Overcoming immunosuppression in bone metastases

Accepted Manuscript Title: Overcoming immunosuppression in bone metastases Authors: Zachary Reinstein, Sahithi Pamarthy, Vinay Sagar, Ricardo Costa, Sarki A. Abdulkadir, Francis J. Giles, Benedito A. Carneiro PII: DOI: Reference:

S1040-8428(17)30179-8 http://dx.doi.org/doi:10.1016/j.critrevonc.2017.05.004 ONCH 2385

To appear in:

Critical Reviews in Oncology/Hematology

Received date: Revised date: Accepted date:

13-3-2017 30-4-2017 9-5-2017

Please cite this article as: Reinstein Zachary, Pamarthy Sahithi, Sagar Vinay, Costa Ricardo, Abdulkadir Sarki A, Giles Francis J, Carneiro Benedito A.Overcoming immunosuppression in bone metastases.Critical Reviews in Oncology and Hematology http://dx.doi.org/10.1016/j.critrevonc.2017.05.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Overcoming immunosuppression in bone metastases Zachary Reinstein1,2,3, Sahithi Pamarthy1,2, Vinay Sagar1,2, Ricardo Costa 1, Sarki A Abdulkadir2,4,5, Francis J Giles 1, Benedito A Carneiro1* 1

Developmental Therapeutics Program, Division of Hematology/Oncology, Feinberg School of Medicine, Chicago, USA. 2 The Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA 3 Department of Microbiology and Molecular Biology, Brigham Young University, Provo, Utah 86402, USA 4 Department of Urology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA 5 Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA

*

Corresponding author: Benedito A Carneiro, MD, MS Developmental Therapeutics Program, Division of Hematology/Oncology, Feinberg School of Medicine, Northwestern University 233 E Superior St Olson Pavilion Chicago, IL 60611 Phone: 312-472-1234 Fax: 312-472-0564 E-mail: [email protected]

Keywords: Bone metastases; immunosuppression; bone metastatic disease; skeletal related events Running title: Immunosuppression in bone metastases Contributions: ZR wrote, reviewed and revised the manuscript and tables, SP wrote, reviewed, and revised the manuscript and figure, VS contributed to the figure and reviewed/revised the manuscript. RC wrote, reviewed, and revised the manuscript. SAA, FJG, BAC conceived, reviewed and revised the manuscript. Acknowledgements: We would like to thank The Woman’s Board of Northwestern Memorial Hospital for their continued support.

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ABSTRACT

Bone metastases are present in up to 70% of advanced prostate and breast cancers and occur at significant rates in a variety of other cancers. Bone metastases can be associated with significant morbidity. The establishment of bone metastasis activates several immunosuppressive mechanisms. Hence, understanding the tumor-bone microenvironment is crucial to inform the development of novel therapies. This review describes the current standard of care for patients with bone metastatic disease and novel treatment options targeting the microenvironment. Treatments reviewed include immunotherapies, cryoablation, and targeted therapies. Combinatorial treatment strategies including targeted therapies and immunotherapies shows promise in pre-clinical and clinical studies to overcome the suppressive environment and improve treatment of bone metastases.

1. Introduction

Prevention and effective treatment of bone metastatic disease can have a significant impact in the outcomes of patients with advanced malignancies. The burden of bone metastatic disease is demonstrated by autopsy studies identifying bone metastases in approximately 70 % of breast and prostate cancer, 30-40 % of non-small cell lung cancer (NSCLC), and 20-35 % of thyroid and kidney cancer-related deaths [1]. It has been estimated that greater than 50 % of patients with bone metastases die within 3 years of diagnosis [2]. Furthermore, bone metastases are associated with skeletal related events (SRE) including pathological fractures, spinal cord compression, and hypercalcemia, which severely compromise patient quality of life (QoL) [3,4]. Even though most patients have concomitant metastases in visceral organs and lymph nodes, there is a subset of patients with bone-predominant metastatic disease that may require a distinct treatment approach compared to, for instance, patients with only pulmonary metastases based on site-specific impact on disease prognosis and, possibly, distinct patterns of response to systemic therapies such as chemotherapy and immunotherapies. Therefore, advancing the understanding of mechanisms involved in the establishment of bone metastases

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can lead to the identification of therapeutic targets with meaningful implication in the management of these patients. This manuscript reviews the challenges in treating bone metastases and highlights emerging therapeutic strategies exploring the interface between the bone microenvironment and immune system with the ultimate goal of fostering the advancement of novel bone-directed therapies.

2. Bone tumor microenvironment 2.1 Bone metastatic niche Tumor cells ‘seed’ the bone marrow in a multistep process [5]. Initially, circulating tumor cells (CTCs) create a pre-metastatic niche with the production of factors that render the bone microenvironment conducive to tumor dissemination [6]. Disseminated tumor cells (DTCs) compete with “hematopoietic stem cell niche” in the bone marrow, thereby creating an “onconiche” where they can proliferate or remain dormant. The bone marrow niche seems to protect DTCs from immune surveillance contributing to resistance to chemotherapy and post treatment relapses by several mechanisms.

