Leukemia Letter supplemental materials - … · Leukemia Letter supplemental materials 2 Supplemental Materials: Chemicals AC220 was purchased from Haoyuan Chemexpress Inc (Shanghai,

Leukemia Letter supplemental materials

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Discovery of a Highly Potent FLT3 Kinase Inhibitor for FLT3-ITD Positive AML Hong Wu1,2,9, Aoli Wang1,2,9, Ziping Qi1,3,9, Xixiang Li1,3,9, Cheng Chen1,3,9, Kailin Yu1,2, Fengming Zou1,3, Chen Hu1,2, Wenchao Wang1,3, Zheng Zhao1,3, Jiaxin Wu1,2, Juan Liu1,2, Xiaochuan liu1,2, Li Wang1,3, Wei Wang1,3, Shanchun Zhang3,4, Richard M. Stone5, Ilene A. Galinsky5, James D. Griffin5, David Weinstock5, Alexandra Christodoulou5, Huiping Wang6,7, Yuanyuan Shen6,7, Zhimin Zhai6,7, Ellen L. Weisberg5*, Jing Liu1,3*, Qingsong Liu1,2,3,8* 1. High Magnetic Field laboratory, Chinese Academy of Sciences, Mailbox 1110, 350

Shushanhu Road, Hefei 230031, Anhui, P. R. China 2. University of Science and Technology of China, P. R. China, Anhui, Hefei, 230036 3. CHMFL-HCMTC target therapy Joint Laboratory, Shushanhu Road, Hefei 230031,

Anhui, P. R. China 4. Hefei Cosource Medicine Technology Co. LTD. Ganquan Road, 358, Hefei, 230031,

Anhui, P.R.China 5. Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical

School, 450 Brookline Ave., Boston, MA 02115, USA. 6. Department of Hematology, the Second Hospital of Anhui Medical University, Hefei,

Anhui 230601, China. 7. Hematology Research Center, Anhui Medical University, Hefei, Anhui 230601,

China. 8. Hefei Science Center, Chinese Academy of Sciences, Shushanhu Road, Hefei 230031,

Anhui, P. R. China 9. These authors contribute equally


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Supplemental Materials: Chemicals

AC220 was purchased from Haoyuan Chemexpress Inc (Shanghai, P.R.China).

Synthesis of CHMFL-FLT3-165

All solvents and reagents were used as obtained. 1H NMR spectra and 13C NMR

spectra were recorded with a Bruker 400 NMR or 850 NMR spectrometer and referenced

to deuterium dimethyl sulfoxide (DMSO-d6). Chemical shifts are expressed in ppm. In

the NMR tabulation, s indicates singlet; d, doublet; t, triplet; q, quartet; m, multiplet; and

br, broad peak. Mass spectra were measured with Agilent 6224 TOF using an ESI source

coupled to an Agilent 1260 Infinity HPLC system operating in reverse mode with an

Agilent Eclipse Plus C18 1.8µm 3.050 mm column. Purification of final compounds

were performed with Agilent 218 preparative system (Eclipse XDB-C18 column, 9.4 x

50 mm, 5 µM) using a gradient of 5-95% acetonitrile in water containing 0.05%

trifluoacetic acid (TFA) over 8 min (10 min run time) at a flow rate of 2 mL/min. The

purity of all compounds were above 95% purity as determined by an Agilent 1260

Infinity HPLC with UV detection at 254 nm.

3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (S1) To a mixture of 1H-pyrazolo[3,4-

d]pyrimidin-4-amine (10 g, 74 mmol) and N-iodo-succinimide (25 g, 111 mmol) in DMF

(100 mL) was stirred at 80 oC for 8 h. The resulting mixture was allowed to cool to room

temperature, and then diluted with 300 mL of water. The precipitate was filtered and

washed with 2×60 mL of saturated aqueous sodium sulfite, 2×100 mL of water,


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respectively and dried under vacuum to give the S1 as a yellow solid(14 g, 73). 1H

NMR (400 MHz, DMSO-d6) δ 8.29 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 156.13 ,

154.69 , 153.35 , 102.79 , 91.06 . TOF LC/MS: (ESI)m/z: 261.9591 [M+H]+.

