# Role of autophagy in modulating tumor cell radiosensitivity: Exploring pharmacological interventions for glioblastoma multiforme treatment

Pierre Bischoff, Jolie Bou-Gharios, Georges Noël, Hélène Burckel

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Pierre Bischoff, Jolie Bou-Gharios, Georges Noël, Hélène Burckel. Role of autophagy in modulating tumor cell radiosensitivity: Exploring pharmacological interventions for glioblastoma multiforme treatment. Cancer/Radiothérapie, 2024, 28 (5), pp.416-423. 10.1016/j.canrad.2024.06.001. hal-05381289

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Review article

# Role of autophagy in modulating tumor cell radiosensitivity: Exploring pharmacological interventions for glioblastoma multiforme treatment

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## Rôle de l'autophagie dans la modulation de la radiosensibilité des cellules tumorales : exploration des interventions pharmacologiques pour le traitement du glioblastome multiforme

Pierre Bischoff<sup>a</sup>, Jolie Bou-Gharios<sup>a, b</sup>, Georges Noël<sup>a, b, c</sup>, Hélène Burckel<sup>a, b, *</sup>

## INFO ARTICLE

Historique de l'article :
Reçu le 12 avril 2024
Reçu sous la forme révisée
le 5 juin 2024
Accepté le 6 juin 2024

Keywords :
Autophagy
Ionizing radiation
Radiosensitization
Glioblastoma

Mots clés :
Autophagie
Rayonnement ionisant
Radiosensibilisation
Glioblastome

## ABSTRACT

Autophagy is an innate cellular process characterized by self-digestion, wherein cells degrade or recycle aged proteins, misfolded proteins, and damaged organelles via lysosomal pathways. Its crucial role in maintaining cellular homeostasis, ensuring development and survival is well established. In the context of cancer therapy, autophagy's importance is firmly recognized, given its critical impact on treatment efficacy. Following radiotherapy, several factors can modulate autophagy including parameters related to radiation type and delivery methods. The concomitant use of chemotherapy with radiotherapy further influences autophagy, potentially either enhancing radiosensitivity or promoting radioresistance. This review article discusses some pharmacological agents and drugs capable of modulating autophagy levels in conjunction with radiation in tumor cells, with a focus on those identified as potential radiosensitizers in glioblastoma multiforme treatment.

© 2024 L'Auteur(s). Published by Elsevier Masson SAS au nom de Société française de radiothérapie oncologique (SFRO). Cet article est publié en Open Access sous licence CC BY (http://creativecommons.org/licenses/by/4.0/).

## RÉSUMÉ

L'autophagie est un processus cellulaire inné caractérisé par l'autodigestion, où les cellules dégradent ou recyclent les protéines âgées ou mal repliées et les organites endommagés via des voies lysosomales. Son rôle crucial dans le maintien de l'homéostasie cellulaire, assurant le développement et la survie, est bien établi. Dans le contexte de la thérapie anticancéreuse, l'importance de l'autophagie est reconnue, du fait de son impact critique sur l'efficacité du traitement. Après la radiothérapie, plusieurs facteurs peuvent moduler l'autophagie, notamment les paramètres liés au type de radiation et aux méthodes de délivrance. L'utilisation concomitante de la chimiothérapie avec la radiothérapie influence également l'autophagie, potentiellement en renforçant la radiosensibilité ou en favorisant la radiorésistance. Cette revue discute de certains agents pharmacologiques et médicaments capables de moduler les niveaux d'autophagie dans les cellules tumorales en combinaison avec les radiations ionisantes, avec un intérêt particulier sur ceux identifiés comme des radiosensibilisateurs potentiels dans le traitement du glioblastome multiforme.

© 2024 L'Auteur(s). Published by Elsevier Masson SAS au nom de Société française de radiothérapie oncologique (SFRO). Cet article est publié en Open Access sous licence CC BY (http://creativecommons.org/licenses/by/4.0/).

