Date of Award

1-1-2016

Document Type

Open Access Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Ranjit S. Bindra

Second Advisor

Roy H. Decker

Abstract

CREATION OF NOVEL INDUCIBLE CELL LINE TO STUDY ALVEOLAR RHABDOMYOSARCOMA. Kaoru Takasaki, Ranjini K. Sundaram, Ranjit S. Bindra. Departments of Therapeutic Radiology and of Pathology, Yale University, School of Medicine, New Haven, CT.

Rhabdomyosarcoma (RMS) is a group of relatively rare mesenchymal malignancies classically associated with skeletal muscle and most frequently seen in the pediatric population. While chemotherapy, radiation, and surgical resection can confer long-term event-free survival rates of up to 85-95%, the prognosis for alveolar rhabdomyosarcoma (ARMS), particularly those positive for either the t(2;13) PAX3-FOXO1 or t(1;13) PAX7- FOXO1 translocation, is significantly worse. As such, the development of therapies that are specific for fusion-positive ARMS cells are of particular clinical interest, and it has been suggested that the concept of synthetic lethality can be exploited to target genes that become essential for cellular survival only in the presence of PAX3/7-FOXO1 expression. A stable tetracycline-inducible PAX3-FOXO1 clonal cell line in a U2OS background is created here in order to study in isolation the effects of the fusion protein on tumor response to novel chemotherapeutic agents. The cell line’s validity as a genetic mimic of PAX3-FOXO1 positive ARMS is verified by testing via qPCR the upregulation of DAPK, FGFR4, GREM1, and MyoD, which serve as proxies for global gene expression changes in fusion-positive ARMS, in the induced vs. uninduced states. DAPK and MyoD consistently demonstrate a statistically significant upregulation, as expected, but data for FGFR4 and GREM1 are inconclusive. Although additional experiments therefore need to be conducted to fully confirm that the inducible cell line indeed exhibits the transcriptional reprogramming seen in PAX3/7-FOXO1 positive ARMS, the data so far support this hypothesis. Ultimately, this cell line will be used in a high-throughput assay to identify chemotherapeutic agents that preferentially kill those with PAX3-FOXO1 expression, with the hopes of improving the poor prognosis that fusion-positive ARMS patients currently face.

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