Metabolism Of All-trans Retinoic Acid Research Articles

Metabolomics reveals dysregulated all-trans retinoic acid and polyunsaturated fatty acid metabolism contribute to PXR-induced hepatic steatosis in mice

Activation of pregnane X receptor (PXR) by xenobiotics has been associated with metabolic diseases. This study aimed to reveal the impact of PXR activation on hepatic metabolome and explore novel mechanisms underlying PXR-mediated lipid metabolism disorder in the liver. Wild-type and PXR-deficient male C57BL/6 mice were used as in vivo models, and hepatic steatosis was induced by pregnenolone-16α-carbonitrile, a typical rodent PXR agonist. Metabolomic analysis of liver tissues showed that PXR activation led to significant changes in metabolites involved in multiple metabolic pathways previously reported, including lipid metabolism, energy homeostasis, and amino acid metabolism. Moreover, the level of hepatic all-trans retinoic acid (ATRA), the main active metabolite of vitamin A, was significantly increased by PXR activation, and genes involved in ATRA metabolism exhibited differential expression following PXR activation or deficiency. Consistent with previous research, the expression of downstream target genes of peroxisome proliferator-activated receptor α (PPARα) was decreased. Analysis of fatty acids by Gas Chromatography-Mass Spectrometer further revealed changes in polyunsaturated fatty acid metabolism upon PXR activation, suggesting inhibition of PPARα activity. Taken together, our findings reveal a novel metabolomic signature of hepatic steatosis induced by PXR activation in mice.

Nano-fenretinide demonstrates remarkable activity in acute promyeloid leukemia cells

Acute promyelocytic leukemia (APL) is characterized by rearrangements of the retinoic acid receptor, RARα, which makes all-trans retinoic acid (ATRA) highly effective in the treatment of this disease, inducing promyelocytes differentiation. Current therapy, based on ATRA in combination with arsenic trioxide, with or without chemotherapy, provides high rates of event-free survival and overall survival. However, a decline in the drug activity, due to increased ATRA metabolism and RARα mutations, is often observed over long-term treatments. Furthermore, dedifferentiation can occur providing relapse of the disease. In this study we evaluated fenretinide, a semisynthetic ATRA derivative, encapsulated in nanomicelles (nano-fenretinide) as an alternative treatment to ATRA in APL. Nano-fenretinide was prepared by fenretinide encapsulation in a self-assembling phospholipid mixture. Physico-chemical characterization was carried out by dinamic light scattering and spectrophotometry. The biological activity was evaluated by MTT assay, flow cytometry and confocal laser-scanning fluorescence microscopy. Nano-fenretinide induced apoptosis in acute promyelocytic leukemia cells (HL60) by an early increase of reactive oxygen species and a mitochondrial potential decrease. The fenretinide concentration that induced 90–100% decrease in cell viability was about 2.0 µM at 24 h, a concentration easily achievable in vivo when nano-fenretinide is administered by oral or intravenous route, as demonstrated in previous studies. Nano-fenretinide was effective, albeit at slightly higher concentrations, also in doxorubicin-resistant HL60 cells, while a comparison with TK6 lymphoblasts indicated a lack of toxicity on normal cells. The results indicate that nano-fenretinide can be considered an alternative therapy to ATRA in acute promyelocytic leukemia when decreased efficacy, resistance or recurrence of disease emerge after protracted treatments with ATRA.

Abstract 266: The effects of retinoid agents and retinoic acid receptor genotype on proliferation, differentiation, and stem cells in colorectal cancer

