Refractory testicular germ cell tumors are highly sensitive to the targeting of polycomb pathway demethylases KDM6A and KDM6B

Refractory testicular germ cell tumors are highly sensitive to the targeting of polycomb pathway demethylases KDM6A and KDM6B | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Refractory testicular germ cell tumors are highly sensitive to the targeting of polycomb pathway demethylases KDM6A and KDM6B Doha Shokry, Mehwish W Khan, Christine Powell, Samantha Johnson, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4986186/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 31 Oct, 2024 Read the published version in Cell Communication and Signaling → Version 1 posted 11 You are reading this latest preprint version Abstract Testicular germ cell tumors (TGCTs) can be treated with cisplatin-based therapy. However, a clinically significant number of cisplatin-resistant patients die from progressive disease as no effective alternatives exist. Curative cisplatin therapy results in acute and life-long toxicities in the young TGCT patient population providing a rationale to decrease cisplatin exposure. In contrast to genetic alterations, recent evidence suggests that epigenetics is a major driving factor for TGCT formation, progression, and response to chemotherapy. Hence, targeting epigenetic pathways with “epidrugs” is one potential relatively unexplored strategy to advance TGCT treatment beyond cisplatin. In this report, we demonstrate for the first time that targeting polycomb demethylases KDM6A and KDM6B with epidrug GSK-J4 can treat both cisplatin-sensitive and -resistant TGCTs. While GSK-J4 had minimal effects alone on TGCT tumor growth in vivo, it dramatically sensitized cisplatin-sensitive and -resistant TGCTs to cisplatin. We validated KDM6A/KDM6B as the target of GSK-J4 since KDM6A/KDM6B genetic depletion had a similar effect to GSK-J4 on cisplatin-mediated anti-tumor activity and transcriptome alterations. Pharmacologic and genetic targeting of KDM6A/KDM6B potentiated or primed the p53-dominant transcriptional response to cisplatin, with also evidence for basal activation of p53. Further, several chromatin modifier genes, including BRD4 , lysine demethylases, chromodomain helicase DNA binding proteins, and lysine methyltransferases, were repressed with cisplatin only in KDM6A/KDM6B-targeted cells, implying that KDM6A/KDM6B inhibition sets the stage for extensive chromatin remodeling of TGCT cells upon cisplatin treatment. Our findings demonstrate that targeting polycomb demethylases is a new potent pharmacologic strategy for treating cisplatin resistant TGCTs that warrants clinical development. Testicular cancer polycomb GSK-J4 GSK-126 preclinical transcriptomics cisplatin chemotherapy resistance epigenetics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Testicular germ cell tumors (TGCTs) are curable with traditional cisplatin-based chemotherapy ( 1 ). However, cisplatin resistance occurs in approximately 15% of metastatic patients, with the majority dying from progressive disease ( 2 ). No effective therapies exist for this patient population, and no clinically effective strategies exist to overcome cisplatin resistance, likely due to a lack of detailed understanding of the mechanisms responsible for cisplatin resistance in TGCTs ( 2 , 3 ). Further, testicular cancer is the most common carcinoma of males ages 15 to 45 and cisplatin-based cures are associated with many toxicities and co-morbidities, including ototoxicity, infertility, neuropathy, and a high risk of secondary cancers ( 4 , 5 ). Hence, cisplatin-sparing strategies for this young patient population are needed. TGCTs are believed to arise from aberrant differentiation of primordial germ cells during development ( 6 ). There is mounting evidence that TGCTs may be a cancer that is especially driven by epigenetic dysfunction, both in terms of etiology and response or resistance to chemotherapy ( 7 , 8 ). This includes a unique pattern of DNA hypomethylation and histone modifications compared to most other tumors due to their embryonic origins at a stage undergoing extensive epigenetic reprogramming, an association with in utero endocrine disruption and environmental stress, and a lack of driver DNA mutations ( 6 , 9 – 11 ). Particularly prominent is a lack of p53 mutations that is a proposed factor for high cisplatin curability. The possible unique reliance on distinct epigenetic drivers suggests that TGCTs may be uniquely sensitive to epigenetic-based therapies ( 7 , 8 ). Two major epigenetic pathways associated with target gene repression are CpG island methylation mediated by DNA methyltransferases DNMT1, DNMT3A, and DNMT3B and the polycomb pathway ( 12 , 13 ). There are two major polycomb repressive complexes (PRCs), PRC1 and PRC2. PRC2 contains the core components EZH2, SUZ12, and EED, while PRC1 is comprised of BMI1, CBX, RIN1A/B, and PCH ( 14 ). EZH2 catalyzes H3K27 trimethylation, which is a docking site for the PRC1 complex that catalyzes monoubiquitination of H2A on K119 ( 14 ). Both modifications repress gene expression. Additionally, the histone demethylases KDM6A and KDM6B remove H3K27 methylation ( 15 ). The role of the PRC2 complex in cancer is complex, with the majority of studies suggesting that EZH2 promotes oncogenesis and is a pharmacologic cancer target ( 16 ). However, there are several examples of documented tumor suppressor functions of PRC2 ( 17 – 19 ). This complexity extends to whether polycomb represses or augments chemotherapy responses ( 20 – 23 ). We and others have shown that TGCT cells are hypersensitive to low doses of hypomethylating agents (HMAs), such as decitabine and guadecitabine, including cisplatin-resistant cells, and that pretreatment with HMAs can reverse cisplatin resistance in cell and mouse xenograft models ( 24 – 28 ). This strategy has been tested clinically with some promising patient responses in small trials ( 29 , 30 ). Furthermore, we developed cisplatin-resistant TGCT isogenic cell models and demonstrated that cisplatin resistance is associated with a coordinated decrease in EZH2, BMI1, and H3K27me3 levels coupled with a bi-directional shift between gene promoter and gene body DNA methylation among multiple gene sets resulting in an upregulation of polycomb target genes and a downregulation of tumor suppressor genes ( 31 – 33 ). A gene signature based on polycomb target genes was also associated with recurrent and progressive disease in TGCT patients ( 32 ). Further, DNMT3B levels were highly upregulated in cisplatin-resistant TGCT cells compared to isogenic parental cells, and DNMT3B-knockdown alone in parental cells was sufficient to induce H3K27me3, EZH2, and BMI1 levels and cisplatin hypersensitivity ( 33 ). This suggests that DNA methylation and polycomb are coordinately regulated in TGCTs to modulate cisplatin sensitivity. The apparent connectedness of DNA methylation and polycomb signaling alterations with cisplatin resistance, coupled with the demonstrated promising preclinical and clinical activity of DNA methylation targeting HMAs in TGCTs, prompted us to examine whether polycomb targeting may be a second epigenetic-based treatment for cisplatin-resistant TGCTs. Genetic and pharmacologic inhibition of polycomb H3K27me3 demethylases KDM6A and KDM6B sensitized cisplatin-resistant and wild-type TGCT cells to cisplatin and produced dramatic synergistic tumor regression in animal models. This was associated with decreased expression of DNMT3B. Transcriptome analysis revealed a robust alteration in gene expression with polycomb demethylase targeting and basal and cisplatin-mediated potentiation of p53 target gene activation and downregulation of multiple chromatin-modifying enzymes in response to cisplatin. Our findings preclinically validate targeting polycomb demethylases KDM6A/B as a potent pharmacologic strategy for treating cisplatin-resistant TGCTs that warrants further preclinical and clinical investigation. 2. Materials and Methods 2.1 Drug treatments and cell viability and proliferation assays All cells were cultured in DMEM (Sigma) with 10% FBS (GeminiBio). The NT2/D1, 833K, and 2102EP cells are human testicular cancer-derived embryonal carcinoma cell lines and colon cancer HCT116, breast cancer MDA-MB-231 and MCF7, and glioblastoma U87-MG cell lines were all purchased from ATCC and authenticated by ATCC with karyotyping and short tandem repeat profiling, as described ( 24 ). Cells were frozen within 1 month of purchase and used within 2 months of resuscitation. Derivation of cisplatin-resistant NT2/D1-A4 and 2102EP-C1 cells was previously described in detail ( 32 ). Cells were treated with the indicated dosages of cisplatin (Sigma) for 6 hours and cells assayed for survival 3 days later. For sequential treatments, cells were pretreated with the EZH2 inhibitor GSK-126 or the KDM6A/KDM6B histone demethylase inhibitor GSK-J4 (both from Selleck Chemicals) for 3 days at doses that alone did not affect viability by more than 5% (0.5 µM and 1.0 µM, respectively) and then treated with cisplatin ( 32 ). To assess cell viability, CellTiter-Glo (Promega) assays were performed. For each cell line, three biological replicates were tested at each concentration, and experiments were repeated at least twice on different days. 2.2 Lentiviral shRNA knockdown Lentiviruses were produced by co-transfecting HEK293 cells with 10 µg of the viral packaging vector pCMV-dR8.2 and envelope vector pCMV-VSV-G (10:1 ratio) and 10 µg of lenti-shRNA or + targeting BMI1 (TRCN0000020155, TRCN0000020156, TRCN0000020157), EZH2 (TRCN0000018365, TRCN0000040074 and TRCN0000040075), KDM6A (TRCN0000107760, TRCN0000107761, TRCN0000107762, TRCN0000107763, TRCN0000107764) and KDM6B (TRCN0000359976, TRCN0000236678, TRCN0000236677, TRCN0000236676, TRCN0000236679), were purchased from Sigma along with pLKO.1-puro empty vector control (SHC001). The HEK293 cell medium was changed 24 hours after transfection and cells were incubated for 48 hours to allow for virus production. After 48 hours, HEK293 medium containing viral particles was filtered and transferred onto 833K, 2102EP, or NT2/D1 cells for 48 hours. Cells were selected with 5 µg/ml puromycin. For dual KDM6A and KDM6B knockdown, a combination of two lentivirus pLKO.1-puro, pLKO.1-Neo (Addgene plasmid #13425), shRNA targeting KDM6A (TRCN0000107764-neo) and shRNA targeting KDM6B (TRCN0000236676) were used. 48-hour post-transduction cells were selected with 5 µg/ml puromycin and 250 µg/ml neomycin. 2.3 Xenograft experiments All animal experiments were approved by the University of Illinois Urbana-Champaign IACUC under protocol 24080. Under this protocol maximum allowable tumor size is 15 mm in any direction, which was not exceeded. For mouse studies, 5–8-week-old male athymic nude mice (Jackson Labs) were injected subcutaneously in the flank with 5 × 10 6 2102EP-C1 and NT2/D1 cells after resuspension in a 50:50 ratio of DMEM/Matrigel (Corning). Once palpable tumors were detected, tumor volume was measured twice weekly with calipers using the formula V= (L × W × W)/2. At a tumor volume of approximately 150 mm 3 (day 1), mice were randomly assigned to vehicle (PBS), GSK-J4, cisplatin, or a combination of cisplatin + GSK-J4 treatments. GSK-J4 was given by intraperitoneal (IP) injection every other day for 6 total injections at 50 mg/kg (days 1, 3, 5, 7, 9 and 11). Cisplatin was given as a single IP injection on day 5 at 7.5 mg/kg for 2102EP-C1 cells and 6.0 mg/kg for NT2/D1 cells. In separate experiments, mice were injected with 5 × 10 6 2102EP-C1-pLKO.1 cells or 2102EP-C1-shKDM6A + shKDM6B (sh6A + 6B) cells. Once tumors reached a volume of 150 mm 3 mice were randomly assigned to a single IP injection of either PBS or 7.5 mg/Kg cisplatin. Body weight was also measured twice weekly. Mice were sacrificed when tumors reached humane endpoints with euthanasia by carbon dioxide followed by cervical dislocation. 2.4 RNA-sequencing RNA was extracted from cisplatin-resistant NT2/D1-A4 and 2102EP-C1 cells pretreated with only 1.0 µM GSK-J4 for 3 days, treated with only 0.5 µM cisplatin for 6 hours, or both. RNA was also extracted from 2102EP-C1-pLKO.1 or 2102EP-C1-shKDM6A + shKDM6B (sh6A + 6B) cells treated with PBS or 0.5 µM cisplatin for 6 hours. In all cases, cells were harvested for RNA 24 hours after cisplatin treatment. RNA was isolated with the RNeasy plus Mini Kit (Qiagen) and RNA sequencing was performed by the Roy J. Carver Biotechnology Center. RNA-Seq libraries were prepared using the TruSeq Stranded mRNA Sample Prep kit. The libraries were sequenced on a HiSeq 4000 using HiSeq 4000 sequencing kit version 1. Initial quality control was performed using FASTQC. Trimmomatic was used to remove low-quality bases from both ends LEADING ≤ 28 and TRAILING ≤ 28, respectively, with a minimum length of 30. The reads in FASTQ format were aligned to human genome assembly NCBI GRCh38.p14 using STAR aligner. Reads were counted and assigned to genes using featureCount. The “Limma” R package was used to identify differentially expressed genes ( 34 ). Genes whose expression was not greater than 0.5 counts per million in at least 2 samples were removed and the resultant filtered expression matrix was TMM-normalized. Benjamini-Hochberg False Discovery Rate (FDR) was used to correct for multiple hypotheses. The “Enhanced Volcano” R package was used to visualize volcano plots. The RNA-seq datasets for the current study have been submitted to the NCBI Database of GEO Datasets under the accession numbers GSEXXXX. 2.5 Downstream enrichment analysis Gene Set Enrichment Analysis (GSEA) from the Broad Institute was performed to identify enriched gene sets ( 35 ). GeneOverlap package from R Bioconductor was used to identify significant gene set overlap between common or exclusive gene expression changes between pLKO.1 control cells (pLKO.1 cisplatin-treated vs pLKO.1 untreated), those genes basally regulated by KDM6A and KDM6B knockdown (sh6A + 6B untreated vs pLKO.1 untreated), and those genes regulated by cisplatin in KDM6A and KDM6B knockdown cells (sh6A + 6B cisplatin-treated vs sh6A + 6B untreated) and C2 gene sets from the MSigDB database ( 35 , 36 ). 2.6 Western analysis and real-time PCR For Western analysis cells were lysed in radioimmune precipitation buffer and separated by SDS-PAGE. Antibodies to actin (MA1-744, Thermo Fisher), DNMT3B (HPA001595, Atlas Antibodies) Ubiquitin H2A-K119 (3240, Cell Signaling Technology), H3K27me3 (9733, Cell Signaling Technology), BMI1 (6964, Cell Signaling Technology), EZH2 (5246, Cell Signaling Technology) and histone H3 (ab1791, Abcam) were used. Total cellular RNA was isolated using the RNeasy Mini Kit (Qiagen), and complementary DNAs (cDNAs) were synthesized using High-capacity cDNA Synthesis Kit (Thermo Fisher Scientific). Quantitative real-time PCR assays were performed with PowerUp™ SYBR™ green master mix (Thermo Fisher Scientific) and the QuantStudio 3 Real-time System (Thermo Fisher Scientific). In all cases gene expression was normalized to β-actin. Primers for RT-PCR will be provided upon request. 