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A BCL-xL/BCL-2 PROTAC effectively clears senescent cells in the liver and reduces MASH-driven hepatocellular carcinoma in mice

Nature Aging volume 5, pages 386–400 (2025)Cite this article

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Abstract

Accumulation of senescent cells (SnCs) plays a causative role in many age-related diseases and has also been implicated in the pathogenesis and progression of metabolic dysfunction-associated steatotic liver disease (MASLD). Senolytics that can selectively kill SnCs have the potential to be developed as therapeutics for these diseases. Here we report the finding that 753b, a dual BCL-xL/BCL-2 proteolysis-targeting chimera (PROTAC), acts as a potent and liver-tropic senolytic. We found that treatment with 753b selectively reduced SnCs in the liver in aged mice and STAM mice in part due to its sequestration in the liver. Moreover, 753b treatment could effectively reduce the progression of MASLD and the development of hepatocellular carcinoma (HCC) in STAM mice even after the mice developed substantial metabolic dysfunction-associated steatohepatitis (MASH) and hepatic fibrosis. These findings suggest that BCL-xL/BCL-2 PROTACs have the potential to be developed as therapeutics for MASLD to reduce MASH-driven HCC.

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Fig. 1: 753b is a potent and broad-spectrum senolytic agent in vitro.
Fig. 2: 753b effectively reduces senescence burden in naturally aged mice in a tissue-specific manner.
Fig. 3: 753b is a more potent senolytic than ABT263 against senescent murine hepatocytes.
Fig. 4: 753b reduces hepatic cellular senescence, steatosis, fibrosis and injury in STAM mice.
Fig. 5: 753b administration reduces tumorigenesis in STAM mice.
Fig. 6: Early treatment (P45–P90) with 753b has no effect on hepatic cellular senescence, steatosis, fibrosis and injury and on HCC development in STAM mice.
Fig. 7: Delayed treatment (P90–P150) with 753b reduces hepatic cellular senescence, steatosis, fibrosis and injury and inhibits HCC development in STAM mice.
Fig. 8: 753b dose not inhibt HCC progression.

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Source Data and Supplementary Information are provided with this paper. All other data are available from the corresponding authors upon reasonable request.

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Acknowledgements

This work was supported by US National Institutes of Health (NIH) grants R01 AG063801 (G.Z. and D.Z.), R01 CA242003 (G.Z. and D.Z.), K01 AA024174 (L.P.) and R01 AA028035 (L.P.) as well as a Children’s Miracle Research Foundation grant awarded to L.P. This research used resources of the Mays Cancer Center Drug Discovery and Structural Biology Shared Resource (NIH P30 CA054174), the Center for Innovative Drug Discovery (CPRIT Core Facility Award RP210208 and NIST Award 60NANB24D117) and the San Antonio Nathan Shock Center (NIH P30 AG013319). We also thank M. Zeeshan for help with senolytic testing in PACs and S. Khan and D. Lyu for their assistance with some of these studies. The experiment involving HTVi of oncogene expression plasmids was assisted by M. McLaughlin and B. Barre.

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Author notes

  1. These authors contributed equally: Yang Yang, Natacha Jn-Simon, Yonghan He.

Authors and Affiliations

  1. Department of Biochemistry and Structural Biology, University of Texas Health Science Center, San Antonio, TX, USA

    Yang Yang & Daohong Zhou

  2. Department of Pharmacology and Therapeutics, College of Medicine, University of Florida, Gainesville, FL, USA

    Yang Yang

  3. Department of Pathology, Tulane University, New Orleans, LA, USA

    Natacha Jn-Simon, Chunbao Sun, Tian Tian, Sreenivasulu Basha, Xian-Ming Yin & Liya Pi