Abstract Understanding how to therapeutically target colonic stem cells (SCs) is important because SC overpopulation drives CRC growth and development. Our goal is to therapeutically sensitize cancer SCs (CSCs) to the differentiation-inducing effects of retinoid agents. We previously found that ALDH specifically marks normal and malignant colonic SCs. We also found that retinoic acid (RA) receptors and other RA signaling components are selectively expressed in ALDH+ SCs, indicating that RA signaling occurs through ALDH+ SCs. Because all-trans retinoic acid (ATRA) is a highly effective treatment for acute promyelocytic leukemia, which is caused by a genetic translocation involving retinoic acid receptor alpha (RARA), we conjecture that the response of RA agents may be effective against solid tumors depending on their pathway genotype. Our bioinformatics analysis on mutations and overexpression of RA signaling component genes in CRC showed that most of the RA pathway genes are altered in human CRCs. Therefore, we hypothesize that the ability of retinoid agents and retinoid metabolism blocking agents to induce differentiation of CSCs depends on the RA pathway genotype of CRC cells.Testing the effect of various retinoid drugs on cell proliferation shows that RA pathway genotype affects the response of RA agents in CRC cell lines. HCT116 and SW480 CRC cell lines, which have RA receptor mutations, displayed resistance to ATRA. In contrast, HT29 CRC cells that have wild-type RA receptors are sensitive to ATRA. All three cell lines however showed similar dose responses to the ATRA metabolizing enzyme, CYP26A1 inhibitor Liarozole. This finding indicates that regardless of RA receptor mutations, inhibition of ATRA metabolism can increase intracellular RA levels in a similar manner on all three cell lines to induce anti-proliferative effects. We used CRISPR-Cas9 gene editing to knock out the RARA gene in HT29 cells and evaluated its response to ATRA compared to HT29 cells. The RARA knockout derived lines showed reduced sensitivity to ATRA treatment and a slower rate of cell division compared to HT29 control cells. Additionally, Nanostring mRNA profiling analysis shows that ATRA treatment of HT29 cells increases expression of RARA, CYP26A1, and KRT20 (differentiation marker); and decreased decreases expression of CSC markers LGR5 and ALDH1A1. Altogether, these findings indicate that upregulated RA signaling can reduce cell proliferation, increase differentiation, and decrease CSC numbers in CRCs. Overall, our findings indicate that colon SCs are regulated by RA signaling mechanisms and dysregulation of RA signaling contributes to the SC overpopulation that drives CRC growth. Thus, therapeutically sensitizing CSCs to differentiation-inducing effects of retinoid agents should provide insight into designing new SC-targeted therapies for CRC. Citation Format: Victoria O. Hunsu, Caroline O. Facey, Bruce M. Boman. The effects of retinoid agents and retinoic acid receptor genotype on proliferation, differentiation, and stem cells in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 266.

A variant in CYP26B1 associated with esophageal squamous cell carcinoma risk by affecting retinoic acid metabolism.

All-trans retinoic acid (ATRA) is the natural and synthetic analogue of vitamin A, playing an essential tumor suppressive role in multiple cancers including the esophageal squamous cell carcinoma (ESCC). Cytochrome P450 family 26 subfamily B member 1 (CYP26B1) exerts a critical regulator of ATRA levels through specific inactivation of ATRA to hydroxylated forms. Our previous exome-wide analyses revealed a rare missense variant in CYP26B1 significantly associated with ESCC risk in the Chinese population. However, it is still unclear whether there are common variants in CYP26B1 affect the susceptibility of ESCC and the tumor promotion role of CYP26B1 in vivo. In this research, we conducted a two-stage case-control study comprised of 5057 ESCC cases and 5397 controls, followed by a series of biochemical experiments to explore the function of CYP26B1 and its common variants in the tumorigenesis of ESCC. Intriguingly, we identified a missense variant rs2241057[A>G] in the fourth exon of CYP26B1 significantly associated with the ESCC risk (combined odds ratio = 1.28; 95% confidence interval = 1.15-1.42; p = 2.96 × 10-6 ). Through further functional analysis, we demonstrated that ESCC cells with the overexpression of rs2241057[G] had a significant lower level of retinoic acid, compared with the overexpression of rs2241057[A] or the control vector. In addition, the CYP26B1 overexpression and knock-out ESCC cells affected cell proliferation rate both in vitro and in vivo. These results highlighted the carcinogenicity of CYP26B1 related to the ATRA metabolism in ESCC risk.

Abstract 5810: The effects of retinoid agents and retinoic acid receptor genotype on proliferation, differentiation, and stem cells in colorectal cancer

