ARTICLE https://doi.org/10.1038/s41467-021-24618-3 OPEN SMARCA4 deficient tumours are vulnerable to KDM6A/UTX and KDM6B/JMJD3 blockade Octavio A. Romero 1,11 ✉, Andrea Vilarrubi 1,11, Juan J. Alburquerque-Bejar 1, Antonio Gomez 2, Alvaro Andrades3,4, Deborah Trastulli 5, Eva Pros 1, Fernando Setien1, Sara Verdura5, Lourdes Farré 6, Juan F. Martín-Tejera6, Paula Llabata 1, Ana Oaknin 7, Maria Saigi 1,8, Josep M. Piulats8, Xavier Matias-Guiu9, Pedro P. Medina 3,4, August Vidal 9,10, Alberto Villanueva 6,10 & Montse Sanchez-Cespedes 1 ✉ 1234567890():,; Despite the genetic inactivation of SMARCA4, a core component of the SWI/SNF-complex commonly found in cancer, there are no therapies that effectively target SMARCA4-deficient tumours. Here, we show that, unlike the cells with activated MYC oncogene, cells with SMARCA4 inactivation are refractory to the histone deacetylase inhibitor, SAHA, leading to the aberrant accumulation of H3K27me3. SMARCA4-mutant cells also show an impaired transactivation and significantly reduced levels of the histone demethylases KDM6A/UTX and KDM6B/JMJD3, and a strong dependency on these histone demethylases, so that its inhibition compromises cell viability. Administering the KDM6 inhibitor GSK-J4 to mice orthotopically implanted with SMARCA4-mutant lung cancer cells or primary small cell carcinoma of the ovary, hypercalcaemic type (SCCOHT), had strong anti-tumour effects. In this work we highlight the vulnerability of KDM6 inhibitors as a characteristic that could be exploited for treating SMARCA4-mutant cancer patients. 1 Cancer Genetics Group, Josep Carreras Leukaemia Research Institute (IJC), Badalona, Barcelona, Spain. 2 Rheumatology Research Group, Vall d’Hebron Research Institute, Barcelona, Spain. 3 Department of Biochemistry and Molecular Biology I. Faculty of Sciences, University of Granada, Granada, Spain. 4 GENYO. Centre for Genomics and Oncological Research: Pfizer, University of Granada, Andalusian Regional Government, Granada, Spain. 5 Genes and Cancer Group, Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute-IDIBELL Barcelona, Barcelona, Spain. 6 Chemoresistance and Predictive Factors Group, Program Against Cancer Therapeutic Resistance (ProCURE), Catalan Institute of Oncology (ICO), Oncobell Program, Bellvitge Biomedical Research Institute (IDIBELL), L’Hospitalet del Llobregat, Barcelona, Spain. 7 Department of Medical Oncology, Vall d’Hebrón Hospital, Barcelona, Spain. 8 Department of Medical Oncology, Catalan Institute of Oncology (ICO), Barcelona, Spain. 9 Department of Pathology, University Hospital of Bellvitge, IDIBELL, CIBERONC, L’Hospitalet del Llobregat, Barcelona, Spain. 10 Xenopat S.L., Parc Cientific de Barcelona (PCB), Barcelona, Spain. 11 These authors contributed equally: Octavio A. Romero, Andrea Vilarrubi. ✉email: oromero@carrerasresearch.org; mscespedes@carrerasresearch.org NATURE COMMUNICATIONS | (2021)12:4319 | https://doi.org/10.1038/s41467-021-24618-3 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24618-3 C hromatin remodelling is one of the epigenetic processes that of MYC (hereafter referred to as MYCamp) with respect to those is commonly disturbed in cancer, mainly through alterations with genetic inactivation of SMARCA4 (hereafter, SMARCA4- in the mammalian switch/sucrose non-fermentable (SWI/ def). The administration of SAHA was more effective at reducing SNF) complex. This complex modifies the structure of the chro- the growth of MYCamp cells than of SMARCA4def cells (mean of matin by the ATP-dependent disruption of DNA–histone interac- half-maximum effective concentrations (EC50) of 0.5 and 1.4 µM, tions at the nucleosomes, thereby activating or repressing gene for each group, respectively) (Fig. 1a, b, Supplementary Fig. 1a). expression. The various functions and components of the SWI/SNF Flow cytometric analysis showed that, in the MYCamp cells, complex have been thoroughly reviewed elsewhere1,2. SAHA blocked cell cycle progression at the G0/G1 or SubG1 Alterations at genes encoding different components of the SWI/ phases and increased apoptosis, whereas no changes were SNF complex are present in a variety of tumour types and are thus observed in SMARCA4def cells (Supplementary Fig. 1b–d; Sup- an important feature of cancer development3. The SMARCA4 (also plementary Fig. 2). These effects were not influenced by lung known as BRG1) gene codes a core catalytic component of the SWI/ cancer histopathological subtypes, since they occurred in NSCLC SNF complex that features a bromodomain and helicase/ATPase and SCLC types. The selective sensitivity to SAHA of MYCamp activity1,2. Our own previous work produced the first evidence that cells was validated using publicly available datasets including SMARCA4 is genetically inactivated in cancer and that SMARCA4 more than 750 cancer cell lines of different origin and genetic deficiency prevents the response to pro-differentiation stimuli in background (Fig. 1c). cancer cells4–6. In lung cancer, SMARCA4 inactivation affects about SAHA increases global histone acetylation, favouring an open one-third of non-small cell lung cancers (NSCLCs) and pre- chromatin structure and promoting transcriptional activation21. ferentially occurs against a background of wild type MYC (either C, Transcriptionally active chromatin domains are characterised by a L or N) or of members of the MYC-axis, such as MAX or MGA4–7. distinct array of histone marks, e.g., H3K27ac, H3K4me1 and This hints at the existence of an important network that connects H3K4me3, whereas H3K27me3 and H2AK119ub are often found at SWI/SNF and MAX/MYC functions. Mutations of SMARCA4 also silent gene loci22,23. We first tested the effects of SAHA in global occur in other types of cancer, notably in the rare and very H3K27ac and H3K27me3, two different marks on the same residue, aggressive small cell carcinoma of the ovary, hypercalcaemic type but with opposite functions22,23. As expected, SAHA triggered an (SCCOHT)8, in which SMARCA4 inactivation has been reported in increase in global H3K27ac in all the cells, while we observed an almost 100% of cases9–11. aberrant accumulation of H3K27me3 in the SMARCA4def cells, The progress made towards understanding the role of chromatin rather than the expected decrease (Fig. 1d). To determine whether remodelling in cancer development highlights the great potential of this was due to a defective SMARCA4, we used H1299 cells, which new epigenetic-based therapeutic strategies. With particular refer- lack SMARCA4 expression owing to an intragenic homozygous ence to SMARCA4, some previous studies have sought the vul- deletion4, and restored wild type SMARCA4 (H1299-wtSMARCA4), nerabilities of SMARCA4-deficient tumours with a view to using a doxycycline-inducible system, as previously reported6. As a exploiting them for cancer treatment. SMARCA4 and SMARCA2 control, we expressed a mutant form that lacked the ATPase domain are mutually exclusive catalytic subunits of the SWI/SNF complex, (p.Glu668_Gln758del)4 (hereafter referred to as H1299-mutS- and the inhibition of SMARCA2 activity appears to be synthetic MARCA4). Administration of SAHA did not affect global lethal in cancer cells carrying SMARCA4-inactivating mutations, an H3K27me3 in the H1299-wtSMARCA4 cells, whereas the H1299- effect that could be explained by paralogue insufficiency12,13. Fur- mutSMARCA4 cells underwent an increase in H3K27me3 con- ther, SWI/SNF-mutant cancer cells with a wild type KRAS back- comitantly with a decrease in global H3K27ac (Fig. 1e). A dominant ground depend on the non-catalytic action of the histone negative function of an overexpressed mutant SMARCA4 protein methyltransferase, EZH214. However, we currently know of no may underlie this effect in the H1299-mutSMARCA4 cells. Thus, the small compounds that are capable of suppressing the ATPase or absence of a functional SMARCA4 induces defects in the dynamics non-catalytic functions of SMARCA2 and EZH2, respectively, so of the H3K27me3 mark, following administration of SAHA. these molecules are not yet suitable for use in therapeutic inter- The net levels of H3K27me3 are dictated by the coordinated ventions. More recently, it has been proposed that cancer cells with action of histone methyltransferases (EZH2) and demethylases an inactive SMARCA4 may be susceptible to CDK4/6 inhibitors15. (KDM6A and KDM6B)23. The administration of SAHA did not On the other hand, components of the SWI/SNF complex bind alter the levels of EZH2 in most SMARCA4def cells (except in the to various nuclear receptors (e.g., oestrogen, androgen, glucocorti- H23 cells which showed an increase), indicating that over- coid and retinoid receptors), thereby adapting the gene expression activation of the methyltransferase activity of EZH2 is unlikely to programmes to the demands of the cell environment16–19. We have account for the defects in H3K27me3 triggered by SAHA in these reported that SMARCA4 is required to promote cell growth inhi- cells (Fig. 1f; Supplementary Fig. 3a). In contrast, treatment with bition triggered by corticoids and retinoids in cancer cells6 and that SAHA alone reduced EZH2 levels in the MYCamp cells, such effects are enhanced by combination with the pan- histone consistent with the fact that SAHA triggers inhibition of cell deacetylase (HDAC) inhibitor suberanilohydroxamic acid cycle progression and increases apoptosis in cancer cells with this (SAHA)20. We observed that MYC-amplified but not SMARCA4- genetic context. These effects were enhanced by the addition of mutant cancer cells were sensitive to these treatments. GSK126, an inhibitor of the enzymatic activity of EZH2. Previous Here, we show that cancer cells that lack SMARCA4 have a studies have shown that GSK126, alone or combined with HDAC defective regulation of H3K27ac/H3K27me3 and exhibit low levels inhibitors, suppress the growth of the small cell carcinoma of the of KDM6s, indicative of a deficient activity of these demethylases. ovary hypercalcaemic type (SCCOHT)24,25. Here, the