2.7 Statistics Student’s t-tests and ANOVA were performed using GraphPad Prism 10. p -values indicative of non-significance ( p > 0.05) and significance ( p ≤ 0.05; * p ≤ 0.01; **, p ≤ 0.001; *** and p ≤ 0.0001; ****) were determined. Mean and standard error of the mean were used to describe sample variability. 3. Results Pharmacologic repression of the polycomb pathway by inhibition of polycomb methylase EZH2 with GSK-126 confers cisplatin resistance to TGCT cells but not other cancer cell types, while polycomb induction by inhibition of polycomb demethylases KDM6A and KDM6B with GSK-J4 sensitizes TGCT cells to cisplatin. We showed previously that multiple isogeneic cisplatin-resistant TGCT cell lines had a reduction in the polycomb repressive mark H3K27me3 and reduced levels of polycomb repressive complex 2 (PRC2) component EZH2 and polycomb repressive 1 (PRC1) component BMI1 with a corresponding induction of polycomb target genes ( 32 ). We also show previously in multiple cell lines and in Fig. 1 , that inhibition of polycomb signaling with the EZH2 inhibitor GSK-J4 confers cisplatin resistance in parental 2102EP TGCT cells, while potentiation of polycomb signaling with an inhibitor of the H3K27me3 demethylases KDM6A and KDM6B, called GSK-J4, confers cisplatin sensitization in cisplatin-resistant 2102EP-C1 TGCT cells ( 32 ). Note, cells were pretreated with GSK-J4 and GSK-126 for 3 days at doses previously established to not affect cell proliferation or viability as single agents ( 32 ). To address whether polycomb has the ability to generally alter the cisplatin sensitivity of cancer cells, we tested the effects of GSK-126 and GSK-J4 on a number of non-TGCT cancer cell lines, including breast cancer cells MCF-7 and MDA-231, colon cancer cells HCT116, and glioblastoma cells U87-MG. GSK-126 and GSK-J4 had minimal effects on cisplatin sensitivity of these cell lines, suggesting that cisplatin sensitivity of TGCT cells may be uniquely altered by polycomb ( Fig. 1 ) . Knockdown of EZH2 and BMI1 confers cisplatin resistance in wild-type TGCT cells, while knockdown of KDM6A/KDM6B sensitizes TGCT cells to cisplatin in vitro . We next tested whether genetic perturbation of the polycomb pathway could alter the cisplatin sensitivity of TGCT cells. Inhibition of polycomb signaling by EZH2- or BMI1-knockdown conferred cisplatin resistance in parental cisplatin-sensitive TGCT 833K, 2102EP, and NT2/D1 cells ( Fig. 2 A-C ) . Note, while BMI1 shRNA decreased BMI1 levels, it did not alter biological target Ub-H2AK119, while EZH2-knockdown did repress H3K27me3 levels as expected ( Fig. 2 B ) . This implies that BMI1 may have a Ub-H2AK119-independent effect on TGCT cells. Reciprocally, induction of polycomb signaling with dual knockdown of KDM6A and KDM6B resulted in cisplatin sensitization in both cisplatin-sensitive NT/2D1 and 2102EP cells and cisplatin-resistant counterparts, NT2/D1-A4 and 2102EP-C1 cells ( Fig. 2 D-H ) . Also, DNMT3B-knockdown sensitized parental TGCT cells to cisplatin and induced BMI1, EZH2, and H3K27me3 levels ( 33 ). Consistent with this interconnected relationship, pharmacologic inhibition of KDM6A/KDM6B with GSK-J4 or KDM6A/KDM6B knockdown repressed expression of DNMT3B in both cisplatin-sensitive and -resistant TGCT cells ( Fig. 4 F-H ) . In prior work, we have shown an interconnected relationship between alterations in DNA methylation mediated by DNMT3B and H3K27me3-mediated polycomb signaling ( 31 , 33 ). For example, DNMT3B is overexpressed in cisplatin-resistant TGCT cells, while H3K27me3 levels are decreased compared to parental cells ( 33 ). Also, DNMT3B-knockdown sensitized parental TGCT cells to cisplatin and induced BMI1, EZH2, and H3K27me3 levels ( 33 ). Consistent with this interconnected relationship KDM6A/KDM6B-knockdown repressed expression of DNMT3B in both cisplatin-sensitive and -resistant TGCT cells ( Fig. 2 D-F ) . H3K27me3 specific histone demethylase inhibitor GSK-J4 and KDM6A and KDM6B dual knockdown dramatically synergizes with cisplatin to promote TGCT inhibition and regression in vivo. To assess whether GSK-J4 could potentiate cisplatin sensitivity to TGCT cells in vivo , we performed xenograft studies with cisplatin-resistant 2102EP-C1 and cisplatin-sensitive NT2/D1 cells. While GSK-J4 alone had minimal effects on TGCT growth, GSK-J4 produced a dramatic synergistic interaction with cisplatin treatment with evidence of tumor regression ( Fig. 3 ) . Mice treated with a single round of GSK-J4 and cisplatin therapy remained tumor-free at the end of the experiment (90 days). Note that the dose of cisplatin was decreased in cisplatin-sensitive NT2/D1 cells in order to observe a potentiation effect with GSK-J4 ( Fig. 3 ) . This suggests that GSK-J4 may not only be able to restore cisplatin sensitivity to resistant cells but may also be a strategy for cisplatin-sparing therapy for cisplatin-sensitive tumors. GSK-J4 or GSK-J4 plus cisplatin had minimal toxicity as assessed by total body weight (Supplemental Figure S1 ) . In contrast, while not as dramatic, EZH2 inhibitor GSK-126 conferred cisplatin resistance in TGCT xenografts (Supplemental Figure S2 ) . To address whether GSK-J4 may have off-target effects, we performed further xenografts with cisplatin-resistant 2102EP-C1 cells after dual knockdown of the intended targets of GSK-J4, KDM6A and KMD6B, and compared the results to control cells. Similar to GSK-J4, KDM6A/ KDM6B-knockdown minimally effected basal tumor growth but dramatically potentiated the effects of cisplatin, again with evidence of tumor regression, and all mice remained tumor-free for over 90 days after a single dose of cisplatin ( Fig. 3 ) . Together these results suggest that pharmacologic and genetic activation of the polycomb pathway in TGCTs has strong cisplatin potentiated effects in vivo with low overall toxicity. The dramatic in vivo effects, as compared to in vitro effects of GSK-J4 and KDM6A/KDM6B-knockdown, suggest that host and/or tumor microenvironment contributions may be occurring during cisplatin sensitization. Transcriptome analysis of GSK-J4 and KDM6A/KDM6B-knockdown cells reveals the importance of basal activation of polycomb and p53 signaling in cisplatin sensitization. To investigate potential mechanisms responsible for the cisplatin sensitization effects of polycomb demethylase targeting in TGCT cells, we performed RNA-seq analysis of cisplatin-resistant NT2/D2-A4 and 2102EP-C1 cells untreated or treated with cisplatin and GSK-J4 alone or in combination and also untreated or cisplatin treated control 2102EP-C1-PLKO.1 and isogeneic dual 2102EP-C1-shKDM6A + 6B-knockdown cells. Note, in cell viability/cytotoxic assays, cells were treated with cisplatin for 6 hours and assayed 3 days later to mimic clinical usage (peak cisplatin plasma concentration over a short amount of time) ( 1 , 2 ). As we have documented before, for transcriptomic analysis, the post-cisplatin time point was shortened to 24 hours to better assess proximal alterations in gene expression not associated with active cell death ( 25 , 37 ). Multidimensional scaling (MDS) plots demonstrated a clear separation of the experimental groups and a tight grouping of biological replicates within groups ( Fig. 4 A ) . Volcano plots and GSEA revealed that cisplatin treatment of cisplatin-resistant NT2/D1-A4, 2102EP-C1, and 2102EP-C1-PLKO.1 control cells had a restricted pattern of gene alterations dominated by upregulated p53 target genes ( Fig. 4 B-C ) , as we have noted previously in transcriptome analysis of TGCT cells treated with cisplatin ( 25 , 37 ). Of note, the degree of transcriptional changes was substantially reduced in comparison to parental cisplatin-sensitive cells (data not shown). In contrast, GSK-J4-pretreated NT2/D1-A4 and 2102EP-C1 cells and untreated 2102EP-C1-shKDM6A + 6B cells had a substantially more robust transcriptional response to cisplatin, again dominated by upregulated p53 target genes ( Fig. 4 B-C ) . Interestingly, GSK-J4 treatments alone and KDM6A/KDM6B-knockdown alone in 2102EP-C1-shKDM6A + 6B cells demonstrated substantial alterations in gene expression. Upregulated genes for GSK-J4 alone treatments were again dominated by p53 target genes ( Fig. 4 C and Fig. 5 A-B ) despite the fact that the GSK-J4 treatments were not toxic or growth-inhibitory in long-term assays (32 and data not shown). This basal p53 target gene effect was less prominent in 2102EP-C1-shKDM6A + 6B cells but was still evident in a narrower subset of p53 target genes ( Fig. 5 C ) . In contrast, downregulated genes had a more diverse gene set enrichment pattern among the experiments, including gene sets involving mRNA splicing, DNA methylation, and histones (Supplemental Table S1 ) . The top 20 gene sets enriched for upregulated and downregulated genes for each experimental arm for all three RNA-seq experiments are provided in Supplemental Table S1 . Additionally, and again consistent with the interconnected relationship between alterations in DNA methylation mediated by DNMT3B and H3K27me3-mediated polycomb signaling ( 31 , 33 ), pharmacologic inhibition of KDM6A/KDM6B with GSK-J4 or KDM6A/KDM6B-knockdown repressed expression of DNMT3B ( Fig. 5 D-E ) . Transcriptome analysis of KDM6A/KDM6B-knockdown cells reveals cisplatin sensitization is associated with alterations in chromatin remodeling genes upon cisplatin treatment. To gain a broader insight into the role of polycomb demethylase targeting in cisplatin sensitization of TGCT cells, we further analyzed the KDM6A/KDM6B-knockdown plus cisplatin treatment in 2102EP-C1 cells experiment by identifying subsets of unique and overlapping upregulated and downregulated genes from three comparisons, those genes regulated by cisplatin in PLKO.1 control cells (PLKO.1 cisplatin-treated vs PLKO.1 untreated), those genes basally regulated by KDM6A/KDM6B-knockdown (shKDM6A + 6B untreated vs PLKO.1 untreated), and those genes regulated by cisplatin in KDM6A/KDM6B-knockdown cells (shKDM6A + 6B cisplatin-treated vs shKDM6A + 6B untreated) ( Fig. 6 A-B ) . Venn diagrams were generated with a cutoff of > 1.2 fold-change with FDR < 0.001 and GeneOverlap analysis with Fisher exact tests were performed against the 5529 curated sets from the Broad MSigDB C2 collection ( Fig. 6 A-B ) . Gene lists from Venn diagram analysis and GeneOverlap results are provided in Supplemental Table S2 and Supplemental Table S3 . Upregulated genes were again dominated by p53 target genes with p53 gene sets enriched for genes commonly upregulated in both PLKO.1 and 2102EP-C1-shKDM6A + 6B cells treated with cisplatin (Group 1), and genes upregulated by cisplatin in both cells but also basally upregulated upon KDM6A/KDM6B-knockdown (Group 2) ( Fig. 6 A ) . This analysis again suggests that targeting polycomb demethylases basally modifies the p53 pathway in TGCT cells to sensitize these cells to cisplatin. Gene sets enriched exclusively for genes upregulated in shKDM6A + 6B cells treated with cisplatin were involved in hypoxia and E-cadherin (CDH1) signaling (Group 3) ( Fig. 6 A ) . Finally, gene sets enriched exclusively for upregulated genes in untreated KDM6A/KDM6B knockdown cells compared to untreated PLKO.1 cells include several related to cancer (Group 4). In contrast, there was strong enrichment for gene sets involved in H3K27me3 and polycomb signaling for gene exclusively downregulated in KDM6A/KDM6B-knockdown cells, as would be expected by knocking down polycomb demethylases (Group 5) ( Fig. 6 B ) . Interestingly, we found a gene set of 272 chromatin-modifying enzymes and proteins ( 38 ) that were only downregulated in 2102EP-C1-shKDM6A + 6B cells treated with cisplatin (Group 6) ( Fig. 6 B-C ) . This included bromodomain protein BRD4, ATP-dependent chromatic remodeler SMARCA4, and cassettes of lysine demethylases (KDMs), chromodomain helicase DNA binding proteins (CHDs), and lysine methyltransferases (KMTs) ( Fig. 6 C ) ( 38 ). This suggests that targeting KDM6A/KDM6B sets the stage for further cisplatin-mediated chromatin remodeling in TGCT cells. Gene sets enriched in genes exclusively downregulated by cisplatin in cisplatin-resistant PLKO.1 cells were related to senescence (Group 7). 4. Discussion Due largely to a dearth of driver mutations in contrast to many solid tumors, there have been no effective targeted therapies developed for TGCTs, which are mainly treated with cisplatin-based chemotherapies developed over 4 decades ago ( 10 , 11 ). While cisplatin has transformed metastatic testicular cancer from a deadly, to in the majority of cases, a curable disease, there are no effective backup therapies for the 15% of cisplatin-refractory/resistant patients who typically die from progressive disease ( 1 , 2 ). Further, curative cisplatin therapy results in acute and life-long toxicities, which are especially pertinent to the adolescent and young adult TGCT patient population ( 4 , 5 ). Strategies to decrease cisplatin exposure would likely lead to improved quality of life for these patients. In contrast to genetic alterations, recent evidence suggests that epigenetics is a major driving factor for TGCT formation, progression, and response or resistance to chemotherapy ( 7 , 8 , 39 ). Hence, targeting epigenetic pathways with “epidrugs” is one potential relatively unexplored strategy to advance TGCT treatment beyond cisplatin. In this report, we preclinically validate targeting polycomb demethylases KDM6A and KDM6B with epidrug GSK-J4 for the treatment of both cisplatin-sensitive and -resistant TGCTs. While GSK-J4 had minimal effects alone on TGCT tumor growth in vivo , it dramatically sensitized cisplatin-sensitive and -resistant TGCTs to cisplatin. We validated KDM6A/KDM6B as the target of GSK-J4 since KDM6A/KDM6B genetic depletion had a remarkably similar effect to GSK-J4 on cisplatin-mediated anti-tumor activity and transcriptome alterations. Pharmacologic and genetic targeting of KDM6A/KDM6B potentiated or primed the p53-dominant transcriptional response to cisplatin, with also evidence for basal activation of p53. Further, several chromatin modifier gene families were repressed with cisplatin only in KDM6A/KDM6B-targeted cells, implying that KDM6A/KDM6B inhibition sets the stage for extensive chromatin remodeling of TGCT cells upon cisplatin treatment. Another interesting finding of our study was the contrast between the dramatic cisplatin sensitization effect of GSK-J4 in vivo compared to cell culture. This suggests that perhaps there is priming of anti-tumor microenvironment and innate host immune mechanisms with GSK-J4 against TGCTs, a premise that is worthy of future study. Several lines of evidence suggest that TGCTs may be particularly driven by epigenetic alterations ( 6 – 9 ). TGCT are thought to derive from aberrantly differentiated primordial germ cells during a stage in development where the male germ line undergoes a dynamic