Journal: Nucleic Acids Research

Article Title: New structural insights into the control of the retinoic acid receptors RAR/RXR by DNA, ligands, and transcriptional coregulators

doi: 10.1093/nar/gkaf967

Figure Lengend Snippet: The interaction of RR–BMS493 bound to DNA with the NCoR NID reveals new features. ( A – D ) The differential interdomain distances in RAR/RXR upon NCoR NID interaction (NRR–DNAs versus RR–DNA). Light green indicates differences in distances for RAR–DBD–LBD, purple represents RXR, dark green corresponds to RAR–DBD/RXR–LBD, and red denotes RXR–DBD/RAR–LBD. Panels are organized by the bound element (panel A: DR0L, panel B: DR1, panel C: DR5, panel D: IR0). ( E and F ) Differential plots of normalized weighted contact densities (Δρ) calculated as Δρ = ρNRR-DNA – ρRR-DNA. Positive Δρ values (blue) represent regions where the contact density increases in the presence of NCoR NID , while negative (red) indicate regions where it decreases. ( E ) The lower-left quadrant corresponds to NRR–DR0L, while the upper-right quadrant represents NRR–DR1. ( F ) The lower-left quadrant corresponds to NRR–DR5, and the upper-right quadrant represents NRR–IR0. ( G ) Illustration comparing apo RR–DNA–BMS493 versus RR–DNA–BMS493 bound to NCoR NID . RAR is depicted in green, RXR is shown in purple, and NCoR NID is represented in gray. The RAR inverse agonist (BMS493) is drawn as a light green circle, and the DNA is shown as brown bars. ( H ) Differential heatmap highlighting structural changes in RAR (apo-NB and DNA bound forms) upon BMS493 and NCoR NID binding. Red and blue shades indicate increased and decreased deuterium incorporation, respectively. Secondary structural elements are shown above each heatmap, where α-helices are represented in lighter colors and labeled with their respective numbering, and β-strands are shown in orange. ( I ) Same structural effects as in panel (H) but on the RXR part of the RAR/RXR heterodimer. ( J ) Illustration comparing NRR with NRR–DNA–BMS493. ( K and L ) Differential heatmaps showing structural changes in RAR and RXR, respectively, based on the conditions illustrated in panel (J). ( M ) HDX differential protections showing NCoRNID-binding effect plotted on the model generated from the crystallographic structure of RARβ/RXR–DR1 . The data correspond to exchange times of 3 h and 10 s (inset)—highlighted on the right side of the heatmaps by colored arrowheads. ( N ) Illustration of the NCoR NID versus the NCoR NID bound to RR–DNAs. ( O ) Differential HDX-MS heatmap of NCoR NID , showing the impact of the RR–DNA–BMS493 interaction on the corepressor protection.

Article Snippet: The ligands AM580, BMS493, and CD3254 were purchased from Tocris Bioscience.

Techniques: Binding Assay, Labeling, Generated

Journal: bioRxiv

Article Title: Phenotypic dissection of epithelial lineages and therapeutic manipulation of differentiation programs in human Adenoid Cystic Carcinomas (ACCs)

doi: 10.1101/2022.01.19.476843

Figure Lengend Snippet: (a) Schematic modeling of the RA signaling pathway, displaying known potentiators (orange) and attenuators (blue) of RA signaling. (b) Comparison of the gene-expression levels of known mediators of RA signaling between myoepithelial-like (CD49f high /KIT neg ; red) and ductal-like (CD49f low /KIT + ; green) cells isolated from bi-phenotypic ACC models. Genes identified as attenuators of RA signaling displayed preferential expression in CD49f high /KIT neg cells, while genes identified as potentiators of RA signaling displayed preferential expression in CD49f low /KIT + cells, as revealed by RNA-seq. Error bars: mean +/- standard deviation. Statistical test: Student’s t-test for paired samples (• p<0.01, *p<0.05, **p<0.01). (c) Heatmap of the mean z-score values for the expression level of genes identified as modulators of RA, as measured across the two ACC populations. (d-g) Establishment and phenotypic characterization of three-dimensional (3D) organoid cultures from human ACCs. Organoids grow as spherical structures (d) , retain cribriform histology (e) and contain both myoepithelial-like and ductal-like populations, as visualized by IHC staining for TP63 (f) and KIT (g) . (h) Analysis by flow cytometry of ACC organoids treated for one week with either DMSO (n=13), ATRA (10 µM; n=10), Bexarotene (10 µM; n=6), BMS493 (10 µM; n=10) or AGN193109 (10 µM; n=6). (i-j) Comparison of the percentage of CD49f high /KIT neg (i) and CD49f low /KIT + (j) cells found in organoids following in vitro treatment with modulators of RA signaling. Treatment with agonists of RA signaling was associated with an increase in the relative percentage of CD49f low /KIT + cells, while treatment with inhibitors of RA signaling was associated with its reduction as compared to DMSO-treated controls. Statistical test: one-way ANOVA with Dunnett’s multiple comparisons test (*p<0.05, **p<0.01, ***p<0.001). (k-p) Representative flow plots and quantification of the relative frequency of myoepithelial-like (CD49f high /KIT neg ) and ductal-like (CD49f low /KIT + ) cells in ACC organoids established from SGTX6 (k-m) or ACCX6 (n-p) PDX lines, treated for one week with either DMSO, ATRA (10 µM) or BMS493 (10 µM), confirming results previously obtained in the ACCX5M1 PDX line. Data correspond to the results of at least two independent experiments (with a minimum of 3 replicates for each treatment condition).

