Acquired NF2 mutation confers resistance to TRK inhibition in an ex vivo LMNA::NTRK1-rearranged soft-tissue sarcoma cell model.

Genomic rearrangements of the neurotrophic receptor tyrosine kinase genes (NTRK1, NTRK2, and NTRK3) are the most common mechanism of oncogenic activation for this family of receptors, resulting in sustained cancer cell proliferation. Several targeted therapies have been approved for tumours harbouring NTRK fusions and a new generation of TRK inhibitors has already been developed due to acquired resistance. We established a patient-derived LMNA::NTRK1-rearranged soft-tissue sarcoma cell model ex vivo with an acquired resistance to targeted TRK inhibition. Molecular profiling of the resistant clones revealed an acquired NF2 loss of function mutation that was absent in the parental cell model. Parental cells showed continuous sensitivity to TRK-targeted treatment, whereas the resistant clones were insensitive. Furthermore, resistant clones showed upregulation of the MAPK and mTOR/AKT pathways in the gene expression based on RNA sequencing data and increased sensitivity to MEK and mTOR inhibitor therapy. Drug synergy was seen using trametinib and rapamycin in combination with entrectinib. Medium-throughput drug screening further identified small compounds as potential drug candidates to overcome resistance as monotherapy or in combination with entrectinib. In summary, we developed a comprehensive model of drug resistance in an LMNA::NTRK1-rearranged soft-tissue sarcoma and have broadened the understanding of acquired drug resistance to targeted TRK therapy. Furthermore, we identified drug combinations and small compounds to overcome acquired drug resistance and potentially guide patient care in a functional precision oncology setting. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Establishment and functional testing of a novel ex vivo extraskeletal osteosarcoma cell model (USZ20-ESOS1).

Extraskeletal osteosarcoma (ESOS) is a rare malignant mesenchymal tumor that originates in the soft tissue. ESOS accounts for less than 1% of all soft tissue sarcomas and exhibits an aggressive behavior with a high propensity for local recurrence and distant metastasis. Despite advances in treatment, the prognosis for ESOS remains poor, with a five-year survival rate of less than 50% and 27% for metastatic patients. Ex vivo models derived from patient samples are critical tools for studying rare diseases with poor prognoses, such as ESOS, and identifying potential new treatment strategies. In this work, we established a novel ESOS ex vivo sarco-sphere model from a metastatic lesion to the dermis for research and functional testing purposes. The ex vivo cell model accurately recapitulated the native tumor, as evidenced by histomorphology and molecular profiles. Through a functional screening approach, we were able to identify novel individual anti-cancer drug sensitivities for different drugs such as romidepsin, miverbresib and to multiple kinase inhibitors. Overall, our new ESOS ex vivo cell model represents a valuable tool for investigating disease mechanisms and answering basic and translational research questions.

Addressing Modern Diagnostic Pathology for Patient-Derived Soft Tissue Sarcosphere Models in the Era of Functional Precision Oncology.

Responses to therapy often cannot be exclusively predicted by molecular markers, thus evidencing a critical need to develop tools for better patient selection based on relations between tumor phenotype and genotype. Patient-derived cell models could help to better refine patient stratification procedures and lead to improved clinical management. So far, such ex vivo cell models have been used for addressing basic research questions and in preclinical studies. As they now enter the era of functional precision oncology, it is of utmost importance that they meet quality standards to fully represent the molecular and phenotypical architecture of patients' tumors. Well-characterized ex vivo models are imperative for rare cancer types with high patient heterogeneity and unknown driver mutations. Soft tissue sarcomas account for a very rare, heterogeneous group of malignancies that are challenging from a diagnostic standpoint and difficult to treat in a metastatic setting because of chemotherapy resistance and a lack of targeted treatment options. Functional drug screening in patient-derived cancer cell models is a more recent approach for discovering novel therapeutic candidate drugs. However, because of the rarity and heterogeneity of soft tissue sarcomas, the number of well-established and characterized sarcoma cell models is extremely limited. Within our hospital-based platform we establish high-fidelity patient-derived ex vivo cancer models from solid tumors for enabling functional precision oncology and addressing research questions to overcome this problem. We here present 5 novel, well-characterized, complex-karyotype ex vivo soft tissue sarcosphere models, which are effective tools to study molecular pathogenesis and identify the novel drug sensitivities of these genetically complex diseases. We addressed the quality standards that should be generally considered for the characterization of such ex vivo models. More broadly, we suggest a scalable platform to provide high-fidelity ex vivo models to the scientific community and enable functional precision oncology.

Unravelling homologous recombination repair deficiency and therapeutic opportunities in soft tissue and bone sarcoma.

