Yiman Liu

Supplementary Figure S1. Generation of a conditional knock-in mouse model for the Enl-T1 mutation. Supplementary Figure S2. Impact of Enl mutation on the peripheral blood and spleen. Supplementary Figure S3. Characterization of Enl-T1 allele expression and concurrent mutations in heterozygous knock-in Enl-T1 mouse model. Supplementary Figure S4. UBC-cre-ERT2/Enl flox-T1/+ mice develop aggressive acute leukemia following tamoxifen treatment. Supplementary Figure S5. Enl mutation leads to expansion of myeloid cells in mice in the leukemic phase. Supplementary Figure S6. Enl mutation leads to the decrease of B220+CD19+ B, CD4+T, CD8+ T cells in mice in the leukemic phase. Supplementary Figure S7. Impact of the Enl mutation on the bone marrow, peripheral blood, spleen, and thymus in mice in the pre-leukemic phase. Supplementary Figure S8. Enl mutation perturbs the normal hematopoietic hierarchy and leads to abnormal expansion of myeloid progenitors in mice in the leukemic phase. Supplementary Figure S9. Enl mutation does not lead to the expansion of myeloid progenitors in mice in the pre-leukemic phase. Supplementary Figure S10. Enl mutation promotes self-renewal properties of HSPCs. Supplementary Figure S11. Enl mutation-induced up- and down-regulated genes are related to distinct biological functions. Supplementary Figure S12. Enl mutation leads to a gain of myeloid differentiation signatures in HSPCs. Supplementary Figure S13. Mutant ENL-induced H3K27ac signals correlate with upregulation of development and inflammation associated transcriptional programs. Supplementary Figure S14. HSPCs gain H3K27me3 during differentiation in wildtype mice. Supplementary Figure S15. Differentiation-associated gain of H3K27me3 is impaired in Enl-mutated hematopoietic cells. Supplementary Figure S16. ENL mutants form condensate at key target genes and increase gene expression in HSPCs. Supplementary Figure S17. Condensate formation property correlates with mutant ENL’s oncogenic function in human CD34+ HSPCs. Supplementary Figure S18. Expression levels of different FLAG-ENL transgenes in LSK cells. Supplementary Figure S19. Condensate formation property correlates with mutant ENL’s oncogenic function in GMP cells. Supplementary Figure S20. Disrupting condensate formation by the H116P mutation reduces ENL-T1-induced increases in chromatin occupancy of FLAG-ENL, H3K27ac, and p300 at a subset of target genes. Supplementary Figure S21. Impact of mutant ENL on leukemia development and condensate formation. Supplementary Figure S22. Small molecule inhibition of the acetyl-binding activity of mutant ENL suppresses chromatin function and Hoxa cluster gene activation in LSK cells. Supplementary Figure S23. Small molecule inhibition of the acetyl-binding activity of mutant ENL impairs its chromatin and transcriptional function in HSPCs. Supplementary Figure S24. Small molecule inhibition of the acetyl-binding activity of mutant ENL inhibits its impact on the self-renewal property in HSPCs.