Bone marrow DTCs from breast cancer lose expression of Ki67 and overexpress ERBB2 that might play a role in resistance to chemotherapy. DTCs at distant sites found after chemotherapy also express cytokeratin heterodimers CK8/CK18 and CK8/CK19 known to inhibit major histocompatibility complex 1 (MHC 1) interactions with CD8+ T cells, a crucial step in immune escape [7]. To overcome natural killer (NK) cell cytotoxicity following loss of MHC, DTCs adopt a stem cell phenotype with reduced expression of NK cell receptor D (NKG2D) and MHC1 polypeptide related sequence (MICA/MICB) [8]. Furthermore, DTCs home to the hematopoietic stem cell (HSC) niche, which is the most hypoxic area of bone marrow. It is known that increased expression of hypoxia induced factor (HIF1  in neoplastic cells enhances a disintegrin and metalloproteinase (ADAM10) which in turn cleaves MICA/MICB leading to suppression of antigen presentation and NK cell cytotoxicity [9]. Therefore, the hypoxic environment of HSC niche in bone marrow contributes to immune escape, stem-like properties of DTCs, and promotes resistance to chemotherapy [10].

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2.2 Osteomimicry It is well established that cancers like breast and prostate exhibit bone tropism and express bone associated genes and proteins, a phenomenon called “osteomimicry", which promotes their dissemination, survival, and proliferation in the bone [11]. Tumor cells homing to bone express molecules like osteonectin, cathepsin-K and connexins that are usually expressed by osteoclasts and osteoblasts. Prostate cancer cells acquire an osteoblast-like phenotype by expressing bone sialoprotein and osteocalccin. RUNX2 is a transcription factor crucial for osteoblast differentation and the expression of RUNX2 by cancer cells marks a key step in osteomimicry. The stem cell pathways Notch and Wnt were shown to be important for activation of hepatocyte growth factor (HGF) and its interaction with Met, another key pathway in osteomimicry [12]. Breast cancer cells also express osteoclast activating factors like parathyroid hormone-related protein (PTHrP) and TNF- to promote RANKL induced osteoclastogenesis [13]. Together, the expression of these molecules promotes colonization to bone, confers a survival advantage and initiates the tumor-bone vicious cycle.

2.3 Tumor-bone vicious cycle Bone is a significant site of metastatic disease because of its large surface area and high vascular supply. Tumor invasion to bone results in bone resorption and formation, a process induced by cancer cells and mediated by osteoblasts and osteoclasts [14]. Certain types of solid tumors metastasize to bone and induce osteolytic (bone destructive) or osteoblastic (bone formative) phenotypes. For example, metastases from prostate cancer frequently form osteoblastic lesions in contrast to the predominant osteolytic lesions associated with breast, lung and kidney cancers [15]. Tumor-derived signaling mediators like WNT, bone morphogenic proteins (BMPs) and transforming growth factor-beta (TGF-) stimulate osteoblast function by activating RUNX and activated transcription factor (ATF) signaling [16-18]. Activated osteoblasts secrete receptor activator of nuclear factor kappa  (RANKL), which binds to its receptor RANK on osteoclast precursors and induces osteoclastogenesis through NF-kB, NFATc1 and C-JUN

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signaling [19]. During the process of resorption, bone cells and the mineralized bone matrix release TGF-, IGF, and Ca2+ promoting tumor growth and release of osteolytic and osteoblastic factors like PTHrP, IL-6 and matrix metalloproteinases (MMPs) [20]. Thus, a “vicious cycle” of tumor-induced bone disease is formed, wherein tumor modifies the bone microenvironment to support its own survival [21].

2.4 Bone microenvironment – cellular and molecular mediators The vast immune cell composition of bone marrow including lymphocytes and myeloid cells exhibit both anti-tumor and tumor-promoting effects on bone metastases [22,23]. During an antitumor response, dendritic cells present tumor specific antigens to activate CD4+ T cells, which in turn activate CD8+ T cells leading to the killing of antigen positive tumor cells [24]. In contrast, activated regulatory T cells (T-Regs) and T helper cells type 17 (Th 17 cells) cause a protumor response through immune suppression and RANKL mediated osteoclast differentiation [25,26]. Additionally, myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) suppress T-cell mediated anti-tumor responses through production of cytokines and angiogenic factors [27,28]. In addition to the immune cells, endothelial cells, fibroblasts, and mesenchymal stem cells are recruited to promote neoangiogenesis and bone metastatic growth [29]. Tumor-derived exosomes were also shown to educate bone marrow progenitors to support tumor growth [30]. Together, the resident and infiltrated stromal cells and their molecular mediators constitute a unique bone metastatic microenvironment that results in sustained immunosuppression contributing to bone induced tumor growth (Figure 1). 2.5 Immune Evasion – mechanisms and implications Cancer cells thrive in the bone owing to their ability to escape immune surveillance by multiple mechanisms [31]. However, the role of the bone immune microenvironment in controlling or promoting metastatic progression remains open to further investigation. During normal physiological conditions, the combined CD4+ and CD8+ T cells constitute < 5% of mononuclear cells and NK cells make up to 1 -2% of lymphocyte populations. It is thought that metastatic

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dissemination selects tumor cells that are resistant to apoptosis and hence are immune to T and NK cell mediated cytotoxicity [32]. Metastatic tumor cells routinely lose the expression of MHC class 1 antigen presentation leading to immune evasion. A majority of metastatic tumor cells target several molecules key to the immune checkpoint pathway to achieve immune suppression. Programmed cell death ligand (PD-L1) is expressed primarily on cancer cells and binds to the programmed cell death (PD)-1 receptor on T cells to inhibit their activation [33]. The recruitment of T-regs that express cytotoxic T lymphocyte associated antigen (CTLA 4), and lymphocyte activation gene-3 (LAG-3) leads to suppression of T cell activity [34]. Cytotoxic T cells and T regs also co-express T cell immunoglobulin and mucin containing domain (TIM 3) with PD-1, key surface membrane immune checkpoint proteins mediating immune suppression [35]. Hypoxic tumor cells secrete high amounts of adenosine, which inhibits T cell function by binding to A2AR, another immune checkpoint protein expressed on T cells [36,37].

