NEK kinases in cell cycle regulation, DNA damage response, and cancer progression

Abstract

The NIMA-related kinase (NEK) family of serine/threonine kinases is essential for the regulation of cell cycle progression, mitotic spindle assembly, and genomic stability. In this review, we explore the structural and functional diversity of NEK kinases, highlighting their roles in both canonical and non-canonical cellular processes. We examine recent preclinical findings on NEK inhibition, showcasing promising results for NEK-targeted therapies, particularly in cancer types characterized by high NEK expression. We discussed the therapeutic potential of targeting NEKs as modulators of cell cycle and DDR pathways, with a focus on identifying strategies to exploit NEK activity for enhanced treatment efficacy. Future research directions are proposed to further elucidate NEK-mediated mechanisms and to develop selective inhibitors that target NEK-related pathways.

Introduction

Discovery and evolution of NEKs

Never in Mitosis A (NIMA)-related kinases (NEKs) belong to a family of serine/threonine kinases initially discovered in Aspergillus nidulans, a filamentous fungus commonly used to study cell cycle regulation [1, 2]. In a 1983 genetic screen, Ron Morris identified a group of mutants that were unable to progress through mitosis. These mutants were named “nim” mutants, short for “never in mitosis,” reflecting their arrest in cell division [1, 2]. Among these, the nimA gene was the first to be characterized [1, 3]. It encodes a serine/threonine kinase that plays a critical role in cell cycle progression [1, 3]. Mutations in nimA arrest cells in the G2 phase of the cell cycle, while its overexpression leads to premature mitotic entry [3, 4]. NIMA degradation is necessary for proper mitosis exit [3, 4].

Beyond mitosis, NIMA is involved in several essential cellular processes. These include chromatin condensation, which ensures the compaction of chromosomes before cell division; centrosome separation, which facilitates the assembly of the mitotic spindle; and spindle formation itself, which ensures the proper segregation of chromosomes [5–7]. NIMA also participates in microtubule nucleation, a process critical for establishing spindle apparatus. NIMA interacts with proteins such as Fin1, a regulator of chromatin and centrosomal function, to ensure accurate mitosis [3, 5–7].

Conservation of NEKs across species

The regulatory roles of NEKs, initially observed in fungi, have evolved and diversified across species [8]. In humans, the NEK family comprises eleven kinases (NEK1 - NEK11), each encoded by distinct genes [7, 9–11]. These proteins contain highly conserved N-terminal kinase domains, which are structurally similar across family members [2, 12]. The conservation of this catalytic domain is essential in maintaining cellular functions [8, 12]. Despite the structural similarity in their kinase domains, NEK proteins are functionally diverse, owing to differences in their non-catalytic regions [11, 12]. This region, often located in the C-terminal domain, contribute to the distinct roles of each NEK by mediating protein-protein interactions, cellular localization, and substrate specificity [11, 13].

Each NEK kinase performs specialized roles across several critical processes; such as cell cycle regulation, DNA damage response, ciliogenesis and immune function [11, 13]. Those functions suggest the expansion of NEK caliber from relatively simple mitotic regulators in fungi to multifunctional proteins that coordinate diverse cellular functions in higher organisms [11, 13, 14]. Given their centrality to cell cycle control and genomic integrity, NEKs have emerged as important players in cancer biology. Their dysregulation is associated with tumor progression, chromosomal instability, and therapeutic resistance; as well as aggravated inflammatory response [15–18]. On the other hand, their inhibition suppresses inflammation; which is an important property of anti-cancer agents, such as synthetic somatostatin analogs, Hsp90 inhibitors and GHRH antagonists [19, 20]

NEK1

NEK1 is one of the most well-characterized NEKs, with roles in DNA damage response and ciliogenesis [21]. It is activated by cellular stress, DNA damage, and oxidative condition [21]. A key upstream regulator is ATR (ataxia-telangiectasia and Rad3-related protein), a central kinase in the DNA damage response (DDR) pathway [22]. ATR activation in response to replication stress or double-strand breaks (DSBs) promotes NEK1 phosphorylation, especially during the G2/M cell cycle checkpoint, ensuring cell survival by delaying mitosis until damage is resolved [22, 23]. Other stress-related signals, such as oxidative stress and environmental insults like IR and UV exposure can also activate NEK1, likely through post-translational modifications including phosphorylation and ubiquitination [21]. Ionizing radiation (IR) and ultraviolet radiation (UV) cause redistribution of NEK1 from the cytoplasm to nuclear foci of damaged DNA [21]. In addition, protein-protein interactions with other kinases – such as NEK9 - or molecular scaffolds; are thought to guide NEK1’s localization and proper activation at specific subcellular compartments [23, 24].

Upon activation by DNA damage or other stress signals, NEK1 can translocate from the cytoplasm to the nucleus, where it forms discrete nuclear foci at sites of DNA damage to coordinate DNA repair processes [25] which may involve P53 [26].

NEK1 regulates formation of primary cilia

NEK1 is involved in the regulation of primary cilia, which serve as sensory organelles and signaling hubs [27], by regulating the stability of centrosomal proteins such as the Von Hippel-Lindau (VHL) [27]. Loss of NEK1 function causes defective ciliogenesis, contributing to ciliopathies such as polycystic kidney disease. Proper cilia function is essential for developmental processes and tissue homeostasis [9, 28].

NEK1 regulates DNA damage response

In DNA repair, this kinase interacts with proteins involved in homologous recombination (HR), such as RAD54, ATR, ATRIP, VDAC1, BRCA1 and HSP90 [27]. Hsp90 inhibitors are tested in clinical trials for cancer therapy and exert anti-inflammatory activities, utilizing unfolded protein response [29–31]. Rad54 is crucial for homologous recombination – mediated double-strand break repair. It transforms the synaptic complex into heteroduplex DNA, thereby enabling DNA polymerases to initiate repair synthesis. The ATPase activity of this repair protein is essential for displacing Rad51 from DNA, enabling the completion of HR [32].

