Supplementary Figure S1. pakt KG1 MOLM14 MV411 HL60 THP1 TAKT Vinculin
Supplementary Figure S2. A. MOLM14 2808 6766 0.5 μm 2257 0.5 μm 4537 4 μm 1379 4 μm 1997 Cells PSYK B. pakt MOLM14 C. MOLM14 psyk TSYK TAKT pakt Vinculin TAKT pmtor pmtor TmTOR TmTOR prps6 TRPS6 GAPDH p4ebp1 0.4% 0.5 μm 4 µm T4EBP1 Vinculin 0 30 60 90 120 24 hr Time (min)
Supplementary Figure S3. A. MOLM14 pakt TAKT psyk TSYK Vinculin 0.01% GDC0941 1.0 μm 0.01% GDC0941 1.0 μm B. MOLM14 p4ebp1 T4EBP1 psyk TSYK Vinculin 0.01% Torin 1 350 nm 0.01% Torin 1 350 nm
Supplementary Figure S4. A. HL60 KG1 MOLM14 MFI: 500 MFI: 613 MFI: 1453 MFI: 3554 MFI: 324 MFI: 174 MFI: 42 MFI: 2748 MFI: 197 MFI: 187 MFI: 52 MFI: 180 prps6 Expression (log) B. HL60 KG1 MOLM14 MFI: 5884 MFI: 12000 MFI: 6605 MFI: 7767 MFI: 6590 MFI: 9631 MFI: 2338 MFI: 8496 MFI: 4985 MFI: 6709 MFI: 534 MFI: 6902 p4ebp1 Expression (log) 0.4% 0.5 μm 4 μm
Supplementary Figure S5. A. HL60 MOLM14 MFI: 4517 MFI: 4666 MFI: 6011 Control shrna SYK shrna #10 MFI: 2332 MFI: 1172 MFI: 2537 SYK shrna #1 MFI: 2131 MFI: 1226 MFI: 1942 TotalSYK Expression (log) B. HL60 MOLM14 Control shrna MFI: 1485 MFI: 6975 MFI: 1395 SYK shrna #10 MFI: 954 MFI: 2484 MFI: 1004 SYK shrna #1 MFI: 936 MFI: 2595 MFI: 758 prps6 Expression (log) C. HL60 MOLM14 Control shrna MFI: 8642 MFI: 12925 MFI: 17022 SYK shrna #10 MFI: 6571 MFI: 9531 MFI: 20921 SYK shrna #1 MFI: 6467 MFI: 9180 MFI: 16590 p4ebp1 Expression (log)
A. Supplementary Figure S6. p4ebp1 MOLM14 T4EBP1 Vinculin 0.01% GDC0941 1.0 μm 0.01% GDC0941 1.0 μm B. MOLM14 p4ebp1 T4EBP1 Vinculin 0.01% Torin1 350 nm 0.01% Torin1 350 nm
Supplementary Figure S7. HL60 RSG2 RSG2 NCF1 NCF1 KIAA0513 KIAA0513 IER3 IER3 EMR3 EMR3 KIAA0913 KIAA0913 CYP4F3 CYP4F3 GOS2 SLC2A3 S100P upregulated genes GOS2 SLC2A3 S100P upregulated genes SERPINA1 SERPINA1 FUCA1 FUCA1 ALOX5 ALOX5 NPC2 NPC2 FCER1G FCER1G NCF2 NCF2 ITGAM ITGAM ITGB2 ITGB2 DSCR2 DSCR2 NPM1 NPM1 PBP PBP KIAA0164 KIAA0164 FLJ12525 MPO TRA1 downregulated genes FLJ12525 MPO TRA1 downregulated genes LOC51659 LOC51659 PRTN3 PRTN3 HSPB1 HSPB1 Differentiation Score Differentiation Score 0.1% 0.5% 4EGI1, 25 um 0.1% 0.5% 4EGI1, 25 um
Supplementary Figure S8. A. 6000 5000 CD11b expression CD14 expression Median of Fluorescence 4000 3000 2000 1000 0 : 4EGI1: HL60 B. HL60 4EGI1 4EGI1 4EGI1 4EGI1 Cells BrdU C. HL60 Control Control 6% 4% Number of Cells 13% 12% 4EGI 4EGI1 4EGI1 4EGI 4% 3% 4EGI 4EGI1 4EGI1 4EGI 12% 11% Active Caspase 3
perk1/2 TERK1/2 Vinculin 4 µm 100 nm PD0325901 500 nm GDC0941 MOLM14 HL60 Supplementary Figure S9.
