Biology subjects

Croucher, D. R.

Publications and source records attributed to Croucher, D. R..

11 recordsLinked to original sources

bioRxiv2025

Inclusion of JNK-independent drugs within multi-agent chemotherapy improves response in relapsed high-risk neuroblastoma

The acquisition of a chemoresistant state underlies poor prognosis in many cancers, including neuroblastoma. We previously demonstrated that heterogeneity in apoptosis induction through c-Jun N-Terminal Kinase (JNK) promotes a form of non-genetic chemoresistance in neuroblastoma observable at both patient and single-cell levels. As the maintenance of this JNK-impaired state in the relapse setting is a significant barrier to the efficacy of many standard-of-care chemotherapy drugs, we combined a mechanistic, mathematical model of JNK activation with a paediatric focused drug screen and identified approved oncology drugs capable of inducing apoptosis in a JNK-independent manner. Functional genomics further revealed that synergy between these JNK-independent drugs and standard-of-care chemotherapies emerged from differential utilisation of apoptotic network components, rather than from their direct mechanistic targets. Efficacy studies with PDX models also confirmed that including a JNK-independent drug within existing chemotherapy backbones significantly improved response in the relapse setting, where new approaches are urgently needed. One Sentence SummaryDrug combinations utilising differential sets of network components can overcome the apoptotic impaired state of relapsed high-risk neuroblastoma.

cancer biology↗

bioRxiv2025

A selective inhibitor of oncogenic JNK signalling perturbs metastatic outgrowth of triple-negative breast cancer through metabolic blockade

Although c-Jun N-terminal Kinase (JNK) represents an attractive anti-cancer target, its pleiotropic functionality limits the use of direct JNK inhibitors. Here, we identify a distinct subcellular pattern of JNK activity as a therapeutic vulnerability in breast cancer, where cytoplasmic JNK activity predicts poor survival outcomes, is elevated in triple-negative breast cancers (TNBC) and is essential for metastatic outgrowth. Mechanistic analyses reveal cytoplasmic JNK acts through multiple mechanisms, with downstream targets involved in cellular metabolism and cytoskeletal regulation. On this basis, we leveraged actin-based phenotypic drug-screening and identified K12, an indirect but selective inhibitor of cytoplasmic JNK that blocks TNBC metastatic outgrowth in vivo. We reveal that K12 inhibits glutaminase-1 and the pyruvate dehydrogenase complex, and that this poly-pharmacology overcomes pyruvate anaplerosis, a known resistance mechanism of existing glutaminase inhibitors. These findings demonstrate the potential of selectively targeting the oncogenic function of JNK, offering new treatment options for early-stage metastatic TNBC.

cancer biology↗

bioRxiv2025

JNK pathway suppression drives resistance to combination endocrine therapy and CDK4/6 inhibition in ER+ breast cancer

PurposeEndocrine therapy in combination with CDK4/6 inhibition doubles the progression-free survival of patients with advanced ER+ breast cancer, but resistance is inevitable, leaving patients with limited treatment options. Experimental DesignWe performed unbiased genome-wide CRISPR/Cas9 knockout screens using ER+ breast cancer cells to identify novel drivers of resistance to combination endocrine therapy (tamoxifen) and CDK4/6 inhibitor (palbociclib) treatment. Screen hits were validated by CRISPR/Cas9 knockout models, mechanistic analyses and evaluation of patient samples. ResultsOur screens identified the inactivation of JNK signalling, including loss of the kinase MAP2K7, as a key driver of combination resistance. We developed multiple CRISPR/Cas9 knockout ER+ breast cancer cell lines (MCF-7 and T-47D) to investigate the effects of MAP2K7, MAPK8 and MAPK9 loss. MAP2K7 knockout increased metastatic burden in vivo and led to impaired JNK-mediated stress responses, as well as promoting cell survival and reducing senescence entry following endocrine therapy and CDK4/6 inhibitor treatment. Mechanistically, this occurred via loss of the AP-1 transcription factor c-JUN, leading to an attenuated response to combination endocrine therapy plus CDK4/6 inhibition. Furthermore, we analysed ER+ advanced breast cancer patient cohorts and found that inactivation of the JNK pathway was associated with increased metastatic burden, and low pJNKT183/Y185 activity correlated with a poorer response to systemic endocrine and CDK4/6 inhibitor therapies. ConclusionsOverall, we demonstrate that suppression of JNK signalling enables persistent growth during combined endocrine therapy and CDK4/6 inhibition. Furthermore, our data provide a pre-clinical rationale to screen patients tumours for JNK signalling deficiency prior to receiving combined endocrine therapy and CDK4/6 inhibition. STATEMENT OF TRANSLATIONAL RELEVANCEResistance to CDK4/6 inhibitors in the context of endocrine therapy resistance presents an urgent clinical challenge for the management of estrogen receptor positive (ER+) breast cancer. However, the mechanisms driving resistance to this therapeutic combination remain poorly understood. Here, we have identified inactivation of JNK signalling, specifically loss of the JNK kinase MAP2K7, as a major determinant of resistance to combined endocrine therapy and CDK4/6 inhibition in ER+ breast cancer. We uncovered that MAP2K7 loss augments tumour survival in vitro and in vivo. This occurs via disruption of activator protein-1 (AP-1) transcription factors, and thus prevents the induction of therapy-induced senescence and JNK-induced stress response. These findings reveal a critical tumour suppressor role for the JNK pathway in ER+ breast cancer, highlighting the importance of identifying patients with deficient JNK signalling and cautioning against the development of JNK inhibitors for this setting.