Nek1 phosphorylates Rad54 at Ser572 specifically in the G2 phase of the cell cycle. This phosphorylation promotes Rad51 removal from chromatin, allowing HR to proceed [32]. Mutations that prevent phosphorylation at this site (Rad54-S572A) impair HR, resulting in unresolved Rad51 foci. In contrast, phospho-mimetic mutations (Rad54-S572E) restore HR but can also prematurely remove Rad51 from stalled replication forks during the S phase, leading to fork degradation [32]. These findings emphasize the necessity for precise temporal regulation of Rad54 phosphorylation to balance HR efficiency and genomic stability.

NEK1 functions as a key upstream regulator of Ataxia Telangiectasia and Rad3-related (ATR) kinase, ensuring that the ATR-ATRIP (ATR-interacting protein) complex is stable and active, ready to respond to DNA replication stress and damage [22]. NEK1’s interaction with ATRIP is essential for ATR’s autophosphorylation at threonine 1989 (Thr1989), which is one of the earliest events during ATR activation after DNA damage [22]. This phosphorylation event allows ATR to engage with downstream effectors such as Chk1, initiating the checkpoint signaling cascade [22]. NEK1 interaction with this complex enables a rapid and effective DNA damage response to replication stress and DNA lesions, ensuring cell survival [22].

Many cancers rely on the ATR-Chk1 pathway to survive DNA replication stress induced by rapid proliferation [33]. Inhibiting NEK1 in cancer cells has been shown to increase DNA damage accumulation, reduce repair efficiency, and sensitize cells to chemotherapy [33, 34]. These findings suggest that targeting the NEK1-ATR axis could enhance the effectiveness of DNA-damaging treatments, such as cisplatin, by preventing the efficient repair of damaged DNA [33, 34].

NEK1 may operate alongside, or independently from ATR, phosphorylating shared substrates based on specific stimuli and cell cycle phases [35]. In NEK1-deficient cells, significant DNA damage, such as chromosomal breaks, occurs quickly, leading to genomic instability [35]. This damage happens independently of ATM and ATR signaling, suggesting that NEK1 acts as a unique mediator in DNA repair pathways [35].

BRCA1 is essential for recognizing, processing, and repairing DNA double-strand breaks (DSBs) [36]. Upon DNA damage, BRCA1 forms a complex with BARD1, and initiates the recruitment of repair proteins to damage sites [36]. This complex regulates end resection and generates the single-stranded DNA required for HR [36]. BRCA1 also facilitates the assembly of RAD51 filaments on these strands, promoting their interaction with a homologous DNA template [36, 37].

HSP90 binds and protects key repair proteins from degradation, ensuring they remain functional during periods of cellular stress [38, 39]. Stabilization of HSP90 enhances the function of this chaperone under conditions of severe genotoxic stress [38, 39], and P53 is involved in the anti-inflammatory effects of Hsp90 inhibitors [40–43].

NEK1 and tumorigenesis

In prostate cancers, elevated levels of NEK1 are associated with increased cancer cell survival, proliferation, and resistance to apoptosis [44]. Furthermore, NEK1 activates and stabilizes YAP1, a protein responsible for the progression of prostate cancer to castration-resistant prostate cancer (CRPC) [45]. CRPC is a more aggressive and treatment-resistant form of prostate cancer that arises after androgen deprivation therapy [45]. This makes the NEK1 / YAP1 axis a potential therapeutic target for inhibiting cancer progression.

Moreover, NEK1’s ability to regulate DNA repair mechanisms enables cancer cells to survive DNA-damaging therapies, such as radiation and chemotherapy [46]. Aberrant NEK1 expression prevents the accumulation of DNA damage, enables the cells to bypass apoptosis and promotes genomic instability [46]. NEK1, implicated in kidney and prostate cancers, is a potential therapeutic target for sensitizing cancer cells to DNA-damaging agents [47].

NEK2

NEK2 is perhaps the most well-studied member of the NEK family in relation to cancer. NEK2 functions by phosphorylating centrosome-associated proteins, promoting centrosome disjunction and spindle assembly [48]. Apart from its mitotic role, NEK2 has also been connected to ciliogenesis [49]. It influences microtubule dynamics, affecting the formation of primary cilia—microtubule-based structures involved in cellular communication [49]. Its involvement in multiple signaling pathways including AKT, Wnt, and EZH2 contributes to cancer cell survival, proliferation, and resistance to treatment [50].

NEK2 contains a coiled-coil domain near its C-terminus that facilitates homodimerization, a necessary step for its activation and localization [11, 51]. Dimerization enhances the autophosphorylation of NEK2 at specific residues that are crucial for its activation and function [51]. The primary residues involved in NEK2 autophosphorylation include Threonine 175 (Thr175) and Serine 213 (Ser213) [51]. These events activate NEK2 and facilitate its role in the cell. Once active, NEK2 phosphorylates various substrates essential for maintaining centrosomal cohesion and regulating mitotic spindle formation [52].

The NEK2 kinase exists in three main isoforms – NEK2A, NEK2B, and NEK2C – which differ in their subcellular localization and functional roles [52, 53]. These isoforms result from alternative splicing and exhibit context-specific behaviors relevant to cell cycle regulation and cancer development [52, 53].

NEK2A is the full-length variant with 445 amino acids. It contains both an N-terminal catalytic domain and a C-terminal non-catalytic domain that plays a role in substrate recognition and dimerization [52]. NEK2A is distributed throughout the nucleus and cytoplasm. Its activity peaks during the G2/M phase to promote centrosome separation and mitotic entry [52]. This isoform undergoes autophosphorylation and forms active homodimers, facilitated by a leucine zipper (LZ) motif. Those events lead to phosphorylation of centrosomal proteins such as C-Nap1 and Rootletin [52].

NEK2B is a shorter variant of NEK2, mainly localized to the cytoplasm [52]. It plays a more restricted role, primarily during cell division, with less involvement in centrosomal functions compared to NEK2A [14, 52]. NEK2B lacks certain regulatory sequences present in NEK2A, which makes it more resistant to degradation by the anaphase-promoting complex (APC/C). This provides NEK2B with a unique role in maintaining mitotic processes, especially during early embryogenesis and specific cancer contexts [14, 52].

