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. 2015 Aug;100(8):e302-6.
doi: 10.3324/haematol.2015.124560. Epub 2015 May 8.

Resistance to ABT-199 induced by microenvironmental signals in chronic lymphocytic leukemia can be counteracted by CD20 antibodies or kinase inhibitors

Rachel Thijssen  1 , Erik Slinger  1 , Katinka Weller  2 , Christian R Geest  2 , Tim Beaumont  3 , Marinus H J van Oers  4 , Arnon P Kater  4 , Eric Eldering  5
Affiliations

VSports在线直播 - Resistance to ABT-199 induced by microenvironmental signals in chronic lymphocytic leukemia can be counteracted by CD20 antibodies or kinase inhibitors

Rachel Thijssen et al. Haematologica. 2015 Aug.
No abstract available

Keywords: ABT-199; CD20 antibodies; chronic lymphocytic leukemia; kinase inhibitors; resistance VSports手机版. .

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"VSports app下载" Figures

Figure 1.
Figure 1.
Bcl-XL is expressed in CLL cells in LN tissue. One representative CLL LN sample out of 4 stained is shown and stained for (A) Isotype control staining and nuclear staining with DAPI in blue, scale-bar represent 10 mm. (B) CD20 in green and Bcl-XL in red and nuclear staining with DAPI in blue (C and D). CLL cells (Online Supplementary Table S1; patients #23–25) were co-cultured with NIH3T3 fibroblasts transfected with empty vector (3T3) (C) or co-cultured with NIH3T3 fibroblasts transfected with hCD40L (3T40L) (D) for three days. After detachment, cytospins were made and stained for CD20, Bcl-XL and DAPI. Imaging was performed using a Leica TCS SP8-X confocal microscope. One CLL sample is shown of a total of three analyzed, scale-bar represent 10 mm. (A and B) Paraffin-embedded LN samples from CLL patients were incubated with primary antibody anti-CD20 (eBioscience, San Diego, CA, USA) and anti-Bcl-XL (Cell Signaling, Boston, MA, USA) and subsequently incubated with Alexa Fluor 488 labeled goat anti-mouse and Alexa Fluor 594 labeled goat anti-rabbit antibodies (Invitrogen, Camarillo, CA, USA) and counterstained with DAPI. Immunofluorescent imaging (40×) was performed using a Leica DMRA fluorescence microscope.
Figure 2.
Figure 2.
CLL cells become resistant to ABT-199 upon CD40 stimulation. CLL cells were cultured on control 3T3 or 3T40L cells in the presence or absence of 25 ng/ml IL-21 or IL-4 for 72 h. (A) Western blot analysis was performed using standard techniques with anti-Mcl-1, anti-Bcl-2, anti-Bcl-XL, anti-Bfl-1 and anti-β-actin antibodies. Blots from two representative CLL samples are shown of a total of seven analyzed (Online Supplementary Table S1; patient #1–3A, 10B, 11). (B) After detachment, cells were incubated with 0.001–10 mM ABT-199 for h 24. Viability was assessed by DiOC6/PI staining. Left panel shows % viable cells, right panel shows specific apoptosis. Results are shown as mean ± SEM, n=8 (3 IgVH mutated, 3 unmutated, 2 IgVH status unknown (Online Supplementary Table S1; patient #4–11) (C) CLL cells (Online Supplementary Table S1; patient #5, 13A, 19) were nucleofected (Amaxa, Koln, Germany) with 3 mg siRNA (Bcl-XL, Mcl-1 and Silencer Select Negative Control) and cultured on 3T40L cells for three days. Left panel: lysates were probed for Bcl-XL, Mcl-1 or actin as a loading control. As a control, lysates of CLL cells on 3T3 are shown. (Right) after nucleofection and culture, cells were incubated with ABT-199 for 24 h, n=3, mean ± SEM. Reagents: anti-Mcl-1 (Cell Signaling), anti-Bcl-2 (Enzo Life Sciences, Raamsdonksveer, The Netherlands), anti-Bcl-XL (BD Biosciences), anti-β-actin (Santa Cruz Biotechnology), polyclonal antibody against Bfl-1 was a kind gift of Jannie Borst (The Netherlands Cancer Institute, Amsterdam, The Netherlands), IL-21 and IL-4 (Gibco, Invitrogen, Bleiswijk, The Netherlands), ABT-199 from Abbvie (Abbott Park, IL, USA), siRNA from obtained from Ambion (ID#1920, ID#8583 and Cat#4390843). Specific apoptosis is defined as [% cell death in treated cells] − [% cell death in medium control] / [% viable cells medium control] × 100.
Figure 3.
Figure 3.
CD40L-induced resistance to ABT-199 can be overcome by combination therapy. (A) CLL cells were co-cultured with 3T3 or 3T40L for three days with 10 μg/mL crosslinked rituximab (RXL) or GA101 as described,, or in combination with indicated concentrations of ABT-199 and analyzed for apoptosis after 24 h. Averaged results are presented as percentage cell death (mean ± SEM) for 6 CLL samples (Online Supplementary Table S1; patient #3B, 5, 7, 10B, 19, 22) for GA101 and 3 CLL samples (Online Supplementary Table S1; patient #3B, 7, 10B) for rituximab. (B) CLL cells were co-cultured in the presence of 0.1 or 1 mM dasatinib and after deatchment were incubated with ABT-199. (n=8 Online Supplementary Table S1; patient #5, 8, 10B-12, 19–21), (C) CLL cells were co-cultured in the presence of 1 mM ibrutinib, 1 mM idelalisib or 30 mM imatinib. After detachment, cells were incubated with ABT-199. (n=8 Online Supplementary Table S1; patient #3B-5, 8, 12, 13B-15). (D) CD40-stimulated CLL cells were co- cultured for 48 h in the presence of dasatinib, imatinib, or ibrutinib as indicated. Lysates were probed for Mcl-1, Bcl-XL and Bfl-1 and actin for loading control. The results are representative of 3 blots. Reagents: GA101 (Roche, Woerden, The Netherlands), dasatinib (Novartis, Basel, Switzerland), ibrutinib (Pharmacyclics, Sunnyvale, CA, USA), idelalisib (Selleckchem, Houston, TX, USA), imatinib (Novartis, Basel, Switzerland). Statistics by Students t-test: *P<0.05;**P<0.01; ***P<0.001.
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