TGF-β Antagonists Research Articles

Oncogenic Calreticulin Induces Immune Escape by Stimulating TGFβ Expression and Regulatory T-cell Expansion in the Bone Marrow Microenvironment.

Increasing evidence supports the interplay between oncogenic mutations and immune escape mechanisms. Strategies to counteract the immune escape mediated by oncogenic signaling could provide improved therapeutic options for patients with various malignancies. As mutant calreticulin (CALR) is a common driver of myeloproliferative neoplasms (MPN), we analyzed the impact of oncogenic CALRdel52 on the bone marrow (BM) microenvironment in MPN. Single-cell RNA sequencing revealed that CALRdel52 led to the expansion of TGFβ1-producing erythroid progenitor cells and promoted the expansion of FoxP3+ regulatory T cells (Treg) in a murine MPN model. Treatment with an anti-TGFβ antibody improved mouse survival and increased the glycolytic activity in CD4+ and CD8+ T cells in vivo, whereas T-cell depletion abrogated the protective effects conferred by neutralizing TGFβ. TGFβ1 reduced perforin and TNFα production by T cells in vitro. TGFβ1 production by CALRdel52 cells was dependent on JAK1/2, PI3K, and ERK activity, which activated the transcription factor Sp1 to induce TGFβ1 expression. In four independent patient cohorts, TGFβ1 expression was increased in the BM of patients with MPN compared with healthy individuals, and the BM of patients with MPN contained a higher frequency of Treg compared with healthy individuals. Together, this study identified an ERK/Sp1/TGFβ1 axis in CALRdel52 MPNs as a mechanism of immunosuppression that can be targeted to elicit T-cell-mediated cytotoxicity. Significance: Targeting the mutant calreticulin/TGFβ1 axis increases T-cell activity and glycolytic capacity, providing the rationale for conducting clinical trials on TGFβ antagonists as an immunotherapeutic strategy in patients with myeloproliferative neoplasms.

Transcriptomic and Proteomic Changes Driving Pulmonary Fibrosis Resolution in Young and Old Mice.

Bleomycin-induced pulmonary fibrosis in mice mimics major hallmarks of idiopathic pulmonary fibrosis. Yet in this model, it spontaneously resolves over time. We studied molecular mechanisms of fibrosis resolution and lung repair, focusing on transcriptional and proteomic signatures and the effect of aging. Old mice showed incomplete and delayed lung function recovery 8 weeks after bleomycin instillation. This shift in structural and functional repair in old bleomycin-treated mice was reflected in a temporal shift in gene and protein expression. We reveal gene signatures and signaling pathways that underpin the lung repair process. Importantly, the downregulation of WNT, BMP, and TGFβ antagonists Frzb, Sfrp1, Dkk2, Grem1, Fst, Fstl1, and Inhba correlated with lung function improvement. Those genes constitute a network with functions in stem cell pathways, wound, and pulmonary healing. We suggest that insufficient and delayed downregulation of those antagonists during fibrosis resolution in old mice explains the impaired regenerative outcome. Together, we identified signaling pathway molecules with relevance to lung regeneration that should be tested in-depth experimentally as potential therapeutic targets for pulmonary fibrosis.

Intratumoral immunotherapy with mRNAs encoding chimeric protein constructs encompassing IL-12, CD137 agonists, and TGF-β antagonists

Intratumoral immunotherapy strategies for cancer based on interleukin-12 (IL-12)-encoding cDNA and mRNA are under clinical development in combination with anti-PD-(L)1 monoclonal antibodies. To make the most of these approaches, we have constructed chimeric mRNAs encoding single-chain IL-12 fused to single-chain fragment variable (scFv) antibodies that bind to transforming growth factor β (TGF-β) and CD137 (4-1BB). Several neutralizing TGF-β agents and CD137 agonists are also undergoing early-phase clinical trials. To attain TGF-β and CD137 binding by the constructions, we used bispecific tandem scFv antibodies (taFvs) derived from the specific 1D11 and 1D8 monoclonal antibodies (mAbs), respectively. Transfection of mRNAs encoding the chimeric constructs achieved functional expression of the proteins able to act on their targets. Upon mRNA intratumoral injections in the transplantable mouse cancer models CT26, MC38, and B16OVA, potent therapeutic effects were observed following repeated injections into the tumors. Efficacy was dependent on the number of CD8+ Tcells able to recognize tumor antigens that infiltrated the malignant tissue. Although the abscopal effects on concomitant uninjected lesions were modest, such distant effects on untreated lesions were markedly increased when combined with systemic PD-1 blockade.

