CHK1 belongs to the Ca²⁺/calmodulin-regulated Ser/Thr kinase family (CAMKK). It is a protein consisting of 476 amino acids with a molecular weight of 54 kDa [18,19]. All homologs are characterized by an N-terminal kinase domain (KD; residues 1–289) and a C-terminal kinase-associated regulatory region (KA1; residues 375–476), which are connected by a flexible linker region (residues 261–340) [20,21] (Fig. 2A).

The KD exhibits sequence homology and structural conservation across different species [20]. KD is characterized by a bilobal structure containing the ATP-binding site at its centre [20]. The two lobes of the KD are linked by β6′ of the hinge region and held together at the lobe interface by an extensive hydrogen-bond network involving β6′, the loop connecting αC and β4 of the N-terminal lobe, and strands β7 and β8 of the C-terminal lobe (Fig. 2A). The KD domain adopts an active conformation in which the ATP binding site, the catalytic residues, and the activation loop are well ordered [20,21]. In contrast to the activation loop of other kinases [22,23] the activation loop of KD does not require phosphorylation.

The KA1 regulatory domain was first described in yeast [24]. Despite the presence of significant structural homology, the degree of sequence identity among species is relatively low [18,25,26]. The conserved features of the KA1 domain include a four-stranded β-sheet flanked by two α-helices that form a concave hydrophobic core (βαββββα motif) [18,27] (Fig. 2A).

A physical interaction has been observed between KD and KA1 [28], suggesting a negative impact of KA1 on the kinase activity of KD. Indeed, isolated KD exhibits over 20-fold greater activity towards various substrates, such as Cdc25C, than the recombinant full-length CHK1 protein [20]. However, the absence of the KA1 region at the C-terminus results in a complete loss of CHK1 biological function. Indeed, in response to DNA damage KA1 positively regulates KD. This has been demonstrated by the fact that upon DNA damage-induced phosphorylation, intramolecular interactions between KD and KA1 are disrupted [18], thereby determining a further activation of CHK1 [20,25,27]. Interestingly, this phosphorylation involves residues located within the activation loop and requires KD activity. This suggests that the process of autophosphorylation plays a role in the regulation of CHK1 function [18,24,28,29,30,31,32] (Fig. 2B).

Ataxia telangiectasia and Rad3-related (ATR) kinase and CHK1 have been demonstrated to mediate cell cycle arrest in response to replication fork stalling or DNA damage [33,34,35,36]. During replication stress (RS) or single-stranded DNA (ssDNA) accumulation, the replication protein A (RPA) coats ssDNA, thereby facilitating ATR recruitment and activation via ATR-interacting protein (ATRIP) [19,37,38,39]. Once localized on the SSDNA, ATR phosphorylates RPA32 at Ser33, followed by cyclin-dependent kinases 1/2 (CDK1/2)-mediated phosphorylation at Ser23 and Ser29 [40]. The activation of ATR is further enhanced by topoisomerase 2-binding protein 1 (TOPBP1) and the RAD9-RAD1-HUS1 (9-1-1) complex [41,42,43]. Furthermore, the Ewing’s tumour-associated antigen 1 (ETAA1) binds to RPA and promotes ATR activation [44,45]. Additionally, the TIMELESS/TIPIN complex enhances the interaction between Clastin and RPA [46]. In the initial phase of the DDR, ATR also phosphorylates histone H2AX at Ser319, leading to the formation of γH2AX, a key marker of DNA damage [47].

The accumulation of the ATR/ATRIP complex is essential to induce the ATR-dependent CHK1 phosphorylation at Ser317 and Ser345, both of which are located within the C-terminal KA1 domain (Table 1) [19,21,39,48,49,50,51,52,53]. Phosphorylation at Ser317 is a critical regulatory event that is primarily mediated by the ATR kinase in response to RS and genotoxic damage. This post-translational modification plays an essential role in activating the CHK1 checkpoint functions, thereby preventing premature mitosis and ensuring proper cell cycle arrest and DNA repair [54]. Specifically, phosphorylation at Ser317 is required for the subsequent phosphorylation of other key residues, including Ser296 and Ser345 (Table 1). Collectively, there residues promote full CHK1 activation during the DDR. Notably, the mutation of Ser317 impairs checkpoint activation and replication fork progression yet does not necessarily affect cell survival in the absence of stress. This observation suggests that this site may have distinct functional roles in addition to DDR [54,55,56,57,58,59,60]. CHK1 phosphorylation at Ser345 facilitates its retention in the nucleus and enhances its stability. This phosphorylation is primarily mediated by the ATR kinase in response to RS and DNA damage and serves as the final and essential modification for full activation of the CHK1 checkpoint functions, including cell cycle arrest and DNA repair [19,61,62]. Indeed, CHK1 phosphorylated at Ser345 accumulates both in the nuclear pool and, more prominently, at chromatin regions containing RPA-coated ssDNA [19,61,63].

