Mark William Richards

Aurora-A kinase (AurA) is a serine/threonine kinase regulated by protein binding partners such as TPX2, which interacts with the Y-pocket of the kinase domain. Although the canonical functions of AurA are mitotic, where it phosphorylates spindle-associated proteins such as TACC3, there is evidence for non-mitotic, chromatin-associated roles. Heterogenous nuclear ribonucleoprotein K (hnRNPK) is a transcriptional regulator and an AurA substrate, how AurA is activated in this pathway is unknown. Their interaction contributes to cancer-associated nuclear accumulation of AurA and has been implicated in driving oncogenic transcription of MYC. Here, we show that hnRNPK promotes AurA autophosphorylation and stimulates AurA activity. The interface was mapped to residues 330-380, an intrinsically disordered region of hnRNPK, and the AurA Y-pocket, using NMR spectroscopy and competitive-binding experiments with mitotic and synthetic AurA-binding partners. AlphaFold3 modelling of the complex predicted Phe339 and Trp345 at the kinase Y-pocket, and site-specific mutagenesis confirmed these as key residues for AurA binding and activation. Our work provides a mechanistic basis for hnRNPK-dependent activation of AurA and efficient phosphorylation of hnRNPK Ser379. Defining the AurA/hnRNPK interface highlights the Y-pocket as a target site for modulation of nuclear AurA, a potentially cancer-specific role of the kinase. Significance Statement: Aurora-A kinase (AurA) has critical functions during mitosis but is increasingly connected with cancer-associated roles in the cell nucleus. One such pathway involves the transcriptional regulator hnRNPK, which functions with AurA to promote oncogene expression, yet how the kinase is activated in this context has remained unclear. We show that an intrinsically disordered region of hnRNPK directly activates AurA through interaction with a regulatory site in the kinase domain. Through defining the molecular basis of this interaction and identifying mutations that disrupt it, we provide a mechanistic explanation for AurA activation in the cell nucleus and establish a framework for understanding how AurA contributes to oncogenic transcription.

Aurora Kinase A (AurA) is an essential mitotic kinase and therapeutic target in cancer. Most protein kinase inhibitors target the conserved ATP-binding pocket, often resulting in poor selectivity and off-target effects. Here, we identify and characterise small synthetic protein binders, Adhirons, as allosteric inhibitors of AurA. Using ‘phage display, we isolated Adhiron reagents that bind a previously uncharacterised site on the αG-helix of the kinase C-lobe. Structural and biochemical analyses revealed that the Adhiron inhibited AurA by modulating the activation loop via this cryptic site, which we designate the T-pocket. In cells, Adhiron expression mimics the effects of small molecule inhibitors of AurA on substrate and auto-phosphorylation, while sparing Aurora kinase B and without impairing TPX2-mediated localisation of AurA to the mitotic spindle. The AurA-inhibitory Adhirons demonstrate remarkable selectivity, potency and affinity, a highly sought-after combination of properties for kinase inhibition facilitating their use as tractable research tools for probing AurA function and as pharmacophore templates for structure-based drug design. Finally, these reagents illustrate a generalisable strategy for targeting allosteric sites across the Kinome. *Jack P Roberts & James Holder contributed equally to this work.

The mitotic spindle is a bipolar array of microtubules, radiating from the poles which each contain a centrosome, embedded in pericentriolar material. Two proteins, ch-TOG and TACC3, have multiple functions at the mitotic spindle due to operating either alone, together, or in complex with other proteins. To distinguish these activities, we need new molecular tools to dissect their function. Here, we present the structure of the α-helical bundle domain of ch-TOG that mediates its interaction with TACC3 and a structural model describing the interaction, supported by biophysical and biochemical data. We have isolated Affimer tools to precisely target the ch-TOG-binding site on TACC3 in live cells, which displace ch-TOG without affecting the spindle localization of other protein complex components. Inhibition of the TACC3–ch-TOG interaction led unexpectedly to fragmentation of the pericentriolar material in metaphase cells and delayed mitotic progression, uncovering a novel role of TACC3–ch-TOG in maintaining pericentriolar material integrity during mitosis to ensure timely cell division.

Summary: Deregulated MYCN is a driver of aggressive pediatric and adult neuroendocrine tumors, but critical oncogenic processes downstream of MYCN remain poorly defined. In neuroblastoma, MYCN interacts with and activates the Aurora-A kinase. Here we show that Aurora-A is a CDK-activating kinase for CDK12 by phosphorylating T893 in the T-loop, thereby enhancing its kinase activity. Aurora-A-dependent activation of CDK12 controls phosphorylation of T4 of RNA polymerase and recruits transcription termination complexes, thereby preventing transcription-replication conflicts. Enhanced crosslinking and immunoprecipitation sequencing reveals that Aurora-A associates with splice sites on nascent RNA. RNA-bound Aurora-A is catalytically inactive. MYCN competes with RNA for binding to Aurora-A and displaces Aurora-A from RNA in cells, promoting its CDK12 kinase activity. Combining Aurora-A and CDK12 inhibition potently suppresses the growth of MYCN -amplified neuroblastoma cells and patient-derived xenografts. Our data demonstrate that an Aurora-A/CDK12-dependent transcription termination pathway is a critical and targetable dependency of MYCN-driven tumors.

