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Blood, Vol. 113, Issue 18, 4403-4413, April 30, 2009
Concurrent up-regulation of BCL-XL and BCL2A1 induces approximately 1000-fold resistance to ABT-737 in chronic lymphocytic leukemia
Blood Vogler et al.
113: 4403
Supplemental materials for: Vogler et al
Files in this Data Supplement:
- Figure S1. Expression of BCL2 proteins in CLL (JPG, 52.9 KB)
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(A) CLL cells from 14 patients were analyzed by western blotting for the indicated BCL2 family proteins and β-actin was used as a loading control. The EC50 values for ABT-737 sensitivity have been assessed by PS externalization and AnnexinV/FITC staining after 4 h of exposure. (B) The ratio of MCL1 to -actin was correlated with the individual EC50 for ABT-737 using linear regression in GraphPad Prism. The p value for MCL1 is p=0.95.
- Figure S2. Combined knockdown of BCL-XL and BCL2A1 is not successful in all patients (JPG, 58.3 KB)
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CLL cells were transfected with a negative control siRNA, a siRNA against BCL-X or BCL2A1 or a combination of siRNAs against BCL2A1 and BCL-X using Amaxa program X-5. Directly after nucleofection, cells were cultured on CD154-expressing cells in the presence of IL-4 (10 ng/ml) for 16 h. After nucleofection and exposure to CD154, CLL cells were removed from CD154-expressing cells and exposed to different concentrations of ABT-737 for 4 h. (A) Apoptosis was assessed as PS externalization and binding of AnnexinV/FITC (n = 8). (B) Efficiency of knockdown was analyzed by western blotting. Nucleofection with combined BCL2A1 and BCL-X siRNA resulted in less efficient downregulation of BCL2A1 as compared to nucleofection with only BCL2A1 siRNA.
- Figure S3. Resistance of CLL to ABT-737 upon CD154 stimulation can be reduced by CDK-inhibitors, HDAC inhibitors or proteasome inhibitors (JPG, 67.5 KB)
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(A–C) CLL cells were cultured on either parental or CD154-expressing L cells in the presence of IL-4 (10 ng/ml) for 1 day. (A) After culture on CD154-expressing cells, CLL cells were treated with 20 µg/ml rituximab (Roche, Welwyn Garden City, UK), 500 ng/ml TRAIL, or 10 µM fludarabine (Sigma-Aldrich Company Ltd.) in combination with different concentrations of ABT-737 for 20 h and apoptosis was assessed by PS externalization and AnnexinV/FITC binding (n = 3). (B and C) After removal of CLL cells from L cells, CLL cells were treated with 10, 50, or 100 µM seliciclib (B) or 10, 100, 500, or 1000 nM trichostatin A (TSA) (C) for 20 h followed by treatment with different concentrations of ABT-737 for 4 h. Apoptosis was determined by PS externalization and AnnexinV/FITC binding (n = 6 for seliciclib, n= 3 for TSA). (D) CLL cells were cultured on either parental or CD154-expressing L cells in the presence of IL-4 (10 ng/ml) for 3 days. After removal of CLL cells from L cells, CLL cells were exposed to different concentrations of ABT-737 combined with bortezomib (10 or 100 nM) (Millenium Pharmaceuticals, Cambridge, MA) or MG132 (100 nM or 1 µM) (Calbiochem). Apoptosis was determined after 20 h by PS externalization and AnnexinV/FITC binding (n = 5).
- Figure S4. BCL2A1 and BCL-XL expression confers resistance to ABT-737 (JPG, 69.7 KB)
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(A) In CLL the BH3-only proteins BIM and PUMA as well as the multidomain BCL2 protein BAK are constitutively bound to BCL2 (left panel). Upon exposure to CD154, expression of NOXA, BCL2A1 and BCL-XL is increased (right panel). Whereas upregulated NOXA is bound by MCL1, no binding partners of BCL2A1 or BCL-XL were identified. (B) Upon exposure to low concentrations of ABT-737 (10–100 nM), either BH3-only proteins (Model A) or BAK (Model B) are displaced from BCL2, resulting in oligomerization of BAK and apoptosis (left panel). Upon exposure to CD154, ABT-737 binds to the newly synthesized free BCL-XL, which acts as a sink and prevents apoptosis (right panel). (C) Besides binding to BCL-XL, high concentrations of ABT-737 (1–10 µM) might bind to free BCL2A1, which can again act as a sink for ABT-737. In addition, BCL2A1 might confer resistance to ABT-737 by sequestering BH3-only proteins (Model A) and/or BAK (Model B) displaced from BCL2 by ABT-737. Thus expression of BCL-XL and BCL2A1 inhibits BAK activation and apoptosis even at higher concentrations of ABT-737.
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