Defects in homologous recombination repair (HRR) in tumors correlate with poor prognosis and metastases development. Determining HRR deficiency (HRD) is of major clinical relevance as it is associated with therapeutic vulnerabilities and remains poorly investigated in sarcoma. Here, we show that specific sarcoma entities exhibit high levels of genomic instability signatures and molecular alterations in HRR genes, while harboring a complex pattern of chromosomal instability. Furthermore, sarcomas carrying HRDness traits exhibit a distinct SARC-HRD transcriptional signature that predicts PARP inhibitor sensitivity in patient-derived sarcoma cells. Concomitantly, HRDhigh sarcoma cells lack RAD51 nuclear foci formation upon DNA damage, further evidencing defects in HRR. We further identify the WEE1 kinase as a therapeutic vulnerability for sarcomas with HRDness and demonstrate the clinical benefit of combining DNA damaging agents and inhibitors of DNA repair pathways ex vivo and in the clinic. In summary, we provide a personalized oncological approach to treat sarcoma patients successfully.

Establishment, characterization and functional testing of two novel ex vivo extraskeletal myxoid chondrosarcoma (EMC) cell models.

Extraskeletal myxoid chondrosarcoma (EMC) is a malignant mesenchymal neoplasm of uncertain differentiation as classified by the WHO Classification of Tumours 2020. Although often associated with pronlonged survival, EMC has high rates of distant recurrences and disease-associated death. EMCs are translocation sarcomas and harbor in > 90% of the cases an NR4A3 rearrangement. The molecular consequences of the NR4A3 gene fusions are not yet fully elucidated as well-characterized ex vivo cell models for EMC are lacking. Patient-derived ex vivo models are important and essential tools for investigating disease mechanisms associated with diseases that are rare, that exhibit poor prognosis and for the identification of potential novel treatment options. We established two novel EMC ex vivo models (USZ20-EMC1 and USZ22-EMC2) for functional testing and research purposes. USZ20-EMC1 and USZ22-EMC2 were established and maintained as sarco-sphere cell models for several months in culture. The cells were molecularly characterized using DNA sequencing and methylation profiling. Both cell models represent their native tumor tissue as confirmed by histomorphology and their molecular profiles, suggesting that native tumor cell function can be recapitulated in the ex vivo models. Using a functional screening approach, novel anti-cancer drug sensitivities including potential synergistic combinations were identified. In conclusion, two novel EMC ex vivo cell models (USZ20-EMC1 and USZ22-EMC2) were successfully established and characterized from native tumor tissues. Both cell models will be useful tools for further investigating disease mechanisms and for answering basic and translational research questions.

A Functional Precision Oncology Approach to Identify Treatment Strategies for Myxofibrosarcoma Patients.

In this era of precision medicine, numerous workflows for the targeting of high-recurrent mutations in common tumor types have been developed, leaving patients with rare diseases with few options. Here, we implement a functional precision oncology approach utilizing comprehensive genomic profiling in combination with high-throughput drug screening, to identify tumor-specific drug sensitivities for patients with rare tumor types such as myxofibrosarcoma. From a patient with a high-grade myxofibrosarcoma, who was enrolled in the Englander Institute for Precision Medicine (EIPM) program, we established patient-derived 3D sarco-spheres and xenograft models for functional testing. In the absence of a large cohort of clinically similar cases, high-throughput drug screening was performed on the patient-derived cells, and compared with two other myxofibrosarcoma lines and a benign fibroblast line to functionally identify tumor-specific drug sensitivities. The addition of functional drug sensitivity testing to complement genomic profiling identified multiple therapeutic options that were further validated in patient derived xenograft models. Genomic analyses detected the frequently known codeletion of the tumor suppressors CDKN2A/B together with the methylthioadenosine phosphorylase (MTAP) and a TP53 E286fs*50 mutation. High-throughput drug screening demonstrated tumor-specific sensitivity to compounds targeting the cell cycle. Based on genomic analysis and high-throughput drug screening, we show that targeting the cell cycle in these tumors is a powerful approach. IMPLICATIONS: This study demonstrates the potential of functional testing to aid clinical decision making for patients with rare or molecularly complex malignancies when combined with comprehensive genomic profiling.

Total Flavonoids of Litchi Seed Attenuate Prostate Cancer Progression Via Inhibiting AKT/mTOR and NF-kB Signaling Pathways.

Litchi seeds have been traditionally used in Chinese herbal formula for urologic neoplasms including prostate cancer (PCa). However, the effective components of Litchi seeds and the mechanisms of their actions on PCa cell growth and metastasis remain unclear. In this study, we investigated the effects and molecular mechanisms of the Total Flavonoid of Litchi Seed (TFLS) in PCa PC3 and DU145 cell lines. We found that TFLS significantly inhibited the PCa cell proliferation, induced apoptosis, and prevented cell migration and invasion. Furthermore, we observed that TFLS upregulated the expression of epithelial biomarker E-cadherin and downregulated mesenchymal biomarker Vimentin. TFLS also increased the expression of cleaved-PRAP and Bax, and decreased the expression of Bcl-2 in both PC3 and DU145 cells. Besides, TFLS inhibited AKT signaling pathway by reducing the phosphorylation of AKT and activities of downstream signal transducers including mTOR, IκBα and NF-kB. Finally, TFLS treated mice exhibited a significant decrease in tumor size without toxicity in major organs in vivo. These results indicated that TFLS could suppress PCa cell growth in vivo and inhibit PCa cell proliferation and metastasis in vitro through induction of apoptosis and phenotypic reversal of EMT, which may be achieved by inhibiting the AKT/mTOR and NF-κB signaling pathways. Taken together, our data provide new insights into the role of TFLS as a novel potent anti-cancer agent for the treatment of PCa.

