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# Targeting SUV4-20H Epigenetic Enzymes Enhances Topoisomerase II Poisoning in Prostate Cancer

Fatima Alhourani, Marine Tauziet, Margaux Ayeul, Pierre Dambrun, Hiba Daher, Julie Patouillard, Benoit Miotto, Aurélie Gennetier, Simon George, Xavier Mialhe, et al.

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Fatima Alhourani, Marine Tauziet, Margaux Ayeul, Pierre Dambrun, Hiba Daher, et al. Targeting SUV4-20H Epigenetic Enzymes Enhances Topoisomerase II Poisoning in Prostate Cancer. Cancer Research, 2025, Online ahead of print. 10.1158/0008-5472.CAN-24-3974. hal-05354376

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Submitted on 7 Nov 2025

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# Targeting SUV4-20H Epigenetic Enzymes Enhances Topoisomerase II Poisoning in Prostate Cancer

Fatima Alhourani ¹,²,³, Marine Tauziet ¹,², Margaux Ayeul ¹,², Pierre Dambrun ¹, Hiba Daher ¹,²,³, Julie Patouillard ¹, Benoit Miotto ⁴, Aurélie Gennetier ¹, Simon George ⁶, Xavier Miahle ⁶, Mona Dergham ³, Florence M. Cammas ¹,²,⁵, Adeline Torro ¹, Diego Tosi¹, Celine Gongora ¹,²,⁵, Philippe Pourquier ¹,², Cyril Ribeyre ²,⁷, Véronique Baldin ¹,²,⁵, Raghida Abou Merhi ³* and Eric Julien ¹,²,⁵,*

1. Institut de Recherche en Cancérologie de Montpellier (IRCM), INSERM U1194, Institut Régional du Cancer (ICM), Montpellier F-34298, France

2. University of Montpellier, Montpellier, F-34090, France

3. Faculty of Sciences, Genomics and Surveillance Biotherapy (GSBT) Laboratory, R. Hariri Campus, Lebanese University, Hadath 1003, Lebanon.

4. Université Paris Cité, Institut Cochin, INSERM, CNRS, Paris, France.

5. Centre National de la Recherche Scientifique (CNRS), F-34293, Montpellier, France

6. MGX-Montpellier GenomiX, University of Montpellier, CNRS, INSERM, Montpellier France

7. Institut of Molecular Genetics of Montpellier (IGMM), CNRS UMR 5535, Montpellier, France

**\* Correspondence:**

Eric JULIEN, PhD, (lead contact)
Institut de Recherche en Cancérologie de Montpellier (IRCM), INSERM U1194, 208 Avenue des Apothicaires, Montpellier F-34298, France
Email: eric.julien@inserm.fr

Raghida Abou Merhi, PhD
Faculty of Sciences, Genomics and Surveillance Biotherapy (GSBT) Laboratory, R. Hariri Campus, Lebanese University, Hadath 1003, Lebanon
Email: raboumerhi@ul.edu.lb

**Running Title**: Chromatin-targeted synergistic strategy in prostate cancer

The authors declare no potential conflicts of interest.
ABSTRACT

Commonly used in cancer therapy, topoisomerase II (TOP2) poisons are designed to stabilize the normally transient DNA TOP2 cleavage complexes in chromatin, leading to deleterious DNA double-strand breaks. TOP2 poisons are often associated with significant side effects, highlighting the need to identify strategies aimed at improving the efficacy of TOP2 poisons in order to lower the required dosage. Here, we demonstrated that inhibiting histone H4-lysine 20 (H4K20) methyltransferases SUV4-20H1 and SUV4-20H2 induced synthetic lethality in combination with the TOP2 poison etoposide in prostate cancer. Remarkably, the loss of the SUV4-20H enzymes, which prevents the conversion of H4K20 mono-methylation to higher methylation states, increased replication fork velocity without impacting prostate cancer cell behavior. However, these apparently innocuous epigenetic changes significantly enhanced the trapping of TOP2 complexes in chromatin and increased DNA damage in response to etoposide. Furthermore, SUV4-20H depletion and the subsequent changes in H4K20 methylation impaired the repair of TOP2-induced DNA breaks by disrupting BRCA1-mediated homologous recombination processes, ultimately leading to extensive cancer cell death and significant inhibition of prostate tumor growth in vivo. Overall, these findings demonstrate that targeting the epigenetic activity of SUV4-20H is a powerful strategy to enhance the efficacy of TOP2 poisons and may represent a therapeutic alternative in prostate cancer, where SUV4-20H2 expression emerges as a potential marker of aggressive disease and high metastatic risk.

