Studies of amplification in rhabdomyosarcoma

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

In recent studies, we commenced an investigation of the inter-relatedness of the PAX3-FOXO1 (P3F) fusion with several key genes that are overexpressed by the different amplicons in FP RMS. In particular, we noted that MYCN is overexpressed as a result of the 2p24 amplicon and MIR17HG is overexpressed as a result of the 13q31 amplicon. In our previous studies, we gathered evidence that MYCN expression is upregulated by P3F, and other previous published data revealed that MIR17HG is upregulated by Myc family proteins, such as MYCN. Based on these previous findings, we wanted to confirm the relationship of P3F, MYCN and MIR17HG in FP RMS cells and develop a system for extending studies of this relationship. Our goal was to set up a dynamic FP RMS cell system in which P3F expression could be inducibly turned off. In particular, we transduced the Rh30 FP RMS line with a construct constitutively expressing guide RNA's corresponding to PAX3 cDNA sequences 5' of the P3F fusion point as well as a construct with the components for doxycycline inducible expression of Cas9. To optimize the performance characteristics of this system, we screened multiple subclones for cells in which doxycycline induces substantial loss of P3F protein expression. Using this system, we then investigated the consequences of inducible loss of P3F expression and found a corresponding loss of MYCN and MIR17HG expression. In addition to decreased expression of the MIR17HG pri-miRNA, there is decreased expression of the six mature miRNA's (miR-17, miR-18, miR-19a, miR-19b, miR-20 and miR-92) processed from the MIR17HG pri-miRNA. Although a subset of the cells in this population show decreased proliferation along with differentiation or cell death, a subpopulation can be recovered that still demonstrates decreased P3F, MYCN and MIR17HG expression. To examine the forward effects of P3F or MYCN action in these cells, we established lentiviral constructs constitutively expressing P3F or MYCN. Transduction of the P3F construct into these cells resulted in increased P3F levels and a corresponding increase in expression of MYCN, the MIR17HG pri-miRNA and the six mature miRNA's. In addition, transduction of the MYCN construct into these cells resulted in increased MYCN levels and a corresponding increase in expression of the MIR17HG pri-miRNA and the six mature miRNA's; however there is no change in P3F expression. These findings are consistent with a model in which P3F induces MYCN expression and MYCN in turn induces MIR17HG expression in FP RMS cells. Therefore, we propose that an important downstream target of both P3F and MYCN in FP RMS is MIR17HG and the associated miRNA's, and also postulate that MIR17HG overexpression may recapitulate certain functional aspects of MYCN and P3F overexpression in FP RMS. To address these issues, we will next compare the phenotypic effects of P3F, MYCN and MIR17HG expression in RMS cells as well as in our human myoblast cell culture system. For this purpose, we have developed a lentiviral construct constitutively expressing MIR17HG, which when transduced into the human myoblasts results in increased expression of the MIR17HG pri-miRNA as well as the six resulting mature miRNA's. For loss of function studies, in addition to the CRSIPR-Cas9 system for inactivating P3F expression, we have also identified guide RNA's corresponding to sequences in MYCN cDNA that mediate effective inactivation of MYCN expression. For loss of function studies involving the miRNA's of the MIR17HG, the number and small size of these effector molecules complicates use of a standard CRISPR-Cas9 system. For this reason, we are developing "sponge" constructs that will bind up specific miRNAs as well as alternative CRISPR systems for inactivating MIR17HG expression.