Abstract 3888: Study of tumor recurrence following loss of PAX3-FOXO1 oncoprotein expression in rhabdomyosarcoma cells

Abstract

Abstract We previously developed a human alveolar rhabdomyosarcoma (RMS) model using immortalized human myoblasts engineered with constitutive MYCN and doxycycline-inducible PAX3-FOXO1 (P3F) expression constructs. These cells formed primary tumors rapidly when injected into mice fed a doxycycline-containing diet. When doxycycline is discontinued to turn off P3F expression, tumors initially regress, and then recurrent tumors without P3F expression form after a variable delay. We propose that P3F is necessary for initial tumor development but is not needed in later stages due to the accumulation of additional molecular changes. We further hypothesize that these novel changes may result in dysregulated expression of one or more P3F target genes. We derived cell lines from the primary and recurrent tumors and confirmed the expression of P3F in primary tumor-derived (PTD) cells and its absence in recurrent tumor-derived (RTD) cells. By limiting dilution, we generated subclones from RTD cell lines to obtain homogeneous populations of cells with P3F-independent transformation. Microarray analysis revealed that the expression profile of RTD subclones was substantially different from that of PTD cells. However, using untreated parental cells as a reference, we found that RTD and PTD cells shared 92 upregulated genes including 14 P3F transcriptional targets; among these 14 genes, FGF8 showed the highest level of upregulation (>30-fold) in RTD cells. Using a CRISPR-Cas9 approach, FGF8 knockout in these RTD cells impaired transforming capability in focus formation assays and tumorigenicity in vivo. Conversely, FGF8 overexpression in non-transformed MYCN-expressing parental myoblasts induced transformation and tumorigenic capability. In medium transfer studies, conditioned media (CM) from RTD cell cultures or from parental cells overexpressing FGF8 induced in vitro transformation in parental cells, whereas CM from FGF8-knockout RTD cells or parental cells did not induce transformation. To further implicate FGF8, immunodepletion of FGF8 from the RTD conditioned media induced a substantial reduction in transforming activity in these medium transfer studies. To examine the role of FGF8 in PTD cells and human alveolar RMS cells (RH30), we used a similar CRISPR-Cas9 approach to knockout FGF8 expression and similarly found impaired in vitro transformation. Finally, FGF8 overexpression in a spontaneous non-transformed RH30 variant with low P3F expression levels resulted in stimulation of transformation in vitro and tumorigenicity in vivo. Together, our results indicate that FGF8, a P3F transcriptional target, is necessary for the oncogenicity of P3F-positive RMS cells and that dysregulated FGF8 expression leads to the development of P3F-independent recurrent RMS tumors. Citation Format: Salah Boudjadi, Thanh Nguyen, Bishwanath Chatterjee, Puspa Pandey, Wenyue Sun, Frederik Barr. Study of tumor recurrence following loss of PAX3-FOXO1 oncoprotein expression in rhabdomyosarcoma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3888.