Fig 2: Response to 5-AZA/VEN therapy in patients with AML can be predicted by MAC scoring in LSC-like cells. A, Schematic representation of the experimental design for B–G. Mononuclear cells of AML patient samples treated first-line with 5-AZA/VEN from three independently processed cohorts (cohort 1: n = 17, cohort 2: n = 18, and vohort 3: n = 24) were stained with surface antibodies, followed by intracellular staining of three BCL2 family proteins. MAC-Score was calculated based on normalized BCL2 family protein expression levels in LSC-like, non-LSC, mature, and total blast cells. B, Expression of BCL2, MCL1, and BCL-xL in LSC-like cells of patients with AML from cohorts 1 and 2 combined and associated 5-AZA/VEN therapy outcome. Protein expression is shown as MFI z-scores. C, MAC-Score in LSC-like cells of patients with AML from cohorts 1 and 2 combined and association to 5-AZA/VEN therapy outcome. D, Comparison of MAC-Score in LSC-like, non-LSC, mature, and total blast cells of patients with AML from cohorts 1 and 2 and association to 5-AZA/VEN therapy outcome. E, EFS of first-line 5-AZA/VEN AML patients from cohorts 1 and 2 combined with above and below median MAC-Score, BCL2 expression, MCL1 expression, or BCL-xL expression in LSC-like cells. F, MAC-Score in LSC-like cells of patients with AML from cohort 3 and associated 5-AZA/VEN therapy outcome. G, EFS of first-line 5-AZA/VEN AML patients from cohort 3 with above (>0.4) and below (<0.4) median MAC-Score in LSC-like cells. H, Schematic representation of the experimental design for I–J. Mononuclear cells of patients with relapsed/refractory AML who received 5-AZA/VEN as a salvage therapy (cohort 4: n = 23) were stained with surface antibodies, followed by intracellular staining of BCL2 family proteins. I, MAC-Score in LSC-like cells of patients with AML from cohort 4 and associated 5-AZA/VEN therapy outcome. J, EFS of salvage-treated 5-AZA/VEN AML patients from cohort 4 with above (>0.4) and below (<0.4) median MAC-Score determined in LSC-like cells. Each dot represents an AML patient sample with the line marking the mean unless specified otherwise. Mann–Whitney test was used to compare groups and log-rank test to compare therapy durations of AML patients. R/R, relapsed/refractory to standard induction. Parts of the figure were created with BioRender.com.

Fig 3: LSC-like cells as defined by functional and transcriptomic parameters are predominantly located in the immature GPR56+ but not in the CD64+CD11b+ mature subpopulation. A, FACS gating strategy for mature, non-LSC, and LSC-like subpopulations. Displayed are AML bulk cells from primitive CD34+ (NPM1-wild-type), CD34- (NPM1-mutated), and monocytic (NPM1-mutated) samples. B, Percentages of mature, non-LSC-like, and LSC-like subpopulations among bulk AML cells in 72 diagnostic AML samples sorted by the frequency of the mature population. C, Schematic overview of the experimental setup for xenotransplantation experiments and RNA sequencing (RNA-seq) of FACS-sorted subpopulations. D, Percentage of human leukemic engraftment obtained from mature, non-LSC, and LSC-like subpopulations of 14 AML samples at endpoints in the bone marrow of NSG mice. Each dot represents an individual mouse with the line marking mean engraftment levels. E, Mean percentage of human engraftment per mouse obtained from mature, non-LSC, and LSC-like subpopulations of 14 AML samples at endpoints in the bone marrow of NSG mice. Each dot represents an individual patient with AML with the line marking mean engraftment levels. Friedmann test was used to compare LSC-like with non–LSC-like and mature subpopulations. F, PCA plot of bulk RNA-seq data from LSC-like, non-LSC, and mature subpopulations from Prim-AML (n = 14) and Mono-AML (n = 9) annotated based on subpopulation and AML subclass. Each dot represents a population from one AML sample. G, LSC17 score in LSC-like, non-LSC, and mature subpopulations from Prim-AML (n = 14) or Mono-AML (n = 9) patient samples. LSC17 score was calculated for each AML sample as the mean expression of the 17 LSC signature genes. H–J, Normalized counts of BCL2 (H), MCL1 (I), and BCL2L1 (J) expression in LSC-like, non-LSC, and mature subpopulations from Prim-AML (n = 14) or Mono-AML (n = 9) patient samples. K, Schematic representation of the experimental setup used in L–O to measure intracellular BCL2, MCL1, and BCL-xL protein expression by flow cytometry. L and M, Mean fluorescence intensity (MFI) of BCL2 in (L) AML bulk and (M) LSC-like, non-LSC, and mature subpopulations from Prim-AML (n = 11) or Mono-AML (n = 7) patient samples. N and O, MFI of MCL1 in (N) AML bulk and (O) LSC-like, non-LSC, and mature subpopulations from Prim-AML (N = 11) or Mono-AML (N = 7) patient samples. P and Q, Representative tSNE plots of (P) AML26 (Prim-AML) and (Q) AML50 (Mono-AML) showing expression of CD64, CD34, GPR56, MCL1, BCL2, and BCL-xL. AML bulk is defined as mononuclear cells from patients with AML after the exclusion of dead cells, doublets, lymphocytes, and nucleated erythrocytes. Two-way ANOVA with Tukey correction for multiple comparisons test was used to compare groups of four, and Mann–Whitney test was used to compare groups of two unless specified otherwise. Each dot represents an AML patient sample with the line marking the mean unless specified otherwise. Fix, fixation; IC-staining, intracellular staining; Mut, mutated; NL, non-LSC; NSG, NOD.Prkdcscid.Il2rgnull; Perm, permeabilization; WT, wild-type. Parts of the figure were created with BioRender.com.