Fig. 3: Inhibition of purine biosynthesis promotes myeloid differentiation of LSCs in vitro.

a The read numbers of sgRNAs against purine biosynthetic genes at day 25 compared to day 10 and input from a whole-genome CRISPR screen in MOLM-13 cells; (n = 5, individual sgRNA). Each dot represents one sgRNA. D, day. b Cancer cell line dependency scores (CERES) of purine biosynthetic genes in AML (n = 20) and other cancers (n = 749) from DepMap. The box plot presents an interquartile range, and the whiskers show a 95% confidence interval. c Competitive growth assays with Cas9-expressing MOLM-13 cells that express sgRNAs against negative control (NC, negative control), positive control (MYC), and genes involved in the purine biosynthetic pathway over 17 days. The percentages of sgRNA-expressing cells were normalized to those on day 4 after transduction. d Flow cytometry histograms of mature myeloid cell markers CD11b and Gr-1 expression in murine LSCs upon treatment with MPA, MMF, or 6-MP (0.1-1 μM) for 24 h. MPA, mycophenolic acid; 6-MP, 6-mercaptopurine. e Wright–Giemsa staining of LSCs showing myeloid differentiation upon treatment with MMF, MPA, and 6-MP (0.1-1 μM) for 24 h. Scale bar, 20 μm. f Fluorescence images showing engulfment of GFP-labeled Streptococcus agalactiae COH1 by LSC-derived myeloid cells after exposure to MMF (1 μM) for 24 h. Scale bar, 20 μm. g Relative abundance of guanosine mono and dinucleotides in LSCs treated with MMF (0.25 μM) or guanosine (100 μM) alone or in combination for 2 h (n = 5, biologically independent samples). h Mean fluorescence intensity (MFI) of mature myeloid cell marker CD11b (left panel) and Gr-1 (right panel) in LSCs treated with MMF (0.25-0.5 μM) alone or in combination with guanosine (100 μM) for 24 h (n = 3, biologically independent samples). i, j Cell cycle (i) and apoptosis (j) analyses of LSCs upon treatment with MMF (0.25-1 μM) alone or in combination with guanosine (100 μM) for 24 h (n = 3, biologically independent samples). k, l Percentage of BFP (present in sgRNA vector) (k) and MFI of CD11b (l) in Cas9-expressing LSCs after the transduction of sgRNA targeting Rosa26, Impdh1, or Impdh2 genes for 4 days (n = 3, biologically independent samples). m MFI of CD11b in a panel of AML cell lines treated with MMF (0.25-0.5 μM) for 3 days (n = 3, biologically independent samples). n Relative MFI of CD11b in PDX samples treated with MMF (1 μM) for 6 days (n = 3, biological samples). All data are represented as mean ± SD. p values in this figure were calculated by unpaired, two-tailed Student’s t-test (b, l and n) or ANOVA with multiple comparisons analysis using Bonferroni correction post hoc analyses (a, g–k and m). See also Supplementary Fig. 3. Source data are provided as a Source Data file.