Recent reports point to the loss of interferon regulatory factor 7 (IRF7), a key mediator of type 1 interferon gene signature, increases the risk of bone metastases [38]. Latent metastatic cells silence Wnt Signaling pathway to enter the quiescent state and escape innate immune responses for extended periods [39]. Similarly, suppression of JAK-STAT pathway impairs NK cell-mediated antitumor activity [40]. Tumor-derived growth factors and cytokines like TGF- and IL-6 recruit cancer-associated fibroblasts (CAFs), which target T-cell activity and also recruit tumor-associated macrophages to promote immune evasion. Activated MDSCs also mediate immune suppression through the production of reactive oxygen species that inhibits T-cell effector functions [27]. In addition to immune checkpoint inhibition, circulating tumor cells aggregate with platelets to escape immune recognition and TNF- related cell death. Supportively, platelets release pro-angiogenic factors that promote tumor metastasis [41]. Although the crosstalk between bone, tumor, and immune cells is well-known, bone metastasis frequently results in resistance to chemotherapy and post-treatment relapses due to immune escape by disseminated tumor cells. Detailed investigations in the field of tumor osteoimmunology are warranted to understand the mechanisms of immunosurveillance in tumor-induced bone disease [42] .

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3. Standard of care treatment of bone metastases

Bone metastases are treated with the same approaches as metastatic disease involving other sites (i.e., chemotherapy, radiation therapy, and immunotherapy) with few exceptions of bonedirected therapy such as Radium-223 approved for metastatic prostate cancer. Osteoclast inhibitors such as bisphosphonates and denosumab are considered supportive therapies used to reduce the risk of SREs. Bisphosphates (i.e., zoledronic acid) stimulate osteoblasts and inhibit osteoclast differentiation and survival by binding to hydroxyapatite in the bone with selective inhibition of farnesyl pyrophosphate synthase in active areas of bone remodeling [43]. Denosumab, a fully human monoclonal antibody, binds to RANKL expressed on the surface of osteoblasts blocking the activation of the receptor (RANK) present on osteoclasts[44]. Denosumab disrupts the homeostasis of osteoblast and osteoclast activation and differentiation leading to a reduction of osteoclasts and osteoclast activity inhibition [45].

Zoledronic acid is usually given on every 4-week schedule however preliminary results from the CALGB 70604 phase 3 non-inferiority trial supported equivalent efficacy of two different dose schedules for the prevention of SRE among patients with metastatic cancer to the bones (i.e., breast cancer, prostate cancer, multiple myeloma) [46]. In light of the results of this trial zoledronic acid may be administered either monthly or every 3 months for patient with metastatic bone disease [47]. Approved by the FDA in 2013, denosumab was shown to significantly decrease skeletal-related events (SRE) compared to zoledronic acid. In addition, denosumab demonstrated fewer adverse events compared to zoledronic acid [48]. More recently, 3420 women with hormone-receptor positive breast cancer receiving adjuvant treatment with aromatase inhibitors were randomized to adjuvant treatment with denosumab vs. placebo every 6 months in the adjuvant denosumab in breast cancer trial (ABCSG-18). The patients who received denosumab had a significantly delayed time to first clinical fracture (HR 0.50, P<0.0001) [49]. After a median follow-up of 4 years, 203 disease free survival (DFS) events were observed in the placebo group, and 167 in the denosumab group (HR 0.81, P=0.051) [50].

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Similar findings have been reported with adjuvant treatment with bisphosphonates [51]. These results suggest that bone-modifying agents not only reduce the incidence of SRE in the adjuvant treatment of breast cancer but may also have anti-tumor activity in a subset of patients with localized breast cancer.

Radiopharmaceuticals have also been used as a targeted therapy to bone metastases. These drugs act by releasing alpha, beta or gamma radiation to target tumor cells as the compounds accumulate in the bone matrix [52]. Both SR-89 and SM- 153 have been approved since the 1990’s and have shown to improve pain from bone metastases and reduce SREs [52]. Radium223 has been approved by the FDA for treatment of symptomatic metastatic CRPC with disease limited to the bones based on improvement in SREs, and pain when compared to SR-89/SM-153 but also was the first radiopharmaceutical to show an increase in overall survival [53]. Combination clinical studies of anti-androgens abiraterone and enzalutamide with Radium-223 and denosumab are ongoing [52].

In cancers with a high incidence of bone metastases, there are 3 classes of immunotherapies that have been approved. The first, immune checkpoint inhibitors include nivolumab (anti-PD1), ipilimumab (anti-CTLA-4), pembrolizumab (anti-PD-1), and atezolizumab (anti-PD-L1). All of these therapies are humanized antibodies against the immune checkpoints [54]. The next class is cytokine therapy which includes interferon , and interleukin-2. Both of these cytokines induce T-cell proliferation [55]. The dendritic cell therapy sipuleucel-T promotes stimulation of prostate cancer antigen specific T-cells [4]. While checkpoint inhibitors and other classes of immunotherapeutics have shown significant efficacy in controlling visceral metastatic disease in several malignancies, their efficacy specifically in bone metastases is not well understood. The bone-metastatic microenvironment displays a unique immune phenotype that could result in a distinct pattern of response to immune therapy when compared to other metastatic sites.

