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Immune checkpoint blockade has been largely unsuccessful for the treatment of bone metastatic castrate-resistant prostate cancer (mCRPC). Here, we report a combinatorial strategy to treat mCRPC using γδ-enriched chimeric antigen receptor (CAR) T cells and zoledronate (ZOL). In a preclinical murine model of bone mCRPC, γδ CAR-T cells targeting prostate stem cell antigen (PSCA) induced a rapid and significant regression of established tumors, combined with increased survival and reduced cancer-associated bone disease. Pretreatment with ZOL, a U.S. Food and Drug Administration–approved bisphosphonate prescribed to mitigate pathological fracture in mCRPC patients, resulted in CAR-independent activation of γδ CAR-T cells, increased cytokine secretion, and enhanced antitumor efficacy. These data show that the activity of the endogenous Vγ9Vδ2 T cell receptor is preserved in CAR-T cells, allowing for dual-receptor recognition of tumor cells. Collectively, our findings support the use of γδ CAR-T cell therapy for mCRPC treatment.

Table S1: Crystallographic data collection and refinement statistics Table S2: Inhibitory activity against other BET bromodomains Table S3: Bromodomain profiling data Fig. S1: Detailed binding interactions of compounds 1 - 5 in BRD4-1 Fig. S2: Comparison of compound 2 and JQ1 in Brd4-1 Fig. S3: Chemical structures of other compounds used in this work. Fig. S4: Statistical evaluation of kinome profiling data using the Gini coefficient Fig. S5: Original flow cytometry data of MM1.S cells Fig. S6: MM1.S cells require stimulation with IL-6 to detect phospho-STAT3 levels Fig. S7: Effect of dual BRD4-kinase inhibitors on MV-4-11 cells. Fig. S8: Time dependent alteration of c-Myc and p21Cip1 levels in MM1.S cells. Fig. S9: Effect of dual BET-kinase inhibitors on ruxolitinib sensitive MPN cell lines Fig. S10: Dose-effect analysis of UKE-1 cells treated with drug combinations Fig. S11: Cell line sensitivity by tissue type

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.

Table S1. UV calibration line parameters for BMMPI quantification. Figure S1. MMP-2 expression in murine and human breast cancer cell lines and BMMPI structure. Figure S2. Selective MMP-2 inhibition by the BMMPIs ML104 and ML115. Figure S3. Impact of BMMPIs on 4T1 bone metastatic breast cancer growth and osteolysis. Figure S4. ML115 is rapidly cleared from the plasma and accumulates in the bone marrow microenvironment over time. effects.Figure S5. The addition of exogenous TGFβ protects breasts cancer cells from BMMPI

Supplementary Data from Osteoclast-Derived Matrix Metalloproteinase-9 Directly Affects Angiogenesis in the Prostate Tumor–Bone Microenvironment

Table S4: Kinome profiling data (Microsoft Excel sheet) Table S5: Cell line screening data (Microsoft Excel sheet)

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.

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.

All Supplementary Figures and Legends

Supplemental Figure 1 and Movie 1. Simulation images from the computational homeostatic basic multicellular unit (BMU) including parallel images of RANKL, TGFβ and bone derived factors including insulin like growth factor of the course of bone resorption and apposition. Supplemental Figure 2. Simulation images from the computational prostate tumorbone microenvironment model including parallel images of RANKL, TGFβ and bone derived factors of the course of 200 days. Supplemental Figure. 3. Plot shows that the behavior of the biological model (blue line) and computational model (black line) produce comparable outputs and show similar trends. Supplemental Figure 4. Determining the importance of MSC recruitment in the computational model. Supplemental Figure 5. Analysis of dosing efficacy in the computational model.

Supplementary Figure Legend from Effect of Ablation or Inhibition of Stromal Matrix Metalloproteinase-9 on Lung Metastasis in a Breast Cancer Model Is Dependent on Genetic Background

Supplementary Figures 1-5 from Osteoclast-Derived Matrix Metalloproteinase-7, but Not Matrix Metalloproteinase-9, Contributes to Tumor-Induced Osteolysis

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]

Normal bone remodeling.

