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A B C Supplemental Figure S1 SET2 HEL92.1.7 BRD4 β-Actin SET2
nM, JQ1 BRD4 β-Actin B C SET2 + 10 µM Pomalidomide, 18 hr HEL92.1.7 SET2 50 250 50 250 nM, ARV825, 18 hr 2.5 10 2.5 10 µM, Pomalidomide, 18 hr BRD4 BRD4 BRD2 BRD2 GAPDH GAPDH
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Fold Change ARV/Control
Supplemental Figure S2 Gene Fold Change ARV/Control P-value Fold Change OTX/Control MYC 0.38 0.0005 0.88 0.286 PIM1 0.14 0.009 0.28 0.058 BCL2 0.32 0.017 0.31 0.057 BCL2L1 (Bcl-xL) 0.33 3.95E-07 0.45 0.0008 LMO2 0.27 0.024 0.073 CTNNB1 1.67 3.44 E-05 1.73 0.0003 CDKN1A 3.21 1.54 0.119 HEXIM1 5.12 5.79E-05 5.32
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Reactome_RNA_Pol II_Transcription Reactome_RNA_Pol II_Transcription
Supplemental Figure S3 A GSEA with REACTOME PATHWAYS ARV-825 over OTX015 over Control Control REACTOME_TRANSLATION 5.01 4.18 REACTOME_3_UTR_MEDIATED_TRANSLATIONAL_REGULATION 4.66 3.49 REACTOME_MRNA_PROCESSING 3.01 2.96 REACTOME_TRANSCRIPTION 3 3.51 REACTOME_MRNA_SPLICING 2.88 2.55 REACTOME_RNA_POL_II_TRANSCRIPTION 2.75 2.63 REACTOME_CELL_CYCLE 4.59 4.06 REACTOME_DNA_REPLICATION 3.18 2.93 REACTOME_CHROMOSOME_MAINTENANCE 3.15 3.28 REACTOME_TELOMERE_MAINTENANCE REACTOME_PACKAGING_OF_TELOMERE_ENDS 3.13 3.77 REACTOME_G1_S_TRANSITION 2.73 2.26 REACTOME_CELL_CYCLE_CHECKPOINTS 2.72 2.47 REACTOME_TCA_CYCLE_AND_RESPIRATORY_ELECTRON_TRANSPORT 4.81 3.75 REACTOME_CHOLESTEROL_BIOSYNTHESIS 3.36 2.04 REACTOME_RESPIRATORY_ELECTRON_TRANSPORT_ATP_SYNTHESIS 4.62 3.6 Transcription/ Translation Cell Cycle Energy and Metabolism B Genes induced by ARV825 Genes depleted by ARV-825 NES=2.75 q< Enrichment Score Normalized Reactome_RNA_Pol II_Transcription Genes induced by OTX015 Genes depleted NES=2.63 q<0.0011 Enrichment Score Normalized Reactome_RNA_Pol II_Transcription C
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Cancer-related pathways Immune system Metabolism Transcription/
Supplemental Figure S4 GSEA with REACTOME PATHWAYS ARV-825 over Control OTX015 over Control REACTOME_SIGNALING_BY_WNT -2.38 -2.2 REACTOME_REGULATION_OF_APOPTOSIS -2.24 -2.15 REACTOME_P53_INDEPENDENT_G1_S_DNA_DAMAGE_CHECKPOINT -2.16 -2.32 REACTOME_GPCR_LIGAND_BINDING -3.73 -3.75 REACTOME_GPCR_DOWNSTREAM_SIGNALING -3.18 -3.37 REACTOME_G_ALPHA_I_SIGNALLING_EVENTS -3.15 -3.5 REACTOME_CHEMOKINE_RECEPTORS_BIND_CHEMOKINES -3.3 REACTOME_IMMUNE_SYSTEM -2.36 -3.36 REACTOME_ACTIVATION_OF_NF_KAPPAB_IN_B_CELLS -2.33 REACTOME_DOWNSTREAM_SIGNALING_EVENTS_OF_B_CELL_RECEPTOR_BCR -2.19 -1.95 REACTOME_CYTOKINE_SIGNALING_IN_IMMUNE_SYSTEM -2.42 REACTOME_TCR_SIGNALING -2.14 -2.06 REGULATION_OF_METABOLIC_PROCESS -3.1 -2.18 REGULATION_OF_CELLULAR_METABOLIC_PROCESS -3.01 -2.17 REACTOME_PEPTIDE_HORMONE_BIOSYNTHESIS -2.89 -1.89 REACTOME_GLYCOPROTEIN_HORMONES -2.84 -1.66 REACTOME_TERMINATION_OF_O_GLYCAN_BIOSYNTHESIS -2.69 REACTOME_PEPTIDE_LIGAND_BINDING_RECEPTORS -2.67 -3.71 REACTOME_NUCLEOTIDE_LIKE_PURINERGIC_RECEPTORS -2.53 -3.05 REACTOME_O_LINKED_GLYCOSYLATION_OF_MUCINS -2.81 REACTOME_TANDEM_PORE_DOMAIN_POTASSIUM_CHANNELS -1.72 REACTOME_METABOLISM_OF_RNA -3.9 -4.45 REGULATION_OF_RNA_METABOLIC_PROCESS -2.54 -2.07 TRANSCRIPTION -3.32 -2.95 REGULATION_OF_GENE_EXPRESSION -3.03 TRANSLATION -2.46 -3.14 REACTOME_REGULATION_OF_MRNA_STABILITY_BY_PROTEINS_THAT_BIND_AU_RICH_ELEMENTS -2.3 -2.4 REACTOME_DESTABILIZATION_OF_MRNA_BY_AUF1_HNRNP_D0 -2.23 REGULATION_OF_TRANSCRIPTION_FROM_RNA_POLYMERASE_II_PROMOTER -1.82 REACTOME_NUCLEAR_RECEPTOR_TRANSCRIPTION_PATHWAY -1.43 Cancer-related pathways Immune system Metabolism Transcription/ Translation
