Integrated Epigenetic Profiling Identifies EZH2 As a Therapeutic Target to Re-Establish Immune Recognition of Leukemia Relapses with Loss of HLA Class II Expression

Background It is becoming increasingly recognized that evasion from immune control represents one of the main drivers of acute myeloid leukemia (AML) relapse after allogeneic hematopoietic cell transplantation (allo-HCT). In particular, alterations in the antigen processing and presentation machinery represent one of the most effective strategies enacted by tumor cells to avoid recognition from T cells. Whereas it is now well recognized that genomic loss of HLA is frequently at the basis of post-transplantation relapse, it was only recently reported that up to 40% of AML relapses display transcriptional downregulation and complete loss of surface expression of HLA class II molecules without any genetic lesion explaining this phenotype (Christopher et al, N Engl J Med, 2018; Toffalori et al, Nat Med, 2019). This led us to investigate the links between epigenetic changes, immune evasion and post-transplantation relapse. Methods Starting from primary AML samples pairwise collected from patients at diagnosis and relapse with non-genomic loss of HLA class II expression, we generated Patients-Derived Xenografts (PDXs) into NOD-SCID γ-chain null mice. Leukemic cells expanded in the mice and their original human counterparts were analyzed for surface expression of selected immune-related markers (HLA class I and II, PD-L1, B7-H3), and characterized for changes in gene expression (by RNA-Seq), DNA methylation profile (by RRBS), histone modifications associated with active promoters (H3K4me3) or regulatory elements (H3K27ac) (by ChIP-seq) and chromatin accessibility (by ATAC-Seq). The results obtained by all these approaches were integrated by Multi-Omics Factor Analysis (MOFA), followed by Gene Set Enrichment Analysis (GSEA). Finally, the immunological effects of epigenetic drugs and recombinant immune-modulatory cytokines on primary and PDX-derived AML samples were tested in ex-vivo short-term cultures on a layer of mesenchymal stromal cells. Results We verified that PDXs faithfully recapitulate immune-related differences between diagnosis and post-transplantation relapse, including loss of expression of HLA class II molecules (Figure 1A). Integration of all the high-throughput technologies by MOFA evidenced that the differences between diagnosis and post-transplantation relapse samples were mostly explained by changes in chromatin accessibility and histone marks, and largely unrelated to the DNA methylation profile (Figure 1B). We documented that the gene sets that emerged upon integrating epigenetic analyses by MOFA matched our previously published immune-related relapse signature (Toffalori et al, Nat Med, 2019) but also and most intriguingly lists of genes known to be targeted by EZH2, the enzymatic subunit of the PRC2 chromatin repressor complex (Figure 1C). These computational analyses were supported by the evidence of a relapse-specific closed chromatin status of HLA class II genes and their regulators (Figure 1D). To revert these epigenetic changes, we inhibited EZH2 with tazemetostat (EPZ-6438), an epigenetic drug currently being tested in early-phase clinical trials for lymphomas, in two AML relapses with non-genomic loss of HLA class II expression. EZH2 inhibition reduced the levels of the repressor mark H3K27me3 (Figure 1E), increased the surface expression of HLA class II molecules on leukemia cells (Figure 1F) and ultimately improved leukemia recognition by CD4+ T cells (Figure 1G). Notably, these effects were even more pronounced when EZH2 inhibition was combined with IFN-g treatment (Figure 1F,G), suggesting synergism between this epigenetic compound and cytokines released by immune cells upon target recognition. Conclusions Our results provide mechanistic insights into epigenetic regulation of HLA class II downregulation in leukemia and a strong therapeutic rationale to test EZH2 inhibition as an innovative strategy for the treatment of AML post-transplantation relapses. Figure 1 Disclosures Vago: GenDx: Research Funding; Moderna Therapeutics: Research Funding.

