How genes are desilenced within mesoscale repressive chromatin is a crucial yet poorly understood phenomenon. Prevailing models posit that methylation of lysine-9 of histone H3 (H3K9me3) engages heterochromatin protein 1 (HP1) to drive chromatin compaction and transcriptional silencing. The erasure of this repressive mark and its replacement with acetyl/acyl groups recruits positive factors such as BRD4/BET to elicit gene transcription. We report that in Friedreich's ataxia, a synthetic gene regulator (SynGR1) drives transcription across repressive chromatin without removal or replacement of H3K9me3 or HP1. By selectively recruiting BRD4/BET into repressive GAA-repeats in frataxin ( FXN ), SynGR1 creates a paradoxical state where gene transcription and repressive chromatin co-exist. Contrary to convention, we find that BRD4 readily partitions into phase separated HP1 condensates in vitro and into HP1 puncta in patient-derived cells, thus presenting a mechanistic explanation for desilencing transcription without the dispersal of mesoscale repressive chromatin. Epigenetic drugs that gate sequential steps in transcription, synergistically stimulate FXN expression while concomitantly increasing, rather than eliminating, repressive H3K9me3 and HP1 levels. More broadly, this study highlights the dynamic nature of repressive chromatin and the context-dependence of epigenetic marks in regulating gene expression.

How genes are desilenced without erasure of repressive chromatin is a poorly understood phenomenon. A dominant mode of repression occurs through methylation of lysine 9 of histone H3 (H3K9me3), a mark that engages heterochromatin protein 1 (HP1) to drive chromatin compaction and transcriptional silencing. The erasure and replacement of this repressive mark with acetyl/acyl groups recruits positive factors such as BRD4/BET to elicit gene transcription. Here we report that, in Friedreich's ataxia, a synthetic gene regulator (SynGR1/SynTEF1) licenses transcription across repressive chromatin without removal or replacement of H3K9me3 or HP1. By recruiting BRD4/BET into repressive GAA repeats in frataxin (FXN), SynGR1 creates a paradoxical state wherein gene transcription and repressive chromatin coexist. Contrary to convention, we find that BRD4 partitions into phase separated HP1 condensates in vitro and into HP1 puncta in patient-derived cells, thus offering a mechanistic explanation for desilencing transcription without the dispersal of mesoscale repressive chromatin. More broadly, our study highlights the dynamic nature of repressive chromatin and the context dependence of epigenetic marks in regulating gene expression.

Retinoic acid (RA) is a standard-of-care neuroblastoma drug thought to be effective by inducing differentiation. Curiously, RA has little effect on primary human tumors during upfront treatment but can eliminate neuroblastoma cells from the bone marrow during post-chemo maintenance therapy—a discrepancy that has never been explained. To investigate this, we treat a large cohort of neuroblastoma cell lines with RA and observe that the most RA-sensitive cells predominantly undergo apoptosis or senescence, rather than differentiation. We conduct genome-wide CRISPR knockout screens under RA treatment, which identify bone morphogenic protein (BMP) signaling as controlling the apoptosis/senescence vs differentiation cell fate decision and determining RA’s overall potency. We then discover that BMP signaling activity is markedly higher in neuroblastoma patient samples at bone marrow metastatic sites, providing a plausible explanation for RA’s ability to clear neuroblastoma cells specifically from the bone marrow, by seemingly mimicking interactions between BMP and RA during normal development.

Drug combinations are essential to modern medicine, but their discovery remains slow and inefficient as experimental complexity expands rapidly with each additional drug tested. Although modern liquid handling systems enable complex and highly customizable experimental designs, a lack of strategies integrating these technologies with combination-specific analytical methods has limited throughput. Here we introduce Combocat, an open-source and streamlined framework that combines acoustic liquid handling protocols with machine learning-based inference to achieve ultrahigh-throughput drug combination screening. Using Combocat, we generate a reference dataset of over 800 unique combinations in a dense 10 × 10 matrix format across multiple cell types, and use this to train a predictive model that accurately infers drug combination effects from sparse data, drastically reducing the number of experimental measurements required. As proof of concept, we screened 9,045 combinations in a neuroblastoma cell line-the largest number of combinations tested in a single cell line to date-achieved using minimal resources. By integrating advanced drug dispensing technologies with predictive computational modeling, Combocat provides a scalable solution to accelerate the discovery of novel drug combinations.

