Identification of a novel senolytic agent, navitoclax, targeting the Bcl-2 family of anti-apoptotic factors

Abstract

Clearing senescent cells extends healthspan in mice. Using a hypothesis-driven bioinformatics-based approach, we recently identified pro-survival pathways in human senescent cells that contribute to their resistance to apoptosis. This led to identification of dasatinib (D) and quercetin (Q) as senolytics, agents that target some of these pathways and induce apoptosis preferentially in senescent cells. Among other pro-survival regulators identified was Bcl-xl. Here, we tested whether the Bcl-2 family inhibitors, navitoclax (N) and TW-37 (T), are senolytic. Like D and Q, N is senolytic in some, but not all types of senescent cells: N reduced viability of senescent human umbilical vein epithelial cells (HUVECs), IMR90 human lung fibroblasts, and murine embryonic fibroblasts (MEFs), but not human primary preadipocytes, consistent with our previous finding that Bcl-xl siRNA is senolytic in HUVECs, but not preadipocytes. In contrast, T had little senolytic activity. N targets Bcl-2, Bcl-xl, and Bcl-w, while T targets Bcl-2, Bcl-xl, and Mcl-1. The combination of Bcl-2, Bcl-xl, and Bcl-w siRNAs was senolytic in HUVECs and IMR90 cells, while combination of Bcl-2, Bcl-xl, and Mcl-1 siRNAs was not. Susceptibility to N correlated with patterns of Bcl-2 family member proteins in different types of human senescent cells, as has been found in predicting response of cancers to N. Thus, N is senolytic and acts in a potentially predictable cell type-restricted manner. The hypothesis-driven, bioinformatics-based approach we used to discover that dasatinib (D) and quercetin (Q) are senolytic can be extended to increase the repertoire of senolytic drugs, including additional cell type-specific senolytic agents.

Keywords: ABT-263; Bcl-2 family; TW-37; dasatinib; quercetin; senescent cells.

Figure 1

Apoptotic pathways. Bcl‐2 family members act upstream of mitochondrial‐mediated apoptosis. Pro‐apoptotic BH3‐only proteins (e.g., Noxa) transiently bind Bax/Bak, facilitating activation, while anti‐apoptotic Bcl‐2 family members (e.g., Bcl‐xl, Bcl‐2, Mcl‐1, Bcl‐w) block Bax/Bak directly. AP20187, which activates the caspase‐8 moiety of the myristoylated membrane‐bound ATTAC fusion protein product of the INK‐ATTAC transgene, is also shown. Both the caspase‐8‐ and Bak/Bax/cytochrome c‐related pathways activate the executioner caspases 3 and 7.

Figure 2

N targets senescent cells. (A) N is more effective in reducing viability (ATPLite) of senescent HUVECs and IMR90 cells than human preadipocytes. Proliferating or senescent cells were exposed to different concentrations of N for 3 days. The red line denotes plating densities at day 0 of senescent and nonsenescent cells, both set to 100%. HUVEC and IMR90 data represent means ± SEM of four replicates at each drug concentration. Preadipocyte data are means ± SEM of four different subjects; P < 0.001, ANOVA. (B) N induces apoptosis in senescent HUVECs and IMR90 cells. HUVECs were treated with N for 12 h, and then caspases 3 and 7 were assayed using a luminescent substrate. N (1000 nM) induced apoptosis in senescent cells by TUNEL assay; P < 0.001, t‐test. (C) The Bcl‐2/Bcl‐xl inhibitor, TW‐37, does not impact the viability of senescent HUVECs, IMR90 cells, or human preadipocytes. Cells were exposed to different concentrations of TW‐37 for 3 days. The red line denotes plating densities on day 0 of senescent (set to 100%) and nonsenescent cells (also set to 100%). HUVEC and IMR90 cell data are means ± SEM of four replicates at each concentration. Preadipocyte data are means ± SEM of four different subjects.

Figure 3

Navitoclax selectively kills senescent Ercc1‐deficient murine embryonic fibroblasts, while TW‐37 is less senolytic. Cultures containing ~ 50% senescent MEFs were exposed to drugs for 48 h prior to analysis of SA‐βGal⁺ cells using C₁₂ FDG and Hoechst staining to determine (A) the ratios of senescent to nonsenescent cells within each of the wells as a function this ratio in vehicle‐treated cells. This indicates that T only eliminates senescent cells at high concentrations. (B) We also determined the ratio of the number of senescent (or proliferating) cells in each well to the number of senescent (or proliferating) cells in the corresponding vehicle‐treated wells (red dotted lines). This indicates that T actually reduces proliferating cell numbers at concentrations below those that reduce senescent cells. The data shown are means ± SD of 3 replicates; **P < 0.01; ***P < 0.001, ANOVA.

Figure 4

Senolytic effects of targeting Bcl‐2 family members by RNA interference. (A) Proliferating or senescent HUVECs, IMR90 cells, or primary human preadipocytes were transfected with siRNAs individually or in combination 2 days before viability analysis (ATPLite). Decreases in targeted mRNAs were confirmed by RT–PCR (Fig. S2). Bcl‐xl siRNA alone or in combination with Bcl‐2 and Bcl‐xl siRNA was senolytic in HUVECs, but not IMR90 cells or preadipocytes. The combination of Bcl‐2, Bcl‐xl, and Bcl‐w siRNAs reflects the targets of N, while Bcl‐2, Bcl‐xl, and Mcl‐1 reflects those of T. HUVEC and IMR90 cell data are means ± SEM of five replicates at each concentration. Preadipocyte data are means ± SEM of four different subjects. *P < 0.05, t‐test. (B) Bcl‐2 family member siRNAs act by inducing apoptosis in targeted senescent cells (caspase‐3&7 activity assay). Bcl‐2 and Bcl‐xl siRNAs induced apoptosis in HUVECs both on their own, in combinations together, or combined with Bcl‐w siRNA. The combination of Bcl‐2, Bcl‐xl, and Bcl‐w siRNAs, reflecting the targets of N, induced apoptosis to a greater extent in senescent than nonsenescent HUVECs, while the combination of Bcl‐2, Bcl‐xl, and Mcl‐1 siRNAs, reflecting actions of T, did not. Data are means ± SEM of three replicates. *P < 0.05, ANOVA.

Figure 5

Bcl‐2 family member proteins in senescent HUVECs, IMR90 cells, and human preadipocytes. Bcl‐2 family proteins were followed after 10‐Gy radiation by immunoblot as cells became senescent as indicated by morphology and p16^{INK 4A}. (A) representative immunoblots. (B) RT–PCR analyses. Means ± SEM of immunoblots from 3 HUVEC and 3 IMR90 cultures and 3 immunoblots from different subjects. *P < 0.05, t‐test.

Figure 6

Combining D + N or D + N + Q did not reduce viability (ATPLite) of senescent preadipocytes more than D alone. Proliferating and senescent preadipocytes were exposed to N + Q plus different concentrations of D for 3 days. Means ± SEM of preadipocytes from 4 subjects; *P < 0.001, ANOVA.