# Acyl-CoA-binding protein as a driver of pathological aging

Léa Montégut, Flavia Lambertucci, Lucas Moledo-Nodar, Carmen Fiuza-Luces, Carlos Rodríguez-López, Jose Antonio Serra-Rexach, Sylvie Lachkar, Omar Motiño, Mahmoud Abdellatif, Sylvère Durand, et al.

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## ► To cite this version:

Léa Montégut, Flavia Lambertucci, Lucas Moledo-Nodar, Carmen Fiuza-Luces, Carlos Rodríguez-López, et al.. Acyl-CoA-binding protein as a driver of pathological aging. Proceedings of the National Academy of Sciences of the United States of America, 2025, 122, 10.1073/pnas.2501584122. hal-05210878

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# Acyl-CoA-binding protein as a driver of pathological aging

Léa Montégut<sup>a,b,1,2</sup> <sup>†</sup>, Flavia Lambertucci<sup>a,b,1</sup>, Lucas Moledo-Nodar<sup>c</sup> <sup>‡</sup>, Carmen Fiuza-Luces<sup>d</sup> <sup>‡</sup>, Carlos Rodríguez-López<sup>e,f</sup> <sup>†</sup>, Jose Antonio Serra-Rexach<sup>e,g</sup> <sup>†</sup>, Sylvie Lachkar<sup>a,b</sup> <sup>‡</sup>, Omar Motiño<sup>a,b,h</sup>, Mahmoud Abdellatif<sup>a,b,lj</sup> <sup>‡</sup>, Sylvère Durand<sup>a,b</sup>, Fanny Aprahamian<sup>a,b</sup> <sup>†</sup>, Vincent Carbonnier<sup>a,b</sup> <sup>‡</sup>, Delphine Le Corre<sup>k</sup>, Sophie Mouillet-Richard<sup>k</sup> <sup>†</sup>, Hui Chen<sup>a,b</sup> <sup>‡</sup>, Allan Sauvat<sup>a,b</sup> <sup>‡</sup>, Yanbing Dong<sup>a,b</sup> <sup>‡</sup>, Sijing Li<sup>a,b</sup> <sup>‡</sup>, Yan Rong<sup>a,b</sup>, Federico Pietrocola<sup>l</sup>, Pierre Laurent-Puig<sup>k,m</sup> <sup>‡</sup>, Carlos López-Otín<sup>a,c,n</sup> <sup>†</sup>, Isabelle Martins<sup>a,b</sup> <sup>‡</sup>, Clea Barcena<sup>c,1,3</sup> <sup>†</sup>, Alejandro Lucia<sup>d,o,1,3</sup>, and Guido Kroemer<sup>a,b,m,1,3</sup> <sup>‡</sup>

Affiliations are included on p. 10.

Edited by Sean Curran, University of Southern California, Los Angeles, CA; received January 21, 2025; accepted June 4, 2025 by Editorial Board Member Peter Tontonoz

The tissue hormone acyl coenzyme A-binding protein (ACBP, encoded by the gene *diazepam-binding inhibitor*, DBI) has been implicated in various facets of pathological aging. Here, we show that ACBP plasma concentrations are elevated in (close-to-)centenarians (mean ± SD age 99.5 ± 4.5 y) commensurate with their health deterioration, correlating with a reduced glomerular filtration rate and a surge in senescence-associated cytokines. ACBP neutralization by means of a monoclonal anti-body (mAb) improved health span in a strain of progeroid mice. In a mouse model of chronic kidney injury induced by cisplatin, anti-ACBP mAb administration counter-acted both histopathological and functional signs of organ failure. ACBP inhibition also prevented the senescence of tubular epithelial cells and glomerular podocytes induced by cisplatin or doxorubicin, respectively, as measurable by the immunohistochemical detection of cyclin-dependent kinase inhibitor 1A (CDKN1A, best known as p21). Senescence was also prevented by anti-ACBP mAb treatment in additional mouse models of accelerated aging. This applied to liver damage induced by a combination of high-fat diet and carbon tetrachloride, where hepatic cells become senescent. Moreover, administration of anti-ACBP mAb prevented natural and doxorubicin-accelerated cardiomyocyte senescence. We performed single-nucleus RNA sequencing to study the transcriptome of hearts that had been exposed to doxorubicin and/or anti-ACBP in vivo. In cardiomyocytes, doxorubicin caused an anti-ACBP-reversible dysregulation of mRNAs coding for cardioprotective proteins involved in autophagy, fatty acid oxidation, mitochondrial homeostasis, and oxidative phosphorylation. Altogether, these findings plead in favor of a broad age-promoting effect of ACBP across different organ systems.

aging | autophagy | chemotherapy | kidney injury | senescence

Acyl coenzyme A-binding protein (ACBP, encoded by the gene *diazepam-binding inhibitor*, DBI) has two major functions (1, 2). As an intracellular protein, it binds to medium-sized activated fatty acids (which are acyl coenzyme A esters) to facilitate their intracellular transport and metabolism (3, 4). As an extracellular protein, it can bind to γ-aminobutyric acid (GABA) type A receptors (GABA<sub>A</sub>R) (5) to allosterically modulate their response to GABA (6, 7). Outside of the central nervous system, ACBP is detectable in the plasma and acts as a hormone which, besides stimulating food intake and lipo-anabolic reactions, inhibits autophagy in multiple cell types including cardiomyocytes, skeletal myocytes, and hepatocytes (8, 9). Given that autophagy is a major antiaging mechanism (10, 11), we speculated that ACBP might accelerate aging through the inhibition of autophagy.

Accumulating evidence suggests that ACBP contributes to aging. Thus, the knockout of ACBP orthologs stimulates autophagic flux and augments lifespan in yeast and nematodes (12–16). In yeast, longevity extension by the *Acb1* knockout is lost upon concomitant deletion of the essential autophagy genes *Atg5* or *Atg7*, indicating that the beneficial effect of ACBP inhibition requires autophagy (16). In humans, ACBP plasma concentrations increase with age and body mass index (BMI), independently from one another (17, 18). In still-healthy individuals, supranormal circulating ACBP levels are associated with future cardiovascular events (such as heart surgery, myocardial infarction, and stroke), a correlation that is independent of BMI or chronological age, suggesting that ACBP can be considered a risk factor for biological aging (16). Accordingly, plasma ACBP levels correlate with dyslipidemia (high levels of free fatty acids and total/low-density-lipoprotein cholesterol, and low levels of high-density lipoprotein cholesterol) and other

## Significance

Knockout of the orthologs of ACBP increases lifespan in yeast and nematodes, indicating that this tissue hormone may participate to the aging process. Accordingly, compared to younger adults, long-lived humans (close-to-centenarians) exhibit high ACBP plasma concentrations that further increase with acute health deterioration requiring hospitalization. In mice, antibody-mediated neutralization of ACBP is sufficient to prevent cellular senescence and organ failure induced by chemotherapeutic cytotoxicants. These findings suggest that ACBP may contribute to aging in mammals.

