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Clinical Significance: Despite initial response to therapies, patients with triple negative breast cancer (TNBC) have the highest risk of metastatic relapse within 5 years of diagnosis. There is an urgent need for better treatments for this deadly disease. To this end, understanding the molecular drivers can reveal novel therapeutic targets. Background: The TNBC oncogenic program is known to be driven by MYC. MYC expression is regulated by bromodomain proteins and several bromodomain inhibitors such as JQ1 have proven effective at inhibiting MYC expression. However, resistance can occur rapidly upon activation of alternative cell survival mechanisms, such as autophagy. TNBC is noted for its elevated basal autophagy. Interestingly, publicly available datasets show that ULK3, an initial effector of the autophagy program, is upregulated in TNBC patients while the opposite is observed for the classical autophagy initiator ULK1. Moreover, TNBC patients with low ULK3 expression levels tend to achieve longer relapse free survival (RFS) than higher expression cohort, in opposition to ULK1. Importantly, ULK3 has not been investigated to date in regulating TNBC cell intrinsic autophagy and no drugs exist that can inhibit ULK3. Methods and Results: Given the importance of MYC and ULK3 in TNBC progression, our team developed a novel class of small molecules, namely dual BRD4/ULK3 inhibitors, that are superior to JQ1 alone in limiting TNBC viability. In vitro data demonstrate that TNBC cells are more sensitive to the cytotoxic activity of the dual inhibitors compared to other breast cancer subtypes. Further investigation through western blot analysis confirms the effectiveness of these novel dual inhibitors in abrogating MYC expression overtime, and completely blocking the autophagy program (measured by downstream ULK3, ULK1, LC3B, p62 protein levels), resulting in cell death. Interestingly, the human cell line SUM159R, that is resistant to BRD4 inhibitor JQ1 and cross-resistant to standard chemotherapeutics (doxorubicin) is also highly sensitive to our dual inhibitors, implying their potential use for the treatment of refractory TNBC disease. Excitingly, and for the first time, using a genetic silencing approach (shULK3 in SUM159), we demonstrated that ULK3 is critical for TNBC autophagy and cell survival. As a proof of concept, we reproduced the same genetic silencing experiment in a different cell line, namely WM1366-LC3BmCherry-GFP, an LC3B conveniently engineered system used to visualize proper autophagosome formation and material degradation through autophagy. We verified that both genetic ablation and pharmacologic inhibition of ULK3 are strategies that lead to cancer cell death. Conclusions: In conclusion, we 1) characterized a novel class of dual BRD4/kinase compounds, capable of inhibiting BRD4 activity and autophagy, and superior to the BRD4 inhibitor, JQ1. 2) showed that ULK3 is over-expressed in TNBC. 3) demonstrated that silencing ULK3 blocks autophagy in TNBC cell lines and results in significantly lower cell viability. 4) found that our dual inhibitors present a double bind for TNBC cells that results in increased cytotoxicity and potentially will be useful for the treatment of resistant TNBC. Clinically, we also posit that our dual inhibitors could be easily administered as a single agent, avoiding the potential complication of pharmacokinetics/pharmacodynamics associated with administering multiple therapies. Citation Format: Marilena Tauro, Tao Li, Conor C Lynch. Dual epigenetic/autophagy inhibition as a novel strategy to tackle triple negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD3-10.

Background: Metastatic castrate resistant prostate cancer (mCRPC) is frequently manifested in the bone, leading to increased morbidity and mortality. We have previously demonstrated that γδ Chimeric Antigen Receptor (CAR) T cells targeting prostate stem cell antigen (PSCA) led to significant regression of established prostate cancer cells in the bone. Regression was further increased by combination with the bisphosphonate zoledronate (ZOL), routinely administered to mCRPC patients to prevent bone loss. To further optimize the use of γδ CAR T cells for mCRPC, we aimed to determine the kinetics of γδ CAR T cell in a mouse model of bone metastatic prostate cancer, either as single treatment or in combination with ZOL. In addition, we identified the sites of systemic ZOL accumulation in an immune competent model that could potentially induce off-target effects of γδ CAR T cells. Methods: Male NSG mice were injected with C4-2B prostate cancer cells expressing PSCA and luciferase in the left tibia, while the right tibia received PBS. ZOL (25 μg/kg) was injected every other day subcutaneously in half of the mice and was discontinued one day prior to administering T cells. When tumors were established, mice received γδ PSCA-CAR T cells, γδ untransduced (UT) T cells or were left untreated. Bone marrow from tumor-bearing tibia, tumor-naive tibia, femur, spleen and blood were recovered at multiple time points, and the number of γδ T cells was analyzed by flow cytometry. To determine systemic ZOL uptake, C57BL/6 mice were injected with PTE-82 prostate cell line in both tibias. One week after injection, mice received ZOL-AF647 every other day for 2 weeks. Bone marrow, liver, kidney, peritoneal lavage, skin-draining lymph nodes, Peyer’s patches and spleen were recovered and analyzed by flow cytometry. Results: γδ CAR T cells showed a rapid accumulation in the bone marrow recovered from tumor-bearing tibias, with 3 times more cells than those from mice treated with γδ UT cells (p=0.0002). The number of γδ T cells peaked at 5 days post infusion and were still detected after 21 days. Increased γδ CAR T cell accumulation was not observed in tumor-free bones or in spleen or blood, suggesting a preferential localization of γδ CAR T cells at tumor sites. Treatment with ZOL did not significantly affect the number or phenotype of γδ T cells accumulated in bone. Analysis of ZOL distribution showed significant uptake by macrophages, specially from liver and peritoneal lavage (p Conclusion: γδ PSCA-CAR T cells accumulate and get activated at tumor sites with limited distribution outside the bone, while their kinetics is not affected by ZOL treatment in the NSG xenograft model. Additional studies will be necessary to determine the impact of ZOL uptake by myeloid cells on γδ CAR T cells migration. Citation Format: Leticia Tordesillas, Junior Cianne, Jeremy S. Frieling, Xiomar Bustos, Conor C. Lynch, Daniel Abate-Daga. Biodistribution of zoledronate and effects on gd PSCA-CAR T cells in a model of bone metastatic prostate cancer [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 1767.

Recurrent metastatic prostate cancer (PC) typically manifests in the skeleton. Despite our knowledge that DTCs give rise to incurable bone metastatic PC, the molecular mechanisms underpinning their entry into dormancy and exit, remain under-investigated. To address this, we developed a novel in vitro model of PC dormancy as determined by increased entry into G0/G1 state (flowcytometry) and increased p38/ERK activity ratio & p27 (immunoblotting). Interestingly, we also observed dormancy entry resulted in the formation of clusters that remained dormant for long periods (21 days) and could be reawakened via serum addition. RNASeq and bioinformatic analysis of control vs. dormant cells revealed PRDM16 to be significantly elevated during entry into dormancy across mouse (RM1) and human cell lines (LAPC4, 22RV1). In vitro, we observed that genetic silencing of PRDM16 leads to a significant reduction in the ability of PC cells to enter dormancy. Analyses show that PRDM16 increases the expression of anti-apoptotic proteins including BCL-2 while inhibiting pro-apoptotic proteins such as BIM and NOXA. Our preliminary data shows that PRDM16 expression in CRPC cells can be induced by BMP7 and conversely decreased by the BMP signaling antagonist, noggin, factors with known critical roles in regulating bone homeostasis. In vivo, we used intrailiac-artery technique to deliver actively growing RM1 cells or dormant RM1 clusters (D-RM1) to the hind limbs (n = 8) of syngeneic mice (C57BL/6). Bioluminescence shows that D-RM1 remains dormant for more than 30 days. Subsequent histological analyses show that D-RM1 home preferentially to the endosteal niche where, compared to RM1 controls, they remain largely negative for ki-67 and cleaved caspase 3 and exhibit high expression of PRDM16. In conclusion, our data points to PRDM16 as a key regulator of survival pathways during PC entry into dormancy. Citation Format: Mostafa Nasr, Tao Li, Ryan T. Bishop, Conor C. Lynch. PRDM16 is key for promoting bone metastatic prostate cancer cell survival during entry into dormancy [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 61.

Osteosarcoma (OS) frequently metastasizes to lung where it greatly contributes to patient mortality. Frustratingly, OS treatment options have remained static due to a paucity of strong pre-clinical evidence to motivate clinical trials. To identify therapeutic targets, we assessed a number of FDA approved therapies for their cytotoxic effects. We found that histone deacetylase (HDAC) inhibitors (panobinostat and romidepsin) limited OS viability at low nM and were highly effective for the treatment of lung metastatic disease in vivo. Although clinically approved, HDAC inhibitors have noted toxicities. Therefore, we next examined the potential mechanisms through which HDAC controlled OS growth. Gene expression analyses on romidepsin treated OS cells revealed key up and downregulated targets. We found that SLC17A7, a vesicular glutamate transporter, was highly and consistently induced in response to romidepsin (greater than 100 fold more than base line) across multiple OS cell lines. Functionally, SLC17A7 traps glutamate in cytoplasmic synaptic vesicles that are then released via exocytosis. Our data show that HDAC inhibition reduces glutamate secretion from OS cells while SLC17A7 overexpression significantly impaired OS viability. Further, we showed that the glutamate export inhibitor, riluzole, significantly impairs the OS growth in vitro and is highly effective for the treatment of lung metastatic OS in vivo. Taken together, we conclude that HDAC1/2 suppression of SLC17A7 ensures high cytoplasmic glutamate levels that can be leveraged for OS growth. We posit that targeting glutamate usage by OS cells using FDA approved inhibitors such as riluzole will be highly effective for the treatment of lung metastatic OS. Citation Format: Niveditha Nerlakanti, Jeremy McGuire, Ryan T. Bishop, Diana Yu, Damon R. Reed, Conor C. Lynch. Delineating the role of vesicular glutamate transporter SLC17A7 in osteosarcoma [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 276.

