# Neutralization of the autophagy-repressive tissue hormone DBI/ACBP (diazepam binding inhibitor, acyl-CoA binding protein) enhances anticancer immunosurveillance

Léa Montégut, Isabelle Martins, Guido Kroemer

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Léa Montégut, Isabelle Martins, Guido Kroemer. Neutralization of the autophagy-repressive tissue hormone DBI/ACBP (diazepam binding inhibitor, acyl-CoA binding protein) enhances anticancer immunosurveillance. Autophagy, 2024, 20, pp.2836 - 2838. 10.1080/15548627.2024.2411854. hal-04889680

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Submitted on 30 Jan 2025

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# Neutralization of the autophagy-repressive tissue hormone DBI/ACBP (diazepam binding inhibitor, acyl-CoA binding protein) enhances anticancer immunosurveillance

Léa Montégut a,b, Isabelle Martins a,b, and Guido Kroemer a,b,c

aCentre de Recherche des Cordeliers, Equipe labellisée par la Ligue contre le cancer, Inserm U1138, Université Paris Cité, Sorbonne Université, Paris, France; bMetabolomics and Cell Biology Platforms, Gustave Roussy Institut, Villejuif, France; cDepartment of Biology, Institut du Cancer Paris CARPEM, Hôpital Européen Georges Pompidou, AP-HP, Paris, France

## ABSTRACT

The plasma concentration of the macroautophagy/autophagy inhibitor DBI/ACBP (diazepam binding inhibitor, acyl-CoA binding protein) increases with aging and body mass index (BMI). Both advanced age and obesity are among the most important risk factors for the development of cancer. We observed that patients with cancer predisposition syndromes due to mutations in *BRCA1*, *BRCA2* and *TP53* exhibit abnormally high plasma DBI/ACBP levels. Additionally, patients without known cancer predisposition syndromes also manifest higher DBI/ACBP levels before imminent cancer diagnosis (within 0–3 years) as compared to age and BMI-matched controls who remain cancer-free. Thus, supranormal plasma DBI/ACBP constitutes a risk factor for later cancer development. Mouse experimentation revealed that genetic or antibody-mediated DBI/ACBP inhibition can delay the development or progression of cancers. In the context of chemoimmunotherapy, DBI/ACBP neutralization enhances tumor infiltration by non-exhausted effector T cells but reduces infiltration by regulatory T cells. This resulted in better cancer control in models of breast cancer, non-small cell lung cancer and sarcoma. We conclude that DBI/ACBP constitutes an actionable autophagy checkpoint for improving cancer immunosurveillance.

**Abbreviation:** BMI, body mass index; CTL, cytotoxic T lymphocyte; DBI, diazepam binding inhibitor, acyl-CoA binding protein; mAb, monoclonal antibody; NSCLC, non-small cell lung cancer; PDCD1/PD-1, programmed cell death 1; scRNA-seq, single-cell RNA sequencing; T~reg~, regulatory T cell.

## ARTICLE HISTORY

Received 8 September 2024
Revised 23 September 2024
Accepted
27 September 2024

## KEYWORDS

Autophagy; cancer;
immunotherapy;
metabolism; obesity

DBI/ACBP (diazepam binding inhibitor, acyl-CoA binding protein) functions as an extracellular autophagy checkpoint. Intriguingly, this protein appears to mediate pro-aging effects, commensurate with the established anti-aging effects of autophagy. Thus, knockout of the genes encoding orthologs of DBI/ACBP in yeast and nematodes causes lifespan extension. In human populations, plasma concentrations of DBI/ACBP increase with age and body mass index (BMI), and both these correlations are statistically independent from each other. Additionally, in a population of healthy volunteers enrolled in a longitudinal follow-up of disease development, elevated plasma levels of DBI/ACBP are observed prior to cardiovascular events. In a similar approach, we examined whether measurements of plasma DBI/ACBP would make it possible to predict the development of cancer, another age-related disease, in still (apparently) healthy individuals [1]. We found that neoplastic disease is preceded by a surge in plasma DBI/ACBP that is significant compared to individuals that are matched for major epidemiological characteristic (such as sex, age, BMI and known risk factors including tobacco abuse) but do not develop cancer (Figure 1A). This observation suggests that DBI/ACBP is not only a biomarker of chronological aging – because it increases with age measured in years –

but also a biomarker of biological aging – because its elevation precedes the development of age-related diseases.