adminis- This forms not only the basis of the refractoriness to SAHA, but tration of GSK126, alone or in combination with SAHA, did not also sensitises the cancer cells to inhibition of the demethylase reduce the proliferation or viability of the SMARCA4def lung activity of KDM6s, heavily compromising their viability. cancer cells (Supplementary Fig. 3b–d), implying that EZH2 activity does not, by itself, cause the refractoriness to cell growth inhibition by SAHA in SMARCA4def lung cancer cells. Results Refractoriness to growth inhibition and increase H3K27me3 by SAHA in SMARCA4def cells. We studied the differential Regulation of KDM6 expression by SMARCA4. The histone response to SAHA in lung cancer cells with oncogenic activation demethylases KDM6A (also known as UTX) and KDM6B (also 2 NATURE COMMUNICATIONS | (2021)12:4319 | https://doi.org/10.1038/s41467-021-24618-3 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24618-3 ARTICLE a b d kDa MYCamp MYCamp SMARCA4def 0 0.5 1 0 0.5 1 SAHA (μM) 17- H3K27ac DMS273 DMS114 3 *** 17- (12) H3K27me3 SAHA EC50 [μM] (16) 45- ACTIN 2 DMS273 H2170 SMARCA4def SCLC H446 H841 17- H3K27ac 1 17- H3K27me3 45- ACTIN H460 H23 0 H1299 H23 SMARCA4def MYCamp H3K27ac 17- (n=6) (n=8) 17- H3K27me3 H157 45- ACTIN H1975 H157 DMS114 NSCLC c 1.0 SMARCA4 CREBBP Log EC50 Vorinostat [μM] H2170 H1299 STK11 0.5 EGFR KRAS RB1 0.0 Vehicle SAHA [1μM] Vehicle SAHA [1μM] MYC -0.5 www.cancerrxgene.org MYCN GDSC2 PANCANCER cell lines e H1299- H1299- f -1.0 wtSMARCA4 mutSMARCA4 24 48 (hrs) SMARCA4def MYCamp 24 48 - - + + - - + + SAHA - - + - - + - - + - - + - - + - - + - - + GSK126 [1μM] kDa - + + + - + + + Dox kDa - + + - + + - + + - + + - + + - + + - + + SAHA [1μM] 17 H3K27ac 185 SMARCA4 H3K27ac 17 H3K27me3 17 EZH2 17 H3K27me3 98 45 ACTIN 45 ACTIN H157 H23 DMS114 H841 H460 H446 DMS273 Fig. 1 SAHA reduces growth and H3K27me3 levels in MYCamp, but not in SMARCA4def cancer cells. a Clonogenic assays of the indicated lung cancer cells, untreated (vehicle) or treated with SAHA. NSCLC, non-small cell lung cancer; SCLC small cell lung cancer. b Distribution and mean of EC50 values for treatment with SAHA in MYCamp (0.5 µM) and SMARCA4def cells (1.4 µM) (from MTT assays for cell viability; Supplementary Fig. 1). Bars show mean ± SD; two-sided unpaired Student’s t-test. Values from two independent experiments per cell line are represented. In box-whisker plots, the horizontal band inside box indicates the median, the bottom and top edges of the box 25th–75th percentiles and the whiskers indicate the min to max. ***P = 0.0002. c Plot with the comparative half maximal effective concentration (EC50) values for treatment with SAHA (vorinostat) in a panel of 758 cancer cell lines (from www.cancerrxgene.org database), according to the presence of selected gene alterations, including amplification of MYC oncogenes and inactivation of SMARCA4. d–f Representative western blots depicting global levels of the indicated proteins and histone marks (H3K27ac and H3K27me3) in the indicated cells following treatment with SAHA (d), in the H1299-wtSMARCA4 and H1299-mutSMARCA4 cell models after induction of SMARCA4 (dox, doxycycline, 1 µg/mL; 72 h), with or without SAHA treatment at 24 and 48 h (e), or in the indicated cells following treatments with SAHA and SAHA plus GSK126 (f). ACTIN, protein-loading control. A Source Data file is available for this figure. known as JMJD3) have H3K27 as a substrate and play a central MYCamp cells, the depletion of SMARCA4 reduced the levels of role in the development of some types of tumours23. Searching both KDM6s (Supplementary Fig. 4e). Finally, the analysis of the the Cancer Cell Line Encyclopedia (CCLE) we found significantly changes in gene expression from publicly available databases lower levels of expression of several histone demethylases (Sanger CRISPR) support the down-regulation of several KDMs, (KDMs), including KDM6A and KDM6B, in SMARCA4def specially KDM6A and KDM6B, following the knockout of compared with MYCamp cells or with LC cells that are wild type SMARCA4 using CRISPR/CAS9 in a panel of human cancer for SMARCA4 and MYC (Fig. 2a and Supplementary Fig. 4a). cell lines (https://depmap.org/portal/depmap/) (Supplementary The basal levels of EZH2 were similar in the different groups Fig. 4f). Collectively, these observations indicate that a functional (Fig. 2b; Supplementary Fig. 4b). We validated our observations SMARCA4 is required to activate KDM6 expression. in a panel of LC cell lines at the mRNA and protein levels in To study the genome-wide effects of wild type and mutant which, with few exceptions, SMARCA4def cells carry lower levels SMARCA4 and of SAHA on the dynamics of H3K27 modifica- of KDM6A and KDM6B, as compared to the MYCamp cells (Fig. tion, we performed chromatin immunoprecipitation followed by 2b, c; Supplementary Fig. 4b). The same observations were made sequencing (ChIP-seq) of SMARCA4, EZH2, H3K27ac and in lung primary tumours (Supplementary Fig. 4c). Further, the H3K27me3, in the H1299 cell model. No peaks were observed ectopic expression of the SMARCA4 wild type (H1299- for SMARCA4 before adding doxycycline, which is consistent wtSMARCA4 cells), triggered an upregulation of KDM6A and with the absence of SMARCA4 in these cells (Fig. 2e; Supple- KDM6B, albeit subtle. An opposite effect was observed for the mentary Fig. 5a). The global occupancy of wild type and mutant mutant (H1299-mutSMARCA4 cells). The levels of EZH2 were SMARCA4 was similar, indicating that ATPase activity does not unaffected (Fig. 2d; Supplementary Fig. 4d). Conversely, in the influence recruitment to the chromatin (Fig. 2e–g; Supplementary NATURE COMMUNICATIONS | (2021)12:4319 | https://doi.org/10.1038/s41467-021-24618-3 | www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24618-3 a 8 KDM6B KDM4B KDM6A b c KDM6B 8 4 MYCamp SMARCA4def 12 * 6 -DMS273 -DMS114 6 **** -H1299 **** 2 8 -H446 -H841 -A427 4 **** 4 -H69 -H82 -H23 Relative expression levels kDa 4 2 2 0 185- -SMARCA4 0 0 0 200- -KDM6B Log RPKMs -2 -2 -2 155- -KDM6A KDM6A 12 4 KDM5A KDM2B 6 KDM1A * 4 98- **** -EZH2 *** 8 4 52- -TUBULIN 2 2 **** 4 17- -H3K27ac 2 0 17- -H3K27me3 0 0 0 45- SMARCA4def MYCamp -ACTIN (n=5) (n=5) 2 -2 2 SMARCA4def (n=31) MYCamp (n=37) Others (n=110) e wtSMARCA4 mutSMARCA4 d SMARCA4 SMARCA4 6 KDM6A 2.5 * KDM6B Relative expression * dox 2.0 dox + dox+/SAHA 4 1.5 1.0 2 n.s n.s EZH2 EZH2 0.5 Read count frequency 0 0.0 - + - + - + - + dox wtSMARCA4 mutSMARCA4 wtSMARCA4 mutSMARCA4 f H3K27Ac H3K27Ac mutSMARCA4 wtSMARCA4 460 28 103 386 13280 (dox+) 3290 11447 2117 14307 H3K27me3 H3K27me3 dox (+) 1907 10136 2590 5035 7008 1871 2160 6719 6007 SMARCA4 H3K27ac EZH2 g wtSMARCA4 mutSMARCA4 wtSMARCA4 mutSMARCA4 [0-25] [0-60] dox- dox+ dox+/SAHA [0-15] [0-7] dox- SMARCA4 dox+ EZH2 dox+/SAHA [0-300] [0-300] H3K27ac dox- dox+ H3K27me3 [0-5] dox+/SAHA [0-5] dox- dox+ dox+/SAHA KDM6B KDM6B KDM6A KDM6A Fig. 5a, b). The H3K27ac deposition at promoters was not overexpressed mutant protein has a dominant negative effect. affected following restitution of SMARCA4 activity (Fig. 2f). H3K27ac marks were present in at least 80% of the promoters However, in the SMARCA4 mutant cells, the H3K27ac peaks bound by SMARCA4 and some also showed EZH2 occupancy were sharper and it was evident a reduction of H3K27ac (Fig. 2f; Supplementary Fig. 6a). The increase in EZH2 binding downstream of the peaks at promoters, in the gene body regions. following expression of the SMARCA4 mutant is consistent with This was in parallel with a strong increase in EZH2 binding to the previous findings that ectopic expression of an SMARCA4- DNA in SMARCA4 mutant-expressing cells (Fig. 2e, g; inactive protein allows the occupancy of PRC1 and PRC2 in CpG Supplementary Fig. 5c). The latter observation suggests that the island promoters throughout the genome26. 4 NATURE COMMUNICATIONS | (2021)12:4319 | https://doi.org/10.1038/s41467-021-24618-3 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24618-3 ARTICLE Fig. 2 SMARCA4 regulates the levels of the KDM6s. a Violin plots comparing levels of the indicated KDMs in SMARCA4def (n = 31), MYCamp (n = 37) and other (wild type for SMARCA4 and for MYC) (n = 110) lung cancer cell lines (from the Cancer Cell Line Encyclopedia—CCLE at cBioportal). RPKMs (reads per million). Bars show mean ± SD. Two-sided unpaired Student’s t-test: ***P < 0.001; ****P < 0.0001. b Western blot depicting endogenous levels of the indicated proteins in lung cancer cell lines. TUBULIN and ACTIN, protein-loading control. c and d Real-time quantitative PCR of KDM6A and KDM6B (relative to ACTB) for comparing mRNA levels, for each individual cell line, among the indicated groups of lung cancer cell lines *P = 0.032 (KDM6B) and *P = 0.016 (KDM6A) (c), or in the H1299-wtSMARCA4 and H1299-mutSMARCA4 cell models (dox, doxycycline, 1 µg/mL; 72 h). *P = 0.029 (KDM6A) and P = 0.032 (KDM6B) (d). In box-whisker plots, the horizontal band inside box indicates the median, the bottom and top edges of the box 25th–75th percentiles and the whiskers indicate the min to max. Four and five biological replicates were included for the determination of the KDM6A and KDM6B levels, respectively, in the H1299 cell model. Bars show mean ± SD. Two-sided unpaired Student’s t-test, e Read count frequency of heatmaps, at ±2 kb regions centred on the transcriptional start site (TSS), of the indicated proteins and conditions in the H1299 cell model (dox, doxycycline, 1 µg/mL and SAHA 1 µM, for 72 h). f Venn diagrams representing overlap of SMARCA4, H3K27ac and EZH2 peaks in the indicated cells and under the stipulated conditions. g Representative snapshots from IGV of ChIP-seq profiles at selected target loci performed in H1299 cell models. A Source Data file is available for this figure. The administration of SAHA reduced the number of dependent manner (Fig. 3c, d) and prevented the SAHA-triggered promoters recruiting SMARCA4 wild type by half, without global decrease in H3K27me3 in both KDM6s-depleted cells