cancer biology↗

bioRxiv2024

PSKH1 kinase activity is differentially modulated via allosteric binding of Ca2+ sensor proteins

Protein Serine Kinase H1 (PSKH1) was recently identified as a crucial factor in kidney development and is overexpressed in prostate, lung and kidney cancers. However, little is known about PSKH1 regulatory mechanisms, leading to its classification as a "dark" kinase. Here, we used biochemistry and mass spectrometry to define PSKH1s consensus substrate motif, protein interactors, and how interactors, including Ca2+ sensor proteins, promote or suppress activity. Intriguingly, despite the absence of a canonical Calmodulin binding motif, Ca2+-Calmodulin activated PSKH1 while, in contrast, the ER-resident Ca2+ sensor of the CREC family, Reticulocalbin-3, suppressed PSKH1 catalytic activity. In addition to antagonistic regulation of the PSKH1 kinase domain by Ca2+ sensing proteins, we identified UNC119B as a protein interactor that activates PSKH1 via direct engagement of the kinase domain. Our findings identify complementary allosteric mechanisms by which regulatory proteins tune PSKH1s catalytic activity, and raise the possibility that different Ca2+ sensors may act more broadly to tune kinase activities by detecting and decoding extremes of intracellular Ca2+ concentrations.

biochemistry↗

bioRxiv2024

Feed-forward stimulation of CAMK2 by the oncogenic pseudokinase PEAK1 generates a therapeutically "actionable" signalling axis in triple negative breast cancer

The PEAK family of pseudokinases, comprising PEAK1-3, are signalling scaffolds that play oncogenic roles in several poor prognosis human cancers, including triple negative breast cancer (TNBC). However, therapeutic targeting of pseudokinases is challenging due to their lack of catalytic activity. To address this, we screened for PEAK1 effectors by affinity purification and mass spectrometry, identifying calcium/calmodulin-dependent protein kinase 2 (CAMK2)D and CAMK2G. PEAK1 promoted CAMK2D/G activation in TNBC cells via a novel feed-forward mechanism involving PEAK1/PLC{gamma}1/Ca2+ signalling and direct binding via a consensus CAMK2 interaction motif in the PEAK1 N-terminus. In turn, CAMK2 phosphorylated PEAK1 to enhance association with PEAK2, which is critical for PEAK1 oncogenic signalling. To achieve pharmacologic targeting of PEAK1/CAMK2, we repurposed RA306, a second generation CAMK2 inhibitor under pre-clinical development for treatment of cardiovascular disease. RA306 demonstrated on-target activity against CAMK2 in TNBC cells and inhibited PEAK1-enhanced migration and invasion in vitro. Moreover, RA306 significantly attenuated TNBC xenograft growth and blocked metastasis in a manner mirrored by CRISPR-mediated PEAK1 ablation. Overall, these studies establish PEAK1 as a critical cell signalling nexus, identify a novel mechanism for regulation of Ca2+ signalling and its integration with tyrosine kinase signals, and identify CAMK2 as a therapeutically actionable target downstream of PEAK1.

