{'fraction_non_alphanumeric': 0.031015408929276966, 'fraction_numerical': 0.022026866851047017, 'mean_word_length': 5.038920369818073, 'pattern_counts': {'":': 0, '<': 6, '<?xml version=': 0, '>': 2, 'https://': 2, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Inhibition of HDACs reduces Ewing sarcoma tumor growth through EWS-FLI1 protein destabilization

Oncogenic transcription factors lacking enzymatic activity or targetable binding pockets are typically considered “undruggable”. An example is provided by the EWS-FLI1 oncoprotein, whose continuous expression and activity as transcription factor are critically required for Ewing sarcoma tumor formation, maintenance, and proliferation. Because neither upstream nor downstream targets have so far disabled its oncogenic potential, we performed a high-throughput drug screen (HTS), enriched for FDA-approved drugs, coupled to a Global Protein Stability (GPS) approach to identify novel compounds capable to destabilize EWS-FLI1 protein by enhancing its degradation through the ubiquitin-proteasome system. The protein stability screen revealed the dual histone deacetylase (HDAC) and phosphatidylinositol-3-kinase (PI3K) inhibitor called fimepinostat (CUDC-907) as top candidate to modulate EWS-FLI1 stability. Fimepinostat strongly reduced EWS-FLI1 protein abundance, reduced viability of several Ewing sarcoma cell lines and PDX-derived primary cells and delayed tumor growth in a xenograft mouse model, whereas it did not significantly affect healthy cells. Mechanistically, we demonstrated that EWS-FLI1 protein levels were mainly regulated by fimepinostat's HDAC activity. Our study demonstrates that HTS combined to GPS is a reliable approach to identify drug candidates able to modulate stability of EWS-FLI1 and lays new ground for the development of novel therapeutic strategies aimed to reduce Ewing sarcoma tumor progression.


Introduction
Despite the recent advances in treatment and survival, development of targeted therapies for paediatric malignancies is still very challenging. This might be due to the rarity of paediatric cancers, which limits the enrolment of large numbers of patients in clinical trials, but also to rarity of mutations that render identification of targetable oncogenic drivers arduous. 1 , 2 Chromosomal rearrangements giving rise to oncogenic fusion genes are more common in childhood cancers than in adult tumors. 3 , 4 The transcribed fusion oncoproteins contribute to cell transformation via deregulation of signalling pathways or ectopic activation of genes that support uncontrolled growth and migration of tumor cells. Because of their tumor specificity and established role in tumorigenesis, these oncoproteins often represent unique and promising targets for therapeutic intervention. [5][6][7] Ewing sarcoma (ES) is a rare and aggressive solid tumor mainly affecting children and adolescents. Despite about 80% of survival for localized ES, survival of children diagnosed with metastatic ES or with tumor relapse remains below 40%, 8 , 9 mainly due to the lack of targeted therapies. Similar to other paediatric cancers, ES is characterized by a silent mutational landscape with EWS-FLI1 fusion gene as the most frequent recurrent mutation. 10 , 11 The EWS-FLI1 (EF) oncoprotein originates from the translocation t(11:22)(q24;q12) which fuses the transactivation domain of EWS encoded by EWSR1 gene to the DNA-binding domain of the ETS family member FLI1. 12 The fusion protein acts as aberrant transcription factor by dysregulating gene expression by binding to either an ETS-family DNA motif or to GGAA repeats to promote tumor development. 13 , 14 Previous studies have demonstrated that targeting EF with antisense oligonucleotides or RNA interference (RNAi) strategies can inhibit Ewing sarcoma tumor growth and viability both in vitro and in vivo. 15 , 16 Hence, EF represents an attractive therapeutic target for Ewing sarcoma therapy.
Since direct targeting of transcription factors is still very challenging, we have previously attempted to target endogenous regulators of EF turnover. Along with this strategy, we identified a deubiquitinating enzyme (DUB), namely ubiquitin specific protease 19 (USP19), as positive regulator of EF stability. USP19 genetic depletion led to diminished EF protein levels, followed by a significant reduction of tumor cell proliferation both in vitro and in vivo. 17 Hence, targeting oncoprotein levels instead of their activity may represent a promising strategy to eradicate monogenically-driven cancers. Because targeting DUBs might limit specificity and, in some cases, lead to toxic effects, here we aimed to directly identify small molecules affecting EF protein stability. Towards this goal, we combined a high-throughput drug screen with a global protein stability 18 approach (HTS-GPS) to monitor EF protein stability upon compound treatment. We uncovered fimepinostat, formerly known as CUDC-907, as a strong destabilizer of EF and responsible of a significantly diminished viability of Ewing sarcoma tumor cells in vitro and delayed tumor growth in vivo .