Supplementary Table 1 from Effect of Ablation or Inhibition of Stromal Matrix Metalloproteinase-9 on Lung Metastasis in a Breast Cancer Model Is Dependent on Genetic Background

Supplementary Figures 1-2 from Tie2 Signaling Regulates Osteoclastogenesis and Osteolytic Bone Invasion of Breast Cancer

Supplementary Figure 1 from Matrix Metalloproteinase 7 Mediates Mammary Epithelial Cell Tumorigenesis through the ErbB4 Receptor

Prostate cancer bone microenvironment.

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

Supplementary Figure 1 from Effect of Ablation or Inhibition of Stromal Matrix Metalloproteinase-9 on Lung Metastasis in a Breast Cancer Model Is Dependent on Genetic Background

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.

Multiple myeloma (MM) is an osteolytic plasma cell malignancy that, despite being responsive to therapies such as proteasome inhibitors, frequently relapses. Understanding the mechanism and the niches where resistant disease evolves remains of major clinical importance. Cancer cell intrinsic mechanisms and bone ecosystem factors are known contributors to the evolution of resistant MM but the exact contribution of each is difficult to define with currentin vitroandin vivomodels. However, mathematical modeling can help address this gap in knowledge. Here, we describe a novel biology-driven hybrid agent-based model that incorporates key cellular species of the bone ecosystem that control normal bone remodeling and, in MM, yields a protective environment under therapy. Critically, the spatiotemporal nature of the model captures two key features: normal bone homeostasis and how MM interacts with the bone ecosystem to induce bone destruction. We next used the model to examine how the bone ecosystem contributes to the evolutionary dynamics of resistant MM under control and proteasome inhibitor treatment. Our data demonstrates that resistant disease cannot develop without MM intrinsic mechanisms. However, protection from the bone microenvironment dramatically increases the likelihood of developing intrinsic resistance and subsequent relapse. The spatial nature of the model also reveals how the bone ecosystem provides a protective niche for drug sensitive MM cells under treatment, consequently leading to the emergence of a heterogenous and drug resistant disease. In conclusion, our data demonstrates a significant role for the bone ecosystem in MM survival and resistance, and suggests that early intervention with bone ecosystem targeting therapies may prevent the emergence of heterogeneous drug resistant MM.

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.

Bone-forming osteoblasts and -resorbing osteoclasts control bone injury repair, and myeloid-derived cells such as monocytes and macrophages are known to influence their behavior. However, precisely how these multiple cell types coordinate and regulate each other over time within the bone marrow to restore bone is difficult to dissect using biological approaches. Conversely, mathematical modeling lends itself well to this challenge. Therefore, we generated an ordinary differential equation (ODE) model powered by experimental data (osteoblast, osteoclast, bone volume, pro- and anti-inflammatory myeloid cells) obtained from intra-tibially injured mice. Initial ODE results using only osteoblast/osteoclast populations demonstrated that bone homeostasis could not be recovered after injury, but this issue was resolved upon integration of pro- and anti-inflammatory myeloid population dynamics. Surprisingly, the ODE revealed temporal disconnects between the peak of total bone mineralization/resorption, and osteoblast/osteoclast numbers. Specifically, the model indicated that osteoclast activity must vary greatly (> 17-fold) to return the bone volume to baseline after injury and suggest that osteoblast/osteoclast number alone is insufficient to predict bone the trajectory of bone repair. Importantly, the values of osteoclast activity fall within those published previously. These data underscore the value of mathematical modeling approaches to understand and reveal new insights into complex biological processes.

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.

