Retinoic Acid Receptor Alpha Research Articles

Neuroplasticity Alterations Induced by Isotretinoin use in RARA Gene Polymorphism is Associated with Increased Risk of Depression and Suicidal Ideation in Patients with Vitamin D Deficiency: A Systematic Review and Meta-Analysis

Rationale: Isotretinoin is widely used for severe acne treatment, but concerns have emerged regarding its potential association with depression and suicidal ideation. Previous studies have produced mixed findings, necessitating a comprehensive evaluation of the evidence. This systematic review and meta-analysis aim to clarify the association between isotretinoin use and the onset of depression or suicidal ideation, focusing on genetic predispositions, particularly in patients with vitamin D deficiency. Objectives: This review assesses the risk of depression and suicidal ideation in patients treated with isotretinoin for severe acne compared to those not using isotretinoin. A secondary focus is placed on identifying specific genetic factors, such as polymorphisms in the RARA and LEP genes, that may predispose patients to these psychiatric side effects. Eligibility Criteria: Randomized controlled trials (RCTs), cohort studies, and case-control studies comparing isotretinoin users to non-users were included. Exclusion criteria included studies that lacked a comparator group or did not report psychiatric outcomes (depression or suicidal ideation). Outcomes: The primary outcome was the incidence of depression or suicidal ideation among isotretinoin users compared to non-users. Secondary outcomes examined the influence of genetic polymorphisms, specifically in the RARA and LEP genes, on the risk of developing psychiatric side effects. Risk of Bias: Risk of bias was assessed using the Cochrane Risk of Bias tool for randomized trials and the Newcastle-Ottawa Scale for observational studies. The overall quality of the included studies was moderate to high. Synthesis Methods: A random-effects meta-analysis was performed to pool odds ratios (ORs) with 95% confidence intervals (CIs). Heterogeneity was assessed using the I² statistic, and sensitivity analyses were conducted to ensure the robustness of the findings. Included Studies: Fifteen studies were included, comprising 3 randomized controlled trials, 7 cohort studies, and 5 case-control studies, with a total of 8,000 isotretinoin users and 10,000 non-users. The studies were conducted in diverse clinical settings with a follow-up range of 6 months to 5 years. Synthesis of Results: The pooled analysis demonstrated a significant association between isotretinoin use and an increased risk of depression (OR: 1.3, 95% CI: 1.1–1.5, p < 0.05). Specific genetic polymorphisms, particularly in the RARA and LEP genes, were identified as potential risk factors for developing depression among isotretinoin users, particularly in patients with concurrent vitamin D deficiency. Conclusions: Isotretinoin use is associated with an increased risk of depression, particularly in individuals with specific genetic polymorphisms such as RARA and LEP. Clinicians should consider conduct precision medicine genetic testing testing for these polymorphisms before initiating isotretinoin treatment and closely monitor patients for psychiatric alterations, mood changes, especially those with with vitamin D deficiency or genetic predisposition to depression

Optical Genome Mapping Reveals Complex and Cryptic Rearrangement Involving PML::RARA Fusion in Acute Promyelocytic Leukemia

Background/objectives: Acute promyelocytic leukemia (APL) is an aggressive subtype of acute myeloid leukemia (AML), characterized by the hallmark translocation t(15;17) resulting in a PML::RARA fusion. Once diagnosed, APL is now considered to be one of the most treatable forms of AML. However, without early detection and treatment, the disease is associated with rapid deterioration and lethal side effects. Methods: We describe a case of diagnostic APL presenting with a normal karyotype, normal RARA break-apart FISH, and unclear, atypical PML/RARA FISH findings. We used optical genome mapping (OGM) to characterize this atypical PML/RARA fusion. Results: OGM allowed for detection of a PML::RARA fusion resulting from a cryptic and complex insertion of PML::RARA into RARA on 17q21.2 whereby a segment of 15q24.1 was inserted into the 17q21.2. The recipient breakpoint of the insertion was at intron 2 of the RARA gene and the donor breakpoint of the insertion was at exon 5/intron 6 of the PML gene. Conclusions: This is the first report of an insertional PML::RARA fusion into the RARA gene on 17q detected by OGM. OGM has demonstrated its utility in a clinical cytogenetics environment, allowing for clearer characterization and diagnosis of various neoplasms.

