Loss of WT1 Drives Adaptive Plasticity in CCDC6-RET Selpercatinib-Resistant Papillary Thyroid Cancer

<b>Background</b>: Papillary Thyroid Cancer (PTC) harboring CCDC6-RET translocation is typically classified as a differentiated epithelial tumor. Although Selpercatinib, a RET-selective drug, was recently approved for use in advanced PTC, the emergence of drug resistance has already been observed. Tumor plasticity, including non-canonical Epithelial-Mesenchymal Transition (EMT) programs, is recognized as a key mechanism underlying drug resistance. The downregulation of the transcription factor Wilms' Tumor 1 (WT1) in cancer is associated with increased motility, invasiveness, and metastatic potential. <b>Methods</b>: In this study, we developed a selpercatinib-resistant PTC-derived cell line, TPC-1-SelpR. Bioinformatic analyses were conducted to study the promoter of the <i>CCDC6-RET</i> gene and the transcriptomic landscape of PTC from RNAseq data. Subsequent real-time PCR, Western blot, and imaging techniques, such as confocal microscopy (CM) and fluorescence microscopy (FM), were employed to study the effects of WT1 loss-of-function following RNAi silencing. <b>Results</b>: In TPC-1-SelpR, WT1 expression appears downregulated compared to its counterpart, TPC-1. Crucially, WT1 silencing induced a context-dependent modulation of the CCDC6-RET driver: while WT1 silencing reduced CCDC6-RET expression in TPC-1, in TPC-1-SelpR, a post-transcriptional compensation of CCDC6-RET was observed. The gene expression of several factors involved in EMT, such as Twist, Vimentin, Integrin beta-1, and Profilin, was rewired in TPC-1-SelpR^{WT1-knockdown}. Although the Vimentin protein product decreased, CM and FM analyses confirmed a reorganization of residual protein: the subcellular redistribution was more dispersed in TPC-1-SelpR^{WT1-knockdown}. Further upregulation of the stemness factor Sox2 over the differentiation factor Sox17 occurred. These molecular changes were associated with higher cell motility of TPC-1-SelpR^{WT1-knockdown}. <b>Conclusions</b>: Collectively, these findings suggest that WT1 is a critical regulator involved in tumor plasticity, thereby supporting selpercatinib resistance.