Abstract Understanding how to therapeutically target colonic stem cells (SCs) is important because SC overpopulation drives CRC growth and development. Our goal is to therapeutically sensitize cancer SCs (CSCs) to differentiation-inducing effects of retinoid agents against CRC. We found that ALDH specifically marks normal and malignant colonic SCs and that retinoic acid (RA) signaling mainly occurs in colonic ALDH+ SCs. That RA signaling occurs through ALDH+ SCs was derived from finding that RA receptors and RA signaling components are selectively expressed in ALDH+ SCs. Indeed, All-transretinoic acid (ATRA) is a highly effective treatment for leukemia (APL) which has a RARA translocation. We conjecture that response of solid tumors to RA agents depends on RA pathway genotype. Our bioinformatics analysis shows mutations often occur in RA receptors in human CRCs. Therefore, we hypothesize that the ability of RA agents and RA metabolism blocking agents to induce differentiation of CSCs depends on RA pathway genotype. Methods: The ability of RA agents (ATRA, 13-cis retinoic acid, 9-cis retinoic acid) and RA metabolism blocking agents (Liarozole, Talarozole) to inhibit proliferation and induce differentiation of CRC cells was evaluated. Nanostring Profiling was used to measure effects of agents on mRNA expression of various markers. Results: Our results show that RA pathway genotype affects the response of RA agents in CRC cell lines. HCT116 and SW480 cells, which have RA receptor mutations, displayed resistance to ATRA. In contrast, HT29 cells that have wild-type RA receptors are sensitive to ATRA. All three cell lines however showed similar responses to the CYP26A1 inhibitor Liarozole. This finding indicates that regardless of RA receptor mutations, inhibition of ATRA metabolism will increase intracellular RA levels. Nanostring profiling analysis shows ATRA treatment of HT29 cells increases expression of RARA, CYP26A1 (ATRA metabolizing enzyme), & KRT20 (differentiation marker), and decreases expression of CSC markers LGR5 & ALDH1A1. Moreover, ATRA treatment of both CRC cell lines decreases ALDH+ cell population size. These findings show upregulated RA signaling can increase differentiation and decrease CSC numbers in CRC. Discussion Our findings indicate that colon SCs are regulated by RA signaling mechanisms and dysregulation of RA signaling contributes to the SC overpopulation that drives CRC growth. Thus, therapeutically sensitizing CSCs to differentiation-inducing effects of RA agents should provide insight into design of new SC-targeted therapies for CRC. Citation Format: Victoria Oluwajuwon Hunsu, Caroline O. Facey, Bruce M. Boman. The effects of retinoid agents and retinoic acid receptor genotype on proliferation, differentiation, and stem cells in colorectal cancer [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 5810.

Computational model for fetal skeletal defects potentially linked to disruption of retinoic acid signaling.

All-trans retinoic acid (ATRA) gradients determine skeletal patterning morphogenesis and can be disrupted by diverse genetic or environmental factors during pregnancy, leading to fetal skeleton defects. Adverse Outcome Pathway (AOP) frameworks for ATRA metabolism, signaling, and homeostasis allow for the development of new approach methods (NAMs) for predictive toxicology with less reliance on animal testing. Here, a data-driven model was constructed to identify chemicals associated with both ATRA pathway bioactivity and prenatal skeletal defects. The phenotype data was culled from ToxRefDB prenatal developmental toxicity studies and produced a list of 363 ToxRefDB chemicals with altered skeletal observations. Defects were classified regionally as cranial, post-cranial axial, appendicular, and other (unspecified) features based on ToxRefDB descriptors. To build a multivariate statistical model, high-throughput screening bioactivity data from >8,070 chemicals in ToxCast/Tox21 across 10 in vitro assays relevant to the retinoid signaling system were evaluated and compared to literature-based candidate reference chemicals in the dataset. There were 48 chemicals identified for effects on both in vivo skeletal defects and in vitro ATRA pathway targets for computational modeling. The list included 28 chemicals with prior evidence of skeletal defects linked to retinoid toxicity and 20 chemicals without prior evidence. The combination of thoracic cage defects and DR5 (direct repeats of 5 nucleotides for RAR/RXR transactivation) disruption was the most frequently occurring phenotypic and target disturbance, respectively. This data model provides valuable AOP elucidation and validates current mechanistic understanding. These findings also shed light on potential avenues for new mechanistic discoveries related to ATRA pathway disruption and associated skeletal dysmorphogenesis due to environmental exposures.

Abstract 6067: Studying the anti-proliferating and differentiating effects of retinoids based on pathway genotype of colorectal cancer cell lines

