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IKAROS and MENIN coordinate therapeutically actionable leukemogenic gene expression in MLL-r acute myeloid leukemia

Abstract

Acute myeloid leukemia (AML) remains difficult to treat and requires new therapeutic approaches. Potent inhibitors of the chromatin-associated protein MENIN have recently entered human clinical trials, opening new therapeutic opportunities for some genetic subtypes of this disease. Using genome-scale functional genetic screens, we identified IKAROS (encoded by IKZF1) as an essential transcription factor in KMT2A (MLL1)-rearranged (MLL-r) AML that maintains leukemogenic gene expression while also repressing pathways for tumor suppression, immune regulation and cellular differentiation. Furthermore, IKAROS displays an unexpected functional cooperativity and extensive chromatin co-occupancy with mixed lineage leukemia (MLL)1–MENIN and the regulator MEIS1 and an extensive hematopoietic transcriptional complex involving homeobox (HOX)A10, MEIS1 and IKAROS. This dependency could be therapeutically exploited by inducing IKAROS protein degradation with immunomodulatory imide drugs (IMiDs). Finally, we demonstrate that combined IKAROS degradation and MENIN inhibition effectively disrupts leukemogenic transcriptional networks, resulting in synergistic killing of leukemia cells and providing a paradigm for improved drug targeting of transcription and an opportunity for rapid clinical translation.

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Fig. 1: IKZF1 (IKAROS) and MTA2 exhibit synthetic lethal interaction with pharmacologic inhibition of MENIN and DOT1L.
Fig. 2: IMiDs effectively target the IKAROS protein for degradation and show therapeutic efficacy in MLL-r AML.
Fig. 3: IKAROS is a core transcriptional regulator in MLL-r AML.
Fig. 4: Combined targeting of MENIN and IKAROS results in synergistic induction of apoptosis and cooperative deregulation of gene expression.
Fig. 5: Inhibition of MENIN–MLL1 protein–protein interaction leads to proteasomal degradation of IKAROS protein.
Fig. 6: Chromatin co-occupancy and proximal protein–protein interactions of IKAROS and the MLL fusion network.
Fig. 7: Combination therapy with VTP-50469 and LEN results in synergistic anti-leukemic activity in vivo.

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Data availability

Raw and analyzed data for ChIP–seq, CUT&RUN-seq, RNA-seq, ATAC-seq and ABC model predictions have been deposited at the National Center for Biotechnology Information Gene Expression Omnibus under the accession number GSE168463; deposited data correspond to Figs. 37 and associated Extended Data Figs 4, 5 and 710. All MS data and search results have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD007862) via the PRIDE partner repository with the dataset identifiers PXD025227, PXD025228, PXD025229, PXD025230, PXD025231, PXD025232, PXD025271 and PXD025272. Additional data that support the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

S.A.A. is supported by National Institutes of Health (NIH) grants CA176745, CA206963, CA204639 and CA066996, by a St. Jude Children’s Research Hospital Research Consortium grant, Curing Kids Cancer and by the Leukemia and Lymphoma Society, USA. B.J.A. received a Career Development fellowship from the Leukemia and Lymphoma Society, USA. J.A.C. is supported by a Ruth L. Kirschstein Postdoctoral Individual National Research Service award (NIH, F32CA250240-02). W.B. is supported by an NIH T32 training grant (5T32HL007574-38-40) and the Wong Family Award in Translational Oncology. S.G. is supported by the Sara Elizabeth O’Brien Trust Fellowship. X.S.L. is supported by the Breast Cancer Research Foundation (BCRF-20-100). E.S.F. is supported by NIH grants CA214608, CA231637 and CA066996 and by a Damon Runyon-Rachleff Innovator award (DRR-50-18). S.N.O. is supported by the Damon Runyon Cancer Research Foundation (DRSG, 26-18). F.P. is supported by a grant from the German Research Foundation (PE 3217/1-1). A.C. received funding from German Cancer Aid (Deutsche Krebshilfe). We are grateful for support from the St. Baldrick’s Foundation to the Pediatric LEAP Consortium. We thank all members of the Armstrong laboratory for invaluable discussions. We thank J. Perry, Y. Pikman and A. Pinilla along with the CPCT for the development and use of human PDX models. We are extremely grateful to A. Pinilla, A. Conway, A. Robichaud, P. Gokhale and the Experimental Therapeutics Core Facility at the DFCI for assistance with mouse studies. We thank J. Healey, J. Gadrey and S. Kitajima for technical support. We thank E. Frank and K. Ross for statistical analyses. We thank E. Fink, A. Guirguis, Q. Sievers and V. Koduri for advice on IKAROS studies and in vivo studies. We thank M. Filipovski for invaluable assistance with CUT&RUN experiments. The content is solely the responsibility of the authors and does not represent the official views of the NIH.

