CDK4 inhibition reduces proliferation and mineralization in MAP2K1+ melorheostosis: opening a pathway to treatment.

Abstract

Melorheostosis is a rare disease where excessive bone overgrowth and exostoses cause deformity and pain. Somatic mutations of MAP2K1 result in hyperactivation of the ERK pathway in osteoblasts and increase in vitro mineralization. Currently, there is no effective treatment. Because melorheostosis-derived osteoblasts show increased expression of cell cycle proliferation-related molecules compared to controls, we hypothesized that rapid progression through the cell cycle contributes to the bony overgrowth. We used an induced pluripotent stem cell (iPSCs) model from unaffected and affected regions of patient skin fibroblasts. We then differentiated iPSCs into induced mesenchymal stromal cells (iMSCs), and then into osteoblasts. Using propidium iodide (PI) mediated cell cycle assay with flow cytometry, we recorded that affected iMSCs (and primary patient osteoblasts) have a higher proportion of proliferative cells than unaffected. We noticed that affected osteoblasts and iMSCs have elevated the expression of phospho-Rb-a crucial molecule for G1 to S phase transition. Immunofluorescence confirmed a significantly higher cell proliferation marker Ki-67 index in affected iMSCs. EdU incorporation assays validated higher percentage of S phase cells in affected populations. We applied the FDA-approved CDK4 inhibitor palbociclib to iMSCs and primary osteoblasts and found significant reduction of phospho-Rb. Palbociclib treated iMSCs and osteoblasts displayed elevated G0/G1 peak on flow cytometry and lowered EdU incorporation, thus confirming blockade of cell cycle progression by limiting S phase entry. Interestingly, palbociclib treatment for an initial 5 d of total 21 d of osteogenic stimulation restricts mineralization in affected cells to a greater extent than unaffected. This suggests that increased proliferation is contributing to the bone growth phenotype in patients. Thus, our data suggest that targeting cell cycle machinery can be a potential therapeutic approach for melorheostosis patients.