Unveiling the transcriptomic landscape and the potential antagonist feedback mechanisms of TGF-β superfamily signaling module in bone and osteoporosis

BackgroundTGF-β superfamily signaling is indispensable for bone homeostasis. However, the global expression profiles of all the genes that make up this signaling module in bone and bone-related diseases have not yet been well characterized.MethodsTranscriptomic datasets from human bone marrows, bone marrow-derived mesenchymal stem cells (MSCs) and MSCs of primary osteoporotic patients were used for expression profile analyses. Protein treatments, gene quantification, reporter assay and signaling dissection in MSC lines were used to clarify the interactive regulations and feedback mechanisms between TGF-β superfamily ligands and antagonists. Ingenuity Pathway Analysis was used for network construction.ResultsWe identified TGFB1 in the ligand group that carries out SMAD2/3 signaling and BMP8A, BMP8B and BMP2 in the ligand group that conducts SMAD1/5/8 signaling have relatively high expression levels in normal bone marrows and MSCs. Among 16 antagonist genes, the dominantly expressed TGF-β superfamily ligands induced only NOG, GREM1 and GREM2 via different SMAD pathways in MSCs. These induced antagonist proteins further showed distinct antagonisms to the treated ligands and thus would make up complicated negative feedback networks in bone. We further identified TGF-β superfamily signaling is enriched in MSCs of primary osteoporosis. Enhanced expression of the genes mediating TGF-β-mediated SMAD3 signaling and the genes encoding TGF-β superfamily antagonists served as significant features to osteoporosis.ConclusionOur data for the first time unveiled the transcription landscape of all the genes that make up TGF-β superfamily signaling module in bone. The feedback mechanisms and regulatory network prediction of antagonists provided novel hints to treat osteoporosis.3vrgH_v5FiqyUZTyTihmKDVideo

Multi-layered regulation of neuroectoderm differentiation by retinoic acid in a primitive streak-like context.

SummaryThe formation of the primitive streak (PS) and the subsequent induction of neuroectoderm are hallmarks of gastrulation. Combining an in vitro reconstitution of this process based on mouse embryonic stem cells (mESCs) with a collection of knockouts in reporter mESC lines, we identified retinoic acid (RA) as a critical mediator of early neural induction triggered by TGFβ or Wnt signaling inhibition. Single-cell RNA sequencing analysis captured the temporal unfolding of cell type diversification, up to the emergence of somite and neural fates. In the absence of the RA-synthesizing enzyme Aldh1a2, a sensitive RA reporter revealed a hitherto unidentified residual RA signaling that specified neural fate. Genetic evidence showed that the RA-degrading enzyme Cyp26a1 protected PS-like cells from neural induction, even in the absence of TGFβ and Wnt antagonists. Overall, we characterized a multi-layered control of RA levels that regulates early neural differentiation in an in vitro PS-like system.

Inhibiting TGFβ Signaling in CAR T-Cells May Significantly Enhance Efficacy of Tumor Immunotherapy

The use of genetically engineered T cells expressing chimeric antigen receptors (CAR) against CD19 or BCMA to treat hematologic malignancies has demonstrated promising results in clinical trials. However, it is possible that the efficacy of CAR T cells could be limited in some clinical indications due to suppressive factors in the tumor microenvironment. Immunosuppressive cytokines (e.g., IL-6, IL-10 and TGFβ) provide a favorable environment for tumor growth and inhibit the tumoricidal activity of the endogenous T cells. In particular, transforming growth factor β (TGFβ) has been shown to suppress T-cell proliferation, inhibit the maturation of T helper cells and reduce their effector functions. Elevated levels of TGFβ have been detected in many human cancers, including multiple myeloma (MM). Here, we sought to modulate the activity of CAR T cell-based immunotherapy for MM by inhibiting TGFβ signaling in BCMA-specific CAR T cells (Budha et al., Cancer Cell, 2012).We evaluated two strategies to modulate the TGFβ pathway: (1) knocking out TGFβ Receptor II (TGFβRII) using CRISPR gene editing and (2) overexpressing a dominant negative TGFβRII in BCMA targeted CAR T cells. TGFβRII modulation inhibited TGFβ-dependent Smad2/3 phosphorylation. TGFβRII-modulated BCMA CAR T cells were insensitive to TGFβ-mediated inhibition of proliferation, cell cytotoxicity and cytokine production, both IFNg and granzyme B, in the context of primary and/or repeat antigen stimulation with BCMA+ve MM cell lines in vitro . We further evaluated the gene expression signatures of BCMA CAR T cells isolated from tumor and spleen in a NSG mouse xenograft model subcutaneously inoculated with myeloma cell line (RPMI8226). TGFβ pathway gene expression changes indicating TGFβ exposure were observed in BCMA CAR T cells isolated from tumor, but not spleen, suggesting that BCMA CAR T cells are encountering a TGFβ-enriched TME. However, both TGFβ pathway modulating strategies prevented the development of the TGFβ-induced gene expression phenotype.Together, these data suggest that inhibition of TGFβRII signaling may potentiate CAR T activity by directly inhibiting immunosuppressive effects of TGFβ in the tumor microenvironment. By specifically targeting TGFβ signaling in CAR T cells, it may be possible to avoid side effects associated with systemic administration of TGFβ antagonists. Gene editing to remove TGFβRII expression or overexpressing dominant negative TGFβ RII could modulate the activity of BCMA CAR T therapy in MM. DisclosuresVong:Juno Therapeutics: Employment, Equity Ownership. Nye:Juno Therapeutics: Employment, Equity Ownership. Hause:Juno Therapeutics: Employment, Equity Ownership. Clouser:Juno Therapeutics: Employment, Equity Ownership. Jones:Juno Therapeutics: Employment, Equity Ownership. Burleigh:Juno Therapeutics: Employment, Equity Ownership. Borges:Editas Medicine: Employment, Equity Ownership. Chin:Editas Medicine: Employment, Equity Ownership. Marco:Editas Medicine: Employment, Equity Ownership. Barrera:Editas Medicine: Employment, Equity Ownership. Da Silva:Editas Medicine: Employment, Equity Ownership. Harbinski:Editas Medicine: Employment, Equity Ownership. Giannoukos:Editas Medicine: Employment, Equity Ownership. Dhanapal:Editas Medicine: Employment, Equity Ownership. Jiang:Juno Therapeutics: Employment, Equity Ownership. Salmon:Juno Therapeutics: Employment, Equity Ownership. Wilson:Editas Medicine: Employment, Equity Ownership. Myer:Editas Medicine: Employment, Equity Ownership. Welstead:Editas Medicine: Employment, Equity Ownership. Bond:Juno Therapeutics: Employment, Equity Ownership. Sather:Juno Therapeutics: Employment, Equity Ownership.