CHK1 can also be phosphorylated at Ser286 and Ser301 by CDK1 (Table 1) [54]. These post-translational modifications facilitate full CHK1 activation, ensuring proper checkpoint function [64]. Following the checkpoint signalling, the Ser345-phosphorylated CHK1 located within nucleoplasm undergoes ubiquitination and proteasomal degradation, a process which is mediated by CRL4–DDB1–CDT2. In contrast, the cytoplasmatic cytoplasmic CHK1 is degraded by CRL1–SKP1–Fbx6 [63].

Although most studies on CHK1 activity focus on the phosphorylation of Ser296, Ser317, and Ser345, it is well established that the phosphorylation of additional residues is also critical for CHK1 regulation (Table 1) [19,49,52,56].

The Ser280 phosphorylation is catalysed by several kinases including the Akt serine/threonine kinase (AKT) [65,66], ribosomal s6 kinase (RSK) [67], proviral integration site for moloney murine leukaemia virus-1 (PIM1) [68], and AMP-activated protein kinase (AMPK) [69]. The functional outcome varies depending on the upstream kinase, cell type, and growth conditions [19]. For instance, during the G0/G1 transition, Ser280 is phosphorylated by the mitogen-activated protein kinase (MAPK)/MAPK-activated protein kinase-1 (also known as 90 kDa RSK or p90RSK) downstream of the Ras–MAPK cascade [39,67], facilitating CHK1 translocation from the cytoplasm to the nucleus and maintaining genome integrity during cell cycle progression [67]. Consequently, nuclear accumulation of the Ser280-phosphorylated CHK1 accelerates the subsequent phosphorylation at Ser296 and Ser345 CHK1 residues [67]. In contrast, AMPK-dependent phosphorylation occurs during glucose deprivation, promoting recognition by β-TrCP proteins, which mediate ubiquitination and degradation of CHK1 [69].

Further CHK1 residues that undergo phosphorylation are Ser286 and Ser301, which are targeted during mitosis by CDK1 [54]. These phosphorylation events promote CHK1 nuclear export at the end of G2 phase, thereby alleviating CHK1-mediated inhibition of CDK1 and supporting mitotic progression [39,70,71,72,73]. Interestingly, both CDK1 and CDK2 can phosphorylate Ser286 and Ser301 during the DDR, in addition to the ATR-dependent phosphorylation of Ser317 and Ser345. This finding suggests that proliferative and checkpoint signals may coexist [59,66,71].

It has been demonstrated that Thr378 and Thr382 are additional sites of CHK1 autophosphorylation [32]. These residues are located within the KA1 domain, in a conserved region associated with ubiquitination and proteasomal degradation [74]. Despite this localisation, the autophosphorylation of Thr378 and Thr382 is associated with both CHK1 activation and degradation. On the one hand, these two residues stabilize the kinase in an active conformation. Conversely, they unveil a region that is recognised by the CRL1–FBX6 complex, which targets the protein for proteasomal degradation [32].