Supplementary Table S1. Genes differentially expressed between Enl-T1 and Enl-WT LSK cells. Supplementary Table S2. Genes differentially expressed between Enl-T1 and Enl-WT GMP cells. Supplementary Table S3. Genes differentially expressed between Enl-T1 and Enl-WT L-GMP cells. Supplementary Table S4. Genes differentially expressed between Enl-T1 and Enl-WT cKit + Mac1+ cells. Supplementary Table S5. Genes differentially expressed between Enl-T1 and Enl-WT cKit-Mac1+ cells. Supplementary Table S6. Shared Enl-T1 up-regulated DEGs in LSK, GMP, and L-GMP cells. Supplementary Table S7. Expression of Hoxa genes in Enl-WT and Enl-T1 hematopoietic populations. Supplementary Table S8. GSEA gene sets used in Supplementary Figure S12. Supplementary Table S9. GSVA score of patients from TARGET-AML database. Supplementary Table S10. H3K27ac peaks in all hematopoietic populations. Supplementary Table S11. H3K27ac differential regions of ENL-T1 versus ENL-WT in LSK cells. Supplementary Table S12. H3K27ac differential regions of ENL-T1 versus ENL-WT in GMP cells. Supplementary Table S13. H3K27ac differential regions of ENL-T1 versus ENL-WT in L-GMP cells. Supplementary Table S14. H3K27ac differential regions of ENL-T1 versus ENL-WT in cKit + Mac1+ cells. Supplementary Table S15. H3K27ac differential regions of ENL-T1 versus ENL-WT in cKit-Mac1+ cells. Supplementary Table S16. T1-UP DEGs associated with H3K27ac T1 gained DRs in all hematopoietic populations. Supplementary Table S17. H3K27ac T1 gained differential regions associated with T1-UP DEGs in all hematopoietic populations. Supplementary Table S18. p300 ChIP-seq normalized signal at T1 gained H3K27ac differential regions in Enl-WT and Enl-T1 GMP, L-GMP cells. Supplementary Table S19. T1 gained H3K27ac differential regions with both p300 UP and associated gene expression UP in Enl-T1 cells for GMP and L-GMP. Supplementary Table S20. T1 gained H3K27ac differential regions with p300 UP and associated gene expression UP in Enl-T1 cells for L-GMP under A-485 treatment. Supplementary Table S21. H3K27me3 peaks in wildtype hematopoietic populations. Supplementary Table S22. Hematopoietic differentiation associated-H3K27me3 peaks. Supplementary Table S23. H3K27me3 peaks in all hematopoietic populations. Supplementary Table S24. H3K27me3 differential regions of ENL-T1 versus ENL-WT in LSK cells. Supplementary Table S25. H3K27me3 differential regions of ENL-T1 versus ENL-WT in GMP cells. Supplementary Table S26. H3K27me3 differential regions of ENL-T1 versus ENL-WT in L-GMP cells. Supplementary Table S27. H3K27me3 differential regions of ENL-T1 versus ENL-WT in cKit + Mac1+ cells. Supplementary Table S28. H3K27me3 differential regions of ENL-T1 versus ENL-WT in cKit-Mac1+ cells. Supplementary Table S29. T1-UP DEGs associated with H3K27me3 lost differential regions in all hematopoietic populations. Supplementary Table S30. Group1 and group2 gene list in cKit + Mac1+ and cKit-Mac1+ cells. Supplementary Table S31. FLAG-ENL peaks in LSK cells expressing the indicated FLAG-ENL transgenes. Supplementary Table S32. FLAG-ENL gained regions of T1 versus WT in LSK cells expressing the indicated FLAG-ENL transgenes. Supplementary Table S33. H3K27ac T1 gained differential regions associated with T1 gained FLAG-ENL DRs in LSK cells expressing the indicated FLAG-ENL transgenes. Supplementary Table S34. Genes differentially expressed between Enl-T1-DMSO and Enl-WT-DMSO LSK cells. Supplementary Table S35. Genes differentially expressed between Enl-T1-DMSO and Enl-WT-DMSO in GMP cells. Supplementary Table S36. Expression of Hoxa genes under Enl-WT-DMSO, Enl-T1-DMSO and Enl-T1-TDI conditions in LSK and GMP cells. Supplementary Table S37. Oligos used in this study. Supplementary Table S38. Antibodies used in this study.

Supplementary Table S1 shows the high-confident NPM1c binding peaks in OCI-AML3 cells.

