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PDF file - 110K, Primer sequences for real-time PCR and RT-PCR

PDF file - 256K, Figure S1. Effects of aCXCL10 on tumor metastasis into lungs and adrenal glands. Figure S2. CXCL10-induced adhesion and survival of B16F10 cells

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BackgroundRadiation therapy (RT) has been demonstrated to generate an in situ vaccination (ISV) effect in murine models and in patients with cancer; however, this has not routinely translated into enhanced clinical response to immune checkpoint inhibition (ICI). We investigated whether the commonly used vaccine adjuvant, monophosphoryl lipid A (MPL) could augment the ISV regimen consisting of combination RT and ICI.Materials/methodsWe used syngeneic murine models of melanoma (B78) and prostate cancer (Myc-CaP). Tumor-bearing mice received either RT (12 Gy, day 1), RT+anti-CTLA-4 (C4, day 3, 6, 9), MPL (20 µg IT injection days 5, 7, 9), RT+C4+MPL, or PBS control. To evaluate the effect of MPL on the irradiated tumor microenvironment, primary tumor with tumor draining lymph nodes were harvested for immune cell infiltration analysis and cytokine profiling, and serum was collected for analysis of antitumor antibody populations.ResultsCombination RT+C4+MPL significantly reduced tumor growth, increased survival and complete response rate compared with RT+C4 in both B78 and Myc-CaP models. MPL favorably reprogrammed the irradiated tumor-immune microenvironment toward M1 macrophage and Th1 TBET+CD4+ T cell polarization. Furthermore, MPL significantly increased intratumoral expression of several Th1-associated and M1-associated proinflammatory cytokines. In co-culture models, MPL-stimulated macrophages directly activated CD8 T cells and polarized CD4 cells toward Th1 phenotype. MPL treatment significantly increased production of Th1-associated, IgG2c antitumor antibodies, which were required for and predictive of antitumor response to RT+C4+MPL, and enabled macrophage-mediated antibody-dependent direct tumor cell killing by MPL-stimulated macrophages. Macrophage-mediated tumor cell killing was dependent on FcγR expression. In metastatic models, RT and MPL generated a systemic antitumor immune response that augmented response to ICIs. This was dependent on macrophages and CD4+ but not CD8+T cells.ConclusionsWe report the potential for MPL to augment the ISV effect of combination RT+C4 through FcγR, macrophage, and TBET+CD4+ Th1 cell dependent mechanisms. To our knowledge, this is the first report describing generation of a CD8+ T cell-independent, Th1 polarized, systemic antitumor immune response with subsequent generation of immunologic memory. These findings support the potential for vaccine adjuvants to enhance the efficacy of in situ tumor vaccine approaches.

Rationale: Clinical interest in combining targeted radionuclide therapies (TRT) with immunotherapies is growing. External beam radiation therapy (EBRT) activates a type 1 interferon (IFN1) response mediated via stimulator of interferon genes (STING), and this is critical to its therapeutic interaction with immune checkpoint blockade. However, little is known about the time course of IFN1 activation after EBRT or whether this may be induced by decay of a TRT source. Methods: We examined the IFN1 response and expression of immune susceptibility markers in B78 and B16 melanomas and MOC2 head and neck cancer murine models using qPCR and western blot. For TRT, we used 90Y chelated to NM600, an alkylphosphocholine analog that exhibits selective uptake and retention in tumor cells including B78 and MOC2. Results: We observed significant IFN1 activation in all cell lines, with peak activation in B78, B16, and MOC2 cell lines occurring 7, 7, and 1 days, respectively, following RT for all doses. This effect was STING-dependent. Select IFN response genes remained upregulated at 14 days following RT. IFN1 activation following STING agonist treatment in vitro was identical to RT suggesting time course differences between cell lines were mediated by STING pathway kinetics and not DNA damage susceptibility. In vivo delivery of EBRT and TRT to B78 and MOC2 tumors resulted in a comparable time course and magnitude of IFN1 activation. In the MOC2 model, the combination of 90Y-NM600 and dual checkpoint blockade therapy reduced tumor growth and prolonged survival compared to single agent therapy and cumulative dose equivalent combination EBRT and dual checkpoint blockade therapy. Conclusions: We report the time course of the STING-dependent IFN1 response following radiation in multiple murine tumor models. We show the potential of TRT to stimulate IFN1 activation that is comparable to that observed with EBRT and this may be critical to the therapeutic integration of TRT with immunotherapies.