producing major changes in global H3K27ac deposition (Supple- (Fig. 3b). mentary Fig. 5a, b). SMARCA4 does not interact directly with the These findings suggest that a deficiency in KDM6B account for DNA, but rather recognises and binds acetylated lysines within the resistance of the SMARCA4def cells to growth inhibition histone H3 and H4 tails27. Given this, the severe reduction of by SAHA. global H3K27ac deposition observed in SMARCA4 mutant- overexpressing cells following SAHA treatment, also shown by Inhibition of KDM6A/B is toxic in SMARCA4def cancer cells. western blot (Fig. 1e), could be the reason for the strong We hypothesised that the low levels and impaired regulation of reduction in the global intensity of the SMARCA4 peaks (Fig. 2e). KDM6s expression and the defects in H3K27 modification may Conversely, the treatment with SAHA prompted the recruitment render SMARCA4def cells particularly susceptible to KDM6s of EZH2 to the DNA and the increase in H3K27me3 deposition inhibition. We tested the effects of GSK-J4 on the growth of our in promoter regions, in mutant and wild type SMARCA4- panel of cancer cells and found that the drug was more toxic in expressing cells. However, the effect in the latter cell type was less the SMARCA4def cells, with a five-fold lower EC50 than in the dramatic, and we attribute this to the slow rate of H3K27me3 MYCamp cells or in the lung cancer cells that are wild type for removal (Fig. 2e; Supplementary Fig. 6a). The observation that both SMARCA4 and MYC (Fig. 4a, b; Supplementary Fig. 8a). SAHA increases EZH2 recruitment and H3K27me3 deposition is The greater sensitivity of the SMARCA4def cancer cells for GSK- somewhat counterintuitive but may be a compensatory effect to J4 is supported by studies available from databases (Supplemen- avoid detrimental high levels of histone acetylation. tary Fig. 8b). For the next stage of the study, we chose to use SMARCA4 was bound to different KDMs, including KDM2B, GSK-J4 at a concentration of 1 µM (Fig. 4a). We depleted KDM4B, KDM6A and KDM6B, among others, in association with SMARCA4 in three MYCamp cells and observed a decrease in the H3K27ac. However, there was also a concomitant increase in EC50 for GSK-J4, which is further evidence that GSK-J4 is more EZH2 occupancy in the SMARCA4 mutant-expressing cells toxic in cancer cells with a non-functional SMARCA4 (Fig. 4c). (Fig. 2g; Supplementary Fig. 6b). These results support the idea We also tested the effects of rescuing SMARCA4 on the response that SMARCA4 regulates the expression of various KDMs, to GSK-J4 using the H1299 cell model. Overexpression of the including KDM6A and KDM6B, through direct promoter mutant SMARCA4 increased sensitivity to GSK-J4 relative to the occupancy. The increase in EZH2 in the promoter of these genes restitution of wild type SMARCA4 (EC50, 0.11 µM versus 0.2 µM) is consistent with a lack of transcriptional activation and even (Supplementary Fig. 8c, d). The toxicity was even greater in the some transcriptional repression of these KDMs in the H1299- H1299-mutSMARCA4 than in the parental H1299 cells, sup- mutSMARCA4 cells (Fig. 2d; Supplementary Fig. 4d). porting the existence of a dominant negative effect of over- expressing a SMARCA4-mutant protein. GSK-J4 is a potent inhibitor of KDM6s but can also suppress KDM6B depletion mimics the response of SMARCA4def cells the activity of other KDMs, so we investigated how the low levels to SAHA. We wondered to what extent the lack of KDM6A or of KDM6s suppress cell viability by depleting KDM6A- and KDM6B regulation is involved in the greater H3K27me3 and KDM6B. Downregulation of KDM6A or KDM6B inhibited the refractoriness to SAHA in the SMARCA4def cells, and whether growth of the SMARCA4-def cells without affecting the MYCamp their relative contributions differ. First, we found that the mRNA cells (Fig. 4d). In a large scale public CRISPR-based genetic screen levels of the KDM6B were inversely correlated with the EC50 to the effect of KDM6s knock down in cell survival of SMARCA4def SAHA (Supplementary Fig. 7a). Next, using shRNAs, we down- cancer cell was also observed (Supplementary Fig. 8e). Moreover, regulated KDM6A and KDM6B expression in different MYCamp the lower levels of KDM6A, and to a lesser extent of KDM6B, in cells (Fig, 3a; Supplementary Fig. 7b), and noted that, mimicking the MYCamp cells, sensitised the cells to the treatment with GSK- the behaviour of the SMARCA4def cells, the reduction in KDM6B J4 (Fig. 4e, f). levels, but not of KDM6A, suppressed the ability to inhibit cell growth (Supplementary Fig. 7c) by SAHA. The depletion of KDM6B, but not KDM6A, also prevented SAHA from decreasing Overexpression of KDM6A and KDM6B in SMARCA4def cells overall levels of H3K27me3 deposition (Fig. 3b), hinting at a reverts sensitivity to KDM6A/B inhibition. Next, we aimed to more widespread role for KDM6B in the global removal of determine whether the increase in the levels of KDM6A and H3K27me3. The administration of the small molecule compound KDM6B will revert the sensitivity of the cells to KDM6A/B GSK-J428, a very specific inhibitor of KDM6A/KDM6B, has inhibition. To do that, we have stably overexpressed the two similar effects, reverting the sensitivity to SAHA in a dose- different KDM6s in a panel of SMARCA4def cells (H1299, H841, NATURE COMMUNICATIONS | (2021)12:4319 | https://doi.org/10.1038/s41467-021-24618-3 | www.nature.com/naturecommunications 5 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24618-3 a b H82 H460 c (MYCamp-cells) kDa -- - + + - - + + GSK-J4 [1μM] shKDM6A shKDM6B + - + - + - + SAHA [1μM] Vehicle SAHA [1μM] SAHA [1μM]/ 17 H3K27me3 GSKJ4 [1μM] kDa shNT #63 #64 #676 #976 H460 52 TUBULIN 155 KDM6A sh#NT 200 KDM6B 17 H3K27me3 DMS273 52 52 TUBULIN TUBULIN sh#63 H460 17 H3K27me3 52 TUBULIN H82 sh#64 17 H3K27me3 52 TUBULIN H2170 sh#676 17 H3K27me3 52 TUBULIN sh#976 d Vehicle 0.5 μM GSK-J4 1 μM GSK-J4 2 μM GSK-J4 Cell [GSK-J4] CI value Line DMS273 H460 H82 100 DMS273 0.5 μM 1.4 1 μM 3.28 % viability H460 0.5 μM 2.07 50 EC50=0.27 μM EC50=0.72 μM EC50=0.68 1 μM 2.96 EC50=0.27 μM EC50=1.25 μM EC50=1.43 EC50=0.68 μM EC50=1.64 μM EC50=2.87 H82 0.5 μM 1.37 EC50=1.61 μM EC50=3.27 μM EC50=4.55 0 -3 -2 -1 0 1 -3 -2 -1 0 1 -3 -2 -1 0 1 1 μM 2.75 Log SAHA concentration [μM] Fig. 3 KDM6B depletion mimics the response of SMARCA4-deficient cells to SAHA. a Western blot depicting levels of KDM6s in H460 cells infected with the non-target short hairpin (shNT) and with different shKDM6A (#63 and #64) and shKDM6B (#676, #976). b Western blot depicting levels of H3K27me3 and cells infected with the shNT, shKDM6A (#63, #64) or shKDM6B (#676, #976) treated with GSK-J4 and/or SAHA for 72 h. c Representative clonogenic assays for the indicated cells and treatments. d Viability of indicated cell lines, measured using MTT assays, 5 days after treatment with increasing concentrations of SAHA and co-treated or not with GSK-J4 at different concentrations. Lines, number of viable cells relative to the untreated cells. Data are presented as mean ± SD from three replicate cell cultures in two experiments. EC50, half-maximal effective concentration. On the right, table presenting the combination index (CI) at the indicated concentrations of GSK-J4 (average CI from two independent experiments). CI < 1, CI = 1 and CI > 1 indicate synergism, additive effect, and antagonism, respectively. A Source Data file is available for this figure. DMS114 and A427) (Fig. 5a). The overexpression of both and the tumours we minced into small fragments and orthoto- KDM6A and KDM6B increased the EC50 to GSK-J4 in all the cell pically implanted into the lungs of another cohort of mice20,29, to lines tested. This effect was stronger after overexpressing the generate the orthotopic tumours. We randomly assigned the KDM6A (Fig. 5b, c). animals, implanted with each of the tumours, to treatment or Furthermore, we simultaneously overexpressed SMARCA4 and vehicle groups of mice. Treatment with GSK-J4 strongly increased KDM6A or KDM6B in the H1299 cells (Fig. 5d) and measured the overall and median survival of the animals implanted with the the effects in cell viability upon treatment with GSK-J4. Wild type SMARCA4def tumours (DMS114X and H841X) relative to their SMARCA4 increased the resistance to GSK-J4 mediated by matched vehicle group, whereas we found no differences between KDM6A and KDM6B overexpression (Fig. 5e, f). vehicle and treated groups in the animals implanted with the Together, these findings imply that the lack of SMARCA4 MYCamp tumours (DMS273X) (Fig. 6a). Remarkably, five of the confers vulnerability to KDM6s inhibition on cancer cells, and mice implanted with the H841X and two of those implanted with that the intrinsically low levels of KDM6s, caused by the defective the DMS114X and treated with GSK-J4 were alive at the end of function of SMARCA4, underpin these effects. the experiment, but had to be sacrificed despite not having respiratory difficulties or other symptoms associated with tumour Anti-tumour effects of GSK-J4 in SMARCA4def lung cancer progression. Our histopathological examination of tumour mas- orthotopic mouse models. We investigated the ability of the ses revealed the existence of large areas of necrosis in the tumours GSK-J4 compound to suppress tumour growth in vivo. To this from GSK-J4-treated mice in comparison with the tumours from end, we first grew two of the SMARCA4def (DMS114 and H841) the vehicle-treated mice (Fig. 6b, c; Supplementary Fig. 9). We and one MYCamp (DMS273) cell lines subcutaneously into the also used immunohistochemistry to determine the changes in the back of the mice (n = 3 mice/cell line). Once the solid tumour levels of H3K27me3 in the tumour samples following treatment had entered the exponential growth phase, mice were euthanized, with GKS-J4. We noted a significant increase in the levels of 6 NATURE COMMUNICATIONS | (2021)12:4319 | https://doi.org/10.1038/s41467-021-24618-3 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24618-3 