wave of DNA methylation erasure. Issues that impact the microenvironment of male germ cell development in utero , including cryptorchidism, hypospadias, impaired spermatogenesis, high estrogen exposure, and exposure to endocrine disrupting chemicals, have been associated with TGCTs ( 40 – 44 ). Germline genetic disorders of sex development associated with fetal androgen insufficiency are also associated with an increased risk of germ cell malignancy ( 45 ). Further, TGCTs have a very low mutational burden and a low frequency of driver oncogenic or tumor-suppressive mutations, especially in nonseminomas ( 10 , 11 ). Evidence from our lab and others has shown that TGCT cells hyperactive p53 during cisplatin responses ( 37 , 46 – 48 ). The current work also suggests an important role for p53 in cisplatin sensitization upon KDM6A/KDM6B-targeting as p53 target gene expression was potentiated and basally activated. Due to their developmental origins, TGCTs may have unique and more open, embryonic stem cell-like chromatin as compared to somatic cell-derived solid tumors, which may make TGCTs uniquely vulnerable to certain epidrugs ( 9 , 49 ). We and others have shown that TGCT cells are exquisitely sensitive to hypomethylating agents (HMAs) at very low doses that are dependent on intrinsically high levels of DNMT3B ( 24 – 28 ). Further, pretreatment with HMAs can restore cisplatin sensitivity to cisplatin-resistant TGCT cells ( 24 – 26 ). Two recent clinical trials suggest that HMAs may have clinical activity in the setting of cisplatin refractory TGCTs ( 29 , 30 ). Utilizing isogenic HMA- and cisplatin-resistant cell lines, we found that sensitivity/resistance to HMAs and cisplatin appear to be mechanistically linked by epigenetic remodeling involving DNA methylation and the polycomb pathway. Namely, there is a common set of polycomb target genes upregulated in cisplatin-resistant TGCT cells due to a shift in DNA methylation linked to high levels of DNMT3B ( 31 – 33 ). Further, DNMT3B genetic targeting induced H3K27me3, EZH2, and BMI1 and resulted in increased sensitivity to cisplatin ( 33 ). The data presented here provides further evidence for this linkage as targeting KDM6A/KDM6B was associated with a decrease in DNMT3B levels along with increasing cisplatin sensitivity. Hence, HMAs and GSK-J4 may be essentially targeting the same pathway vulnerability in TGCTs, with GSK-J4 having perhaps the theoretical advantage of being less genotoxic compared to HMAs, which incorporate into DNA and form protein adducts with DNMTs. The precise mechanism for how DNMT3B and polycomb are linked to regulate cisplatin sensitivity of TGCTs will require further study. This report suggests that GSK-J4 may have cisplatin-sensitization properties for cisplatin-sensitive as well as cisplatin-resistant TGCT patients. The role of polycomb in cancer is complex ( 19 ). In solid tumors, polycomb has mainly been associated with oncogenesis and poor outcomes, which has spurred the clinical development of EZH2 inhibitors ( 50 ). However, loss-of-function PRC2 mutations also occur in a subset of tumor types, including malignant peripheral nerve sheath tumors (MPNSTs), pediatric gliomas, and T-cell acute lymphoblastic leukemia ( 17 – 19 ). This highlights the complex role of polycomb in tumorigenesis. This complexity extends to whether polycomb-mediated epigenetic changes are associated with cancer drug resistance ( 20 – 23 ). Targeting polycomb demethylases has not been well developed clinically for cancer therapy compared to EZH2 targeting, with a limited number of preclinical reports of GSK-J4 having anti-tumor activity ( 15 , 51 ). To our knowledge, GSK-J4 has not entered the clinic. Whether GSK-J4 or other KDM6A/KDM6B inhibitors have acceptable toxicity profiles will be important to ascertain. It is noteworthy that in our studies GSK-J4 and KDM6A/KDM6B-targeting had minimal effect alone, suggesting that a therapeutic window may exist for combination therapy in those tumors like TGCTs that already have a heightened sensitivity to cisplatin. 5. Conclusions The biology of testicular germ cell tumors appears to be especially driven by epigenetic mechanisms suggesting that they may be highly sensitive to epidrugs. Our findings preclinically validate targeting polycomb demethylases KDM6A/KDM6B as a potent pharmacologic strategy for treating cisplatin-resistant TGCTs that warrants further preclinical and clinical investigation. Abbreviations CHDs; chromodomain helicase DNA binding proteins, GSEA; gene set enrichment analysis, HMAs; DNA hypomethylating agents, KDMs; lysine demethylases. KMTs; lysine methyltransferases, MPNSTs; malignant peripheral nerve sheath tumors, PRCs; polycomb repressive complexes,PRC1; polycomb repressive 1,PRC2; polycomb repressive complex 2, TGCTs; testicular germ cell tumors Declarations Conflict of Interest Statement: The authors declare no potential conflicts of interest. Ethics approval and consent to participate. Not applicable Consent for publication Not applicable Availability of data and materials The RNA-seq datasets generated and/or analyzed during the current study are available in the NCBI Database of GEO Datasets under the accession number XXXXX. All other data generated or analyzed during this study are available from the corresponding authors on reasonable request. Competing Interests The authors declare that they have no competing interests. Funding This work was supported by the National Institutes of Health grants R01-CA211875 (MJS) and DOD PRCRP Impact Award W81XWH2110903 (MJS) and DOD Breakthrough Award BC221269 (MJS), NIEHS 5T32ES007326-25 (RIB), and a UIUC block grant (DS). Author contributions DS, ZF, SJF, MHP, MDV, JPS, MJS, and RS performed the conceptualization; DS, CP, SJ, BCR, RIB, ZS, MPH, and RS performed the methodology; DS and RS performed the validation; DS, MWK, ZF, MJS, and RS were involved in formal analysis; DS, MWK, CP, SJ, BRC, ZS, and RS performed the investigation; DS, MWK, ZF, and RS performed data curation; DS and MJS wrote the original draft preparation; RIB, SJF, MHP, MDV, JPS, MJS, and RS reviewed and edited the manuscript; DS and RS performed visualization; SJF, MJS, and RS were involved in supervision; SJF, MJS, and RS and were involved in project administration; DS, RIB, SJF, MJS, and RS contributed to funding acquisition. All authors reads and approved the final manuscript. Acknowledgments We dedicate this study to the memory of Dr. Sarah J. Freemantle, a valued scientist and cherished colleague and mentor. We would like to thank members of the Roy J. Carver Biotechnology Center at the University of Illinois, including Dr. Alvaro Hernandez and Chris Wright, for RNA sequencing. References Adra N, Einhorn LH. Testicular cancer update. Clin Adv Hematol Oncol. 15(5):386-96, 2017. Feldman DR, Patil S, Trinos MJ, Carousso M, Ginsberg MS, Sheinfeld J, Bajorin DF, Bosl GJ, Motzer RJ. Progression-free and overall survival in patients with relapsed/refractory germ cell tumors treated with single-agent chemotherapy: endpoints for clinical trial design. Cancer 118:981-6, 2012. Singh R, Fazal Z, Freemantle SJ, Spinella MJ. Mechanisms of cisplatin sensitivity and resistance in testicular germ cell tumors. Cancer Drug Resist. 2019;2(3):580-594. Fung C, Sesso HD, Williams AM, Kerns SL, Monahan P, Abu Zaid M, Feldman DR, Hamilton RJ, Vaughn DJ, Beard CJ, Kollmannsberger CK, Cook R, Althouse S, Ardeshir-Rouhani-Fard S, Lipshultz SE, Einhorn LH, Fossa SD, Travis LB. Multi-institutional assessment of adverse health outcomes among North American testicular cancer survivors after modern cisplatin-based chemotherapy. J Clin Oncol. 2017;35(11):1211-22. Shrem NS, Wood L, Hamilton RJ, Kuhathaas K, Czaykowski P, Roberts M, Matthew A, Izard JP, Chung P, Nappi L, Jones J, Soulières D, Aprikian A, Power N, Canil C. Testicular cancer survivorship: Long-term toxicity and management. Can Urol Assoc J. 2022 Aug;16(8):257-272. Lobo J, Gillis AJM, Jerónimo C, Henrique R, Looijenga LHJ. Human germ cell tumors are developmental cancers: Impact of epigenetics on pathobiology and clinic. Int J Mol Sci. 20(2). pii: E258, 2019. Singh R, Fazal Z, Freemantle SJ, Spinella MJ. Between a Rock and a Hard Place: An Epigenetic-Centric View of Testicular Germ Cell Tumors. Cancers (Basel). 2021 Mar 25;13(7):1506. Nicu AT, Medar C, Chifiriuc MC, Gradisteanu Pircalabioru G, Burlibasa L. Epigenetics and Testicular Cancer: Bridging the Gap Between Fundamental Biology and Patient Care. Front Cell Dev Biol. 2022 Apr 8;10:861995. Rijlaarsdam MA, Looijenga LH. An oncofetal and developmental perspective on testicular germ cell cancer. Semin Cancer Biol. 29:59-74, 2014. Litchfield, K.; Summersgill, B.; Yost, S.; Sultana, R.; Labreche, K.; Dudakia, D.; Renwick, A.; Seal, S.; Al-Saadi, R.; Broderick, P.; et al. Whole-exome sequencing reveals the mutational spectrum of testicular germ cell tumours. Nat. Commun. 2015, 6, 5973. Shen, H.; Shih, J.; Hollern, D.P.; Wang, L.; Bowlby, R.; Tickoo, S.K.; Thorsson, V.; Mungall, A.J.; Newton, Y.; Hegde, A.M.; et al. Integrated molecular characterization of testicular germ cell tumors. Cell Rep. 2018, 23, 3392–3406. Lee AV, Nestler KA, Chiappinelli KB. Therapeutic targeting of DNA methylation alterations in cancer. Pharmacol Ther. 2024 Jun;258:108640. doi: 10.1016/j.pharmthera.2024.108640. Epub 2024 Apr 1. PMID: 38570075. German B, Ellis L. Polycomb Directed Cell Fate Decisions in Development and Cancer. Epigenomes. 2022 Sep 6;6(3):28. Guo Y, Wang GG. Modulation of the high-order chromatin structure by Polycomb complexes. Front Cell Dev Biol. 2022 Oct 5;10:1021658. Abu-Hanna J, Patel JA, Anastasakis E, Cohen R, Clapp LH, Loizidou M, Eddama MMR. Therapeutic potential of inhibiting histone 3 lysine 27 demethylases: a review of the literature. Clin Epigenetics. 2022 Aug 1;14(1):98. An R, Li YQ, Lin YL, Xu F, Li MM, Liu Z. EZH1/2 as targets for cancer therapy. Cancer Gene Ther. 2023 Feb;30(2):221-235. doi: 10.1038/s41417-022-00555-1. Epub 2022 Nov 11. PMID: 36369341. Mohammad F, Weissmann S, Leblanc B, Pandey DP, Højfeldt JW, Comet I, Zheng C, Johansen JV, Rapin N, Porse BT, Tvardovskiy A, Jensen ON, Olaciregui NG, Lavarino C, Suñol M, de Torres C, Mora J, Carcaboso AM, Helin K. EZH2 is a potential therapeutic target for H3K27M-mutant pediatric gliomas. Nat Med. 2017;23(4):483-92. Epub 2017/03/07. doi: 10.1038/nm.4293. PubMed PMID: 28263309. Pekmezci M, Cuevas-Ocampo AK, Perry A, Horvai AE. Significance of H3K27me3 loss in the diagnosis of malignant peripheral nerve sheath tumors. Mod Pathol. 2017;30(12):1710-9. Epub 2017/08/05. doi: 10.1038/modpathol.2017.97. PubMed PMID: 28776579. Wang J, Wang GG. No easy way out for EZH2: Its pleiotropic, noncanonical effects on gene regulation and cellular function. Int J Mol Sci. 2020;21(24). Epub 2020/12/18. doi: 10.3390/ijms21249501. PubMed PMID: 33327550. Samaržija I, Tomljanović M, Novak Kujundžić R, Trošelj KG. EZH2 Inhibition and Cisplatin as a Combination Anticancer Therapy: An Overview of Preclinical Studies. Cancers (Basel). 2022 Sep 29;14(19):4761. doi: 10.3390/cancers14194761. PMID: 36230683; PMCID: PMC9561994. Wang Q, Chen X, Jiang Y, Liu S, Liu H, Sun X, Zhang H, Liu Z, Tao Y, Li C, Hu Y, Liu D, Ye D, Liu Y, Wang M, Zhang X. Elevating H3K27me3 level sensitizes colorectal cancer to oxaliplatin. J Mol Cell Biol. May 8, 2019. Hu S, Yu L, Li Z, Shen Y, Wang J, Cai J, Xiao L, Wang Z. Overexpression of EZH2 contributes to acquired cisplatin resistance in ovarian cancer cells in vitro and in vivo. Cancer Biol. Ther. 10, 788–795. 2010. Zhu Z, Tang J, Wang J, Duan G, Zhou L, Zhou X. MiR-138 acts as a tumor suppressor by targeting EZH2 and enhances cisplatin-induced apoptosis in osteosarcoma cells. PLoS ONE 11, 2016. Beyrouthy MJ, Garner KM, Hever MP, Freemantle SJ, Eastman A, Dmitrovsky E, Spinella MJ. High DNA methyltransferase 3B expression mediates 5-aza-deoxycytidine hypersensitivity in testicular germ cell tumors. Cancer Res 69:9360-6, 2009. Biswal BK, Beyrouthy MJ, Hever-Jardine MP, Armstrong D, Tomlinson CR, Christensen BC, Marsit CJ, Spinella MJ. Acute hypersensitivity of pluripotent testicular cancer-derived embryonal carcinoma to low-dose 5-aza deoxycytidine is associated with global DNA Damage-associated p53 activation, anti-pluripotency and DNA demethylation. PLoS One 7:e53003, 2012. Albany C, Hever-Jardine MP, von Herrmann KM, Yim CY, Tam J, Warzecha JM, Shin L, Bock SE, Curran BS, Chaudhry AS, Kim F, Sandusky GE, Taverna P, Freemantle SJ, Christensen BC, Einhorn LH, Spinella MJ. Refractory testicular germ cell tumors are highly sensitive to the second generation DNA methylation inhibitor guadecitabine. Oncotarget 8:2949-59, 2017. Wongtrakoongate P, Li J, Andrews PW. Aza-deoxycytidine induces apoptosis or differentiation via DNMT3B and targets embryonal carcinoma cells but not their differentiated derivatives. Br J Cancer 110:2131-8, 2014. Oing C, Verem I, Mansour WY, Bokemeyer C, Dyshlovoy S, Honecker F. 5-azacitidine exerts prolonged pro-apoptotic effects and overcomes cisplatin-resistance in non-seminomatous germ cell tumor cells. Int J Mol Sci. 20. pii: E21, 2018. Albany C, Fazal Z, Singh R, Bikorimana E, Adra N, Hanna NH, Einhorn LH, Perkins SM, Sandusky GE, Christensen BC, Keer H, Fang F, Nephew KP, Spinella MJ. A phase 1 study of combined guadecitabine and cisplatin in platinum refractory germ cell cancer. Cancer Med. 2021 Jan;10(1):156-163. Crabb SJ, Danson S, Catto JWF, Hussain S, Chan D, Dunkley D, Downs N, Marwood E, Day L, Saunders G, Light M, Whitehead A, Ellis D, Sarwar N, Enting D, Birtle A, Johnson B, Huddart R, Griffiths G. Phase I Trial of DNA Methyltransferase Inhibitor Guadecitabine Combined with Cisplatin and Gemcitabine for Solid Malignancies Including Urothelial Carcinoma (SPIRE). Clin Cancer Res. 2021 Apr 1;27(7):1882-1892. Fazal Z, Singh R, Fang F, Bikorimana E, Baldwin H, Corbet A, Tomlin M, Yerby C, Adra N, Albany C, Lee S, Freemantle SJ, Nephew KP, Christensen BC, Spinella MJ. Hypermethylation and global remodelling of DNA methylation is associated with acquired cisplatin resistance in testicular germ cell tumours. Epigenetics. 2020:1-14. Singh R, Fazal Z, Corbet AK, Bikorimana E, Rodriguez JC, Khan EM, Shahid K, Freemantle SJ, Spinella MJ. Epigenetic remodeling through downregulation of polycomb repressive complex 2 mediates chemotherapy resistance in testicular germ cell tumors. Cancers. 11(6). pii: E796, 2019. Singh R, Fazal Z, Bikorimana E, Boyd RI, Yerby C, Tomlin M, Baldwin H, Shokry D, Corbet AK, Shahid K, Hattab A, Freemantle SJ, Spinella MJ. Reciprocal epigenetic remodeling controls testicular cancer hypersensitivity to hypomethylating agents and chemotherapy. Mol Oncol. 2022 Feb;16(3):683-698. Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, Smyth GK. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015 Apr 20;43(7):e47. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, Pomeroy SL, Golub TR, Lander ES, Mesirov JP. