Multifaceted role of GCN2 in tumor adaptation and therapeutic targeting

Highlights

• •A thorough review of research on GCN2 has been conducted, offering a detailed overview of its multifaceted impacts on cellular processes.
• •Through pan-cancer analysis, further investigation was carried out on the diverse roles of GCN2 in various tumor prognoses, exploring its relationship with immune responses and potential effects on immunotherapy outcomes.
• •A comprehensive survey of current GCN2 inhibitors was performed, compiling findings from both in vitro and in vivo studies.

Abstract

Tumor cells voraciously consume nutrients from their environment to facilitate rapid proliferation, necessitating effective strategies to manage nutrient scarcity during tumor growth and progression. A pivotal regulatory mechanism in this context is the Integrated Stress Response (ISR), which ensures cellular homeostasis under conditions such as endoplasmic reticulum stress, the unfolded protein response, and nutrient deprivation. Within the ISR framework, the kinase GCN2 is critical, orchestrating a myriad of cellular processes including the inhibition of protein synthesis, the enhancement of amino acid transport, autophagy initiation, and angiogenesis. These processes collectively enable tumor survival and adaptation under nutrient-limited conditions. Furthermore, GCN2-mediated pathways may induce apoptosis, a property exploited by specific therapeutic agents. Leveraging extensive datasets from TCGA, GEO, and GTEx projects, we conducted a pan-cancer analysis to investigate the prognostic significance of GCN2 expression across diverse cancer types. Our analysis indicates that GCN2 expression significantly varies and correlates with both adverse and favorable prognoses depending on the type of cancer, illustrating its complex role in tumorigenesis. Importantly, GCN2 also modulates the tumor immune microenvironment, influencing immune checkpoint expression and the functionality of immune cells, thereby affecting immunotherapy outcomes. This study highlights the potential of targeting GCN2 with specific inhibitors, as evidenced by their efficacy in preclinical models to augment treatment responses and combat resistance in oncology. These findings advocate for a deeper exploration of GCN2′s multifaceted roles, which could pave the way for novel targeted therapies in cancer treatment, aiming to improve clinical outcomes.

Introduction

To maintain rapid growth under nutrient-poor conditions, tumor cells modify their metabolic pathways to reconcile the heightened need for biomass production with energy generation [1]. A key regulatory mechanism in this adaptation is the Integrated Stress Response (ISR), which maintains cellular equilibrium under various stresses such as endoplasmic reticulum stress, the unfolded protein response, and nutrient shortages [2]. The ISR initiates through the phosphorylation of eIF-2α at serine‑51, controlled by four specific kinases: heme-regulated initiation factor-2α kinase, dsRNA-activated protein kinase (PKR), PKR-like endoplasmic reticulum kinase, and general control nonderepressible 2 (GCN2) [2]. Among these, GCN2, encoded by the EIF2AK4 gene, is essential, serving as a direct link between nutrient availability and protein stability[3].

GCN2 activation occurs primarily during amino acid scarcity and features a multi-domain structure, including a standard eukaryotic protein kinase catalytic domain, a histidyl-tRNA synthetase-like domain, and a C-terminal ribosome domain [[4], [5], [6]]. When active, GCN2 phosphorylates eIF2α, reducing overall protein synthesis but enhancing the translation of specific mRNAs, such as ATF4. ATF4 is crucial in regulating the expression of genes that maintain cellular homeostasis, affect protein metabolism, and manage responses to various physiological and pathological conditions [7].

Recent research underscores GCN2′s role in mediating interactions between different pathways within tumors, highlighting its potential as a cancer therapy target. This review offers a detailed analysis of GCN2′s role in cancer, exploring its mechanisms across different tumor types and stress conditions, and includes a pan-cancer analysis of GCN2. We also review existing GCN2 inhibitors and their preclinical evaluations. Significantly, GCN2 not only impacts tumor cells but also influences the tumor immune microenvironment, emphasizing the importance of understanding GCN2′s roles to develop effective cancer treatments, including immunotherapies.

GCN2′s key role in the protection of cancer cells

GCN2 kinase has emerged as a critical regulator in cancer biology, playing a pivotal role in cellular adaptation to environmental and metabolic stressors [8]. As a crucial sensor of amino acid availability, GCN2 orchestrates a comprehensive response to nutrient deprivation, a common challenge faced by rapidly proliferating tumor cells. GCN2 preserves cellular homeostasis by modulating protein synthesis [[9], [10], [11]], upregulating amino acid transporters [10,[12], [13], [14]], promoting autophagy [15,16] and enhancing the efficiency of scarce amino acids for protein synthesis [17]. When cells face MYC-induced proteotoxicity [18,19] or other forms of nutrient scarcity, GCN2′s activation promotes angiogenesis [20,21], oversees mitochondrial quality control [[22], [23], [24]] and boosts ribosome biogenesis [25,26]. These functions underscore GCN2’s pivotal role in preserving cellular stability and promoting tumorigenesis.

Maintaining cellular homeostasis

Reducing global protein synthesis

GCN2 plays a crucial role in cellular adaptation to nutrient stress by modulating protein synthesis and amino acid metabolism [[9], [10], [11]]. Under conditions of amino acid scarcity, GCN2 enhances ATF4 expression, which subsequently induces the production of the stress response protein Sestrin2. This protein plays a crucial role in maintaining mTORC1 repression by preventing its localization to the lysosome, ultimately leading to a significant reduction in global protein synthesis [27]. This adaptation is vital for maintaining stable amino acid levels, crucial for tumor cell survival. For instance, in human colorectal carcinoma cells, deprivation of external arginine activates the GCN2 pathway, reducing protein synthesis through mTOR signaling inhibition[9]. Similarly, in hepatocellular carcinoma, restricting arginine availability decreases mRNA and protein levels, but these effects are reversible by inhibiting GCN2, which then promotes cellular senescence [10]. Proline deprivation in melanoma cells also activates GCN2, reducing protein synthesis [11]. Furthermore, treatment with ixazomib in certain cancer cells leads to amino acid depletion, GCN2 activation, and subsequent suppression of protein synthesis, demonstrating how GCN2 can respond to different metabolic stresses to regulate tumor survival [28] (Fig. 1).

Fig. 1.

GCN2′s Protective Mechanism in Tumor Cells. GCN2 activation can be triggered by various stressors, including proteotoxicity due to MYC overactivation, impaired protein degradation from VCP/P97 inhibition, or amino acid scarcity caused by certain experimental conditions or drugs. This leads to the accumulation of uncharged tRNA, which activates GCN2. Energy deficits, indicated by inhibited mitochondrial ATP synthase or LDHA, as well as reduced expression of MIC60 in the mitochondrial inner membrane or TRAP1 under nutrient-deficient conditions, can also activate GCN2. Once activated, GCN2 enhances cell survival through several pathways: it inhibits the mTOR pathway, decreases ribosome formation, and phosphorylates eIF2α to reduce protein synthesis. Additionally, GCN2 activates the MEK/ERK and AKT pathways. It activates genes downstream of ATF4, improving the cell's ability to acquire amino acids and survive. GCN2 also regulates mitosis to maintain genomic stability, avoids IFN-induced cellular damage by downregulating IFNAR1, and may decrease apoptosis by inhibiting the JNK/P38 pathway, thereby promoting cell survival.

