Identify and characterize therapeutic targets against cancer stemness and chemotherapy resistance in rhabdomyosarcoma

Embryonal rhabdomyosarcoma (ERMS) is one of the most common and devastating pediatric soft tissue sarcomas. While localized disease can be controlled with multi-modal therapies, there remains no effective therapy for patients with relapsed or metastatic disease. The tumor propagating cells (TPCs) possess stem cell-like characteristics, including the capacity for self-renewal, and are thought to be responsible for initiating treatment-resistant relapse and metastasis of some cancer types. Several studies have demonstrated the therapeutic promise of targeted therapy against these stem cell-like properties. ERMS has been demonstrated to follow a cancer stem cell model with molecularly defined markers. We have previously isolated ERMS cells resistant to vincristine, a chemotherapy agent commonly used to treat RMS patients. ERMS cells resistant to chemotherapy agents have been shown to exhibit increased stemness and upregulation of stem cell markers. However, whether targeting cancer stemness will enhancing killing of RMS cells resistant to chemotherapeutic agents remains to be investigated. In addition, essential genes and pathways that drive RMS resistance to chemotherapy agents remain to be identified. Protein kinases are prime drug targets, as their aberrant activities are frequently essential for driving cancer initiation and progression. However, the kinases that could be used for therapeutic targeting against cancer stemness in RMS have not been identified. We previously performed a comprehensive siRNA kinome screen, utilizing a in vitro self-renewal assay as a functional readout cancer stemness. Of 714 kinases targeted, we identified 11 candidate genes essential for self-renewal of ERMS cells in vitro. Seven of these 11 targets have selective inhibitors against. Our hypotheses are (1) Selected kinases that regulate stem cell-like features of RMS can be targeted to enhance killing of tumor cells refractory to chemotherapeutic agents and (2) selected genes and pathways that are differentially regulated in chemotherapy-resistant RMS cells can be targeted therapeutically. We will test the hypothesis by first using the CRISPR/Cas9 genetic targeting strategy and treatment with selective inhibitors to characterize the loss-of-function effects of 7 candidate kinases on tumor cell growth and self-renewal in vitro and in vivo. We will also assess whether targeted disruption of top kinase candidate by CRISPR/Cas9 and treatment with selective inhibitor can enhance killing of chemotherapy-resistant ERMS cells. Finally, we will perform RNA sequencing analysis in vincristine-resistant ERMS cells to identify additional targetable genes and pathways that are differentially regulated compared to non-resistant cells. The druggable targets against cancer stemness and chemotherapy resistance as identified in the proposed study represent novel therapeutic targets to improve survival outcomes of ERMS patients with relapsed or treatment-refractory disease.