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005 Oct 25;102(43):15545-50. Shen L & Sinai ISoMaM (2020) GeneOverlap: test and visualize gene overlaps. R package version 1.26.0. http://shenlab-sinai.github.io/shenlab-sinai/ Kerley-Hamilton JS, Pike AM, Li N, DiRenzo J and Spinella MJ. A p53-dominant transcriptional response to cisplatin in testicular germ cell tumor-derived human embryonal carcinoma. Oncogene. 24:6090-100, 2005. Liberzon A, Subramanian A, Pinchback R, Thorvaldsdóttir H, Tamayo P, Mesirov JP. Molecular signatures database (MSigDB) 3.0. Bioinformatics. 2011 Jun 15;27(12):1739-40. Nicu AT, Ionel IP, Stoica I, Burlibasa L, Jinga V. Recent Advancements in Research on DNA Methylation and Testicular Germ Cell Tumors: Unveiling the Intricate Relationship. Biomedicines. 2024 May 8;12(5):1041. doi: 10.3390/biomedicines12051041. PMID: 38791003; PMCID: PMC11117643. Bräuner, E.V.; Lim, Y.H.; Koch, T.; Uldbjerg, C.S.; Gregersen, L.S.; Pedersen, M.K.; Frederiksen, H.; Petersen, J.H.; Coull, B.A.; Andersson, A.M.; et al. Endocrine disrupting chemicals and risk of testicular cancer: A systematic review and meta-analysis. J. Clin. Endocrinol. Metab. 2021, 106, e4834–e4860. Sharma, A.; Mollier, J.; Brocklesby, R.W.K.; Caves, C.; Jayasena, C.N.; Minhas, S. Endocrine-disrupting chemicals and male reproductive health. Reprod. Med. Biol. 2020, 19, 243–253. Skakkebaek, N.E.; Rajpert-De Meyts, E.; Buck Louis, G.M.; Toppari, J.; Andersson, A.M.; Eisenberg, M.L.; Jensen, T.K.; Jørgensen, N.; Swan, S.H.; Sapra, K.J.; et al. Male reproductive disorders and fertility trends: Influences of environment and genetic susceptibility. Physiol. Rev. 2016, 96, 55–97. Selvi, I.; Ozturk, E.; Yikilmaz, T.N.; Sarikaya, S.; Basar, H. Effects of testicular dysgenesis syndrome components on testicular germ cell tumor prognosis and oncological outcomes. Int. Braz. J. Urol. 2020, 46, 725–740. Hanson, H.A.; Anderson, R.E.; Aston, K.I.; Carrell, D.T.; Smith, K.R.; Hotaling, J.M. Subfertility increases risk of testicular cancer: Evidence from population-based semen samples. Fertil. Steril. 2016, 105, 322–328.e321. Trabert, B.; Zugna, D.; Richiardi, L.; McGlynn, K.A.; Akre, O. Congenital malformations and testicular germ cell tumors. Int. J. Cancer 2013, 133, 1900–1904. Gutekunst M, Oren M, Weilbacher A, Dengler MA, Markwardt C, Thomale J, Aulitzky WE and van der Kuip H. p53 hypersensitivity is the predominant mechanism of the unique responsiveness of testicular germ cell tumor (TGCT) cells to cisplatin. PLoS One . 6:e19198, 2011. Duale N, Lindeman B, Komada M, Olsen AK, Andreassen A, Soderlund EJ and Brunborg G. Molecular portrait of cisplatin induced response in human testis cancer cell lines based on gene expression profiles. Mol Cancer. 6:53, 2007. Song W, Wang J, Yang Y, Jing N, Zhang X, Chen L and Wu J. Rewiring drug-activated p53-regulatory network from suppressing to promoting tumorigenesis. J Mol Cell Biol. 4:197-206, 2012. Van Mierlo G, Dirks RAM, De Clerck L, Brinkman AB, Huth M, Kloet SL, Saksouk N, Kroeze LI, Willems S, Farlik M, Bock C, Jansen JH, Deforce D, Vermeulen M, Déjardin J, Dhaenens M, Marks H Integrative proteomic profiling reveals PRC2-dependent epigenetic crosstalk maintains ground-state pluripotency. Cell Stem Cell 24, 123–137, 2019. Liu Y, Yang Q. The roles of EZH2 in cancer and its inhibitors. Med Oncol. 2023 May 6;40(6):167. Dalpatraj N, Naik A, Thakur N. GSK-J4: An H3K27 histone demethylase inhibitor, as a potential anti-cancer agent. Int J Cancer. 2023 Sep 15;153(6):1130-1138. Additional Declarations No competing interests reported. Supplementary Files SupplementalTableS1.xlsx Supplemental Table 1. The top 20 gene sets from GSEA, as determined by NES, enriched for upregulated and downregulated genes for each experimental arm in Figure 4. SupplementalTableS2.xlsx Supplemental Table 2. Gene lists corresponding to Venn Diagrams of Figure 6. P53 target genes in TGCT cells as previously highlighted in Kerley et al (37) are in highlighted text. SupplementalTableS3.xlsx Supplemental Table 3. The top 20 gene sets from GeneOverlap analysis, as determined by p value, enriched for Group 1 through Group 7 comparisons of Figure 6. FigureS1.jpg Supplemental Figure 1. Body weight measurements for xenograft studies of Figure 3. FigureS2.jpg Supplemental Figure 2. Xenograft study of combination of EZH2 inhibitor GSK-126 and cisplatin in 2012EP cells. Cite Share Download PDF Status: Published Journal Publication published 31 Oct, 2024 Read the published version in Cell Communication and Signaling → Version 1 posted Editorial decision: Revision requested 01 Oct, 2024 Reviews received at journal 25 Sep, 2024 Reviews received at journal 18 Sep, 2024 Reviewers agreed at journal 04 Sep, 2024 Reviewers agreed at journal 04 Sep, 2024 Reviewers agreed at journal 04 Sep, 2024 Reviewers agreed at journal 03 Sep, 2024 Reviewers invited by journal 02 Sep, 2024 Editor assigned by journal 29 Aug, 2024 Submission checks completed at journal 29 Aug, 2024 First submitted to journal 27 Aug, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Cells were assayed for viability 3 days later. U87-MG glioblastoma, HCT116 colon cancer, and MDA-MB-231 and MCF7 breast cancer cells were treated similarly, except cisplatin dosages were higher due to less inherent sensitivity to cisplatin compared to TGCT cells.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4986186/v1/d4b2fb2b56937e8960bc0977.jpg"},{"id":66944375,"identity":"ac56f934-29f6-4b43-9182-62567420e33c","added_by":"auto","created_at":"2024-10-18 09:36:18","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2424446,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown of polycomb components EZH2 and BMI1 confer cisplatin resistance in TGCT cells, while knockdown of polycomb demethylases KDM6A/KDM6B sensitizes TGCT cells to cisplatin\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e(A) RT-PCR demonstrating efficient knockdown of EZH2 and BMI1 in TGCT cells NT2/D1, 2102EP, and 833K. (B) Western blot demonstrating efficient knockdown of EZH2 and BMI1 in 2012EP and 833K cells and repression of H3K27me3 levels with EZH2 knockdown. (C) Cell proliferation and viability assays of control pLKO.1 and EZH2- and BMI1-knockdown cells treated with cisplatin. (D,E) RT-PCR demonstrating single and dual KDM6A- and KDM6B-knockdown in 2102EP, 2012EP-C1, NT2/D1, and NT2/D1-A4 cells and repression of DNMT3B expression upon KDM6A/KDM6B-knockdown. (F) Western blot demonstrating induction of H3K27me3 and repression of DNMT3B expression with single and dual knockdown of KDM6A and KDM6B in 2102EP-C1 cells. (G,H) Cell proliferation and viability assays of control PLK pLKO.1 and KDM6A/KDM6B-knockdown cells treated with cisplatin.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4986186/v1/b767deb8d300fa152822b766.jpg"},{"id":66946378,"identity":"3f2096ca-4fdf-4f41-87df-1f637bb6602f","added_by":"auto","created_at":"2024-10-18 09:44:18","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3334199,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKDM6A/KDM6B inhibitor GSK-J4 and dual KDM6A/KDM6B-knockdown sensitize human TGCT tumor xenografts to cisplatin. \u003c/strong\u003e(A) Schematic of GSK-J4, cisplatin, or GSK-J4 + cisplatin treatment schedule for cisplatin-resistant 2102EP-C1 and cisplatin-sensitive NT2/D1 cells xenografts (left) and schematic of cisplatin treatment schedule for KDM6A/KDM6B dual knockdown or control 2102EP-C1 cells (right). (B-J) Depicted are tumor volume, percent change in tumor volume from the day prior to treatment initiation (day 0), and survival of mouse xenografts for 2102EP-C1 and NT2/D1 tumors treated with GSK-J4, cisplatin, or GSK-J4 + cisplatin (left) or xenograft tumors of KDM6A/KDM6B dual knockdown or control 2102EP-C1 cells (right). TV, tumor volume.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4986186/v1/8075e44104b4d7e4461178ee.jpg"},{"id":66946381,"identity":"4ab5724f-d110-41e4-94a3-f16ebe2e7426","added_by":"auto","created_at":"2024-10-18 09:44:18","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3403336,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptome analysis of GSK-J4 treated and KDM6A/KDM6B knockdown cells reveals the importance of basal activation of gene expression and p53 signaling in cisplatin sensitization. \u003c/strong\u003e(A) Multidimensional scaling (MDS) plots of the three RNA-seq experiments NT2/D1-A4 and 2102EP-C1 cells treated with GSK-J4, cisplatin, or GSK-J4 + cisplatin and KDM6A/KDM6B dual knockdown or control 2102EP-C1 cells treated with cisplatin. (B) Enhanced volcano plots of the three RNA-seq experiments. For the first two experiments, each treatment (cisplatin, GSK-J4, or the combination) is compared to untreated vehicle control. For the third experiment, PLK control + cisplatin, untreated sh6A+6B, and sh6A+6B + cisplatin cells are compared to untreated PLK cells. Significant cutoff is fold-change \u0026gt;1.3 and FDR \u0026lt; 0.05. The number of up- and downregulated genes are provided. (C) Gene set enrichment analysis (GSEA) results corresponding to the volcano plot comparisons for upregulated genes. Top 5 gene sets as determined by normalized enrichment score (NES) from the MSigDB C2 collection are provided. P53 target gene collections are highlighted. The top 20 gene sets enriched for upregulated and downregulated genes for each experimental arm for all three RNA-seq experiments are provided in \u003cstrong\u003eSupplemental Table S1\u003c/strong\u003e. Red text highlights p53 target gene sets, including KERLEY_RESPONSE_TO_CISPLATIN, which we previously identified to be a p53 dominate gene set in cisplatin treated NT2/D1 cells (37).\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4986186/v1/66bd1f24774c628192559d95.jpg"},{"id":66944371,"identity":"3c57e58d-67c8-4b65-a4b4-5ac0e39a3223","added_by":"auto","created_at":"2024-10-18 09:36:18","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":986327,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePolycomb demethylase targeting potentiates p53 target gene activation and represses DNMT3B expression in TGCT cells. \u003c/strong\u003e\u0026nbsp;(A,B,C) Expression of select p53 target genes across the 4 experimental arms of the three RNA-seq experiments of Figure 4. (D,E,F) DNMT3B expression across the 4 experimental arms of the three RNA-seq experiments of Figure 4.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4986186/v1/159cff0dd279e7959f0f4ed8.jpg"},{"id":66947185,"identity":"96271d3c-25d6-47ab-b210-793cc8e6dd22","added_by":"auto","created_at":"2024-10-18 09:52:18","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4124873,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptome analysis of KDM6A/KDM6B-knockdown cells reveals cisplatin sensitization is associated with alterations in chromatin remodeling genes upon cisplatin treatment. \u003c/strong\u003e(A) Venn diagram comprised of comparing genes upregulated in PLKO.1 cisplatin-treated vs PLKO.1 untreated, sh6A/KDM6B untreated vs PLKO.1 untreated, and sh6A/KDM6B cisplatin vs sh6A/6B untreated. Also depicted are gene set enrichment analysis (GSEA) results corresponding to indicated comparison groups. Top 10 gene sets from GeneOverlap analysis as determined by p value from the MSigDB C2 collection are provided. P53 target gene collections are highlighted. malignant peripheral nerve sheath tumors (MPNSTs), B) Venn diagram comprised of comparing genes downregulated in PLKO.1 cisplatin-treated vs PLKO.1 untreated, sh6A/6B untreated vs PLKO.1 untreated, and sh6A/6B cisplatin-treated vs sh6A/6B untreated. Also depicted are GeneOveralp results corresponding to indicated comparison groups. Top 10 gene sets as determined by p value from the MSigDB C2 collection are provided. The REACTOME_CHROMATIN_MODIFYING_ENZYMES gene set is highlighted. (C) Expression of select chromatin-modifying enzymes and proteins across the 4 experimental arms of the RNA-seq experiment. P53 target gene sets are in red text, H3K27me3/polycomb gene sets are in blue text, REACTOME_CHROMATIN_MODIFYING_ENZYMES gene set is in green text.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4986186/v1/a9e96ea7bb3e93e679b205d7.jpg"},{"id":68207155,"identity":"3fbba69e-341e-4185-8740-9c3178758786","added_by":"auto","created_at":"2024-11-04 16:35:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16424922,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4986186/v1/c997ad71-3f85-4ac1-b66c-4c0911e70fa6.pdf"},{"id":66949039,"identity":"9b6abd1e-cad4-4584-bad4-65619ec89e0b","added_by":"auto","created_at":"2024-10-18 10:00:18","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":26437,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Table\u003c/strong\u003e \u003cstrong\u003e1\u003c/strong\u003e. The top 20 gene sets from GSEA, as determined by NES, enriched for upregulated and downregulated genes for each experimental arm in Figure 4.\u003c/p\u003e","description":"","filename":"SupplementalTableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4986186/v1/3c6be13031aa4b41f013f218.xlsx"},{"id":66944370,"identity":"1f9eab8b-9f80-4e00-b71d-65f527828bbf","added_by":"auto","created_at":"2024-10-18 09:36:17","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":69618,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Table 2.\u003c/strong\u003e Gene lists corresponding to Venn Diagrams of Figure 6. P53 target genes in TGCT cells as previously highlighted in Kerley et al (37) are in highlighted text.\u003c/p\u003e","description":"","filename":"SupplementalTableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4986186/v1/d1ee417634619dd02cafc833.xlsx"},{"id":66947184,"identity":"6abd0eaf-db46-4591-bc40-643d9caac881","added_by":"auto","created_at":"2024-10-18 09:52:18","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":18119,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Table 3\u003c/strong\u003e. The top 20 gene sets from GeneOverlap analysis, as determined by p value, enriched for Group 1 through Group 7 comparisons of Figure 6.\u003c/p\u003e","description":"","filename":"SupplementalTableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4986186/v1/8c2917fe44a4d22089f17253.xlsx"},{"id":66944376,"identity":"b7d80520-e942-4921-8757-7afb93f3b7f2","added_by":"auto","created_at":"2024-10-18 09:36:18","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":700006,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Figure 1. \u003c/strong\u003eBody weight measurements for xenograft studies of Figure 3.\u003c/p\u003e","description":"","filename":"FigureS1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4986186/v1/a86f976186fb6318d46e486a.jpg"},{"id":66944379,"identity":"4b5287b1-0536-45aa-842b-0179f5b7c4e1","added_by":"auto","created_at":"2024-10-18 09:36:18","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":393436,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Figure 2. \u003c/strong\u003eXenograft study of combination of EZH2 inhibitor GSK-126 and cisplatin in 2012EP cells.\u003c/p\u003e","description":"","filename":"FigureS2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4986186/v1/6374809b5313ec5a9b71e9d4.