Abstract Background: Our overarching goal is to therapeutically sensitize human colorectal cancer stem cells to differentiation-inducing effects of retinoid agents. Tumorigenesis in colorectal cancer (CRC) results from the acquisition of mutations in a colonic stem cell which transforms it into a cancer stem cell (CSC). There are no curative treatments for advanced CRC due to the resistance of CSCs to chemotherapy and/or radiation. All-trans retinoic acid (ATRA) is known to be a greatly effective treatment for acute promyelocytic leukemia (APL) which is caused by translocation in the retinoic acid receptor alpha (RARA) gene. We conjecture that the response of retinoic acid (RA) agents may have effects on solid tumors depending on their pathway genotype. Results from our bioinformatics analysis on mutations and overexpression of RA signaling component genes in CRC showed that the majority of RA pathway genes are overexpressed in CRC. Therefore, we hypothesize that the ability of retinoid and retinoid metabolism blocking agents to induce differentiating effects in CSCs depends on the RA pathway genotype expressed in CRC cells. Methods: The ability of RA agents (ATRA, 13-cis retinoic acid, 9-cis retinoic acid) and RA metabolism blocking agents (Liarozole and Talarozole) to inhibit proliferation and induce differentiation on CRC cell lines with different mutations in RA signaling were evaluated. Additionally, Nanostring Profiling was used to measure the effects of the agents on mRNA expression of CSC markers, indicators of cell differentiation, and RA receptors. Results: Our results show that the RA pathway genotype affects the response of RA agents in CRC cell lines. Both HCT116 and SW480 CRC cell lines, which are both mutant in RARA and RARA and RXRG respectively displayed resistance to ATRA. HT29 CRC cells express wild- type RA receptors and are sensitive to ATRA. All three cell lines showed similar dose responses to the CYP26A1 inhibitor Liarozole. This indicates that regardless of RA receptor mutations, inhibition of ATRA metabolism increases intracellular RA levels in a similar manner on all 3 cell lines. Nanostring Profiling shows that ATRA treatment of HT29 cells induces: an increase in the expression of RARA, CYP26A1 (ATRA metabolizing enzyme) and KRT20 (differentiation marker); and a decrease in the expression of CSC markers LGR5 and ALDH1A1. This indicates an active RA signaling pathway, induced differentiation, and a decrease in stem cells. Discussion: Overall, our findings indicate that colon SCs are regulated by RA signaling mechanisms and dysregulation of RA signaling contributes to the SC overpopulation that drives CRC growth. Therefore, further investigation on how to therapeutically sensitize human colonic CSCs to the differentiation-inducing effects of retinoid agents should provide insight into designing new SC-targeted therapies for CRC. Citation Format: Victoria O. Hunsu, Caroline O. Facey, Lynn M. Opdenaker, Bruce M. Boman. Studying the anti-proliferating and differentiating effects of retinoids based on pathway genotype of colorectal cancer cell lines [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 6067.

Cardiac retinoic acid levels decline in heart failure.

Although low circulating levels of the vitamin A metabolite, all-trans retinoic acid (ATRA), are associated with increased risk of cardiovascular events and all-cause mortality, few studies have addressed whether cardiac retinoid levels are altered in the failing heart. Here, we showed that proteomic analyses of human and guinea pig heart failure (HF) were consistent with a decline in resident cardiac ATRA. Quantitation of the retinoids in ventricular myocardium by mass spectrometry revealed 32% and 39% ATRA decreases in guinea pig HF and in patients with idiopathic dilated cardiomyopathy (IDCM), respectively, despite ample reserves of cardiac vitamin A. ATRA (2 mg/kg/d) was sufficient to mitigate cardiac remodeling and prevent functional decline in guinea pig HF. Although cardiac ATRA declined in guinea pig HF and human IDCM, levels of certain retinoid metabolic enzymes diverged. Specifically, high expression of the ATRA-catabolizing enzyme, CYP26A1, in human IDCM could dampen prospects for an ATRA-based therapy. Pertinently, a pan-CYP26 inhibitor, talarozole, blunted the impact of phenylephrine on ATRA decline and hypertrophy in neonatal rat ventricular myocytes. Taken together, we submit that low cardiac ATRA attenuates the expression of critical ATRA-dependent gene programs in HF and that strategies to normalize ATRA metabolism, like CYP26 inhibition, may have therapeutic potential.

The Effects of Voriconazole on Metabolism of All-Trans Retinoic Acid in the Treatment of Acute Promyelocytic Leukemia: A Case Report

All-trans retinoic acid (ATRA) is a derivative of vitamin A and is the mainstay treatment of acute promyelocytic leukemia (APL). Hypercalcemia is a rare yet important side-effect of ATRA, especially when it is used concomitantly with a medication that impedes its metabolism by inhibiting cytochrome P-450 in the liver and thus increasing the duration of exposure to ATRA. Azole antifungal drugs such as voriconazole are frequently used in patients undergoing chemotherapy due to a high incidence of fungal infections. These medications inhibit two vital enzymes of cytochrome P-450, CYP2C9 and CYP3A4, potentiating the effects of ATRA on calcium metabolism. We present a case of a nine-year-old girl who underwent chemotherapy with all-trans retinoic acid for acute promyelocytic leukemia. The patient was given an anti-fungal cover with voriconazole for extensive fungal chest infection simultaneously. She was found to have asymptomatic hypercalcemia on routine follow-up during the consolidation phase. Both medications were stopped. Subsequently, she was admitted to the ward and managed conservatively with hydration. Serum calcium levels were returned to normal within six days after stopping the combination of ATRA and voriconazole. We underscore that the use of anti-fungal medications should be limited while using ATRA. However, strict monitoring must be done when a combination of these drugs is started, if necessary.