  4. Department of Pharmacodynamics, College of Pharmacy, University of Florida, Gainesville, FL, USA

    Yonghan He & Daohong Zhou

  5. Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, FL, USA

    Peiyi Zhang, Wanyi Hu & Guangrong Zheng

  6. Advanced Magnetic Resonance Imaging and Spectroscopy Facility, University of Florida, Gainesville, FL, USA

    Huadong Zeng

  7. Department of Cell Systems and Anatomy, University of Texas Health Science Center, San Antonio, TX, USA

    Araceli S. Huerta & Lu-Zhe Sun

  8. Department of Medicine, University of Texas Health Science Center, San Antonio, TX, USA

    Robert Hromas

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Contributions

L.P. and D.Z. made equal contributions to developing concepts and strategies in this study. Methodologies and techniques used in this study were mainly carried out by Y.Y., N.J.-S., Y.H., C.S., T.T., S.B. and L.P. Additional MRI for liver cancer imaging was performed by C.S., H.Z. and L.P. Synthesis and characterization of 753b was done by P.Z. and W.H., under the supervision of G.Z. Characterization of 753b senolytic activity in vitro and in naturally aged mice was done by Y.Y. and Y.H., under the supervision of D.Z. The STAM mouse model studies were done by N.J.-S., C.S., T.T. and S.B., under the supervision of L.P., and Y.Y. contributed to some of the analyses of hepatic senescence and fibrosis, under the supervision of D.Z. HTVi-induced HCC mouse model study was done by A.S.H., under the supervision of L.-Z.S. Writing of the original draft was performed by Y.Y., Y.H., L.P. and D.Z. X.-M.Y. and R.H. were involved in experimental design and data interpretation. Review and editing of the paper was performed by all authors.

Corresponding authors

Correspondence to Liya Pi or Daohong Zhou.

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Competing interests

Y.Y., Y.H., P.Z., W.H., G.Z., L.P. and D.Z. are inventors on patents for the use of BCL-xL PROTACs as anti-tumor agents and senolytics. R.H., G.Z. and D.Z. are cofounders of and have equity in Dialectic Therapeutics, which develops BCL-xL/2 PROTACs to treat cancer. The other authors declare no competing interests.

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Nature Aging thanks Thomas Bird and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Evaluation of 753b-induced degradation of the BCL-2 family proteins in WI-38 cells and 753b senolytic activity against renal epithelial cells (RECs), human umbilical vein endothelial cells (HUVECs), and preadipocytes (PACs) in vitro.

A. Representative western blotting images of the levels of BCL-xL, BCL-2, BCL-w, MCL-1, and von Hippel-Lindau (VHL) in NC WI-38 cells after they were treated with increasing concentrations of 753b in a cell culture for 24 h. B. Densitometric analyses of BCL-xL, BCL-2, BCL-w, and MCL-1 expression in NC WI-38 cells from A are presented. DC50, drug concentration causing 50% degradation of protein of interest; Dmax, the maximum level of degradation of protein of interest. C. The levels of VHL, BCL-xL, BCL-2, BCL-w, and MCL-1 in NC and IR-SnC WI-38 cells and human platelets (PLTs) from three donors (P1-3) were detected by western blotting. Similar results from NC and IR-SnC WI-38 cells were observed in a separate assay. D-E. Cell viability analyses show that 753b is more potent than ABT263 against IR-SnC and REP-SnC REC (D) and HUVEC (E) but less toxic to their non-senescent counterparts. The viability of NC, IR-SnC and REP-SnC REC and HUVEC was determined 72 h after treatment with increasing concentrations of ABT263 and 753b. EC50, half-maximal effective concentration. The data presented are mean ± SD (n = 6 technical replicates) of a representative assay. EC50, half-maximal effective concentration. F & G. Cell viability analyses show that 753b is not senolytic, but dasatinib and quercetin (D + Q) are, against IR-SnC PAC. The viability of IR-SnC PAC was determined 72 h after treatment with increasing concentrations of ABT263 and 753b (F), or with vehicle (VEH), low D + Q (1 μM D plus 20 μM Q) and high D + Q (10 μM D plus 200 μM Q) (G). The data presented are mean ± SD (n = 3 technical replicates) of a representative assay. β-actin was used as a loading control in A and C.

Source data

Extended Data Fig. 2 753b has no effect on the levels of Cdkn2a expression in the lung, kidney and fat tissues but reduces hepatic expression of SASP factors in naturally aged mice.

A. The levels of Cdkn2a mRNA in the lung, kidney and inguinal fat from untreated young mice and naturally aged mice treated with VEH and 753b. B. The levels of Cxcl12, Ccl5, Ccl2, Cxcl10, Mmp3, Mmp13, Il6, Il1a, Tnfa, and Tnfsf11 in the liver tissue from untreated young mice and naturally aged mice treated with VEH and 753b. The data are presented as means ± SEM (n = 7, 6, and 7 mice per group for young mice, VEH- and 753b-treated aged mice, respectively) and were analyzed by one-way ANOVAs with Šídák’s multiple comparisons test or Tukey’s multiple comparisons test.

Source data

Extended Data Fig. 3 753b reduces splenic expression of SASP factors in naturally aged mice.