The mitotic spindle is a bipolar array of microtubules, radiating from the poles which each contain a centrosome, embedded in pericentriolar material. Two proteins, ch-TOG and TACC3, have multiple functions at the mitotic spindle due to operating alone, together or in complex with other proteins. To distinguish these activities, we need new molecular tools to dissect their function. Here, we present the structure of the α -helical bundle domain of ch-TOG that mediates its interaction with TACC3 and a structural model describing the interaction, supported by biophysical and biochemical data. We have isolated Affimer tools to precisely target the ch-TOG-binding site on TACC3 in live cells, which displace ch-TOG without affecting the spindle localization of other protein complex components. Inhibition of the TACC3–ch-TOG interaction led unexpectedly to fragmentation of the pericentriolar material in metaphase cells following the formation of a bipolar spindle and delayed mitotic progression; uncovering a novel role of TACC3–ch-TOG in maintaining pericentriolar material integrity during mitosis to ensure timely cell division.

The myc family of proteins (c-, N- and L-myc) are transcription factors (TFs) responsible for maintaining the proliferative program in cells. They consist of a C-terminal domain that mediates heterodimerisation with Max and DNA binding, and an N-terminal disordered region culminating in the transactivation domain (TAD). The TAD participates in many protein–protein interactions, notably with kinases that promote stability (Aurora-A) or degradation (ERK1, GSK3) via the ubiquitin-proteasome system. Structural characterization of the TAD of N-myc, is very limited, with the exception of a crystal structure of Aurora-A bound to a helical region of N-myc. We probed the structure, dynamics and interactions of N-myc TAD using nuclear magnetic resonance (NMR) spectroscopy following its complete backbone assignment enabled by a truncation approach. Chemical shift analysis revealed that N-myc has two regions with clear helical propensity: one region within Trp77–Glu86 and the second between Ala122–Glu132. These regions also have more restricted ps–ns motions than the rest of the TAD, and, along with another known interaction site (myc box I), have comparatively high transverse ( R ₂ ) ¹⁵ N relaxation rates, indicative of slower timescale dynamics and/or chemical exchange. Collectively these features suggest differential propensities for structure and interaction, either internal or with binding partners, across the TAD. Solution studies on the interaction between N-myc and Aurora-A revealed a previously uncharacterised binding site. The specificity and kinetics of sequential phosphorylation of N-myc by ERK1 and GSK3 were characterised using NMR and showed no significant structural changes through the rest of the TAD. When doubly phosphorylated on residues Ser62 and Thr58, N-myc formed a robust interaction with the Fbxw7–Skp1 complex. Our study provides foundational insights into N-myc TAD dynamics and a backbone assignment that will underpin future work on the structure, dynamics, interactions and regulatory post-translational modifications of this key oncoprotein.

Fascin-1-mediated actin-bundling activity is central to the generation of plasma membrane protrusions required for cell migration. Dysregulated formation of cellular protrusions is observed in metastatic cancers, where they are required for increased invasiveness, and is often correlated with increased Fascin-1 abundance. Therefore, there is interest in generating therapeutic Fascin-1 inhibitors. We present the identification of Nb 3E11, a nanobody inhibitor of Fascin-1 actin-bundling activity and filopodia formation. The crystal structure of the Fascin-1/Nb 3E11 complex reveals the structural mechanism of inhibition. Nb 3E11 occludes an actin-binding site on the third β-trefoil domain of Fascin-1 that is currently not targeted by chemical inhibitors. Binding of Nb 3E11 to Fascin-1 induces a conformational change in the adjacent domains to stabilize Fascin-1 in an inhibitory state similar to that adopted in the presence of small-molecule inhibitors. Nb 3E11 could be used as a tool inhibitor molecule to aid in the development of Fascin-1 targeted therapeutics.