Author information

Authors and Affiliations

Authors

Contributions

S.A.A. supervised the study. S.A.A., B.J.A., J.A.C. and W.B. conceived the study and performed data analysis. C.H. and Q.Z. performed bioinformatic analysis. B.J.A., J.A.C., W.B., R.P.N., S. Parvin and K.A.D. performed experiments. A.C.P.T., S. Perlee, Y.J.K., J.A.H. and N.A.E. provided technical assistance. A.C., S.N.O., F.P., H.R., G.M.M., A.L., E.S.F., Y.P. and X.S.L. contributed to critical experimental planning and resources. B.J.A. and S.A.A. wrote the first draft of the manuscript. All authors contributed to editing subsequent manuscript drafts.

Corresponding author

Correspondence to Scott A. Armstrong.

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Competing interests

S.A.A. has been a consultant and/or shareholder for Neomorph, Imago BioSciences, Vitae–Allergan Pharma, Cyteir Therapeutics, C4 Therapeutics, Accent Therapeutics and Mana Therapeutics. S.A.A. is an inventor on a patent application related to MENIN inhibition WO/2017/132398A1. S.A.A. has received research support from Janssen, Novartis, Syndax and AstraZeneca. X.S.L. is a cofounder, board member, SAB and consultant of GV20 Oncotherapy and its subsidiaries; an SAB of 3DMedCare; a consultant for Genentech; and a stockholder of Abbott Laboratories, Amgen Inc, Johnson & Johnson, Merck & Co, Inc. and Pfizer, Inc.; and receives sponsored research funding from Takeda and Sanofi. G.M.M. is a shareholder of Syndax Pharmaceuticals. B.J.A. is a former employee of the Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia, and receives proceeds from royalties and milestone payments related to the BCL2 inhibitor ABT-199 (venetoclax). E.S.F. is an equity holder and scientific advisor for Civetta Therapeutics, Jengu Therapeutics (board of directors) and Neomorph; a shareholder in C4 Therapeutics; and a consultant to EcoR1 Capital, Sanofi, Astellas, Deerfield and RA Capital. The Fischer laboratory receives or has received research funding from Novartis, Astellas, Deerfield and Ajax. All other authors declare no potential conflicts of interest.

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Extended data

Extended Data Fig. 1 Chromatin remodeling complexes modulate the cellular response to therapeutic targeting of MLL-r driven gene expression.

a, Proliferation assay conducted in real-time during the functional genomic screen showing cell number over time. Data represent mean +/−SEM (n = 4). b, Volcano plots depicting Wald p-value and beta value calculated using MAGeCK MLE for VTP-50469 and EPZ-5676, comparing the vehicle-treated and drug-treated state on Day 14. c, Gene set testing comparing the behaviour of group 2 (resistance) and group 4 (synthetic lethal) genetic hits between each screen. Family-wise error rate p-value determined by GSEA computational method. d, Expanded panels of CRISPR/Cas9-based competition assays targeting either IKZF1 or MTA2 monitoring sgRNA-RFP expression over time in Cas9-expressing in 4 MLL-r human AML cell lines (MOLM13, MV4;11, OCI-AML2 and THP-1), with and without concurrent treatment with the MENIN inhibitor, VTP-50469. e, CRISPR/Cas9-based competition assays targeting IKZF1 monitoring sgRNA-RFP expression over time in 4 non-MLL-fusion human AML cell lines (IMSM2, OCI-AML3, U937 and HL60) expressing Cas9. IMSM2 and OCI-AML3 carry the NPM1c mutation.