Alternatively Activated Macrophages Are the Primary Retinoic Acid-Producing Cells in Human Decidua.

In situ production and metabolism of all-trans retinoic acid (RA) in decidual tissue are critically important for endometrial stromal differentiation, embryo implantation, and healthy placentation. However, the cellular source(s) of RA in this tissue has yet to be determined. To identify the primary RA-producing cells in human term decidua, we isolated cells from decidua basalis of delivered placenta and quantified cellular retinal dehydrogenase (RALDH) activity, a major biosynthetic enzyme whose activity determines the synthesis of RA from retinol, using an Aldefluor assay and flow cytometry. RA production in decidual tissue and sorted cell subpopulations was evaluated by liquid chromatography-tandem mass spectrometry. CD14+ cells (macrophages/monocytes) showed > 4-fold higher RALDH activity than stromal cells (CD10+), T cells (CD3+), or non-T lymphocytes (CD3-negative). CD11c+ cells that did not co-express CD14 showed about one-third the RALDH activity of their CD14 co-expressing counterparts. The highest RALDH activity was found in "alternatively activated" M2 macrophages delineated by the simultaneous expression of CD14 and CD163. The greater RA synthesizing capacity of M2 versus CD14+CD163-ve (M1) cells was confirmed by direct quantitation of RA biosynthesis from retinol. RA levels in whole decidua were correlated with M2 cell density but not with stromal cell (CD10+) number, the major cell type comprising the decidua. These results identified M2 monocyte/macrophages as the primary source of RA in human term decidua. This finding may have implications for certain pregnancy complications that are known to be associated with reduced numbers of decidual M2 cells.

The Effect of Arsenic Trioxide on All-trans Retinoic Acid Binding to Human Serum Albumin.

Tretinoin or All-trans retinoic acid (ATRA) is an efficient medication in leukemia treatment. Arsenic trioxide (ATO) significantly improves the effectiveness of ATRA. In this study, the effect of ATO on ATRA binding to human serum albumin (HSA) was investigated. Fluorescence and UV-Vis spectroscopy and equilibrium dialysis technique were used to determine ATRA binding to HSA in the presence and absence of ATO and of two compounds, warfarin and ibuprofen, specific for binding to HSA sites I and II, respectively ("site markers"). The association constants for ATRA binding and the number of binding sites as well as the thermodynamic parameters of complex formation, were obtained at different temperatures. Fluorescence results showed a static quenching mechanism for ATRA binding to HSA. The calculated thermodynamic parameters revealed that the binding reaction is a spontaneous and exothermic process and also that hydrogen bonds and van der Waals forces have a central role in the binding of ATRA to HSA. Competitive experiments showed that none of markers seriously prevents ATRA binding to HSA. Interestingly, the fluorescence and equilibrium dialysis data showed that ATO increases the binding of ATRA to HSA, and converts the binding mode of ATRA from mainly hydrogen bonding to include hydrophobic interactions as well. These results suggest that ATO can prevent the metabolism of ATRA and keep it in the blood for longer by increasing the binding of ATRA to HSA.

Effect of inhibition of 15-PGDH on Kras-driven tumor expansion through all-trans retinoic acid metabolism in the pancreas.

e16197Background: Chronic inflammation has a crucial role in cancer development and the progression of various tumors, including pancreatic ductal adenocarcinoma (PDAC). The arachidonate cascade is...

A fourth subtype of retinoic acid receptor-related orphan receptors is activated by oxidized all-trans retinoic acid in medaka (Oryzias latipes)

BackgroundThe three known subtypes of the retinoic acid receptor-related orphan receptor (ROR) have been implicated in the control of immunity, brain function, and circadian rhythm in mammals. Here, we demonstrate by phylogenetic analysis that there were originally four subtypes of RORs in vertebrates. One of the novel ror paralogs, rord1 (rorca in the Ensembl database), is conserved among teleosts, but absent in mammals. Using medaka (Oryzias latipes) as a model teleost, we evaluated the expression pattern of this gene, its transactivational properties for endogenic chemicals, and its ability to activate the promoters of putative target genes.ResultsIn eyes, the transcript of rord1 was expressed at higher levels during the day than at night. Interestingly, cholesterol derivatives, which are well-known ligands for mammalian RORs, did not efficiently promote transcriptional activity via RORd1. Thus we sought to identify the ligands that regulate the transcriptional activity of RORd1 using a luciferase reporter cell-based screening system. Using this system, we identified two metabolites of all-trans retinoic acid (ATRA), 4OH-ATRA and 4-keto ATRA, as potential ligands of RORd1. Moreover, RORd1 activated the promoter of cyp26a1 in a 4OH-ATRA -dependent manner.ConclusionsA novel ror subtype, rord has two paralogs, rord1 and rord2, in teleost. Rord1 mRNA is highly abundant in the eyes of medaka during light periods, suggesting that rord1 expression is involved in the regulation of circadian rhythm. We identified two ATRA metabolites, 4OH-ATRA and 4 K–ATRA, as endogenous candidate ligands of RORd1. We also show that 4-oxygenated ATRA metabolites have the potential to activate cyp26a1, the metabolic enzyme of ATRA. Our results support the notion that RORd1 is involved in the metabolism of ATRA in medaka.