cancer biology↗

bioRxiv2021

Bisphosphonate drugs have actions outside the skeleton and inhibit the mevalonate pathway in alveolar macrophages

Bisphosphonates drugs target the skeleton and are used globally for the treatment of common bone disorders. Nitrogen-containing bisphosphonates act by inhibiting the mevalonate pathway in bone-resorbing osteoclasts but, surprisingly, also appear to reduce the risk of death from pneumonia. We overturn the long-held belief that these drugs act only in the skeleton and show that a fluorescently-labelled bisphosphonate is internalised by alveolar macrophages and peritoneal macrophages in vivo. Furthermore, a single dose of a nitrogen-containing bisphosphonate (zoledronic acid) in mice was sufficient to inhibit the mevalonate pathway in tissue-resident macrophages, causing the build-up of a mevalonate metabolite and preventing protein prenylation. Importantly, one dose of bisphosphonate enhanced the immune response to bacterial endotoxin in the lung and increased the level of cytokines and chemokines in bronchoalveolar fluid. These studies suggest that bisphosphonates, as well as preventing bone loss, may boost immune responses to infection in the lung and provide a mechanistic basis to fully examine the potential of bisphosphonates to help combat respiratory infections that cause pneumonia.

pharmacology and toxicology↗

bioRxiv2020

Resistance to platinum chemotherapy in lung adenocarcinoma is driven by a non-genetic, cell-cycle dependent mechanism

We previously used a pulse-based in vitro assay to unveil targetable signalling pathways associated with innate cisplatin resistance in lung adenocarcinoma (Hastings et al., 2020). Here we advanced this model system and identified a non- genetic mechanism of resistance that drives recovery and regrowth in a subset of cells. Using RNAseq and a suite of biosensors to track single cell fates both in vitro and in vivo, we identified that early S phase cells have a greater ability to maintain proliferative capacity, which correlated with reduced DNA damage over multiple generations. In contrast, cells in G1, late S or those treated with PARP/RAD51 inhibitors, maintained higher levels of DNA damage and underwent prolonged S/G2 phase arrest and senescence. Combined with our previous work, these data indicate that there is a non-genetic mechanism of resistance in lung adenocarcinoma that is dependent on the cell cycle stage at the time of cisplatin exposure.

cell biology↗

bioRxiv2020

A cryptic tubulin-binding domain links MEKK1 to microtubule remodelling

The MEKK1 protein is a pivotal kinase activator of responses to cellular stress. Activation of MEKK1 can trigger various responses, including mitogen activated protein (MAP) kinases, NF-{kappa}B signalling, or cell migration. Notably, MEKK1 activity is triggered by microtubule-targeting chemotherapies, amongst other stressors. Here we show that MEKK1 contains a previously unidentified tumour overexpressed gene (TOG) domain. The MEKK1 TOG domain binds to tubulin heterodimers--a canonical function of TOG domains--but is unusual in that it appears alone rather than as part of a multi-TOG array, and has structural features distinct from previously characterised TOG domains. MEKK1 TOG demonstrates a clear preference for binding curved tubulin heterodimers, which exist in soluble tubulin and at sites of microtubule polymerisation and depolymerisation. Mutations disrupting tubulin-binding lead to destabilisation of the MEKK1 protein in cells, and ultimately a decrease in microtubule density at the leading edge of polarised cells. We also show that MEKK1 mutations at the tubulin-binding interface of the TOG domain recur in patient derived tumour sequences, suggesting selective enrichment of tumour cells with disrupted MEKK1-microtubule association. Together, these findings provide a direct link between the MEKK1 protein and tubulin, which is likely to be relevant to cancer cell migration and response to microtubule-modulating therapies. SIGNIFICANCE STATEMENTThe protein kinase MEKK1 activates stress response pathways in response to various cellular stressors, including chemotherapies that disrupt dynamics of the tubulin cytoskeleton. Filip[c]ik et al., show that MEKK1 contains a previously uncharacterised domain that can preferentially bind to the curved tubulin heterodimer--which is found at sites of microtubule assembly and disassembly. Mutations that interfere with MEKK1-tubulin binding disrupt microtubule networks in migrating cells and are enriched in patient-derived tumour sequences. These results suggest that MEKK1-tubulin binding may be relevant to cancer progression, and the efficacy of microtubule-disrupting chemotherapies that require the activity of MEKK1.