Background: Sequencing of immune checkpoint inhibitors (ICI) and external beam radiation therapy (EBRT) for cancer treatment has been studied, but the optimal sequencing has yet to be determined. While some studies have noted therapeutic advantages of priming the tumor immune microenvironment with EBRT prior to ICI, others have described the benefit of modulating the tumor infiltrating lymphocyte (TIL) population with ICI before EBRT. Targeted radionuclide therapy (TRT) approaches allow investigation of how irradiation by a tumor-targeted radionuclide and differences in emission type, linear energy transfer, and dose rate affect optimal timing of ICI administration. NM600 is an alkylphosphocholine analog selectively taken up by tumors capable of chelating numerous radionuclides for comparative studies. Objective: We use two immunologically cold tumor models, MOC2 head and neck squamous cell carcinoma and B78 melanoma, to describe the influence of dose rate on type I interferon (IFN1) signaling and the effect of ICI and 90Y-, 177Lu-, and 225Ac-NM600 TRT sequences on tumor response. Methods: 90Y, 177Lu, or 225Ac were added to culture media in activities estimated using GEANT4 Monte Carlo to deliver 12 Gy to the cell monolayer. qPCR was performed on cDNA from cells irradiated with EBRT, 90Y, 177Lu, or 225Ac, and harvested on days 1, 3, or 7. In vivo dosimetry was performed using the Monte Carlo-based RAPID platform utilizing serial PET/CT or SPECT/CT imaging and/or longitudinal biodistribution. Differences over time (days 4, 7, 14, 21, 28 after RT) in TIL and systemic immune cell populations were measured by flow cytometry following no treatment, 12 Gy EBRT, or 90Y-, 177Lu-, or 225Ac-NM600 in MOC2 tumors. Mice bearing B78 tumors received 1.5 Gy 90Y-, 177Lu-, or 225Ac-NM600, or no radiation on day 1 +/- ICI (anti-CTLA4 + anti-PDL1) on days -3/0/3 (early), 4/7/10 (middle), or 11/14/17 (late). Mice were monitored for tumor growth and survival. Results: TRT and EBRT induced IFN1 responses in MOC2 cells. MOC2 cells treated every 24h with EBRT-matched 90Y/225Ac dose rates led to upregulation of IFN1-associated Ifnb1 and Mx1, mimicking radionuclide-induced responses. Increased tumor CD8/Treg ratios and decreased Tregs were observed at day 7 following all RT forms in MOC2 tumors. Long half-life 225Ac-NM600 (90Y: 65h; 177Lu: 161h; 225Ac: 240h) induced similar TIL changes at day 21. For 1.5 Gy 90Y-, 177Lu-, and 225Ac-NM600, B78 tumor growth delay and statistically significant overall survival benefit over respective TRT monotherapy and control groups was observed with early (day -3/0/3) dual ICI administration. Conclusions: These studies demonstrate novel immunomodulatory effects of α- and β- emitting TRT and the capacity to achieve substantial antitumor responses with appropriate TRT + ICI sequencing. These results may inform clinical trial design of TRT + ICI regimens for patients with metastatic cancers. Citation Format: Caroline P. Kerr, Joseph J. Grudzinski, Carolina A. Ferreira, David Adam, Julia Sheehan-Klenk, Amber M. Bates, Won Jong Jin, Ohyun Kwon, Justin C. Jagodinsky, Maria Powers, Raghava N. Sriramaneni, Paul A. Clark, Luke Zangl, Thanh Phuong T. Nguyen, Anatoly N. Pinchuk, Cynthia Choi, Christopher F. Massey, Reinier Hernandez, Bryan Bednarz, Jamey P. Weichert, Zachary S. Morris. Impact of sequencing of immune checkpoint blockade and targeted radionuclide therapy on murine tumor response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2828.

Molecular and cellular effects of radiotherapy on tumor microenvironment (TME) can help prime and propagate antitumor immunity. We hypothesized that delivering radiation to all tumor sites could augment response to immunotherapies. We tested an approach to enhance response to immune checkpoint inhibitors (ICIs) by using targeted radionuclide therapy (TRT) to deliver radiation semiselectively to tumors. NM600, an alkylphosphocholine analog that preferentially accumulates in most tumor types, chelates a radioisotope and semiselectively delivers it to the TME for therapeutic or diagnostic applications. Using serial 86Y-NM600 positron emission tomography (PET) imaging, we estimated the dosimetry of 90Y-NM600 in immunologically cold syngeneic murine models that do not respond to ICIs alone. We observed strong therapeutic efficacy and reported optimal dose (2.5 to 5 gray) and sequence for 90Y-NM600 in combination with ICIs. After combined treatment, 45 to 66% of mice exhibited complete response and tumor-specific T cell memory, compared to 0% with 90Y-NM600 or ICI alone. This required expression of STING in tumor cells. Combined TRT and ICI activated production of proinflammatory cytokines in the TME, promoted tumor infiltration by and clonal expansion of CD8+ T cells, and reduced metastases. In mice bearing multiple tumors, combining TRT with moderate-dose (12 gray) external beam radiotherapy (EBRT) targeting a single tumor augmented response to ICIs compared to combination of ICIs with either TRT or EBRT alone. The safety of TRT was confirmed in a companion canine study. Low-dose TRT represents a translatable approach to promote response to ICIs for many tumor types, regardless of location.