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Refractory testicular germ cell tumors are highly sensitive to the targeting of polycomb pathway demethylases KDM6A and KDM6B","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eTesticular germ cell tumors (TGCTs) are curable with traditional cisplatin-based chemotherapy (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). However, cisplatin resistance occurs in approximately 15% of metastatic patients, with the majority dying from progressive disease (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). No effective therapies exist for this patient population, and no clinically effective strategies exist to overcome cisplatin resistance, likely due to a lack of detailed understanding of the mechanisms responsible for cisplatin resistance in TGCTs (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Further, testicular cancer is the most common carcinoma of males ages 15 to 45 and cisplatin-based cures are associated with many toxicities and co-morbidities, including ototoxicity, infertility, neuropathy, and a high risk of secondary cancers (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Hence, cisplatin-sparing strategies for this young patient population are needed.\u003c/p\u003e \u003cp\u003eTGCTs are believed to arise from aberrant differentiation of primordial germ cells during development (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). There is mounting evidence that TGCTs may be a cancer that is especially driven by epigenetic dysfunction, both in terms of etiology and response or resistance to chemotherapy (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). This includes a unique pattern of DNA hypomethylation and histone modifications compared to most other tumors due to their embryonic origins at a stage undergoing extensive epigenetic reprogramming, an association with \u003cem\u003ein utero\u003c/em\u003e endocrine disruption and environmental stress, and a lack of driver DNA mutations (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Particularly prominent is a lack of p53 mutations that is a proposed factor for high cisplatin curability. The possible unique reliance on distinct epigenetic drivers suggests that TGCTs may be uniquely sensitive to epigenetic-based therapies (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTwo major epigenetic pathways associated with target gene repression are CpG island methylation mediated by DNA methyltransferases DNMT1, DNMT3A, and DNMT3B and the polycomb pathway (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). There are two major polycomb repressive complexes (PRCs), PRC1 and PRC2. PRC2 contains the core components EZH2, SUZ12, and EED, while PRC1 is comprised of BMI1, CBX, RIN1A/B, and PCH (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). EZH2 catalyzes H3K27 trimethylation, which is a docking site for the PRC1 complex that catalyzes monoubiquitination of H2A on K119 (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Both modifications repress gene expression. Additionally, the histone demethylases KDM6A and KDM6B remove H3K27 methylation (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). The role of the PRC2 complex in cancer is complex, with the majority of studies suggesting that EZH2 promotes oncogenesis and is a pharmacologic cancer target (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). However, there are several examples of documented tumor suppressor functions of PRC2 (\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). This complexity extends to whether polycomb represses or augments chemotherapy responses (\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe and others have shown that TGCT cells are hypersensitive to low doses of hypomethylating agents (HMAs), such as decitabine and guadecitabine, including cisplatin-resistant cells, and that pretreatment with HMAs can reverse cisplatin resistance in cell and mouse xenograft models (\u003cspan additionalcitationids=\"CR25 CR26 CR27\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). This strategy has been tested clinically with some promising patient responses in small trials (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Furthermore, we developed cisplatin-resistant TGCT isogenic cell models and demonstrated that cisplatin resistance is associated with a coordinated decrease in EZH2, BMI1, and H3K27me3 levels coupled with a bi-directional shift between gene promoter and gene body DNA methylation among multiple gene sets resulting in an upregulation of polycomb target genes and a downregulation of tumor suppressor genes (\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). A gene signature based on polycomb target genes was also associated with recurrent and progressive disease in TGCT patients (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Further, DNMT3B levels were highly upregulated in cisplatin-resistant TGCT cells compared to isogenic parental cells, and DNMT3B-knockdown alone in parental cells was sufficient to induce H3K27me3, EZH2, and BMI1 levels and cisplatin hypersensitivity (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). This suggests that DNA methylation and polycomb are coordinately regulated in TGCTs to modulate cisplatin sensitivity.\u003c/p\u003e \u003cp\u003eThe apparent connectedness of DNA methylation and polycomb signaling alterations with cisplatin resistance, coupled with the demonstrated promising preclinical and clinical activity of DNA methylation targeting HMAs in TGCTs, prompted us to examine whether polycomb targeting may be a second epigenetic-based treatment for cisplatin-resistant TGCTs. Genetic and pharmacologic inhibition of polycomb H3K27me3 demethylases KDM6A and KDM6B sensitized cisplatin-resistant and wild-type TGCT cells to cisplatin and produced dramatic synergistic tumor regression in animal models. This was associated with decreased expression of DNMT3B. Transcriptome analysis revealed a robust alteration in gene expression with polycomb demethylase targeting and basal and cisplatin-mediated potentiation of p53 target gene activation and downregulation of multiple chromatin-modifying enzymes in response to cisplatin. Our findings preclinically validate targeting polycomb demethylases KDM6A/B as a potent pharmacologic strategy for treating cisplatin-resistant TGCTs that warrants further preclinical and clinical investigation.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Drug treatments and cell viability and proliferation assays\u003c/h2\u003e \u003cp\u003eAll cells were cultured in DMEM (Sigma) with 10% FBS (GeminiBio). The NT2/D1, 833K, and 2102EP cells are human testicular cancer-derived embryonal carcinoma cell lines and colon cancer HCT116, breast cancer MDA-MB-231 and MCF7, and glioblastoma U87-MG cell lines were all purchased from ATCC and authenticated by ATCC with karyotyping and short tandem repeat profiling, as described (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Cells were frozen within 1 month of purchase and used within 2 months of resuscitation. Derivation of cisplatin-resistant NT2/D1-A4 and 2102EP-C1 cells was previously described in detail (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCells were treated with the indicated dosages of cisplatin (Sigma) for 6 hours and cells assayed for survival 3 days later. For sequential treatments, cells were pretreated with the EZH2 inhibitor GSK-126 or the KDM6A/KDM6B histone demethylase inhibitor GSK-J4 (both from Selleck Chemicals) for 3 days at doses that alone did not affect viability by more than 5% (0.5 \u0026micro;M and 1.0 \u0026micro;M, respectively) and then treated with cisplatin (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). To assess cell viability, CellTiter-Glo (Promega) assays were performed. For each cell line, three biological replicates were tested at each concentration, and experiments were repeated at least twice on different days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Lentiviral shRNA knockdown\u003c/h2\u003e \u003cp\u003eLentiviruses were produced by co-transfecting HEK293 cells with 10 \u0026micro;g of the viral packaging vector pCMV-dR8.2 and envelope vector pCMV-VSV-G (10:1 ratio) and 10 \u0026micro;g of lenti-shRNA or +\u0026thinsp;targeting BMI1 (TRCN0000020155, TRCN0000020156, TRCN0000020157), EZH2 (TRCN0000018365, TRCN0000040074 and TRCN0000040075), KDM6A (TRCN0000107760, TRCN0000107761, TRCN0000107762, TRCN0000107763, TRCN0000107764) and KDM6B (TRCN0000359976, TRCN0000236678, TRCN0000236677, TRCN0000236676, TRCN0000236679), were purchased from Sigma along with pLKO.1-puro empty vector control (SHC001). The HEK293 cell medium was changed 24 hours after transfection and cells were incubated for 48 hours to allow for virus production. After 48 hours, HEK293 medium containing viral particles was filtered and transferred onto 833K, 2102EP, or NT2/D1 cells for 48 hours. Cells were selected with 5 \u0026micro;g/ml puromycin. For dual KDM6A and KDM6B knockdown, a combination of two lentivirus pLKO.1-puro, pLKO.1-Neo (Addgene plasmid #13425), shRNA targeting KDM6A (TRCN0000107764-neo) and shRNA targeting KDM6B (TRCN0000236676) were used. 48-hour post-transduction cells were selected with 5 \u0026micro;g/ml puromycin and 250 \u0026micro;g/ml neomycin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Xenograft experiments\u003c/h2\u003e \u003cp\u003e All animal experiments were approved by the University of Illinois Urbana-Champaign IACUC under protocol 24080. Under this protocol maximum allowable tumor size is 15 mm in any direction, which was not exceeded. For mouse studies, 5\u0026ndash;8-week-old male athymic nude mice (Jackson Labs) were injected subcutaneously in the flank with 5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e 2102EP-C1 and NT2/D1 cells after resuspension in a 50:50 ratio of DMEM/Matrigel (Corning). Once palpable tumors were detected, tumor volume was measured twice weekly with calipers using the formula V= (L \u0026times; W \u0026times; W)/2. At a tumor volume of approximately 150 mm\u003csup\u003e3\u003c/sup\u003e (day 1), mice were randomly assigned to vehicle (PBS), GSK-J4, cisplatin, or a combination of cisplatin\u0026thinsp;+\u0026thinsp;GSK-J4 treatments. GSK-J4 was given by intraperitoneal (IP) injection every other day for 6 total injections at 50 mg/kg (days 1, 3, 5, 7, 9 and 11). Cisplatin was given as a single IP injection on day 5 at 7.5 mg/kg for 2102EP-C1 cells and 6.0 mg/kg for NT2/D1 cells. In separate experiments, mice were injected with 5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e 2102EP-C1-pLKO.1 cells or 2102EP-C1-shKDM6A\u0026thinsp;+\u0026thinsp;shKDM6B (sh6A\u0026thinsp;+\u0026thinsp;6B) cells. Once tumors reached a volume of 150 mm\u003csup\u003e3\u003c/sup\u003e mice were randomly assigned to a single IP injection of either PBS or 7.5 mg/Kg cisplatin. Body weight was also measured twice weekly. Mice were sacrificed when tumors reached humane endpoints with euthanasia by carbon dioxide followed by cervical dislocation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 RNA-sequencing\u003c/h2\u003e \u003cp\u003eRNA was extracted from cisplatin-resistant NT2/D1-A4 and 2102EP-C1 cells pretreated with only 1.0 \u0026micro;M GSK-J4 for 3 days, treated with only 0.5 \u0026micro;M cisplatin for 6 hours, or both. RNA was also extracted from 2102EP-C1-pLKO.1 or 2102EP-C1-shKDM6A\u0026thinsp;+\u0026thinsp;shKDM6B (sh6A\u0026thinsp;+\u0026thinsp;6B) cells treated with PBS or 0.5 \u0026micro;M cisplatin for 6 hours. In all cases, cells were harvested for RNA 24 hours after cisplatin treatment. RNA was isolated with the RNeasy plus Mini Kit (Qiagen) and RNA sequencing was performed by the Roy J. Carver Biotechnology Center. RNA-Seq libraries were prepared using the TruSeq Stranded mRNA Sample Prep kit. The libraries were sequenced on a HiSeq 4000 using HiSeq 4000 sequencing kit version 1. Initial quality control was performed using FASTQC. Trimmomatic was used to remove low-quality bases from both ends LEADING\u0026thinsp;\u0026le;\u0026thinsp;28 and TRAILING\u0026thinsp;\u0026le;\u0026thinsp;28, respectively, with a minimum length of 30. The reads in FASTQ format were aligned to human genome assembly NCBI GRCh38.p14 using STAR aligner. Reads were counted and assigned to genes using featureCount. The \u0026ldquo;Limma\u0026rdquo; R package was used to identify differentially expressed genes (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Genes whose expression was not greater than 0.5 counts per million in at least 2 samples were removed and the resultant filtered expression matrix was TMM-normalized. Benjamini-Hochberg False Discovery Rate (FDR) was used to correct for multiple hypotheses.\u003c/p\u003e \u003cp\u003eThe \u0026ldquo;Enhanced Volcano\u0026rdquo; R package was used to visualize volcano plots. The RNA-seq datasets for the current study have been submitted to the NCBI Database of GEO Datasets under the accession numbers GSEXXXX.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Downstream enrichment analysis\u003c/h2\u003e \u003cp\u003eGene Set Enrichment Analysis (GSEA) from the Broad Institute was performed to identify enriched gene sets (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). GeneOverlap package from R Bioconductor was used to identify significant gene set overlap between common or exclusive gene expression changes between pLKO.1 control cells (pLKO.1 cisplatin-treated vs pLKO.1 untreated), those genes basally regulated by KDM6A and KDM6B knockdown (sh6A\u0026thinsp;+\u0026thinsp;6B untreated vs pLKO.1 untreated), and those genes regulated by cisplatin in KDM6A and KDM6B knockdown cells (sh6A\u0026thinsp;+\u0026thinsp;6B cisplatin-treated vs sh6A\u0026thinsp;+\u0026thinsp;6B untreated) and C2 gene sets from the MSigDB database (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Western analysis and real-time PCR\u003c/h2\u003e \u003cp\u003eFor Western analysis cells were lysed in radioimmune precipitation buffer and separated by SDS-PAGE. Antibodies to actin (MA1-744, Thermo Fisher), DNMT3B (HPA001595, Atlas Antibodies) Ubiquitin H2A-K119 (3240, Cell Signaling Technology), H3K27me3 (9733, Cell Signaling Technology), BMI1 (6964, Cell Signaling Technology), EZH2 (5246, Cell Signaling Technology) and histone H3 (ab1791, Abcam) were used. Total cellular RNA was isolated using the RNeasy Mini Kit (Qiagen), and complementary DNAs (cDNAs) were synthesized using High-capacity cDNA Synthesis Kit (Thermo Fisher Scientific). Quantitative real-time PCR assays were performed with PowerUp\u0026trade; SYBR\u0026trade; green master mix (Thermo Fisher Scientific) and the QuantStudio 3 Real-time System (Thermo Fisher Scientific). In all cases gene expression was normalized to β-actin. Primers for RT-PCR will be provided upon request.