Beyond the Surface: Uncovering the PML::RARA Fusion in Cytogenetically Cryptic and FISH-Negative Acute Promyelocytic Leukemia- A Case Report and Review of the Literature

Abstract Introduction/Objective Chromosomal t(15;17)(q22;q21), resulting in a fusion between the PML gene on chromosome 15q22 and the RARA gene on chromosome 17q21, is the cytogenetic hallmark of acute promyelocytic leukemia (APL). Conventional karyotyping, along with fluorescence in situ hybridization (FISH), can detect the PML/RARA rearrangements in up to 98% of APL cases. Reverse transcriptase-polymerase chain reaction (RT-PCR) is often used to confirm the initial diagnosis. APL with PML::RARA fusion, which is negative with FISH and conventional karyotyping approaches but positive with RT-PCR, is extremely rare. Herein, we report a de novo APL case that was positive by RT-PCR but negative by cytogenetics and FISH studies. A comprehensive review of the literature was also performed. Methods/Case Report A 23-year-old male presented to the ED for left upper extremity weakness 2 days after post- dental cleaning. Upon admission, he developed intracranial hemorrhage, necessitating urgent decompressive hemicraniectomy. Blood smear revealed promyelocytes; flow cytometric analysis showed a CD13+, CD33+, MPO+, CD117+, CD34-, and HLA-DR- abnormal cell population. STAT interphase FISH analyses for PML/RARA fusion and RARA break-apart were negative. A diagnosis of APL was made based on abnormal promyelocytes and characteristic flow cytometry findings. ATRA was immediately administrated. However, the post-operative course was complicated by significant coagulopathy, pancytopenia, and multi-organ failure, and the patient expired 7 days later. RT-PCR returned positive for PML::RARA long fusion transcript; the conventional cytogenetics study was uninformative. The clinical, pathological, and genetic features of 31 cases of FISH/Cytogenetics-negative APL harboring cryptic rearrangements of PML-RARA, detected by RT-PCR in the literature, have been reviewed. Various detection methods were also discussed. Results (if a Case Study enter NA) N/A Conclusion Conventional cytogenetics, FISH and RT-PCR analysis are complementary studies for all clinical suspected APL cases. The clinicopathological characteristics also play critical roles in APL diagnosis when genetic confirmation of the PML::RARA fusion is unavailable.

Biomineralized Nuclear Receptor Protein-Selection Mass Spectrometry for the Discovery of Drugs and Toxics.

Protein-selection mass spectrometry is cost-effective for the discovery of drugs and toxics. Nuclear receptors (NRs) are major targets for pharmaceuticals and endocrine-disrupting chemicals and are, thus, widely used as "bait" proteins. However, their application is limited due to the tendency to lose protein activity during cold storage. To address this problem, we introduced a novel biomineralization-based approach to preserve activity in NRs, exemplified by human retinoic acid receptor alpha (hRARα), a target for cancer and leucocythemia therapy. Since information on the coordination chemistry of metal ion and NR protein complexes is almost unavailable, we applied peptide mapping analysis for the first time for the rational design of his-hRARα-Co phosphate nanobiomaterial with high bioactivity. This nanobiomaterial successfully captured hRARα bioactive chemicals from a Chinese herb and environmental water and discovered an unsaturated fatty acid, (±)-(9Z,11E)-13-hydroxy-9,11-octadecadienoic acid ((±)13-HODE), which exhibited strong hRARα antagonistic activity.

Retinoic acid receptor alpha expression in endometrioid heterotopias and the endometrium of patients with endometriosis

BACKGROUND: Vitamin A is a fat-soluble compound, which is a biologically inactive food form and undergoes transformation by cytosolic alcohol dehydrogenases and microsomal retinol dehydrogenases into retinaldehyde, followed by oxidation by retinaldehyde dehydrogenases 1, 2 and 3 into retinoic acid. All-trans-retinoic acid has been identified as an active metabolite of retinoic acid, indirectly participating in various processes: differentiation, proliferation and apoptosis. Based on a number of studies, the role of retinoic acid and its receptors in various diseases has been proven. For example, the destruction of the RAR-β gene by the integration of the hepatitis B virus into its loci can be a factor in the development of human hepatocellular carcinoma. Subsequently, it was described that the characteristic point of interruption of translocation t(15:17), observed in acute promyelocytic leukemia, was located at the locus encoding retinoic acid receptor-α. As a result, in patients with acute promyelocytic leukemia, complete remission of the disease could often be achieved by treatment with high doses of all-trans-retinoic acid. Existing data on the ability of retinol to influence the processes of differentiation, proliferation, and apoptosis indicate that retinol may be involved in the pathogenesis of endometriosis, but the mechanisms of its effect on the disease are subject to further study. AIM: The aim of this study was to evaluate the retinoic acid receptor alpha expression in endometrioid heterotopias and the endometrium of patients with different endometriosis stages according to the Revised American Society for Reproductive Medicine classification. MATERIALS AND METHODS: This was a prospective study to determine the retinoic acid receptor alpha expression in the endometrium and endometrioid heterotopias of patients with endometriosis (n = 28) and of the control group (n = 10). RESULTS: The retinoic acid receptor alpha expression in endometrioid heterotopias was lower compared to the eutopic endometrium of patients with endometriosis and the endometrium of patients in the control group by 3.4 and two times, respectively (p 0.001; the Kruskal – Wallis method). CONCLUSIONS: The data obtained indicate the involvement of retinol in the pathogenesis of endometriosis. Despite the relative increase in retinol level in the peripheral blood and peritoneal fluid in patients with endometriosis compared to the control group, shown in our previous studies, the reduced retinoic acid receptor alpha expression in endometrioid heterotopias does not allow for full binding of vitamin A to the receptor and is accompanied by its improper metabolism.

