RARγ Agonist Research Articles

CD1530, selective RARγ agonist, facilitates Achilles tendon healing by modulating the healing environment including less chondrification in a mouse model.

Heterotopic ossification (HO) in Achilles tendon often arises due to endochondral ossification during the healing process following trauma. Retinoic acid receptor γ (RARγ) plays a critical role in this phenomenon. This study aims to elucidate the therapeutic effects of CD1530, an RARγ selective agonist, along with the contributing cells, in Achilles tendon healing, utilizing a cell lineage tracing system. Local injection of CD1530 facilitated histological tendon healing by inhibiting chondrification in a mouse Achilles rupture model. Resident Scleraxis (Scx)+ cells in Achilles tendon were not found to be actively involved in HO or tendon healing following injury. Instead, these processes were primarily driven by tendon stem/progenitor cells (TSPC)-like cells. Furthermore, an in vitro assay revealed that CD1530 attenuated inflammation in injured Achilles tendon-derived tendon fibroblasts (iATF) and inhibited the chondrogenesis of iATF. This dual effect suggests the potential of CD1530 in effectively modulating the healing environment during tendon healing. Together, the present study demonstrated that the local administration of CD1530 accelerated tendon healing by modulating the healing environment, including reducing chondrification via targeting TSPC-like cells in a mouse Achilles tendon rupture model. These results suggest that CD1530 may have the potential to be a novel tendon therapy that offers benefits via the inhibition of chondrogenesis.

Analysis of the Actions of RARγ Agonists on Growing Osteochondromas in a Mouse Model.

The actions of the retinoic acid nuclear receptor gamma (RARγ) agonist, palovarotene, on pre-existing osteochondromas were investigated using a mouse multiple osteochondroma model. This approach was based on the knowledge that patients often present to the clinic after realizing the existence of osteochondroma masses, and the findings from preclinical investigations are the effects of drugs on the initial formation of osteochondromas. Systemic administration of palovarotene, with increased doses (from 1.76 to 4.0 mg/kg) over time, fully inhibited tumor growth, keeping the tumor size (0.31 ± 0.049 mm3) similar to the initial size (0.27 ± 0.031 mm3, p = 0.66) while the control group tumor grew (1.03 ± 0.23 mm3, p = 0.023 to the drug-treated group). Nanoparticle (NP)-based local delivery of the RARγ agonist also inhibited the growth of osteochondromas at an early stage (Control: 0.52 ± 0.11 mm3; NP: 0.26 ± 0.10, p = 0.008). Transcriptome analysis revealed that the osteoarthritis pathway was activated in cultured chondrocytes treated with palovarotene (Z-score = 2.29), with the upregulation of matrix catabolic genes and the downregulation of matrix anabolic genes, consistent with the histology of palovarotene-treated osteochondromas. A reporter assay performed in cultured chondrocytes demonstrated that the Stat3 pathway, but not the Stat1/2 pathway, was stimulated by RARγ agonists. The activation of Stat3 by palovarotene was confirmed using immunoblotting and immunohistochemistry. These findings suggest that palovarotene treatment is effective against pre-existing osteochondromas and that the Stat3 pathway is involved in the antitumor actions of palovarotene.

Molecular Interactions of Selective Agonists and Antagonists with the Retinoic Acid Receptor γ.

All-trans retinoic acid (ATRA), the major active metabolite of all-trans retinol (vitamin A), is a key hormonal signaling molecule. In the adult organism, ATRA has a widespread influence on processes that are crucial to the growth and differentiation of cells and, in turn, the acquisition of mature cell functions. Therefore, there is considerable potential in the use of retinoids to treat diseases. ATRA binds to the retinoic acid receptors (RAR) which, as activated by ATRA, selectively regulate gene expression. There are three main RAR isoforms, RARα, RARβ, and RARγ. They each have a distinct role, for example, RARα and RARγ regulate myeloid progenitor cell differentiation and hematopoietic stem cell maintenance, respectively. Hence, targeting an isoform is crucial to developing retinoid-based therapeutics. In principle, this is exemplified when ATRA is used to treat acute promyelocytic leukemia (PML) and target RARα within PML-RARα oncogenic fusion protein. ATRA with arsenic trioxide has provided a cure for the once highly fatal leukemia. Recent in vitro and in vivo studies of RARγ have revealed the potential use of agonists and antagonists to treat diseases as diverse as cancer, heterotopic ossification, psoriasis, and acne. During the final drug development there may be a need to design newer compounds with added modifications to improve solubility, pharmacokinetics, or potency. At the same time, it is important to retain isotype specificity and activity. Examination of the molecular interactions between RARγ agonists and the ligand binding domain of RARγ has revealed aspects to ligand binding that are crucial to RARγ selectivity and compound activity and key to designing newer compounds.