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Statistics\u003c/h2\u003e \u003cp\u003eStudent\u0026rsquo;s t-tests and ANOVA were performed using GraphPad Prism 10. \u003cem\u003ep\u003c/em\u003e-values indicative of non-significance (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) and significance (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05; * \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.01; **, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.001; *** and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.0001; ****) were determined. Mean and standard error of the mean were used to describe sample variability.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e \u003cb\u003ePharmacologic repression of the polycomb pathway by inhibition of polycomb methylase EZH2 with GSK-126 confers cisplatin resistance to TGCT cells but not other cancer cell types, while polycomb induction by inhibition of polycomb demethylases KDM6A and KDM6B with GSK-J4 sensitizes TGCT cells to cisplatin.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe showed previously that multiple isogeneic cisplatin-resistant TGCT cell lines had a reduction in the polycomb repressive mark H3K27me3 and reduced levels of polycomb repressive complex 2 (PRC2) component EZH2 and polycomb repressive 1 (PRC1) component BMI1 with a corresponding induction of polycomb target genes (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). We also show previously in multiple cell lines and in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, that inhibition of polycomb signaling with the EZH2 inhibitor GSK-J4 confers cisplatin resistance in parental 2102EP TGCT cells, while potentiation of polycomb signaling with an inhibitor of the H3K27me3 demethylases KDM6A and KDM6B, called GSK-J4, confers cisplatin sensitization in cisplatin-resistant 2102EP-C1 TGCT cells (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Note, cells were pretreated with GSK-J4 and GSK-126 for 3 days at doses previously established to not affect cell proliferation or viability as single agents (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). To address whether polycomb has the ability to generally alter the cisplatin sensitivity of cancer cells, we tested the effects of GSK-126 and GSK-J4 on a number of non-TGCT cancer cell lines, including breast cancer cells MCF-7 and MDA-231, colon cancer cells HCT116, and glioblastoma cells U87-MG. GSK-126 and GSK-J4 had minimal effects on cisplatin sensitivity of these cell lines, suggesting that cisplatin sensitivity of TGCT cells may be uniquely altered by polycomb \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eKnockdown of EZH2 and BMI1 confers cisplatin resistance in wild-type TGCT cells, while knockdown of KDM6A/KDM6B sensitizes TGCT cells to cisplatin\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eWe next tested whether genetic perturbation of the polycomb pathway could alter the cisplatin sensitivity of TGCT cells. Inhibition of polycomb signaling by EZH2- or BMI1-knockdown conferred cisplatin resistance in parental cisplatin-sensitive TGCT 833K, 2102EP, and NT2/D1 cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C\u003cb\u003e)\u003c/b\u003e. Note, while BMI1 shRNA decreased BMI1 levels, it did not alter biological target Ub-H2AK119, while EZH2-knockdown did repress H3K27me3 levels as expected \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. This implies that BMI1 may have a Ub-H2AK119-independent effect on TGCT cells. Reciprocally, induction of polycomb signaling with dual knockdown of KDM6A and KDM6B resulted in cisplatin sensitization in both cisplatin-sensitive NT/2D1 and 2102EP cells and cisplatin-resistant counterparts, NT2/D1-A4 and 2102EP-C1 cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-H\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlso, DNMT3B-knockdown sensitized parental TGCT cells to cisplatin and induced BMI1, EZH2, and H3K27me3 levels (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Consistent with this interconnected relationship, pharmacologic inhibition of KDM6A/KDM6B with GSK-J4 or KDM6A/KDM6B knockdown repressed expression of DNMT3B in both cisplatin-sensitive and -resistant TGCT cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eF-H\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn prior work, we have shown an interconnected relationship between alterations in DNA methylation mediated by DNMT3B and H3K27me3-mediated polycomb signaling (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). For example, DNMT3B is overexpressed in cisplatin-resistant TGCT cells, while H3K27me3 levels are decreased compared to parental cells (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Also, DNMT3B-knockdown sensitized parental TGCT cells to cisplatin and induced BMI1, EZH2, and H3K27me3 levels (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Consistent with this interconnected relationship KDM6A/KDM6B-knockdown repressed expression of DNMT3B in both cisplatin-sensitive and -resistant TGCT cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-F\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eH3K27me3 specific histone demethylase inhibitor GSK-J4 and KDM6A and KDM6B dual knockdown dramatically synergizes with cisplatin to promote TGCT inhibition and regression\u003c/b\u003e \u003cb\u003ein vivo.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo assess whether GSK-J4 could potentiate cisplatin sensitivity to TGCT cells \u003cem\u003ein vivo\u003c/em\u003e, we performed xenograft studies with cisplatin-resistant 2102EP-C1 and cisplatin-sensitive NT2/D1 cells. While GSK-J4 alone had minimal effects on TGCT growth, GSK-J4 produced a dramatic synergistic interaction with cisplatin treatment with evidence of tumor regression \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Mice treated with a single round of GSK-J4 and cisplatin therapy remained tumor-free at the end of the experiment (90 days). Note that the dose of cisplatin was decreased in cisplatin-sensitive NT2/D1 cells in order to observe a potentiation effect with GSK-J4 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. This suggests that GSK-J4 may not only be able to restore cisplatin sensitivity to resistant cells but may also be a strategy for cisplatin-sparing therapy for cisplatin-sensitive tumors. GSK-J4 or GSK-J4 plus cisplatin had minimal toxicity as assessed by total body weight \u003cb\u003e(Supplemental Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e)\u003c/b\u003e. In contrast, while not as dramatic, EZH2 inhibitor GSK-126 conferred cisplatin resistance in TGCT xenografts \u003cb\u003e(Supplemental Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo address whether GSK-J4 may have off-target effects, we performed further xenografts with cisplatin-resistant 2102EP-C1 cells after dual knockdown of the intended targets of GSK-J4, KDM6A and KMD6B, and compared the results to control cells. Similar to GSK-J4, KDM6A/ KDM6B-knockdown minimally effected basal tumor growth but dramatically potentiated the effects of cisplatin, again with evidence of tumor regression, and all mice remained tumor-free for over 90 days after a single dose of cisplatin \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Together these results suggest that pharmacologic and genetic activation of the polycomb pathway in TGCTs has strong cisplatin potentiated effects \u003cem\u003ein vivo\u003c/em\u003e with low overall toxicity. The dramatic \u003cem\u003ein vivo\u003c/em\u003e effects, as compared to \u003cem\u003ein vitro\u003c/em\u003e effects of GSK-J4 and KDM6A/KDM6B-knockdown, suggest that host and/or tumor microenvironment contributions may be occurring during cisplatin sensitization.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTranscriptome analysis of GSK-J4 and KDM6A/KDM6B-knockdown cells reveals the importance of basal activation of polycomb and p53 signaling in cisplatin sensitization.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate potential mechanisms responsible for the cisplatin sensitization effects of polycomb demethylase targeting in TGCT cells, we performed RNA-seq analysis of cisplatin-resistant NT2/D2-A4 and 2102EP-C1 cells untreated or treated with cisplatin and GSK-J4 alone or in combination and also untreated or cisplatin treated control 2102EP-C1-PLKO.1 and isogeneic dual 2102EP-C1-shKDM6A\u0026thinsp;+\u0026thinsp;6B-knockdown cells. Note, in cell viability/cytotoxic assays, cells were treated with cisplatin for 6 hours and assayed 3 days later to mimic clinical usage (peak cisplatin plasma concentration over a short amount of time) (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). As we have documented before, for transcriptomic analysis, the post-cisplatin time point was shortened to 24 hours to better assess proximal alterations in gene expression not associated with active cell death (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMultidimensional scaling (MDS) plots demonstrated a clear separation of the experimental groups and a tight grouping of biological replicates within groups \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Volcano plots and GSEA revealed that cisplatin treatment of cisplatin-resistant NT2/D1-A4, 2102EP-C1, and 2102EP-C1-PLKO.1 control cells had a restricted pattern of gene alterations dominated by upregulated p53 target genes \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-C\u003cb\u003e)\u003c/b\u003e, as we have noted previously in transcriptome analysis of TGCT cells treated with cisplatin (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Of note, the degree of transcriptional changes was substantially reduced in comparison to parental cisplatin-sensitive cells (data not shown). In contrast, GSK-J4-pretreated NT2/D1-A4 and 2102EP-C1 cells and untreated 2102EP-C1-shKDM6A\u0026thinsp;+\u0026thinsp;6B cells had a substantially more robust transcriptional response to cisplatin, again dominated by upregulated p53 target genes \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-C\u003cb\u003e)\u003c/b\u003e. Interestingly, GSK-J4 treatments alone and KDM6A/KDM6B-knockdown alone in 2102EP-C1-shKDM6A\u0026thinsp;+\u0026thinsp;6B cells demonstrated substantial alterations in gene expression. Upregulated genes for GSK-J4 alone treatments were again dominated by p53 target genes \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eC \u003cb\u003eand\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B\u003cb\u003e)\u003c/b\u003e despite the fact that the GSK-J4 treatments were not toxic or growth-inhibitory in long-term assays (32 and data not shown). This basal p53 target gene effect was less prominent in 2102EP-C1-shKDM6A\u0026thinsp;+\u0026thinsp;6B cells but was still evident in a narrower subset of p53 target genes \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. In contrast, downregulated genes had a more diverse gene set enrichment pattern among the experiments, including gene sets involving mRNA splicing, DNA methylation, and histones \u003cb\u003e(Supplemental Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e)\u003c/b\u003e. The top 20 gene sets enriched for upregulated and downregulated genes for each experimental arm for all three RNA-seq experiments are provided in \u003cb\u003eSupplemental Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAdditionally, and again consistent with the interconnected relationship between alterations in DNA methylation mediated by DNMT3B and H3K27me3-mediated polycomb signaling (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e), pharmacologic inhibition of KDM6A/KDM6B with GSK-J4 or KDM6A/KDM6B-knockdown repressed expression of DNMT3B \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-E\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTranscriptome analysis of KDM6A/KDM6B-knockdown cells reveals cisplatin sensitization is associated with alterations in chromatin remodeling genes upon cisplatin treatment.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo gain a broader insight into the role of polycomb demethylase targeting in cisplatin sensitization of TGCT cells, we further analyzed the KDM6A/KDM6B-knockdown plus cisplatin treatment in 2102EP-C1 cells experiment by identifying subsets of unique and overlapping upregulated and downregulated genes from three comparisons, those genes regulated by cisplatin in PLKO.1 control cells (PLKO.1 cisplatin-treated vs PLKO.1 untreated), those genes basally regulated by KDM6A/KDM6B-knockdown (shKDM6A\u0026thinsp;+\u0026thinsp;6B untreated vs PLKO.1 untreated), and those genes regulated by cisplatin in KDM6A/KDM6B-knockdown cells (shKDM6A\u0026thinsp;+\u0026thinsp;6B cisplatin-treated vs shKDM6A\u0026thinsp;+\u0026thinsp;6B untreated) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B\u003cb\u003e)\u003c/b\u003e. Venn diagrams were generated with a cutoff of \u0026gt;\u0026thinsp;1.2 fold-change with FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and GeneOverlap analysis with Fisher exact tests were performed against the 5529 curated sets from the Broad MSigDB C2 collection \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B\u003cb\u003e)\u003c/b\u003e. Gene lists from Venn diagram analysis and GeneOverlap results are provided in \u003cb\u003eSupplemental Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e and \u003cb\u003eSupplemental Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e\u003c/b\u003e. Upregulated genes were again dominated by p53 target genes with p53 gene sets enriched for genes commonly upregulated in both PLKO.1 and 2102EP-C1-shKDM6A\u0026thinsp;+\u0026thinsp;6B cells treated with cisplatin (Group 1), and genes upregulated by cisplatin in both cells but also basally upregulated upon KDM6A/KDM6B-knockdown (Group 2) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. This analysis again suggests that targeting polycomb demethylases basally modifies the p53 pathway in TGCT cells to sensitize these cells to cisplatin. Gene sets enriched exclusively for genes upregulated in shKDM6A\u0026thinsp;+\u0026thinsp;6B cells treated with cisplatin were involved in hypoxia and E-cadherin (CDH1) signaling (Group 3) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Finally, gene sets enriched exclusively for upregulated genes in untreated KDM6A/KDM6B knockdown cells compared to untreated PLKO.1 cells include several related to cancer (Group 4).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn contrast, there was strong enrichment for gene sets involved in H3K27me3 and polycomb signaling for gene exclusively downregulated in KDM6A/KDM6B-knockdown cells, as would be expected by knocking down polycomb demethylases (Group 5) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Interestingly, we found a gene set of 272 chromatin-modifying enzymes and proteins (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e) that were only downregulated in 2102EP-C1-shKDM6A\u0026thinsp;+\u0026thinsp;6B cells treated with cisplatin (Group 6) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-C\u003cb\u003e)\u003c/b\u003e. This included bromodomain protein BRD4, ATP-dependent chromatic remodeler SMARCA4, and cassettes of lysine demethylases (KDMs), chromodomain helicase DNA binding proteins (CHDs), and lysine methyltransferases (KMTs) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). This suggests that targeting KDM6A/KDM6B sets the stage for further cisplatin-mediated chromatin remodeling in TGCT cells. Gene sets enriched in genes exclusively downregulated by cisplatin in cisplatin-resistant PLKO.1 cells were related to senescence (Group 7).