Proteomic Profiling of Medullary Thyroid Cancer Identifies CAPN1 as a Key Regulator of NF1 and RET Fueled Growth

<b><i>Background:</i></b> Medullary thyroid cancer (MTC) is a frequently metastatic tumor of the thyroid that develops from the malignant transformation of C-cells. These tumors most commonly have activating mutations within the RET or RAS proto-oncogenes. Germline mutations within RET result in C-cell hyperplasia, and cause the MTC pre-disposition disorder, multiple endocrine neoplasia, type 2A (MEN2A). Single-agent therapies for MTC, including vandetanib (VAN) and cabozantinib for all MTCs and selpercatinib (SEL) for RET-mutated MTC, lead to partial responses but are not curative. <b><i>Methods:</i></b> To identify new therapeutic targets for MTC, we conducted proteomic profiling of normal C-cells, MTC cells, pre-malignant MEN2A patient samples, and MTC tumors. <b><i>Results:</i></b> From this analysis, we identified CAPN1, a member of the CALPAIN (CAPN) family endopeptidases, as widely upregulated in MTC samples. We found that short hairpin RNA-mediated depletion of CAPN1 or inhibitors of CAPN1 significantly reduced MTC cell growth, colony formation, and xenograft tumor growth <i>in vivo</i>. In addition, we show that CAPN1 inhibitors synergize with VAN and SEL <i>in vitro</i>, maximizing apoptosis. Mechanistic experiments implicate CAPN1 in inhibiting neurofibromin, encoded by NF1, and CAPN1 inhibitors stabilize NF1 protein levels and diminish downstream RAS/RET activation of AKT and ERK. <b><i>Conclusions:</i></b> Our data suggest that increased CAPN1 levels support RET and RAS-fueled growth by reducing NF1 levels. We find that combinatorial therapies between CAPN1 inhibitors and VAN or SEL show maximal efficacy in MTC cells.