Building on the regulatory mechanisms of GCN2, MYC, a pivotal oncogene in cancer metabolism, drives cell growth and proliferation by manipulating similar metabolic pathways. MYC indirectly activates GCN2 kinase by accumulating uncharged tRNAs, leading to ATF4 upregulation [29]. ATF4, working in tandem with MYC, orchestrates the expression of key genes involved in amino acid and protein synthesis, including 4E-BP1. This gene acts as a translation inhibitor, helping to balance protein synthesis and mitigate proteotoxic stress, a common consequence of MYC overexpression [18]. Inhibition of GCN2, achieved through pharmacological agents, increases protein synthesis rates and MYC expression in APC-deficient cells, leading to MYC-induced cell death [19]. This complex interplay highlights the critical role of GCN2 and MYC in regulating protein synthesis and stress response pathways, which are fundamental for the survival and proliferation of cancer cells.

Upregulating of amino acid transporter gene expression

GCN2′s role extends to the regulation of over 60 solute carrier (SLC) genes crucial for amino acid transport, thereby preserving the availability of these vital nutrients for tumor expansion [12]. This regulation ensures the continuous availability of vital nutrients necessary for tumor growth and expansion. In various cancers such as osteosarcoma [13], prostate cancer (PC) [12], hepatocellular carcinoma (HCC) [10], and breast cancer [14], GCN2′s regulatory effect on SLC genes is crucial. In PC, GCN2′s significance is underscored by its influence on a broad array of SLC transporter genes critical especially 4F2 for tumor growth [12]. In osteosarcoma 143B cells, the knockout of ASCT2, key for glutamine uptake, activates GCN2, leading to upregulation of SNAT1 to compensate for ASCT2′s reduced functionality. Silencing GCN2 in these cells results in decreased cell proliferation [13]. In HCC, GCN2 responds to arginine deprivation by upregulating the SLC7A1 transporter, which is essential for arginine uptake and, consequently, cell survival [10]. Similarly, in HeLa cells, kynurenine presence hinders cystine uptake but activates GCN2, enhancing ATF4 translation and SLC7A11 expression, increasing cystine acquisition [14]. These examples highlight GCN2’s sophisticated mechanisms in adapting to amino acid deprivation across different cancer types, illustrating its integral role in maintaining cellular metabolism and promoting cancer cell survival under nutrient-limited conditions.

Regulating autophagy

Amino acids and autophagy share a reciprocal relationship, whereby scarcity of amino acids triggers a signaling cascade that reduces their demand while simultaneously enhancing their intracellular synthesis, recycling, and influx. The GCN2 signaling pathway is central to this process, especially in the regulation of stress-induced expression of autophagy-related genes [5]. Through the activation of its kinase and the downstream transcription factor ATF4, GCN2 significantly increases the transcription of key autophagy proteins. These proteins are essential for various stages of autophagy, including the synthesis (such as ATG5 and ATG12), maturation (like LC3), and turnover of autophagosomes, in response to amino acid deprivation [15]. Additionally, the inhibition of proteins such as HSP70 and VCP/p97 can lead to the activation of GCN2, which plays a protective role in cancer cells by enhancing autophagy and metabolic turnover, thus supporting cellular survival under nutrient-stressed conditions [15,16]. It's important to note that merely increasing gene expression does not automatically activate autophagy. mTORC1 serves as the primary acute regulator of autophagy [30]. A crucial initial step in autophagy is the activation of ULK1. When nutrients are abundant, mTORC1 phosphorylates ULK1, thus preventing its activation by AMPK, which is a key promoter of autophagy [31]. Therefore, the activation of autophagy by GCN2 may be partially achieved through its inhibition of the mTOR pathway [27].

Balancing amino acid utilization

In addition to reducing protein synthesis, promoting autophagy, and enhancing amino acid transport, GCN2 plays a pivotal role in rerouting scarce amino acids from various metabolic pathways to protein synthesis, particularly under conditions of amino acid scarcity. This function is critical for sustaining cell survival and functionality in environments lacking nutrients. Tryptophan, an essential amino acid and the least abundant of those encoded by the genetic code, fulfills roles beyond its basic function as a protein building block [32]. Trp-degrading enzymes are commonly expressed in various cancers because the metabolites of Trp, like IDO1 and TDO2, play significant roles in the tumor's immune evasion tactics [33]. Consequently, cancer cells need to manage the allocation of Trp between its conversion into metabolic products and its use in protein synthesis. In specific cancers such as breast cancer, colon cancer, and B-cell lymphoma, Trp deprivation specifically elevates cytoplasmic WARS protein levels without affecting nuclear or extracellular WARS expression, a response driven by the GCN2-ATF4 axis. According to computational models, a shortage of Trp funnels it towards WARS utilization [17].

Promoting angiogenesis

Amino acid deprivation triggers a notable increase in angiogenesis through upregulated expression of vascular endothelial growth factor (VEGF), facilitated by the GCN2-ATF4 pathway. As tumors face limitations in their blood supply, their need for oxygen and essential nutrients, such as glucose and amino acids, intensifies. An adaptive strategy employed by tumors is to reinitiate their blood supply through the angiogenic switch [34,35]. This phenomenon has been observed in various cancer cell types, including head and neck squamous carcinoma, human oral squamous carcinoma cells, glioblastoma cells, breast cancer cells, and lung carcinoma cells. In these cells, a significant elevation in VEGF expression occurs in response to amino acid deprivation, mediated by GCN2 activation [20,21]. Additionally, studies have shown that the knockdown of GCN2 in tumor cells leads to reduced tumor growth and angiogenesis in vivo, underscoring the pivotal role of the GCN2/ATF4 pathway in enabling tumor adaptation and growth under nutrient-deprived conditions [20].

Mitochondria and GCN2 activation

Mitochondria are pivotal at every stage of oncogenesis, from the initiation of malignant transformation to the progression of metastasis. They not only provide energy but also regulate key survival mechanisms under nutrient stress and therapeutic interventions. The GCN2 kinase pathway is central to these processes, activating complex adaptation strategies in cancer cells facing oxidative damage, nutrient deficiency, and therapeutic stress. In specific cancers, such as colon [36] and gastric cancers [37,38], mitochondrial signals trigger GCN2 activation, which leads to a preferential shift towards glycolysis, reflecting a strategic modulation of energy production to support cellular proliferation and survival. For instance, the inhibition of ATP synthase in colon cancer cells induces a shift to glycolysis through GCN2 activation, showcasing adaptability in energy metabolism [36].

GCN2 is a crucial mediator in mitochondrial quality control. It responds to energy deficits by activating and coordinating mitochondrial-associated genes, such as TNF receptor-associated protein 1 (TRAP1). TRAP1, a member of the mitochondrial HSP90 family, is instrumental in mitigating oxidative stress and preventing apoptosis [39]. When nutrient levels are low, the activation of TRAP1 further stimulates the GCN2-eIF2α pathway, leading to the upregulation of ATF4. This, in turn, boosts the expression of its target genes, BiP/Grp78 and xCT, enhancing the cell's ability to cope with ER stress and oxidative damage [22,23,40]

Furthermore, the role of Lactate dehydrogenase isoform A (LDHA) in cancer cells illustrates another layer of metabolic adaptation. Under hypoxic conditions, LDHA facilitates survival by converting pyruvate to lactate, reducing dependence on mitochondrial oxidation [41]. Inhibition of LDHA can also activate the GCN2-ATF4 pathway, which then enhances the uptake of glutamine and other essential amino acids through the expression of SLC1A5, activating key survival pathways like mTORC1 [23].

