Subtype-Specific and Structure Variation-Induced Chromatin Spatial Reorganization in Acute Myeloid Leukemia [Hi-C]

Acute myeloid leukemia (AML) is a set of heterogeneous myeloid malignancies hallmarked by mutations in epigenetic modifiers, transcription factors and kinases that can cause epigenetic reshaping. It is unclear whether those mutations drive chromatin 3D structure alteration and contribute to oncogenic dysregulation in AML. By performing Hi-C and whole genome sequencing in 21 primary AML and healthy donors’ samples, we identified recurrent AML- or subtype-specific alteration of compartments, TADs, and chromatin loops. To study the impact on gene regulation, we performed RNA-Seq, ATAC-Seq and CUT&TAG for CTCF, H3K27ac, and H3K27me3 in the same samples. We observed dysregulation of many AML-related genes, represented by MYCN, MEIS1, WT1, ERG, MYC GATA3, BCL11B and IKZF2, intimately linked to the recurrent gain of loops and switch of compartment or TAD, alongside acquisition of AML-specific enhancer or repressor. Further, we profiled structure variations using WGS and Hi-C data to reconstruct the cancer 3D genome, by which we identified structure variation-induced neo-loops and enhancer-hijacking events. Furthermore, through conducting whole genome bisulfite sequencing in patient samples, we found altered methylation correlated with A/B compartment switch, and loss of CTCF insulation due to hypermethylation, leading to extensive gain of loops in AML. By treating the AML cells with DNA hypomethylation agent 5-azacytidine, the altered chromatin structure and gene expression can be restored, with switched compartment reverted and gained loops dissociated, alongside compromised AML cell proliferation, overall providing insights into AML treatment through therapeutic restoration of chromatin structure. We performed Hi-C and RNA-seq in 18 primary luekmiea samples and purified PBMC from 3 healthy controls. We also performed Hi-C in the following 5 conditions: Kasumi-1 treated wih DMSO for 48 hours, Kasumi-1 treated with 2uM 5-azacytidine for 48 hours and 0.5uM 5-azacytidine for 12 days, HL-60 treated with DMSO for 48 hours and HL-60 treated with 1uM 5-azacytidien for 48 hours . We also performed RNA-seq with two biological replicates each in Kasumi-1 cells treated with DMSO (48h) and Kasumi-1 cells treated with 2uM 5-azacytidine (48h). We performed whole genome sequencing on 18 leukemia samples, whole geome bisulfite sequencing in 10 AML samples and 2 controls, ATAC-seq in 3 controls and 15 leukemia samples, and CUT&TAG for H3K27ac and H3K27me3 in 2 controls and 11 leukemia samples, and CUT&TAG for CTCF in 2 controls and 8 leukemia samples. The raw data files have not been included due to privacy concerns.