Knowing that DBI/ACBP neutralization can prevent anthracycline-induced cardiac aging, we decided to explore the potential pathophysiological role of DBI/ACBP in carcinogenesis, a process that is notoriously age-related. For this we first compared mammalian carcinogenesis in mice that lacked DBI/ACBP due to the inducible knockout of the *Dbi* gene and in control mice. Mammalian carcinogenesis induced by a combination of synthetic progesterone and DNA damage is affected by the *dbi* knockout in a peculiar fashion. While the appearance of tumors is not influenced by DBI/ACBP, the *dbi* knockout did slow down the growth of such tumors. Because the neoformation of mammary carcinomas is under immunosurveillance by NK (but not T) cells, while tumor progression of established cancers is under immunosurveillance by T (but not NK) cells, we concluded that DBI/ACBP might affect T lymphocyte-dependent immunosurveillance. Accordingly, the growth of a transplantable mammary carcinoma cell line could be retarded by DBI/ACBP inhibition using a monoclonal antibody (mAb), but only if this mAb is combined with another one targeting PDCD1/PD-1 (programmed cell death 1), which is a T cell-relevant immune

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<figure><img src="image_9.png" /><figcaption>**Figure 1.** Evidence in favor of the procarcinogenic and cancer immunosurveillance-suppressive action of DBI/ACBP. (A) Imminent cancer diagnosis in patients with high DBI/ACBP levels. (B) Delayed cancer development in mice in which DBI/ACBP is inhibited. (C) Improved outcome of chemoimmunotherapy in the context of DBI/ACBP neutralization. (D) Signs of local immune activation in tumors from mice treated with chemoimmunotherapy combined with anti-dbi/acbp antibody. Icons by servier (https://smart.servier.com/), licensed under CC-BY 3.0 unported, were used and modified to generate the figure.</figcaption></figure>

checkpoint. We also found that urethane-induced lung carcinogenesis (which is under T cell-dependent control) is retarded when neutralizing autoantibodies against DBI/ACBP are induced. The orthotopic growth of a non-small cell lung cancer (NSCLC) is also reduced when DBI/ACBP is inhibited, and this effect is lost in athymic mice lacking thymus-derived T cells. Altogether, these results suggest that DBI/ACBP neutralization stimulates T cell-dependent cancer immunosurveillance (Figure 1B).

Driven by this conclusion, we wondered whether anti-DBI/ACBP mAb might be useful in the therapeutic setting, i.e., in the treatment of established cancers. For this, we combined the anti-DBI/ACBP mAb with chemoimmunotherapy (which is the current standard of care for many cancers, i.e., chemotherapy together with immunotherapy targeting PDCD1) against two types of cancers, namely NSCLC and cutaneous sarcoma. Orthotopic NSCLCs were generated by intravenous injection of luciferase-expressing TC-1 cells in immunocompetent C57BL/6 mice and then monitored by bioluminescence imaging of the thorax. Orthotopic sarcomas were induced by

subcutaneous injection of MCA205 cells and then monitored by palpation and caliper-based measurements in the same mouse strain. Mice bearing either of the two tumor types similarly responded to chemoimmunotherapy, and this response was further improved by combination with the anti-DBI/ACBP mAb (Figure 1C). Hence, it appears that DBI/ACBP neutralization enhances the therapeutic efficacy of chemoimmunotherapy.

In a final twist, we decided to explore the mechanisms through which anti-DBI/ACBP mAb improves therapeutic outcome. For this we compared the phenotype of MCA205 fibrosarcoma-infiltrating T lymphocytes in four different groups of mice, namely, (i) animals receiving control treatments only (i.e., vehicle and isotype control antibodies), (ii) those receiving chemoimmunotherapy only (i.e., oxaliplatin plus anti-PDCD1 antibody), (iii) mice receiving anti-DBI/ACBP only, and (iv) tumor bearers treated with the combination of chemoimmunotherapy and anti-DBI/ACBP. We then characterized the T cell infiltrate of the tumors using two complementary methods, namely, (i) multi-color immunofluorescence phenotyping by high-dimensional
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cytometry, and (ii) single-cell transcriptomics by means of RNA sequencing (scRNA-seq). Both methods convergently led to similar conclusions. The combination treatment (chemoimmunotherapy + DBI/ACBP blockade) is the most efficient in increasing infiltration by cytotoxic T lymphocytes (CTL) and T helper cells, but decreasing regulatory T cells ($T_{reg}$), hence improving the local CTL: Treg ratio (Figure 1D). Moreover, DBI/ACBP blockade increases the chemoimmunotherapy-driven surge of activated T helper cells but reduces the surge in exhausted CD8$^+$ T cells, as defined by combinations of T cell exhaustion markers. At the transcriptomic level, DBI/ACBP neutralization reduces the abundance of RNA coding for CTL effector molecules (e.g., GZMB [granzyme B]), a decrease in inhibitory immune checkpoint molecules (PDCD1 and CTLA4 [cytotoxic T-lymphocyte associated protein 4]) and an increase in stimulatory immune checkpoint markers (ICOS, CD28) on a per-cell basis across several T cell subpopulations. Most interestingly, all T lymphocyte subpopulations detectable by scRNA-seq exhibit an anti-DBI /ACBP mAb-induced upregulation of *Lmna*, which encodes lamin A/C, an obligatory T cell activation marker that is also endowed with potent antiaging effects.