Additionally, in the context of cellular architecture, Mic60’s role in sustaining mitochondrial structure is vital for regulating energy dynamics and apoptosis resistance [42]. In prostatic adenocarcinoma, low expression of Mic60 triggers compensatory responses that include GCN2/Akt kinase signaling, essential for maintaining cell viability. Targeting Mic60-deficient tumors with GCN2 inhibitors has shown promise in inducing apoptosis and inhibiting tumor proliferation [24].

This integration of GCN2 signaling with mitochondrial quality control processes links nutrient availability to mitochondrial health, providing a comprehensive framework for understanding the interplay between metabolic demands and cellular adaptation strategies. This insight offers a rich landscape of potential therapeutic targets, highlighting the importance of mitochondrial quality control in determining cellular fate under various stress conditions.

GCN2 and ribosome biogenesis

In the dynamic landscape of cancer cell proliferation, metabolic reprogramming is a pivotal strategy that enhances biomass production, supporting rapid cell growth and division [43]. This shift in metabolism is closely associated with the upregulation of ribosome biogenesis (RiBi), a crucial process for synthesizing the proteins necessary for cellular expansion. GCN2 plays a key role as a modulator of RiBi and cellular metabolic adaptation, particularly in response to the varying nutrient conditions encountered by cancer cells.

GCN2 exerts a dual regulatory effect on ribosome biogenesis in colon cancer cells. Under nutrient scarcity, it curtails the transcription of 47S pre-rRNA, thereby preventing irreversible nucleolar stress and conserving resources essential for maintaining cellular viability during metabolic stress. Conversely, in conditions where nutrients are plentiful, GCN2 activates the transcription of 47S pre-rRNA, facilitating the rapid proliferation typical of cancer cells. This nuanced regulation highlights the potential of combining GCN2 inhibitors with RNA Pol I inhibitors to effectively disrupt cancer cell survival strategies. By shifting their state from cell cycle arrest to apoptosis, these inhibitors can exploit the cells’ dependency on the prevailing nutritional environment [25].

In pancreatic cancer cells, particularly those with activated K-Ras mutations, there is a notable adaptability to nutrient-poor environments, which often involves the scavenging of extracellular proteins to sustain tumor growth. While GCN2 does not directly participate in the scavenging process, it is integral to enabling growth under these challenging conditions(26).

It achieves this through two primary mechanisms: firstly, by modulating translation initiation to prevent ribosome stalling—a common issue in amino acid-depleted conditions [26]. Phosphorylation of eIF2α inhibits the initiation of translation[44], which leads to a decrease in the number of ribosomes actively engaged in translation. Under conditions of leucine deprivation, cells with a GCN2 knockout (GCN2 KO) demonstrate a shift towards polysomes. This accumulation of polysomes in GCN2 KO cells is likely due to ribosome stalling [45]. This indicates that GCN2 is essential for adjusting ribosomal dynamics in response to fluctuations in amino acid availability, thereby preventing the inefficient buildup of stalled ribosomes that occurs in its absence. This modulation is crucial for optimizing the use of limited amino acids for protein synthesis, effectively shifting the bottleneck in translation from elongation to initiation. Secondly, GCN2 selectively enhances the synthesis of proteins necessary for cellular catabolism, such as lysosomal hydrolases, while downregulating the production of anabolic proteins, including those involved in ribosome assembly. This selective synthesis strategy is exemplified by the enzyme cathepsin L, which under GCN2 regulation, demonstrates a high turnover rate in pancreatic cancer cells, indicating its pivotal role in cellular adaptation to nutrient scarcity [26].

Key regulator of mitosis and genomic stability

In the realm of mitosis, GCN2 ensures accurate chromosome alignment and segregation. It achieves this by inhibiting PP1 phosphatases through direct phosphorylation [46]. This inhibition is crucial as it prevents the premature dephosphorylation of mitotic substrates, thus maintaining the integrity of chromosome segregation. This action helps mitigate chromosomal errors and avoid delays in mitosis.

The interaction between GCN2 and mitotic regulation is pivotal in maintaining genomic stability, which is crucial for preventing the propagation of genetic errors in cell division. This role of GCN2 highlights its potential as a target in cancer therapy. Specifically, the combination of GCN2 inhibition with Aurora A kinase inhibitors could amplify therapeutic efficacy, offering a promising strategy for enhancing the effectiveness of cancer treatments [46]. This approach underscores the importance of GCN2 in cancer biology and its potential to improve therapeutic outcomes by ensuring the accuracy and stability of mitotic

Interconnected pathways for ensuring cell survival

In the complex cellular response to amino acid limitation, GCN2 emerges as a crucial sensor, although it is not the only one involved. Research using GCN2-deficient mouse embryo fibroblast cells has demonstrated that GCN2 is essential for the activation of the MEK/ERK pathway under amino acid scarcity. This is particularly evident in hepatocellular carcinoma, where phosphorylation of eIF2 by GCN2 is necessary for the activation of MEK, demonstrating a GCN2-dependent pathway that leads to further phosphorylation of eIF2 and triggers the MEK/ERK signaling cascade [47]. Similar mechanisms have been observed in lung cancer cells, where inhibition of the VCP/p97 complex resulted in the activation of GCN2, which in turn led to ERK activation [16].

Moreover, the inhibition of GCN2, when combined with asparagine depletion through treatments like ASNase, activates the MAPK pathways. This dual mechanism of action induces cell death by triggering both apoptotic and cell survival signals, illustrating a therapeutic potential in leveraging these pathways [48]. In non-small cell lung cancer (NSCLC) cells, deprivation of specific amino acids such as glutamine, arginine, methionine, and lysine activates the AKT pathway through the GCN2/ATF4/REDD1 axis. This leads to the subsequent activation of mTORC2, which promotes a cell survival signal under amino acid scarcity [49]. This complex interplay between GCN2 and other signaling pathways accentuates the intricacy of cellular responses to amino acid limitation and underscores the pivotal role of GCN2 in maintaining cellular homeostasis and adapting to metabolic stress.

Other mechanisms

GCN2′s role in modulating the cellular response to interferons (IFNs) highlights its contribution to the complex interplay between cellular stress responses and tumor immune evasion. Specifically, in melanoma cells under amino acid deficiency, GCN2 mediates the downregulation of IFNAR1, a key receptor for Type 1 IFNs. This mechanism potentially enables tumors to circumvent the anti-tumorigenic effects of endogenous IFNs, suggesting a pathway that could be targeted therapeutically to enhance IFN responsiveness and combat immune evasion [50]. Additionally, GCN2’s role extends into metabolic regulation, as evidenced by its interaction with ethylmalonic encephalopathy protein 1, which is significant in the metastasis of triple-negative breast cancer. This interaction leads to the activation of the GCN2-EIF2α-ATF4 pathway, implicating GCN2 in the regulation of tumor metastasis and offering a novel perspective on targeting metabolic pathways in cancer treatment [51].