Multiple myeloma (MM) is an incurable disease. Classical chemotherapeutics including bortezomib, melphalan, lenalidomide and thalidomide have greatly enhanced survival times. Unfortunately, patients typically relapse and become refractory with an average survival of 5 years post-diagnosis. Our emerging studies demonstrate a novel role for ULK3 in regulating autophagy in MM, a key program that sustains cell survival under times of stress and has been implicated as a major mechanism of proteasome inhibitor (PI) resistance. MM is known to be highly dependent on autophagy and, currently, specific ULK3 inhibitors are lacking. We posit that by targeting this marker in chemotherapy resistant MM patients, we can circumvent alternative metabolic routes and resensitize to standard of care proapoptotic therapy. We performed RNASeq analysis of CD138+ MM cells derived from patients across the disease stages spectrum (n=815) to confirm the role of ULK3 in disease progression and resistance to chemotherapy. We developed novel inhibitors SG3014/MA9060 that target multiple kinases including ULK3 (EC50 90nM) as well as BRD4. BRD4 is a known driver of MYC and its expression is increased in refractory MM. The BRD4 inhibitor, JQ1, effectively impairs the tumorigenic potential of MM but resistance has also been noted. We determined the efficacy of MA9060 for the treatment of CD138+ MM isolated from naive and refractory patients using a novel ex vivo high throughput platform developed at Moffitt.ULK3 is highly associated with MM stage of the disease. Refractory MM patients have increased autophagy activity with significantly higher expression of ULK3 in refractory patients and in drug resistant cell lines (immunoblotting U266 vs U266-PSR; RPMI-8226 vs RPMI-8226-B25; ABNL vs V10 resistant cells).Genetic ablation of ULK3 by siRNA in U266 and 8226 cell lines results in rapid cessation of the downstream autophagy proteins (ULK1, ATG13, pATG13) and MM cell death within 72h of transduction. Increased concentrations of autophagy inhibitors MA9060/SG3014 progressively decreased CMYC and ULK3 levels, as measured by immunoblotting in U266 cells. In vivo preclinical model of U266Luc tail vein injection proved our drugs are highly effective in reducing tumor dissemination and extending overall survival (CTRL untreated n=65 days vs MA9060 n=110). Importantly, we noted no overt toxicity and protected effect against myeloma-induced bone disease. This novel class of drug works synergistically with PI and can re-sensitize PI resistant disease to these effective therapies. We also show by EMMA ex vivo platform that MA9060 is highly effective for the treatment of CD138+ MM cells isolated from patients with refractory disease.ULK3 represents a novel target for treatment of MM refractory disease. Our dual inhibitors can increase overall survival in vivo and ex vivo, therefore we expect to quickly translate our novel molecules to the clinic. Citation Format: Marilena Tauro, Tao Li, Mark Meads, Praneeth R. Sudalagunta, Raghunandan R. Alugubelli, Nicholas J. Lawrence, Ernst Schonbrunn, Harshani Lawrence, Kenneth H. Shain, Conor C. Lynch. Novel autophagy inhibitory strategies to overcome chemotherapy resistance in multiple myeloma [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 452.

Bone metastatic castrate resistant prostate cancer (CRPC) generates tumors hallmarked by extensive bone formation due to enhanced mesenchymal stem cell (MSC)-osteoblast differentiation. These lesions are at present incurable and substantially diminish patient quality of life, but exciting preclinical studies by our group have shown that these metastases are highly sensitive to gamma delta (γδ) T cell-based CAR-T therapies. However, the impacts of CAR-T cell therapies on the bone microenvironment are presently unknown. Because MSCs are an important stromal component of the cancer-bone microenvironment with emerging immunoregulatory activities, we have recently focused on characterizing the interactions between γδ CAR-T cells and the MSC compartment and their implications for bone metastatic CRPC. Our data show that: 1) γδ CAR-T cells or conditioned media (CM) did not significantly alter MSC viability, 2) MSCs were robustly chemotactic to CM derived from tumor-activated γδ CAR-T cells, 3) MSCs treated with tumor-activated γδ CAR-T CM showed a significant induction of ICAM-1 (9.12-fold, P=0.00640) compared to controls, 4) ICAM-1 silencing significantly reduced γδ CAR-T educated MSC migratory ability, 5) tumor-activated γδ CAR-T CM differentially regulated key genes involved in MSC-osteoblast differentiation, a finding supported by Alizarin red S staining and alkaline phosphatase functional assays, and 6) tri-cultures of MSCs, CRPC cells, and γδ CAR-T cells demonstrate that MSCs significantly enhanced γδ CAR-T mediated CRPC killing. Taken together, we have shown that γδ CAR-T cells promote MSC recruitment in an ICAM-1 dependent manner, and MSCs enhance γδ CAR-T targeted CRPC killing. Reciprocally, γδ CAR-T can promote MSC commitment to osteoblast lineage without impacting their viability. These data are the first to interrogate how bone stromal components regulate γδ CAR-T mediated killing in the context of bone metastatic CRPC. We are currently investigating the mechanisms underpinning the bi-directional interplay between these cell populations. Citation Format: Jeremy S. Frieling, Xiomar Bustos, Ryan T. Bishop, Leticia Tordesillas, Daniel Abate-Daga, Conor C. Lynch. Elucidating reciprocal interactions between bone marrow stromal cells and a novel γδ CAR T cell therapy in the context of bone metastatic prostate cancer. [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 3992.

Multiple myeloma promotes systemic skeletal bone disease that greatly contributes to patient morbidity. Resorption of type I collagen–rich bone matrix by activated osteoclasts results in the release of sequestered growth factors that can drive progression of the disease. Matrix metalloproteinase-13 (MMP13) is a collagenase expressed predominantly in the skeleton by mesenchymal stromal cells (MSC) and MSC-derived osteoblasts. Histochemical analysis of human multiple myeloma specimens also demonstrated that MMP13 largely localizes to the stromal compartment compared with CD138+ myeloma cells. In this study, we further identified that multiple myeloma induces MMP13 expression in bone stromal cells. Because of its ability to degrade type I collagen, we examined whether bone stromal–derived MMP13 contributed to myeloma progression. Multiple myeloma cells were inoculated into wild-type or MMP13–null mice. In independent in vivo studies, MMP13–null mice demonstrated significantly higher overall survival rates and lower levels of bone destruction compared with wild-type controls. Unexpectedly, no differences in type I collagen processing between the groups were observed. Ex vivo stromal coculture assays showed reduced formation and activity in MMP13–null osteoclasts. Analysis of soluble factors from wild-type and MMP13–null MSCs revealed decreased bioavailability of various osteoclastogenic factors including CXCL7. CXCL7 was identified as a novel MMP13 substrate and regulator of osteoclastogenesis. Underscoring the importance of host MMP13 catalytic activity in multiple myeloma progression, we demonstrate the in vivo efficacy of a novel and highly selective MMP13 inhibitor that provides a translational opportunity for the treatment of this incurable disease.Significance:Genetic and pharmacologic approaches show that bone stromal–derived MMP13 catalytic activity is critical for osteoclastogenesis, bone destruction, and disease progression.

Background: Osteosarcoma (OS) frequently metastasizes to lung and the prognosis for these patients is grim with 5-year overall survival rates as low as 30%. There has been little change in the treatment paradigm for this disease and new targeted therapies are an urgent and unmet clinical need. Previously, we showed that the epigenetic regulators, histone deacetylase 1 and 2 (HDAC1 and 2) are key mediators of OS progression and lung metastasis and that the HDAC1/2 selective inhibitor (romidepsin) significantly prevented OS lung metastasis in vivo. Yet, inhibition of HDACs by romidepsin has noted toxicities in humans and here we define the potential mechanisms through which HDAC1/2 mediate their effects in a bid to identify additional therapeutic targets. Methods: Human OS cell lines were treated with vehicle or romidepsin for 24h before undergoing RNA/protein isolation for microarray/proteomics to identify differentially regulated genes. Standard siRNA, proliferation, migration and molecular analyses were preformed to investigate the role of candidate genes in OS malignant behavior. Results: Microarray/proteomics analyses on romidepsin treated OS cells identified neuropilin-1 (NRP1), a membrane bound co-receptor for vascular endothelial growth factor, to be highly downregulated in response to romidepsin; ( Conclusions: HDAC1/2 contributes to OS growth potentially via the upregulation of NRP1, a therapeutically actionable target. Citation Format: Niveditha Nerlakanti, Jeremy Mcguire, Diana Yu, Damon Reed, Conor C. Lynch. Epigenetic upregulation of neuropilin-1 promotes osteosarcoma progression and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 988.

Multiple myeloma (MM) is a largely incurable cancer characterized by the expansion of plasma cells in the bone marrow. A key component of MM growth involves the establishment of a “vicious cycle” of enhanced bone resorption and tumor growth, resulting in extensive bone destruction and osteolytic lesions. Although there are many approved therapies that contribute to the long-term survival of patients, MM ultimately become refractory to treatment. The evolution of resistance is in part due to the bone ecosystem, which protects MM cells during treatment through interactions between the tumor cells and mesenchymal stem cells and cytokines secreted during bone resorption. The acquisition of refractory disease has also been attributed to intrinsic drug resistance, which allows for MM clones with inherent resistance to become the dominant population due to treatment-imposed selective pressures. While both the protective bone microenvironment and intrinsic drug resistance contribute to refractory disease, the role of the bone microenvironment in the development of cell intrinsic resistance has not been established and remains difficult to assess using current in vitro and in vivo methods alone. However, integration of cancer and evolutionary biology with computational modeling allows a unique insight into the spatiotemporal aspects of myeloma evolution and how treatment impacts the evolutionary dynamics of the disease. To explore these evolutionary dynamics, we developed a hybrid agent-based model that incorporates key cellular species that drive normal bone remodeling and, in the context of cancer, create an environment that provides protection. We use published data as well as our own to calibrate parameters such as the bone mineralization rate and to compare cell population dynamics to model outputs. Furthermore, we incorporate spatial data from immunofluorescence imaging of bone to motivate model assumptions on the localization of dividing and dying MM cells. We show that our model captures two key features: normal bone homeostasis, and the myeloma-bone vicious cycle. We then examine how the bone ecosystem impacts the growth dynamics of MM cells and the degradation of bone under standard of care treatments such as bortezomib, an anticancer therapy. Specifically, we test how the spatial distribution of the cells and factors that provide protection, as well as the timing and dose of treatment, impacts treatment response. Our data demonstrates that resistant disease cannot develop without myeloma intrinsic mechanisms, however, protection from the bone microenvironment dramatically increases the likelihood of intrinsic resistance developing. This model provides a foundation to explore how ecological and evolutionary dynamics in the bone ecosystem and MM contribute to drug resistance and tumor growth which ultimately has the potential to help improve treatment strategies. Citation Format: Anna K. Miller, Ryan T. Bishop, Tao Li, Conor C. Lynch, David Basanta. Dissecting the role of the bone ecosystem and intrinsic resistance in the evolution of refractory multiple myeloma [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr A031.

