Bcl-2, Bcl-xL, and Bcl-w are not equivalent targets of ABT-737 and navitoclax (ABT-263) in lymphoid and leukemic cells by Delphine Mérino, Seong L. Khaw, Stefan P. Glaser, Daniel J. Anderson, Lisa D. Belmont, Chihunt Wong, Peng Yue, Mikara Robati, Belinda Phipson, Walter D. Fairlie, Erinna F. Lee, Kirsteen J. Campbell, Cassandra J. Vandenberg, Suzanne Cory, Andrew W. Roberts, Mary J. C. Ludlam, David C. S. Huang, and Philippe Bouillet Blood Volume 119(24):5807-5816 June 14, 2012 ©2012 by American Society of Hematology

High Bcl-2 expression correlates with sensitivity to ABT-263. High Bcl-2 expression correlates with sensitivity to ABT-263. The level of expression of 10 Bcl-2 family members in 39 NHL cell lines was assayed by microarray. (A) The heat-map represents the mRNA expression levels of Bcl-2 family genes. Red represents high expression, green low expression. ABT-263-DX is the linear predictor of cell line resistance against ABT-263 (see “Microarray and predictive model”). The bottom panel displays ABT-263 IC50 values, presence of the t(14:18) translocation (IGH:BCL2) or amplification of Bcl-2 (> 3 copies) and ABC subtype classification. (B) The coefficients of the Bcl-2 family genes have been represented as a linear predictor. The value of each coefficient represents its relative contribution to the predictive signature. (C) In patients treated with R-CHOP and CHOP,33 a low ABT-263-DX value correlated with poor prognosis. The corresponding hazard ratios and P values were calculated using Cox regressing model fitting the ABT-263-DX value as a continuous variable. Delphine Mérino et al. Blood 2012;119:5807-5816 ©2012 by American Society of Hematology

High Bcl-2 expression protects lymphoid cells against dexamethasone or etoposide, but sensitizes to ABT-737/263. High Bcl-2 expression protects lymphoid cells against dexamethasone or etoposide, but sensitizes to ABT-737/263. BM transitional B cells from WT and vavP–Bcl-2 transgenic mice were isolated and cultured in the presence of (A) dexamethasone, (B) etoposide, (C) ABT-737, or ABT-263 at the indicated doses for 24 hours. (D) BM transitional B cells from vavP–Mcl-1 mice are resistant to ABT-737. Survival data are shown for transitional B cells (n = 3 mice for each genotype) and are the results of 3 independent experiments, each performed in triplicate. Experiments depicted in panels C and D were performed concurrently. Values represent mean ± SEM. Delphine Mérino et al. Blood 2012;119:5807-5816 ©2012 by American Society of Hematology

Bim plays an important role in the response to ABT-737. Bim plays an important role in the response to ABT-737. (A) Thymocytes and BM-derived B cells from wt, Bim−/−, Noxa−/−, Puma−/−, Bmf−/−, Bax−/−, Bak−/−, or Bax−/−Bak−/− mice were cultured in the absence or presence of increasing doses of ABT-737, and their viability determined after 24 hours. EC50 values are represented as a heat map. Blue represents sensitivity, and red, resistance. (B) The hematopoietic system of lethally irradiated mice was reconstituted with WT, Bim−/−, Noxa−/−, Puma−/−, or Bax−/−Bak−/− fetal liver cells retrovirally transduced with an empty vector or a vector encoding Bcl-2. Eight weeks after reconstitution, the viability of the resulting cells in response to treatment with several doses of ABT-737 was analyzed in culture as described in Figure 2; n = 3 mice per group, 3 independent experiments (except for Bax−/−Bak−/− n = 1 because of the difficulty to obtain viable animals). Values represent the mean of EC50 ± SEM, detailed in supplemental Table 2. Delphine Mérino et al. Blood 2012;119:5807-5816 ©2012 by American Society of Hematology

