in vivo. Our data highlight multiple functions of PRAME during lymphomagenesis and provide a preclinical rationale for synergistic therapies combining epigenetic reprogramming with PRAME-targeted therapies.

TMEM30A loss-of-function mutations drive lymphomagenesis and confer therapeutically exploitable vulnerability in B-cell lymphoma.

Transmembrane protein 30A (TMEM30A) maintains the asymmetric distribution of phosphatidylserine, an integral component of the cell membrane and 'eat-me' signal recognized by macrophages. Integrative genomic and transcriptomic analysis of diffuse large B-cell lymphoma (DLBCL) from the British Columbia population-based registry uncovered recurrent biallelic TMEM30A loss-of-function mutations, which were associated with a favorable outcome and uniquely observed in DLBCL. Using TMEM30A-knockout systems, increased accumulation of chemotherapy drugs was observed in TMEM30A-knockout cell lines and TMEM30A-mutated primary cells, explaining the improved treatment outcome. Furthermore, we found increased tumor-associated macrophages and an enhanced effect of anti-CD47 blockade limiting tumor growth in TMEM30A-knockout models. By contrast, we show that TMEM30A loss-of-function increases B-cell signaling following antigen stimulation-a mechanism conferring selective advantage during B-cell lymphoma development. Our data highlight a multifaceted role for TMEM30A in B-cell lymphomagenesis, and characterize intrinsic and extrinsic vulnerabilities of cancer cells that can be therapeutically exploited.

Single-Cell Transcriptome Analysis Reveals Disease-Defining T-cell Subsets in the Tumor Microenvironment of Classic Hodgkin Lymphoma.

Hodgkin lymphoma is characterized by an extensively dominant tumor microenvironment (TME) composed of different types of noncancerous immune cells with rare malignant cells. Characterization of the cellular components and their spatial relationship is crucial to understanding cross-talk and therapeutic targeting in the TME. We performed single-cell RNA sequencing of more than 127,000 cells from 22 Hodgkin lymphoma tissue specimens and 5 reactive lymph nodes, profiling for the first time the phenotype of the Hodgkin lymphoma-specific immune microenvironment at single-cell resolution. Single-cell expression profiling identified a novel Hodgkin lymphoma-associated subset of T cells with prominent expression of the inhibitory receptor LAG3, and functional analyses established this LAG3+ T-cell population as a mediator of immunosuppression. Multiplexed spatial assessment of immune cells in the microenvironment also revealed increased LAG3+ T cells in the direct vicinity of MHC class II-deficient tumor cells. Our findings provide novel insights into TME biology and suggest new approaches to immune-checkpoint targeting in Hodgkin lymphoma. SIGNIFICANCE: We provide detailed functional and spatial characteristics of immune cells in classic Hodgkin lymphoma at single-cell resolution. Specifically, we identified a regulatory T-cell-like immunosuppressive subset of LAG3+ T cells contributing to the immune-escape phenotype. Our insights aid in the development of novel biomarkers and combination treatment strategies targeting immune checkpoints.See related commentary by Fisher and Oh, p. 342.This article is highlighted in the In This Issue feature, p. 327.

Targeting Hypoxia-Induced Carbonic Anhydrase IX Enhances Immune-Checkpoint Blockade Locally and Systemically.

Treatment strategies involving immune-checkpoint blockade (ICB) have significantly improved survival for a subset of patients across a broad spectrum of advanced solid cancers. Despite this, considerable room for improving response rates remains. The tumor microenvironment (TME) is a hurdle to immune function, as the altered metabolism-related acidic microenvironment of solid tumors decreases immune activity. Here, we determined that expression of the hypoxia-induced, cell-surface pH regulatory enzyme carbonic anhydrase IX (CAIX) is associated with worse overall survival in a cohort of 449 patients with melanoma. We found that targeting CAIX with the small-molecule SLC-0111 reduced glycolytic metabolism of tumor cells and extracellular acidification, resulting in increased immune cell killing. SLC-0111 treatment in combination with immune-checkpoint inhibitors led to the sensitization of tumors to ICB, which led to an enhanced Th1 response, decreased tumor growth, and reduced metastasis. We identified that increased expression of CA9 is associated with a reduced Th1 response in metastatic melanoma and basal-like breast cancer TCGA cohorts. These data suggest that targeting CAIX in the TME in combination with ICB is a potential therapeutic strategy for enhancing response and survival in patients with hypoxic solid malignancies.