Background. Despite medical advances, currently there is no treatment for breast to bone metastasis. The progression of bone metastatic breast cancer is critically dependent on interactions with the surrounding microenvironment. Therefore, identifying the underpinning molecular mechanisms is vital for the development of new therapies. Rationale. Gene expression analysis and validation in human and murine specimens of bone metastases revealed matrix metalloproteinases, such as MMP-2, are highly expressed in the bone metastatic microenvironment. Genetic ablation of MMP-2 demonstrated the importance of this MMP in driving the growth of the osteolytic bone metastatic breast cancer by regulating the bioavailability of transforming growth factor β (TGFβ). These data support the rationale for the development of a highly specific MMP-2 inhibitor for the eradication of active bone metastatic breast cancer. Methods. We utilized a novel chemical approach to synthesize bone seeking MMP inhibitors (BMMPIs) on a bisphosphonic backbone, with specificity for MMP-2 in the nanomolar range (IC50=140 nM). In vitro, we tested the effect of BMMPIs at varying doses (1nM-100μM) on the viability of the major cellular components of the cancer-bone microenvironment, namely breast cancer cells (PyMT, 4T1), osteoblasts (MC3T3) and osteoclasts (primary monocytes and RAW 264.7). In vivo, mice were intratibially inoculated with either luciferase expressing 4T1 or PyMT (1x105) cells. Mice (n=10/group) then received vehicle, zoledronate (1 mg/kg) or BMMPIs (1 mg/kg). Tumor growth was determined via luminescence quantitation. Cancer induced bone disease was measured ex vivo by μCT, Xray and histomorphometry. MMP activity in vivo and ex vivo was determined via specific activatable MMP probes. Pharmacokinetic and pharmacodynamic studies were performed. Plasma and bone marrow supernatants were collected from PyMT-R221A tumor bearing mice treated with ML115 (5mg/Kg) at 0.25, 0.5, 1, 2, 4, 8, 24 hours and three weeks (n=3 mice/time point). Currently, we are investigating the BMMPIs ability to impact the metastatic process through an in vivo model of intracardiac inoculation. Results. BMMPIs significantly impacted the viability of breast cancer cells and osteoclasts in vitro (p ML115 is rapidly cleared from the plasma and accumulates selectively in the bone marrow microenvironment over time. Conclusions. MMP-2 specific BMMPIs prevent bone metastatic breast cancer growth by impacting cancer cell viability and cancer induced osteolysis. Given that bisphosphonates are well tolerated in the clinical setting, we predict that BMMPIs could be translated to the clinical setting for the treatment and eradication of bone metastatic breast cancer. Citation Format: Tauro M, Laghezza A, Tortorella P, Soliman HH, Lynch CC. Tackling bone metastatic breast cancer growth with novel bone-seeking matrix metalloproteinase-2 inhibitors [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-06-02.

// Gemma Shay 1 , Marilena Tauro 1 , Fulvio Loiodice 2 , Paolo Tortorella 2 , Daniel M. Sullivan 3 , Lori A. Hazlehurst 4 and Conor C. Lynch 1 1 Tumor Biology Department, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA 2 Department of Pharmacy and Pharmaceutical Sciences, Universita degli Studi di Bari “A. Moro”, Bari, Italy 3 Blood and Marrow Transplantation and Cellular Immunology Department, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA 4 Hematopoietic Malignancy and Transplantation Program, West Virginia University, Morgantown, WV, USA Correspondence to: Conor C. Lynch, email: // Keywords : bone marrow microenvironment, multiple myeloma, skeletal malignancies, matrix metalloproteinases, bone targeted MMP inhibitors Received : February 09, 2017 Accepted : April 27, 2017 Published : May 23, 2017 Abstract Multiple myeloma is a plasma cell malignancy that homes aberrantly to bone causing extensive skeletal destruction. Despite the development of novel therapeutic agents that have significantly improved overall survival, multiple myeloma remains an incurable disease. Matrix metalloproteinase-2 (MMP-2) is associated with cancer and is significantly overexpressed in the bone marrow of myeloma patients. These data provide rationale for selectively inhibiting MMP-2 activity as a multiple myeloma treatment strategy. Given that MMP-2 is systemically expressed, we used novel “bone-seeking” bisphosphonate based MMP-2 specific inhibitors (BMMPIs) to target the skeletal tissue thereby circumventing potential off-target effects of MMP-2 inhibition outside the bone marrow-tumor microenvironment. Using in vivo models of multiple myeloma (5TGM1, U266), we examined the impact of MMP-2 inhibition on disease progression using BMMPIs. Our data demonstrate that BMMPIs can decrease multiple myeloma burden and protect against cancer-induced osteolysis. Additionally, we have shown that MMP-2 can be specifically inhibited in the multiple myeloma-bone microenvironment, underscoring the feasibility of developing targeted and tissue selective MMP inhibitors. Given the well-tolerated nature of bisphosphonates in humans, we anticipate that BMMPIs could be rapidly translated to the clinical setting for the treatment of multiple myeloma.

During September 16th-20th 2016, metastasis experts from around the world convened for the 16th Biennial Congress of the Metastasis Research Society and 12th National Congress of the Chinese Tumor Metastasis Society in Chengdu, China to share most current data covering basic, translational, and clinical metastasis research. Presentations of the more than 40 invited speakers of the main congress and presentations from the associated Young Investigator Satellite Meeting are summarized in this report by session topic. The congress program also included three concurrent short talk sessions, an advocacy forum with Chinese and American metastatic patient advocates, a 'Meet the Professors Roundtable' session for young investigators, and a 'Meet the Editors' session with editors from Cancer Cell and Nature Cell Biology. The goal of integrating expertise and exchanging the latest findings, ideas, and practices in cancer metastasis research was achieved magnificently, thanks to the excellent contributions of many leaders in the field.

Bone metastasis is common during breast cancer progression. Matrix metalloproteinase-2 (MMP-2) is significantly associated with aggressive breast cancer and poorer overall survival. In bone, tumor- or host-derived MMP-2 contributes to breast cancer growth and does so by processing substrates, including type I collagen and TGFβ latency proteins. These data provide strong rationale for the application of MMP-2 inhibitors to treat the disease. However, in vivo, MMP-2 is systemically expressed. Therefore, to overcome potential toxicities noted with previous broad-spectrum MMP inhibitors (MMPIs), we used highly selective bisphosphonic-based MMP-2 inhibitors (BMMPIs) that allowed for specific bone targeting. In vitro, BMMPIs affected the viability of breast cancer cell lines and osteoclast precursors, but not osteoblasts. In vivo, we demonstrated using two bone metastatic models (PyMT-R221A and 4T1) that BMMPI treatment significantly reduced tumor growth and tumor-associated bone destruction. In addition, BMMPIs are superior in promoting tumor apoptosis compared with the standard-of-care bisphosphonate, zoledronate. We demonstrated MMP-2–selective inhibition in the bone microenvironment using specific and broad-spectrum MMP probes. Furthermore, compared with zoledronate, BMMPI-treated mice had significantly lower levels of TGFβ signaling and MMP-generated type I collagen carboxy-terminal fragments. Taken together, our data show the feasibility of selective inhibition of MMPs in the bone metastatic breast cancer microenvironment. We posit that BMMPIs could be easily translated to the clinical setting for the treatment of bone metastases given the well-tolerated nature of bisphosphonates. Mol Cancer Ther; 16(3); 494–505. ©2017 AACR.

Simple Summary Using a first-principles approach, we demonstrate how standard-of-care therapies for bone metastatic prostate cancer (BMPCa) patients can be optimized with the use of routine measurements to significantly delay the evolution of resistant disease, potentially extending overall patient survival. Abstract Background: Bone metastatic prostate cancer (BMPCa), despite the initial responsiveness to androgen deprivation therapy (ADT), inevitably becomes resistant. Recent clinical trials with upfront treatment of ADT combined with chemotherapy or novel hormonal therapies (NHTs) have extended overall patient survival. These results indicate that there is significant potential for the optimization of standard-of-care therapies to delay the emergence of progressive metastatic disease. Methods: Here, we used data extracted from human bone metastatic biopsies pre- and post-abiraterone acetate/prednisone to generate a mathematical model of bone metastatic prostate cancer that can unravel the treatment impact on disease progression. Intra-tumor heterogeneity in regard to ADT and chemotherapy resistance was derived from biopsy data at a cellular level, permitting the model to track the dynamics of resistant phenotypes in response to treatment from biological first-principles without relying on data fitting. These cellular data were mathematically correlated with a clinical proxy for tumor burden, utilizing prostate-specific antigen (PSA) production as an example. Results: Using this correlation, our model recapitulated the individual patient response to applied treatments in a separate and independent cohort of patients (n = 24), and was able to estimate the initial resistance to the ADT of each patient. Combined with an intervention-decision algorithm informed by patient-specific prediction of initial resistance, we propose to optimize the sequence of treatments for each patient with the goal of delaying the evolution of resistant disease and limit cancer cell growth, offering evidence for an improvement against retrospective data. Conclusions: Our results show how minimal but widely available patient information can be used to model and track the progression of BMPCa in real time, offering a clinically relevant insight into the patient-specific evolutionary dynamics of the disease and suggesting new therapeutic options for intervention. Trial registration: NCT # 01953640. Funding: Funded by an NCI U01 (NCI) U01CA202958-01 and a Moffitt Team Science Award. CCL and DB were partly funded by an NCI PSON U01 (U01CA244101). AA was partly funded by a Department of Defense Prostate Cancer Research Program (W81XWH-15-1-0184) fellowship. LC was partly funded by a postdoctoral fellowship (PF-13-175-01-CSM) from the American Cancer Society.

Background. Breast to bone metastasis is a common event during breast cancer progression. The resultant lesions are painful and currently, despite medical advances, are incurable. The progression of bone metastatic breast cancer is critically dependent on interactions with the surrounding microenvironment. Therefore, identifying the underpinning molecular mechanisms is vital for the development of new therapies. Rationale. Gene expression analysis and validation in human and murine specimens of bone metastases revealed matrix metalloproteinases, such as MMP-2, are highly expressed in the bone metastatic microenvironment. Genetic ablation of MMP-2 demonstrated the importance of this MMP in driving the growth of the osteolytic bone metastatic breast cancer by regulating the bioavailability of transforming growth factor β (TGFβ). These data support the rationale for the development of a highly specific MMP-2 inhibitor for the eradication of active bone metastatic breast cancer. Methods. Given that previous broad-spectrum MMP inhibitor (MMPI) trials were unsuccessful due to dose limiting systemic side effects, we utilized a novel chemical approach to synthesize bone seeking MMP inhibitors (BMMPIs) on a bisphosphonic backbone, with specificity for MMP-2 in the nanomolar range (IC50=140 nM). In vitro, we tested the effect of BMMPIs at varying doses (1nM-100μM) on the viability of the major cellular components of the cancer-bone microenvironment, namely breast cancer cells (PyMT, 4T1), osteoblasts (MC3T3) and osteoclasts (primary monocytes and RAW 264.7). In vivo, mice were intratibially inoculated with either luciferase expressing 4T1 or PyMT (1x105) cells. Mice (n=10/group) then received vehicle, zoledronate (1 mg/kg) or BMMPIs (1 mg/kg). Tumor growth was determined via luminescence quantitation. Cancer induced bone disease was measured ex vivo by μCT, Xray and histomorphometry. MMP activity in vivo and ex vivo was determined via specific activatable MMP probes. Results. BMMPIs significantly impacted the viability of breast cancer cells and osteoclasts in vitro (p Conclusions. MMP-2 specific BMMPIs prevent bone metastatic breast cancer growth by impacting cancer cell viability and cancer induced osteolysis. Given that bisphosphonates are well tolerated in the clinical setting, we predict that BMMPIs could be translated to the clinical setting for the treatment and eradication of bone metastatic breast cancer. Citation Format: Tauro M, Laghezza A, Tortorella P, Lynch CC. Treatment of skeletal metastatic breast cancer with bone seeking matrix metalloproteinase inhibitors. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-16-02.