Source data

Extended Data Fig. 2 Using CRISPR/Cas9 to genetically target IKZF1 and MTA2 and validate genomic screen findings.

a, Combined analysis of sgRNA depletion across samples, depicted in Extended Data Fig. 1d, showing enhanced sgRNA depletion in the presence of the MENIN-inhibitor, VTP-50469, despite the disadvantage of drug-induced cell cycle arrest. Data represent mean + /−SEM with p-value by two-tail t-test evaluated for n = 4 cell lines and n = 6 sgRNAs targeting IKZF1 or n = 4 sgRNAs targeting MTA2. b, Western blot analysis for MTA2 protein in sgRNA-RFP sorted cells comparing MTA2-targeted bulk cell populations with non-targeting control and ACTIN used as a loading control. c, Western blot analysis for IKAROS protein in sgRNA-RFP sorted cells comparing IKZF1-targeted bulk cell populations with non-targeting control and ACTIN used as loading control. d, Analysis of annexin V staining (apoptosis) and CD11b/CD14 (monocytic differentiation) using flow cytometry 7 days following CRISPR/Cas9-mediated deletion of either IKZF1 or MTA2. Representative experiment is shown. e, Colony forming assay comparing MOLM13 cells with sgRNA targeting Luciferase (Non-Targeting) control versus two different sgRNAs targeting IKZF1 (n = 4 per sgRNA), representative colony morphology is shown. f, Relative IC50 for proliferation assays, pertaining to Fig. 1j, comparing MOLM13 cells with sgRNA targeting Luciferase (Non-Targeting control) and two sgRNAs targeting IKZF1 (n = 3 per sgRNA) upon treatment with MENIN inhibition. g, Apoptosis assay in MOLM13 and MV4;11 cell lines testing the impact of sgRNAs targeting Luciferase (NT control) versus two sgRNAs targeting IKZF1 on the response to MENIN inhibition with VTP-50469, using doses indicated. Apoptosis was assessed by DAPI exclusion (viability) and annexin V staining with 50,000 cells analysed per sample. Representative example of multiple independent experiments.

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Extended Data Fig. 3 IMiDs effectively target IKAROS protein for degradation in human MLL-r AML.

a, Western blot analysis for IKAROS, CK1α and MENIN protein following treatment of MV4;11 human MLL-r AML cell line for 5 hours with increasing doses of THAL, LEN, POM and CC220, using ACTIN as a loading control. b, Western blot analysis for IKAROS and CK1α following treatment of OCI-AML2 human MLL-r AML cell line for 5 hours with increasing doses of THAL, LEN, POM and CC220, using ACTIN as a loading control. c, LEN and CC220 dose-response curves on: Day 9 of treatment for the NPM1-mutant (NPM1c) IMSM2 human AML cell line cell (Absolute IC50 267.0 nM [95% CI 189.6 to 381.0] for LEN, 17.3 nM [95% CI 9.3 to 37.8] for CC220) and Day 18 of treatment for the NPM1-mutant (NPM1c) OCI-AML3 human AML cell line (Absolute IC50 4626 nM [95% CI could not be calculated] for LEN, 57.7 nM [95% CI 33.1 to 101.5] for CC220). Data represent mean + /−SEM with absolute IC50 indicated (n = 3). d, Scatterplot for MS determination of IMiD substrates 5 hours after drug treatment in the MV4;11 and OCI-AML2 cell lines. Data represent the log-fold change in abundance and log10(p-value). p-value determined by moderated t-test as implemented by the Bioconductor Limma package.