Abstract PO5-24-12: IRX5010, a Highly Selective RARγ Nuclear Receptor Agonist Compound Inhibits In Vivo Growth of Murine EMT-6 Triple Negative Breast Cancer and Human JIMT-1Her2+ Breast Cancer By Induction of Tumor-Infiltrating Effector Memory T-cells

Abstract Research Objectives and Rationale: Previously, RARγ agonism has been shown to play an essential role in CD8 T-cell-mediated immunity to infectious pathogens. However, we found no previous reports on effects of RARγ agonists on in vivo tumor growth of triple negative or Her2+ breast cancers. Here we explored whether RARγ could play a critical role in T-cell-mediated immunity in these breast cancers, using potent highly selective novel RARγ agonist compounds. Experimental methods and results: We synthesized and screened compounds for selective transactivation of RAR γ in reporter assays. Three highly potent compounds, demonstrating highly selective RARγ transactivation at less than one nanomolar concentrations, were selected for further evaluation of potential anti-cancer activities in vitro. None of the three evaluated compounds demonstrated significant inhibitory effects on in vitro growth of EMT-6 triple negative breast cancer cells at concentrations of 20-100 millimolar, indicating that the three compounds have insignificant direct inhibitory effects on EMT-6 cancer cell growth in vitro. We then examined the effects of one of the compounds in vivo in a syngeneic mouse model of EMT-6 triple negative breast cancer. Treatment with the most potent of the three compounds, IRX5010, demonstrated dose response inhibition of in vivo growth of EMT-6 tumors. Inhibition of tumor growth at day 17 relative to vehicle was 59% and 69% respectively in mice treated with IRX5010 at 10 or 25 mg/kg/day. A cohort treated with 50 mg/kg/day was sacrificed early on day 12 due to excessive weight loss and deaths of some of the animals. This cohort experienced a 73% inhibition of tumor growth relative to vehicle. Gross necropsies of animals in the 25 and 50 mg/kg/day dose groups did not show observable pathologic findings in any major organ. Flow cytometry was performed on freshly excised tumors on the day of termination of the in-life portion of the study in the 10 and 25 mg/kg/day dose groups. Tumors from mice treated with IRX5010 had increased intratumoral numbers of Total CD45+ cells; Total CD3+, Total CD4+, and Total CD8+ T-cells per mg of tumor tissue, relative to vehicle controls. IRX5010 treated mice also had increased intratumoral CD4+IL-17+ T-helper cells; and increased CD4+CD62L- and CD8+CD62L- T-effector memory T-cells per mg of tumor tissue relative to vehicle-treated control mice. We also conducted an experiment of treatment with IRX5010 in mice transplanted with human immune cells (to provide a human immune system) to examine its effects on xenografted human Her2+ JIMT-1 cancer cells. In this model, treatment with IRX5010 at 10 mg/kg/day resulted in a 17% inhibition of tumor growth relative to vehicle control over 26 days of treatment. Flow cytometric analysis showed that tumors from the IRX5010 treated mice contained increased numbers per mg of tumor tissue of intratumoral human CD8+ T-cells, most of which were CD8+ CD7- CD45RA+ terminally differentiated effector memory phenotype T-cells. Conclusions: Treatment with IRX5010, a potent and highly selective RARγ nuclear receptor agonist compound demonstrated substantial treatment effects relative to vehicle control on inhibition of growth of murine triple negative breast cancer in vivo, despite having essentially no growth inhibitory effect on these cells in vitro. Treatment of xenografted human Her2+ tumors in mice with humanized immune systems resulted in a moderate inhibition of Her2+ breast cancer growth relative to vehicle controls. In both models, treatment with IRX5010 induced tumor infiltrating T-lymphocytes of largely effector memory phenotypes, demonstrating an effect on the tumor immune microenvironment. These data identify RARγ and RARγ agonists as a new treatment target and novel treatment modality for breast cancers, especially for the highly unmet need for new and better treatments for triple negative breast cancer. Citation Format: Martin Sanders, Mary Topalovski, Vidyasagar Vuligonda. IRX5010, a Highly Selective RARγ Nuclear Receptor Agonist Compound Inhibits In Vivo Growth of Murine EMT-6 Triple Negative Breast Cancer and Human JIMT-1Her2+ Breast Cancer By Induction of Tumor-Infiltrating Effector Memory T-cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-24-12.

A novel retinoic acid receptor-γ agonist antagonizes immune checkpoint resistance in lung cancers by altering the tumor immune microenvironment

All-trans-retinoic acid (ATRA), the retinoic acid receptors (RARs) agonist, regulates cell growth, differentiation, immunity, and survival. We report that ATRA-treatment repressed cancer growth in syngeneic immunocompetent, but not immunodeficient mice. The tumor microenvironment was implicated: CD8+ T cell depletion antagonized ATRA’s anti-tumorigenic effects in syngeneic mice. ATRA-treatment with checkpoint blockade did not cooperatively inhibit murine lung cancer growth. To augment ATRA’s anti-tumorigenicity without promoting its pro-tumorigenic potential, an RARγ agonist (IRX4647) was used since it regulates T cell biology. Treating with IRX4647 in combination with an immune checkpoint (anti-PD-L1) inhibitor resulted in a statistically significant suppression of syngeneic 344SQ lung cancers in mice—a model known for its resistance to checkpoints and characterized by low basal T cell and PD-L1 expression. This combined treatment notably elevated CD4+ T-cell presence within the tumor microenvironment and increased IL-5 and IL-13 tumor levels, while simultaneously decreasing CD38 in the tumor stroma. IL-5 and/or IL-13 treatments increased CD4+ more than CD8+ T-cells in mice. IRX4647-treatment did not appreciably affect in vitro lung cancer growth, despite RARγ expression. Pharmacokinetic analysis found IRX4647 plasma half-life was 6 h in mice. Yet, RARα antagonist (IRX6696)-treatment with anti-PD-L1 did not repress syngeneic lung cancer growth. Together, these findings provide a rationale for a clinical trial investigating an RARγ agonist to augment check point blockade response in cancers.