Small molecule library screening and HTS-flow cytometry
Cells of a clone of SKNMC hPGK-DsRed-P2A-TagBFP-EF were seeded in 384-well plates in 20μl of medium at 4000 cells/well. After one day, medium was replaced with drugs pre-diluted in culture medium. Two drug libraries were screened in triplicates: 1) FDA-approved library purchased from NEXUS-ETH (Zurich, Switzerland) with 2'486 drugs and 2) targeted compound library purchased from Selleckchem with 204 compounds. We screened the NEXUS library at the final concentrations of 0.5 and 5μM and the Selleckchem library at 0.5μM. The following controls were included in each 384-well screen plate: medium only, DMSO, CHX and MG-132. The following day, cells were detached with accutase at 37 °C and 5% CO 2 and resuspended in PBS to a final volume of 30μl. Fluorescence intensities of DsRed and TagBFP were measured by using the automated High Throughput Sampler (HTS) LSRFortessa TM flow cytometer (BD Bioscience). The high-throughput mode was used to measure 10μl of sample per well.

Fluorescence microscopy
SKNMC cells with stable doxycycline-inducible 3xflag-EF wt and K380R were seeded in black 96-well plates at 10000 cells/well with or without doxycycline. The next day cells were treated with the drugs of interest for one day. Cells were stained with 20mM Hoechst (ThermoFisher Scientific AG) and 2μg/ml propidium iodide and incubated at 37 °C and 5% CO 2 for 30min. Fluorescence was acquired with an Operetta CLS TM high content analyser (PerkinElmer), and data was analysed with the Harmony 4.5 software.
To assess localization of TagBFP-EF and DsRed, cells were seeded in chamber slides, fixed with 4% PFA (Carl Roth) for 15 min followed by 5 min incubation with 0.1M glycine in PBS, permeabilized for 15 min with 0.1% Triton X-100 in PBS and blocked with 4% horse serum in 0.1% Triton X-100/PBS. All steps were carried out at room temperature. Slides were then mounted with Dako Glycergel mounting medium covered with a coverslip and analysed by fluorescence microscopy.
Mouse xenograft experiments 4 * 10 6 SKNMC cells were injected subcutaneously into the flank of 6to 12-weeks old male NOD-Scid il2rg-/-(NSG) mice. Mice with tumor volumes between 20 and 80 mm 3 were assigned into two groups: vehicle and fimepinostat (75 mg/kg). Fimepinostat was prepared in 30% captisol (Cydex) + 1 N NaOH. Right before oral administration, pH was adjusted (5 < pH > 9) with 1 N HCl. Mice were treated 4 days/week for 4 weeks. Tumor growth was assessed by caliper measurements, and the volumes were calculated using the formula V = (4/3) π r3 with r = (d1 + d2)/4. Body weight and condition were assessed once and twice a day, respectively, for the duration of the study. Mice were sacrificed when tumor volume reached 1000 mm 3 .
For additional methods, see supplementary materials and methods and supplementary Table S1.

A fluorescent-based reporter approach measures stability of EF at the single cell level
To monitor stability of EF in live cells, we first established the GPS system in SKNMC Ewing sarcoma cell line. We engineered a lentiviral reporter construct in which the expression cassette is driven by one unique promoter and permits simultaneous expression of two fluorescent proteins from a single mRNA molecule, using a porcine teschovirus-1 2A self-cleaving (P2A) peptide sequence 19 ( Fig. 1 A). In this system, red fluorescent protein (DsRed) serves as internal control, whereas the blue fluorescent protein (TagBFP) was expressed as fusion protein with EF. Since the fluorescent proteins per se are very stable and DsRed and TagBFP-EF will be expressed in a constant ratio translated from the same mRNA, the fluorescence ratio TagBFP/DsRed directly measures stability of EF protein.
We established a SKNMC-based Ewing sarcoma cell model stably and uniformly expressing TagBFP-EF and DsRed by single cell sorting using FACS (DsRed-TagBFP-EF). Both fluorescence proteins were highly expressed ( Fig. 1 B, left panel) and TagBFP-EF readily detectable by Western blot ( Fig. 1 B, right panel). Hence, this cell model allows to determine the fluorescence ratio between control and candidate protein with high accuracy.