Abstract 1645: A new TGF-b pathway reporter mouse for analysis of TGF-β signaling in normal homeostasis and cancer

Abstract TGF-βs play a central regulatory role in maintaining homeostasis and coordinating response to injury in the adult animal. Consequently, dysregulation of TGF-β signaling has been implicated in many pathological states, including cancer. Several TGF-β pathway antagonists are now in early phase clinical oncology trials. However, surprisingly little is known about when and where the TGF-β pathway is activated in the adult animal. To address this need, we have generated a TGF-β pathway reporter mouse in which the expression of eGFP is driven by an artificial enhancer element consisting of 6 repeats of a strong Smad3 binding element from the distal region of the JunB promoter (S3 × 6>GFP reporter). This reporter was >10x more sensitive in vitro than the commonly used CAGA12-based reporter. The reporter construct was knocked into the mouse ROSA26 locus using CRISPR technology and a founder line was derived. Whole body fluorescent imaging of an adult female mouse highlighted TGF-β pathway activation in the gastrointestinal tract, lymph nodes, costal cartilage, brown adipose tissue, brain ventricles and choroid plexi, among other tissues. Since high dose pharmacologic inhibition of the TGF-β pathway has previously been associated with cardiac valvulopathy in preclinical toxicology studies, we immunostained heart sections from the reporter mouse for GFP and observed high endogenous TGF-β pathway activation in the atrioventricular valve leaflets. Quantitative IVIS fluorescent imaging of isolated organs confirmed TGF-β pathway activation in many different tissues in the normal adult mouse, with the pancreas showing the highest level of endogenous activation. Interestingly, the level of TGF-β pathway activation in different tissues was highly correlated with the frequency of inactivating mutations in TGF-β pathway components in tumors from the corresponding tissue in humans. This observation suggests that a high level of TGF-β pathway activation in normal tissues may reflect a non-redundant tumor suppressor role for the TGF-β pathway in maintaining homeostasis in those tissues. To specifically address the activation state of the TGF-β pathway during tumorigenesis, we intercrossed the S3 × 6>GFP reporter mouse with the MMTV-PyVT mouse model of metastatic breast cancer. Reporter activity was strongly upregulated in mammary tumors when compared with the surrounding mammary gland. Using cells cultured from the primary tumors, we confirmed that the reporter signal in the tumor cells can be blocked by small molecule antagonists of the TGF-β pathway. This reporter mouse should be a useful tool to assess the cellular location and extent of TGF-β pathway activation during tumor development, and the impact of TGF-β antagonists on TGF-β signaling in tumors and normal tissues. Citation Format: Yu-an Yang, Christina Stuelten, Youngjae Bahn, Ji-Hyeon Lee, Madhu Gargesha, Hibret Adissu, Mark Simpson, Caroline S. Hill, Sushil G. Rane, Lalage M. Wakefield. A new TGF-b pathway reporter mouse for analysis of TGF-β signaling in normal homeostasis and cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1645.

The Outcome of TGFβ Antagonism in Metastatic Breast Cancer Models In Vivo Reflects a Complex Balance between Tumor-Suppressive and Proprogression Activities of TGFβ.

TGFβs are overexpressed in many advanced cancers and promote cancer progression through mechanisms that include suppression of immunosurveillance. Multiple strategies to antagonize the TGFβ pathway are in early-phase oncology trials. However, TGFβs also have tumor-suppressive activities early in tumorigenesis, and the extent to which these might be retained in advanced disease has not been fully explored. A panel of 12 immunocompetent mouse allograft models of metastatic breast cancer was tested for the effect of neutralizing anti-TGFβ antibodies on lung metastatic burden. Extensive correlative biology analyses were performed to assess potential predictive biomarkers and probe underlying mechanisms. Heterogeneous responses to anti-TGFβ treatment were observed, with 5 of 12 models (42%) showing suppression of metastasis, 4 of 12 (33%) showing no response, and 3 of 12 (25%) showing an undesirable stimulation (up to 9-fold) of metastasis. Inhibition of metastasis was immune-dependent, whereas stimulation of metastasis was immune-independent and targeted the tumor cell compartment, potentially affecting the cancer stem cell. Thus, the integrated outcome of TGFβ antagonism depends on a complex balance between enhancing effective antitumor immunity and disrupting persistent tumor-suppressive effects of TGFβ on the tumor cell. Applying transcriptomic signatures derived from treatment-naïve mouse primary tumors to human breast cancer datasets suggested that patients with breast cancer with high-grade, estrogen receptor-negative disease are most likely to benefit from anti-TGFβ therapy. Contrary to dogma, tumor-suppressive responses to TGFβ are retained in some advanced metastatic tumors. Safe deployment of TGFβ antagonists in the clinic will require good predictive biomarkers.

