(A) Lineage hierarchy in acute myeloid leukemia based on EMBLEM and prior genetic information. mtDNA mutations reveals pHSC clonal heterogeneity. The clonal precursor of the leukemic stem cell is not the clone with most representation in the pHSC pool, but rather the clone with epigenomic bias towards the leukemic regulatory program, as depicted by related color schemes. (B) EMBLEM deconvolutes AML clonal heterogeneity. Heteroplasmic mtDNA mutations in three cell populations from patients SU070 are shown. Mutations sites (in rows) in each FACS-sorted cell population (in columns) are shown, with size of each circle representing its VAF. Several mtDNA mutations (sites shown in purple) are detected in pHSCs and transmitted to LSCs and blasts, confirming those pHSC clones at the apex of leukemia lineage. LSCs accumulated additional mtDNA mutations (sites shown in green) and are transmitted to leukemic blasts in patient SU070. Allele frequency, sequencing depth and annotation of the variant allele are shown in Figure 2—figure supplement 1 and Supplementary file 2. (C) Same plot as (B) shown for patient SU353. In addition to the shared mtDNA mutations in pHSCs, LSCs, and blasts (purple), two pHSCs-specific mtDNA mutations are also detected (yellow). Allele frequency, sequencing depth and annotation of the variant allele are shown in Figure 2—figure supplement 1 and Supplementary file 2. (D) Heteroplasmic mutations in single pHSCs from one patient reveal clonal heterogeneity. Each column is a mtDNA nucleotide position; each row is one cell. Blue color indicates the presence of the mtDNA variant. Shown are cells with any mtDNA mutation detected, or cells with more than 40X coverage of the mitochondrial genome without any detected mutation(pHSC with WT mtDNA). The number of cells in each clonotype are indicated on the right. (E) Landscape of single-cell chromatin accessibility of blood progenitor and leukemic cells in patient SU353. tSNE map using bias-corrected deviations from chromatin accessibility showing cluster of AML blasts, LSCs, pHSCs and normal HSC, colored by cell types. (F) Chromatin accessibility of the FOS:JUN binding motif across the same single cells. tSNE map colored by deviation z-score for motif associated to FOS:JUN, the most variable TF motif. (G) pHSC clones possess distinct epigenomic signatures. Clone 1 that gives rise to the AML has a chromatin accessibility profile that more resembles LSCs and leukemic blasts. ‘WT’ pHSC refers to the pHSC with WT mtDNA. Clonotype information from EMBLEM is overlaid on the tSNE map defined by TF motif deviations, and colored by different lineal sub-populations defined by mtDNA mutations. (H) Quantitation of distinct single-cell chromatin accessibility at FOS:JUN motifs among different pHSC clones defined by EMBLE. Clone 1 pHSCs tend to down regulate FOS:JUN accessibility, while clone 2 pHSC shows substantially greater cell-to-cell variability. pHSCs with no detectable mtDNA variants and normal HSCs are shown for comparison. TF deviation of single cells (black dots) is shown on the distribution box-plot. The statistical significant were indicated by ‘*” when p<0.05, ‘**” when p<0.01(Wilcoxon rank-sum test).