8795 Impact of Retinoic Acid Receptor Alpha Conditional Knockout on Ovarian Follicle Development in the Mouse

Abstract Disclosure: Z. Bogin: None. A. Keisker: None. A. Urbanowski: None. C. White: None. J.L. Kipp: None. The ovaries are the primary endocrine and reproductive organs in females. The ovarian follicle is the basic structural and functional unit of the ovary. When there is abnormal development in ovarian follicles, infertility and reproductive diseases such as polycystic ovarian syndrome (PCOS), premature ovarian failure (POF), and ovarian cancers may occur. Retinoic acid (RA), a biologically active derivative of vitamin A, is a signaling molecule that plays a vital role in various physiological processes, including embryonic development, tissue differentiation, and reproduction. We have shown that RA stimulates granulosa cell proliferation through a cell signaling cascade involving Retinoic Acid Receptors (RARs), which have three isoforms alpha, beta, and gamma. In this study, we characterized a new retinoic acid receptor alpha conditional knockout (RARA cKO) mouse model with RARA deleted in granulosa cells during embryonic development. The body weight and uterus weight of the RARA cKO mice were decreased at week 7 but returned to normal at week 15 and 12 months. The animals were either sub-fertile or infertile with phenotypic evidence of POF. In addition, a variety of ovarian pathologies were observed, including the presence of hemosiderin, increased cyst formation, and abnormal follicle counts. Overall, this study provides evidence of the involvement of retinoic acid signaling, mediated by RARA, in ovarian development and fertility. This study will help to better understand the prevention and treatment of ovarian diseases. Presentation: 6/2/2024

8795 Impact of Retinoic Acid Receptor Alpha Conditional Knockout on Ovarian Follicle Development in the Mouse

Abstract Disclosure: Z. Bogin: None. A. Keisker: None. A. Urbanowski: None. C. White: None. J.L. Kipp: None. The ovaries are the primary endocrine and reproductive organs in females. The ovarian follicle is the basic structural and functional unit of the ovary. When there is abnormal development in ovarian follicles, infertility and reproductive diseases such as polycystic ovarian syndrome (PCOS), premature ovarian failure (POF), and ovarian cancers may occur. Retinoic acid (RA), a biologically active derivative of vitamin A, is a signaling molecule that plays a vital role in various physiological processes, including embryonic development, tissue differentiation, and reproduction. We have shown that RA stimulates granulosa cell proliferation through a cell signaling cascade involving Retinoic Acid Receptors (RARs), which have three isoforms alpha, beta, and gamma. In this study, we characterized a new retinoic acid receptor alpha conditional knockout (RARA cKO) mouse model with RARA deleted in granulosa cells during embryonic development. The body weight and uterus weight of the RARA cKO mice were decreased at week 7 but returned to normal at week 15 and 12 months. The animals were either sub-fertile or infertile with phenotypic evidence of POF. In addition, a variety of ovarian pathologies were observed, including the presence of hemosiderin, increased cyst formation, and abnormal follicle counts. Overall, this study provides evidence of the involvement of retinoic acid signaling, mediated by RARA, in ovarian development and fertility. This study will help to better understand the prevention and treatment of ovarian diseases. Presentation: 6/2/2024

Targeting CNS myeloid infiltrates provides neuroprotection in a progressive multiple sclerosis model

Demyelination and axonal injury in chronic-progressive Multiple Sclerosis (MS) are presumed to be driven by a neurotoxic bystander effect of meningeal-based myeloid infiltrates. There is an unmet clinical need to attenuate disease progression in such forms of CNS-compartmentalized MS. The failure of systemic immune suppressive treatments has highlighted the need for neuroprotective and repair-inducing strategies. Here, we examined whether direct targeting of CNS myeloid cells and modulating their toxicity may prevent irreversible tissue injury in chronic immune-mediated demyelinating disease. To that end, we utilized the experimental autoimmune encephalomyelitis (EAE) model in Biozzi mice, a clinically relevant MS model. We continuously delivered intracerebroventricularly (ICV) a retinoic acid receptor alpha agonist (RARα), as a potent regulator of myeloid cells, in the chronic phase of EAE. We assessed disease severity and performed pathological evaluations, functional analyses of immune cells, and single-cell RNA sequencing on isolated spinal CD11b+ cells. Although initiating treatment in the chronic phase of the disease, the RARα agonist successfully improved clinical outcomes and prevented axonal loss. ICV RARα agonist treatment inhibited pro-inflammatory pathways and shifted CNS myeloid cells toward neuroprotective phenotypes without affecting peripheral infiltrating myeloid cell phenotypes, or peripheral immunity. The treatment regulated cell-death pathways across multiple myeloid cell populations and suppressed apoptosis, resulting in paradoxically marked increased neuroinflammatory infiltrates, consisting mainly of microglia and CNS / border-associated macrophages. This work establishes the notion of bystander neurotoxicity by CNS immune infiltrates in chronic demyelinating disease. Furthermore, it shows that targeting compartmentalized neuroinflammation by selective regulation of CNS myeloid cell toxicity and survival reduces irreversible tissue injury, and may serve as a novel disease-modifying approach.