Abstract 1549: Undesirable metastasis-stimulating effects of anti-TGF-β therapy in breast cancer models involve interference with tumor cell-autonomous tumor suppressive activity

Abstract TGF-βs are central regulators of normal tissue homeostasis and response to stress and injury. Dysregulation of this pathway occurs frequently in cancer, and overexpression of TGF-βs in many advanced human tumors correlates with metastasis and poor prognosis. Preclinical studies have shown that TGF-β antagonists can slow tumor progression, largely by reactivating anti-tumor immunity, and there are now >45 ongoing or completed early phase clinical oncology trials using TGF-β antagonists. However, TGF-βs also have tumor suppressive activities, although these are conventionally considered to be restricted to the early stages of tumorigenesis. Most preclinical studies have focused on a very small number of widely used tumor models distributed across many different disease sites. Here we have assembled a panel of twelve immunocompetent mouse allograft models of metastatic breast cancer to capture some of the heterogeneity of the human disease within one disease histology. We find heterogeneous responses of these models to treatment with a neutralizing anti-TGF-β antibody, using lung metastatic burden as the clinically relevant endpoint. Anti-TGF-β antibody treatment inhibited metastasis in 5 models (InhibMet), had no effect in 4 models (NoEff) and showed an undesirable stimulatory effect on metastasis in 3 models (StimMet). As seen previously, the therapeutic effect in the InhibMet models was dependent on an intact immune system. However, the metastasis stimulatory effect in the StimMet models was immune-independent. Transcriptomic analysis of untreated primary tumors showed that InhibMet models had highest expression of a cytotoxic T-cell signature, and higher levels of infiltrating CD8 T-cells were detected by immunohistochemistry, suggesting that a therapeutic response to TGF-β antagonism may depend on some level of pre-existing immune response. In the MVT1 StimMet model, blockade of TGF-β signaling in the tumor cell using a dominant negative TGF-β receptor increased metastasis, suggesting that TGF-β antagonists can interfere with metastasis-suppressing effects of TGF-β on the tumor parenchyma. Consistent with this finding, the StimMet models showed higher expression of an independently-derived TGF-β gene signature that specifically reflects tumor suppressive responses to TGF-β in the tumor cell. In vitro, TGF-β suppressed clonogenicity and tumorsphere formation in all StimMet models but not InhibMet models, suggesting that suppressive effects of TGF-β may target the cancer stem cell population in the StimMet models. Thus our data suggest that contrary to dogma, tumor suppressive effects of TGF-β may still be active in some cases of advanced metastatic disease. Good predictive biomarkers will be essential to exclude patients who might experience deleterious responses to anti-TGF-β therapy. Note: This abstract was not presented at the meeting. Citation Format: Yu-an Yang, Howard Yang, Binwu Tang, Alex Wu, Nellie Moshkovich, Maxwell Lee, Lalage Wakefield. Undesirable metastasis-stimulating effects of anti-TGF-β therapy in breast cancer models involve interference with tumor cell-autonomous tumor suppressive activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1549.

Abstract 1768: An anti-TGF-β1/2 antibody that spares TGF-β3 retains full anti-tumor efficacy and generates an improved metabolic profile

Abstract TGF-β family members are overexpressed in many advanced cancers and correlate with metastasis and poor prognosis. Based on encouraging preclinical data, therapeutics that target the TGF-β pathway are now in early phase clinical trials in oncology. While the three isoforms of TGF-β have essentially identical activities in vitro, there is relatively little known about how they might differ in vivo. Using a panel of twelve mouse allograft models of metastatic breast cancer, we showed that while TGF-β1 protein was consistently up-regulated in mammary tumors compared with normal mammary gland, the opposite was true for TGF-β3. Furthermore, in human breast cancer high TGF-β3 mRNA or protein expression was associated with better outcome, particularly in estrogen-receptor positive breast cancers. Collectively the data suggest that TGF-β1 and TGF-β3 may have opposing effects on breast cancer progression. Using an antibody that selectively neutralizes only TGF-β1 and TGF-β2, we explored the effect of sparing TGF-β3 on therapeutic outcome in the 4T1 and TSAE1 models of metastatic breast cancer. While the TGFβ1,2 antibody and two pan-TGF-β antibodies had similar efficacy against the metastasis endpoint in these very aggressive models, transcriptomic analysis of primary tumors after two weeks of antibody therapy suggested that sparing TGF-β3 might have positive effects on the metabolic profile of treated animals. To address this issue directly, we showed that mice without tumors had a significantly improved glucose tolerance following treatment with anti-TGF-β1/2 antibodies for 2-3 weeks when compared with mice treated with pan-TGF-β antibodies. Addressing potential human relevance, we showed that high expression of transcripts that were selectively upregulated in the primary tumors when TGF-β3 was spared correlated with good outcome in human breast cancer. The data suggest that use of isoform-selective TGF-β antagonists may offer advantages over the use of pan-TGF-β blocking agents for the treatment of breast cancer. Citation Format: Yu-an Yang, Srividya Vasu, Howard Yang, Maxwell P. Lee, Sushil Rane, Amer M. Mirza, Lalage M. Wakefield. An anti-TGF-β1/2 antibody that spares TGF-β3 retains full anti-tumor efficacy and generates an improved metabolic profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1768.

