Role of p21 positive senescent cells in radiation-induced skeletal injury and repair

Project Summary/Abstract Skeletal deterioration and related fracture risk is exacerbated in the elderly cancer survivors receiving radiation treatment (RTx), affecting independent living, and reducing quality of life. Cellular senescence, one of the major pathways induced following RTx-induced DNA damage, is characterized by a pro-inflammatory senescence associated secretory phenotype (SASP, consisting of chemokines, cytokines, growth factors, matrix degrading enzyme, etc), and is mainly regulated by cyclin dependent kinase inhibitors (CDKi’s) p16Ink4a and p21Cip1. Till recently, it was understood that p16Ink4a and p21Cip1 co-expressed in all senescent cells and regulated their function interchangeably. This theory however did not match with the expression pattern of p16Ink4a and p21Cip1 post-RTx or in aging. Using gene expression and RNA in situ hybridization studies, we have recently shown that cells express p21 or p16Ink4a in unique populations of bone marrow cells, osteoblasts, and osteocytes independent of the expression of either senescence marker. Only a small proportion of cells express both p16Ink4a and p21Cip1. Furthermore, we have compelling preliminary data using mass cytometry of bone cells, which allowed us to visualize these independent unique populations of p16Ink4a and p21Cip1 expressing cells without any coexpression of SASP, suggesting a physiological function of these CDKis. Interestingly, in a parallel analysis in radiated bones, we identified elevated levels of Cd11b+p21+ myeloid cell population, which was accompanied by expression of several SASP factors, thus allowing us to characterize these p21+ cells as p21+ senescent (p21+SEN) cells. In a recent seminal finding, using transgenic mice harboring transgenes that enable the selective elimination of either p16Ink4a or p21Cip1 expressing cells, the elimination of p21+SEN cells in the p21-ATTAC [apoptosis through targeted activation of caspase] mice, but not the elimination of p16+SEN cells in the p16-INK- ATTAC mice, could mitigate most of the RTx-related adverse events in bone in young mice. Whether this approach of clearance of p21+SEN cells will work to alleviate RTx-related bone deterioration in old mice, which have a pre-existing high burden of senescent cells, remains to be seen. Based on our compelling preliminary data, we will test our central hypothesis that: “Acute generation of p21+SEN cells mediate RTx-related skeletal deterioration and BMSC dysfunction, but targeted early clearance of p21+SEN cells can alleviate RTx-related chronic skeletal deterioration and promote fracture healing”. To test our central hypothesis, our aims are: (aim 1)To identify key mechanisms that are involved in RTx-related skeletal deterioration following early clearance of p21+SEN cells in young and aged mice, (aim 2): To assess bone architectural changes following cell specific clearance (using our novel Cre-LoxP mice, p21-LOX-ATTAC) of p21+Cd11b+SEN myeloid cells and (aim3): To assess if prior clearance of senescent cells pharmacologically or by genetic clearance of p21+SEN cells will promote fracture healing. The project will address questions related to basic biology of aging and role of p21 in skeletal cells and lay the groundwork to support the idea that an early intervention could prove effective to counter adverse changes from RTx, and to alleviate chronic skeletal deterioration and reduce the risk of potential fractures.