This article is a PNAS Direct Submission. S.P.C. is a guest editor invited by the Editorial Board.

Copyright © 2025 the Author(s). Published by PNAS. This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

¹L.M., F.L., C.B., A.L., and G.K. contributed equally to this work.

²Present address: Department of Immunology and Immunotherapy, Icahn School of Medicine at Mount Sinai, New York, NY 10029.

³To whom correspondence may be addressed. Email: barcenaclea@uniovi.es, alejandro.lucia@universidadeEuropea.es, or kroemer@orange.fr.

This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/ pnas.2501584122/-/DCSupplemental.

Published July 7, 2025.
cardiometabolic risk factors such as hyperglycemia and hypertension (16). Several population studies investigating plasma biomarkers of human longevity have linked higher plasma levels of ACBP to reduced lifespan and/or health span in people under 90. In the American Cardiovascular Health Study (N = 3,067), where plasma ACBP was measured in individuals aged approximately 75 y, elevated levels were associated with a lower likelihood of survival to 90 y (OR = 0.84, 95% confidence interval [95% CI] = [0.78;0.91]) (19). Similarly, plasma proteomics data from the UK Biobank (N = 47,600; ages 40 to 70), showed that higher ACBP levels correlated with an increased risk of multiple age-related comorbidities, including type 2 diabetes (HR = 1.20, 95% CI = [1.16; 1.25]), ischemic heart disease (HR = 1.13, 95% CI = [1.09; 1.17]), and chronic obstructive pulmonary disease (HR = 1.15, 95% CI = [1.10; 1.20]). Elevated ACBP levels were also linked to a higher risk of all-cause mortality within 15 y after blood sampling (HR = 1.14, 95% CI = [1.11; 1.18]) (20). Finally, in the Atherosclerosis Risk in Communities (ARIC) study (N = 4,189; mean age 57.8 ± 5.0 y), higher plasma ACBP levels were associated with a greater risk of physical frailty two decades after blood sampling (21).

Of note, in mice, neutralization of ACBP protects the heart from ischemia-induced necrosis in an autophagy-dependent fashion, meaning that the beneficial effect of a monoclonal antibody (mAb) specific for ACBP is lost upon cardiomyocyte-specific knockout of *Atg7* (9). Moreover, ACBP neutralization prevents cardiomyopathy induced by the chemotherapeutic doxorubicin (DOXO), a cardiotoxic anthracycline that is well known to trigger premature cardiac aging in childhood cancer survivors (22–24). We also reported that apparently still healthy individuals who are diagnosed with malignant disease within 3 y after blood sampling exhibit higher ACBP plasma concentrations than controls matched for age, BMI, sex, and common cancer risk factor, while ACBP inhibition reduces carcinogenesis and tumor progression in mice (25). These findings support the idea that ACBP may be causally involved in the aging process and in major chronic diseases.

Based on these premises, we sought to confirm the potential proaging effects of ACBP in exceptionally aged humans. We found that plasma ACBP levels were higher in centenarians than in middle-aged volunteers and were further exacerbated with health deterioration in the former, correlating with reduced renal clearance. In turn, ACBP neutralization improved health span in a murine model of accelerated aging and prevented loss of renal function induced by DNA-damaging agents in preclinical models, an effect that was coupled with the reduction of cellular senescence. We observed comparable antisenescence effects of ACBP neutralization in models of accelerated senescence affecting other organs, including the liver and heart.

## Results

**ACBP Plasma Levels in Older Adults Differing in Their Health Status.** Plasma samples collected from (close-to-)centenarians (mean ± SD age 100.5 ± 1.4 y, range 98 to 103) living in a nursing home contained approximately three times higher ACBP levels than healthy adults aged 30 to 48 y. The ACBP concentrations increased to even higher levels in (close-to-)centenarians (98.5 ± 5.8 y, range 90 to 108) who were hospitalized due to the acute disease exacerbation (Fig. 1 *A* and *B*). The diverse range of diseases presented by this cohort resulted in an average duration of stay of 11 d, with 24 % of the patients dying during hospitalization (SI Appendix, Fig. S1A and Table S1). In this cohort population of exceptionally aged individuals, plasma ACBP positively correlated with the Charlson Comorbidity Index (CCI) (Fig. 1C),

a geriatric tool assessing the clinical status to predict mortality risk (26). Reflective of their stable (vs. degrading) health condition, the CCI was lower in the nursing home group than in the hospitalized patients (SI Appendix, Fig. S1B). Accordingly, high ACBP correlated with an unfavorable neutrophil-lymphocyte ratio (NLR), which is a universal poor prognosis factor (27) (SI Appendix, Fig. S1C). Moreover, ACBP negatively correlated with the estimated glomerular filtration rate (eGFR, Fig. 1D and SI Appendix, Fig. S1 D and E), in accordance with a recent study reporting an association between ACBP and renal dysfunction in a cohort of younger (<80 y) individuals (28).

Mass spectrometric metabolomics identified a positive correlation between ACBP and several metabolites (SI Appendix, Fig S2 A and B). Threonic acid, a metabolite of vitamin C, was the sole metabolite that correlated with ACBP in both the subcohort of (close-to-)centenarians from the nursing home and the hospitalized subcohort (SI Appendix, Figs. S2C and S3). Metabolites associated with the catabolism of polyamines (such as mono- and diacetylated spermine and spermidine) correlated with ACBP in the aggregate of the two subcohorts of (close-to-)centenarians, as well as within the hospitalized subcohort (SI Appendix, Fig. S2 A and B).