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Extended Data Fig. 4 IKAROS degradation perturbs diverse cellular pathways.

a, RNA-seq dot-plot with linear regression showing z-score for differential gene expression correlating LEN (5 μM) and CC220 (1 μM) treatment for 3 days. b, RNA-seq volcano plot for MV4;11 treated with LEN for 6 days. c, GSEA results for ‘oncogenic signatures’ for RNA-seq data on Day 3. Dot plot is shown of the log10 false discovery rate (FDR) q-value and normalized enrichment score (NES) as determined by GSEA computational method. d, GSEA results for ‘Hallmarks’ signatures. Dot plot is shown of the log10 false discovery rate (FDR) q-value and normalized enrichment score (NES) as determined by the GSEA computational method. e, Bar code plots created using the GSEA tool for putative MLL-fusion target genes with selected genes from leading edge analysis indicated with adj-p-value<0.05 in MOLM13 or MV4;11. Normalized enrichment score and family-wise error rate p-value as determined by GSEA computational method. f, Gene expression z-score heatmap for selected genes within immune regulatory and inflammatory pathways under treatment with CC220 (n = 3) compared to DMSO-treated control (n = 3). g, Venn diagram for genes displaying > 2-fold change in expression following CC-220 treatment comparing cell lines. Gene number and percentage overlap is indicated. Selected genes deregulated in both cell lines are indicated. h, Correlation of IKAROS-bound gene TSSes, determined by ChIP-seq, with RNA-seq changes. Bar code plot showing gene expression changes following treatment with CC220 (3 days) for the top 500 genes bound by IKAROS (by peak enrichment over background). Selected genes from leading edge analysis indicated. Normalized enrichment score and family-wise error rate p-value determined by GSEA computational method. i, Correlation of IKAROS-bound enhancers, determined by ChIP-seq and the ABC prediction tool, with RNA-seq gene expression changes. Bar code plots display gene expression changes following treatment with CC-220 (3 days) for the top 500 genes predicted to be regulated by IKAROS-bound enhancers (by peak enrichment over background at enhancers). Normalized enrichment score and family-wise error rate p-value determined by GSEA computational method. Venn diagram for overlap between gene expression change and genes predicted to be regulated by the top 1000 IKAROS-bound enhancers.

Extended Data Fig. 5 IKAROS CUT&RUN motif foot printing.

a, IKZF1 DNA-binding motifs. b, Foot printing for CTCF protein over the CTCF motif for each cell line; used as a positive control for motif detection. Result for CUT&RUNTools de novo DREME motif detection is shown with highly significant discovery of the known CTCF motif. MEME and E-value is indicated. c, CUT&RUNTools foot printing for IKAROS and IgG control over the IKZF1 motif in the MOLM13 cell line. Cut site probability distribution as determined by CUT&RUNTools. d, CUT&RUNTools foot printing for IKAROS over the JUN:FOS motif (centrally bound), CEBPα motif (centrally bound), SPI/Pu.1 motif (centrally bound) and the RUNX motif (non-centrally bound), as indicated, in the MOLM13 cell line. Cut site probability distribution as determined by CUT&RUNTools.

Extended Data Fig. 6 Combined targeting of MENIN and IKAROS.

