Relapse associated aberrant DNA hypermethylation in AML post-allogeneic stem cell transplantation Free

Introduction

Allogeneic stem cell transplantation (SCT) remains a cornerstone of curative therapy for acute myeloid leukemia (AML), yet relapse occurs in up to 50% of cases. While clinical and molecular predictors are well studied, there are limited studies describing the DNA methylation landscape of relapsed AML following transplantation. Given the clinical efficacy of hypomethylating agents in this setting, we postulated that aberrant DNA hypermethylation may be linked to gene suppression and immune escape as a mechanism contributing to post-SCT relapse.

Methods

We conducted reduced representation bisulfite sequencing (RRBS) for single nucleotide resolution of DNA methylation and RNA-seq using bone marrow mononuclear cells (BMMC) from 7 AML patients at 4 timepoints: diagnosis, post-chemotherapy remission, post-SCT remission, and relapse post-SCT (Study 1). In a second independent cohort (Study 2), we performed RRBS and myeloid-panel next-generation sequencing (NGS) using BMMC from 12 AML patients at diagnosis and relapse (6 post-SCT and 6 post-chemotherapy). Diagnosis and relapse samples with high blast counts were selected (median 87%) to minimize the effects of cellular heterogeneity. The mean CpG coverage was 2.8 million per sample with a minimum cutoff of 10 reads for individual CpGs. Differential methylation was defined as a ≥25% difference in methylation of CpGs within 3 kb of transcriptional start sites. Mean differentially methylated cytosines (DMCs) was calculated per million CpGs sequenced to allow for different sequencing depth across samples.

Results

Study 1 enabled intra-patient comparison with normal BM cells (post-chemotherapy remission versus diagnosis and post-SCT remission versus post-SCT relapse) to identify DMCs and correlate with gene expression changes. Post-SCT relapse samples had 1,032 DMCs (625 hypermethylated and 407 hypomethylated). Reactome pathway analysis of the 334 nearest genes of the 625 hypermethylated DMCs revealed pathways related to development and signaling. In contrast, AML at diagnosis had only 20 DMCs compared with post-chemotherapy remission.

Study 2 allowed us to compare the impact of treatment (SCT vs chemotherapy) on the changes in methylation status from diagnosis to relapse. We hypothesized that post-SCT samples would have a distinct methylation profile enriched for immune pathways. Overall, there were 1,162 DMCs per million CpGs sequenced (78% hypermethylated) in post-SCT relapse samples compared with diagnosis. A similar number of DMCs (2,264 DMCs per million CpGs sequenced) were observed in post-chemotherapy relapse samples compared with diagnosis. However, there was 10-fold more shared genes (present in at least 4 of the 6 samples) within 3kb of DMCs in post-SCT relapse samples (371 compared with 32 in post-chemotherapy relapse samples). We used the 7 paired diagnosis and post-SCT relapse samples of Study 1 to independently validate 224 of those 371 (60%) commonly shared genes. The 224 genes nearest to the DMCs (present in at least 8 of 13 post-SCT relapses) were enriched for developmental and lineage-specification pathways, including developmental biology, gastrulation and regulation of beta-cell development. Gene set enrichment analysis (GSEA) using an immune specific gene set revealed post-SCT relapse was enriched for hypermethylation in gene sets associated with B cell stimulation, myeloid activation and inflammatory or vaccine-related immune responses. In contrast, chemotherapy relapse showed no significant enrichment (FDR q value >0.25) and exhibited a more heterogeneous and attenuated immune methylation profile.

We performed NGS on 13 diagnosis and post-SCT relapse to determine if patients without clonal evolution may exhibit higher methylation. However, mean normalized CpG methylation was not significantly different in patients with acquired mutations at post-SCT relapse compared to those with stable mutations (mean 3,360 DMCs per million CPGs sequenced vs mean 1,396 DMCs per million CpGs sequenced, p = 0.71, Mann–Whitney U test).

Conclusion

Despite the small number of cases examined, we found that DNA hypermethylation was more pronounced and coordinated in AML relapse after SCT than chemotherapy. These epigenetic changes may contribute to a stem-like phenotype and immune evasion, independent of gene mutations. These findings support the rationale for exploring hypomethylating agents as part of post-SCT surveillance or pre-emptive relapse therapy.