Jingyu Xiang

Hedgehog signaling inhibition blocks growth of resistant tumors through effects on tumor microenvironment.

Hedgehog (Hh) signaling is implicated in bone development and cellular transformation. Here we show that inhibition of Hh pathway activity inhibits tumor growth through effects on the microenvironment. Pharmacologic inhibition of the Hh effector Smoothened (Smo) increased trabecular bone in vivo and inhibited osteoclastogenesis in vitro. In addition, enhanced Hh signaling due to heterozygosity of the Hh inhibitory receptor Patched (Ptch1(+/-)) increased bone resorption, suggesting direct regulation of osteoclast (OC) activity by the Hh pathway. Ptch1(+/-) mice had increased bone metastatic and subcutaneous tumor growth, suggesting that increased Hh activation in host cells promoted tumor growth. Subcutaneous growth of Hh-resistant tumor cells was inhibited by LDE225, a novel orally bioavailable SMO antagonist, consistent with effects on tumor microenvironment. Knockdown of the Hh ligand Sonic Hh (SHH) in these cells decreased subcutaneous tumor growth and decreased stromal cell production of interleukin-6, indicating that tumor-derived Hh ligands stimulated tumor growth in a paracrine fashion. Together our findings show that inhibition of the Hh pathway can reduce tumor burden, regardless of tumor Hh responsiveness, through effects on tumor cells, OCs, and stromal cells within the tumor microenvironment. Hh may be a promising therapeutic target for solid cancers and bone metastases.

The ADP receptor P2RY12 regulates osteoclast function and pathologic bone remodeling

The adenosine diphosphate (ADP) receptor P2RY12 (purinergic receptor P2Y, G protein coupled, 12) plays a critical role in platelet aggregation, and P2RY12 inhibitors are used clinically to prevent cardiac and cerebral thrombotic events. Extracellular ADP has also been shown to increase osteoclast (OC) activity, but the role of P2RY12 in OC biology is unknown. Here, we examined the role of mouse P2RY12 in OC function. Mice lacking P2ry12 had decreased OC activity and were partially protected from age-associated bone loss. P2ry12-/- OCs exhibited intact differentiation markers, but diminished resorptive function. Extracellular ADP enhanced OC adhesion and resorptive activity of WT, but not P2ry12-/-, OCs. In platelets, ADP stimulation of P2RY12 resulted in GTPase Ras-related protein (RAP1) activation and subsequent αIIbβ3 integrin activation. Likewise, we found that ADP stimulation induced RAP1 activation in WT and integrin β3 gene knockout (Itgb3-/-) OCs, but its effects were substantially blunted in P2ry12-/- OCs. In vivo, P2ry12-/- mice were partially protected from pathologic bone loss associated with serum transfer arthritis, tumor growth in bone, and ovariectomy-induced osteoporosis: all conditions associated with increased extracellular ADP. Finally, mice treated with the clinical inhibitor of P2RY12, clopidogrel, were protected from pathologic osteolysis. These results demonstrate that P2RY12 is the primary ADP receptor in OCs and suggest that P2RY12 inhibition is a potential therapeutic target for pathologic bone loss.

Noncompetitive modulation of the proteasome by imidazoline scaffolds overcomes bortezomib resistance and delays mm tumor growth in vivo

Multiple myeloma (MM) is a malignant disorder of differentiated B-cells for which standard care involves the inhibition of the proteasome. All clinically used proteasome inhibitors, including the chemotherapeutic drug bortezomib, target the catalytic active sites of the proteasome and inhibit protein proteolysis by competing with substrate binding. However, nearly all (~97%) patients become intolerant or resistant to treatments within a few years, after which the average survival time is less than 1 year. We describe herein the inhibition of the human proteasome via a noncompetitive mechanism by the imidazoline scaffold, TCH-13. Consistent with a mechanism distinct from that of competitive inhibitors, TCH-013 acts additively with and overcomes resistance to bortezomib. Importantly, TCH-013 induces apoptosis in a panel of myeloma and leukemia cell lines, but in contrast, normal lymphocytes, primary bone marrow stromal cells (hBMSC), and macrophages are resistant to its cytotoxic effects. TCH-013 was equally effective in blocking MM cell growth in co-cultures of MM cells with hBMSC isolated from CD138 negative bone marrow (BM) samples of MM patients. The cellular activity translated well in vivo where TCH-013 delayed tumor growth in an MM xenograft model to a similar extent as bortezomib.

Antagonizing Integrin β3 Increases Immunosuppression in Cancer.