Bone metastatic prostate cancer significantly impacts patient quality of life and overall survival, and despite available therapies, it is presently incurable with an unmet need for improved treatment options. As mediators of tumor progression, matrix metalloproteinases (MMPs) can degrade extracellular matrix components and regulate growth factor and cytokine bioactivity. Depending on tissue context, MMPs can either promote or inhibit tumorigenesis. Therefore, it is essential to study individual MMPs in specific cancer contexts and microenvironments to support the design and application of selective MMP inhibitors. Here we report that tumor-derived MMP-3 contributes to bone metastatic prostate cancer progression via intrinsic and extrinsic routes. MMP-3 ablation in prostate cancer cell lines significantly reduced in vitro growth combined with lowered AKT and ERK phosphorylation and total VEGFR1 and FGFR3 protein levels. In vivo, MMP-3 ablated tumors grew at a slower rate and were significantly less vascularized. Quantitative PCR analyses of wild type and MMP-3 silenced prostate cancer cells also demonstrate downregulation of a wide array of angiogenic factors. The extrinsic role for MMP-3 in angiogenesis was supported by in vitro endothelial tube formation assays where the lack of MMP-3 in prostate cancer conditioned media resulted in slower rates of tube formation. Taken together, our results suggest that tumor-derived MMP-3 contributes to prostate cancer growth in bone. These data indicate that selective inhibition of MMP-3 and/or targeting MMP generated products could be efficacious for the treatment of prostate to bone metastases.

Myeloid-derived monocyte and macrophages are key cells in the bone that contribute to remodeling and injury repair. However, their temporal polarization status and control of boneresorbing osteoclasts and bone-forming osteoblasts responses is largely unknown. In this study, we focused on two aspects of monocyte/macrophage dynamics and polarization states over time: 1) the injury-triggered pro- and anti-inflammatory monocytes/macrophages temporal profiles, 2) the contributions of pro-versus anti-inflammatory monocytes/macrophages in coordinating healing response. Bone healing is a complex multicellular dynamic process. While traditional in vitro and in vivo experimentation may capture the behavior of select populations with high resolution, they cannot simultaneously track the behavior of multiple populations. To address this, we have used an integrated a coupled ordinary differential equations (ODEs)-based framework describing multiple cellular species to in vivo bone injury data in order to identify and test various hypotheses regarding bone cell populations dynamics. Our approach allowed us to infer several biological insights including, but not limited to,: 1) anti-inflammatory macrophages are key for early osteoclast inhibition and pro-inflammatory macrophage suppression, 2) pro-inflammatory macrophages are involved in osteoclast bone resorptive activity, whereas osteoblasts promote osteoclast differentiation, 3) Pro-inflammatory monocytes/macrophages rise during two expansion waves, which can be explained by the anti-inflammatory macrophages-mediated inhibition phase between the two waves. In addition, we further tested the robustness of the mathematical model by comparing simulation results to an independent experimental dataset. Taken together, this novel comprehensive mathematical framework allowed us to identify biological mechanisms that best recapitulate bone injury data and that explain the coupled cellular population dynamics involved in the process. Furthermore, our hypothesis testing methodology could be used in other contexts to decipher mechanisms in complex multicellular processes.Author SummaryMyeloid-derived monocytes/macrophages are key cells for bone remodeling and injury repair. However, their temporal polarization status and control of bone-resorbing osteoclasts and boneforming osteoblasts responses is largely unknown. In this study, we focused on two aspects of monocyte/macrophage population dynamics: 1) the injury-triggered pro- and anti-inflammatory monocytes/macrophages temporal profiles, 2) the contributions of pro-versus anti-inflammatory monocytes/macrophages in coordinating healing response. In order to test various hypotheses regarding bone cell populations dynamics, we have integrated a coupled ordinary differential equations-based framework describing multiple cellular species to in vivo bone injury data. Our approach allowed us to infer several biological insights including: 1) anti-inflammatory macrophages are key for early osteoclast inhibition and pro-inflammatory macrophage suppression, 2) pro-inflammatory macrophages are involved in osteoclast bone resorptive activity, whereas osteoblasts promote osteoclast differentiation, 3) Pro-inflammatory monocytes/macrophages rise during two expansion waves, which can be explained by the anti-inflammatory macrophages-mediated inhibition phase between the two waves. Taken together, this mathematical framework allowed us to identify biological mechanisms that recapitulate bone injury data and that explain the coupled cellular population dynamics involved in the process.

