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Targeting BCL2 with venetoclax for DH-DPL
Treatment with venetoclax augments the accumulation of PP2A B56a in BCL2 in DP-DHL cells
Immunoprecipitation and western blot analyses of Karpas231 and OCI-Ly8 cells showed that BCL2 was sub- stantially associated with BIM at steady state (Figure 6A). Likewise, BCL2 was bound to PP2A B56a (Figure 6A). We also confirmed that PP2A B56a was bound to MCL1 in both DH-DPL cell lines (data not shown). We then evaluat- ed alterations of these associations after exposure to vene- toclax. Within 60 min after treatment with venetoclax, total protein levels of BIM bound to BCL2 were decreased similarly in Karpas231 and OCI-Ly8 cells (Figure 6B). In contrast, the proportion of PP2A B56a bound to BCL2 was slightly increased (Figure 6B). Although BIM protein levels decreased gradually following exposure to venetoclax, total levels of BCL2 bound to BIM were decreased at the early phase of venetoclax treatment in both DH-DPL cell lines (Figure 6B). These results indicate that venetoclax not only disrupts the association between BCL2 and BIM, but also augments the accumulation of PP2A B56a in BCL2.
Exposure to 200 nM of venetoclax induces apoptosis in primary DH-DPL cells
We also evaluated the apoptotic sensitivity of primary DH-DPL cells to venetoclax in vitro. The fresh bone mar- row specimen was obtained from one patient with DH- DPL (UPN3) and exposed to 200 nM of venetoclax. Examination of the bone marrow revealed massive infil- tration of blastoid cells with a mature B-cell phenotype [terminal deoxynucleotidyl transferase (TdT)-negative and
A
BCL6-positive] (Figure 7A). Immunohistochemistry con- firmed that MYC, BCL2, and MCL1 were highly expressed in the neoplastic cells (Figure 7A). Flow cytom- etry clearly detected the apoptotic change 24 h after expo- sure to venetoclax (Figure 7B). Almost all the blastoid DH- DPL cells were positive for annexin V (Figure 7B). Consistent with the results in Karpas231 and OCI-ly8 cells, the primary DH-DPL cells were highly susceptible to low concentrations of venetoclax.
Discussion
Since DH-DPL is a rapidly progressing disease and often refractory to even intensive therapies, patients with this lymphoma have dismal survival outcomes unlike those with any other type of DLBCL.2,3,11 Hence, it is urgently necessary to develop effective treatments for DH-DPL. Appropriate balances between pro-survival and pro-apop- totic BCL2 family proteins are usually disrupted in GCB- like DLBCL including DH-DPL, in which the overexpres- sion of pro-survival proteins, including BCL2, MCL1, and BCL-xL, likely confers resistance to chemotherapeutic agents.10,12,15 In this study, we found that BCL2 seems to play crucial roles in anti-apoptotic activities in DH-DPL, and further verified that venetoclax is still a promising agent for this type of lymphoma. The present DH-HGBL cases, mostly corresponding to DH-DPL, showed lower levels of MCL1 expression, associated with resistance to venetoclax, compared with other GCB-like DLBCL cases. In addition, the frequent nuclear localization of MCL1 in
B
Figure 4. Alteration of apoptotic sensitivity to venetoclax in com- bination with S63845. (A) The proportion of annexin-V-positive cells was counted using flow cytometry in triplicate. Addition of 100 nM of S63845 to 200 nM of venetoclax led to a clear increase of annexin V-positive cells in the BJAB line, indicating that S63845 and venetoclax had a synergic effect on induc- ing apoptosis. (B) In contrast, 100 nM of S63845 or 200 nM of venetoclax did not show even an additive effect in Karpas231, OCI-Ly8, and SU-DHL1 cells. DMSO: dimethyl sulfoxide.
haematologica | 2019; 104(7)
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