<footnote><sup>a</sup> Radiobiology Laboratory, Institut de cancérologie Strasbourg Europe (ICANS), 3, rue de la Porte-de-l'Hôpital, 67000 Strasbourg, France</footnote>

<footnote><sup>b</sup> Laboratory of Engineering, Informatics and Imaging (ICube), Integrative multimodal imaging in healthcare (Imis), UMR 7357, université de Strasbourg, 4, rue Kirschleger, 67000 Strasbourg, France</footnote>

<footnote><sup>c</sup> Department of Radiation Oncology, Institut de cancérologie Strasbourg Europe (ICANS), Unicancer, 17, rue Albert-Calmette, 67200 Strasbourg, France</footnote>

<footnote>* Corresponding author. Radiobiology Laboratory, Institut de cancérologie Strasbourg Europe (ICANS), 3, rue de la Porte-de-l'Hôpital, 67000 Strasbourg, France. Adresse e-mail : h.burckel@icans.eu (H. Burckel).</footnote>
# 1. Introduction

Autophagy, an intracellular process initially described by de Deter et al., involves self-digestion through organelles degradation and long-lived or damaged proteins by lysosomes [1]. Its intrinsic involvement in various diseases, including cancer, has been extensively investigated by physiologists and pathologists [2-5]. Autophagy may have a dual role in disease progression, exerting both beneficial and detrimental effects influenced by intrinsic and extrinsic factors. Therefore, pharmacological modulation of autophagy offers new therapeutic prospects [6-8].

In cancer treatment, autophagy, along with apoptosis, are considered as forms of programmed cell death significantly affecting the efficacy of chemotherapy or radiotherapy (RT) [9-14]. Unlike apoptosis, the activation of autophagy can result in either a cytotoxic or a cytotoxic effect [9]. This duality may be seen as the “Holy Grail”, an ideal yet challenging way in which normal cells are shielded from radiation or cytotoxic drugs, while cancerous cells are targeted. However, the reality is intricate, as autophagy’s occurrence and outcomes are contingent upon not only cell normality or abnormality status, but also on various extracellular factors. Given this biphasic nature, autophagy has been described in the literature as “Janus”, a “Trojan horse”, “a friend or a foe”, and a “double-edged sword” [15-19].

Therefore, the involvement of autophagy in cancer initiation, progression, and treatment strategies has sparked sustained interest, particularly in glioblastoma multiforme (GBM), the most aggressive primary brain tumor [16,17,20-28]. Indeed, in GBM, autophagy exhibits dual effects, potentially promoting or inhibiting tumor growth and progression, thereby offering promising avenues for therapeutic intervention [15,29-31].

# 2. Radiation-induced autophagy

## 2.1. Molecular mechanisms

Significant advancements have been achieved in understanding the molecular mechanisms underlying autophagy in response to ionizing radiation (IR), as well as its role in promoting radiosensitivity [19,32-36]. Primarily, two main factors contribute to the induction of autophagy by IR: DNA damage and the stimulation of endoplasmic reticulum stress (ERS). An abnormal increase of reactive oxygen species (ROS) production, resulting from IR, emerges as a common trigger for autophagy activation stemming from both factors.

## 2.2. DNA damage

Autophagy can be triggered in response to DNA damage, particularly induced by double-strand breaks (DSBs), similar to apoptotic processes [37]. Autophagy actively influences the DNA repair through diverse mechanisms, including impairment of homologous repair and non-homologous end joining processes [38]. Additionally, autophagy facilitates DNA repair by recycling specific proteins involved in the repair machinery. However, beyond a certain threshold of DNA degradation, autophagy may lead to cell death [39]. For instance, in the presence of heavy-damaged DNA, autophagic cell death serves as an alternative when apoptosis is unattainable [37]. AMP-activated protein kinase (AMPK) acts as a key genomic stress sensor in response to DNA damage, influencing both apoptotic and autophagic pathways [40]. Furthermore, mitochondrial dysfunction activates AMPK, promoting cell survival by orchestrating the autophagic degradation of damaged mitochondria, thereby shielding cells from IR-induced toxicity [41,42].

