Hypothesis: Targeting the pRb pathway with a focus on immunomodulatory effects offers a novel method to improve treatment outcomes in children with metastatic osteosarcoma.
Rationale: Our preliminary evidence showing antigen presentation in osteoblasts is pRb-dependent, coupled with studies demonstrating that pRb-activating CDK4/6 inhibitors trigger anti-tumor immunity, support the idea that interrogating pRb’s role in antigen presentation in osteoblasts and tumors could enhance osteosarcoma immunotherapy. To achieve this goal, two Specific Aims are proposed:
Aim 1. Test the role of pRb in modulating expression of antigen presentation genes in osteosarcoma cells in culture. Our preliminary studies indicate that pRb-null cells have impaired expression of antigen presentation genes, and conversely, CDK6 inactivation impairs osteosarcoma growth in vivo but not in culture. To validate a role of the pRb pathway in anti-tumor immunity in osteosarcoma, we will (1) assess the expression of antigen presentation genes in immortal and transformed osteoblasts from WT, pRb-null, and CDK6K43M mice with and without CDK4/6 inhibitors that have been shown to modulate antigen presentation and anti-tumor immunity in breast cancer. (2) We will test two modes of modulating pRb targets in the absence of pRb. (a) we will treat cells with Hh pathway agonists and antagonists, as we have found that this can restore osteoblast cell fate in pRb-null cells, and (b) we will inhibit expression of KDM5a genetically and pharmacologically, as this has been shown to activate Runx2-dependent gene expression programs in the absence of pRb.
Aim 2. Interrogate the role of the pRb pathway in tumor immunity in osteosarcoma in vivo. We will assay tumor growth in syngeneic mice implanted with pRb-null or wild-type control osteosarcoma cell lines as in Aim 1, with and without CDK4/6 inhibitors, hedgehog pathway agonists/antagonists, or KDM5 inhibitors. Tumors/implantation sites will be assayed histologically and by single cell sequencing to determine the composition of immune infiltrates and the tumor microenvironment. Combined, these experiments will provide significant guidance on methods to improve immunotherapy of both pRb-null and pRb-sufficient osteosarcoma.