Regulation of pH by Carbonic Anhydrase 9 Mediates Survival of Pancreatic Cancer Cells With Activated KRAS in Response to Hypoxia.

BACKGROUND & AIMS: Most pancreatic ductal adenocarcinomas (PDACs) express an activated form of KRAS, become hypoxic and dysplastic, and are refractory to chemo and radiation therapies. To survive in the hypoxic environment, PDAC cells upregulate enzymes and transporters involved in pH regulation, including the extracellular facing carbonic anhydrase 9 (CA9). We evaluated the effect of blocking CA9, in combination with administration of gemcitabine, in mouse models of pancreatic cancer. METHODS: We knocked down expression of KRAS in human (PK-8 and PK-1) PDAC cells with small hairpin RNAs. Human and mouse (KrasG12D/Pdx1-Cre/Tp53/RosaYFP) PDAC cells were incubated with inhibitors of MEK (trametinib) or extracellular signal-regulated kinase (ERK), and some cells were cultured under hypoxic conditions. We measured levels and stability of the hypoxia-inducible factor 1 subunit alpha (HIF1A), endothelial PAS domain 1 protein (EPAS1, also called HIF2A), CA9, solute carrier family 16 member 4 (SLC16A4, also called MCT4), and SLC2A1 (also called GLUT1) by immunoblot analyses. We analyzed intracellular pH (pHi) and extracellular metabolic flux. We knocked down expression of CA9 in PDAC cells, or inhibited CA9 with SLC-0111, incubated them with gemcitabine, and assessed pHi, metabolic flux, and cytotoxicity under normoxic and hypoxic conditions. Cells were also injected into either immune-compromised or immune-competent mice and growth of xenograft tumors was assessed. Tumor fragments derived from patients with PDAC were surgically ligated to the pancreas of mice and the growth of tumors was assessed. We performed tissue microarray analyses of 205 human PDAC samples to measure levels of CA9 and associated expression of genes that regulate hypoxia with outcomes of patients using the Cancer Genome Atlas database. RESULTS: Under hypoxic conditions, PDAC cells had increased levels of HIF1A and HIF2A, upregulated expression of CA9, and activated glycolysis. Knockdown of KRAS in PDAC cells, or incubation with trametinib, reduced the posttranscriptional stabilization of HIF1A and HIF2A, upregulation of CA9, pHi, and glycolysis in response to hypoxia. CA9 was expressed by 66% of PDAC samples analyzed; high expression of genes associated with metabolic adaptation to hypoxia, including CA9, correlated with significantly reduced survival times of patients. Knockdown or pharmacologic inhibition of CA9 in PDAC cells significantly reduced pHi in cells under hypoxic conditions, decreased gemcitabine-induced glycolysis, and increased their sensitivity to gemcitabine. PDAC cells with knockdown of CA9 formed smaller xenograft tumors in mice, and injection of gemcitabine inhibited tumor growth and significantly increased survival times of mice. In mice with xenograft tumors grown from human PDAC cells, oral administration of SLC-0111 and injection of gemcitabine increased intratumor acidosis and increased cell death. These tumors, and tumors grown from PDAC patient-derived tumor fragments, grew more slowly than xenograft tumors in mice given control agents, resulting in longer survival times. In KrasG12D/Pdx1-Cre/Tp53/RosaYFP genetically modified mice, oral administration of SLC-0111 and injection of gemcitabine reduced numbers of B cells in tumors. CONCLUSIONS: In response to hypoxia, PDAC cells that express activated KRAS increase expression of CA9, via stabilization of HIF1A and HIF2A, to regulate pH and glycolysis. Disruption of this pathway slows growth of PDAC xenograft tumors in mice and might be developed for treatment of pancreatic cancer.