**Statement of Significance**: Targeting the chromatin-modifying activities of the SUV4-20H enzyme family offers a promising epigenetic strategy to enhance the potency of the topoisomerase-2 poison etoposide in prostate cancer therapy.
INTRODUCTION

The human type-II topoisomerase isoforms α and β (TOP2α and TOP2β, respectively) are essential nuclear enzymes required for removing DNA topological constraints that arise during DNA replication, transcription and mitosis [1,2]. TOP2 enzymes act as transient cleavage-complex intermediates (TOP2ccs) capable of generating reversible double strand DNA breaks (DSBs) by cleaving the DNA phosphodiester backbone [1]. These TOP2ccs complexes consist of covalent phosphotyrosyl bonds of TOP2 monomers at the 5' end of the breaks in both strands. After passing another molecule of DNA through the break site, TOP2 re-ligates the ends and subsequently dissociates from DNA [3]. While TOP2ccs are normally reversible and short-lived under physiological conditions, they can be stabilized and trapped by specific poisons, such as etoposide or mitoxantrone [3,4]. These poisons inhibit the re-ligation step of the reaction, leading to irreversible and cytotoxic TOP2-linked DNA breaks [5]. Therefore, TOP2 poisons are commonly used in oncology to treat a wide range of malignancies, such as lung, breast, testicular and castration-resistant prostate cancer [4,6,7]. However, despite their relative effectiveness, the use of TOP2 poisons is often associated with significant side effects, including long-term toxicities, secondary malignancies, and dose-limiting cardiotoxicity [6,8]. Thus, identifying alternative strategies aimed at mitigating the dosage of TOP2 poisons while increasing their effectiveness is an important issue from both fundamental and clinical perspectives.

The methylation of histone H4 at lysine 20 (H4K20) is an abundant cell-cycle regulated epigenetic modification that can exist as three distinct states: mono-(H4K20me1), di-(H4K20me2) or tri-methylated (H4K20me3) [9,10]. The lysine methyltransferase SET8 is the sole enzyme responsible for H4K20me1, which can be
subsequently converted to H4K20me2 and H4K20me3 by the lysine
methyltransferases SUV4-20H1 and SUV4-20H2, collectively referred to as SUV4-
20Hs [9]. Through their ability to induce H4K20me2 and H4K20me3 signals, SUV4-
20H enzymes have been associated with the regulation of heterochromatin, DNA
replication and repair mechanisms [11–15]. Yet, SUV4-20H2 knockout mice exhibit no
apparent phenotype, suggesting that this enzyme has non-essential functions during
development and in normal adult tissues [16]. In contrast, SUV4-20H1 knockout leads
to developmental defects and perinatal lethality, likely due to widespread chromatin
alterations [16].

Although our understanding of the physiological functions of SUV4-20H
enzymes remains limited, several reports have suggested a pathological role for these
enzymes in cancer [17]. While SUV4-20H1 depletion can drive malignant
transformation in TP53-deficient adult stem cells [18], its expression has also been
shown to inhibit tumor growth in a mouse glioblastoma xenograft model [19]. However,
SUV4-20H1 is also amplified and overexpressed in other cancer types [17],
suggesting a more complex pathological role than previously appreciated. Similarly,
reduced SUV4-20H2 expression is observed in breast cancer and correlates with
EGFR resistance in lung cancers [20–22], while its overexpression promotes
hepatocarcinoma progression [23]. Interestingly, however, targeting both SUV4-20H1
and SUV4-20H2 sensitizes liver cancer cells to Poly(ADP-ribose) polymerase (PARP)
inhibition, suggesting that these enzyme might constitute attractive targets for new
anti-cancer strategies [23].

Herein, we identify SUV4-20H2 as a marker of aggressiveness in prostate
cancer, independently of SUV4-20H1 expression. Importantly, SUV4-20H2 and
SUV4-20H1 are not essential for the survival or proliferation of prostate cancer cells.
However, simultaneous inhibition or loss of both SUV4-20H enzymes and the resulting alterations of H4K20me levels across the genome accelerate replication fork velocity. Although seemingly innocuous, these chromatin alterations specifically enhance the trapping of TOP2α cleavage complexes in response to etoposide, particularly in metastatic prostate cancer cell lines. Furthermore, the loss of SUV4-20H enzymatic activity and the associated changes in H4K20 methylation impair the repair of TOP2αcc-induced DNA breaks by disrupting homologous recombination mechanisms. This, in turn, leads to extensive cancer cell death and effectively halts tumor growth in vivo.
MATERIALS & METHODS