Supplementary Figure S10. A. MOLM14 MFI: 3645 MFI: 917 MFI: 3391 MFI: 533 prps6 Expression (log) Rapamycin 10 nm MOLM14 MFI: 8815 MFI: 6982 MFI: 13100 MFI: 10600 p4ebp1 Expression (log) B. 40 35 HL60 **** Differentiation Score 30 25 20 15 10 5 *** **** **** **** *** 0 45 40 0.25% Rapa 12.5 nm Rapa 25 nm Rapa 50 nm Rapa 100 nm 4EGI1 25 µm 4EGI1 40 µm **** ns * ** *** **** not significant p < 0.05 p < 0.01 p < 0.001 p < 0.0001 Differentiation Score 35 30 25 20 15 10 ns ns * ** **** 5 0 0.25% Rapa 12.5 nm Rapa 25 nm Rapa 50 nm Rapa 100 nm 4EGI1 25 µm 4EGI1 40 µm
Supplementary Table S1. Differentiation Signature (32Gene Signature) GENE NAME RefSeq PROBE SEQUENCE RGS2 NM_002923 5'TAATACGACTCACTATAGGGCTTTAATCTCAATCAATACAAATCAGGGAATAGGTGGTCTGAAC3' 5'PhosGTGGTGTCTCACTCTGAAAATCCCTTTAGTGAGGGTTAAT3' NCF1 NM_000265 5'TAATACGACTCACTATAGGGCTTTATCAATACATACTACAATCATGGACGCCGAGGGCAGCCCC3' 5'PhosGACCCCTGTCCAGCGCGGCTTCCCTTTAGTGAGGGTTAAT3' KIAA0513 NM_014732 5'TAATACGACTCACTATAGGGTACACTTTATCAAATCTTACAATCCACCAGTATCTTCTCTGTTG3' 5'Phos/CATTTTTGCAATCTTGTGTCTCCCTTTAGTGAGGGTTAAT3' IER3 NM_003897 5'TAATACGACTCACTATAGGGTACATTACCAATAATCTTCAAATCGCTGTCACGGAGCGACTGTC3' 5'PhosGAGATCGCCTAGTATGTTCTTCCCTTTAGTGAGGGTTAAT3' EMR3 NM_032571 5'TAATACGACTCACTATAGGGCAATTCAAATCACAATAATCAATCGATGGGTCCTGACTCAAAAC3' 5'PhosCCAGTGAGGGGGATGTTTTTTCCCTTTAGTGAGGGTTAAT3' KIAA0913 NM_015037 5'TAATACGACTCACTATAGGGTAATCTTCTATATCAACATCTTACTGGGAGGGGGCGTTGGGTGG3' 5'PhosCCTCTGGTATTTATTTGGCATCCCTTTAGTGAGGGTTAAT3' CYP4F3 NM_000896 5'TAATACGACTCACTATAGGGATACTTCATTCATTCATCAATTCATCTGGATTTTCTATCTATTC3' 5'PhosCATGTTGGACCAATACCACATCCCTTTAGTGAGGGTTAAT3' PSMG1 NM_003720 5'TAATACGACTCACTATAGGGTCAATTACCTTTTCAATACAATACAACAACCGAATATAGTACAC3' 5'PhosGACCTTCCTGCAGCAGTTCTTCCCTTTAGTGAGGGTTAAT3' NPM1 NM_002520 5'TAATACGACTCACTATAGGGCTTTTCAATTACTTCAAATCTTCATGATAGGACATAGTAGTAGC3' 5'PhosGGTGGTCAGACATGGAAATGTCCCTTTAGTGAGGGTTAAT3' PEBP1 NM_002567 5'TAATACGACTCACTATAGGGTTACTCAAAATCTACACTTTTTCAGAAAAGCTGGTCTGGAGTTG3' 5'PhosCTGAATGTTGCATTAATTGTTCCCTTTAGTGAGGGTTAAT3' ANP32E NM_030920 5'TAATACGACTCACTATAGGGAAAGTTGAGTATTGATTTGAAAAGATATTTTGTAGAAGTTTTCG3' 5'PhosGTCCTATTTTAATGCTTTTGTCCCTTTAGTGAGGGTTAAT3' BCLAF1 