The levels of Il1b, Serpine1, Mmp3, Mmp13, Cxcl12, Ccl5, Ccl2, Cxcl10, Il6, Il1a, Tnfa, and Tnfsf11 in the spleens from untreated young mice and naturally aged mice treated with VEH and 753b. The data are presented as means ± SEM (n = 8, 6, and 7 mice per group for young mice, VEH- and 753b-treated aged mice, respectively) and were analyzed by one-way ANOVAs with Šídák’s multiple comparisons test or Tukey’s multiple comparisons test.

Source data

Extended Data Fig. 4 Characterization of SnCs in the liver from STAM mice and additional evaluations of 753b treatment on STAM mice.

A. A cartoon indicates distribution of zone 1, 2, and 3 hepatocytes in liver lobules along with blood flow across the periportal to pericentral axis. Periportal hepatocytes are in zone 1 that consists of portal veins, hepatic arteries, and bile ducts. B-C. SA-β-gal staining was combined with immunohistochemistry to characterize types of SnCs in the livers from STAM mice 8 weeks after STZ and 4 weeks after HFD. Antibodies against the pericentral hepatocyte marker Cyp2E1 (B), periportal hepatocyte marker GP6Cα (C), hepatocyte marker HNF4α (D), and biliary epithelial cell marker CK19 (E) were used for the stainings. Representative images of the stainings are presented on the left (scale bar = 100 µm) and higher magnification images of the marked area on the left images are presented on the right for C-E. Data presented in A-E are from one representative experiment and three independent experiments were performed with similar results. F. The levels of Cdkna1 mRNA in the tumor free liver tissues from VEH-treated and 753b-treated STAM mice on P150. The data are presented as means ± SEM (n = 5 mice/group) and were analyzed by a two-tailed, unpaired Student’s t-test. G. The levels of selected SASP mRNA in the tumor free liver tissues from STAM mice on P150. The data are presented as means ± SEM (n = 2 and 3 mice for VEH and 753b group, respectively) and were analyzed by a two-tailed, unpaired t-tests. H. Photo of reprentative VEH-treated and 753b-treated STAM mice on P150. I. Whole body weight of STAM mice on P150. Data are presented as means ± SEM (n = 5 mice per group) and were analyzed by a two-tailed, unpaired Student’s t-test. J. Blood levels of glucose in VEH- and 753b-treated STAM mice after IP injection of insulin one week before the termination of the experiment on P150. Data are presented as means ± SEM (n = 5 mice per group) and analyzed by two-way ANOVA.

Source data

Extended Data Fig. 5

Diagram illustrating the time-dependent progression of NAFLD and development of HCC in STAM mice and different 753b treatment schedules and their effects on HCC development and progression.

Extended Data Fig. 6 The effects of early and delayed treatments with 753b on the selective markers of hepatic inflammation, necroptosis, and macrophase activation in the livers from STAM mice.

A. The levels of Ccl2, Ccl5, Mlkl, Ripk3, and Itgax/Cd11c mRNA in the tumor free liver tissues from STAM mice on P150 after receiving earlier VEH or 753b treatment as shown in Fig. 6a. The data are presented in A as means ± SEM (n = 5 mice/group) and were analyzed by a two-tailed, unpaired Student’s t-tests. B. Western blotting image of αSMA and Type 1 procollagen in the tumor free liver tissues (left panel), and that of Gpc3 expression in the whole liver tissues (right panel), from STAM mice on P150 after receiving delayed VEH or 753b treatment as shown in Fig. 7a. C. The levels of Ccl2, Ccl5, Il6, Serpine1, Mmp3, Mmp13, Mlkl, Ripk3, and Itgax/Cd11c mRNA in the tumor free liver tissues from STAM mice on P150 after receiving delayed VEH or 753b treatment as shown in Fig. 7a. The data are presented in C as means ± SEM (n = 5 mice/group) and were analyzed by a two-tailed, unpaired Student’s t-tests.

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Extended Data Fig. 7 753b is not cytotoxic to HCC cells in vitro.

A. Cell viability of human HCC cells, HepG2 and Huh7, 72 h after treatment with increasing concentrations of ABT263 and 753b in cell culture. EC50, half-maximal effective concentration. The data presented are mean ± SD (n = 6 technical replicates) of a reprentative assay. Similar results were observed in two additional assays. B. Representative western blotting images of BCL-xL, BCL-2, BCL-w and MCL-1 in HepG2 cells after they were treated with increasing concentrations of ABT263 and 753b for 16 h. β-actin was used as a loading control.

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Yang, Y., Jn-Simon, N., He, Y. et al. A BCL-xL/BCL-2 PROTAC effectively clears senescent cells in the liver and reduces MASH-driven hepatocellular carcinoma in mice. Nat Aging 5, 386–400 (2025). https://doi.org/10.1038/s43587-025-00811-7

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