**Analysis of prostate cancer patient data.** Gene expression values from the Prostate Cancer and PanCancer TCGA cohorts were retrieved from the Firebrowse website reporting processed TCGA data (http://firebrowse.org) [24,25]. Expression values from normal (N=52) and prostate cancer (N=497) specimen were determined by RNA sequencing and expressed as mRNA gene expression. They were quantified using the RSEM package; https://github.com/deweylab/RSEM). Genomic and clinical data analyzed are freely available, re-usable and anonymized, and were retrieved from the cBioPortal for Cancer Genomics portal. The following detailed parameters were extracted: patient age, recurrent structural variants and mutations, neoplasm disease lymph node stage, Gleason score, fraction genome altered, MSI MANTIS score, aneuploidy score, Buffa and Winter hypoxia score. Gene expression values from GSE21032 [26] and GSE35988 [27] datasets were collected from the GEO repository (https://www.ncbi.nlm.nih.gov/gds) to assess gene expression changes in primary, local and metastatic prostate tumors. Statistical significances were calculated using Mann-Withney U test.

**Cell culture.** All cancer cell lines were purchased from the ATCC and routinely tested for *Mycoplasma* contamination using MycoAlert detection kit from Lonza. Prostate cell lines were authenticated via short tandem Repeats (STR) via Eurofins. Cells were used in experiments with early passages (<10) from thawing. DU145 (ATCC, RRID: CVCL_0105), 786-O (ATCC, RRID: CVCL_1051), HCT116 (ATCC, RRID: CVCL_0291), MCF7 (ATCC, RRID: CVCL_0031), MDA-MB231 (ATCC, RRID: CVCL_0062), A549 (ATCC, RRID: CVCL_0023) cell lines were grown in Dulbecco's
modified Eagle's medium (DMEM) + Glutamax (Gibco). Caki (ATCC, RRID: CVCL_0234) cell lines were grown in McCoy's 5A medium. BT549 (ATCC, RRID: CVCL_1092), H358 (ATCC, RRID: CVCL_0414), LnCaP (ATCC, RRID: CVCL_4788), 22RV1 (ATCC, RRID: CVCL_1045), and PC3 (ATCC, RRID: CVCL_0035) cell lines were grown in Roswell Park Memorial Institute medium (RPMI 1640) + Glutamax (Gibco 61870-010). Culture medium was supplemented with 10% decomplemented fetal bovine serum (FBS) (Eurobio/Sigma-Aldrich) and 1% penicillin/streptomycin. Cells were incubated at 37°C, 5% CO2.

**Drugs and antibiotic.** The following reagents were used: A-196 (MedChemexpress, HY100201), Etoposide (Sigma-Aldrich, E1383), Camptothecin (Sigma-Aldrich, C9911), Mitoxantrone (Sigma-Aldrich, M6545), Doxorubicin (Sigma-Aldrich, D1515), Pyridostatin (MedChem express, HY-15176A), Cazabitaxel (Sigma-Aldrich, SML2487), Olaparib (MedChemexpress, HY-10162), Puromycin (InvivoGen, ant-pr-1, RRID: AB_2895955).

**SUV4-20H1 and SUV4-20H2 KO cellular models.** Knock-out of SUV4-20H1 and SUV4-20H2 were generated by CRISPR-Cas9 technology in prostate DU145 cell line. Briefly, DU145 cells were transfected using Jet-PEI with the plasmid pSpCAS9-2A-GFP and the lentiviral vector U6-gRNA2A-TagBFP expressing BFP and guide RNAs targeting SUV4-20H1 (HS5000028803) or SUV4-20H2 (HS5000023102). The guide RNAs were purchased from the Sanger Whole genome CRISPR arrayed library (Merck). Two days after transfection, cells were sorted by flow cytometry for GFP and BFP expression and positive clones were grown for three weeks without selection for Cas9 and RNA guides expression. The cellular clones inactivated for SUV4-20H1 or
SUV4-20H2 were then identified by western blotting using antibodies against H4K20 methylation, SUV4-20H1 and SUV4-20H2. The inactivation of SUV4-20H1 or SUV4-27 20H2 encoding genes was then confirmed by DNA sequencing. The double KO SUV4-20H1 and SUV4-20H2 cells were generated by transfection of SUV4-20H2-KO DU145 cell line with the guide RNA targeting SUV4-20H1.

