1. Volume 36, Issue 6, Pages 660-673.e11 (December 2019)
A Menin-MLL Inhibitor Induces Specific Chromatin Changes and Eradicates Disease in Models of MLL-Rearranged Leukemia 
Andrei V. Krivtsov, Kathryn Evans, Jayant Y. Gadrey, Benjamin K. Eschle, Charlie Hatton, Hannah J. Uckelmann, Kenneth N. Ross, Florian Perner, Sarah N. Olsen, Tara Pritchard, Lisa McDermott, Connor D. Jones, Duohui Jing, Ali Braytee, Diego Chacon, Eric Earley, Brian M. McKeever, David Claremon, Andrew J. Gifford, Heather J. Lee, Beverly A. Teicher, John E. Pimanda, Dominik Beck, Jennifer A. Perry, Malcolm A. Smith, Gerard M. McGeehan, Richard B. Lock, Scott A. Armstrong 
Cancer Cell 
Volume 36, Issue 6, Pages e11 (December 2019)
DOI: /j.ccell
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2. Cancer Cell 2019 36, 660-673.e11DOI: (10.1016/j.ccell.2019.11.001)
Copyright © 2019 Elsevier Inc. Terms and Conditions

3. Figure 1 Development of VTP50469
(A) Molecules 1 to 4 in medicinal chemistry development of VTP Dotted lines indicate VTP50469 potential interactions with Y276 and W341 of Menin.
(B) Crystal structure of VTP50469 bound in Menin pocket.
(C) Table summarizing VTP50469 IC50 concentrations in MLL-r and control cell lines obtained in cell proliferation experiments. IC50 mean ± SD.
(D) Western blot analysis of size fractionated nuclear lysates from DMSO- or VTP50469-treated MOLM13 cells with Menin antibody.
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4. Figure 2 Gene Expression Changes Induced by VTP50469
(A) Volcano plot of RNA-seq data obtained from MOLM13 cells treated with VTP Selected MLL fusion target genes are labeled.
(B) Heatmap of top 20 most suppressed genes after 2- and 7-day VTP50469 treatment in MOLM13 cells. MLL fusion target genes are in red.
(C–E) GSEA of gene expression changes in MOLM13 cells treated with VTP50469 compared with (C) 113 MLL-AF9 target genes; (D) 198 genes suppressed by MI-389 in MV4;11 cells; and (E) 96 genes suppressed by treatment with EPZ5676 in MOLM13 cells.
(F) DMSO-normalized relative expression of 17 genes that lose expression >3-fold after 7 days EPZ5676 (1 μM) or VTP50469 (330 nM) treatment in MOLM13 cells. The table shows gene expression changes at 2 and 7 days.
(G) PCA clustering of gene expression in MOLM13 cells after 2 or 7 days of treatment with VTP50469 or EPZ5676.
(H) Volcano plot of RNA-seq data obtained from MOLM13-Cas9 cells infected with MEN1 or control sgRNA at day 6. Selected MLL fusion targets genes are labeled.
(I) Venn diagram depicting the overlap of genes that are suppressed >2-fold by either VTP50469 treatment (7 days) or MEN1 knockout (6 days).
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5. Figure 3 VTP50469 Remodels MLL Fusion Chromatin-Associated Complexes
(A and B) Menin, MLL1n, DOT1L, and H3K79me2 ChIP-seq data in MOLM13 cells treated with VTP50469 or DMSO were plotted as (A) heatmap/tornado plots of transcription start site (TSS) ±3 kb and (B) average/meta-plots of 113 MLL-AF9 targets TSS ±3 kb.
(C) Gene tracks of Menin, MLL1n, DOT1L, and H3K79me2 ChIP-seq signals (rpm) at selected MLL-AF9 target genes in MOLM13 cells treated with VTP50469.
(D) Gene tracks of MLL1n CUT&RUN signal on selected MLL-AF9 target genes in control (CTRL) and MEN1 knockout MOLM13 cells.
(E) Venn diagram depicting the overlap in genes that lose MLL1n occupancy (>2-fold) after VTP50469 treatment or CRISPR deletion of MEN1.
(F) Scatterplot depicting correlation of MLL1n and Menin gene occupancy after VTP50469 treatment in MOLM13 cells. VTP50469/DMSO ratios shown as log10 values. Gene expression levels are indicated in the heatmap.
(G) Scatterplot depicting correlation of MLL1n and DOT1L gene occupancy after 3-day treatment with VTP50469 in MOLM13 cells. VTP50469/DMSO ratios shown as log10 values. H3K79me2 levels at genes are indicated in the heatmap.
(H) GSEA of gene expression changes in MOLM13 cells treated with VTP50469 for 7 days compared with genes that lose Menin, MLL1n, or DOT1L chromatin occupancy.
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6. Figure 4 Establishing an Effective VTP50469 Dose In Vivo
(A) Kaplan-Meyer survival curves of NSG mice engrafted with MV4;11 cells and dosed with VTP50469 as indicated. Green bar indicates the 28-day treatment period (n = 10).
(B) PK/PD assessment of MEIS1 expression in MV4;11 cells engrafted in nude rats compared with VTP50469 plasma concentration after dosing using implanted micropumps.
(C) %hCD45+ cells in the peripheral blood (PB) of NSG mice transplanted with MLL-r AML PDX (68552 or 40315) on either control or 0.1% VTP50469 mouse chow. Bars represent SD.
(D and G) %hCD45+ cells in the bone marrow (BM) and spleens (SP) of mice implanted with (D) MLL-r AML PDX or (G) MLLwt/wt (IHD2 R132H) AML and dosed for 28 days with 0.1% VTP50469 chow or control diet. Bars represent the median; the t test was used to calculate the p value.
(E and H) Spleen weights from mice implanted with (E) MLL-r AML PDX or (H) MLLwt/wt (IHD2 R132H) AML and dosed as described in (D). Bars represent the median; the t test was used to calculate the p value.
(F and I) Fluorescence-activated cell sorting assessment of CD11b surface expression in BM isolated from mice implanted with (F) MLL-r AML PDX or (I) MLLwt/wt (IHD2 R132H) AML and dosed as described in (D). Bars represent the median, the t test was used to calculate the p value.
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7. Figure 5 In Vivo Activity of VTP50469 in MLL-r B-ALL PDX Models
(A) %hCD45+ over time in the PB of NSG mice transplanted with an MLL-r B-ALL PDX (MLL-2) and treated with the indicated doses of VTP50469 (BID). Treatment was administered for the first 28 days.
(B) Kaplan-Meier survival analysis of the mice treated in (A). Vertical notches indicate non-leukemia-related events.
(C and D) Postmortem analysis of %hCD45 in the BM (C) and spleen (D) of mice treated with control or VTP50469 (120 mg/kg) at day 0, day 28 or event (whichever occurred sooner). Bars represent the median; ****p <
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8. Figure 6
VTP50469 Increases Survival of Mice Engrafted with MLL-r B-ALL PDXs
(A) H&E staining of the BM of naive and MLL-2-engrafted mice treated with control or VTP50469 at day 0, 28, and 328. Boxes indicate the %hCD45+. Scale bars, 10 μm.
(B) Swimmer plot of event-free survival (EFS) of individual mice engrafted with 8 MLL-r B-ALL PDXs (MLL-1, 2, 3, 5, 6, 7, 8, and 14) and a BCR-ABL1 B-ALL PDX (ALL-56) treated with VTP50469 (120 mg/kg BID for 28 days). Hatched area indicates 28-day treatment period. Blue blocks on the end of the lanes indicate mice that were euthanized for non-leukemia-related events. Red blocks on the end of the lanes indicated mice that are ongoing in the study.
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9. Figure 7
VTP50469 Treatment Suppresses MLL Fusion Target Genes in MLL-r ALL PDX Cells In Vivo
(A) Heatmap representation of gene expression of 145 MLL-AF4 target genes expressed in MLL-r B-ALL PDX cells isolated from mice after 28-day dosing with control or VTP50469 chow.
(B) GSEA of gene expression changes from PDX cells treated compared with MLL-AF4 target genes; VTP50469-sensitive genes in RS4;11 cells that lose occupancy by Menin, MLL1n, or DOT1L.
(C) PCA of single-cell gene expression in MLL-r ALL PDX after a 28-day treatment with VTP50469 or vehicle using genes that lose MLL1n chromatin occupancy.
(D) Heatmap representation of the 27 genes defined in (C).
Cancer Cell  , e11DOI: ( /j.ccell )
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10. Cancer Cell 2019 36, 660-673.e11DOI: (10.1016/j.ccell.2019.11.001)
Copyright © 2019 Elsevier Inc. Terms and Conditions

