Next-generation retinoid X receptor agonists increase ATRA signaling in organotypic epithelium cultures and have distinct effects on receptor dynamics.

Retinoid X receptors (RXRs) are nuclear transcription factors that partner with other nuclear receptors to regulate numerous physiological processes. Although RXR represents a valid therapeutic target, only a few RXR-specific ligands (rexinoids) have been identified, in part due to the lack of clarity on how rexinoids selectively modulate RXR response. Previously, we showed that rexinoid UAB30 potentiates all-trans-retinoic acid (ATRA) signaling in human keratinocytes, in part by stimulating ATRA biosynthesis. Here, we examined the mechanism of action of next-generation rexinoids UAB110 and UAB111 that are more potent in vitro than UAB30 and the FDA-approved Targretin. Both UAB110 and UAB111 enhanced ATRA signaling in human organotypic epithelium at a 50-fold lower concentration than UAB30. This was consistent with the 2- to 5- fold greater increase in ATRA in organotypic epidermis treated with UAB110/111 versus UAB30. Furthermore, at 0.2 µM, UAB110/111 increased the expression of ATRA genes up to 16-fold stronger than Targretin. The less toxic and more potent UAB110 also induced more changes in differential gene expression than Targretin. Additionally, our hydrogen deuterium exchange mass spectrometry analysis showed that both ligands reduced the dynamics of the ligand-binding pocket but also induced unique dynamic responses that were indicative of higher affinity binding relative to UAB30, especially for Helix 3. UAB110 binding also showed increased dynamics towards the dimer interface through the Helix 8 and Helix 9 regions. These data suggest that UAB110 and UAB111 are potent activators of RXR-RAR signaling pathways but accomplish activation through different molecular responses to ligand binding.

UAB30, a novel RXR agonist, decreases tumorigenesis and leptomeningeal disease in group 3 medulloblastoma patient-derived xenografts.

BACKGROUND: Group 3 tumors account for approximately 25-30% of medulloblastomas and have the worst prognosis. UAB30 is a novel synthetic rexinoid shown to have limited toxicities in humans and significant efficacy in the pediatric neuroectodermal tumor, neuroblastoma. We hypothesized that treatment with UAB30 would decrease tumorigenicity in medulloblastoma patient-derived xenografts (PDXs). METHODS: Three group 3 medulloblastoma PDXs (D341, D384 and D425) were utilized. Cell viability, proliferation, migration and invasion assays were performed after treatment with UAB30 or 13-cis-retinoic acid (RA). Cell cycle analysis was completed using flow cytometry. A flank model, a cerebellar model, and a model of leptomeningeal metastasis using human medulloblastoma PDX cells was used to assess the in vivo effects of UAB30 and RA. RESULTS: UAB30 treatment led to cell differentiation and decreased medulloblastoma PDX cell viability, proliferation, migration and invasion and G1 cell cycle arrest in all three PDXs similar to RA. UAB30 and RA treatment of mice bearing medulloblastoma PDX tumors resulted in a significant decrease in tumor growth and metastasis compared to vehicle treated animals. CONCLUSIONS: UAB30 decreased viability, proliferation, and motility in group 3 medulloblastoma PDX cells and significantly decreased tumor growth in vivo in a fashion similar to RA, suggesting that further investigations into the potential therapeutic application of UAB30 for medulloblastoma are warranted.

A novel retinoid X receptor agonist, UAB30, inhibits rhabdomyosarcoma cells in vitro.

BACKGROUND: While patients with early-stage rhabdomyosarcoma (RMS) have seen steady improvement in prognosis over the last 50 y, those with advanced-stage or high-grade disease continue to have a dismal prognosis. Retinoids have been shown to cause growth suppression and terminal differentiation in RMS cells, but the toxicities associated with retinoic acid limit its use. Rexinoids provide an alternative treatment approach to retinoic acid. Rexinoids primarily bind the retinoid X receptor with minimal retinoic acid receptor binding, the entity responsible for many of the toxicities of retinoid therapies. UAB30 is a novel rexinoid with limited toxicities. We hypothesized that UAB30 would lead to decreased cell survival in RMS. MATERIALS AND METHODS: Two RMS cell lines, one embryonal (RD) subtype and one alveolar (St. Jude Cancer Research Hospital 30) subtype, were used. Cells were treated with UAB30, and cytotoxicity, proliferation, mobility, and apoptosis were evaluated. RESULTS: UAB30 significantly decreased RMS tumor cell viability and proliferation. Invasion, migration, and attachment-independent growth were reduced following UAB30 treatment. UAB30 also resulted in apoptosis and G1 cell cycle arrest. UAB30 affected both the alveolar and embryonal RMS cell lines in a similar fashion. CONCLUSIONS: The results of these studies suggest a potential therapeutic role for the low-toxicity synthetic retinoid X receptor selective agonist, UAB30, in RMS treatment.