All-Trans Retinoic Acid-Responsive LGR6 Is Transiently Expressed during Myogenic Differentiation and Is Required for Myoblast Differentiation and Fusion.

All-trans retinoic acid (ATRA) promotes myoblast differentiation into myotubes. Leucine-rich repeat-containing G-protein-coupled receptor 6 (LGR6) is a candidate ATRA-responsive gene; however, its role in skeletal muscles remains unclear. Here, we demonstrated that during the differentiation of murine C2C12 myoblasts into myotubes, Lgr6 mRNA expression transiently increased before the increase in the expression of the mRNAs encoding myogenic regulatory factors, such as myogenin, myomaker, and myomerger. The loss of LGR6 decreased the differentiation and fusion indices. The exogenous expression of LGR6 up to 3 and 24 h after the induction of differentiation increased and decreased the mRNA levels of myogenin, myomaker, and myomerger, respectively. Lgr6 mRNA was transiently expressed after myogenic differentiation in the presence of a retinoic acid receptor α (RARα) agonist and an RARγ agonist in addition to ATRA, but not in the absence of ATRA. Furthermore, a proteasome inhibitor or Znfr3 knockdown increased exogenous LGR6 expression. The loss of LGR6 attenuated the Wnt/β-catenin signaling activity induced by Wnt3a alone or in combination with Wnt3a and R-spondin 2. These results indicate that LGR6 promotes myogenic differentiation and that ATRA is required for the transient expression of LGR6 during differentiation. Furthermore, LGR6 expression appeared to be downregulated by the ubiquitin-proteasome system involving ZNRF3.

Abstract 1836: Combining a novel retinoic acid receptor-γ agonist with immune checkpoint blockade represses lung cancer growth in vivo

Abstract All-trans-retinoic acid (ATRA), a pan-agonist for retinoic acid receptors (RARs), regulates diverse cellular functions including growth, differentiation and immune function. We report here that ATRA-treatment represses tumor growth in syngeneic, immunocompetent but not in immunodeficient mice. Tumor immune microenvironment was implicated since depletion of cytotoxic T lymphocytes antagonized these effects in syngeneic mice. Combining ATRA with immune checkpoint blockade did not inhibit lung cancer growth in mice. We sought to augment retinoid anti-tumor effects without affecting its pro-tumorigenicity. We previously reported that CD38 mediated resistance to checkpoint blockade in murine 344SQ lung cancer cells via RARα transcriptional activation of CD38 expression. Yet, combining the RARα antagonist (IRX6696) with anti-PD-L1 did not augment anti-tumorigenicity in transplanted 344SQ cells in syngeneic mice. Prior work implicated RARγ in regulating T cell response. Combining the novel RARγ agonist (IRX4647) with anti-PD-L1 statistically-significantly repressed 344SQ lung cancer cell growth in syngeneic mice. This line is relatively resistant to checkpoint blockade. Immunofluorescent analysis of these treated tumors revealed that combined IRX4647 and anti-PD-L1 treatments reduced CD38 expression in the tumor stroma relative to IRX4647 or anti-PD-L1 treatment alone. Statistically-significantly elevated helper (CD4+) T cells were detected in treated tumors along with increased IL-5 and IL-13 expression observed in plasma and tumors. These cytokines can activate helper T cells, altering lung cancer growth. These microenvironment effects were associated with in vivo anti-tumorigenicity. IRX4647-treatment did not appreciably alter in vitro growth of lung cancer cells although retinoid receptors expression profiles were affected. Pharmacokinetic study of IRX4647 found its plasma half-life was 6 hours. Combining an RARγ agonist with immune checkpoint blockade exerted superior anti-neoplastic efficacy against lung cancer versus an ATRA-based regimen. Given these findings we propose exploring activity of this RARγ agonist with an optimal checkpoint inhibitor in a lung cancer clinical trial. Citation Format: Cheng-Hsin Wei, Lu Huang, Blair Kreh, Xiuxia Liu, Liliya Tyutyunyk-Massey, Zibo Chen, Mi Shi, Vidyasagar Vuligonda, Martin Sanders, Serguei Kozlov, King Chan, Amir Horowitz, Mary Carrington, Patrick Hwu, Weiyi Peng, Ethan Dmitrovsky, Xi Liu. Combining a novel retinoic acid receptor-γ agonist with immune checkpoint blockade represses lung cancer growth in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1836.

