Fig 2: Transient interaction between GR and an activated RAR/RXR complex represses CYP26A1 expression(A–C) HepG2 cells were treated 30 min with 0.1% DMSO or 1 nM RA ± 100 nM Dex, followed by crosslinking, lysis, and sonication for ChIP. Chromatin samples were reacted with RARa (A), RAR? (B), or GR (C) antibodies. DNA samples obtained by reverse-crosslinking were used for qPCR with primers designed to scan (i) the GRE + RARE of the PCK1 promoter (positive control), (ii) RARE1, and (iii) RARE2/3/4 in the CYP26A1 promoter.(D) Co-immunoprecipitation without over-expression. HepG2 were treated with 0.1% DMSO or 1 nM RA ± 100 nM Dex. A set of treatment groups was treated with crosslinker DSG. Isolated protein samples were immunoprecipitated with RARa antibody. The precipitated complex was resolved by western blotting using a GR antibody. See also SF7, SF8.

Fig 3: Osimertinib-refractory cancer cells are sensitive to pan-RAR inhibition. A, Schematic representation of the treatment of PC9- and H1650-derived Tr-BrM cells with vehicle, osimertinib alone, a pan-RAR antagonist (AGN194310) alone, or AGN194310 in combination with osimertinib. At 25 days after tumor cell injection, after confirmation of metastatic signal, mice were administered long-term treatment with either (i) vehicle (Veh), (ii) AGN194310 (Pan-RARi; 0.5 mg/kg body weight/day), (iii) osimertinib (Osi; 5 mg/kg body weight/day), or (iv) AGN194310 (0.5 mg/kg body weight/day) plus osimertinib (5 mg/kg body weight/day) by oral gavage 5 days per week until the endpoint (7 weeks after tumor cell injection). B,Ex vivo photon flux of posttreatment brains from the experiment described in A was determined at endpoint by bioluminescence imaging. The photon-flux scale is indicated on the right. C, Violin plots depicting normalized photon flux of brains imaged ex vivo from the mice described in B. The normalized photon flux for brain tissue was calculated by dividing the photon flux from brain collected ex vivo by the total photon flux at day 0 (i.e., the day of injection) and multiplying that value by 100. Data are presented as mean values ± SEM. P values were determined by a two--tailed, unpaired Mann–Whitney test. For PC9-Tr-BrM mice: n = 8 for vehicle; n = 8 for osimertinib; n = 7 for pan-RARi; and n = 7 for osimertinib plus pan-RARi. For H1650-Tr-BrM mice: n = 12 for vehicle; n = 8 for osimertinib; n = 10 for pan-RARi; and n = 6 for osimertinib plus pan-RARi. ns, P value not significant. D, Representative images of CK7 IHC on posttreatment brain sections collected at endpoint from the experiment described in A. Scale bars, 200 μm for top (PC9-Tr-BrM) and 100 μm for bottom (H1650-Tr-BrM). E, Quantitative analysis of the percentage of CK7-immunostained brain sections covered by metastasis (Met) shown in D. Data are presented as mean values ± SEM. P values were determined by a two-tailed, unpaired Mann–Whitney test. For PC9-Tr-BrM mice: n = 11 for vehicle; n = 9 for osimertinib; n = 4 for pan-RARi; and n = 5 for osimertinib plus pan-RARi. For H1650-Tr-BrM mice: n = 6 for vehicle; n = 6 for osimertinib; n = 9 for pan-RARi; and n = 6 for osimertinib plus pan-RARi. F, Schematic representation of the experiment testing the effect of RAR gene knockdown on brain metastasis development. Mice were injected with either PC9- or H1650-derived Tr-BrM cells with one of two sets of shRNA-mediated stable dual knockdown of RARα and RARγ (sh-RARα + γ), or control shRNA (sh-Con), via intracardiac injection. Experiments involving shRNA set #1 are shown in G–J, whereas experiments involving shRNA set #2 are shown in Supplementary Fig. S6L–S6O. Mice were euthanized 7 weeks after tumor cell injection, and brains were collected for analysis. G,Ex vivo photon flux of posttreatment brains from the experiment described in F was determined at endpoint by bioluminescence imaging. The photon-flux scale is indicated on the right. H, Violin plots depicting normalized photon flux of brains imaged ex vivo from the mice represented in G. The normalized photon flux for brain tissue was calculated by dividing the photon flux from brain collected ex vivo by the total photon flux at day 0 (i.e., the day of injection) and multiplying that value by 100. Data are presented as mean values ± SEM. P values were determined by a two--tailed, unpaired Mann–Whitney test. For PC9-Tr-BrM: n = 7 for sh-Con; n = 5 for sh-RAR. For H1650-Tr-BrM: n = 8 for sh-Con; n = 6 for sh-RAR. I, Representative images of CK7 IHC on posttreatment brain sections collected at endpoint from the experiment described in F. Scale bars, 100 μm for all images. J, Quantitative analysis of the percentage of CK7-immunostained brain sections covered by metastasis shown in I. Data are presented as mean values ± SEM. P values were determined by a two-tailed, unpaired Mann–Whitney test. For PC9-Tr-BrM: n = 10 for sh-Con; n = 10 for sh-RAR. For H1650-Tr-BrM: n = 5 for sh-Con; n = 5 for sh-RAR.

