Kim, Kitai

Abstract Reprogramming of aged donor tissue cells into induced pluripotent stem cells (A-iPSC) preserved the epigenetic memory of aged-donor tissue, defined as genomic instability and poor tissue differentiation in our previous study. The unbalanced expression of RNA exosome subunits affects the RNA degradation complex function and is associated with geriatric diseases including premature aging and cancer progression. We hypothesized that the age-dependent progressive subtle dysregulation of EXOSC2 (exosome component 2) causes the aging traits (abnormal cell cycle and poor tissue differentiation). We used embryonic stem cells as a tool to study EXOSC2 function as the aging trait epigenetic memory determined in A-iPSC because these aging traits could not be studied in senesced aged cells or immortalized cancer cells. We found that the regulatory subunit of PP2A phosphatase, PPP2R5E, is a key target of EXOSC2 and this regulation is preserved in stem cells and cancer.

Abstract Background Chromosomal translocation has been detected in many human cancers including gliomas and is considered a driving force in tumorigenesis. Co-deletion of chromosome arms 1p and 19q is a hallmark for oligodendrogliomas. On the molecular level, 1p/19q co-deletion results from t(1;19)(q10;p10), which leads to the concomitant formation of a hybrid chromosome containing the 1q and 19p arms. A method to generate 1p/19q co-deletion is lacking, which hinders the investigation of how 1p/19q co-deletion contributes to gliomagenesis. Methods We hypothesized that chromosomal translocation, such as t(1;19)(q10;p10) resulting in the 1p/19q co-deletion, may be induced by simultaneously introducing DNA double-strand breaks (DSBs) into chromosomes 1p and 19q using CRISPR/Cas9. We developed a CRISPR/Cas9-based strategy to induce t(1;19)(q10;p10) and droplet digital PCR (ddPCR) assays to detect the hybrid 1q/19p and 1p/19q chromosomes. Results After translocation induction, we detected both 1p/19q and 1q/19p hybrid chromosomes by PCR amplification of the junction regions in HEK 293T, and U-251 and LN-229 glioblastoma cells. Sequencing analyses of the PCR products confirmed DNA sequences matching both chromosomes 1 and 19. Furthermore, the 1p/19q hybrid chromosome was rapidly lost in all tested cell lines. The 1q/19p hybrid chromosome also become undetectable over time likely due to cell survival disadvantage. Conclusion We demonstrated that t(1;19)(q10;p10) may be induced by CRISPR/Cas9-mediated genomic editing. This method represents an important step toward engineering the 1p/19q co-deletion to model oligodendrogliomas. This method may also be generalizable to engineering other cancer-relevant translocations, which may facilitate the understanding of translocation roles in cancer progression.