Abstract 7042: Role of Myc family proteins in the growth and oncogenic transformation of fusion-positive rhabdomyosarcoma

Abstract Fusion-positive rhabdomyosarcoma (FP RMS) is a pediatric soft tissue cancer associated with the myogenic lineage and characterized by a PAX::FOXO1 fusion gene, either PAX3::FOXO1 or PAX7::FOXO1. Our previous studies demonstrated that a human myoblast model of FP RMS requires MYCN as well as PAX3::FOXO1 for oncogenic transformation. The MYC family proteins including MYC, MYCN or MYCL are often amplified or overexpressed in different cancers, and contribute to regulation of oncogenic properties. This study aims to investigate the expression of these Myc family proteins in FP RMS cell lines and their role in FP RMS growth and oncogenic transformation. The CRISPR-Cas9 system was utilized to knock down specific genes in these cell lines, and then focus formation, colony formation, and population growth were used to assess growth and oncogenic activity. Using western blot studies, the FP RMS cell lines were divided into three groups based on the expression profile of Myc proteins: MYC-dominant, MYCN-dominant, and MYC/MYCN-codominant. In contrast, there is no significant expression of MYCL in any of these FP RMS cell lines. As a positive control, knockdown of the specific PAX::FOXO1 gene in each cell line abrogated growth and oncogenicity in all three subsets of FP RMS cell lines. In MYC-dominant CW9019 cells, MYCN knockdown had a small effect on growth and oncogenicity while MYC knockdown markedly decreased these parameters and double knockdown of MYC and MYCN had an even stronger inhibitory effect. In contrast, MYCN knockdown in MYCN-dominant RH5 cells substantially reduced growth and transformation while MYC knockdown did not alter these activities and double knockdown of MYC and MYCN generated effects comparable to MYCN knockdown. Finally, knockdown of either MYC or MYCN only partially diminished growth and oncogenicity in MYC/MYCN-codominant FP RMS cell lines including RH30, RH41, and RH28, whereas double knockdown of MYC and MYCN had a stronger inhibitory effect. These findings indicate that MYC and MYCN are functionally redundant in most FP RMS cell lines whereas MYC or MYCN are functionally dominant in a smaller subset of FP RMS lines. Moreover, expression of these Myc family proteins contributes to regulation of growth and oncogenic transformation in FP RMS cell lines. Citation Format: Pawan Kumar Raut, Bishwanath Chatterjee, Hana Kim, Frederic G. Barr. Role of Myc family proteins in the growth and oncogenic transformation of fusion-positive rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7042.