**Measure of cell proliferation and sensitivity to drug treatments.** Cell viability upon drug treatments as indicated was evaluated using Resazurin (Alamar Blue) (Merck, R7017) and MTT (Merck, M2128) viability assays and cell proliferation and growth rate were measured with the Incucyte cell-by-cell analysis module following the manufacturer recommendations (Sartorius).

**Drug combination screening.** For drug combination screening, prostate cancer cell lines were treated with increasing concentration of each drug alone or in combination for three days. The percentage of viable and dead cells were then evaluated using the Celigo Imaging Cytometer (RRID/SCR_018808) after Hoechst 3342 (ThermoFisher Scientific, H1399) and propidium iodide (PI) (Sigma-Aldrich, P4170) staining and following the manufacture recommendations (Nexcelom). The interaction between the drugs tested in vitro was quantified with a concentration matrix test, in which increasing concentration of each single drug were assessed with all possible combinations of the other drugs. For each combination, the percentage of expected growing cells in the case of effect independence was calculated according to the Bliss equation: $fuc=fuAfuB$, where fuc is the expected fraction of cells unaffected by the drug combination in the case of effect independence, and fuA and fuB are the fractions of cells unaffected by treatment A and B, respectively. The difference between the fuc
value and the fraction of living cells in the cytotoxicity test was considered as an estimation of the interaction effect, with positive values indicating synergism and negative values antagonism. The combination index and the efficiency index were calculated based on these values using R software.

**Measure of drug synergy and statistical analysis.** Cell growth was evaluated using the sulforhodamine B (SRB) assay. Briefly, 1000 cells/well were seeded in 96-well plates. After 24 hours, drugs were added in serial dilution. Cells were incubated for 96 hours, after which, they were fixed in 10% trichloroacetic acid solution (SIGMA), and stained with 0.4 % SRB (SIGMA) solution in 1% acetic acid (FLUKA). The plate was washed three times with 1% acetic acid and fixed SRB was dissolved in 10 mmol/L Tris-Base solution (Trizma®base SIGMA). The absorbance was read at 560 nm using the PHERAstar FS plate reader (BMG LABTECH). The percentage of living cells after incubation with each drug alone or in combination was then calculated. The interaction between the drugs tested in vitro was investigated with a concentration matrix test, in which increasing concentration of each single drug were assessed with all possible combinations of the other drugs [28]. For each combination, the percentage of expected growing cells in the case of effect independence was calculated according to the Bliss equation: $fu_c = fu_Afu_B$, where $fu_c$ is the expected fraction of cells unaffected by the drug combination in the case of effect independence, and $fu_A$ and $fu_B$ are the fractions of cells unaffected by treatment A and B, respectively. The differences between the $fu_c$ value and the fraction of living cells in the cytotoxicity test was considered as an estimation of the interaction effect, with positive values indicating synergism and negative values antagonism. According to the approach proposed by Lehar, a point-by-point calculation of the expected values in case of
absence of interaction effect is performed over all the matrix concentration combinations in order to obtain an expected-value matrix. Then, a combination index, C.I., is calculated as follows: $C.I.=\ln A \ln B \sum A, B (M_0-M_E)$; where $M_0$ and $M_E$ are the matrices of the observed and expected values, respectively, and $f_A$ and $f_B$ are the dilution factors for the drugs A and B. The CI is a positive-gated, effect-weighted volume over the lack of interaction effect (i.e. Bliss independence), adjusted for variable dilution factors $f_A$ and $f_B$. The efficacy Index (E.I.) is calculated as follow: $E.I. = \ln A \ln B \sum A, B M_0$; where $M_0$ is the number of dead cells. Combination index represent the level of synergistic effect between two drugs, and the Efficacy Index represent the effect of both drugs on the cell mortality. The two indexes are then plotted in order to visualize the drug combination the most synergistic and efficient.