3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (S2) To a mixture of 3-iodo-

1H-pyrazolo[3,4-d]pyrimidin-4-amine (10 g, 38.3 mmol) and DMF/H2O (150 mL, v/v,

3/2) was added (4-phenoxyphenyl)boronic acid (9.8 g, 45.9 mmol), K3PO4 (12.1 g, 57.4

mmol) and Pd(dppf)Cl2 (1.4 g, 1.9 mmol). The reaction mixture was placed into an oil

bath preheated to 120 °C, with stirring at this temperature for 12 h under argon. The

resulting mixture was allowed to cool to room temperature, then was diluted with 150 mL

of water. The precipitate was filtered, and dried under the vacuum. The crude product

was recrystallized with MeOH to afford S2 as a off-white color solid (7.2 g, 62). 1H

NMR (400 MHz, DMSO-d6) δ 13.57 (s, 1H), 8.23 (s, 1H), 7.68 (d, J = 7.8 Hz, 2H), 7.45

(t, J = 7.2 Hz, 2H), 7.26 – 7.08 (m, 5H). 13C NMR (101 MHz, DMSO-d6) δ 158.59,

157.53, 156.77, 156.57, 155.26, 144.41, 130.54, 130.51, 128.93, 124.24, 119.44, 119.38,

97.46. TOF LC/MS: (ESI) m/z: 304.1217 [M+H]+.

tert-butyl(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-

yl)cyclohexyl)carbamate(S3) To a mixture of triphenyl phosphine (5.1 g, 19.8 mmol) in

THF (300 mL) was added diisopropyl azodicarboxylate (3.9 mL, 19.8 mmol) at 0 °C,

The reaction mixture was stirred at this temperature for 0.5 h under argon, then added

tert-butyl(4-hydroxycyclohexyl)carbamate (4.2 g, 19.8 mmol), the reaction mixture was

stirred at 0 °C for 0.5 h, then S2 (3 g, 9.9 mmol) was added. The reaction mixture was

allowed to room temperature with stirring for 12 h. The resulting mixture was then

concentrated to afford the crude product, which was purified by flash chromatography

(eluting 0%-2% MeOH in dichloromethane) to provide S3 as a light foam (3.4 g, 69%).

1H NMR (400 MHz, DMSO-d6) δ 8.25 (s, 1H), 7.70 (d, J = 7.3 Hz, 2H), 7.44 (t, J = 7.1

Hz, 2H), 7.15 (dd, J = 16.9, 8.0 Hz, 5H), 4.74 (s, 1H), 3.60 (s, 1H), 2.28 (d, J = 10.4 Hz,

2H), 1.70 (d, J = 24.4 Hz, 6H), 1.39 (d, J = 12.7 Hz, 9H). 13C NMR (214 MHz, DMSO-

d6) δ 158.62, 157.41, 156.84, 155.85, 153.95, 143.05, 130.58, 130.54, 128.73, 124.18,


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119.47, 119.35, 97.78, 77.88, 54.31, 49.14, 31.08, 28.88, 27.33. TOF LC/MS: (ESI) m/z:

501.2695 [M+H]+.

