Yanhe Wen

Table S1. Read normalized ratios of ChIPseq signal of untreated versus dTAG-13 treated OCI-AML3-NPM1c-FKBP12 cells.Table S2. Summary of NPM1c and NPM1 Chromatin Binding Targets.Table S3. Patient info for patient derived xenograft (PDX) models used.Table S4. ChIPseq reads per kilobase (rpk) data summary of PDX samples with NPM1c-Ab and NPM1-Ab.Table S5. RNAseq differential expression data of OCI-AML3-NPM1c-FKBP12 treated with dTag-13 for 3 or 24 h and OCI-AML3 cells treated with Selinexor for 6 h.Table S6. SLAMseq differential expression data of OCI-AML3-NPM1c-FKBP12 cells treated with dTag-13 for 15, 30, 60, 120 or 180 minutes.Table S7. PROseq differentially transcribed genes in OCI-AML3-NPM1c-FKBP12 cells treated with dTag-13 for 30 or 60 minutes and OCI-AML3 cells treated with VTP-50469 for 48 hours.Table S8. CRISPR tiling screen with NPM1 WT specific guides (blue) and NPM1c specific guides (red).Table S9. ChIPseq rpk values for CRM1 and HA ChIPs in 293T cells.Table S10. RNAseq differential gene expression analysis in 293T cells after overexpression of NPM1c-HA compared to all other samples (MIG, NPM1wt-HA and AS1-HA).Table S11. Fold change in ChIPseq differential peak interval signal in OCI-AML3 cells treated with Selinexor (100 nM, 6 hours) or VTP-50469 (330 nM, 96 hours).Table S12. Oligos used in this study.Table S13. Antibodies used in this study.Table S14. Summary of total and mapped reads of ChIPseq samples.

Supplementary Figure 1. NPM1c degradation leads to differentiation of OCI-AML3 cells.Supplementary Figure 2. NPM1c chromatin targets identified in OCI-AML3-NPM1c-FKBP12 cells and validated by targeted degradation.Supplementary Figure 3. Motif analysis reveals developmental transcription factor binding sites at NPM1c bound regions.Supplementary Figure 4. NPM1c chromatin targets are highly expressed in patient samples of NPM1c AML.Supplementary Figure 5. NPM1c degradation has rapid effects on transcription at it target genes.Supplementary Figure 6. Transcriptional machinery is lost from NPM1c targets after 1 hour of NPM1c degradation.Supplementary Figure 7. CRIPSR domain scan reveals specific dependency of NPM1 mutant cells on the AS2 region.Supplementary Figure 8. The AS2 region affects NPM1c localization in 293T and OCI-AML3 cells.Supplementary Figure 9. Selinexor treatment disrupts NPM1c chromatin binding.Supplementary Figure 10. NPM1c cooperates with MLL1 to drive oncogenic gene expression.Supplementary Figure 11. NPM1c is selectively lost from chromatin in response to Menin inhibitor treatment.Supplementary Figure 12. Menin-MLL inhibition leads to selective loss of NPM1c target gene expression.

The t(11;22) translocation encodes the EWS::FLI1 fusion oncoprotein which is the primary driver of Ewing sarcoma. EWS::FLI1 creates unique, de novo pathogenic enhancers that drive gene expression and are a central mechanism of oncogenesis. Which chromatin regulatory proteins are critical to this mechanism is understudied. Here, we perform a comparative analysis of the function of the chromatin complexes MLL3/4 and p300/CBP in EWS::FLI1-mediated gene regulation. Using EWS::FLI1 degradation models, we define a subset of EWS::FLI1-sensitive enhancers whose activity correlates with p300/CBP function. We perturb both chromatin complexes to establish that in contrast to MLL3/4, p300/CBP is a critical regulator of EWS::FLI1-driven enhancer activity and downstream gene expression. We also show that p300/CBP small-molecule inhibition decelerates tumor growth in vivo. Our work highlights the context-dependent nature of chromatin protein activity at oncogenic enhancers and reveals p300/CBP as an important regulator of Ewing sarcoma.

