A BCL-xL/BCL-2 PROTAC effectively clears senescent cells in the liver and reduces MASH-driven hepatocellular carcinoma in mice
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Collaborative orchestration of BH3-only proteins governs Bak/Bax-dependent hepatocyte apoptosis under antiapoptotic protein-deficiency in mice
The fine-tuned balance between anti-apoptotic Bcl-2 family proteins, such as Bcl-xL and Mcl-1, and pro-apoptotic Bcl-2 family proteins, like Bak and Bax, is crucial for maintaining hepatocyte integrity. BH3-only proteins, including Bid, Bim, Puma, Noxa, Bad, Bmf, Bik and Hrk, serve as apoptosis initiators. They are activated by various stimuli, which leads to Bak/Bax activation. We previously reported that Bid and Bim contributed to hepatocyte apoptosis through Bak/Bax activation in the absence of anti-apoptotic proteins Bcl-xL and/or Mcl-1. However, the comprehensive involvement of all eight BH3-only proteins in Bak/Bax-dependent hepatocyte apoptosis remains unclear. Puma disruption suppressed hepatocyte apoptosis in hepatocyte-specific Bcl-xL or Mcl-1 knockout (Bcl-xLΔHep/ΔHep or Mcl-1ΔHep/ΔHep) mice. Disruption of Bid and Bim partially prevented lethality in Mcl-1ΔHep/+ Bcl-xLΔHep/ΔHep mice, although severe hepatocyte apoptosis persisted, which was suppressed by additional Puma disruption. However, hepatocyte apoptosis was still induced compared to that in Mcl-1ΔHep/+ Bcl-xLΔHep/ΔHep BaxΔHep/ΔHep Bak−/− mice. Triple disruption of Bid, Bim and Puma did not prevent induction of hepatocyte apoptosis in tamoxifen-induced Mcl-1iΔHep/iΔHep Bcl-xLiΔHep/iΔHep mice. Primary hepatocytes, isolated from Mcl-1fl/fl Bcl-xLfl/fl Bid−/− Bim−/− Puma−/− mice and immortalized, underwent apoptosis with doxycycline-dependent Cre recombination. Among the remaining five BH3-only proteins, Bik and Hrk were not expressed in these cells, and Noxa knockdown, but not Bad or Bmf knockdown, reduced apoptosis. Noxa disruption alleviated hepatocyte apoptosis in Mcl-1ΔHep/ΔHep mice and tamoxifen-induced Mcl-1iΔHep/iΔHep Bcl-xLiΔHep/iΔHep Bid−/− Bim−/− Puma−/− mice, prolonging survival. Apoptosis persisted in immortalized primary hepatocytes isolated from Mcl-1fl/fl Bcl-xLfl/fl Bid−/− Bim−/− Puma−/− Noxa−/− mice where doxycycline-dependent Cre recombination was induced, but was completely suppressed by Bak/Bax knockdown, while Bad or Bmf knockdown had no effect. In conclusion, among the eight BH3-only proteins, Puma and Noxa, alongside Bid and Bim, contributed to Bak/Bax-dependent hepatocyte apoptosis, but not indispensably, in the absence of Mcl-1 and Bcl-xL.
A1AT dysregulation of metabolically stressed hepatocytes by Kupffer cells drives MASH and fibrosis
Metabolic dysfunction-associated steatohepatitis (MASH) is associated with the activation of Kupffer cells (KCs) and hepatic stellate cells, at which point a metabolically stressed hepatocyte becomes integral to the progression of the disease. We observed a significant reduction in the level of alpha-1-antitrypsin (A1AT), a hepatocyte-derived secreted factor, in both patients with MASH and mice fed a fast-food diet (FFD). KC-mediated hepatic inflammation, most notably IL-1β, led to the transcriptional inhibition of A1AT by HNF4α. In quintuple Serpina1a–e knockout mice, ablation of A1AT worsened MASH through increased activity of proteinase 3 (PR3), a proinflammatory protease produced by F4/80hi/CD11blow/TIM4−/CCR2+ monocyte-derived KCs (MoKCs). Conversely, A1AT restoration or PR3 inhibition mitigated MASH progression. A PR3-bound cytokine array identified IL-32 as a key factor associated with MASH. Combining IL-32 with SERPINA1, the gene encoding A1AT, synergistically predicted patients at risk of MASH through univariate logistic regression analysis. Furthermore, in vivo overexpression of IL-32γ alleviated MASH induced by FFD. However, additional knockout of A1AT increased PR3 activity, consequently abolishing the anti-MASH effects of IL-32γ. Blocking PR3-mediated IL-32γ cleavage via the V104A mutation sustained its protective actions, while the PR3-cleaved C-terminal fragment activated KCs. Additionally, after cleavage, the antifibrogenic effect of IL-32γ is lost, resulting in a failure to prevent the activation of hepatic stellate cells. This study highlights the critical role of hepatocyte-derived A1AT in the PR3/IL-32γ axis during MASH development. Strategies to correct A1AT dysregulation, such as A1AT supplementation or PR3 inhibition with sivelestat, may offer protection against the development and progression of MASH and fibrosis.