1-(4-aminocyclohexyl)-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine(S4)

To a solution of S3 (3.4 g, 6.8 mmol) in EtOAc (5 mL) was added 4 N HCl in EtOAc (15

mL). The reaction mixture was stirred at room temperature for 1 h. After complete

conversion of the starting material, excess EtOAc was removed dunder vacuum. The

residue was added EtOAc and water. The water layer was basified with 2 N NaHCO3

solution and extracted with EtOAc (2×100 mL). The organic layers were then washed

with water followed by brine. The organic layer was dried over sodium sulfate, filtered,

and concentrated to provide S4 as a light foam (2.2 g, 85%). 1H NMR (400 MHz,

DMSO-d6) δ 8.24 (s, 1H), 7.68 (d, J = 7.4 Hz, 2H), 7.43 (t, J = 7.1 Hz, 2H), 7.28 – 7.07

(m, 5H), 4.69 (s, 1H), 3.12 (s, 1H), 2.39 (d, J = 9.8 Hz, 2H), 1.69 (s, 6H). 13C NMR (214

MHz, DMSO-d6) δ 158.62, 157.43, 156.80, 155.81, 153.89, 142.98, 130.58, 130.54,

128.73, 124.21, 119.44, 119.40, 97.79, 55.19, 44.98, 31.97, 26.46. TOF LC/MS: (ESI)

m/z: 401.2082. [M+H]+.

(R)-2-amino-N-(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-

yl)cyclohexyl)-4-methylpentanamide hydrochloride(CHMFL-FLT3-165) To a solution of

S4 (40 mg, 0.1 mmol) in DMF (2 mL) was added (R)-2-((tert-butoxycarbonyl)amino)-4-

methylpentanoic acid(23 mg, 0.1 mmol), HATU (38 mg, 0.1 mmol) and DIEA (13 mg,

0.1 mmol). The resulting mixture was stirred for 30 min. Then it was diluted with EtOAc

(80 mL), washed with water (20 mL×3) and brine (30 mL). The organic layers were dried

anhydrous sodium sulfate , concentrated and purified by HPLC to afford (50 mg) to the

title compound as white solid. Then the solid was dissovled in 4 N HCl in EtOAc (5 mL).

The resulting mixture was stirred at room temperature for 1 h. Excess EtOAc was

removed under the vacuum to afford the title compound as white solid (35 mg, 43%). 1H

NMR (400 MHz, DMSO-d6) δ 8.25 (s, 1H), 7.95 (d, J = 6.3 Hz, 1H), 7.68 (d, J = 7.3 Hz,

2H), 7.45 (t, J = 7.1 Hz, 2H), 7.18 (td, J = 14.3, 7.9 Hz, 5H), 4.76 (s, 1H), 3.90 (s, 1H),

3.23 (s, 1H), 2.22 (s, 2H), 1.94 – 1.63 (m, 8H), 1.40 (d, J = 6.3 Hz, 1H), 1.24 (s, 2H),

0.88 (dd, J = 15.4, 6.0 Hz, 6H). 13C NMR (214 MHz, DMSO-d6) δ 175.70, 158.63,


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157.47, 156.81, 155.90, 154.00, 143.13, 130.60, 130.57, 128.67, 124.23, 119.46, 119.40,

97.85, 54.42, 53.34, 44.95, 29.01, 27.43, 24.70, 23.64, 22.45. TOF LC/MS: (ESI) m/z:

514.3319. [M+H]+.

Cell lines and cell culture

The human cancer cell lines SKM-1, SU-DHL-2, U2932, JVM-2, Namalwa were

purchased from the CoBioer Biosciences CO., LTD (Nan jing, China). The FLT3-ITD-

expressing lines MV4-11, MOLM-13 and MOLM14 were provided by Dr. Scott

Armstrong, Dana Farber Cancer Institute (DFCI), Boston, MA. The wt FLT3-expressing

AML line, NB4 (KRAS A18D), was obtained from Dr. Gary Gilliland. MOLM-13,

MOLM-14, NB4, SKM-1, SU-DHL-2, U2932, JVM-2, Namalwa and FLT3 mutant

isogenic BaF3 cells lines were cultured in RPMI 1640 media (Corning, USA) with 10%

fetal bovine serum (FBS) and and supplemented with 2% L-glutamine 1%

penicillin/streptomycin. MV4-11 was cultured in IMDM media (Cornig, USA) with 10%

FBS and supplemented with 2% L-glutamine and 1% pen/strep. All cell lines were

maintained in culture media at 37ºC with 5% CO2.

