Hypothesis

In senescent cells, protein aggregates function as a protective sink that sequesters pro‑apoptotic BH3‑only proteins (e.g., BIM, PUMA, NOXA) through hydrophobic interactions with amyloid‑like surfaces. This sequestration raises the threshold for mitochondrial outer membrane permeabilization (MOMP) by limiting the availability of BH3‑only activators that can neutralize anti‑apoptotic Bcl‑2 family members. The senescent‑cell‑specific upregulation of the NRF2‑Bcl‑2 transcriptional axis not only boosts anti‑apoptotic Bcl‑2, Bcl‑xL and MCL‑1 but also drives chaperone‑mediated aggregation, coupling proteostasis stress to survival dependence. Consequently, disrupting aggregates should liberate sequestered BH3‑only proteins, lower the apoptotic priming threshold, and render senescent cells exquisitely sensitive to Bcl‑2/MCL‑1 inhibitor combinations at sub‑toxic doses.

Mechanistic Rationale

1. Aggregate as BH3‑only scaffold – Amyloid and tau fibrils expose conserved hydrophobic grooves that can bind the BH3 α‑helix of pro‑apoptotic proteins with micromolar affinity, as shown for synthetic amyloid peptides binding BIM (see analogous studies on Aβ‑BIM interactions). In senescent cells, the burden of misfolded proteins promotes formation of such aggregates, effectively sequestering BH3‑only molecules away from Bcl‑2/MCL‑1.
2. NRF2‑Bcl‑2 axis amplifies dependence – NRF2 directly transactivates Bcl‑2, Bcl‑xL and MCL‑1 promoters [4] while also upregulating HSP70 and DNAJB6 chaperones that facilitate aggregate formation [3]. This creates a feed‑where: higher NRF2 activity → more anti‑apoptotic Bcl‑2 proteins + more aggregates → greater BH3‑only sequestration → heightened reliance on Bcl‑2/MCL‑1 for survival.
3. Synthetic lethality prediction – If aggregates are dissolved (e.g., by a disaggregase such as HSP110/HSP70/HSP40 combo or by enhancing autophagic flux), freed BH3‑only proteins will competitively inhibit Bcl‑2/MCL‑1, lowering the dose of navitoclax (Bcl‑2/Bcl‑xL inhibitor) and an MCL‑1 inhibitor (e.g., S63845) required to trigger MOMP.

Experimental Plan

• Step 1: Validate sequestration – Isolate detergent‑resistant aggregates from senescent HUVECs and IMR90 fibroblasts (induced by irradiation or oncogenic RAS). Perform co‑immunoprecipitation and mass spectrometry to detect bound BH3‑only proteins. Compare levels in proliferating vs senescent cells.
• Step 2: Measure liberation – Treat senescent cells with an aggregate‑disrupting agent (e.g., low‑dose spermidine to enhance autophagy or a small‑molecule HSP70 activator). Assess cytosolic BH3‑only protein release by western blot and mitochondrial priming using BH3 profiling.
• Step 3: Test synergy – Combine aggregate disruptors with navitoclax + MCL‑1 inhibitor at varying concentrations. Quantify senolytic efficacy via SA‑β‑gal loss, Annexin V/PI staining, and clonogenic survival. Expect a left‑shift in dose‑response curves (EC50 reduction ≥5‑fold) relative to inhibitor alone.
• Step 4: In vivo validation – Use aged mice with inducible p16‑3MR reporter. Treat with aggregate disruptor plus low‑dose navitoclax + MCL‑1 inhibitor. Assess senescent cell burden (p16‑3MR luciferase) and frailty indices.

Falsifiability

If aggregate disruption does not increase cytosolic BH3‑only levels, or if it fails to sensitize senescent cells to Bcl‑2/MCL‑1 inhibition (no synergistic senolysis at reduced doses), the hypothesis is refuted. Conversely, observing BH3‑only liberation and dose‑synergistic senolysis would support the model that senescent cells rely on aggregate‑mediated sequestration of pro‑apoptotic factors as a linchpin of their survival, offering a precision‑senolytic strategy that exploits the proteostasis‑apoptosis nexus.