CHK1 plays two seemingly opposing functions: cell cycle arrest and proliferation. These are highly context-dependent and modulated, at least in part, by the mitogen-activated protein kinase (MAPK)/p90RSK pathway. The MAPK/p90RSK acts downstream of the Ras pathway [13,67]. The process of cell proliferation depends upon the timely reception of extracellular growth factor signals, which are primarily transmitted through two core pathways downstream of receptor tyrosine kinases (RTKs). The phosphatidylinositol 3-kinase (PI3-K)/Akt/protein kinase B (PKB) pathway and the Ras/MAPK cascade, composed of Raf/MAPK kinase (MEK)1/2/extracellular signal-regulated kinase (ERK)1/2, are two such pathways [116,117]. p90RSK, a Ser/Thr kinase, acts downstream of this latter pathway. Upon growth factor stimulation, p90RSK is phosphorylated by ERK1/2 on multiple residues by several kinases and thereby activated. The activation of p90RSK results in the phosphorylation of CHK1 at Ser280, thereby promoting its nuclear retention [67], where it undergoes further phosphorylation at Ser296 and Ser345 [61,72,118] (Fig. 3). As outlined above, the phosphorylation of Ser296 and Ser345 residues is essential for the DDR. Interestingly, cell exposure to DNA-damaging agents such as ultraviolet (UV) light, ionising radiation (IR), and hydroxyurea (HU) also induces the p90RSK-dependent phosphorylation of CHK1 at Ser280, which is subsequently phosphorylated at Ser296 and Ser345 to promote the DDR [119,120]. This finding indicates a convergence between the ATR-CHK1 and p90RSK pathways, thereby facilitating nuclear CHK1 accumulation. This is imperative for preserving genomic stability during periods of cell proliferation. The Ras/MAPK/RSK axis is frequently found to be overexpressed in a variety of human cancers. Therefore, its interaction with CHK1 represents a pivotal nexus that connects oncogenic signalling with genome maintenance and metabolic regulation [119,120].

CHK1 is an antioxidant regulator. Indeed, CHK1 is directly involved in nuclear reactive oxygen species (ROS) sensing, particularly H₂O₂ [121]. CHK1 senses nuclear H₂O₂ through the oxidation of the conserved Cys408 residue, which is located in KA1. Oxidation of Cys408 activates CHK1 kinase activity. Once activated, CHK1 phosphorylates the single-stranded DNA-binding protein (SSBP1) and prevents it from localising to the mitochondria. This CHK1-dependent pathway is pivotal for regulating the flow of signals between the nucleus and the mitochondria. It also plays a key role in reducing nuclear ROS levels, thereby contributing to the maintenance of cellular redox homeostasis [121]. Importantly, this pathway contributes to platinum-based chemotherapies’ resistance in ovarian cancer models. Importantly, this links CHK1-mediated ROS sensing and regulation to chemoresistance mechanisms [121].

CHK1 also maintains dNTP homeostasis and supports the function of ribonucleotide reductase (RNR), which is the rate-limiting enzyme in dNTP synthesis under cellular redox stress [122]. RNR tightly controls the size of the dNTP pool, and disruption to its enzymatic activity results in dNTP insufficiency, RS, and S-phase accumulation [123,124,125]. Mammalian RNR consists of two homodimers, RRM1 and RRM2. RRM1 levels remain relatively constant throughout the cell cycle, but RRM2 accumulates during S phase and declines as cells progress into mitosis [126]. RNR activity requires electron transfer from both internal and external redox systems to sustain the reduction of ribonucleotides to deoxyribonucleotides. Disruption to this process impairs redox cycling, depletes dNTP pools, and exacerbates RS [127]. During RS, transcription of RRM2 is induced in an E2F1-dependent manner downstream of the ATR/CHK1 pathway [128,129]. Furthermore, UV-induced DNA damage relocates RRM1 and RRM2 from the cytoplasm to the nucleus, a process that depends on ATR/CHK1 signalling [122]. Depletion or inhibition of the thioredoxin (Trx) system, which normally keeps RNR subunits in a reduced functional state [130,131], enhances RRM1 oxidation. This slows replication fork progression due to an imbalance in dNTPs. In this context, CHK1 is activated downstream of ATR, leading to E2F1-dependent upregulation of RRM2 (i.e., the regulatory subunit of RNR). This compensates for the dNTP insufficiency caused by RRM1 oxidation [122]. CHK1 signalling also promotes the nuclear relocation of the two RNR components following DNA damage, thereby ensuring continued dNTP synthesis under stressful conditions. This mechanism supports DNA replication and genome stability, even in the face of oxidative conditions that threaten replication fork progression. Inhibiting CHK1 in Trx1-depleted contexts leads to reduced RRM2 expression, decreased tyrosyl radical activity, and limited RRM1 oxidation, which in turn impairs replication. CHK1 is therefore vital for safeguarding against RS by maintaining an equilibrium in the levels of dNTPs, facilitating their synthesis, and regulating the activity of redox-sensitive proteins within the nucleus. This makes it an indirect yet pivotal antioxidant regulator in situations of genotoxic and oxidative stress [122].