GCN2 plays a crucial role in protecting cancer cells by modulating various cellular processes. It responds to environmental stress and amino acid scarcity, maintaining cellular homeostasis through mechanisms such as regulation of protein synthesis and promotion of autophagy. GCN2′s involvement in key processes like angiogenesis, mitochondrial quality control, and ribosome biogenesis underscores its pivotal role in supporting tumor growth and resistance to therapy. Understanding GCN2’s multifaceted role in cancer biology could pave the way for new therapeutic strategies, potentially leading to more effective treatments for various cancers.

GCN2 in drug resistance

GCN2 plays a critical role in cellular adaptation to stress conditions, such as amino acid deprivation and oxidative damage, by regulating key genes like ASNS [52] and xCT [40,53]. This regulation allows cells to survive in challenging environments, potentially leading to drug resistance. Specifically, GCN2 reduces protein synthesis, preventing the accumulation of misfolded proteins and thereby reducing proteotoxic stress. Additionally, GCN2 induces the expression of the 19S subunit of the proteasome, enhancing the cell's capability to degrade damaged proteins. This mechanism contributes to resistance against proteasome inhibitors [54,55], highlighting its significance in the cellular stress response (Fig. 2).

Fig. 2.

Mechanisms of GCN2-mediated drug resistance. The GCN2-ATF4 axis mediates the expression of the ASNS gene, forming a crucial resistance mechanism against ASNase. In the presence of cisplatin, which increases cellular ROS levels, resistance is facilitated by the SLC7A11 transporter, also regulated by the GCN-ATF4 axis, that helps in overcoming oxidative stress. In treatments involving proteasome inhibitors (PIs), GCN2 alleviates amino acid scarcity by reducing protein synthesis while concurrently inhibiting the upregulation of the 19S subunit, contributing to PI resistance. Additionally, the GCN2-ATF4 axis enhances the expression of autophagy-related genes such as ATG3, ATG5, ATG12, and LC3B, helping cells evade apoptosis induced by HAP70 inhibitors. Apatinib's inhibition of the GLS1 gene leads to glutamine deficiency in cells, which activates the GCN2-ATF4 axis, resulting in the upregulation of SLC1A5 and ASNS to compensate for glutamine shortages. Furthermore, GCN2 inhibits JNK-mediated apoptosis, promoting resistance to various chemotherapeutic agents including epirubicin, paclitaxel, vemurafenib, and FGFR1 inhibitors, showcasing its broad impact on drug resistance.

L-asparaginase (ASNase)

The exploration of amino acid metabolism as a therapeutic strategy has revealed promising avenues for developing innovative cancer treatments. At the forefront of this approach is ASNase, which plays a crucial role in managing acute lymphoblastic leukemia (ALL) by depleting asparagine [56]. The effectiveness of ASNase is significantly enhanced or diminished by the modulation of asparagine synthetase (ASNS) expression, which is regulated through the GCN2/ATF4 pathway, highlighting a crucial target for intervention [52]. GCN2 inhibition has shown potential to increase the sensitivity of cancer cells to ASNase, especially in those cells with low baseline levels of ASNS, demonstrating efficacy both in laboratory and clinical settings(48). MYC, a known contributor to cancer metabolism, activates GCN2 kinase, leading to increased ASNS expression and subsequently contributing to ASNase resistance in ALL [18]. Additionally, the inhibition of Bruton's tyrosine kinase, either genetically or pharmacologically with agents like ibrutinib, can disrupt c-Myc function. This disruption reduces the production of uncharged tRNAs and diminishes the activation of the GCN2-ATF4-ASNS pathway, showing significant potential to improve treatment outcomes across various ALL genomic subtypes.

In the context of acute myeloid leukemia (AML), sensitivity to asparagine depletion is notably less compared to ALL, largely due to differential ASNS expression [57].While ASNase holds potential for AML therapy, particularly in combination with methotrexate or high-dose cytarabine, its efficacy generally falls short of what is observed in ALL [58,59]. However, combining ASNase with GCN2 inhibitors has been shown to lower the IC50 values for ASNase in AML cells, suggesting that GCN2 plays a key role in the resistance mechanism to ASNase in AML. This combination approach also showed similar reductions in IC50 values in pancreatic cancer cells [48].

Furthermore, the pharmacological inhibition of the ASNS gene, for instance, with Bisabosqual A (Bis A), has been shown to synergistically enhance the therapeutic potential of ASNase against non-small cell lung cancer (NSCLC). However, this may activate the GCN2-ASNS pathway, indicating potential negative feedback mechanisms at play [60]. This complex interplay between ASNase, GCN2 inhibition, and ASNS expression underscores the nuanced dynamics of amino acid metabolism in cancer therapy, offering a rich landscape for the development of more effective treatments.

Chemotherapeutic agents

Enhanced synthesis of glutathione (GSH), a major intracellular antioxidant, is a common adaptation among cisplatin-resistant tumor cells, posing a significant challenge to the efficacy of cisplatin-based chemotherapy. In gastric cancer, resistance is often associated with elevated expression of SLC7A11 (xCT), a critical enzyme in GSH synthesis. This enzyme is upregulated via the GCN2 pathway, serving to counteract the cytotoxic effects of cisplatin and promote detoxification processes(40, 53). Moreover, Growth Differentiation Factor 15 (GDF15) plays a pivotal role in cisplatin resistance. It is markedly overexpressed in resistant cells and activates the GFRAL-GCN2-eIF2α-ATF4-xCT signaling pathway, which leads to increased intracellular production of GSH. This upsurge in GSH levels effectively neutralizes the cytotoxic impact of cisplatin, enabling cancer cells to thrive despite the presence of the drug [40,61].

In the context of breast cancer, cells that are resistant to other chemotherapeutic agents such as epirubicin or paclitaxel show a similar reliance on the GCN2 pathway for survival. Silencing GCN2 in these cells significantly reduces their clonogenic capacity, which is associated with the upregulation of JNK and PERK expression and activity. This regulatory change is linked to increased expression of FOXO3, a transcription factor that plays a crucial role in cellular stress responses and apoptosis [62].

Proteasome inhibitors

The sensitivity of multiple myeloma (MM) cells to proteasome inhibitors (PIs) is primarily due to their role in disrupting the breakdown of toxic misfolded proteins, which accumulate because of high-level immunoglobulin production inherent to this cancer type [63]. In the treatment of MM, PIs are notably effective at initiating endoplasmic reticulum (ER) stress and subsequent apoptotic signaling pathways [64]. However, the effectiveness of PIs can be compromised by the concurrent activation of a GCN2-dependent amino acid response (AAR) in MM cells [55]. This response may potentially foster resistance to PIs, as it helps cells adapt to the induced amino acid scarcity. Additionally, PI resistance in MM has been correlated with reduced expression of 19S proteasome subunits [54]. Observations of decreased mRNA levels for several 19S subunits in cells recovering from PI treatment might indicate the early stages of resistance development, or possibly reflect the presence of cells that inherently exhibit lower 19S subunit expression before treatment begins [54,55]. Furthermore, inhibiting GCN2 markedly enhances the expression levels of 19S subunits in recovering cells, suggesting a potential association between GCN2 inhibition and the expression of these subunits, thereby possibly impacting PI resistance mechanisms in MM cells.