**RNA sequencing.** Total RNA from DU145 cells treated or not with A-196 for 10 days was extracted using the Monarch Total RNA Miniprep Kit (Biolabs), and treated with DNase I treatment to remove genomic DNA contamination. RNA sequencing was performed at MGX Montpellier GenomicX, Montpellier, France. RNA-Sequencing library was prepared using a Stranded mRNA Prep Ligation kit from Illumina. The validation of the libraries was performed by quantifying the concentration and the size of fragments of DNA on Fragment Analyzer (High Sensitivity NGS kit) as well as by qPCR (ROCHE Light Cycler 480). The clustering and sequencing steps were carried out on an Illumina NovaSeq 6000 using the SBS (Sequence by Synthesis) technique using the NovaSeq Reagent Kits. Image analysis and base calling were performed using the NovaSeq Control Software and the Real-Time Analysis component (Illumina). Demultiplexing was performed using Illumina's conversion software (bcl2fastq, v2.20.0.422). The quality of the raw data was assessed using FastQC (v0.11.9) from the Babraham Institute and the
Illumina software SAV (Sequencing Analysis Viewer, v1.1.23). FastQ Screen (v0.15.0) was used to identify potential contamination. The alignment program TopHat2 (v2.1.1), with Bowtie (v2.4.5) [29] was used to align the RNA-Seq reads to the human genome (GRCh38, from Ensembl), whose index was built (bowtie2-build) using a set of gene model annotations (gff file downloaded from NCBI on 2022/01/04). For alignment, the TopHat2 was set with the option –library-type fr-firststrand, in accordance to the kit used for library construction, and with options –read-mismatch 6 –read-gap-length –read-edit-dist 6. Otherwise, default TopHat2 options have been used. SAMtools (v1.13) was used to sort and index the alignment files. Then, gene counting was performed with featureCounts (v2.0.3) [30]. As the data is from a strand-specific assay, the reads have to be mapped to the opposite strand of the gene (‘-s 2’ option). Before statistical analysis, genes with less than 15 reads (cumulating all the analysed samples) were filtered out. Differentially expressed genes were identified using the Bioconductor [4] package DESeq2 (v1.32.0) [5] (R v4.1.1). Data were normalized using the DESeq2 normalization method with a false discovery rate threshold of 0.01 and log FC threshold of 0.69 (50% increase in gene expression). Genes with adjusted p-value below 1% (according to the FDR method from Benjamini-Hochberg) were called differentially expressed (DE). functional enrichment analysis and functional annotation of gene lists were performed with home-made R scripts and DAVID web software from NIH. RNAseq Data have been deposited in the public NIH (SRA) database and available with the accession reference PRJNA1162501.

**DNA fiber assays.** DNA was labeled for 30 minutes with 25µM 5-iodo-20-deoxyuridine (IdU) (Sigma-Aldrich, 17125) and washed with medium, then treated with 50µM 5-chloro-20-deoxyuridine (CldU) (Sigma-Aldrich, C6891) for 30 minutes. After labelling,
cells were collected with trypsin and resuspended in PBS at 500 cells/µL. Cells were lysed with spreading buffer (0.5% SDS, 200 mM Tris-HCl, pH 7.5, and 50 mM EDTA) and DNA fibers were stretched onto glass slides and left to air dry then fixed in methanol/acetic for 10 minutes. After fixation, DNA was denatured with 2.5 M HCl for 1h 30 minutes, and slides were blocked with 6%BSA PBS 0.1% Tween-20 for 1 h. Then, slides were incubated with primary anti-BrdU/IdU (BD Biosciences, 347580, RRID: AB_395210) and anti-CldU (Origene, TA190126, RRID: AB_2895955) for 45 minutes (1/100) followed with secondary antibodies Alexa 488 antimouse (Invitrogen, A11017, RRID: AB_2534084) and anti-rat-Cy3 (Jackson Immuno-research, 712-166153, RRID: AB_2340669) respectively for 30 minutes (1/100) in PBS 0.1% TritonX-100 at 37°. Slides were mounted with ProLong Diamond antifade (Invitrogen). Fibers were visualized and imaged by Zeiss Axio Imager M2 upright microscope (Zeiss) equipped with an Apochromat 40X objective (NA 1.4, immersion oil). Replication track lengths were analyzed using ImageJ software (RRID:SCR_003070). Statistical significance was performed using GraphPad Prism (version 10.4.2 (633), RRID: SCR_002798) by an unpaired t-test. ns: p-value > 0.05; * p-value < 0.05 ; ** p-value < 0.01 ; *** : p-value < 0.001.

**Colony formation assays.** Cells were seeded at 50,000 cells/well in 12-well plate. After 24h, cells were treated with combinational concentrations of A-196 and etoposide, Mitoxantrone or Camptothecin for 72h. Following treatment, drugs with medium were removed and cells were harvested and resuspended in fresh medium at 200 cells/ml. Cells were seeded in new 12-well plates and incubated at 37°C for 14 days to allow the formation of colonies. After 14 days, medium was removed, and cells were fixed with methanol for 20 minutes then washed and incubated with crystal violet staining solution
for 5 minutes in order to stain the colonies. The number of colonies was counted and
statistical significance was evaluated using GraphPad Prism by an unpaired t-test. ns:
p-value > 0.05; * p-value < 0.05 ; ** p-value < 0.01 ; *** p-value < 0.001.