Molecular and Genetic Characterization of MHC Deficiency Identifies EZH2 as Therapeutic Target for Enhancing Immune Recognition.

We performed a genomic, transcriptomic, and immunophenotypic study of 347 patients with diffuse large B-cell lymphoma (DLBCL) to uncover the molecular basis underlying acquired deficiency of MHC expression. Low MHC-II expression defines tumors originating from the centroblast-rich dark zone of the germinal center (GC) that was associated with inferior prognosis. MHC-II-deficient tumors were characterized by somatically acquired gene mutations reducing MHC-II expression and a lower amount of tumor-infiltrating lymphocytes. In particular, we demonstrated a strong enrichment of EZH2 mutations in both MHC-I- and MHC-II-negative primary lymphomas, and observed reduced MHC expression and T-cell infiltrates in murine lymphoma models expressing mutant Ezh2 Y641. Of clinical relevance, EZH2 inhibitors significantly restored MHC expression in EZH2-mutated human DLBCL cell lines. Hence, our findings suggest a tumor progression model of acquired immune escape in GC-derived lymphomas and pave the way for development of complementary therapeutic approaches combining immunotherapy with epigenetic reprogramming. SIGNIFICANCE: We demonstrate how MHC-deficient lymphoid tumors evolve in a cell-of-origin-specific context. Specifically, EZH2 mutations were identified as a genetic mechanism underlying acquired MHC deficiency. The paradigmatic restoration of MHC expression by EZH2 inhibitors provides the rationale for synergistic therapies combining immunotherapies with epigenetic reprogramming to enhance tumor recognition and elimination.See related commentary by Velcheti et al., p. 472.This article is highlighted in the In This Issue feature, p. 453.

Double-Hit Gene Expression Signature Defines a Distinct Subgroup of Germinal Center B-Cell-Like Diffuse Large B-Cell Lymphoma.

PURPOSE: High-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH) has a poor outcome after standard chemoimmunotherapy. We sought to understand the biologic underpinnings of HGBL-DH/TH with BCL2 rearrangements (HGBL-DH/TH- BCL2) and diffuse large B-cell lymphoma (DLBCL) morphology through examination of gene expression. PATIENTS AND METHODS: We analyzed RNA sequencing data from 157 de novo germinal center B-cell-like (GCB)-DLBCLs, including 25 with HGBL-DH/TH- BCL2, to define a gene expression signature that distinguishes HGBL-DH/TH- BCL2 from other GCB-DLBCLs. To assess the genetic, molecular, and phenotypic features associated with this signature, we analyzed targeted resequencing, whole-exome sequencing, RNA sequencing, and immunohistochemistry data. RESULTS: We developed a 104-gene double-hit signature (DHITsig) that assigned 27% of GCB-DLBCLs to the DHITsig-positive group, with only one half harboring MYC and BCL2 rearrangements (HGBL-DH/TH- BCL2). DHITsig-positive patients had inferior outcomes after rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone immunochemotherapy compared with DHITsig-negative patients (5-year time to progression rate, 57% and 81%, respectively; P < .001), irrespective of HGBL-DH/TH- BCL2 status. The prognostic value of DHITsig was confirmed in an independent validation cohort. DHITsig-positive tumors are biologically characterized by a putative non-light zone germinal center cell of origin and a distinct mutational landscape that comprises genes associated with chromatin modification. A new NanoString assay (DLBCL90) recapitulated the prognostic significance and RNA sequencing assignments. Validating the association with HGBL-DH/TH- BCL2, 11 of 25 DHITsig-positive-transformed follicular lymphomas were classified as HGBL-DH/TH- BCL2 compared with zero of 50 in the DHITsig-negative group. Furthermore, the DHITsig was shared with the majority of B-cell lymphomas with high-grade morphology tested. CONCLUSION: We have defined a clinically and biologically distinct subgroup of tumors within GCB-DLBCL characterized by a gene expression signature of HGBL-DH/TH- BCL2. This knowledge has been translated into an assay applicable to routinely available biopsy samples, which enables exploration of its utility to guide patient management.