Chemical inducers of proximity (CIPs) stabilize biomolecular interactions, often causing an emergent rewiring of cellular biochemistry. While rational design strategies can expedite the discovery of heterobifunctional CIPs, monovalent, molecular glue-like CIPs have relied predominantly on serendipity. Envisioning a prospective approach to discover molecular glues for a pre-selected target, we hypothesized that pre-existing ligands could be systematically decorated with chemical modifications to empirically discover protein-ligand surfaces that are tuned to cooperatively engage another protein interface. Here, we used sulfur(VI)-fluoride exchange (SuFEx)-based high-throughput chemistry (HTC) to install 3,163 structurally diverse chemical building blocks onto ENL and BRD4 ligands and then screened the crude products for degrader activity. This revealed dHTC1, a potent, selective, and stereochemistry-dependent degrader of ENL. It recruits CRL4 ^CRBN to ENL through an extended interface of protein-protein and protein-ligand contacts, but only after pre-forming the ENL:dHTC1 complex. We also characterized two structurally distinct BRD4 degraders, including dHTC3, a molecular glue that selectively dimerizes the first bromodomain of BRD4 to SCF ^FBXO3 , an E3 ligase not previously accessible for chemical rewiring. Altogether, this study introduces HTC as a facile tool to discover new CIPs and actionable cellular effectors of proximity pharmacology.

Supplementary Figure 1. NPM1c degradation leads to differentiation of OCI-AML3 cells.Supplementary Figure 2. NPM1c chromatin targets identified in OCI-AML3-NPM1c-FKBP12 cells and validated by targeted degradation.Supplementary Figure 3. Motif analysis reveals developmental transcription factor binding sites at NPM1c bound regions.Supplementary Figure 4. NPM1c chromatin targets are highly expressed in patient samples of NPM1c AML.Supplementary Figure 5. NPM1c degradation has rapid effects on transcription at it target genes.Supplementary Figure 6. Transcriptional machinery is lost from NPM1c targets after 1 hour of NPM1c degradation.Supplementary Figure 7. CRIPSR domain scan reveals specific dependency of NPM1 mutant cells on the AS2 region.Supplementary Figure 8. The AS2 region affects NPM1c localization in 293T and OCI-AML3 cells.Supplementary Figure 9. Selinexor treatment disrupts NPM1c chromatin binding.Supplementary Figure 10. NPM1c cooperates with MLL1 to drive oncogenic gene expression.Supplementary Figure 11. NPM1c is selectively lost from chromatin in response to Menin inhibitor treatment.Supplementary Figure 12. Menin-MLL inhibition leads to selective loss of NPM1c target gene expression.

The dysregulation of developmental and stem cell–associated genes is a common phenomenon during cancer development. Around half of patients with acute myeloid leukemia (AML) express high levels of HOXA cluster genes and MEIS1. Most of these AML cases harbor an NPM1 mutation (NPM1c), which encodes for an oncoprotein mislocalized from the nucleolus to the cytoplasm. How NPM1c expression in hematopoietic cells leads to its characteristic gene-expression pattern remains unclear. Here, we show that NPM1c directly binds to specific chromatin targets, which are co-occupied by the histone methyltransferase KMT2A (MLL1). Targeted degradation of NPM1c leads to a rapid decrease in gene expression and loss of RNA polymerase II, as well as activating histone modifications at its targets. We demonstrate that NPM1c directly regulates oncogenic gene expression in collaboration with the MLL1 complex and define the mechanism by which MLL1–Menin small-molecule inhibitors produce clinical responses in patients with NPM1-mutated AML.Significance:We uncovered an important functional role of mutant NPM1 as a crucial direct driver of oncogenic gene expression in AML. NPM1c can bind to chromatin and cooperate with the MLL complex, providing the first functional insight into the mechanism of Menin–MLL inhibition in NPM1c leukemias.See related article by Wang et al., p. 724.This article is highlighted in the In This Issue feature, p. 517

The dysregulation of developmental and stem cell–associated genes is a common phenomenon during cancer development. Around half of patients with acute myeloid leukemia (AML) express high levels of HOXA cluster genes and MEIS1. Most of these AML cases harbor an NPM1 mutation (NPM1c), which encodes for an oncoprotein mislocalized from the nucleolus to the cytoplasm. How NPM1c expression in hematopoietic cells leads to its characteristic gene-expression pattern remains unclear. Here, we show that NPM1c directly binds to specific chromatin targets, which are co-occupied by the histone methyltransferase KMT2A (MLL1). Targeted degradation of NPM1c leads to a rapid decrease in gene expression and loss of RNA polymerase II, as well as activating histone modifications at its targets. We demonstrate that NPM1c directly regulates oncogenic gene expression in collaboration with the MLL1 complex and define the mechanism by which MLL1–Menin small-molecule inhibitors produce clinical responses in patients with NPM1-mutated AML.Significance:We uncovered an important functional role of mutant NPM1 as a crucial direct driver of oncogenic gene expression in AML. NPM1c can bind to chromatin and cooperate with the MLL complex, providing the first functional insight into the mechanism of Menin–MLL inhibition in NPM1c leukemias.See related article by Wang et al., p. 724.This article is highlighted in the In This Issue feature, p. 517