Overcoming Xenoantigen Immunity to Enable Cellular Tracking and Gene Regulation with Immune-competent "NoGlow" Mice.

The ability to temporally regulate gene expression and track labeled cells makes animal models powerful biomedical tools. However, sudden expression of xenobiotic genes [e.g., GFP, luciferase (Luc), or rtTA3] can trigger inadvertent immunity that suppresses foreign protein expression or results in complete rejection of transplanted cells. Germline exposure to foreign antigens somewhat addresses these challenges; however, native fluorescence and bioluminescence abrogates the utility of reporter proteins and highly spatiotemporally restricted expression can lead to suboptimal xenoantigen tolerance. To overcome these unwanted immune responses and enable reliable cell tracking/gene regulation, we developed a novel mouse model that selectively expresses antigen-intact but nonfunctional forms of GFP and Luc, as well as rtTA3, after CRE-mediated recombination. Using tissue-specific CREs, we observed model and sex-based differences in immune tolerance to the encoded xenoantigens, illustrating the obstacles of tolerizing animals to foreign genes and validating the utility of these "NoGlow" mice to dissect mechanisms of central and peripheral tolerance. Critically, tissue unrestricted NoGlow mice possess no detectable background fluorescence or luminescence and exhibit limited adaptive immunity against encoded transgenic xenoantigens after vaccination. Moreover, we demonstrate that NoGlow mice allow tracking and tetracycline-inducible gene regulation of triple-transgenic cells expressing GFP/Luc/rtTA3, in contrast to transgene-negative immune-competent mice that eliminate these cells or prohibit metastatic seeding. Notably, this model enables de novo metastasis from orthotopically implanted, triple-transgenic tumor cells, despite high xenoantigen expression. Altogether, the NoGlow model provides a critical resource for in vivo studies across disciplines, including oncology, developmental biology, infectious disease, autoimmunity, and transplantation. SIGNIFICANCE: Multitolerant NoGlow mice enable tracking and gene manipulation of transplanted tumor cells without immune-mediated rejection, thus providing a platform to investigate novel mechanisms of adaptive immunity related to metastasis, immunotherapy, and tolerance.

Dormant tumors circumvent tumor-specific adaptive immunity by establishing a Treg-dominated niche via DKK3.

Approximately 30% of breast cancer survivors deemed free of disease will experience locoregional or metastatic recurrence even up to 30 years after initial diagnosis, yet how residual/dormant tumor cells escape immunity elicited by the primary tumor remains unclear. We demonstrate that intrinsically dormant tumor cells are indeed recognized and lysed by antigen-specific T cells in vitro and elicit robust immune responses in vivo. However, despite close proximity to CD8+ killer T cells, dormant tumor cells themselves support early accumulation of protective FoxP3+ T regulatory cells (Tregs), which can be targeted to reduce tumor burden. These intrinsically dormant tumor cells maintain a hybrid epithelial/mesenchymal state that is associated with immune dysfunction, and we find that the tumor-derived, stem cell/basal cell protein Dickkopf WNT signaling pathway inhibitor 3 (DKK3) is critical for Treg inhibition of CD8+ T cells. We also demonstrate that DKK3 promotes immune-mediated progression of proliferative tumors and is significantly associated with poor survival and immunosuppression in human breast cancers. Together, these findings reveal that latent tumors can use fundamental mechanisms of tolerance to alter the T cell microenvironment and subvert immune detection. Thus, targeting these pathways, such as DKK3, may help render dormant tumors susceptible to immunotherapies.

Trastuzumab/pertuzumab combination therapy stimulates antitumor responses through complement-dependent cytotoxicity and phagocytosis.