NM_001077440 5'TAATACGACTCACTATAGGGTTCACTTTTCAATCAACTTTAATCAAATAACTCACTGATACCTG3' 5'PhosCGTTAACATACTTTGTTTTGTCCCTTTAGTGAGGGTTAAT3' LAS1L NM_031206 5'TAATACGACTCACTATAGGGCAATTTCATCATTCATTCATTTCATGGGAGACAGCCTGGATCAG3' 5'PhosCCACATCAACTCAGTTGTCCTCCCTTTAGTGAGGGTTAAT3' MPO NM_000250 5'TAATACGACTCACTATAGGGCTTTCTACATTATTCACAACATTATTCCTCACCCTGATTTCTTG3' 5'PhosCTTATTCACTGAAGTTCTCCTCCCTTTAGTGAGGGTTAAT3' HSP90B1 NM_003299 5'TAATACGACTCACTATAGGGCTATCTTCATATTTCACTATAAACGGAGAGACTTGTTTTGGATG3' 5'PhosCCCCCTAATCCCCTTCTCCCTCCCTTTAGTGAGGGTTAAT3' GINS2 NM_016095 5'TAATACGACTCACTATAGGGCTTTCAATTACAATACTCATTACAGCCAACAATGCTGACCGGTG3' 5'PhosCTTATCCTCTAAGCCCTGATTCCCTTTAGTGAGGGTTAAT3' RRP7A NM_015703 5'TAATACGACTCACTATAGGGTCATTTCACAATTCAATTACTCAACCTCAATGCAAAAGCCCTTG3' 5'PhosCTGGCAACGAAAAAGCCTCATCCCTTTAGTGAGGGTTAAT3' PRTN3 NM_002777 5'TAATACGACTCACTATAGGGTCATTTCAATCAATCATCAACAATCTTCGTGATCTGGGGATGTG3' 5'PhosCCACCCGCCTTTTCCCTGACTCCCTTTAGTGAGGGTTAAT3' HSPB1 NM_001537 5'TAATACGACTCACTATAGGGTACACTTTAAACTTACTACACTAAAAATCCGATGAGACTGCCGC3' 5'PhosCAAGTAAAGCCTTAGCCTGGTCCCTTTAGTGAGGGTTAAT3' G0S2 NM_015714 5'TAATACGACTCACTATAGGGCTATTACACTTTAAACATCAATACTAGAACTGACCTACCACAAG3' 5'PhosCATCCACCAAAGGAGTTTGGTCCCTTTAGTGAGGGTTAAT3' SLC2A3 NM_006931 5'TAATACGACTCACTATAGGGCTTTCTATTCATCTAAATACAAACACTTCATGTCAACTTTCTGG3' 5'PhosCTCCTCAAACAGTAGGTTGGTCCCTTTAGTGAGGGTTAAT3' S100P NM_005980 5'TAATACGACTCACTATAGGGCTTTCTATCTTTCTACTCAATAATTCACAGATTCCTGGCAGAGC3' 5'PhosCATGGTCCCAGGCTTCCCAATCCCTTTAGTGAGGGTTAAT3' SERPINA1 NM_000295 5'TAATACGACTCACTATAGGGAATCATACTCAACTAATCATTCAATTACATTTACCCAAACTGTC3' 5'PhosCATTACTGGAACCTATGATCTCCCTTTAGTGAGGGTTAAT3' FUCA1 NM_000147 5'TAATACGACTCACTATAGGGAATCTACACTAACAATTTCATAACGGAAAAGGCTTACCAGGCTG3' 5'PhosCTATGGTCAACTCTTCAGAATCCCTTTAGTGAGGGTTAAT3' ALOX5 NM_000698 5'TAATACGACTCACTATAGGGCTACTATACATCTTACTATACTTTCTCAGCATTTCCACACCAAG3' 5'PhosCAGCAACAGCAAATCACGACTCCCTTTAGTGAGGGTTAAT3' NPC2 NM_006432 5'TAATACGACTCACTATAGGGATACTAACTCAACTAACTTTAAACCAGAAACTGAGCTCCGGGTG3' 5'PhosGCTGGTTCTCAGTGGTTGTCTCCCTTTAGTGAGGGTTAAT3' FCER1G NM_004106 5'TAATACGACTCACTATAGGGTCATTTACCAATCTTTCTTTATACCCAGGAACCAGGAGACTTAC3' 