Use of loss of function RNA interference screen to evaluate TNK-2 as a factor involved in rhabdomyosarcoma cell growth.

Abstract

9525 Background: Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue tumor. Embryonal (ERMS) and alveolar rhabdomyosarcoma (ARMS) represent different genetic alterations: 11p15 LOH in ERMS and PAX3 (or PAX7)-FOXO1 translocation in ARMS. Patients with metastatic or recurrent disease are essentially non-curable. To find novel therapeutic approaches for these patients, we sought to identify genes necessary for survival of both subtypes of RMS cells using a loss of function shRNA screen. We identified TNK-2 (ACK-1), a non-receptor protein tyrosine kinase known to interact with Cdc-42, a rho family GTPase critical for growth and differentiation, and possibly p130Cas, a scaffold protein involved in regulating growth and development. TNK-2 has not previously been linked to RMS. Methods: RD and RH30 cell lines (ERMS and ARMS respectively) engineered to tightly regulate gene expression via the bacterial tetracycline repressor were infected by a doxycycline regulated retroviral shRNA library. Each shRNA in the library contains a 60 nucleotide barcode, allowing abundance of shRNAs to be measured before and after doxycycline-induced expression. Missing barcodes were interpreted as genes required for cell survival. This approach showed that the TNK-2 gene was required for the survival of RH30 cells to a greater extent than RD cells. To confirm this finding, specific shRNA sequences targeting this gene were delivered to engineered RD and RH30 cell lines, We evaluated cell growth in vitro during 11 days using MTS assay. Results: As expected, the shRNA targeting TNK-2 inhibited cell growth by 21-40% in RH30 cells and by 19-37% in RD cells (compared to non-doxycycline induced control) with a maximum effect on day 6. Expression of a non-targeting shRNA showed less than 6% decrease in cell number. Conclusions: The use of functional genetics to identify novel critical pathways for RMS growth and survival has identified TNK-2 as a critical factor. This non-receptor protein tyrosine kinase has not previously been identified with these tumors and work is ongoing to determine whether this pathway may identify novel therapeutic opportunities. No significant financial relationships to disclose.