Apurinic/Apyrimidinic Endonuclease 1 Induced Genomic Instability Causes T-Cell Acute Lymphoblastic Leukemia in Zebrafish

Genomic instability is not only a hallmark of cancer, but potentially a primary mechanism for its occurrence. DNA repair mechanisms play a protective role during DNA damage induced by both normal metabolic activities and environmental factors such as reactive oxygen species (ROS), UV light and γ-irradiation. Genes related to DNA repair are usually considered as tumor suppressors. However, incomplete repair may induce severe genomic instability, leading eventually to transformation.

Apurinic/apyrimidinic endonuclease 1 (APEX1), a gene involved in DNA repair with an important role in the base excision repair pathway, leads to transformation of normal cells in vitro. To investigate the role of APEX1 in tumor initiation in vivo, we generated a novel transgenic zebrafish model to overexpress APEX1 in fish. Specifically, pDestTol2A2_ubi:loxP-EGFP-loxP-APEX1-mCherry plasmid was injected into single cell embryos derived from the TP53 mutant line Tp53^M214K/M214K to generate a stable conditional inducible transgenic zebrafish line: Tg:APEX1^fl/-mCherry Tp53^M214K/M214K. To activate APEX1 expression in vivo, this line was mated with Tg:HSP70-Cre^+/+ fish. Compound zebrafish Tg: APEX1^fl/- mCherry,Tp53^+/M214K,HSP70-Cre^+/- carrying a Cre-activatable APEX1 knock-in allele were heated at 24hpf, and induction of APEX1 expression was monitored by downstream reporter - mCherry expression. Ten to twelve months post-fertilization, Tg:APEX1^fl/- mCherry,Tp53^+/M214K,HSP70-Cre^+/- fish developed abnormal swelling. Flow cytometry analysis of fish kidney marrow and peripheral blood showed dramatically increased precursor populations in scatter analysis. Histopathologic analysis showed that multiple organs were infiltrated with malignant lymphoblastic cells. None of the control fish Tg: GFP,Tp53^+/M214K,HSP70-Cre^+/- developed tumors during their life span.  Zebrafish with T-ALL have heterozygous Tp53^+/M214K background, but the expression of p21, mdm2 and bax in Tp53^+/M214K fish is exactly the same as in Tp53^+/+ fish; and Tp53^+/M214Kzebrafish themselves have not developed tumors during their life span. RNA from lymphoblastic cells was evaluated by qRT-PCR and showed increased expression of CD3, LCK and Tal indicating a T-cell acute lymphoblastic leukemia/lymphoma (T-ALL). We have performed whole genomic DNA sequencing in extracted DNA from fish tumor cells and compared it with their normal counterpart and observed multiple copy number changes and mutations.  We have now begun to see the development of other tumors in other organs including the eye and the testis. To uncover the molecular mechanism of tumorigenesis induced by APEX1, we have performed Mass Spectrometry analysis on APEX1 pulled down from 293T and AG08498 cells ectopically expressing APEX1. Besides verified binding proteins, such as PCNA, we also identified Ku70 and Ku80 binding to APEX1 directly. Further immunoflurescent staining and confocal microscopy of 293T cells also found co-localization of APEX1 and Ku70/Ku80. Those two proteins initiate Non-Homologous End Joining (NHEJ) repair and start the error-prone double strand repair and DNA damage. These results indicate that excessive repair activity may induce DNA damage and genomic instability.

In summary, this is the first demonstration where overexpression of a DNA repair gene is responsible for induction of genomic instability leading to malignant transformation. It provides new insight into the process of tumorigenesis and development of both therapy as well as preventive strategies.