Abstract Retinoic acid (RA) is a standard-of-care neuroblastoma drug used during post-chemo consolidation therapy. Based on clinical trials from the 90s, RA benefits 10-15% of patients. It is widely believed the anti-cancer activity of RA is due to retinoid-induced differentiation of cancer cells, conclusions largely attributable to observations in cell culture. However, given RA is typically used in the minimal residual disease setting, this mechanism has never been definitively proved in patients. To better understand RA’s activity, we deployed several new technologies. First, we conducted genome-wide CRISPR modifier screens in RA-treated hyper-sensitive neuroblastoma cell lines. Surprisingly, we found that, in these cells, RA primarily decreased cell viability via apoptosis or senescence, rather than differentiation—activities in which the CRISPR screens strongly implicated bone morphogenetic protein (BMP) signaling. Using ChIP-seq and RNA-seq we showed these behaviors were mediated by the coordinated gene regulatory actions of RARA and BMP-family SMAD transcription factors. Notably, interactions between BMP signaling and RA are well established in developmental biology, where BMP signaling can tip cell fate decisions between differentiation, apoptosis, and senescence upon exposure to naturally occurring RA, behaviors that can seemingly be maintained in neuroblastoma cells. Next, we assessed the correlations between RA IC50 and the expression of all (∼20,000) genes in a panel of 19 cell lines. Remarkably, SMAD9, a critical downstream transcription factor of the BMP pathway, was the #1 most correlated gene with RA IC50 (R = -0.92, P = 4.2 × 10-6), suggesting a highly generalizable relationship between BMP signaling and RA response. By modulating SMAD9, or other components of BMP signaling, we could promote apoptosis/senescence and sensitize cells to RA. We then performed large-scale drug combination screens of RA and the drug FK506, which can amplify BMP signaling activity. RA exhibited a synergistic effect with FK506 in all 10 neuroblastoma cell lines we tested, very strikingly in some cases, suggesting it could be possible to pharmacologically amplify the activity of RA in patients. Finally, using published single-cell RNA-seq data from neuroblastoma patient samples, we found BMP signaling activity is relatively low in primary tumors, but much higher in disseminated metastatic neuroblastoma cells in the bone marrow. We confirmed this trend using immunofluorescence staining in 6 paired primary patient samples. This site-specific variability in BMP signaling activity provides the first reasonable explanation for RA’s curious clinical activity, whereby it has little effect on bulky established tumors, but has been shown to clear disseminated metastatic cells from the bone marrow during consolidation therapy. Overall, our study revealed that BMP signaling controls neuroblastoma cell fate and sensitivity to RA and that this observation is consistent with the unique clinical behaviors of this drug. Citation Format: Min Pan, Yinwen Zhang, William C. Wright, Hyeong-Min Lee, Richard H. Chapple, Xueying Liu, Jonathan Low, Duane Currier, Allister J. Loughran, Michael A. Dyer, Shondra M. Pruett, Burgess Freeman III, Taosheng Chen, Brian J. Abraham, Elizabeth Stewart, John Easton, Paul Geeleher. BMP signaling determines neuroblastoma sensitivity to retinoic acid by directing cell fate [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr B005.

Combination chemotherapy is crucial for successfully treating cancer. However, the enormous number of possible drug combinations means discovering safe and effective combinations remains a significant challenge. To improve this process, we conduct large-scale targeted CRISPR knockout screens in drug-treated cells, creating a genetic map of druggable genes that sensitize cells to commonly used chemotherapeutics. We prioritize neuroblastoma, the most common extracranial pediatric solid tumor, where ~50% of high-risk patients do not survive. Our screen examines all druggable gene knockouts in 18 cell lines (10 neuroblastoma, 8 others) treated with 8 widely used drugs, resulting in 94,320 unique combination-cell line perturbations, which is comparable to the largest existing drug combination screens. Using dense drug-drug rescreening, we find that the top CRISPR-nominated drug combinations are more synergistic than standard-of-care combinations, suggesting existing combinations could be improved. As proof of principle, we discover that inhibition of PRKDC, a component of the non-homologous end-joining pathway, sensitizes high-risk neuroblastoma cells to the standard-of-care drug doxorubicin in vitro and in vivo using patient-derived xenograft (PDX) models. Our findings provide a valuable resource and demonstrate the feasibility of using targeted CRISPR knockout to discover combinations with common chemotherapeutics, a methodology with application across all cancers.