a, Annexin V staining (apoptosis) by flow cytometry following 6 days treatment with VTP-50469, LEN, CC220 and combination (MOLM13). b, Cell cycle analysis: histogram for DAPI stain and bar graphs displaying cell cycle stage (%) are shown for each condition. Representative experiment shown. (MOLM13) c, CD11b/CD14 expression measured by flow cytometry 6 days following treatment with VTP-50469, LEN, CC220 and combination (MOLM13). Representative experiment shown. d, Dose-response curves for LEN and CC220, with and without VTP-50469 (8 nM), compared to VTP-50469 alone (dotted line) measured by Cell-Titer Glo in MV4;11 for synergy studies. e, Chou-Talalay synergy analysis for VTP-50469 in combination with LEN or CC-220 (MV4;11). Chou-Talalay Combination Index (CI) plots (left panel) and normalized isobolograms (right panel) are shown for each IMiD-VTP-50469 combination (day 6). Line of additivity is shown (red). f, CC-220 and MENIN inhibitor dose response curves (day 6) with and without overexpression of non-degradable IKAROS (Q146H) in MOLM13. Data represent mean + /−SD with non-linear regression curve fit shown. g, IMiD and MENIN inhibitor apoptosis after 6 days treatment, with and without overexpression of a wild-type IKAROS or non-degradable IKAROS mutant (Q146H) in MOLM13. h, Scatterplots for mass spectrometry (MS) results on MOLM13, MV4;11 and OCI-AML2 cell lines after 5 days pre-treatment with VTP-50469 and then treatment with THAL, LEN, POM and CC-220 (iberdomide), for 5 h. Data represent the log-fold change in abundance and log10(p-value). p-value determined by moderated t-test as implemented by the Bioconductor Limma package. i, BH3-profiling in MV4;11 under drug treatments indicated. Cytochrome C (Cyt C) release was measured. Data represent mean + /−SEM (n = 3). j, Western blot analysis for BCL2, MCL1 and BCL-XL protein following treatment of OCI-AML2 human MLL-r AML cell line for 72 hours with LEN, EPZ-5676, VTP-50469 and each combination, using GAPDH as a loading control.

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Extended Data Fig. 7 MENIN and IKAROS display overlapping functions in regulation of gene expression.

a, Number of differentially expressed genes (greater than 2-fold change and adjusted p-value <0.05) from RNA-seq data for MOLM13 and MV4;11 cell lines treated with CC220 1 μM (n = 3), VTP-50469 50 nM (n = 3) and the combination (n = 3) for 3 days. b, Heatmap showing DESeq2 statistical z-score for all genes deregulated under treatment with VTP-50469 alone across all other treatment groups. c, Heatmap of DESeq2 statistical z-score for MENIN and IKAROS co-regulated gene expression. Showing genes with shared or additive regulation. Selected genes are indicated. d, Cell surface expression of FLT3 as measured by flow cytometry in the MOLM13 cell line. Cells were pre-treated for 2 days with CC220 or DMSO control and then VTP-50469 was added for 24 hours. e, CRISPR screen beta scores from MAGeCK MLE comparing sgRNA representation at day 0 compared to day 14 in the DMSO-treated control samples for the MOLM13 cell line. Negative beta scores for MYC, FLT3, RPA3 (common essential gene), RPS14, and BCL2 are indicated. f, Gene expression for IKZF1 as determined by RNA-seq in MOLM13 and MV4;11, indicating log-2-fold change value and adjusted p-value determined using DESeq2. g, Western blot analysis for IKAROS protein in the MOLM13 cell line following 5 days treatment with either VTP-50469 or iberdomide/CC220, followed by rescue treatment with bortezomib (BTZ) for 6 hours, using GAPDH as a loading control. Performed in the presence of the broad-spectrum caspase inhibitor, QV-D-OPH, to prevent cell death. h, Tornado plots depicting global IKAROS chromatin binding at TSSes, as determined by CUT&RUN in the MOLM13 cell line.

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Extended Data Fig. 8 IKAROS protein interactome in MLL-r AML.

