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# Cytoplasmic HMGB2 orchestrates CALR translocation in the course of immunogenic cell death

Peng Liu, Liwei Zhao, Oliver Kepp, Guido Kroemer

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Peng Liu, Liwei Zhao, Oliver Kepp, Guido Kroemer. Cytoplasmic HMGB2 orchestrates CALR translocation in the course of immunogenic cell death. OncoImmunology, 2024, 13 (1), pp.2421028. 10.1080/2162402x.2024.2421028. hal-04889518

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Submitted on 15 Jan 2025

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# Cytoplasmic HMGB2 orchestrates CALR translocation in the course of immunogenic cell death

Peng Liu<sup>a,b</sup>, Liwei Zhao<sup>a,b</sup>, Oliver Kepp<sup>D,a,b</sup>, and Guido Kroemer<sup>a,b,c</sup>

<sup>a</sup>Centre de Recherche des Cordeliers, Equipe Labellisée par la Ligue Contre le Cancer, Université de Paris Cité, Sorbonne Université, Inserm U1138, Institut Universitaire de France, Paris, France; <sup>b</sup>Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Center, Villejuif, France; <sup>c</sup>Department of Biology, Institut du Cancer Paris CARPEM, Paris, France

## ABSTRACT

A recent in vitro study showed that pharmacological inhibition of the nuclear export receptor XPO1 suppresses oxaliplatin-induced nuclear release of HMGB1 and HMGB2, as well as the translocation of CALR to the plasma membrane. Moreover, cell-targeted-HMGB2 protein potently induced CALR exposure, even in the absence of oxaliplatin.

## ARTICLE HISTORY

Received 17 October 2024

Revised 20 October 2024

Accepted 21 October 2024

## KEYWORDS

Adjuvanticity; Calreticulin; checkpoint blockade; Immunotherapy

## Main text

The manner in which cells undergo cell death has a profound impact on the ensuing immune response. Cell death can be perceived as tolerogenic, neutral or immunogenic depending on its molecular characteristics. In cancer therapy, the induction of immunogenic cell death (ICD) has been shown to improve patient outcomes and to synergize with immune checkpoint inhibitors (ICIs) targeting the CTLA-4 or PD-1/PD-L1 axes.¹

Mechanistically, ICD promotes both a surge in adjuvanticity and an increase in the antigenicity of cancer cells.² The rise in adjuvanticity is driven by cellular stress pathways associated with ICD, which are integrated by eukaryotic translation initiation factor 2 subunit 1 (EIF2S1, commonly known as eIF2α). This process orchestrates cellular responses that trigger the controlled release of danger-associated molecular patterns (DAMPs) by tumor cells acting on pattern recognition receptors expressed in the immune compartment.³ Notable ICD-associated DAMPs include ATP, which acts as a chemoattractant, and annexin A1 (ANXA1), which serves as a homing signal for tumor-infiltrating dendritic cells (DCs). Additionally, calreticulin (CALR) is exposed on the cell surface, acting as an ‘eat-me’ signal for the uptake of tumor antigens by dendritic cells (DCs).⁴ Type I interferons (IFNs) produced by tumor cells undergoing ICD enhance chemokine release, which promotes the recruitment of T lymphocytes to the tumor microenvironment.⁵ High-mobility group box 1 (HMGB1), released during ICD, plays a crucial role in DC maturation, enhancing their ability to process and present antigens to naïve T cells.⁶,⁷ This, in turn, ignites adaptive anticancer immune responses, including cytotoxic T lymphocyte-mediated tumor lysis and the generation of immune memory cells, altogether increasing the therapeutic efficacy of conventional treatments, while sensitizing the patient for subsequent ICI.

Mechanistically, the process of DAMP emission during ICD is facilitated by autophagy, liberating energy resources and secreting vesicular ATP through pannexin 1 (PANX1) channels as well as through a defined pattern of ER stress which triggers the *de novo* synthesis and simultaneous translocation of CALR to the plasma membrane.⁸ HMGB1 release was shown to be a two-step process that involves (i) the translocation of nuclear HMGB1 to the cytoplasm and (ii) its release through the ruptured plasma membrane.