## 2.3. Endoplasmic reticulum stress

Endoplasmic reticulum stress (ERS) arises as a response to disturbances in the normal physiological functions of the endoplasmic reticulum, induced by various factors, notably ROS or exposure to adverse physical and/or chemical conditions [43]. The unfolded protein response assumes a central role in ERS and can be activated by IR, spanning from ultraviolet to high linear energy transfer (LET) particles such as carbon ions. Excessive activation of ERS can result in cell death either through apoptosis or via interconnected pathways with autophagy [44-46]. In addition, ERS is observed in cells exhibiting deficiencies in the mitochondrial apoptotic pathway [47]. Therefore, pharmacological modulators targeting ERS provide an alternative mean to modulate the autophagic response to IR [12].

# 3. Pharmacological strategies to modulate autophagy in GBM in combination with IR

The intrinsic molecular and cellular heterogeneity of GBM, along with dysregulations in cell death pathways, confer resistance to conventional chemotherapy and RT [48]. Despite advancements in treatments, GBM prognosis remains exceedingly unfavorable [49,50]. First-line treatment of GBM typically involves temozolomide (TMZ) in combination with RT, sometimes supplemented with other pharmaceutical agents. Regardless of the timing of administration relative to RT, these drugs potentiate cell death through several mechanisms, such as DNA adducts formation hindering DNA damage repair and inducing diverse programmed cell death modalities, including autophagy. In fact, numerous drugs modulating autophagy, either inhibiting or activating it, have been identified. Their efficacy and toxicity depend on their ability to influence the stress response of GBM cells [51-53]. New therapeutic approaches are developed often through experimental studies involving GBM cell lines, with a notable focus on the prevalent usage of the human-derived U-87 MG cell line. The impact of various chemotherapeutic agents and drugs on autophagy induction has been documented. Therefore, in the following section, we will describe different clinical pharmaceutical drugs that affect autophagy, with a special focus on their combination with IR and their role on the sensitization of GBM to RT.

## 3.1. Alkylating agents

### 3.1.1. Temozolomide

Extensive scientific literature has investigated the molecular mechanisms underlying the induction of autophagy by temozolomide in GBM [54,55]. However, the independent or combined cytotoxic effects of TMZ and IR remain a subject of debate [56,57]. While TMZ induces cell death in malignant cells through apoptosis, autophagy, and senescence pathways, the possibility of senescent GBM cells escaping the tumor and contributing to recurrence is raised [58]. Furthermore, the question of whether TMZ and IR act independently or influence the autophagy machinery, potentially accelerating or inhibiting the dynamics or flux of autophagy remains unresolved [59]. Nonetheless, other categories of drugs capable of modulating autophagy may be associated with TMZ and RT combination.

### 3.1.2. Platinum derivatives

While alkylating-like agents such as platinum derivatives have been used to target GBM, the intrinsic resistance of GBM cells to these drugs has been hindered their efficacy [49]. However, to enhance therapeutic outcomes in GBM with newer platinum-derived compounds, innovative strategies are imperative [60-64]. For instance, experiments involving oxaliplatin, a
platinum derivative recognized for its increased DNA-targeting efficiency compared to cisplatin, were conducted in conjunction with high-LET radiation. Results demonstrated that combining oxaliplatin with fast neutrons significantly decreased the viability of U-87 MG cells through induction of autophagy. Thus, the combination of oxaliplatin with similar radiation modalities could emerge as a promising strategy for treating GBM [65].

### 3.2. PI3K/Akt/mTOR inhibitors

The mammalian target of rapamycin (mTOR) is as a crucial regulator in signaling pathways controlling autophagy induced by IR or other genotoxic agents [14,35,66,67]. Operating as a serine/threonine kinase, mTOR is an integral constituent of mTOR complex 1 (mTORC1). In malignant cells, mTOR frequently undergoes aberrations attributed to genetic or epigenetic alterations in upstream regulators across diverse pathways [53]. Dysregulated mTORC1 activity is closely linked with cancer progression and compromises the autophagic response of malignant cells to IR. Thus, targeting mTOR by specific inhibitors has given rise to the development of a new class of anticancer drugs, including RAD001 (everolimus) [66,68] and ABTL0812 [69].

