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V.M. Smith et al.
mutations), BN2 (BCL6 fusion and NOTCH2 mutations), N1 (NOTCH1 mutations) and EZB (EZH2 mutations and BCL2 translocations) as recently described.8 An initial comparison of different selective BH3-mimetics indicated that A1331852 was more potent than A1155463, and S63845 displayed significantly higher potency than A1210477 (Figure 1B-D, Online Supplementary Figure S1, Table 1).
DLBCL cell lines displayed highly heterogeneous responses to BH3-mimetics (Figure 1B-D). RIVA, U2932 and OCI-LY1 cells responded primarily to ABT-199, indi- cating a dependency on BCL-2 for survival. In contrast, RCK8, SUDHL8 and MedB1 cells were highly sensitive to A1331852, demonstrating BCL-XL dependency. Notably, these three cell lines displayed sensitivity to low nanomo- lar/picomolar concentrations of A1331852, with half maximal effective conentrations (EC50) of 0.0006, 0.005 and 0.002 mM, respectively, highlighting its potency in cellular systems. Susceptibility to S63845 was more homogeneous than that to ABT-199 or A1331852, with ten of the 18 cell lines responding to less than 3 mM. The most sensitive cell line in our panel was SUDHL10 (EC50 0.006 mM), which was previously described to be resist- ant to BH3-mimetics.23
Most cell lines were primarily sensitive to one specific BH3-mimetic, indicating firstly that in each cell line one particular BCL-2 family protein was functionally most dominant and, secondly and unexpectedly, that expres- sion of the other anti-apoptotic BCL-2 proteins could not prevent induction of apoptosis. However, four cell lines (OCI-LY1, RIVA, SUDHL8 and TMD8) were sensitive to multiple inhibitors. Notably, five of the 18 cell lines did not respond to any inhibitor at submicromolar concentra- tions (OCI-LY10, Pfeiffer, OCI-LY3, Karpas-1106 and HBL1) (Table 1).
BH3-profiling using XXa1_Y4eK may predict sensitivity to A1331852
To confirm that BCL-XL and MCL-1 are important ther- apeutic targets in DLBCL, we utilized a genetic approach to silence BCL-XL or MCL-1. Knockdown of BCL-XL by siRNA was sufficient to induce apoptosis in RCK8, SUDHL8 and MedB1 cells but not in the BCL-2-depen- dent RIVA or U2932 cells, whereas knockdown of MCL- 1 was sufficient to induce apoptosis in SUDHL10, TMD8 and U2946 cells but not in BCL-XL-dependent MedB1 cells, which correlated with susceptibility to A1331852 and S63845, respectively (Figure 2A-D). BH3-profiling may serve as a surrogate assay to investigate priming in tumor samples.24 To examine whether BH3-profiling may predict the sensitivity to BH3-mimetics in DLBCL, per- meabilized cells were exposed to BH3-peptides from BIM, which binds to all anti-apoptotic BCL-2 proteins, BAD, which binds to BCL-2 and BCL-XL, and the engi- neered peptide XXa1_Y4eK, which binds with high affin- ity selectively to BCL-XL.18 All tested cell lines displayed a dose-dependency towards BIM (Figure 2E). Both RIVA and RCK8 cells also responded to BAD and XXa1_Y4eK, congruent with a dependency on BCL-2 and/or BCL-XL for survival. In contrast, the MCL-1-dependent cell line SUDHL10 did not respond to BAD or XXa1_Y4eK, as observed in previous studies.23 Next, we asked whether the response to XXa1_Y4eK may correlate with the sen- sitivity to A1331852 in a larger panel of cell lines. The EC50 for A1331852 displayed a significant correlation with
the response to XXa1_Y4eK (P<0.001), indicating that BH3-profiling could serve as a biomarker to predict responses to BH3-mimetics provided that specific and potent peptides, such as XXa1_Y4eK, are available (Figure 2F).
BCL-2 protein expression was highly variable but only partially associated with sensitivity to BH3-mimetics
Next, we aimed to understand the heterogeneity in the response to BH3-mimetics in the panel of DLBCL cell lines. Western blot analysis revealed that the expression of BCL-2 proteins was highly variable (Figure 2G, Online Supplementary Figure S2A). Several of the cell lines have genetic alterations involving BCL2 e.g. t(14;18)(q32.3;q21.3) chromosomal translocation or gene amplifications (Table 1). Quantification of protein expres- sion indicated that gene alterations of BCL2 correlated partially with high protein expression (Online Supplementary Figure S2B). Although there was a tendency for cells with genetic alterations of BCL2 to be more sen- sitive to ABT-199, as reported previously,25 this difference was not statistically significant (Online Supplementary Figure S2C). Of note, although SUDHL4 and SUDHL6 cells are reported to contain missense mutations of BCL2, which may prevent antibody recognition.26 BCL-2 protein expression was detectable with the antibody used in our study.
The highest expression of BCL-XL was detected in RCK8, SUDHL8 and MedB1 cells, which were most sen- sitive to A1331852. Expression of MCL-1 was more homogeneous, with all cell lines expressing detectable MCL-1 protein and the highest expression being in the MCL1 amplified U2946 cells.27 The pore-forming BCL-2 proteins BAK and BAX were expressed in all cell lines while BH3-only protein expression was highly variable (Figure 2G).
To test whether susceptibility to BH3-mimetics was associated with the levels of expression of their targeted BCL-2 proteins, the EC50 values were correlated with BCL-2 protein expression. Linear regression analysis showed a significant correlation between the response to ABT-199 and expression of BCL-2, but this appeared to be driven by the very high or very low BCL-2-expressing cell lines. Sensitivity to ABT-199 also correlated signifi- cantly with the ratio of BCL-2 to MCL-1 expression (Online Supplementary Figure S3A). Although the cell lines with highest sensitivity to A1331852 expressed BCL-XL strongly, the correlation of BCL-XL expression and sensi- tivity to A1331852 was not statistically significant, which may be explained by several cell lines expressing BCL-XL strongly but nevertheless being resistant to A1331852 (HBL1, Pfeiffer and Karpas-1106). Susceptibility to A1331852 was more strongly correlated with the ratio of BCL-XL expression to a combined expression of the other anti-apoptotic proteins BCL-2 and MCL-1, although the resistant Pfeiffer and Karpas-1106 cells still displayed a high ratio and made this correlation weak (R2=0.23) (Online Supplementary Figure S3B). Sensitivity to S63845 did not correlate with expression of its target MCL-1 (Online Supplementary Figure S3C) but, as described previ- ously,15 did to some extent inversely correlate with expression of BCL-XL. In addition, we found a significant correlation of S63845 sensitivity with the ratio of MCL-1 to BIM expression.
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