5'PhosGAGACTCTGAAGCATGAGAATCCCTTTAGTGAGGGTTAAT3' NCF2 NM_000433 5'TAATACGACTCACTATAGGGCTACAAACAAACAAACATTATCAAAAGGGCACGAGAGAGTCTTC3' 5'PhosCAGGTACTGATCCTGTTTCTTCCCTTTAGTGAGGGTTAAT3' ITGAM NM_000632 5'TAATACGACTCACTATAGGGTACACTTTCTTTCTTTCTTTCTTTGGTTTCCTTCAGACAGATTC3' 5'PhosCAGGCGATGTGCAAGTGTATTCCCTTTAGTGAGGGTTAAT3' ITGB2 NM_000211 5'TAATACGACTCACTATAGGGTACACAATCTTTTCATTACATCATAGAAATCCAGTTATTTTCCG3' 5'PhosCCCTCAAAATGACAGCCATGTCCCTTTAGTGAGGGTTAAT3' GAPDH NM_002046 5'TAATACGACTCACTATAGGGTCATTCATATACATACCAATTCATATCTCCCCTCCTCACAGTTG3' 5'PhosCCATGTAGACCCCTTGAAGATCCCTTTAGTGAGGGTTAAT3' HNRNPAB NM_031266 5'TAATACGACTCACTATAGGGTCAATCATCTTTATACTTCACAATGCCTGGACCTGTGGACCCTG3' 5'PhosGTTGTAAAGAGTAAATTGTATCCCTTTAGTGAGGGTTAAT3'
Supplementary Table S2. Primary Patient Sample Characteristics Patient Diagnosis Cytogenetics 1 New diagnosis M5AML 46 XY 2 Progressive MDS (RAEB2) 46 XY 3 New diagnosis AML with monocytic differentiation 46,XY,t(11;17)(q23;q12) 4 New diagnosis mixed phenotype acute leukemia (T/myeloid) 49,XX,4,10,19[8]/46,XX [cp12] 5 Relapsed AML 47,XY,11[15]/47,XY,del(11)(q11q21)[3]/46,XY[2]
Supplementary Table S3. Significance values for viability comparisons in Figure 4A. Oneway analysis of variance p VALUE Bonferroni's Multiple Comparison Test Day 3 Day 6 HL60 Control shrna x Control shrna GDC0941, 1000nM *** *** HL60 Control shrna x Control shrna Torin 1, 250nM *** *** HL60 Control shrna x Control shrna 4EGI1, 25µM *** *** HL60 Control shrna x SYK shrna#1 *** *** HL60 Control shrna x SYK shrna#1 GDC0941, 1000nM *** *** HL60 Control shrna x SYK shrna#1 Torin 1, 250nM *** *** HL60 Control shrna x SYK shrna#1 4EGI1, 25µM *** *** HL60 Control shrna GDC0941, 1000nM x SYK shrna#1 GDC0941, 1000nM *** *** HL60 Control shrna Torin 1, 250nM x SYK shrna#1 Torin 1, 250nM *** *** HL60 Control shrna 4EGI1, 25µM x SYK shrna#1 4EGI1, 25µM *** *** HL60 SYK shrna#1 x SYK shrna#1 GDC0941, 1000nM *** *** HL60 SYK shrna#1 x SYK shrna#1 Torin 1, 250nM ** *** HL60 SYK shrna#1 x SYK shrna#1 4EGI1, 25µM *** *** Day 3 Day 6 Control shrna x Control shrna GDC0941, 1000nM *** *** Control shrna x Control shrna 4EGI1, 25µM *** *** Control shrna x SYK shrna#1 *** *** Control shrna x SYK shrna#1 GDC0941, 1000nM *** *** Control shrna x SYK shrna#1 4EGI1, 25µM *** *** Control shrna GDC0941, 1000nM x SYK shrna#1 *** *** Control shrna GDC0941, 1000nM x SYK shrna#1 GDC0941, 1000nM *** *** Control shrna 4EGI1, 25µM x SYK shrna#1 *** *** Control shrna 4EGI1, 25µM x SYK shrna#1 4EGI1, 25µM *** *** SYK shrna#1 x SYK shrna#1 GDC0941, 1000nM ns *** SYK shrna#1 x SYK shrna#1 4EGI1, 25µM ns *** Control shrna x Control shrna Torin 1, 50nM *** *** Control shrna x SYK shrna#1 *** *** Control shrna x SYK shrna#1 Torin 1, 50nM *** *** Control shrna Torin 1, 50nM x SYK shrna#1 Torin 1, 50nM *** *** SYK shrna#1 x SYK shrna#1 Torin 1, 50nM *** *** ns not significant ** p<0.01 *** p<0.001
Supplementary Figure Legends Supplementary Figure S1. AKT Activation in AML cell lines. Basal levels of phosphorylated AKT in six untreated AML cell lines. AKT activation was assessed by immunoblotting to pakt (Ser473). Supplementary Figure S2. inhibits SYK and modulates AKT and mtor activation. (a) Cells from two AML cell lines were treated for 24 hours with versus and SYK phosphorylation at Tyr525/526 evaluated by intracellular flow cytometry. (b) Western blot of pakt (Ser473) and pmtor (Ser2448) in MOLM14 treated with for 6 hours. (c) Cells were treated with 4 µm for various time points, as indicated, and analyzed for psyk (Tyr525/526), pakt (Ser473), pmtor (Ser2448), prps6 (Ser240/244) and p4ebp1 (Thr37/46). The xaxis denotes expression (log scale) for each protein of interest, and each is labeled with the median fluorescent intensity (MFI) per condition. Supplementary Figure S3. Feedback on SYK is not observed with inhibitors of PI3K or mtorc1/2. (a) MOLM14 and cells were treated with GDC0941 or vehicle for 24 hours prior to western blot analysis with pakt Ser (473) or psyk (Tyr525/526). (b) MOLM14 and cells were treated with Torin 1 or vehicle for 24 hours prior to western blot analysis with p4ebp1 (Thr37/46) or p SYK (Tyr525/526). For psyk analysis, cell extracts were first immunoprecipitated with antibody to SYK before western blotting with psyk.
