Functional analysis of recurrent CDC20 promoter variants in human melanoma

Small nucleotide variants in non-coding regions of the genome can alter transcriptional regulation, leading to changes in gene expression which can activate oncogenic gene regulatory networks. Melanoma is heavily burdened by non-coding variants, representing over 99% of total genetic variation, including the well-characterized TERT promoter mutation. However, the compendium of regulatory non-coding variants is likely still functionally under-characterized. We developed a pipeline to identify hotspots, i.e. recurrently mutated regions, in melanoma containing putatively functional non-coding somatic variants that are located within predicted melanoma-specific regulatory regions. We identified hundreds of statistically significant hotspots, including the hotspot containing the TERT promoter variants, and focused on a hotspot in the promoter of CDC20. We found that variants in the promoter of CDC20, which putatively disrupt an ETS motif, lead to lower transcriptional activity in reporter assays. Using CRISPR/Cas9, we generated an indel in the CDC20 promoter in human A375 melanoma cell lines and observed decreased expression of CDC20, changes in migration capabilities, increased growth of xenografts, and an altered transcriptional state previously associated with a more proliferative and less migratory state. Overall, our analysis prioritized several recurrent functional non-coding variants that, through downregulation of CDC20, led to perturbation of key melanoma phenotypes

Autologous humanized PDX modeling for immuno-oncology recapitulates features of the human tumor microenvironment

BACKGROUND: Interactions between immune and tumor cells are critical to determining cancer progression and response. In addition, preclinical prediction of immune-related drug efficacy is limited by interspecies differences between human and mouse, as well as inter-person germline and somatic variation. To address these gaps, we developed an autologous system that models the tumor microenvironment (TME) from individual patients with solid tumors. METHOD: With patient-derived bone marrow hematopoietic stem and progenitor cells (HSPCs), we engrafted a patient\u27s hematopoietic system in MISTRG6 mice, followed by transfer of patient-derived xenograft (PDX) tissue, providing a fully genetically matched model to recapitulate the individual\u27s TME. We used this system to prospectively study tumor-immune interactions in patients with solid tumor. RESULTS: Autologous PDX mice generated innate and adaptive immune populations; these cells populated the TME; and tumors from autologously engrafted mice grew larger than tumors from non-engrafted littermate controls. Single-cell transcriptomics revealed a prominent vascular endothelial growth factor A (VEGFA) signature in TME myeloid cells, and inhibition of human VEGF-A abrogated enhanced growth. CONCLUSIONS: Humanization of the interleukin 6 locus in MISTRG6 mice enhances HSPC engraftment, making it feasible to model tumor-immune interactions in an autologous manner from a bedside bone marrow aspirate. The TME from these autologous tumors display hallmarks of the human TME including innate and adaptive immune activation and provide a platform for preclinical drug testing

Autologous humanized PDX modeling for immuno-oncology recapitulates features of the human tumor microenvironment.

BACKGROUND: Interactions between immune and tumor cells are critical to determining cancer progression and response. In addition, preclinical prediction of immune-related drug efficacy is limited by interspecies differences between human and mouse, as well as inter-person germline and somatic variation. To address these gaps, we developed an autologous system that models the tumor microenvironment (TME) from individual patients with solid tumors. METHOD: With patient-derived bone marrow hematopoietic stem and progenitor cells (HSPCs), we engrafted a patient\u27s hematopoietic system in MISTRG6 mice, followed by transfer of patient-derived xenograft (PDX) tissue, providing a fully genetically matched model to recapitulate the individual\u27s TME. We used this system to prospectively study tumor-immune interactions in patients with solid tumor. RESULTS: Autologous PDX mice generated innate and adaptive immune populations; these cells populated the TME; and tumors from autologously engrafted mice grew larger than tumors from non-engrafted littermate controls. Single-cell transcriptomics revealed a prominent vascular endothelial growth factor A (VEGFA) signature in TME myeloid cells, and inhibition of human VEGF-A abrogated enhanced growth. CONCLUSIONS: Humanization of the interleukin 6 locus in MISTRG6 mice enhances HSPC engraftment, making it feasible to model tumor-immune interactions in an autologous manner from a bedside bone marrow aspirate. The TME from these autologous tumors display hallmarks of the human TME including innate and adaptive immune activation and provide a platform for preclinical drug testing

Geraniol ameliorates testicular damage in diabetic rats via inhibition of redox parameters and mitochondrial-mediated apoptosis

Background: Diabetes is a disease characterized by high levels of reactive oxygen species that trigger a series of events that culminate in programmed cell death. Terpenoids from plants used in traditional Chinese medicine have preventive and therapeutic potential. However, it is not known if geraniol, a monoterpene will prevent testicular damage in diabetic rats. Thus, this study investigated the antioxidant, and antiapoptotic effects of geraniol on testicular damage in diabetic male Wistar rats. Also, the interactions between geraniol with rat liver F1 ATPase and oxidized rat cytochrome c were investigated. Methods: A single intraperitoneal dose of streptozotocin (60 mg/kg) was used to induce diabetes in rats. Male Wistar rats were divided into 4 groups - CT (control), DA (Diabetic, 60 mg/kg streptozotocin), GE (Geraniol-treated, 60 mg/kg streptozotocin + 200 mg/kg geraniol), and GL (Glibenclamide-treated, 60 mg/kg streptozotocin + 5 mg/kg Glibenclamide). Rats were sacrificed by cervical dislocation under anesthesia after 21 days of treatment. Testicular superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), cytochrome c, caspase 9 and caspase 3, and testosterone (testes and plasma) were determined spectrophotometrically. The interactions between geraniol, and glibenclamide, with rat liver F1-ATPase (1MAB) and oxidized rat cytochrome c (5FD4) were assessed by molecular docking. Results: The results showed that SOD, and CAT levels were significantly (p<0.05) lowered in the DA group. Significant increases in SOD, CAT, and GSH levels were observed in the testes of rats in the GE and GL groups. Also, cytochrome c, caspase 9, and caspase 3 levels in rat testes were significantly lowered in the GE and GL groups when compared to the DA group. Furthermore, testosterone levels in the plasma and testes of rats in the GE and GL groups were significantly (p<0.05) increased in the treated groups when compared to the DA group. The binding affinities of geraniol for 1MAB (-5.4 Kcal/mol) and 5DF4 (-6.1 Kcal/mol) were comparable to that of glibenclamide (-5.5 Kcal/mol; -7.5 Kcal/mol, respectively). Conclusion: This study shows that geraniol has the potential to ebb testicular damage in diabetic rats through the inhibition of oxidative stress, caspase activation, rat liver F1-ATPase, and oxidized rat cytochrome c. This study suggests the use of geraniol-rich diet in traditional Chinese medicine