Genetic profiling of MYC and BCL2 in diffuse large B-cell lymphoma determines cell-of-origin-specific clinical impact.

The clinical significance of MYC and BCL2 genetic alterations in diffuse large B-cell lymphoma (DLBCL), apart from translocations, has not been comprehensively investigated using high-resolution genetic assays. In this study, we profiled MYC and BCL2 genetic alterations using next-generation sequencing and high-resolution SNP array in 347 de novo DLBCL cases treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) at the British Columbia Cancer Agency. Cell-of-origin (COO) subtype was determined by Lymph2Cx digital gene expression profiling. We showed that the incidence of MYC/BCL2 genetic alterations and their clinical significance were largely dependent on COO subtypes. It is noteworthy that the presence of BCL2 gain/amplification is significantly associated with poor outcome in activated B-cell-like and BCL2 translocation with poor outcome in germinal center B-cell subtypes, respectively. Both have prognostic significance independent of MYC/BCL2 dual expression and the International Prognostic Index (IPI). Furthermore, the combination of BCL2 genetic alterations with IPI identifies markedly worse prognostic groups within individual COO subtypes. Thus, high-resolution genomic assays identify extremely poor prognostic groups within each COO subtype on the basis of BCL2 genetic status in this large, uniformly R-CHOP-treated population-based cohort of DLBCL. These results suggest COO subtype-specific biomarkers based on BCL2 genetic alterations can be used to risk-stratify patients with DLBCL treated with immunochemotherapy.

Genome-Wide Profiles of Extra-cranial Malignant Rhabdoid Tumors Reveal Heterogeneity and Dysregulated Developmental Pathways.

Malignant rhabdoid tumors (MRTs) are rare lethal tumors of childhood that most commonly occur in the kidney and brain. MRTs are driven by SMARCB1 loss, but the molecular consequences of SMARCB1 loss in extra-cranial tumors have not been comprehensively described and genomic resources for analyses of extra-cranial MRT are limited. To provide such data, we used whole-genome sequencing, whole-genome bisulfite sequencing, whole transcriptome (RNA-seq) and microRNA sequencing (miRNA-seq), and histone modification profiling to characterize extra-cranial MRTs. Our analyses revealed gene expression and methylation subgroups and focused on dysregulated pathways, including those involved in neural crest development.

MEF2B mutations in non-Hodgkin lymphoma dysregulate cell migration by decreasing MEF2B target gene activation.

Myocyte enhancer factor 2B (MEF2B) is a transcription factor with mutation hotspots at K4, Y69 and D83 in diffuse large B-cell lymphoma (DLBCL). To provide insight into the regulatory network of MEF2B, in this study, we analyse global gene expression and DNA-binding patterns. We find that candidate MEF2B direct target genes include RHOB, RHOD, CDH13, ITGA5 and CAV1, and that indirect target genes of MEF2B include MYC, TGFB1, CARD11, MEF2C, NDRG1 and FN1. MEF2B overexpression increases HEK293A cell migration and epithelial-mesenchymal transition, and decreases DLBCL cell chemotaxis. K4E, Y69H and D83V MEF2B mutations decrease the capacity of MEF2B to activate transcription and decrease its' effects on cell migration. The K4E and D83V mutations decrease MEF2B DNA binding. In conclusion, our map of the MEF2B regulome connects MEF2B to drivers of oncogenesis.