Abstract 3122: Negative correlation of single-cell PAX3:FOXO1 expression with tumorigenicity in rhabdomyosarcoma

Abstract

Abstract Background: Rhabdomyosarcomas (RMS) are phenotypically and functionally heterogeneous. Expression of the fusion oncogene PAX3:FOXO1 (P3F) was previously shown to differ between individual tumor cells and fluctuate over time. Methods: In mouse Myf6Cre+/-,Pax3:Foxo1+/+,p53-/- RMS tumors, expression of P3F is directed by the Pax3 promoter and coupled to an eYFP fluorescent marker, which is activated as a second cistron downstream from an encephalomyocarditis virus-derived internal ribosome entry site. Low passage Myf6Cre+/-,Pax3:Foxo1+/+,p53-/- mouse RMS cell lines and primary human RMS cultures were used to study the functional impact of variable P3F expression at the cellular level in RMS. Results: The Myf6Cre+/-,Pax3:Foxo1+/+,p53-/- mouse RMS cell pool contains cells expressing different levels of YFP, correlating with variable P3F expression. Single-cell PCR was used to demonstrate substantial cell-to-cell variability in P3F expression in the human RMS cell pool. Myf6Cre+/-,Pax3:Foxo1+/+,p53-/- mouse RMS cells were then sub-fractionated by fluorescence-activated cell sorting (FACS) to discriminate YFPhigh/P3Fhigh and YFPlow/P3Flow cell subsets. YFPlow/P3Flow mouse RMS cells included 87% G0/G1 cells and reorganized their actin cytoskeleton to produce a cellular phenotype characterized by more efficient adhesion and migration. This translated into higher tumor-propagating cell frequencies of YFPlow/P3Flow compared to YFPhigh/P3Fhigh cells after injection into the extremity muscles of immunocompromised mice. We also observed higher clonal activity of YFPlow/P3Flow compared to YFPhigh/P3Fhigh cells in vitro. Both YFPlow/P3Flow and YFPhigh/P3Fhigh cells gave rise to mixed clones in vitro, consistent with fluctuations in P3F expression over time. Finally, exposure to the anti-tropomyosin compound TR100 disrupted the cytoskeleton and reversed enhanced migration and adhesion of YFPlow/P3Flow RMS cells. Conclusions: Our observations indicate that therapies aimed at eliminating P3Fhigh cells by targeting the fusion oncogene may not cure the disease. Moreover, dynamic expression of PAX3:FOXO1 at the single cell level may result in adaptive plasticity, allow RMS cells to adapt to environmental challenges and provide them with a critical advantage during tumor progression. Citation Format: Carla Regina, Geoffroy Andriuex, Sina Angenendt, Michaela Schneider, Manching Ku, Marie Follo, Marco Wachtel, Eugene Ke, Ken Kikuchi, Anton G. Henssen, Beat W. Schäfer, Melanie Boerries, Amy J. Wagers, Charles Keller, Simone Hettmer. Negative correlation of single-cell PAX3:FOXO1 expression with tumorigenicity in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3122.