ARTICLE a **** b SMARCA4def-cells MYCamp-cells 3 n.s DMS114 H1299 H841 H460 H82 **** DMS273 EC50 GSK-J4 [uM] Vehicle (12) (8) 2 GSK-J4 [1μM] (14) 1 SMARCA4def (n=7) MYCamp (n=6) Others (n=4) 0 c 120 H82 H69 H460 % viability 80 shNT shSMARCA4#1 40 shSMARCA4#4 shNT (EC50 2.6 μM) shNT (EC50 0.7 μM) shNT (EC501.3 μM) #1 (EC50 0.7 μM) #1 (EC50 0.2 μM) #1 (EC50 0.9 μM) #2 (EC50 0.4 μM) #2 (EC50 0.3 μM) #2 (EC50 1 μM) 0 -3 -2 -1 0 1 -3 -2 -1 0 1 -3 -2 -1 0 1 Log GSK-J4 concentration [μM] d shKDM6A shKDM6B e shKDM6A NT shKDM6B shNT #63 shNT #676 shNT #63 #64 #676 #976 #64 #976 H1299 120 H82 SMARCA4def-cells % viability H82 80 H841 40 DMS114 shNT (EC50 1.7μM) shNT (EC50 1.7μM) #63 (EC50 0.8μM) #676 (EC50 0.9μM) #64 (EC50 0.2μM) #976 (EC50 0.4μM) 0 MYCamp-cells 120 H460 H460 H82 % viability 80 DMS273 40 shNT(EC50 1.4μM) shNT (EC50 1.2μM) #63 (EC50 0.6μM) #676 (EC50 0.97μM) #64 (EC50 0.2μM) #976 (EC50 0.8μM) 0 f * DMS273 DMS273 #63 120 3 **** #64 EC50 GSK-J4 [μM] #676 % viability 2 80 #976 H82 40 1 H460 shNT(EC50 1.5μM) shNT (EC50 2.4 μM) #63 (EC50 1.4 μM) #676 (EC50 2.2 μM) DMS273 #64 (EC50 0.8μM) #976 (EC50 1.5 μM) 0 0 shNT shKDM6A shKDM6B -3 -2 -1 0 1 -3 -2 -1 0 1 Log GSK-J4 concentration [μM] Fig. 4 SMARCA4def cells are vulnerable to KDM6s inhibition. a Distribution and mean of half-maximal effective concentration (EC50) values for GSK-J4 (MTT assays. Supplementary Fig. 6a) in the indicated groups of cells. Values, from each cell line and from two independent experiments are represented. Two-sided unpaired Student’s t-test. ****P < 0.0001. In box-whisker plots, the horizontal band inside box indicates the median, the bottom and top edges of the box 25th–75th percentiles and the whiskers indicate the min to max. b Representative clonogenic assays for the indicated cells and treatments. c Viability of the indicated cells infected with a short hairpin non-target (shNT) control or with two shRNAs targeting SMARCA4 (#1 and #4), measured using MTT assays, after treatment with increasing concentrations of GSK-J4 for 5 days. Lines, number of viable cells relative to untreated cells. Data are presented as mean and SD from three replicates and two experiments. d Representative clonogenic assays for the indicated cells infected with shNT, shKDM6A (#63, #64) or shKDM6B (#676, #976). e Viability of the indicated cell lines, measured using MTT assays, infected with a non-target (shNT) control or with two shRNAs targeting KDM6A or KDM6B after treatment with increasing concentrations of GSK-J4 for 5 days. Lines, number of viable cells relative to the untreated cells. Error bars, mean ± SD from triplicates. f Distribution and mean of the EC50 from two independent experiments per each of the three LC cell lines. Lines show mean ± SD. P-values were calculated using paired two-tailed Student’s t test. *P = 0.015; ****P < 0.0001; ns, not significant. A Source Data file is available for this figure. H3K27me3 in all the tumours treated with GSK-J4, suggesting a very aggressive and rare type of ovarian cancer that features that the compound had effectively reached the tumours (Fig. 6d). inactivation of SMARCA4 in almost all cases9–11. Here, we gen- erated patient-derived orthotopic xenografts (PDOXs) using the GSK-J4 reduces tumour growth in mice implanted with primary tumours of two SCCOHT patients (OVA250 and SMARCA4def SCCOHT. As previously mentioned, SCCOHT is OVA259), by orthotopically implanting the tumour in the mouse NATURE COMMUNICATIONS | (2021)12:4319 | https://doi.org/10.1038/s41467-021-24618-3 | www.nature.com/naturecommunications 7 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24618-3 a c A427 0.8 EC50 GSK-J4 [μM] kDa EV KDM6A EV KDM6A EV KDM6A EV KDM6A H841 155 KDM6A ** 0.6 DMS114 48 ACTIN EV KDM6B EV KDM6B EV KDM6B EV KDM6B 0.4 * 200 KDM6B 0.2 48 ACTIN H1299 H841 DMS114 A427 0.0 EV KDM6A KDM6B B b EV (EC50 0.21 μM) KDM6A (EC50 0.59 μM) EV (EC50 0.21 μM) KDM6B (EC50 0.44 μM) e EV (EC50 0.27 μM) SMARCA4(EC50 0.64 μM) % viability y 80 80 80 % viability 40 40 40 H1299 H841 H841 0 0 0 -3 -2 -1 0 1 -3 -2 -1 0 1 -3 -2 -1 0 1 EV (EC50 0.27 μM) KDM6A (EC50 1.22 μM) Log GSK-J4 concentration [μM] SMARCA4/KDM6A (EC50 1.52 μM) % viability 80 d f SMARCA4/KDM6A SMARCA4/KDM6B 2.0 40 EC50 GSK-J4 [μM] SMARCA4 **** H1299 1.5 *** 0 **** -3 -2 -1 0 1 EV (EC50 0.27 μM) kDa 1.0 *** KDM6B (EC50 0.81 μM) 185 ** SMARCA4 SMARCA4/KDM6B (EC50 1.23 μM) 200 KDM6B 0.5 % viability 155 80 KDM6A 0.0 SMARCA4 EV KDM6A KDM6B SMARCA4/KDM6A SMARCA4/KDM6B 52 TUBULIN 40 H1299 0 -3 -2 -1 0 1 Log GSK-J4 concentration [μM] Fig. 5 Overexpression of KDM6s reverts sensitivity to KDM6s inhibition. a Western blot depicting levels of ectopic KDM6s and KDM6B in the indicated SMARCA4def cells overexpressing the indicated proteins. EV Empty vector. b Example of viability of H841 cell line, measured using MTT assays, 5 days after treatment with increasing concentrations of GSK-J4. Lines, number of viable cells relative to the untreated cells. Data are presented as mean ± SD from three replicate cell cultures in two experiments. EC50, half-maximal effective concentration. c Distribution and mean of the EC50 from two independent experiments per each of the three LC cell lines. Lines show mean ± SD. P-values were calculated using paired two-tailed Student’s t test. **P = 0.0017; *P = 0.031. d Western blot depicting levels of KDM6s and SMARCA4 in the H1299 cells overexpressing the indicated proteins. e Viability of indicated cell lines, measured using MTT assays, 5 days after treatment with increasing concentrations of GSK-J4. Lines, number of viable cells relative to the untreated cells. Data are presented as mean ± SD from three replicate cell cultures in two experiments. f Distribution and mean of the EC50 from three independent experiments. Lines show mean ± SD. P-values were calculated using paired two-tailed Student’s t test. **P = 0.0016; ***P = 0.0002; ****P < 0.0001; A Source Data file is available for this figure. ovary30. We used the PDOXs, in their first pass, to derive primary capability, exclusively in the OVA250L and OVA259L cells, cancer cell cultures (OVA250L and OVA259L). We confirmed whereas SAHA did not affect cell growth (Fig. 7b, c). The levels of the presence of biallelic inactivating mutations at SMARCA4 and induced cleavage at poly-(ADP-ribose) polymerase 1 (PARP1) the lack of protein in the two patients’ tumour cells and PDOXs were increased in the SMARCA4def cells indicating apoptosis (Fig. 7a; Supplementary Fig. 10a). First, we tested the effects of the (Supplementary Fig. 10b). It was puzzling to observe that the SAHA and GSK-J4 compounds in the primary cultures and OVA250L and OVA259L cells had extremely low levels of global included, as a reference, two commercial epithelial ovarian car- H3K27ac, even after treatment with SAHA. Similar to what cinoma cell lines (OVCAR-3 and OVCAR-8), which are wild type happened in the lung cancer cells, the global basal levels of for SMARCA4 (https://cancer.sanger.ac.uk/cell_lines). Treatment H3K27me3 were increased after administration of SAHA with GSK-J4 strongly suppressed cell viability and clonogenic (Fig. 7d). 8 NATURE COMMUNICATIONS | (2021)12:4319 | https://doi.org/10.1038/s41467-021-24618-3 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24618-3 ARTICLE a MYCamp-cells SMARCA4def-cells SMARCA4def-cells 100 100 100 Vehicle Vehicle Vehicle Survival probability (%) GSK-J4(50mg/kg) GSK-J4(50mg/kg) GSK-J4(50mg/kg) 75 75 75 50 50 50 25 25 25 DMS273X DMS114X H841X 0 0 0 0 15 30 45 0 15 30 45 60 75 90 15 30 45 60 75 90 Days after treatment Days after treatment Days after treatment Group Median Group Median survival Group Median survival (days) (days) survival (days) Vehicle (n=4) 24 Vehicle (n=6) 20.5 Vehicle (n=6) 41.5 GSK-J4 (n=4) 24 GSK-J4 (n=12) 43 GSK-J4 (n=12) 62.5 P=n.s P<0.01 P<0.0001 b c 100 **** *** DMS273X DMS114X H841X 75 Necrosis (%) LU HE Vehicle 50 LU 25 n.s 0 VH GSK VH GSK VH GSK GSK-J4 HE DMS273X DMS114X H841X LU d Vehicle GSK-J4 LU DMS114X Vehicle H841X GSK-J4 DMS273X Vehicle GSK-J4 P-value Low 8 2 High 1 7 ** Next, we investigated the influence of GSK-J4 treatment on the reduction in the size and weight of the tumours from mice treated growth of the OVA250 tumour in vivo. We orthotopically with the GSK-J4 inhibitor in the OVA250X model (Fig. 7f). implanted primary tumours, either treatment-naïve or derived Although the differences did not reach statistical significance, from mice previously treated with cisplatin (CDDP) (see the histological examination revealed the presence of a few viable “Methods” section for further details), in the ovary of female nude tumour cells in the tumours from mice treated with GSK-J4. mice to generate the OVA250X and OVA250XR tumours, These tissues contained a large amount of fibrosis, instead of the respectively (Fig. 7e; Supplementary Fig. 10c–f). We observed a necrosis observed in the lung cancer orthotopic model, possibly NATURE COMMUNICATIONS | (2021)12:4319 | https://doi.org/10.1038/s41467-021-24618-3 | www.nature.com/naturecommunications 9 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24618-3 Fig. 6 GSK-J4 induces tumour regression of SMARCA4def lung tumours in vivo. a Kaplan–Meier curves showing overall survival for GSK-J4-treated compared with vehicle control groups of each indicated orthotopically implanted mice model. Panels below, number of mice (n) and mean survival times for each group of treatment and cell line. P-values from the two-sided log-rank (Mantel–Cox) test of the plots are included. **P < 0.01; ****P < 0.0001. n.s., not significant. b Representative sections of haematoxylin and eosin (H&E) staining of tumours from the indicated cells, from mice treated with either GSK-J4 or vehicle. Upper panels, tumour regions are marked within circles. Scale bars, 2.5 mm. The pink areas inside the tumours indicate necrosis. HE heart, LU lung. Bottom panels, representative sections, at higher magnification, of tumours from the indicated cells and treatments. Scale bars, 50 µm. c Quantification