High-throughput single-cell joint RNA and DNA methylation mapping of Acute Myeloid Leukemia

PROJECT SUMMARY DNA methylation (5mC) is an essential epigenetic mechanism crucial for the establishment and maintenance of cell function. Dysregulation of DNA methylation leads to various diseases, such as hematological malignancies. In acute myeloid leukemia (AML), aberrations in DNA methylation patterns are a central feature, often arising from mutations in epigenetic regulators such as DNMT3A and TET2. These mutations disrupt the normal epigenetic programming of hematopoietic stem cells (HSCs), leading to aberrant gene expression and methylation patterns that are characteristic of AML. However, the intricate interplay between DNA methylation and gene regulation, especially in a cell-type-specific manner, remains largely unknown, limiting our understanding of the epigenetic mechanisms underlying cancer initiation and progression. Recent advances in single-cell technologies have been revolutionizing our understanding of tissue structure and disease progression. However, current single-cell 5mC mapping techniques are either low throughput (96 or 384 cells per assay), low data quality, or without paired scRNA-seq information, limiting their application in cancers. To bridge the gap, we seek to develop a scalable and cost-effective single-cell multi-omic profiling method (SHARE-ME-seq) that jointly captures gene expression, chromatin accessibility, and DNA methylation at the single-cell level, in a larger effort to make a substantive leap in AML treatment by identifying and targeting methylation-driven pathways. In Aim 1, we will develop and optimize SHARE-ME-seq in cell line mixture and mouse brain samples, focusing on improving the data quality and throughput of the assay. We expect SHARE-ME-seq to achieve a throughput of 1 million cells in a single assay with sensitivity rivaling current state-of-the-art methods and 100 times lower cost. In Aim 2, we will apply SHARE-ME-seq to bone marrow samples from 4 healthy donors and 8 AML donors with TET2 or DNMT3A mutations, generating ~360k high-quality multi-omic single-cell profiles. By further integrating single-cell transcriptome and chromatin accessibility data that is measured simultaneously with DNA methylation, we will ask i) how DNA methylation, chromatin accessibility, and RNA co-define hematopoietic cell type and cell state and ii) how DNA methylation contributes to lineage priming in AML with different mutations. Together, these experiments enable genome-wide unbiased identification of 5mC-associated regulatory elements, transcription factors, and candidate genes in AML providing unprecedented insights into the regulatory mechanisms underlying cancer cell plasticity and metastasis. This approach is broadly applicable to various cancers and primary tissue types, holding the promise of unveiling new therapeutic targets in malignancies and potentially formulating approaches to counteract the cancers.