Mechanisms and therapeutic targeting of osteoimmune functions of RANKL in breast cancer

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Complexities in treating breast cancer (BCa) with bone metastasis are aggravated by a vicious protumorigenic pathology involving a shift in skeletal homeostasis towards aggressive osteoclast activity and polarization of myeloid cells, favoring M2 macrophage (MФ) and myeloid-derived suppressor cell (MDSC) accumulation as key mediators of immunosuppression. In addition to the tumor cells, protumorigenic myeloid cells contribute to the cascade by expressing checkpoint ligands, blunting antitumor functions of effector T cells. Hence, a better understanding of key signaling mechanisms that alter skeletal and immune homeostasis towards protumorigenic functions will enable the designing of new combination therapies targeting this biphasic effect. In this pursuit, we have identified that in addition to robust activation of osteoclast precursors, receptor activator of nuclear factor kappa-Β ligand (RANKL) plays an important role as an osteoimmune link in MФ polarization and programmed death-ligand 1 (PD-L1) expression. We identified that elevated RANKL from BCa cells induce paracrine effects on differentiation of monocytes to immunosuppressive M2 MФ in a spatiotemporal manner. Preliminary studies presented in this application indicate that whereas RANKL canonical autocrine signaling via RANK activates a feed-forward loop in BCa cells, non-canonical RANKL signaling enhances PD-L1 expression in M2 MФ and MDSCs via the leucine-rich repeat containing G-protein coupled receptor (Lgr4). Based on our published and preliminary findings, the overarching goal of this proposal is to expand our understanding on the pleiotropic mechanisms of RANKL in BCa immunosuppression and bone damage, and to test the potential of combining a novel osteoprotegerin (OPG) cell therapy without interfering in TNF-related apoptosis-inducing ligand (TRAIL) function, with checkpoint blockade and chemotherapies, to reverse tumor- associated pathology in the immune and skeletal systems. We recently adopted a protein structure-based engineering approach and identified a critical domain on OPG for TRAIL binding and successfully developed and validated in vivo an OPG variant (OPGY49R) that retains RANKL binding, but lacks TRAIL binding. Preliminary studies, directly comparing a cell-based, single-application OPGY49R treatment with multiple applications of a neutralizing RANKL mAb therapy indicated systemically stable levels of OPGY49R from a single injection and a significant decrease in CD8+ T cell exhaustion, compared to RANKL mAb treatment. More importantly, OPGY49R greatly decreased metastasis of primary tumors in vivo, demonstrating its potential advantage over the RANKL mAb, denosumab, which failed to delay bone metastasis or disease recurrence in patients with high-risk early- stage BCa in a recent international double-blinded randomized placebo-controlled, phase 3 study (D-CARE). This proposal will test this novel, biologically driven combination therapy approach by using immunocompetent mouse models of BCa, as applicable to both pre-metastatic and metastatic disease.