of necrotic areas. Mean ± SD are indicated for each group. The number of tumours, each from a different individual, were n = 4 in each VH and GSK-J4 treated groups for the DMS273X mice; n = 4 in the VH and n = 5 in the GSK-J4 groups, for the DMS114X mice and n = 4 in the VH and n = 6 in the GSK-J4 groups, for the H841X mice. Two-sided unpaired Student’s t-test. ***P = 0.0006; ****P < 0.0001. VH vehicle. d Representative immunostaining of H3K27me3 in tumours from the indicated cells and treatments. Scale bars, 25 µm. Below, distribution of H3K27me3 staining among tumours (three tumours per cell line and condition) from the DMS273X, H841X and DMS114X tumours treated with vehicle or GSK-J4. Low (intensity values 1 and 2); high (intensity values 3 and 4) (Supplementary Table 6). Two-sided Fisher’s Exact test. *P < 0.05. A Source Data file is available for this figure. because the OVA250X was derived from a primary tumour and refractoriness of these cells to the appropriate modification of not from cancer cell lines (Fig. 7g). Similar to what was observed gene expression upon different stimuli. in the lung cancer mouse models, the GSK-J4 treatment triggered Despite the high degree of sequence similarity in the catalytic an increase in H3K27me3 (Fig. 7h). Further, the levels of caspase domain of KDM6A and KDM6B, these two enzymes have also 3, indicative of apoptosis were increased in the SMARCAdef some specific roles32. KDM6A is mainly associated with the tumours in those mice treated with GSK-J4 (Fig. 7h). Next, we demethylation of H3K27me3 at the transcriptional start sites of generated the OVA250XR tumours to determine the benefits of the HOX genes upon differentiation stimuli, whereas KDM6B is GSK-J4 in tumours that have been pre-treated with CDDP, the involved in inflammation and other, general physiological standard treatment for SCCOHT. The morphology of the processes32. Furthermore, KDM6A, but not KDM6B, is respon- OVA250XR tumours was similar with that of the primary sible for Kabuki syndrome (KS), an infrequent, inherited disease tumour and of the OVA250X (Supplementary Fig. 10e). The that is characterised by neurological, endocrine and autoimmune OVA250XR showed greater refractoriness to CDDP than did disorders33. Here, we found that a deficiency in KDM6B is OVA250X (Supplementary Fig. 10f). The treatment with GSK-J4 responsible for the refractoriness to SAHA in SMARCA4def cells. significantly decreased tumour growth in the OVA250XR. Taken Further, a deficiency in KDM6B also accounts for the global together, these observations demonstrate that the KDM6s increase in H3K27me3 upon administration of SAHA, which is inhibitor could constitute a therapeutic option for SCCOHT consistent with a broader role for KMD6B in H3K27me3 patients even after they have become resistant to CDDP deposition than that of KDM6A34. Non-catalytic activities have (Supplementary Fig. 10g–i). been proposed for the KDM6s which may also account for some of these differences32. Discussion Considering its potential clinical applicability, the most rele- Here, we present evidence that cancer cells carrying oncogenic vant finding presented here is the great vulnerability of the MYC are susceptible to growth inhibition by treatment with the SMARCA4def cells to KDM6s inhibition, which was evident in HDAC inhibitor, SAHA. HDAC inhibitors, including SAHA, cell culture and in mouse models with orthotopic transplants of have come to be recognised as biologically active compounds of lung cancer cells and SCCOHTs. Their frequency and poor value for treating cancers, although their use is currently limited prognosis mean that the use of GSK-J4 or similar compounds can to some haematological malignancies31. Our current findings have a great impact on the treatment of SMARCA4-mutant indicate that the pre-selection of patients with tumours in which tumours. Likewise, SCCOHT is an aggressive carcinoma with any of the MYC family of genes have been genetically activated rhabdoid characteristics and, though infrequent overall, pre- will have better response rates to SAHA, which suggests that dominantly affects young women8–11. Our current results, which SAHA could be used to treat neuroblastomas and lung cancers, show that GSK-J4 strongly suppresses its growth in vivo, among other types of cancer, in which the MYC genes are emphasises the huge potential of KDM6s inhibitors in treating amplified. this disease, which otherwise has very limited treatment options Second, we found that lung or ovarian cancer cells with inac- and, consequently, a dismal prognosis. Currently, there is only tivated SMARCA4 not only were refractory to the growth sup- limited, preclinical information about the use of KDM6s inhibi- pression triggered by SAHA, but also aberrantly accumulated tors in cancer treatment. Anti-tumorigenic activities of GSK-J4 H3K27me3 following the administration of this inhibitor. The have been shown in some leukaemias and in gliomas with H3F3A levels of global H3K27ac were low in the SMARCA4def cells, a mutations, both of which are attributable to the inhibition of characteristic that was accentuated in the SCCOHT cells. We KDM6B35,36. In our case, the depletion of either KDM6A or ruled out a central role for EZH2 methyltransferase activity in the KDM6B affected the viability of SMARCA4def cells, suggesting refractoriness to SAHA in these cells, although the dependency that, in this context, the depletion of either of them cannot be on a non-catalytic role for EZH2 cannot be completely dis- compensated. A limiting amount of KDM6s, due the lack of counted, as it is known to affect the survival of the SWI/SNF- SMARCA4-mediated transactivation and regulation, seems to mutant cancer cells14. Additionally, we demonstrate that the underlie the toxicity to the KDM6 inhibitor, GSK-J4, in SMAR- transactivation of several lysine demethylases (KDMs), including CA4def cells. It has also been shown that the KDM6s enhance the KDM6A and KDM6B, is impaired in cells that lack SMARCA4, accessibility of the SWI/SNF complex to H3 and promote chro- leading to a downregulation of basal KDM6s. This, coupled with matin remodelling37,38 which may also contribute to the strong inability of these cells to modulate the levels of EZH2 expression dependency of SMARCA4def cells, which rely only in the in response to SAHA and, in keeping with other SMARCA2-dependent SWI/SNF complex, on these demethylases knowledge6,16–20, is evidence that defective chromatin remodel- for survival. Despite our current findings about the toxicity of the ling in SMARCA4def cells promotes a closed chromatin structure GSK-J4 compound in SMARCA4def cells, the effects could be and a transcriptionally rigid scenario that maintains the broader, and cancer cells in which other members of the SWI/ 10 NATURE COMMUNICATIONS | (2021)12:4319 | https://doi.org/10.1038/s41467-021-24618-3 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24618-3 ARTICLE a b SMARCA4def SMARCA4wt c OVA259L c.1521C>A, pTyr507* OVA250L OVA259L OVCAR-8 100 Vehicle % viability OVA250L c.2935C>T; pArg979* 50 SAHA [1μM] OVA259L EC50= 0,26 μM OVA250L EC50= 0,07 μM OVCAR-3 EC50= 1,7 μM GSKJ4 OVCAR-8 EC50= 1,4 μM [1μM] 0 OVA250L c.3412_3413delinsA; -3 -2 -1 0 1 pLeu1138Serfs*2 Log GSK-J4 concentration [μM] OVCAR-8 OVA250L OVA259L d - + - + - + - + - + - + -SAHA kDa - - + + - - + + - - + + -GSK-J4 e Primary cell Culture 17- -H3K27ac OVA250L 17- -H3K27me3 Vehicle (n=5) 52- -TUBULIN GSK-J4 (n=7) f -GSK-J4 -Vehicle OVA250X P=n.s Ovary P=n.s 450 4 Tumor volume (mm3) Vehicle (n=3) Tumor weight (gr) 3 300 2 SCCOHT CCDP (n=3) OVA250XR patient CDDP pretreated 150 1 GSK-J4 (n=5) 0 0 Orthotopic implants Orthotopic implants Vehicle GSK-J4 Vehicle GSK-J4 (Ovary) (Ovary) g I Vehicle h Vehicle GSK-J4 II H3K27me23 GSK-J4 I II **** Tumor fibrosis (%) 100 CASPASE 3 75 I II 50 25 0 Vehicle GSK-J4 SNF-complex or of related pathways are genetically inactivated, Genomic DNA and total RNA were extracted by standard protocols. One primary may also be vulnerable to this inhibitor. lung cancer cell line PCD11 was derived from malignant pleural effusions39. Two primary cancer cell lines cultures were derived from orthoxenografts/PDOXs generated in nude mice from two primary tumours of two SCCOHT patients Methods (OVA250L and OVA259L) that were obtained from Bellvitge Hospital and the Cancer cell cultures. The cell lines NCI-H841, NCI-H23, NCI-H82, NCI-H69, Catalan Institute of Oncology (ICO) with the approval of the Ethical Committee NCI-H157, NCI-H1975, NCI-H2170, NCI-H460, NCI-H1963, NCI-H446, (CEIC Bellvitge). Ethical and legal protection guidelines of human subjects, DMS114, NCI-H727, NCI-H228, NCI-H128 and NCI-H1299 cell lines are from including informed consent, were followed. Fresh orthoxenografts/PDOXs grown the American Type Culture Collection (ATCC). The DMS273 cell line is from the in the mouse ovaries were collected when mice were sacrificed at passage #1, then European Collection of Authenticated Cell Cultures (ECACC). The Lu165 cells minced with sterile scalpels. Single cells and clumps were transferred to cell culture were obtained from the RIKEN Cell Bank (Japan). The cells were grown under plates and maintained in DMEM supplemented with 10% FBS plus 50 U/mL recommended conditions and maintained at 37 °C in a humidified atmosphere of penicillin and 50 mg/mL streptomycin under standard culture conditions. When 5% CO2/95% air. The cell lines were authenticated by genotyping for TP53 and cell colonies with epithelial cell morphology were observed, cells were trypsinized other known mutations. All cell lines used in this study were mycoplasma-free. and expanded. Both primary cell lines were considered established after >6 NATURE COMMUNICATIONS | (2021)12:4319 | https://doi.org/10.1038/s41467-021-24618-3 | www.nature.com/naturecommunications 11 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24618-3 Fig. 7 GSK-J4 reduces cancer cell viability of SCCOHT orthotopically implanted in mice. a Chromatogram