Defining the communication between agonist and coactivator binding in the retinoid X receptor α ligand binding domain.

Retinoid X receptors (RXRs) are obligate partners for several other nuclear receptors, and they play a key role in several signaling processes. Despite being a promiscuous heterodimer partner, this nuclear receptor is a target of therapeutic intervention through activation using selective RXR agonists (rexinoids). Agonist binding to RXR initiates a large conformational change in the receptor that allows for coactivator recruitment to its surface and enhanced transcription. Here we reveal the structural and dynamical changes produced when a coactivator peptide binds to the human RXRα ligand binding domain containing two clinically relevant rexinoids, Targretin and 9-cis-UAB30. Our results show that the structural changes are very similar for each rexinoid and similar to those for the pan-agonist 9-cis-retinoic acid. The four structural changes involve key residues on helix 3, helix 4, and helix 11 that move from a solvent-exposed environment to one that interacts extensively with helix 12. Hydrogen-deuterium exchange mass spectrometry reveals that the dynamics of helices 3, 11, and 12 are significantly decreased when the two rexinoids are bound to the receptor. When the pan-agonist 9-cis-retinoic acid is bound to the receptor, only the dynamics of helices 3 and 11 are reduced. The four structural changes are conserved in all x-ray structures of the RXR ligand-binding domain in the presence of agonist and coactivator peptide. They serve as hallmarks for how RXR changes conformation and dynamics in the presence of agonist and coactivator to initiate signaling.

Src is a novel potential off-target of RXR agonists, 9-cis-UAB30 and Targretin, in human breast cancer cells.

9-cis-UAB30 (UAB30) and Targretin are well-known retinoid X receptor (RXR) agonists. They were highly effective in decreasing the incidence of methylnitrosourea (MNU)-induced mammary cancers. However, whether the anti-mammary cancer effects of UAB30 or Targretin originate from the activation of RXR is unclear. In the present study, we hypothesized that UAB30 and Targretin not only affect RXR, but likely influence one or more off-target proteins. Virtual screening results suggest that Src is a potential target for UAB30 and Targretin that regulates extracellular matrix (ECM) molecules and cell motility and invasiveness. In vitro kinase assay data revealed that UAB30 or Targretin interacted with Src and attenuated its kinase activity. We found that UAB30 or Targretin substantially inhibited invasiveness and migration of MCF-7 and SK-BR-3 human breast cancer cells. We examined the effects of UAB30 and Targretin on the expression of matrix metalloproteinases (MMP)-9, which are known to play an essential role in tumor invasion. We show that activity and expression of MMP-9 were decreased by UAB30 or Targretin. Western blot data showed that UAB30 or Targretin decreased AKT and its substrate molecule p70(s6k), which are downstream of Src in MCF-7 and SK-BR-3 cells. Moreover, knocking down the expression of Src effectively reduced the sensitivity of SK-BR-3 cells to the inhibitory effects of UAB30 and Targretin on invasiveness. Taken together, our results demonstrate that UAB30 and Targretin each inhibit invasion and migration by targeting Src in human breast cancer cells.

Novel second-generation rexinoid induces growth arrest and reduces cancer cell stemness in human neuroblastoma patient-derived xenografts.