NEK2C is primarily localized within the nucleus and may have roles in nuclear events associated with the cell cycle and functions to regulate chromatin dynamics and DNA damage response pathways, suggesting non-centrosomal roles [52–54].

NEK2 regulates centrosome dynamics

NEK2 regulates centrosome cohesion and separation through interaction with centrosomal proteins such as C-Nap1 and Rootletin [55]. These proteins regulate the formation of bipolar spindles [55]. It phosphorylates C-Nap1 at the onset of mitosis, initiating centrosome disjunction. During interphase, C-Nap1 holds centrosomes together through a fibrous linker, preventing premature separation [14, 56]. Phosphorylation by NEK2 weakens this cohesion, allowing centrosomes to migrate to opposite poles of the cell to form the bipolar spindle necessary for chromosome segregation [56, 57].

Elevated NEK2 expression levels drive excessive phosphorylation of C-Nap1, disrupting centrosome cohesion. Dysregulation of the NEK2-C-Nap1 axis contributes to centrosome amplification [58, 59]. This feature, commonly seen in malignant cells, results in the formation of multipolar spindles, which increase the likelihood of erroneous chromosome segregation and tumor progression [58, 59]. In parallel, Rootletin, a coiled-coil protein, is essential for maintaining centrosome cohesion and organizing the fibrous linker between centrosomes [60]. This protein forms fibrous tethers that connect the two centrosomes, maintaining their cohesion. NEK2 phosphorylates Rootletin, to initiate the disassembly of these tethers at the onset of mitosis [59, 60]. Those events event enable the centrosomes to separate and migrate to opposite poles of the cell, forming the bipolar spindle required for mitosis.

Dysfunctional NEK2-Rootletin interaction promotes centrosomal abnormalities [59, 61]. Elevated NEK2 activity in tumors phosphorylates Rootletin excessively, causing untimely centrosome disjunction and amplifying centrosome numbers [59, 60]. This results in the formation of defective spindles and erroneous chromosome segregation, fostering aneuploidy and genomic instability, which are considered hallmarks of aggressive cancer behavior [61, 62].

NEK2 and tumorigenesis

NEK2 contributes to tumorigenesis by promoting centrosome disjunction and amplifying chromosomal instability. This instability leads to aneuploidy and accelerates tumor progression [63]. Overexpression or hyperactivation of NEK2 can drive oncogenesis by disrupting normal cell cycle control, promoting chromosomal instability, and enhancing proliferation [64]. Studies have shown that NEK2 acts as a critical driver of tumorigenesis in multiple cancers, including breast, lung, liver, and lymphoma [64, 65].

NEK2 overexpression contributes to chemotherapy resistance through the activation of oncogenic pathways such as the AKT and Wnt/β-catenin signaling cascades [63, 66]. In colorectal cancer, this kinase supports tumor survival and resistance to radiation by modulating the Wnt pathway, promoting cell growth and preventing apoptosis [66–68]. Furthermore, in glioblastoma, the protein physically interacts with EZH2, a histone methyltransferase, stabilizing its expression and enabling the maintenance of tumor cell stemness and growth, even under stress conditions like radiation exposure [9, 69–71]. In models of diffuse large B-cell lymphoma (DLBCL), inhibiting NEK2 induces G2/M arrest and apoptosis, suggesting that tumors with high NEK2 expression are highly dependent on its activity for survival [72, 73].

NEK2 increases ABC transporter activity, contributing to multidrug resistance. Studies show that knocking down NEK2 resensitizes cancer cells to drugs like paclitaxel, bortezomib, and doxorubicin [63, 74]. NEK2 can also influence tumor immune microenvironment in pancreatic cancers by stabilizing PD-L1, an immune checkpoint protein that enables cancer cells to evade immune surveillance [74]. NEK2 phosphorylates PD-L1, preventing its degradation and enhancing immune resistance [74]. In the tumor microenvironment, NEK2 expression correlates with the deregulation of apoptosis and DNA damage repair, leading to enhanced survival of cancer cells under therapeutic stress [75].

NEK2 as a therapeutic target

Due to its critical role in cancer, NEK2 has become a promising therapeutic target. Preclinical studies have demonstrated that inhibiting NEK2 using small-molecule inhibitors effectively impairs cancer cell proliferation and sensitizes tumors to chemotherapy [52, 72]. NBI-961, a selective NEK2 inhibitor, has shown efficacy in DLBCL models by inducing NEK2 degradation and causing apoptosis in cancer cells while sensitizing them to doxorubicin and vincristine [72].

Targeting NEK2 in combination therapies may also overcome resistance mechanisms. For example, NEK2 knockdown sensitizes glioblastoma cells to radiation by destabilizing EZH2, reducing tumor growth [69]. In cervical cancer, NEK2 inhibition impairs the Wnt/β-catenin pathway, decreasing cell survival and enhancing therapeutic efficacy [76]. These findings suggest that NEK2 inhibitors could synergize with existing therapies to improve patient outcomes in multiple cancer types.

RNAi-mediated knockdown of NEK2 in experimental models of breast cancer and cholangiocarcinoma demonstrated significant tumor suppression, reduced proliferation, and enhanced survival in mice [63, 77]. Gene expression analyses indicate that NEK2 is overexpressed across a wide range of cancers, making it a potential pan-cancer biomarker [25]. High NEK2 expression correlates with adverse clinical outcomes, suggesting its utility as a prognostic marker [25, 78]. An expanding body of evidence underscores NEK2’s relevance across cancers, positioning it not only as a prognostic marker but also as a therapeutic possibility in modern cancer treatment strategies.

NEK3

NEK3, located on chromosome 13, has garnered attention for its non-canonical roles beyond cell cycle; including cell motility, differentiation, and tumor progression [9, 21]. NEK3 possesses a kinase domain located at its N-terminus, responsible for its catalytic activity, and a non-catalytic region at the C-terminus that regulates localization and substrate interaction [21]. Unlike many members of the family that undergo autophosphorylation, NEK3 activation relies on phosphorylation by other upstream kinases and specific cellular signals such as prolactin and growth factors [25, 79].

