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

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SURVEILLANCE AND AGEING PHENOTYPES Article 02 November 2022 TARGETING SENESCENT HEPATOCYTES FOR TREATMENT OF METABOLIC DYSFUNCTION-ASSOCIATED STEATOTIC LIVER DISEASE AND MULTI-ORGAN

DYSFUNCTION Article Open access 28 March 2025 ANTAGONIZING THE IRREVERSIBLE THROMBOMODULIN-INITIATED PROTEOLYTIC SIGNALING ALLEVIATES AGE-RELATED LIVER FIBROSIS VIA SENESCENT CELL KILLING

Article 11 May 2023 DATA AVAILABILITY Source Data and Supplementary Information are provided with this paper. All other data are available from the corresponding authors upon reasonable

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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. AUTHOR INFORMATION Author notes * These authors contributed equally: Yang Yang, Natacha Jn-Simon, Yonghan He. AUTHORS AND AFFILIATIONS * Department of

Biochemistry and Structural Biology, University of Texas Health Science Center, San Antonio, TX, USA Yang Yang & Daohong Zhou * Department of Pharmacology and Therapeutics, College of

Medicine, University of Florida, Gainesville, FL, USA Yang Yang * Department of Pathology, Tulane University, New Orleans, LA, USA Natacha Jn-Simon, Chunbao Sun, Tian Tian, Sreenivasulu

Basha, Xian-Ming Yin & Liya Pi * Department of Pharmacodynamics, College of Pharmacy, University of Florida, Gainesville, FL, USA Yonghan He & Daohong Zhou * Department of Medicinal

Chemistry, College of Pharmacy, University of Florida, Gainesville, FL, USA Peiyi Zhang, Wanyi Hu & Guangrong Zheng * Advanced Magnetic Resonance Imaging and Spectroscopy Facility,

University of Florida, Gainesville, FL, USA Huadong Zeng * Department of Cell Systems and Anatomy, University of Texas Health Science Center, San Antonio, TX, USA Araceli S. Huerta & 

Lu-Zhe Sun * Department of Medicine, University of Texas Health Science Center, San Antonio, TX, USA Robert Hromas Authors * Yang Yang View author publications You can also search for this

author inPubMed Google Scholar * Natacha Jn-Simon View author publications You can also search for this author inPubMed Google Scholar * Yonghan He View author publications You can also

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can also search for this author inPubMed Google Scholar * Wanyi Hu View author publications You can also search for this author inPubMed Google Scholar * Tian Tian View author publications

You can also search for this author inPubMed Google Scholar * Huadong Zeng View author publications You can also search for this author inPubMed Google Scholar * Sreenivasulu Basha View

author publications You can also search for this author inPubMed Google Scholar * Araceli S. Huerta View author publications You can also search for this author inPubMed Google Scholar *

Lu-Zhe Sun View author publications You can also search for this author inPubMed Google Scholar * Xian-Ming Yin View author publications You can also search for this author inPubMed Google

Scholar * Robert Hromas View author publications You can also search for this author inPubMed Google Scholar * Guangrong Zheng View author publications You can also search for this author

inPubMed Google Scholar * Liya Pi View author publications You can also search for this author inPubMed Google Scholar * Daohong Zhou View author publications You can also search for this

author inPubMed Google Scholar 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. ETHICS DECLARATIONS 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. PEER REVIEW PEER REVIEW INFORMATION _Nature Aging_ thanks Thomas Bird and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. ADDITIONAL

INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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; _D_max, 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. Source data 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.

Source data SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supplementary Figs. 1 and 2, Supplementary Tables 1–6 and Supplementary Materials. REPORTING SUMMARY SOURCE DATA SOURCE DATA

FIG. 1 Unprocessed western blots. SOURCE DATA FIG. 3 Unprocessed western blots. SOURCE DATA FIG. 4 Unprocessed western blots. SOURCE DATA FIG. 5 Unprocessed western blots. SOURCE DATA

EXTENDED DATA FIG. 1 Unprocessed western blots. SOURCE DATA EXTENDED DATA FIG. 6 Unprocessed western blots. SOURCE DATA EXTENDED DATA FIG. 7 Unprocessed western blots. SOURCE DATA FIG. 1

Statistical source data. SOURCE DATA FIG. 2 Statistical source data. SOURCE DATA FIG. 3 Statistical source data. SOURCE DATA FIG. 4 Statistical source data. SOURCE DATA FIG. 5 Statistical

source data. SOURCE DATA FIG. 6 Statistical source data. SOURCE DATA FIG. 7 Statistical source data. SOURCE DATA FIG. 8 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 1 Statistical

source data. SOURCE DATA EXTENDED DATA FIG. 2 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 3 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 4 Statistical source data.

SOURCE DATA EXTENDED DATA FIG. 6 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 7 Statistical source data. RIGHTS AND PERMISSIONS Springer Nature or its licensor (e.g. a society or

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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).

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