Abstract A009: Expression of Myc family proteins contributes to the growth and oncogenic transformation of fusion-positive rhabdomyosarcoma

Abstract Background and Hypothesis: Fusion-positive rhabdomyosarcoma (FP RMS) is a pediatric soft tissue cancer associated with the skeletal muscle lineage and defined by the 2;13 and 1;13 chromosomal translocations. These chromosomal translocations break within the PAX3 or PAX7 and FOXO1 genes and generate a PAX::FOXO1 fusion gene, either PAX3::FOXO1 or PAX7::FOXO1. Our previous studies showed that a human myoblast model of FP RMS depends on both MYCN and PAX3::FOXO1 for oncogenic transformation. The Myc family genes MYC and MYCN are frequently amplified or overexpressed in various cancers and play a critical role in the regulation of oncogenic properties. This study aims to investigate the expression of MYC and MYCN in FP RMS cell lines and their role in FP RMS growth and oncogenic transformation. Study Design and methods: The protein expression of MYC, MYCN, and PAX3::FOXO1 (or PAX7::FOXO1) was evaluated by Western blotting in FP RMS cell lines. The CRISPR-Cas9 system was applied to knock down these genes in the FP RMS cell lines, and then population growth, colony formation, and focus formation were used to determine growth and oncogenic transformation. Results and Conclusion: The FP RMS cell lines were classified into three groups based on the protein expression profile of MYC and MYCN: MYC-dominant, MYCN-dominant, and MYC/MYCN-codominant. As a positive control, knockdown of the specific PAX::FOXO1 gene in each cell line markedly attenuated the growth and oncogenic transformation 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 significantly diminished these parameters, and double knockdown of MYC and MYCN produced an even stronger inhibitory effect. In contrast, knockdown of MYCN in MYCN-dominant RH5 cells substantially reduced growth and transformation while MYC knockdown had no effect in these activities and double knockdown of MYC and MYCN produced effects comparable to MYCN knockdown. Finally, knockdown of either MYC or MYCN only partially inhibited growth and oncogenic behavior in MYC/MYCN-codominant FP RMS cell lines including RH30, RH41, and RH28, whereas double knockdown of MYC and MYCN generated a stronger inhibitory effect. These findings demonstrate that MYC and MYCN are functionally redundant in most FP RMS cell lines in terms of oncogenic behavior, and MYC or MYCN are functionally dominant in a smaller subset of FP RMS lines. Moreover, expression of MYC and MYCN contributes to the growth and oncogenic transformation in FP RMS cell lines. Relevance and Importance: The Myc family proteins MYC and MYCN are differentially expressed in FP RMS cell lines and are considered as critical factors for regulating the growth and oncogenicity in these cell lines. Citation Format: PAWAN K. RAUT, Bishwanath Chatterjee, Hana Kim, Shabir Zargar, Frederic G. Barr. Expression of Myc family proteins contributes to the growth and oncogenic transformation of fusion-positive rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Fusion-Positive Cancer: From Discovery to Therapy; 2026 Jan 13-15; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(1_Suppl):Abstract nr A009.