BackgroundCurrent clinical trials are using radiation therapy (RT) to enhance an antitumor response elicited by high-dose interleukin (IL)-2 therapy or immune checkpoint blockade (ICB). Bempegaldesleukin (BEMPEG) is an investigational CD122-preferential IL-2 pathway agonist with prolonged in vivo half-life and preferential intratumoral expansion of T effector cells over T regulatory cells. BEMPEG has shown encouraging safety and efficacy in clinical trials when used in combination with PD-1 checkpoint blockade. In this study, we investigated the antitumor effect of local RT combined with BEMPEG in multiple immunologically ‘cold’ tumor models. Additionally, we asked if ICB could further enhance the local and distant antitumor effect of RT+BEMPEG in the setting of advanced solid tumors or metastatic disease.MethodsMice bearing flank tumors (B78 melanoma, 4T1 breast cancer, or MOC2 head and neck squamous cell carcinoma) were treated with combinations of RT and immunotherapy (including BEMPEG, high-dose IL-2, anti(α)-CTLA-4, and α-PD-L1). Mice bearing B78 flank tumors were injected intravenously with B16 melanoma cells to mimic metastatic disease and were subsequently treated with RT and/or immunotherapy. Tumor growth and survival were monitored. Peripheral T cells and tumor-infiltrating lymphocytes were assessed via flow cytometry.ResultsA cooperative antitumor effect was observed in all models when RT was combined with BEMPEG, and RT increased IL-2 receptor expression on peripheral T cells. This cooperative interaction was associated with increased IL-2 receptor expression on peripheral T cells following RT. In the B78 melanoma model, RT+BEMPEG resulted in complete tumor regression in the majority of mice with a single ~400 mm3tumor. This antitumor response was T-cell dependent and supported by long-lasting immune memory. Adding ICB to RT+BEMPEG strengthened the antitumor response and cured the majority of mice with a single ~1000 mm3B78 tumor. In models with disseminated metastasis (B78 primary with B16 metastasis, 4T1, and MOC2), the triple combination of RT, BEMPEG, and ICB significantly improved primary tumor response and survival.ConclusionThe combination of local RT, BEMPEG, and ICB cured mice with advanced, immunologically cold tumors and distant metastasis in a T cell-dependent manner, suggesting this triple combination warrants clinical testing.

Brain metastases develop in over 60% of advanced melanoma patients and negatively impact quality of life and prognosis. In a murine melanoma model, we previously showed that an in situ vaccination (ISV) regimen, combining radiation treatment and intratumoral (IT) injection of immunocytokine (IC: anti-GD2 antibody fused to IL2), along with the immune checkpoint inhibitor + anti-CTLA-4, robustly eliminates peripheral flank tumors but only has modest effects on co-occurring intracranial tumors. In this study, we investigated the ability of low-dose radiation to the brain to potentiate anti-tumor immunity against a brain tumor when combined with ISV + anti-CTLA-4. B78 (GD2(+), immunologically “cold”) melanoma tumor cells were implanted into the flank and the right striatum of the brain in C57BL/6 mice. Flank tumors (50–150 mm(3)) were treated following a previously optimized ISV regimen [radiation (12 Gy × 1, treatment day 1), IT-IC (50 lg daily, treatment days 6–10), and + anti-CTLA-4 (100 lg, treatment days 3, 6, 9)]. Mice that additionally received whole-brain radiation treatment (WBRT, 4 Gy × 1) on day 15 demonstrated significantly increased survival compared to animals that received ISV + anti-CTLA-4 alone, WBRT alone or no treatment (control) (P < 0.001, log-rank test). Timing of WBRT was critical, as WBRT administration on day 1 did not significantly enhance survival compared to ISV + anti-CTLA-4, suggesting that the effect of WBRT on survival might be mediated through immune modulation and not just direct tumor cell cytotoxicity. Modest increases in T cells (CD8(+) and CD4(+)) and monocytes/macrophages (F4/80(+)) but no changes in FOXP3(+) regulatory T cells (Tregs), were observed in brain melanoma tumors with addition of WBRT (on day 15) to ISV + anti-CTLA-4. Cytokine multiplex immunoassay revealed distinct changes in both intracranial melanoma and contralateral normal brain with addition of WBRT (day 15) to ISV + anti-CTLA-4, with notable significant changes in pro-inflammatory (e.g., IFNγ, TNFα and LIX/CXCL5) and suppressive (e.g., IL10, IL13) cytokines as well as chemokines (e.g., IP-10/CXCL10 and MIG/CXCL9). We tested the ability of the alkylphosphocholine analog, NM600, to deliver immunomodulatory radiation to melanoma brain tumors as a targeted radionuclide therapy (TRT). Yttrium-86 ((86)Y) chelated to NM600 was delivered intravenously by tail vein to mice harboring flank and brain melanoma tumors, and PET imaging demonstrated specific accumulation up to 72 h at each tumor site (~12:1 brain tumor/brain and ~8:1 flank tumor/muscle). When NM600 was chelated to therapeutic β-particle-emitting (90)Y and administered on treatment day 13, T-cell infiltration and cytokine profiles were altered in melanoma brain tumor, like that observed for WBRT. Overall, our results demonstrate that addition of low-dose radiation, timed appropriately with ISV administration to tumors outside the brain, significantly increases survival in animals co-harboring melanoma brain tumors. This observation has potentially important translational implications as a treatment strategy for increasing the response of tumors in the brain to systemically administered immunotherapies.