a, Binary overlap percentage between all IKAROS peaks enriched 5-fold over background, genome-wide, with each of the other 3 factors, MEIS1 (at 15FE), MENIN (at 5FE) and MLL1 (at 5FE) according ChIP-seq data. b, IGV tracks depicting binding of IKAROS, MENIN, MLL1 and MEIS1, as determined by ChIP-seq, in the region of the TNF gene. Promoters and enhancers, predicted from the ABC tool, are indicated with genomic location indicated (Hg19). c, IGV tracks depicting binding of IKAROS, MENIN, MLL1 and MEIS1 at the MYC and FLT3 genes as determined by ChIP-seq. Promoters and selected enhancers, predicted from the ABC tool, are indicated with genomic location indicated (Hg19). d, Schematic diagrams of protein domains and experimental workflow of BioID system. IKAROS, MEIS1, HOXA10 and ZNF692 proteins were tagged with the biotin ligase BirA*, the left side of the protein schematic diagram denotes N-terminal tagging and the right-side, C-terminal. MV4;11 and MOLM13 cells expressing BioID constructs, 8 total cell lines, were cultured separately. ZF = Zinc finger domain, HBD = homeobox domain, LFQ = label free quantitation, LC-MS/MS = liquid chromatography coupled tandem mass spectrometry. e, Western Blot analysis of the expression of BirA* and HA tagged fusion proteins in MV4;11 and MOLM13 cells, using total Histone H3 as a loading control. f, Venn diagram displaying the number of proteins identified in label free quantitation analysis of the MOLM13 IKAROS, MEIS1 and HOXA10 BioID LC-MS/MS data. All proteins included in Venn diagram have a > 2-fold enrichment over the ZNF692 control. g, List of proteins common to IKAROS, MEIS1, HOXA10 BioID identified in LFQ analysis having >2-fold enrichment over the ZNF692 control in the MV4;11 and MOLM13 cell lines.

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Extended Data Fig. 9 IKAROS and MENIN co-reside within an isolatable protein complex.

a, IGV ChIP-seq tracks for IKAROS, MENIN, MEIS1 and MLL1 at selected gene TSSes. Tracks are derived from the same ChIP-seq experiments depicted in Fig. 6 and Extended Data Fig. 8. b, Direct protein Co-IP using IKAROS as the bait and probed for MENIN, HOXA9, MEIS1, MTA2 and IKAROS in the MV4;11, OCI-AML2 and MOLM13 cell lines using high salt nuclear extraction and detergent wash. c, Direct protein Co-IP using MLL1 and MENIN as the bait and probed for MENIN and IKAROS, in the MV4;11, OCI-AML2 and MOLM13 cell lines using high salt nuclear extraction and detergent wash. d, Direct protein Co-IP using IKAROS as the bait and probed for MENIN, MLL1, IKAROS and MTA2 in the MV4;11 and THP-1 cell lines using benzonase-treated nuclear extract and high glycerol wash. e, Direct protein Co-IP using MLL1 (rabbit antibody) and MENIN (rabbit antibody) as the bait and probed for MLL1, MENIN and rabbit IKAROS antibody (rIKAROS), in the MV4;11 and THP-1 cell lines using benzonase-treated nuclear extract and high glycerol wash. Rabbit immunoglobulin is seen overlying the IKAROS Western blot bands (indicated in the figure) due to the use of an anti-rabbit secondary antibody. f, Direct protein Co-IP using IKAROS as the bait, with and without prior VTP-50469 treatment (250 nM for 48 hr), and probed for MENIN, MTA2, and IKAROS, in the THP-1 cell line.

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Extended Data Fig. 10 Combination therapy with VTP-50469 and LEN results in additive anti-leukemic activity in vivo.