Oncogenic Ras/RSK activation is closely associated with the interaction of CHK1 with phosphoglycerate mutase 1 (PGAM1), a glycolytic enzyme that catalyses the interconversion of 3-phosphoglycerate (3-PG) to 2-phosphoglycerate (2-PG) [132,133]. Beyond its canonical enzymatic role, PGAM1 contributes to physiological homeostasis as it undergoes diverse post-translational modifications (e.g., phosphorylation, acetylation, and ubiquitination) in response to environmental cues and stress [134,135,136]. In cancer cells, PGAM1 enhances glycolytic flux through its interaction with CHK1, particularly under conditions of oncogenic Ras activation. CHK1 binds PGAM1, thereby stabilising it enzymatically and transcriptionally, resulting in an increase in the expression of glycolytic genes (e.g., hexokinase 2, glucose-6-phosphatase isomerase, aldolase A, and pyruvate kinase). This increase in glycolytic activity and lactate production, in turn, creates an immunosuppressive acidic TME that is essential for the Warburg effect that accompanies cancerous proliferation [133] (Fig. 4). Of note, PGAM1 activity also modulates mitochondrial function [137,138] and the pentose phosphate pathway (PPP) [139,140,141]. Indeed, 3-BP, the substrate of PGAM1, can act as an inhibitor of the 6-phosphoglycerate dehydrogenase (G6PD), thereby reducing the nucleotide synthesis reaction in the PPP [142,143]. Indeed, the PPP plays a pivotal role in the cellular response to ROS by generating NADPH, which in turn supports antioxidant defences. This implies that the interaction between CHK1 and PGAM1 may also be involved in the defence against oxidative stress (see paragraph 6.2) [106,107,108].

CHK1 physically interacts with pyruvate kinase isoform M2 (PKM2), the rate-limiting enzyme in glycolysis that converts phosphoenolpyruvate to pyruvate while generating ATP. In cancer cells, PKM2 is predominantly present in a low-activity dimeric form that promotes the Warburg effect. This configuration diverts upstream glycolytic intermediates to biosynthetic pathways such as the PPP for NADPH/ribose production. This supports nucleotide synthesis, maintains redox balance against ROS, and facilitates rapid proliferation [144]. In cardiomyocytes, the interaction between CHK1 and PKM2 triggers PKM2 phosphorylation, thereby stimulating glycolysis and the PKM2-mediated transcription of genes involved in proliferative processes [80]. Despite the absence of quantifiable data concerning the impact of CHK1 on PKM2 interaction on the progression of the cell cycle through the various phases, it has been documented that CHK1:PKM2 interaction induces progression from G1 to S phase [80].

Acetylation, a prevalent post-translational modification of mitochondrial proteins, frequently results in the loss of protein function [145,146]. The acetylation of mitochondrial proteins is primarily governed by sirtuin family members, particularly SIRT3 [147]. SIRT3 has been identified as a pivotal modulator of mitochondrial redox and inflammation, operating as a primary mitochondrial deacetylase [148,149]. This is due to the fact that SIRT3 is responsible for the deacetylation of some subunits of the respiratory chain complex I. It is noteworthy that SIRT3 is transcriptionally activated by NF-κB and relocates to mitochondria through the phosphorylation by CDK1 [150]. In the context of cardiomyocytes, CHK1 has been observed to phosphorylate SIRT3, thereby activating its deacetylase function. The inhibition of CHK1 by AZD7762 results in a decrease in the acetylation levels of the respiratory chain I subunits, owing to a concomitant decrease in the activated SIRT3 [12]. Consequently, this results in a disruption of the respiratory chain [12]. This highlights the CHK1-dependent cross-talk between mitochondrial protein acetylation and bioenergetics [146,148].

