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Supplementary Figure from EML4-ALK Variant 3 Promotes Mitotic Errors and Spindle Assembly Checkpoint Deficiency Leading to Increased Microtubule Poison Sensitivity

Western blots quantification

Prognostic effect of MAD2L1 in second line cohort. Kaplan Meier curve for overall survival (OS) in patients treated with vinorelbine second line (n=3) stratified according to MAD2L1 expression

Figure S2 shows Hsp70 inhibition does not block clustering of acentrosomal spindle poles.

Supplementary figure legends

Cancer cells frequently possess extra amplified centrosomes clustered into two poles whose pseudo-bipolar spindles exhibit reduced fidelity of chromosome segregation and promote genetic instability. Inhibition of centrosome clustering triggers multipolar spindle formation and mitotic catastrophe, offering an attractive therapeutic approach to selectively kill cells with amplified centrosomes. However, mechanisms of centrosome clustering remain poorly understood. Here, we identify a new pathway that acts through NIMA-related kinase 6 (Nek6) and Hsp72 to promote centrosome clustering. Nek6, as well as its upstream activators polo-like kinase 1 and Aurora-A, targeted Hsp72 to the poles of cells with amplified centrosomes. Unlike some centrosome declustering agents, blocking Hsp72 or Nek6 function did not induce formation of acentrosomal poles, meaning that multipolar spindles were observable only in cells with amplified centrosomes. Inhibition of Hsp72 in acute lymphoblastic leukemia cells resulted in increased multipolar spindle frequency that correlated with centrosome amplification, while loss of Hsp72 or Nek6 function in noncancer-derived cells disturbs neither spindle formation nor mitotic progression. Hence, the Nek6–Hsp72 module represents a novel actionable pathway for selective targeting of cancer cells with amplified centrosomes. Cancer Res; 77(18); 4785–96. ©2017 AACR.

EML4-ALK is an oncogenic fusion protein present in approximately 5% of non–small cell lung cancers (NSCLC). Alternative breakpoints in the gene encoding EML4 result in distinct variants that are linked to markedly different patient outcomes. Patients with EML4-ALK variant 3 (V3) respond poorly to ALK inhibitors and have lower survival rates compared with patients with other common variants, such as V1. Here, we use isogenic Beas-2B bronchial epithelial cell lines expressing EML4-ALK V1 or V3, as well as ALK-positive NSCLC patient cells that express V1 (H3122 cells) or V3 (H2228 cells), to show that EML4-ALK V3 but not V1 leads to hyperstabilized K-fibers in mitosis, as well as errors in chromosome congression and segregation. This is consistent with our observation that EML4-ALK V3 but not V1 localizes to spindle microtubules and that wild-type EML4 is a microtubule stabilizing protein. In addition, cells expressing EML4-ALK V3 exhibit loss of spindle assembly checkpoint control that is at least in part dependent on ALK catalytic activity. Finally, we demonstrate that cells expressing EML4-ALK V3 have increased sensitivity to microtubule poisons that interfere with mitotic spindle assembly, whereas combination treatment with paclitaxel and clinically approved ALK inhibitors leads to a synergistic response in terms of reduced survival of H2228 cells. Implications: This study suggests that combining the microtubule poison, paclitaxel, with targeted ALK inhibitors may provide an effective new treatment option for patients with NSCLC with tumors that express the EML4-ALK V3 oncogenic fusion.

The human retinal pigment epithelial RPE-1 cell line immortalized with hTERT retains a stable karyotype with a modal chromosome number of 46 and has been widely used to study physiological events in human cell culture systems. To facilitate inducible knock-out or knock-in experiments in this cell line, we have modified the AAVS1 locus to harbour a DNA fragment encoding ERT2-Cre-ERT2 fusion protein under regulation of a Tet-On expression system. In the generated cell line, active Cre recombinase was induced by simple addition of doxycycline and tamoxifen to the culture medium. As proof of concept, we successfully introduced an oncogenic point mutation to the endogenous KRAS gene locus of this cell line. The cell line will serve as a powerful tool to conduct functional analyses of human genes.