Next, we validated whether changes in the ratio TagBFP/DsRed would effectively correlate with changes in EF stability. For this, we introduced a stop codon in the expression cassette downstream of the TagBFP sequence to prevent expression of EF (DsRed-TagBFP-STOP, Supplementary Fig.S1A) and generated a second cell clone. We confirmed the presence of DsRed and the absence of any exogenous EF in this second model by Western Fig. 1. Fluorescence-based reporter approach to measure EF protein stability. A, Schematic representation of the fluorescence reporter construct hPGK-DsRed-P2A-TagBFP-EWS-FLI1. The presence of a P2A peptide enables ribosomal skipping and production of independent proteins from a unique mRNA molecule in a constant ratio: DsRed and TagBFP fused to EF. TagBFP/DsRed fluorescence ratio is determined by flow cytometry and represents a measure for EF protein stability. B, Left panel, a clonal population of SKNMC cells stably expressing DsRed and TagBFP was established by single cell sorting. Right panel, Western blot analysis of endogenous EF and TagBFP-EF expression in sorted SKNMC, detected with an anti-FLI1 antibody. C, Validation of the fluorescence-based reporter to measure EF protein stability. On the left, SKNMC clonal populations expressing the reporters DsRed-TagBFP-EF and DsRed-TagBFP-STOP were analyzed by Western blotting using anti-FLI1 and anti-mCherry antibodies. On the right, the two cell populations were treated with DMSO, 0.5μM MG-132, cycloheximide (CHX) or chloroquine (CQ) for 24h. TagBFP/DsRed ratios were measured by flow cytometry and analyzed with FlowJo. D, Workflow of the HTS-GPS method. SKNMC DsRed-TagBFP-EF cells were treated with two drug libraries for 24h: 1) FDA-approved drug library with 2'486 drugs at the concentrations of 0.5 and 5μM and 2) targeted compounds library with 204 small molecules at the concentration of 0.5μM. EF stability was determined by HTS-flow cytometry. Increased and decreased TagBFP/DsRed ratio compared to DMSO-treated cells correlate with stabilized and destabilized EF, respectively. E, EF protein stability screen. TagBFP mean fluorescence intensities (y-axis) are plotted versus DsRed intensities (x-axis) as percentage for each drug treatment. Top candidates that shifted TagBFP/DsRed ratio > 25% (dashed lines) compared to DMSO-treated cells were grouped in categories according to their molecular mechanism. HTS, high-throughput drug screen; GPS, Global Protein Stability; EF, EWS-FLI1. blot, in contrast to expression of both endogenous and exogenous EF in SKNMC DsRed-TagBFP-EF ( Fig. 1 C, left panel). We incubated both newly established cell lines with 0.5μM of the proteasome inhibitor MG-132, the lysosome inhibitor chloroquine (CQ) and the protein synthesis inhibitor cycloheximide (CHX) for 24h ( Fig. 1 C, right panel). Incubation with MG-132 positively shifted the TagBFP/DsRed ratio of DsRed-TagBFP-EFexpressing cells, whereas no change was observed upon lysosomal inhibition, hence confirming that EF is a substrate of the proteasome system with high turnover rate, as previously described. 20 Incubation with CHX shifted the ratio to the left, indicating diminished EF protein levels. In contrast, the TagBFP/DsRed ratio of DsRed-TagBFP-STOP cells did not vary neither in presence of MG-132 nor CHX, indicating that changes of the fluorescence ratio indeed specifically monitor alterations of EF protein stability.
To validate the functionality of EF protein fused to TagBFP, we transduced wild type and a pool of several clones of DsRed-TagBFP-EF SKNMC cells with a doxycycline inducible shRNA directed towards the 3'-untranslated part of the transcript targeting endogenous EF only. 48h after dox induction we observed knockdown of endogenous EF both at protein (5-fold down, Supplementary Fig. S1B) and mRNA (3.3-fold down, Supplementary Fig.  S1C) levels. Ectopic expression of EF rescues expression of both repressed (LOX: 2.9-fold lower in ectopic EF-expressing cells compared to wild type cells with knockdown of endogenous EF) and activated (NR0B1 10-fold higher in ectopic EF-expressing cells compared to wild type cells with knockdown of endogenous EF) target genes ( Supplementary Fig. S1D). Further, knockdown of endogenous EF in wild type cells resulted in only 50% viable cells after 48h of dox induction compared to untreated cells, while overexpression of the ectopic oncoprotein fused to TagBFP fully rescued cell viability ( Supplementary Fig. S1E). Hence, ectopically expressed TagBFP-EF demonstrated to be functional despite its fusion to the TagBFP part. We also confirmed the mainly nuclear localization of the TagBFP-EF by fluorescence microscopy, whereas the independent DsRed protein showed a diffuse localization in both nucleus and cytoplasm ( Supplementary Fig. S1F).
These experiments suggest that we successfully established a functional system to reliably monitor EF fusion protein levels.

Protein stability screen identifies small molecule modulators of EF stability
Next, we screened two drug libraries, namely (1) a library composed of 2'486 FDA-approved drugs and (2) a library composed of 204 handpicked targeted compounds. TagBFP/DsRed ratio was measured by highthroughput sampler (HTS)-flow cytometry, which allows automated realtime detection of the two fluorescent proteins at the level of single live cells. Compounds that selectively affect EF stability are expected to alter the TagBFP/DsRed fluorescence intensity ratio, whereby an increased ratio is expected to correlate with EF stabilization, in contrast to a diminished ratio which would monitor lower oncoprotein levels ( Fig. 1 D).
