|
|
Blood, Vol. 111, Issue 7, 3723-3734, April 1, 2008
Potentiation of antileukemic therapies by the dual PI3K/PDK-1 inhibitor, BAG956: effects on BCR-ABL– and mutant FLT3-expressing cells
Blood Weisberg et al.
111: 3723
Supplemental materials for: Weisberg et al
Files in this Data Supplement:
- Table S1. Panel of human leukemia call lines used in random screening of BAG956 (PDF, 26 KB)
- Figure S1. BAG956 treatment (3-day) of Kasumi-1 (JPG, 12.8 KB)
-
Characteristics of AML and ALL cell lines tested in manuscript: C1498 is a mouse (C57BL/6J) acute myeloid leukemia line. SUP-B15 is an acute lymphoblastic leukemia (B lymphoblast) line. This line was was derived from malignant cells collected from the bone marrow of an 8 year old child with Philadelphia chromosome positive B cell ALL. The cells express multiple B lineage markers, but do not express T cell markers. The cells are positive for the beta-2-microglobulin, Leu12, My7 (CD13), OKT9 (CD71), OKT10 (CD38) and CALLA (CD10) antigens. They are are negative for CB1, Leu 1 (CD5), Leu2 (CD8), Leu3 (CD4), Leu4 (CD3), Leu5 (CD2), Leu6 (CD1a), Leu9, Leu M1 (CD15), My9 (CD33), surface immunoglobulin (sIg −) and Epstein-Barr virus. KG-1 is an acute myelogenous leukemia line derived from the bone marrow of a 59-year-old Caucasian male with erythroleukemia that evolved into acute myelogenous leukemia . The stemline chromosome number is near-diploid, with the 2S component occurring at 1.8%. Five markers (constitutive markers) were common to most, if not all, metaphases analyzed. Modal chromosome number is 47 (or 46 plus a small metacentric chromosome which is smaller than the G1 group chromosome). Normal chromosomes 5, 7, 8, 12 and 17 were monosomic, and others were disomic. The Y chromosome is detected in the Q-banded preparations. KG-1 cells spontaneously differentiate to granulocyte and macrophage like cells. They show a good response to colony stimulating factor (CSF). The line is EBNA negative (EBNA−). GDM-1 is a myelomonoblastic leukemia line. The GDM-1 cell line was established in 1980 from the peripheral blood of a patient with a Philadelphia chromosome negative myeloproliferative disorder, after transformation to acute myelomonoblastic leukemia. The cells lack B- and T-cell surface markers including T-associated antigens, E-rosetting capacity, surface and intracytoplasmic immunoglobulins. They are non-specific esterase positive. The cells are phagocytic for latex beads and iron particles. Exposure to phorbol 12-myristate 13-acetate (TPA) increases the phagocytic activity. TPA also induces macrophage-like differentiation. The cells are negative for Epstein-Barr virus nuclear antigen (EBNA−). This cell line is a model system for studying myelomonocytic disorders and leukemias. Kasumi-1 is an acute myeloblast leukemia line from the peripheral blood of a 7-year old juvenile. This is a leukemic cell line with an 8;21 chromosome translocation. This translocation juxtaposes the AML1 with ETO (or MTG8) gene, giving rise to the fusion gene AML1-ETO (also known as AML1-MTG or RUNX1-CBF2T1), hence the cells produce chimeric AML1-ETO protein. This protein down-regulates CEBPA mRNA, protein and DNA binding activity, which is crucial for the differentiation of granulocytes. The cell line was established from the peripheral blood of an acute myeloid leukemia (AML) patient. The cells are positive for myeloperoxidase showing a morphology of myeloid maturation. In proliferation assay the cells in culture showed response to interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor (G-CSF), and granulocytemacrophage CSF (GM-CSF), but not to IL-1 or IL-5. Neither granulocytic nor eosinophilic maturation was observed in the in vitro liquid culture by the addition of dimethyl sulfoxide, G-CSF, or IL-5, respectively. Induction of macrophagelike cells was seen by the addition of phorbol ester. Proliferation is inhibited by 1,25S-(OH)2-16,23-diene-26-F3-10-nor D3.
- Figure S2. Effects of BAG956 and rapamycin, alone and together (JPG, 131 KB)
-
(A) Effects on MOLM14 cell cycle progression following 24 hr of treatment. (B) Effects of BAG956 and rapamycin, alone and together, on Ba/F3.p210 cell cycle progression following 24 hr of treatment.
- Figure S3 (JPG, 169 KB)
-
(A) Comparison of three-day treatments of MOLM14 with BAG956 as a single agent (250, 500 nM), rapamycin as a single agent (2.5, 5 nM), and the combination of BAG956 and rapamycin at the respective concentrations. (B) Upper panel: Annexin/pi staining: Three-day treatment of 32D.p210−luc+ cells with vehicle or BAG956 (100 nM). Lower panel: Proliferation study: Three-day treatment of 32D.p210−luc+ cells with vehicle or BAG956 (100, 1000nM).
- Figure S4. Treatment of primary bone marrow CML (blast crisis) patient samples (JPG, 26.8 KB)
-
(A) Treatment for 24 hours with imatinib. (B) Treatment of patient samples shown in (A) with BAG956 for 24 hours.
- Figure S5. Treatment of AML patient samples with BAG956 for 48 and 72 hours (JPG, 25.6 KB)
-
- Figure S6. Treatment of Ba/F3 cells expressing a variety of oncogenic tyrosine kinases (JPG, 35.2 KB)
-
(A) Treatment with rapamycin. (B) Treatment with BAG956.
- Figure S7. Immunoblots showing effects of BAG956 and rapamycin, alone and together, on p-S5 ribosomal protein in Ba/F3.p210 and parental Ba/F3 cells (JPG, 33.9 KB)
-
- Figure S8. Immunoblot showing effect of BAG956 on PI3K/Akt signaling in MOLM14 cells following 2 hr of treatment (JPG, 20.5 KB)
-
- Figure S9. Percent spleen weights for in vivo imaging experiment #1, which investigated the effects of the combination of BAG956 (100mg/kg) and nilotinib (20mg/kg) (JPG, 18.6 KB)
-
The percent spleen weight of one mouse receiving both BAG956 and nilotinib (which died a week earlier than the rest) was not included in the calculation of average percent spleen weight for the drug combination group.
|
|