In head and neck squamous cell carcinoma (HNSCC) tumors that over-expresses huEGFR, the anti-EGFR antibody, cetuximab, antagonizes tumor cell viability and sensitizes to radiation therapy. However, the immunologic interactions between cetuximab and radiation therapy are not well understood. We transduced two syngeneic murine HNSCC tumor cell lines to express human EGFR (MOC1- and MOC2-huEGFR) in order to facilitate evaluation of the immunologic interactions between radiation and cetuximab. Cetuximab was capable of inducing antibody-dependent cellular cytotoxicity (ADCC) in MOC1- and MOC2-huEGFR cells but showed no effect on the viability or radiosensitivity of these tumor cells, which also express muEGFR that is not targeted by cetuximab. Radiation enhanced the susceptibility of MOC1- and MOC2-huEGFR to ADCC, eliciting a type I interferon response and increasing expression of NKG2D ligands on these tumor cells. Co-culture of splenocytes with cetuximab and MOC2-huEGFR cells resulted in increased expression of IFNγ in not only NK cells but also in CD8+ T cells, and this was dependent upon splenocyte expression of FcγR. In MOC2-huEGFR tumors, combining radiation and cetuximab induced tumor growth delay that required NK cells, EGFR expression, and FcγR on host immune cells. Combination of radiation and cetuximab increased tumor infiltration with NK and CD8+ T cells but not regulatory T cells. Expression of PD-L1 was increased in MOC2-huEGFR tumors following treatment with radiation and cetuximab. Delivering anti-PD-L1 antibody with radiation and cetuximab improved survival and resulted in durable tumor regression in some mice. Notably, these cured mice showed evidence of an adaptive memory response that was not specifically directed against huEGFR. These findings suggest an opportunity to improve the treatment of HNSCC by combining radiation and cetuximab to engage an innate anti-tumor immune response that may prime an effective adaptive immune response when combined with immune checkpoint blockade. It is possible that this approach could be extended to any immunologically cold tumor that does not respond to immune checkpoint blockade alone and for which a tumor-specific antibody exists or could be developed.

C-X-C motif chemokine receptor 3 (CXCR3) is a G protein-coupled receptor for three ligands which are C-X-C motif chemokine 9 (CXCL9), CXCL10, and CXCL11 [1]. Previously we have reported that CXCL10 promotes pro-inflammatory cytokine expression, and forms positive feedback loop [2,3]. In the present study, we described mRNA expression of CXCL9 and CXCL11 under CXCL10 stimuli in the presence or absence of CXCR3 antagonist, JN-2 in bone marrow-derived macrophages (BMMs) and CD4+ T cells. In addition, we examined pro-inflammatory cytokine expression under CXCL9 or CXCL11 stimuli in BMMs and CD4+ T cells. Keywords: CXCR3, CXCL9, CXCL11, Cytokine