After treating cells for 24h with 0.5μM compounds and monitoring the fluorescence ratios, results were depicted as TagBFP (y-axis) versus DsRed (x-axis) mean fluorescence intensities ( Fig. 1 E). Compounds altering the fluorescence ratio by more than 25% were considered candidates affecting EF protein stability. All the proteasome inhibitors included as positive controls were successfully identified as stabilizers by this procedure, while two protein synthesis inhibitors were found to significantly reduce the TagBFP/DsRed ratio and therefore to destabilize EF. Among the small molecule destabilizers, we also identified cytarabine that was previously reported to decrease EF protein levels. 21 In total, the screen identified 56 compounds diminishing the fluorescence ratio ≤ 25% (Supplementary Table S2). When classifying these hits according to their molecular mechanisms, we identified 23 antineoplastic drugs, 8 HDAC inhibitors, 8 PI3K/mTOR/Akt/IGFR-1 inhibitors, 1 dual HDAC and PI3K inhibitor, 3 cytoskeletal signalling inhibitors, 3 cell cycle inhibitors, 3 cardiac stimulants, 2 anthelmintic compounds, 2 protein synthesis inhibitors, 2 MAPK inhibitors and 1 STAT inhibitor.

Fimepinostat strongly reduces EF protein stability and viability of Ewing sarcoma cell lines and patient-derived xenograft-derived cells (PDCs)
Because we were interested to identify novel compounds that may destabilize EF via specific signalling pathways, we concentrated further validation onto HDAC and PI3K/mTOR/Akt/IGFR-1 pathway inhibitors, as they were among the most enriched classes of candidates (8 out of 15 HDACi, 8 out of 36 PI3K/mTOR/Akt/IGFR-1 inhibitors and 1 out of 1 dual HDACi-PI3Ki).
Among them, the only dual HDAC and PI3K inhibitor fimepinostat, displayed the most promising effect as it diminished endogenous EF protein levels by 85% in ES-01 PDC, 60% in SKNMC and 70% in A673 ES cell lines after one day treatment at nanomolar IC 50 concentrations ( Fig. 2 A) and it strongly reduced viability of 3 ES cell lines and 4 PDCs with IC 50 ranging between 5 and 42.7nM after 72h ( Fig. 2 B). EF expression in all cell models tested was assessed by western blot ( Fig. 2 C). All eight HDACi and two PI3K/mTOR pathway inhibitor candidates confirmed to reduce endogenous EF protein levels and cell viability in both SKNMC and A673 cell lines at nanomolar or low micromolar concentrations (Supplementary Fig.  S2A-S2B). However, fimepinostat was the only candidate that remarkably affected viability of non-malignant cells with only at least ten-fold higher concentrations compared to ES cells with IC 50 of 518nM in fibroblasts MRC5 and 1248nM in mesenchymal stromal cells MSCs. Hence, these results emphasize the validity of fimepinostat as top candidate that unveiled a prominent therapeutic window between cancer cells and normal fibroblasts and mesenchymal stromal cells. When increasing the concentration of fimepinostat to 500nM, we measured a diminished expression of EF transcript by 30% in SKNMC and 60% in A673, compared to a reduction of EF protein levels of 70% in SKNMC and 95% in A673 (Supplementary Fig. S2C and S2D, left panel). Despite the slight reduction of EWS wild type mRNA following fimepinostat treatment in SKNMC, A673 and MRC5 cells, its protein levels remained unaffected (Supplementary Fig. S2C and S2D, right panel). Hence, these data suggest that fimepinostat may act on the EWS promoter and affect regulation of both EWS wild type and EF transcripts. However, while EWS wild type protein stability is preserved, the fusion oncoprotein is much more strongly affected, which suggests a major influence of fimepinostat on its protein stability.
Fimepinostat also strongly inhibited the growth of CADO-ES cells expressing the EWS-ERG oncoprotein with an IC 50 of 5nM and reduced the fusion protein stability in a dose-dependent manner ( Fig. 2 D).
We also included a Ewing-like sarcoma primary cell culture expressing the CIC-DUX4 oncoprotein. 22 In contrast to all other ES cells tested, these primary cells were less sensitive to fimepinostat with IC 50 of 296.7nM, suggesting that fimepinostat treatment results in specific reduction of EWS-FLI1 and EWS-ERG protein levels, with a mechanism that does not affect other fusion oncoproteins.
To underline this observation, we treated cells for three days with 75nM fimepinostat and measured cell viability. Interestingly, viability of all ES cells tested was clearly reduced (relative mean viability ranging from 5 to 33%), except for the Ewing-like sarcoma cells (relative mean viability of 67%), whereas non-tumorigenic cell lines were even less affected (relative mean viability ranging from 74 to 94%) ( Fig. 2 E).
Taken together, our results clearly demonstrate the validity of the HTS-GPS method to identify protein destabilizers, since most of the candidate hits could be successfully confirmed to destabilize endogenous EF protein.