Two HER2-specific mAbs, trastuzumab and pertuzumab (T+P), combined with chemotherapy comprise standard-of-care treatment for advanced HER2+ breast cancers (BC). While this antibody combination is highly effective, its synergistic mechanism-of-action (MOA) remains incompletely understood. Past studies have suggested that the synergy underlying this combination occurs through the different mechanisms elicited by these antibodies, with pertuzumab suppressing HER2 heterodimerization and trastuzumab inducing antitumor immunity. However, in vivo evidence for this synergy is lacking. In this study, we found that the therapeutic efficacy elicited by their combination occurs through their joint ability to activate the classical complement pathway, resulting in both complement-dependent cytotoxicity and complement-dependent cellular phagocytosis of HER2+ tumors. We also demonstrate that tumor C1q expression is positively associated with survival outcome in HER2+ BC patients and that complement regulators CD55 and CD59 were inversely correlated with outcome, suggesting the clinical importance of complement activity. Accordingly, inhibition of C1q in mice abolished the synergistic therapeutic activity of T+P therapy, whereas knockdown of CD55 and CD59 expression enhanced T+P efficacy. In summary, our study identifies classical complement activation as a significant antitumor MOA for T+P therapy that may be functionally enhanced to potentially augment clinical therapeutic efficacy.

Runx2 Deficiency in Osteoblasts Promotes Myeloma Resistance to Bortezomib by Increasing TSP-1-Dependent TGFß1 Activation and Suppressing Immunity in Bone Marrow.

Multiple myeloma is a plasma cell malignancy that thrives in the bone marrow (BM). The proteasome inhibitor bortezomib is one of the most effective first-line chemotherapeutic drugs for multiple myeloma; however, 15% to 20% of high-risk patients do not respond to or become resistant to this drug and the mechanisms of chemoresistance remain unclear. We previously demonstrated that multiple myeloma cells inhibit Runt-related transcription factor 2 (Runx2) in pre- and immature osteoblasts (OB), and that this OB-Runx2 deficiency induces a cytokine-rich and immunosuppressive microenvironment in the BM. In the current study, we assessed the impact of OB-Runx2 deficiency on the outcome of bortezomib treatment using OB-Runx2+/+ and OB-Runx2-/- mouse models of multiple myeloma. In vitro and in vivo experiments revealed that OB-Runx2 deficiency induces multiple myeloma cell resistance to bortezomib via the upregulation of immunosuppressive myeloid-derived suppressor cells (MDSCs), downregulation of cytotoxic T cells, and activation of TGFß1 in the BM. In multiple myeloma tumor-bearing OB-Runx2-/- mice, treatment with SRI31277, an antagonist of thrombospondin-1 (TSP-1)-mediated TGFß1 activation, reversed the BM immunosuppression and significantly reduced tumor burden. Furthermore, treatment with SRI31277 combined with bortezomib alleviated multiple myeloma cell resistance to bortezomib-induced apoptosis caused by OB-Runx2 deficiency in cocultured cells and produced a synergistic effect on tumor burden in OB-Runx2-/- mice. Depletion of MDSCs by 5-fluorouracil or gemcitabine similarly reversed the immunosuppressive effects and bortezomib resistance induced by OB-Runx2 deficiency in tumor-bearing mice, indicating the importance of the immune environment for drug resistance and suggesting new strategies to overcome bortezomib resistance in the treatment of multiple myeloma.

IL26, a Noncanonical Mediator of DNA Inflammatory Stimulation, Promotes TNBC Engraftment and Progression in Association with Neutrophils.

IL26 is a unique amphipathic member of the IL10 family of cytokines that participates in inflammatory signaling through a canonical receptor pathway. It also directly binds DNA to facilitate cellular transduction and intracellular inflammatory signaling. Although IL26 has almost no described role in cancer, our in vivo screen of inflammatory and cytokine pathway genes revealed IL26 to be one of the most significant inflammatory mediators of mammary engraftment and lung metastatic growth in triple-negative breast cancer (TNBC). Examination of human breast cancers demonstrated elevated IL26 transcripts in TNBC specimens, specifically in tumor cells as well as in Th17 CD4+ T cells within clinical TNBC specimens. IL26 did not have an autocrine effect on human TNBC cells, but rather its effect on engraftment and growth in vivo required neutrophils. IL26 enhanced mouse-derived DNA induction of inflammatory cytokines, which were collectively important for mammary and metastatic lung engraftment. To neutralize this effect, we developed a novel IL26 vaccine to stimulate antibody production and suppress IL26-enhanced engraftment in vivo, suggesting that targeting this inflammatory amplifier could be a unique means to control cancer-promoting inflammation in TNBC and other autoimmune diseases. Thus, we identified IL26 as a novel key modulator of TNBC metastasis and a potential therapeutic target in TNBC as well as other diseases reliant upon IL26-mediated inflammatory stimulation. SIGNIFICANCE: These findings identify IL26 as a unique, clinically relevant, inflammatory amplifier that enhances TNBC engraftment and dissemination in association with neutrophils, which has potential as a therapeutic target. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/15/3088/F1.large.jpg.