Chemical or genetic Pin1 inhibition exerts potent anticancer activity against hepatocellular carcinoma by blocking multiple cancer-driving pathways

Hepatocellular carcinoma (HCC) is one of the most prevalent and malignant cancers with high inter- and intra-tumor heterogeneity. A central common signaling mechanism in cancer is proline-directed phosphorylation, which is further regulated by the unique proline isomerase Pin1. Pin1 is prevalently overexpressed in human cancers including ~70% of HCC, and promotes tumorigenesis by activating multiple cancer-driving pathways. However, it was challenging to evaluate the significance of targeting Pin1 in cancer treatment until the recent identification of all-trans retinoic acid (ATRA) as a Pin1 inhibitor. Here we systematically investigate functions of Pin1 and its inhibitor ATRA in the development and treatment of HCC. Pin1 knockdown potently inhibited HCC cell proliferation and tumor growth in mice. ATRA-induced Pin1 degradation inhibited the growth of HCC cells, although at a higher IC50 as compared with breast cancer cells, likely due to more active ATRA metabolism in liver cells. Indeed, inhibition of ATRA metabolism enhanced the sensitivity of HCC cells to ATRA. Moreover, slow-releasing ATRA potently and dose-dependently inhibited HCC growth in mice. Finally, chemical or genetic Pin1 ablation blocked multiple cancer-driving pathways simultaneously in HCC cells. Thus, targeting Pin1 offers a promising therapeutic approach to simultaneously stop multiple cancer-driving pathways in HCC.

Design, synthesis, and biological evaluation of amide imidazole derivatives as novel metabolic enzyme CYP26A1 inhibitors

All-trans-retinoic acid (ATRA) as a physiological metabolite of vitamin A is widely applied in the treatment of cancer, skin, neurodegenerative and autoimmune diseases. CYP26A1 enzyme, induced by ATRA in liver and target tissues, metabolizes ATRA into 4-hydroxyl-RA. Inhibition of CYP26A1 metabolic enzyme represents a promising strategy for discovery of new specific anticancer agents.Herein, we describe the design, synthesis and biological evaluation of a series of new amide imidazole derivatives as retinoic acid metabolism blocking agents (RAMBAs) toward CYP26A1 enzyme. First, based on the recent theoretical models (Sun et al., J. Mol. Graph. Model., 2015, 56, 10–19) a series of RAMBAs with novel scaffolds were designed using fragment-based drug discovery approach. Subsequently, the new RAMBAs were synthesized and evaluated for their biological activities. All the compounds demonstrated appropriate enzyme activities and cell activities. The promising inhibitors 20 and 23 with IC50 value of 0.22μM and 0.46μM toward CYP26A1, respectively, were further evaluated for CYP selectivity and the metabolic profile of ATRA. Both compounds 20 and 23 showed higher selectivity for CYP26A1 over other CYPs (CYP2D6, CYP3A4) when compared to liarozole. They also showed better inhibitory activities for the metabolism of ATRA when also compared to liarozole. These studies further validated the pharmacophore and structure–activity relationship models obtained about CYP26A1 inhibitors and highlighted the promising activities of the new series of CYP26A1 inhibitors designed from such models. They also paved the way for future development of those candidates as potential drugs.