**Proteins extraction and immunoblotting.** For immunoblot analysis, cells washed with phosphate-buffered saline (PBS) were lysed in 2X SDS Sample Buffer (10 mM Tris-HCl, pH6.8, 5% glycerol, 1% SDS, 1 mM DTT, 0.5% β-mercapto-ethanol) at 95°C for 10 minutes. After measuring protein quantity by Bradford, equal amounts of protein were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with one of the following antibodies: anti-H4K20me0 (Abcam, #227804, RRID: AB_2892991), H4K20me1 1/1000 (Cell Signaling Technology, #9724, RRID: AB_1264176), H4K20me2 1/1000 (Cell Signaling Technology, #9759, RRID: AB_330951), H4K20me3 1:1000 (Cell Signaling Technology, #5737, RRID: AB_10828431), Histone H4 1:1000 (Cell Signaling Technology, #2935, RRID: AB_1147658), Histone H3 (Cell Signaling Technology, #4499, RRID: AB_10544537), SUV4-20H1 1:1000, SUV4-20H2 1:1000, Tubulin 1:20000 (Sigma T5168, RRID: AB_477579), DNA-PK 1:1000 (Invitrogen, PA5-29023, RRID: AB_2546499), phospho-S2056 DNA-PK 1:1000 (Cell Signaling Technology, #6876, RRID not found), ATM 1/1000 (Cell Signaling Technology, #2873, RRID: AB_2062659), phospho-ATM S1981 1/1000 (Cell Signaling Technology, #13050, RRID: AB_2798100), ATR 1/1000 (Cell Signaling Technology, #13934, RRID: AB_2798347), phospho-T1989 ATR 1:1000 (Cell Signaling Technology, #30632, RRID: AB_2798992), RPA32 1/1000 (Cell Signaling Technology, #35869, RRID: AB_2799086), phospho-S4/S8 RPA 1:1000 (Cell Signaling Technology, #54762, RRID: AB_2799471), anti-BRCA1 (Cell Signaling Technology, #9010S, RRID: AB_2228244), anti-BARD1 (Sigma PLA0074, RRID not found). Secondary antibodies used are the following: Anti-rabbit
1:10000 (Cell Signaling Technology, #7074, RRID: AB_2099233) and anti-mouse
1:10000 (Cell Signaling Technology, #7076, RRID: AB_330924) coupled to HRP. Protein
bands were visualized on X-ray film by electro-chemiluminescence (Immobilon Western
HRP Substrate, WBKLS0500, Millipore). The quantification of signal was performed
using ImageJ software (RRID:SCR_003070). Statistical significance was evaluated
using GraphPad Prism (version 10.4.2 (633), RRID: SCR_002798) software by an
unpaired t-test. ns: p-value > 0.05; *: p-value < 0.05; ** p-value < 0.01; ***: p-value <
0.001.