Sorafenib suppresses TGF-β responses by inducing caveolae/lipid raft-mediated internalization/degradation of cell-surface type II TGF-β receptors: Implications in development of effective adjunctive therapy for hepatocellular carcinoma

Sorafenib is the only FDA approved drug for the treatment of advanced hepatocellular carcinoma (HCC) and other malignancies. Studies indicate that TGF-β signalling is associated with tumour progression in HCC. Autocrine and paracrine TGF-β promotes tumour growth and malignancy by inducing epithelial-mesenchymal transition (EMT). Sorafenib is believed to antagonize tumour progression by inhibiting TGF-β-induced EMT. It improves survival of patients but HCC later develops resistance and relapses. The underlying mechanism of resistance is unknown. Understanding of the molecular mechanism of sorafenib inhibition of TGF-β-induced signalling or responses in HCC may lead to development of adjunctive effective therapy for HCC. In this study, we demonstrate that sorafenib suppresses TGF-β responsiveness in hepatoma cells, hepatocytes, and animal liver, mainly by downregulating cell-surface type II TGF-β receptors (TβRII) localized in caveolae/lipid rafts and non-lipid raft microdomains via caveolae/lipid rafts-mediated internalization and degradation. Furthermore, sorafenib-induced downregulation and degradation of cell-surface TβRII is prevented by simultaneous treatment with a caveolae disruptor or lysosomal inhibitors. On the other hand, sorafenib only downregulates cell-surface TβRII localized in caveolae/lipid rafts but not localized in non-lipid raft microdomains in hepatic stellate cells. These results suggest that sorafenib inhibits TGF-β signalling mainly by inducing caveolae/lipid raft-mediated internalization and degradation of cell-surface TβR-II in target cells. They may also imply that treatment with agents which promote formation of caveolae/lipid rafts, TGF-β receptor kinase inhibitors (e.g., LY2157299) or TGF-β peptide antagonists (by liver-targeting delivery) may be considered as effective adjunct therapy with sorafenib for HCC.

Abnormal scar identification with spherical-nucleic-acid technology.

The accurate diagnosis of scar type and severity relies on histopathology of biopsied tissue, which is invasive and time-consuming, causes discomfort and may exacerbate scarring. Here, we show that imaging nanoprobes for the live-cell detection of intracellular messenger RNA (mRNA) (also known as NanoFlares) enable measurements of the expression of connective tissue growth factor (CTGF) as a visual indicator of hypertrophic scars and keloids. During cell culture, NanoFlares enabled the distinction of hypertrophic and keloidal fibroblasts from normal fibroblasts, and the detection of changes in CTGF expression resulting from the regulatory effects of transforming growth factor-β (TGF-β) agonists and TGF-β antagonists. We also applied the NanoFlares topically to the skin of live mice and rabbits, and to ex vivo human skin models. Transepidermal penetration of the NanoFlares enabled the visual and spectroscopic quantification of underlying abnormal fibroblasts on the basis of CTGF mRNA expression. Our proof-of-concept studies of topically applied NanoFlare technology as a means of biopsy-free scar diagnosis may eventually inform therapeutic decisions on the basis of the mRNA-expression patterns of skin disorders.

Abstract 3741: Antimetastatic activity of ISTH0047, a potent and selective TGF-beta 2 antisense oligonucleotide, in syngeneic lung metastatic model of mouse 4T1 mammary carcinoma