Abstract While retinoic acid (RA) has been successfully used for leukemia treatment for decades, the attempt to treat solid tumors with RA remains challenging, with less than 10% of neuroblastoma (NB) patients achieving complete remission when treated with RA alone. It has been believed the anti-cancer activity of retinoids is due to retinoid-induced terminal differentiation, however, what determines cell response or whether differentiation is the main effect of RA has never been fully understood. To better understand RA’s activity, we conducted genome-wide CRISPR modifier screens in RA-treated hyper-sensitive NB cell lines. Surprisingly, we found that, in these cells, RA primarily decreased cell viability via apoptosis or senescence, rather than differentiation—activities in which the CRISPR screens strongly implicated bone morphogenetic protein (BMP) signaling. BMP activation promoted apoptosis/senescence and sensitized cells to RA. Conversely, BMP inhibitors and SMAD9 (a critical transcription factor of the BMP pathway) knockout enhanced RA’s ability to induce differentiation but reduced cell sensitivity to RA. Our ChIP-seq and RNA-seq data showed these behaviors were mediated by the coordinated gene regulatory actions of RARA and BMP-family SMAD transcription factors. Furthermore, in a panel of 19 cell lines we screened with RA, we assessed the correlations between RA IC50 and the expression of all (~20,000) genes (publicly available data from the GDSC and Depmap). Remarkably, SMAD9 was the number 1 and number 10 most correlated gene, respectively, with RA IC50 (ranked by Pearson correlation coefficient; GDSC R = -0.92, P = 4.2 × 10−6; Depmap R = -0.81, P = 4.8 × 10−4). We also performed comprehensive large-scale drug screens in these cell lines with combinations of RA and a BMP activator FK506. RA exhibited a synergistic effect with FK506 in all the NB cell lines, very strikingly in some cell lines. All these data suggest BMP signaling is generally required for RA sensitivity and BMP activators are promising candidates in combination with RA to treat NB. Using published bulk and single cell RNA-seq data from NB patient samples, we found BMP signaling activity is relatively low in primary tumors, but well maintained in disseminated NB cells derived from bone marrow. This explains why RA is clinically used as a maintenance therapy and can only successfully treat the minimal residual disease and suggests that tumor microenvironment is a critical factor in determining cell response to RA. Overall, our study revealed that BMP controls NB cell fate and sensitivity in RA treatment. Notably, interactions between BMP signaling and RA are well established in developmental biology, where BMP signaling can tip cell fate decisions between differentiation, apoptosis, and senescence upon exposure to endogenous RA. Our data suggest that this developmental process can be maintained in NB cells, unveiling a novel mode of anti-neoplastic action. Citation Format: Min Pan, Yinwen Zhang, William C. Wright, Hyeong-Min Lee, Richard H. Chapple, Xueying Liu, Jonathan Low, Duane Currier, Allister J. Loughran, Dyer A. Dyer, Shondra M. Pruett, Burgess Freeman, Taosheng Chen, Brian J. Abraham, Elizabeth Stewart, John Easton, Paul Geeleher. BMP signaling determines neuroblastoma cell fate and sensitivity to retinoic acid [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 151.

UBTF tandem duplications (UBTF-TDs) have recently emerged as a recurrent alteration in pediatric and adult acute myeloid leukemia (AML). UBTF-TD leukemias are characterized by a poor response to conventional chemotherapy and a transcriptional signature that mirrors NUP98-rearranged and NPM1-mutant AMLs, including HOX-gene dysregulation. However, the mechanism by which UBTF-TD drives leukemogenesis remains unknown. In this study, we investigated the genomic occupancy of UBTF-TD in transformed cord blood CD34+ cells and patient-derived xenograft models. We found that UBTF-TD protein maintained genomic occupancy at ribosomal DNA loci while also occupying genomic targets commonly dysregulated in UBTF-TD myeloid malignancies, such as the HOXA/HOXB gene clusters and MEIS1. These data suggest that UBTF-TD is a gain-of-function alteration that results in mislocalization to genomic loci dysregulated in UBTF-TD leukemias. UBTF-TD also co-occupies key genomic loci with KMT2A and menin, which are known to be key partners involved in HOX-dysregulated leukemias. Using a protein degradation system, we showed that stemness, proliferation, and transcriptional signatures are dependent on sustained UBTF-TD localization to chromatin. Finally, we demonstrate that primary cells from UBTF-TD leukemias are sensitive to the menin inhibitor SNDX-5613, resulting in markedly reduced in vitro and in vivo tumor growth, myeloid differentiation, and abrogation of the UBTF-TD leukemic expression signature. These findings provide a viable therapeutic strategy for patients with this high-risk AML subtype.