Fig 4: ChIP assays highlight the human-specific binding site of RARα/RXRα complexes on the TBX1 promoter region. (A) Schema showing novel (human-specific) and putative RAR/RXR binding sites (i.e. RARE) on the human TBX1 promoter region. (B) The ChIP assays demonstrated that recruitment of RARα/RXRα complexes onto one of the novel RARE sites of the human TBX1 promoter (−1884–1873 bp) was augmented at Days 3 (D3) and 6 (D6) in CM differentiation of WT hESCs. *P < 0.01 and **P < 0.0001 vs. IgG (negative control). (C) The ChIP assays using STRA6-KO cells demonstrated that RARα/RXRα complexes were also recruited onto the identified RARE site of the human TBX1 promoter (−1884–1873 bp) at Days 3 and 6 in CM differentiation but to a much lesser degree compared with that in WT cells in (B). #P < 0.01 vs. IgG and vs. D6_RARα/RXRα in WT (B). ##P < 0.001 vs. IgG and vs. D3_RARα/RXRα in WT (B). (D) The ChIP assays using only the anti-RARα antibody demonstrated that recruitment of RARα onto the identified RARE site of the human TBX1 promoter (−1884–1873 bp) was augmented at Days 3 and 6 in CM differentiation of WT hESCs again. *P < 0.01 and **P < 0.0001 vs. IgG. (E) Western blotting analysis for expression of TBX1 protein with GAPDH protein (a loading control) in WT cells at Days 0, 3, 6, and 12 in hESC-CM differentiation. (F) Quantitative results in (E). *P < 0.01 vs. Day 0. (G) Comparison of expression of TBX1 protein between WT and STRA6-KO cells at Days 4 and 6 in hESC-CM differentiation with or without treatment with retinoic acid (RA, 0.5 μM) during Days 3–7. GAPDH was used as a loading control. (H) Quantitative results in (G). NT, normal treatment (without adding RA). *P < 0.01 between the NT-administered and RA-co-administered WT cells at Days 4 and 6, respectively. **P < 0.05 between the NT-administered and RA-co-administered STRA6-KO cells at Days 4 and 6, respectively. ***P < 0.01 between WT and STRA6-KO cells under the same treatment conditions at Days 4 and 6, respectively. (I) Comparison of expression of TBX1 protein between WT and TBX1 promoter-mutant (Mt) cells at Days 4 and 6 in hESC-CM differentiation with or without treatment with RA (0.5 μM) during Days 3–7. GAPDH was used as a loading control. (J) Quantitative results in (I). *P < 0.01 between the NT-administered and RA-co-administered WT cells at Days 4 and 6, respectively. #P = not significant between the NT-administered and RA-co-administered TBX1 promoter-mutant cells at Days 4 and 6, respectively. Differences between groups were examined with one-way ANOVA followed by Tukey–Kramer post hoc test.

Fig 5: CDK9 suppresses RARα protein expression and physically interacts with RARα.a Quantitative proteomic analysis and volcano plot of Hut78 cell proteins upon 23 (GT-02897) treatment, with P values derived from Wilcoxon rank-sum test. b Western blotting of indicated proteins in Hut78 under 23 (GT-02897) treatment. Western blot analysis of indicated proteins in HH and Hut78 (c) or 293T (d) cells after CDK9 depletion via lentiviruses. e A doxycycline (Dox)-inducible knockdown system was applied in HH and Hut78 cells, followed by Western blotting post Dox treatment. f Volcano plot revealing differentially expressed genes in Hut78 post CDK9 depletion, with P values derived by Wilcoxon rank-sum test. g Q-PCR assessment of CDK9 and RARA levels in Hut78 cells after CDK9 depletion. h Western blot analysis of proteins in Hut78 after CDK9 depletion, with or without MG132 treatment. i 293T cells were co-transfected with RARα and CDK9 constructs, followed by co-immunoprecipitation (Co-IP) and Western blot analysis. j GST-tagged CDK9 was incubated with HA-RARα from the WCL of 293T cells transfected with 3 × HA-tagged RARα, followed by GST pulldown and Western blot analysis. WCL: whole cell lysate. k Schematic diagram of full-length CDK9 protein (top). GST pulldown assay was performed using GST-tagged CDK9 and WCL of 293T cells transfected with 3 × HA-tagged RARα. l Lentiviral infection of Hut78 with shNC or shCDK9-1, followed by transfection of EV, 3 × Flag-tagged WT, T186A, and S347A CDK9, and Western blotting of indicated proteins. m Western blotting of indicated proteins in Hut78 post CDK9 depletion or Flavopiridol treatment. n Q-PCR analysis of RARA in Hut78 cells treated with Flavopiridol. o Schematic diagram of full-length RARα protein (top). Co-transfection of 3×Flag-tagged CDK9 with full-length or mutant RARα constructs in 293T, followed by Co-IP and immunoblotting (bottom). Results in (g, n) represent biologically independent experiments of n = 3, with P values calculated by two-tailed Student’s t-test. Data are presented as mean ± SEM, with source data provided as a Source Data file. b–e, h–m, o n = 3, independent experiments, a representative example is shown. The samples derive from the same experiment each but different gels for CDK9, β-actin and another for RARα (b, d, e, h), for RARα, β-actin and another for CDK9 (c), for HA-RARα, β-actin and another for Flag-CDK9 (i), for RARα, β-actin and another for Flag-CDK9 (l) were processed in parallel. Band intensities in (b–e, h, l, m) were analyzed and compared using Image J. Relative densitometric values are provided below the blot images.