Integrin β3 is critical for tumor invasion, neoangiogenesis, and inflammation, making it a promising cancer target. However, preclinical and clinical data of integrin β3 antagonists have demonstrated no benefit or worse outcomes. We hypothesized that integrin β3 could affect tumor immunity and evaluated tumors in mice with deletion of integrin β3 in macrophage lineage cells (β3KOM). β3KOM mice had increased melanoma and breast cancer growth with increased tumor-promoting M2 macrophages and decreased CD8(+) T cells. Integrin β3 antagonist, cilengitide, also enhanced tumor growth and increased M2 function. We uncovered a negative feedback loop in M2 myeloid cells, wherein integrin β3 signaling favored STAT1 activation, an M1-polarizing signal, and suppressed M2-polarizing STAT6 activation. Finally, disruption of CD8(+) T cells, macrophages, or macrophage integrin β3 signaling blocked the tumor-promoting effects of integrin β3 antagonism. These results suggest that effects of integrin β3 therapies on immune cells should be considered to improve outcomes. Cancer Res; 76(12); 3484-95. ©2016 AACR.

CXCR4 Protein Epitope Mimetic Antagonist POL5551 Disrupts Metastasis and Enhances Chemotherapy Effect in Triple-Negative Breast Cancer

The SDF-1 receptor CXCR4 has been associated with early metastasis and poorer prognosis in breast cancers, especially the most aggressive triple-negative subtype. In line with previous reports, we found that tumoral CXCR4 expression in patients with locally advanced breast cancer was associated with increased metastases and rapid tumor progression. Moreover, high CXCR4 expression identified a group of bone marrow–disseminated tumor cells (DTC)-negative patients at high risk for metastasis and death. The protein epitope mimetic (PEM) POL5551, a novel CXCR4 antagonist, inhibited binding of SDF-1 to CXCR4, had no direct effects on tumor cell viability, but reduced migration of breast cancer cells in vitro. In two orthotopic models of triple-negative breast cancer, POL5551 had little inhibitory effect on primary tumor growth, but significantly reduced distant metastasis. When combined with eribulin, a chemotherapeutic microtubule inhibitor, POL5551 additively reduced metastasis and prolonged survival in mice after resection of the primary tumor compared with single-agent eribulin. Hypothesizing that POL5551 may mobilize tumor cells from their microenvironment and sensitize them to chemotherapy, we used a “chemotherapy framing” dosing strategy. When administered shortly before and after eribulin treatment, three doses of POL5551 with eribulin reduced bone and liver tumor burden more effectively than chemotherapy alone. These data suggest that sequenced administration of CXCR4 antagonists with cytotoxic chemotherapy synergize to reduce distant metastases. Mol Cancer Ther; 14(11); 2473–85. ©2015 AACR.

HTLV-1 viral oncogene HBZ induces osteolytic bone disease in transgenic mice

Adult T-cell leukemia/lymphoma (ATL) is an aggressive T cell malignancy that occurs in HTLV-1 infected patients. Most ATL patients develop osteolytic lesions and hypercalcemia of malignancy, causing severe skeletal related complications and reduced overall survival. The HTLV-1 virus encodes 2 viral oncogenes, Tax and HBZ. Tax, a transcriptional activator, is critical to ATL development, and has been implicated in pathologic osteolysis. HBZ, HTLV-1 basic leucine zipper transcription factor, promotes tumor cell proliferation and disrupts Wnt pathway modulators; however, its role in ATL induced osteolytic bone loss is unknown. To determine if HBZ is sufficient for the development of bone loss, we established a transgenic Granzyme B HBZ (Gzmb-HBZ) mouse model. Lymphoproliferative disease including tumors, enlarged spleens and/or abnormal white cell counts developed in two-thirds of Gzmb-HBZ mice at 18 months. HBZ positive cells were detected in tumors, spleen and bone marrow. Importantly, pathologic bone loss and hypercalcemia were present at 18 months. Bone-acting factors were present in serum and RANKL, PTHrP and DKK1, key mediators of hypercalcemia and bone loss, were upregulated in Gzmb-HBZ T cells. These data demonstrate that Gzmb-HBZ mice model ATL bone disease and express factors that are current therapeutic targets for metastatic and bone resident tumors.

