Fig 2: 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 3: 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 4: Targetability of mutant RARα.a Transcriptional activities (relative to RA-treated WT) of partially activatable mutants S287L and N299H after treatment with alternative RAR agonists (1 µM). b Cell viabilities of partially activatable mutants S287L and N299H after treatment with alternative RAR agonists (1 µM). N = 3, error bar = SD. *p < 0.5, ****p < 0.0001. c Transcriptional activities (relative to RA-treated WT) of non-activatable mutants F286del and L409R after treatment with alternative RAR agonists (1 µM). d Cell viabilities of non-activatable mutants F286del and L409R after treatment with alternative RAR agonists (1 µM). N = 3, error bar = SD.

Fig 5: RAR expression in macrophages.(A) Representative western blots of RARα (antibody clone E6Z6K), RARβ (antibody clone B-12), and RARγ (antibody clone G-1) in RAW, BMDMs and peritoneal (Per) macrophages. (B) Intensity of RARβ signal divided by relative ponceau stain intensity. One-way ANOVA with Tukey’s was conducted where * indicates p < 0.05. n = 4 independent experiments. Data are expressed as means ± SD.