EML4-ALK is an oncogenic fusion protein that accounts for approximately 5% of NSCLC cases. Targeted inhibitors of ALK are the standard of care treatment, often leading to a good initial response. Sadly, some patients do not respond well, and most will develop resistance over time, emphasizing the need for alternative treatments. This review discusses recent advances in our understanding of the mechanisms behind EML4-ALK-driven NSCLC progression and the opportunities they present for alternative treatment options to ALK inhibitor monotherapy. Targeting ALK-dependent signalling pathways can overcome resistance that has developed due to mutations in the ALK catalytic domain, as well as through activation of bypass mechanisms that utilise the same pathways. We also consider evidence for polytherapy approaches that combine targeted inhibition of these pathways with ALK inhibitors. Lastly, we review combination approaches that use targeted inhibitors of ALK together with chemotherapy, radiotherapy or immunotherapy. Throughout this article, we highlight the importance of alternative breakpoints in the EML4 gene that result in the generation of distinct EML4-ALK variants with different biological and pathological properties and consider monotherapy and polytherapy approaches that may be selective to particular variants.

Mesothelioma is a universally lethal cancer lacking effective therapy. The spindle poison vinorelbine exhibits clinical activity in the relapsed setting, and in preclinical models requires BRCA1 to initiate apoptosis. However, the mechanisms underlying this regulation and the clinical implications have not been explored. Here, we show that BRCA1 silencing abrogated vinorelbine-induced cell-cycle arrest, recruitment of BUBR1 to kinetochores, and apoptosis. BRCA1 silencing led to codepletion of MAD2L1 at the mRNA and protein levels consistent with its status as a transcriptional target of BRCA1. Silencing of MAD2L1 phenocopied BRCA1 and was sufficient to confer resistance to vinorelbine. This was recapitulated in cell lines selected for resistance to vinorelbine, which acquired loss of both BRCA1 and MAD2L1 expression. Following ex vivo vinorelbine in 20 primary tumor explants, apoptotic response rate was 59% in BRCA1/MAD2L1-positive explants compared with 0% in BRCA1/MAD2L1-negative explants. In 48 patients, BRCA1 and/or MAD2L1 loss of expression was not prognostic; however, in a subset of patients treated with vinorelbine, survival was shorter for patients lacking BRCA1/MAD2L1 expression compared with double-positive patients (5.9 vs. 36.7 months, P = 0.03). Our data implicate BRCA1/MAD2L1 loss as a putative predictive marker of resistance to vinorelbine in mesothelioma and warrant prospective clinical evaluation.

EML4–ALK is an oncogenic fusion present in ∼5% of non-small cell lung cancers. However, alternative breakpoints in the EML4 gene lead to distinct variants of EML4–ALK with different patient outcomes. Here, we show that, in cell models, EML4–ALK variant 3 (V3), which is linked to accelerated metastatic spread, causes microtubule stabilization, formation of extended cytoplasmic protrusions and increased cell migration. EML4–ALK V3 also recruits the NEK9 and NEK7 kinases to microtubules via the N-terminal EML4 microtubule-binding region. Overexpression of wild-type EML4, as well as constitutive activation of NEK9, also perturbs cell morphology and accelerates migration in a microtubule-dependent manner that requires the downstream kinase NEK7 but does not require ALK activity. Strikingly, elevated NEK9 expression is associated with reduced progression-free survival in EML4–ALK patients. Hence, we propose that EML4–ALK V3 promotes microtubule stabilization through NEK9 and NEK7, leading to increased cell migration. This represents a novel actionable pathway that could drive metastatic disease progression in EML4–ALK lung cancer.

Cancer cells frequently possess extra amplified centrosomes clustered into two poles whose pseudo-bipolar spindles exhibit reduced fidelity of chromosome segregation and promote genetic instability. Inhibition of centrosome clustering triggers multipolar spindle formation and mitotic catastrophe, offering an attractive therapeutic approach to selectively kill cells with amplified centrosomes. However, mechanisms of centrosome clustering remain poorly understood. Here, we identify a new pathway that acts through NIMA-related kinase 6 (Nek6) and Hsp72 to promote centrosome clustering. Nek6, as well as its upstream activators polo-like kinase 1 and Aurora-A, targeted Hsp72 to the poles of cells with amplified centrosomes. Unlike some centrosome declustering agents, blocking Hsp72 or Nek6 function did not induce formation of acentrosomal poles, meaning that multipolar spindles were observable only in cells with amplified centrosomes. Inhibition of Hsp72 in acute lymphoblastic leukemia cells resulted in increased multipolar spindle frequency that correlated with centrosome amplification, while loss of Hsp72 or Nek6 function in noncancer-derived cells disturbs neither spindle formation nor mitotic progression. Hence, the Nek6–Hsp72 module represents a novel actionable pathway for selective targeting of cancer cells with amplified centrosomes. Cancer Res; 77(18); 4785–96. ©2017 AACR.