Runx2 Deficiency in Osteoblasts Promotes Myeloma Progression by Altering the Bone Microenvironment at New Bone Sites.

Multiple myeloma is a plasma cell malignancy that thrives in the bone marrow (BM), with frequent progression to new local and distant bone sites. Our previous studies demonstrated that multiple myeloma cells at primary sites secrete soluble factors and suppress osteoblastogenesis via the inhibition of Runt-related transcription factor 2 (Runx2) in pre- and immature osteoblasts (OB) in new bone sites, prior to the arrival of metastatic tumor cells. However, it is unknown whether OB-Runx2 suppression in new bone sites feeds back to promote multiple myeloma dissemination to and progression in these areas. Hence, we developed a syngeneic mouse model of multiple myeloma in which Runx2 is specifically deleted in the immature OBs of C57BL6/KaLwRij mice (OB-Runx2-/- mice) to study the effect of OB-Runx2 deficiency on multiple myeloma progression in new bone sites. In vivo studies with this model demonstrated that OB-Runx2 deficiency attracts multiple myeloma cells and promotes multiple myeloma tumor growth in bone. Mechanistic studies further revealed that OB-Runx2 deficiency induces an immunosuppressive microenvironment in BM that is marked by an increase in the concentration and activation of myeloid-derived suppressor cells (MDSC) and the suppression and exhaustion of cytotoxic CD8+ T cells. In contrast, MDSC depletion by either gemcitabine or 5-fluorouracil treatment in OB-Runx2-/- mice prevented these effects and inhibited multiple myeloma tumor growth in BM. These novel discoveries demonstrate that OB-Runx2 deficiency in new bone sites promotes multiple myeloma dissemination and progression by increasing metastatic cytokines and MDSCs in BM and inhibiting BM immunity. Importantly, MDSC depletion can block multiple myeloma progression promoted by OB-Runx2 deficiency.Significance: This study demonstrates that Runx2 deficiency in immature osteoblasts at distant bone sites attracts myeloma cells and allows myeloma progression in new bone sites via OB-secreted metastatic cytokines and MDSC-mediated suppression of bone marrow immunity.

CD47 blockade augmentation of trastuzumab antitumor efficacy dependent on antibody-dependent cellular phagocytosis.

The HER2-specific monoclonal antibody (mAb), trastuzumab, has been the mainstay of therapy for HER2+ breast cancer (BC) for approximately 20 years. However, its therapeutic mechanism of action (MOA) remains unclear, with antitumor responses to trastuzumab remaining heterogeneous and metastatic HER2+ BC remaining incurable. Consequently, understanding its MOA could enable rational strategies to enhance its efficacy. Using both murine and human versions of trastuzumab, we found its antitumor activity dependent on Fcγ receptor stimulation of tumor-associated macrophages (TAMs) and antibody-dependent cellular phagocytosis (ADCP), but not cellular cytotoxicity (ADCC). Trastuzumab also stimulated TAM activation and expansion, but did not require adaptive immunity, natural killer cells, and/or neutrophils. Moreover, inhibition of the innate immune ADCP checkpoint, CD47, significantly enhanced trastuzumab-mediated ADCP and TAM expansion and activation, resulting in the emergence of a unique hyperphagocytic macrophage population, improved antitumor responses, and prolonged survival. In addition, we found that tumor-associated CD47 expression was inversely associated with survival in HER2+ BC patients and that human HER2+ BC xenografts treated with trastuzumab plus CD47 inhibition underwent complete tumor regression. Collectively, our study identifies trastuzumab-mediated ADCP as an important antitumor MOA that may be clinically enabled by CD47 blockade to augment therapeutic efficacy.

Runx2 Suppression by miR-342 and miR-363 Inhibits Multiple Myeloma Progression.

