Supplemental file accompanying the paper:
Effective concentration of a multi-kinase inhibitor within bone marrow correlates with in vitro cell killing in therapy-resistant chronic myeloid leukemia
Authors:Chaofeng Mu1, *, Xiaoyan Wu1,5 *, Helen Ma2, Wenjing Tao2, Guodong Zhang1, Xiaojun Xia1, Jianliang Shen1, Junhua Mai1, Tong Sun1, Xiaoping Sun3, Ralph B. Arlinghaus2, Haifa Shen1,4
Affiliations:
[1] Department of Nanomedicine, Houston Methodist Research Institute, 6670 Bertner Ave., Houston, Texas 77030
[2] Department of Translational Molecular Pathology, [3] Department of Laboratory Medicine, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, Texas 77030
[4] Department of Cell and Developmental Biology, Weill Cornell Medical College, New York, New York 10065
[5]Current address: Department of Pediatric Hematology, Union Hospital, Tongji medical college, Huazhong University of Science and Technology, Wuhan, China 430022
[*] These authors contributed equally to this work.
Correspondence should be addressed to: Haifa Shen, Department of Nanomedicine, Houston Methodist Research Institute, 6670 Bertner Ave., Houston, Texas 77030. Phone: 713-441-7321; Fax: 7134413655; Email:
Supplementary Methods
Cell proliferation and colony formation assay
Bone marrow cells from C3H mice were transduced with Mig1-GFP retrovirus in the presence of 8 µg/mL polybrene. GFP-positive bone marrow cells were purified by flow cytometry with a BD FACSAria II Cell Sorter (BD Sciences) for cell proliferation and colony formation assays.
To measure cell proliferation, GFP-positive bone marrow cells were seeded in 6-well plates, and incubated with increasing concentrations of TG101209. Drug was washed out by centrifugation after 2 hours of treatment, and cells were resuspended in the growth medium supplemented with 10 ng/mL recombinant murine IL-3, 20 ng/mL recombinant murine IL-6, and 100 ng/mL recombinant murine stem cell factor (M-SCF) and seeded in a 96-well plate at a seeding density of 4,000 cells/well. Cell proliferation was measured 48 hours later using a cell counting kit-8 (CCK8) viability assay (Dojindo Molecular Technologies Inc.).
To measure colony formation, bone marrow cells pretreated with TG101209 for 2 hours were plated in the semi-solid Methocult M3231 Medium (STEMCELL Technologies) supplemented with 10 ng/mL recombinant murine IL-3, 20 ng/mL recombinant murine IL-6, and 100 ng/mL recombinant murine stem cell factor (M-SCF). Number of colonies was counted under the microscope after 8 days of incubation at 37oC and 5% CO2.
Inhibition of protein phosphorylation in primary cells from CML patients
Primary CML cells were isolated from peripheral blood of blast crisis CML patients who met the clinical criteria of imatinib-resistance. All samples were obtained with informed consent as part of a clinical protocol approved by the M.D. Anderson Cancer Center. CML blasts were cultured for 8 hours in RPMI 1640 medium and then treated with increasing concentrations of TG101209 for 2 hours. Cell lysates were applied for Western blot analysis.
Supplementary Figures
Supplementary Figure 1.32Dp210T315I cells are resistant to dasatinib treatment. 32Dp210WT and 32Dp210T315I cells were treated with dasatinib for 2 hours before cells were harvested for Western blot analysis on protein phosphorylation.
Supplementary Figure 2.Inhibition of Aurora B kinase activity with barasertib causes G2/M arrest and cell apoptosis. (A) Western blot analysis on dose-dependent inhibition of histone H3 (ser10) phosphorylation in 32Dp210WT and 32Dp210T315I cells after barasertib treatment for 2 hours. (B and C) Cell cycle analysis on 32Dp210T315I cells treated with barasertib for 2 hours followed by incubation in growth medium for 24 or 48 hours.