Antibodies and immunoblotting

The following antibodies were purchased from Cell Signaling Technology (Danvers,

MA): FLT3 (8F2) Rabbit mAb(#3462), Phospho-FLT3 (Tyr589/591) (30D4) Rabbit

mAb(#3464), NF-κB p65 (D14E12) XP® Rabbit mAb(#8242), Phospho-NF-κB p65

(Ser536) (93H1) Rabbit mAb(#3033), Stat5 Antibody(#9363), Phospho-Stat5 (Tyr694)

(C71E5) Rabbit mAb(#9314), c-Myc (D84C12) XP® Rabbit mAb(#5605), Akt (pan)

(C67E7) Rabbit mAb(#4691), Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb(#4060),

Phospho-Akt (Thr308) (244F9) Rabbit mAb(#4056), p44/42 MAPK (Erk1/2) (137F5)

Rabbit mAb(#4695),Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E)

XP® Rabbit mAb(#4370),GAPDH (D16H11) XP® Rabbit mAb. Antibodies were used

at 1:1000. Cells were lysed for 30 min in lysis buffer supplemented with protease/

phosphatase inhibitor cocktail (Cell Signaling Technology). Lysates were cleared by

centrifugation at 13,000 g at 4 ℃ for 10 min, and protein concentrations were determined


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by BCA. Lysates were subjected to electrophoresis through 10% or 15% gel and

immobilized on the nitrocellulose membranes.

FLT3wt and FLT3-ITD protein purification

FLT3 wt cytoplasmic fragment 564-993 with his tag was cloned into baculovirus

expression vector pFASTHT-A. The recombinant bacmid was transfected into SF9 by

Cellfectin (Invitrogen). High titer viral stocks were obtained by two rounds of

amplification of the virus. The protein was expressed by infecting SF9 cells with high

titer viral stocks for 48h. Cells were harvested and re-suspended in lysis buffer (50mM

Tris pH 7.5, 150mM NaCl, and 1mM PMSF). The cells were lysed by ultra sonication

and the cell debris was removed by ultracentrifugation. The supernatant was incubated

with Ni-affinity beads (GE). The beads were then washed by lysis buffer containing 50-

250mM imidazole. The elute was loaded to sephedex 75. The protein was concentrated to

1mg/ml and aliquots were frozen and stored at -80℃. The protein was used for ADP-Glo

assay.

FLT3-ITD sequence was amplified from the cDNA of MV-4-11 cell line and

cloned into baculovirus expression vector pFASTHT-A. The recombinant bacmid was

transfect into SF9 by Cellfectin (Invitrogen). High titer viral stocks were obtained by two

rounds amplifies of the virus. The protein was expressed by infecting SF9 cells with high

titer viral stocks for 48h. Cells were harvested and resuspended in lysis buffer (50mM

Tris pH 7.5, 150mM NaCl, and 1mM PMSF). The cells were lysed by ultrasonication and

the cell debris was removed by ultracentrifugation. The supernatant was incubated with

Ni-affinity beads (GE). The beads were then washed by lysis buffer containing 50-

250mM imidazole. The elute was loaded to sephedex 75. The protein was concentrated to

1mg/ml and aliquots were frozen and stored at -80℃. The protein was used for ADP-Glo

assay.