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eDue largely to a dearth of driver mutations in contrast to many solid tumors, there have been no effective targeted therapies developed for TGCTs, which are mainly treated with cisplatin-based chemotherapies developed over 4 decades ago (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). While cisplatin has transformed metastatic testicular cancer from a deadly, to in the majority of cases, a curable disease, there are no effective backup therapies for the 15% of cisplatin-refractory/resistant patients who typically die from progressive disease (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Further, curative cisplatin therapy results in acute and life-long toxicities, which are especially pertinent to the adolescent and young adult TGCT patient population (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Strategies to decrease cisplatin exposure would likely lead to improved quality of life for these patients. In contrast to genetic alterations, recent evidence suggests that epigenetics is a major driving factor for TGCT formation, progression, and response or resistance to chemotherapy (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Hence, targeting epigenetic pathways with \u0026ldquo;epidrugs\u0026rdquo; is one potential relatively unexplored strategy to advance TGCT treatment beyond cisplatin.\u003c/p\u003e \u003cp\u003eIn this report, we preclinically validate targeting polycomb demethylases KDM6A and KDM6B with epidrug GSK-J4 for the treatment of both cisplatin-sensitive and -resistant TGCTs. While GSK-J4 had minimal effects alone on TGCT tumor growth \u003cem\u003ein vivo\u003c/em\u003e, it dramatically sensitized cisplatin-sensitive and -resistant TGCTs to cisplatin. We validated KDM6A/KDM6B as the target of GSK-J4 since KDM6A/KDM6B genetic depletion had a remarkably similar effect to GSK-J4 on cisplatin-mediated anti-tumor activity and transcriptome alterations. Pharmacologic and genetic targeting of KDM6A/KDM6B potentiated or primed the p53-dominant transcriptional response to cisplatin, with also evidence for basal activation of p53. Further, several chromatin modifier gene families were repressed with cisplatin only in KDM6A/KDM6B-targeted cells, implying that KDM6A/KDM6B inhibition sets the stage for extensive chromatin remodeling of TGCT cells upon cisplatin treatment. Another interesting finding of our study was the contrast between the dramatic cisplatin sensitization effect of GSK-J4 \u003cem\u003ein vivo\u003c/em\u003e compared to cell culture. This suggests that perhaps there is priming of anti-tumor microenvironment and innate host immune mechanisms with GSK-J4 against TGCTs, a premise that is worthy of future study.\u003c/p\u003e \u003cp\u003eSeveral lines of evidence suggest that TGCTs may be particularly driven by epigenetic alterations (\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). TGCT are thought to derive from aberrantly differentiated primordial germ cells during a stage in development where the male germ line undergoes a dynamic wave of DNA methylation erasure. Issues that impact the microenvironment of male germ cell development \u003cem\u003ein utero\u003c/em\u003e, including cryptorchidism, hypospadias, impaired spermatogenesis, high estrogen exposure, and exposure to endocrine disrupting chemicals, have been associated with TGCTs (\u003cspan additionalcitationids=\"CR41 CR42 CR43\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Germline genetic disorders of sex development associated with fetal androgen insufficiency are also associated with an increased risk of germ cell malignancy (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Further, TGCTs have a very low mutational burden and a low frequency of driver oncogenic or tumor-suppressive mutations, especially in nonseminomas (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Evidence from our lab and others has shown that TGCT cells hyperactive p53 during cisplatin responses (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). The current work also suggests an important role for p53 in cisplatin sensitization upon KDM6A/KDM6B-targeting as p53 target gene expression was potentiated and basally activated.\u003c/p\u003e \u003cp\u003eDue to their developmental origins, TGCTs may have unique and more open, embryonic stem cell-like chromatin as compared to somatic cell-derived solid tumors, which may make TGCTs uniquely vulnerable to certain epidrugs (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). We and others have shown that TGCT cells are exquisitely sensitive to hypomethylating agents (HMAs) at very low doses that are dependent on intrinsically high levels of DNMT3B (\u003cspan additionalcitationids=\"CR25 CR26 CR27\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Further, pretreatment with HMAs can restore cisplatin sensitivity to cisplatin-resistant TGCT cells (\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Two recent clinical trials suggest that HMAs may have clinical activity in the setting of cisplatin refractory TGCTs (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Utilizing isogenic HMA- and cisplatin-resistant cell lines, we found that sensitivity/resistance to HMAs and cisplatin appear to be mechanistically linked by epigenetic remodeling involving DNA methylation and the polycomb pathway. Namely, there is a common set of polycomb target genes upregulated in cisplatin-resistant TGCT cells due to a shift in DNA methylation linked to high levels of DNMT3B (\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Further, DNMT3B genetic targeting induced H3K27me3, EZH2, and BMI1 and resulted in increased sensitivity to cisplatin (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). The data presented here provides further evidence for this linkage as targeting KDM6A/KDM6B was associated with a decrease in DNMT3B levels along with increasing cisplatin sensitivity. Hence, HMAs and GSK-J4 may be essentially targeting the same pathway vulnerability in TGCTs, with GSK-J4 having perhaps the theoretical advantage of being less genotoxic compared to HMAs, which incorporate into DNA and form protein adducts with DNMTs. The precise mechanism for how DNMT3B and polycomb are linked to regulate cisplatin sensitivity of TGCTs will require further study.\u003c/p\u003e \u003cp\u003eThis report suggests that GSK-J4 may have cisplatin-sensitization properties for cisplatin-sensitive as well as cisplatin-resistant TGCT patients. The role of polycomb in cancer is complex (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). In solid tumors, polycomb has mainly been associated with oncogenesis and poor outcomes, which has spurred the clinical development of EZH2 inhibitors (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). However, loss-of-function PRC2 mutations also occur in a subset of tumor types, including malignant peripheral nerve sheath tumors (MPNSTs), pediatric gliomas, and T-cell acute lymphoblastic leukemia (\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). This highlights the complex role of polycomb in tumorigenesis. This complexity extends to whether polycomb-mediated epigenetic changes are associated with cancer drug resistance (\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Targeting polycomb demethylases has not been well developed clinically for cancer therapy compared to EZH2 targeting, with a limited number of preclinical reports of GSK-J4 having anti-tumor activity (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). To our knowledge, GSK-J4 has not entered the clinic. Whether GSK-J4 or other KDM6A/KDM6B inhibitors have acceptable toxicity profiles will be important to ascertain. It is noteworthy that in our studies GSK-J4 and KDM6A/KDM6B-targeting had minimal effect alone, suggesting that a therapeutic window may exist for combination therapy in those tumors like TGCTs that already have a heightened sensitivity to cisplatin.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eThe biology of testicular germ cell tumors appears to be especially driven by epigenetic mechanisms suggesting that they may be highly sensitive to epidrugs. Our findings preclinically validate targeting polycomb demethylases KDM6A/KDM6B as a potent pharmacologic strategy for treating cisplatin-resistant TGCTs that warrants further preclinical and clinical investigation.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCHDs; chromodomain helicase DNA binding proteins, GSEA; gene set enrichment analysis, HMAs; DNA hypomethylating agents, KDMs; lysine demethylases. KMTs; lysine methyltransferases, MPNSTs; malignant peripheral nerve sheath tumors, PRCs; polycomb repressive complexes,PRC1; polycomb repressive 1,PRC2; polycomb repressive complex 2, TGCTs; testicular germ cell tumors\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of Interest Statement: \u0026nbsp;\u003c/strong\u003eThe authors declare no potential conflicts of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe RNA-seq datasets generated and/or analyzed during the current study are available in the NCBI Database of GEO Datasets under the accession number XXXXX. \u0026nbsp;All other data generated or analyzed during this study are available from the corresponding authors on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Institutes of Health grants R01-CA211875 (MJS) and DOD PRCRP Impact Award W81XWH2110903 (MJS) and DOD Breakthrough Award BC221269 (MJS), NIEHS 5T32ES007326-25 (RIB), and a UIUC block grant (DS).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDS, ZF, SJF, MHP, MDV, JPS, MJS, and RS performed the conceptualization; DS, CP, SJ, BCR, RIB, ZS, MPH, and RS performed the methodology; DS and RS performed the validation; DS, MWK, ZF, MJS, and RS were involved in formal analysis; DS, MWK, CP, SJ, BRC, ZS, and RS performed the investigation; DS, MWK, ZF, and RS performed data curation; DS and MJS wrote the original draft preparation; RIB, SJF, MHP, MDV, JPS, MJS, and RS reviewed and edited the manuscript; DS and RS performed visualization; SJF, MJS, and RS were involved in supervision; SJF, MJS, and RS and were involved in project administration; DS, RIB, SJF, MJS, and RS contributed to funding acquisition. \u0026nbsp;All authors reads and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe dedicate this study to the memory of Dr. Sarah J. Freemantle, a valued scientist and cherished colleague and mentor. We would like to thank members of the Roy J. Carver Biotechnology Center at the University of Illinois, including Dr. Alvaro Hernandez and Chris Wright, for RNA sequencing.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdra N, Einhorn LH. Testicular cancer update. Clin Adv Hematol Oncol. 15(5):386-96, 2017.\u003c/li\u003e\n\u003cli\u003eFeldman DR, Patil S, Trinos MJ, Carousso M, Ginsberg MS, Sheinfeld J, Bajorin DF, Bosl GJ, Motzer RJ. Progression-free and overall survival in patients with relapsed/refractory germ cell tumors treated with single-agent chemotherapy: endpoints for clinical trial design. Cancer 118:981-6, 2012.\u003c/li\u003e\n\u003cli\u003eSingh R, Fazal Z, Freemantle SJ, Spinella MJ. Mechanisms of cisplatin sensitivity and resistance in testicular germ cell tumors. Cancer Drug Resist. 2019;2(3):580-594.\u003c/li\u003e\n\u003cli\u003eFung C, Sesso HD, Williams AM, Kerns SL, Monahan P, Abu Zaid M, Feldman DR, Hamilton RJ, Vaughn DJ, Beard CJ, Kollmannsberger CK, Cook R, Althouse S, Ardeshir-Rouhani-Fard S, Lipshultz SE, Einhorn LH, Fossa SD, Travis LB. Multi-institutional assessment of adverse health outcomes among North American testicular cancer survivors after modern cisplatin-based chemotherapy. J Clin Oncol. 2017;35(11):1211-22.\u003c/li\u003e\n\u003cli\u003eShrem NS, Wood L, Hamilton RJ, Kuhathaas K, Czaykowski P, Roberts M, Matthew A, Izard JP, Chung P, Nappi L, Jones J, Souli\u0026egrave;res D, Aprikian A, Power N, Canil C. Testicular cancer survivorship: Long-term toxicity and management. Can Urol Assoc J. 2022 Aug;16(8):257-272. \u003c/li\u003e\n\u003cli\u003eLobo J, Gillis AJM, Jer\u0026oacute;nimo C, Henrique R, Looijenga LHJ. Human germ cell tumors are developmental cancers: Impact of epigenetics on pathobiology and clinic. Int J Mol Sci. 20(2). pii: E258, 2019.\u003c/li\u003e\n\u003cli\u003eSingh R, Fazal Z, Freemantle SJ, Spinella MJ. Between a Rock and a Hard Place: An Epigenetic-Centric View of Testicular Germ Cell Tumors. Cancers (Basel). 2021 Mar 25;13(7):1506. \u003c/li\u003e\n\u003cli\u003eNicu AT, Medar C, Chifiriuc MC, Gradisteanu Pircalabioru G, Burlibasa L. Epigenetics and Testicular Cancer: Bridging the Gap Between Fundamental Biology and Patient Care. Front Cell Dev Biol. 2022 Apr 8;10:861995.\u003c/li\u003e\n\u003cli\u003eRijlaarsdam MA, Looijenga LH. An oncofetal and developmental perspective on testicular germ cell cancer. Semin Cancer Biol. 29:59-74, 2014.\u003c/li\u003e\n\u003cli\u003eLitchfield, K.; Summersgill, B.; Yost, S.; Sultana, R.; Labreche, K.; Dudakia, D.; Renwick, A.; Seal, S.; Al-Saadi, R.; Broderick, P.; et al. Whole-exome sequencing reveals the mutational spectrum of testicular germ cell tumours. Nat. Commun. 2015, 6, 5973.\u003c/li\u003e\n\u003cli\u003eShen, H.; Shih, J.; Hollern, D.P.; Wang, L.; Bowlby, R.; Tickoo, S.K.; Thorsson, V.; Mungall, A.J.; Newton, Y.; Hegde, A.M.; et al. Integrated molecular characterization of testicular germ cell tumors. Cell Rep. 2018, 23, 3392\u0026ndash;3406.\u003c/li\u003e\n\u003cli\u003eLee AV, Nestler KA, Chiappinelli KB. Therapeutic targeting of DNA methylation alterations in cancer. Pharmacol Ther. 2024 Jun;258:108640. doi: 10.1016/j.pharmthera.2024.108640. Epub 2024 Apr 1. PMID: 38570075.\u003c/li\u003e\n\u003cli\u003eGerman B, Ellis L. Polycomb Directed Cell Fate Decisions in Development and Cancer. Epigenomes. 2022 Sep 6;6(3):28.\u003c/li\u003e\n\u003cli\u003eGuo Y, Wang GG. Modulation of the high-order chromatin structure by Polycomb complexes. Front Cell Dev Biol. 2022 Oct 5;10:1021658.\u003c/li\u003e\n\u003cli\u003eAbu-Hanna J, Patel JA, Anastasakis E, Cohen R, Clapp LH, Loizidou M, Eddama MMR. Therapeutic potential of inhibiting histone 3 lysine 27 demethylases: a review of the literature. Clin Epigenetics. 2022 Aug 1;14(1):98.\u003c/li\u003e\n\u003cli\u003eAn R, Li YQ, Lin YL, Xu F, Li MM, Liu Z. EZH1/2 as targets for cancer therapy. Cancer Gene Ther. 2023 Feb;30(2):221-235. doi: 10.1038/s41417-022-00555-1. Epub 2022 Nov 11. PMID: 36369341.