CHK1 plays a multifaceted role in tumour immunity, including enhancing immune surveillance and modulating immune cell infiltration and immune escape mechanisms within the TME. CHK1 is a key player in immune evasion mechanisms. Its increased expression in numerous types of cancer is clearly linked to the infiltration of various immune cells, including B cells, CD4⁺ and CD8⁺ T cells, macrophages, neutrophils, and dendritic cells, in several types of cancers [167]. This allows tumour cells to evade immune surveillance and resist immune-based therapies. Tumour cells use high CHK1 activity for the following reasons: (i) minimize exposure to or presentation of immunogenic tumour antigens, making them less visible to the immune system; (ii) promote TME acidification and consequently modulate infiltration and function of immune cells, decreasing recruitment or suppressing activity of cytotoxic lymphocytes; and (iii) promote expression of immunosuppressive molecules (e.g., PD-L1) on cancer cell surfaces, thereby inhibiting effective T-cell responses [1,167,168,169,170,171]. Accordingly, meta-analyses link high CHK1 expression to poor prognosis and low immune infiltration (e.g., reduced T-cells in pan-cancer) [172].

CHK1 inhibition enhances antitumor immunity primarily via DDR-dependent mechanisms. The pharmacological inhibition of CHK1 promotes cancer immune evasion reversal primarily through the DNA damage accumulation, which leads to cytosolic DNA release that in turn activates the cyclic GMP-AMP synthase-stimulator of IFN genes (cGAS-STING) pathway [173]. Activation of the cGAS-STING pathway leads to downstream signalling resulting in the phosphorylation and activation of the IFN regulatory factor 3 (IRF3), a key transcription factor that drives the expression of IFNs and inflammatory cytokines [174,175] (Fig. 5). The cGAS-STING pathway serves as a central regulator of DDR inhibitors driven immune activation, promoting Signal Transducer and Activator of Transcription 1/TNF-Related Apoptosis Inducing Ligand (STAT1/TRAIL)-dependent signalling and the direct stimulation of anti-tumour immune effector cells [173] (Fig. 5). Recent evidence indicates that, beyond the canonical immune signalling function, cGAS-STING and IRF3 also modulate DNA repair, checkpoint activation, and cell death [176,177]. Of note, the combination of radiotherapy and CHK1 inhibition seems to represent a therapeutic strategy for immunotherapy in ARID1A-deficient tumours, overcoming the current challenges associated with immune checkpoint blockers in colorectal cancer patients [172].

iCHK1 combined with HU (a well-known RS inducer) promotes pro-inflammatory cytokines and chemokines expression. Moreover, it induces immunogenic cell death that in turn promotes an adaptive anti-cancer cytotoxic response [178,179,180,181,182]. Notably, CHK1 inhibition activates caspase-1 via non-apoptotic pathways [104]. Caspase-1, a protease activated by inflammation, plays a pivotal role in processing and releasing key pro-inflammatory cytokines such as interleukin (IL)-1β and IL-18 [183], as well as activating inflammasomes [184,185]. Following CHK1 inhibition with MK-8776, transcriptomic analysis revealed significant upregulation of the IL1B transcript, highlighting the activation of inflammatory pathways and immune signalling [104]. MK-8776 also modulates a variety of other pathways that are important for the inflammatory response, such as the TREM1 and IL-17A axes, NRF-2-mediated oxidative stress, IL-8, neuroinflammation, HIF-1α-dependent signalling, and ferroptosis [104]. These changes confirm the immunomodulatory potential of iCHK1, which enhances cytokine and chemokine expression via classical mediators such as nuclear factor-κB (NF-κB) and cytosolic DNA sensors like the cGAS-STING pathway [104,186,187].