Abstract Transforming growth factor beta (TGF-β) isoforms are the primary mediators for TGF-β signaling via TGF-β receptors and downstream phosphorylation/dephosphorylation cascade. TGF-β is associated with a wide range of biological processes in oncology, including tumor cell invasion, migration, angiogenesis, immunosuppression, as well as regulation of tumor stem cell properties. Mouse 4T1 mammary carcinoma cell line is a transplantable tumor cell line that is highly tumorigenic and invasive and, unlike most tumor models, can spontaneously metastasize from the primary tumor in the mammary gland to multiple distant sites including lymph nodes, blood, liver, lung, brain, and bone. Considering rather challenging preclinical evaluation of antitumor activity in tumor models, mouse 4T1 mammary carcinoma model has been widely used in the literature for evaluation of TGF-β antagonists. In this report, we describe the efficacy of ISTH0047 - a potent and selective TGF-β2 antisense oligonucleotide - in murine 4T1 primary tumors and lung metastasis following tumor cell injection into the mammary fat pad (orthotopic tumor model) of syngeneic Balb/c mice. Consistent with literature data generated with other classes of TGF-β antagonists (e.g., small-molecule kinase inhibitors, antibodies or TGF-β trap agents), although limited antitumor activity was demonstrated on primary tumor growth, marked and statistically significant decrease of lung metastasis number was observed upon subcutaneous administrations of ISTH0047. In addition, side by side comparison with murine surrogates of CTLA-4 or PD-1 antibodies indicated similar efficacy of all test items on lung metastasis in this model. Taken together, these encouraging results pave the way for in-depth preclinical evaluation of both ‘seed and soil’ theory and efficacy of combination regimen (immunomodulation) for better clinical outcome. Citation Format: Katja Wosikowski, Kathy Hasenbach, Jutta Petschenka, Diana Barea Roldan, Sebastian Kreiter, Ugur Sahin, Guillaume Serin, Julie-Orlane Redon, Marc Hillairet de Boisferon, Francis Bichat, Hanna Kohonen, Michel Janicot. Antimetastatic activity of ISTH0047, a potent and selective TGF-beta 2 antisense oligonucleotide, in syngeneic lung metastatic model of mouse 4T1 mammary carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3741.

Abstract B13: Targeting the TGF-β pathway in breast cancer: Insights from preclinical studies

Abstract Overexpression of transforming growth factor-βs (TGF-βs) correlates with metastasis and poor prognosis in many advanced cancers, and TGF-βs have pro-oncogenic effects on nearly every cell type in the ecosystem of advanced tumors. Based on these observations and encouraging results in preclinical models, strategies to block TGF-β signaling are in early phase clinical oncology trials. To date however, preclinical studies supporting the development of anti-TGF-β therapeutics in cancer have focused around a few well-characterized mouse models which do not capture the heterogeneity of the human disease. To address this issue for breast cancer, we have assembled a panel of transplantable mouse models of metastatic mammary cancer that can be used in fully immunocompetent hosts, so as to preserve any immune contributions to therapeutic efficacy. Hormone receptor status, histopathological characteristics and intrinsic subtype of the models were assessed. Using this panel of models, we tested the efficacy of a pan-TGF-β neutralizing antibody (1D11) in inhibiting lung metastasis. We observed therapeutic efficacy or trend to efficacy in 5 models (InhibMet), no effect in 2 models (NoEff) and an undesirable stimulation or trend to stimulation of metastasis in 4 models (StimMet). This heterogeneity in therapeutic responses suggests that it will be critical to develop good predictive biomarkers for patient selection in clinical trials using TGF-β antagonists. Since the model panel provides an excellent platform for biomarker discovery, we have performed transcriptomics on untreated primary tumors across the panel, as well as completing full exome gDNA sequencing and copy number variant analysis on the tumor cell lines. The relationship between response-to-therapy and mutation load will be presented. Analyses of the untreated primary tumors show that the tumor transcriptomes segregate by response to anti-TGF-β therapy in principal component analysis, after removal of mouse strain as a confounding factor. Encouragingly for biomarker development, this observation suggests that there are major pre-existing differences in the biology of InhibMet and StimMet primary tumors before they are treated. A gene signature generated from the differentially expressed gene list was strongly associated with outcome in a metaanalysis of human breast cancer datasets, suggesting human relevance. Ingenuity Pathway Analysis of differentially-expressed genes indicates that InhibMet models are characterized by higher TGF-β pathway activation, higher angiogenesis, poor immune cell infiltration/activation, and other markers of tumor aggressiveness such as higher tumor cell proliferation and survival. Interestingly, the higher TGF-β pathway activation that was strongly predicted in the InhibMet tumors by the transcriptomic analyses (p=5.8e-25) was not evident from a multipronged quantitative proteomics assessment of activation of canonical Smad signaling, or non-canonical signaling through Akt, Erk, Jnk or p38MAPK pathways. Thus steady-state signaling by TGF-β in tumors in vivo may represent the integrated sum of the activities of more downstream signaling pathways than were captured in this analysis, or additional information about the intracellular localization of the phospho-forms of the signaling proteins and/or the identity of interacting proteins may be necessary to fully assess the activation state of the pathway. Despite these caveats, the data suggest that TGF-β antagonists will have therapeutic efficacy in more aggressive, poor prognosis breast cancers, and suggest that it will be possible to generate molecular signatures that predict the therapeutic response. Citation Format: Yu-an Yang, Kathleen Flanders, Jin-qui Chen, Anand S. Merchant, Howard Yang, Maxwell P. Lee, Lalage M. Wakefield. Targeting the TGF-β pathway in breast cancer: Insights from preclinical studies. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B13.