Supplementary Figure 3.Targeted TG101209 enriches in bone marrow. Mice bearing 32Dp210T315I leukemia (n=4) were dosed p.o. with free TG101209 at 100 mg/kg (free TG) or i.v. with targeted TG101209 at 20 mg/kg and 40 mg/kg. Mice were euthanized at the 2 h, 4 h, and 6 h time points in the free TG101209 treatment group, and at the 0.5 h, 2 h, and 6 h time points in the targeted TG101209 treatment group. Drug concentrations in major organs were measured and calculated.
Supplementary Figure 4.TG101209 prevents cytokine-dependent resistance to ponatinib in 32Dp210T315I cells. (A) Western blot analysis on protein phosphorylation in 32Dp210T315I cells treated with ponatinib and/or TG101209 (TG) with or without interleukin 3 (IL-3) stimulation. (B) Dose-dependent inhibition of cell growth after a 2-hour treatment with TG101209 followed by incubation in growth medium with or without IL-3 for 48 hours. (C) Dose-dependent inhibition of cell growth after 2 hours of treatment with ponatinib followed by incubation in growth medium with or without IL-3 for 48 hours.
Supplementary Figure 5. Treatment with targeted TG101209 significantly reduces leukemic cells in peripheral blood. Percentages of GFP-positive tumor cells in peripheral blood of leukemic mice (n = 5) on (A) day 15 and (B) day 25 are shown. *: p < 0.05; **: p < 0.01.
Supplementary Figure 6. Treatment withimatinib and targeted TG101209 significantly extends survival of mice bearing p210WT leukemia. NOD-scid mice were transplanted with bone marrow cells overexpressing p210WT. Mice were divided into 5 groups (n = 10) 7 days after bone marrow transplantation, and treated with the indicated agents. In the mock control group, NOD-scid mice were transplanted with bone marrow cells overexpressing GFP only, and were treated with targeted TG101209 at 40 mg/kg. Kaplan-Meier plot displays animal survival in post-treatment leukemic mice. *: p < 0.05; **: p < 0.01.
Supplementary Figure 7. TG101209 treatment has minimum impact on viability and colony formation ability of bone marrow cells. (A) Dose-dependent cell viability of C3H bone marrow cells after a 2-hour treatment with TG101209 followed by incubation in growth medium for 48 hours. (B) Colony formation of bone marrow cells after a 2-hour treatment with TG101209 followed by incubation in the semi-solid Methocult M3231 medium for 8 days. CFU: colony forming unit. (C) Representative pictures of colonies in the Methocult M3231 medium on day 8.
Supplementary Figure 8.TG101209 treatment effectively inhibits protein kinases in BCR-ABL-dependent and -independent pathways in leukemia cells isolated from patients with blast crisis CML.CML cells were treated with TG101209 for 2 hours before they were harvested for expression analysis.
Table S1. Characterization of untargeted and targeted TG101209 micelles
Formulation / Feeding Ratio / Size (nm) / Zeta Potential (mV) / Drug Loading (%)Untargeted micelles / 8% / 29.92 / - 4.37 / 5.93
Targeted micelles / 8% / 32.54 / - 38.2 / 6.20
Table S2. Pharmacokinetic parameters of TG101209 formulations
Formulation / Dose (mg/kg) / Route / AUC(µg/ml h) / MRT (h) / Vss (L/kg) / CL (L/h/kg) / BA (%)
Untargeted / 40 / i.v. / 22.20 / 3.21 / 0.116 / 0.036 / ---
Targeted / 20 / i.v. / 17.82 / 4.80 / 0.108 / 0.022 / ---
Targeted / 40 / i.v. / 21.56 / 3.30 / 0.122 / 0.037 / ---
Free TG / 100 / p.o. / 14.28 / 4.16 / --- / --- / 22.7
AUC: Area under curve, MRT: Mean residence time, Vss: Apparent distribution volume, CL: Clearance, BA: Bioavailability
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