Article Snippet: On the day of in vivo administration, single-use aliquots were thawed, and BMS493 was further diluted to a concentration of 2 mg/mL in DPBS supplemented with 0.15M hydroxypropyl β-cyclodextrin (HP-β-CD; Cayman Chemicals 16169), for a total volume of 0.5 mL per dose (1 mg/dose).

Techniques: Expressing, Isolation, RNA Sequencing Assay, Standard Deviation, Immunohistochemistry, Flow Cytometry, In Vitro

Journal: bioRxiv

Article Title: Phenotypic dissection of epithelial lineages and therapeutic manipulation of differentiation programs in human Adenoid Cystic Carcinomas (ACCs)

doi: 10.1101/2022.01.19.476843

Figure Lengend Snippet: (a-b) Treatment with all-trans retinoic acid (ATRA), an agonist of RA signaling, induces a dose-dependent increase in the percentage of CD49f low /KIT + cells in 3D organoid cultures established from the ACCX5M1 PDX line. The increase in the percentage of CD49f low /KIT + cells is already observed at the 0.1-1 µM concentration range (a), and becomes progressively more prominent as the concentration is raised to the 10-100 µM concentration range (b). (c-d) Treatment with either BMS493 or AGN19310, two inverse agonists (i.e., inhibitors) of RA signaling, results in a profound reduction in the percentage of CD49f low /KIT + cells in 3D organoid cultures established from either the ACCX5M1 (c; BMS43) or SGTX6 (d; AGN193109) PDX lines, even when administered at low doses (1 µM). Organoid cultures were treated for one week and analyzed by flow cytometry (n=3 wells/condition). Changes in the percentage of CD49f low /KIT + cells were tested for statistical significance using one-way ANOVA with Dunnett’s multiple comparisons test against untreated (NT) or DMSO-treated controls (ns = non-significant, *p<0.05, **p<0.01, ***p<0.001).

Article Snippet: On the day of in vivo administration, single-use aliquots were thawed, and BMS493 was further diluted to a concentration of 2 mg/mL in DPBS supplemented with 0.15M hydroxypropyl β-cyclodextrin (HP-β-CD; Cayman Chemicals 16169), for a total volume of 0.5 mL per dose (1 mg/dose).

Techniques: Concentration Assay, Flow Cytometry

Journal: bioRxiv

Article Title: Phenotypic dissection of epithelial lineages and therapeutic manipulation of differentiation programs in human Adenoid Cystic Carcinomas (ACCs)