ACBP plasma levels discriminated (close-to-)centenarians in the two subcohorts (nursing home residents versus acutely hospitalized patients) better than all inflammatory markers detectable using a commercial proximity extension assay (Fig. 1E). A positive correlation with hepatocyte growth factor (HGF), oxidized low-density lipoprotein receptor 1 (OLR1) and vascular endothelial growth factor (VEGFA), which are three factors associated with cellular senescence and aging (29–31), was observed within both subcohorts, in line with a possible proaging function of ACBP (Fig. 1 *F* and *G*).

Intrigued by these observations, we investigated the role of ACBP in various mouse models of pathological aging.

**Health Span-Improving Effects of ACBP Neutralization in A Mouse Model of Progeria.** Mice lacking the zinc metallopeptidase *Zmpste24* (genotype *Zmpste24*⁻¹) develop an accelerated aging phenotype that resembles the human Hutchinson-Gilford progeroid syndrome (HGPS) (32, 33). Indeed, in affected humans, a range of diseases extending from mandibuloacral dysplasia type B to restrictive dermopathy are caused by mutations in the orthologue *ZMPSTE24*, which codes for a protease required for the processing of the nuclear envelope protein lamin A (34). The resulting accumulation of prelamin A then drives premature aging phenotypes. *Zmpste24*⁻¹ mice display a reduced lifespan and health span (32, 33). Several anatomical and functional alterations typically observed in the model were attenuated when *Zmpste24*⁻¹ mice were treated from the young adult stage (8 to 10 wk) to 30 wk of age with anti-ACBP mAb (Fig. 2 *A* and *B* and SI Appendix, Fig. S4). Anatomically, anti-ACBP-treated animals showed a reduction in kyphosis severity (Fig. 2 *C* and *F* and SI Appendix, Fig S5), with less variability observed in male animals. A correction of splenic atrophy was observed in both male and female mice (Fig. 2D and SI Appendix, Fig. S6). ACBP-neutralized individuals displayed trends for improvement in blood parameters related to health, including increased hematocrit and hemoglobin levels (Fig. 2E), and lower blood urea nitrogen (BUN), a marker of improved kidney function, in female animals (SI Appendix, Fig S7). ACBP neutralization also partially suppressed the premature deterioration in skin/fur quality, dental dysplasia, as well as spasmodic movements when the animals were suspended by the tail from 20 wk of age were also partially restored, with minor sex-specific differences, in the
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** ACBP is elevated in (close-to-)centenarians and increases further with disease.(A) A cohort of N = 37 participants over 90 y old was recruited at their nursing home during a "normal day", i.e., in a context of relative functional autonomy (N = 16), or during episodes of acute hospitalization at the Gregorio Marañón General University Hospital (N = 21). They were matched in silico to healthy young volunteers from the DESIR cohort. (B) Plasma ACBP levels measured in the (close-to-)centenarians are higher, as compared to young controls, and are highest in those who belong to the hospital group. (C) Plasma ACBP levels are positively correlated with the CCI. (D) Plasma ACBP levels are negatively correlated with glomerular filtration rate (GFR), an indicator of renal clearance. (E) ACBP, along with a pool of 12 proinflammatory cytokines (out of the 40 plasma cytokines measured by proximity extension assay), is higher in the patients from the hospital group. (F) Hierarchically clustered correlation matrix highlights a cluster of inflammatory cytokines positively correlating with ACBP within all patients.(G) Correlation analysis of the hospital and nursing home groups separately shows that three plasma cytokines consistently positively correlate with ACBP: vascular endothelial growth factor A (VEGFA), oxidized low-density lipoprotein receptor 1 (OLR1) and hepatocyte growth factor (HGF). Comparisons between groups were performed by one-way ANOVA for plasma levels of ACBP between the three groups and by the Wilcoxon signed-rank test followed by FDR correction for multiple comparisons for comparisons of log2-normalized values of plasma cytokines. Pearson's product-moment correlation coefficients and the corresponding asymptotic P-values were used for the heatmap and Venn diagram. ACBP: acyl-CoA-binding protein; CCL(2-19): C-C motif chemokines (2-19); CSF1: macrophage colony-stimulating factor; CSF2: granulocyte-macrophage colony-stimulating factor; CSF3: granulocyte colony-stimulating factor; CXCL(8-12): C-X-C motif chemokine ligand (8-12); EGF: epidermal growth factor; FLT3LG: Fms-related tyrosine kinase 3 ligand; IFNG: interferon gamma; IL(2-27): interleukin (2-17); LTA: lymphotoxin-alpha; MMP(1/12): matrix metalloproteinase-(1/12); OSM: oncostatin M: TGFA: transforming growth factor alpha; TNF: tumor necrosis factor; TNFSF10: TNF-related apoptosis-inducing ligand; TNFSF12: TNF ligand superfamily member 12.</figcaption></figure>

animals injected with ACBP-neutralizing antibody (Fig. 2 *F* and *G*). Finally, *Zmpste24*-deficient animals, which display low levels of motor activity (35) showed improved walking ability when treated with anti-ACBP mAb (Fig. 2*F* and *SI Appendix*, Fig. S4 and Movie S1).

Overall, these results support the idea that systemic ACBP neutralization can attenuate premature aging at the whole-body level.

**Antisenescence Effects of Anti-ACBP Antibody on the Kidney.**
Intrigued by the inverse correlation between ACBP plasma concentrations and eGFR in (close-to-)centenarians, we decided to study the impact of ACBP on a preclinical model of accelerated kidney aging. The chemotherapeutic agent

cis-diamminedichloroplatinum(II) (best known as cisplatin) was injected repeatedly at low doses into adult mice (age 12 wk) to induce tubular damage (36) (Fig. 3A). Mice were subjected to chronic cisplatin treatment (cumulative dose: 32 mg/kg body weight, injected intraperitoneally [i.p.] over 4 wk) to induce accelerated aging or were injected with vehicle alone (PBS). Mice were additionally administered a murine monoclonal ACBP (mAb)-neutralizing antibody (anti-ACBP; 5 mg/kg body weight, injected i.p. weekly, 24 h before cisplatin or vehicle) or an isotype IgG2a control (16). When compared to its isotype control, anti-ACBP mAb prevented the cisplatin-induced surge in BUN, reflecting renal dysfunction (37) (Fig. 3B) and a drop in estimated GFR (eGFR) (38, 39) that was prevented by the
A

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C57Bl/6J, Zmpste24⁻/⁻
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Female or male

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Spine curvature (mm)