**Chromatin fractionation.** Cells were resuspended with Buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.34 M Sucrose, 10% Glycerol, 1 mM DTT, 0.1% TritonX-100, and protease inhibitors (Roche/Merck, Complete EDTA-free protease inhibitor cocktail reference :11697498001) and incubated for 2 minutes on ice to extract soluble proteins. After centrifugation, supernatant containing soluble proteins was collected and cells were washed with the Buffer A without 0.1% TritonX-100. Then, nuclei were resuspended with Buffer B (3 mM EDTA, 0.2 mM EGTA) and incubated for 30 minutes with occasional vortex on ice to extract nuclear soluble proteins. After centrifugation, supernatant containing soluble proteins was collected and combined with the first fraction of soluble proteins. Chromatin-enriched pellets were then washed with the Buffer B and, after centrifugation, were dissolved in 2X SDS Sample Buffer (10 mM Tris-HCl, pH6.8, 5% glycerol, 1% SDS, 1 mM DTT, 0.5% β-mercaptoethanol) and resolved by SDS-PAGE before immunoblotting.
**Immunofluorescence and Microscopy analysis.** Cells were seeded and treated after 24h with A-196 4µM for 72h. Then, cells were transferred on coverslips with maintenance of A-196 and grown to 50% confluency to be treated with etoposide for 24h. Following this treatment, cells were washed with medium and a fresh medium was added with or without the maintenance of A-196. At the indicated time, cells were pre-extracted with ice-cold PBS containing 0.1% Triton-X100 for 1-2 minutes and fixed with 4% paraformaldehyde (Thermo Scientific, 043368.9M) diluted in PBS for 15 minutes at room temperature (RT) after 0, 6, 24, and 48hr of etoposide removal. After fixation, cells were 34 permeabilized with a solution of PBS with 0.25% Triton-X100 for 10 minutes at RT. Then, cells were incubated for 1h at RT in PBS solution complemented with 5% BSA and 0.1% Tween20 prior to incubation overnight at 4°C with the following primary antibodies: γ-H2AX 1:500 (Millipore, JBW301, RRID: AB_309864), 53BP1 1:500 (Cell Signaling Technology, #4937, RRID: AB_10694558), RPA70 1:100 (Cell Signaling, #2267, RRID: AB_2180506), RAD51 1:500 (Millipore, PC130, RRID: AB_2238184) and BRCA1 1:500 (Cell Signaling Technology, #9010S, RRID: AB_2228244). On the following day, cells were washed with PBS solution containing 0.5% Tween-20 and incubated with appropriate secondary mouse and rabbit antibodies coupled to Alexa Fluor 568 or 488 (Thermo Fisher Scientific, RRID: AB_143165, AB_143157) diluted (1:1000) in PBS solution with 5% BSA and 0.1% Tween-20 for 2 hours at RT. DNA was counterstained with 0.1 µg/ml of DAPI (DNA intercalator, 4',6-Diamidino2-Phenylindole, D1306, Invitrogen, RRID: AB_2629482). Coverslips were mounted with ProLong Diamond Antifade (Invitrogen, P36990). Images were acquired with an ORCA-Flash4.0 LT+ Digital CMOS camera (C1140-42U30, Hamamatsu) controlled by Zen acquisition software (Zeiss) using Zeiss Axio Imager M2 upright microscope (Zeiss) equipped with an Apochromat 63X objective
(NA 1.4, immersion oil). Images were prepared using Zen software (RRIB: SCR_013672) and nuclear foci were quantified using CellProfilerTM (RRID: SCR_007358) software. Representative images were prepared using Photoshop software (Adobe, RRID: SCR_014199). Statistical significance was evaluated using GraphPad Prism (version 10.4.2 (633), RRID: SCR_002798) by an unpaired t-test or two-tailed Student t-test. ns: p-value > 0.05; \* p-value < 0.05; \*\* p-value < 0.01; \*\*\* p-value < 0.001.

**Flow cytometry analysis.** Prior fixation, cells were incubated with BrdU (300µM, Sigma-Aldrich B5002, RRID: AB_476811) for 1 hour. Cells were then fixed with an ice-cold 70% ethanol solution and permeabilized with 0,2% Triton X-100 for 10min. DNA was then denatured with 0,2N HCl before incubation with BrdU antibody (1:200, BD, Biosciences, RRID: AB_395210) diluted in PBS solution with 0,2% Tween20 and 1% BSA for 1 hour at RT followed by 1 hour incubation with an FITC-conjugated antibody (BD, Biosciences; 1:300, RRID: AB_393789). DNA was then counterstained by 12 hours incubation with 2 µg/ml 7-Amino-Actinomycin D (7AAD, Sigma-Aldrich, RRID: AB_2336794) in presence of RNAse (100 µg/ml). FACS acquisition were carried out with Gallios Flow Cytometer (Beckman Coulter, RRID:SCR_019639). FACS data were analyzed using FlowJo software (BD Biosciences, RRID: SCR_008520). Statistical significance was evaluated using GraphPad Prism software by an unpaired t-test. ns: p-value > 0.05; \*: p-value < 0.05; \*\* p-value < 0.01; \*\*\* p-value < 0.001.

**Pulse Field Gel Electrophoresis.** Cells were seeded and treated after 24h with A-196 4µM for 72h. Then, cells were transferred in new plates with maintenance of A-196 and grown to 50% confluency to be treated with etoposide for 3 hours. After
treatment, cells were collected with trypsinization and washed once with PBS. 106 cells were then melted in 0.5% agarose insert. After solidification, these inserts were incubated in lysis buffer (100 mM EDTA pH8, 0.2% Sodium deoxycholate (DOC), 1% Sodium lauryl sarcosine, 1 mg/ml Proteinase K) at 37 °C for 48h and then washed 2-4 times with the following buffer: 20 mM Tris-HCl, pH 8, 50 mM EDTA pH 8. Before loading onto a 0.9% agarose prepared in 0.25X TBE, DNA is separated by pulsed-field gel electrophoresis for 24 hours (Biometra Rotaphor 8 System, 23h; interval: 30-5 s log; angle: 120-110 linear; voltage: 180-120 V log, 13°C). The gel was subsequently stained with ethidium bromide for analysis. The quantification was performed using ImageLab software (RRID: SCR_014210). Statistical significance was evaluated using GraphPad Prism software by an unpaired t-test. ns: p-value > 0.05; * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001.

