Vemurafenib-resistance via de novo RBM genes mutations and chromosome 5 aberrations is overcome by combined therapy with palbociclib in thyroid carcinoma with BRAFV600E

Abstract

PurposePapillary thyroid carcinoma (PTC) is the most frequent endocrine tumor. BRAFV600E 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 BRAFWT/V600E–positive PTC patient-derived cells with P16-/- (CDKN2A-/-).Experimental DesignFollowing 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.ResultsVemurafenib-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 BRAFWT/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 BRAFWT/V600E mutation.ConclusionsCritically, our findings suggest for the first time that targeting BRAFWT/V600E and CDK4/6 represents a novel therapeutic strategy to treat vemurafenib-resistant or vemurafenib-naïve radioiodine-refractory BRAFWT/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 BRAFV600E inhibitor.

Targeting Non-Oncogene Addiction: Focus on Thyroid Cancer

“…Moreover, the treatment of thyroid cancer cells with the CDK4/6 inhibitor palbociclib (PD-0332991) induces a consistent antiproliferative effect [76]. In keeping with our results, other groups have confirmed the efficacy of palbociclib in in vitro and in vivo preclinical models of TC, also in combination with a PI3K/mTOR inhibitor to bypass mechanisms of therapy resistance [89][90][91]. On the whole, we are confident that the evidence that cyclin D1 represents TC cell vulnerability may prompt preclinical and clinical studies employing CDK4/6 inhibitors for treatment of TC.…”

Dissecting Mechanisms of Melanoma Resistance to BRAF and MEK Inhibitors Revealed Genetic and Non-Genetic Patient- and Drug-Specific Alterations and Remarkable Phenotypic Plasticity

“…Interestingly, we have shown that activation of AKT in resistant cell lines might be connected with acquired mutations in a gene encoding RBMX, an RNA-binding protein protecting cells against DNA damage and acting as a tumor suppressor [92,93]. Mutations in RBMX were recently reported in papillary thyroid carcinoma cells resistant to vemurafenib [94], and knock-down of wild-type RBMX caused an increase of p-AKT protein level in naïve cancer cells. Several de novo mutations (G379R, Y357H, R339G, S337N, S303 frameshift) in RBMX that span the RNA-interacting domain of RBMX [92] were found in our study, but only one of them S303fs+/− was present in resistant melanoma cell lines with elevated activity of AKT (21_TRAR, 28_TRAR, 29_PLXR).…”

Intratumoral Genetic Heterogeneity in Papillary Thyroid Cancer: Occurrence and Clinical Significance

“…Additional tools to identify resistant subclones, imply their analysis in cellular systems, which also allows the testing for different and possible novel therapeutic options. As far as TC concerns, Antonello et al expanded a sub-population of cells with primary resistance to vemurafenib and found that they harbor amplification of chromosome 5 and mutations in RBM genes which are crucial for genome stability during cell division [78]. A combined therapeutic approach using BRAF V600E and CDK4/6 inhibitors was able to induce apoptosis in both naïve and vemurafenib-resistant cells, indicating that this combined therapy could be tested in a clinical trial of advanced TC patients.…”