Ligandomes obtained from different HLA-class II-molecules are homologous for N- and C-terminal residues outside the peptide-binding cleft

Human CD4+ T lymphocytes play an important role in inducing potent immune responses. T cells are activated and stimulated by peptides presented in human leucocyte antigen (HLA)-class II molecules. These HLA-class II molecules typically present peptides of between 12 and 20 amino acids in length. The region that interacts with the HLA molecule, designated as the peptide-binding core, is highly conserved in the residues which anchor the peptide to the molecule. In addition, as these peptides are the product of proteolytic cleavages, certain conserved residues may be expected at the N- and C-termini outside the binding core. To study whether similar conserved residues are present in different cell types, potentially harbouring different proteolytic enzymes, the ligandomes of HLA-DRB1*03:01/HLA-DRB > 1 derived from two different cell types (dendritic cells and EBV-transformed B cells) were identified with mass spectrometry and the binding core and N- and C-terminal residues of a total of 16,568 peptides were analysed using the frequencies of the amino acids in the human proteome. Similar binding motifs were found as well as comparable conservations in the N- and C-terminal residues. Furthermore, the terminal conservations of these ligandomes were compared to the N- and C-terminal conservations of the ligandome acquired from dendritic cells homozygous for HLA-DRB1*04:01. Again, comparable conservations were evident with only minor differences. Taken together, these data show that there are conservations in the terminal residues of peptides, presumably the result of the activity of proteases involved in antigen processing.

HLA and microtubule-associated protein tau H1 haplotype associations in anti-IgLON5 disease

ObjectivesWe investigated the associations with HLA and microtubule-associated protein tau (MAPT) H1 haplotype in anti-IgLON5 disease, a recently identified disorder characterized by gait instability, brainstem dysfunction, and a prominent sleep disorder in association with IgLON5 antibodies and pathologic findings of a novel neuronal-specific tauopathy.MethodsWe compared the HLA alleles and MAPT H1/H1 genotype of 35 patients with anti-IgLON5 with healthy controls. The on-line server tool NetMHCIIpan 3.1 was used to predict the IgLON5 peptide binding to HLA Class II molecules.ResultsThe HLA-DRB1*10:01-DQB1*05:01 haplotype was overrepresented in patients with anti-IgLON5 disease (OR = 54.5; 95% CI: 22.2–133.9, p < 0.0001). In addition, HLA-DQA was genotyped in 27 patients, and 25 (92.6%) of them had DQ molecules composed by DQA1*01 and DQB1*05 chains compared with 148/542 (27.3%) controls (OR = 43.9; 95% CI: 10.4–185.5, p < 0.0001). Patients DRB1*10:01 positive developed more frequently sleep or bulbar symptoms than those carrying other HLA alleles (70.0% vs 26.7%; p = 0.011). Prediction algorithms identified 2 IgLON5 peptides (1 located in the signal sequence) that showed strong binding to HLA-DRB1*10:01 and other HLA-DRB1, but not to HLA-DQA and HLA-DQB molecules. The MAPT H1/H1 homozygous genotype was present in 20/24 (83.3%) anti-IgLON5 Caucasian patients compared with 54/116 (46.5%) healthy controls (p = 0.0007).ConclusionsThe robust association of anti-IgLON5 disease with distinct HLA Class II molecules supports a primary autoimmune origin. The significant association of MAPT H1 haplotype also suggests that an underlying neurodegenerative process could be involved in anti-IgLON5 disease.

Relapse Following Allogeneic Hematopoietic Cell Transplantation for Acute Myeloid Leukemia Apparently Due to Somatic Cell Evolution via Epigenetic Variation and Immune Selection

In this brief commentary, I discuss a recently published study that documents the role of immune escape in relapse of acute myeloid leukemia (AML) after hematopoietic cell transplantation (HCT). Of particular interest, the mechanism identified by the authors for the ability of the malignant cells to evade destruction by host T cells is the loss of cell surface expression of HLA class II molecules based on processes other than mutation. The authors labeled this mechanism for altered cell surface display of HLA class II antigens “epigenetic.” This study should be of strong interest for immunologists, oncologists and even specialists in infectious diseases for several reasons. First, the results extend the range of examples for which epigenetic mechanisms can play a critical role in resistance to therapy in oncology or infectious disease. Second, findings relating to decreased cell surface display of HLA class II molecules motivate investigation of novel approaches using cytokines to increase the numbers of HLA class II proteins on malignant myeloid cell membranes and reduce the extent of immune escape by these cells. Third, the data presented suggest experimental directions intended to clarify detailed molecular mechanisms underlying the cases of AML post-HCT relapse and raise questions relating to why some mechanisms of somatic cell evolution and not others are operative in different clinical settings.