Background: Radiation (RT) activates a type 1 interferon (IFN1) response and this is critical to the effect of RT in priming a response to immune checkpoint blockade (ie anti-CTLA4). However, little is known about the time course of this effect. Clinical interest in utilizing systemically administered targeted radionuclide therapy agents (TRT) is growing as these agents can be used to target multiple sites of disease including micro-metastases. It is unclear how IFN1 activation induced by continuous delivery of RT during exponential decay of a TRT source will compare to that induced following instantaneous external beam RT (EBRT). Here we report the time course of IFN1 response following RT in vitro and in vivo. Methods: For in vitro studies, we utilized murine models of melanoma (B16, B16 STING knockout, B78), and head and neck squamous cell carcinoma (MOC2). EBRT was prescribed to 2.5 Gy, 12 Gy, or 3 fractions of 8 Gy. For in vivo studies, syngeneic C57BL/6 mice were engrafted with either B78 or MOC2 cells on the flank and RT was delivered when mean tumor size was 100-150 mm3. EBRT was prescribed to 2.5 Gy or 12 Gy. For TRT, we used 90Y conjugated to NM600, an alkylphosphocholine analog that exhibits selective uptake and retention in tumor cells of nearly any type, including B78 and MOC2. Tumor-specific dosimetry for 90Y-NM600 was determined using sequential 86Y-NM600 PET/CT imaging (3h, 24h, 48h, 72h) and a Monte Carlo based dose calculation platform. TRT was prescribed to a cumulative absorbed dose of 2.5 Gy or 12 Gy. Following delivery of RT, cells or tumors were harvested at 1d, 7d, and 14d post RT and RNA was isolated. Gene expression of Ifn-β and IFN response elements was quantified by qPCR and normalized to untreated controls. Results: We observed significant IFN1 activation in all cell lines, with peak activation in B78, B16, and MOC2 cell lines occurring 7, 7, and 1 days, respectively, following RT for all doses. This effect was STING-dependent. Select IFN response genes remained upregulated at 14 days following RT. IFN1 activation following STING agonist treatment in vitro was identical to RT suggesting time course differences between cell lines were mediated by STING pathway kinetics and not DNA damage susceptibility. In vivo delivery of EBRT and TRT to B78 and MOC2 tumors resulted in a comparable time course and magnitude of IFN1 activation. In the MOC2 model, the combination of 90Y-NM600 and anti-CTLA-4 therapy reduced tumor growth and prolonged survival compared to single agent therapy. Conclusions: We report the time course of the STING-dependent IFN1 response following RT in multiple murine tumor models. We show the potential of TRT to stimulate IFN1 activation that is comparable to that observed with EBRT of equivalent cumulative dose. Further evaluation of the timing and magnitude of IFN1 response following EBRT and TRT may be critical to the optimal integration with immunotherapies. Citation Format: Justin C. Jagodinsky, Amber M. Bates, Reinier Hernandez, Joseph J. Grudzinski, Ian R. Marsh, Ishan Chakravarty, Ian S. Arthur, Luke M. Zangl, Ryan J. Brown, Erin J. Nystuen, Sarah E. Emma, Caroline Kerr, Won Jong Jin, Peter M. Carlson, Jonathan W. Engle, Eduardo Aluicio-Sarduy, Todd E. Barnhart, Trang Le, KyungMann Kim, Bryan P. Bednarz, Jamey P. Weichert, Ravi B. Patel, Zachary S. Morris. Temporal analysis of type 1 interferon activation in tumor cells following external beam radiotherapy or targeted radionuclide therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3060.

Notch signaling plays a central role in various cell fate decisions, including skeletal development. Recently, Notch signaling was implicated in osteoclast differentiation and maturation, including the resorption activity of osteoclasts. However, the specific involvement of notch signaling in resorption activity was not fully investigated. Here, we investigated the roles of Notch signaling in the resorption activity of osteoclasts by use of the gamma-secretase inhibitor dibenzazepine (DBZ). Attenuating Notch signaling by DBZ suppressed the expression of NFATc1, a master transcription factor for osteoclast differentiation. However, overexpression of a constitutively active form of NFATc1 did not fully rescue the effects of DBZ. DBZ suppressed the autophosphorylation of PYK2, which is essential for the formation of the podosome belt and sealing zone, with reduced c-Src/PYK2 interaction. We found that RANKL increases PYK2 activation accompanied by increased NICD2 production in osteoclasts. Overexpression of NICD2 in osteoclasts rescued DBZ-mediated suppression of resorption activity with promotion of PYK2 autophosphorylation and microtubule acetylation. Consistent with the in vitro results, DBZ strongly suppressed bone destruction in an interleukin-1-induced bone loss model. Collectively, these results demonstrate that Notch2 in osteoclasts plays a role in the control of resorption activity via the PYK2-c-Src-microtubule signaling pathway.