depicting changes in SMARCA4, at the genomic DNA level, in the SCCOHTs of two patients. The alterations were biallelic, confirming the complete inactivation of SMARCA4. A normal reference DNA is also included. b Representative clonogenic assays for the indicated cells and treatments. c Percentage of viable cells of the indicated cells, measured using MTT assays, after treatment with increasing concentrations of GSK-J4 for 5 days. Lines show the number of viable cells relative to the total number at 0 h. Error bars, mean ± SD from three replicates. EC50 half-maximal effective concentration. d Western blot of the endogenous levels of the indicated proteins and cancer cells. TUBULIN, protein-loading control (48 h treatments, except 24 h in the GSK-J4 for the OVA250L cells). e Schematic representation of different models and treatments in mice developed from the OVA-250 tumour. SCCOHT small cell carcinoma of the ovary, hypercalcemic type. CDDP cisplatin. Figure schematics were generated using https://smart.servier.com/ (https://creativecommons.org/licenses/by/3.0/). f Left panel, gross pathological photographs at necropsy of the ovarian tumours that arose in mice treated with vehicle (n = 5) or GSK-J4 (n = 7). Right panels, volume and weight of each tumour. Two- sided unpaired Student’s t-test. n.s. not significant. g Representative sections of H&E staining of tumours from the group of mice treated as indicated (n = 5 in each group). Pink areas inside the tumours indicate fibrosis. Scale bars, 2.5 mm; Lower panels, magnification of the areas indicated (rectangle); scale bar, 50 µm (above) and 25 µm (below); Lower right panel, quantification of fibrotic areas. Two-sided unpaired Student’s t-test. Error bars, mean ± SD from replicates. ****P < 0.0001. h Representative immunostaining of H3K27me3 and of CASPASE-3 for OVA250X tumours treated with vehicle or GSK-J4. Scale bars, 25 µm. The quantification of the immunostaining is provided in Supplementary Table 6. A Source Data file is available for this figure. passages in vitro. Specific informed consent was obtained from all patients for 5–7 days with the indicated concentrations of each compound (SAHA, GSK-J4, tumour implantation into mice, and the study was approved by the IDIBELL Ethics GSK-126) or combinations. For the assays, 10 µL of a solution of 5 mg/mL MTT Committee (No. AAALAC-1155). [3-(4,5)-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide] (Sigma Chemi- cal Co.) was added. After incubation for 3 h at 37 °C, the medium was discarded, the formazan crystals that had formed were dissolved in 100 µL of lysis buffer (50% Antibodies and western blots. The following primary antibodies were used for N-N-dimethylformamide in H2O, 20% SDS, 2.5% glacial acetic acid, NaOH 5 mol/ western blots: anti-TUBULIN, T6199 mouse (1/10,000, Sigma Aldrich, St. Louis, L, pH 4.7), and absorbance was measured at 596 nm. Results are presented as the MO, USA); anti-Beta-ACTIN, 13854 (1/20,000 Sigma Aldrich); anti-SMARCA4 median of at least two independent experiments performed in triplicate for each 49360S (1:1000, Cell Signaling Technology); anti-EZH2 5246S (1:1000, Cell Sig- cell line and for each condition. For EC50 calculations, cells were treated with each naling); anti-H3K27ac D5E4 (1:1000, Cell Signaling) anti-H3K27me3 07-449 drug and their various combinations for 5 days. Estimates of EC50 were derived (1:1000, Cell Signaling); anti-UTX (KDM6A) D3Q1I (1:1000, Cell Signaling); anti- from the dose response curves. To assess the drug concentration effect and to KDM6B (JMJD3) #3457 (1:1000, Cell Signaling) for western blots or anti-KDM6B calculate the combination index (CI), cells were plated in 96-well plates and (JMJD3) ab38113 (Abcam) for immunostaining (see also Supplementary Table 1). incubated with a concentration of SAHA ranging from 0.07 to 10 μM (0.07, 0.15, For western blots, whole-cell lysates were collected in a buffer containing 2% SDS 0.3, 0.6, 1.25, 2.5, 5.0 and 10 μM), and the same for GSK-J4 for 5 days. MTT assays 50 mM Tris–HCl (pH 7.4), 10% glycerol and protease inhibitor cocktail (Roche were performed and the EC50 was determined by fitting the dose-response curve Applied Science). Protein concentrations were determined using a Bio-Rad DC utilising the CompuSyn software. The CI values for each dose and the corre- Protein Assay kit (Life Science Research). Equal amounts of lysates (20 µg) were sponding effect level were calculated. The CI offers a quantitative definition for separated by SDS–PAGE and transferred to a nitrocellulose membrane that was drug combinations in which CI < 1, CI = 1 and CI > 1 indicate synergism, an blocked with 5% nonfat dry milk. Membranes were incubated with the primary additive effect, and antagonism, respectively. antibody overnight at 4 °C, then washed before incubation with species-appropriate For clonogenic assays, the plates were seeded with 5000 cells from each cell line, IRDye 680CW (925-68022) or IRDye 800CW (925-32213) fluorescent secondary then treated with SAHA (1 µM), GSK-J4 (1 µM) or GSK-126 (1 µM) for 5 days. antibodies (1:10,000 LI-COR, NE, USA) for 1 h at room temperature. Membrane Cells were stained with crystal violet solution (0.5% Crystal Violet in 25% of imaging was performed on the Odyssey Li-Cor CLx i software: Image Studio methanol). Lite v5.2. Quantitative RT-PCR. To assess mRNA levels of the KDMs in different cells qPCR ChIP-sequencing. For ChIP, cells were grown in P-150 cm cell dishes and fixed analysis was performed. 1 μg of total RNA was reverse-transcribed using Super- with 1% methanol-free formaldehyde (Thermo Scientific) for 10 min at room Script™ II reverse transcriptase (Invitrogen) and Random primers (Promega), temperature, then quenched by 125 mmol/L glycine for 15 min at room tem- according to the manufacturers’ instructions. qRT-PCR was performed in a perature, washed with ice-cold PBS twice and centrifuged at 200 × g, 4 °C for 5 min. Quantstudio Real-Time PCR (Quantstudio Design & Analysis v1.5.1) instrument The pellet was resuspended in 1 mL of cell lysis buffer (10 mmol/L Tris–HCl, 10 using SYBR Green PCR Master Mix (Applied Biosystems). Three biological mmol/L NaCl, 0.5% NP-40, protease inhibitor) and kept at 4 °C, rotating for 30 replicates were carried out. Primer sequences are provided in the Supplementary min. After centrifugation, the pellet was resuspended in 1 mL of nuclear lysis buffer Table 2. (1% SDS, 10 mmol/L EDTA, 50 mmol/L Tris-HCl pH 8.0, protease inhibitor) and kept at 4 °C for 60 min. After another centrifugation, the lysate was sonicated with Treatments and shRNAs. Chemicals were obtained from the following sources: a Covaris M220 instrument to yield chromatin fragments of an average size of SAHA, suberoyl anilide hydroxamic acid (Cayman Chemical Company, Ann 0.25–1.00 kb, and then frozen at −20 °C for 30 min. The chromatin was thawed on Arbour, MI, USA); GSK-J4 (Shelleckchem); GSK-126 (Cayman Chemical Com- ice and centrifuged at 2500 × g. For each ChIP reaction, 60 μL of Magna ChIP™ pany). shRNAs against SMARCA4, KDM6A and KDM6B were purchased from Protein A + G Magnetic Beads (Merck Millipore) was used in accordance with the SIGMA-MISSION (LentiExpressTM Technology, Sigma-Aldrich) as a glycerol manufacturer’s protocol. Before addition of the sheared chromatin to the beads, stock of five pLKO plasmids carrying specific shRNA sequences. A non-target Triton X-100 and Na-deoxycholate was added to a final concentration of 10% each. shRNA (shNT) (Sigma MISSION shRNA non-mammalian control SHC002) was 1% of the chromatin volume was used for input. At least two independent ChIP used as a control. The lentiviruses were generated within the 293T packaging cells. experiments were performed. Oligonucleotide sequences are provided in the Supplementary Table 3. Immunoprecipitated chromatin was deep-sequenced in the Genomics Unit of the Centre for Genomic Regulation (CRG, Barcelona, Spain) using the Illumina HiSeq 2500 system and HiSeq Control Software (HCS) 2.2.68 software (Illumina). Flow cytometry. The cells were collected with PBS and the cell suspension was Briefly, library preparation included end-repair, generation of dA overhangs, transferred into the tubes, containing 70% ethanol, and fixed for 2 h. Cells were cen- adapter ligation, size selection and removal of non-ligated adapters by agarose gene trifuged, ethanol was decanted and cells were suspended in 5 mL PBS, and then treated electrophoresis and amplification (18 cycles) before loading the samples into the with 1 mL propidium iodide (PI)/Triton X-100 staining solution (PI/Triton X-100 sequencer. staining solution with RNase A, freshly made (to 10 mL of 0.1% (v/v) Triton X-100 For ChIP-sequencing data analysis, reads were aligned to the human reference (Sigma) in PBS add 2 mg DNase-freeRNase A (Sigma) and 200 μl of 1 mg/mL PI) for genome hg38, using Bowtie v1.2.2, with default parameters and disallowing multi- 30 min at room temperature. Flow cytometry experiments for detecting PI emission mapping (–m 1)40. PCR duplicates were removed using PICARD (http:// were performed in BD FACSCanto II Cell Analyzer (Becton, Dickinson) and the data broadinstitute.github.io/picard/). Ambiguous and multi-mapped reads were analysis was performed using DNA content frequency histogram deconvolution soft- discarded. Peaks were called using MACS2 v2.1.141. To avoid false positives, peaks ware (FACSDiva v6.1.2). The visualisation graphs were prepared with FlowJo software were discarded if they were present in the ChIP-seq of SMARCA4 in the version 7.6. SMARCA4-deficient cells. Genomic peak annotation was performed with the R package ChIPpeakAnno v3.15, considering the region ±2 kb around the TSS as the Cell growth analysis and calculation of EC50 and CI. For cell viability assays, cell promoter42. All analyses considered peaks overlapping with promoter regions, lines were incubated in 96-well plates. Before harvesting, cells were treated for unless otherwise specified. Peak lists were then transformed to gene target lists. 