Prolactin activates NEK3 through the prolactin receptor (PRL-R) dimerization [13, 25]. The ensuing conformational change activates the receptor’s intracellular domain [13, 80]. PRL-R associates with the Janus kinase 2 (JAK2), a non-receptor tyrosine kinase [80, 81]. JAK2 activation stimulates multiple pathways, including the mitogen activated protein kinase (MAPK) / extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway [81]; and has been involved in cancer promotion [82, 83]. ERK1/2 phosphorylates NEK3 at threonine 165 (Thr165) [84]. This modification alters NEK3’s conformation, promoting its interaction with cytoskeletal components necessary for cell motility and adhesion turnover [84]. Upon prolactin receptor activation, NEK3 promotes the recycling of specific membrane proteins, such as ion channels and growth factor receptors, which are essential for cellular signaling and homeostasis [9, 85]. This regulation ensures that receptors are properly re-expressed on the membrane, maintaining cellular responsiveness. The effects of NEK3 in the GHRH receptor and its splice variants [19] are unknown and warrant investigation.

NEK3 localization is dynamic and it shuttles between the cytoplasm and nucleus based on the cellular context and stimuli [2, 3, 7, 86]. It often interacts with scaffold proteins like the Rac-GEFs (guanine nucleotide exchange factors), to influence cytoskeletal rearrangements critical for cell motility [86]. NEK3’s most well-characterized function is the regulation of microtubules and actin filaments [87]. It is involved in the formation of lamellipodia—sheet-like cellular protrusions involved in migration [3, 87, 88]. Phosphorylated NEK3 enhances focal adhesion disassembly and actin reorganization, facilitating cellular migration [3]. In cancer cells, such as breast cancer, this motility supports invasion and metastatic spread by enabling rapid adhesion turnover [89–91].

NEK4

NEK4, located on chromosome 3, consists of an N-terminal kinase domain, responsible for catalytic activity, and a C-terminal regulatory domain [9, 21]. The regulatory domain contains nuclear localization sequences that facilitate its movement to the nucleus, where it participates in DNA damage repair (DDR) [3, 9]. This kinase is also involved in essential processes like cell cycle checkpoint control, apoptosis, and cytoskeletal rearrangement [3]. NEK4 is expressed in various isoforms, each with distinct cellular roles [92]. The isoforms, such as NEK4.1 and NEK4.2, exhibit different interaction networks, contributing to their specific functions in diverse cellular contexts, including mRNA splicing and DDR [85, 92].

Activation and signaling pathways

Unlike kinases that activate through autophosphorylation, NEK4 is primarily activated in response to cellular stress signals, including those from DNA damage [25, 85]. It becomes active through phosphorylation by upstream kinases and interactions with DDR complexes, such as DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and Ku70/Ku80 [85, 92]. Upon DNA damage, NEK4 plays a crucial role in stabilizing DNA-PK complexes at double-strand break (DSB) sites [85, 93]. This stabilization is essential for the efficient repair of DSBs [94, 95]. NEK4 activity promotes the phosphorylation of histone H2AX, a key marker of DNA damage response [85, 96, 97]. This process ensures that the repair machinery is properly recruited and activated at the sites of damage, facilitating effective DNA repair [96, 97]. NEK4 colocalizes with other DDR proteins, including MDC1, PARP-1, and H2AX, promoting repair processes through non-homologous end joining (NHEJ) and other repair mechanisms [92].

NEK4’s activation also links to other key cellular processes like mRNA splicing [21, 92]. The kinase localizes to nuclear speckles—regions rich in splicing factors—and influences the processing of transcripts required for proper cell cycle arrest and repair during stress conditions [21, 92].

NEK4 in microtubule dynamics and cytoskeletal regulation

NEK4 plays a crucial role in regulating microtubule stability, which is essential for both cell division and intracellular transport [25, 98]. Knockdown studies in breast cancer cells (e.g., MCF-7) show that NEK4 deficiency sensitizes cells to microtubule-disrupting agents like taxol and vincristine, suggesting its role in stabilizing the cytoskeleton [98, 99]. Similar to other NEK family members, NEK4 affects microtubule dynamics by coordinating with proteins involved in mitotic spindle assembly and intracellular trafficking [24, 98, 99].

NEK4 and tumorigenesis

Dysregulation of NEK4 has been implicated in tumorigenesis, primarily through its impact on cell cycle checkpoints, apoptosis and DDR pathways [24, 98, 100]. Defective NEK4-mediated repair processes allow the accumulation of DNA mutations, contributing to tumor progression [92, 101]. Furthermore, its involvement in microtubule stability may suggest that NEK4 dysfunction can disrupt mitotic processes, promoting aneuploidy and cancer cell survival [98, 99]. Studies suggest that NEK4’s influence extends beyond direct DDR. They regulate epithelial-to-mesenchymal transition, mRNA splicing and alternative transcript production to enhance tumor progression and metastasis [92, 93, 100].

NEK5

The role of NEK5, located on chromosome 3, has evolved beyond its canonical roles in cell cycle regulation, contributing to processes such as neuronal differentiation, mitochondrial homeostasis, and muscle function [85, 102, 103]. NEK5 displays a kinase domain at the N-terminus, which confers its enzymatic activity, while the C-terminal region facilitates interactions with other proteins and regulates subcellular localization [85, 103]. This kinase is dynamically localized, shuttling between the cytoplasm, mitochondria, and nucleus, depending on cell type and stimuli [85, 104]. NEK5 has been observed to accumulate in mitochondria under metabolic stress and it can localize to the nucleus to participate in transcriptional regulation [85, 104].

NEK5’s activation diverges from classical autophosphorylation mechanisms, relying on phosphorylation by upstream kinases and contextual cellular signals [25, 105]. NEK5 interacts with cyclin-dependent kinases (CDKs) and AMP-activated protein kinase (AMPK) for its activation, particularly during conditions that require metabolic adaptation or mitochondrial remodeling [104, 106]. In response to metabolic stress (e.g., low ATP levels), AMPK phosphorylates NEK5 to support mitochondrial fission and adaptation [24, 85, 104]. This ensures that mitochondrial dynamics align with energy demands, preventing dysfunction [24, 85, 104].