doi: 10.1101/2022.01.19.476843

Figure Lengend Snippet: (a-l) Organoids established from the ACCX6 PDX line were treated with DMSO, ATRA (10 µM) or BMS493 (10 µM) and subsequently analyzed by IHC. Treatment with ATRA resulted in an expansion of KIT + cells (g) , while treatment with BMS493 resulted in a complete loss of KIT expression (k) , associated with a dramatic change in the organoids’ morphology, characterized by the appearance of amorphous, eosin-rich deposits at their center (i) . Neither ATRA or BMS493 appeared to upregulate MKI67 expression in either cell population (d, h, l). Scale bars = 100 µm. (m-n) Schematic workflow of prospective experiments aimed at testing the effects of RA signaling on purified populations of myoepithelial-like and ductal-like cells: CD49f high /KIT neg and CD49f low /KIT + cells were sorted in parallel from the same ACCX5M1 tumors and cultured for one week as 2D monolayers in the presence of either ATRA (10 µM) or BMS493 (10 µM). (o) Treatment with ATRA did not reduce the viability of either CD49f high /KIT neg or CD49f low /KIT + cells (viability assay: alamarBlue; technical replicates: n=3; error bars: mean +/- standard deviation; statistical test: Student’s t-test, two-tailed; ns = non-significant, *p<0.05, **p<0.01, ***p<0.001). (p) Treatment with ATRA caused CD49f high /KIT neg cells to change phenotype and become undistinguishable from CD49f low /KIT + cells by flow cytometry. (q) Schematic modeling of the effects of RA agonism on the cell composition of ACC organoids: myoepithelial-like cells are stimulated to differentiate into ductal-like cells. (r) Treatment with BMS493 had a minor effect on the viability of CD49f high /KIT neg cells, but caused the death of the majority CD49f low /KIT + cells. (s) Upon visual inspection by conventional microscopy, CD49f low /KIT + cells treated with BMS493 appeared fragmented. Scale bar = 100 µm. (t) Schematic modeling of the effects of RA inhibition on the cell composition of ACC organoids: ductal-like cells are selectively killed.

Article Snippet: On the day of in vivo administration, single-use aliquots were thawed, and BMS493 was further diluted to a concentration of 2 mg/mL in DPBS supplemented with 0.15M hydroxypropyl β-cyclodextrin (HP-β-CD; Cayman Chemicals 16169), for a total volume of 0.5 mL per dose (1 mg/dose).

Techniques: Expressing, Purification, Cell Culture, Viability Assay, Standard Deviation, Two Tailed Test, Flow Cytometry, Microscopy, Inhibition

Journal: bioRxiv

Article Title: Phenotypic dissection of epithelial lineages and therapeutic manipulation of differentiation programs in human Adenoid Cystic Carcinomas (ACCs)

doi: 10.1101/2022.01.19.476843

Figure Lengend Snippet: (a-o) Analysis of organoid morphology and histology, following treatment with either activators (ATRA; direct agonist) or inhibitors (BMS493; inverse agonist) of RA signaling. Organoids were established from a PDX line with bi-phenotypic histology (ACCX5M1) and treated for one week with either DMSO (a-e), 10 µM ATRA (f-j) or 10 µM BMS493 (k-o). Scale bars = 100 µm. Treatment with ATRA did not change organoid morphology (f), but increased the number of KIT + cells (i), as visualized by immunohistochemistry (IHC). Treatment with BMS493 caused a dramatic change in organoid morphology, characterized by the appearance of dense areas in the organoid centers, when observed using bright-field microscopy (k, arrowheads). When organoids were stained with hematoxylin and eosin (H&E), these areas consisted of an eosin-rich material, with apoptotic nuclei (l, arrowheads). (p-q) Analysis by flow cytometry of cell cycle distribution in organoids established from ACCX5M1 (p) and ACCX6 (q) PDX lines, following 1 week of treatment with either DMSO, ATRA (10 µM) or BMS493 (10µM). (r-s) Treatment with either ATRA or BMS493 did not increase the percentage of cells in the G2/M phase of the cell-cycle in ACCX5M1 (r) and ACCX6 (s) organoids. Experiments included at least three replicates (n=3 wells/condition). Error bars: mean +/- standard deviation. Differences in the percentage of cells in the G2/M phase were tested for statistical significance using one-way ANOVA with Dunnett’s multiple comparisons test (ns=non-significant, *p<0.05).

Article Snippet: On the day of in vivo administration, single-use aliquots were thawed, and BMS493 was further diluted to a concentration of 2 mg/mL in DPBS supplemented with 0.15M hydroxypropyl β-cyclodextrin (HP-β-CD; Cayman Chemicals 16169), for a total volume of 0.5 mL per dose (1 mg/dose).

Techniques: Immunohistochemistry, Microscopy, Staining, Flow Cytometry, Standard Deviation

Journal: bioRxiv

Article Title: Phenotypic dissection of epithelial lineages and therapeutic manipulation of differentiation programs in human Adenoid Cystic Carcinomas (ACCs)

