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Supplemental Figure 1
A
B
panobinostat
OCI-AML3
Supplemental Figure 2
Control 20 50 nM BC2059, 24 hrA
B
OCI-AML3
Control 20 50 100 nM BC2059, 48 hr
Annexin V-FITC
TO-PRO-3 iodide
HL-60
Control 50 100 nM BC2059, 48 hr
Annexin V-FITC
TO-PRO-3 iodide
C
D
Primary FLT3-ITD
AML
Propidium iodide
Control 20 50 100 nM BC2059, 48 hrE
Supplemental Figure 2
Supplemental Figure 3
A
D
β-Actin
MCL1
p27
OCI-AML3
0 20 50 100 nM, BC2059, 24 h
BIMEL
BIML
BIMS
B
β-Actin
p27
BIMEL
BIML
BIMS
MV4-11
0 20 50 100 nM, BC2059, 24 h
C
Primary FLT3-ITD AML
0 20 50 100 nM, BC2059, 24 h
β-Actin
p27
BIMEL
BIML
BIMS
β-Catenin
20 nMBC2059
100 nMBC2059
Control
DAPITBL1 β-CateninMerge TBL1/β-Catenin
MV4-11 cells
β-catenin DAPI Merge
CD34+ Primary FLT3-ITD AML cells
Control
50 nM BC2059
B
A
C
β-catenin DAPI Merge
CD34+ FLT3 WT Primary AML cells
Control
100 nM BC2059,16 hours
Primary AML cells
TBL1 β Catenin Merge DAPI
Control
100 nM BC2059
20 nM BC2059
D
Supplemental Figure 4
Supplemental Figure 5
A B
C D
Probe Set ID Fold-Change Gene Title Gene Symbol205943_at 10.8824 tryptophan 2,3-dioxygenase TDO2204103_at 10.6149 chemokine (C-C motif) ligand 4 CCL4216834_at 10.3006 regulator of G-protein signaling 1 RGS1201739_at 9.68812 serum/glucocorticoid regulated kinase 1 SGK1215078_at 7.01567 superoxide dismutase 2, mitochondrial SOD2205114_s_at 6.90463 chemokine (C-C motif) ligand 3 CCL3
204472_at 6.23896GTP binding protein overexpressed in skeletal muscle GEM
205476_at 5.13516 chemokine (C-C motif) ligand 20 CCL20212724_at 4.6973 Rho family GTPase 3 RND3201041_s_at 4.671 dual specificity phosphatase 1 DUSP1220054_at 4.66513 interleukin 23, alpha subunit p19 IL23A243296_at 4.53019 Nicotinamide phosphoribosyltransferase NAMPT212099_at 4.39901 ras homolog gene family, member B RHOB206025_s_at 4.19698 tumor necrosis factor, alpha-induced protein 6 TNFAIP61554997_a_at 4.16341 prostaglandin-endoperoxide synthase 2 PTGS2214038_at 3.89781 chemokine (C-C motif) ligand 8 CCL8207433_at 3.84862 interleukin 10 IL10230170_at 3.71956 oncostatin M OSM216598_s_at 3.60761 chemokine (C-C motif) ligand 2 CCL2201466_s_at 3.53626 jun proto-oncogene JUN227697_at 3.51644 suppressor of cytokine signaling 3 SOCS3219371_s_at 3.40553 Kruppel-like factor 2 (lung) KLF2206115_at 3.37739 early growth response 3 EGR3207850_at 3.32973 chemokine (C-X-C motif) ligand 3 CXCL3204533_at 3.30282 chemokine (C-X-C motif) ligand 10 CXCL10203665_at 3.2795 heme oxygenase (decycling) 1 HMOX1209774_x_at 3.20119 chemokine (C-X-C motif) ligand 2 CXCL2204748_at 3.15079 prostaglandin-endoperoxide synthase 2 PTGS2
Supplemental Table 1: Genes with greater than 3 fold induction in OCI-AML3 cells following treatment with 100 nM of BC-2059 for 8 hours.