Fimepinostat significantly alters expression of the EF target gene signature
To evaluate which pathways may be primarily affected by fimepinostat in Ewing sarcoma cells, we performed RNAseq experiments with ES-01 cells after treatment with fimepinostat at its IC 50 concentration for ten and twenty hours. Albeit we observed a strong reduction of EF protein levels (60% after 10h and 75% after 20h, Fig. 3 A, left panel), its transcriptional expression was not significantly altered at these two time points (10% after 10h and 20% after 20h, Fig. 3 A, right panel).
RNAseq identified 6027 genes that were differentially expressed after 10h treatment with fimepinostat and 7597 after 20h, with a marked increase of upregulated genes between the two time points compared to downregulated genes (after 10h, 3256 genes were up-and 2771 downregulated, while after 20h transcript levels of 4452 and 3145 genes were altered, respectively, Fig. 3 B and Supplementary Fig. S3A).
Treatment with fimepinostat affected expression of 30.3% of EF target genes after 10h (265 upregulated and 309 downregulated; false discovery rate ≤0.01, |fold change| ≥ 1.5, Fig. 3 C), and 37.2% after 20h (364 upand 341 downregulated; false discovery rate ≤0.01, |fold change| ≥ 1.5, Supplementary Fig. S3B). Gene Set Enrichment Analysis (GSEA) performed with the pre-ranked list of all sequenced genes after 10h treatment was able to identify the described EF signatures and confirmed that genes normally upregulated in Ewing family tumors (EFT) 23 and in cells engineered to express EF 24 were downregulated upon treatment with fimepinostat ( Fig. 3 D, left panel). Beside the EF signature, we also identified already described HDAC and mTOR inhibition signatures and confirmed that HDACi and mTORi-regulated genes were indeed down-or upregulated upon fimepinostat treatment [25][26][27] ( Fig. 3 D, right panel and Supplementary  Fig. S3C). Gene ontology (GO) analysis of significantly downregulated genes after 10h treatment (false discovery rate ≤ 0.01, fold change ≤ -1.5) was performed using the Metascape online platform ( https://metascape.org/ ) and confirmed enrichment of genes upregulated in EFT 28 or upon overexpression of EF 24 (Supplementary Fig. S3D).
Together, our findings demonstrate that fimepinostat significantly reverts the oncogenic transcriptional activity of EF on its target genes by destabilization of the fusion oncoprotein.

Fimepinostat treatment reduces viability of tumor cells through enhanced degradation of EF
Next, we aimed to elucidate whether destabilization of EF by fimepinostat is the main trigger that suppresses viability of tumor cells. Toward this, we used SKNMC cells expressing a doxycycline inducible exchange system. These cells express a doxycycline inducible shRNA targeting endogenous EF and simultaneously overexpress either wild type or K380R mutant 3xflag-EF. K380 was previously demonstrated to regulate EF protein turnover by the ubiquitin-proteasome system (UPS), with the mutation K380R preventing its ubiquitination and stabilizing the oncoprotein 20 ( Fig. 4 A). After doxycycline treatment for 48h, endogenous EF was largely exchanged with ectopic wild type or mutant 3xflag-EF. Importantly, co-treatment with 40nM and 80nM fimepinostat during the last 24h reduced the ectopic wild type protein levels (by 20% and 50%, respectively) but not the mutant, whose protein levels remained unchanged ( Fig. 4 B).
To correlate EF protein levels with cell viability, we measured the fraction of dead cells by fluorescence microscopy after fimepinostat treatment of cells expressing either wild type or mutant EF. Strikingly, expression of mutant EF significantly reduced the number of dead cells compared to wild type protein upon exposure to increasing concentrations of fimepinostat ( Supplementary Fig.S4A).
In contrast, overexpression of the mutant EF was not able to significantly rescue viability of cells after treatment with vincristine and etoposide ( Fig. 4 D) which are antineoplastic drugs used in the clinics that do not directly affect EF protein stability (Supplementary Fig. S4B).
These data support the notion that fimepinostat plays a relevant role to impair Ewing sarcoma tumor progression via reduced EF protein abundance.

Inhibition of specific HDACs reduces EF stability and viability of tumor cells
Fimepinostat was developed by covalently linking two pharmacophores, one targeting HDACs and the other one PI3K ( Fig. 5 A). Therefore, we sought to assess the effect of individual inhibition of HDAC and PI3K signalling pathways on Ewing sarcoma cell viability and on EF protein stability. We treated SKNMC and A673 cells with increasing concentrations of the HDAC inhibitor quisinostat (JNJ-26481585) and the PI3K inhibitor pictilisib (GDC-0941) alone and in combination, in comparison to fimepinostat. Quisinostat alone strongly reduced Ewing sarcoma cell viability while pictilisib showed less effects ( Fig. 5 B). The combination of quisinostat with pictilisib did not seem to significantly enhance the effect of quisinostat, suggesting a predominant dependence of Ewing sarcoma cells on balanced histone deacetylase activity. Fimepinostat more predominantly reduced EF protein stability compared to either quisinostat or pictilisib alone (Supplementary Fig. S5A).