4. Emerging Therapies for Bone Metastases

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4.1 Targeting the “Vicious Cycle”

Advances in targeted therapies have led to the approval of multiple small molecule inhibitors that target both tumor cells and the microenvironment. For the purposes of this review, the focus will be those targeted therapies, approved and in development, that specifically affect the tumor-bone microenvironment and immune suppression.

The "vicious cycle" of bone metastases involves the feedback loop between the bone-derived growth factors (RANKL, PTHrP, IL-1, IL-6, and IL-8) and tumor-derived osteolytic factors (TGF-, FGF, PDGF) [56]. Multiple therapies are being developed to target this cycle.

Osteoprotegerin (OPG) is a natural inhibitor of RANKL, and consequently, inhibits osteoclastogenesis. Yet, multiple experiments studying OPG administered systemically versus OPG produced by tumor cells at various concentrations showed conflicting results in regards to tumor progression and bone resorption [57-62]. Ryser et al devised a mathematical model of the OPG absorption gradient alongside a model of bone remodeling to determine the effects of OPG at different concentrations and demonstrated the optimal concentrations of OPG for therapeutic effect [63]. Additional research is necessary for validation and development of therapies.

Multiple strategies have been developed to inhibit TGF-with the most recent successes seen in small molecule inhibitors (galunisertib) and vaccinations (belagenpumatucel-L and gemogenovatucel-T) [64]. OS benefit was seen in phase II trials of galunisertib and belagenpumatucel-L. In addition, both of these treatments reduced TGF-and overall metastatic burden [65,66]. While bone metastases were not specifically measured in these studies, due to the crucial role of TGF- in the vicious cycle, these results should merit research in patients with bone metastases.

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51 is an integrin that binds to fibronectin in the ECM and regulates tumor invasion and angiogenesis as mediated by (MMP-1) [67]. Thus 51 inhibition of the receptor in fibronectin prevents metastases and angiogenesis, especially in regards to the vicious cycle of osteolytic lesions [68]. Ac-PHSCN-NH2, a small peptide inhibitor showed no objective responses but showed progression-free survival in breast cancer patients for up to 14 months [69]. A second generation Ac-PhScN-NH2 inhibitor containing D-isomers of histidine (h) and cysteine (c)—is over 100,000-fold more potent at blocking basement membrane invasion by two breast cancer cells lines. Monotherapy in a mouse model reduced intratibial colony progression by almost 80 %[68].

P62 is a multi-functional protein that plays a significant anti-inflammatory role as well as induction of RANKL-stimulated osteoclastogenesis [70,71]. P62 is overexpressed in tumor tissues but not in normal tissue, making it an attractive target [72]. Because of this and the dual roles of P62, inhibition has been seen as a promising therapy to increase immune function in the tumor microenvironment and treatment of osteolytic lesions [72]. Three studies in animal models looking at the effectiveness of a DNA vaccine targeted against P62 saw preliminary antitumor activity with responses in tumor size and suppression of metastases. In addition, increased lymphocyte infiltration and suppression of osteoporosis was observed [71,73,74]. It has been hypothesized that in osteolytic tumors, these dual effects may prove this P62 vaccine highly effective [72].

Cabozantinib is a multi-targeted tyrosine kinase inhibitor which inhibits MET, vascular endothelial growth factor receptor 2 (VEGFR-2) as well as other tyrosine kinases including RET, KIT, AXL and FLT3, approved for medullary thyroid cancer and renal cell carcinoma [75]. MET is particularly overexpressed in bone metastases [76]. It has also been investigated in mCRPC after it showed significant inhibition of osteoblastic and osteolytic lesions in a mouse xenograft model [76]. Results in two, phase II trials also showed promising results, displaying an increase of progression-free survival from 5.9 weeks to 23.9 weeks compared to placebo, 63% bone scan response and pain palliation in 57% of patients [77,78]. These promising results led to two

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large-scale phase III trials known at COMET I/II. Although an improvement in patient free survival (PFS) was noted, unfortunately, no overall survival advantage was observed in the larger cohort [79,80]. However, in the METEOR trial investigating cabozantinib as a second-line therapy in advanced renal cell carcinoma, OS survival benefit was observed when compared to everolimus [81]. Most interesting was that in patients with bone metastases, the hazard ratio was significantly reduced when compared to patients with no metastases. Choueiri et al hypothesize that this effect is due to cabozantinib specific action against bone metastases based on its ability to inhibit Met signaling, but further research is warranted before cabozantinib used as a specific bone-targeted therapy [81,82].

