Abstract 3536: Role of Myc family proteins in fusion-positive rhabdomyosarcoma

Abstract

Abstract Fusion-positive rhabdomyosarcoma (FP RMS) is an aggressive pediatric malignancy characterized by the presence of a PAX3-FOXO1 (P3F) or PAX7-FOXO1 fusion gene. Previous RNA expression studies have shown that these FP RMS tumors also express high levels of MYCN and lower levels of MYC. Our recent studies of FP RMS tumors revealed an inverse relationship between MYCN and MYC RNA expression in FP RMS tumors. Western blot studies of FP RMS cell lines and patient-derived xenograft (PDX) tumors identified tumors with high level MYCN and MYC protein expression as well as tumors with high level protein expression of either MYC or MYCN. To model the interaction of P3F with Myc family proteins in FP RMS, we generated human Dbt myoblasts with doxycycline-inducible P3F (iP3F) with or without constitutively expressed MYCN. We previously observed that doxycycline-treated human Dbt myoblasts engineered with iP3F and constitutive MYCN expression (Dbt-MYCN-iP3F) form foci in vitro and rapidly form tumors in vivo, while doxycycline-treated Dbt myoblasts engineered with only iP3F expression (Dbt-iP3F) do not form foci in vitro and form tumors in vivo at a slower rate. Western blot and qRT-PCR analysis of the parental and tumor-derived (TD) Dbt-MYCN-iP3F lines revealed high MYCN and low MYC expression. In contrast, Dbt-iP3F parental lines express moderate levels of MYC and very low levels of MYCN whereas iP3F TD lines show increased MYC and MYCN expression, though this level of MYCN expression is considerably less than that seen in Dbt-MYCN-iP3F lines. Using CRISPR-Cas9 technology to knockdown MYCN or MYC, we found a primary dependence on MYCN in both Dbt-MYCN-iP3F parental and TD lines as indicated by loss of focus formation following MYCN knockdown while MYC knockdown does not interfere with oncogenicity. In contrast, oncogenicity in Dbt-iP3F TD lines is primarily dependent upon MYC whereas MYCN knockdown results in a modest and variable effect on oncogenicity. To elucidate the role of Myc proteins on the expression of P3F target genes, we measured expression of several transcriptional targets in these parental and TD lines by RNA sequencing and qRT-PCR. Several target genes (such as FGFR4) showed comparable upregulation by P3F in both parental and TD lines with or without constitutive MYCN expression. In contrast, a few target genes (such as FGF8) were stimulated by P3F at low levels in Dbt-iP3F parental lines and were stimulated at much higher levels in Dbt-iP3F TD lines as well as in both Dbt-MYCN-iP3F parental and TD lines. This finding indicates that some P3F target genes do not require high MYCN or MYC expression whereas other P3F target genes can only be maximally stimulated by P3F in the presence of high levels of MYCN and/or MYC. We postulate that the dependence of FP RMS on high level expression of a Myc family protein may be explained by the need to stimulate expression of one or more of the P3F target genes in this latter category. Citation Format: Bishwanath Chatterjee, Salah Boudjadi, Puspa Raj Pandey, Hana Kim, Wenyue Sun, Frederic G. Barr. Role of Myc family proteins in fusion-positive rhabdomyosarcoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3536.