CHK1 inhibition enhances immunity also via DDR-independent inflammatory signalling. As previously reported, CHK1 promotes glycolysis and lactate production in cancer cells by binding to PGAM1 and PKM2, thereby enhancing glycolytic flux and supporting the Warburg effect. This leads to elevated extracellular lactate secretion, which dissociates into lactic acid and lowers the pH in the TME to acidic levels (pH 6.5–6.8). Acidic conditions suppress CD8⁺ T cell cytotoxicity and IFN gamma (IFNγ) signatures IFNγ signalling. Indeed, upon internalization of lactic acid through the SMCT2 transporter, CD4⁺ helper T cells and CD8⁺ cytotoxic T cells undergo phosphofructokinase inhibition and hexokinase downregulation, resulting in the inhibition of glycolysis and in the reduction of cell motility. In turn, T cells lose their responsiveness to chemokines and no longer migrate throughout the body [188]. This results in the inhibition of antitumor immune responses and the activation of potent, negative regulators of innate and adaptive immune cells [189]. Pharmacological CHK1 inhibition disrupts this process by blocking CHK1:PGAM1 interaction in cancer cells, reducing lactate efflux, normalizing pH, and restoring T-cell function via decreased PD-L1 on tumours and enhanced effector infiltration [190].

Immune checkpoints are essential immunoregulatory molecules that regulate the immune system, helping to maintain balance and prevent inappropriate immune responses under normal conditions. The Programmed cell death-1 (PD-1)/Programmed cell death-ligand 1 (PD-L1) axis is a fundamental immune checkpoint pathway that facilitates tumour immune evasion and represents a target for therapeutic intervention in cancer immunotherapy. PD-1 is an immune receptor that is expressed on activated CD4⁺ T cells and CD8⁺ T cells as well as on B cells in the periphery [191,192,193,194]. When PD-1 binds to PD-L1, it effectively blocks the immune escape of cancer cells by halting T cell proliferation and IFNγ production [191,195]. CHK1 plays a crucial role in regulating PD-L1 expression in tumour cells through its involvement in the DDR. Indeed, CHK1 regulates the transcriptional expression of PD-L1 by the canonical IRF1/STAT1/3 transcriptional pathway, and ATR stabilizes PD-L1 in a proteasome-dependent manner [155,196,197] (Fig. 5). This mechanism allows tumour cells to evade immune detection by increasing PD-L1 levels, which suppresses cytotoxic T-cell activity. Importantly, inhibiting CHK1 disrupts this pathway, reducing PD-L1 expression and potentially enhancing the efficacy of immune checkpoint therapies targeting the PD-1/PD-L1 axis. Thus, CHK1 represents a promising target for combination cancer therapies, thanks to its capability to link DDR to immune evasion by modulating PD-L1 expression. This makes it clear that CHK1 is key to overcoming tumour immune resistance [155,197,198].

The inhibition of CHK1 results in the accumulation of DNA damage in tumour cells, which in turn promotes the expression of more immunogenic antigens. This enhanced immune recognition can be achieved by increasing the presentation of tumour antigens or changing the expression of immunosuppressive molecules on the surface of tumour cells [169]. Combining iCHK1 with immune checkpoint inhibitors (such as PD-1/PD-L1 antagonists) has been shown to significantly boost anti-tumour immune responses. This is likely because CHK1 inhibition increases DNA damage and enhances immunogenicity in cancer cells [199]. Such enhanced immune recognition can be achieved by increasing the presentation of tumour antigens or changing the expression of immunosuppressive molecules on the surface of tumour cells. This dual strategy aims to maximize therapeutic efficacy by simultaneously targeting cancer cells through two complementary mechanisms [169].

The concomitant targeting of two parallel pathways regulating cell proliferation can result in a synergistic effect of the administered drugs [214]. In the case of iCHK1, their impact on pathways involved in DNA damage control is well established. Moreover, the evidence presented in this review highlights iCHK1’s influence on several metabolic pathways, suggesting its potential use in combination with metabolic pathway inhibitors. This approach would be particularly important given the high toxicity of iCHK1 in humans [215,216]. Therefore, considering the results of the clinical trials on iCHK1 outlined above, it is strongly recommended that these drugs be combined with agents that directly or indirectly target mechanisms regulating cell proliferation, in a manner analogous to those controlled by CHK1. This approach is essential to ensure that iCHK1 treatment doses can be reduced, thereby minimising adverse side effects in patients, specifically those of a haematological nature [215].

The data discussed above indicate that several iCHK1 have entered early-phase clinical trials over the past two decades, where they have been evaluated either as monotherapy or, more commonly, in combination with DNA-damaging agents in Phase I/II studies. Importantly, none of these compounds has progressed successfully to Phase III clinical development, underscoring the nascent but evolving state of iCHK1 development [200,201,202].