The adoptive transfer of genetically engineered T cells expressing a chimeric antigen receptor (CAR) has shown remarkable results against B cell malignancies. This immunotherapeutic approach has advanced and expanded rapidly from preclinical models to the recent approval of CAR-T cells to treat lymphomas and leukemia by the Food and Drug Administration (FDA). Ongoing research efforts are focused on employing CAR-T cells as a therapy for other cancers, and enhancing their efficacy and safety by optimizing their design. Here we summarize modifications in the intracellular domain of the CAR that gave rise to first-, second-, third- and next-generation CAR-T cells, together with the impact that these different designs have on CAR-T cell biology and function. Further, we describe how the structure of the antigen-sensing ectodomain can be enhanced, leading to superior CAR-T cell signaling and/or function. Finally we discuss how tissue-specific factors may impact the clinical efficacy of CAR-T cells for bone and the central nervous system, as examples of specific indications that may require further CAR signaling optimization to perform in such inhospitable microenvironments.

EphA2 receptor tyrosine kinase (RTK) is highly expressed in breast tumor cells across multiple molecular subtypes and correlates with poor patient prognosis. In this study, the potential role of EphA2 in this clinically relevant phenomenon is investigated as metastasis of breast cancer to bone is a major cause of morbidity and mortality in patients. It was found that the EphA2 function in breast cancer cells promotes osteoclast activation and the development of osteolytic bone disease. Blocking EphA2 function molecularly and pharmacologically in breast tumors reduced the number and size of bone lesions and the degree of osteolytic disease in intratibial and intracardiac mouse models, which correlated with a significant decrease in the number of osteoclasts at the tumor–bone interface. EphA2 loss of function in tumor cells impaired osteoclast progenitor differentiation in coculture, which is mediated, at least in part, by reduced expression of IL‐6. EPHA2 transcript levels are enriched in human breast cancer bone metastatic lesions relative to visceral metastatic sites; EphA2 protein expression was detected in breast tumor cells in bone metastases in patient samples, supporting the clinical relevance of the study's findings. These data provide a strong rationale for the development and application of molecularly targeted therapies against EphA2 for the treatment of breast cancer bone metastatic disease. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Bone metastatic prostate cancer (PCa) promotes mesenchymal stem cell (MSC) recruitment and their differentiation into osteoblasts. However, the effects of bone-marrow derived MSCs on PCa cells are less explored. Here, we report MSC-derived interleukin-28 (IL-28) triggers prostate cancer cell apoptosis via IL-28 receptor alpha (IL-28Rα)-STAT1 signaling. However, chronic exposure to MSCs drives the selection of prostate cancer cells that are resistant to IL-28-induced apoptosis and therapeutics such as docetaxel. Further, MSC-selected/IL-28-resistant prostate cancer cells grow at accelerated rates in bone. Acquired resistance to apoptosis is PCa cell intrinsic, and is associated with a shift in IL-28Rα signaling via STAT1 to STAT3. Notably, STAT3 ablation or inhibition impairs MSC-selected prostate cancer cell growth and survival. Thus, bone marrow MSCs drive the emergence of therapy-resistant bone metastatic prostate cancer yet this can be disabled by targeting STAT3., The effects of bone-marrow derived MSCs on prostate cancer cells remain unknown. Here the authors show that MSC-derived IL-28 induces prostate cancer cell apoptosis via IL-28Rα-STAT1 signalling, while acquired resistance to apoptosis is associated with a shift in IL-28Rα signalling via STAT1 to STAT3.

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.