Small molecules

In breast cancer, the overexpression of Hsp70 is a common adaptation that enables cells to manage misfolded proteins that would otherwise lead to apoptosis or significant dysfunction in normal cells [15]. GCN2 is pivotal in the resistance of breast cancer cells to Hsp70 inhibitors (MAL3–101), with its inhibition significantly reducing the survival of resistant cells. This suggests that breast cancer cells rely on GCN2- induced autophagy as a survival mechanism, particularly when proteostasis is challenged by Hsp70 inhibition, showcasing a sophisticated adaptive strategy [15]. In the context of melanoma and colorectal cancer cells harboring the BRAF mutation, short-term treatment with BRAF kinase inhibitors, such as vemurafenib, leads to the activation of the GCN2 kinase. This activation induces ATF4, a critical factor that may enhance cell survival in the face of BRAF inhibition, illustrating how cancer cells adapt to targeted therapies [65].

Further, in human non-small cell lung cancer (NSCLC) cells, Apatinib, a tyrosine kinase inhibitor targeting VEGFR-2, disrupts glutamine metabolism. It achieves this by inhibiting GLS1 and activating the amino acid response (AAR) pathway through GCN2/eIF2α/ATF4. This leads to overexpression of ATF4, which increases the expression of SLC1A5 and ASNS, promoting glutamine consumption and metabolization, another example of adaptive metabolic reprogramming in response to therapy [66]. Additionally, GCN2 plays a significant role in drug resistance to treatments such as fibroblast growth factor receptor 1 (FGFR1) inhibitors. In T-ALL cells, resistance to FGFR1 inhibitors arises from the activated GCN2-ATF4 pathway, which sustains mTORC1 activation, contributing to ongoing drug resistance [67].

These insights into the modulation of amino acid metabolism and stress response pathways, particularly through the GCN2 kinase, are crucial in understanding the efficacy and resistance mechanisms of various cancer treatments. The activation of GCN2 and its downstream effects on ATF4 expression and nutrient transporter regulation are central to the cellular response to these therapies. Targeting these pathways offers promising strategies for enhancing the effectiveness of cancer treatments and overcoming drug resistance, underscoring the importance of continued research in this area to improve patient outcomes across a broad spectrum of malignancies.

GCN2 induced cell death

GCN2 does not always serve as a protective mechanism for cell survival. In certain scenarios, such as when the stress response to amino acid deprivation (leucine, cystine, or glutamine) becomes uncontrolled, it can initiate pathways leading to programmed cell death [21,[68], [69], [70]] (Fig. 3). This underlines the complex roles of GCN2 in cancer biology and emphasizes the importance of GCN2-mediated mechanisms in enhancing the efficacy of specific pharmacological agents.

Fig. 3.

Mechanisms of apoptosis modulated by GCN2. GCN2 triggers programmed cell death through the GCN2-ATF4-CHOP pathway when nutrient deprivation is irreversible, a process also implicated in some drug-induced cell deaths. Additionally, deprivation of leucine activates the GCN2 pathway, which represses Sterol Regulatory Element-Binding Protein 1C (SREBP1C) and impacts the expression of Fatty Acid Synthase (FASN), essential for lipid biosynthesis and critical in cancer cell growth. Deprivation of cystine elevates CHAC1 gene expression via the GCN2-ATF4 axis, leading to glutathione (GSH) degradation, which intensifies oxidative stress and results in cell death. Lack of glutamine increases susceptibility to TRAIL-induced apoptosis by upregulating TRAIL receptor 2 (TRAIL-R2) through the GCN2-ATF4 pathway, triggering apoptosis when these cells encounter TRAIL. Certain pharmacological agents such as cortisone activate GCN2, fostering the formation of stress granules (SGs) and inducing cell death. Methotrexate (MTX) stimulates GCN2, enhancing HMGB1 expression, whose release into the extracellular space can initiate immunogenic cell death (ICD). Furthermore, MTX and doxorubicin (DOXO) promote CALR gene expression through the GCN2-ATF4 pathway, increasing levels of calreticulin (CRT). CRT's translocation to the cell surface serves as an "eat me" signal, enabling ICD. This intricate interaction of GCN2 with cellular stress responses highlights its potential as a therapeutic target in cancer treatment.

In A549/8 lung carcinoma cells, reduced glutamine levels disrupt cell cycle distribution and significantly induce apoptosis/necrosis. This effect is driven by a GCN2/eIF2α-dependent mechanism that increases CHOP and GADD34 mRNA expression, highlighting glutamine's critical role in cell survival and the severe impact of its deprivation [21]. Similarly, glutamine deprivation in TNBC cells increases their susceptibility to TRAIL-induced apoptosis, linked to the upregulation of TRAIL receptor 2 (TRAIL-R2/DR5) and the downregulation of FLICE-inhibitory protein (FLIP), with GCN2 kinase activation playing a role in this regulatory mechanism [68].

In breast cancer cells, the absence of leucine markedly reduces cell viability and proliferation and induces apoptosis. This process is regulated by the GCN2 pathway, which influences the expression of Fatty Acid Synthase (FASN) by repressing Sterol Regulatory Element-Binding Protein 1C (SREBP1C), crucial in lipid biosynthesis and cancer cell growth [69]. In triple-negative breast cancer (TNBC) cells, cystine deprivation activates the GCN2-eIF2α-ATF4 signaling cascade, leading to increased CHAC1 expression. The degradation of glutathione (GSH) by CHAC1 heightens oxidative stress, leading to cell death through mechanisms such as necroptosis and ferroptosis, underscoring TNBC cells' vulnerability to cystine deficits and oxidative damage(70).

Apoptosis modulated by GCN2 is crucial for the action of specific drugs, with ATF4-mediated gene transcription being a common pathway. The GCN2-ATF4-CHOP axis is central to apoptosis induced by agents such as borrelidin [71], pegylated-human-arginase I [72], proline-competitive phosphoribosylpyrophosphate synthetase inhibitors [73], and Na+/K+ATPase ligands [74], which ultimately lead to cell death. Moreover, drugs like methotrexate (MTX) [75,76] and doxorubicin [75] have been shown to induce apoptosis in melanoma and prostate cancer cells by exploiting the eIF2α phosphorylation pathway via GCN2 and PERK. This induction not only compromises cancer cell survival but also triggers an immunogenic response, potentially enhancing therapeutic efficacy by activating the immune system against tumor cells. This involves GCN2 activation, facilitating the translocation of calreticulin (CRT), an endoplasmic reticulum resident protein, to the cell surface [75,76], and the extracellular release of the high mobility group box-1 (HMGB1) protein [76], key events in initiating immunogenic cell death (ICD) in melanoma and prostate cancers. Additionally, MTX treatment leads to the activation of dendritic cells in mice, which is diminished when GCN2 is silenced [76].

Cytoplasmic stress granules (SGs) are non-membrane-bound cellular aggregates that form under various stress conditions, including those induced by vinca alkaloids such as vinorelbine (VRB). In U2OS cells, cortisone increases VRB-induced cell death and reduces the population of cells trapped in mitotic catastrophe, mediated by the core SG proteins G3BP1 and G3BP2. Cortisone affects SG dynamics and the integrated stress response (ISR) signal transduction pathways, ultimately influencing cell viability and death via GCN2 [77].

Chronic activation of the ISR can lead to apoptosis, suggesting that sustained activation of the GCN2 pathway might also inhibit tumor growth [78]. HC-7366, a novel orally bioavailable activator of GCN2 kinase, has demonstrated efficacy in this regard. HC-7366 monotherapy showed significant tumor growth inhibition in preclinical cancer models, including colorectal (78–95 % inhibition), head and neck (33 % regression), sarcoma (80 % inhibition), prostate (65 % inhibition), fibrosarcoma (84 % inhibition), and acute myeloid leukemia (98 % inhibition) [79,80].