Haptoglobin (Hp), a type of acute-phase protein, is known to have a systemic anti-inflammatory function and to modulate inflammation by directly affecting immune cells, such as T cells, dendritic cells, and macrophages. However, the effects of Hp on osteoclast differentiation are not well studied, even though osteoclast precursor cells belong to a macrophage-monocyte lineage. In this study, we found that the bone volume was reduced, and the number of osteoclasts was increased in Hp-deficient mice compared with wild-type mice. Moreover, our in vitro studies showed that Hp inhibits osteoclastogenesis by reducing the protein level of c-Fos at the early phase of osteoclast differentiation. We revealed that Hp-induced suppression of c-Fos was mediated by increased IFN-β levels. Furthermore, Hp stimulated IFN-β via a TLR4-dependent mechanism. These results demonstrate that Hp plays a protective role against excessive osteoclastogenesis via the Hp–TLR4–IFN-β axis.

Platelet-derived growth factor receptor (PDGFR) signaling has been shown to inhibit osteogenesis. However, therapeutic efficacy of inhibiting PDGF signaling to bone regeneration in vivo and the specific mechanisms by which PDGFR signaling inhibits osteogenic differentiation remain unclear. In the present study, we examined the osteogenic effect of inhibiting PDGFR using trapidil, a PDGFR antagonist, in vivo and in vitro, and evaluated its mechanisms. A rat calvarial defect model was analyzed by micro-computed tomography and histology to determine the pro-osteogenic effect of trapidil in vivo. In addition, primary mouse calvarial osteoblast precursors were cultured in osteogenic differentiation medium with trapidil to study the mechanisms. Trapidil greatly promoted bone regeneration in a rat calvarial defect model and osteogenic differentiation of calvarial osteoblast precursors. For the mechanisms, trapidil induced phosphorylation of Smad1/5/9 and mitogen-activated protein kinase (MAPK) leading to enhance expression of Runx2, crucial transcription factor for osteogenesis. The pro-osteogenic effects of trapidil were inhibited by LDN193189, specific inhibitor of bone morphogenetic protein (BMP) receptor, ALK2 and ALK3, and by depletion of ALK3, and treatment with noggin, an antagonist of BMPs. Moreover, trapidil showed a synergistic effect with BMP2 on osteogenic differentiation. In conclusion, trapidil induced BMPR activity through upregulation of BMP signaling, leading to promoted osteogenesis in vitro and in vivo. Attenuated BMPR activity may be involved in the inhibition of osteogenesis by PDGFR signaling.

Cytoskeletal changes in osteoclasts such as formation of actin ring is required for bone-resorbing activity. The tyrosine kinase Src is a key player in massive cytoskeletal change of osteoclasts, thereby in bone destruction. In order for Src to be activated, trafficking to the inner plasma membrane via myristoylation is of importance. A previous study reported that myristoleic acid derived from myristic acid, inhibited N-myristoyl-transferase, an essential enzyme for myristoylation process. This prompted us to investigate whether myristoleic acid could affect osteoclastogenesis. Indeed, we observed that myristoleic acid inhibited RANKL-induced osteoclast formation in vitro, especially, at later stages of differentiation. Myristoleic acid attenuated the tyrosine phosphorylation of c-Src and Pyk2, which associates with Src, by RANKL. When myristoleic acid was co-administered with soluble RANKL into mice, RANKL-induced bone loss was substantially prevented. Bone dissection clearly revealed that the number of multinucleated osteoclasts was significantly diminished by myristoleic acid. On the other hand, myristoleic acid treatment had little or no influence on early osteoclast differentiation markers, such as c-Fos and NFATc1, and proteins related to cytoskeletal rearrangement, including DC-STAMP, integrin αv and integrin β3 in vitro. Taken together, our data suggest that myristoleic acid is capable of blocking the formation of large multinucleated osteoclasts and bone resorption likely through suppressing activation of Src and Pyk2.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that is characterized by uncontrolled joint inflammation and destruction of bone and cartilage. Previous studies have shown that C-X-C motif chemokine 10 (CXCL10) has important roles in RA development and that blocking CXCL10 expression effectively inhibits arthritis progression in animal models. However, clinical study using anti-CXCL10 monoclonal antibody (MDX-1100) to block CXCL10 expression in patients with RA did not show significant effectiveness. Therefore, we turned our attention to C-X-C motif chemokine receptor 3 (CXCR3), which is a receptor for CXCL9, CXCL10, and CXCL11, to treat RA. In the present study, administration of JN-2, our newly developed CXCR3 antagonist, ameliorated the progression of arthritis in a collagen-induced arthritis animal model. JN-2 also inhibited CXCR3-induced cell migration and pro-inflammatory cytokine expression of bone marrow–derived macrophages and CD4+ T cells in vitro. In addition, we found that CXCL10 formed an auto-amplification loop through activation of NFκB. Furthermore, Phosphorylation of p65 at serine 536 played an important role in the auto-amplification of CXCL10. Overall, the present results demonstrated that JN-2 decreased inflammation by inhibiting CXCR3-enhanced cell migration and pro-inflammatory cytokine expression, which then ameliorated arthritis progression.

