1. Acquired mutations in BCL2 family proteins conferring resistance to the BH3 mimetic ABT-199 in lymphoma
by Vicente Fresquet, Melissa Rieger, Carlo Carolis, Maria J. García-Barchino, and Jose A. Martinez-Climent
Blood
Volume 123(26):
June 26, 2014
©2014 by American Society of Hematology

2. Acquired mutations in the BH3 domain of Bcl2 conferring resistance to ABT-199.
Acquired mutations in the BH3 domain of Bcl2 conferring resistance to ABT-199. (A) Scheme showing the generation of the resistant murine model. A detailed description is provided in “Methods.” (B) Cell lines were incubated with the BH3 mimetics for 48 hours; cell proliferation was measured using the MTS assay. Ly2Bcl2-6 and Ly2Bcl2-9 resistant clones did not show activation of the mitochondrial apoptotic pathway upon exposure to the BH3 mimetics, as shown by the annexin V-FITC measurement and the lack of caspases 3 and 9 cleavage. A representative example from 3 independent experiments is shown. (C) Treatment with 1 µM ABT-199 induced activation of the mitochondrial apoptotic pathway in sensitive but not in resistant cells. Expression analysis of Bcl2 family members by western blot (D) or quantitative real-time PCR (E). (F) Resistant cell lines cultured without ABT-199 (labeled as SA) remained resistant to the BH3 mimetics. (G-H) The protein expression profile in the SA cells was similar to those of the sensitive cell lines. (C-G) Representative data from 1 of 3 independent experiments performed in triplicate (mean ± SD, where indicated). (I) Sequence of Bcl2 identified 2 de novo missense mutations at the same codon (F101) located within the BH3 domain. These mutations were found in the ectopic Bcl2 mouse gene but not in the endogenous mouse Bcl2. Arrows indicate the nucleotide change.
Vicente Fresquet et al. Blood 2014;123:
©2014 by American Society of Hematology

3. Mutation in the transmembrane domain of BAX abolished cell death induced by ABT-199.
Mutation in the transmembrane domain of BAX abolished cell death induced by ABT-199. (A) Scheme showing the generation of HBL2R cells. A detailed description is provided in “Methods.” (B) Both HBL2 and HBL2R cells cultured without ABT-199 for 4 weeks (labeled as HBL2SA) were resistant to the BH3 mimetics. (C) HBL2R cells did not show activation of the mitochondrial apoptotic pathway induced by the BH3 mimetics, as shown by the annexin V-FITC FACS and the lack of caspases 3 and 9 cleavage. Experiments were performed in duplicate. (D) Kaplan-Meier overall survival curves for Rag2−/−IL2γc−/− mice transplanted with HBL2 or HBL2R cells. (E) A representative image showing differences in the tumor volume in mice inoculated with HBL2 and HBL2R cells and treated with ABT-199 (marked with arrows). (F-G) Expression profiling of BCL2 family proteins did not show changes between HBL2 and HBL2SA cells. Shown are representative data from 1 of 3 independent experiments. (H) Cells with acquired resistance to ABT-199 showed a missense mutation in BAX transmembrane domain (G179E). Arrows indicate the base change. cHBL2R, cDNA; gHBL2R, genomic DNA.
Vicente Fresquet et al. Blood 2014;123:
©2014 by American Society of Hematology

4. Functional evaluation of Bcl2 and BAX mutations.
Functional evaluation of Bcl2 and BAX mutations. (A) ABT-199 was unable to bind F101C and F101L Bcl2 mutant proteins. (B) Bcl2 mutations had no impact on Bcl2 binding to Bim in both ABT-199–sensitive and –resistant lymphomas. Immunoprecipitation experiments were performed in duplicate. (C) Ectopic expression of WT BAX (but not G179E) in HBL2R cells restored ABT-199 sensibility. Impact of ABT-199 on cell viability (left). Induction of apoptosis with and without ABT-199 treatment (right). Experiments were performed in triplicate. (D) IF analysis showing that ABT-199 induced BAX translocation from cytoplasm to mitochondria in HBL2 but not in HBL2R cells, as shown by colocalization of BAX and mitotraker (Mito). Experiments were performed in duplicate: a representative example is shown. Quantification values of colocalization BAX and mitotraker IF images are provided in the main text. DAPI, 4,6 diamidino-2-phenylindole. (E) Subcellular localization of BAX performed by cell fractionation experiments showed that mutant BAX remained cytoplasmic after ABT-199 treatment. In addition, BAX G179E mutation prevented release of cytochrome C to cytoplasm. Experiments were performed in duplicate.
Vicente Fresquet et al. Blood 2014;123:
©2014 by American Society of Hematology

5. Proposed model of acquired resistance to ABT-199 in experimental models of human and mouse lymphoma.
Proposed model of acquired resistance to ABT-199 in experimental models of human and mouse lymphoma. Mutations in the BH3 domain of BCL2 abolish ABT-199 binding and thus block apoptosis. Mutation in BAX transmembrane domain prevents mitochondrial translocation of BAX and thus impedes apoptosis.
Vicente Fresquet et al. Blood 2014;123:
©2014 by American Society of Hematology