Identification of TNK2 as a critical kinase in rhabdomyosarcoma through a loss of function shRNA screen.

Abstract

9511 Background: Rhabdomyosarcoma is the most common pediatric soft tissue sarcoma. Embryonal rhabdomyosarcomas (ERMS) are characterized by 11p15 LOH while alveolar rhabdomyosarcomas harbor a translocation between PAX3 or PAX7 and FOXO1. Relapsed or metastatic disease has a 5-year survival rate of 25%. Methods: We sought to identify critical genes for rhabdomyosarcoma cell growth and survival. We performed a loss-of-function shRNA screen where a library of 15,000 shRNAs was introduced in RH30 (ARMS) and RD (ERMS) cells engineered to express the bacterial tetracycline repressor in a tet-on system. Relative abundance of cells containing a certain shRNA was determined using specific barcodes. We subtracted “hits” that are common to 4 lymphoma cell-lines to eliminate common survival pathways. Forty genes were associated with rhabdomyosaroma cell growth, of which 15 cause greater suppression in ARMS. We performed a subsequent validation of these 15 genes in multiple ARMS and ERMS cell lines using three different shRNA sequences using lentiviral constructs. In addition, we validated our results in an independent siRNA screen of the protein kinome in RMS cells. Results: TNK2 (tyrosine kinase, non-receptor 2) was identified as tyrosine kinase involved in rhabdomyosarcoma cell growth independently in the inducible shRNA screen and in the non-inducible siRNA platform. This molecule has been involved in downstream signaling from EGFR and integrins. Amplification of the TNK2 gene, located at 3q29, has been described in breast, prostate and lung cancer. More recently, a siRNA screen identified it as a potential therapeutic target in Ewing’s sarcoma. We confirmed TNK2 expression across patient tumor samples in a publically available database; where patients with high TNK2 expression showed lower overall survival. TNK2 knock down resulted in decreased rhabdomyosarcoma cell growth in vitro and in orthotopic xenografts. We confirmed specificity of our findings with a rescue experiment. Conclusions: We identified TNK2 as a potential therapeutic target in rhabdomyosarcoma using a loss-of-function shRNA screen and subsequent validation. Further work is ongoing to fully characterize the molecular mechanisms involved in these findings.