RESUMO
An in-house genotyping facility should aim to be more cost-effective than outsourced service and more reliable than genotyping performed by short-term employees or students of individual research groups. Reliable genotyping allows efficient and economical management of mice colonies and promotes accurate and reproducible research results. Here we provide a detailed description of our approach to establishing a genotyping core facility, relying on automated PCR assembly and high-resolution melting (HRM) analysis (first derivative). The workflow we devised was tightly managed by purpose-designed applications developed using MATLAB App Designer that allowed straightforward work planning, ensured sample tracking throughout the process, and provided a platform for reliable data analysis and generation of genotyping reports. We successfully transitioned PCR product analysis of more than 250 different target genes from standard gel electrophoresis to the more advanced HRM analysis. About 23% of the target genes required a redesign of primers to adapt to our protocol. The process was highly universal, and only 2% of the target genes required deviation from the standard PCR method to a more restricted protocol that reduces the amplification of nonspecific products. We currently run more than 1,000 PCR reactions weekly, of samples taken at weaning or experimental endpoint, and assemble a large variety of target genes in every PCR plate. We also showed that genotyping of blastocytes instead of embryos can serve as quality control of cryopreservation. Thus, our genotyping protocol promotes the 3Rs (Replacement, Reduction, and Refinement) principles. Our refined genotyping process facilitates cost-effective colony management, replaces tissue types as well as traditional methods with advanced ones, and provides reliable results in a timely manner. MATLAB codes and related data are available in supplementary materials and online.
RESUMO
Diffuse midline gliomas (DMGs) are aggressive and fatal pediatric tumors of the central nervous system that are highly resistant to treatments. Lysine to methionine substitution of residue 27 on histone H3 (H3-K27M) is a driver mutation in DMGs, reshaping the epigenetic landscape of these cells to promote tumorigenesis. H3-K27M gliomas are characterized by deregulation of histone acetylation and methylation pathways, as well as the oncogenic MYC pathway. In search of effective treatment, we examined the therapeutic potential of dual targeting of histone deacetylases (HDACs) and MYC in these tumors. Treatment of H3-K27M patient-derived cells with Sulfopin, an inhibitor shown to block MYC-driven tumors in vivo, in combination with the HDAC inhibitor Vorinostat, resulted in substantial decrease in cell viability. Moreover, transcriptome and epigenome profiling revealed synergistic effect of this drug combination in downregulation of prominent oncogenic pathways such as mTOR. Finally, in vivo studies of patient-derived orthotopic xenograft models showed significant tumor growth reduction in mice treated with the drug combination. These results highlight the combined treatment with PIN1 and HDAC inhibitors as a promising therapeutic approach for these aggressive tumors.
Diffuse midline gliomas (DMGs) are among the most aggressive and fatal brain cancers in children. They are often associated with changes in histones, the proteins that control gene activity and give chromosomes their structure. Most children with DMGs, for example, share the same anomaly in their histone H3 protein (referred to as the H3-K27M mutation). This change affects how small chemical tags called methyl and acetyl groups can be added onto histone 3, which in turn alters the way the protein can switch genes on and off. As a result, tumours start to develop. One potential therapeutic strategy against DMGs is to use histone deacetylase inhibitors (HDACi), a promising type of drugs which inhibits the enzymes that remove acetyl groups from histones. Patients can develop resistance to HDACi, however, highlighting the need to explore other approaches. One possibility is to treat patients with several types of drugs, each usually targeting a distinct biological process that contributes to the emergence of cancer. This combined approach can have multiple benefits; the drugs potentially amplify each other's effect, for example, and it is also less likely for cells to become resistant to more than one compound at the time. In addition, each drug in the combination can be used in a lower dose to reduce side effects and benefit patients. DMG tumour cells often feature higher activity levels of a protein known as MYC, which can contribute to the growth of the tumour. Algranati, Oren et al. therefore set out to test whether combining an HDACi known as Vorinostat with a drug that blocks MYC activity (Sulfopin) can act as an effective treatment for this cancer. Tumour samples from eight DMG patients were treated with either Sulfopin alone, or Sulfopin in association with Vorinostat. Cells exposed to both drugs were less likely to survive, and additional genetic experiments showed that the combined treatment had resulted in pathways that promote tumour development being blocked. When both Sulfopin and Vorinostat were administered to mice made to grow human DMG tumors, the animals showed a greater reduction in tumor growth. Treatment options for DMG are usually limited, with chemotherapy often being ineffective and surgery impossible. The work by Algranati, Oren et al. suggests that combining HDACi and drugs targeting the MYC pathway is a strategy that should be examined further to determine whether clinical applications are possible.