12 NATURE COMMUNICATIONS | (2021)12:4319 | https://doi.org/10.1038/s41467-021-24618-3 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24618-3 ARTICLE For heatmap and intensity plot representation of ChIP-seq signal, bedgraph files sacrificed on day 21 of treatment, and their ovaries dissected out and weighed. were generated using the makeUCSCfile function in HOMER with default Representative fragments were either frozen in nitrogen or fixed and processed for parameters normalising for differences in sample library size, and BigWig files were paraffin embedding. generated using the function bedGraphToBigWig from UCSC. Heatmaps were derived using the functions computeMatrix, in a window of ±2 kb around the Histopathology and immunostaining. For histological analysis, tumours were centre in the TSS, and plotHeatmap from deepTools43 (version 3.5). fixed and embedded in paraffin. Necrosis/fibrosis were morphological assessed after staining with haematoxylin and eosin (H&E), using standard protocols, and Construction of expression vectors and infections. The complete KDM6A then examined by light microscopy in a blinded fashion. For immunostainings, 4- cDNA (NM_021140.4) was PCR-amplified, from a retrotranscribed human RNA μm-thick paraffin-embedded sections of lung and ovarian tumour samples were pool (Agilent Technologies, Santa Clara, CA, USA) using Phusion High-Fidelity deparaffinized overnight at 62 °C and then immersed in xylene. Samples were DNA Polymerase (Thermo Scientific, Waltham, MA, USA) following standard rehydrated and, after microwaving with Tris/EDTA pH 9.0 for antigen retrieval, protocols. The cDNA fragments were cloned into the pLVX-IRES-ZSGreen vector endogenous peroxidase was inhibited with a 3% hydrogen peroxide solution, (Clontech), between the XhoI and NotI endonuclease restriction sites, following the blocked in 10% goat serum and incubated with primary antibodies overnight at 4 ° manufacturer’s instructions. For the KDM6B, the complete KDM6B coding region C (Supplementary Table 1). HRP-conjugated polyclonal goat (anti-mouse and anti- (ENSG00000132510.10) was PCR-amplified, from a human DNA pool (Agilent rabbit) secondary antibodies (NeoStain ABC Kit, NeoBiotech) were used in 1-h Technologies, Santa Clara, CA, USA) using Phusion High-Fidelity DNA Poly- incubations at room temperature. Labelling detection was done using an merase (Thermo Scientific, Waltham, MA, USA), following standard protocols. ImmPACT DAB Peroxidase (HRP) Substrate kit (Vector Laboratories, Burlingame, The fragment was cloned into the pCDNA4/TO vector (Invitrogen), between the CA, USA), and tissue sections were counterstained with haematoxylin. Once KpnI and XhoI endonuclease restriction sites, following the manufacturer’s dehydrated in an ethanol battery and cleared in xylene for 1 h, samples were instructions. The KDM6A and KDM6B inserts were then Sanger-sequenced. All mounted with coverslips with DPX mounting medium (Merck Millipore, Darm- the primers used are listed in Supplementary Tables 4 and 5. For KDM6A ectopic stadt, Germany). Sections were evaluated under a Leica DM1000 microscope by expression, the cells were infected with lentiviruses derived from the pLVX-IRES- two independent observers in a blind fashion. Areas of necrosis/fibrosis were ZSGreen vector and then selected by sorting GFP positive cells, using an BD quantified using Photoshop (version 2021). The scoring criteria for determining FACSArea (Becton, Dickinson). For KDM6B ectopic expression, the cells of H3K27me3 staining were based on the staining intensity (four categories, 1–4) interest were transfected with a pCDNA4/TO-KDM6B construct and clones (Supplementary Table 6). The mean of values from three independent evaluators expressing KDM6B were selected with Zeocin (InvivoGen) and pooled. was determined. Generation of orthotopic tumour models and treatments. Male and female Statistical analysis. Student’s t-tests, EC50 calculations, Kaplan–Meier estimates athymic nu/nu mice (ENVIGO) aged 4–5 weeks were maintained in a sterile and log-rank (Mantel–Cox) test were performed using Prism software (GraphPad environment before use in the lung cancer orthotopic experiments. Female athymic Prism 9). Values of P <5% were considered statistically significant. The statistical nu/nu mice (ENVIGO), 4–6 weeks old, were used for the ovarian cancer orthotopic methods used for each analysis are specified in the figure legends. studies. The animals were housed in individually ventilated cages on a 12-h light–dark cycle at 21–23 °C and 40–60% humidity. Mice were allowed free access Reporting summary. Further information on research design is available in the Nature to an irradiated diet and sterilised water. All animal experiments were approved by Research Reporting Summary linked to this article. the IDIBELL Ethical Committee under protocol 9111 approved by the Government of Catalonian, AAALAC accredited Unit 1155, and performed in accordance with guidelines stated in the International Guiding Principles for Biomedical Research Data availability Involving Animals, developed by the Council for International Organizations of The ChIP-seq raw data obtained in this study has been uploaded to the Gene Expression Medical Sciences (CIOMS). To generate orthotopic lung tumour xenografts the cell Omnibus-GEO (NCBI), under accession number GSE155129. Uncropped western blot lines were injected subcutaneously into the back of the mice (n = 3 mice/cell line). images are also present in a Source Data file. Databases/Datasets used in the study were: Once the solid tumour had entered the exponential growth phase, mice were cBioPortal for Cancer Genomics, Cancer Cell Line Encyclopedia-CCLE (https://www. sacrificed, the tumour was isolated under sterile conditions, and the non-necrotic cbioportal.org/study/summary?id=ccle_broad_2019), COSMIC-Catalogue of Somatic areas were selected and minced in small fragments of 2–3 mm3. These were then Mutations in Cancer (https://cancer.sanger.ac.uk/cell_lines), DepMap Portal-Cancer orthotopically implanted in the lung parenchyma20,29. On day 15, the mice were Dependency Map (https://depmap.org/portal/achilles/), Genomics of Drug Sensitivity in randomised and intraperitoneally treated with GSK-J4 (50 mg/kg/day for each Cancer database (https://www.cancerrxgene.org/compound/Vorinostat/1012/overview/ mouse) or corresponding vehicle only. For the lung orthotopic models, in most ic50, https://depmap.org/portal/compound/GSK-J4?tab=dependency). The authors cases the animals were sacrificed when they displayed serious respiratory difficulty, declare that other data supporting the findings of this study are provided in the which was subsequently confirmed to be associated with lung tumour growth. Supplementary Information/Source Data file. Source data are provided with this paper. Orthoxenografts or PDOXs of SCCOHT were generated. The primary tumour specimens for the two primary SCCOHT samples were freshly obtained at Hospital Universitario de Bellvitge (Hospitalet de Llobregat, Barcelona, Spain). The study Received: 30 October 2020; Accepted: 23 June 2021; was approved by the Institutional Review Board, and written informed consent was obtained from both patients. The orthotopic ovarian tumours were engrafted in mice, following an established protocol30. Briefly, non-necrotic tissue pieces (2–3 mm3) from resected carcinoma were selected and placed in DMEM (BioWhittaker) supplemented with 10% FBS and penicillin/streptomycin at room temperature. Under isofluorane-induced anaesthesia, animals were subjected to a lateral References laparotomy, their ovaries exposed, and tumour pieces anchored to the ovary 1. Clapier, C. R. & Cairns, B. R. The biology of chromatin remodeling complexes. surface with prolene 7.0 sutures. Tumour growth was monitored 2 or 3 times per Annu. Rev. Biochem. 78, 273–304 (2009). week and when the tumour had reached a sufficient size, it was harvested, cut into 2. Mohrmann, L. & Verrijzer, C. P. Composition and functional specificity of small fragments, and transplanted into between two and four new animals. SWI2/SNF2 class chromatin remodeling complexes. Biochim. Biophys. Acta Engrafted tumours (named OVA250X) at early mouse passages were cut into 6–8 1681, 59–73 (2005). mm3 pieces and stored in liquid nitrogen in a cryopreservation solution of 90% FBS 3. Romero, O. A. & Sanchez-Cespedes, M. The SWI/SNF genetic blockade: and 10% dimethyl sulfoxide, awaiting subsequent implantation. effects in cell differentiation, cancer and developmental diseases. Oncogene 33, To generate the cisplatin-resistant ovarian xenograft mouse model, 2681–2689 (2014). orthotopically engrafted OVA250X tumours at passage#1 were allowed to grow (n 4. Medina, P. P. et al. Frequent BRG1/SMARCA4-inactivating mutations in = 3 mice) until palpable intra-abdominal masses were noted. Cisplatin was i.v.- human lung cancer cell lines. Hum. Mutation 29, 617–622 (2008). administered to the animals (cycle #1, 3, 5 mg/kg dose) for 3 consecutive weeks 5. Rodriguez-Nieto, S. et al. Massive parallel DNA pyrosequencing analysis of the (days 0, 7 and 15; cycle#1 of treatment) (Supplementary Fig. 9b). Post-cisplatin tumor suppressor BRG1/SMARCA4 in lung primary tumors. Hum. Mutation tumours at relapse were harvested and engrafted in new animals. This process of 32, E1999–E2017 (2011). cisplatin treatment was repeated up to four times by treating tumour-bearing mice 6. Romero, O. A. et al. The tumour suppressor and chromatin-remodelling with stepwise-incremental doses of cisplatin: cycle #2, 4 mg/kg; cycle #3, 5 mg/kg factor BRG1 antagonizes Myc activity and promotes cell differentiation in and cycle #4, 5 mg/kg (see Supplementary Fig. 9b). Cisplatin-resistant tumours human cancer. EMBO Mol. Med. 4, 603–616 (2012). were obtained (OVA250XR). At doses higher than 3.5 mg/kg, signs of cisplatin 7. Romero, O. A. et al. MAX inactivation in small cell lung cancer disrupts MYC- induced some toxicity that were ameliorated by 2 days administration of saline SWI/SNF programs and is synthetic lethal with BRG1. Cancer Discov. 