Abstract A016: Polyploid giant cancer cells secrete Interferon-beta to induce chemoresistant cell states in fusion-negative rhabdomyosarcoma

Abstract

Fusion-negative rhabdomyosarcoma (FN-RMS) is a childhood muscle cancer where relapse and refractory disease lead 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 remain unknown. 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 Dendra+ CSCs in vitro and in xenografts by direct conversion of differentiated cells into CSCs. This latter phenomenon was completely unexpected and suggested that CSC hierarchies are far less rigid than previously suggested. Indeed, time-lapse imaging verified direct reprograming of differentiated cells into CSCs. In concert with the overall elevation of CSCs, we also noted the production of a previously ill-defined therapy-induced cell type called polyploid giant cancer cells (PGCCs). These polyploid cells showed a high degree of DNA damage, marked by γH2AX (Ser139) phosphorylation, and subsequent activation of the cGAS/STING/Interferon-b pathway. Mechanistically, we identified interferon-b (IFN-b) as the secreted factor emanating from damaged PGCCs that directly reprograms adjacent cells into therapy-resistant CSCs. Importantly, CGAS-STING-IFN pathway inhibitory drugs could sensitize FN-RMS to chemotherapy. IHC and RNA-sequencing comparison of paired patient tumors pre- and post-therapy showed an elevation in the overall number of IFN+ cells, which correlated with the overall CSC signature and the creation of PGGCs after therapy. Taken together, these findings reveal that FN-RMS utilizes paracrine secreted type I interferons emanating from therapy-induced PGCCs to elevate the CSC population and suggests a far less rigid hierarchy of cell states in FN-RMS.