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B A C Supplemental Figure S5 PD-CD34+ sAML PD-CD34+ sAML Control
Gene Fold Change ARV/Control P-value Fold change OTX/Control MYC 0.0529 0.045 0.1122 0.0523 IL7R 0.127 0.0292 0.1398 0.0305 CDK6 0.3262 0.0499 0.531 0.1059 BCL2L1 0.1595 0.0087 0.5545 0.0360 STAT5A 0.5931 0.0154 0.5911 0.0152 LMO2 0.2263 0.0053 0.8172 0.1932 BCL2 0.0898 0.083 0.3771 0.172 CTNNB1 2.697 0.0299 2.061 0.087 CDKN1A 5.667 0.0199 1.60 0.040 Control ARV hrs Control OTX015 8 hrs 1.2 -1.2 0.0 Down genes 1516 Down genes 685 Up genes 1436 Up genes 713 C PD, CD34+ sAML Down-regulated genes PD, CD34+ sAML Up-regulated genes
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D E F G * * * * * * * * * * * † † † † † † † † Supplemental Figure S5
8 hrs
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A B SET2 Supplemental Figure S6 Control 250 nM OTX015, 18 hrs -1.6 1.6
c-KIT Axl MUC1 Connexin-43 -1.6 1.6 0.0 Gys pS641 p90 RSK pT573 MAPK pT202/Y204 PKCβ2 pS660 Cl. Caspase 7 Cl. Caspase 3 Histone H3 H2AX pS140 Poly ADP Ribose
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A B Supplemental Figure S7 SET2 + 10 µM Pomalidomide, 16 hr 50 250 50
50 250 50 250 nM, ARV-825, 16 hr c-Myc PIM1 Bcl-xL CDK4 p21 GAPDH B
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A B PD-CD34+ sAML cells - - + - + Supplemental Figure S8 CD123 CD244
Low Mid High CD123 CD244 CD90 TIM3Fc B BRD4 c-Myc p21 Low Mid High Marker expression p-Rb Control - - + - + 250 nM ARV-825 250 nM OTX015
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Supplemental Figure S9 Vehicle vs OTX015 p= 0.021
Vehicle vs ARV p= 0.163
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A B D C Supplemental Figure S10 SET2 ARV-825 (nM) ruxolitinib (nM) Fa
CI Value 10 50 0.526 12 60 0.624 14 70 0.631 15 75 0.618 16 80 0.628 18 90 0.693 20 100 0.788 HEL92.1.7 ARV-825 (nM) ruxolitinib (nM) Fa CI Value 40 200 0.161 0.973 60 300 0.219 100 500 0.424 120 600 0.528 140 700 0.73 160 800 0.763 1000 0.787 D C PD CD34+ sAML ARV-825 (nM) ruxolitinib (nM) Fa CI Value 50 500 0.308 60 600 0.339 70 700 0.286 80 800 0.309 90 900 0.381 100 1000 0.556 PD CD34+ sAML ARV-825 (nM) ruxolitinib (nM) Fa CI value 5 1000 0.134 0.2999 20 0.111 50 0.1541 100 0.168 0.1078 250 0.179 0.0994
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E F G H Supplemental Figure S10 PD CD34+ sAML ARV-825 (nM)
ruxolitinib (nM) Fa CI value 2.5 500 0.421 5 0.608 20 0.812 1000 0.412 0.756 0.0327 0.856 PD CD34+ sAML ARV-825 (nM) ruxolitinib (nM) Fa CI value 5 1000 0.191 0.8348 20 0.237 50 0.298 100 0.305 250 0.326 500 0.34 G H PD CD34+ sAML ARV-825 (nM) ruxolitinib (nM) Fa CI value 10 500 0.396 20 0.435 50 0.39 100 0.401 PD CD34+ sAML ARV-825 (nM) ruxolitinib (nM) Fa CI value 20 1000 0.422 50 0.532 100 0.566 250 0.653 500 0.668
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