This file contains 16 Supplemental tables and 14 Supplemental Figures, as well as numerous NMR traces, synthetic chemistry schemes, mass spectra, tables, multiple graphics related to gene expression profiles, dose response curves, x-ray structures, westerns, etc.

Figure S1 shows siRNA screening in neuroblastoma cells. Figure S2 shows the dentification of ciclopirox that targets KDM4B. Figure S3 shows the CPX effect on other histone methyl marks. Figure S4 shows that CPX targets the Myc pathway. Figure S5 shows that CPX inhibits oxidative phosphorylation. Figure S6 shows the CPX effect on BE2C xenografts with KDM4B knockdown. Table S1 shows the sequences of the focused KDM siRNA library. Table S2 shows the primers for real time PCR.

Degradation of defective mitochondria is an essential process to maintain cellular homeostasis and it is strictly regulated by the ubiquitin-proteasome system (UPS) and lysosomal activities. Here, using genome-wide CRISPR and small interference RNA screens, we identified a critical contribution of the lysosomal system in controlling aberrant induction of apoptosis following mitochondrial damage. After treatment with mitochondrial toxins, activation of the PINK1-Parkin axis triggered a BAX- and BAK-independent process of cytochrome c release from mitochondria followed by APAF1 and caspase 9–dependent apoptosis. This phenomenon was mediated by UPS-dependent outer mitochondrial membrane (OMM) degradation and was reversed using proteasome inhibitors. We found that the subsequent recruitment of the autophagy machinery to the OMM protected cells from apoptosis, mediating the lysosomal degradation of dysfunctional mitochondria. Our results underscore a major role of the autophagy machinery in counteracting aberrant noncanonical apoptosis and identified autophagy receptors as key elements in the regulation of this process.

Drug combinations are the basis of treatment for modern diseases but arriving at successful combination therapies is fraught with challenges. Decades ago, the limited number of drugs represented a tractable candidate list from which to design combination experiments. However, the current pool of single-agent drugs to potentially combine is far too large to brute-force screen, and purely computational predictions have performed poorly. Suitable screening methods are needed, but the design of experimental approaches has proven to be highly complex; researchers need to carefully balance many variables such as appropriate drug concentration ranges, number of doses, inclusion of replicates, and throughput. Perhaps the most significant obstacle facing these studies is the approach to data analysis, where conflicting definitions of synergy and unintuitive metrics serve to confuse researchers and render largely uninterpretable results. Collectively, these challenges hamper the progress of drug combination research and ultimately translational impact. To overcome these limitations, we have developed a fully self-contained framework to handle both the experimental design and analysis of drug combination experiments. Our method, called Combocat, provides a straightforward way to test and analyze any number of drug combinations and samples, and is suitable for high-throughput. Combocat provides a high-resolution of concentration combinations compared to most current approaches. This is automated by common instruments and uses scripts included within our protocol. Through careful template design, we were able to include 3 replicates of each 10x10 matrix, single-agent drugs, and controls - all within a single 384-well plate. We found our method to work robustly with varying sample types (Human cancer, bacteria, fungi), and readouts. After data generation, files can simply be dragged into our Combocat analysis tool directly. We provide a free, web-based software suite to fully automate the analysis after data collection. The Combocat web tool is intuitive and facilitates interactive exploration of synergy. It also provides a rich array of information such as dose-response curves, IC50 values, synergy matrices, ranked hit plots, and more. Data normalization, synergy algorithms, scoring functions, and other complex calculations are run swiftly and automatically in the background with no need for user input. Notably, we employ statistical testing by taking advantage of experimental replicates, which is a feature we found lacking in most methods. We use a well-documented synergy metric but also decided to formulate our own Combocat score which considers statistical measurements and assay quality. The Combocat score provides an easy interpretation of results and facilitates quick identification of top hits. Collectively, our platform will be used to enhance and expedite the selection of effective drug combinations. Citation Format: William C. Wright, Min Pan, Hyeong-Min Lee, Gregory A. Phelps, Jonathan Low, Duane Currier, Richard E. Lee, Taosheng Chen, Paul Geeleher. Combocat: A high-throughput framework for drug combination studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1907.