Osteosarcoma is an orphan disease with only 900 patients diagnosed each year in US. Most patients are adolescents with small number of older patients. The 5-survival rates for those with metastatic disease is only 30%. Frustratingly, there has been little change in treatment options (chemotherapy, surgery and radiotherapy) due to the difficulty of conducting clinical trials in this rare but deadly cancer. To address this, strong pre-clinical trial information is required to motivate clinical trial design. In vitro and in vivo, we identified that the pan-HDAC inhibitor, panobinostat is highly effective in limiting osteosarcoma growth and metastasis. Panobinostat is approved for the treatment of other malignancies but has noted toxicities. Therefore, to identify therapeutic targets, we explored the HDACs responsible for driving osteosarcoma metastasis and the specific genes through which those HDACs mediated their effects. Our data shows that HDAC1 and HDAC2 are key for osteosarcoma survival using siRNA approaches. Use of the HDAC1/2 selective inhibitor romidepsin in vitro and in vivo supported this finding. To identify targets being regulated by HDAC1/2, we performed microarray and proteomics analyses on romidepsin treated osteosarcoma cells. We found and validated independently that, SLC17A7, a metabolic vesicular glutamate transporter to be highly induced in response to romidepsin treatment; >100 fold more than base line in multiple osteosarcoma cell lines. Functionally, SLC17A7 allows entry of glutamate into the synaptic vesicles from cytoplasm and releases the glutamate from the cells via exocytosis. In our studies, we have seen that romidepsin treatment leads to reduced glutamate levels in the conditioned media and cell death indicating key roles for SLC17A7 in driving osteosarcoma progression and metastasis. In conclusion, our data shows that HDAC1/2 suppression of SLC17A7 is a novel means of promoting osteosarcoma progression and metastasis. Citation Format: Niveditha Nerlakanti, Jeremy McGuire, Diana Yu, Damon R. Reed, Conor C. Lynch. Epigenetic silencing of SLC17A7 promotes osteosarcoma growth [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-044.

Castration-resistant prostate cancer (CRPC) recurs after androgen deprivation therapy (ADT) and is incurable. Reactivation of androgen receptor (AR) signaling in the low androgen environment of ADT drives CRPC. This AR activity occurs through a variety of mechanisms, including up-regulation of AR coactivators such as VAV3 and expression of constitutively active AR variants such as the clinically relevant AR-V7. AR-V7 lacks a ligand-binding domain and is linked to poor prognosis. We previously showed that VAV3 enhances AR-V7 activity to drive CRPC progression. Gene expression profiling after depletion of either VAV3 or AR-V7 in CRPC cells revealed arginine vasopressin receptor 1a (AVPR1A) as the most commonly down-regulated gene, indicating that this G protein–coupled receptor may be critical for CRPC. Analysis of publicly available human PC datasets showed that AVPR1A has a higher copy number and increased amounts of mRNA in advanced PC. Depletion of AVPR1A in CRPC cells resulted in decreased cell proliferation and reduced cyclin A. In contrast, androgen-dependent PC, AR-negative PC, or nontumorigenic prostate epithelial cells, which have undetectable AVPR1A mRNA, were minimally affected by AVPR1A depletion. Ectopic expression of AVPR1A in androgen-dependent PC cells conferred castration resistance in vitro and in vivo. Furthermore, treatment of CRPC cells with the AVPR1A ligand, arginine vasopressin (AVP), activated ERK and CREB, known promoters of PC progression. A clinically safe and selective AVPR1A antagonist, relcovaptan, prevented CRPC emergence and decreased CRPC orthotopic and bone metastatic growth in mouse models. Based on these preclinical findings, repurposing AVPR1A antagonists is a promising therapeutic approach for CRPC.

Heightened matrix metalloproteinase (MMP) activity has been noted in the context of the tumor microenvironment for many years, and causal roles for MMPs have been defined across the spectrum of cancer progression. This is primarily due to the ability of the MMPs to process extracellular matrix (ECM) components and to regulate the bioavailability/activity of a large repertoire of cytokines and growth factors. These characteristics made MMPs an attractive target for therapeutic intervention but notably clinical trials performed in the 1990s did not fulfill the promise of preclinical studies. The reason for the failure of early MMP inhibitor (MMPI) clinical trials that are multifold but arguably principal among them was the inability of early MMP-based inhibitors to selectively target individual MMPs and to distinguish between MMPs and other members of the metzincin family. In the decades that have followed the MMP inhibitor trials, innovations in chemical design, antibody-based strategies, and nanotechnologies have greatly enhanced our ability to specifically target and measure the activity of MMPs. These advances provide us with the opportunity to generate new lines of highly selective MMPIs that will not only extend the overall survival of cancer patients, but will also afford us the ability to utilize heightened MMP activity in the tumor microenvironment as a means by which to deliver MMPIs or MMP activatable prodrugs.

Metastatic castrate resistant prostate cancer (mCRPC) is responsible for the majority of prostate cancer deaths with the median survival after diagnosis being 2 years. The metastatic lesions often arise in the skeleton, and current treatment options are primarily palliative. Using guidelines set forth by the National Comprehensive Cancer Network (NCCN), the medical oncologist has a number of choices available to treat the metastases. However, the sequence of those treatments is largely dependent on the patient history, treatment response and preferences. We posit that the utilization of personalized computational models and treatment optimization algorithms based on patient specific parameters could significantly enhance the oncologist's ability to choose an optimized sequence of available therapies to maximize overall survival. In this perspective, we used an integrated team approach involving clinicians, researchers, and mathematicians, to generate an example of how computational models and genetic algorithms can be utilized to predict the response of heterogeneous mCRPCs in bone to varying sequences of standard and targeted therapies. The refinement and evolution of these powerful models will be critical for extending the overall survival of men diagnosed with mCRPC.

In prostate to bone metastases, the "vicious cycle" paradigm has been traditionally used to illustrate how metastases manipulate the bone forming osteoblasts and resorbing osteoclasts in order to yield factors that facilitate growth and establishment. However, recent advances have illustrated that the cycle is far more complex than this simple interpretation. In this review, we will discuss the role of exosomes and hematopoietic/mesenchymal stem/stromal cells (MSC) that facilitate the establishment and activation of prostate metastases and how cells including myeloid-derived suppressor cells, macrophages, T cells, and nerve cells contribute to the momentum of the vicious cycle. The increased complexity of the tumor-bone microenvironment requires a system level approach. The evolution of computational models to interrogate the tumor-bone microenvironment is also discussed, and the application of this integrated approach should allow for the development of effective therapies to treat and cure prostate to bone metastases.

Osteosarcoma (OS) is the most common bone malignancy in adolescents. At the time of initial diagnosis, approximately 15% of the patients present with metastatic disease in lungs. Prognosis for this cohort is grim with 5-year overall survival rates at 30%. Frustratingly, these statistics have changed little over the past three decades, in part due to the lack of progress in identifying effective therapies and difficulty in coordinating robust clinical trials in this relatively rare malignancy. To address this, we previously screened 54 FDA approved drugs against 5 different OS cell lines and identified the histone deacetylase inhibitor (HDAC), panobinostat (Pano) as having potent effects in vitro. Here, we explore whether Panobinostat is effective in preventing multiple stages of OS progression in vivo using the K7M2 and SAOS2LM7 pre-clinical models of the disease. Pano prevents the growth of primary OS and lung metastasis. Mice were intratibially inoculated with K7M2-Luc cells and then treated with vehicle (n=11) or panobinostat (n=10; 10 mg/kg for all in vivo studies). Tumor growth was measured by bioluminescence (RLU). The clinical endpoint was 1x106RLU (IVIS-200). Primary OS growth was significantly slower in the panobinostat treated group (p Citation Format: Niveditha Nerlakanti, Jeremy McGuire, Diana Yu, Damon R. Reed, Conor C. Lynch. HDAC inhibition significantly reduces primary and lung metastatic osteosarcoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2016.

The bromodomain and extra-terminal (BET) family of proteins are important epigenetic regulators of critical oncogenes, including c-Myc. JQ1 is the most potent BRD4 inhibitor, currently used in several clinical trials and effective in preventing multiple myeloma progression in vivo. Currently, clinical data is showing that patients often develop acquired resistance, indicating that single agent therapies targeting BRD4 such as JQ1 may not provide durable therapeutic responses. To overcome this limitation, we have developed a new class of dual inhibitors, capable to simultaneously target BRD4 and a panel of tyrosine kinases highly expressed in cancer including JAK2, FLT3, RET, FGFR1, ULK1 and ULK3. A preliminary in vitro screening identified SG3-014 as the lead compound, showing BRD4 and c-Myc inhibition activity similar to JQ1, but additionally, it is capable of inhibiting kinases that are over-activated in cancer and responsible for cytokine-mediated drug resistance pathways. In vitro assays on MM cell lines reveals higher SG3-014 sensitivity (5TGM1, 0.85μM; U266, 0.99μM) compared to JQ1 (5TGM1, 4.7μM; U266, 16μM). In vivo studies using 1x106 5TGM1-Luc cells demonstrate that SG3-014 treatment (25mg/Kg) significantly contributes to overall survival compared to JQ1 and the vehicle control cohort (n=10/group) (median survival CTRL=40.5; SG3-014=50.5; JQ1=46 days). Post-study analyses demonstrated a significant reduction in myeloma induced bone disease in the SG3-014 treated mice as measured by X-ray/ μCT/ histomorphometry. Clinical datasets identified ULK3 as the candidate kinase of SG3-014 targeting activity and correlated its expression with MM disease stages. Since ULK3 is involved in cellular senescence and autophagy regulation, a key mechanism by which several cancer cell lines protect themselves against apoptosis and become resistant to standard treatments, we explored the role of SG3-014 inhibition in the autophagy pathway. Preliminary data confirm that SG3-014 is an autophagy inhibitor and, as opposed to JQ1, downregulates ATG proteins level, leading to dysfunctional autophagosome formation. We noted that within 6 h, SG3-014 completely shut down autophagy with decrease levels of ULK1, beclin-1, ATG16L-1, ATG12, ATG3, p62 and increased LC3I/II ratios (additionally confirmed by microscopy and flow cytometry). These effects were not noted with JQ1. Further investigation is clarifying the mechanism by which SG3-014 inhibitory activity on ULK3 is responsible for autophagy mediated cancer cell death. In order to validate this target in the progression of the human disease, our future directions will analyze ex vivo multiple myeloma patient samples and examine the efficacy of the inhibitor in CD138 isolated multiple myeloma cells derived from patient biopsies. We propose dual BRD4/autophagy inhibition as a resilient strategy to prevent multiple myeloma by-pass of single targeted therapies. Citation Format: Marilena Tauro, Muhammad Ayaz, Harshani R. Lawrence, Nicholas J. Lawrence, Kenneth H. Shain, Ernst Schonbrunn, Conor C. Lynch. Dual BET/kinase inhibitors as a novel strategy for the treatment of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 359.