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D

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Normalized spleen weight

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F

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G

**Fig. 2.** ACBP neutralization improves health span in the Zmpste24⁻/⁻ murine progeria model. (A) In-house bred C57Bl/6 N mice deficient for the Zmpste24 metalloprotease gene were randomized at 8 wk of age to be treated either with anti-ACBP (n = 15 females, n = 9 males) or mouse IgG2a isotype control (n = 13 females, n = 9 males) monoclonal antibodies (2.5 mg/kg i.p.), both administered i.p. twice weekly. Functional evaluation was performed after 21 wk of treatment. (B) Representative pictures of the general state of the animals at 26 wk of age. (C) Representative images of computed tomography scanner (CT-scan) from mice of the different groups, and associated measurements of the spine curvatures. (D) Representative images of spleen morphology and associated measurement of the spleen weights in the different groups, normalized by total body mass. (E) Blood measurements of hematocrit (Hct) and calculated hemoglobin concentration (cHgb). (F) Scores of severity for kyphosis, teeth damage, hair loss, loss of walking ability were assessed blindly. The results presented are pooled from two independent mouse cohorts. (G) Spasm prevalence within each group, as assessed by a 5-s tail suspension test. The effect of anti-ACBP on each of the functional criteria was assessed either separately for males and females or in the total cohort of mice by the Wilcoxon rank-sum test or t test depending on variable normality.

anti-ACBP mAb (Fig. 3C). The kidneys of mice receiving vehicle, cisplatin, isotype control mAb, and/or anti-ACBP mAb were stained with hematoxylin/eosin (HE, for histopathological examination), periodic acid-Schiff (PAS, which labels basal membranes of glomeruli and the brush borders of tubuli), and

trichrome Masson (which visualizes collagen fibers). Renal tissue damage and fibrosis induced by cisplatin were both partially suppressed by ACBP neutralization (Fig. 3 D–H).

Cisplatin-induced renal damage leads to cellular senescence characterized by the nuclear expression of cyclin-dependent
A

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C57BI/6J
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**Fig. 3.** Renal damage induced by chronic administration of cisplatin is dampened by ACBP-neutralizing monoclonal antibodies. (A) Chronic kidney disease was induced in adult male C57BI/6 J mice by 4 wk administrations of cisplatin (8 mg/kg i.p., compared to vehicle-injected controls, CTR). In half the animals of the cohort, a monoclonal antibody against ACBP was administered on the day prior to the first cisplatin injection (Anti-ACBP, 5 mg/kg i.p., compared to isotype-injected controls) and maintained weekly for 8 wk. (B and C) At the end of the experiment, blood urea nitrogen (BUN) was measured, and the estimated glomerular filtration rate (eGFR) was calculated from the blood parameters. (D and E) Representative images of hematoxylin-eosin (HE) and periodic acid-Schiff (PAS) staining of kidney sections show the structural changes induced by cisplatin. (F) Pathological scoring of the renal damage was performed on a scale from 0 to 4 based on tubular necrosis, brush border loss, tubular dilation, tubular cast formation, widening of the interstitium, degeneration, and inflammation by two independent examiners, and the average score was calculated for each mouse. (G and H) Masson's trichrome staining was used to quantify renal fibrosis by automatic segmentation of the blue color. Statistical comparisons were done by linear mixed modeling of each measurement (eGFR, pathological score, % fibrotic area), testing the interaction of two fixed effects (cisplatin and anti-ACBP) and taking mouse identity and technical parameters (examiner, histological section, field) as random effects. *P*-values were computed for the chosen pairwise contrasts with the estimated marginal means method.

kinase inhibitor 1A (CDKN1A, best known as p21) in tubular and peritubular cells, which can be detected by immunohistochemistry (36). Anti-ACBP mAb strongly reduced this histopathological sign of tubular senescence (Fig. 4 A and B), commensurate with a reduction in immunoblot-detectable p21 accumulation in renal extracts (Fig. 4 C and D). We also examined the kidneys of the animals treated with doxorubicin

(DOXO, cumulative dose: 20 mg/kg body) and/or anti-ACBP mAb (Fig. 4E), knowing that DOXO can induce accelerated senescence of podocytes (40, 41). As compared to vehicle-treated controls, the glomeruli of kidneys from DOXO-treated animals contained more p21-positive cells, and this surge in senescent cells was completely prevented by anti-ACBP mAb (Fig. 4F and G).
A

Tubuli p21 IHC

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Glomeruli p21 IHC

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C57BI/6J
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Heart p21 IHC

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Liver p21 IHC

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L

<figure><img src="image_38.png" /><figcaption>**Fig. 4.** The organ-protective functions of ACBP neutralization are accompanied by a reduction of the senescence marker p21 in various preclinical models. (A) Representative images of the immunohistochemical (IHC) detection of p21 (CDKN1A: cyclin-dependent kinase inhibitor 1A) positive nuclei in kidney sections from cisplatin-treated mice, and (B) the corresponding quantifications show that the cisplatin-induced increase in p21 nuclear staining is reduced by anti-ACBP administration. (C) Representative images of the immunoblotting of protein lysates from full kidneys. (D) Quantification of the p21 signal after normalization by the stably expressed glyceraldehyde-3-phosphate dehydrogenase (GAPDH) loading control, relative to control group expression levels. (E) Experimental scheme of a model of anthracycline-induced accelerated aging, in which mice were injected weekly with doxorubicin (5 mg/kg, i.p., compared to PBS vehicle-injected controls) with or without anti-ACBP monoclonal antibody (5 mg/kg, i.p., compared to isotype-injected controls) for four cycles. Similar to the results obtained with cisplatin, p21 signal augments in the kidney glomeruli (F and G) and heart cardiomyocytes (H and I) upon doxorubicin treatment and is completely abrogated by anti-ACBP. (J) Experimental scheme of chronic liver damage induced by prolonged administration of western diet (WD) and carbon tetrachloride (CCl₄), compared to regular chow diet (RCD) and vehicle (olive oil) injections. (K and L) Nuclear staining of p21 in hepatocytes by IHC is increased in this model of liver damage, and reversed by anti-ACBP treatment. The interaction between the effect of organ-damaging treatments (cisplatin, doxorubicin, or WD + CCl₄) and anti-ACBP treatment was evaluated by two-way ANOVA, followed by estimation of the marginal means for pairwise comparisons.</figcaption></figure>
Thus, it appears that ACBP neutralization exerts nephroprotective effects that are accompanied by the suppression of senescence in various renal cell types.