Additionally, protein-protein interactions assist NEK5 activation. NEK5 transiently associates with mitochondrial proteins such as Drp1 (Dynamin-related protein 1) and scaffold proteins within mitochondrial complexes, which enhance its kinase activity by promoting substrate recognition [24, 85, 105]. Once active, NEK5 plays a key role in several interconnected pathways involving mitochondrial dynamics, cytoskeletal rearrangement, and cell survival [85].

Regulation of mitochondrial fission

Mitochondria rely on a balance between fission and fusion processes to maintain cellular health and energy production [107, 108]. Dysregulated mitochondrial fission is linked to several diseases, including neurodegenerative disorders [107, 108]. NEK5 interacts with Drp1, a primary factor involved in mitochondrial fission. Drp1 is recruited to mitochondrial membranes to constrict and divide mitochondria [85, 104]. This control mechanism is essential for proper mitochondrial function and quality control, including selective removal of damaged mitochondria through mitophagy [85].

Alterations in NEK5 expression or mutations in its kinase domain can lead to imbalances in mitochondrial dynamics, contributing to mitochondrial dysfunction [85, 104]. Since these disruptions are increasingly recognized in the pathology of neurodegenerative diseases and cancers, targeting NEK5 could offer a potential therapeutic option aimed at restoring mitochondrial homeostasis [109–111].

Neuronal differentiation and cytoskeletal regulation

In neurons, NEK5 contributes to microtubule stability through interacting with tubulin-associated proteins [25, 104, 112]. This promotes the formation of axons and dendrites, essential for neuronal outgrowth [112]. NEK5’s downstream signaling also influences the formation of actin filaments, enhancing cellular migration in both neurons and other motile cells [113]. Dysregulation of these pathways has been linked to neurodegenerative diseases [114, 115].

NEK5 interacts with sarcomeric proteins to regulate muscle cell differentiation and contraction [85, 102, 103]. In myoblasts, NEK5 activity promotes differentiation into mature myotubes by enhancing cytoskeletal reorganization and myofibril alignment [102]. This process is vital for maintaining proper muscle function and regeneration after injury [116].

Cellular stress response and survival

NEK5 contributes to maintaining mitochondrial DNA (mtDNA) integrity, which is essential for proper mitochondrial function and energy production [85, 103]. Optimal mtDNA maintenance ensures the stability of oxidative phosphorylation and prevents accumulation of reactive oxygen species (ROS) [117, 118]. Excessive ROS levels disrupt the redox balance, impairing mitochondrial efficiency and promoting oxidative stress, which can lead to cellular dysfunction and inflammation [85, 117, 119–121].

Elevated ROS and mitochondrial stress have been associated with neurodegenerative diseases, metabolic disorders, and cancer [119, 122, 123]. Mitochondrial dysfunction often releases damage-associated molecular patterns (DAMPs), which trigger inflammatory responses through NLRP3 inflammasome and other innate immune mechanisms [124–126].

NEK5 and tumorigenesis

NEK5’s influence extends to tumor cell survival [104, 113, 127]. Activated NEK5 ensures efficient mitochondrial function in cancer cells, enabling them to thrive in low-nutrient or hypoxic environments [103]. By promoting mitochondrial fission and protecting against oxidative stress, NEK5 contributes to chemoresistance and enhanced tumor growth [103, 128].

NEK6

NEK6, located on chromosome 9, is structurally composed of a kinase domain responsible for its catalytic function, with specific residues critical for substrate binding and phosphorylation [129–131]. Unlike many other members of the NEK family, NEK6 is not regulated by autophosphorylation but it is activated by upstream kinases - such as NEK9 - and through cellular stress-related cues [129, 132].

NEK6 is primarily involved in the progression of the mitotic phase, specifically regulating spindle assembly and ensuring accurate chromosomal alignment and segregation [132, 133]. The kinase becomes active in the G2/M phase of the cell cycle, where it modulates the stability of kinetochore-microtubule attachments [133, 134]. This process ensures proper metaphase-anaphase transition and helps prevent chromosomal instability, an essential function for healthy cell division [133, 134].

Cellular stress and antioxidant defense

Beyond its role in mitosis, NEK6 has emerged as a critical player in stress responses, especially in cancer cells [24, 135]. NEK6 regulates redox balance and mitochondrial activity by modulating the levels of reactive oxygen species [85, 135]. In cells such as those found in prostate cancer models, NEK6 maintains intracellular ROS homeostasis by promoting the expression of key antioxidant proteins, including superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2), and peroxiredoxin 3 (PRDX3) [135]. These antioxidants mitigate oxidative damage and support cell survival under stress conditions [136, 137].

In experimental models where NEK6 is depleted, cells exhibit higher ROS levels, increased mitochondrial dysfunction, and heightened sensitivity to DNA damage [135]. This loss induces apoptosis by reducing anti-apoptotic proteins such as Bcl-2, a critical regulator of mitochondrial integrity [135]. These findings indicate that NEK6 provides protection, enabling cancer cells to withstand oxidative stress and resist cell death.

DNA damage response

NEK6 contributes to the DNA damage response by modulating the activity of stress-responsive pathways, such as checkpoint kinases Chk1 and Chk2, and c-Jun N-terminal kinase (JNK) [138–140]. JNK activation triggers downstream signaling cascades essential for cell cycle arrest and apoptosis following severe DNA damage [140–142]. NEK6 also influences the activation of ATM (ataxia telangiectasia mutated) and the phosphorylation of histone H2AX (γH2AX), two key markers involved in the recognition and repair of DNA double-strand breaks [143, 144].

Furthermore, NEK6 regulates the nuclear localization of NF-κB2, a transcription factor involved in inflammation and cell survival [135]. By promoting NF-κB2 activation, NEK6 promotes cellular resilience in cancer cells, particularly under oxidative stress [135]. This pro-survival mechanism is often exploited by cancer cells to resist chemotherapy and continue proliferating despite DNA damage [135].