Bone metastatic prostate cancer is painful and incurable. We believe that understanding the interactions of tumor cells and the surrounding stromal cells may provide new therapeutic options. Bone metastatic prostate cancer promotes mesenchymal stem cell (MSC) recruitment and differentiation into osteoblasts, leading to the clinically often-observed osteogenic phenotype. However, the reciprocal roles of bone- marrow derived MSCs on prostate cancer cells are less explored. Here, we discovered that MSCs significantly suppress prostate cancer growth in vitro and in vivo. Mechanistically, we show that MSC-derived interleukin-28 (IL-28) promotes prostate cancer cell apoptosis via the IL-28 receptor alpha (IL-28Rα). However, chronic exposure to MSCs yields prostate cancer cell populations that are resistant to IL-28 induced apoptosis and therapeutics such as docetaxel. In vivo, we observed that MSC selected prostate cancer cells rather than being suppressed, grow at a significantly enhanced rate in bone. The increase in apoptosis resistance is accompanied by a shift in downstream signaling of the IL-28Rα from STAT1 to STAT3. Reduction of STAT3 levels or pharmacological inhibition (STI-201) significantly reduces the growth of MSC selected prostate cancer cells in vitro and in vivo. Collectively, these data shed light on how MSCs in the bone marrow microenvironment initially protect against prostate cancer cell establishment but in doing so, help select for aggressive, apoptosis resistant bone metastatic prostate cancer cells that are more aggressive. Given that the majority of prostate to bone metastases are positive for STAT3 expression and activity, our data provide rationale for the therapeutic targeting of STAT3 in bone metastatic prostate cancer. Citation Format: Jeremy J. Mcguire, Leah Cook, Jeremy Frieling, Ayaz Muhammad, Harshani Lawrence, Nicholas Lawrence, Conor Lynch. Mesenchymal stem cell-derived interleukin-28 can drive the selection of apoptosis resistant bone metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1992.

Bone metastasis is a frequent complication in advanced prostate cancer, with the resultant lesions significantly contributing to patient morbidity and mortality. While next generation hormone ablation therapies and bone protecting bisphosphonates alleviate these symptoms, the disease remains incurable, and new therapeutic approaches are of urgent need. In this regard, we have focused on tapping the tumor-seeking potential of chimeric antigen receptor (CAR) T cells. CAR T cells have shown remarkable anti-tumor responses in hematologic malignancies, but unique physical and biological challenges have hindered their activity in solid tumors. Interestingly, patients treated with bisphosphonates such as zoledronate exhibit enhanced recruitment and activation of the γδ subset of T cells in bone due intracellular accumulation of isopentenyl pyrophosphate (IPP) phosphoantigen. Whereas conventional CAR T cell therapies utilize αβ T cells, we posit that designing γδ CAR T cells could improve homing to bone metastases when administered with bisphosphonates. First, to test the impact of prostate specific stem cell antigen (PSCA)-specific γδ CAR T cells against bone metastatic prostate cancer in vivo, NSG mice (n=10) were intratibially injected with PSCA/luciferase-expressing C4-2B (2x105) castrate resistant prostate cancer cells. Tumors were allowed to establish for 10 days and then randomized into control or γδ CAR T (1.5x107 via tail vein) groups. Subsequent bioluminescent imaging indicated a rapid and significant (p=0.0006) regression of tumors in the γδ CAR T cell group, leading to increased overall survival (5/5 γδ CAR T vs. 0/5 control after 68 days, p=0.0002). Ex vivo bone morphometry analysis also demonstrated the significant protective effect of γδ CAR T associated bone disease. To determine whether bisphosphonates could further enhance the homing of γδ CAR T to bone, NSG mice (n=30) were intratibially injected with C4-2B (2x105), and randomized into control and zoledronate (30µg/kg) groups. After 10 days, mice received γδ T cells (3x106). Subsets were sacrificed at 1, 3, and 5 days post-T cell administration, and peripheral blood, tibia bone marrow, and spleens isolated. CD3-Vδ2 flow cytometry indicated increased γδ T cells in the tibia bone marrow from zoledronate groups (Day 1=61%, Day 3=32%, and Day 5=57%). Furthermore, decreased tumor growth rates were observed in the zoledronate group, suggesting that increased homing of γδ T cells induced anti-tumor effects. Our data to date demonstrate that γδ CAR T cells significantly mitigate bone metastatic prostate cancer and associated bone disease. Further, bisphosphonates (already used in the clinical setting) enhance the homing of γδ CAR T cells to the tumor-bone microenvironment. We posit that γδ CAR T will be an effective immunotherapy approach for the treatment of men with incurable bone metastatic prostate cancer. Citation Format: Jeremy Steven Frieling, Maria Cecilia Ramello, Ismahene Benzaid, Emiliano Roselli, Chen Hao Lo, Conor C. Lynch, Daniel Abate-Daga. γδ CAR T-cell therapy significantly mitigates bone metastatic castrate-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 934.

Synergistic action of kinase and BET bromodomain inhibitors in cell killing has been reported for a variety of cancers. Using the chemical scaffold of the JAK2 inhibitor TG101348, we developed and characterized single agents which potently and simultaneously inhibit BRD4 and a specific set of oncogenic tyrosine kinases including JAK2, FLT3, RET, and ROS1. Lead compounds showed on-target inhibition in several blood cancer cell lines and were highly efficacious at inhibiting the growth of hematopoietic progenitor cells from patients with myeloproliferative neoplasm. Screening across 931 cancer cell lines revealed differential growth inhibitory potential with highest activity against bone and blood cancers and greatly enhanced activity over the single BET inhibitor JQ1. Gene drug sensitivity analyses and drug combination studies indicate synergism of BRD4 and kinase inhibition as a plausible reason for the superior potency in cell killing. Combined, our findings indicate promising potential of these agents as novel chemical probes and cancer therapeutics. Mol Cancer Ther; 16(6); 1054–67. ©2017 AACR.

Bone metastasis is a common event during breast cancer progression. Recently, mesenchymal stem cells (MSCs) have been implicated in the metastasis of primary mammary cancer. Given that bone is the native environment for MSCs, we hypothesized MSCs facilitate the homing of circulating mammary cancer cells to the bone. To test this hypothesis, we examined in vitro whether bone derived MSCs from FVB mice could influence the migration of syngeneic murine mammary cancer cell lines derived from the polyoma virus middle-T (PyMT) model of mammary gland tumorigenesis. Our data show that conditioned media derived from MSCs significantly enhanced the migration of PyMT mammary cancer cell lines. Analysis of conditioned media using a cytokine array revealed the presence of numerous cytokines in the MSC conditioned media, most notably, the murine orthologs of CXCL1 and CXCL5 that are cognate ligands of the CXCR2 receptor. Further investigation identified that: (1) CXCL1, CXCL5 and CXCR2 mRNA and protein were expressed by the MSCs and PyMT cell lines and; (2) neutralizing antibodies to CXCL1, CXCL5 and CXCR2 or a CXCR2 small molecule inhibitor (SB265610) significantly abrogated the migratory effect of the MSC conditioned media on the PyMT cells. Therefore, in vitro evidence demonstrates that bone derived MSCs play a role in the migration of mammary cancer cells, a conclusion that has potential implications for breast to bone metastasis in vivo.

Background: Advanced prostate cancer is treated by androgen deprivation, but despite initial responses most tumors inevitably recur. The recurrent disease is termed castration-resistant prostate cancer (CRPC) and is characterized by active androgen receptor (AR) signaling despite decreased androgen levels. AR activity in CRPC occurs through a variety of mechanisms including upregulation of AR coactivators, such as Vav3, and expression of constitutively active AR variants, which lack the ligand binding domain and are linked to poor prognosis. We previously demonstrated that PC cells are growth inhibited by depletion of either Vav3 or the clinically prevalent AR variant, AR-V7 (1). Gene expression profiling in CRPC cells revealed that arginine vasopressin receptor 1a (AVPR1a) was significantly reduced by depletion of either Vav3 or AR-V7. AVPR1a is a G protein-coupled receptor that has not been previously linked to PC. Analysis of publicly available datasets showed that the AVPR1a gene exhibits a significant increase in copy number in human CRPC specimens and AVPR1a mRNA is upregulated in aggressive PC (2-4). Methods: To explore the role of AVPR1a in PC cells, we selectively depleted AVPR1a in a panel of prostate cancer and prostate epithelial cell lines and examined cell survival and proliferation in culture and soft agar, as well as cell migration and invasion. Conversely, we overexpressed AVPR1a in an androgen-dependent PC cell line, LNCaP, and examined growth in vitro and in vivo in androgen-depleted conditions. We investigated AVPR1a antagonism in two distinct in vivo models of CRPC progression and growth using an effective and safe-in-humans AVPR1a antagonist, relcovaptan. Results: We showed that depletion of AVPR1a greatly inhibited the proliferation of multiple CRPC cell lines but had less to no effect on androgen-dependent PC cells or nontumorigenic prostate epithelial cells. Depletion of AVPR1a also decreased CRPC cell anchorage-independent growth. Conversely, overexpression of AVPR1a in androgen-dependent LNCaP cells conferred castration resistance in vitro and in vivo. Similar to depletion of AVPR1a, treatment of PC cells with the AVPR1a antagonist relcovaptan decreased CRPC anchorage-independent growth. AVPR1a depletion or antagonism was also effective at decreasing CRPC metastatic properties: migration and invasion. In a preclinical xenograft model of progression to castration resistance, we found that relcovaptan halted tumor growth and stabilized circulating levels of a clinical tumor progression marker, prostate specific antigen (PSA). We evaluated relcovaptan efficacy on end-stage metastatic CRPC growth. C4-2B CRPC cells were injected into the tibias of castrated mice to model tumor growth and bone remodeling. Relcovaptan significantly inhibited tumor growth and prevented cancer cell-induced bone remodeling. Conclusion: AVPR1a is a promising new therapeutic target against CRPC. We propose that AVPR1a antagonists may be repurposed for PC therapy. References: 1. Peacock SO, et al. Mol Endocrinol 2012;12:1967-79. 2. Chandran UR, et al. BMC Cancer 2007;7:64. 3. Tamura K, et al. BMC Cancer 2007;67:5117-25. 4. Beltran H, et al. Nat Med 2016;22;298-305. Citation Format: Ning Zhao, Stephanie Peacock, Chen Hao Lo, Laine Heidman, Fiorella Magani, Meghan Rice, Ann Greene, Yushan Zhang, Yehia Daaka, Conor Lynch, Kerry Burnstein. Preclinical evaluation of an arginine vasopressin receptor 1A (AVPR1A) antagonist in castration-resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A063.

