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A. Uchida et al.
cause increased sensitivity to venetoclax in DH-DPL cells. Therefore, phosphorylation at Ser70 in BCL2 at steady state is less likely to be a common mechanism of resist- ance to venetoclax in these DH-DPL cells. We further found that venetoclax altered the levels of expression of MCL1 in both DH-DPL cell lines. The stability of MCL1 is mainly controlled by its phosphorylation status.38,39 Phosphorylated MCL1 can be an easy target of an E3 ubiq- uitin ligase complex and is mostly driven to proteasomal degradation.38,39 PP2A dephosphorylates MCL1 and pre- vents interaction between MCL1 and the complex.38,39 A previous study showed that PP2A inhibition dramatically decreased the protein levels of MCL1 within 2 h in Burkitt lymphoma cells.39 Although we confirmed that PP2A B56a is bound to MCL1 in both Karpas231 and OCI-Ly8 cells, the association was unchanged after exposure to veneto- clax (data not shown). However, total amounts of PP2A B56a, which should be bound to MCL1, were clearly decreased 3 h after exposure to venetoclax. Therefore, a considerable part of MCL1 protein may be phosphorylat- ed and disassembled within 3 h after exposure to veneto- clax in the two DH-DPL cell lines. We further verified that 200 nM of venetoclax was sufficient to induce cell death even in primary DH-DPL cells, despite these cells showing relatively abundant MCL1 expression. Venetoclax
achieves and maintains plasma exposure levels of approx- imately 4 μM at daily doses ranging from 400 mg to 1200 mg in patients with chronic lymphocytic leukemia or lym- phomas.25,40 Prolonged exposure to very low concentra- tions of venetoclax may raise new issues about resistance due to the expression of other BCL2 family proteins such as BFL1.14 However, we believe that venetoclax should kill primary DH-DPL cells at daily doses recommended in patients with chronic lymphocytic leukemia.
In conclusion, DH-DPL cells seem to be dependent mainly on BCL2 for survival and relatively low concentra- tions of venetoclax effectively induced apoptosis regard- less of MCL1 expression. Venetoclax not only disrupts the BCL2-BIM interaction, but also leads to dephosphoryla- tion of BCL2 and further downregulates MCL1 expres- sion, probably through modulation of PP2A B56a activity in DH-DPL cells. Although further investigation is needed for clinical application, targeting BCL2 with venetoclax is a promising strategy for the treatment of DH-DPL.
Acknowledgments
This study was not supported by any grant. We thank Dr. Masao Seto (Kurume University, Kurume, Japan) for providing OCI-Ly8 cells. We also thank Mr. Manabu Kubota for a tech- nical assistant on immunohistochemistry.
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