NEK6 as a therapeutic target

NEK6 involvement in antioxidant defenses, DDR pathways, and cancer survival mechanisms highlights its importance in both normal and pathological contexts. Targeting NEK6 offers therapeutic potential in cancers and other inflammatory diseases that rely on redox balance. NEK6 overexpression can inhibit the expression of the wild-type p53 gene, activate the STAT3 signaling pathway, and block the TGF-β/Smad signaling pathway [145–147]. This aberrant expression could also induce early resistance of tumor cells to anticancer drugs like camptothecin and doxorubicin [148]. Remarkably, P53 has been involved in anti-inflammatory processes [42, 149, 150]

NEK6 is upregulated in esophageal adenocarcinoma [151]. In hepatocellular carcinoma cell lines, NEK6 overexpression correlates with tumor viability and progression [152, 153]. Suppression of NEK6 expression inhibited migration, progression and invasiveness of these cancers in pre-clinical studies [153, 154].

In castration-resistant prostate cancer (CRPC), the overexpression of NEK6 correlates with cancer aggressiveness, enhancing tumor cell proliferation, survival, and resistance to chemotherapeutic agents such as cisplatin [135]. Inhibiting NEK6 in these cancer cells leads to reduced mitochondrial function, higher ROS levels, increased DNA damage, and sensitization to chemotherapy, making it a promising target for cancer therapy [135].

In preclinical experimental models, targeting NEK6 with chemical inhibitors has demonstrated promising outcomes. An ATP site-directed inhibitor of NEK6, ((5Z)-2-hydroxy-4-methyl-6-oxo-5-[(5-phenylfuran-2-yl) methylidene]-5,6-dihydropyridine-3-carbonitrile), exhibited synergistic effect with cisplatin and paclitaxel in ovarian cancer cells while also showing antiproliferative activity against human cancer cell lines [155].

NEK7

NEK7, located on chromosome 1, plays essential roles in cell division and inflammation [133, 156] and it is predominantly expressed during the late stages of the cell cycle - specifically during mitosis - where it regulates spindle organization and chromosomal segregation [157, 158]. The structure of NEK7 includes a kinase domain which facilitate substrate phosphorylation, and it is uniquely known for being activated by interaction with NEK9 rather than through autophosphorylation [159, 160].

NEK7’s involvement in the mitotic process is crucial for ensuring proper spindle assembly and microtubule-kinetochore attachments [157, 158]. This function minimizes chromosomal instability, preventing tumorigenesis [157, 158].

Elevated NEK7 expression levels have been associated with aggressive phenotypes in several cancers; including gallbladder and liver cancer, as well as glioblastomas. It promotes proliferation and survival by facilitating the proper completion of mitosis [98, 161, 162]. Clinical studies suggest that tumors with higher NEK7 levels show poor prognosis and increased chemoresistance, indicating its potential as a prognostic marker in cancer therapy [98, 163, 164].

NEK7 and inflammation

NEK7 plays a pivotal role in inflammation by regulating the NLRP3 inflammasome, a multiprotein complex involved in the release of the pro-inflammatory cytokines IL-1β and IL-18 [165–167]. Activation of the NLRP3 inflammasome by NEK7 is tightly linked to potassium efflux and cellular stress [165, 166, 168]. In cancers, this inflammatory axis may promote tumor progression by creating a microenvironment conducive to cancer cell survival and immune evasion [169, 170].

NEK7 in DNA damage response

Emerging evidence highlights NEK7’s involvement in DNA damage response, adding another layer to its importance in cancer biology. By phosphorylating telomere-associated proteins like TRF1, NEK7 helps maintain telomere integrity and protect against oxidative damage [171]. This function supports cellular homeostasis, particularly in rapidly dividing cancer cells exposed to genotoxic stress [171, 172]. The stabilization of telomeres by NEK7 enhances tumor cell longevity, contributing to therapeutic resistance in cancers treated with DNA-damaging agents [171, 172].

Therapeutic modulation of NEK7

NEK7 modulation can disrupt both its mitotic and inflammatory roles, suggesting therapeutic potential in oncology and chronic inflammatory diseases [161, 162]. Inhibiting NEK7 reduces the proliferation of glioblastoma and gallbladder cancer cells while enhancing apoptosis and sensitizing these cells to chemotherapy [161, 162]. Additionally, small molecule inhibitors targeting the NEK7-NLRP3 interaction show promise in mitigating colitis, neuroinflammation, and stroke-associated pneumonia [170, 173]. Continued research into selective NEK7 inhibitors may unlock new treatment strategies for cancers and chronic inflammatory diseases.

NEK8

NEK8 is located on chromosome 17 and functions primarily as a ciliary kinase, participating in essential cellular processes, including DNA damage response and mitotic regulation.

NEK8 in DNA damage response

NEK8 modulates key steps in the homologous recombination repair pathway, particularly during replication stress and double strand break repair [174]. NEK8 is essential for facilitating the formation of RAD51 foci at DNA damage sites, a crucial step in the HR pathway [174]. Formation of this foci indicates the recruitment of HR machinery to DNA damage site and promotes accurate DSB repair by using homologous sequences as repair templates [174, 175]. NEK8-deficient cells exhibit significant reductions in RAD51 foci, which compromises HR efficiency and results in genomic instability under stress conditions [174].

In addition to HR regulation, NEK8 plays a critical role in safeguarding stalled replication forks [174]. NEK8 regulates the protection of replication forks from degradation mediated by MRE11 [174]. In NEK8-deficient cells, DNA fiber analysis reveals that replication forks are more susceptible to collapse, as nascent DNA strands are increasingly degraded upon fork stalling [174]. This fork degradation leads to a buildup of single-stranded DNA, which further elevates the risk of genome instability. These vulnerable sites are prone to breaks and mis-repair, resulting in chromosomal instability [174].