4.2 Targeting immunosupressive cells

As previously noted, TAMs play a crucial immunosuppressive role in the tumor microenvironment. Multiple drugs are being used or are in development to target TAMs. Interestingly, bisphosphates, like zoledronic acid, which inhibit osteoclasts to prevent bone reabsorption in osteolytic lesions, also play a similar role on TAMs [56]. Macrophages are of the same lineage as osteoclasts, and multiple studies point to zoledronic acid causing a change of TAM phenotype from the pro-tumoral M2 to the anti-tumoral M1 [56]. PLX3397 is another promising multi-targeted tyrosine kinase inhibitor in development that can target TAMs. PLX3397 inhibits colony stimulating factor 1 (CSF-1) which is responsible for recruiting immunosuppressive cells including M2 macrophages and MDSCs to the bone microenvironment [83]. Current preclinical data shows its efficacy in inhibiting the recruitment of immunosuppressive myeloid cells as well as improving the immune response in combination with other therapies [83-85]. Similarly, Bindarit targets chemokine (C-C motif) ligand 2 (CCL2), another cytokine responsible for macrophage recruitment. Preclinical studied have shown responses in animal models as well as the prevention of the establishment of the tumorstromal niche inherent to bone metastases [86,87].

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Tasquinimod is second generation, quinoline-3-carboxamide, oral small molecule inhibitor that blocks the interactions of S100A9, a calcium and zinc binding protein involved in many inflammatory response pathways. It targets the tumor microenvironment by inhibition of angiogenesis and immune suppression mediated by TAMs, and prevention of the establishment of the bone-metastatic niche [88,89]. A phase II, randomized, double-blind trial of tasquinimod in mCRPC documented improvement of progression-free survival (PFS) (7.6 vs. 3.3 months, P=.0042) especially among patients with bone metastases (PFS 8.8 months) [90]. These results supported a phase III trial randomizing 1,245 patients with mCRPC to receive tasquinimod or placebo that showed a significant improvement of rPFS (7.0 vs. 4.4 months, HR= 0.64, P=0.001). No overall survival benefit was observed which decreased enthusiasm for further research with this drug [91].

Fibroblast are the key component of the immunosuppressive tumor stroma, therefore inhibitors targeting fibroblasts have significant therapeutic potential. Fibroblasts in idiopathic pulmonary fibrosis highly resemble cancer-associated fibroblasts (CAFs) with their expression of FAP. Thus, two recently approved drugs used to treat idiopathic pulmonary fibrosis, pirfenidone, and nintedanib, are being used to target CAFs in the tumor microenvironment, but results remain unpublished [36].

Although no current drugs have been developed to exclusively target immunosuppressive cells in the bone-tumor stroma, many current targeted therapies have off-target effects against immunosuppressive MDSCs and Tregs. TKIs like suntinib and imatinib block the STAT3 and IDO pathways. By inhibiting these pathways, maturation of MDSCs or Tregs is also inhibited thereby reducing their numbers in the bone-tumor microenvironment [92-95]. Bevacizumab, a VEGF antibody, plays a similar role by shifting monocyte development towards DCs instead of MDSCs in the bone marrow [96]. mTOR inhibitors like rapamycin and temsirolimus also act on the IDO pathway which in turn impairs the homeostasis and subsequent effectiveness of Tregs [97-99]. In addition, many of these target therapies have shown immunostimulating properties by acting on DCs and cytotoxic T-cells [100]. All of these techniques demonstrate a new treatment

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paradigm of not just targeting the tumor cells, but also targeting the tumor-supportive microenvironment. Clinical studies have reflected this paradigm shift, with many studies exploring combinatorial approaches.

4.3 Increasing the Immune Response in Bone Metastases

In contrast to small molecule inhibitors, modulation of the immune system is also being investigated as a viable treatment option of bone metastases. Due to the unique immunoenvironment in bone metastases, adoptive cell transfer (ACT), vaccines, chimeric antigen receptor (CAR) therapies and other immunotherapies have been modified in order to increase efficacy in the bone metastatic microenvironment with promising results in various cancer models.

Methods to increase T-cell invasion into solid tumors have been developed and implemented for several years. Currently, adoptive cell transfer is only approved for melanoma and leukemia, but many other techniques are being developed to increased T-cell infiltration and efficacy with or without ex vivo expansion for bone metastases.

4.3.1 Adoptive cell transfer

Adoptive cell transfer (ACT) has been defined as “the administration of tumor-specific lymphocytes (obtained from the patient (autologous) or from a donor (allogeneic)) following a lymphodepleting preparative regimen” [101]. It holds promise for treatment of bone metastases, yet studies have shown that even after ACT T-cells in metastases still face heavy immunosuppression [102]. The most important method to improve the efficacy of ACT is lymphodepletion through whole-body irradiation or cytotoxic chemotherapy. Two studies of ACT in solid tumors saw complete response only after lymphodepletion protocols [103,104].

4.3.2 Dendritic Cell Therapy (Vaccines)

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Dendritic cells are the primary promoter of a tumor-specific T-cell response through antigen presentation. Thus, several methods have been developed to improve on the immunogenicity of DCs in order to improve the anti-tumoral T-cell response and overcome bone metastatic immunosuppression.

With the recent approval of sipuleucel-T in prostate cancer, DC-based vaccines have been extensively researched to treat solid tumors and bone metastases. Vaccines can either be derived from (1) antigenic peptides of a tumor-associated antigen (TAA), like sipuleucel-T (2) whole tumor cells or (3) mRNA of TAAs [105]. In comparison with vaccines derived from antigenic peptides, those derived from whole tumor cells (either lysates or fusions with DCs) are significantly more immunogenic. These therapies are applicable to all patients, regardless of HLA-type [105]. In addition, multiple, polyclonal TAA responses have been demonstrated with long-term immunity and prevention of immune evasion [106,107]. mRNA-based vaccines have also been shown to be superior to antigenic peptide-based vaccines [108].