The classical HDAC family contains in total 11 highly conserved enzymes which are divided into three classes. They are involved in different biological processes by deacetylating lysine residues both on histone and non-histone proteins. 29 , 30 All HDACs are expressed in Ewing sarcoma patient samples with HDAC 1, 2, and 6 displaying the highest expression level (Supplementary Fig. S5B). Fimepinostat potently inhibits HDAC 1, 2, 3, 6, 10 and 11. 31 To identify the HDACs whose inhibition affect Ewing sarcoma cells viability, we used a panel of HDAC is with a diverse and well-characterized isoform selectivity spanning all HDAC classes ( Fig. 5 C). We profiled their effect on EF stability in SKNMC DsRed-TagBFP-EF cells by flow cytometry after one day treatment with 50 and 500nM. TagBFP/DsRed fluorescence ratio upon each compound treatment was normalized to DMSO-treated cells, whose ratio was set to 100%. Class I (HDAC1/2/3/8), IIb (HDAC6/10) and IV (HDAC11) inhibitors were the most potent, whereas no destabilization of EF was seen upon inhibition of Class IIa (HDAC4/5/7/9) or Class III (Sirtuin) ( Fig. 5 D, left panel). The selective inhibition of Class I HDACs with BG45, domatinostat, mocetinostat and tacetinalide, or in combination with inhibitors of either HDAC6 or HDAC10 of Class IIb, named ACY-738 and chidamide, respectively, had only little effect on EF destabilization, whereas concomitant inhibition of Class I, IIb and IV with fimepinostat, panobinostat, quisinostat and dacinostat reduced TagBFP/DsRed ratios to less than 70% at 500nM compared to DMSO-treated cells, hence most potently reduced EF levels after only one day treatment. Selective inhibition of HDAC11 with elevenostat also had only moderate effect on EF stability ( Fig. 5 D, right panels).
Thus, these results imply that inhibition of HDACs has a more prominent role to impair Ewing sarcoma tumor cell viability than inhibition of PI3K, and that Class I, IIb and IV HDACs are essential to maintain EF protein levels.
To further explore the importance of the balance between acetylation and deacetylation activity on EF stability, we made use of a PROTAC small molecule directed against the acetyltransferases CREB-binding protein (CBP) and p300 named dCBP-1 32 to test for possible rescue of EF degradation. We treated SKNMC with 100 and 500nM dCBP-1 alone and in combination with 500nM fimepinostat. Selective degradation of p300/CBP alone slightly increased EF protein levels compared to DMSO-control and fully restored the oncoprotein stability when combined with fimepinostat ( Fig. 5 E). Despite further reduction of EF transcript levels after dCBP-1 and fimepinostat treatment, compared to either treatment alone ( Fig. 5 F, left panel), EF protein levels were maintained ( Fig. 5 F, right panel), suggesting that fimepinostat interferes with EF stability also at the post-transcriptional level. To assess the cell phenotype, we measured cell proliferation 48h after with fimepinostat together with dCBP-1 ( Supplementary Fig. S5C and S5D). In line with a visible increase in EF protein stability, degradation of p300/CBP efficiently rescued cell viability in presence of fimepinostat (shift by 2.4-fold of IC 50 ). Hence, our results suggest that a general reduction in acetylation restores EF protein stability, either directly or indirectly.

Fimepinostat delays tumor growth in a Ewing sarcoma xenograft mouse model
Having confirmed the potent antiproliferative activity of fimepinostat in vitro , we next aimed to assess drug response in vivo. We subcutaneously Fig. 4. EF K380R preserves stability of the oncoprotein and rescues viability of Ewing sarcoma cells in presence of fimepinostat. A, Doxycycline-inducible exchange cell lines. Ectopic 3xflag-EF wt is poly-ubiquitylated at K380 and degraded through the proteasome. Ectopic expression of the mutant 3xflag-EF K380R prevents its poly-ubiquitylation and degradation through the proteasome. B, SKNMC inducible EF exchange cell lines were incubated with 0.1μg/ml doxycycline for a total of 48h. 24h after dox incubation cells were additionally treated with DMSO, 40nM or 80nM fimepinostat for 24h. Endogenous and exogenous EF protein levels were analyzed by Western blotting using an anti-FLI1 antibody. Below, quantification of 3xflag-EF and 3xflag-EF K380R proteins over GAPDH compared to DMSO control was performed with ImageJ. n = 3; mean ± SEM. * p < 0.1, * * * p < 0.001, ns = not significant by one-way ANOVA. C and D, Inducible EF exchange cell lines. Quantification of dead versus total number of cells in % by fluorescence microscopy after a total of 48h induction with 0.1μg/ml dox and 24h treatment with 40, 80 and 500nM fimepinostat (C) and 50, 100nM vincristine and 0.5, 5μM etoposide (D). Measurements were performed with an Operetta CLS high-content analyzer and quantified with the Harmony 4.5 software. n = 3; mean ± SEM. * * p < 0.01, ns = not significant by multiple t test. Dox, doxycycline; EF, EWS-FLI1. . Lower