Structural requirement of RARγ agonism through computational aspects

RARγ is a therapeutic target for many skin diseases and has potential in cancer treatment. In the current study, we put forward a comprehensive structure-activity relationship study of third and fourth generations of RARγ agonists, addressing multiple crystal structures of RARγ complexes and approved drugs. Adapalene and Trifarotene, through hybrid strategies including protein contacts Atlas analysis, molecular docking, dynamics simulations, MM-GBSA, ASM, and pharmacophore modeling. Our result revealed crucial amino acids Arg267, Ser278, Phe288, Phe230, Met272, Leu271, and Leu268 within the RARγ pocket, as well as pharmacophore features such as two hydrophobic groups, two aromatic rings, and negative ionic features, which are essential for the binding of RARγ agonists. Based on this study, the binding mechanism of RARγ agonists was elucidated, which will be helpful for the rational design of new RARγ agonists for skin diseases and cancer treatment. In this study, Schrödinger suite 2021-2 with OPLS_4 force field, Discovery Studio program 3.0, LigandScout 4.3, and PyMOL are utilized in the investigation.

Retinoic Acid Receptor Gamma (RARγ) Promotes Cartilage Destruction through Positive Feedback Activation of NF-κB Pathway in Human Osteoarthritis.

Osteoarthritis (OA) is a severe inflammation-related disease which leads to cartilage destruction. The retinoic acid receptor gamma (RARγ) has been indicated to be involved in many inflammation processes. However, the role and mechanism of RARγ in cartilage destruction caused by inflammation in OA are still unknown. Here, we demonstrated that the RARγ was highly expressed in chondrocytes of OA patients compared with healthy people and was positively correlated with the damage degree of cartilage in OA. Cytokine TNF-α promoted the transcription and expression of RARγ through activating the NF-κB pathway in OA cartilage. In addition, the overexpression of RARγ resulted in the upregulation of matrix degradation and inflammation associated genes and downregulation of differentiation and collagen production genes in human normal chondrocyte C28/I2 cells. Mechanistically, overexpression of RARγ could increase the level of p-IκBα and p-P65 to regulate the expression of downstream genes. RARγ and IκBα also could interact with each other and had the same localization in C28/I2 cells. Moreover, the SD rats OA model induced by monosodium iodoacetate indicated that CD437 (RARγ agonist) and TNF-α accelerated the OA progression, including more severe cartilage layer destruction, larger knee joint diameter, and higher serum ALP levels, while LY2955303 (RARγ inhibitor) showed the opposite result. RARγ was also highly expressed in OA group and even higher in TNF-α group. In conclusion, RARγ/NF-κB positive feedback loop was activated by TNF-α in chondrocyte to promote cartilage destruction. Our data not only propose a novel and precise molecular mechanism for OA disease but also provide a prospective strategy for the treatment.

S290: ATRA CAN CORRECT DEFECTIVE HIF-1Α/S1P AXIS-MEDIATED CYTOSKELETAL REORGANIZATION IN PROPLATELET FORMATION OF ITP

Background: Bone marrow physiological hypoxia plays a crucial role in haematopoietic stem cell homeostasis. Hypoxia inducible factor (HIF) is central to mediating the cellular response to hypoxia. HIF-1α expression was decreased in the bone marrow of patients with immune thrombocytopenia (ITP), and HIF-1α activation was shown to enhance megakaryopoiesis in mice. Recent studies suggest that “inside-out” signalling by S1P in megakaryocytes (MKs) plays a critical role in proplatelet formation (PPF) (Blood, 2013; J EXP MED, 2012). Our previous data indicated that impaired PPF contributed to the development of thrombocytopenia in ITP. To further explore the underlying mechanism of impaired PPF in ITP, we found that HIF-1α/S1P axis-mediated cytoskeletal reorganization was defective in the PPF of ITP. All-trans retinoic acid (ATRA), which has been shown to be a promising treatment option for ITP patients in our clinical studies (Blood, 2021; Lancet haematology, 2017; Lancet haematology, 2021), could restore cytoskeletal reorganization and correct impaired PPF. Aims: This study aimed to explore the role of hypoxia inducible factor-1α (HIF-1α) in proplatelet formation (PPF) and the underlying mechanisms of ATRA treatment in ITP patients. Methods: Thirty consecutive patients with newly diagnosed ITP and 30 healthy donors were included in our study. MKs were isolated from bone marrow samples. Targeted and untargeted metabolomic profiling through metabolomic analysis was performed to explore the relationship between the metabolome and ITP. Confocal microscopy and transmission electron microscopy were used to observe the PPF and cytoskeleton structure of ITP MKs. An ITP mouse model was established to observe the therapeutic effects of ATRA in the PPF. Results: In the present study, we observed that MKs displayed altered cytoskeletal reorganization and impaired proplatelet formation (PPF) in ITP patients. Targeted and untargeted metabolite profiling revealed a decreased sphingosine-1-phosphate (S1P) level in ITP. Downregulated sphingosine kinase 2 (SPHK2) expression in MKs accounted for the low level of S1P in ITP. S1P is essential for S1P receptor 1 (S1PR1) and Rac1 activation, Src family kinases (SFKs) activity, and subsequent cytoskeletal reorganization and PPF regulation. Moreover, we demonstrated that HIF-1α mediated SPHK2 activation and S1P production. Decreased HIF-1α levels were found in the MKs of patients with ITP, contributing to impaired PPF. We then investigated the effect of ATRA on PPF in ITP patients. ATRA upregulated HIF-1α and SPHK2 expression, increased S1P production and corrected impaired PPF in vitro. In an ITP mouse model, ATRA alleviated thrombocytopenia and restored cytoskeletal reorganization. ATRA corrected impaired PPF by upregulating HIF-1α expression. The exposure of ITP MKs to selective RARα (AM580) or RARγ (BMS961) agonists did not change PPF. However, the treatment of ITP MKs with a selective RARβ agonist (CD2314) significantly increased PPF. Furthermore, we found that the effect of ATRA on enhancing PPF in ITP MKs was reversed in the presence of an RARβ antagonist (CD2665). These data suggest that impaired PPF in ITP MKs is corrected by ATRA in a RARβ-dependent manner. Summary/Conclusion: Together, our data show that the HIF-1α/S1P axis mediates altered cytoskeletal reorganization and impaired PPF in ITP and suggest that ATRA correction of impaired PPF is a potential mechanistic explanation for the clinical efficacy of ATRA in ITP.