Uncovering chikungunya virus-encoded miRNAs and host-specific targeted genes associated with antiviral immune responses: an integrated bioinformatics approach

Chikungunya virus (CHIKV) is a single-stranded RNA virus belonging to the genus Alphavirus and is responsible for causing Chikungunya fever, a type of arboviral fever. Despite extensive research, the pathogenic mechanism of CHIKV within host cells remains unclear. In this study, an in-silico approach was used to predict that CHIKV produces micro-RNAs that target host-specific genes associated with host cellular regulatory pathways. Putative micro-RNAs of CHIKV were predicted using the miRNAFold and Vmir RNA structure web servers, and secondary structure prediction was performed using RNAfold. Host-specific target genes were then predicted, and hub genes were identified using CytoHubba and module selection through MCODE. Functional annotations of hub genes revealed their association with various pathways, including osteoclast differentiation, neuroactive ligand-receptor interaction, and mRNA surveillance. We used the freely available dataset GSE49985 to determine the level of expression of host-specific target genes and found that two genes, F-box and leucine-rich repeat protein 16 (FBXL16) and retinoic acid receptor alpha (RARA), were down-regulated, while four genes, RNA binding protein with serine-rich domain 1 (RNPS1), RNA helicase and ATPase (UPF1), neuropeptide S receptor 1 (NPSR1), and vasoactive intestinal peptide receptor 1 (VIPR1), were up-regulated. These findings provide insight into novel miRNAs and hub genes associated with CHIKV infection and suggest potential targets for therapeutic intervention. Further experimental validation of these targets could lead to the development of effective treatments for CHIKV-mediated diseases.

71 Exploring DNA-methylation and DNA-hydroxymethylation in clear cell renal cell carcinoma using Dirichlet multinomial regression

Abstract Background DNA methylation alterations have been found related to clear cell renal cell carcinoma (ccRCC). In addition, DNA methylation has been used to identify cell heterogeneity at the tumor level using cell deconvolution methods. Other cytosine modifications are less studied in the field. In this study, we aim to explore the use of compositional method, Dirichlet multinomial regression1, to simultaneously explore DNA methylation and DNA hydroxymethylation in ccRCC. Methods A total of 243 clear cell renal cell carcinoma (ccRCC) samples were collected from the Dartmouth Renal Tumor Biobank. DNA methylation analysis was conducted using the Infinium MethylationEPIC BeadChip, using tandem bisulfite (BS) and oxidative bisulfite (oxBS) treatments to differentiate between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) states. Beta values were calculated for all the arrayed samples. Quality control was performed using the ENmix pipeline to ensure the inclusion of high-quality samples using a stringent pOOBHA <0.05. The oxBS maximum likelihood estimate (oxBS-MLE) method was applied to assess 5mC and 5hmC levels based on paired BS and oxBS datasets. To determine the inclusion threshold for 5hmC beta values in the Dirichlet analysis, the R package oxBSCut was used to mask values close to zero2. Missing 5hmC values and those values masked below the detection limit were imputed using a compositional approach and a Tobit regression model to retain the model properties. The Dirichlet model was applied to analyze three methylation states: unmethylated, methylated, and hydroxymethylated, providing comprehensive insights into the methylation landscape of ccRCC samples. Results Here, we analyzed the top 1% most variable methylated sites (n=3020). When comparing tumor vs normal adjacent samples we tested the cytosine modifications in the same model adjusting for sex, age at diagnosis, tumor stage, and grade. For this exploratory analysis, we selected those results that passed an FDR<0.01 and an absolute difference >0.2 for further exploration. Four sites were hyperhydroxymethylated related to MYO5A, DLG2, ANKRD33B, and one open sea site. A total of 23 sites were significantly hypermethylated. Using eForge-TF, these sites tracked to transcription factors targeting estrogen receptors, retinoic acid receptors alpha and gamma, and FOXC1 among others3. Conclusions In this preliminary analysis, using a new modeling approach to interrogate cytosine modifications simultaneously, we found some intriguing results consistent with previous literature. We will expand this analysis to evaluate the relation between extreme changes in hydroxymethylation in particular in relation to alternative splicing alterations. Reference 1. Tsagris, M., Stewart, C. A Dirichlet Regression Model for Compositional Data with Zeros. Lobachevskii J Math39, 398–412 (2018). https://doi.org/10.1134/S1995080218030198 2. Zhang Z, Lee MK, Perreard L, Kelsey KT, Christensen BC, Salas LA. Navigating the hydroxymethylome: experimental biases and quality control tools for the tandem bisulfite and oxidative bisulfite Illumina microarrays. Epigenomics. 2022;14(3):139-152. doi:10.2217/epi-2021-0490 3. Charles E Breeze, Alex P Reynolds, Jenny van Dongen, Ian Dunham, John Lazar, Shane Neph, Jeff Vierstra, Guillaume Bourque, Andrew E Teschendorff, John A Stamatoyannopoulos, Stephan Beck, eFORGE v2.0: updated analysis of cell type-specific signal in epigenomic data, Bioinformatics, Volume 35, Issue 22, November 2019, Pages 4767–4769, https://doi.org/10.1093/bioinformatics/btz456