EML4-ALK is an oncogenic fusion present in ∼5% lung adenocarcinomas. However, distinct EML4-ALK variants differ in the length of the EML4 microtubule-associated protein encoded within the fusion and are associated with a poorly understood variability in disease progression and therapeutic response. Here, we show that EML4-ALK variant 3, which is linked to accelerated metastatic spread and worse patient outcome, causes microtubule stabilization, formation of extended cytoplasmic protrusions, loss of cell polarity and increased cell migration. Strikingly, this is dependent upon the NEK9 kinase that interacts with the N-terminal region of EML4. Overexpression of wild-type EML4, as well as constitutive activation of NEK9, also perturbs cell morphology and accelerates cell migration in a manner that requires the downstream kinase NEK7 but not ALK activity. Moreover, elevated NEK9 is associated in patients with EML4-ALK V3 expression, as well as reduced progression-free and overall survival. Hence, we propose that EML4-ALK V3 promotes microtubule stabilization through recruitment of NEK9 and NEK7 to increase cell migration and that this represents a novel actionable pathway that drives disease progression in lung cancer.

EML4 is a microtubule-associated protein that promotes microtubule stability. We show here that EML4 is distributed as punctate foci along the microtubule lattice in interphase but exhibits reduced association with spindle microtubules in mitosis. Microtubule sedimentation and cryo-electron microscopy and 3D reconstruction reveal that EML4 binds via its basic N-terminal domain to the acidic C-terminal tails of α- and β-tubulin on the microtubule surface. The mitotic kinases Nek6 and Nek7 can phosphorylate EML4 N-terminal domain at S144 and S146 in vitro, and depletion of these kinases leads to increased EML4 binding to microtubules in mitosis. An S144A-S146A double mutant not only binds inappropriately to mitotic microtubules but also interferes with chromosome congression. Meanwhile, constitutive activation of Nek6 or Nek7 reduces EML4 association with interphase microtubules. Together, these data support a model in which Nek6 and Nek7-dependent phosphorylation promotes dissociation of EML4 from microtubules in mitosis thereby altering microtubule dynamics to enable chromosome congression.

Hsp72 is a member of the 70-kD heat shock family of molecular chaperones (Hsp70s) that comprise a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD) connected by a linker that couples the exchange of adenosine diphosphate (ADP) for adenosine triphosphate (ATP) with the release of the protein substrate. Mitotic phosphorylation of Hsp72 by the kinase NEK6 at Thr66 located in the NBD promotes the localization of Hsp72 to the mitotic spindle and is required for efficient spindle assembly and chromosome congression and segregation. Here, we determined the crystal structure of the Hsp72 NBD containing a genetically encoded phosphoserine at position 66. This revealed structural changes that stabilized interactions between subdomains within the NBD. ATP binding to the NBD of unmodified Hsp72 resulted in the release of substrate from the SBD, but phosphorylated Hsp72 retained substrate in the presence of ATP. Mutations that prevented phosphorylation-dependent subdomain interactions restored the connection between ATP binding and substrate release. Thus, phosphorylation of Thr66 is a reversible mechanism that decouples the allosteric connection between nucleotide binding and substrate release, providing further insight into the regulation of the Hsp70 family. We propose that phosphorylation of Hsp72 on Thr66 by NEK6 during mitosis promotes its localization to the spindle by stabilizing its interactions with components of the mitotic spindle.

Hsp72 is a novel mitotic substrate of Nek6, and, together, these proteins play an essential role in assembly of robust mitotic spindles capable of efficient chromosome congression through K-fiber recruitment of the ch-TOG and TACC3 complex., Hsp70 proteins represent a family of chaperones that regulate cellular homeostasis and are required for cancer cell survival. However, their function and regulation in mitosis remain unknown. In this paper, we show that the major inducible cytoplasmic Hsp70 isoform, Hsp72, is required for assembly of a robust bipolar spindle capable of efficient chromosome congression. Mechanistically, Hsp72 associates with the K-fiber–stabilizing proteins, ch-TOG and TACC3, and promotes their interaction with each other and recruitment to spindle microtubules (MTs). Targeting of Hsp72 to the mitotic spindle is dependent on phosphorylation at Thr-66 within its nucleotide-binding domain by the Nek6 kinase. Phosphorylated Hsp72 concentrates on spindle poles and sites of MT–kinetochore attachment. A phosphomimetic Hsp72 mutant rescued defects in K-fiber assembly, ch-TOG/TACC3 recruitment and mitotic progression that also resulted from Nek6 depletion. We therefore propose that Nek6 facilitates association of Hsp72 with the mitotic spindle, where it promotes stable K-fiber assembly through recruitment of the ch-TOG–TACC3 complex.