HC-7366 treatment activated the ISR in tumors from treated mice, as evidenced by the induction of ATF4 target genes such as ASNS, PSAT1, and JUN [80]. Additionally, the treatment promoted the expression of the proapoptotic protein PUMA and led to a reduction in the levels of HIF1α and HIF2α. Metabolomic analysis of tumors treated with HC-7366 revealed significant changes in several metabolites associated with amino acid metabolism, oxidative stress, the urea cycle, and pyrimidine biosynthesis, indicating a broad metabolic impact on tumor cells [80].

In breast cancer models, HC-7366 has shown consistent anti-metastatic efficacy, reducing lung metastases by approximately 75 % across various studies. This anti-tumor effect is associated with a significant decrease in Ly6G+ polymorphonuclear myeloid-derived suppressor cells in the lungs, spleen, and blood, suggesting that HC-7366 may exert its anti-metastatic effects through modulation of the tumor immune microenvironment [81].These findings indicate that activating the GCN2 pathway with HC-7366 could be an effective strategy for cancer therapy.

In summary, the exploration of GCN2′s role in cancer therapy reveals a complex interplay between cellular stress responses and tumor cell viability. Modulating GCN2 and related pathways offers a multifaceted approach to cancer treatment, from direct induction of apoptosis to enhancement of immunogenic responses. As research progresses, the development of targeted therapies exploiting GCN2′s unique role in cellular stress responses holds promise for advancing cancer treatment strategies, highlighting the need for ongoing investigation into these mechanisms.

Role of GCN2 in pan-cancer

GCN2 is increasingly recognized for its potential in clinical applications, particularly in the treatment of various cancers such as papillary renal cell carcinoma (PRCC), head and neck Squamous cell carcinoma (HNSCC), and pancreatic ductal adenocarcinoma (PDAC) [[82], [83], [84]]. In PRCC, high GCN2 expression correlates with aggressive disease features, including larger tumor sizes, advanced TNM stages, high Fuhrman grades, and lymph node metastasis. This overexpression establishes GCN2 as a critical prognostic marker, associated with lower overall and progression-free survival rates [82]. Studies also show a significant prevalence of GCN2 in HNSCC, where most tumors exhibit moderate to strong expression levels, underscoring its role in tumor biology [83]. Further research into PDAC has identified a subtype with constitutive GCN2 activation, enhancing ATF4 signaling and increasing asparagine production. This suggests a novel metabolic dependency where exogenous asparagine might conserve aspartate reserves under stress, highlighting GCN2 inhibitors as promising therapeutic avenues [84].

Analysis of the TCGA dataset reveals high GCN2 expression in tumor tissues across 13 cancer types. To address the lack of normal adjacent tissue samples in some cancers, we merged GTEx normal tissue data with TCGA data. This merged dataset included 29 cancer types, with GCN2 expression notably high in 26 of these, indicating its significant role in tumor pathogenesis (Fig. 4A). In the TCGA dataset, high GCN2 expression correlates with poor prognosis in KIRC but with better prognosis in LGG and MESO (Fig. 4B). Additional analysis using the GEO database revealed that high GCN2 expression is associated with poor prognosis in AML, colon cancer, and pancreatic cancer, but with better prognosis in lung cancer, MM, and ovarian cancer, suggesting that GCN2′s impact on prognosis varies by tumor type (Fig. 4C).

Fig. 4.

GCN2 Pan-Cancer Analysis. GCN2 shows high expression in 13 cancer types according to the TCGA database. A combined analysis with the GTEx database indicates that GCN2 is highly expressed in 26 out of 29 cancer types (A). Within the TCGA dataset, high GCN2 expression correlates with poor prognosis in kidney renal clear cell carcinoma (KIRC) but predicts better outcomes in brain lower grade glioma (LGG) and mesothelioma (MESO) (B). Further studies using the GEO database demonstrate that elevated GCN2 levels are associated with poorer prognosis in acute myeloid leukemia (AML), colon cancer, and pancreatic cancer, while they are linked to better prognosis in lung cancer, multiple myeloma (MM), and ovarian cancer, highlighting the variable impact of GCN2 across different tumor types (C). Analysis of immune cell infiltration across 33 cancer types reveals that in certain cancers, GCN2 is positively correlated with immunosuppressive cells such as Tregs (D). Additionally, in the analysis of immune checkpoints, GCN2 expression consistently shows positive correlations with the expression of most immune checkpoints (E). For a detailed methodology of the pan-cancer analysis, please refer to Appendix 1. Cancer types: Adrenocortical Carcinoma (ACC), Bladder Urothelial Carcinoma (BLCA), Breast Invasive Carcinoma (BRCA), Cervical Squamous Cell Carcinoma and Endocervical Adenocarcinoma (CESC), Cholangiocarcinoma (CHOL), Colon Adenocarcinoma (COAD), Diffuse Large B-Cell Lymphoma (DLBC), Esophageal Carcinoma (ESCA), Glioblastoma Multiforme (GBM), Head and Neck Squamous Cell Carcinoma (HNSC), Kidney Chromophobe (KICH), Kidney Renal Clear Cell Carcinoma (KIRC), and others. Key immune checkpoint-related transcripts: Sialic Acid Binding Ig Like Lectin 15 (SIGLEC15), Indoleamine 2,3-Dioxygenase 1 (IDO1), Cluster of Differentiation 274 also known as PD-L1 (CD274), Hepatitis A Virus Cellular Receptor 2 also known as TIM-3 (HAVCR2), Programmed Cell Death Protein 1 also known as PD-1 (PDCD1), Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA4), Lymphocyte Activation Gene 3 (LAG3), and Programmed Cell Death 1 Ligand 2 also known as PD-L2 (PDCD1LG2).

Further analysis revealed a negative correlation between GCN2 and various immune cells in 33 cancer types, including T cell NK, T cell CD4+ central memory, T cell CD4+ effector memory, T cell CD4+ Th1, and B cell plasma. In some cancers, GCN2 positively correlates with immunosuppressive cells such as Tregs (Fig. 4D). This correlation may stem from GCN2′s role in sensing amino acid deprivation, such as tryptophan depletion—a common feature in the tumor microenvironment. This depletion can trigger a cellular stress response, leading to G1 phase cell cycle arrest in T cells, mediated by GCN2 activation. Thus, GCN2 may contribute to the immunosuppressive environment in tumors by impairing the proliferation and function of effector T cells while potentially favoring Treg expansion [85].