Mitochondrial priming and response to BH3 mimetics in “one-two punch” senogenic-senolytic strategies
A one-two punch sequential regimen of senescence-inducing agents followed by senolytic drugs has emerged as a novel therapeutic strategy in cancer. Unfortunately, cancer cells undergoing therapy-induced senescence (TIS) vary widely in their sensitivity to senotherapeutics, and companion diagnostics to predict the response of TIS cancer cells to a specific senolytic drug are lacking. Here, we hypothesized that the ability of the BH3 profiling assay to functionally measure the mitochondrial priming state—the proximity to the apoptotic threshold—and the dependencies on pro-survival BCL-2 family proteins can be exploited to inform the sensitivity of TIS cancer cells to BH3-mimetics. Replicative, mitotic, oxidative, and genotoxic forms of TIS were induced in p16-null/p53-proficient, BAX-deficient, and BRCA1-mutant cancer cells using mechanistically distinct TIS-inducing cancer therapeutics, including palbociclib, alisertib, doxorubicin, bleomycin, and olaparib. When the overall state of mitochondrial priming and competence was determined using activator peptides, the expected increase in overall mitochondrial priming was an exception rather than a generalizable feature across TIS phenotypes. A higher level of overall priming paralleled a higher sensitivity of competent TIS cancer cells to BCL-2/BCL-xL- and BCL-xL-targeted inhibitors when comparing TIS phenotypes among themselves. Unexpectedly, however, TIS cancer cells remained equally or even less overally primed than their proliferative counterparts. When sensitizing peptides were used to map dependencies on anti-apoptotic BCL-2 family proteins, competent TIS cancer cells appeared to share a dependency on BCL-xL. Furthermore, regardless of senescence-inducing therapeutic, stable/transient senescence acquisition, or genetic context, all TIS phenotypes shared a variable but significant senolytic response to the BCL-xL-selective BH3 mimetic A1331852. These findings may help to rethink the traditional assumption of the primed apoptotic landscape of TIS cancer cells. BCL-xL is a conserved anti-apoptotic effector of the TIS BCL2/BH3 interactome that can be exploited to maximize the efficacy of “one-two punch” senogenic-senolytic strategies.
Cross-talk of inflammation and cellular senescence: a new insight into the occurrence and progression of osteoarthritis
Osteoarthritis (OA) poses a significant challenge in orthopedics. Inflammatory pathways are regarded as central mechanisms in the onset and progression of OA. Growing evidence suggests that senescence acts as a mediator in inflammation-induced OA. Given the lack of effective treatments for OA, there is an urgent need for a clearer understanding of its pathogenesis. In this review, we systematically summarize the cross-talk between cellular senescence and inflammation in OA. We begin by focusing on the mechanisms and hallmarks of cellular senescence, summarizing evidence that supports the relationship between cellular senescence and inflammation. We then discuss the mechanisms of interaction between cellular senescence and inflammation, including senescence-associated secretory phenotypes (SASP) and the effects of pro- and anti-inflammatory interventions on cellular senescence. Additionally, we focus on various types of cellular senescence in OA, including senescence in cartilage, subchondral bone, synovium, infrapatellar fat pad, stem cells, and immune cells, elucidating their mechanisms and impacts on OA. Finally, we highlight the potential of therapies targeting senescent cells in OA as a strategy for promoting cartilage regeneration.
VDAC2 and Bak scarcity in liver mitochondria enables targeting hepatocarcinoma while sparing hepatocytes
Differences between normal tissues and invading tumors that allow tumor targeting while saving normal tissue are much sought after. Here we show that scarcity of VDAC2, and the consequent lack of Bak recruitment to mitochondria, renders hepatocyte mitochondria resistant to permeabilization by truncated Bid (tBid), a Bcl-2 Homology 3 (BH3)-only, Bcl-2 family protein. Increased VDAC2 and Bak is found in most human liver cancers and mitochondria from tumors and hepatic cancer cell lines exhibit VDAC2- and Bak-dependent tBid sensitivity. Exploring potential therapeutic targeting, we find that combinations of activators of the tBid pathway with inhibitors of the Bcl-2 family proteins that suppress Bak activation enhance VDAC2-dependent death of hepatocarcinoma cells with little effect on normal hepatocytes. Furthermore, in vivo, combination of S63845, a selective Mcl-1 inhibitor, with tumor-nectrosis factor-related, apoptosis-induncing ligand (TRAIL) peptide reduces tumor growth, but only in tumors expressing VDAC2. Thus, we describe mitochondrial molecular fingerprint that discriminates liver from hepatocarcinoma and allows sparing normal tissue while targeting tumors.
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