**Antisenescence Effects of Anti-ACBP Antibody on the Heart and Liver.** DOXO is well known for its cardiotoxicity, which constitutes the dose-limiting side effect of anthracycline-based chemotherapies (42). As a result, we investigated nuclear p21 expression in cardiomyocytes, a phenomenon that was upregulated by DOXO and fully prevented by anti-ACBP mAb (Fig. 4 H and I). ACBP neutralization also reduced cardiomyocyte p21 expression in vehicle-only treated animals, suggesting that ACBP inhibition may quell natural heart senescence as well.

We next resorted to a model of hepatic senescence induced by a combination of western diet (WD) and liver damage caused by repeated $CCl_4$ injections in the context of anti-ACBP mAb injections (or IgG isotype controls) (Fig. 4J). WD combined with $CCl_4$ induced a significant surge in the frequency of p21-positive hepatic cells that was strongly inhibited by ACBP neutralization (Fig. 4 K and L).

We conclude that the antisenescence effects of ACBP neutralization extend to various cell types including cardiomyocytes and hepatocytes.

**Cardioprotective Effects of ACBP Neutralization in Doxorubicin-Induced Cardiac Aging.** To further explore the apparent cardioprotective effects of ACBP neutralization at the molecular level we performed single nucleus RNA sequencing (snRNA-Seq) on heart ventricles from each of the groups of mice (treated with vehicle or DOXO, isotype control antibody, and/or anti-ACBP mAb) allowing for the identification of a diverse array of cell types composing the heart (Fig. 5A and SI Appendix, Fig. S8 A and B). Among the relatively abundant cell types (> 500 cells across all 8 samples), the transcriptome of cardiomyocytes was strongly affected by the treatment (Fig. 5B). Volcano plots allowed to compare DOXO-treated cardiomyocytes with their vehicle-treated control equivalents, as well as DOXO-treated cardiomyocytes from mice receiving anti-ACBP mAb with controls receiving the isotype alone (Fig. 5C). This led to the identification, in this particular cluster of cardiomyocytes, of differentially expressed genes (DEGs) that are up- or downregulated by DOXO only with isotype control antibody (thresholds: $P < 0.05$, $≥ 1.5$-fold change), but not in the presence of anti-ACBP ("reversible DEGs"), as well as genes that are modulated by the combination of DOX plus ACBP neutralization ("specific DEGs") (Fig. 5D).

Gene ontology (GO) and Kyoto encyclopedia of genes and genome (KEGG) term analyses focusing on these categories revealed that reversible DEGs were linked to alterations in metabolic pathways associated with energy metabolism (such as fatty acid oxidation and oxidative phosphorylation) while specific DEGs were mostly linked to heart muscle contractility and response to cardiomyopathies (Fig. 5 E and F). More specifically, DOXO induced the significant downregulation of the mRNAs coding for aquaporin 7 (*Aqp7*) and pyruvate dehydrogenase kinase 4 (*Pdk4*), and ACBP neutralization fully reversed these effects (SI Appendix, Table S2). In DOXO-treated mice, anti-ACBP mAb upregulated additional cardioprotective genes (that are not downregulated by DOXO alone), as exemplified by acyl-CoA thioesterases 1 (*Acot1*) and 2 (*Acot2*), Bcl-2-like protein 1 (*Bcl2l1*, best known as Bcl-X$_L$), FK506-binding protein 5 (*Fkbp5*), and flavin containing monooxygenase 2 (*Fmo2*) (SI Appendix, Fig. S9A and Table S2), as also visible as a dot plot (SI Appendix, Fig. S9B).

In sum, anti-ACBP mAb induced the upregulation of several cytoprotective genes that might explain the capacity of this treatment to reverse the cardiotoxicity of DOXO.

## Discussion

The results obtained on ACBP plasma concentrations in (close-to-)centenarians must be inscribed into accumulating evidence indicating that ACBP accelerates natural aging in model organisms (such as yeast and nematodes) (12–16) as well as pathological aging in various mouse models of cardiac, hepatic, and respiratory diseases (43, 44). In this exceptionally old population, we found that ACBP plasma levels positively correlated with CCI, a geriatric tool used for assessing comorbidities and predicting future mortality. Of note, in the nursing home group, the CCI values remained relatively low (mean 1.1 ± 0.5), as compared to hospitalized patients (mean 3.2 ± 0.8), reflecting that these “stable” centenarians typically postpone the onset of major noncommunicable diseases compared to the general population. Importantly, plasma levels of ACBP were particularly elevated in (close-to-)centenarians who were acutely hospitalized due to disease exacerbation compared to relatively healthy nursing home residents of the same age, suggesting that both age and disease (or disease exacerbation) independently contribute to an increase in ACBP concentrations. Experiments in mice with a genetic defect (*Zmpste24*$^{-/}$) causing premature aging revealed that ACBP neutralization can improve various parameters related to health span. In this model of progeria, long-term administration of anti-ACBP mAb was able to partially reverse the degradation of various functional and molecular health parameters including kyphosis, walking ability, splenic atrophy, and hematocrit. Despite some sex differences, often observed in progeria models, these results suggest that ACBP may contribute to the age-related deterioration of general health.

In humans, ACBP elevations inversely correlated with eGFR, perhaps reflecting the capacity of the kidney to clear this protein from the circulation, as has been suggested (28). However, based on our observations in mouse models, it is also possible that ACBP constitutes a pathogenic factor that compromises renal function in aging. Indeed, neutralization of ACBP attenuated the histopathological and functional signs of kidney damage induced by cisplatin in mice, including a reduction in eGFR. This pleads in favor of a potential feedforward loop in which ACBP contributes to chronic kidney failure, which in turn favors an accumulation in circulating ACBP.

In (close-to-)centenarians, ACBP concentrations correlated with a variety of proinflammatory cytokines. These included chemokines (CCL4, best known as macrophage inflammatory protein 1 beta, and CCL7, also named monocyte-chemotactic protein 3), the macrophage-tropic factor CSF1, the profibrotic transforming growth factor alpha (TGFA), as well as the proscenest factors HGF, OLR1, and VEGFA. Driven by this latter observation, we measured cellular senescence in several preclinical models and found that ACBP neutralization can block the surge of p21-positive cells in various organs (heart, kidney, and liver) responding to different toxins ($CCl_4$, cisplatin, or DOXO). Importantly, spontaneous senescence of cardiomyocytes was also suppressed by repeated injections of anti-ACBP antibody, pleading in favor of a rather broad proscenest effect of ACBP.

