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

乳腺癌骨转移治疗的复杂性因恶性促肿瘤发生而加剧。 病理学涉及骨骼稳态向侵袭性破骨细胞活性的转变和 骨髓细胞，有利于M2巨噬细胞（M β）和髓源性抑制细胞（MDSC）的积累， 免疫抑制介质。除肿瘤细胞外，促肿瘤发生的髓样细胞也有助于肿瘤的发生。 级联通过表达检查点配体，钝化效应T细胞的抗肿瘤功能。因此，更好的 了解改变骨骼和免疫稳态的关键信号机制， 这些功能将使得能够设计针对这种双相效应的新的组合疗法。在这一过程中，我们 已经发现，除了破骨细胞前体的强烈激活外，核因子受体激活剂 κ-B配体（RANKL）作为骨免疫连接在骨髓基质细胞极化和程序化中起重要作用， 死亡配体1（PD-L1）表达。我们发现BCa细胞RANKL升高诱导旁分泌效应， 对单核细胞以时空方式分化为免疫抑制性M2 M β的影响。初步研究 本申请中提出的结果表明，尽管RANKL通过RANK的经典自分泌信号激活了一种新的细胞因子， BCa细胞中的前馈回路，非经典RANKL信号传导增强M2 M细胞中的PD-L1表达， MDSC通过富含亮氨酸重复的G蛋白偶联受体（Lgr 4）。 根据我们已发表的初步研究结果，本提案的总体目标是扩大我们的 了解RANKL在BCa免疫抑制和骨损伤中的多效性机制， 测试结合一种新的骨保护素（OPG）细胞疗法而不干扰TNF相关的 凋亡诱导配体（TRAIL）功能，检查点阻断和化疗，以逆转肿瘤- 免疫和骨骼系统的相关病理学。我们最近采用了基于蛋白质结构的 工程方法，并确定了一个关键领域的OPG的TRAIL结合，并成功地开发 并在体内验证了保留RANKL结合但缺乏TRAIL结合的OPG变体（OPGY 49 R）。初步 研究，直接比较基于细胞的单次应用OPGY 49 R治疗与多次应用 中和RANKL mAb治疗表明单次注射后OPGY 49 R的全身稳定水平， 与RANKL mAb治疗相比，CD 8 + T细胞耗竭显著降低。更重要的是，OPGY 49 R 大大降低了体内原发性肿瘤的转移，证明了其优于RANKL的潜在优势。 单克隆抗体，地舒单抗，未能延迟骨转移或疾病复发的高风险早期- 在最近的一项国际双盲、随机、安慰剂对照、III期研究（D-CARE）中， 这项提案将测试这种新的，生物驱动的联合治疗方法，通过使用免疫活性 BCa小鼠模型，适用于转移前和转移性疾病。

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.