Supplemental Information

Supplemental Materials and Methods

Cell Lines

All cell lines were routinely cultured at 37°C in 5% CO2 in RPMI-1640 medium with 2mM L-glutamine (Invitrogen), 10% fetal bovine serum (FBS, Gemini Bio-Products) and penicillin/streptomycin (Invitrogen). Ba/F3 cells were supplemented with 1 ng/mL recombinant IL-3. Stably transfected Ba/F3-FLT3/ITD (Ba/F3-ITD) cells, Ba/F3-FLT3/PM (Ba/F3-PM) cells and drug resistant Ba/F3-ITD cells were obtained as previously described (33). Ba/F3 cells were from ATCC.

Reagents and antibodies

Recombinant human interleukin-3 (IL-3) was purchased from Pepro Tech, Inc. D-luciferin was purchased from Gold Biotechnology. Alpha-1 acid glycoprotein (AGP) and human serum albumin (HSA) were purchased from Sigma (catalog #G9885 and A9511, respectively). BALB/c mouse plasma was purchased from Innovative Research, Inc. The FLT3 S-18 antibody was from Santa Cruz Biotechnology, 4G10 phosphotyrosine mouse monoclonal antibody and recombinant protein A-agarose were from Upstate Biotechnology, and Annexin V-APC and anti-human CD135-PE (FLT3) antibodies were from BD Pharmingen. Phospho-MAPK (#9101), phospho-STAT5 (#9351), phospho-AKT (#9271), MAPK (#9102), STAT5 (#9363) and AKT (#9272) antibodies were from Cell Signaling Technologies, Inc. Goat anti-rabbit horseradish peroxidase antibody and the enhanced chemiluminescence (ECL) kit were from GE Healthcare and Amersham Biosciences, respectively. For fluorescence Western blot detection, Alexa Fluor-680 goat anti-mouse (Invitrogen) and IRDye 800 goat anti-rabbit IgG (Rockland Immunochemicals) secondary antibodies were used.

Growth Inhibition

Cells were seeded at a density of 1 ´ 105/mL to 2.5 ´ 105/mL in the presence or absence of inhibitor for 48h. Cell proliferation was measured using the MTT cell proliferation assay according to the manufacturer’s instructions (Roche Applied Science). Proliferation half maximal inhibitory concentrations (IC50s) were calculated by linear regression analysis of optical density (OD) measured at 570 nm, relative to DMSO control (CalcuSyn, Biosoft).

Mice

Female BALB/C mice (age 6-8 weeks) were obtained from Jackson Laboratories. All mice were housed in microisolator cages in a pathogen-free animal facility. All animal procedures were conducted in accordance with the policy of the Johns Hopkins University School of Medicine Animal Care and Use Committee.

Supplemental Tables

Supplemental Table 1. Proliferation IC50 for treatment of FLT3 activating mutant cells

Cell Line / TTT-3002 IC50 (nM) / AC220 IC50 (nM)
Ba/F3-ITD / <0.25 / <0.25
Ba/F3-D835Y / 4.1 / >100
Ba/F3-D835H / 9.9 / >100
Ba/F3-D835V / 0.6 / >100
Ba/F3-D835F / 0.4 / >100
Ba/F3-D835E / 1.6 / 33.4
Ba/F3-D835L+K / 6.7 / >100
Ba/F3-D835A / 1.3 / >100
Ba/F3-I836S / 9.0 / >100
Ba/F3-I836T / 9.7 / 63.3
Ba/F3-I836L+D / 11.7 / >100
Ba/F3-D593∆ / 2.8 / >100
Ba/F3-V579A / 6.2 / >100

Proliferation half maximal inhibitory concentration (IC50, nM) at 48 h, measured by MTT assay as in Figure 1A.

Supplemental Table 2. FLT3 mutational status and proliferation IC50 of AML patient samples

Patient ID / Age / Gender / ITD mutation / D835 mutation / TKI treatment at relapse / TTT-3002 IC50 (nM) / Sorafenib IC50 (nM) / AC220 IC50 (nM)
AML 1 / 67 / M / + / - / n/a / 19 / 17 / 8
Relapse AML 1 / 36 / M / + / + / Sorafenib / 3 / >50 / 32
Relapse AML 2 / 66 / F / + / + / AC220 / 8 / >50 / 19
Relapse AML 3 / 63 / M / - / + / Sorafenib / 7 / >50 / >50

Proliferation half maximal inhibitory concentration (IC50, nM) at 48 h, measured by MTT assay as in Figure 5B.

Supplemental Table 3. Phospho-FLT3 IC50 in plasma culture conditions

Culture Condition / TTT-3002 IC50 (nM) / CEP701 IC50 (nM)
RPMI with 10% FBS / 0.5 ± 0.02 / 2.8 ± 1.0
HUMAN PLASMA / 7.0 ± 0.5 / >50#
MOUSE PLASMA / 1.8 ± 0.8 / 16.8 ± 7.8

Phospho-FLT3 half maximal inhibitory concentration (IC50, nM) measured after 1 hour drug exposure in RPMI-1640 with 10% FBS, 100% human plasma or 100% mouse plasma, relative to DMSO control, as in Figure 6A, B. Data represent average ± SD.

# IC50 for CEP701 previously reported as 0.7µM in human plasma (7).

Supplemental Figures

Supplemental Figure 1. TTT-3002 inhibits downstream targets of FLT3 signaling in Ba/F3-D835Y/ITD cells. Inhibition of phospho-STAT5 (pSTAT5) and phospho-MAPK (pMAPK) in Ba/F3-D835Y/ITD cells by TTT-3002, CEP-701, sorafenib, AC220 or PKC412, each at 20nM. Fraction of phospho-protein/total protein, relative to DMSO, is indicated below each blot.

Supplemental Figure 2. TTT-3002 and CEP-701 demonstrate minimal protein binding interference due to human serum albumin. A, Ba/F3-ITD cells were cultured in RPMI-1640 supplemented with 10% FBS, human serum albumin (HSA, 0-4.0 g/dL) and TTT-3002 (5nM) or CEP-701 (20nM) for 1 h, and inhibition of pFLT3 was measured by Western blotting. Fraction of pFLT3/FLT3 relative to DMSO control is indicated below each blot. Results representative of three independent experiments. B, Endogenous AGP concentration in pediatric and adult AML patient plasma and normal adult plasma by radial immunodiffusion assay. Error bars represent average of triplicate assays ± SD. C, Endogenous HSA concentration in pediatric and adult AML patient plasma and normal adult plasma by radial immunodiffusion assay. Error bars represent average of triplicate assays ± SD.

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