Biology subjects

Skoda, J.

Publications and source records attributed to Skoda, J..

7 recordsLinked to original sources

bioRxiv2026

Receptor tyrosine kinase inhibitors trametinib and axitinib effectively target the neoplastic component of giant cell tumor of bone in vitro

Background: Giant cell tumor of bone (GCTB) is an intermediate osteolytic neoplasm characterized by a high rate of local recurrence. The tumor comprises two main cell populations: neoplastic stromal cells and osteoclast-like giant cells. Current adjuvant therapies primarily target osteoclast-like giant cells but have limited activity against the neoplastic stromal cells that drive tumor growth and recurrence. Here, we sought to identify actionable vulnerabilities in GCTB stromal cells by profiling druggable receptor tyrosine kinases (RTKs) and associated signaling pathways and evaluating their pharmacological inhibition in patient-derived in vitro models. Methods: RTK activation and downstream signaling were assessed using phospho-protein arrays and western blotting in therapy-naive and denosumab-treated GCTB patient samples and in patient-derived GCTB cell lines, representing clinically relevant models of the neoplastic component. Tyrosine kinase inhibitors (TKIs) targeting activated pathways were evaluated in patient-derived GCTB cell lines cultured under both two-dimensional (2D) and three-dimensional (3D) conditions. Effects on cell viability, proliferation, cell cycle distribution, and cell death were assessed using MTT and XTT assays, flow cytometry, live-cell imaging, and confocal microscopy. Results: Several activated RTKs, including EGFR, IGF-IR, InsR, PDGFR, and PDGFR{beta}, together with downstream signaling proteins MEK1/2 and ERK1/2, were identified as potential therapeutic targets. Among the inhibitors tested, trametinib, a MEK1/2 inhibitor, and axitinib, a multi-target inhibitor of VEGFR1-3, PDGFR{beta}, and c-Kit, demonstrated the strongest antiproliferative and cytotoxic effects, markedly reducing cell viability and proliferation while promoting cell death in vitro. Importantly, trametinib showed selective activity against GCTB stromal cells compared with healthy controls, suggesting a potential therapeutic window. Conclusions: This study identifies trametinib and axitinib as promising therapeutic candidates for targeting the neoplastic component of GCTB. Our findings provide a rationale for further preclinical and clinical evaluation of RTK-directed therapies to complement current treatments and specifically target the neoplastic GCTB stromal cells underlying tumor persistence and recurrence.

cancer biology↗

bioRxiv2026

Novel cumate-inducible models of MYC-driven neuroblastoma enable unconfounded mitochondrial synthetic lethality screens

Oncogenic MYC transcription factors profoundly alter cellular programs, imposing dependencies that can be therapeutically exploited in MYC-driven cancers such as high-risk neuroblastoma. However, dissecting such synthetic lethal vulnerabilities using controlled, tunable gene expression within a uniform genetic background remains challenging. Widely used tetracycline-regulated systems rely on inducers known to perturb mitochondrial function, introducing significant off-target effects that may confound interpretation. To overcome this limitation, we established novel cumate (p-isopropylbenzoate)-inducible neuroblastoma models that enable physiologically unbiased regulation of MYC(N) expression. Functional validation demonstrated that cumate itself does not induce off-target effects on neuroblastoma cell viability, mitochondrial membrane potential, morphology, proteostasis, or stress signaling, even at the highest recommended dose. The developed SHEP-CuO-MYC and -MYCN models show efficient, titratable, and reversible upregulation of c-MYC and N-MYC, respectively, recapitulating the expression levels observed in MYC(N)-amplified neuroblastoma. As a proof-of-concept, we applied these models to mechanistically validate the recently proposed mitoribosomal synthetic lethality, providing fully unbiased evidence that elevated c-MYC/N-MYC levels sensitize neuroblastoma cells to inhibitors of mitochondrial gene expression. Although impairing mitochondrial translation activated mitochondrial integrated stress response in both MYC-on and MYC-off states, it led to dramatic MYC downregulation coupled with enhanced caspase-dependent cell death in MYC-on cells. These findings reveal that MYC(N) overexpression confers a selective, proliferation-independent mitochondrial vulnerability that can be therapeutically targeted by repurposing well-tolerated mitochondrial ribosome-targeting antibiotics. Collectively, our models provide a robust platform for studying the MYC-mitochondria interplay and can be directly adapted for drug repurposing screens targeting mitochondrial dependencies in neuroblastoma and, potentially, other MYC-driven tumors. HIGHLIGHTSO_LICumate shows no inducer-associated mitochondrial or cytotoxic off-target effects C_LIO_LICumate-inducible MYC models enable mechanistic studies of mitochondrial synthetic lethality C_LIO_LIMYC overexpression drives neuroblastoma sensitivity to mitochondrial translation inhibition C_LIO_LICommon ribosomal antibiotics trigger caspase-dependent cell death in MYC-driven tumor cells C_LIO_LIContext-specific MYC downregulation links mitochondrial stress to MYC synthetic lethality C_LI

cancer biology↗

bioRxiv2024

Depletion of chemoresponsive mitochondrial fission mediator DRP1 does not mitigate sarcoma resistance

