Cdk8 inhibition as a therapeutic intervention for ischemic fracture healing

University Of Michigan At Ann Arbor, Ann Arbor MI

Investigators

Abstract

Abstract: Investigators: Christina Capobianco (PhD Student), Kurt Hankenson, DVM, MS, PhD (Sponsor), Tristan Maerz, PhD, (Co-sponsor). Contributors : Annemarie Lang, PhD, Craig Duvall, PhD, Rhima Coleman, PhD. Background: Nonunion fractures, defined by a failure to heal for greater than nine months are physically and psychologically debilitating. Fractures accompanied by compromised or damaged vasculature are five times more likely to go on to nonunion, resulting in decreased fracture callus formation accompanied by increased cell death and fibrosis. Under ischemic fracture conditions, Cdk8 emerged as a top differentially upregulated gene in the PDGFRɑ+ fibroblast population. CDK8 acts as a post-transcriptional mediator for various target genes, including those of the HIF1ɑ and TGFβ signaling pathways, which are highly relevant to cartilage formation and fracture biology. We demonstrated that inhibition of CDK8 improved fracture callus size and bone volume in ischemic fractures in mice as well as chondrogenesis in human mesenchymal stem cells (hMSC), corroborating its role as a negative regulator of chondrogenesis and presenting a therapeutic avenue to improve ischemic fracture callus formation that has not yet been explored. The central objective of this proposal is to establish the role of CDK8 in impaired chondrogenesis and to develop a PDGFRɑ fibroblast-targeting nanoparticle to inhibit CDK8 in the fibroblasts that are bone and cartilage progenitors, promoting chondrogenesis in the context of ischemic fracture healing. Specific Aims: Aim 1) Characterize the impact of CDK8 on chondrogenic differentiation; Aim 2) Demonstrate that PDGFRɑ+ fibroblast-targeting nanoparticles delivering a CDK8 inhibitor improve ischemic fracture healing. Research Plan: Aim 1) The effect of CDK8 on chondrogenesis will be evaluated through CDK8 gain and loss of function in hMSC chondrogenic pellets. Proteomic analysis and bulk RNA sequencing of the pellets will identify alterations in HIF1a and TGFβ signaling pathways as well as permit an unbiased assessment of alternative mechanisms of action that may be occurring via CDK8. CDK8 overexpression and inhibition will be performed in a fracture hematoma model and chondrogenic genes as well as those shown to be perturbed in hMSC will be analyzed via RT-qPCR and immunofluorescent staining. Aim 2) We will fabricate and characterize a PDGFRɑ- antibody bound nanoparticle encapsulating the Cdk8 inhibitor SNX631. C57 Bl/6 mice will receive the SNX631- PDGFRɑ nanoparticle at time of ischemic fracture to assess the effect of targeted and sustained Cdk8 inhibition on ischemic fracture callus formation. To demonstrate translatability to human cells, hMSC pellets will be treated with the nanoparticles and expression of chondrogenic markers will be assessed. Conclusion: Completion of the proposed aims will lead to better understanding of CDK8’s role in chondrogenesis and advancement of therapeutic treatment for ischemic fracture patients to improve their quality of life.