Shanthakumar Tyavanagimatt

Antitumor effects of BFC1108 in an orthotopic breast cancer model. A, BFC1108 inhibits growth of high Bcl-2–expressing MDA-MB-231 TNBC cells in vivo. A total of 106 MDA-MB-231/Bcl-2 cells were implanted in the mammary fat pad of NOD.SCID mice (n = 8 per group). Once palpable tumors were detected, mice were randomized and treated with vehicle or BFC1108 at 100 mg/kg twice a week by intraperitoneal route. Tumor measurements were made with digital calipers twice a week. *, P < 0.05 from day 18 onward. B, The body weights of mice treated with vehicle or BFC1108 in A were measured twice a week. C, Induction of apoptosis of tumor cells by BFC1108 in vivo. Immunofluorescence was performed on frozen tumor sections to determine apoptosis by TUNEL staining. D, BFC1108 induces conformation change of Bcl-2 in tumor tissues. Bcl-2 conformational change was detected by staining with Bcl-2 BH3 antibody. E, Bax activation by BFC1108. Activated Bax was detected using Bax 6A7 antibody in tumor tissues from animals treated with vehicle or BFC1108. F, Activation of caspase-3 by BFC1108. Activated caspase-3 was detected using cleaved caspase-3 antibody in tumor tissues from animals treated with vehicle or BFC1108.

Bioluminescent imaging of data in Figure 7 for individual mouse treated with vehicle or BFC1108 is shown.

Interaction of BFC1108 with Bcl-2. A, Limited proteolysis of Bcl-2 loop domain in the presence of BFC1108. Purified GST-tagged Bcl-2 loop domain and GST only control were incubated with 50 µmol/L BFC1108. The proteolysis pattern of loop domain was determined at the indicated times upon coincubation with trypsin. B, BFC1108 stabilizes and increases melting temperature (Tm) of Bcl-2 full-length protein. Thermal unfolding of Bcl-2 full-length protein in the presence of BFC1108 was monitored by SYPRO Orange fluorescence. Z-tag and no protein control samples were included to confirm BFC1108 specific interaction with Bcl-2. Two-way ANOVA with Sidak multiple comparisons post hoc test, **, P < 0.01; ****, P < 0.0001. C, BFC1108 stabilizes and increases Bcl-2 loop domain melting temperature (Tm). Thermal unfolding of Bcl-2 loop domain in the presence of BFC1108 monitored by SYPRO Orange fluorescence. One-way ANOVA with Dunnett multiple comparisons post hoc test, ****, P < 0.0001. D, MDA-MB-231/Bcl-2, H460 cells were exposed to BFC1108 at 10 µmol/L concentration for 48 hours in a medium containing 10% serum. Change in conformation of Bcl-2 was determined by using Bcl-2 BH3 antibody followed by flow cytometric analysis.

Inhibition of breast cancer lung metastasis by BFC1108. A, BFC1108 suppresses the growth of lung metastasis in vivo. LMD-231 cells (200,000) stably expressing luciferase were injected into the tail vein of 6-week-old nude mice. Lung metastasis was detected in 2 weeks. Mice were randomized and were treated four times a week with vehicle or 100 mg/kg BFC1108 by intraperitoneal route (n = 8 for vehicle treatment and n = 9 for BFC1108 treatment). Bioluminescent imaging was performed once a week and quantified. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Bioluminescent imaging data for individual mouse treated with vehicle or BFC1108 are shown in Supplementary Fig. S2. B, Representative mouse image from vehicle and BFC treatment is shown. C, Treatment with BFC1108 does not alter body weight of mice. Body weights were measured each time the mice were imaged. D, Suppression of lung metastasis by BFC1108. H&E staining showed increased number of tumor cells in lung tissue of mice with vehicle treatment, compared with BFC1108 treatment, at the end of the study. E, BFC1108 reduces proliferation of lung metastatic TNBC cells. Detection of Ki-67 indicating proliferating tumor cells in lung tissue from vehicle and BFC1108-treated mice.

Inhibition of breast cancer lung metastasis by BFC1108. A, BFC1108 suppresses the growth of lung metastasis in vivo. LMD-231 cells (200,000) stably expressing luciferase were injected into the tail vein of 6-week-old nude mice. Lung metastasis was detected in 2 weeks. Mice were randomized and were treated four times a week with vehicle or 100 mg/kg BFC1108 by intraperitoneal route (n = 8 for vehicle treatment and n = 9 for BFC1108 treatment). Bioluminescent imaging was performed once a week and quantified. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Bioluminescent imaging data for individual mouse treated with vehicle or BFC1108 are shown in Supplementary Fig. S2. B, Representative mouse image from vehicle and BFC treatment is shown. C, Treatment with BFC1108 does not alter body weight of mice. Body weights were measured each time the mice were imaged. D, Suppression of lung metastasis by BFC1108. H&E staining showed increased number of tumor cells in lung tissue of mice with vehicle treatment, compared with BFC1108 treatment, at the end of the study. E, BFC1108 reduces proliferation of lung metastatic TNBC cells. Detection of Ki-67 indicating proliferating tumor cells in lung tissue from vehicle and BFC1108-treated mice.