Recent proteome and transcriptome profiling of Alzheimer's disease (AD) brains reveals RNA splicing dysfunction and U1 small nuclear ribonucleoprotein (snRNP) pathology containing U1-70K and its N-terminal 40-KDa fragment (N40K). Here we present a causative role of U1 snRNP dysfunction to neurodegeneration in primary neurons and transgenic mice (N40K-Tg), in which N40K expression exerts a dominant-negative effect to downregulate full-length U1-70K. N40K-Tg recapitulates N40K insolubility, erroneous splicing events, neuronal degeneration and cognitive impairment. Specifically, N40K-Tg shows the reduction of GABAergic synapse components (for example, the GABA receptor subunit of GABRA2) and concomitant postsynaptic hyperexcitability that is rescued by a GABA receptor agonist. Crossing of N40K-Tg and the 5xFAD amyloidosis model indicates that the RNA splicing defect synergizes with the amyloid cascade to remodel the brain transcriptome and proteome, deregulate synaptic proteins and accelerate cognitive decline. Thus, our results support the contribution of U1 snRNP-mediated splicing dysfunction to AD pathogenesis. Splicing dysfunction has been observed in Alzheimer's disease but it remains unclear whether splicing defects have a causal role. Here the authors generate a mouse model with perturbed U1 snRNP activity, recapitulating RNA splicing defects, neuron hyperexcitability, neurodegeneration and synergy with the amyloid cascade when crossed with 5xFAD mice.

Disruption of antagonism between SWI/SNF chromatin remodelers and polycomb repressor complexes drives the formation of numerous cancer types. Recently, an inhibitor of the polycomb protein EZH2 was approved for the treatment of a sarcoma mutant in the SWI/SNF subunit SMARCB1, but resistance occurs. Here, we performed CRISPR screens in SMARCB1-mutant rhabdoid tumor cells to identify genetic contrib-utors to SWI/SNF-polycomb antagonism and potential resistance mechanisms. We found that loss of the H3K36 methyltransferase NSD1 caused resistance to EZH2 inhibition. We show that NSD1 antagonizes poly -comb via cooperation with SWI/SNF and identify co-occurrence of NSD1 inactivation in SWI/SNF-defective cancers, indicating in vivo relevance. We demonstrate that H3K36me2 itself has an essential role in the acti-vation of polycomb target genes as inhibition of the H3K36me2 demethylase KDM2A restores the efficacy of EZH2 inhibition in SWI/SNF-deficient cells lacking NSD1. Together our data expand the mechanistic under-standing of SWI/SNF and polycomb interplay and identify NSD1 as the key for coordinating this transcrip-tional control.

Survival in high-risk pediatric neuroblastoma has remained around 50% for the last 20 years, with immunotherapies and targeted therapies having had minimal impact. Here, we identify the small molecule CX-5461 as selectively cytotoxic to high-risk neuroblastoma and synergistic with low picomolar concentrations of topoisomerase I inhibitors in improving survival in vivo in orthotopic patient-derived xenograft neuroblastoma mouse models. CX-5461 recently progressed through phase I clinical trial as a first-in-human inhibitor of RNA-POL I. However, we also use a comprehensive panel of in vitro and in vivo assays to demonstrate that CX-5461 has been mischaracterized and that its primary target at pharmacologically relevant concentrations, is in fact topoisomerase II beta ( TOP2B ), not RNA-POL I. This is important because existing clinically approved chemotherapeutics have well-documented off-target interactions with TOP2B, which have previously been shown to cause both therapy-induced leukemia and cardiotoxicity—often-fatal adverse events, which can emerge several years after treatment. Thus, while we show that combination therapies involving CX-5461 have promising anti-tumor activity in vivo in neuroblastoma, our identification of TOP2B as the primary target of CX-5461 indicates unexpected safety concerns that should be examined in ongoing phase II clinical trials in adult patients before pursuing clinical studies in children.