Abstract 4094: Neutralizing anti-TGF-β antibodies elicit heterogeneous therapeutic responses in a panel of murine metastatic breast cancer models

Abstract Overexpression of transforming growth factor-βs (TGF-βs) correlates with metastasis and poor prognosis in many advanced cancers, and TGF-βs have pro-oncogenic effects on nearly every cell type in the ecosystem of advanced tumors. Based on these observations and encouraging results in preclinical models, strategies to block TGF-β signaling are in early phase clinical oncology trials. To date however, preclinical studies supporting the development of anti-TGF-β therapeutics in cancer have focused around a few well-characterized mouse models which do not capture the heterogeneity of the human disease. To assess the generalizability of these findings within a given tumor type, we have assembled a panel of transplantable mouse models of metastatic breast cancer. Using this panel of models, we tested the efficacy of a pan-TGF-β neutralizing antibody (1D11), against the metastatic endpoint using fully immunocompetent mouse hosts to capture the contribution of anti-tumor immune responses. We observed therapeutic efficacy or trend to efficacy in 5 models (InhibMet), no effect in 2 models (NoEff) and an undesirable stimulation or trend to stimulation of metastasis in 4 models (StimMet). This heterogeneity in therapeutic responses suggests that it will be critical to develop good predictive biomarkers for patient selection in clinical trials using TGF-β antagonists. Plausible candidate biomarkers suggested by the existing literature, such as p53 mutation status, claudin-low status, or TGF-β protein expression, did not correlate with therapeutic response, so we applied integrated genomic discovery approaches to the panel. We find significant differences in patterns of gene expression, gene polymorphism/mutation and copy number variation between the StimMet and InhibMet models. Notably, transcriptomic analyses of the untreated primary tumors show that these segregate by response to therapy in principal component analysis, after removal of mouse strain and tumor origin (spontaneous vs genetically-engineered) as factors. Analysis of differentially-expressed genes suggests that InhibMet models are characterized by higher TGF-β pathway activation, higher angiogenesis, poor immune cell infiltration/activation, and other markers of tumor aggressiveness such as higher tumor cell proliferation and survival. The data point to fundamental differences in tumor biology between the two classes of model and suggest that it will be possible to generate biomarkers that predict therapeutic response to TGF-β pathway antagonists. Citation Format: Yu-an Yang, Kathleen C. Flanders, Binwu Tang, Miriam R. Anver, Anand Merchant, Howard Yang, Maxwell Lee, Scott Lonning, John M. McPherson, Lalage M. Wakefield. Neutralizing anti-TGF-β antibodies elicit heterogeneous therapeutic responses in a panel of murine metastatic breast cancer models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4094. doi:10.1158/1538-7445.AM2015-4094

Differential Proteome Analysis Identifies TGF-β-Related Pro-Metastatic Proteins in a 4T1 Murine Breast Cancer Model.

Transforming growth factor-β (TGF-β) has a dual role in tumorigenesis, acting as either a tumor suppressor or as a pro-oncogenic factor in a context-dependent manner. Although TGF-β antagonists have been proposed as anti-metastatic therapies for patients with advanced stage cancer, how TGF-β mediates metastasis-promoting effects is poorly understood. Establishment of TGF-β-related protein expression signatures at the metastatic site could provide new mechanistic information and potentially allow identification of novel biomarkers for clinical intervention to discriminate TGF-β oncogenic effects from tumor suppressive effects. In the present study, we found that systemic administration of the TGF-β receptor kinase inhibitor, SB-431542, significantly inhibited lung metastasis from transplanted 4T1 mammary tumors in Balb/c mice. The differentially expressed proteins in the comparison of lung metastases from SB-431542 treated and control vehicle-treated groups were analyzed by a quantitative LTQ Orbitrap Velos system coupled with stable isotope dimethyl labeling. A total of 36,239 peptides from 6,694 proteins were identified, out of which 4,531 proteins were characterized as differentially expressed. A subset of upregulated proteins in the control group was validated by western blotting and immunohistochemistry. The eukaryotic initiation factor (eIF) family members constituted the most enriched protein pathway in vehicle-treated compared with SB-43512-treated lung metastases, suggesting that increased protein expression of specific eIF family members, especially eIF4A1 and eEF2, is related to the metastatic phenotype of advanced breast cancer and can be down-regulated by TGF-β pathway inhibitors. Thus our proteomic approach identified eIF pathway proteins as novel potential mediators of TGF-β tumor-promoting activity.

Abstract 3126: Renca RCC syngeneic model to evaluate efficacy of novel antisense oligonucleotides targeting TGF-β isoforms