Interaction with ATR and CDK2 Pathways

NEK8’s role in DDR extends to interactions with major cell cycle and checkpoint proteins, specifically ATR and CDK2 [176]. ATR is a critical regulator of the replication stress response and coordinates checkpoint control in S-phase to prevent entry into mitosis with unresolved DNA damage [177]. NEK8 interacts with ATR and its co-factors, ATRIP and CHK1, forming a regulatory network that facilitates checkpoint activation and fork stabilization [176].

Notably, NEK8 influences CDK2 activity through its association with cyclin A, linking cell cycle progression and checkpoint control [178, 179]. Loss of NEK8 results in elevated cyclin A/CDK2 activity, which can exacerbate DNA damage by promoting premature entry into S-phase without adequate repair [178, 180].

NEK8 in disease and cancer therapeutics

Disruptions to NEK8 activity result in aberrant mitotic spindle assembly and ciliopathies, such as nephronophthisis and polycystic kidney disease (PKD) [24, 98, 176]. These diseases are marked by defective ciliary function and structural abnormalities [24, 98, 176]. In nephronophthisis, a form of chronic kidney disease, mutations in NEK8 impair ciliary function, disrupting epithelial cell polarity and fluid homeostasis, leading to cyst formation [181, 182]. These mutations are also implicated in left–right asymmetry defects during embryogenesis, highlighting the developmental significance of this kinase [182, 183].

Research has linked abnormal NEK8 expression with several cancers, including gliomas, breast cancer, and gastric cancer, where NEK8 overexpression correlates with poor patient outcomes [105, 179]. Preclinical studies indicate that NEK8 contributes to resistance against certain chemotherapeutic agents by reinforcing DDR pathways, thereby enabling tumor cells to evade apoptosis [105]. NEK8 inhibition decreased tumor progression through increased DNA damage and mitotic arrest, while enhancing the efficacy of chemotherapy drugs [179, 184, 185].

NEK9

NEK9 plays extensive roles in cell cycle regulation, particularly in the G2/M transition and mitotic spindle organization [13, 59, 133]. Structurally, NEK9 contains an N-terminal kinase domain responsible for its catalytic function, while its C-terminal region, which includes a dimerization domain, facilitates interactions with NEK6 and NEK7, among other cellular partners [133, 186]. NEK9 activation depends on its phosphorylation by upstream kinases, and it subsequently phosphorylates downstream substrates critical for cellular division and response to DNA damage [186].

Polo-like kinase 1 (Plk1), a master regulator of mitotic entry, activates NEK9 by phosphorylating it at specific sites, triggering a signaling cascade that involves NEK6 and NEK7 [133, 186]. This activation occurs primarily in the G2 phase and is sustained into mitosis, where NEK9 plays a pivotal role in organizing the mitotic spindle [133, 186]. Activated NEK9 phosphorylates downstream kinases - particularly NEK6 and NEK7 - as well as microtubule - associated motor proteins (e.g. Eg5, also known as kinesin-5) which collectively control early centrosome separation [133].

NEK9 regulates centrosome dynamics

NEK9 interacts with essential centrosomal proteins including γ-Tubulin [187]. This is a component of the microtubule-organizing centers (MTOCs) involved in chromosome segregation by nucleating and stabilizing microtubules essential for the mitotic and meiotic spindles [188, 189]. This tubulin isoform localizes primarily to the centrosomes in somatic cells and to spindle poles in cells that undergo acentrosomal division, such as oocytes [188, 189]. By serving as a core part of the γ-tubulin ring complex (γ-TuRC), γ-tubulin initiates the formation of microtubule minus-ends, organizing the spindle poles and providing structural integrity for spindle assembly [188, 189]. NEK9 recruits γ-tubulin to MTOCs to enhance structural stability and bipolarity of spindles; and promote correct chromosome segregation [187].

DNA damage response and replication stress

In addition to its canonical functions in mitosis, NEK9 is also involved in the DNA damage and replication stress response [133]. Under replication stress conditions, NEK9 promotes the interaction of NEK6 and NEK7 with DNA repair machinery and helps maintain replication fork stability [133]. NEK9 plays a critical role in the cellular response to replication stress by maintaining genome stability through its interactions with checkpoint signaling pathways [190]. Under replication stress, NEK9 helps stabilize replication forks and prevent fork collapse, which otherwise would lead to DNA double-strand breaks [190].

NEK9 facilitates CHK1 activation in response to replication blocks, supporting the ATR-CHK1 pathway’s role in safeguarding stalled replication forks [190]. When NEK9 is depleted, cells exhibit heightened sensitivity to replication stress due to inadequate activation of CHK1 [190–192]. This lack of checkpoint signaling exacerbates DNA damage and compromises cell viability, suggesting that NEK9 is essential for an effective response to replication stress [190–192].

By stabilizing replication forks and promoting DNA repair processes, NEK9 helps prevent chromosomal instability. This is a critical feature in preventing tumorigenesis and sustaining genome integrity under replication stress conditions [190, 192].

Implications in cancer and disease

Alterations in NEK9 expression or mutations affecting its kinase activity have been associated with cancers [98, 193] and its activation can support DNA repair processes that allow cancer cells to withstand treatment – induced genotoxic stress [9, 133]. In several cancer types, including breast and colorectal cancers, NEK9 is often overexpressed, and correlates with increased cellular proliferation, resistance to apoptosis, and enhanced metastatic potential [98, 194].

NEK9 directly influences cell motility and cytoskeletal reorganization in gastric cancer by phosphorylating ARHGEF2, which activates RhoA, a key regulator of cell movement [195, 196]. In these cancers, increased NEK9 and GP130 expression levels promotes metastasis through the IL-6/STAT3 pathway [195]. RNAi and small-molecule inhibitors of NEK9 function disrupts mitotic processes and sensitizes cancer cells to chemotherapeutic agents that induce DNA damage [197, 198]. Both NEK2 and NEK9 habe been involved in barrier regulation and inflammation [17].

NEK10

NEK10 has gained attention for its role in DNA damage response, cell cycle regulation, and implications in cancer biology [98, 105, 163, 199]. It possesses a unique structure, with a central kinase domain flanked by regulatory domains that facilitate interactions with multiple proteins involved in cellular stress responses [98, 105, 163, 199].