4, containing 5% glucose. Mice were transplanted with fragments of OVA250X and 292–303 (2014). OVA250XR tumours, and when tumours reached a homogeneous palpable size 8. Matias-Guiu, X. et al. Human parathyroid hormone related protein in ovarian were randomly allocated into the treatment groups (n = 3-7 mice/group): (i) Placebo; (ii) GSK-J4 (50 mg/kg) and (iii) cisplatin (3.5 mg/kg); drugs were small cell carcinoma. An immunohistochemical study. Cancer 73, 1878–1881 administered once a day, 5 days per week, for 4 consecutive weeks. Animals were (1994). NATURE COMMUNICATIONS | (2021)12:4319 | https://doi.org/10.1038/s41467-021-24618-3 | www.nature.com/naturecommunications 13 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24618-3 9. Jelinic, P. et al. Recurrent SMARCA4 mutations in small cell carcinoma of the 37. Miller, S. A., Mohn, S. E. & Weinmann, A. S. Jmjd3 and UTX play a ovary. Nat. Genet. 46, 424–426 (2014). demethylase-independent role in chromatin remodeling to regulate T-box 10. Witkowski, L. et al. Germline and somatic SMARCA4 mutations characterize family member-dependent gene expression. Mol. Cell 40, 594–605 (2010). small cell carcinoma of the ovary, hypercalcemic type. Nat. Genet. 46, 438–443 38. Narayanan, R. et al. Loss of BAF (mSWI/SNF) complexes causes global (2014). transcriptional and chromatin state changes in forebrain development. Cell 11. Ramos, P. et al. Small cell carcinoma of the ovary, hypercalcemic type, displays Rep. 13, 1842–1854 (2015). frequent inactivating germline and somatic mutations in SMARCA4. Nat. 39. Pros, E. et al. Genome-wide profiling of non-smoking-related lung cancer cells Genet. 46, 427–429 (2014). reveals common RB1 rearrangements associated with histopathologic 12. Oike, T. et al. A synthetic lethality-based strategy to treat cancers harboring a transformation in EGFR-mutant tumors. Ann. Oncol. 31, 274–282 (2020). genetic deficiency in the chromatin remodeling factor BRG1. Cancer Res. 73, 40. Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory- 5508–5518 (2013). efficient alignment of short DNA sequences to the human genome. Genome 13. Hoffman, G. R. et al. Functional epigenetics approach identifies BRM/ Biol. 10, R25 (2009). SMARCA2 as a critical synthetic lethal target in BRG1-deficient cancers. Proc. 41. Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, Natl Acad. Sci. USA 111, 3128–3133 (2014). R137 (2008). 14. Kim, K. H. et al. SWI/SNF-mutant cancers depend on catalytic and non- 42. Zhu, L. et al. ChIPpeakAnno: a Bioconductor package to annotate ChIP-seq catalytic activity of EZH2. Nat. Med. 21, 1491–1496 (2015). and ChIP-chip data. BMC Bioinforma. 11, 237 (2010). 15. Xue, Y. et al. SMARCA4 loss is synthetic lethal with CDK4/6 inhibition in 43. Ramírez, F. et al. deepTools2: a next generation web server for deep- non-small cell lung cancer. Nat. Commun. 10, 557 (2019). sequencing data analysis. Nucleic Acids Res. 44, w160–w165 (2016). 16. Chiba, H., Muramatsu, M., Nomoto, A. & Kato, H. Two human homologues of Saccharomyces cerevisiae SWI2/SNF2 and Drosophila brahma are transcriptional coactivators cooperating with the estrogen receptor and the Acknowledgements retinoic acid receptor. Nucleic Acids Res. 22, 1815–1820 (1994). The authors thank Isabel Bartolessis (Cancer Genetics Group) at IJC for technical assis- 17. De la Serna, I. L., Carlson, K. A. & Imbalzano, A. N. Mammalian SWI/SNF tance. This work was supported by the Spanish Ministry of Economy and Competitivity- complexes promote MyoD-mediated muscle differentiation. Nat. Genet. 27, MINECO (grant number SAF-2017-82186R, to M.S.-C., and grant PI19/01320 to A. 187–190 (2001). Villanueva) and from the Fundación Científica of the Asociación Española Contra el 18. Chi, T. H. et al. Sequential roles of Brg1, the ATPase subunit of BAF Cancer (AECC) (grant number GCB14142170MONT) to M.S.-C. A. Villanueva is also chromatin remodeling complexes, in thymocyte development. Immunity 19, funded by the Department of Health of the Generalitat de Catalunya (2014SGR364). O.A. 169–182 (2003). R. received a Juan de la Cierva postdoctoral contract (grant No. IJCI-2016-28201, until 19. Seo, S., Richardson, G. A. & Kroll, K. L. The SWI/SNF chromatin remodeling November 2019) and an AECC research contract (INVES19045ROME from December protein Brg1 is required for vertebrate neurogenesis and mediates 2019). A. Vilarrubi, P.L. and A.A. are supported by pre-doctoral contracts from the transactivation of Ngn and NeuroD. Development 132, 105–151 (2005). Spanish MINECO (FPI-fellowship: PRE2018-084624, BES-2015-072204 and FPU17/ 20. Romero, O. A. et al. Sensitization of retinoids and corticoids to epigenetic 00067). M.S. was supported by a Rio Hortega contract from the Instituto de Salud drugs in MYC-activated lung cancers by antitumor reprogramming. Oncogene Carlos III (CM17/00180). L.F. received a European Union Horizon 2020 research and 36, 1287–1296 (2017). innovation programme under the Marie Sklodowska-Curie Actions grant agreement, 21. Richon, V. M., Sandhoff, T. W., Rifkind, R. A. & Marks, P. A. Histone deacetylase number 799850. inhibitor selectively induces p21WAF1 expression and gene-associated histone acetylation. Proc. Natl Acad. Sci. USA 97, 10014–10019 (2000). Author contributions 22. Tessarz, P. & Kouzarides, T. Histone core modifications regulating O.A.R. and M.S.-C. conceived and designed the study; An.V., Al.V., A.G., A.A., D.T., nucleosome structure and dynamics. Nat. Rev. Mol. Cell Biol. 15, 703–708 E.P., J.J.A.-B., F.S., P.L. and S.V. performed the experiments and analysed data with (2014). assistance from L.F., J.F.M.-T., M.S.; A.O., J.M.P., X.M.-G., P.P.M. and Au.V. provided 23. Black, J. C., Van Rechem, C. & Whetstine, J. R. Histone lysine methylation essential reagents and intellectual input. O.A.R. and M.S.-C. coordinated the project, dynamics: establishment, regulation, and biological impact. Mol. Cell 48, interpreted results, wrote the manuscript and supervised the project. 491–507 (2012). 24. Chan-Penebre, E. et al. Selective killing of SMARCA2- and SMARCA4- deficient small cell carcinoma of the ovary, hypercalcemic type cells by Competing interests inhibition of EZH2: in vitro and in vivo preclinical models. Mol. Cancer Ther. Al.V. and Au.V. are co-founders of Xenopat S.L. The remaining authors declare no 16, 850–860 (2017). competing interests. 25. Wang, Y. et al. Histone deacetylase inhibitors synergize with catalytic inhibitors of EZH2 to exhibit antitumor activity in small cell carcinoma of the ovary, hypercalcemic type. Mol. Cancer Ther. 17, 2767–2779 (2018). Additional information Supplementary information The online version contains supplementary material 26. Hodges, H. C. et al. Smarca4 ATPase mutations disrupt direct eviction of available at https://doi.org/10.1038/s41467-021-24618-3. PRC1 from chromatin. Nat. Genet. 49, 282–288 (2017). 27. Chandrasekaran, R. & Thompson, M. Polybromo-1-bromodomains bind Correspondence and requests for materials should be addressed to O.A.R. or M.S-C. histone H3 at specific acetyl-lysine positions. Biochem. Biophys. Res. Commun. 355, 661–665 (2007). Peer review information Nature Communications thanks the anonymous reviewer(s) for 28. Kruidenier, L. et al. A selective jumonji H3K27 demethylase inhibitor their contribution to the peer review of this work. Peer reviewer reports are available. modulates the proinflammatory macrophage response. Nature 488, 404–408 (2012). Reprints and permission information is available at http://www.nature.com/reprints 29. Ambrogio, C. et al. Combined inhibition of DDR1 and Notch signaling is a therapeutic strategy for KRAS-driven lung adenocarcinoma. Nat. Med. 22, Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in 270–277 (2016). published maps and institutional affiliations. 30. Vidal, A. et al. Lurbinectedin (PM01183), a new DNA minor groove binder, inhibits growth of orthotopic primary graft of cisplatin-resistant epithelial ovarian cancer. Clin. Cancer Res. 18, 5399–5411 (2012). 31. Li, Y. & Seto, E. HDACs and HDAC inhibitors in cancer development and Open Access This article is licensed under a Creative Commons therapy. Cold Spring Harb. Perspect. Med. 6, a026831 (2016). Attribution 4.0 International License, which permits use, sharing, 32. Arcipowski, K. M., Martinez, C. A. & Ntziachristos, P. Histone demethylases adaptation, distribution and reproduction in any medium or format, as long as you give in physiology and cancer: a tale of two enzymes, JMJD3 and UTX. Curr. Opin. appropriate credit to the original author(s) and the source, provide a link to the Creative Genet. Dev. 36, 59–67 (2016). Commons license, and indicate if changes were made. The images or other third party 33. Bogershausen, N. & Wollnik, B. Unmasking Kabuki syndrome. Clin. Genet. material in this article are included in the article’s Creative Commons license, unless 83, 201–211 (2013). indicated otherwise in a credit line to the material. If material is not included in the 34. Gozdecka, M. et al. UTX-mediated enhancer and chromatin remodeling article’s Creative Commons license and your intended use is not permitted by statutory suppresses myeloid leukemogenesis through noncatalytic inverse regulation of regulation or exceeds the permitted use, you will need to obtain permission directly from ETS and GATA programs. Nat. Genet. 50, 883–894 (2018). the copyright holder. To view a copy of this license, visit http://creativecommons.org/ 35. Ntziachristos, P. et al. Contrasting roles of histone 3 lysine 27 demethylases in licenses/by/4.0/. acute lymphoblastic leukaemia. Nature 514, 513–517 (2014). 36. Hashizume, R. et al. Pharmacologic inhibition of histone demethylation as a therapy for pediatric brainstem glioma. Nat. Med. 20, 1394–1396 (2014). © The Author(s) 2021 14 NATURE COMMUNICATIONS | (2021)12:4319 | https://doi.org/10.1038/s41467-021-24618-3 | www.nature.com/naturecommunications