Sensitization of vav-Bcl-2 lymphocytes to ABT-737 requires the binding of Bim to Mcl-1. Sensitization of vav-Bcl-2 lymphocytes to ABT-737 requires the binding of Bim to Mcl-1. (A) Western blot analysis of NHL cell lines having higher or lower levels of Bcl-2 RNA by microarray analysis (Figure 1). Those expressing higher levels of Bcl-2 protein are sensitive to ABT-263 and express higher levels of Bim. (B) Expression of Bcl-2 and Bim in vavP–Bcl-2/Bim+/+, vavP–Bcl-2/BimB/B, vavP–Bcl-2/BimN/N, and vavP–Bcl-2/BimP/P thymocytes was determined by Western blotting. (C) Thymocyte lysates were prepared and the protein complexes were analyzed by immunoprecipitation and Western blotting. (D) Affinities of Bim and Noxa BH3 peptides for mouse Bcl-2 and mouse Mcl-1 were determined by solution competition assay using a Biacore optical biosensor. Numbers in brackets represent standard deviations for n = 2 to 4 experiments. (E) The sensitivity of BM-derived B cells and thymocytes of the indicated genotypes to ABT-737 was measured and data are represented in a heat map as described in Figure 2. Results represent the mean of at least 3 independent experiments per genotype (detailed in supplemental Table 1). Restricting the binding specificity of Bim to that of Bad renders all cell types resistant to ABT-737, regardless of Bcl-2 overexpression. As a control, the BH3 mutations in Bim did not affect the resistance of vavP-Mcl-1–transgenic cells to ABT-737–induced cell death (vavP–Mcl-1/BimB/B and vavP–Mcl-1/BimN/N). (F) Thymocytes from vavP–Bcl-2/Bim+/+ mice were treated with ABT-737 (1μM) for 5 hours before Bim immunoprecipitation. The composition of the protein complexes was analyzed by Western blotting. Delphine Mérino et al. Blood 2012;119:5807-5816 ©2012 by American Society of Hematology

Bcl-xL and Bcl-w overexpression protects against ABT-737 induced cell death. Bcl-xL and Bcl-w overexpression protects against ABT-737 induced cell death. (A) WT fetal liver cells retrovirally transduced with pMIG–Bcl-2, pMIG–Bcl-xL, pMIG–Bcl-w, pMIG-Mcl1, or control pMIG vectors were transplanted into lethally irradiated recipient mice (n = 5 per group). Lymphocytes were harvested 8 weeks later and treated in culture with ABT-737. The reminder of the results are shown as a heat map in supplemental Figure 3A. (B) Overexpression of Bcl-2, Bcl-xL, Bcl-w, or Mcl-1 in lymphocytes is accompanied by an increase in Bim as assessed by Western blotting. For a reason that is unclear, the Flag-tagged version of Mcl-1 is very difficult to detect with our anti-Flag antibody, but is detected with the anti–Mcl-1 antibody as a slower moving band. (C) Murine DO11.10 T hybridoma cells stably transfected with pEF–mBcl-2, pEF–mBcl-xL or control pEF vectors were challenged with increasing concentrations of ABT-737 for 24 hours. (D) Increase in Bim levels on overexpression of Bcl-2 or Bcl-xL in DO11.10 cells as revealed by Western blotting. (E) Mcl-1fl/fl/Eμ-Myc lymphoma cells retrovirally transduced with pMIG–Bcl-2, pMIG–Bcl-xL, pMIG–Mcl-1 or control pMIG vectors were treated for 3 days with 4-OHT to remove endogenous Mcl-1 before being exposed to ABT-737. These experiments have been reproduced using an independent Mcl-1fl/fl/Eμ-Myc lymphoma line. (F) The corresponding levels of Mcl-1, Bcl-2, Bcl-xL, and Bim were determined by Western blotting. Delphine Mérino et al. Blood 2012;119:5807-5816 ©2012 by American Society of Hematology

ABT-737 exhibits greater potency at displacing BimSΔC from Bcl-2 than from Bcl-xL or Bcl-w. ABT-737 exhibits greater potency at displacing BimSΔC from Bcl-2 than from Bcl-xL or Bcl-w. Flp-In T-REx 293 cells with single copy integration of an inducible bicistronic construct expressing mCherry-BimSΔC and eGFP-Bcl-2, Bcl-xL or Bcl-w were imaged live every 25 minutes on treatment of ABT-737 at a 10-point dose range from 5nM to 100μM. (A) Displacement of mCherry-BimSΔC from the mitochondria was measured at 350 minutes after compound addition and normalized to DMSO controls at t = 0. (B) EC50 values were estimated by fitting mitochondrial displacement of mCherry-BimSΔC in response to ABT-737 to sigmoid curves and averaged for 2 to 4 biologic replicates. P values comparing Bcl-2 with Bcl-xL and Bcl-w were .0009 and .028, respectively. Error bars represent SE, and P values were calculated using a Student t test. Delphine Mérino et al. Blood 2012;119:5807-5816 ©2012 by American Society of Hematology