Specific patterns of mitochondrial dynamics have been repeatedly reported to promote drug resistance in cancer. However, whether targeting mitochondrial fission- and fusion-related proteins could be leveraged to combat multidrug-resistant pediatric sarcomas is poorly understood. Here, we demonstrated that the expression and activation of the mitochondrial fission mediator DRP1 are affected by chemotherapy exposure in common pediatric sarcomas, i.e., rhabdomyosarcoma and osteosarcoma. Unexpectedly, decreasing DRP1 activity through stable DRP1 knockdown did not attenuate sarcoma drug resistance or affect growth rate or the mitochondrial network morphology. The minimal impact on sarcoma cell physiology, combined with the upregulation of fission adaptor proteins (MFF and FIS1) detected in rhabdomyosarcoma cells, indicates that an alternative DRP1-independent mitochondrial fission mechanism may efficiently compensate for the lack of DRP1 activity. By exploring the upstream mitophagy and mitochondrial fission regulator, AMPK1, we found that markedly reduced AMPK1 levels are sufficient to maintain AMPK signaling capacity without affecting chemosensitivity. Collectively, our findings challenge the direct involvement of DRP1 in pediatric sarcoma drug resistance and highlight the complexity of yet-to-be-characterized noncanonical regulators of mitochondrial dynamics. SUMMARY BLURBThe mitochondrial fission mediator DRP1 levels and activation are modulated upon chemotherapy exposure, yet depleting DRP1 does not restore chemosensitivity in the most common pediatric sarcomas.

cancer biology↗

bioRxiv2024

Dysregulation of the p53 pathway provides a therapeutic target in aggressive pediatric sarcomas with stem-like traits

Pediatric sarcomas are bone and soft tissue tumors that often exhibit high metastatic potential and refractory stem-like phenotypes, resulting in poor outcomes. Aggressive sarcomas frequently harbor a disrupted p53 pathway. However, whether sarcoma stemness is associated with abrogated p53 function and might be attenuated via p53 reactivation remains unclear. Here, we show that highly tumorigenic stem-like sarcoma cells exhibit dysregulated p53, making them vulnerable to drugs that restore wild-type p53 activity. Immunohistochemistry of mouse xenografts and human tumor tissues revealed that p53 dysregulations together with enhanced expression of the stemness-related transcription factors SOX2 or KLF4 are crucial features in pediatric osteosarcoma, rhabdomyosarcoma, and Ewings sarcoma development. p53 dysregulation appears to be an important step for sarcoma cells to acquire a fully stem-like phenotype, and p53-positive pediatric sarcomas exhibit a high frequency of early metastasis. Importantly, p53 signaling reactivation via MDM2/MDMX inhibition selectively induces apoptosis in aggressive stem-like Ewings sarcoma cells while sparing healthy fibroblasts. Collectively, our results suggest that restoration of canonical p53 activity provides a promising strategy for improving the treatment of pediatric sarcomas with unfavorable stem-like traits. HIGHLIGHTSO_LISOX2 and KLF4 are crucial factors in pediatric sarcoma tumorigenesis C_LIO_LIDysregulated p53 pathway predisposes sarcoma cells to acquire stem-like features C_LIO_LIp53 positivity is associated with early metastasis in pediatric sarcoma patients C_LIO_LIRestoring wild-type p53 signaling selectively kills stem-like Ewings sarcoma cells C_LI

cancer biology↗

bioRxiv2023

Melanotransferrin Functions as a Pro-Oncogenic WNT Agonist: A Yin-Yang Relationship in Melanoma with the WNT Antagonist and Metastasis Suppressor, NDRG1