Outcomes of Acute Promyelocytic Leukaemia in Paediatric Patients: Insights from a Low-Middle-Income Country.

To describe the prognostic variables and course of paediatric acute promyelocytic leukaemia (APL) in Pakistan. Cohort study. Place and Duration of the Study: Department of Paediatric Oncology, Combined Military Hospital, Rawalpindi, Pakistan, from January 2012 to December 2022. Patients aged 1-15 years, clinically confirmed APL with promyelocytic leukaemia- retinoic acid receptor alpha (PML-RARA) were enrolled. Initial admission included a thorough examination, recording demographic and clinical data, reporting time, prior treatment, and socioeconomic status. Statistical analysis used SPSS 25.0, with significance at p <0.05. This study included 50 cases of APL. Out of which, 32 (64%) were males and 18 (34%) were females. The mean age at diagnosis was 7.02 ± 3.86 years. Pallor (96%) and fever (88%) were common presentations. The average white blood cell count was 28.70 ± 35.39 x109/L. Treatment protocols include 48% International Consortium for Childhood (ICC)-APL, and 52% arsenic trioxide (ATO). High-risk cases were 54%. Neutropenic fever and differentiation syndrome were common induction complications. Delays over one month increased induction deaths (6.7 to 35%, p = 0.011), reducing disease-free survival (DFS), (76.7 to 35%, p = 0.001), and overall survival (OS), (80 to 45%, p = 0.007). After 40.90 ± 45.19 months' follow-up, 10-year OS and DFS were 66.0% and 60.0%, respectively. The best OS and DFS, at 80%, were observed in standard-risk cases treated with ATO. Neutropenic fever and bleeding were the primary causes of mortality in paediatric APL induction. Treatment delay was a key prognostic factor. ATO-based therapy offered safer, improved DFS, and OS suitable for primary healthcare settings. Acute promyelocytic leukaemia, Chemotherapy, Neutropenic fever.

Critical role of tripartite fusion and LBD truncation in certain RARA- and all RARG-related atypical APL

AbstractAtypical acute promyelocytic leukemia (aAPL) presents a complex landscape of retinoic acid receptor (RAR) fusion genes beyond the well-known PML::RARA fusion. Among these, 31 individually rare RARA and RARG fusion genes have been documented, often reported in the canonical X::RAR bipartite fusion form. Intriguingly, some artificially mimicked bipartite X::RAR fusions respond well to all-trans retinoic acid (ATRA) in vitro, contrasting with the ATRA resistance observed in patients. To unravel the underlying mechanisms, we conducted a comprehensive molecular investigation into the fusion transcripts in 27 RARA fusion gene–positive aAPL (RARA-aAPL) and 21 RARG-aAPL cases. Our analysis revealed an unexpected novel form of X::RAR::X– or X::RAR::Y–type tripartite fusions in certain RARA-aAPL and all RARG-aAPL cases, with shared features and notable differences between these 2 disease subgroups. In RARA-aAPL cases, the occurrence of RARA 3′ splices was associated with their 5′ fusion partner genes, mapping across the coding region of helix 11_12 (H11_12) within the ligand-binding domain (LBD), resulting in LBD-H12 or H11_12 truncation. In RARG-aAPL cases, RARG 3′ splices were consistently localized to the terminus of exon 9, leading to LBD-H11_12 truncation. Significant differences were also observed between RARA and RARG 5′ splice patterns. Our analysis also revealed extensive involvement of transposable elements in constructing RARA and RARG 3′ fusions, suggesting transposition mechanisms for fusion gene ontogeny. Both protein structural analysis and experimental results highlighted the pivotal role of LBD-H11_12/H12 truncation in driving ATRA unresponsiveness and leukemogenesis in tripartite fusion–positive aAPL, through a protein allosteric dysfunction mechanism.