Abstract LB518: Osteochondromas respond and its development is inhibited by retinoic acid nuclear receptor gamma agonist treatment

Abstract Osteochondromas (OC) are cartilage-capped tumors that arise near growing physis and are considered the most common benign bone tumor in children. They can lead to skeletal deformity, pain, loss of motion and neurovascular compression. Occasionally, malignant degeneration can occur. OC is thought to arise by mysregulation of chondrogenesis or chondrocyte differentiation, leading to ectopic cartilage formation. OC can occur as a solitary or multiple OC (MO). MO can occur from a hereditary cancer syndrome in which EXT1 and EXT2 are the major causative genes. Currently, treatment is limited to surgical resection only. There are no available FDA-approved therapies for MO. Previous translational research suggested that retinoic acid nuclear receptor gamma agonist (RARγ) suppresses ectopic cartilage formation including OC in rodent models. These preclinical studies led to a clinical trial to study the efficacy and safety of Palovarotene, a RARγ agonist, for the systemic treatment of MO (NCT03442985). The clinical trial was terminated due to concerns of skeletal toxicity observed in pediatric patients. To overcome this adverse action, we should elucidate the molecular action of RARγ agonists on osteochondromas and refine the treatment regimen. The purposes of our study are to examine the mechanism in which RARγ elicits osteochondroma growth and to determine whether the responsiveness to RARγ agonists is different between OC and growth plate. Treatment of RARγ agonists on mouse chondrocytes stimulated cell death induced by hydrogen peroxide (H2O2). In addition, human OC explants treated with RARγ agonists contained more apoptotic cells than vehicle-treated OC explants. OCs were induced in AcanCreER; Ext1e2neofl/e2neofl MO mouse model via tamoxifen induction at P5. After two weeks, mice were treated with Palovarotene (1.76 mg/Kg) or corn oil, and 24hrs after treatment, forelimbs were harvested. In-situ hybridization analysis revealed that Palovarotene-treated OC had much more transcripts of Cyp26b1 (retinoid target gene) as compared with the corn oil-treated OC and that OCs had significantly higher response to Palovarotene compared to the adjacent growth plate. When Palovarotene (1.76 mg/Kg, daily) treatment continued for 2 weeks, OC development was greatly suppressed compared to vehicle control in the wrists and ribs as determined by histological staining and radiological assessments, whereas the Palovarotene-treated growth plate did not show histological abnormality. Together, these findings indicate that OC have higher sensitivity to RARγ agonist compared to the growth plate and RARγ agonists rapidly inhibits OC development without a significant negative effect on growth plate and may exert anti-tumor function on OC by inducing cell death via apoptosis. Citation Format: Sonia Garcia, Hongying Tian, John Herzenberg, Joshua Abzug, Vincent Ng, Ashley Cellini, Masahiro Iwamoto, Motomi Enomoto-Iwamoto. Osteochondromas respond and its development is inhibited by retinoic acid nuclear receptor gamma agonist treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB518.

Abstract LB518A: Osteochondromas respond and its development is inhibited by retinoic acid nuclear receptor gamma agonist treatment