A recent article titled "HMGB2-induced calreticulin translocation required for immunogenic cell death and ferroptosis of cancer cells are controlled by the nuclear exporter XPO1" published by Ian A. Blair and colleagues in *Communications Biology* reported that the ICD inducing agent oxaliplatin triggers the extracellular release of nuclear HMGB1 and HMGB2 from A549 and HCT116 cancer cells, both of which can be reversed with the synthetic small-molecule selinexor, which inhibits the nuclear export receptor exportin 1 (XPO1)⁹ (Figure 1A-C). In contrast, cisplatin, which is unable to induce CALR exposure, exclusively triggers the release of HMGB1 but not that of HMGB2,¹⁰ suggesting that HMGB2 might be important for ICD-relevant signaling pathways.

The study by Ian A. Blair's group⁹ further revealed that selinexor prevents oxaliplatin-mediated calreticulin (CALR) translocation to the plasma membrane (Figure 1C). In HCT116 colon cancer cells, selinexor inhibited the oxaliplatin-induced reduction of the antioxidant regulator Nrf2, Fe(II) accumulation, lipid peroxidation, and resulting ferroptosis.⁹ This suggests that XPO1 is required for oxaliplatin-induced ferroptosis. It remains to be determined which proteins must be exported via XPO1 from the nucleus to trigger ferroptosis. Of note, a cell-targeted form of the recombinant HMGB2 protein (CT-HMGB2), which can cross the plasma membrane due to the

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<figure><img src="image_11.png" /><figcaption>**Figure 1.** Implication of XPO1 and HMGB2 in the translocation of endoplasmic reticulum-sessile CALR to the plasma membrane. A,B comparison of the untreated state (A) of cancer cells with that of oxaliplatin-treated cells (B). In oxaliplatin treated cells, nuclei release high mobility group box 1 and 2 (HMGB1 and HMGB2) into the cytoplasm. Moreover, calreticulin (CALR) translocates from the endoplasmic reticulum to the cell surface. C,D pharmacological manipulation of XPO1 and HMGB1 affects the CALR exposure pathway. Selinexor, an inhibitor of nuclear export receptor exportin1 (XPO1), inhibits the nuclear exodus of HMGB1 and HMGB2 as well as CALR exposure (C). CALR exposure can be induced by treatment with cell targeted (CT)-HMGB2 protein (D).</figcaption></figure>

presence of a twin-arginine targeting (TAT)-peptide motif, induced translocation of CALR even in the absence of oxaliplatin, suggesting a key role for HMGB2 in CALR exposure⁹ (Figure 1D). However, it has not been determined whether these CT-HMGB2 effects are coupled to cytotoxic effects. Moreover, a control experiment comparing CT-HMGB1 to CT-HMGB2 is missing.

Altogether, these results point at a critical role of HMGB2 in the immunogenicity of cancer cell death, underscoring that changes in nuclear homeostasis can cause far-reaching immunological consequences. Along these lines it is important to mention that transcription inhibitors such as dactinomycin are prime ICD inducers.¹¹ In sum, further mechanistic studies are needed to elucidate the precise molecular pathway linking the nuclear HMGB2 release to CALR exposure. Moreover, considering the present study, the clinical use of selinexor in anticancer therapy should be critically reviewed.

## Disclosure statement

No potential conflict of interest was reported by the author(s).

## Funding

Figures have been created with a licensed version of BioRender.com. O. K. receives funding by Association pour la recherche sur le cancer (ARC) and Institut National du Cancer (INCa). G.K. is supported by the Ligue contre le Cancer (équipe labellisée); Agence National de la Recherche (ANR) – Projets blancs; AMMICa US23/CNRS UMS3655; ARC; Cancéropôle Ile-de-France; Fondation pour la Recherche Médicale (FRM); a donation by Elior; Equipex Onco-Pheno-Screen (ANR 21-ESRE-0028); European Joint Programme on Rare Diseases (EJPRD); European Research Council Advanced Investigator Award [ERC-2021-ADG, ICD-Cancer, Grant No. 101052444], European Union Horizon 2020 Projects Oncobiome, Prevalung [grant No. 101095604] and Crimson (No. 101016923); Fondation Carrefour; INCa; Institut Universitaire de France; LabEx Immuno-Oncology (ANR-18-IDEX-0001); a Cancer Research ASPIRE Award from the Mark Foundation; the RHU Immunolife; Seerave Foundation; SIRIC Stratified Oncology Cell

DNA Repair and Tumor Immune Elimination (SOCRATE); and SIRIC Cancer Research and Personalized Medicine (CARPEM).

## Author contributions

PL and LZ collected data, designed display items and edited the manuscript. GK and OK wrote the manuscript.

## Data availability statement

All data that led to the conclusions in this manuscript have been referenced and all sources have been described.

## ORCID

Oliver Kepp http://orcid.org/0000-0002-6081-9558

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