We found that ACBP neutralization can upregulate cardioprotective genes in the context of DOXO-induced cardiac damage. Thus, an ACBP-specific mAb reversed the DOXO-induced downregulation of two major cardioprotective genes, *Pdk4* and *Aqp7*.
<figure><img src="image_39.png" /></figure>

Heart snRNAseq
Clusters

<figure><img src="image_40.png" /></figure>

<figure><img src="image_41.png" /></figure>

<figure><img src="image_42.png" /></figure>

<figure><img src="image_43.png" /></figure>

<figure><img src="image_44.png" /></figure>

<figure><img src="image_45.png" /></figure>

<figure><img src="image_46.png" /></figure>

Reversible pathways

<figure><img src="image_47.png" /></figure>

<figure><img src="image_48.png" /></figure>

**Fig. 5.** Single-nuclei RNA sequencing analysis of the reversion of the doxorubicin-induced cardiac damage upon ACBP neutralization. Nuclei were extracted from ventricles from mice treated with chronic administration of doxorubicin with or without anti-ACBP (scheme in Fig. 4E) and submitted to single-nuclei RNA-sequencing (snRNA-seq, N = 2 per experimental condition). (A) 2D projection of the first two dimensions from the Uniform Manifold Approximation Projection (UMAP) of the 14 clusters, corresponding to cell populations, were identified based on their nuclear transcription profiles. (B) In the top 8 abundant clusters, the amplitude of transcriptional changes induced by DOXO was quantified as the number of differentially expressed genes (DEGs, Wilcoxon test *P* < 0.05 and fold-change > 1.5) normalized by the number of cells in the clusters. (C) Volcano plots show the transcriptional changes induced in the cardiomyocytes cluster by DOXO alone or by the combination of anti-ACBP with DOXO, both compared to control conditions. (D) Venn diagram comparison of the corresponding DEGs was used to define three categories of DEGs: genes that are differentially expressed (DE) by DOXO but not anymore when combining it to anti-ACBP ("reversible DEGs"), genes that are specifically DE in the anti-ACBP + DOXO combination ("specific DEGs"), and genes that are DE by DOXO no matter the monoclonal antibody treatment ("common DEGs"). (E and F) Pathway enrichment analysis of the reversible DEGs and specific DEGs show enrichment of metabolic and cardioprotective pathways from the Gene Ontology (Bioprocesses terms, abbreviated G) and Kyoto Encyclopedia of Genes and Genomes (KEGG, abbreviated K) databases. Numbers to the right of each pathway name correspond to the number of upregulated (green) and downregulated (red) genes from the pathway found in the DEGs list.
Pdk4 is known to be downregulated in hypertrophic rat hearts (45), and in aged mouse cardiomyocytes compared to those of young mice, in particular under ischemia/reperfusion (46, 47), as well as in Notch3-deficient mice with hypertrophic cardiomyopathy (48). Pdk4 is upregulated by several cardioprotective interventions including hypertrophic preconditioning (49), administration of palmitoleic acid (50) or retinoic acid receptor agonists (51) to mice, and endurance training in rats (52). Transgenic cardiac-specific overexpression Pdk4 protects against HFD-induced myocyte lipid accumulation in mice (53). Thus, a large body of literature suggests that PDK4 has cardioprotective effects that are likely mediated by the inhibition of pyruvate dehydrogenase, switching cardiac bioenergetics from glycolysis to beta-oxidation. Similarly, Aqp7 has been documented to be cardioprotective. Thus, AQP7 mRNA is downregulated in hearts from patients with ischemic cardiomyopathy-induced heart failure (54). In mice, knockout of Aqp7 exacerbates infarction induced by ischemia (55), providing experimental proof for a positive role for AQP7 in the maintenance of heart function.

Beyond these two factors, there are additional mRNA species induced by ACBP neutralization that are not downregulated by DOXO but that nevertheless might play a role in cardioprotection. These include *Acot1* and 2, two acyl-CoA thioesterases that contribute to fatty acid oxidation, knowing that *Acot1* overexpression attenuates cardiac dysfunction in diabetic mice (56); *Bcl2l1* (also known as Bcl-X~L~), which inhibits apoptosis (57); DNA-damage-inducible transcript 4 (*Ddit4*, also known as Redd1), which induces autophagy via mammalian target of rapamycin complex 1 (mTORC1) inhibition (58); FK506-binding protein 5 (*Fkbp5*), an autophagy inducer (59, 60) that is downregulated in human heart failure (61); flavin containing monooxygenase 2 (*Fmo2*), which prevents cardiac fibrosis (62); hydroxymethylglutaryl CoA synthase-2 (*Hmgcs2*), which is the rate-limiting enzyme in ketogenesis (63); and potassium calcium-activated channel subfamily N member 2 (*Kcnn2*), which orthologue is reduced in patients with atrial fibrillation and concomitant heart failure (64). Based on these results, we speculate that ACBP neutralization induces multipronged alterations in the cardiomyocyte-specific transcriptome that contribute to cardioprotection.

**Limitations of This Study.** At this point, the mechanisms that account for the senescence-suppressive effect of anti-ACBP mAb remain to be elucidated. It is possible that they involve the induction of autophagy, one of the cellular effects of ACBP neutralization (2, 8, 9). Indeed, there are several examples of autophagy inducers that have senolytic effects and hence eliminate senescent cells from tissues. This has been well documented for inhibitors of BCL2-like proteins including BCL2 itself and BCL2L1, cardiac glycosides, and 4,4'-dimethoxychalcone, which all induce autophagy and also have potent senolytic effects (65–70). Similarly, the senolytic combination of dasatinib (a tyrosine kinase inhibitor used to treat certain types of leukemia) and the natural flavonoid quercetin potently induces autophagy to protect against diabetic kidney disease (71). Inhibition of autophagy is sufficient to stimulate senescence, as this has been documented for several cell types including hepatocytes (72, 73), keratinocytes (74), microglia (75), satellite cells from the skeletal muscle (76), trophoblast cells (77), and kidney tubular epithelial cells (78). Hence, it appears plausible that autophagy induction contributes to the senescence-suppressive effects of ACBP neutralization (16, 79). Beyond this speculation, it is also possible that ACBP subverts the immune system-mediated clearance of senescent cells, commensurate with

its capacity to block (potentially autophagy-dependent) cancer immunosurveillance (25, 80). Hence, future investigation must distinguish between these possibilities and clarify the hierarchical relationship between ACBP neutralization, autophagy modulation, and senolysis. Moreover, the contribution of ACBP to human aging must be validated in future interventional studies targeting ACBP or its receptor.