Novel techniques have been developed to better activate bone-marrow DCs for an anti-tumor response. Two methods using carbon black nanoparticles and antigens with mannosylated dendrimers were shown to significantly activate bone marrow DCs and increase T-cell stimulating capacity of DCs up to two fold [109,110]. Just as exosomes play a crucial role in establishing the bone metastatic niche [30]. DC-derived exosomes are capable of establishing an anti-tumoral T-cell response [111]. Multiple pre-clinical studies showed the high potency of DC-derived exosome-based therapy, including the eradication of established tumors in a mouse model [112,113]. Three phase I trials in metastatic melanoma, non-small cell lung cancer, and colorectal cancer showed high tolerability to the treatment, with only grade 1 or 2 adverse reactions. Stable disease and partial responses were observed, but due to the modest efficacy of the treatment, only one phase 2 trial is being studied [114-116]. The lower efficacy may be a limitation of the TAA used and not the exosome therapy itself. With newly identified TAAs,

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these therapies may provide a highly effective and highly tolerated immunotherapy for bone metastases.

DCs can also be directly targeted through various treatments. These include sialidase [117], Toll-like receptor agonists (resiquimod, imiquimod, poly ICLC), and another DC-targeted adjuvant, Montanide. These treatments cause a wide range of effects including activation of lymphocytes, macrophages and DCs, while time transforming the tumor microenvironment by directly inducing apoptosis of tumor cells [118-123][124]. Clinical trials have not studied bone metastases directly, but described significant responses suggesting that DC-targeted treatments could interfere with the tumor-bone microenvironment [123,125-128].

4.3.3 CARs

CARs have also been targeted directly against the microenvironment. CARs targeted against cancer-associated fibroblasts (CAFs) present in bone metastases have seen significant efficacy. It is known that depletion of fibroblast activation protein (FAP)+ CAF reverses immunosuppression and activates T-cells [129,130]. Therefore, vaccines and CARs that target FAP have shown anti-tumor activity. Yet, when combined with vaccination or direct targeting of TAAs, the effect has been shown to be synergistic— decreasing tumor volume and increasing survival in mouse models [131-133]. Interestingly, a bispecific antibody against FAP and death receptor 5 (DR5) has shown to induce apoptosis in the non-functional cell death pathway. Most remarkable of this study was the low toxicity and complete regression of tumors when combined with irinotecan or doxorubicin in patient-derived xenograft models [134].

CARs engineered in conjunction with chemokine receptors have shown higher affinity towards the tumor microenvironment. Specifically, when chemokine (C-C motif) receptor (CCR) 2, was co-engineered with a CAR, T-cells were better trafficked to multiple types of tumor metastases [135-137]. Interestingly, the ligand for CCR2 is CCL2, which we have already shown has been implicated with macrophage recruitment and the establishment of the bone-stromal niche [86].

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For homing specificity to prostate bone metastases, the chemokine stromal cell-derived factor (SDF)-1 also known as Chemokine (C-X-C motif) ligand (CXCL) 12 has been identified [138-140]. By engineering its receptor, chemokine C-X-C motif receptor (CXCR) 4, into CAR T cells, precise trafficking to prostatic bone metastases could be ensured [141].

Additionally, Bispecific T-cell Engagers (BiTEs) are also chimeric proteins with two scFVs to bind to CD3 on T-cells and to a surface antigen on tumor cells. BiTEs have been developed to target tumor-associated antigens in prostate cancer, melanoma, lung cancer and various other cancers [142]. BiTEs hold promise in the treating bone metastases as they are not directly affected by the immunosuppression. Although these proteins can be quickly produced and show low toxicities, they generally have less efficacy when compared to CAR therapies due to the inability to induce T-cell memory [141].

4.3.4 Improving T cell efficacy through release of TAAs

Cryoablation of skeletal metastases has been shown to bring significant pain palliation [143]. In addition to directly killing tumor tissue, cryoablation augments the immune response by the rapid release of TAAs through necrotic tumor cells [144-148]. After these responses had been observed, other studies were conducted using immune adjuvants, and subsequent improvements of tumor immunity were detected [148-150]. In these studies, complete suppression of tumor growth was observed in most mouse models. Yet, the mechanism of resistance in the few tumors that failed to respond remained unstudied until recently. The expansion of activated leukocyte cell adhesion molecule (ALCAM) positive cells was identified as the immunosuppressant mechanism in these models. When an anti-ALCAM antibody, combined with an anti-CTLA antibody and cryoablation was performed, this resistance was completely reversed, with objective responses observed in 100% of mice, and complete responses observed in 93% of mice studied [151]. These studies show that not only can cryoablation drive a significant anti-tumor response, but when resistance to these treatments

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occurs, xenograft studies suggest that through the implementation of an anti-ALCAM antibody, this immunosuppression can almost be completely reversed.

In the case of prostate cancer, anti-androgen therapy by abiraterone and enzalutamide caused apoptotic release of TAAs and improved the sensitivity of T-cell-mediated lysis of tumor cells [152]. Radiation therapy is another suggested mechanism of antigen release to improve T-cell response [141].