In conclusion, four key arguments plead in favor of a major contribution of the age-associated, autophagy inhibitor DBI/ACBP to carcinogenesis and the failure of cancer immunosurveillance. First, in patients, elevations in DBI/ACBP plasma concentrations precede imminent cancer diagnosis. Second, in mice, inhibition of DBI/ACBP delays cancer development induced by carcinogens or orthotopic injection of malignant cells. Third, DBI/ACBP neutralization ameliorates the outcome of cancer treatments with chemotherapy and immunotherapy targeting PDCD1. Fourth, anti-DBI /ACBP mAb favorably influences the composition and functional state of tumor-infiltrating T lymphocytes. Thus, anti-DBI/ACBP mAb joins an expanding list of autophagy inducers (including the small molecules aspirin, 3,4-dimethoxychalcone, spermidine and thiofrepton) that mediate T cell-dependent anticancer effects.

## Disclosure statement

IM is a consultant of Osasuna Therapeutics. GK has been holding research contracts with Daiichi Sankyo, Eleor, Kaleido, Lytix Pharma, PharmaMar, Osasuna Therapeutics, Samsara Therapeutics, Sanofi, Sutro, Tollys, and Vascage. GK is on the Board of Directors of the Bristol Myers Squibb Foundation France. GK is a scientific co-founder of everImmune, Osasuna Therapeutics, Samsara Therapeutics and Therafast Bio. GK is in the scientific advisory boards of Hevolution, Institut Servier, Longevity Vision Funds and Rejuveron Life Sciences. GK is the inventor of patents covering therapeutic targeting of aging, cancer, cystic fibrosis and metabolic disorders. GK's brother, Romano Kroemer, was an employee of Sanofi and now consults for Boehringer-Ingelheim. GK's wife, Laurence Zitvogel, has held research contracts with Glaxo Smyth Kline, 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 cofounder 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.

## Funding

GK is supported by the Ligue contre le Cancer (équipe labellisée); Agence National de la Recherche (ANR-22-CE14-0066 VIVORUSH, ANR-23-CE44-0030 COPPERMAC, ANR-23-R4HC-0006 Ener-LIGHT); Association pour la recherche sur le cancer (ARC); Cancéropôle Ile-de-France; Fondation pour la Recherche Médicale (FRM); a donation by Elior; European Joint Programme on Rare Diseases (EJPRD) Wilsonmed; European Research Council Advanced Investigator Award (ERC-2021-ADG, Grant No. 101052444; project acronym: ICD-Cancer, project title: Immunogenic cell death (ICD) in the cancer-immune dialogue); The ERA4 health Cardinoff Grant Ener-LIGHT; European Union Horizon 2020 research and innovation programmes Oncobiome (grant agreement number: 825410, Project Acronym: ONCOBIOME, Project title: Gut OncoMicrobiome Signatures [GOMS] associated with cancer incidence, prognosis and prediction of treatment response, Prevalung (grant agreement number 101095604, Project Acronym: PREVALUNG EU, project title: Biomarkers affecting the transition from cardiovascular disease to lung cancer: towards stratified interception), Neutrocure (grant agreement number 861878 : Project Acronym: Neutrocure; project title: Development of “smart” amplifiers of reactive oxygen species specific to aberrant polymorphonuclear neutrophils for treatment of inflammatory and autoimmune diseases, cancer and myeloablation); National support managed by the Agence Nationale de la Recherche under the France 2030 programme (reference number 21-ESRE-0028, ESR/Equipex + Onco-Pheno-Screen); Hevolution Network on Senescence in Aging (reference HF-E Einstein Network); Institut National du Cancer (INCa); Institut Universitaire de France; LabEx Immuno-Oncology ANR-18-IDEX-0001; a Cancer Research ASPIRE Award from the Mark Foundation; PAIR-Obésité INCa_1873, the RHUs Immunolife and LUCA-pi (ANR-21-RHUS-0017 and ANR-23-RHUS-0010, both dedicated to France Relance 2030); Seerave Foundation; SIRIC Cancer Research and Personalized Medicine (CARPEM, SIRIC CARPEM INCa-DGOS-Inserm-ITMO Cancer_18006 supported by Institut National du Cancer, Ministère des Solidarités et de la Santé and INSERM). This study contributes to the IdEx Université de Paris Cité ANR-18-IDEX-0001. Views and opinions expressed are those of the author(s) only and do not necessarily reflect those of the European Union, the European Research Council or any other granting authority. Neither the European Union nor any other granting authority can be held responsible for them.

## ORCID

Léa Montégut http://orcid.org/0000-0003-4778-4757

Isabelle Martins http://orcid.org/0000-0003-0885-613X

Guido Kroemer http://orcid.org/0000-0002-9334-4405

## Reference

[1] Montégut L, Liu P, Zhao L, et al. Acyl-coenzyme a binding protein (ACBP) - a risk factor for cancer diagnosis and an inhibitor of immunosurveillance. Mol Cancer. 2024;23:187.