left and right panel, TagBFP/DsRed ratios measured by HTS-flow cytometry after incubation with fimepinostat, panobinostat, quisinostat, dacinostat and elevenostat, and analyzed with FlowJo. E, SKNMC cells were treated with 100 and 500nM dCBP-1 and 4h later fimepinostat (500nM) was added alone or combined. After one day treatment, cells were lysed, and proteins were immunoblotted with the indicated antibodies. F, Quantification of EF mRNA and protein normalized with GAPDH. n = 3; mean ± SEM. * p < 0.1, * * p < 0.01, ns = not significant by ordinary one-way ANOVA. injected NOD-Scid (NSG) mice with SKNMC cells and after tumors became palpable assigned mice to different treatment groups with similar mean tumor size at start (the vehicle group had a mean tumor volume at start of 30,6 mm 3 and the fimepinostat treatment group of 37 mm 3 ). Mice were treated 4 days per week for a total of 4 weeks with either vehicle or fimepinostat (75 mg/kg) ( Fig. 6 A). While tumors in the vehicle group continuously grew and reached the mean maximum volume (1000 mm 3 ) after 29 days, the mean of tumor volumes in the group receiving fimepinostat at this time point was reduced by half (500 mm 3 ) ( Fig. 6 B). This is also reflected in the survival of mice, since nearly half of the animals treated with fimepinostat were still living at this time point ( Fig. 6 C). We did not observe any significant weight loss during the course of the experiment. To assess EF protein levels in xenograft tumors, we isolated tumors after 4 weeks of treatment and performed immunohistochemical analysis using the same FLI1 antibody as above. We indeed observed a diminished signal in the treated compared to the control tumors ( Fig. 6 D). In addition, treated tumors displayed larger unstained areas that may represent fibrotic and/or necrotic areas. This is also in line with cleaved caspase-3 staining which was higher in treated tumors versus controls. Western blot analysis with lysates from xenograft tumors confirmed destabilization of EF and of its target gene c-Myc, together with increased histone acetylation upon treatment with fimepinostat ( Fig. 6 E).
The data demonstrate that fimepinostat is able to reduce EF protein levels and slow tumor growth also in vivo . However, reduced stability of EF in Ewing sarcoma cells (EF-low cells) may correlate with an increased migratory status of tumor cells. 33 , 34 Hence, we investigated the effect of fimepinostat on cell migration directly in a spheroid invasion assay in Ewing sarcoma SKNMC cells (Supplementar y Figure S6). We obser ved that treatment with a low concentration of fimepinostat (50nM) indeed increased SKNMC cells dissemination from spheroids, compared to DMSOtreated cells. This confirms the action of fimepinostat as on-target for EF degradation since increased migration was previously shown for EF-low cells. Nevertheless, treatment with 500nM and 1μM fimepinostat reduced cell migration, which emphasizes the ability of this drug to reduce both proliferation and invasiveness of Ewing sarcoma.

Discussion
Here, we identified fimepinostat as an attractive drug candidate that destabilizes EF oncoprotein and potently reduces Ewing sarcoma tumor growth both in vitro and in vivo , while preserving non-tumor cell lines. RNA-sequencing revealed a significant influence of fimepinostat on the transcriptional activity of EF with almost one third of its target genes being differentially expressed following short incubation with fimepinostat. To underscore the clinical relevance of fimepinostat for Ewing sarcoma therapy, the exchange of wild type EF with its more stable mutant rescued tumor cell viability despite treatment with fimepinostat, hence demonstrating a surprising specificity of the drug to act via enhanced degradation of EF.
Targeting stability of a driver oncogene is an attractive therapeutic strategy that can contribute to fight cancer. EF has the potent ability to drive oncogenicity, a fact which encourages studies to better understand the mechanisms regulating its turnover, consequently allowing identification of small molecules capable to reduce its levels. The E3 ligase TRIM8 35 and three deubiquitinases, USP7, 36 USP19, 17 and recently OTUD7A, 37 have been reported as dependencies in Ewing sarcoma. TRIM8 depletion or overexpression lead to low or high EF protein levels, respectively, which are both toxic to Ewing sarcoma cells, 35 while genetic depletion of USP19 and pharmacologic inhibition of OTUD7A have been demonstrated to reduce Ewing sarcoma growth. 17 , 37 Hence, these results underscore the importance of modulating stability of EF as innovative strategy in Ewing sarcoma therapy. In addition, the small molecule YK-4-279 interferes with the interaction of EF with RNA helicase A and thereby efficiently affects both EF activity and ES cell proliferation. 38 Based on these data the derivative drug TK-216 is now tested in a clinical trial in relapsed or refractory Ewing sarcoma patients.