The EMLs are a conserved family of microtubule-associated proteins (MAPs). The founding member was discovered in sea urchins as a 77-kDa polypeptide that co-purified with microtubules. This protein, termed EMAP for echinoderm MAP, was the major non-tubulin component present in purified microtubule preparations made from unfertilized sea urchin eggs [J. Cell Sci. (1993) 104, 445–450; J. Cell Sci. (1987) 87(Pt 1), 71–84]. Orthologues of EMAP were subsequently identified in other echinoderms, such as starfish and sand dollar, and then in more distant eukaryotes, including flies, worms and vertebrates, where the name of ELP or EML (both for EMAP-like protein) has been adopted [BMC Dev. Biol. (2008) 8, 110; Dev. Genes Evol. (2000) 210, 2–10]. The common property of these proteins is their ability to decorate microtubules. However, whether they are associated with particular microtubule populations or exercise specific functions in different microtubule-dependent processes remains unknown. Furthermore, although there is limited evidence that they regulate microtubule dynamics, the biochemical mechanisms of their molecular activity have yet to be explored. Nevertheless, interest in these proteins has grown substantially because of the identification of EML mutations in neuronal disorders and oncogenic fusions in human cancers. Here, we summarize our current knowledge of the expression, localization and structure of what is proving to be an interesting and important class of MAPs. We also speculate about their function in microtubule regulation and highlight how the studies of EMLs in human diseases may open up novel avenues for patient therapy.

Summary Mitosis is controlled by multiple protein kinases, many of which are abnormally expressed in human cancers. Nek2, Nek6, Nek7, and Nek9 are NIMA-related kinases essential for proper mitotic progression. We determined the atomic structure of Nek7 and discovered an autoinhibited conformation that suggests a regulatory mechanism not previously described in kinases. Additionally, Nek2 adopts the same conformation when bound to a drug-like molecule. In both structures, a tyrosine side chain points into the active site, interacts with the activation loop, and blocks the αC helix. Tyrosine mutants of Nek7 and the related kinase Nek6 are constitutively active. The activity of Nek6 and Nek7, but not the tyrosine mutant, is increased by interaction with the Nek9 noncatalytic C-terminal domain, suggesting a mechanism in which the tyrosine is released from its autoinhibitory position. The autoinhibitory conformation is common to three Neks and provides a potential target for selective kinase inhibitors.

Nek6 and Nek7 are members of the NIMA-related serine/threonine kinase family. Previous work showed that they contribute to mitotic progression downstream of another NIMA-related kinase, Nek9, although the roles of these different kinases remain to be defined. Here, we carried out a comprehensive analysis of the regulation and function of Nek6 and Nek7 in human cells. By generating specific antibodies, we show that both Nek6 and Nek7 are activated in mitosis and that interfering with their activity by either depletion or expression of reduced-activity mutants leads to mitotic arrest and apoptosis. Interestingly, while completely inactive mutants and small interfering RNA-mediated depletion delay cells at metaphase with fragile mitotic spindles, hypomorphic mutants or RNA interference treatment combined with a spindle assembly checkpoint inhibitor delays cells at cytokinesis. Importantly, depletion of either Nek6 or Nek7 leads to defective mitotic progression, indicating that although highly similar, they are not redundant. Indeed, while both kinases localize to spindle poles, only Nek6 obviously localizes to spindle microtubules in metaphase and anaphase and to the midbody during cytokinesis. Together, these data lead us to propose that Nek6 and Nek7 play independent roles not only in robust mitotic spindle formation but also potentially in cytokinesis.

