Abstract B007: Therapy-associated paracrine signaling alters the cell states in Rhabdomyosarcoma

Fusion-negative rhabdomyosarcoma (FN-RMS) is a childhood muscle cancer where relapse and refractory disease leads to poor outcomes. Our group has recently shown that FN-RMS is a cancer stem cell (CSC) disease and that these CD44+/CD90+/EGFR+ cells likely contribute to therapy-resistance, yet the precise mechanisms governing self-renewal and tumor regrowth after stress are not known. To better visualize and describe CSCs in the context of therapy response, we developed a fluorescent reporter using the EGFR promoter to drive expression of a photoconvertible-nuclear localized protein (H2B-Dendra) that specifically marks FN-RMS CSCs. Photoconversion and live cell imaging confirmed that the CSCs were largely quiescent under normal growth conditions. Yet, following stress, CSCs could re-enter the cell cycle and self-renew. Moreover, a single dose of chemotherapy was also able to elevate the overall number of CSCs by (1) inducing cell cycle re-entry and (2) by direct conversion of differentiated cells into CSCs. This latter phenomenon was completely unexpected and suggested that CSC hierarches are far less rigid than previously suggested. Indeed, time-lapse imaging verified direct reprograming of differentiated cells into CSCs. Mechanistically, we identified interferon-b (IFN-b) as the secreted factor emanating from damaged cells to directly reprogram cells into therapy-resistant CSCs. Indeed, the CGAS-STING-IFN pathway drove local paracrine-mediated reprogramming of CSCs and small molecule pathway inhibitors could sensitize FN-RMS to chemotherapy. Taken together, these findings reveal that FN-RMS utilizes paracrine expressed type I interferons to elevate the CSC population in response to therapy and suggests a far less rigid hierarchy of cell states in FN-RMS. Citation Format: Sabateeshan Mathavarajah, Tiffany C Eng, Anna Luciano, Elisa J Quantin, Devika D Kannambadi, Shuze Wang, Alexander D Weissman, Jihee Lee, Yueyang Wang, Yun Wei, Sara G Danielli, Michael A Marconi, David M Langenau. Therapy-associated paracrine signaling alters the cell states in Rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_2):Abstract nr B007.