PROTAC-Mediated Dual Degradation of BCL-xL and BCL-2 Is a Highly Effective Therapeutic Strategy in Small-Cell Lung Cancer

Abstract

BCL-xL and BCL-2 are validated therapeutic targets in small-cell lung cancer (SCLC). Targeting these proteins with navitoclax (formerly ABT263, a dual BCL-xL/2 inhibitor) induces dose-limiting thrombocytopenia through on-target BCL-xL inhibition in platelets. Therefore, platelet toxicity poses a barrier in advancing the clinical translation of navitoclax. We have developed a strategy to selectively target BCL-xL in tumors, while sparing platelets, by utilizing proteolysis-targeting chimeras (PROTACs) that hijack the cellular ubiquitin proteasome system for target ubiquitination and subsequent degradation. In our previous study, the first-in-class BCL-xL PROTAC, called DT2216, was shown to have synergistic antitumor activities when combined with venetoclax (formerly ABT199, BCL-2-selective inhibitor) in a BCL-xL/2 co-dependent SCLC cell line, NCI-H146 (hereafter referred to as H146), in vitro and in a xenograft model. Guided by these findings, we evaluated our newly developed BCL-xL/2 dual degrader, called 753b, in three BCL-xL/2 co-dependent SCLC cell lines and the H146 xenograft models. 753b was found to degrade both BCL-xL and BCL-2 in these cell lines. Importantly, it was considerably more potent than DT2216, navitoclax, or DT2216 + venetoclax in reducing the viability of BCL-xL/2 co-dependent SCLC cell lines in cell culture. In vivo, 5 mg/kg weekly dosing of 753b was found to lead to significant tumor growth delay, similar to the DT2216 + venetoclax combination in H146 xenografts, by degrading both BCL-xL and BCL-2. Additionally, 753b administration at 5 mg/kg every four days induced tumor regressions. At this dosage, 753b was well tolerated in mice, without observable induction of severe thrombocytopenia as seen with navitoclax, and no evidence of significant changes in mouse body weights. These results suggest that the BCL-xL/2 dual degrader could be an effective and safe therapeutic for a subset of SCLC patients, warranting clinical trials in future.

Keywords: BCL-2; BCL-xL; PROTAC; apoptosis; small-cell lung cancer.

Figure 1

753b degrades both BCL-xL and BCL-2 leading to apoptosis in SCLC cells. (a) Chemical structures of DT2216 and 753b. Linker in 753b is highlighted in red. (b–d) Immunoblot analyses of BCL-X_L, BCL-2, BCL-w, MCL-1, caspase 3 (C3), cleaved caspase 3 (CC3), PARP and cleaved (c)-PARP in SCLC H146 (b), H211 (c), and H1059 cells (d) after they were treated with indicated concentrations of 753b for 24 h. The β-tubulin was used as an equal loading control. (e–g) Densitometric analysis of BCL-xL and BCL-2 immunoblots in H146 (e), H211 (f), and H1059 cells (g) showing DC₅₀ and D_max values for each protein. DC_50, concentration required to degrade 50% of the protein; D_max, maximum degradation in percentage; nd, not determined.

Figure 2

753b is more potent than DT2216 or navitoclax to kill BCL-X_L/2-dependent SCLC cells. (a–e) Viability of H146 (a), H211 (b), H1059 (c), and H378 (d) SCLC cells, and WI38 normal lung fibroblasts (e), after they were treated with increasing concentrations of 753b, DT2216, or navitoclax for 72 h. (f) IC₅₀ values for 753b, DT2216, and navitoclax in SCLC cell lines and WI38 cells are tabulated.

Figure 3

753b showed comparable or enhanced efficacy as compared to DT2216 + venetoclax combination to kill BCL-xL/2-dependent SCLC cells. (a–c) Viability of H146 (a), H211 (b), and H1059 cells (c) after they were treated with increasing concentrations of DT2216, venetoclax, or their 1:1 combination. IC₅₀ values for the individual agents and combinations are tabulated. (d–f) Viability of H146 (d), H211 (e), and H1059 cells (f) after they were treated with increasing concentrations of 753b, venetoclax, or their 1:1 combination. IC₅₀ values for the individual agents and combinations are tabulated.

Figure 4

753b is more potent than DT2216 and similar to DT2216 + venetoclax for inhibiting growth of BCL-X_L/2-dependent H146 xenograft tumors in mice. (a) Tumor volume changes in H146 xenografts after treatment with vehicle, DT2216 (15 mg/kg, weekly i.e., q7d, i.p.), a combination of DT2216 with venetoclax (50 mg/kg, 5 days a week, p.o.), or 753b (5 mg/kg, q7d, i.p.). Data are presented as mean ± SEM (n = 7, 6, 6, and 7 mice in vehicle, DT2216, DT2216 + venetoclax, and 753b groups, respectively, at the start of treatment). When the biggest tumor dimension reached 1.5 cm, the mice were sacrificed in accordance with IACUC protocol, and the remaining mice continued to be treated for up to 104 days. Tumor volume changes are shown up to post-treatment day 88, when 5 or more mice were alive in each treatment group. **** p < 0.0001, ns: not significant as determined by one-way ANOVA and Dunnett’s multiple comparisons test at post-treatment day 32. (b) Kaplan-Meier survival analysis of mice as treated in (a). Survival time was recorded at the tumor endpoint, i.e., biggest tumor dimension of 1.5 cm or more. The median survival time is shown on the right. *** p < 0.001, ns: not significant as determined by two-sided Student’s t-test. (c) Mouse body weight changes in H146 xenografts after treatment as in (a). Data are presented as mean ± SEM. (d) Immunoblot analysis of BCL-xL, BCL-2, and MCL-1 in H146 xenograft tumors two days after last treatment with vehicle, DT2216, DT2216 + venetoclax (DT + VEN), or 753b (n = 3 mice per group) as in (a). (e) Densitometric analysis of immunoblots in (d). ** p < 0.01, *** p < 0.001 compared to vehicle as determined by one-way ANOVA and Dunnett’s multiple comparisons test.

Figure 5

753b induces regressions of larger H146 xenograft tumors in mice. (a) Tumor volume changes in H146 xenografts after treatment with vehicle or 753b (5 mg/kg, every four days i.e., q4d, i.p.). Data are presented as mean ± SEM (n = 5 mice). ** p < 0.01 compared to vehicle as determined by two-sided Student’s t-test. (b), Tumor weights at the end of experiment in (a). Data are presented as mean ± SEM (n = 5 mice). *** p < 0.001 compared to vehicle as determined by two-sided Student’s t-test. (c) The images of excised tumors from (a). (d) Mouse body weight changes in H146 xenografts after treatment as in (a).