We previously reported that agonist anti-TRAILR mAbs (HGS-ETR1) induced cell death of myeloma cells through both the extrinsic and intrinsic pathway. Although caspase 8 activation was fully required to initiate death signaling, it was unclear whether intrinsic pathway played or not a major role in apoptosis execution. In the current work, we evaluated the contribution of the intrinsic pathway in a large panel of HMCL (n=10). To address this question, we monitored mitochondrial depolarization and the contribution of caspase 9. Apoptosis and mitochondrial polarization were both evaluated by flow cytometry. Kinetic analysis of mitochondrial polarization (JC-1) shows that all mitochondria were fully depolarized after only 4h following HGS-ETR1 mAb addition: the depolarization started early, as soon as 1h. In good agreement with mitochondrial depolarization, we observed that specific inhibitor of caspase 9 activity (z-LEHD-fmk) always prevented at least 50% of death (and up to 90%). Our data show that in all cell lines evaluated, activation of the intrinsic pathway was required for cell death even with high concentrations of mAb. We previously showed that anti-TRAILR apoptosis was always associated with an early and massive Mcl-1 cleavage (

Menoret et al
Blood
2006
,
108
:
1346
). In myeloma cells, Mcl-1 is physically associated with Bim and thus prevents Bim to induce mitochondrial cell death (Gomez-Bougie EJI 2004, 34;3156). By immunoprecipitation, we show that, in the presence of HGS-ETR1 mAb, Mcl-1/Bim complexes were disrupted and Bim was free to activate Bax. Bim is also found to be complexed to both Bcl-XL and Bcl-2 in viable cells. During anti-TRAILR mAb induced apoptosis, Bcl-2 and Bcl-XL were also cleaved and full length Bcl-XL, as Mcl-1, fully disappeared indicating that BcL-XL decrease probably participated to the increase of free Bim. Furthermore, immunoprecipitation of Bax showed that Bax was associated with Bcl-2 (and not with Mcl-1 or Bcl-XL) and this association was reduced in apoptotic conditions. These data show that apoptosis induced by anti-TRAILR mAb mainly involved the intrinsic pathway through disruption of both anti-apoptotic/Bim and Bcl-2/bax complexes. This result suggests that the association of anti-TRAILR mAb with melphalan or bortezomib would not synergistically increase anti-TRAILR mAb efficiency and that anti-TRAILR mAb should be rather combined with drugs acting independently of the intrinsic pathway.

Topics:
agonists, apoptosis, myeloma cells, monoclonal antibodies, bcl-xl protein, depolarization, caspase-9, complex, immunoprecipitation, bortezomib

Disclosures: No relevant conflicts of interest to declare.

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2008, The American Society of Hematology
2008
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