doi: 10.1101/2022.01.19.476843

Figure Lengend Snippet: (a-b) Analysis by flow cytometry of two PDX lines representative of human ACCs with solid histology (ACCX9, ACCX11) revealing a ductal-like, mono-phenotypic (CD49f low /KIT + ) cell composition. (c-f) IHC analysis of KIT and TP63 expression ACCX9 and ACCX11, showing ubiquitous expression of the ductal-specific marker KIT (c,e) and loss of the myoepithelial-specific marker TP63 (d,f) . Scale bars = 50 µm. (g) Principal component analysis (PCA) of RNA-seq data from human ACCs, in which results from two PDX lines with solid histology (ACCX9, ACCX11) are combined with 5 autologous pairs of CD49f high /KIT neg and CD49f low /KIT + cells populations from bi-phenotypic PDX lines (ACCX5M1, ACCX6, ACCX14, ACCX22, SGTX6). (h) Hierarchical clustering of RNA-sequencing data from human ACCs, based on the expression levels of the top 100 genes identified as differentially expressed between CD49f high /KIT neg and CD49f low /KIT + cells. Solid ACCs cluster with CD49f low /KIT + cells from bi-phenotypic tumors, irrespective of the method used to analyze their transcriptional profile. (i-j) Upon visual inspection by conventional microscopy, ACCX9 organoids cultured for one week in the presence of BMS493 (10 µM) display widespread cell fragmentation, in contrast to organoids cultured with DMSO alone. (k) Quantification of organoid viability using the alamarBlue HS assay, confirming the cytotoxic activity of BMS493 (10 µM) against PDX lines with solid histology (ACCX9, ACCX11). Error bars: mean +/- standard deviation; p-values: two-tailed Student’s t-tests; n=3 replicates/condition.

Article Snippet: On the day of in vivo administration, single-use aliquots were thawed, and BMS493 was further diluted to a concentration of 2 mg/mL in DPBS supplemented with 0.15M hydroxypropyl β-cyclodextrin (HP-β-CD; Cayman Chemicals 16169), for a total volume of 0.5 mL per dose (1 mg/dose).

Techniques: Flow Cytometry, Expressing, Marker, RNA Sequencing Assay, Microscopy, Cell Culture, Activity Assay, Standard Deviation, Two Tailed Test

Journal: bioRxiv

Article Title: Phenotypic dissection of epithelial lineages and therapeutic manipulation of differentiation programs in human Adenoid Cystic Carcinomas (ACCs)

doi: 10.1101/2022.01.19.476843

Figure Lengend Snippet: (a) Individual growth curves of ACCX9 tumors treated with DMSO (gray) or BMS493 (magenta). Cross symbols (†) denote two animals who were sacrificed early, due to deterioration of their health condition. (b) Growth rates of ACCX9 tumors treated with either DMSO (n=6) or BMS493 (n=7). Two treatment cohorts are identified by different symbols (circles = cohort 1; triangles = cohort 2). Differences in mean growth rates were statistically significant (Welch’s t-test; **p<0.01). Black symbols denote the two animals who were sacrificed because of a deterioration of their health condition. (c) Individual animal weights over the course of in vivo treatment with BMS493 (†: animals sacrificed due to deterioration of health conditions). (d) Individual growth curves of ACCX11 tumors treated with DMSO (gray) or BMS493 (magenta). (e) Growth rates of ACCX11 tumors treated with either DMSO (n=4) or BMS493 (n=5). Differences in mean growth rates were statistically significant (Welch’s t-test; **p<0.01). (f) Individual animal weights over the course of in vivo treatment with BMS493. (g) Individual growth curves of ACCX5M1 tumors treated with DMSO (gray) or BMS493 (magenta). Cross symbols (†) denote two animals who either were sacrificed early due to deterioration of their general health or who died at the final time point. (h) Growth rates of ACCX5M1 tumors treated with either DMSO (n=6) or BMS493 (n=6). Two treatment cohorts are identified by different symbols (circles = cohort 1; triangles = cohort 2). Differences in mean growth rates were statistically significant (Welch’s t-test; *p<0.05). Black symbols denote the two animals who either were sacrificed early due to deterioration of their general health or died at the final time point. (i) Individual animal weights over the course of treatment (†: animals sacrificed or found dead).

Article Snippet: On the day of in vivo administration, single-use aliquots were thawed, and BMS493 was further diluted to a concentration of 2 mg/mL in DPBS supplemented with 0.15M hydroxypropyl β-cyclodextrin (HP-β-CD; Cayman Chemicals 16169), for a total volume of 0.5 mL per dose (1 mg/dose).