Experimental investigations involving RAD001 have explored its effects in combination with fast neutrons and TMZ. This triple combination amplified their concerted action by inducing irreversible DNA damage, thereby increasing autophagy-mediated cellular death. Such a trimodal therapy, which integrates high-LET radiation, conventional anticancer agents, and molecular-targeted agents, holds promise in overcoming GBM resistance to RT [70,71]. Similarly, ABTL0812, an orally administrated small molecule currently undergoing phase II clinical trials, exhibits significant anticancer properties [72]. This alpha-hydroxylated unsaturated fatty acid has demonstrated efficacy in impairing tumor growth and improving disease-free survival in orthotopic human GBM xenografted mice models [73]. Its most potent effects were observed when combined with IR and TMZ. The molecular mechanisms of this drug involved inhibiting Akt/mTORC1 and activating ERS via the unfolded protein response, ultimately leading to the induction of autophagic cell death [69,73,74].

It is noteworthy to mention that the variability in GBM responses to IR is partly influenced by the presence of glioma-initiating cells (GICs) within the tumor. Rapamycin, the prototypical mTOR inhibitor, induces the differentiation of human GICs and increases their radiosensitivity through autophagic mechanisms [66]. Transient exposure of GICs to rapamycin and IR inhibits their capacity to propagate as intracerebral GBM in nude mice, thereby impeding tumor development. Hence, stimulating autophagic cell death in GICs through mTOR inhibition could potentially pave the way for a molecular-targeted treatment of GBM [75].

### 3.3. Poly (ADP-ribose) polymerase inhibitors

The nuclear enzyme poly (ADP-ribose) polymerase-1 (PARP-1) holds a pivotal role in the repair of potentially lethal DNA DSBs. Its involvement in DNA repair processes, particularly in the context of RT, where it decreases the efficacy of IR, emphasizes its significance. Thus, PARP inhibitors generally exhibit potent radiosensitizing properties [76-79]. Examples of such inhibitors include olaparib and niraparib, both of which are currently ongoing clinical trials in combination with RT and TMZ, demonstrating documented clinical benefit [78,80,81]. However, the therapeutic efficacy of PARP-1 inhibitors has been questioned, as recent investigations suggest their capacity to promote autophagy and cellular senescence [82].

### 3.4. Antimetabolite agent

Gemcitabine, an antimetabolite agent, has undergone recent preclinical investigations for its efficacy in GBM, yielding controversial results. This controversy is attributed to various side effects, including its limited half-life and the development of chemoresistance [83]. In prior phase II studies, this nucleoside analog was investigated as a radiosensitizer for newly diagnosed GBM, showing favorable outcomes in patients who received oral TMZ, RT, and gemcitabine [84]. Ultrastructural analysis in an experimental model revealed gemcitabine's ability to induce significant autophagy in C6 glioma cells [85]. Moreover, gemcitabine demonstrated its potential to augment the effects of IR on pancreatic tumor cell lines, resulting in synergistic toxicity both in vitro and in vivo. The blockade of autophagy by 3-metyl-adenine (3-MA) reduced cytotoxic activity, indicating autophagy's contribution in post-treatment cell death and the amplification of DNA damage, consequently increasing the cytotoxicity. The combined treatment resulted in a synergistic increase in apoptosis, underscoring the crucial role of autophagy activation not only in pancreatic cancer treatment but also in addressing other types of malignant tumors [68].

### 3.5. Modulation of EGFR

The epithelial growth factor receptor (EGFR) is a member of the ErbB family of tyrosine kinase transmembrane receptors, playing a crucial role in mediating intracellular signaling pathways that promote tumor growth and progression. Its genetic mutations and overexpression are characteristic indicators in GBM, underlining their significant contribution in tumor radioresistance [66]. The potential of EGFR blockade to radiosensitize tumors has been demonstrated in many clinical trials across various cancer types [79]. Investigations of the involvement of EGFR in autophagy were conducted using GBM cell lines where EGFR was silenced, resulting in diminished cell migration and increased radiosensitivity. Interestingly, evidence of the pro-death role of autophagy in enhancing radiosensitivity was observed. Silencing the autophagic gene ATG7 restored clonogenic capacity of irradiated cells, indicating autophagy's contribution to the radiosensitization of GBM cells [86]. In clinical trials, the combined administration of nimotuzumab, a humanized monoclonal antibody targeting EGFR, with fractionated RT resulted in a significant increase in median survival compared to RT alone [87]. Therefore, modulation of EGFR activity in combination with RT and TMZ represents a promising therapeutic strategy for GBM and other tumors characterized by EGFR dysregulation [88].

