Vemurafenib-resistance via de novo RBM genes mutations and chromosome 5 aberrations is overcome by combined therapy with palbociclib in thyroid carcinoma with BRAF(V600E)

PURPOSE: Papillary thyroid carcinoma (PTC) is the most frequent endocrine tumor. BRAF(V600E) represents the PTC hallmark and is targeted with selective inhibitors (e.g. vemurafenib). Although there have been promising results in clinical trials using these inhibitors, most patients develop resistance and progress. Tumor clonal diversity is proposed as one mechanism underlying drug resistance. Here we have investigated mechanisms of primary and secondary resistance to vemurafenib in BRAF(WT/V600E)–positive PTC patient-derived cells with P16(-/-) (CDKN2A(-/-)). EXPERIMENTAL DESIGN: Following treatment with vemurafenib, we expanded a sub-population of cells with primary resistance and characterized them genetically and cytogenetically. We have used exome sequencing, metaphase chromosome analysis, FISH and oligonucleotide SNP-microarray assays to assess clonal evolution of vemurafenib-resistant cells. Furthermore, we have validated our findings by networks and pathways analyses using PTC clinical samples. RESULTS: Vemurafenib-resistant cells grow similarly to naïve cells but are refractory to apoptosis upon treatment with vemurafenib, and accumulate in G2-M phase. We find that vemurafenib-resistant cells show amplification of chromosome 5 and de novo mutations in the RBM (RNA-binding motifs) genes family (i.e. RBMX, RBM10). RBMX knockdown in naïve-cells contributes to tetraploidization, including expansion of clones with chromosome 5 aberrations (e.g. isochromosome 5p). RBMX elicits gene regulatory networks with chromosome 5q cancer-associated genes and pathways for G2-M and DNA damage-response checkpoint regulation in BRAF(WT/V600E)-PTC. Importantly, combined therapy with vemurafenib plus palbociclib (inhibitor of CDK4/6, mimicking P16 functions) synergistically induces stronger apoptosis than single agents in resistant-cells and in anaplastic thyroid tumor cells harboring the heterozygous BRAF(WT/V600E) mutation. CONCLUSIONS: Critically, our findings suggest for the first time that targeting BRAF(WT/V600E) and CDK4/6 represents a novel therapeutic strategy to treat vemurafenib-resistant or vemurafenib-naïve radioiodine-refractory BRAF(WT/V600E)-PTC. This combined therapy could prevent selection and expansion of aggressive PTC cell sub-clones with intrinsic resistance, targeting tumor cells either with primary or secondary resistance to BRAF(V600E) inhibitor.