The chemokine CXCL10 and its receptor CXCR3 play a role in breast cancer metastasis to bone and osteoclast activation. However, the mechanism of CXCL10/CXCR3-induced intracellular signaling has not been fully investigated. To evaluate CXCL10-induced cellular events in the mouse breast cancer cell line 4T1, we developed a new synthetic CXCR3 antagonist JN-2. In this study, we observed that secretion of CXCL10 in the supernatant of 4T1 cells was gradually increased during cell growth. JN-2 inhibited basal and CXCL10-induced CXCL10 expression and cell motility in 4T1 cells. Treatment of 4T1 cells with CXCL10 increased the expression of P65, a subunit of the NF-κB pathway, via activation of the NF-κB transcriptional activity. Ectopic overexpression of P65 increased CXCL10 secretion and blunted JN-2-induced suppression of CXCL10 secretion, whereas overexpression of IκBα suppressed CXCL10 secretion. These results indicate that the CXCL10/CXCR3 axis creates a positive feedback loop through the canonical NF-κB signaling pathway in 4T1 cells. In addition, treatment of osteoblasts with conditioned medium from JN-2-treated 4T1 cells inhibited the expression of RANKL, a crucial cytokine for osteoclast differentiation, which resulted in an inhibitory effect on osteoclast differentiation in the co-culture system of bone marrow-derived macrophages and osteoblasts. Direct intrafemoral injection of 4T1 cells induced severe bone destruction; however, this effect was suppressed by the CXCR3 antagonist via downregulation of P65 expression in an animal model. Collectively, these results suggest that the CXCL10/CXCR3-mediated NF-κB signaling pathway plays a role in the control of autonomous regulation of CXCL10 and malignant tumor properties in breast cancer 4T1 cells.

Histone deacetylase (HDAC) enzymes play important roles in physiological and pathological processes by catalyzing the deacetylation of lysine residues in histone and non-histone proteins. Inhibition of HDACs has emerged as an attractive therapeutic strategy for various diseases including cancer and inflammatory diseases. We recently found that MS-275, a class I-specific HDAC inhibitor, exhibits an anabolic effect on bone through promoting expression of alkaline phosphatase in osteoblasts. MS-275 has also been suggested to inhibit inflammatory bone destruction, but the underlying mechanisms are still poorly understood. In this study, we investigated the effects and mechanism of action of MS-275 on osteoclast differentiation and activation. We found that MS-275 inhibits osteoclast differentiation in coculture of osteoblasts and bone marrow cells without affecting expression of receptor activator of NF-κB ligand (RANKL), a key cytokine for osteoclast differentiation, in osteoblasts. MS-275 inhibited RANKL-mediated osteoclast differentiation from its precursors by suppressing RANKL-induced expression of c-Fos, a crucial transcription factor for osteoclastogenesis. The inhibitory effect of MS-275 on osteoclast differentiation was blunted by ectopic overexpression of c-Fos. In addition to osteoclast differentiation, MS-275 decreased bone resorbing activity of mature osteoclasts. Consistent with the in vitro effects, MS-275 decreased osteoclast number and bone destruction in IL-1-induced mouse calvarial bone destruction model. Taken together, our results demonstrate that MS-275 suppresses bone destruction by inhibiting osteoclast differentiation and activation, suggesting a potential therapeutic value of MS-275 for bone disorders associated with increased bone resorption.

Bone is a preferred site of metastasis from breast cancer, and increased osteoclast activity is implicated in breast cancer outgrowth in the bone microenvironment. Our previous observation of an anti-osteoclastic activity of Trolox, a vitamin E analog, led us to investigate whether Trolox inhibits bone metastasis and osteolysis caused by breast cancer. Administration of Trolox markedly inhibited osteolytic bone metastasis in an experimental metastasis model by intracardiac injection of 4T1 breast cancer cells. Trolox inhibited proliferation of 4T1 cells in the bone marrow but not in the mammary fat pad. In addition, Trolox could reduce tumor burden, osteolysis, and prostaglandin E2 (PGE2) production induced by direct inoculation of 4T1 cells into the marrow cavity of the tibia. Trolox decreased the migratory and invasive activities of 4T1 cells via PGE2-dependent and independent mechanisms. It also inhibited the ability of 4T1 cells to stimulate the expression of receptor activator of nuclear factor-κB ligand (RANKL), a key cytokine for osteoclast differentiation factor, in osteoblasts. In addition, Trolox suppressed RANKL expression in osteoblasts induced by soluble factors from 4T1 cells. Furthermore, Trolox suppressed 4T1 cell-induced osteoclast differentiation in the co-culture of bone marrow cells and osteoblasts via both PGE2-dependent and independent mechanisms. Taken together, these results suggest that Trolox inhibits breast cancer cell-induced osteoclast differentiation and the invasive behavior of cancer cells through PGE2-dependent and independent mechanisms, thereby suppressing osteolytic bone metastasis of breast cancer.