Techniques: In Vivo

Journal: bioRxiv

Article Title: Phenotypic dissection of epithelial lineages and therapeutic manipulation of differentiation programs in human Adenoid Cystic Carcinomas (ACCs)

doi: 10.1101/2022.01.19.476843

Figure Lengend Snippet: (a ) Schematic of the BMS493 dosing regimen utilized for the in vivo treatment of solid ACC models (40 mg/kg doses, i.p., 3 times/week x 3 weeks). Schematic was created with BioRender.com. (b-e) Comparison of tumor growth kinetics, expressed as either fold-increases in mean tumor volumes or growth rates, between mice treated with BMS493 and mice treated with the drug’s vehicle alone (DMSO), following subcutaneous engraftment with solid ACC models (ACCX9: b-c ; ACCX11: d-e ). (f) Schematic of the BMS493 dosing regimen utilized for the in vivo treatment of a bi-phenotypic ACC model (40 mg/kg doses, i.p., 4 times/week x 3 weeks). (g-h) Comparison of tumor growth kinetics, expressed as either fold-increases in tumor volumes or growth rates, between mice treated with BMS493 and mice treated with the drug’s vehicle alone (DMSO), following subcutaneous engraftment with a bi-phenotypic ACC model (ACCX5M1). Differences between mean fold-increases in tumor volume were tested for statistical significance using a two-tailed Student’s t-test (ns = non-significant, *p<0.05, **p<0.01, ***p<0.001). Differences between mean growth rates were tested for statistical significance using a two-tailed Welch’s t-test. Growth rates were calculated assuming exponential kinetics. Error bars: mean +/- standard deviation.

Article Snippet: On the day of in vivo administration, single-use aliquots were thawed, and BMS493 was further diluted to a concentration of 2 mg/mL in DPBS supplemented with 0.15M hydroxypropyl β-cyclodextrin (HP-β-CD; Cayman Chemicals 16169), for a total volume of 0.5 mL per dose (1 mg/dose).

Techniques: In Vivo, Two Tailed Test, Standard Deviation

Journal: bioRxiv

Article Title: Epithelial sensing of vitamin A shapes intestinal antimicrobial defense

doi: 10.64898/2026.03.08.710399

Figure Lengend Snippet: (A) Vitamin A-derived retinol is metabolized to RA, which activates retinoic acid receptors (RARs). RARs bind target gene promoters to drive RA-dependent transcription. (B) HT-29 cells (human intestinal epithelial cells) were treated with the RAR antagonist BMS493 or the RAR agonist Ch55 and simultaneously stimulated overnight with RA and IL-22. REG3G transcripts were quantified by qPCR. Each data point represents an independent experimental replicate (n=6 per group). (C) HCT-116, a transfection-competent human intestinal epithelial cell line expressing RARA and RARG , was treated for 24 hours with an siRNA targeting either gene and then stimulated overnight with retinol and IL-22. REG3G transcripts were quantified by qPCR. Each data point represents an independent experimental replicate (n=4 per group). (D) IEC-specific disruption of RAR signaling using a dominant-negative RAR (dnRAR) knock-in allele. dnRAR mice harbor a loxP -flanked STOP cassette upstream of a dominant-negative RAR open reading frame. The dnRAR is derived from a mutant human RARα (RAR403) lacking the ligand-dependent transactivation domain and functions as a pan-RAR inhibitor. Crossing dnRAR mice with Villin-Cre transgenic mice excises the STOP cassette in IECs, resulting in IEC-selective expression of dnRAR and inhibition of RAR signaling. (E) qPCR analysis of Reg3g expression in small intestines of conventional dnRAR fl/fl (n=12) and dnRAR IEC (n=17) mice from five litters, and germ-free wild-type mice (n=21). (F) Immunofluorescence microscopy of REG3G in small intestines of dnRAR fl/fl and dnRAR IEC mice. Sections were stained for REG3G and counterstained with DAPI. Scale bar, 100 μ m. Images are representative of at least three fields per sample and two independent experiments (three littermates per group). (G) Mean fluorescence intensities of at least 150 villi from the images represented in (F) were quantified across at least two mice of each genotype. RAR, retinoic acid receptor; RA, retinoic acid; IEC, intestinal epithelial cell; REG3G, regenerating islet-derived protein 3γ; siRNA, small interfering RNA; dnRAR, dominant negative retinoic acid receptor; Conv, conventional; GF, germ-free. Means ± SEM are plotted; *p < 0.05; **p < 0.01; ***p<0.001; ns, not significant by Mann-Whitney test. See also .

Article Snippet: Pharmacologic modulation of retinoic acid receptor (RAR) activity was performed using the pan-RAR antagonist BMS493 (Fisher Scientific) or the pan-RAR agonist Ch55 (Tocris).

Techniques: Derivative Assay, Transfection, Expressing, Disruption, Dominant Negative Mutation, Knock-In, Mutagenesis, Transgenic Assay, Inhibition, Immunofluorescence, Microscopy, Staining, Fluorescence, Small Interfering RNA, MANN-WHITNEY