In multiple myeloma, abnormal plasma cells accumulate and proliferate in the bone marrow. Recently, we observed that Runx2, a bone-specific transcription factor, is highly expressed in multiple myeloma cells and is a major driver of multiple myeloma progression in bone. The primary goal of the present study was to identify Runx2-targeting miRNAs that can reduce tumor growth. Expression analysis of a panel of miRNAs in multiple myeloma patient specimens, compared with healthy control specimens, revealed that metastatic multiple myeloma cells express low levels of miR-342 and miR-363 but high levels of Runx2. Reconstituting multiple myeloma cells (CAG) with miR-342 and miR-363 reduced the abundance of Runx2 and the expression of metastasis-promoting Runx2 target genes RANKL and DKK1, and suppressed Runx2 downstream signaling pathways Akt/ß-catenin/survivin, which are required for multiple myeloma tumor progression. Intravenous injection of multiple myeloma cells (5TGM1), stably overexpressing miR-342 and miR-363 alone or together, into syngeneic C57Bl/KaLwRij mice resulted in a significant suppression of 5TGM1 cell growth, decreased osteoclasts and increased osteoblasts, and increased antitumor immunity in the bone marrow, compared with mice injected with 5TGM1 cells expressing a miR-Scramble control. In summary, these results demonstrate that enhanced expression of miR-342 and miR-363 in multiple myeloma cells inhibits Runx2 expression and multiple myeloma growth, decreases osteolysis, and enhances antitumor immunity. Thus, restoring the function of Runx2-targeting by miR-342 and miR-363 in multiple myeloma cells may afford a therapeutic benefit by preventing multiple myeloma progression.Implications: miR-342 and miR-363-mediated downregulation of Runx2 expression in multiple myeloma cells prevents multiple myeloma progression. Mol Cancer Res; 16(7); 1138-48. ©2018 AACR.

Adipocyte-Lineage Cells Support Growth and Dissemination of Multiple Myeloma in Bone.

Multiple myeloma (MM) cells reside in the bone marrow microenvironment and form complicated interactions with nonneoplastic, resident stromal cells. We previously found that aggressive MM cells shift osteoblast progenitors toward adipogenesis. In addition, adipocytes are among the most common cell types in the adult skeleton; both mature adipocytes and preadipocytes serve as endocrine cells that secrete a number of soluble molecules into the microenvironment. Therefore, we used a combination of in vivo and in vitro methods to test the hypothesis that an increase in adipocyte lineage cells feeds back to promote MM progression. The results of this study revealed that bone marrow from patients with MM indeed contains increased preadipocytes and significantly larger mature adipocytes than normal bone marrow. We also found that preadipocytes and mature adipocytes secrete many molecules important for supporting MM cells in the bone marrow and directly recruit MM cells through both monocyte chemotactic protein-1 and stromal cell-derived factor-1α. Co-culture experiments found that preadipocytes activate Wnt signaling and decrease cleaved caspase-3, whereas mature adipocytes activate ERK signaling in MM cells. Furthermore, mature adipocyte conditioned medium promotes MM growth, whereas co-culture with preadipocytes results in enhanced MM cell chemotaxis in vitro and increased tumor growth in bone in vivo. Combined, these data reveal the importance of preadipocytes and mature adipocytes on MM progression and represent a unique target in the bone marrow microenvironment.

Heparanase promotes myeloma progression by inducing mesenchymal features and motility of myeloma cells.

Bone dissemination and bone disease occur in approximately 80% of patients with multiple myeloma (MM) and are a major cause of patient mortality. We previously demonstrated that MM cell-derived heparanase (HPSE) is a major driver of MM dissemination to and progression in new bone sites. However the mechanism(s) by which HPSE promotes MM progression remains unclear. In the present study, we investigated the involvement of mesenchymal features in HPSE-promoted MM progression in bone. Using a combination of molecular, biochemical, cellular, and in vivo approaches, we demonstrated that (1) HPSE enhanced the expression of mesenchymal markers in both MM and vascular endothelial cells; (2) HPSE expression in patient myeloma cells positively correlated with the expression of the mesenchymal markers vimentin and fibronectin. Additional mechanistic studies revealed that the enhanced mesenchymal-like phenotype induced by HPSE in MM cells is due, at least in part, to the stimulation of the ERK signaling pathway. Finally, knockdown of vimentin in HPSE expressing MM cells resulted in significantly attenuated MM cell dissemination and tumor growth in vivo. Collectively, these data demonstrate that the mesenchymal features induced by HPSE in MM cells contribute to enhanced tumor cell motility and bone-dissemination.

