Fig 2: 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.

Fig 3: Analysis of apoptosis in ABCB7-deficient pro-B cells.(A, B) Intracellular flow cytometry analysis of Bcl-xL (B) and Mcl-1 (C) expression in Fr. C cells (B220+ CD19+ CD43+ BP-1+) from wild-type (WT) and Mb1-cre ABCB7 conditional knockout (cKO) mice. Quantification of MdFI is shown on the right of each plot. Isotype controls are shown in gray. Offset histograms are representative of at least three independent experiments (total of 4–10 mice/group). (C) Flow cytometry analysis of Annexin V binding and fixable viability dye (FVD) labeling of Fr. B-C’ cells from WT and Mb1-cre ABCB7 cKO mice. Quantification of the proportion of each fraction that are dead (Annexin V+ FVD+) is shown on the right. Contour plots are representative of four independent experiments (total of 8–10 mice/group). (D) Quantification of the proportion of each fraction that are apoptotic (Annexin V+ FVD-) from (C). (E) Quantification of the proportion of each fraction that are apoptotic (Annexin V+ FVD-) after 16 hr in culture. Data represent three independent experiments (total of six mice/group). (A–E) Error bars represent SEM, and p-values are indicated above the data. Statistics were obtained by using an unpaired Student’s t-test.

Fig 4: Most malignant cell lines are resistant to BH3 mimetics independently of p53 status(A) TP53WT cell lines were treated with the indicated concentrations of venetoclax (Bcl-2 inhibitor, N = 2), A-1331852 (Bcl-XL inhibitor, N = 3) or S-63845 (Mcl-1 inhibitor, N = 3) for 24 h. Viability was measured by flow cytometry using Mito-O/To-Pro-3 staining.(B) TP53WT and TP53KO clones deriving from Nalm-6 (N = 3), OCI-Ly10 (N = 2), LME-1 (N = 3), and PGA-1 (n = 1) were incubated with S-63845 for 24 h. Viability was assessed by flow cytometry using Mito-O/To-Pro-3 staining. Data are presented as mean ± SEM and differences were analyzed with two-way ANOVA with Tukey’s multiple comparison test (∗p < 0.05, ∗∗p < 0.01).(C) PGA-1 TP53WT and TP53KO clones were cultured in complete media for 48 h, RNA was extracted and expression of TP53 transcriptional targets and apoptosis-related genes was investigated using RT-MLPA. Data were averaged and plotted as Z score using the ComplexHeatmap R package.

Fig 5: Glutamine deprivation sensitizes malignant B cells of different origin to BH3 mimetics(A) PGA-1 TP53 WT and TP53 KO clones were cultured in compete or Gln- for 48 h, RNA was extracted and expression of TP53 transcriptional targets and apoptosis-related genes was investigated using RT-MLPA. Data were averaged and plotted as fold change to complete media in a heatmap using the ComplexHeatmap R package (N = 2). Crosses indicate missing data.(B) Bcl-2 family members expression was analyzed by flow cytometry in PGA-1 TP53 WT and TP53 KO cells (N = 2).(C) PGA-1 and OCI-Ly10 cells with different p53 status were incubated in Gln-for 16 h and subsequently treated with A-1331852 (Bcl-XL inhibition) (PGA-1 WT N = 6, the rest N = 3).(D) Parental PGA-1 cells were incubated in Gln-for 16 h and subsequently treated with venetoclax (Bcl-2 inhibition, N = 2) or S-63845 (Mcl-1 inhibition, N = 2) for 24 h.(E) PBMCs from TP53+/+ and TP53−/− CLL patients were cultured ex vivo in unstimulated conditions in complete media (Us) or on top of CD40L-expressing 3T3 fibroblasts to stimulate CD40 signaling in the presence or absence of Gln. Percentage of leukemic cells (CD5+CD19+) and CLL activation (CD19+CD95+) were measured 24 h post-stimulation. At that time, a fraction of CLL cells was harvested and treated with venetoclax for additional 24h. Viability was assessed by Mito-O/To-Pro-3 staining (TP53+/+ N = 7, TP53−/− N = 3). Data are presented as mean ± SEM and differences were analyzed with two-way ANOVA with Tukey’s multiple comparison test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).