Mutation in Retinoic X Receptor-γ Is a Possible Mechanism of All-Trans Retinoic Acid Resistance in Acute Promyelocytic Leukemia(APL): Identifying Genetic Changes Related to Drug Resistance in APL Using Whole Exome Sequencing

BackgroundAcute promyelocytic leukemia (APL) has the best prognosis among acute myeloid leukemia (AML). However, a subset of APL patients is not cured with all-trans retinoic acid (ATRA) combined with anthracycline-based cytotoxic chemotherapy. Some mechanisms such as increased ATRA metabolism have been suggested to acquired resistance to ATRA. However genetic mechanism of ATRA resistance has not been characterized at all. Hence, in this study, we tried to reveal genetic alterations attributable to ATRA resistance.MethodsFirst, we performed whole exome sequencing (WES) using DNA of three APL patients who showed resistance to ATRA based treatment. These included two patients who failed to achieve complete remission (CR) after induction chemotherapy, and one patient who experienced relapse after CR despite of consolidation treatment. DNA extracted from bone marrow at the time of diagnosis was used for analysis, while DNA extracted from saliva at the time of CR was used as germline control. Calling of single nucleotide variants (SNV) was performed using internal pipeline called Adiscan (http://www.syntekabio.com). Annotation was performed using Polyphen-2. SNV’s found by WES were validated by direct Sanger sequencing. The frequency of those validated SNV’s was defined in a separate APL cohort.ResultsWe identified 34 somatic non-synonymous SNV’s in three patients. Polyphen-2 algorithm predicted 9 among 34 SNV’s to damage protein function. Sanger sequencing revealed 8 over 9 SNV’s to be validated. These validated SNV’s include RXRG M77R, N6AMT2 A78S, TXNDC15 D198E, B3GALTL A444T, RBBP8NL E182G, TNPO3 L173W, BHMT M185I and ADAMTS5 G85D. When these 8 SNV’s were genotyped in a separate cohort, none of these SNV’s was found in the APL cohort composed of 30 ATRA sensitive patients, suggesting these SNV’s would be truly related to ATRA resistance in APL. Especially, when a simulation using amber molecular dynamics was performed, we observed 1) Increase in hydrogen bonding, 2) Decreased helix folding structure, 3) Decreased energy state in RXRG mutant case.ConclusionsWES identified eight SNV’s which were unique in ATRA resistant cases. Among those mutations, RXRG could be a promising nonsynonymous mutation that explains the genetic mechanism of ATRA resistance. DisclosuresNo relevant conflicts of interest to declare.

Effect of 1,25-dihydroxyvitamin D on all-trans retinoic acid metabolism and interleukin-4 signaling in porcine alternatively activated lung macrophages. (IRM7P.493)

Abstract All-trans retinoic acid (ATRA), the most active vitamin A (VA) metabolite, and 1,25-dihydroxyvitamin D (VD3), the most active form of vitamin D (VD), and interleukin-4 (IL-4) each positively regulate the development of alternatively activated macrophages (AAM) however, their interactions are unknown. We have previously shown that VA, ATRA, and IL-4 have differing mechanisms for sustaining a Th2-type AAM response. Herein, we demonstrate that VD3 regulates the contribution of ATRA and IL-4 inducible signals to this system. While ATRA (10-7 M) increases mRNA expression of retinol producing dehydrogenase/reductase (SDR family) member, 4 fold, VD3 (10-7 M) decreases its expression 3-fold and additively increases expression of retinal producing, Aldehyde dehydrogenase 1 family, member A2, and dehydrogenase/reductase (SDR family) member 9 by 5 fold when present along with ATRA. Conversely, VD3 decreases IL-4 (10 ng/ml) -induced chemokine (C-C motif) ligand 17 by 4 fold, and C-type lectin CD209 antigen by 60 fold. These results suggest that VD3 increases expression of genes involved in ATRA anabolism while decreasing expression of IL-4 responsive genes. In conclusion, VD3 may regulate the development of AAM by increasing levels of ATRA and decreasing the effect of IL-4, thereby supporting an alternate signal for AAM development.

Ablation of DHRS3 is associated with alterations in retinoic acid metabolism and signaling and with defects in developmental programming in mice (39.8)

Background: The levels of the signaling molecule all‐trans‐retinoic acid (ATRA) are tightly controlled at the level of enzymes involved in its synthesis and degradation. Previous studies have implicated the retinaldehyde reductase DHRS3 in establishing a negative feedback mechanism to limit the formation of ATRA during zebrafish development.Objective: Here, we investigate the physiological role of DHRS3 in the regulation of embryonic levels of ATRA by characterizing a mouse deficient in this enzyme.Results: Lack of DHRS3 leads to increased levels of ATRA and decreased levels of retinol and retinyl esters in Dhrs3‐/‐ embryos. Furthermore, accumulation of excess ATRA in Dhrs3‐/‐ embryos is accompanied by alterations in ATRA signaling and defects in developmental programming. Dhrs3‐/‐ embryos die late in gestation and display defects in cardiac outflow tract formation and in atrial and ventricular septation. In addition, Dhrs3‐/‐ embryos also exhibit defects in skeletal development and palatogenesis. The extensive effects of Dhrs3 ablation are also consistent with its widespread expression pattern. Our studies also reveal that Dhrs3 is expressed in many embryonic and adult tissues in which ATRA metabolism and ATRA signaling play important roles, and its expression is responsive to vitamin status.Conclusion: Our studies demonstrate that DHRS3 is a pivotal enzyme in the regulation of ATRA formation during embryogenesis, and we establish the Dhrs3‐/‐ mouse as a new model to study the pathogenic mechanisms of ATRA‐induced embryopathy.Grant Funding Source: Supported by NIH grants: P20 GM103420‐10 (A.R.M), HHSN272202000046C (M.A.K.), R01DE016082 (P.A.T.)