However, it is important to highlight that the effects of GCN2 on the tumor immune microenvironment remain controversial. T cells lacking GCN2 exhibit proliferation defects during activation [86], and CD8+ T cells devoid of GCN2 display impaired anti-tumor immunity in a murine glioma model [87]. Interestingly, activating effector CD8+ T cells with the GCN2 agonist halofuginone (halo) in vitro enhances their amino acid starvation response, oxidative metabolism, and effector functions. Mechanistically, autophagy and the CD98-mTOR signaling axis are critical downstream mediators of the phenotypes induced by halo treatment. The adoptive transfer of halo-treated CD8+ T cells into tumor-bearing mice leads to robust tumor control and curative responses. In vivo, halo-treated T cells, in conjunction with agonistic 4–1BB antibodies, suppress tumor growth in immune therapy-resistant mouse models. Crucially, treating human CD8+ T cells with halo also results in similar metabolic and functional reprogramming [88]. In immune checkpoint analysis, GCN2 expression correlated positively with the expression of most immune checkpoints (Fig. 4E). For instance, in LAML, patients with high GCN2 expression generally showed high expression of CD274, HAVCR2, PDCD1LG2, and SIGLEC15. In COAD patients, CD274, CTLA4, HAVCR2, PDCD1LG2, and TIGIT were positively correlated with GCN2. In PAAD, all immune checkpoints except SIGLEC15 were positively correlated with GCN2. These findings suggest that GCN2 plays a significant role in the tumor immune microenvironment and may influence responses to certain immunotherapies.

GCN2 inhibitors

Recent advancements in cancer therapy have emphasized the potential of various GCN2 inhibitors (Table 1), particularly Triazolo[4,5-d] pyrimidines (TAP), for their potent efficacy in targeting GCN2 within cancer cells. Notable compounds such as GCN2-IN-1 (A-92) [24,46,77,[89], [90], [91], [92], [93]], TAP20 [94,95], GCN2iA(48), GCN22iB [10,12,48,55,96,97] and Compounds 1 and 2 [98] have demonstrated significant inhibition of GCN2, marking a pivotal development in targeted cancer treatment strategies. Additional inhibitors include GCN2-IN-6 [14,99,100], a sulfonamide derivative, and broad-spectrum effects observed with small molecule drugs like GZD824 [101], further expanding the arsenal against cancer through diverse mechanisms of action.

Table 1.

Results of In Vivo and In Vitro Preclinical Studies on GCN2 Inhibitors.

Drug	Chemical structure	IC50 in Enzyme assay	IC50 in cell assay	IC50 of other targets	In vitro experiments				In vivo experiments				Ref
					Cell line	concentration	Main observation	Special	Mouse model	Dosage	Main observation	Special
TAP20 (A245)		17 nm	0.29–0.56um	GSK3α IC50 at 1.7 nm; GSK3β IC50 at 12 nm; CDK9/cyclinD1 IC50 at 180nm	143B/HCC1806	3um	Reduced the proliferation of ASCT2 KO HCC1806 cells when combined with concurrent silencing of SNAT1 or LAT1.	N.A	N.A	N.A	N.A	N.A	94
					MDA-MB-231/MDA-MB-468/HPAFII/SKOV3/OVCAR3	>1 µM	Suppressed GCN2.	N.A	N.A	N.A	N.A	N.A	95
					143B	1–3um	Dramatically reduced matrix-dependent invasion.	N.A	N.A	N.A	N.A	N.A	95
					MB-231/HPAF-II	3um	Inhibitors of proteostasis, MEK–ERK pathway, pan-CDK inhibitors, flavopiridol, and seliciclib, were potently synergistic with TAP20.	N.A	N.A	N.A	N.A	N.A	95
AST-0513		15nm	N.A	N.A	SNU-1041	2 μM	Induced cell cycle arrest and apoptosis under amino acid deprivation conditions.	N.A	Mice	N.A	Adverse effects on organs were not observed in mice.	N.A	83
					Detroit-562/FaDu/SNU-1041/SNU-1066/SNU-1076	1um	Inhibits the GCN2-ATF4 pathway, significant anti-proliferation effect.	N.A	N.A	N.A	N.A	N.A	83
GCN2-IN-1		<0.3um	0.3–3um	N.A	Hela	0.63–10μM	Effectively suppressed GCN2 activity, led to a decrease in basal eIF2α phosphorylation levels.	At low concentrations ranging from 0 to 0.31 μM, there is a paradoxical increase in GCN2 phosphorylation without noticeable activation of downstream signaling pathways. At concentrations between 10 and 40 μM, a dose-dependent increase in eIF2α phosphorylation was observed, which was associated with the activation of PERK kinase.	N.A	N.A	N.A	N.A	89
					Mic60-low PC3	10um	Inhibited proliferation and induced Annexin V-associated apoptosis and caspase-dependent cell death, particularly in Mic60-silenced PC3 cells compared to control cultures.	N.A	N.A	N.A	N.A	N.A	24
					U20s	4um	Blocked the assembly of SGs induced by mRNA transfection.	N.A	N.A	N.A	N.A	N.A	90
					U20S	4um	Blocked the assembly of SGs induced by Cortisone.	N.A	N.A	N.A	N.A	N.A	77
					HeLa	2um	Disrupts the timing and phosphorylation levels of crucial mitotic regulators, leading to improper chromosome alignment, chromosome missegregation, an increased incidence of tripolar spindles, and delayed mitotic progression. This effect is significantly enhanced when combined with Aurora A inhibition, resulting in exacerbated mitotic abnormalities and increased cell death.	N.A	N.A	N.A	N.A	N.A	46
					A549	10um	Simultaneous use PERK inhibitor rescued the negative effect of CuET on translation.	N.A	N.A	N.A	N.A	N.A	93
					MDA-MB 231	1um	Reduced mutp53 protein levels.	N.A	N.A	N.A	N.A	N.A	91
GCN2-IN-6		1.8nm	9.3nm	PERK IC50 at 230nm	CCRF-CEM	9.3um	Antiproliferative activity in combination with asparaginase.	N.A	CCRF-CEM ALL xenograft model	3 mg kg-1	Suppression of GCN2 pathway activation with asparaginase treatment.	N.A	99
					OCI-LY	7.5um	Inhibited cell proliferations, combined treatment with SIRT3 inhibitor (YC8–02) yielded significantly greater anti-lymphoma effect.	N.A	N.A	N.A	N.A	N.A	100
					Karpas	10um	Inhibited cell proliferations, combined treatment with SIRT3 inhibitor (YC8–02) yielded significantly greater anti-lymphoma effect.	N.A	N.A	N.A	N.A	N.A	100
					Hela	0.5um	Reverse the SLC7A11 expression induced by KYN hinders cystine uptake.	N.A	N.A	N.A	N.A	N.A	14
Triazolo[4,5-d]pyrimidine derivatives Compound 1		47.6nm	N.A	PKR IC50 at 119.3nm	National Cancer Institute NCI-60 Tumor Cell Screening Program	10um	Both 1 and 2 displayed cytotoxic activity against cells within the different types of cancers tested. Leukemia and breast cancer cell lines comprised the only cancer groups that were uniformly sensitive to both 1 and 2, with the greatest sensitivity seen in the leukemia SR cell line.	N.A	N.A	N.A	N.A	N.A	98
Triazolo[4,5-d]pyrimidine derivatives Compound 2		18.6nm	N.A	PKR IC50 at 39.9nm	National Cancer Institute NCI-60 Tumor Cell Screening Program	10um		N.A	N.A	N.A	N.A	N.A	98
GCN2iA		4nm	N.A	GSK3β>75 % inhibition at 1um	U20S	1um	Suppressed GCN2 and eIF2α phosphorylation along with ATF4 expression in amino acid-deficient conditions.	Unfavorable pharmacokinetic profile in vivo.	N.A	N.A	N.A	N.A	97
					CCRF-CEM/MOLT-4/SU86.86/AsPC-1	1um	Enhance sensitivity to ASNase.	N.A	N.A	N.A	N.A	N.A	97
GCN2iB		2.4nm	N.A	MAP2K5/STK10/ZAK >95 % inhibition at 1μmol/L	CCRF-CEM/MV-4–11/SU.86.86	1um	Combined treatment with ASNase showed antiproliferative effect.	In the absence of external stress, low concentrations (10–100 nm) have the potential to activate GCN2.	CCRF-CEM ALL xenograft model	10 mg kg-1 twice daily	Cotreated with ASNase suppressed GCN2 pathway, elicited potent antitumor activity.	No weight loss	97
					LNCaP/C4–2B/22Rv1/PC-3	500 nM to 10µM	Significantly impeded the proliferation.	N.A	MV-4–11 and SU.86.86 xenografts	10 mg kg-1 twice daily	Robust antitumor activity of the combination of GCN2iB and ASNase.	SU.86.86 model showed a modest cachectic body weight reduction	97
					LNCaP/C4–2B/22Rv1/PC-3	2um	Decreased the expression of SLC genes, led to a reduction in GCN2 activation.	N.A	Disseminated ALL xenograft model (MOLT-3).	10 mg kg-1 twice daily	The combination of ASNase and GCN2 inhibitor (GCN2iB) provided a survival advantage compared to the vehicle-treated control.	No weight loss	97
					Clone cell line from pancreatic tumors from KPC mice with constitutively active GCN2 metabolic	1/2/5 um	Sufficient to reduce the expression of ATF4. Inhibiting the proliferation of a sensitive clone that acquires oligomycin resistance from co-culturing with a resistant clone.	N.A	Cancer cell (LNCaP/22Rv1)/ patient derived androgen-sensitive tumor xenograft models	30 mg kg-1 twice daily for durations between 2 and 6 weeks	Pronounced anti-tumor efficacy across all four models.	No weight loss	12
					HCC	1um	Increased EdU incorporation, reduced accumulation of p21, decreased expression of SLC7A1, lowered SESTRIN2/SESN2 mRNA and protein levels, activated mTORC1, and enhanced autophagy.	N.A	Subcutaneous SNU-398 xenograft tumors in mice	10 mg kg-1	Combined treatment with arginine deprivation induced the expression of BCL2 and SASP-associated genes, as well as β-galactosidase staining in xenograft tumors. Additionally, when used in conjunction with ABT-263, this treatment significantly reduced tumor growth in arginine-restricted mice, specifically in the SNU-398 tumor model.	No weight loss	12
					HCC/ PC-3/SKLMS/MDA-MB-231	1um	Combined treatment of Arg-deprivation with GCN2iB and the pan-BCL2 inhibitor ABT-263 significantly increased the number of apoptotic cells.	N.A	Hepa55.1c subcutaneous xenografts	10 mg kg-1	Combined with ABT-263 further decreased tumor growth in Arg restricted mice.	No weight loss	84
					HT-1080	1um	Reverse CHAC1 induction and the corresponding cell death in response to cystine deprivation.	N.A	HepaMP-9–1/SNU-398 xenografts	10 mg kg-1	Combined Arg deprivation harboring tumors and also developed senescent cells.	No weight loss	10
					RPMI- 8226/OPM2/ NCI-H929	1um	Enhanced the carfilzomib-induced reduction on day7.	N.A	HepaMP-9–1 xenografts	10 mg kg-1	Increased levels of VEGF.	No weight loss	55
					RPMI-8226	1um	After CFZ treatment on day 5, deregulated DDIT3, ATF3, CHAC1, SESN2, SLC7A11, and TRIB. Increased 20S and particularly 19S proteasome-subunit transcript levels.	N.A	N.A	N.A	N.A	N.A	55
GZD824		0.3–1uM	N.A	BCR/ABL IC50 at 0.34 nM	HT-1080	2um	Prevented ATF4 induction and phosphorylation of GCN2 at T899 during halofuginone stress.	At low concentrations (approximately 0.1 μM), GZD824 induces eIF2α phosphorylation and ATF4 expression in a GCN2-dependent manner under normal nutrient conditions. However, at higher concentrations (0.3–1 μM), GZD824 inhibits ATF4 induction even under GCN2-activating stress conditions.	N.A	N.A	N.A	N.A	101