biochemistry↗

bioRxiv2020

A zebrafish functional genomics model to investigate the role of human A20 variants in vivo

Germline loss-of-function variation in TNFAIP3, encoding A20, has been implicated in a wide variety of autoinflammatory and autoimmune conditions, with acquired somatic missense mutations linked to cancer progression. Furthermore, human sequence data reveals that the A20 locus contains ~400 non-synonymous coding variants which are largely uncharacterised. The growing number of A20 coding variants with unknown function, but potential clinical impact, poses a challenge to traditional mouse-based approaches. Here we report the development of a novel functional genomics approach that utilises the new A20-deficient zebrafish (Danio rerio) model to investigate the impact of TNFAIP3 genetic variants in vivo. Similar to A20-deficient mice, A20-deficient zebrafish are hyper-responsive to inflammatory triggers and exhibit spontaneous early lethality. While ectopic addition of human A20 rescued A20-null zebrafish from lethality, missense mutations at two conserved A20 residues, S381A and C243Y reversed this protective effect. Ser381 represents a phosphorylation site important for enhancing A20 activity that is abrogated by its mutation to alanine, or by a C243Y mutation that associates with human autoimmune disease. These data reveal an evolutionarily conserved role for A20, but also demonstrate how a zebrafish functional genomics pipeline can be utilized to investigate the in vivo significance of medically relevant TNFAIP3 gene variants. This approach could be utilised to investigate genetic variation for other conserved genes.

genetics↗

bioRxiv2019

Dynamic analysis of pulsed cisplatin identifies effectors of resistance in lung adenocarcinoma

Identification of clinically viable strategies for overcoming resistance to platinum chemotherapy in lung adenocarcinoma has been hampered by inappropriately tailored in vitro assays of drug response. Therefore, using a pulse model that closely recapitulates the in vivo pharmacokinetics of platinum therapy, we profiled cisplatin-induced signalling, DNA damage and apoptotic responses across a panel of lung adenocarcinoma cell lines. By coupling this data with real-time, single cell imaging of cell cycle and apoptosis, we show that TP53 mutation status influenced the mode of cisplatin induced cell cycle arrest, but could not predict cisplatin sensitivity. In contrast, P70S6K-mediated signalling promoted resistance by increasing p53/p63 and p21 expression, reducing double-stranded DNA breaks and apoptosis. Targeting P70S6K sensitised both TP53 wildtype and null lines to cisplatin, but not TP53 mutant lines. In summary, using in vitro assays that mimic in vivo pharmacokinetics identified P70S6K as a robust mediator of cisplatin resistance and highlighted the importance of considering somatic mutation status when designing patient-specific combination therapies.

cancer biology↗

bioRxiv2019

Mechanism of preferential complex formation by Apoptosis Signal-regulating Kinases

Apoptosis signal-regulating kinases (ASK1-3) are activators of the P38 and JNK MAP kinase pathways. ASK1-3 form oligomeric complexes known as ASK signalosomes that initiate signalling cascades in response to diverse stress stimuli. Here we demonstrate that oligomerization of ASK proteins is driven by previously uncharacterised sterile-alpha motif (SAM) domains that reside at the C-terminus of each ASK protein. SAM domains from ASK1-3 have distinct behaviours: ASK1 forms unstable oligomers, ASK2 is predominantly monomeric, and the ASK3 SAM domain forms a stable oligomer even at low concentration. In contrast to their isolated behaviour, the ASK1 and ASK2 SAM domains preferentially form a stable heterocomplex. The crystal structure of the ASK3 SAM domain, small-angle X-ray scattering, and mutagenesis suggests that ASK3 oligomers and ASK1-ASK2 complexes form discrete quasi-helical rings, via the mid-loop-end-helix interface. Preferential ASK1-ASK2 binding is consistent with mass spectrometry showing that full-length ASK1 forms heterooligomeric complexes incorporating high levels of ASK2. Accordingly, disruption of SAM domain-association impairs ASK activity in the context of electrophilic stress induced by 4-hydroxy-2-nonenal. These findings provide a structural template for how ASK proteins assemble foci to drive inflammatory signalling, and reinforce that strategies targeting ASK kinases should consider the concerted actions of multiple ASK family members.

biochemistry↗