Project Summary/Abstract Children with Neurofibromatosis Type 1 (NF1) are predisposed to cancer but can also present with unilateral bowing of the tibia that often progresses to fracture. Unlike the robust bone healing of most children, bone healing in these patients fails, leading to invasive and repeated surgeries, and often amputation of the leg. Research focused on identifying the cause of this refractory bone healing revealed that Nf1 deficiency in bone mesenchymal progenitors blunts their osteogenic potential and triggers chronic and uncontrolled RAS/ERK signaling, although blocking this pathway failed to rescue osteoprogenitor differentiation and the delayed bone healing of mice lacking Nf1 is osteoprogenitors. It has now become clear that multiple signaling pathways and compensatory mechanisms are involved in the abnormal differentiation of Nf1- null osteoprogenitors, and that targeting them to promote bone union in children with NF1 will be very challenging. This proposal steers away from this strategy, thanks to the observation of early senescence in Nf1-null osteoprogenitors, which should make them sensitive to “senolytic” drugs. Our hypothesis is thus that eliminating Nf1-null osteoprogenitors - rather than correcting their multiple defects – with senolytics will promote bone union in children with NF1 pseudarthrosis. We propose to determine if clearance of Nf1-null senescent osteoprogenitors, genetically or pharmacologically, improves the poor healing potential of mice lacking Nf1 in osteoprogenitors, and whether the senescence associated secretory phenotype (SASP) from these cells alters the behavior of lineages contributing to bone repair. A second major premise of this work is the observation that mice deficient for Nf1 selectively in adult osteoprogenitors lose muscle mass. This finding and other in vitro and in vivo results led us to challenge the current view of a muscle-intrinsic defect and to address the hypothesis that bone-derived factor(s) from Nf1-null osteoprogenitors contribute to the muscle weakness observed in children with NF1, which could also be alleviated by the clearance of Nf1-null osteoprogenitors with senolytics. Overall, this work has the potential to improve the clinical management of NF1 pseudarthrosis with a strategy that will pharmacologically clear faulty Nf1-null osteoprogenitors and relieve their inhibitory influence on other cells contributing to bone repair, thus restoring robust bone regeneration. Mechanistically, this study may support senescence in Nf1-null osteoprogenitors as a mechanism to prevent the spread of RAS-induced mutational damage and preclude potential malignant transformation at the cost of a deleterious bystander effect on bone repair and muscle function.