We conclude that in various preclinical models, the neutralization of ACBP prevents senescence, which is one of the principal mechanisms of aging. These findings support the idea that ACBP could be targeted in future research for the treatment of aging and age-related conditions.

## Materials and Methods

### Human Cohort.

**Sample collection and handling.** This study received ethical approval (Comité de ética de la investigación con medicamentos del Hospital General Universitario Gregorio Marañón, Madrid, Spain; reference #BNC09/2023). Upon written informed consent from the participants, blood samples were collected in i) a Hospital in the city of Madrid (Spain) (Hospital General Universitario Gregorio Marañón), for the subcohort of [close-to-)centenarians who were acutely hospitalized and ii) four different nursing homes of the same city, for the subcohort of (close-to-)centenarians showing a relatively well-preserved health and functional status and, as opposed to the patient's subcohort, no acute disease exacerbation. Blood parameters were analyzed following the local routine (SI Appendix, Fig. S1C) and estimated glomerular filtration rate (eGFR) was calculated with the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) 2021 equation (81). For molecular analysis, a fraction of heparinized blood was kept for up to 90 min on ice prior to plasma separation. Plasma was then aliquoted and stored at -80 °C, each left untouched until thawed for ACBP ELISA quantification, metabolomic, and proximity extension assay proteomic profiling. To compare the plasma levels of (close-to-)centenarians with relevant controls, we matched the participants included in this cohort with healthy 30-to-50-y-old controls from a previously published study (16).

**Animal Models.** Mice were housed in 12 h light/dark cycles under a temperature-controlled environment and fed ad libitum. Animal experiments were conducted following the recommendations from the Federation of European Laboratory Animal Science Associations (FELASA) and approved by the local ethics committee (protocols #35132-202202021617318 v5, #27952-2020111215128045 v4, #34495-202201012222248 v3, #52922, and #PROAE38_2023).

**Progeroid mouse model.** 8-to-10-wk-old Zmpste24⁻¹⁻¹ mice (33) were either injected with a mouse monoclonal IgG2a antibody directed against ACBP (Anti-ACBP, Fred Hutch Antibody Technology, clone 7a) or the matching isotype control (BioXCell, clone 2A3, #BE0089) twice per week (2.5 mg/kg bodyweight, intraperitoneal, i.p.) for 22 wk.

**Cisplatin-induced kidney damage.** Chronic administration of cisplatin to a cumulative dose of 32 mg/kg, spread over 4 wk injections, was used to evaluate kidney damage after 8 wk (36, 82). Anti-ACBP or the matching isotype control (5 mg/kg i.p.) was injected once per week, on the day immediately prior to each cisplatin injection.

**Doxorubicin-induced renal and cardiac damage.** 8-wk-old females were randomized to receive either anti-ACBP or the matching isotype control at 5 mg/kg i.p., and, on the next day, freshly dissolved doxorubicin (DOXO, 5 mg/kg i.p., Sigma #D1515) or saline vehicle, both administered once per week for 4 wk. Animals were killed 1 wk after the last injection of DOXO, a time point at which functional cardiac damage was previously shown to be attenuated by anti-ACBP (16). Left ventricles were either fixed for histological analysis or snap-frozen for less than a week before being processed according to the manufacturer's instruction for single-nuclei isolation and sequencing.

**Model of liver damage.** 8-wk-old males received a regular chow diet (RCD, Safe, Augy, France; #A04) and water or a western-style chow diet (WD, a high-fat, high-fructose, and high-cholesterol diet, Inotiv, Indianapolis, IN; #MD.120528) along with high-sugar water (23.1 g/L fructose plus 18.9 g/L glucose), both ad libitum. $Cl_4$ (Sigma Aldrich, Saint-Louis, MO; #289116) was diluted in olive oil (Sigma;
#01514) and injected i.p. at a dose of 2 mL/kg weekly for 4 wk (79). Control animals were solely injected i.p. with olive oil vehicle. After 16 wk of diet, anti-ACBP or the matching isotype control was regularly administrated for 4 wk at 5 mg/kg i.p., twice a week.

**Functional and molecular analyses.** Details about sample processing and methods for the functional and molecular evaluation are available as a *SI Appendix*.

**Data, Materials, and Software Availability.** sn-RNAseq data have been deposited in GEO (GSE286549) (83). Patient data will not be shared because the exceptional longevity of the patients included makes them easy to identify.

**ACKNOWLEDGMENTS.** Immunohistochemical analysis of p21 was performed by the Histopathology Facility from Institute for Research in Biomedicine - IRB Barcelona. We thank the platform for "Pathologie expérimentale et translationnelle" at Gustave Roussy, as well as the Cordeliers Histology, Imaging, Cytometry and Spatial-omics platform, whose members conducted all other histopathological stainings. We thank the team of Isabelle Cremer in Cordeliers Research Center for giving us access their automated nuclei counter. We also thank Agustín Brea Pastor from the University of Oviedo for his help with the computer tomographic imaging process. G.K. receives grants by the Ligue contre le Cancer (équipes labellisées); Institut National du Cancer (INCa); European Union Horizon 2020 research and innovation programmes Oncobiome (grant agreement number: 825410), Prevaling (Grant agreement number 101095604), Agence Nationale de la Recherche under the France 2030 programme (reference number 21-ESRE-0028, ESR/Equipex + Onco-PhenOScreen); INCa; Agence Nationale de la Recherche (ANR), i.e., French Ministry of Health PIA5, ANR, program RHUS "ANR-21-RHUS-0017" IMMUNOLIFE and ANR-23-RHUS-0010 LUCA-pi; Seerave Foundation, an European Research Council Advanced Investigator Award (ERC-2021-ADG, Grant No. 101052444; project acronym: ICD-Cancer) and Site de recherche intégrée sur le cancer (SIRIC) Cancer Research and Personalized Medicine (CARPEM, SIRIC CARPEM INCa-DGOS-Inserm-ITMO Cancer_18006 supported by INCa, Ministère des Solidarités et de la Santé et INSERM). This study contributes to the IdEx Université de Paris Cité ANR-18-IDEX-0001. L.M-N. is supported by the FPI program (PRE2021-100065) funded by MCIN/AEI/10.13039/501100011033. C.B. is supported by the Ramon y Cajal program (RYC2021-031291-I) funded by MICIN/AEI/10.13039/50100011033 and cofunded by European Union NextGenerationEU/PRTR. C.R-L is supported by the Biomedical Research Networking Centre on Frailty and Healthy Aging (CIBERFES, CB16/10/00314). Research by C.F.L. is funded by Instituto de Salud Carlos III (ISCIII) and cofunded by the European Union through projects PI20/00645, PI23/00396, and FORT23/00023, and by the MCIN/AEI/10.13039/501100011033 and «Next Generation EU»/PRTR (project CNS2023-144144). We thank Rosalba Gestión (nursing homes of Sevilla La Nueva, Madrid, Spain). M.A. acknowledges support from the Medical University of Graz (Start Fund, Flagship Project VASC-HEALTH and the excellence cluster MetAGE), BioTechMed-Graz (Young Researcher Group), the Austrian Science Fund (FWF; DOI: 10.55776/P34926

