CDK4 phosphorylation status and rational use for combining CDK4/6 and BRAF/MEK inhibition in advanced thyroid carcinomas

Background: CDK4/6 inhibitors (CDK4/6i) have been established as standard treatment against advanced Estrogen Receptor-positive breast cancers. These drugs are being tested against several cancers, including in combinations with other therapies. We identified the T172-phosphorylation of CDK4 as the step determining its activity, retinoblastoma protein (RB) inactivation, cell cycle commitment and sensitivity to CDK4/6i. Poorly differentiated (PDTC) and anaplastic (ATC) thyroid carcinomas, the latter considered one of the most lethal human malignancies, represent major clinical challenges. Several molecular evidence suggest that CDK4/6i could be considered for treating these advanced thyroid cancers. Methods: We analyzed by two-dimensional gel electrophoresis the CDK4 modification profile and the presence of T172-phosphorylated CDK4 in a collection of 98 fresh-frozen tissues and in 21 cell lines. A sub-cohort of samples was characterized by RNA sequencing and immunohistochemistry. Sensitivity to CDK4/6i (palbociclib and abemaciclib) was assessed by BrdU incorporation/viability assays. Treatment of cell lines with CDK4/6i and combination with BRAF/MEK inhibitors (dabrafenib/trametinib) was comprehensively evaluated by western blot, characterization of immunoprecipitated CDK4 and CDK2 complexes and clonogenic assays. Results: CDK4 phosphorylation was detected in all well-differentiated thyroid carcinomas (n=29), 19/20 PDTC, 16/23 ATC and 18/21 thyroid cancer cell lines, including 11 ATC-derived ones. Tumors and cell lines without phosphorylated CDK4 presented very high p16^CDKN2A levels, which were associated with proliferative activity. Absence of CDK4 phosphorylation in cell lines was associated with CDK4/6i insensitivity. RB1 defects (the primary cause of intrinsic CDK4/6i resistance) were not found in 5/7 tumors without detectable phosphorylated CDK4. A previously developed 11-gene expression signature identified the likely unresponsive tumors, lacking CDK4 phosphorylation. In cell lines, palbociclib synergized with dabrafenib/trametinib by completely and permanently arresting proliferation. These combinations prevented resistance mechanisms induced by palbociclib, most notably Cyclin E1-CDK2 activation and a paradoxical stabilization of phosphorylated CDK4 complexes. Conclusion: Our study supports further clinical evaluation of CDK4/6i and their combination with anti-BRAF/MEK therapies as a novel effective treatment against advanced thyroid tumors. Moreover, the complementary use of our 11 genes predictor with p16/KI67 evaluation could represent a prompt tool for recognizing the intrinsically CDK4/6i insensitive patients, who are potentially better candidates to immediate chemotherapy.