Biochemcial kinase assay

The ADP-Glo™ kinase assay (Promega, Madison, WI) was used to screen CHMFL-

FLT3-165 for its BTK (4 ng/μL), C-KIT (5 ng/μL) inhibition effects. The kinase reaction

system contains 4.95 μL protein, 0.55 μL of serially diluted CHMFL-FLT3-165, and 5.5


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μL substrate Poly (4:1 Glu, Tyr) peptide (0.2 μg/μL) (Promega, Madison, WI) with 100

μM ATP (Promega, Madison, WI). The reaction in each tube was started immediately by

adding ATP and kept going for an hour under 37°C. After the tube cooled for 5 minutes

at room temperature, 5 μL solvent reactions were carried out in a 384-well plate. Then 5

μL of ADP-Glo™ reagent was added into each well to stop the reaction and consume the

remaining ADP within 40 minutes. At the end, 10 μL of kinase detection reagent was

added into the well and incubated for 30 minutes to produce a luminescence signal.

Luminescence signal was measured with an automated plate reader (Perkin-Elmer

Envision) and each measurement was performed in triplicate.

A fluorescence resonance energy transfer-based Z′-LYTE kinase assay kit−Tyr 2 peptide

(Invitrogen, Carlsbad, CA) was used to evaluate the IC50 value of CHMFL-FLT3-165

for inhibition of FLT3 and FLT3-ITD kinase. The reaction was performed on a 384-well

plate with a 10-μL reaction volume per well containing 2 μM Tyr 2 peptide substrate in

reaction buffer, and 2.5μL FLT3-WT or FLT3-ITD kinase with a serial 3-fold dilution of

CHMFL-FLT3-165 (2.5μL, 10 μM to 0.5nM). The final reaction concentration of ATP

was 500 μM. After 1-h incubation, a reaction was developed and terminated, and the

fluorescence measured with an automated plate reader (SpectraMax I3, MD, USA). A

dose-response curve was fitted using Prism 5.0 (GraphPad Software Inc., San Diego,

CA).ATP competitive assay

ADP-GloTM assay kits from Promega Corporation were used according to instructions.

CHMFL-FLT3-165 was generally prepared with 1:3 serial dilutions for 4 concentrations

(100 nM, 50nM, 20nM, and 10nM); 6 concentrations were used (1 mM to 10 μM) for

ATP competition experiments. The kinase reaction was performed with 1× kinase

reaction buffer (40 nM Tris base pH 7.5, 20 mM MgCl2, 0.4 mM DTT), 0.1 mg/ml BSA,

distilled H2O, substrate and FLT3 or FLT3-ITD kinases in a total assay volume of 5 µL

following the manufacturer’s protocol. Reactions in each well were started immediately

by adding ATP and kept going for half an hour under 37°C. After the plate cooled for

5 minutes at room temperature, 5 μL of ADP-Glo reagent was added into each well to

stop the reaction and consume the remaining ADP within 40 minutes. At the end, 10 μL

of kinase detection reagent was added into the well and incubated for 1 hour to produce a


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luminescence signal. Luminescence was measured using an Envision multilabel plate

reader (PerkinElmer, Buckinghamshire, UK) with an integration time of 1 sec per well.

Proliferation studies

Cells were grown in 96-well culture plates (3000/well). The compounds of various

concentrations were added into the plates, DMSO concentrations were kept constant and

did not exceed 0.1% of the total volume. For IL-3 rescue assay, FLT3 mutant isogenic

BaF3 cells lines were treated with increasing doses of HMFL-FLT3-165 with or without

Recombinant interleukin 3 (IL-3) 1ng/mL. Cell proliferation was determined after

treatment with compounds for 72 hours. Cell viability was measured using the CellTiter–

Glo assay (Promega, USA), according to the manufacturer’s instructions, and

luminescence was measured in a multi-label reader (Envision, PerkinElmer, USA). Data

were normalized to control groups (DMSO) and represented by the mean of three

independent measurements with standard error <20%. GI50 values were calculated using

Prism 5.0 (GraphPad Software, San Diego, CA).

Colony formation assay

1 mL of 3 % agarose combined with 1 mL MOLM-13, MOLM-14 or MV4-11 growth

media was used as the bottom agar in a 6-well plate. 800 cells in 1.8 ml growth media

was combined with 0.2 mL of 3% agarose solution and plated on top of the bottom layer.