Probe Set IDFold-
Change Gene Title Gene Symbol
220184_at -3.31242 Nanog homeobox NANOG
204834_at -2.8117 fibrinogen-like 2 FGL2
223044_at -2.80649solute carrier family 40 (iron-regulated transporter), member 1 SLC40A1
213436_at -2.74949 cannabinoid receptor 1 (brain) CNR1
207063_at -2.47669 non-protein coding RNA 185 NCRNA00185
204301_at -2.33592kelch repeat and BTB (POZ) domain containing 11 KBTBD11
238900_at -2.32845major histocompatibility complex, class II, DR beta 1 HLA-DRB1
240429_at -2.28028 zinc finger protein 546 ZNF546
206978_at -2.27337 chemokine (C-C motif) receptor 2 CCR2
228055_at -2.21045 napsin B aspartic peptidase pseudogene NAPSB
217047_s_at -2.21015 family with sequence similarity 13, member A FAM13A
233555_s_at -2.20734 sulfatase 2 SULF2
207794_at -2.13184 chemokine (C-C motif) receptor 2 CCR2
227804_at -2.12735 TLC domain containing 1 TLCD1
219714_s_at -2.11454calcium channel, voltage-dependent, alpha 2/delta subunit 3 CACNA2D3
217546_at -2.11094 metallothionein 1M MT1M
229545_at -2.10518 fermitin family member 1 FERMT1
211341_at -2.09286 POU class 4 homeobox 1 POU4F1
207761_s_at -2.06641 methyltransferase like 7A METTL7A
218858_at -2.06145 DEP domain containing 6 DEPDC6
207728_at -2.03694 activating transcription factor 7 interacting protein ATF7IP
213725_x_at -2.02015 xylosyltransferase I XYLT1
Supplemental Table 2: Genes with greater than 2 fold depletion in OCI-AML3 cells following treatment with 100 nM of BC-2059 for 8 hours
Network Associated Network Functions Score
1 Cellular Movement, Hematological System Development and Function, Immune Cell Trafficking 31
2 Cell Morphology, Endocrine System Disorders, Gastrointestinal Disease 24
3 Neurological Disease, Organismal Injury and Abnormalities, Molecular Transport 21
4 Cellular Development, Cellular Growth and Proliferation, Hematological System Development and Function
21
Supplemental Table 3: Ingenuity Pathway Analysis of the 4 most perturbed networks in OCI-AML3 cells following treatment with 100 nM of BC-2059 for 8 hours.
OCI-AML3
BC2059 PS Fa CI(nM) (nM) 60 6 0.566 1.043 70 7 0.701 0.961 75 7.5 0.811 0.812 80 8 0.836 0.811 90 9 0.9 0.735
CA
HL-60
BC2059 PS Fa CI(nM) (nM) 60 6 0.52 1.027 70 7 0.66 0.792 75 7.5 0.771 0.578 80 8 0.756 0.653 90 9 0.867 0.442
B
Supplemental Figure 6
MV4-11
BC2059 PS Fa CI(nM) (nM)30 3 0.474 0.96335 3.5 0.641 0.61240 4 0.746 0.45250 5 0.942 0.128
*
*
Control
DAPI Merge
10 nM PS, 16 h
OCI-AML3 cells
TBL1
20 nM PS, 16 h
50 nM PS, 16h
A
β-Actin
TBL1
OCI-AML3
0 10 20 50 nM, PS, 16 h
B
Supplemental Figure 7
BC2059 (nM)
PS (nM)
Fa CI
50 25 0.391 0.545
60 30 0.44 0.453
70 35 0.441 0.525
75 37.5 0.453 0.515
80 40 0.527 0.322
90 45 0.498 0.446
100 50 0.512 0.449
BC2059 (nM)
PS (nM)
Fa CI
50 25 0.47 0.993
60 30 0.625 0.624
70 35 0.611 0.773
75 37.5 0.668 0.644
80 40 0.653 0.735
90 45 0.694 0.761
100 50 0.512 0.449
AML #1 AML #2
Supplemental Figure 8