Abstract Transforming growth factor beta (TGF-β represents a family of cytokines, which function as the primary mediators for TGF-β signaling via TGF-β receptor type II (TβIIR) and both non-canonical and canonical downstream signaling pathways. TGF-β is associated with a wide range of biological processes in oncology, including tumor cell invasion, migration, angiogenesis, immunosuppression, as well as regulation of tumor stem cell properties. Hence, optimal preclinical evaluation of efficacy of TGF-β antagonists is challenging. Isarna Therapeutics has designed and developed selective and potent LNA-modified antisense oligonucleotides targeting the various TGF-β isoforms. In order to adequately evaluate selected preclinical development candidates, Oncodesign has developed customized experimental Renca tumor models in syngeneic and/or immunodeficient mice. The Renca cell line was established from a murine transplantable renal adenocarcinoma of spontaneous origin, and has been used under various experimental conditions: (1) subcutaneous tumor model by inoculating cells into the flanks of the animals; (2) the pulmonary metastatic tumor model by an intravenous injection of cells into the tail vein; and (3) the orthotopic tumor model by injecting cells into the renal subcapsule (and subsequent pulmonary metastasis). Outcome of this development program and preliminary results for selected TGF-β antisense oligonucleotides will be presented and discussed. Citation Format: Hanna Korhonen, Julie-Orlane Redon, Damien France, Guillaume Serin, Francis Bichat, Frank Jaschinski, Katja Wosikowski, Michel Janicot. Renca RCC syngeneic model to evaluate efficacy of novel antisense oligonucleotides targeting TGF-β isoforms. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3126. doi:10.1158/1538-7445.AM2014-3126

An integrated genomic approach identifies persistent tumor suppressive effects of transforming growth factor-β in human breast cancer.

IntroductionTransforming growth factor-βs (TGF-βs) play a dual role in breast cancer, with context-dependent tumor-suppressive or pro-oncogenic effects. TGF-β antagonists are showing promise in early-phase clinical oncology trials to neutralize the pro-oncogenic effects. However, there is currently no way to determine whether the tumor-suppressive effects of TGF-β are still active in human breast tumors at the time of surgery and treatment, a situation that could lead to adverse therapeutic responses.MethodsUsing a breast cancer progression model that exemplifies the dual role of TGF-β, promoter-wide chromatin immunoprecipitation and transcriptomic approaches were applied to identify a core set of TGF-β-regulated genes that specifically reflect only the tumor-suppressor arm of the pathway. The clinical significance of this signature and the underlying biology were investigated using bioinformatic analyses in clinical breast cancer datasets, and knockdown validation approaches in tumor xenografts.ResultsTGF-β-driven tumor suppression was highly dependent on Smad3, and Smad3 target genes that were specifically enriched for involvement in tumor suppression were identified. Patterns of Smad3 binding reflected the preexisting active chromatin landscape, and target genes were frequently regulated in opposite directions in vitro and in vivo, highlighting the strong contextuality of TGF-β action. An in vivo-weighted TGF-β/Smad3 tumor-suppressor signature was associated with good outcome in estrogen receptor-positive breast cancer cohorts. TGF-β/Smad3 effects on cell proliferation, differentiation and ephrin signaling contributed to the observed tumor suppression.ConclusionsTumor-suppressive effects of TGF-β persist in some breast cancer patients at the time of surgery and affect clinical outcome. Carefully tailored in vitro/in vivo genomic approaches can identify such patients for exclusion from treatment with TGF-β antagonists.

SMAD4 mutations causing Myhre syndrome result in disorganization of extracellular matrix improved by losartan

Myhre syndrome (MS, MIM 139210) is a connective tissue disorder that presents with short stature, short hands and feet, facial dysmorphic features, muscle hypertrophy, thickened skin, and deafness. Recurrent missense mutations in SMAD4 encoding for a transducer mediating transforming growth factor β (TGF-β) signaling are responsible for MS. We found that MS fibroblasts showed increased SMAD4 protein levels, impaired matrix deposition, and altered expression of genes encoding matrix metalloproteinases and related inhibitors. Increased TGF-β signaling and progression of aortic root dilation in Marfan syndrome can be prevented by the antihypertensive drug losartan, a TGF-β antagonists and angiotensin-II type 1 receptor blocker. Herein, we showed that losartan normalizes metalloproteinase and related inhibitor transcript levels and corrects the extracellular matrix deposition defect in fibroblasts from MS patients. The results of this study may pave the way toward therapeutic applications of losartan in MS.

P7-004 Histamine inhibits differentiation of skin fibroblasts into myofibroblasts

【Background】 Histamine (HA) and TGF-β, major mediators secreted by mast cells are involved in skin inflammation and systemic sclerosis. However, it remains largely unclear how the respective signaling are integrated to achieve development of skin fibrosis. 【Objectives】 Investigate the effect of HA signaling during myofibroblasts differentiation. 【Methods】 Skin fibroblasts (SFBs) were stimulated with TGF-β, HA, or HA receptor antagonists. Expression of α smooth muscle actin (αSMA), HA receptors, and Smads were analyzed at mRNA and protein. 【Results】 Expression of H1-receptor (H1R) and H2-receptor (H2R) were detectable in SFBs at both mRNA and protein level. Interestingly, addition of HA inhibited αSMA expression induced by TGF-β which was antagonized by H1R antagonist but not H2R antagonist. Correspondingly, phosphorylated Smad2 was induced by TGF-β stimulation whereas inhibited its phosphorylation after HA treatment. 【Conclusions】 HA inhibited differentiation of SFBs into myofibroblast induced by TGF-β in a H1R dependent manner, which emphasized the cirtical functions of H1R in the development of skin fibrosis. Thus, H1R may be a therapeutic target for skin fibrosis.