Fig. 5: Npm1c and Flt3-ITD leukemia abrogates normal chromatin factor function to maintain leukemic fitness through enforcing differentiation blockade.
From: In vivo screening characterizes chromatin factor functions during normal and malignant hematopoiesis
a, UMAP projection of single-cell transcriptomes from Npm1c and Flt3-ITD primary leukemia. The color-coded clusters correspond to cells with specific signatures: leukemic stem cells (LSC, green), granulocyte-like (Gra.1 and Gra.2, Gran-perturbed, blue), erythroid-like (Ery.1 and Ery.2, red) and basophil-like (Baso-like, yellow). The analysis integrates datasets from six different Perturb-seq experiments. b, mRNA-derived and CITE-seq-derived expression of lineage makers in the differentiated leukemia subpopulations. c,d, Clonogenic (c) and proliferation (d) assays for differentiated leukemia subpopulations, isolated according to the strategy in Extended Data Fig. 9e. Colonies were counted after 7 days of culture in methylcellulose. Proliferation and clonogenic values were obtained from n = 4 biologically independent experiments. ****P < 0.0001 (two-way analysis of variance (ANOVA)). The error bars are the s.e.m., the midpoints show the mean. e, Enrichment analyses of specific chromatin factor knockouts across differentiated leukemia subpopulations. Dot color and size relate to the log2(OR) and the percentage of significant enrichments versus NTCs, respectively. The analysis is based on measurements for two sgRNAs per chromatin factor target. All values are shown in Supplementary Table 6. Gra.P1 and Gra.P2, granulocyte-like. f, Perturbed growth curves for leukemic chromatin factor knockouts. The assay measures the change in the proportion of blue fluorescent protein (BFP) BFP sgRNA-expressing cells over time, n = 4 biologically independent experiments. All ***P < 0.001, except for Smarcd1 versus NTC (day 9) where ***P = 0.0009 (two-way ANOVA). The error bars are the s.e.m., the midpoints show the mean. g, FACS analysis of mega-erythroid (CD55) and myeloid (CD11b, Gr1) surface differentiation markers in leukemia cells depleted for cBAF (Smarcb1 and Smarcd2 knockout) and MLL (Kmt2a knockout) components. h, FACS analysis of myeloid surface differentiation markers (CD11b, Gr1) in leukemia cells treated with increasing doses of Men1 inhibitor (revumenib). Raw data can be found in Supplementary Data 1.