NEK10 in cell cycle regulation and mitotic integrity

NEK10 interacts with P53 and other DDR proteins to regulate cell cycle progression. In response to cellular stress, NEK10 promotes damage repair by enhancing cell cycle checkpoints activity at the G2/M transition [21, 199]. This kinase also interacts with the RAF1/MEK/ERK signaling axis. Under UV-induced stress, NEK10 forms a complex with RAF1 and MEK1/2, which in turn phosphorylates ERK. This signaling is crucial for cells’ response to environmental stressors and involves [85, 199, 200].

NEK10 in DNA damage response and p53 regulation

NEK10 affects DDR through its regulation of the tumor suppressor protein P53 [201]. NEK10 can phosphorylate P53 at tyrosine 327 (Y327), which is critical for P53’s activation and stabilization in response to DNA damage [201]. This phosphorylation event promotes the transcriptional activity of P53, enhancing the expression of downstream target genes, such as P21 [201]. P21 mediates cell cycle arrest to provide the cells with time to repair DNA lesions and prevent the propagation of tumorigenic mutations [202, 203].

In cancers where NEK10 is downregulated or dysfunctional, P53 activation is often impaired, compromising cell’s ability to respond to DNA damage effectively. Such impairment promotes genomic instability, contributing to cancer progression and resistance to genotoxic therapies [201, 204]. Loss of NEK10 expression or function has been observed in various cancers, including breast and lung cancers, where it correlates with poor prognosis and increased tumor aggressiveness [201, 205].

NEK11

NEK11 regulates DNA replication checkpoints, preventing premature mitotic entry under replication stress [206]. Following DNA damage, NEK11 associates with CDC25A phosphatase to enhance checkpoint activation [207]. This kinase phosphorylates CDC25A, promoting its degradation and thus preventing premature cell cycle progression that could result in genomic instability [207]. Additionally, NEK11 interacts with ATR to stabilize stalled replication forks [206, 207]. This prevents their collapse and the accumulation of chromosomal aberrations [177, 206, 207]. In colorectal cancers, NEK11 dysregulation is linked to altered sensitivity to chemotherapeutics that induce DNA replication stress [25, 105, 163, 208]. Targeting this NEK11 may enhance therapeutic intervention in cancers characterized by genomic instability and DDR pathway dependencies.

Conclusions

NEKs are multifunctional kinases essential for cell cycle regulation, genomic stability, and cancer progression. Their functional specificity, driven by conserved kinase domains and diverse regulatory regions, allows cancer cells to exploit these kinases for survival and proliferation. Many NEKs are overexpressed in aggressive cancers, driving chromosomal instability and proliferation, correlating with poor prognosis and chemoresistance. Targeting NEKs with RNAi or small-molecule inhibitors has shown promise in preclinical disease models. Moreover, those kinases hold potential as important biomarkers and therapeutic targets and may offer new opportunities for improving treatment outcomes.

Future perspectives:

Exploring specific molecular mechanisms underlying the effect of NEKs will fully leverage their potential as therapeutic targets. For instance, delineating the role of these kinases in DDR pathways could reveal new ways to target cancer cells reliant on them for survival. Future studies could focus on the development of isoform-specific NEK inhibitors that minimize off-target effects. Furthermore, combination of NEK inhibitors with FDA-approved chemotherapy or other agents (Hsp90 inhibitors, GHRH antagonists or synthetic somatostatin analogs) might be proven beneficial in cancer treatment.

Highlights.

NEK kinases are involved in cell cycle regulation, motility and permeability

NEK kinases are involved in inflammation and tumor pregression

NEK inhibition suppresses experimental cancers

NEK inhibition may represent a promising strategy to counteract abnormal cell proliferation

Abbreviations:

NIMA

Never in mitosis A

NEK

NIMA-related kinase

DDR

DNA Damage Response

RNAi

RNA interference

MTOC

Microtubule-Organizing Center

GVBD

Germinal Vesicle Breakdown

DSB

Double-Strand Break

ERK

Extracellular Signal-Regulated Kinase

Plk1

Polo-Like Kinase 1

CDK

Cyclin-Dependent Kinase

ATR

Ataxia Telangiectasia and Rad3-related protein

TRF1

Telomeric Repeat-binding Factor 1

NLRP3

NOD-, LRR- and pyrin domain-containing protein 3

HR

Homologous Recombination

MRE11

Meiotic Recombination 11

PKD

Polycystic Kidney Disease

γ-TuRC

Gamma-Tubulin Ring Complex

CHK1

Checkpoint Kinase 1

ARHGEF2

Rho Guanine Nucleotide Exchange Factor 2

RhoA

Ras Homolog Family Member A

GP130

Glycoprotein 130

RAF1

Rapidly Accelerated Fibrosarcoma 1

MEK

Mitogen-Activated Protein Kinase/ERK Kinase

ATRIP

ATR-Interacting Protein

VDAC1

Voltage-Dependent Anion Channel 1

BRCA1

Breast Cancer Gene 1

HSP90

Heat Shock Protein 90

Chk1

Checkpoint Kinase 1

YAP1

Yes-Associated Protein 1

ATM

Ataxia Telangiectasia Mutated

BARD1

BRCA1-Associated RING Domain Protein 1

CRPC

Castration-Resistant Prostate Cancer

DNA

Deoxyribonucleic Acid

AKT

Protein Kinase B

Wnt

Wingless/Integrated Signaling Pathway

EZH2

Enhancer of Zeste Homolog 2

APC/C

Anaphase-Promoting Complex/Cyclosome

C-Nap1

Centrosomal Nek2-Associated Protein 1

DLBCL

Diffuse Large B-Cell Lymphoma

PRDX3

Peroxiredoxin 3

Bcl-2

B-Cell Lymphoma 2

JNK

c-Jun N-terminal Kinase

H2AX

H2A Histone Family Member X

GHRH

Growth Hormone Releasing Hormone

Hsp90

Heat Shock Protein 90