### 3.6. Bcl-2 protein inhibitors

The resistance of malignant cells to apoptosis frequently hampers the effectiveness of RT in various cancer types. The resistance is exacerbated often by the upregulation of anti-apoptotic Bcl-2 subfamily proteins. Targeting these endogenous apoptosis pathways with Bcl-2 inhibitors has emerged as a pharmacological strategy to potentiate the anticancer effect of RT. Small-molecule inhibitors of Bcl-2, such as gossypol and obatoclax, have been evaluated as radiosensitizers. For example, polyphenolic compounds gossypol (AT101) and apogossypolone (ApoG2), derived from cottonseeds, exhibit potent inhibition of Bcl-2. ApoG2 has demonstrated radiosensitization of nasopharyngeal carcinoma cells by inducing autophagy rather than apoptosis [89]. This radiosensitizing effect extends to other cancers, including those with dysregulated apoptotic signaling like GBM. Autophagic cell death induced by ApoG2 enhances the action of TMZ in malignant glioma [90]. The cytotoxic effects are attributed to mitochondrial
dysfunctions and repair of DSBs [91,92]. Moreover, ApoG2 and AT101 have been observed to increase radiosensitivity in GBM cells [93,94]. Recent promising results were achieved in patients with gastroesophageal carcinoma treated with the association of docetaxel, fluorouracil and RT [95]. However, it is important to note that AT101 can also initiate Bcl-2-dependent cytoprotective autophagy, as reported in several types of cancer, such as blader cancer [96]. Likewise, obatoclax, another Bcl-2 inhibitor, can overcome resistance to histone deacetylase (HDAC) inhibitors such as SAHA or LBH589, which are employed as radiosensitizers in patient-derived glioma stem-like cells. Independently, obatoclax strongly induced autophagy [97]. When combined with IR and SAHA, obatoclax increases caspases activity, and enhances autophagy. This triple combination results in substantial mitochondrial stress and caspase-independent cell death characterized by defective autophagy, contributing to its efficacy in killing cancer cells. Necroptosis, facilitated by autophagy, emerges as an additional contributor to the observed cytotoxicity [98].

### 3.7. STAT3 inhibitor

In GBM, the aberrant regulation of signal transducer and activator of transcription 3 (STAT3), a key intracellular signaling protein, is frequently observed and correlates with the malignant phenotype. Inhibiting STAT3 using WP1066, an analog of caffeic acid, radiosensitized GBM cells, likely through the activation of the mitochondrial apoptotic pathway. Coadministration of IR and WP1066 increases autophagic flux. Treatment with 3-MA or ATG5 siRNA, which inhibits autophagy, enhances the radiosensitizing effect of WP1066, resulting in an increased population of apoptotic cells. This dual inhibition strategy holds promise in overcoming the radioresistance in GBM, as demonstrated by clinical investigations using GBM clinical specimens [99–101].

### 3.8. HMG-CoA inhibitor

As a member of statins class, pitavastatin inhibits 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, thereby reducing blood cholesterol levels. Additionally, it acts as a potent inhibitor of nuclear factor-kappa B (NFkB), whose constitutive activation is associated with protection against apoptosis [102]. In radioresistant head and neck cancer cells, pitavastatin inhibits the mevalonate pathway, enhancing radiosensitivity and reducing clonogenic potential [103]. Although pitavastatin alone did not inhibit the growth of three human GBM cell lines, its combination with IR led to an increased cell death. Autophagy is induced across all cell lines following IR, regardless of their radiosensitivity. However, pitavastatin-treated or untreated cells do not exhibit apoptosis post-IR. Treatment with 3-MA, abolishes the cytotoxic effect of this combination, indicating the involvement of autophagic cell death in the antitumor mechanism. However, in oral and colon cancer cell lines, Pitavastatin has been recently shown to induce apoptosis by blocking autophagic flux [104].