As single agents, iCHK1 demonstrated measurable antitumour activity, particularly in molecularly defined subsets of tumours characterized by defects in the DDR, high levels of RS, or TP53 mutations. However, despite the mechanistic rationale and some clinical responses, the overall efficacy of monotherapy has been modest and frequently constrained by dose-limiting hematologic toxicities. A more promising strategy has emerged from combination regimens that exploit synthetic lethality or augment DNA damage beyond repair thresholds.

Significant challenges remain in the clinical advancement of iCHK1. Monotherapy efficacy appears restricted to specific subgroups of patients. Combination regimens represent the most extensively explored approach to date. CHK1 inhibition synergizes with DNA-damaging chemotherapeutics by abrogating cell cycle checkpoints, thereby enhancing cytotoxicity. Indeed, SRA737 combination with gemcitabine, GDC-0575 combination with gemcitabine, and prexasertib in combination with platinum-based agents like cisplatin have demonstrated improved activity compared to monotherapy. Optimal synchronization of CHK1 inhibition with DNA-damaging therapies requires further refinement, including studies evaluating sequential versus concurrent dosing strategies [215,216]. However, these combinations also exacerbate toxicity, particularly hematologic suppression, highlighting the importance of optimized scheduling and dose modulation to achieve a tolerable therapeutic index in patients [200,201,210].

Across clinical trials, the most prominent adverse effects reported are hematologic toxicities and, more rarely, cardiotoxicity [218]. In early studies, high-grade neutropenia/leukopenia and anaemia were among the most frequently observed adverse events, together with other dose-limiting toxicities that ultimately led to the discontinuation of clinical development programs [246,247]. The mechanistic basis of these CHK1-dependent hematologic toxicities is closely linked to the essential role of CHK1 in haematopoiesis. Indeed, CHK1 activity has been shown to be critical for normal blood cell development and homeostasis and to be indispensable for the survival and expansion of hematopoietic stem and progenitor cells, as pharmacological inhibition or genetic loss of CHK1 in these cells induces apoptosis and irreversible loss of haematopoiesis [100]. Given that hematopoietic progenitors are among the most rapidly proliferating normal cell populations, CHK1 inhibition in this compartment is likely to contribute directly to the clinically relevant hematologic toxicities observed in patients treated with iCHK1. In conclusion, available evidence indicates that the failure of iCHK1 to progress to Phase III clinical trials is primarily due to hematologic toxicities arising from the essential biological functions of CHK1, rather than from kinase-independent CHK1 activities.

iCHK1 are specifically designed to block the kinase activity of the protein, and only limited evidence supports physiological roles of CHK1 that are independent of its catalytic function. For instance, CHK1-dependent replication fork progression on damaged DNA through interaction with PCNA has been reported to occur independently of its kinase domain, suggesting the existence of non-catalytic functions in certain replication-associated contexts [248]. However, these kinase-independent activities appear to be highly specialized, observed in specific experimental systems, and do not represent the dominant functions of CHK1 in normal cell cycle regulation. Importantly, such non-kinase roles have not been linked to clinical toxicity or to differences in therapeutic window in humans and remain mechanistic observations at the cellular level rather than drivers of dose-limiting toxicities in patients [248].

Ongoing preclinical and clinical studies will further illuminate the role of CHK1 in cancer therapy and help refine treatment strategies, leading to improved outcomes for patients with advanced malignancies. A crucial component for the advancement of iCHK1 in clinical practice is the rigorous identification of biomarker(s), which could address the use of iCHK1 towards specific patient cohorts and enable personalized treatment regimens while minimizing adverse side effects [218].

Although an in-depth discussion of potential biomarkers for the use of iCHK1 in clinical trials is beyond the scope of the present work, here we have highlighted the role of CHK1 as a metabolic and immunological regulator, showing the mechanistic interaction among CHK1 and metabolic and immunomodulatory pathways (e.g., PGAM-glycolysis; cGAS/STING). Thus, it is tempting to speculate that proteins involved in metabolic and immune response could serve as selective biomarkers for patient stratification that could address tailored strategies for cancer co-treatments.