Studies of amplification in rhabdomyosarcoma

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In past studies of gene amplification in fusion-positive (FP) rhabdomyosarcoma (RMS), we identified MYCN and MIR17HG as key targets of the 2p24 and 13q31 amplicons, respectively. MYCN encodes a member of the Myc family of transcription factors and MIR17HG generates six microRNA's (miRNA's). In our recent studies, we investigated the relationship of the PAX3-FOXO1 (P3F) fusion with MYCN and MIR17HG. To link these genes in a common pathway, we note that previous studies in my laboratory showed that MYCN is a transcriptional target upregulated by P3F, and other published studies revealed that Myc family proteins, such as MYCN, contribute to the upregulation of MIR17HG. Based on these findings, we hypothesized a scenario in which P3F upregulates MYCN, which in turn upregulates MIR17HG. To investigate the validity of this scenario, we used two cell culture-based systems. In the first system, we engineered two FP RMS cell lines (RH30 and RH41) with an inducible Cas9 and constitutively expressing PAX3-directed gRNA's, thereby permitting inducible knockout of P3F. In each RMS line, P3F knockout resulted in a substantial decrease in MYCN expression. Analysis of the six mature miRNA's of the miR-17-92 cluster (miR-17, miR-18, miR-19a, miR-19b, miR-20 and miR-92) processed from MIR17HG also demonstrated a substantial decrease in expression of these miRNA's following P3F knockout. In rescue experiments, lentiviral transduction of a P3F construct (modified to be resistant to knockout) into these P3F knockout cells increased expression of MYCN and the miRNA's of the miR-17-92 cluster. Of interest, recent experiments with lentiviral transduction of MYCN into the P3F knockout cells suggests that MYCN may only restore a lower level of miR-17-92 expression than the level restored by P3F rescue. These findings suggest that P3F may have regulatory effects on MIR17HG (and the miR-17-92 cluster) that are not fully recapitulated by MYCN. In a second system, we used the human myoblast line Dbt along with inducible P3F and constitutive MYCN constructs to assess the effects of P3F and/or MYCN on this miRNA cluster. It should be noted that P3F only induces a small increase in MYCN expression in these myoblasts so that this system permits P3F and MYCN to be independently assessed without significant effects on each other's expression. In these experiments, P3F alone does not significantly upregulate miRNA expression in the myoblasts whereas MYCN expression induces a small increase in expression of each of the six miRNA's. In contrast, the combination of P3F and MYCN results in a substantial increase in expression of each of the six miRNA's. Therefore, there appears to be a synergistic effect between P3F and MYCN so that P3F does not entirely act through upregulation of MYCN. Although MYCN may be necessary for P3F to upregulate the six microRNA's, MYCN is not sufficient to fully upregulate these targets. To assess the phenotypic effects of high versus low level miR-17-92 expression, we developed two experimental systems. In one system, we transduced a lentiviral construct constitutively expressing the miR-17-92 cluster into derivatives of the human Dbt myoblast system, resulting in increased expression of the six corresponding miRNA's with or without increased expression of MYCN or P3F. For loss of function studies, we engineered human RMS cell lines to express an inducible Cas9-KRAB-MeCP2 construct and a construct constitutively expressing six gRNA's targeting the MIR17HG 5' regulatory region. In these cells, the modified Cas9 protein acts as a transcriptional repressor and down-regulates MIR17HG expression, resulting in very low levels of the six miRNA's of the miR-17-92 cluster. Now that these two systems have been fully developed and implemented, we will assay the phenotypic changes associated with increased miR-17-92 expression in Dbt myoblasts and decreased miR-17-92 expression in human RMS cells.