BFC1108 suppresses viability of multiple cancer cell types. A, Bcl-2–selective inhibition of clonogenic survival of MDA-MB-231 cells. MDA-MB-231/Vector and MDA-MB-231/Bcl-2 cells were exposed to 10 µmol/L concentration in medium containing 10% FBS for 48 hours and the colony formation was determined after 2 weeks. B, Quantification of colony formation data shown in A. ***, P < 0.001. C, Bcl-2–dependent induction of apoptosis by BFC1108. MDA-MB-231 cells with high or low Bcl-2 expression were treated with 10 µmol/L BFC1108 in a medium containing 10% FBS for 48 hours and apoptosis was determined by annexin V staining. Data from one representative experiment is shown. Refer to Supplementary Fig. S1 for additional independent experiments. D, BFC1108 inhibits viability of breast cancer cells with Bcl-2 expression. The indicated breast cancer cells were treated for 48 hours, and viability was measured using CellTiter-Glo assay. Data were analyzed by two-way ANOVA and Dunnett multiple comparison post hoc test, **, P < 0.001; ****, P < 0.00001. Bottom panel indicates Bcl-2 RMA-normalized mRNA expression from the Cancer Cell Line Encyclopedia (50). E, BFC1108 suppresses viability of multiple cancer cell types with minimal effects on MCF10A nontransformed breast epithelial cells. Cells were treated for 48 hours in 10% serum containing medium and cell viability was determined. ***, P < 0.001.

Interaction of BFC1108 with Bcl-2. A, Limited proteolysis of Bcl-2 loop domain in the presence of BFC1108. Purified GST-tagged Bcl-2 loop domain and GST only control were incubated with 50 µmol/L BFC1108. The proteolysis pattern of loop domain was determined at the indicated times upon coincubation with trypsin. B, BFC1108 stabilizes and increases melting temperature (Tm) of Bcl-2 full-length protein. Thermal unfolding of Bcl-2 full-length protein in the presence of BFC1108 was monitored by SYPRO Orange fluorescence. Z-tag and no protein control samples were included to confirm BFC1108 specific interaction with Bcl-2. Two-way ANOVA with Sidak multiple comparisons post hoc test, **, P < 0.01; ****, P < 0.0001. C, BFC1108 stabilizes and increases Bcl-2 loop domain melting temperature (Tm). Thermal unfolding of Bcl-2 loop domain in the presence of BFC1108 monitored by SYPRO Orange fluorescence. One-way ANOVA with Dunnett multiple comparisons post hoc test, ****, P < 0.0001. D, MDA-MB-231/Bcl-2, H460 cells were exposed to BFC1108 at 10 µmol/L concentration for 48 hours in a medium containing 10% serum. Change in conformation of Bcl-2 was determined by using Bcl-2 BH3 antibody followed by flow cytometric analysis.

Cancer cells exploit the expression of anti-apoptotic protein Bcl-2 to evade apoptosis and develop resistance to therapeutics. High levels of Bcl-2 leads to sequestration of pro-apoptotic proteins causing the apoptotic machinery to halt. In this study, we report discovery of a small molecule, BFC1108 (5-chloro-N-(2-ethoxyphenyl)-2-[(4-methoxybenzyol)amino]benzamide), which targets Bcl-2 and converts it into a pro-apoptotic protein. The apoptotic effect of BFC1108 is not inhibited, but rather potentiated, by Bcl-2 overexpression. BFC1108 induces a conformational change in Bcl-2, resulting in the exposure of its BH3 domain both in vitro and in vivo. BFC1108 suppresses the growth of triple-negative breast cancer xenografts with high Bcl-2 expression and inhibits breast cancer lung metastasis. This study demonstrates a novel approach to targeting Bcl-2 using BFC1108, a small molecule Bcl-2 functional converter that effectively induces apoptosis in Bcl-2–expressing cancers.Significance:We report the identification of a small molecule that exposes the Bcl-2 killer conformation and induces death in Bcl-2–expressing cancer cells. Selective targeting of Bcl-2 and elimination of cancer cells expressing Bcl-2 opens up new therapeutic avenues.

Bcl-2 dependent apoptotic effects of BFC1108. Independent experimental replicates of data in Figure 2C are shown.

Bioluminescent imaging of data in Figure 7 for individual mouse treated with vehicle or BFC1108 is shown.