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DOI: 10.55776/COE14), and the European Commission (H2020-MSCA-IF). M.A. and G.K. received funding from FWF (DOI: 10.55776/16931) and ANR (Ener-LIGHT consortium) under the umbrella of the Partnership Fostering a European Research Area for Health (ERA4Health) (GA N° 101095426 of the EU Horizon Europe Research and Innovation Programme. O.M. is supported by the "Beatriz Galindo Junior" Program from the Spanish Ministry of Universities (BG22/00104). Research by A.L. and C.F.L. is funded by Wereld Kanker Onderzoek Fonds as part of the World Cancer Research Fund International grant program (IIG_FULL_2021_007).

Author affiliations: "Team Metabolism, Cancer & Immunity, Centre de Recherche des Cordeliers, Equipe Labellisée par la Ligue contre le Cancer, Université Paris Cité, Sorbonne Université, Inserm U1138, Institut Universitaire de France, Paris 76006, France; ^Metabolomics and Cell Biology Platforms, Gustave Roussy Institute, Villejuif 94808, France; "Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Oncología, Universidad de Oviedo, Oviedo 33007, Spain; ^Physical Exercise and Pediatric Cancer Research Group, Research Institute of the Hospital, Madrid 28041, Spain; ^Centro de Investigación Biomédica en Red de Fragilidad y Envejecimiento Saludable, Instituto de Salud Carlos III, Madrid 28029, Spain; ^Department of Geriatrics, Hospital General Universitario Gregorio Marañón, Health Research Institute Gregorio Marañón, Madrid 28007, Spain; ^School of Medicine, Department of Medicine, Universidad Complutense, Madrid 28040, Spain; ^Unidad de Excelencia, Instituto de Biomedicina y Genética Molecular, Universidad de Valladolid-Centro Superior de Investigaciones Científicas, Valladolid 47003, Spain; ^BioTechMed-Graz, Graz 8010, Austria; ^Division of Cardiology, Medical University of Graz, Graz 8010, Austria; ^Team Personalized Medicine, Pharmacogenomics and Therapeutic Optimization, Centre de Recherche des Cordeliers, Equipe Labellisée par la Ligue contre le Cancer, Université Paris Cité, Sorbonne Université, Inserm U1138, Institut Universitaire de France, Paris 75006, France; ^Department of Cell and Molecular Biology, Karolinska Institute, Solna, Stockholm 17177, Sweden; "Institut du Cancer Paris, Cancer Research for Personalized Medicine, Assistance Publique-Hôpitaux de Paris Centre, Hôpital Européen Georges Pompidou, Paris 75015, France; "Facultad de Ciencias de la Vida y la Naturaleza, Universidad Nebrija, Madrid 28248, Spain; and ^Department of Sport Sciences, Faculty of Medicine, Health and Sports, Universidad Europea de Madrid, Madrid 28670, Spain

Author contributions: L. Montégut, F.L., L.M.-N., C.F.-L., C.R.-L., J.A.S.-R., O.M., S.M.-R., C.L.-O., I.M., C.B., A.L., and G.K. designed research; L. Montégut, F.L., L.M.-N., C.F.-L., C.R.-L., J.A.S.-R., S. Lachkar, O.M., M.A., S.D., F.A., D.L.C., S.M.-R., H.C., Y.D., S. Li, Y.R., and C.B. performed research; A.S., F.P., and P.L-P. contributed new reagents/analytic tools; L. Montégut, F.L., L.M.-N., S.D., F.A., and V.C. analyzed data; and L. Montégut and G.K. wrote the paper.

Competing interest statement: I.M. consults for Osasuna Therapeutics. G.K. has been holding research contracts with Daiichi Sankyo, Eeleor, Kaleido, Lytix Pharma, PharmaMar, Osasuna Therapeutics, Samsara Therapeutics, Sanofi, Sutro, Tollys and Vascage. G.K. is on the Board of Directors of the Bristol Myers Squibb Foundation France. G.K. is a scientific co-founder of everImmune, Osasuna Therapeutics, Samsara Therapeutics and Therafast Bio; is in the scientific advisory boards of Evolution, Institut Servier, Longevity Vision Funds and Rejuveron Life Sciences; and is the inventor of patents covering therapeutic targeting of aging, cancer, cystic fibrosis and metabolic disorders. G.K.'s brother, Romano Kroemer, was an employee of Sanofi and now consults for Boehringer-Ingelheim. G.K.'s wife, Laurence Zitvogel, has held research contracts with GSK, Incyte, Lytix, Kaleido, Innovate Pharma, Daiichi Sankyo, Pilege, Merus, Transgene, 9 m, Tusk and Roche, was on the Board of Directors of Transgene, is a co-founder of everImmune and holds patents covering the treatment of cancer and the therapeutic manipulation of the microbiota. The funders had no role in the design of the study, in the writing of the manuscript or in the decision to publish the results, L.M. O.M., M.A., I.M. and G.K. are involved in patents dealing with the therapeutic utility of ACBP neutralization. G.K. has been holding research contracts with Daiichi Sankyo, Eeleor, Kaleido, Lytix Pharma, PharmaMar, Osasuna Therapeutics, Samsara Therapeutics, Sanofi, Sutro, Tollys and Vascage.

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