Abstract Osteochondromas (OC) are cartilage-capped tumors that arise near growing physis and are considered the most common benign bone tumor in children. They can lead to skeletal deformity, pain, loss of motion and neurovascular compression. Occasionally, malignant degeneration can occur. OC is thought to arise by mysregulation of chondrogenesis or chondrocyte differentiation, leading to ectopic cartilage formation. OC can occur as a solitary or multiple OC (MO). MO can occur from a hereditary cancer syndrome in which EXT1 and EXT2 are the major causative genes. Currently, treatment is limited to surgical resection only. There are no available FDA-approved therapies for MO. Previous translational research suggested that retinoic acid nuclear receptor gamma agonist (RARγ) suppresses ectopic cartilage formation including OC in rodent models. These preclinical studies led to a clinical trial to study the efficacy and safety of Palovarotene, a RARγ agonist, for the systemic treatment of MO (NCT03442985). The clinical trial was terminated due to concerns of skeletal toxicity observed in pediatric patients. To overcome this adverse action, we should elucidate the molecular action of RARγ agonists on osteochondromas and refine the treatment regimen. The purposes of our study are to examine the mechanism in which RARγ elicits osteochondroma growth and to determine whether the responsiveness to RARγ agonists is different between OC and growth plate. Treatment of RARγ agonists on mouse chondrocytes stimulated cell death induced by hydrogen peroxide (H2O2). In addition, human OC explants treated with RARγ agonists contained more apoptotic cells than vehicle-treated OC explants. OCs were induced in AcanCreER; Ext1e2neofl/e2neofl MO mouse model via tamoxifen induction at P5. After two weeks, mice were treated with Palovarotene (1.76 mg/Kg) or corn oil, and 24hrs after treatment, forelimbs were harvested. In-situ hybridization analysis revealed that Palovarotene-treated OC had much more transcripts of Cyp26b1 (retinoid target gene) as compared with the corn oil-treated OC and that OCs had significantly higher response to Palovarotene compared to the adjacent growth plate. When Palovarotene (1.76 mg/Kg, daily) treatment continued for 2 weeks, OC development was greatly suppressed compared to vehicle control in the wrists and ribs as determined by histological staining and radiological assessments, whereas the Palovarotene-treated growth plate did not show histological abnormality. Together, these findings indicate that OC have higher sensitivity to RARγ agonist compared to the growth plate and RARγ agonists rapidly inhibits OC development without a significant negative effect on growth plate and may exert anti-tumor function on OC by inducing cell death via apoptosis. Citation Format: Sonia Garcia, Hongying Tian, John Herzenberg, Joshua Abzug, Vincent Ng, Ashley Cellini, Masahiro Iwamoto, Motomi Enomoto-Iwamoto. Osteochondromas respond and its development is inhibited by retinoic acid nuclear receptor gamma agonist treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB518A.

Retinoid Agonists in the Targeting of Heterotopic Ossification.

Retinoids are metabolic derivatives of vitamin A and regulate the function of many tissues and organs both prenatally and postnatally. Active retinoids, such as all trans-retinoic acid, are produced in the cytoplasm and then interact with nuclear retinoic acid receptors (RARs) to up-regulate the transcription of target genes. The RARs can also interact with target gene response elements in the absence of retinoids and exert a transcriptional repression function. Studies from several labs, including ours, showed that chondrogenic cell differentiation and cartilage maturation require (i) the absence of retinoid signaling and (ii) the repression function by unliganded RARs. These and related insights led to the proposition that synthetic retinoid agonists could thus represent pharmacological agents to inhibit heterotopic ossification (HO), a process that recapitulates developmental skeletogenesis and involves chondrogenesis, cartilage maturation, and endochondral ossification. One form of HO is acquired and is caused by injury, and another severe and often fatal form of it is genetic and occurs in patients with fibrodysplasia ossificans progressiva (FOP). Mouse models of FOP bearing mutant ACVR1R206H, characteristic of most FOP patients, were used to test the ability of the retinoid agonists selective for RARα and RARγ against spontaneous and injury-induced HO. The RARγ agonists were found to be most effective, and one such compound, palovarotene, was selected for testing in FOP patients. The safety and effectiveness data from recent and ongoing phase II and phase III clinical trials support the notion that palovarotene may represent a disease-modifying treatment for patients with FOP. The post hoc analyses showed substantial efficacy but also revealed side effects and complications, including premature growth plate closure in some patients. Skeletally immature patients will need to be carefully weighed in any future regulatory indications of palovarotene as an important therapeutic option in FOP.

Dysregulated retinoic acid signaling in airway smooth muscle cells in asthma.

Vitamin A deficiency has been shown to exacerbate allergic asthma. Previous studies have postulated that retinoic acid (RA), an active metabolite of vitamin A and high-affinity ligand for RA receptor (RAR), is reduced in airway inflammatory condition and contributes to multiple features of asthma including airway hyperresponsiveness and excessive accumulation of airway smooth muscle (ASM) cells. In this study, we directly quantified RA and examined the molecular basis for reduced RA levels and RA-mediated signaling in lungs and ASM cells obtained from asthmatic donors and in lungs from allergen-challenged mice. Levels of RA and retinol were significantly lower in lung tissues from asthmatic donors and house dust mite (HDM)-challenged mice compared to non-asthmatic human lungs and PBS-challenged mice, respectively. Quantification of mRNA and protein expression revealed dysregulation in the first step of RA biosynthesis consistent with reduced RA including decreased protein expression of retinol dehydrogenase (RDH)-10 and increased protein expression of RDH11 and dehydrogenase/reductase (DHRS)-4 in asthmatic lung. Proteomic profiling of non-asthmatic and asthmatic lungs also showed significant changes in the protein expression of AP-1 targets consistent with increased AP-1 activity. Further, basal RA levels and RA biosynthetic capabilities were decreased in asthmatic human ASM cells. Treatment of human ASM cells with all-trans RA (ATRA) or the RARγ-specific agonist (CD1530) resulted in the inhibition of mitogen-induced cell proliferation and AP-1-dependent transcription. These data suggest that RA metabolism is decreased in asthmatic lung and that enhancing RAR signaling using ATRA or RARγ agonists may mitigate airway remodeling associated with asthma.