Cells were maintained in a humidified 5% CO2 incubator at 37°C for 15 days, and

continuously treated with 1:10 serially diluted CHMFL-FLT3-165 (10 µM to 1 nM) in a

soft agar medium. On the 15th day, the numbers of colonies in each well were counted

and each measurement was performed in triplicate.

Cell cycle analysis

MOLM13, MOLM14, MV4-11 cells were treated with serially diluted CHMFL-FLT3-

165, AC220 for 24 hours. The cells were fixed in 70% cold ethanol and incubated at

−20 °C overnight then stained with PI/RNase staining buffer (BD Pharmingen). Flow


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cytometry was performed using a FACS Calibur (BD), and results were analyzed by

ModFit software.

Signaling pathway effect examination MOLM13, MOLM14 ,MV4-11 cells were treated

with DMSO, serially diluted CHMFL-FLT3-165,AC220 for 4h. Cells were then washed

in PBS and lysed in cell lysis buffer. FLT3,Phospho-FLT3(Tyr589/591), AKT, Phospho-

AKT Ser473, Phospho-AKT Thr308, STAT5, Phospho- STAT5 (Tyr694),NF-ΚB-P65,

Phospho-NF-ΚB-P65(Ser536), ERK,Phospho-p44/42MAPK(Erk1/2)

(Thr202/Tyr204),C-Myc and GAPDH antibody (Cell signaling Technology) were used

for immunoblotting.

Apoptosis effect examination

MOLM13, MOLM14 cells were treated with serially diluted CHMFL-FLT3-165, AC220

for 24 hours. MV4-11 cells were treated with serially diluted CHMFL-FLT3-165, AC220

for 48 hours. Cells were then washed in PBS and lysed in cell lysis buffer. PARP,

Caspase-3, GAPDH antibody (Cell signaling Technology) were used for immunoblotting.

Molecular Modeling

Firstly, we prepared the FLT3 DFG-in conformation, which was obtained by a DGF-

peptide modification based on a released FLT3 protein structure with DFG-out peptide

conformation(PDB id 1RJB). Detailedly the preparation was to carry out a short

molecular dynamics (1.0 ns) with the restrains on DFG peptide to induce a DFG-in mode,

here the restrained molecular dynamics was run using Tinker4.2 with amber99 force field.

Then from the trajectory of FLT3 DFG-in conformations, a conformation with the

minimum RMSD compared with the PDB 1RJB was used as our FLT3 DFG-in

conformation. Then CHMFL-FLT3-165 was docked into the FLT3 DFG-in conformation.

In the docking protocol, the Lamarchian genetic algorithm (GA) and the default

parameters of GA were applied except that the population size was set to 100 and the rate

of gene mutation was set to 0.2. The docked complexes were then clustered and sorted


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with the binding energy, and the binding model with the lowest binding energy was used

to further reveal the binding mechanism.

Human AML primary cells

Mononuclear cells were isolated from samples from AML patients identified as harboring

mutant FLT3. Cells were tested in liquid culture (DMEM, supplemented with 20% FBS)

in the presence of different concentrations of inhibitors. All blood and bone marrow

samples from AML patients were obtained under approval of the Dana Farber Cancer

Institute Institutional Review Board. The ethics committees approved the consent

procedure. All studies were performed with ACUC approved protocols at DFCI.

Purification of CD34+ bone marrow cells

Fresh human bone marrow samples was obtained with agreement from healthy people

(The Second Hospital of Anhui Medical University, China). After density gradient

centrifugation, CD34+ cells were purified using immunomagnetic beads according to the

manufacturers’ instructions (Human CD34+ selection kit, Miltenyi Biotec, Bergisch-

Gladbach, Germany). The purity of CD34+ cells was >80%.