\u003c/li\u003e\n\u003cli\u003eMohammad F, Weissmann S, Leblanc B, Pandey DP, H\u0026oslash;jfeldt JW, Comet I, Zheng C, Johansen JV, Rapin N, Porse BT, Tvardovskiy A, Jensen ON, Olaciregui NG, Lavarino C, Su\u0026ntilde;ol M, de Torres C, Mora J, Carcaboso AM, Helin K. EZH2 is a potential therapeutic target for H3K27M-mutant pediatric gliomas. Nat Med. 2017;23(4):483-92. Epub 2017/03/07. doi: 10.1038/nm.4293. PubMed PMID: 28263309.\u003c/li\u003e\n\u003cli\u003ePekmezci M, Cuevas-Ocampo AK, Perry A, Horvai AE. Significance of H3K27me3 loss in the diagnosis of malignant peripheral nerve sheath tumors. Mod Pathol. 2017;30(12):1710-9. Epub 2017/08/05. doi: 10.1038/modpathol.2017.97. PubMed PMID: 28776579.\u003c/li\u003e\n\u003cli\u003eWang J, Wang GG. No easy way out for EZH2: Its pleiotropic, noncanonical effects on gene regulation and cellular function. Int J Mol Sci. 2020;21(24). Epub 2020/12/18. doi: 10.3390/ijms21249501. PubMed PMID: 33327550.\u003c/li\u003e\n\u003cli\u003eSamaržija I, Tomljanović M, Novak Kujundžić R, Tro\u0026scaron;elj KG. EZH2 Inhibition and Cisplatin as a Combination Anticancer Therapy: An Overview of Preclinical Studies. Cancers (Basel). 2022 Sep 29;14(19):4761. doi: 10.3390/cancers14194761. PMID: 36230683; PMCID: PMC9561994.\u003c/li\u003e\n\u003cli\u003eWang Q, Chen X, Jiang Y, Liu S, Liu H, Sun X, Zhang H, Liu Z, Tao Y, Li C, Hu Y, Liu D, Ye D, Liu Y, Wang M, Zhang X. Elevating H3K27me3 level sensitizes colorectal cancer to oxaliplatin. J Mol Cell Biol. May 8, 2019.\u003c/li\u003e\n\u003cli\u003eHu S, Yu L, Li Z, Shen Y, Wang J, Cai J, Xiao L, Wang Z. Overexpression of EZH2 contributes to acquired cisplatin resistance in ovarian cancer cells in vitro and in vivo. Cancer Biol. Ther. 10, 788\u0026ndash;795. 2010.\u003c/li\u003e\n\u003cli\u003eZhu Z, Tang J, Wang J, Duan G, Zhou L, Zhou X. MiR-138 acts as a tumor suppressor by targeting EZH2 and enhances cisplatin-induced apoptosis in osteosarcoma cells. PLoS ONE 11, 2016.\u003c/li\u003e\n\u003cli\u003eBeyrouthy MJ, Garner KM, Hever MP, Freemantle SJ, Eastman A, Dmitrovsky E, Spinella MJ. High DNA methyltransferase 3B expression mediates 5-aza-deoxycytidine hypersensitivity in testicular germ cell tumors. Cancer Res 69:9360-6, 2009.\u003c/li\u003e\n\u003cli\u003eBiswal BK, Beyrouthy MJ, Hever-Jardine MP, Armstrong D, Tomlinson CR, Christensen BC, Marsit CJ, Spinella MJ. Acute hypersensitivity of pluripotent testicular cancer-derived embryonal carcinoma to low-dose 5-aza deoxycytidine is associated with global DNA Damage-associated p53 activation, anti-pluripotency and DNA demethylation. PLoS One 7:e53003, 2012.\u003c/li\u003e\n\u003cli\u003eAlbany C, Hever-Jardine MP, von Herrmann KM, Yim CY, Tam J, Warzecha JM, Shin L, Bock SE, Curran BS, Chaudhry AS, Kim F, Sandusky GE, Taverna P, Freemantle SJ, Christensen BC, Einhorn LH, Spinella MJ. Refractory testicular germ cell tumors are highly sensitive to the second generation DNA methylation inhibitor guadecitabine. Oncotarget 8:2949-59, 2017.\u003c/li\u003e\n\u003cli\u003eWongtrakoongate P, Li J, Andrews PW. Aza-deoxycytidine induces apoptosis or differentiation via DNMT3B and targets embryonal carcinoma cells but not their differentiated derivatives. \u003cem\u003eBr J Cancer\u003c/em\u003e 110:2131-8, 2014.\u003c/li\u003e\n\u003cli\u003eOing C, Verem I, Mansour WY, Bokemeyer C, Dyshlovoy S, Honecker F. 5-azacitidine exerts prolonged pro-apoptotic effects and overcomes cisplatin-resistance in non-seminomatous germ cell tumor cells. \u003cem\u003eInt J Mol Sci. \u003c/em\u003e20. pii: E21, 2018.\u003c/li\u003e\n\u003cli\u003eAlbany C, Fazal Z, Singh R, Bikorimana E, Adra N, Hanna NH, Einhorn LH, Perkins SM, Sandusky GE, Christensen BC, Keer H, Fang F, Nephew KP, Spinella MJ. A phase 1 study of combined guadecitabine and cisplatin in platinum refractory germ cell cancer. Cancer Med. 2021 Jan;10(1):156-163.\u003c/li\u003e\n\u003cli\u003eCrabb SJ, Danson S, Catto JWF, Hussain S, Chan D, Dunkley D, Downs N, Marwood E, Day L, Saunders G, Light M, Whitehead A, Ellis D, Sarwar N, Enting D, Birtle A, Johnson B, Huddart R, Griffiths G. Phase I Trial of DNA Methyltransferase Inhibitor Guadecitabine Combined with Cisplatin and Gemcitabine for Solid Malignancies Including Urothelial Carcinoma (SPIRE). Clin Cancer Res. 2021 Apr 1;27(7):1882-1892.\u003c/li\u003e\n\u003cli\u003eFazal Z, Singh R, Fang F, Bikorimana E, Baldwin H, Corbet A, Tomlin M, Yerby C, Adra N, Albany C, Lee S, Freemantle SJ, Nephew KP, Christensen BC, Spinella MJ. Hypermethylation and global remodelling of DNA methylation is associated with acquired cisplatin resistance in testicular germ cell tumours. Epigenetics. 2020:1-14.\u003c/li\u003e\n\u003cli\u003eSingh R, Fazal Z, Corbet AK, Bikorimana E, Rodriguez JC, Khan EM, Shahid K, Freemantle SJ, Spinella MJ. Epigenetic remodeling through downregulation of polycomb repressive complex 2 mediates chemotherapy resistance in testicular germ cell tumors. Cancers. 11(6). pii: E796, 2019.\u003c/li\u003e\n\u003cli\u003eSingh R, Fazal Z, Bikorimana E, Boyd RI, Yerby C, Tomlin M, Baldwin H, Shokry D, Corbet AK, Shahid K, Hattab A, Freemantle SJ, Spinella MJ. Reciprocal epigenetic remodeling controls testicular cancer hypersensitivity to hypomethylating agents and chemotherapy. Mol Oncol. 2022 Feb;16(3):683-698.\u003c/li\u003e\n\u003cli\u003eRitchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, Smyth GK. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015 Apr 20;43(7):e47.\u003c/li\u003e\n\u003cli\u003eSubramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, Pomeroy SL, Golub TR, Lander ES, Mesirov JP. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005 Oct 25;102(43):15545-50. \u003c/li\u003e\n\u003cli\u003eShen L \u0026amp; Sinai ISoMaM (2020) GeneOverlap: test and visualize gene overlaps. R package version 1.26.0. http://shenlab-sinai.github.io/shenlab-sinai/\u003c/li\u003e\n\u003cli\u003eKerley-Hamilton JS, Pike AM, Li N, DiRenzo J and Spinella MJ. A p53-dominant transcriptional response to cisplatin in testicular germ cell tumor-derived human embryonal carcinoma. \u003cem\u003eOncogene.\u003c/em\u003e 24:6090-100, 2005.\u003c/li\u003e\n\u003cli\u003eLiberzon A, Subramanian A, Pinchback R, Thorvaldsd\u0026oacute;ttir H, Tamayo P, Mesirov JP. Molecular signatures database (MSigDB) 3.0. Bioinformatics. 2011 Jun 15;27(12):1739-40.\u003c/li\u003e\n\u003cli\u003eNicu AT, Ionel IP, Stoica I, Burlibasa L, Jinga V. Recent Advancements in Research on DNA Methylation and Testicular Germ Cell Tumors: Unveiling the Intricate Relationship. Biomedicines. 2024 May 8;12(5):1041. doi: 10.3390/biomedicines12051041. PMID: 38791003; PMCID: PMC11117643.\u003c/li\u003e\n\u003cli\u003eBr\u0026auml;uner, E.V.; Lim, Y.H.; Koch, T.; Uldbjerg, C.S.; Gregersen, L.S.; Pedersen, M.K.; Frederiksen, H.; Petersen, J.H.; Coull, B.A.; Andersson, A.M.; et al. Endocrine disrupting chemicals and risk of testicular cancer: A systematic review and meta-analysis. J. Clin. Endocrinol. Metab. 2021, 106, e4834\u0026ndash;e4860.\u003c/li\u003e\n\u003cli\u003eSharma, A.; Mollier, J.; Brocklesby, R.W.K.; Caves, C.; Jayasena, C.N.; Minhas, S. Endocrine-disrupting chemicals and male reproductive health. Reprod. Med. Biol. 2020, 19, 243\u0026ndash;253.\u003c/li\u003e\n\u003cli\u003eSkakkebaek, N.E.; Rajpert-De Meyts, E.; Buck Louis, G.M.; Toppari, J.; Andersson, A.M.; Eisenberg, M.L.; Jensen, T.K.; J\u0026oslash;rgensen, N.; Swan, S.H.; Sapra, K.J.; et al. Male reproductive disorders and fertility trends: Influences of environment and genetic susceptibility. Physiol. Rev. 2016, 96, 55\u0026ndash;97.\u003c/li\u003e\n\u003cli\u003eSelvi, I.; Ozturk, E.; Yikilmaz, T.N.; Sarikaya, S.; Basar, H. Effects of testicular dysgenesis syndrome components on testicular germ cell tumor prognosis and oncological outcomes. Int. Braz. J. Urol. 2020, 46, 725\u0026ndash;740.\u003c/li\u003e\n\u003cli\u003eHanson, H.A.; Anderson, R.E.; Aston, K.I.; Carrell, D.T.; Smith, K.R.; Hotaling, J.M. Subfertility increases risk of testicular cancer: Evidence from population-based semen samples. Fertil. Steril. 2016, 105, 322\u0026ndash;328.e321.\u003c/li\u003e\n\u003cli\u003eTrabert, B.; Zugna, D.; Richiardi, L.; McGlynn, K.A.; Akre, O. Congenital malformations and testicular germ cell tumors. Int. J. Cancer 2013, 133, 1900\u0026ndash;1904.\u003c/li\u003e\n\u003cli\u003eGutekunst M, Oren M, Weilbacher A, Dengler MA, Markwardt C, Thomale J, Aulitzky WE and van der Kuip H. p53 hypersensitivity is the predominant mechanism of the unique responsiveness of testicular germ cell tumor (TGCT) cells to cisplatin. \u003cem\u003ePLoS One\u003c/em\u003e. 6:e19198, 2011.\u003c/li\u003e\n\u003cli\u003eDuale N, Lindeman B, Komada M, Olsen AK, Andreassen A, Soderlund EJ and Brunborg G. Molecular portrait of cisplatin induced response in human testis cancer cell lines based on gene expression profiles.\u003cem\u003e Mol Cancer.\u003c/em\u003e 6:53, 2007.\u003c/li\u003e\n\u003cli\u003eSong W, Wang J, Yang Y, Jing N, Zhang X, Chen L and Wu J. Rewiring drug-activated p53-regulatory network from suppressing to promoting tumorigenesis.\u003cem\u003e J Mol Cell Biol.\u003c/em\u003e 4:197-206, 2012.\u003c/li\u003e\n\u003cli\u003eVan Mierlo G, Dirks RAM, De Clerck L, Brinkman AB, Huth M, Kloet SL, Saksouk N, Kroeze LI, Willems S, Farlik M, Bock C, Jansen JH, Deforce D, Vermeulen M, D\u0026eacute;jardin J, Dhaenens M, Marks H Integrative proteomic profiling reveals PRC2-dependent epigenetic crosstalk maintains ground-state pluripotency. Cell Stem Cell 24, 123\u0026ndash;137, 2019.\u003c/li\u003e\n\u003cli\u003eLiu Y, Yang Q. The roles of EZH2 in cancer and its inhibitors. Med Oncol. 2023 May 6;40(6):167.\u003c/li\u003e\n\u003cli\u003eDalpatraj N, Naik A, Thakur N. GSK-J4: An H3K27 histone demethylase inhibitor, as a potential anti-cancer agent. Int J Cancer. 2023 Sep 15;153(6):1130-1138.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-communication-and-signaling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccas","sideBox":"Learn more about [Cell Communication and Signaling](http://biosignaling.biomedcentral.com/)","snPcode":"12964","submissionUrl":"https://submission.nature.com/new-submission/12964/3","title":"Cell Communication and Signaling","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Testicular cancer, polycomb, GSK-J4, GSK-126, preclinical, transcriptomics, cisplatin, chemotherapy resistance, epigenetics","lastPublishedDoi":"10.21203/rs.3.rs-4986186/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4986186/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTesticular germ cell tumors (TGCTs) can be treated with cisplatin-based therapy. However, a clinically significant number of cisplatin-resistant patients die from progressive disease as no effective alternatives exist. Curative cisplatin therapy results in acute and life-long toxicities in the young TGCT patient population providing a rationale to decrease cisplatin exposure. In contrast to genetic alterations, recent evidence suggests that epigenetics is a major driving factor for TGCT formation, progression, and response to chemotherapy. Hence, targeting epigenetic pathways with \u0026ldquo;epidrugs\u0026rdquo; is one potential relatively unexplored strategy to advance TGCT treatment beyond cisplatin. In this report, we demonstrate for the first time that targeting polycomb demethylases KDM6A and KDM6B with epidrug GSK-J4 can treat both cisplatin-sensitive and -resistant TGCTs. While GSK-J4 had minimal effects alone on TGCT tumor growth in vivo, it dramatically sensitized cisplatin-sensitive and -resistant TGCTs to cisplatin. We validated KDM6A/KDM6B as the target of GSK-J4 since KDM6A/KDM6B genetic depletion had a similar effect to GSK-J4 on cisplatin-mediated anti-tumor activity and transcriptome alterations. Pharmacologic and genetic targeting of KDM6A/KDM6B potentiated or primed the p53-dominant transcriptional response to cisplatin, with also evidence for basal activation of p53. Further, several chromatin modifier genes, including \u003cem\u003eBRD4\u003c/em\u003e, lysine demethylases, chromodomain helicase DNA binding proteins, and lysine methyltransferases, were repressed with cisplatin only in KDM6A/KDM6B-targeted cells, implying that KDM6A/KDM6B inhibition sets the stage for extensive chromatin remodeling of TGCT cells upon cisplatin treatment. Our findings demonstrate that targeting polycomb demethylases is a new potent pharmacologic strategy for treating cisplatin resistant TGCTs that warrants clinical development.\u003c/p\u003e","manuscriptTitle":"Refractory testicular germ cell tumors are highly sensitive to the targeting of polycomb pathway demethylases KDM6A and KDM6B","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-18 09:36:13","doi":"10.21203/rs.3.rs-4986186/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-01T21:39:05+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-25T14:00:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-19T00:41:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"123043322430806199514558056221860839898","date":"2024-09-05T00:29:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"116917472967192104945071170309479020523","date":"2024-09-04T22:26:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"191605102834210765609566557249444746196","date":"2024-09-04T19:34:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"212892785208677116370465808433986426416","date":"2024-09-03T08:26:57+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-02T15:53:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-29T12:31:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-29T12:30:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Communication and Signaling","date":"2024-08-27T17:21:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-communication-and-signaling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccas","sideBox":"Learn more about [Cell Communication and Signaling](http://biosignaling.biomedcentral.com/)","snPcode":"12964","submissionUrl":"https://submission.nature.com/new-submission/12964/3","title":"Cell Communication and Signaling","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4f02b91a-65fc-484f-bcb8-30a1063e9491","owner":[],"postedDate":"October 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-11-04T16:26:18+00:00","versionOfRecord":{"articleIdentity":"rs-4986186","link":"https://doi.org/10.1186/s12964-024-01912-3","journal":{"identity":"cell-communication-and-signaling","isVorOnly":false,"title":"Cell Communication and Signaling"},"publishedOn":"2024-10-31 16:20:13","publishedOnDateReadable":"October 31st, 2024"},"versionCreatedAt":"2024-10-18 09:36:13","video":"","vorDoi":"10.1186/s12964-024-01912-3","vorDoiUrl":"https://doi.org/10.1186/s12964-024-01912-3","workflowStages":[]},"version":"v1","identity":"rs-4986186","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4986186","identity":"rs-4986186","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}