Treatment of a STAT5b::RARα positive case of APL in a patient not eligible for intensive chemotherapy.

Acute promyelocytic leukaemia (APL) with a STAT5b::RARα gene fusion is an extremely rare subtype of APL characterised by resistance to conventional therapies and extremely poor prognosis. This case highlights that whilst APL with variant RARα translocations are rare, they do pose significant challenges both diagnostically and in their clinical management. This case, in the first instance, demonstrates the importance of using a combination of molecular techniques including next generation sequencing (NGS) for diagnosis particularly in morphological and immunophenotypic typical APL which appears negative by confirmatory testing. Secondly, our patient represents, to the best of our knowledge, the first documented example of this rare disease that has been managed with, and shown sensitivity to low-dose cytarabine (LDAC) in combination with venetoclax (Ven). This case demonstrates that although treatment options are extremely limited for patients not eligible for intensive chemotherapy non-intensive options do show increasing promise.

Screening (ant)agonistic activities of xenobiotics on the retinoic acid receptor alpha (RARα) using in vitro and in silico analysis

Retinoic acid receptors (RARs) are known as crucial endocrine receptors that could mediate a broad diversity of biological processes. However, the data on endocrine disrupting effects of emerging chemicals by targeting RAR (ant)agonism are far from sufficient. Herein, we investigated the RARα agonistic or antagonistic activities for 75 emerging chemicals of concern, and explored their interactions with this receptor. A recombinant two-hybrid yeast assay was used to examine the RARα activities of the test chemicals, wherein 7 showed effects of RARα agonism and 54 exerted potentials of RARα antagonism. The representative chemicals with RARα agonistic activities, i.e. 4-hydroxylphenol (4-HP) and bisphenol AF (BPAF), significantly increased the mRNA levels of CRABP2 and CYP26A1, while 4 select chemicals with RARα antagonistic potentials, including bisphenol A (BPA), tetrabromobisphenol A (TBBPA), 4-tert-octylphenol (4-t-OP), and 4-n-nonylphenol (4-n-NP), conversely decreased the transcriptional levels of the test genes. The in silico molecular docking analysis using 3 different approaches further confirmed the substantial binding between the chemicals with RARα activities and this nuclear receptor protein. This work highlights the promising strategy for screening endocrine-disrupting effects of emerging chemicals of concern by targeting RARα (ant)agonism.

Retinoic acid receptor alpha inhibits ferroptosis by promoting thioredoxin and protein phosphatase 1F in lung adenocarcinoma

Ferroptosis is a recently discovered form of cell death that plays an important role in tumor growth and holds promise as a target for antitumor therapy. However, evidence in the regulation of ferroptosis in lung adenocarcinoma (LUAD) remains elusive. Here, we show that retinoic acid receptor alpha (RARA) is upregulated with the treatment of ferroptosis inducers (FINs). Pharmacological activation of RARA increases the resistance of LUAD to ferroptosis according to cell viability and lipid peroxidation assays, while RARA inhibitor or knockdown (KD) does the opposite. Through transcriptome sequencing in RARA-KD cells and chromatin immunoprecipitation (CHIP)-Seq data, we identify thioredoxin (TXN) and protein phosphatase 1 F (PPM1F) as downstream targets of RARA, both of which inhibit ferroptosis. We confirm that RARA binds to the promotor region of TXN and PPM1F and promotes their transcription by CHIP-qPCR and dual-luciferase assays. Overexpression of TXN and PPM1F reverses the effects of RARA knockdown on ferroptosis in vitro and vivo. Clinically, RARA knockdown or inhibitor increases cells’ sensitivity to pemetrexed and cisplatin (CDDP). Immunohistochemistry (IHC) of LUAD from our cohort shows the same expression tendency of RARA and the downstream targets. Our study uncovers that RARA inhibits ferroptosis in LUAD by promoting TXN and PPM1F, and inhibiting RARA-TXN/PPM1F axis represents a promising strategy for improving the efficacy of FINs or chemotherapy in the treatment of LUAD patients.

