Fig. 6: Inhibition of purine biosynthesis reduces LSC gene expression.

a Volcano plot showing the genes downregulated, upregulated, and not significantly affected by MMF treatment in LSCs. Differentially Expressed Genes (DEGs) were identified using adjusted p < 0.05, and log₂(fold change) >1 or <−1. b Venn diagrams showing the number of genes downregulated by MMF compared to control, and genes upregulated by MMF plus guanosine compared to MMF treatment alone. c Gene set enrichment analysis (GSEA) plot showing negative enrichment of MLL-AF9 target genes²⁵ in MMF-treated versus control LSCs. d, e GSEA plots showing positive enrichment of MLL-AF9 target genes²⁵ (d) and LSC gene signature³³ (e) in LSCs treated with MMF and guanosine versus those treated only with MMF. f Top 30 Gene Ontology (GO) terms for genes downregulated by MMF compared to control, as determined by DAVID functional annotation analysis. g GSEA plot showing negative enrichment of genes directly regulated by MLL-AF9²⁴ in CX-5461-treated versus control LSCs. h Volcano plot showing altered chromatin accessibility of LSCs induced by MMF (0.25 μM) treatment for 16 h. i, j Motif analysis of MMF-induced regions with increased (i) and reduced (j) chromatin accessibility in LSCs by ATAC-seq. k Peaks of the altered chromatin accessibility in LSCs induced by MMF, overlapped with or without menin binding. l Meta profiles of ATAC-seq data from control and MMF-treated LSCs near genes downregulated by MMF. m Immunoblots of NF-YA, menin, LEDGF, and GAPDH on separate membranes in LSCs treated with control vehicle, MMF (250-1000 nM) or CX-5461 (25-100 nM) for 16 h. A representative blot from at least two independent experiments is shown. n Immunoblots of menin and LEDGF in LSCs treated with control, MMF (0.5 μM), or CX-5461 (100 nM) for 16 h subjected to immunoprecipitation using either IgG or anti-menin antibodies. *, a non-specific band. A representative blot from at least two independent experiments is shown. o ChIP-qPCR analysis of menin occupation at the Hoxa9 promoter in LSCs treated with control, MMF (500 nM), CX-5461 (100 nM), or VTP-50469 (250 nM) (n = 3, biologically independent samples). p MFI of myeloid differentiation marker CD11b in MOLM-13 cells expressing vector control or 3Ty1-PCE, with or without treatment of VTP-50469 (125 nM), MMF (250 nM), and CX-5461 (100 nM) for 4 days (left panel). Immunoblots on the right show the protein level of 3Ty1-PCE and GAPDH on separate membranes (n = 3, biologically independent samples). q The effects of VTP-50469 (125 nM), MMF (250 nM), and CX-5461 (100 nM) on MOLM-13 cells expressing 3Ty1-PCE after 4 days (n = 3, biologically independent samples). r MFI of myeloid differentiation marker CD11b in THP-1 cells expressing vector control or Hoxa9-p2a-Meis1, with or without treatment of MMF (0.5-1 μM) for 4 days (n = 3, biologically independent samples). All data are represented as mean ± SD. p values in this figure were calculated by permutation test (c–e and g), hypergeometric test (f, i and j), unpaired, two-tailed student t-test (p and q) and ANOVA with multiple comparisons analysis using Dunnett’s (o) or Bonferroni correction (p, q and r) post hoc analyses. See also Supplementary Fig. 6. Source data are provided as a Source Data file.