AbstractInhibition of members of the bromodomain and extraterminal (BET) family of proteins has proven a valid strategy for cancer chemotherapy. All BET identified to date contain two bromodomains (BD; BD1 and BD2) that are necessary for recognition of acetylated lysine residues in the N-terminal regions of histones. Chemical matter that targets BET (BETi) also interact via these domains. Molecular and cellular data indicate that BD1 and BD2 have different biological roles depending upon their cellular context, with BD2 particularly associated with cancer. We have therefore pursued the development of BD2-selective molecules both as chemical probes and as potential leads for drug development. Here we report the structure-based generation of a novel series of tetrahydroquinoline analogs that exhibit >50-fold selectivity for BD2 versus BD1. This selective targeting resulted in engagement with BD-containing proteins in cells, resulting in modulation of MYC proteins and downstream targets. These compounds were potent cytotoxins toward numerous pediatric cancer cell lines and were minimally toxic to nontumorigenic cells. In addition, unlike the pan BETi (+)-JQ1, these BD2-selective inhibitors demonstrated no rebound expression effects. Finally, we report a pharmacokinetic-optimized, metabolically stable derivative that induced growth delay in a neuroblastoma xenograft model with minimal toxicity. We conclude that BD2-selective agents are valid candidates for antitumor drug design for pediatric malignancies driven by the MYC oncogene.Significance:This study presents bromodomain-selective BET inhibitors that act as antitumor agents and demonstrates that these molecules have in vivo activity towards neuroblastoma, with essentially no toxicity.

Pregnane X receptor (PXR) is activated by chemicals to transcriptionally regulate drug disposition and possibly decrease drug efficacy and increase resistance, suggesting therapeutic value for PXR antagonists. We previously reported the antagonist SPA70 and its analog SJB7, which unexpectedly is an agonist. Here, we describe another unexpected observation: mutating a single residue (W299A) within the PXR ligand-binding domain converts SPA70 to an agonist. After characterizing wild-type and W299A PXR activity profiles, we used molecular dynamics simulations to reveal that in wild-type PXR, agonists stabilize the activation function 2 (AF-2) helix in an “inward” position, but SPA70 displaces the AF-2. In W299A, however, SPA70 stabilizes the AF-2 “inward”, like agonists. We validated our model by predicting the antagonist SJC2 to be a W299A agonist, which was confirmed experimentally. Our work correlates previously unobserved ligand-induced conformational changes to PXR cellular activity and, for the first time, reveals how PXR antagonists work.

Mitochondria are vital organelles that coordinate cellular energy homeostasis and have important roles in cell death. Therefore, the removal of damaged or excessive mitochondria is critical for maintaining proper cellular function. The PINK1-Parkin pathway removes acutely damaged mitochondria through a well-characterized mitophagy pathway, but basal mitochondrial turnover occurs via distinct and less well-understood mechanisms. Here we report that the MEKK3-MEK5-ERK5 kinase cascade is required for mitochondrial degradation in the absence of exogenous damage. We demonstrate that genetic or pharmacological inhibition of the MEKK3-MEK5-ERK5 pathway increases mitochondrial content by reducing lysosome-mediated degradation of mitochondria under basal conditions. We show that the MEKK3-MEK5-ERK5 pathway plays a selective role in basal mitochondrial degradation but is not required for non-selective bulk autophagy, damage-induced mitophagy, or restraint of mitochondrial biogenesis. This illuminates the MEKK3-MEK5-ERK5 pathway as a positive regulator of mitochondrial degradation that acts independently of exogenous mitochondrial stressors.

Histone lysine demethylases (KDMs) play critical roles in oncogenesis and therefore may be effective targets for anticancer therapy. Using a time-resolved fluorescence resonance energy transfer demethylation screen assay, in combination with multiple orthogonal validation approaches, we identified geldanamycin and its analog 17-DMAG as KDM inhibitors. In addition, we found that these Hsp90 inhibitors increase degradation of the alveolar rhabdomyosarcoma (aRMS) driver oncoprotein PAX3-FOXO1 and induce the repressive epigenetic mark H3K9me3 and H3K36me3 at genomic loci of PAX3-FOXO1 targets. We found that as monotherapy 17-DMAG significantly inhibits expression of PAX3-FOXO1 target genes and multiple oncogenic pathways, induces a muscle differentiation signature, delays tumor growth and extends survival in aRMS xenograft mouse models. The combination of 17-DMAG with conventional chemotherapy significantly enhances therapeutic efficacy, indicating that targeting KDM in combination with chemotherapy may serve as a therapeutic approach to PAX3-FOXO1-positive aRMS.