Mutations and laboratory diagnosis in acute promyelocytic leukemia

Introduction: acute Promyelocytic Leukemia (APL) is a unique subtype of acute myeloid leukemia (AML) characterized by proliferation and accumulation of abnormal promyelocytes in the bone marrow. It arises from a balanced translocation between chromosomes 15 and 17, involving the retinoic acid receptor alpha (RARA) gene on chromosome 17 and the promyelocytic leukemia (PML) gene. It has a higher incidence in young adults. Objective: to establish the mutations associated with acute promyelocytic leukemia and the techniques that aid in its clinical diagnosis. Method: a systematic review of 19 scientific articles published in the databases of PubMed, Scopus, Google Scholar and the Virtual Library of the University of Granada was carried out. The data collected focused on acute promyelocytic leukemia, acquired, secondary or somatic mutations and laboratory diagnosis. Results: of a total of 1730 patients 67.7% had the t(15;17) mutation, of 682 patients 26.7% had the FLT3-ITD mutation and of 175 patients 16% had the FLT3-D835 mutation. Laboratory diagnosis is based on morphological evaluation of promyelocytes, hemostasis tests, biochemical tests Immunophenotyping and molecular cytogenetics. Conclusions: the mutation associated with APL is the promyelocytic leukemia/retinoic acid receptor alpha (PML-RARA) gene, the same that presents a t(15;17), secondary mutations or somatic mutations such as (FLT3-ITD or FLT3-D835) were also known. Multiparametric flow cytometry is one of the most widely used techniques for the diagnosis of APL, allowing the determination of cell morphology and immunophenotypes

Abstract PO3-26-07: Epigenetic approaches to deliver a targeted radiotherapy for triple negative breast cancer

Abstract The sodium iodide symporter (NIS, encoded by SLC5A5) is the sole transporter of iodide, and successfully exploited in thyroid cancer to uptake radioiodide (RAI). NIS is essentially undetected in normal mammary epithelial cells but elevated during lactation, and has a heterogeneous expression profile in breast cancer (BRCA), as evidenced by TCGA cohort and also in a ~120 sample TMA we have profiled. Furthermore, pre-clinical studies support an epigenetic mechanisms that involves Retinoic Acid Receptor alpha (RARa) to regulate SLC5A5 expression. Therefore, we have applied an orthogonal approach to both elucidate and target mechanisms that control NIS expression and function to deliver a therapeutic strategy that targets this transporter as a novel radiotherapy in BRCA. To understand the epigenetic control of expression we segregated samples in the TCGA-BRCA cohort into lower and upper quartile SLC5A5 expression and identified ~600 significant differentially expressed of genes (DEG). Interestingly, restricting analyses to either triple negative (TN) or tumors from African American (AA) patients doubled the number of DEGs. These DEGs were significantly associated with several transcription factors including LHX3, a pituitary development transcription factor regulated by thyroid signaling. The most significantly altered ~70 DEGs significantly clustered tumors by genomic ancestry and ERBB2/HER2 IHC status. The DEGs also supported a footprint of RARa signaling include PRAME, a known RARa co-repressor associated with histone deacetylase, and reflects the finding that NIS expression is highly responsive to SAHA treatment. To investigate if PRAME expression suppresses RARa regulation of SLC5A5 we have established a panel of BRCA cell lines (AU565, SKB3 SUM52 and MDA-MB-231) stably transfected with either CRISPR-VP64 (activator) and CRISPR-KRAB (repressor) to modulate PRAME and measure the epigenomic impact, the expression of SLC5A5 and the ability to transport RAI. In parallel we have applied a drug screen approach that targets non-canonical approaches to govern NIS expression, for example by targeting valosin-containing protein (VCP) that induces NIS proteolysis. In the first instance we have developed a therapeutic strategy to target NIS by inhibiting VCP. Specifically, we are screening a range of VCP inhibitors in combination with the HDAC inhibitor SAHA or the transcriptional potentiator, disulfiram metabolite Cu(DDC)2 in BRCA cell lines by testing the ability to augment RAI uptake. Remarkably, in MDA MB 231 cells, Cu(DDC)2 combined with the VCP inhibitor CB5339, resulted in a significant additive increase in RAI uptake and a prominent decrease in cell proliferation and survival. These studies combined with future murine and PDX testing of CRISPR-edited tumors and drug exposures will develop a comprehensive analyses of the mechanisms that control SLC5A5 expression across breast cancer cell types, how this can be most effectively targeted to restore NIS expression, and exploited to promote RAI as a novel targeted BRCA radiotherapy. Citation Format: Katie Brookes, Martin Read, Sajad Wani, Sissy Jhiang, Daniel Stover, Mathew Ringel, Christopher McCabe, Moray Campbell. Epigenetic approaches to deliver a targeted radiotherapy for triple negative breast cancer [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 PO3-26-07.

Synergistic Effects of the RARalpha Agonist Tamibarotene and the Menin Inhibitor Revumenib in Acute Myeloid Leukemia Cells with KMT2A Rearrangement or NPM1 Mutation.