### 3.9. USP-1 inhibitor

Pimozide, a neuroleptic agent, acts as a specific inhibitor targeting ubiquitin-specific protease-1 (USP-1), a protein highly expressed in GBM. Its efficacy in GBM is augmented by its capacity to induce selective autophagy and penetrate the blood brain barrier [105]. Initially identified for its ability to enhance cell death in breast cancer cells when combined with IR, Pimozide has shown efficacy in reducing the proliferation of GBM cancer cell lines by inducing apoptosis [106,107]. Pimozide mediates its effects by suppressing the phosphorylation of STAT3 and inhibiting the expression of anti-apoptotic markers like c-Myc. Interestingly,

it also decreases spheroid formation by inhibiting the dopamine D2 receptor in U-87 MG cells, leading to autophagy-dependent cell death [15,108–110]. Thus, pimozide represents a promising candidate for therapeutic protocols aimed at reinforcing the radiosensitization of GBM.

### 3.10. Additional autophagy modulators

#### 3.10.1. Quinoline-based drugs

Historically, both chloroquine (CQ) and hydroxychloroquine (HCQ) have been primarily prescribed for malaria prevention and treatment. Moreover, these agents exhibit significant anticancer properties, either alone or in combination with RT [111,112]. The mechanisms underlying these effects involve the induction apoptosis, ERS, and the inhibition of autophagy, attributed to their lysosomotropic characteristics. This characteristic interferes with lysosome acidification, a critical step for autophagosomes degradation. Evaluation of various CQ derivatives has demonstrated efficacy in killing GBM cells resistant to TMZ [113]. CQ can also counteract cellular senescence induced by TMZ and enhance the radiosensitivity of GBM cells by inhibiting autophagy and activating apoptosis in GICs [58,114]. This effect involves destabilization of the lysosomal membrane, ultimately leading to cell death by necrosis [115]. However, CQ does not alter the intrinsic radiosensitivity of various cancer cell lines (colorectal, mammary and cervical adenocarcinoma, lung, and prostate carcinoma, etc.) irradiated with a single dose up to 8 Gy [116]. Additionally, CQ induces vessel normalization and improves chemotherapeutic drug delivery through an autophagic-independent mechanism. This effect is achieved by reducing the hypoxic radioresistant cell population within the tumor rather than affecting the intrinsic radiosensitivity of cancer cells. Additionally, CQ inhibits autophagy induced by high-LET carbon ions in sarcoma cell lines, enhancing the radiosensitization through aggravation of ERS-related apoptosis [117].

#### 3.10.2. Silver and gold nanoparticles

Nanostructured materials are currently employed across a range of medical applications, notably as radiosensitizers [118]. Gold nanoparticles (GNPs) have been reported for their capacity to improve the efficacy of multi-targeted treatments on glioma cells [119–122]. Similarly, silver nanoparticles (AgNPs) have demonstrated radiosensitizing properties in GBM cells via the induction of autophagic cell death [123].

Significantly, in hypoxic cells, the presence of AgNPs elicited a greater induction of autophagy compared to normoxic cells [124]. Subsequently, this elevated autophagic activity resulted in heightened apoptosis through signaling pathways such as ERK and JNK. Specific inhibitors that target these proteins result in reduced levels of autophagy in cells treated with AgNPs and IR. Particularly, ROS are found to be crucial in the mechanism of action of AgNPs on radiation-induced autophagy. Inhibiting autophagy by 3-MA increased ROS levels in GBM cells treated with AgNPs, thereby enhancing the apoptotic response [18]. Comparatively, when combined with IR, AgNPs demonstrate an upregulation of autophagy compared to GNPs. Copper cysteamine nanoparticles have also been shown to be effective as radiosensitizers in the treatment of colorectal carcinoma, inducing apoptosis and autophagy in target cells [125]. Overall, these findings provide a scientific basis for the utilization of metallic nanoparticles in cancer treatment, with a growing interest on the integral role of autophagy in RT [119].

