NEW YORK – Through a single-cell, multi-omic analysis, researchers homed in on a transcription factor that regulates genes associated with poorer survival in mixed-phenotype acute leukemia.
Researchers led by Stanford University's William Greenleaf sought to identify pathological molecular features of mixed-phenotype acute leukemia by first analyzing the single-cell transcriptomic and epigenetic profiles of healthy blood cells during their development. Once they established profiles of those healthy cells, they examined how the profiles of leukemic cells compared. As they reported Monday in Nature Biotechnology, the researchers uncovered both common and patient-specific regulatory features. In particular, they found that transcription factor RUNX1 regulates leukemia-specific genes that are associated with poorer survival.
"These results demonstrate how integrative, multi-omic analysis of single cells within the framework of normal development can reveal both distinct and shared molecular mechanisms of disease from patient samples," Greenleaf and his colleagues wrote in their paper.
Mixed-phenotype acute leukemia exhibits features of both acute myeloid leukemia and acute lymphoblastic leukemia and, as such, is marked by features of multiple hematopoietic lineages.
For their analysis, the researchers first performed droplet-based cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) and single-cell assay for transposase-accessible chromatin by sequencing (scATAC-seq) of more than 35,000 healthy bone marrow and peripheral blood mononuclear cells. With this, they generated multi-omic maps of hematopoiesis. They validated the maps and found them to reflect the "essential phenotypic, transcriptomic, and epigenetic features of blood development."
After establishing these maps, the researchers developed a framework to analyze signatures of hematopoietic development at the single-cell level. With this, they then sought to examine how those signatures differed between healthy and leukemic cells. They assayed thousands of single cells from mixed-phenotype acute leukemia (MPAL) samples using both CITE-seq and scATAC-seq and identified 4,616 genes that were differentially upregulated and 72,196 significantly upregulated peaks. They projected these single-cell analyses onto their hematopoietic maps to find epigenetic and gene expression diversity and grouped the cells into broad hematopoietic development compartments.
When they compared the leukemic programs of these MPAL hematopoietic compartments to data from previous studies of leukemia, the researchers found both similarities and differences between MPAL and AML leukemic programs, which they postulated could account for the poor response of MPALs to AML treatments.
The researchers also focused on the transcription factors that might regulate these leukemia programs. They found that RUNX1 motifs were enriched among certain MPAL subsets. RUNX1, they noted, is a frequently mutated gene in hematological malignancies, and they uncovered 732 genes regulated by a RUNX1-containing distal element in at least two MPAL subsets.
Additionally, CD69 — which has been linked to lymphocyte activation through JAK-STAT signaling and lymphocyte retention in lymphoid organs — was differentially upregulated in nearly every MPAL subset.
When the researchers then stratified patients from The Cancer Genome Atlas with AML based on their RUNX1-target genes, they found that those with a high RUNX1-target gene signature had a significant decrease in survival. Based on this, the researchers concluded that RUNX1 acts as a potential oncogene in MPAL and possibly AML, as it regulates malignant genes associated with poorer overall survival.
"We anticipate that similar approaches will be used in future studies to … enable molecular dissection of molecular dysfunction in pathogenic cellular subtypes, with the ultimate goal of identifying personalized therapeutic targets through integrative single-cell molecular characterization," the researchers added in their paper.