Proteins of the echinoderm microtubule (MT)-associated protein (EMAP)-like (EML) family contribute to formation of the mitotic spindle and interphase MT network. EML1-4 consist of Trp-Asp 40 (WD40) repeats and an N-terminal region containing a putative coiled-coil. Recurrent gene rearrangements in non-small cell lung cancer (NSCLC) fuse EML4 to anaplastic lymphoma kinase (ALK) causing expression of several oncogenic fusion variants. The fusions have constitutive ALK activity due to self-association through the EML4 coiled-coil. We have determined crystal structures of the coiled-coils from EML2 and EML4, which describe the structural basis of both EML self-association and oncogenic EML4-ALK activation. The structures reveal a trimeric oligomerization state directed by a conserved pattern of hydrophobic residues and salt bridges. We show that the trimerization domain (TD) of EML1 is necessary and sufficient for self-association. The TD is also essential for MT binding; however, this property requires an adjacent basic region. These observations prompted us to investigate MT association of EML4-ALK and EML1-ABL1 (Abelson 1) fusions in which variable portions of the EML component are present. Uniquely, EML4-ALK variant 3, which includes the TD and basic region of EML4 but none of the WD40 repeats, was localized to MTs, both when expressed recombinantly and when expressed in a patient-derived NSCLC cell line (H2228). This raises the question of whether the mislocalization of ALK activity to MTs might influence downstream signalling and malignant properties of cells. Furthermore, the structure of EML4 TD may enable the development of protein-protein interaction inhibitors targeting the trimerization interface, providing a possible avenue towards therapeutic intervention in EML4-ALK NSCLC.

Proteins of the echinoderm microtubule-associated protein (EMAP)-like (EML) family contribute to formation of the mitotic spindle and interphase microtubule network. They contain a unique hydrophobic EML protein (HELP) motif and a variable number of WD40 repeats. Recurrent gene rearrangements in nonsmall cell lung cancer fuse EML4 to anaplastic lymphoma kinase (ALK), causing expression of several fusion oncoprotein variants. We have determined a 2.6-A crystal structure of the representative ∼70-kDa core of EML1, revealing an intimately associated pair of β-propellers, which we term a TAPE (tandem atypical propeller in EMLs) domain. One propeller is highly atypical, having a discontinuous subdomain unrelated to a WD40 motif in place of one of its blades. This unexpected feature shows how a propeller structure can be assembled from subdomains with distinct folds. The HELP motif is not an independent domain but forms part of the hydrophobic core that joins the two β-propellers. The TAPE domain binds α/β-tubulin via its conserved, concave surface, including part of the atypical blade. Mapping the characteristic breakpoints of each EML4-ALK variant onto our structure indicates that the EML4 TAPE domain is truncated in many variants in a manner likely to make the fusion protein structurally unstable. We found that the heat shock protein 90 (Hsp90) inhibitor ganetespib induced degradation of these variants whereas others lacking a partial TAPE domain were resistant in both overexpression models and patient-derived cell lines. The Hsp90-sensitive EML4-ALK variants are exceptions to the rule that oncogenic fusion proteins involve breakpoints in disordered regions of both partners.