RARγ activation sensitizes human myeloma cells to carfilzomib treatment through the OAS-RNase L innate immune pathway

AbstractProteasome inhibitors (PIs) such as bortezomib (Btz) and carfilzomib (Cfz) are highly efficacious for patients with multiple myeloma (MM). However, relapses are frequent, and acquired resistance to PI treatment emerges in most patients. Here, we performed a high-throughput screen of 1855 Food and Drug Administration (FDA)-approved drugs and identified all-trans retinoic acid (ATRA), which alone has no antimyeloma effect, as a potent drug that enhanced MM sensitivity to Cfz-induced cytotoxicity and resensitized Cfz-resistant MM cells to Cfz in vitro. ATRA activated retinoic acid receptor (RAR)γ and interferon-β response pathway, leading to upregulated expression of IRF1. IRF1 in turn initiated the transcription of OAS1, which synthesized 2-5A upon binding to double-stranded RNA (dsRNA) induced by Cfz and resulted in cellular RNA degradation by RNase L and cell death. Similar to ATRA, BMS961, a selective RARγ agonist, could also (re)sensitize MM cells to Cfz in vitro, and both ATRA and BMS961 significantly enhanced the therapeutic effects of Cfz in established MM in vivo. In support of these findings, analyses of large datasets of patients' gene profiling showed a strong and positive correlation between RARγ and OAS1 expression and patient's response to PI treatment. Thus, this study highlights the potential for RARγ agonists to sensitize and overcome MM resistance to Cfz treatment in patients.

RAR Gamma Activation Sensitizes Human Myeloma Cells to Carfilzomib Treatment through OAS-RNase L Innate Immune Pathway

Proteasome inhibitors (PIs) such as bortezomib (Btz) and carfilzomib (Cfz) kill MM cells by disrupting the degradation of misfolded proteins, presumably derived from high-level immunoglobulin production, which provides an appealing explanation for why MM cells are so uniquely sensitive to PIs. However, relapses are frequent and acquired resistance to PI treatment emerges in most patients. Therefore, identifying novel and safe drugs overcoming PI resistance in MM will aid in chemo-(re)sensitization, reducing PI-induced side effects, and maximizing the outcomes of PI therapy. Here we performed a high-throughput screen of 1855 FDA-approved drugs (Figure 1) and identified all-trans retinoic acid (ATRA), a classic pan-retinoic acid receptor (RAR) agonist used successfully to treat acute promyelocytic leukemia (APL), as a potent drug that enhanced MM sensitivity to Cfz-induced cytotoxicity and re-sensitized Cfz-resistant MM cells to Cfz in vitro. To determine which RARs are important for ATRA enhancement of Cfz-induced apoptosis in MM, esiRNAs of RARs for knocking down RARs and selective agonists of RARs were used in Cfz-treated MM cells. We identified that RARγ activation is important for ATRA sensitizing MM cells to Cfz treatment. To determine which signaling pathways are involved in ATRA-treated MM cells, gene-profiling data analysis of Cfz- versus ATRA+Cfz-treated MM cells was performed and real-time PCR was used to validate the microarray results. We found that ATRA treatment activated IFN-β response pathway (Figure 2), leading to upregulated expression of IRF1 and OAS1-3. Interestingly, similar to ATRA, IFN-β, which alone did not induce MM apoptosis, enhanced Cfz-induced MM cell apoptosis. Furthermore, using RNA integrity assay and dsRNA detection assay, we identified that ATRA treatment elevated the expression of OASs, which synthesized 2-5A upon binding to dsRNA induced by Cfz and resulted in cellular RNA degradation by RNase L and cell death (Figure 3). By knocking down each gene of OAS1-3, we demonstrated that OAS1 is essential for ATRA to sensitize MM cells to Cfz-induced apoptosis. Furthermore, we determined the impact and significance of RARγ and OAS1 in human MM pathogenesis and drug response by analyzing the gene-profiling data of 264 MM patients from Mulligan et al. datasets and 1,143 MM patients from MMRF coMMpass study IA13. In support of these findings, analyses of the large patient's gene-profiling datasets showed a strong and positive correlation between RARγ and OAS1 expression and patient's response to PI treatment (Figure 4). Finally, BMS961, a selective RARγ agonist, similar to ATRA, could also (re)sensitize MM cells to Cfz in vitro, and both ATRA and BMS961 significantly enhanced the therapeutic effects of Cfz in established MM in vivo (Figure 5). Thus, this study highlights the potential for RARγ agonists to sensitize MM and overcome MM resistance to Cfz treatment in patients. Disclosures No relevant conflicts of interest to declare.

Retinoic acid receptor \u03b3 activation promotes differentiation of human induced pluripotent stem cells into esophageal epithelium

BackgroundThe esophagus is known to be derived from the foregut. However, the mechanisms regulating this process remain unclear. In particular, the details of the human esophagus itself have been poorly researched. In this decade, studies using human induced pluripotent stem cells (hiPSCs) have proven powerful tools for clarifying the developmental biology of various human organs. Several studies using hiPSCs have demonstrated that retinoic acid (RA) signaling promotes the differentiation of foregut into tissues such as lung and pancreas. However, the effect of RA signaling on the differentiation of foregut into esophagus remains unclear.MethodsWe established a novel stepwise protocol with transwell culture and an air–liquid interface system for esophageal epithelial cell (EEC) differentiation from hiPSCs. We then evaluated the effect of all-trans retinoic acid (ATRA), which is a retinoic acid receptor (RAR)α, RARβ and RARγ agonist, on the differentiation from the hiPSC-derived foregut. Finally, to identify which RAR subtype was involved in the differentiation, we used synthetic agonists and antagonists of RARα and RARγ, which are known to be expressed in esophagus.ResultsWe successfully generated stratified layers of cells expressing EEC marker genes that were positive for lugol staining. The enhancing effect of ATRA on EEC differentiation was clearly demonstrated with quantitative reverse transcription polymerase chain reaction, immunohistology, lugol-staining and RNA sequencing analyses. RARγ agonist and antagonist enhanced and suppressed EEC differentiation, respectively. RARα agonist had no effect on the differentiation.ConclusionWe revealed that RARγ activation promotes the differentiation of hiPSCs-derived foregut into EECs.