Supplementary Figure S4. Chemical inhibition of SYK leads to more consistent inhibition of RPS6 than 4EBP1 when assessed by intracellular phosphoflow cytometry. Four AML cell lines were treated with either vehicle () or (0.5 μm and 4 μm) for 24 hours and levels of (a) RPS6 and (b) 4E BP1 phosphorylation were assessed by intracellular phosphoflow cytometry. Phosphorylation of RPS6 at the Ser240/244 site was inhibited across all cell lines with treatment, but the effects on 4EBP1 T37/46 phosphorylation were more variable. The histograms for each condition are representative of at least two biological replicates across up to 3 repeated experiments. The xaxis denotes expression (log scale) for each protein of interest, and each is labeled with the median fluorescent intensity (MFI) per condition. Supplementary Figure S5. Genetic inhibition of SYK leads to more consistent alterations of RPS6 than 4EBP1 when assessed by phosphoflow cytometry. RPS6 and 4EBP1 phosphorylation were assessed in three AML cell lines 72 hours after lentiviral infection with two SYKdirected shrnas or a control shrna. (a) Immunostaining for totalsyk shows that expression was decreased with both hairpins in each cell line. (b) RPS6 phosphorylation at the Ser240/244 site was consistently inhibited in all cell lines tested with SYKdirected shrnas. (c) The effects of SYKdirected shrna on 4EBP1 phosphorylation were subtle or absent. The histograms for each condition are representative of at least two biological replicates across two SYKdirected shrnas. The xaxis denotes expression (log scale) for each protein of interest and each is labeled with the median fluorescent intensity (MFI) per condition.
Supplementary Figure S6. Effects of GDC0941 and Torin 1 treatment on 4EBP1 phosphorylation in AML. Two AML cell lines treated for 24 hours with vehicle versus (a) GDC0941 or (b) Torin 1. Activated 4E BP1 was assessed by immunoblotting to p4ebp1 (Thr37/46). Supplementary Figure S7. Inhibition by and 4EGI1 shows variable effects on individual gene expression. HL60 and were grown in the presence of, 4EGI1 or 4EGI1 for 3 days at which time, GEHTS analysis was performed. The heat maps are derived from the resulting rowwise comparisons of the performance of individual genes in the signature. Red color indicates a relative increase in gene expression, whereas, blue indicates a relative decrease. Supplementary Figure S8. Combined treatment with 4EGI1 and induces differentiation and inhibits proliferation. HL60 and cell lines were treated with 4 μm, 25 μm 4EGI1 or a combination of these two molecules for 5 days before flow analysis of (a) myeloid differentiation markers (CD11b and CD14), (b) BrdU incorporation, and (c) apoptosis induction marker (active caspase 3). Supplementary Figure S9. MEK inhibition blocks MAPK activation in RASmutated AML cell lines. AML cells were treated with vehicle or 4 µm alone, or in combination with the MEK inhibitor PD0325901
(100 nm) or the PI3K inhibitor GDC0941 (500 nm) for 24 hours before western blot analysis with perk1/2 (Thr202/Tyr204). Supplementary Figure S10. Effects of rapamycin on RPS6 and 4EBP1 activation and differentiation in AML. (a) Two AML cell lines were treated with either vehicle () or 10 nm rapamycin for 24 hours and levels of RPS6 and 4EBP1 phosphorylation were assessed by intracellular phosphoflow cytometry. The histograms for each condition are representative of at least two biological replicates across up to 3 repeated experiments. The xaxis denotes expression (log scale) for each protein of interest, and each is labeled with the median fluorescent intensity (MFI) per condition. (b) Cells were treated with rapamycin or 4EGI1 for 3 days and the Differentiation Score was determined. Data was analyzed and Pvalues determined as described in Figure 5. Statistical differences between all chemicallytreated samples and treated samples are displayed as Pvalue symbols.
Supplementary Table Legends Supplementary Table S1. Differentiation signature (32gene signature). Listed are the gene names, Ref Seq numbers, Luminex LUA tags, and the probe sequence for the 32 myeloid differentiation signature genes. Supplementary Table S2. Primary patient AML/MDS sample information including clinical diagnosis and cytogenetics. Supplementary Table S3. Significance values for viability comparisons in Figure 4A. P value symbols are indicated for the various pairwise comparison tests. All comparisons for HL60 are from the same experiment. The comparisons for derive from two separate experiments. The first 9 rows show comparisons for treated with GDC0941 and 4EGI1, and the last 5 rows show comparisons from Torin 1 treatment.