PI3Ka inhibition blocks osteochondroprogenitor specification and the hyper-inflammatory response to prevent heterotopic ossification

Heterotopic ossification (HO) occurs following mechanical trauma, burns, or congenitally in patients suffering from fibrodysplasia ossificans progressiva (FOP). Recently, we demonstrated that inhibitors of phosphatidyl-inositol 3-kinase alpha (PI3Ka), including the already marketed BYL719/Alpelisib/Piqray, may become a useful therapy for patients undergoing HO. In this study, we have confirmed these results and optimized the timing of administration of BYL719. We found that BYL719 effectively prevents HO when administered even up to three to seven days after injury. We demonstrate in cell cultures and in a mouse model of HO, that the major actions of BYL719 are on-target effects through inhibition of PI3Ka, without directly affecting ACVR1/ALK2 kinase activity. In vivo, we found that lack of PI3Ka in progenitors at injury sites is sufficient to prevent HO. Moreover, time course assays in HO lesions demonstrate that BYL719 not only blocks osteochondroprogenitor specification, but also reduces the inflammatory response. BYL719 inhibits the migration, the proliferation, and the expression of pro-inflammatory cytokines in monocytes and mast cells, suggesting that BYL719 hampers the hyper-inflammatory status of HO lesions. As part of this study, we have conducted bulk RNA sequencing using human mesenchymal stem cells (hMSCs) expressing ACVR1/ALK2-R206H to assess molecular effectors driving the therapeutic potential of BYL719 in FOP. Altogether, these results highlight the potential of PI3Ka inhibition as a safe and effective therapeutic strategy for HO. Overall design: We performed bulk RNA-sequencing using human fetal bone-marrow derived mesenchymal stem cells (hMSCs) transduced with lentiviral particles containing ACVR1-R206H or ACVR1-WT. These cells were serum starved for 16 hours before pre-stimulation with BYL719 (1uM) and subsequent Activin A (50ng/ml) or BMP6 (50ng/ml) stimulation for 1 hour. The control cells were not treated with BYL719 or ligand, but with DMSO and ligand vehicle, respectively, instead (all groups n=4).