Understanding the Action of RARγ Agonists on Human Osteochondroma Explants.

Osteochondromas are cartilage-capped growths located proximate to the physis that can cause skeletal deformities, pain, limited motion, and neurovascular impingement. Previous studies have demonstrated retinoic acid receptor gamma (RARγ) agonists to inhibit ectopic endochondral ossification, therefore we hypothesize that RARγ agonists can target on established osteochondromas. The purpose of this study was to examine the action of RARγ agonist in human osteochondromas. Osteochondroma specimens were obtained during surgery, subjected to explant culture and were treated with RARγ agonists or vehicles. Gene expression analysis confirmed the up-regulation of RARγ target genes in the explants treated with NRX 204647 and Palovarotene and revealed strong inhibition of cartilage matrix and increased extracellular matrix proteases gene expression. In addition, immunohistochemical staining for the neoepitope of protease-cleaved aggrecan indicated that RARγ agonist treatment stimulated cartilage matrix degradation. Interestingly, cell survival studies demonstrated that RARγ agonist treatment stimulated cell death. Moreover, RNA sequencing analysis indicates changes in multiple molecular pathways due to RARγ agonists treatment, showing similarly to human growth plate chondrocytes. Together, these findings suggest that RARγ agonist may exert anti-tumor function on osteochondromas by inhibiting matrix synthesis, promoting cartilage matrix degradation and stimulating cell death.

Selective Agonists of Nuclear Retinoic Acid Receptor Gamma Inhibit Growth of HCS-2/8 Chondrosarcoma Cells.

Chondrosarcoma is the second most common primary bone sarcoma. Treatment of chondrosarcoma is limited to surgery due to radiation and chemotherapy resistance of this cancer. An ideal treatment for chondrosarcoma would be a well-tolerated, minimally invasive local or systemic treatment modality to halt or slow tumor growth prior to resection of local, unresectable local, or metastatic disease. Palovarotene, an agonist of nuclear retinoic acid receptor γ (RARγ) has shown therapeutic action for treatment of heterotopic ossification and osteochondroma without serious adverse effects in animal models. We hypothesized that selective agonists of RARγ would have an inhibitory effect on chondrosarcoma. All human chondrosarcoma specimens expressed RARγ as determined by immunohistochemical staining. The ΗCS-2/8 chondrosarcoma cell line, established from low-grade human chondrosarcoma, was used to examine the actions of RARγ agonists. In ΗCS2/8 pellet cultures, RARγ agonist treatment reduced the mass size and significantly decreased total glycosaminoglycan, protein amounts, and gene expression levels of cartilage matrix molecules when compared with control groups. Systemic treatment with RARγ agonists significantly inhibited the growth of ΗCS-2/8 cell transplants in vivo. Furthermore, local injection of RARγ agonist-loaded poly-lactic acid nanoparticles induced regression of the mass size of the transplants. Histologic analysis demonstrated that RARγ agonist treatment inhibited cell proliferation activity and stimulated encapsulation of the tumor. These findings indicate that RARγ agonists, including palovarotene, may have an anti-tumor effect on low-grade chondrosarcomas. © 2019Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:1045-1051, 2020.

Inhibitory effect of retinoic acid receptor agonists on in vitro chondrogenic differentiation.

Retinoic acid (RA), an active metabolite of vitamin A, plays pivotal roles in a wide variety of biological processes, such as body patterning, organ development, and cell differentiation and proliferation. RA signaling is mediated by nuclear retinoic acid receptors, α, β, and γ (RARα, RARβ, and RARγ). RA is a well-known regulator of cartilage and skeleton formation and RARs are also essential for skeletal growth and hypertrophic chondrocyte-specific gene expression. These important roles of RA and RARs in chondrogenesis have been widely investigated using in vivo mouse models. However, few reports are available on the function of each subtype of RARs on in vitro chondrocyte differentiation. Here, we examined the effect of specific agonists of RARs on chondrogenic differentiation of ATDC5 and C3H10T1/2 cells. Subtype-specific RAR agonists as well as RA decreased the expressions of chondrogenic differentiation marker genes and inhibited chondrogenic differentiation, which was accompanied with morphological change to spindle-shaped cells. Among RAR agonists, RARα and RARγ agonists revealed a strong inhibitory effect on chondrogenic differentiation. RARα and RARγ agonists also hampered viability of ATDC5 cells. These observations suggested that RARα and RARγ are dominant receptors of RA signaling that negatively regulate chondrogenic differentiation.