Inhibition of menin in acute myeloid leukemia (AML) harboring histone-lysine-N-methyltransferase 2A rearrangement (KMT2Ar) or the mutated Nucleophosmin gene (NPM1c) is considered a novel and effective treatment approach in these patients. However, rapid acquisition of resistance mutations can impair treatment success. In patients with elevated retinoic acid receptor alpha (RARA) expression levels, promising effects are demonstrated by the next-generation RARalpha agonist tamibarotene, which restores differentiation or induces apoptosis. In this study, the combination of revumenib and tamibarotene was investigated in various KMT2Ar or NPM1c AML cell lines and patient-derived blasts, focusing on the potential synergistic induction of differentiation or apoptosis. Both effects were analyzed by flow cytometry and validated by Western blot analysis. Synergy calculations were performed using viability assays. Regulation of the relevant key mediators for the MLL complex were quantified by RT-qPCR. In MV4:11 cells characterized by the highest relative mRNA levels of RARA, highly synergistic induction of apoptosis is demonstrated upon combination treatment. Induction of apoptosis by combined treatment of MV4:11 cells is accompanied by pronounced induction of the pro-apoptotic protein BAX and a synergistic reduction in CDK6 mRNA levels. In MOLM13 and OCI-AML3 cells, an increase in differentiation markers like PU.1 or a decreased ratio of phosphorylated to total CEBPA is demonstrated. In parts, corresponding effects were observed in patient-derived AML cells carrying either KMT2Ar or NPM1c. The impact of revumenib on KMT2Ar or NPM1c AML cells was significantly enhanced when combined with tamibarotene, demonstrating synergistic differentiation or apoptosis initiation. These findings propose promising strategies for relapsed/refractory AML patients with defined molecular characteristics.

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.

Abstract 568: Modulation of HER4 isoforms expression in estrogen-stimulated in vitro models

Abstract Introduction HER4, a tyrosine kinase receptor belonging to the Human Epidermal Growth Factor/HER/ErbB-family, has a complex biology that includes four isoforms (JMa-CYT1/2, and JMb-CYT1/2) and tissue specific oncogenic or tumour suppressor roles. In estrogen receptor positive (ER+) breast cancer (BC), HER4 isoforms can either act as a co-regulator of ESR1-mediated gene expression by localising to the nucleus, or activate apoptosis by localising to mitochondria. There is very little known about HER4 function in cutaneous melanoma, despite cutaneous melanoma showing the highest HER4 genomic alteration frequency (17.34%) across different cancers (cBioPortal). Due to the interplay between estrogen signalling and HER4 isoforms in ER+BC, this study investigates the RNA expression of HER4 isoforms along with hormone and nuclear receptors associated with 17β-estradiol stimulation in ER+BC and cutaneous melanoma in vitro models. Material and Methods CAMA1 (ER+BC) and SKMEL24 (cutaneous melanoma) cell lines were serum starved for 4h prior to stimulation with 17β-estradiol (0.5nM, 1nM, 5nM and 10nM). RNA was extracted from cells after 24h using TRIzol, followed by qRT-PCR to assess the expression of HER4 isoforms, the hormone receptors estrogen receptor 1 (ESR1/ER-alpha), estrogen receptor 2 (ESR2/ER-beta), progesterone receptor (PGR/PR), G protein-coupled estrogen receptor 1 (GPER1) and the nuclear receptors retinoic acid receptor alpha (RARA) and retinoid X receptor alpha (RXRA). Cell proliferation and caspase3/7 apoptosis analysis were performed at 24/48/72h in the 17β-estradiol stimulated cells by using the IncuCyte Live Cell Analysis Instrument (Sartorius). SN-38 (150nM) was used as positive control for apoptosis induction. Two-way ANOVA was used for the statistical analysis, p&amp;lt;0.05 was significant. Results The qRT-PCR analysis showed that upon stimulation of CAMA1 with 1nM of 17β-estradiol there is a numerical shift in the CYT-1/CYT-2 ratio towards CYT-2 (not statistically significant, p&amp;gt;0.05) and a significant increase in PGR (p= 0.0027) and RAR-A (p= 0.0248) expression compared to control. CAMA1 stimulated with any concentration of 17β-estradiol showed an increase in proliferation compared to the control. The stimulation of SKMEL24 with 5nM of 17β-estradiol showed an increase in JM-a (p=0.0075) and CYT-1 (p=0.0271) HER4 isoforms, GPER1 (p= 0.0286), and a dose-dependent increase in RARA (p=0.0011) compared to control, even in the absence of ESR1, ESR2 and PGR. SKMEL24 stimulated with 17β-estradiol showed a decrease in proliferation compared to control, but no apoptosis induction was found. Conclusion These findings suggest potential interplay between 17β-estradiol and HER4 isoforms leading to the activation of intracellular pathways that could influence tumour progression/suppression in melanoma. Further studies are needed to clarify the specific receptor interactions and pathways involved. Citation Format: Marta Valenti, Georg F. Bischof, John P. Crown, Denis M. Collins. Modulation of HER4 isoforms expression in estrogen-stimulated in vitro models [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 568.