Tumor-associated macrophages, particularly those with an anti-inflammatory phenotype, have long been implicated in the progression of primary solid malignancies, including prostate cancer. Metastatic prostate cancer typically manifests in the bone where it induces painful osteogenic lesions that are incurable. Bone is naturally rich in myeloid-derived macrophages whose temporal polarization into pro- (M1) and anti-inflammatory (M2) phenotypes is critical for regulating the bone repair program mediated by bone-resorbing osteoclasts and bone-building osteoblasts. However, the dynamics of macrophage polarization in the context of bone metastatic prostate cancer are underexplored and difficult to address with traditional biologic approaches. To address the role of macrophage polarization in the context of bone metastatic prostate cancer, we first investigated the macrophage polarization temporal dynamics in normal bones and analyzed macrophage plasticity in vivo subsequent to intratibial injury. Bone marrows were isolated at several time points and profiled by flow cytometry for pro- and anti-inflammatory monocyte macrophage content. Contralateral tibias were analyzed for bone volume, osteoblast and osteoclast numbers. We then designed a mathematical model describing the different cell population dynamics in the bones. Generation of the model required testing a number of assumptions regarding macrophage polarization behavior. For example, it is unknown whether M1 resolves at the initial stages of bone injury repair through death or by repolarizing into M2. For each aspect, competing mechanistic assumptions were proposed and simulated by sets of ODEs. The best-fitting assumptions for each aspect were integrated into a single comprehensive ODE model to fully describe the dynamics of the bone resident cell populations during bone remodeling. This experimentally validated model is now being used to address how bone resident cells respond to metastatic prostate cancer cells. In conclusion, we have generated an ODE model that describes macrophage polarization over time during bone repair and how pro- and anti-inflammatory macrophages interact with bone stromal cells. The mathematical model predictions are in agreement with our biologic experiments in vivo and will allow us to interrogate how macrophage polarization impacts the behavior of metastatic prostate cancer cells in the bone microenvironment. Citation Format: Etienne A. Baratchart, Chen Hao Lo, Conor Lynch, David Basanta. Dynamic modeling of macrophage plasticity in bone metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4260.

Bone metastatic breast cancer is currently incurable and will be evident in more than 70% of patients that succumb to the disease. Understanding the factors that contribute to the progression and metastasis of breast cancer can reveal therapeutic opportunities. Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes whose role in cancer has been widely documented. They are capable of contributing to every step of the metastatic cascade, but enthusiasm for the use of MMP inhibition as a therapeutic approach has been dampened by the disappointing results of clinical trials conducted more than 20 years ago. Since the trials, our knowledge of MMP biology has expanded greatly. Combined with advances in the selective targeting of individual MMPs and the specific delivery of therapeutics to the tumor microenvironment, we may be on the verge of finally realizing the promise of MMP inhibition as a treatment strategy. Here, as a case in point, we focus specifically on MMP-2 as an example to show how it can contribute to each stage of breast-cancer-to-bone metastasis and also discuss novel approaches for the selective targeting of MMP-2 in the setting of the bone-cancer microenvironment.

In human prostate to bone metastases and in a novel rodent model that recapitulates prostate tumor–induced osteolytic and osteogenic responses, we found that osteoclasts are a major source of the proteinase, matrix metalloproteinase (MMP)-9. Because MMPs are important mediators of tumor-host communication, we tested the effect of host-derived MMP-9 on prostate tumor progression in the bone. To this end, immunocompromised mice that were wild-type or null for MMP-9 received transplants of osteolytic/osteogenic-inducing prostate adenocarcinoma tumor tissue to the calvaria. Surprisingly, we found that that host MMP-9 significantly contributed to prostate tumor growth without affecting prostate tumor–induced osteolytic or osteogenic change as determined by microcomputed tomography, microsingle-photon emission computed tomography, and histomorphometry. Subsequent studies aimed at delineating the mechanism of MMP-9 action on tumor growth focused on angiogenesis because MMP-9 and osteoclasts have been implicated in this process. We observed (a) significantly fewer and smaller blood vessels in the MMP-9 null group by CD-31 immunohistochemistry; (b) MMP-9 null osteoclasts had significantly lower levels of bioavailable vascular endothelial growth factor-A164; and (c) using an aorta sprouting assay, conditioned media derived from wild-type osteoclasts was significantly more angiogenic than conditioned media derived from MMP-9 null osteoclasts. In conclusion, these studies show that osteoclast-derived MMP-9 affects prostate tumor growth in the bone microenvironment by contributing to angiogenesis without altering prostate tumor–induced osteolytic or osteogenic changes. Mol Cancer Res; 8(4); 459–70. ©2010 AACR.

By analogy, the study of metastasis is like a group of blind people studying an elephant. Each describes the pachyderm based on the part (s)he touches, but none comprehends the whole elephant because his/her exposure is limited. Likewise, the complexity of metastasis can only be appreciated when one

Matrix metalloproteinases (MMP) are a family of enzymes with a myriad of functions. Lately, we have come to realize that broad-spectrum inhibition of these enzymes, as was tried unsuccessfully in multiple phase III trials in cancer patients, is likely unwise given the protumorigenic and antitumorigenic functions of various family members. Here, we used the multistage mammary tumor model MMTV-PyVT to investigate roles for either MMP7 or MMP9 in tumor progression. We found no effect of genetic ablation of MMP7 or MMP9 on the multifocal tumors that developed in the mammary glands. Lack of MMP7 also had no effect on the development of lung metastases, suggesting that MMP7 is irrelevant in this model. In contrast, MMP9 deficiency was associated with an 80% decrease in lung tumor burden. The predominant cellular source of MMP9 was myeloid cells, with neutrophils being the largest contributor in tumor-bearing lungs. Experimental metastasis assays corroborated the role of host-derived MMP9 in lung metastasis and also facilitated determination of a time frame most relevant for the MMP9-mediated effect. The lung tumors from MMP9-deficient mice showed decreased angiogenesis. Surprisingly, the antimetastatic outcome of MMP9 ablation seemed to be dependent on strain. Only mice that had genetic background derived from C57BL/6 showed reduced metastasis, whereas mice fully of the FVB/N background showed no significant effect. These strain-specific responses were also observed in a study using a highly selective pharmacologic inhibitor of MMP9 and thus suggest that responses to MMP inhibition are controlled by genetic differences. [Cancer Res 2008;68(15):6251–9]

To delineate the role of matrix metalloproteinase 7 (MMP7) in mammary tumorigenesis, MMP7 was expressed in the normal murine mammary gland cell line, c57MG. MMP7 markedly enhanced the growth rate of the c57MG cells in three-dimensional culture and promoted tumor formation in vivo. Subsequent investigation showed that MMP7 (a) up-regulated ErbB4 receptor levels, (b) solubilized the ErbB4 receptor cognate ligand heparin-bound epidermal growth factor, and (c) mediated the proteolytic processing of ErbB4 to yield a soluble intracellular domain (ICD) that localized to the cytoplasm and the nucleus. Furthermore, overexpression of the ErbB4 ICD in the c57MG cell line recapitulated the proliferative effects of MMP7 in vitro and in vivo. These data indicate a novel mechanism for mammary epithelial cell transformation by MMP7. [Cancer Res 2007;67(14):6760–7

Systemic therapy has improved osteosarcoma event-free and overall survival, but 30–50% of patients originally diagnosed will have progressive or recurrent disease, which is difficult to cure. Osteosarcoma has a complex karyotype, with loss of p53 in the vast majority of cases and an absence of recurrent, targetable pathways. In this study, we explored 54 agents that are clinically approved for other oncologic indications, agents in active clinical development and others with promising preclinical data in osteosarcoma at clinically achievable concentrations in 5 osteosarcoma cell lines. We found significant single-agent activity of multiple agents and tested 10 drugs in all permutations of two-drug combinations to define synergistic combinations by Chou and Talalay analysis. We then evaluated order of addition to choose the combinations that may be best to translate to the clinic. We conclude that the repurposing of chemotherapeutics in osteosarcoma by using an in vitro system may define novel drug combinations with significant in vivo activity. In particular, combinations of proteasome inhibitors with histone deacetylase inhibitors and ixabepilone and MK1775 demonstrated excellent activity in our assays.

A limited number of in vivo models that rapidly assess bone development or allow for the study of tumor progression in a closed in vivo environment exist. To address this, we have used bone tissue engineering techniques to generate a murine in vivo bone bioreactor. The bioreactor was created by implanting an osteoconductive hydroxyapatite scaffold pre-loaded with saline as a control or with bone morphogenetic protein-2 (BMP-2) to the murine femoral artery. Control and BMP-2 bioreactors were harvested and histologically assessed for vascularization and bone formation at 6 and 12 weeks post implantation. BMP-2 significantly enhanced the formation of osteoid within the bioreactor in comparison to the controls. To test the in vivo bone bioreactor as a model of tumor: bone interaction, FVB mice were implanted with control or BMP-2 treated bioreactors. After 6 weeks, an osteolytic inducing mammary tumor cell line tagged with luciferase (PyMT-Luc) derived from the polyoma virus middle T (PyMT) model of mammary tumorigenesis was delivered to the bioreactor via the femoral artery. Analysis of luciferase expression over time demonstrated that the presence of osteoid in the BMP-2 treated bioreactors significantly enhanced the growth rate of the PyMT-Luc cells in comparison to the control group. These data present a unique in vivo model of ectopic bone formation that can be manipulated to address molecular questions that pertain to bone development and tumor progression in a bone environment.

