Juha K. Rantala

MYCN amplification drives replication stress in high-risk neuroblastoma, yet how MYCN-amplified tumour cells tolerate this stress to sustain proliferation remains poorly understood. Here we show that FBXL12, an SCF ubiquitin ligase substrate receptor that targets the Fanconi anaemia protein FANCD2 for degradation at replication forks, as well as the broader Fanconi anaemia and replication stress transcriptional program are elevated in high-risk and MYCN-amplified neuroblastoma. High FBXL12 expression independently predicts poor survival across neuroblastoma patient cohorts. FBXL12 loss stabilizes FANCD2 on chromatin, elevates ATR-dependent replication stress signalling and DNA damage during S phase, and impairs proliferation of MYCN-amplified neuroblastoma cells in vitro and in vivo. Mechanistically, MYCN directly engages the FBXL12-FANCD2 complex and antagonises FBXL12-mediated degradation of FANCD2 at replication forks, revealing that the oncogenic driver of replication stress also actively preserves the chromatin-bound FANCD2 pool required to tolerate it. Beyond S phase, FBXL12 loss disrupts FANCD2-dependent mitotic DNA synthesis and transmits unresolved replication intermediates into daughter cells. FBXL12-deficient cells consequently show transcriptional activation of MYC target gene, ATR, and mTOR signalling programs, and this pathway-concordant state confers differential sensitivity to ATR, and mTOR-targeting compounds, nominating candidate therapeutic strategies for this disease subset. Together, these findings define a MYCN-FBXL12-FANCD2 axis as a clinically relevant vulnerability in high-risk neuroblastoma. HIGHLIGHTS: FBXL12 is upregulated in high-risk neuroblastoma and predicts poor survival FBXL12 loss traps FANCD2 on chromatin and drives S-phase DNA damage MYCN antagonises FBXL12-mediated degradation of FANCD2 at replication forks FBXL12 loss impairs mitotic DNA synthesis and damages daughter cells FBXL12-deficient cells show ATR/mTOR activation and drug sensitivity

Background: Bladder cancer (BC) is the tenth most common cancer and the ninth leading cause of cancer death worldwide. BC has high rates of treatment failure, so alternate approaches are needed to personalise treatments to individual patients in order to improve outcomes from this disease. One method that could provide actionable results to influence clinical decisions on alternative treatments is ex vivo drug screening. Methods: We explored the feasibility of using ex vivo drug screening directly on patient tumour tissue from transurethral resection of the bladder tumours (TURBT) and cystectomies. We screened 38 BC patients investigating drug sensitivities to 15 agents, including standard of care treatments and some more exploratory compounds. In addition, we investigated ex vivo sensitivity and resistance over the 15 compounds and annotated common mutational profiles. We saw high methodological success (41/54 samples, 75.9%), in clinically useful timeframes (4 days) and identified distinct drug and tumour clusters. Results: Here, we show that drug resistance is associated with aggressive clinical features, mutation burden, and differs with individual gene mutations. Cross-resistance between agents is common. Cisplatin-resistant tumours differ by mutational profiles and include those with multi-drug resistance and those sensitive to alternative agents. Observed clinical responses match our ex vivo response (5/6 patients, 83.3%). Proliferative responses are observed to some receptor tyrosine kinase inhibitors, cautioning against their unselected widespread use. Conclusions: Ex vivo drug screening identifies drug clusters of patients’ tumours that could potentially respond to standard of care and alternative therapies. Our approach offers a platform to potentially individualise treatments, especially in drug-resistant tumours.

Phosphodiesterase 3 A (PDE3A) is a well-characterized enzyme that plays a crucial role in various cellular processes, including cAMP-mediated signaling, CREB-mediated induction of p21 and signaling through protein kinases A and G. PDE3A has also been suggested as an inflammation-associated stemness gene which upon interaction with protein SLFN12, leads to blocked protein translation and induction of apoptosis. PDE3A has been found to be highly expressed in several human cancer types including sarcomas, pancreatic ductal adenocarcinoma, non-small cell lung cancer and melanoma. PDE3A has thus emerged as a potential therapeutic cancer target. However, to fully understand the functional role and validate target potential of PDE3A in different human cancers, further research is needed. We report here a pan-cancer ex vivo study of PDE3A protein expression across 24 different cancer types represented by 59 different molecular subtypes correlated with ex vivo drug screening of PDE3A-SLFN12 molecular glue anagrelide in 250 patient derived functional tumor models. Results of the study identify highest PDE3A expression in melanomas and across different histological subtypes of sarcomas. Correlating with the expression profile of PDE3A, anagrelide was found to display best therapeutic potential in sarcomas with high protein expression of both PDE3A and SLFN12, though sarcoma heterogeneity warrants further subtype-specific validation.

DNA replication is tightly regulated to ensure a single round of chromosome duplication per cell division. DNA licensing restricts origin firing to once-per-cell-cycle while aberrant licensing promotes re-replication and genome instability. Here, we investigate the mechanisms that protect genome integrity following re-replication induced by depletion of the licensing inhibitor Geminin. We find that re-replicating cells require FANCD2 to prevent genome instability. FANCD2 is rapidly recruited to chromatin upon Geminin loss, where it limits unrestrained fork progression and prevents single strand DNA gap accumulation and fork breakage. Genome-wide analyses reveal that upon re-replication, FANCD2 localizes to early origins within highly transcribed regions prone to accumulate R-loops and enriched in early replicating fragile sites. Importantly, reducing transcription and R-loops alleviates re-replication-induced genome fragility, whereas PARP inhibition exacerbates it. Our study uncovers a role for FANCD2 in safeguarding genome integrity during re-replication, offering avenues for selective targeting of cancer cells.

