Gene fusion dependence in acute myeloid leukemia Ewing sarcoma, and ependymoma cell lines

Authors

Aisha Pasha

Document Type

Article

Publication Date

Summer 2022

Keywords

JGM

JAX Location

In: Student Reports, Summer 2022, The Jackson Laboratory

Sponsor

Nathaniel Jillette, Patrick Ng, Ph.D. and Ching Lau, M.D., Ph.D.

Abstract

Ewing sarcoma, pediatric acute myeloid leukemia (AML), and ependymoma (EPN) are cancers primarily found in children and all demonstrate a prevalence of fusion genes. Fusion genes can arise from chromosomal translocations and impact normal protein production. Four cell lines and their specific fusion genes were studied. The Ewing sarcoma cell lines, A673 and SK-NEP-1, have a fusion between exon 7 of EWSR1 and exon 6 of FLI1 and between exon 7 of EWSR1 and exon 5 of FLI1, respectively. The AML cell line, M-07e, has a fusion between exon 11 of CBFA2T3 and exon 3 of GLIS2. The EPN cell line, BXD-1425EPN, has a fusion between exon 4 of ZFTA and exon 3 of RELA. The aim of this study was to determine if cancer cell viability is dependent on the presence of fusion genes. First, each cell line underwent a validation process involving RT-PCR and Sanger sequencing to confirm the presence of the fusion. Then, the validated fusion site was targeted using siRNA. qRT-PCR, Western blotting, and Cell Titer-Glo 2.0 Cell Viability assay were performed to quantify the effects the siRNA had on each fusion gene. The fusion genes in the AML and EPN cell lines were unable to be silenced; however, the Ewing sarcoma cell lines were transfected successfully and cell viability in the SK-NEP-1 samples treated with siRNA targeting the fusion decreased. The results of this study demonstrated that the EWSR1- FLI1 fusion gene is necessary for SK-NEP-1 cell line survival.