#### 3.10.3. Curcumin

Curcumin, a polyphenolic compound extracted from the root of Curcuma loga, commonly known as turmeric, exhibits broad utility in traditional medicine for addressing various diseases [126]. Its antitumor properties have been well documented across several
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Overview of some pharmacological strategies for modulating autophagy in glioblastoma. Combining autophagy modulators with ionizing radiation might offer an effective approach to enhance the radiosensitivity of glioblastoma multiforme. Created with BioRender.com</figcaption></figure>

cancer types, particularly in GBM [127,128]. A notable advantage of curcumin is its reduced toxicity, rendering it suitable for adjuvant therapy. Its mechanisms of action against malignant cells include cell cycle arrest, apoptosis induction and the stimulation of autophagy, possibly through inhibition of the PI3K-Akt/mTOR pathway [129–131]. While synergistic effects have been described in vitro when combining curcumin with IR, such effects have yet to be confirmed in clinical trials [132,133].

**4. Conclusion and prospects for future research**

In this review, we have discussed the intricate involvement of autophagy in cancer therapeutics, with a particular emphasis on GBM. Autophagy exhibits a dual role, exerting influence on the fate of malignant cells by either promoting their death or facilitating their survival. This inherent complexity has fostered intensive investigations aimed at identifying novel pharmacological compounds or repurposing existing drugs capable of modulating autophagy in response to RT (Fig. 1). Compounds such as chloroquine, PARP-1 inhibitors, mTOR inhibitors, USP-1 inhibitors and silver nanoparticles hold promise as potential adjuvant treatments for GBM. Conversely, efforts are directed towards enhancing the cytoprotective properties of autophagy to reduce the adverse effects of radiation on normal cells.

Rather than viewing autophagy activation as a mere process of “battle damage repair”, autophagy is now recognized as a sophisticated metabolic process that addresses cellular damage induced by IR. However, the potential of autophagy activation to protect cells from radiation’s harmful effects has been underappreciated. Oncologists often view cytoprotective autophagy negatively, attributing it to treatment resistance rather than recognizing its potential

benefits. Recent findings suggest that combining autophagy inhibitors with inducers may hold promise for various cancer types [8].

Nevertheless, uncertainties persist regarding the precise role of autophagy in radiation-induced cell death [134]. The main question remaining concerns its interplay with cellular senescence and whether it represents a final stage of cell death or a preliminary step preceding apoptosis, necroptosis or autosis, a distinct form of cell death [36,135,136]. The presence of autophagy markers in dying cells does not definitively establish the involvement of autophagy in that specific cell death type [39]. Moreover, certain forms of autophagy-dependent cell death may occur without the presence of classical autophagy markers [136].

The effectiveness of apoptotic signaling in the presence of extreme autophagy remains unclear, likening autophagy's role to that of the Loch Ness monster due to its elusive nature in cell destruction [137]. To elucidate its contribution to the cell death process, specific criteria must be identified to determine whether autophagy acts as a “cell killer, an accomplice, or only a bystander in the cell death process” [42]. Currently, predictive markers for autophagy and suitable assays to monitor its modulation by drugs or radiation in patients are still lacking, but their development would facilitate the design of more specific autophagy inhibitors or activators for cancer treatment [138].

In the context of RT, autophagy inhibitors could potentially protect healthy tissues from IR or eliminate cancer cells through a tumor-specific immune response. Understanding the mode of cell death is crucial as the immune system’s clearance of dead or dying cells impacts RT outcomes [139,140]. Future therapies may combine RT with immunotherapy, particularly integrating immune checkpoint inhibitors [125,139]. Finally, combining RT
with molecular-targeted drugs that interfere with cell death shows promise in cancer therapy, especially for the sensitization of GBM to first-line treatments [29,141].

## Author contributions

PB: review of literature and manuscript writing and conceptualization; JBG: review of literature, editing and review of manuscript; GN: editing and review of manuscript; HB: manuscript conceptualization, validation of data, editing and review of manuscript.

## Funding

This study has received financial support from Institut de cancérologie Strasbourg Europe.

## Disclosure of interests

The authors did not disclose their relationships/activities/interests.

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