Supplementary Figure S1 is associated with Figure1 and it shows the NPM1-WT binds to the rDNA arrays and NPM1c binds to non-repetitive genomic regions. Supplementary Figure S2 is associated with Figure1 and it shows NPM1c’s chromatin binding and association with gene expression. Supplementary Figure S3 is associated with Figure2 and shows NPM1c regulates the transcription of its target genes with BRU-seq. Supplementary Figure S4 is associated with Figure2 and it shows the characterization of NPM1c condensate with biochemical assay and imaging assay. Supplementary Figure S5 is associated with figure 3. The figure shows NPM1c and chromatin landacpe dymanics during dTag-13 treatment and wash-off. Supplementary Figure S6 is associated with figure 4. It shows the supplemental data of HOXB8-NPM1c-knock-in model. Supplementary figure S7 is associated with figure 5. It shows the supplemental data of XPO1's binding to chromatin in various leukemia cell lines and normal HSPCs. Supplementary Figure S8 is associated with figure 6. It shows the supplemental information of synergy between Menin and XPO1 inhibitor in the NPM1c AML cell line model.

Supplementary Table S3 shows the differential expressed genes in NPM1c-koncked in HOXB8 cell lines vs parental cells

Supplementary Table S2 shows the differential expressed genes in dTAG-13 vs DMSO treatment at 24hr in OCI-AML3-NPM1c-degron2 cells.

Supplemenary Table S4 shows the differential expressed genes in 25nM Selinexorvs DMSO treatment at 24hr in OCI-AML3-NPM1c-degron2 cells.

Supplementary Table S5 shows all the oligos used in this study

Nucleophosmin (NPM1) is a ubiquitously expressed nucleolar protein with a wide range of biological functions. In 30% of acute myeloid leukemia (AML), the terminal exon of NPM1 is often found mutated, resulting in the addition of a nuclear export signal and a shift of the protein to the cytoplasm (NPM1c). AMLs carrying this mutation have aberrant expression of the HOXA/B genes, whose overexpression leads to leukemogenic transformation. Here, for the first time, we comprehensively prove that NPM1c binds to a subset of active gene promoters in NPM1c AMLs, including well-known leukemia-driving genes—HOXA/B cluster genes and MEIS1. NPM1c sustains the active transcription of key target genes by orchestrating a transcription hub and maintains the active chromatin landscape by inhibiting the activity of histone deacetylases. Together, these findings reveal the neomorphic function of NPM1c as a transcriptional amplifier for leukemic gene expression and open up new paradigms for therapeutic intervention.Significance:NPM1 mutation is the most common mutation in AML, yet the mechanism of how the mutant protein results in AML remains unclear. Here, for the first time, we prove mutant NPM1 directly binds to active chromatin regions and hijacks the transcription of AML-driving genes.See related article by Uckelmann et al., p. 746.This article is highlighted in the In This Issue feature, p. 517

Aberrant chromatin-associated condensates have emerged as drivers of transcriptional dysregulation in cancer. Although extensive studies have elucidated intrinsic protein sequence features governing their formation, how extrinsic factors within the chromatin environment modulate their assembly and pathogenic function remains poorly understood. Gain-of-function mutations in the histone acetylation reader ENL, found in pediatric leukemia and Wilms tumor, drive oncogenesis by inducing condensate formation at highly selective genomic loci. Here, we uncover a critical role for locally produced gene transcripts in reinforcing the nucleation, chromatin engagement, and oncogenic activity of ENL mutant condensates. Mutant ENL binds RNA in part through a conserved basic patch within its YEATS domain, and this interaction enhances condensate formation both in vitro and across diverse cellular contexts. Using a chemically inducible condensate displacement and re-nucleation system, we show that blocking ENL–RNA interactions or transcription impairs condensate reformation at endogenous targets. RNA interactions preferentially enhance mutant ENL occupancy at top-bound, condensate-permissive loci, leading to increased transcriptional bursting and robust gene activation at the single-cell and single-allele level. In mouse models, disrupting ENL–RNA interactions reduces condensate formation and oncogenic transcription in hematopoietic stem and progenitor cells, thereby suppressing ENL mutant-driven leukemogenesis. Together, these findings demonstrate that locally produced RNA transcripts can promote locus-specific nucleation of pathogenic condensates on chromatin, which in turn drive persistent and hyperactive transcription of oncogenic targets and lead to tumorigenesis.