Three Heritable T Cell Malignancy Models Established Using a Forward Genetic Mutagenesis Screen in Zebrafish.

Abstract T cell cancers are common in pediatric oncology, and include the clinical entities T cell acute lymphoblastic leukemia (T-ALL) and the Non-Hodgkin Lymphoma, T cell lymphoblastic lymphoma (T-LBL). These diseases carry worse prognoses than their B cell counterparts, and the regimens used in their treatment confer significant short- and long-term morbidities. Unlike many pediatric malignancies, which have characteristic chromosomal translocations, most T cell-derived cancers carry no such cytogenetic hallmark. Because such straightforward oncogenic lesions are typically absent, insights into the genetic underpinnings and molecular pathogenesis of these T cell diseases are also less comprehensive. To address these deficiencies, we have performed a forward genetic screen for T cell malignancies using the vertebrate model organism, zebrafish (Danio rerio). Although others have used transgenic zebrafish to show that known mammalian oncogenes can induce zebrafish leukemias, to date, this powerful animal model has not been used as a discovery tool to elucidate novel genetic mutations underlying de novo T cell malignancies. Zebrafish share ontogeny with other vertebrates, and possess an adaptive immune system strikingly similar to mammals. However, unlike other vertebrate models, zebrafish are amenable to forward genetic approaches, where animals are randomly mutagenized, and then screened using high-throughput strategies to detect rarely-occurring phenotypes. Using a zebrafish line with T lymphocyte-specific expression of green fluorescent protein (GFP), we have conducted a germline mutagenesis screen to identify animals that develop T cell malignancy. Thus far, we have isolated three independent lines with a heritable predisposition to developing T cell cancers. In all three lines, age of onset, patterns of spread, and morphologic appearance resemble human T-ALL/-LBL. In addition, we have compiled histologic and immunohistochemical evidence further characterizing these diseases. Analysis of T cell receptor (TCR) β gene sequences from malignant tissue has verified the monoclonality of malignant cells, based on identical VDJ rearrangements. Malignant cells have also been successfully transplanted serially into sub-lethally irradiated allogeneic recipients, using as few as 2500 donor cells to transfer disease. Furthermore, we have shown the malignancies themselves to be radiation sensitive, like their human correlates. Soon after radiation-induced remissions are achieved, however, high rates of aggressive disease relapse are seen, modeling recurrent human T-ALL/-LBL. In summary, we have identified three independent zebrafish mutant lines, which each recapitulate human T lymphocyte malignancy, and that are all heritably transmissible from one generation to the next. The positional cloning of the underlying genetic mutation of each line will provide exciting candidates for study in these human diseases. Moreover, using these lines as subjects in ongoing investigations, we are examining the genomic, epigenetic, and transcriptional profiles of these malignant cell populations, comparing pre- and post-transplant samples, pre- and post-irradiation isolates, and following other accepted and investigational therapeutic interventions. Finally, these lines are now poised for utilization in additional high-throughput screening approaches, including forward genetic suppressor and enhancer mutagenesis projects, as well as small molecule drug screens. Collectively, these new vertebrate models of T-ALL/-LBL should provide vital experimental platforms for studies of this important class of human cancers.