Interestingly, some drugs exhibit different effects on GCN2 at varying concentrations. For example, at low concentrations, GCN2iB can paradoxically activate GCN2, enhancing the phosphorylation of eIF2 and ATF4 expression in HEK293 cells, illustrating its potential as a nuanced therapeutic agent for modulating stress responses [48]. Similarly, GCN2-IN-1 exhibits a dual action in HeLa cells, where it paradoxically increases GCN2 phosphorylation at low doses without affecting downstream signals, while higher doses suppress GCN2, reducing eIF2α phosphorylation and further activating PERK kinase to trigger the ISR [89]. Extensive research on GCN2 inhibitors has significantly advanced their potential in cancer therapy by elucidating their impact on cancer cell behavior and aiding in the development of targeted treatments. However, their clinical application is still evolving. Primarily designed for in vitro use, in vivo studies are limited to specific compounds like GCN2iB, AST-0513, and GCN2-IN-6. Issues regarding their specificity and potential off-target effects remain prominent. Although these inhibitors effectively block ISR under amino acid scarcity in vitro, their effect on GCN2′s role in cell proliferation is unclear. The promising prospects of combination therapies involving GCN2 inhibitors warrant further exploration.

Conclusion

GCN2 plays diverse roles in tumor cells depending on the context, primarily recognized for its protective functions in tumor cells under stress. While inhibiting GCN2 alone does not significantly impact tumor cell growth, its influence on downstream gene expressions has been linked to mechanisms of drug resistance across various cancers. This insight suggests that a combined approach targeting GCN2 could provide an effective strategy to counter drug resistance in cancer therapy.

Recent studies have further explored the pro-apoptotic pathways activated by GCN2, illuminating how this kinase might be harnessed to induce tumor cell death. Several clinical trials are currently investigating the activation of GCN2 through small molecule drugs such as HC-7366 (NCT06234605, NCT05121948, NCT06285890) and NXP800 (NCT05226507). HC-7366 is being tested in combination with Azacitidine and Venetoclax for treating AML and with Belzutifan for renal cell carcinoma. Meanwhile, NXP800 is under evaluation as a monotherapy for advanced ovarian cancer. In the field of immunotherapy, the role of GCN2 is drawing attention due to its significant impact on immune cell functions. Targeting GCN2 to boost the efficacy of immune cell therapies presents a promising path for advancing cancer treatment.

In conclusion, GCN2 is a multifaceted kinase responsive to cellular stress, influencing tumor progression, treatment resistance, and potentially leading to cell death under certain circumstances. Understanding its complex role in cancer biology is crucial for developing targeted therapeutic strategies that exploit its unique functions. Continued research into GCN2 could unlock new possibilities for cancer treatment, particularly in overcoming drug resistance and enhancing the efficacy of immunotherapies.

Funding

This study was funded by the Hangzhou municipal biomedical and health industry development support special science and technology project, with grant numbers 2021WJCY096, 2021WJCY243, 2021WJCY245, and 2021WJCY246.

CRediT authorship contribution statement

Can Chen: Writing – original draft, Validation, Conceptualization. Yaping Xie: Writing – original draft, Conceptualization. Shenxian Qian: Validation, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.