In the last years, a variety of different methods have been developed to target fusion transcription factor-driven tumors. 39 Here, we employed a high-throughput screen and a GPS reporter system to monitor stability of EF protein since the fluorescent proteins themselves have a very long halflife. Relying on TagBFP/DsRed fluorescence ratio as measure of EF stability, we identified the dual HDAC and PI3K inhibitor fimepinostat producing a remarkable antitumor response. It reduced Ewing sarcoma cell proliferation at low nanomolar concentrations and significantly delayed tumor growth in a xenograft mouse model.
To dissect the roles of HDAC and PI3K inhibition by the small molecule fimepinostat, we treated tumor cells with single HDAC and PI3K inhibitors, both alone and in combination. Albeit our data show a more potent reduction of viability by HDAC inhibition compared to PI3Ki, treatment with fimepinostat still reduced tumor cell viability stronger than the combination of the single inhibitors. Thus, the fusion of two small molecules in one structure may have generated a chimeric compound that does not completely recapitulate the properties of the individual molecules. For example, previous findings suggest that fimepinostat can also downregulate MYC 40 and suppress the RAF/MEK/MAPK signalling pathway via HDAC inhibition. 31 Hence, multiple inhibitory activities within one molecule might prevent activation of compensatory mechanisms and potentially result in a greater and more durable effect.
A previous study showed that HDAC inhibition by panobinostat or vorinostat reduced EF mRNA and, as result, its protein abundance. This would exclude that these HDAC inhibitors could affect fusion protein stability directly. 41 Likewise, it was demonstrated that PI3K inhibition with BEZ235 affected EF gene expression via the EWS promoter, which resulted in reduced mRNA and protein levels. 42 In contrast, our study shows that fimepinostat can also reduce stability of exogenous wild type EF proteins, both when fused to TagBFP or flag-tagged. Hence, we suggest that the chimeric small molecule fimepinostat may be able to destabilize EF protein via alternative mechanisms than transcriptional regulation.
Our observation that EF stability is completely rescued following degradation of p300/CBP in presence of fimepinostat emphasizes a counter intuitive mechanism in ES cells compared to other tumor models. Hypothetically, increased acetylation by HDAC inhibition leads to diminished EF protein stability, whereas co-treatment with a selective degrader of p300/CBP restores its stability. Despite a significant reduction of EF mRNA levels in presence of both dCBP-1 and fimepinostat, p300 inhibition leads to a clear stabilization of EF protein levels both in presence and absence of fimepinostat. Overall, this suggests a mechanism where acetylation modulates the stability of EF either directly or indirectly.
Acetylation might regulate the function of EF at different levels. P300/CBP proteins are known to exert their enzymatic function as lysine acetyltransferase to a large number of proteins including DNA-binding transcription factors and other chromatin regulators. 43 Despite the fact that evidence for direct acetylation of EF still remains unresolved, 44 this posttranslational modification may play a role in regulating its stability. Indeed, acetylation of wild type Fli1 by p300/CBP has been shown to impair DNA binding and decrease protein stability. 45 , 46 However, we cannot exclude indirect effects of HDAC inhibitors on transcription that can contribute to diminish EF levels.
Recent studies have shown that EF expression levels can influence tumorigenic phenotypes. High levels of the fusion protein have been associated with a more proliferative cellular state, whereas low levels with a higher propensity to migrate, invade and metastasize. 33 Fimepinostat represses Ewing sarcoma growth and reduces cell migration at higher concentrations through destabilization of EF protein, but also targets HDAC and PI3K signalling pathways, 31 which have been reported as vulnerabilities in Ewing sarcoma. 47 , 48 The PI3K/Akt/mTOR pathway is often deregulated in Ewing sarcoma with genetic alterations observed in PIK3CA, PIK3R1 and PTEN, which play an important role in tumor progression. 48 , 49 Inhibition of HDAC activity largely affects proliferation and survival of Ewing sarcoma cells, alone or in combination with DNA damaging agents, through a variety of pathways that include induction of apoptosis, cell cycle arrest, prevention of tumor invasion and metastasis. 47 , 50-53 Clinical trials have been testing HDAC inhibitors for the treatment of young patients with recurrent or refractory solid tumors. 54 , 55 Hence, fimepinostat can provide simultaneous and sustained inhibition of multiple oncogenic pathways in Ewing sarcoma, as well as reduce levels and transcriptional activity of EF, which jointly may reduce the ability of tumor cells to disseminate.
In the current study we identified EF destabilizers with a reliable method that can be applied to unveil more small molecule destabilizers of other oncoproteins driving different types of cancer. We showed that fimepinostat significantly reduces EF stability and delays tumor growth. Fimepinostat is currently being tested in children and young adults with relapsed or refractory solid tumors ( https://clinicaltrials.gov/ ). Hence, our study may encourage to test fimepinostat in a clinical trial as a therapeutic alternative strategy for Ewing sarcoma therapy.