Heat shock proteins (HSPs) are ATP-dependent molecular chaperones which aid folding of nascent polypeptides, maintain proteins in unstable conformations and prevent protein denaturation. These functions are essential in many biological contexts, including assembly and disassembly of macromolecular complexes, trafficking of proteins and regulation of enzyme activity [1]. Mitotic cell division is particularly complex involving rapid changes in cytoskeletal and organelle architecture. One would therefore expect it to be highly dependent on HSPs; however, much remains to be learnt about the roles of HSPs in mitosis. In a recent study, we discovered that Hsp72, an inducible cytoplasmic isoform of the Hsp70 family, is essential to build a mitotic spindle capable of efficient chromosome congression and segregation [2]. Firstly, Hsp72 contributes to generation of stable kinetochore (K)-fibres. K-fibres are bundles of microtubules that connect the spindle poles with the kinetochores and are essential for chromosome movement. Upon depletion of Hsp72 or addition of an Hsp70 inhibitor, cells exhibited reduced K-fibres. This was coincident with misaligned chromosomes, metaphase delay and a strongly active spindle assembly checkpoint. The loss of K-fibres was not caused by reduction in microtubule nucleation, but rather failure to recruit the K-fibre-stabilising proteins, ch-TOG and TACC3. Hsp72 localises to spindle poles and spindle fibres in a similar manner to ch-TOG and TACC3. Furthermore, when TACC3 was immunoprecipitated from cells treated with the Hsp70 inhibitor, association with its partner, ch-TOG, was reduced. Together, this suggests that Hsp72 stabilises K-fibres by facilitating assembly of ch-TOG and TACC3 into a complex that can then serve to bundle K-fibre microtubules [3]. Secondly, abrogation of Hsp72 function led to reduced interpolar distances, reduced astral microtubules and misoriented spindles. Astral microtubules attach the spindle to the cell cortex to maintain its shape and position. These data indicate that Hsp72 has additional functions in astral microtubule organization or cortical attachment. This is likely to be independent of the ch-TOG-TACC3 complex as this is not thought to be required for astral microtubule function. Importantly, whilst Hsp72 depletion and chemical inhibition of Hsp70 give similar phenotypes, there are some clear differences. This can be explained by that fact that chemical inhibition blocks the activity of all Hsp70 isoforms and it is possible that other Hsp70 isoforms have roles in mitosis. On the other hand, chemical inhibition does not remove the protein and so the different phenotypes may result from Hsp72 having mitotic functions as a scaffold that are independent of its catalytic activity. Hsp72 is phosphorylated in mitosis by Nek6, a protein kinase that is also required for robust spindle assembly [4, 5]. Nek6 phosphorylates Hsp72 on T66, a residue that sits within the nucleotide-binding domain just upstream of the catalytic lysine (K71). Mislocalization of a T66A mutant and failure of Hsp72 to associate with the spindle in the absence of Nek6 indicate that phosphorylation mediates localisation of Hsp72 to the spindle. Furthermore, expression of the T66A mutant resulted in reduced K-fibres, whilst the T66E mutant could rescue the K-fibre defects and loss of ch-TOG/TACC3 recruitment to K-fibres seen upon either Hsp72 or Nek6 depletion. However, understanding the mechanism through which phosphorylation regulates Hsp72 will require molecular insights on Hsp72 interactions with its mitotic partners. Interestingly, a phosphospecific antibody revealed that Hsp72 phosphorylated on T66 was not only localised to the spindle apparatus, but also enriched on spindle poles and kinetochores. This begs the question of whether phosphorylated Hsp72 has additional, perhaps distinct, substrates at the kinetochore. The absence of total Hsp72 may lead to loss of function of these proteins as well, exacerbating the K-fibre assembly and chromosome congression defects. Similarly, problems in cortical attachment of microtubules could contribute to loss of astral microtubules and spindle orientation defects. It will thus be important to ascertain whether (phosphorylated) Hsp72 may stabilize the plus ends of microtubules that contact kinetochores and the cell cortex. Besides their homeostatic function, HSPs have an important role in protecting cells from the proteotoxic stress that can arise in different pathological states [6]. These include protein-folding disorders, autoimmune diseases and cancer. In cancer, the induced expression of Hsp70 due to proteotoxic stress promotes cell survival and tumor progression. As cancer cells undergo frequent mitotic division, Hsp70 inhibitors could have therapeutic value in targeting the proliferating tumour tissue. Whilst it has been frustratingly difficult to develop potent and selective inhibitors of Hsp70 itself [7], drugging upstream regulators, such as Nek6, offers an alternative approach. In contrast to cancer, Hsp70 proteins are beneficial to the patient in slowing the onset of neurodegenerative disorders, such as Alzheimer's, Huntington's or Parkinson's disease. Here, they promote removal of misfolded proteins through autophagy or proteasomal degradation. However, as neuronal cells are post-mitotic and do not undergo division, anti-cancer therapies specifically targeting the mitotic form of Hsp70 would have the advantage of not precipitating the onset or progression of neurodegenerative diseases.

The mitotic spindle is the apparatus upon which chromosomes are segregated during cell division. We have discovered new roles for two members of the NIMA-related kinase (NEK) family in different molecular processes of spindle assembly. Moreover, loss of these proteins leads to segregation errors that drive cancer progression.

Genetic screens for cell division cycle mutants in the filamentous fungus Aspergillus nidulans led to the discovery of never-in-mitosis A (NIMA), a serine/threonine kinase that is required for mitotic entry. Since that discovery, NIMA-related kinases, or NEKs, have been identified in most eukaryotes, including humans where eleven genetically distinct proteins named NEK1 to NEK11 are expressed. Although there is no evidence that human NEKs are essential for mitotic entry, it is clear that several NEK family members have important roles in cell cycle control. In particular, NEK2, NEK6, NEK7 and NEK9 contribute to the establishment of the microtubule-based mitotic spindle, whereas NEK1, NEK10 and NEK11 have been implicated in the DNA damage response. Roles for NEKs in other aspects of mitotic progression, such as chromatin condensation, nuclear envelope breakdown, spindle assembly checkpoint signalling and cytokinesis have also been proposed. Interestingly, NEK1 and NEK8 also function within cilia, the microtubule-based structures that are nucleated from basal bodies. This has led to the current hypothesis that NEKs have evolved to coordinate microtubule-dependent processes in both dividing and non-dividing cells. Here, we review the functions of the human NEKs, with particular emphasis on those family members that are involved in cell cycle control, and consider their potential as therapeutic targets in cancer.