Mapping Pharmacological Network of Multi-Targeting Litchi Ingredients in Cancer Therapeutics.

Considerable pharmacological studies have demonstrated that the extracts and ingredients from different parts (seeds, peels, pulps, and flowers) of Litchi exhibited anticancer effects by affecting the proliferation, apoptosis, autophagy, metastasis, chemotherapy and radiotherapy sensitivity, stemness, metabolism, angiogenesis, and immunity via multiple targeting. However, there is no systematical analysis on the interaction network of "multiple ingredients-multiple targets-multiple pathways" anticancer effects of Litchi. In this study, we summarized the confirmed anticancer ingredients and molecular targets of Litchi based on published articles and applied network pharmacology approach to explore the complex mechanisms underlying these effects from a perspective of system biology. The top ingredients, top targets, and top pathways of each anticancer function were identified using network pharmacology approach. Further intersecting analyses showed that Epigallocatechin gallate (EGCG), Gallic acid, Kaempferol, Luteolin, and Betulinic acid were the top ingredients which might be the key ingredients exerting anticancer function of Litchi, while BAX, BCL2, CASP3, and AKT1 were the top targets which might be the main targets underling the anticancer mechanisms of these top ingredients. These results provided references for further understanding and exploration of Litchi as therapeutics in cancer as well as the application of "Component Formula" based on Litchi's effective ingredients.

The effect of inhibition of host MreB on the infection of thermophilic phage GVE2 in high temperature environment.

In eukaryotes, the manipulation of the host actin cytoskeleton is a necessary strategy for viral pathogens to invade host cells. Increasing evidence indicates that the actin homolog MreB of bacteria plays key roles in cell shape formation, cell polarity, cell wall biosynthesis, and chromosome segregation. However, the role of bacterial MreB in the bacteriophage infection is not extensively investigated. To address this issue, in this study, the MreB of thermophilic Geobacillus sp. E263 from a deep-sea hydrothermal field was characterized by inhibiting the MreB polymerization and subsequently evaluating the bacteriophage GVE2 infection. The results showed that the host MreB played important roles in the bacteriophage infection at high temperature. After the host cells were treated with small molecule drug A22 or MP265, the specific inhibitors of MreB polymerization, the adsorption of GVE2 and the replication of GVE2 genome were significantly repressed. The confocal microscopy data revealed that MreB facilitated the GVE2 infection by inducing the polar distribution of virions during the phage infection. Our study contributed novel information to understand the molecular events of the host in response to bacteriophage challenge and extended our knowledge about the host-virus interaction in deep-sea vent ecosystems.

The role of interactions between bacterial chaperone, aspartate aminotransferase, and viral protein during virus infection in high temperature environment: the interactions between bacterium and virus proteins.

BACKGROUND: The life cycle of a bacteriophage has tightly programmed steps to help virus infect its host through the interactions between the bacteriophage and its host proteins. However, bacteriophage-host protein interactions in high temperature environment remain poorly understood. To address this issue, the protein interaction between the thermophilic bacteriophage GVE2 and its host thermophilic Geobacillus sp. E263 from a deep-sea hydrothermal vent was characterized. RESULTS: This investigation showed that the host's aspartate aminotransferase (AST), chaperone GroEL, and viral capsid protein VP371 formed a linearly interacted complex. The results indicated that the VP371-GroEL-AST complex were up-regulated and co-localized in the GVE2 infection of Geobacillus sp. E263. CONCLUSIONS: As reported, the VP371 is a capsid protein of GVE2 and the host AST is essential for the GVE2 infection. Therefore, our study revealed that the phage could use the anti-stress system of its host to protect the virus reproduction in a high-temperature environment for the first time.

[Chinese traditional medicine recognition by support vector machine (SVM) terahertz spectrum].

Identification is very important for the development of Chinese traditional medicines. In recent years, rapid progress in ultrafast laser technology provides a steady and available source for,terahertz pulses generation, which greatly promotes the development of THz spectroscopy and imaging technique. SVM is a method for recognition of two kinds of samples. Appling SVM to the identification of Chinese traditional medicines through THz spectrum is a new way. The experiment on three groups of Chinese traditional medicines (zhigancao and shengancao, nanchaihu and beichaihu, shandougen and beidougen) was studied. The THz frequency spectrum and absorptivity were obtained and used to construct the feature space of Chinese traditional medicines. Three kinds of SVM were build, which used three kinds of kernel functions. By comparison, a model of BP artificial neural network was constructed. The result of using three kinds of SVM and BP artificial neural network to identify the Chinese traditional medicines showed that both methods have good prediction ability, but obviously the effect of SVM is better than BP artificial neural network for small samples. Using SVM in terahertz spectrum is a efficacious way for classification of Chinese traditional medicines.