11. Cancer Cell 2019 36, 660-673.e11DOI: (10.1016/j.ccell.2019.11.001)
Copyright © 2019 Elsevier Inc. Terms and Conditions

12. Cancer Cell 2019 36, 660-673.e11DOI: (10.1016/j.ccell.2019.11.001)
Copyright © 2019 Elsevier Inc. Terms and Conditions

13. Cancer Cell 2019 36, 660-673.e11DOI: (10.1016/j.ccell.2019.11.001)
Copyright © 2019 Elsevier Inc. Terms and Conditions

14. Cancer Cell 2019 36, 660-673.e11DOI: (10.1016/j.ccell.2019.11.001)
Copyright © 2019 Elsevier Inc. Terms and Conditions

15. Cancer Cell 2019 36, 660-673.e11DOI: (10.1016/j.ccell.2019.11.001)
Copyright © 2019 Elsevier Inc. Terms and Conditions

16. Cancer Cell 2019 36, 660-673.e11DOI: (10.1016/j.ccell.2019.11.001)
Copyright © 2019 Elsevier Inc. Terms and Conditions

17. Cancer Cell 2019 36, 660-673.e11DOI: (10.1016/j.ccell.2019.11.001)
Copyright © 2019 Elsevier Inc. Terms and Conditions

18. Cancer Cell 2019 36, 660-673.e11DOI: (10.1016/j.ccell.2019.11.001)
Copyright © 2019 Elsevier Inc. Terms and Conditions

19. Cancer Cell 2019 36, 660-673.e11DOI: (10.1016/j.ccell.2019.11.001)
Copyright © 2019 Elsevier Inc. Terms and Conditions

20. Cancer Cell 2019 36, 660-673.e11DOI: (10.1016/j.ccell.2019.11.001)
Copyright © 2019 Elsevier Inc. Terms and Conditions