The ability to rapidly assess the efficacy of therapeutic strategies for incurable bone metastatic prostate cancer is an important and urgent need. Pre-clinical in vivo models provide insights yet are limited in their capacity to interrogate temporal multi-cellular interactions occurring in the cancer-bone microenvironment. Computational and mathematical models that integrate and, are validated experimentally, can overcome this limitation and accelerate biological research. Here, we describe how a biologically driven discrete continuum hybrid cellular automaton (HCA) model approach can dissect the pleiotropic effects of inhibiting putative therapeutic targets such as, TGFβ. Using our HCA model, we tested five different therapeutic doses delivered in two therapeutic windows. In silico results predict that TGFβ inhibition, applied prior to tumor seeding acts by directly impacting prostate cancer cell viability but also by simultaneously restricting osteoclast formation and unexpectedly, promoting osteoblast differentiation. This effect was dependent on the prostate cancer cell expression of TGFβ receptors. In silico predictions were validated with two independent in vivo models of bone metastatic prostate cancer (PAIII and C4-2B). Using immunohistochemical information from human bone metastatic prostate cancer samples, we also demonstrate how the HCA can be used to predict the evolution of heterogeneous disease in response to applied therapies. Collectively, these data underscore the power of a combined HCA/biological approach in optimizing the efficacy of applied therapies and measuring their impact on bone metastatic prostate cancer. Citation Format: Leah Cook, Arturo Araujo, Julio Pow-Sang, Mikalai Budzevich, David Basanta, Conor C. Lynch. Predictive computational modeling to define effective treatment strategies for bone metastatic prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A13.

A paucity of therapeutic options is available to treat men with metastatic castration-resistant prostate cancer (mCRPC). However, recent developments in our understanding of the disease have resulted in several new therapies that show promise in improving overall survival rates in this patient population.Agents approved for use in the United States and those undergoing clinical trials for the treatment of mCRPC are reviewed. Recent contributions to the understanding of prostate biology and bone metastasis are discussed as well as how the underlying mechanisms may represent opportunities for therapeutic intervention. New challenges to delivering effective mCRPC treatment will also be examined.New and emerging treatments that target androgen synthesis and utilization or the microenvironment may improve overall survival rates for men diagnosed with mCRPC. Determining how factors derived from the primary tumor can promote the development of premetastatic niches and how prostate cancer cells parasitize niches in the bone microenvironment, thus remaining dormant and protected from systemic therapy, could yield new therapies to treat mCRPC. Challenges such as intratumoral heterogeneity and patient selection can potentially be circumvented via computational biology approaches.The emergence of novel treatments for mCRPC, combined with improved patient stratification and optimized therapy sequencing, suggests that significant gains may be made in terms of overall survival rates for men diagnosed with this form of cancer.

Bone metastasis will impact most men with advanced prostate cancer. The vicious cycle of bone degradation and formation driven by metastatic prostate cells in bone yields factors that drive cancer growth. Mechanistic insights into this vicious cycle have suggested new therapeutic opportunities, but complex temporal and cellular interactions in the bone microenvironment make drug development challenging. We have integrated biologic and computational approaches to generate a hybrid cellular automata model of normal bone matrix homeostasis and the prostate cancer-bone microenvironment. The model accurately reproduces the basic multicellular unit bone coupling process, such that introduction of a single prostate cancer cell yields a vicious cycle similar in cellular composition and pathophysiology to models of prostate-to-bone metastasis. Notably, the model revealed distinct phases of osteolytic and osteogenic activity, a critical role for mesenchymal stromal cells in osteogenesis, and temporal changes in cellular composition. To evaluate the robustness of the model, we assessed the effect of established bisphosphonate and anti-RANKL therapies on bone metastases. At approximately 100% efficacy, bisphosphonates inhibited cancer progression while, in contrast with clinical observations in humans, anti-RANKL therapy fully eradicated metastases. Reducing anti-RANKL yielded clinically similar results, suggesting that better targeting or dosing could improve patient survival. Our work establishes a computational model that can be tailored for rapid assessment of experimental therapies and delivery of precision medicine to patients with prostate cancer with bone metastases. Cancer Res; 74(9); 2391–401. ©2014 AACR.

The matrix metalloproteinases (MMPs) are important in tumour cell invasion and metastasis in many common cancers. However, relatively few studies have investigated the role of MMPs and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), in leukaemia cell invasion. This study examined two leukaemia cell lines, K562 and HL-60 and showed that the K562 cell line was four times more invasive than the HL-60 cell line. The expression of MMP-2, matrilysin (MMP-7), MMP-9, TIMP-1, TIMP-2 and TIMP-3 was analysed. Both cell lines produced similar amounts of MMP-2, MMP-9 and TIMP-2. The K562 cells expressed more TIMP-1 than the HL-60 cells and neither cell line expressed TIMP-3. Interestingly, only the K562 cells expressed matrilysin suggesting a potential role for matrilysin in leukaemia cell invasion. In vitro invasion assays performed in the presence of a matrilysin blocking antibody showed a 40% reduction in invasive ability. This data suggests that matrilysin plays an important role in leukaemia cell invasion.

Introduction: Bone metastatic prostate cancer is common, incurable and, hallmarked by extensive osteoblast driven bone formation that greatly impacts the patient's quality of life. To generate new cures a better understanding of the cellular and molecular mechanisms governing prostate cancer-bone interaction are required. Mesenchymal stem cells (MSCs) give rise to multiple cell types including osteoblasts. The bone is a natural reservoir for MSCs and therefore we reasoned that MSCs contribute to the prostate cancer progression and pathophysiology in bone. Rationale/Hypothesis: Our in vitro data identified that MSCs are recruited to prostate cancer derived signals and that human and murine specimens of bone metastatic prostate cancer have an increased amount of MSCs compared tumor naïve controls (α-smooth muscle actin positive). Based on this data, we hypothesized that MSCs contribute to prostate cancer induced osteogenesis. Methodology: Primary MSCs isolated from bone and characterized as CD29+, SCA-1+, CD45- and differentiation capacity were utilized. To mimic bone metastatic prostate cancer, we used an in vivo mouse model (PAIII) of osteogenic bone metastatic prostate cancer. Mice were intratibially inoculated with PAIIIs, PAIIIs/MSCs (1:1), or MSCs (n=8/group, 1x104total cells). Tumor growth was measured at time points using luminescence imaging. Subsequently the tibias were collected for imaging and histological analysis. In vitro, cleaved caspase-3, immunoblot and immunofluorescence were employed to identify mechanisms of MSC-prostate cancer interaction. A resistant clone of the PaIII cell line was derived by multiple rounds of treatment with MSC conditioned media and subsequently used for comparison in vitro and in vivo. Results: Surprisingly, we initially observed that MSCs suppressed PAIII prostate cancer growth in-vivo compared to PAIII alone at day 11 (p Conclusions: Our data identify MSCs as a major component of osteogenic bone metastatic prostate cancers. Prostate cancer cells promote MSC differentiation into osteoblasts that in turn contributes to the osteogenic nature of the disease. Conversely, MSCs promote the evolution of apoptosis resistant clones that ultimately contribute to tumor progression. Understanding the molecular mechanisms underpinning prostate cancer-MSC interaction could yield exciting therapeutic opportunities to eradicate incurable bone metastatic prostate cancer. Citation Format: Jeremy McGuire, Leah Cook, Jeremy Frieling, Conor Lynch. Mesenchymal stem cells in the prostate cancer-bone microenvironment increase osteogenesis and generate apoptotic resistant cancer cells. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C18.

Background: Prostate cancer is the second most common malignancy in American men and causes ∼27540 deaths per annum. microRNAs (miRNAs) are a class of short non-coding RNAs that regulate the expression of protein-coding genes via either mRNA degradation or translational repression. These small molecules are known to be aberrantly expressed in prostate cancer (PCa) and possess both tumor suppressive and oncogenic properties. miR-125 is a highly conserved miRNA family comprised of three homolog members hsa-miR-125a, hsa-miR-125b-1, and hsa-miR-125b-2. Members of this family have been attributed both disease suppressing and promoting functions in certain contexts however their role in PCa has yet to be fully elucidated. Methods: miRNA expression was evaluated in a publicly available dataset (GSE21032) comprised of 28 normal, 98 primary PCa and 13 metastasis samples. miR-125 expression was assessed in a panel of 8 prostate cell lines comprising PrEC prostate epithelial cells, RWPE-1, BPH1, DU145, LNCaP, C4-2B, PC3, and PC3-2M cell lines using the Qiagen miScript system. MirWalk was used to identify predicted targets of the miRNAs. miR-125 expression was transiently inhibited in LNCaP, C4-2B, PC3, and PC3-2M cell lines using miScript miRNA inhibitors and miRNA and target mRNA expression, cell proliferation and migration were measured post-transfection. Results: Analysis of human data revealed miR-125a-3p was significantly up-regulated in metastasis samples compared to both primary PCa and normal tissues (log fold change (FC) 1.743, p-value 9.01E-08 logFC = 1.536 p-value = 8.80E-08 respectively). Next, we validated miR-125 family member expression in prostate cancer cell lines compared to PrEC and RWPE-1 prostate epithelial cells. Most notably, we observed that miR-125b-2 expression was significantly higher in all prostate cancer cell lines. Inhibition of miR-125b-2, significantly reduced the proliferation of the LNCaP, C4-2B, and PC3-2M cell lines. Moreover, inhibition of this miRNA significantly impaired prostate cancer cell migration by 43.8% (p-value = 0.003). Using MirWalk, we examined potential targets for regulation by miR-125b-2 and identified the potent tumor suppressor Quaking family as top hit. Our results show that inhibition of miR-125b-2 significantly enhanced the expression of Quaking family members (1.79 fold). Conclusions: Our results demonstrate that the miR-125 family of miRNAs plays a role in regulating prostate cancer cell proliferation and migration, potentially by mitigating Quaking activity. Citation Format: Sinead T. Aherne, Fiona O’Neill, Stephen F. Madden, Martin Clynes, Conor C. Lynch. miR-125 family of miRNAs mediates prostate cancer cell proliferation and migration. [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 1091.