Trapidil, a platelet-derived growth factor antagonist, was originally developed as a vasodilator and anti-platelet agent and has been used to treat patients with ischemic coronary heart, liver, and kidney disease. In this study, we investigated the effects of trapidil on osteoclastogenesis and elucidated the possible mechanism of action of trapidil. Trapidil strongly inhibited osteoclast formation in co-cultures of bone marrow cells and osteoblasts without affecting receptor activator of NF-κB ligand (RANKL) or osteoprotegerin expression in osteoblasts. In addition, trapidil suppressed RANKL-induced osteoclast formation from osteoclast precursors. Trapidil reduced RANKL-induced expression of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), a master transcription factor for osteoclastogenesis, without affecting the expression of c-Fos that functions as a key upstream activator of NFATc1 during osteoclastogenesis. Ectopic expression of a constitutively active form of NFATc1 reversed the anti-osteoclastogenic effect of trapidil, indicating that NFATc1 is a critical target of the anti-osteoclastogenic action of trapidil. RANKL-induced calcium oscillation and Pim-1 expression, which are required for NFATc1 induction and osteoclastogenesis, were abrogated by trapidil. Consistent with the in vitro results, trapidil had a potent inhibitory effect on osteoclast formation and bone resorption induced by interleukin-1 in an animal model. Taken together, our data demonstrate that trapidil abrogates RANKL-induced calcium oscillation and Pim-1 expression required for NFATc1 induction, thereby inhibiting osteoclastogenesis.

Amplification of the chemokines CXCL10 and RANKL has been suggested to promote osteoclast differentiation and osteolytic bone metastasis, but a function for endogenous CXCL10 in these processes is not well established. In this study, we show that endogenous CXCL10 is critical to recruit cancer cells to bone, support osteoclast differentiation and promote for the formation of osteolytic bone metastases. Neutralizing CXCL10 antibody reduced migration of cancer cells expressing the CXCL10 receptor CXCR3, and loss of CXCR3 or CXCL10 decreased bone tumor burden in vivo. Bone colonization augmented host production of CXCL10, which was required for cancer growth and subsequent osteolysis. Direct interactions between cancer cells and macrophages further stimulated CXCL10 production from macrophages. Growth of bone metastases required CXCL10-stimulated adhesion of cancer cells to type I collagen as well as RANKL-mediated osteoclast formation. Together, our findings show that CXCL10 facilitates trafficking of CXCR3-expressing cancer cells to bone, which augments its own production and promotes osteoclastic differentiation. CXCL10 therefore may represent a therapeutic target for osteolytic bone metastasis. Cancer Res; 72(13); 3175–86. ©2012 AACR.

Objective Osteoclasts are bone-resorbing multinucleated cells derived from the monocyte/macrophage lineage during normal and pathological bone turnover. Recently, several studies revealed that alpha-tocopheryl succinate (αTP-suc) have demonstrated potent anti-cancer activities in vitro and in vivo. However, the effects of αTP-suc on osteoclast formation and bone resorption remain unknown. Thus, in this study, we examined the effects of αTP-suc on osteoclast differentiation and bone resorbing activity in inflammatory bone loss model. Methods Osteoclast differentiation assay was performed by cocultures of mouse bone marrow cells and calvarial osteoblasts in culture media including interleukin-1 (IL-1). Osteoclasts were stained for tartrate-resistant acid phosphatase (TRAP). The level of receptor activator of nuclear factor-kappaB ligand (RANKL) mRNA was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). ICR mice were administered an intraperitoneal injections of αTP-suc or dimethyl sulfoxide (DMSO) 1 day before the implantation of a freeze-dried collagen sponge loaded with phosphate-buffered saline (PBS) or IL-1 over the calvariae and every other day for 7 days. The whole calvariae were obtained and analyzed by micro-computed tomography (CT) scanning, and stained for TRAP. Results αTP-suc inhibits osteoclast formation in cocultures stimulated by IL-1 and decreased the level of expression of RANKL mRNA in osteoblasts. In addition, administered intraperitoneal injections of αTP-suc prevented IL-1-mediated osteoclast formation and bone loss in vivo. Conclusion Our findings suggest that αTP-suc may have therapeutic value for treating and preventing bone-resorptive diseases, such as osteoporosis.