INTRODUCTION: The poor therapeutic efficacy seen with current treatments for neuroblastoma may be attributed to stem cell-like cancer cells (SCLCCs), a subpopulation of cancer cells associated with poor prognosis and disease recurrence. Retinoic acid (RA) is a differentiating agent used as maintenance therapy for high-risk neuroblastoma but nearly half of children treated with RA relapse. We hypothesized that 6-Methyl-UAB30 (6-Me), a second-generation rexinoid recently developed with a favorable toxicity profile compared to RA, would reduce cancer cell stemness in human neuroblastoma patient-derived xenografts (PDXs). METHODS: Cells from three neuroblastoma PDXs were treated with 6-Me and proliferation, viability, motility, and cell-cycle progression were assessed. CD133 expression, sphere formation, and mRNA abundance of stemness and differentiation markers were evaluated using flow cytometry, in vitro extreme limiting dilution analysis, and real-time PCR, respectively. RESULTS: Treatment with 6-Me decreased proliferation, viability, and motility, and induced cell-cycle arrest and differentiation in all three neuroblastoma PDXs. In addition, 6-Me treatment led to decreased CD133 expression, decreased sphere-forming ability, and decreased mRNA abundance of Oct4, Nanog, and Sox2, indicating decreased cancer cell stemness. CONCLUSIONS: 6-Me decreased oncogenicity and reduced cancer cell stemness of neuroblastoma PDXs, warranting further exploration of 6-Me as potential novel therapy for neuroblastoma.

Novel retinoic acid derivative induces differentiation and growth arrest in neuroblastoma.

INTRODUCTION: Retinoic acid (RA) is a differentiating agent utilized as maintenance therapy for high-risk neuroblastoma (NB), but associated toxicities limit its use. We have previously shown that a non-toxic, novel rexinoid, 9-cis-UAB30 (UAB30), decreased NB cell proliferation and in vivo tumor growth. A second generation, mono-methylated compound, 6-Methyl-UAB30 (6-Me), has been recently designed having greater potency compared with UAB30. In the current study, we hypothesized that 6-Me would inhibit NB cell proliferation and survival and induce differentiation and cell-cycle arrest. METHODS: Proliferation and viability were measured in four human NB cell lines following treatment with UAB30 or 6-Me. Cell-cycle was analyzed and tumor cell stemness was evaluated with extreme limiting dilution assays and immunoblotting for expression of stem cell markers. A xenograft murine model was utilized to study the effects of 6-Me in vivo. RESULTS: Treatment with 6-Me led to decreased proliferation and viability, induced cell cycle arrest, and increased neurite outgrowth, indicating differentiation of surviving cells. Furthermore, treatment with 6-Me decreased tumorsphere formation and expression of stem cell markers. Finally, inhibition of tumor growth and increased animal survival was observed in vivo following treatment with 6-Me. CONCLUSION: These results indicate a potential therapeutic role for this novel rexinoid in neuroblastoma treatment.

The retinoid X receptor agonist, 9-cis UAB30, inhibits cutaneous T-cell lymphoma proliferation through the SKP2-p27kip1 axis.

BACKGROUND: Bexarotene (Targretin®) is currently the only FDA approved retinoid X receptor (RXR) -selective agonist for the treatment of cutaneous T-cell lymphomas (CTCLs). The main side effects of bexarotene are hypothyroidism and elevation of serum triglycerides (TGs). The novel RXR ligand, 9-cis UAB30 (UAB30) does not elevate serum TGs or induce hypothyroidism in normal subjects. OBJECTIVES: To assess preclinical efficacy and mechanism of action of UAB30 in the treatment of CTCLs and compare its action with bexarotene. METHODS: With patient-derived CTCL cell lines, we evaluated UAB30 function in regulating growth, apoptosis, cell cycle check points, and cell cycle-related markers. RESULTS: Compared to bexarotene, UAB30 had lower half maximal inhibitory concentration (IC50) values and was more effective in inhibiting the G1 cell cycle checkpoint. Both rexinoids increased the stability of the cell cycle inhibitor, p27kip1 protein, in part, through targeting components involved in the ubiquitination-proteasome system: 1) decreasing SKP2, a F-box protein that binds and targets p27kip1 for degradation by 26S proteasome and 2) suppressing 20S proteasome activity (cell line-dependent) through downregulation of PSMA7, a component of the 20S proteolytic complex in 26S proteasome. CONCLUSIONS: UAB30 and bexarotene induce both early cell apoptosis and suppress cell proliferation. Inhibition of the G1 to S cell cycle transition by rexinoids is mediated, in part, through downregulation of SKP2 and/or 20S proteasome activity, leading to increased p27kip1 protein stability. Because UAB30 has minimal effect in elevating serum TGs and inducing hypothyroidism, it is potentially a better alternative to bexarotene for the treatment of CTCLs.