Dual-therapy with αvβ3-targeted Sn2 lipase-labile fumagillin-prodrug nanoparticles and zoledronic acid in the Vx2 rabbit tumor model

Fumagillin, an unstable anti-angiogenesis mycotoxin, was synthesized into a stable lipase-labile prodrug and incorporated into integrin-targeted lipid-encapsulated nanoparticles (αvβ3-Fum-PD NP). Dual anti-angiogenic therapy combining αvβ3-Fum-PD NP with zoledronic acid (ZA), a long-acting osteoclast inhibitor with proposed anti-angiogenic effects, was evaluated. In vitro, αvβ3-Fum-PD NP reduced (P<0.05) endothelial cell viability without impacting macrophage viability. ZA suppressed (P<0.05) macrophage viability at high dosages but not endothelial cell proliferation. 3D MR neovascular imaging of rabbit Vx2 tumors showed no effect with ZA, whereas αvβ3-Fum-PD NP alone and with ZA decreased angiogenesis (P<0.05). Immunohistochemistry revealed decreased (P<0.05) microvascularity with αvβ3-Fum-PD NP and ZA and further microvascular reduction (P<0.05) with dual-therapy. In vivo, ZA did not decrease tumor macrophage numbers nor cancer cell proliferation, whereas αvβ3-Fum-PD-NPs reduced both measures. Dual-therapy with ZA and αvβ3-Fum-PD-NP may provide enhanced neo-adjuvant utility if macrophage ZA uptake is increased. From the Clinical Editor: Although anti-angiogenesis is one of the treatment modalities in the fight against cancer, many cancers become resistant to VEGF pathway inhibitors. In this article, the authors investigated the use of dual therapy using fumagillin, integrin-targeted lipid-encapsulated nanoparticles (αvβ3- Fum-PD NP) and zoledronic acid (ZA), in both in-vitro and in-vivo experiments. This combination approach may provide an insight to the design of future drugs against cancers.

Evaluating acetate metabolism for imaging and targeting in multiple myeloma.

Purpose: We hypothesized that in multiple myeloma cells (MMC), high membrane biosynthesis will induce acetate uptake in vitro and in vivo. Here, we studied acetate metabolism and targeting in MMC in vitro and tested the efficacy of 11C-acetate–positron emission tomography (PET) to detect and quantitatively image myeloma treatment response in vivo. Experimental design: Acetate fate tracking using 13C-edited-1H NMR (nuclear magnetic resonance) was performed to study in vitro acetate uptake and metabolism in MMC. Effects of pharmacological modulation of acetate transport or acetate incorporation into lipids on MMC cell survival and viability were assessed. Preclinical mouse MM models of subcutaneous and bone tumors were evaluated using 11C-acetate-PET/CT imaging and tissue biodistribution. Results: In vitro, NMR showed significant uptake of acetate by MMC and acetate incorporation into intracellular metabolites and membrane lipids. Inhibition of lipid synthesis and acetate transport was toxic to MMC, while sparing resident bone cells or normal B cells. In vivo, 11C-acetate uptake by PET imaging was significantly enhanced in subcutaneous and bone MMC tumors compared with unaffected bone or muscle tissue. Likewise, 11C-acetate uptake was significantly reduced in MM tumors after treatment. Conclusions: Uptake of acetate from the extracellular environment was enhanced in MMC and was critical to cellular viability. 11C-Acetate–PET detected the presence of myeloma cells in vivo, including uptake in intramedullary bone disease. 11C-Acetate–PET also detected response to therapy in vivo. Our data suggested that acetate metabolism and incorporation into lipids was crucial to MM cell biology and that 11C-acetate–PET is a promising imaging modality for MM. Clin Cancer Res; 23(2); 416–29. ©2016 AACR.

Abstract 1114: Combination of a novel CXCR4 antagonist with chemotherapy reduces breast cancer bone metastatic tumor burden