Background: Carriage of the ancestral APOE ε4 allele confers a risk of developing Alzheimer's and coronary artery disease, but its persistence in human populations also suggests some potential survival advantages. To date it remains unclear whether APOE ε4 carriage independently associates with a better or worse long-term cardiac phenotype. Purpose: Using data from the 1946 National Survey of Health and Development (NSHD) birth cohort, we investigated whether APOE ε4 carriage associates with adverse or beneficial left ventricular (LV) size and function parameters by echocardiography in older age. Methods: Based on the presence or absence of APOE ε4, genotypes were divided into: non-APOE ε4 (ε2ε2, ε2ε3, ε3ε3), heterozygous-APOE ε4 (ε2ε4 and ε3ε4) and homozygous-APOE ε4 (ε4ε4). Echocardiographic data at 60–64 years included: left ventricular ejection fraction (LV EF), E/e', systolic and diastolic LV posterior wall and interventricular septal thickness (LVPWTs/d, IVSs/d), and body-surface area indexed LV mass (LVmassi) and myocardial contraction fraction (MCFi). Generalized linear models explored associations between APOE ε4 genotypes as exposures and echocardiographic biomarkers as outcomes. As a combination of gene variants, APOE ε genotype is expected to be an instrumental variable and therefore unconfounded. Thus, Model 1 was unadjusted. To obtain more precise regression estimates, Model 2 was adjusted for factors associated with the outcome, namely sex and socio-economic position (SEP). To explore the mechanistic pathway downstream of APOE ε genotype but upstream of the echocardiographic outcomes, subsequent models were adjusted for mediators as follows: Model 3 for body mass index, Model 4 for the presence of cardiovascular disease (CVD), Model 5 for diabetes, Model 6 for high cholesterol and Model 7 for hypertension. Results: 1464 participants were included. Compared to non-APOE ε4 and homozygous groups, heterozygous-APOE ε4 individuals had similar cardiac phenotypes in terms of EF, E/e', LVPWTs/d, IVSs/d and LVmassi but had a 7% higher MCFi 95% confidence interval [CI]: 1%-13%, p=0.017) which persisted even after adjustment for sex and SEP (95% CI 1%-12%, p=0.026) that was attenuated to 6% after adjustment for CVD (95% CI 0–13% p=0.050) and hypertension (95% CI 1–13% p=0.022). Conclusion: The heterozygous-APOE ε4 state associates with improved myocardial shortening in older age resulting in greater LV stroke volume generation per 1 mL of myocardium. As we found no association between APOE ε4 carriage and LVPWTs/d, IVSs/d or LVmassi, MCFi enhancement may be mediated by improved myocardial energetics and contractility, with calcium and androgens potentially implicated, rather than through pathological ventricular thickening. Although a dose relationship is normally expected with ε4 carriage, any benefit from increased energetics and contractility is likely to be counterbalances by the higher risk of CVD and cardiovascular risk factors. Funding Acknowledgement: Type of funding sources: Public grant(s) – National budget only. Main funding source(s): UK Medical Research Council British Heart Foundation

Variants of the oncogenic EML4-ALK fusion protein contain a similar region of ALK encompassing the kinase domain, but different portions of EML4. Here, we show that EML4-ALK V1 and V3 proteins form cytoplasmic foci that contain components of the MAPK, PLCγ and PI3K signalling pathways. The ALK inhibitors ceritinib and lorlatinib dissolve these foci and EML4-ALK V3 but not V1 protein re-localises to microtubules, an effect recapitulated in a catalytically inactive EML4-ALK mutant. Mutations that promote a constitutively active ALK stabilise the cytoplasmic foci even in the presence of these inhibitors. In contrast, the inhibitor alectinib increases foci formation of both wild-type and catalytically inactive EML4-ALK V3 proteins, but not a Lys-Glu salt bridge mutant. We propose that EML4-ALK foci formation occurs as a result of transient association of stable EML4-ALK trimers mediated through an active conformation of the ALK kinase domain. Our results demonstrate the formation of EML4-ALK cytoplasmic foci that orchestrate oncogenic signalling and reveal that their assembly depends upon the conformational state of the catalytic domain and can be differentially modulated by structurally divergent ALK inhibitors.

Amplification of MYCN is the driving oncogenic change in a subset of high-risk neuroblastomas. The MYCN protein and the Aurora-A kinase form a complex during the S phase that stabilizes MYCN. Here we show that MYCN activates Aurora-A on chromatin, which phosphorylates histone H3 at serine 10 in the S phase, promotes the deposition of histone H3.3 and suppresses R-loop formation. Inhibition of Aurora-A induces transcription–replication conflicts and activates ataxia telangiectasia and Rad3-related (ATR) kinase, which limits double-strand break accumulation upon Aurora-A inhibition. Combined inhibition of Aurora-A and ATR kinases induces rampant tumor-specific apoptosis and tumor regression in mouse models of neuroblastoma, leading to permanent eradication in a subset of mice. The therapeutic efficacy is due to both tumor cell-intrinsic and immune cell-mediated mechanisms. We propose that targeting the ability of Aurora-A to resolve transcription–replication conflicts is an effective therapy for MYCN-driven neuroblastoma.