Tumor xenograft model

5 weeks old female Balb/c-nu mice were purchased from the Shanghai Experimental

Center, Chinese Science Academy (Shanghai, China). All animals were housed in a

specific pathogen-free facility and used according to the animal care regulations of Hefei

Institutes of Physical Science Chinese Academy of Sciences. Prior to implantation, cells

were harvested during exponential growth. 5 million MV4-11 cells in 1640 were

formulated as a 1:1 mixture with Matrigel (BD Biosciences) and injected into the

subcutaneous space on the right flank of Balb/c-nu mice. Daily oral administration was

initiated when tumors had reached a size of 200 to 400 mm3. Animals were then

randomized into treatment groups of 6 or 7 mice each for efficacy studies. CHMFL-


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FLT3-165 was delivered daily in a HKI solution (0.5% Methocellulose/0.4% Tween 80

in ddH2O) by orally gavage. A range of doses of CHMFL-FLT3-165 or their vehicles

were administered, as indicated in figure legends. Body weight and tumor growth were

measured daily after CHMFL-FLT3-165 treatment. Tumor volumes were calculated as

follows: tumor volume (mm3)＝[(W2 × L)/2] in which width (W) is defined as the

smaller of the two measurements and length (L) is defined as the larger of the two

measurements.

Histological examination.

Tumor tissues were fixed in 10% neutral-buffered formalin and embedded in paraffin.

Six-micron tissue section were prepared, deparaffinized, dehydrated, and then stained

with hematoxylin and eosin (H&E) using routine methods. Commercially available

primary antibody to human Ki-67 (ZSGB-BIO, Beijing, China) was used for Ki-67

staining. After heat-induced antigen retrieval, formalin-fixed and paraffin-embedded

tumor tissue sections were stained with primary antibody overnight at 4℃. The slides

were subsequently incubated with ImmPRES anti-mouse Ig (Vector Laboratories,

Burlingame, CA) at room temperature for 30 min, stained with peroxidase substrate 3, 3’-

diaminobenzidine chromogen (Vector Laboratories), and finally counterstained with

hematoxylin. TUNEL staining was assessed using In Situ Cell Death Detection Kit (POD)

(Roche, Mannheim, Germany) according to the manufacturer’s instructions.

MV4-11 engraftment model generation

NOD-SCID mice were irradiated (2 Gy) 24 hours before tail vein injection of 5 million

MV4-11 cells in 0.2 mL 1640. CHMFL-FLT3-165 or vehicle treatments were initiated

daily by oral gavage 3 weeks after cell inoculation. Mice were monitored daily and were

euthanized when moribund or at early signs of hind limb paralysis.

FACS detection of drug efficacy

Single cell suspensions from bone marrow, peripheral blood, spleen and lymph node of

MV4-11 engrafted NOD-SCID mice were prepared, and red blood cells were lysed.


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Single-cell suspensions were blocked and incubated with PE-conjugated HLA-ABC

(G46-2.6). Samples were acquired by a BD FACScalibur cytometer and analyzed by

FlowJo software. Appropriate isotype-matched, irrelevant control mAbs were used to

determine the level of background staining. The antibodies were purchased from BD

Biosciences (San Diego, USA).

Supplemental table 1: CHMFL-FLT3-165 anti-proliferation effect against FLT3 wt/mutants engineered isogenic BaF3 cell lines Cells CHMFL-FLT3-165

(GI50: µM) Parental BaF3 2.2 TEL-FLT3-BaF3 0.008 FLT-ITD-BaF3 0.0003 FLT3-D835Y-BaF3 0.021 FLT3-D835H-BaF3 0.003 FLT3-D835V-BaF3 0.012 FLT3-ITD-D835Y-BaF3 0.073 FLT3-ITD-F691L-BaF3 0.067 FLT3-K663Q-BaF3 0.84


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Supplemental Table 2: IL-3 rescue experiments on FLT3 wt/mutants isogenic BaF3 cells Cell lines CHMFL-FLT3-165

