Journal: JNCI Journal of the National Cancer Institute

Article Title: Targeting Primitive Chronic Myeloid Leukemia Cells by Effective Inhibition of a New AHI-1–BCR-ABL–JAK2 Complex

doi: 10.1093/jnci/djt006

Figure Lengend Snippet: Effects of TG101209 (TG) in combination with tyrosine kinase inhibitors (TKIs) on multiple subsets of CD34 + chronic myeloid leukemia (CML) cells. A) CD34 + CML cells (n = 3) were cultured for 72 hours in the presence of imatinib (IM), nilotinib (NL), dasatinib (DA), or TG alone or a combination of TKI with TG, and the phenotypes of cells present within the viable (AnnexinV - /7-aminoactinomysin [7-AAD - ]) gate were then determined by fluorescence-activated cell sorting based on their expression of CD34 and CD38. The total number of viable cells within each subpopulation was then calculated based on viable cell counts at 72 hours and compared with the starting number of cells within each subpopulation (baseline), represented by the red line . B–C) CD34 + CML cells (n = 2) ( B ) from newly diagnosed chronic-phase patients and CD34 + normal bone marrow (n = 3) ( C ) were plated in standard colony-forming cell (CFC) assays after 3 ( left ) or 6 ( right ) days of prior incubation in suspension cultures with 5 µM IM, 150nM DA, 5 µM NL, or 100nM TG alone or in combination. Colonies produced in the CFC assays were counted after 12 to 14 days of further incubation, and the numbers obtained are expressed as a percentage of values obtained in control assays without drug added. Data are means, and error bars represent 95% confidence intervals of triplicate measurements. P values were calculated using methods appropriate to fitting linear mixed models.

Article Snippet: CML cell lines (K562, K562 IMR, K562 lenti-AHI-1, K562 SH4-bulk, and BV173) were cultured in Roswell Park Memorial Institute 1640 media with 10% fetal bovine serum plus 100U/mL penicillin and 0.1mg/mL streptomycin (STEMCELL Technologies).

Techniques: Cell Culture, Fluorescence, FACS, Expressing, Incubation, Suspension, Produced, Control

Journal: JNCI Journal of the National Cancer Institute

Article Title: Targeting Primitive Chronic Myeloid Leukemia Cells by Effective Inhibition of a New AHI-1–BCR-ABL–JAK2 Complex

doi: 10.1093/jnci/djt006

Figure Lengend Snippet: Effects of oral administration of TG101209 (TG) and imatinib (IM) on elimination of chronic myeloid leukemia (CML) BV173 cells and survival of leukemic mice in immunodeficient mice. A) BV173 cells were cultured with 1.0 µM IM, 0.5 µM TG, IM plus TG, or no drug for 3 days, and the progeny recovered from 2.5×10 6 initial cells were then injected intravenously into NOD/SCID–interleukin 2 receptor γ–chain-deficient (NSG) mice (n = 6 mice per condition). The left panel shows fluorescence-activated cell sorting profiles of engrafted human CD19/20 + cells detected in bone marrow aspirates of representative mice obtained 3 weeks posttransplant. The percentage of human CD19/20 + BV173 cells detected in the bone marrow of mice examined 3 weeks after injection is shown in the right panel . Data are means, and error bars represent 95% confidence intervals of six measurements per condition. B) Survival curve for recipients of BV173 cells (2.5×10 6 per mouse; n = 6 mice per group) treated by oral gavage beginning at 2 weeks posttransplant with vehicle, IM (50mg/kg), TG (60mg/kg), and IM (50mg/kg) plus TG (60mg/kg) twice a day for 2 weeks. Statistically significantly prolonged survival was observed in mice receiving the combination treatment ( left panel ). Body weights of mice in each treated group were measured, as indicated in the right panel . Log-rank tests were used to compare median survival of different groups (n = 6 mice per group), and P values were calculated using a two-sided Student t test. C) Model of the mechanism by which Abelson helper integration site 1 (AHI-1)–BCR-ABL and AHI-1–Janus kinase 2 (JAK2) interactions regulate constitutive activation of BCR-ABL and JAK2/ signal transducer and activator of transcription 5 (STAT5), resulting in increased leukemic stem cell proliferation, survival and maintenance and reduced tyrosine kinase inhibitor (TKI) response of these cells. Targeting both BCR-ABL and JAK2 activities to destabilize this complex perturbs these biological properties. IL-3 = interleukin 3; IL-3R = interleukin 3 receptor.

Article Snippet: CML cell lines (K562, K562 IMR, K562 lenti-AHI-1, K562 SH4-bulk, and BV173) were cultured in Roswell Park Memorial Institute 1640 media with 10% fetal bovine serum plus 100U/mL penicillin and 0.1mg/mL streptomycin (STEMCELL Technologies).

Techniques: Cell Culture, Injection, Fluorescence, FACS, Activation Assay

Journal: JNCI Journal of the National Cancer Institute

Article Title: Targeting Primitive Chronic Myeloid Leukemia Cells by Effective Inhibition of a New AHI-1–BCR-ABL–JAK2 Complex

doi: 10.1093/jnci/djt006

Figure Lengend Snippet: Elimination of chronic myeloid leukemia (CML) leukemic stem cells capable of long-term production of BCR-ABL + cells in immunodeficient mice using combined exposure to TG101209 (TG) and imatinib (IM). A) CD34 + cells from three CML patients were cultured with 1.0 µM IM, 0.1 µM TG, IM plus TG, or without treatment for 3 days. Cells recovered were injected intravenously into NOD/SCID–interleukin 2 receptor γ–chain-deficient (NSG) mice (n = 3 mice per condition per patient sample). After 4, 8, 12, and 16 weeks, bone marrow (BM) aspirates were obtained, and the presence of human CD45 + and CD34 + cells was measured. B) Fluorescence-activated cell sorting profiles of regenerated human myeloid cells (CD45/CD14/15/33/66b + ) in the BM of representative mice analyzed 12 weeks posttransplant. C) The percentage of human myeloid (CD45/CD14/15/33/66b + ) and lymphoid cells (CD45/CD19/20 + ) in the BM of representative mice analyzed 4 weeks posttransplant. D) BCR-ABL transcript levels measured by quantitative reverse transcription polymerase chain reaction in fluorescence-activated cell sorting–purified human CD45 + cells obtained at 4 and 12 weeks posttransplant from the BM of mice injected with cells treated with inhibitors and those without. Data are means, and error bars represent 95% confidence intervals of six to nine measurements per condition. P values were calculated using a two-sided Student t test.

Article Snippet: CML cell lines (K562, K562 IMR, K562 lenti-AHI-1, K562 SH4-bulk, and BV173) were cultured in Roswell Park Memorial Institute 1640 media with 10% fetal bovine serum plus 100U/mL penicillin and 0.1mg/mL streptomycin (STEMCELL Technologies).

Techniques: Cell Culture, Injection, Fluorescence, FACS, Reverse Transcription, Polymerase Chain Reaction, Purification