Prostate cancer frequently metastasizes to bone, resulting in increased risk of fractures and severe pain that significantly impacts patient quality of life. Bone metastatic prostate cancer is currently incurable with standard of care therapies being mainly palliative. In bone, prostate cancer generates extensive osteogenic lesions by promoting osteoblast activity. Osteoblasts are specialized bone forming cells derived from mesenchymal stem cells. In a bid to find new molecular targets through which prostate cancer induces osteogenesis, we incubated naïve MSCs with osteogenic bone metastatic prostate cancer cell line C4-2B conditioned media. Gene expression analysis revealed a 3-fold increase in expression of the TGFâ co-receptor, betaglycan. Given the critical role of TGFâ in the context of bone metastatic disease, we examined the role of betaglycan in MSC behavior. To this end, we utilized shRNA to knockdown betaglycan gene expression in mouse bone marrow-derived primary MSCs. Reduced betaglycan expression in knockdown (KD) MSCs had little to no impact on the expression of TGFâ signaling receptors, TGFâRI and TGFâRII, or pan-TGFâ ligand expression. However, using PAI promoter activity, a readout for TGFâ signaling, we observed a 3-fold increase in luminescence in KD-MSCs compared to controls. Likewise, phosphorylated Smad 2 was significantly increased in Betaglycan KD-MSCs in response to TGFâ treatment, collectively demonstrating increased TGFâ signaling in MSCs with reduced betaglycan expression. Using modified Boyden chamber assays, we found that 50% more Betaglycan-KD MSCs migrated towards recombinant TGFâ than controls (100 KD-MSCs cells migrated towards recombinant TGFâ vs. 50 control cells); this change was reversed with the addition of recombinant betaglycan. Using osteogenic differentiation assay, we found that Betaglycan KD-MSC differentiation was reduced by approximately 70% compared to control MSCs as measured by alizarin red staining. Addition of recombinant TGFâ further suppressed differentiation in both KD-MSCs and Control MSCs. This phenomenon was rescued by the addition of recombinant betaglycan, suggesting that MSC-derived betaglycan is critical for driving the osteogenic program. We are currently determining the impact of betaglycan on MSC behavior in the tumor bone microenvironment using in vivo models of bone metastatic prostate cancer. Collectively, these findings suggest that prostate cancer-induced MSC osteogenic programs are regulated in part via the induction of betaglycan. Citation Format: Leah M. Cook, Jeremy J. McGuire, Conor C. Lynch. Betaglycan-mediated regulation of mesenchymal stromal cell behavior in the prostate tumor-bone microenvironment. [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 749.

Myeloma cells promote osteolysis and suppress osteogenesis in the bone microenvironment by regulating osteoclasts (OCL) and osteoblasts (OBL), respectively. Patients often suffer painful, osteoporotic bone status as diseases progresses. Bone consists of 90% type-I collagen, which is degraded by OCL derived cathepsin K during regular bone remodeling. While bisphosphonates delay pathological fracture, they have little impact on overall survival; therefore, development of new therapies is crucial. Matrix metalloproteinase-13 (MMP-13) is an enzyme involved in type-I collagen degradation and resorption. Immunohistochemical staining of a panel of human myeloma biopsies (n = 15) show high MMP-13 expression predominantly in bone stroma and to a lesser extent in the myeloma compartment. Consistent with previous reports, MMP-13 expression localizes to mesenchymal stromal cells (MSC) and OBLs rather than OCLs. Further supporting these observations, analysis of publically available data sets revealed upregulation of MMP-13 expression in MSCs co-cultured with myeloma cells (1.81 LogFC, p To test our hypothesis, we generated immune-compromised MMP-13-null mice on a C57BL/6 background for ex vivo and in vivo studies. Mouse myeloma cell line, 5TGM1, homes to the skeleton and progresses upon tail vein injection in this model. Our in vivo studies showed that 5TGM1-bearing MMP-13-null mice had significantly improved overall survival compared to wild-type controls (Mean 39 vs. 43 days; p Citation Format: Chen Hao Lo, Gemma Shay, Conor C. Lynch. Host-derived MMP-13 mediates multiple myeloma-induced osteolysis. [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 3282.

Background Breast to bone metastases frequently induce a “vicious cycle” in which osteoclast mediated bone resorption and proteolysis results in the release of bone matrix sequestered factors that drive tumor growth. While osteoclasts express numerous proteinases, analysis of human breast to bone metastases unexpectedly revealed that bone forming osteoblasts were consistently positive for the proteinase, MMP-2. Given the role of MMP-2 in extracellular matrix degradation and growth factor/cytokine processing, we tested whether osteoblast derived MMP-2 contributed to the vicious cycle of tumor progression in the bone microenvironment. Methodology/Principal Findings To test our hypothesis, we utilized murine models of the osteolytic tumor-bone microenvironment in immunocompetent wild type and MMP-2 null mice. In longitudinal studies, we found that host MMP-2 significantly contributed to tumor progression in bone by protecting against apoptosis and promoting cancer cell survival (caspase-3; immunohistochemistry). Our data also indicate that host MMP-2 contributes to tumor induced osteolysis (μCT, histomorphometry). Further ex vivo/in vitro experiments with wild type and MMP-2 null osteoclast and osteoblast cultures identified that 1) the absence of MMP-2 did not have a deleterious effect on osteoclast function (cd11B isolation, osteoclast differentiation, transwell migration and dentin resorption assay); and 2) that osteoblast derived MMP-2 promoted tumor survival by regulating the bioavailability of TGFβ, a factor critical for cell-cell communication in the bone (ELISA, immunoblot assay, clonal and soft agar assays). Conclusion/Significance Collectively, these studies identify a novel “mini-vicious cycle” between the osteoblast and metastatic cancer cells that is key for initial tumor survival in the bone microenvironment. In conclusion, the findings of our study suggest that the targeted inhibition of MMP-2 and/or TGFβ would be beneficial for the treatment of bone metastases.

We investigated the effects of the matrix metalloproteinase 13 (MMP13)-selective inhibitor, 5-(4-{4-[4-(4-fluorophenyl)-1,3-oxazol-2-yl]phenoxy}phenoxy)-5-(2-methoxyethyl) pyrimidine-2,4,6(1H,3H,5H)-trione (Cmpd-1), on the primary tumor growth and breast cancer-associated bone remodeling using xenograft and syngeneic mouse models. We used human breast cancer MDA-MB-231 cells inoculated into the mammary fat pad and left ventricle of BALB/c Nu/Nu mice, respectively, and spontaneously metastasizing 4T1.2-Luc mouse mammary cells inoculated into mammary fat pad of BALB/c mice. In a prevention setting, treatment with Cmpd-1 markedly delayed the growth of primary tumors in both models, and reduced the onset and severity of osteolytic lesions in the MDA-MB-231 intracardiac model. Intervention treatment with Cmpd-1 on established MDA-MB-231 primary tumors also significantly inhibited subsequent growth. In contrast, no effects of Cmpd-1 were observed on soft organ metastatic burden following intracardiac or mammary fat pad inoculations of MDA-MB-231 and 4T1.2-Luc cells respectively. MMP13 immunostaining of clinical primary breast tumors and experimental mice tumors revealed intra-tumoral and stromal expression in most tumors, and vasculature expression in all. MMP13 was also detected in osteoblasts in clinical samples of breast-to-bone metastases. The data suggest that MMP13-selective inhibitors, which lack musculoskeletal side effects, may have therapeutic potential both in primary breast cancer and cancer-induced bone osteolysis.

Breast to bone metastasis is a common occurrence in the majority of patients with advanced breast cancer. The metastases are often incurable and are associated with bone destruction and high rates of morbidity. Understanding the underlying mechanisms of how metastatic tumor cells induce bone destruction is critically important. We previously reported that Tie2, a receptor tyrosine kinase, is significantly increased in human breast cancer tissues compared with normal and benign breast tumors and regulates tumor angiogenesis. In this study, we identify a new function of Tie2 in osteoclastogenesis and osteolytic bone invasion of breast cancer. Tie2 is present in hematopoietic stem/precursor cells. Genetic deletion of Tie2 or neutralization of Tie2 function using soluble Tie2 receptor impaired osteoclastogenesis in an embryonic stem cell differentiation assay. In contrast, deletion of Tie2 has no effect on osteoblastogenesis. As CD11b myeloid cells have the potential to become osteoclasts and Tie2 is present in a certain population of these cells, we isolated Tie2+ and Tie2− myeloid cells. We observed a significant reduction of osteoclastogenesis in Tie2− compared with Tie2+ CD11b cells. Consistently, neutralization of Tie2 activity in vivo significantly inhibited osteolytic bone invasion and tumor growth in a mammary tumor model, which correlated with a significant reduction of osteoclasts and tumor angiogenesis. Collectively, these data reveal a direct and novel role of Tie2 signaling in osteoclast differentiation. These findings identify Tie2 as a therapeutic target for controlling not only tumor angiogenesis but also osteolytic bone metastasis in breast cancer. Cancer Res; 70(7); 2819–28

Perturbations in cell-cell contact machinery occur frequently in epithelial cancers and result in increased cancer cell migration and invasion. Previously, we demonstrated that MMP-7, a protease implicated in mammary and intestinal tumor growth, can process the adherens junction component E-cadherin. This observation leads us to test whether MMP-7 processing of E-cadherin could directly impact cell proliferation in nontransformed epithelial cell lines (MDCK and C57MG). Our goal was to investigate the possibility that MMP-7 produced by cancer cells may have effects on adjacent normal epithelium. Here, we show that MMP-7 processing of E-cadherin mediates, (1) loss of cell-cell contact, (2) increased cell migration, (3) a loss of epithelial cell polarization and (4) increased cell proliferation via RhoA activation. These data demonstrate that MMP-7 promotes epithelial cell proliferation via the processing of E-cadherin and provide insights into the molecular mechanisms that govern epithelial cell growth.

The matrix metalloproteinases MMP-2, MMP-3, MMP-7, MMP-9, and MMP-13 are highly expressed in the tumor-bone microenvironment, and, of these, MMP-7 and MMP-9 were found to be localized to bone-resorbing osteoclasts in human breast-to-bone metastases. In a bid to define the roles of host-derived MMP-7 and MMP-9 in the tumor-bone microenvironment, the tibias of MMP-7 and MMP-9 null mice were injected with osteolytic luciferase–tagged mammary tumor cell lines. Our data show that osteoclast-derived MMP-7 significantly contributes to tumor growth and tumor-induced osteolysis whereas osteoclast-derived MMP-9 had no effect on these processes. MMP-7 is capable of processing a number of nonmatrix molecules to soluble active forms that have profound effects on cell-cell communication, such as RANKL, a crucial mediator of osteoclast precursor recruitment and maturation. Therefore, the ability of osteoclast-derived MMP-7 to promote RANKL solubilization in the tumor-bone microenvironment was explored. Results revealed that levels of soluble RANKL were significantly lower in the MMP-7 null mice compared with wild-type (WT) controls. In keeping with this observation, MMP-7 null mice had significantly fewer osteoclast numbers at the tumor-bone interface compared with the WT controls. In summary, we propose that the solubilization of RANKL by MMP-7 is a potential mechanism through which MMP-7 mediates mammary tumor–induced osteolysis. Our studies indicate that the selective inhibition of MMP-7 in the tumor-bone microenvironment may be of benefit for the treatment of lytic breast-to-bone metastases. [Cancer Res 2009;69(16):6747–55]