GI50(μM) BaF3-tel-FLT3 0.017

BaF3-tel-FLT3+IL-3 1.5

BaF3-FLT3-ITD ＜0.0003

BaF3-FLT3-ITD+IL-3 1.3

BaF3-FLT3-D835Y 0.006

BaF3-FLT3-D835Y+IL-3 1.7

WT-BaF3 1.6

Supplemental Table 3: CHMFL-FLT3-165 anti-proliferation effect against FLT3-ITD/wt cancer cell lines Cells CHMFL-FLT3-165

(GI50: µM) PKC412

MOLM13 0.003 0.003 MOLM14 0.008 0.002 MV4-11 0.002 0.009 SKM-1 1.6 0.11 NB4 2.4 0.43 SU-DHL-2 3.2 0.2 U2932 1.6 0.33 Namalwa 1.6 0.77 Supplemental Table 4: Kinome wide selectivity profiling of CHMFL-FLT3-165 See separate Excel file. Supplemental Table 5: Patients primary cells information Sample ID Gender Age Genetic information


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AML1 F 59

<5% bone marrow blasts; 2.6K WBC count; crit: 30; 1% peripheral blasts; previous therapy: 3+7 chemotherapy; cytogenetics: normal; mutations: IDH2 (5%), RUNX1 (15%), SRSF2 (16.8%), FLT3-ITD (24 aa).

AML2 M 69

90% bone marrow blasts; 23K WBC count; crit: 24; 5% peripheral blasts; previous therapy: azacytidine, cytarabline, high dose Ara-c; cytogenetics: normal; mutations: SRSF2 (54%), ASXL1 (46%), RUNX1 (39.4%), TET2 (ins) (46%), TET2 (point mutation) (2.8%), TET2 (del) (3.5%), FLT3-ITD (51 aa).

AML3 F 66

75% bone marrow blasts; 5K WBC count; crit: 24; 13% peripheral blasts; previous therapy: azacytidine; cytogenetics: trisomy 21, ring chromosome 18; mutations:NPM1 (41%), DMT3 mutation (45%), IDH1 (2.5%), PHF6 (32%), FLT3-ITD (66 aa, 18 aa).

AML4 F 68

5-10% bone marrow blasts; 3.9K WBC count; crit: 34; 0% peripheral blasts; previous therapy: 3+7 chemotherapy, sorafenib, high dose Ara-c, mitoxantrone/etoposide/cytarabine, allogeneic stem cell transplant; cytogenetics: normal; mutations: STAG2 (14.2%), TET2 (5.5%).

Supplemental Fig. 1 Molecular modeling of CHMFL-FLT3-165 on FLT3 kinase


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Supplemental Fig. 2: Clonogenic formation assay of CHMFL-FLT3-165 with FLT3-ITD positive AML cancer cell lines


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Supplemental Fig. 3: CHMFL-FLT3-165 inhibitory activity against FLT3 wt/ITD and c-KIT kinase in TEL transfused BaF3 cells

Supplemental Fig. 4 ADP-Glo assay of CHMFL-FLT3-165 against BTK kinase

CHMFL-FLT3-165

-2 0 2 40

40

80

120

BTK IC50=193nM

Concentration log [nM]

Rel

ativ

e ac

tivi

ty (

%)


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Supplemental Fig. 5: FACS detection of the purity of CD34+ bone marrow cells

Supplemental Fig. 6: Immunohistochemistry stain of CHMFL-FLT3-165 on MV4-11 inoculated mouse xenograft model


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Supplemental Fig. 7: body weight monitoring of CHMFL-FLT3-165 in vivo treatment

Documents

Leukemia Letter supplemental materials - … · Leukemia Letter supplemental materials 2 Supplemental Materials: Chemicals AC220 was purchased from Haoyuan Chemexpress Inc (Shanghai,