Molecular and Genetic Characterization of MHC Deficiency Identifies EZH2 As a Therapeutic Target for Restoring MHC Expression in Diffuse Large B-Cell Lymphoma

Introduction: Among the tumor immune escape mechanisms described to date, alterations in the expression of major histocompatibility complex (MHC) molecules play a crucial role in the development of diffuse large B-cell lymphoma (DLBCL). Although the frequency of loss of MHC expression differs between ABC- and GCB-DLBCL cell of origin (COO) subtypes, distinct genetic alterations and molecular features that affect MHC expression and the composition of immune cells in the tumor microenvironment remain ill-defined. Here, we aimed to uncover the biologic and genomic basis underlying acquired loss of MHC expression.

Method: We analyzed biopsies from 347 patients newly diagnosed with de novo DLBCL and uniformly treated with R-CHOP in British Columbia. We performed targeted resequencing, SNP6.0 array and RNAseq for genetic analyses. Immunohistochemical (IHC) staining of MHC-I and -II was performed on tissue microarrays (n=332). COO was assigned by the Lymph2Cx assay in 323 cases (183 GCB, 104 ABC and 36 unclassifiable). Immune cell composition was assessed by IHC, flow cytometry and gene expression profiling (GEP)-based deconvolution of cellular signatures.

To experimentally confirm decreased MHC expression induced by EZH2 mutation, we measured surface MHC-I and -II expression on tumor B cells using EZH2Y641/BCL2 mouse model which was previously established (Beguelin et al, Cancer Cell 2013). We also treated human DLBCL cells harboring EZH2 mutation and wild type using EZH2 inhibitor (EPZ-6438), and evaluated their surface MHC-I and -II expression.

Results: Loss of MHC-I and -II expression was observed in 43% and 28% of DLBCL cases, respectively. MHC-II loss of expression was significantly associated with the reduction of tumor-infiltrating lymphocytes (TILs), especially CD4 positive T-cells (FOXP3⁺ cells, PD-1⁺ cells, and CD4⁺ naïve and memory T-cells), and cytolytic activity (GZMB and PRF1 mRNA expression) in GCB-DLBCL (all; p<0.001), but not in ABC-DLBCL. MHC-II-negativity was associated with unfavorable prognosis only in GCB-DLBCL (5-year time-to-progression; 59% vs 79%, p=0.007), whereas there was no prognostic impact of MHC-I expression in either subtype, suggesting a link between loss of MHC-II expression and reduced immune surveillance leading to poor prognosis, specifically in GCB-DLBCL.

We next performed GEP using RNAseq separately in each COO subtype. Interestingly, only four genes (HLA-DMA, DRA, DPA1 and CD74) were differentially expressed according to MHC-II expression (FDR<0.001) in ABC-DLBCL. By contrast, a total of 641 genes were differentially expressed in GCB-DLBCL. Of importance, a dark zone (DZ) B-cell signature was strongly enriched in MHC-II-negative GCB-DLBCL cases (FDR<0.001), suggesting that MHC-II deficiency defines the tumor originated from DZ of the germinal center.

Correlative genetic analysis revealed that, as expected, mutations of CIITA and RFXAP were detected more frequently in MHC-II-negative GCB-DLBCL (p=0.01 and 0.003, respectively). Strikingly, CD83 mutations, which elevate and stabilize MHC-II expression in centrocytes of the light zone (LZ), were significantly enriched in MHC-II positive GCB-DLBCL (p= 0.008), suggesting that these mutations affecting the antigen presentation machinery are selectively acquired in GCB-DLBCL tumors to further reduce and increase the surface MHC-II expression.

Genetic analysis also highlighted that EZH2 mutations were most significantly enriched in MHC-II-negative as well as MHC-I-negative GCB-DLBCL cases (both, p<0.001). Indeed, 77% of EZH2 mutated cases demonstrated loss of either MHC-I and/or MHC-II expression on the tumor cells. Notably, we found significantly lower MHC-I and MHC-II expression in high-grade lymphomas of EZH2 mutant Vav-BCL2 transgenic mice compared to EZH2 wildtype control tumors. Furthermore, of potential clinical relevance, in-vitro EZH2 inhibition significantly restored MHC-I and MHC-II gene expression as well as protein expression in EZH2-mutated human DLBCL cells, but not EZH2 wild type tumor cells.

Conclusion: Our findings provide important implications for understanding the cancer biology underlying acquired loss of MHC expression. The restoration of MHC expression by EZH2 inhibitors suggests a novel approach of epigenetically enhancing tumor recognition and eradication in combination with immune therapies.