Matricellular proteins as regulators of cancer metastasis to bone.

Metastasis is the major cause of death in cancer patients, and a frequent site of metastasis for many cancers is the bone marrow. Therefore, understanding the mechanisms underlying the metastatic process is necessary for future prevention and treatment. The tumor microenvironment is now known to play a role in the metastatic cascade, both at the primary tumor and in metastatic sites, and includes both cellular and non-cellular components. The extracellular matrix (ECM) provides structural support and signaling cues to cells. One particular group of molecules associated with the ECM, known as matricellular proteins, modulate multiple aspects of tumor biology, including growth, migration, invasion, angiogenesis and metastasis. These proteins are also important for normal function in the bone by regulating bone formation and bone resorption. Recent studies have described a link between some of these proteins and metastasis of various tumors to the bone. The aim of this review is to summarize what is currently known about matricellular protein influence on bone metastasis. Particular attention to the contribution of both tumor cells and non-malignant cells in the bone has been given.

Myeloma cell-derived Runx2 promotes myeloma progression in bone.

The progression of multiple myeloma (MM) is governed by a network of molecular signals, the majority of which remain to be identified. Recent studies suggest that Runt-related transcription factor 2 (Runx2), a well-known bone-specific transcription factor, is also expressed in solid tumors, where expression promotes both bone metastasis and osteolysis. However, the function of Runx2 in MM remains unknown. The current study demonstrated that (1) Runx2 expression in primary human MM cells is significantly greater than in plasma cells from healthy donors and patients with monoclonal gammopathy of undetermined significance; (2) high levels of Runx2 expression in MM cells are associated with a high-risk population of MM patients; and (3) overexpression of Runx2 in MM cells enhanced tumor growth and disease progression in vivo. Additional studies demonstrated that MM cell-derived Runx2 promotes tumor progression through a mechanism involving the upregulation of Akt/ß-catenin/Survivin signaling and enhanced expression of multiple metastatic genes/proteins, as well as the induction of a bone-resident cell-like phenotype in MM cells. Thus, Runx2 expression supports the aggressive phenotype of MM and is correlated with poor prognosis. These data implicate Runx2 expression as a major regulator of MM progression in bone and myeloma bone disease.

Heparanase inhibits osteoblastogenesis and shifts bone marrow progenitor cell fate in myeloma bone disease.

A major cause of morbidity in patients with multiple myeloma is the development and progression of bone disease. Myeloma bone disease is characterized by rampant osteolysis in the presence of absent or diminished bone formation. Heparanase, an enzyme that acts both at the cell-surface and within the extracellular matrix to degrade polymeric heparan sulfate chains, is upregulated in a variety of human cancers including multiple myeloma. We and others have shown that heparanase enhances osteoclastogenesis and bone loss. However, increased osteolysis is only one element of the spectrum of myeloma bone disease. In the present study, we hypothesized that heparanase would also affect mesenchymal cells in the bone microenvironment and investigated the effect of heparanase on the differentiation of osteoblast/stromal lineage cells. Using a combination of molecular, biochemical, cellular and in vivo approaches, we demonstrated that heparanase significantly inhibited osteoblast differentiation and mineralization, and reduced bone formation in vivo. In addition, heparanase shifts the differentiation potential of osteoblast progenitors from osteoblastogenesis to adipogenesis. Mechanistically, this shift in cell fate is due, at least in part, to heparanase-enhanced production and secretion of the Wnt signaling pathway inhibitor DKK1 by both osteoblast progenitors and myeloma cells. Collectively, these data provide important new insights into the role of heparanase in all aspects of myeloma bone disease and strongly support the use of heparanase inhibitors in the treatment of multiple myeloma.

Total synthesis of 7-deoxy-6-O-methylfusarentin featuring a chelation-controlled 1,3-Reetz-Keck-type allylation.

The total synthesis of 7-deoxy-6-O-methylfusarentin (1) and a formal synthesis of 7-deoxy-6,8-O-dimethylfusarentin (2) has been successfully achieved in 10 steps. The described tactic underscores a diastereoselective strategy which incorporates a single acyclic reaction based on the initial stereocenter by means of a 1,3-chelation-controlled Reetz-Keck-type allylation.