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Targeting BCL2 with venetoclax for DH-DPL
washed three times with ice-cold lysis buffer and boiled for 3 min with 20 μL/tube of loading buffer. The supernatant was subjected to western blot analysis.
Statistical analysis
Differences in positive rates of MYC and BCL2 family proteins between DH-HGBL and other GCB-like DLBCL were calculated using a two-tailed Mann-Whitney U test and Student t test. P val- ues <0.05 were considered statistically significant. All analyses were performed using EZR (Saitama Medical Center, Jichi Medical University, v.1.33).31
Results
MCL1 overexpression is less frequently observed in DH-HGBL
We initially evaluated the protein expression of MYC and BCL2 family members in clinical samples. A total of 27 cases were analyzed because they had minimal data and specimens for immunohistochemical and cytogenetic evaluations (Online Supplementary Table S1). Immunohistochemistry and fluorescence in situ hybridiza- tion analyses using MYC split and IGH-BCL2 fusion probes confirmed that these clinical samples comprised eight DH-HGBL cases harboring both MYC and BCL2 gene rearrangements and 19 GCB-like DLBCL cases. Of course, all DH-HGBL cases showed the GCB phenotype, and seven of the eight cases corresponded to DH-DPL (Online Supplementary Table S1). Among the 19 GCB-like DLBCL cases, five showed overexpression of both MYC and BCL2 proteins and were regarded as non-DH DPL (Online Supplementary Table S1). Although immunohisto- chemistry clearly detected overexpression of both MYC and BCL2 proteins more frequently in DH-HGBL than in other GCB-like DLBCL (Mann-Whitney U test, P=0.028 in MYC and P=0.013 in BCL2), differences between DH- HGBL and non-DH DPL were not seen (Figure 1A). Among the BCL2 family proteins, immunhistochemistry clearly confirmed the expression of BCL2, MCL1 and BIM, but rarely detected BAX regardless of the groups (Figure 1A,B, and Online Supplementary Figure S1). There were no differences in the rates of positivity for BIM and BAX among the three groups, whereas MCL1 overexpres- sion was detected significantly more frequently in other GCB-like DLBCL than in DH-HGBL (Mann-Whitney U test, P=0.005) (Figure 1A). This significance was also observed between DH-HGBL and non-DH DPL (Student t test, P=0.019) (Figure 1A). Concerning the staining pattern for MCL1, we found differences between DH-HGBL and other GCB-like DLBCL including non-DH DPL. All DH- HGBL cases showed the cytoplasmic staining pattern, whereas two of five non-DH DPL (40%) and six of 14 other GCB-like DLBCL cases (43%) exhibited the nuclear staining pattern (Figure 1B, Online Supplementary Table S1). These results suggest that the intrinsic anti-apoptotic activities in DHL-HGBL or DH-DPL may be dependent on BCL2 rather than MCL1.
Karpas231 and OCI-Ly8 are DH-DPL cells
We next performed in vitro investigations to characterize DH-DPL cells. Fluorescence in situ hybridization analyses confirmed that all four GCB-like DLBCL-derived cell lines have the 8q24/MYC translocation (Figure 2A). MYC was fused to IGH in BJAB, SU-DHL10, and OCI-Ly8 (Figure
2A). In addition to the rearrangement, IGH-BCL2 fusion resulting from t(14;18)(q32;q21) was detected in SU- DHL10, Karpas231, and OCI-LY8 (Figure 2A). Western blot analysis showed that the four lines express BCL6, MYC, BRD4, MCL1, BCL-xL, BIM, BAD, BAK, and BAX at a variety of levels (Figure 2B). Among the pro-apoptotic BH3-only proteins, BIM was abundantly expressed in each cell line (Figure 2B). The results were consistent with those from clinical samples. In contrast, protein expression levels of BCL-xL and MCL1 were relatively low in SU- DHL10 and OCI-Ly8, respectively (Figure 2B). Although SU-DHL10 has the IGH-BCL2 fusion, we failed to detect BCL2 protein expression in this line (Figure 2B). Also, NOXA was not detected in BJAB cells (Figure 2B). These results indicate that BJAB represents non-DH DPL cells and that SU-DHL10, Karpas231 and OCI-Ly8 are regarded as DH-HGBL cells. Furthermore, Karpas231 and OCI-Ly8 correspond to DH-DPL cells having relatively abundant BCL2 proteins, a considerable part of which is phosphory- lated at Ser70.
Exposure to 200 nM of venetoclax effectively induces apoptosis in DH-DPL cells
We subsequently evaluated the growth inhibitory effect and apoptotic sensitivity to JQ-1 and three BH3 mimetics in each cell line. Except for the BJAB cell line, JQ-1 similar- ly suppressed cell proliferation in a dose-dependent man- ner, while the inhibitory effects of BH3 mimetics were dif- ferent among the four lines (Figure 3A). Venetoclax effec- tively suppressed proliferation of Karpas231 and OCI-Ly8 cells even at low concentrations (20 nM and 200 nM), whereas this agent had no effect on either BJAB or SU- DHL10 cells (Figure 3A). In contrast, S63845 clearly showed an inhibitory effect at low concentrations (10 nM and 100 nM) only in SU-DHL10 cells (Figure 3A). Although A-1155463 inhibits cell growth at levels less than 1 μM in sensitive cells,28,32 at nanomolar levels this agent failed to suppress the proliferation of any cell line (Figure 3A).
Flow cytometry detected apoptotic changes 24 h after exposure to these drugs. Although 50 μM of JQ-1 effi- ciently suppressed proliferation of SU-DHL10, Karpas231, and OCI-Ly8 cells, the exposure was insufficient to induce apoptosis in the two DH-DPL-cell lines. Approximately 70% of SU-DHL10 cells were positive for annexin V, whereas the positive rates were limited to around 18~40% in Karpas231 and OCI-Ly8 cells (Figure 3B). Exposure to 200 nM of venetoclax effectively led to apop- totic changes in Karpas231 and OCI-Ly8 cells. More than 80% of the cells were positive for annexin V in both DH- DPL-cell lines (Figure 3B). Conversely, exposure to 100 nM of S63845 induced cell death only in SU-DHL10. Although around 60% of the cells were annexin V-positive, more than 90% of SU-DHL10 cells were stained with 7-AAD (Figure 3B). In this study, exposure to 1 μM of A-1155637 did not induce even modest cell death in each cell line (Figure 3B). These results indicate that anti-apoptotic activities in the four lines were less related to BCL-xL.
We further evaluated the alteration of apoptotic sensi- tivity to venetoclax in combination with S63845. In BJAB cells, the addition of S63845 to venetoclax led to a clear increase in annexin V-positive cells, indicating that S63845 and venetoclax had a synergic effect on inducing apopto- sis (Figure 4A). In contrast, S63845 did not show even an additive effect in Karpas231, OCI-Ly8, and SU-DHL1 cells
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