Zirpoli, Sara (2025) Novel combinatorial strategies for Anaplastic Thyroid Carcinoma. [Tesi di dottorato]

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Abstract

Anaplastic Thyroid Carcinoma (ATC) represents a rare, aggressive, and therapy-resistant endocrine malignancy. Although the FDA has approved the combination of the BRAF inhibitor Dabrafenib and the MEK inhibitor Trametinib for BRAFV600E-positive ATCs, its clinical benefit is limited by the development of resistance. Furthermore, there are no systematic treatments for patients with non-BRAF mutations. Thus, there is a clear need for novel, effective, and rational therapeutic approaches for ATCs. This thesis pursued two main aims: overcoming resistance to MAPK-targeted therapies and exploiting cellular metabolism as a therapeutic target. The dysregulation of the MAPK signaling pathway is a hallmark of most ATCs. A major limitation of targeted therapies against this pathway is the development of resistance, often promoted by the recruitment of the tyrosine phosphatase SHP2 downstream RTKs, which reactivates the pathway. Here, we aimed to test the therapeutic potential of the addition of SHP2 inhibitor SHP099 to MAPK inhibitors (MAPKi) Trametinib and Trametinib/Dabrafenib in RAS- and BRAF mutant ATC cell lines, respectively. The addition of SHP099 fully or partially reverted MAPK reactivation, enhanced cytotoxicity in both 2D and 3D cell cultures, impaired cell migration and sphere-forming efficiency, and increased the expression of the sodium iodide symporter (NIS), thereby improving iodide uptake. Moreover, we generated Trametinib-resistant RAS-mutant ATC cell lines, which exhibited an immunosuppressive phenotype, epithelial-to mesenchymal transition (EMT)- and stem-like features, and increased SHP2 phosphorylation levels. In these resistant cells, SHP099 treatment inhibited EMT, stemness, and cell viability, while increasing NIS expression and iodide uptake. In addition, when combined with Trametinib, SHP099 significantly inhibited cell proliferation and viability, suggesting a re-sensitization to Trametinib. Another promising therapeutic strategy is targeting metabolic reprogramming of cancer cells. ATC cells display a significant increase in the expression of genes involved in One carbon metabolism (1CM), essential for synthesizing nucleotides, glutathione and glycine. Previous data showed that inhibiting 1CM affects ATC cell proliferation by depleting the purine pool. Nucleotides shortage induces replicative stress and triggers cell cycle checkpoints. Given that most ATCs lack functional TP53, we hypothesized that combining 1CM inhibition with G2/M checkpoint inhibition could be a viable synthetic lethality strategy. Our findings demonstrated a significant synergistic decrease in cell viability in both 2D and 3D cultures following their simultaneous inhibition. More specifically, we observed an initial replication stress, followed by an increase of γH2AX phosphorylation and eventually of cleaved PARP levels, indicating significant DNA damage that ultimately led to cell death. In conclusion, both strategies demonstrated significant therapeutic potential and represent promising combinatorial approaches for ATC treatment.

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