a, Immunohistochemistry for human CD45 antigen on bone marrow sections (sternum) on PDX/CBAM-68552 following 3 weeks treatment with vehicle, LEN 50 mg/kg daily, VTP-50469 0.1% rodent diet and the drug combination. b, Analysis of differentiation in circulating leukaemia cells using CD11b and CD14 expression assessed by flow cytometry in CBAM-68552 after 3 weeks drug treatment. Percentage of double-positive cells is indicated. Representative examples from individual mice are shown. c, Assessment of apoptosis in human CD45-positive cells from bone marrow of drug-treated mice using the CBSK-17D model following 2 weeks drug treatment. The percentage of total viable, human cells are indicated. Representative examples from individual mice are shown. d, Quantitation of apoptotic cells from bone marrow of mice transplanted with the CBSK-17D PDX model following 2 weeks drug treatment. Data represent mean+/−SEM with p-value by unpaired two-tail t-test. e, RNA-seq heatmap from PDX/CBAM-68552 following 3 weeks treatment with vehicle, LEN 50 mg/kg daily, VTP-50469 0.1% rodent diet and the drug combination. Heatmap displaying RNA-Seq DESeq2 statistical z-score for the co-regulated gene network between VTP-50469 and LEN f, RNA-seq heatmap displaying DESeq2 statistical z-score for genes detected in the ‘granulocyte’ pathway as additively deregulated using QIAGEN Ingenuity Pathway Analysis (IPA) analysis. g, Bar code plots using the gene set for HOXA9 regulated genes under each drug treatment from RNA-seq in vivo using the PDX/CBAM-68552 model. Normalized enrichment score and family-wise error rate p-value determined by the GSEA computational method. h, Gene set testing for reported MLL-fusion gene targets for LEN and VTP-50469. Family-wise p-value and normalized enrichment score (NES) determined by the GSEA computational method. i, IPA upstream regulator analysis. Graph displays selected activation z-scores for detected pathways with p-value <0.05. j, Measurement of peripheral blood circulating human CD45 positive cells from PDX mice transplanted with the DFAM-16835 PDX model (NPM1c) after two weeks of drug treatment in vivo. Data represent mean + /−SEM with p-value determined using unpaired, two-tail t-test.

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Supplementary information

Reporting Summary

Supplementary Tables 1–8

Supplementary Table 1. MAGeCK MLE and MAGeCKFlute computational pipeline output results. Results are listed for MLE calculations for EPZ-5676, VTP-50469 and a combined analysis of EPZ-5676 and VTP-50469. P values were determined using the MAGeCK MLE algorithm (see Methods for details). Supplementary Table 2. Global MS proteomic analysis for IMiD neo-substrate profiles in the MOLM-13, MV4;11 and OCI-AML2 cell lines with and without VTP-50469 pretreatment. P values were determined by moderated t-tests as implemented by the Bioconductor limma package (see Methods for details). Supplementary Table 3. Next-generation RNA-seq of MV;411 and MOLM-13 cell lines following treatment of MV;411 and MOLM-13 cell lines with LEN at 5 μM, CC220 at 1 μM, VTP-50469 at 50 nM and the two combinations for 72 h. Data are also listed for the MV4;11 cell line treated for 6 d with LEN at 5 μM. Data are also listed for the CBAM-68552 PDX model treated in vivo for 3 weeks with LEN at 50 mg per kg daily, VTP-50469 at 0.1% in the rodent diet and the combination. Results from the DESeq2 computational pipeline are listed. P values for the RNA-seq analysis were derived using the Bioconductor package DESeq2 (see Methods for details). Supplementary Table 4. ChIP–seq analysis peak calling and factor co-occupancy for IKAROS, MENIN, MEIS1 and MLL1 in the MV4;11 and MOLM-13 cell lines. The presence of a peak at the indicated genomic locus is indicated by a ‘Y’. Peaks falling within ±1 kb of a TSS are indicated in the ‘TSS’ column. Peaks overlapping an enhancer, predicted by the ABC model, are indicated in the ‘enhancer’ column. Supplementary Table 5. Oligonucleotide sequences used in the methodology are listed. Supplementary Table 6. Gene lists used in the presented data analysis are listed. Supplementary Table 7. Spectral counting for proteins detected by LC–MS/MS for IKAROS, MEIS1 and HOXA10 BioID systems in MV;411 and MOLM-13 cell lines. Supplementary Table 8. LFQ for proteins detected by LC–MS/MS for IKAROS, MEIS1, HOXA10 and ZNF692 BioID systems in MV;411 and MOLM-13 cell lines.

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Aubrey, B.J., Cutler, J.A., Bourgeois, W. et al. IKAROS and MENIN coordinate therapeutically actionable leukemogenic gene expression in MLL-r acute myeloid leukemia. Nat Cancer 3, 595–613 (2022). https://doi.org/10.1038/s43018-022-00366-1

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