The outcome of patients with metastatic osteosarcoma has not improved since the introduction of chemotherapy in the 1970s. Development of therapies targeting the metastatic cascade is a tremendous unmet medical need. The Wnt signaling pathway has been the focus of intense investigation in osteosarcoma because of its role in normal bone development. Although the role of Wnt signaling in the pathogenesis of osteosarcoma is controversial, there are several reports of dickkopf-1 (DKK-1), a Wnt signaling antagonist, possibly playing a pro-tumorigenic role. In this work we investigated the effect of anti-DKK-1 antibodies on the growth and metastasis of patient-derived osteosarcoma xenografts. We were able to detect human DKK-1 in the blood of tumor-bearing mice and found a correlation between DKK-1 level and tumor proliferation. Treatment with the anti-DKK-1 antibody, BHQ880, slowed the growth of orthotopically implanted patient-derived osteosarcoma xenografts and inhibited metastasis. This effect was correlated with increased nuclear beta-catenin staining and increased expression of the bone differentiation marker osteopontin. These findings suggest that Wnt signaling is anti-tumorigenic in osteosarcoma, and support the targeting of DKK-1 as an anti-metastatic strategy for patients with osteosarcoma.

PURPOSE. This study determines if δ-opioid receptor agonist (i.e. SNC-121)-induced epigenetic changes via regulation of histone deacetylases (HDACs) for retinal ganglion cell (RGC) neuroprotection in glaucoma model. METHODS. Intraocular pressure was raised in rat eyes by injecting 2M hypertonic saline into the limbal veins. SNC-121 (1 mg/kg; i.p.) was administered to the animals for 7 days. Retinas were collected at days 7 and 42, post-injury followed by measurement of HDAC activities, mRNA, and protein expression by enzyme assay, quantitative real-time PCR (qRT-PCR), Western blotting, and immunohistochemistry. RESULTS. The visual acuity, contrast sensitivity, and pattern electroretinograms (ERGs) were declined in ocular hypertensive animals, which were significantly improved by SNC-121 treatment. Class I and IIb HDACs activities were significantly increased at days 7 and 42 in ocular hypertensive animals. The mRNA and protein expression of HDAC 1 was increased by 1.33 ± 0.07-fold and 20.2 ± 2.7%, HDAC 2 by 1.4 ± 0.05-fold and 17.0 ± 2.4%, HDAC 3 by 1.4 ± 0.06-fold and 17.4 ± 3.4%, and HDAC 6 by 1.5 ± 0.09-fold and 15.1 ± 3.3% at day 7, post-injury. Both the mRNA and protein expression of HDACs were potentiated further at day 42 in ocular hypertensive animals. HDAC activities, mRNA, and protein expression were blocked by SNC-121 treatment at days 7 and 42 in ocular hypertensive animals. CONCLUSIONS. Data suggests that class I and IIb HDACs are activated and upregulated during early stages of glaucoma. Early intervention with δ-opioid receptor activation resulted in the prolonged suppression of class I and IIb HDACs activities and expression, which may, in part, play a crucial role in RGC neuroprotection.

The main objective of this study was to determine the inhibition of pro-inflammatory cytokines and their associated signaling molecules by δ-opioid receptor activation by a selective ligand, SNC-121 in chronic rat glaucoma model. Intraocular pressure was raised in rat eyes by injecting 2 M hypertonic saline into the limbal veins. SNC-121 (1 mg/kg; i. p) or Stattic (5 mg/kg; i. p) was administered in Brown Norway rats daily for 7 days. The mRNA expression of IL-1β, TNF-α, Fas, IL-6, leukemia inhibitory factor, and IFN-γ was increased significantly in the retina of ocular hypertensive animals at day 7, post injury. Administration of SNC-121 (1 mg/kg; i. p. injection) for 7 days (once a day) completely inhibited the increase in the mRNA and protein expression of pro-inflammatory cytokines. Mechanistically, we provide data showing a significant increase in the phosphorylation of STAT3 at tyrosine 705 whereas a moderate but significant increase in the total STAT3 protein expression was also seen in the retina of ocular hypertensive animals. Data illustrated that SNC-121 administration completely abrogated ocular hypertension-induced increase in STAT3Y705 phosphorylation. Interestingly, acetylation of STAT3 at lysine 685 (AcK685) was reduced in ocular hypertensive animals and subsequently increased significantly by SNC-121 treatment. Stattic, a selective STAT3 inhibitor, administration resulted in a complete attenuation in the production of IL-1β and IL-6 in ocular hypertensive animals. In conclusion, δ-opioid receptor activation suppressed the phosphorylation of STAT3 at tyrosine 705 and increased acetylation at lysine 686 and these posttranslational modifications can regulate the production of some but not all pro-inflammatory cytokines in response to glaucomatous injury.

Immunologically “cold” tumors are not responsive to current immunotherapies. The impact of T-cell activation with immune checkpoint inhibition or vaccines is limited in these cancers by a lack of intertumoral T cells or an “exhausted” T-cell phenotype, which are frequently induced by abundant immune-suppressive cues from intertumoral innate immune cells. Tumor-associated macrophages (TAMs) are a major cell population of the tumor microenvironment and play a key role in promoting tumor progression in many solid organ cancers. During cancer progression, tumoral and microenvironmental cues educate TAMs towards a M2-phenotype that nurtures cancer stem cells, accelerates metastasis, and confers resistance to chemotherapy. Current drug development efforts targeting TAMs primarily focus an unselective systemic inhibition of macrophage recruitment to disease sites or modulation of their immuno-cytokine profile. RP-182 is a 10mer striapathic peptide with immunomodulatory function that was derived from a biophysical homology screen of carbohydrate recognition domain patterns and naturally occurring host defense peptides (HDP) and innate defense regulators (IDR). We demonstrate that RP-182 specifically and effectively binds to the mannose receptor MRC1/CD206 expressed on M2-like macrophages, induces a conformational switch of the receptor, and activates in human and murine M2-like macrophages a program of phagocytosis, autophagy, and apoptosis. In genetically engineered murine models of pancreas cancer RP-182 suppressed tumor growth, extended survival, and improved antitumor immunity, findings also seen in other syngeneic cancer models that include variety of murine and human (PDX) cancer models. RP-182 enhanced the effects of chemo- and immune checkpoint therapy. In conclusion, the RP-182 peptide depletes the immune-evasive M2 population of TAMs via engagement with the M2-specific lectin receptor CD206. This is associated with improved clinical outcome in transgenic mice with pancreas cancer. Similarly, RP-182 selectively reduces immune-suppressive M2-like macrophages, a finding also made in a noncancerous murine fibrosis model. Collectively, preclinical findings show that HDPs/IDRs derived from biophysical homology studies effectively reduce innate immune cell subpopulations with therapeutic merit in cancerous and nonmalignant diseases. Citation Format: Rushikesh Sable, Jesse Jaynes, Michael Ronzetti, Wendy Guzman, Zachary Knotts, Natalia de Val, Juan Morgan, Clayton Yates, Baljinnyam Bolormaa, Udo Rudloff. Precision targeting of M2-like macrophages by the innate defense regulator RP-182 in pancreatic cancer and noncancerous diseases [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B49.