A persistent mystery in the melanoma field has been the function of one of the first melanoma tumor antigens characterized, namely p97 (melanotransferrin; MTf). While MTf expression increases melanoma cell proliferation, migration, and tumorigenesis, the molecular mechanism responsible is unknown. On the other hand, N-myc down-stream regulated gene 1 (NDRG1) is a potent metastasis suppressor and WNT antagonist. Expression of NDRG1 in melanoma cells suggests a role in inhibiting metastasis, with this study investigating MTfs role in oncogenic signaling. We demonstrate MTf acts as a pro-oncogenic WNT agonist, which down-regulates NDRG1, while silencing MTf increases NDRG1 expression. In contrast, silencing NDRG1 increases MTf expression. These observations demonstrate a bidirectional negative feedback loop and "Yin-Yang" relationship between MTf and NDRG1. Mechanistically, MTf was directly associated with the WNT co-receptor, lipoprotein-receptor 6 (LRP6), and increased total LRP6 expression, activated p-LRP6 (Ser1490), {beta}-catenin, and activated {beta}-catenin (Ser552) levels, with MTf expression inducing their nuclear accumulation. Additionally, MTf expression increased downstream WNT targets, namely cyclin D1 and c-Myc, with c-Myc down-regulating NDRG1 expression. Silencing c-Myc prevented the Yin-Yang relationship between NDRG1 and MTf, indicating c-Myc played a key role in their inverse regulation. Melanoma patient specimens demonstrated that a low NDRG1/MTf ratio was significantly (p = 0.008) associated with lower survival and metastasis. Chemotherapeutic agents that up-regulated NDRG1 depressed MTf and nuclear LRP6 and potently inhibited melanoma xenograft growth in vivo. This study demonstrates MTf acts as a WNT agonist, with a Yin-Yang relationship being observed with the WNT antagonist, NDRG1.

cancer biology↗

bioRxiv2023

Inhibiting mitochondrial translation overcomes multidrug resistance in MYC-driven neuroblastoma via OMA1-mediated integrated stress response

High-risk neuroblastoma remains a clinically challenging childhood tumor with a 5-year survival of only 50%. Tumors overexpressing N-MYC or c-MYC oncoproteins define a group of MYC-driven high-risk neuroblastoma with the most dismal outcomes, mainly caused by treatment failure due to the emergence and regrowth of multidrug-resistant cancer cells. Specific mitochondrial processes have been implicated in the maintenance of aggressive stem-like phenotypes in various cancers. We have recently identified a novel mitochondria-mediated mechanism of neuroblastoma multidrug resistance. However, the potential of pharmacological targeting of mitochondria to overcome therapy resistance and stemness in neuroblastoma remains unclear. Here, we show that c-MYC/N-MYC-driven multidrug-resistant neuroblastoma cells are highly vulnerable to cell death induced by the inhibition of mitochondrial translation. In contrast with normal fibroblasts, doxycycline (DOXY)-mediated inhibition of mitochondrial ribosomes efficiently impaired the survival of neuroblastoma cells regardless of their multidrug resistance and stem-like phenotypes. Mechanistically, inhibiting mitochondrial translation induced the mitochondrial stress-activated integrated stress response (ISR) via the OMA1-eIF2 axis, which preceded neuroblastoma cell death. Strikingly, several oncoproteins associated with poor neuroblastoma prognosis, including c-MYC and N-MYC, were markedly downregulated upon ISR activation. Comparing models of various neuroectodermal tumors and normal fibroblasts, we identified high levels of phosphorylated c-MYC and N-MYC (indicating their activity and rapid turnover) as a factor that predetermines susceptibility of neuroblastoma cells to DOXY-induced cell death. Neuroblastoma cells failed to develop significant DOXY resistance over a long-term repeated (pulsed) selection pressure, further demonstrating mitochondrial protein balance as a clinically relevant vulnerability of cancer cells that rely on high MYC activity. Together, our findings provide insight into mitochondrial retrograde regulatory networks in the context of MYC dependence and demonstrate the mitochondrial translation machinery as a promising therapeutic target in multidrug-resistant MYC-driven neuroblastoma.

cancer biology↗

bioRxiv2021

REDUCED ER-MITOCHONDRIA CONNECTIVITY PROMOTES NEUROBLASTOMA MULTIDRUG RESISTANCE

Most cancer deaths result from progression of therapy resistant disease, yet our understanding of this phenotype is limited. Cancer therapies generate stress signals that act upon mitochondria to initiate apoptotic programs. We isolated mitochondria from neuroblastoma cell lines obtained from children at diagnosis and after relapse following failed therapy, and profiled responses to tBid and Bim, death effectors activated by therapeutic stress. Mitochondria from post-relapse models had markedly attenuated cytochrome c release (surrogate for apoptotic commitment) in comparison with patient-matched diagnostic models. Mitochondrial DNA content, size, and shape did not differ consistently. However, we used electron microscopy to identify reduced endoplasmic reticulum-mitochondria contacts (ERMCs) as correlated with therapy resistance. ERMCs form microdomains for the transfer of Ca2+ to mitochondria. We confirmed reduced Ca2+ transfer in resistant cells, with restoration by re-opposing ERMCs via genetically-encoded linkers. However, reduced Ca2+ transfer was not present in all ERMC-reduced cancers with therapy resistance, supporting Ca2+-independent mechanisms. Genetically or biochemically reducing ERMCs in therapy sensitive tumors phenocopied resistance, validating these inter-organelle contacts as physiologic regulators of apoptosis. Our work confirms the importance of ERMCs in stress signaling and provides a previously unrecognized mechanism for cancer cell resistance that is not exclusive to other contributors.

cancer biology↗