Abstract B257: Identification of new CYP26 inhibitors with efficacy in breast cancer xenograft models.

Abstract Retinoids, such as the prototypical retinoid all trans retinoic acid (ATRA), have been used successfully in the treatment of acute promyelocytic leukemia (APL) patients. They have also been tested for efficacy in solid tumors, including breast cancer. Despite promising preclinical data, the clinical success of using ATRA for breast cancer patients has been limited so far. We hypothesize that this is largely due to three factors: (1) a lack of understanding of the patient population that is likely to respond to retinoid therapy, (2) a significant induction of retinoic acid metabolism with retinoid therapy, resulting in sub-optimal retinoid exposure in tumors and (3) the narrow therapeutic index of ATRA. Several recent studies indicate that breast cancer cells that express the Retinoic Acid Receptor Alpha (RARA) are more likely to respond to retinoic acid than cells without RARA expression. This provides a rationale for selection of patients that are likely to benefit from retinoid therapy in future clinical trials. The cytochrome P450 enzyme CYP26 is the key enzyme involved in ATRA metabolism. CYP26A1 expression is elevated in breast cancer tissue compared to normal tissue and CYP26A1 expression is negatively correlated with patient survival. Angion has identified orally bioavailable small molecule inhibitors of CYP26. Angion's lead inhibitor blocks retinoid metabolism in tumor cells and enhances serum and tumor retinoic acid levels. The compound shows robust single agent efficacy in several murine breast cancer xenograft models. By elevating endogenous serum and tumor retinoic acid levels in a physiological range, CYP26 inhibition therapy is likely to result in a better therapeutic index than systemic retinoid therapy. The identification of orally bioavailable, potent, and selective CYP26 inhibitors eliminates two major drawbacks of systemic retinoid therapy, and– in combination with new insights in selecting patients that are more likely to respond to retinoid therapy– may provide a new impetus for evaluation of retinoic acid signaling modulators as a treatment for breast cancer patients. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B257. Citation Format: Bert Oehlen, Gaifeng Ma, Siobhan McCormack, Dong Sung Lim, Jim Tarrant, Xiaokang Zhu, Bijoy Panicker, Itzhak D. Goldberg. Identification of new CYP26 inhibitors with efficacy in breast cancer xenograft models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B257.

The retinaldehyde reductase DHRS3 is essential for preventing the formation of excess retinoic acid during embryonic development

Oxidation of retinol via retinaldehyde results in the formation of the essential morphogen all-trans-retinoic acid (ATRA). Previous studies have identified critical roles in the regulation of embryonic ATRA levels for retinol, retinaldehyde, and ATRA-oxidizing enzymes; however, the contribution of retinaldehyde reductases to ATRA metabolism is not completely understood. Herein, we investigate the role of the retinaldehyde reductase Dhrs3 in embryonic retinoid metabolism using a Dhrs3-deficient mouse. Lack of DHRS3 leads to a 40% increase in the levels of ATRA and a 60% and 55% decrease in the levels of retinol and retinyl esters, respectively, in Dhrs3(-/-) embryos compared to wild-type littermates. Furthermore, accumulation of excess ATRA is accompanied by a compensatory 30-50% reduction in the expression of ATRA synthetic genes and a 120% increase in the expression of the ATRA catabolic enzyme Cyp26a1 in Dhrs3(-/-) embryos vs. controls. Excess ATRA also leads to alterations (40-80%) in the expression of several developmentally important ATRA target genes. Consequently, Dhrs3(-/-) embryos die late in gestation and display defects in cardiac outflow tract formation, atrial and ventricular septation, skeletal development, and palatogenesis. These data demonstrate that the reduction of retinaldehyde by DHRS3 is critical for preventing formation of excess ATRA during embryonic development.