Background: Bone is the most common site of metastasis for patients with breast cancer. Tumor cells migrate to and reside in the protective bone marrow microenvironment niche through adhesive interaction between tumor CXCR4 and stromal CXCL12 (SDF1). CXCL12 is produced by activated osteoblasts, bone marrow and lung stromal cells, and endothelial cells. Nearly 60% of breast cancers express CXCR4 and this is associated with decreased survival. We hypothesized that a Protein Epitope Mimetic (PEM) POL5551, a novel CXCR4 antagonist, will limit the extent of tumor metastasis by disrupting stromal-mediated protection from cytotoxic chemotherapy and in turn may prolong survival. Approach: In vitro, POL5551 had no direct cytotoxic activity and did not reduce proliferation of CXCR4+ MDA-MB-231 or 4T1 osteolytic breast cancer cell lines. However, in an in vitro scratch-wound assay POL5551 inhibited migration of MDA-MB-231 cells. In a Gaussia luciferase (GLuc) complementation model in MDA-MB-231 cells, the interaction of CXCL12 with CXCR4, but not CXCR7, was blocked by low nanomolar concentrations of POL5551. At 20 mg/kg administered from day 10 post inoculation, POL5551 displayed no single agent activity on primary tumor xenografts, and in combination with eribulin there was no synergistic effect on primary tumor xenografts. However, continuation of treatment with POL5551 after surgical tumor removal decreased tumor metastasis and prolonged survival to 58 days compared to control at 45 days and eribulin alone at 51 days. Also, POL5551 showed effects on immune infiltration to the primary tumor. To test the hypothesis that CXCR4 antagonism disrupts the protective bone marrow niche in which tumor cells reside and sensitizes them to cytotoxic chemotherapy, we employed a “framing dosing strategy”. In a second model of metastasis produced by intracardiac injection of MDA-MB-231 cells, POL5551 (20 mg/kg, s.c.) was administered to mice with bone metastases 4 hours before and 4 & 18 hours after eribulin chemotherapy (0.2 mg/kg, i.v.). While bone is the predominant site of metastasis with these cell lines, lung and liver metastasis also occurs. POL5551 in combination with eribulin decreased bone tumor burden more than eribulin alone (reduction in leg bone & liver tumor burden, versus control respectively: eribulin alone 58% & 75%, n.s.; POL5551+eribulin: 89% & 86%, P Conclusion: These preclinical data support a synergism of CXCR4 antagonism during chemotherapy for the treatment of metastatic breast cancer. These and additional data support initiation of a Phase I clinical trial to evaluate a related PEM CXCR4 antagonist in combination with eribulin chemotherapy in patients with metastatic breast cancer who have failed at least 2 lines of therapy. Note: This abstract was not presented at the meeting. Citation Format: Jingyu Xiang, Michelle A. Hurchla, Kathryn Luker, Garry Douglas, Barbara Romagnoli, Eric Chevalier, Michael Bauer, Johann Zimmermann, Klaus Dembowsky, Gary Luker, Katherine N. Weilbaecher. Combination of a novel CXCR4 antagonist with chemotherapy reduces breast cancer bone metastatic tumor burden. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1114. doi:10.1158/1538-7445.AM2014-1114

Abstract 3285: HTLV-1 viral oncogene Hbz induces leukemia with osteolytic bone involvement in mice

Adult T-cell leukemia/lymphoma (ATL) develops in a subset of patients infected with the HTLV-1 virus. Most ATL patients become refractory to chemotherapy and have a median survival time of 6 months. Although uncommon in hematologic malignancies, 80% of ATL patients develop osteolytic lesions and hypercalcemia of malignancy. Bone resident and metastatic tumors release paracrine factors that modulate the bone microenvironment to facilitate disease progression and decrease survival. HTLV-1 encodes 2 viral oncogenes, Tax and Hbz. Tax is critical to ATL development and regulates tumor growth and proliferation in part through trans-activation of NFκB and CREB. We have previously shown Tax expression driven by the Granzymbe B promoter is sufficient for the development of leukemia/lymphoma with osteolytic lesions and hypercalcemia. We and others have shown that Tax alters the expression of paracrine factors that modulate the bone microenvironment through effects on bone forming osteoblasts (OB) and bone resorping osteoclasts (OC). Tax is expressed in early lymphocyte transformation with low expression in advanced ATL. HBZ is expressed early in lymphocyte transformation and throughout ATL progression. We hypothesize that in ATL cells, HTLV-1 viral oncogenes Tax and Hbz cooperate to modulate bone metabolism in a paracrine manner to enhance ATL tumor growth and progression. Mice with Granzyme B driven Hbz expression (T and NK cells) develop leukemia/lymphoproliferative disease in lymph nodes correlating with increased spleen weight. We found that lymphoproliferative disease is also present in the bone marrow. Hbz mice have decreased trabecular bone at 18 months by microCT and radiographic analysis. These data suggest Hbz can alter bone metabolism. Future studies will define the effects of Hbz on bone formation, OB and OC specific effects and tumor progression. Understanding HTLV-1 oncogene modulation of the bone microenvironment will uncover critical pathways in tumor/bone cross talk enabling the development of novel targeted therapies for ATL patients. Citation Format: Alison Esser, Dan Rauch, Jingyu Xiang, John Harding, Nicole Kohart, Patrick Green, Stefan Niewiesk, Thomas Rosol, Lee Ratner, Katherine Weilbaecher. HTLV-1 viral oncogene Hbz induces leukemia with osteolytic bone involvement in mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3285.