Some modest improvement in immunotherapy response can be seen by increasing the number of tumor-infiltrating lymphocytes. In bone metastases, aberrant angiogenic activity promotes the proliferation of tumor cells, especially in bone metastases. Yet, due to the low quality of blood vessels, T-cells are unable to proliferate in the microenvironment [141,153]. Although anti-vascular endothelial growth factor (VEGF) treatments are meant to completely inhibit the formation of new vasculature, when used in lower doses, normalization of vasculature occurs, chemokine T-cell attractants are upregulated, and consequent T-cell infiltration to metastases occurs [153]. A synergistic reduction of tumor growth was observed when anti-angiogenic therapy was combined with ACT [154,155]. In addition, acute bacterial inflammation, in this case, directed at the prostate, increases TILs in prostate cancer [156].

Although anti-CTLA-4 and anti-PD-1 antibodies are already approved as immune checkpoint inhibitors, it has been shown that blocking TGF- and TIM-3 improves the T-cell responses. In the case of TIM-3, antibody blockade prevents exhaustion of not only T-cells but also NK-cells [102,124].

4.3.5 Oncolytic Viruses

Oncolytic viruses specifically target certain tumor cells while promoting an immune response upon cell lysis [157]. Talimogene laherparepvec, or T-Vec, is currently the only approved oncolytic virus for treatment of melanoma [158]. Vesicular stomatitis virus (VSV) is another

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virus that is currently being studied for treatment of pancreatic cancer and leukemia [159,160]. VSV is very sensitive to IFN- response and therefore does not proliferate well in normal tissues or neoplastic tissues that do have an IFN- response[161]. One of the proposed pathways of acquired immune resistance in tumors is a defective Janis Kinase 1 (JAK1) pathway, which in turn prevents and IFN- response of antigen presentation [162]. Multiple studies in pancreatic cancer and head and neck cancer have shown that JAK 1 inhibition overcomes resistance to VSV [161,163]. Although it has not been studied, this proposed mechanism suggests that tumor cells that have acquired resistance to immune checkpoint therapies might be susceptible to infection and subsequent lysis by VSV. This provides another method to overcome resistance to immunotherapies and severe immunosuppression in the tumor-bone microenvironment

Conclusion Bone metastases are associated with significant morbidity and detrimental impact on overall outcomes of patients with several malignancies. This manuscript summarized the complexity of tumor-bone microenvironment and discussed potential therapeutic targets. Advancing the understanding of bone immune environment can inform treatment strategies including combinatorial strategies with checkpoint inhibitors.

18

Figure 1: Depiction of Bone-Tumor Immunology A) Establishment of the “Onco-niche”. B) Representation of the positive feedback mechanism of the tumor-bone vicious cycle. C) Endogenous and therapeutic anti-tumor responses. D) Pro-tumor mechanisms from the immune system.

19

Table 1. Standard of care treatment of bone metastases Treatment

SRE Treatments Zoledronic acid

denosumab

Radium-223

Immunotherapies nivolumab

Mechanism

Effect on Bone metastases

Notes

Refs

inhibition of farnesyl pyrophosphate synthase mAb against RANKL

Inhibition of Osteolysis

Action against TAMs as well Approved in adjuvant use

[164]

Localizes to bone, releases alpha radiation

Reduces osteoclast activity Cytotoxic to tumor cells by inducing dsDNA breaks

Anti-PD-1 mAb

ipilimumab

Anti-CTLA-4 mAb

pembrolizumab

Anti-PD-1 mAb

atezolizumab

Anti-PD-L1 mAB

Sipuleucel-T

Dendritic cells stimulated with GM-CSF and PAP

[164]

Offers less myelosuppression due to shorter range of alpha radiation

[52]

Currently unstudied in bone metastases Currently unstudied in bone metastases Currently unstudied in bone metastases Currently unstudied in bone metastases Unknown

[100]

20

Table 2. Emerging Targeted Therapies for Bone Metastases Treatment Mechanism Effect on Bone Outcomes metastases Tumor-targeted Osteoprotegerin Natural Inhibits Conflicting inhibitor of osteoclastogenesis outcomes RANKL and subsequent depending on bone reabsorption dose and type of administration Cabozantinib TKI, inhibits inhibition of PFS- 5.9 to 23.9 MET, VEGFR- osteoblastic and months, pain 2, RET,KIT, osteolytic lesions in palliation in 57%, AXL, FLT3 xenografts bone scan response in 63% AP12009 NucelotideReverses tumorNo bone (trabedersen) based, blocks mediated immune metastases data production suppression and reported prevented of TGF-2 metastases galunisertib Small Inhibit the boneIncreased OS, molecule tumor viscous cycle especially in inhibitor of patients with low TGF- levels of TGF- belagenpumatucel- Vaccine with Target tumor cells in OS benefits in L four TGF-b2- bone patients with prior antisense microenvironment chemotherapy or generadiotherapy that secrete TGF- modified, irradiated, allogeneic NSCLC cell lines Abiraterone + Androgen Shown to inhibit significantly prednisone synthesis progression of increase palliative inhibitor via prostatic lesions in benefit and CYP17A1 the bone decrease SREs inhibition P62 DNA Vaccine P62 in Decreases tumor Suppression of plasmid size, suppresses osteoporosis administered metastases,

Notes

Refs

New models have shown optimal dosages, need to be tested

[57,63]

OS improvement not seen in phase III trial

[75-82]

[165,166]

No specific end points measured with bone metastases No specific end points measured with bone metastases

[64,65]

Only for use in mCRPC, SoC

[168]

Currently only studied in animal models

[70-72,74]

[64,66,167]