In many solid cancer types, surgery alone could be a sufficient first therapy option for a significant number of cancer patients. However, there are currently no diagnostic solutions to identify patients who could be stratified to surgery alone. To identify a biomarker predicting cancer surgery response, candidate biomarkers were studied in a non-metastatic head and neck squamous cell carcinoma (nmHNSCC) cohort well representative of the HPV-negative patient population. LIMA1 immunohistochemistry (IHC) with specificity-validated antibodies outperformed all other biomarkers in multivariable survival analyses of patients with nmHNSCC ( n = 128, HR 2.10, P = 0.006). The prognostic effect was selective to LIMA1-alpha isoform IHC detection in patients who had received surgical therapy ( n = 184, HR 2.39, P > 0.001). Strikingly, our real-world validation results, using two prospectively collected cohorts ( n = 15 and n = 86), demonstrate that none of the LIMA1 negative patients died of HNSCC during the follow-up. Collectively, we report here the discovery of a diagnostic LIMA1-alpha IHC assay for HPV-negative HNSCC patient stratification to surgery-only therapy. Application of LIMA1 detection in routine nmHNSCC diagnostics would revolutionize the clinical management of HNSCC patients.

B cell lymphomas (BCL) are a heterogenous group of hematologic malignancies, with over 70 identified subtypes. Current first line treatment for all subtypes is rituximab and chemotherapy regimen (R-CHOP); of the treated patients, up to 40-60% relapse or have refractory disease, with typically higher mortality rates. Therefore, more efficient treatment, but also improved ex vivo research models are needed to study the treatment response and resistance. To overcome the expensive and time-consuming process of drug discovery and development, we aim to combat the lymphoma heterogeneity with repurposed drugs, especially focusing on cationic amphiphilic drugs (CADs) that damage cancer cell lysosomes. Lysosomes are central organelles in nutrient sensing and cellular metabolism. Malignant transformation rewires cellular metabolism to boost growth. In turn, cancer cells increase lysosomal production and activity to meet the metabolic needs. Our current results indicate that targeting the reported cancer cell lysosomal ‘weakness’ with repurposed CADs Ebastine and Penfluridol prevents pro-survival cell signaling, and suppresses long-term tumor outgrowth in B lymphoma cell lines, patient blood B cells, and in patient lymph node-derived organoids. We have developed a novel patient-derived lymphoma organoid model as an ex vivo drug assay platform from patients’ own cancer tissue. With combining the lymphoma organoid model and repurposed drugs, this study provides a simpler and cost-effective, yet translationally relevant clinical solution for personalized medicine and improved treatment of BCL patients.

Integrin-dependent adhesion to the extracellular matrix (ECM) mediates mechanosensing and signaling in response to altered microenvironmental conditions. In order to provide tissue- and organ-specific cues, the ECM is composed of many different proteins that temper the mechanical properties and provide the necessary structural diversity. Despite most human tissues being soft, the prevailing view from predominantly in vitro studies is that increased stiffness triggers effective cell spreading and activation of mechanosensitive signaling pathways. To address the functional coupling of ECM composition and matrix rigidity on compliant substrates, we developed a matrix spot array system to screen cell phenotypes against different ECM mixtures on defined substrate stiffnesses at high resolution. We applied this system to both cancer and normal cells and surprisingly identified ECM mixtures that support stiffness-insensitive cell spreading on soft substrates. Employing the motor-clutch model to simulate cell adhesion on biochemically distinct soft substrates, with varying numbers of available ECM–integrin–cytoskeleton (clutch) connections, we identified conditions in which spreading would be supported on soft matrices. Combining simulations and experiments, we show that cell spreading on soft is supported by increased clutch engagement on specific ECM mixtures and even augmented by the partial inhibition of actomyosin contractility. Thus, “stiff-like” spreading on soft is determined by a balance of a cell’s contractile and adhesive machinery. This provides a fundamental perspective for in vitro mechanobiology studies, identifying a mechanism through which cells spread, function, and signal effectively on soft substrates.

Despite advances in cancer genomics and the increased use of genomic medicine, metastatic cancer is still mostly an incurable and fatal disease. With diminishing returns from traditional drug discovery strategies, and high clinical failure rates, more emphasis is being placed on alternative drug discovery platforms, such as ex vivo approaches. Ex vivo approaches aim to embed biological relevance and inter-patient variability at an earlier stage of drug discovery, and to offer more precise treatment stratification for patients. However, these techniques also have a high potential to offer personalised therapies to patients, complementing and enhancing genomic medicine. Although an array of approaches are available to researchers, only a minority of techniques have made it through to direct patient treatment within robust clinical trials. Within this review, we discuss the current challenges to ex vivo approaches within clinical practice and summarise the contemporary literature which has directed patient treatment. Finally, we map out how ex vivo approaches could transition from a small-scale, predominantly research based technology to a robust and validated predictive tool. In future, these pre-clinical approaches may be integrated into clinical cancer pathways to assist in the personalisation of therapy choices and to hopefully improve patient experiences and outcomes.