21

created antibody response Stromal-targeted Zoledronic acid

PLX3397

Multitargeted TKI, inhibits CSF1

Bindarit

Inhibits CCL2

Tasquinimod

Blocks S100A9

Pirfenidone, and nintedanib Ac-PhScN-NH2 inhibitor

Target CAFs

Sunitinib

Imatinib

increased TIL, and decreases osteoclastogenesis

Acts on TAMs, change from M2 to M1 phenotype Prevents recruitment of M2 TAMs/MDSCs, improves immune response Prevents macrophage recruitment, Inhibits establishment of tumor-stromal niche inhibits angiogenesis, immunomodulates through TAMs, prevents of the establishment of the bone-metastatic niche

Significantly decreased tumor volume with ACT

Preclinical data only

[83,85,169]

Reduced metastasis formation

Animal models only

[86,87]

patients with bone metastases 8.8 months vs 3.4 months (placebo) PFS, no OS benefit seen

No further research after failed phase III trial

[90,91]

No data published

[36]

2nd generation is 100,000x more potent. 1st gen saw 14 months PFS Co-administered with CEA vaccine

[67,68]

51 inhibition through a small peptide TKI, blocks STAT3 and IDO pathways

prevents metastases and angiogenesis, due to vicious cycle of osteolytic lesions

reduced intratibial colony progression by almost 80 % in mouse model

Decreases MDSCs, Tregs. Increased TIL

TKI, blocks STAT3 and IDO pathways

activates CD8+ T cells, induces Treg apoptosis

Reduced tumor volume, increased OS in mouse model, No change in tumor burden in RCC patients Increased antiIn mouse models tumor response only with immunotherapies

[92,93]

[94,95]

22

Bevacizumab

Anti-VEGF mAB

Prevents dysfunction of DC into MDSCs

Temsirolimus

mTOR inhibitor, acts on IDO pathway Tetravalent FAP-DR5 Antibody

Activates CD8+ T cells, inhibits Tregs

Ex vivo DCs from MM patients functioned normally Better PFS in RCC patients

targets CAFs and defective apoptosis pathway on tumor cells

Stable disease and Low toxicities complete tumor regression with doxorubicin in vivo

[134]

Specific targeting of ACT to solid tumor DCs activated against a variety of TAAs Increase antigen uptake and activate DCs

More direct administration of ACT to bone metastases Better and more specific activation of DCs in bone microenvironment

Limited research in rat models

[170]

Significant increase of bone-marrow DC activation

Increased effectiveness of other vaccine based therapies

Endocytosis of exosomes for polyepitopic antigen presentation and response Exogenous sialic acid removal

Strong anti-tumoral T-cell response

High tolerability, with stable disease and partial responses in metastatic disease

RG7386

Immune-targeted Chitosan thermogels

Vaccines via whole tumor lysates/fusions

carbon black nanoparticles & Antigens with mannosylated dendrimers DC-derived exosomes

Sialidase

Toll-like receptor agonists (resiquimod, imiquimod, poly ICLC)

Increased maturation and stimulation of autologous T-cells Activation of Alone or in macrophages combination with DCs and vaccines, decrease other immune suppression lymphocytes in tumor

Increased survival among patients with metastatic disease

At lower doses normalization of vasculature, increased TIL

[96,153]

[97-99]

Multiple trials done, see 97

[106,107]

[109,110]

Only one phase II trial done, may be more effective with different TAA

Increased tumorcell apoptosis in murine model Dramatic results seen specifically with poly ICLC with complete tumor regression

[111-113]

[117]

Considered adjuvants, Montanide also acts similarly

[118-123]

23

microenvironment, can directly induce apoptosis of tumor cells Increase CAR T-cell proliferation and cytotoxicity Overcomes immunosuppression in tumor microenvironment

CRISPR-CaS9 edited CAR T-cells

PD-1 knockout

TRUCKs

IL-12 secreting CAR T-cells

CAR T-cells with chemokine receptors

CCR2 and CXCR4 receptors engineered into CAR Tcells Targets CAFs

Better T-cell trafficking to tumor metastases, specifically prostatic metastases for CXCR4 Reverses immunosuppression, activates T-cells against TAAs

Bispecific T-cell Engagers

Target TAA and CD3 for T-cell activation

Cryoablation

Direct destruction of metastases through liquid-cooled probes

Increased T-cell cytotoxcity in metastatic disease, unaffected by tumor immunosupression Rapid necrotic release of TAA induces immune response throughout body

Vesicular stomatitis virus

Oncolytic virus, highly sensitive to IFN- response

Anti-FAP CAR Tcells

Targets tumors with defective JAK1 pathway associated with immune resistance

No study results posted

NCT02793856

Increased efficacy of CAR T-cells, decreased immunosupression of bone-derived stroma cells Dramatic increase of TIL including in bone microenvironment

[171-173]

Synergy with vaccination, decreasing tumor volume and OS in mouse model Less efficacy compared to CARs due to inability to induce T-cell memory With anti-ALCAM antibody, complete response in 100% of mouse models, significant pain palliation in patients Currently unstudied

CXCR4 can also be [136therapeutic target, 141,174] but inhibition increases osteoclastogenesis [129-133]

Low toxicities, easily produced

[141,142]

Use of Anti-CTLA antibody and adjuvants also improves response

[144150,175]

Could be next-line treatment after failure of immunotherapies

[158-163]

24

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