**DNA repair assays by flow cytometry.** Homologous recombination (HR) and nonhomologous end-joining (NHEJ) U2OS reporter cells were grown in Dulbecco's modified Eagle's medium (DMEM) + Glutamax (Gibco). Reporter cells were treated with DMSO or A-196 (4 mM) during 4 days before transfection with 5 ug of plasmid encoding the I-Scel endonuclease and 0.5 ug of plasmid encoding for histone H2B-mcherry using Jet-PEI reagent. Still maintaining DMSO or A-196 treatment, cells were collected 36 hours after transfection and analyzed for mcherry and GFP fluorescence using FACS ARIA-III (BD Biosciences) and FlowJo software (BD Biosciences). Immuoblots analysis were used to H4K20 methylation changes in A196 treated cells prior to plasmid transfection and I-Scel expression prior to FACS analysis. DNA repair efficiency was calculated based on the GFP/mCherry ratio and according to the levels in control
(DMSO) un-transfected cells. The results are presented as mean ± s.d. from four independent experiments with *p*-values determined with unpaired t-test.

**Immuno-Complex of Enzyme (ICE) assay.** Topoisomerase-DNA covalent cleavage complexes were isolated from parental DU145 and SUV4-20H1/SUV4-20H2 double knockout (DKO) cells using the Immuno Complex of Enzyme (ICE) assay as previously described [31]. Cells were pre-treated with 4 µM A-196 for 4 days prior to seeding at 200,000 cells per well in 6-well plates and maintained in A-196 throughout the experiment. Cells were then treated with either etoposide (1 or 5 µM for 3 hours) or camptothecin (CPT, 1 µM for 1 hour) before harvesting by scraping six wells per condition. Collected cells were centrifuged at 1,500 g for 5 minutes and pellets were then lysed directly in 2 mL of 1% sarkosyl, followed by Dounce homogenization. Lysates were layered onto step gradients consisting of 2 mL each of cesium chloride (CsCl) solutions with densities of 1.82, 1.72, 1.50, and 1.45 g/mL and centrifuged at 165,000 g for 17 hours at 20°C using a Beckman SW41Ti rotor. DNA-containing fractions were collected from the bottom of the tubes using a fraction collector, pooled, quantified by agarose gel 37 electrophoresis, normalized for DNA content, and diluted 1:1 with 25 mM sodium phosphate buffer (pH 6.5) prior to slot blotting onto Immobilon-P membranes using a slot-blot vacuum manifold, with three different DNA input concentrations per sample. Topoisomerase-DNA adducts were detected by immunoblotting using primary antibodies against Top1 (BD Pharmingen, reference 556597, RRID: AB_396986; 1:1000 in PBS-T with 5% milk), Top2α (Santa Cruz, reference sc-365916, RRID: AB_2238324; 1:1000 in TBS-T with 5% BSA), and Top2β (Santa Cruz, reference sc-13059, RRID: AB_628129 1:250 in PBS-T with 5% milk). Membranes were incubated overnight with primary antibodies at 4°C, washed three
times with PBS-T, and then incubated for 1 hour at room temperature with appropriate HRP-conjugated secondary antibodies. After three additional washes, membranes were developed using ECL RevelBlot Intense substrate (Ozyme, OZYB002), and chemiluminescent signals were detected and quantified using a LI-COR Odyssey Fc Imaging System (Li-Cor, RRID:SCR_023227).

**Heparin-based extraction of DNA-tightly bound proteins.** Cells were plated and treated after 24h with A-196 4µM for 72h. Then, cells were transferred into a 6-well plate with maintenance of A-196 and grown to 50% confluency to be treated with etoposide for 3h. For immunoblotting analysis, cells were harvested with trypsin, and washed with cold PBS. After gentle centrifugation, pellets were resuspended in lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.5% IGEPAL CA-630, 2X HALT Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, 78442) complemented with 100 U/mL Heparin (Sigma-Aldrich, H3393) for 15 minutes on ice. After the incubation, lysates were centrifuged at 15000 rpm for 5 minutes at 4°C and pellets were resuspended again with lysis buffer. To facilitate the migration of the protein in immunoblotting, two cycles of sonication were performed to degrade the DNA present in the extracts using Bioruptor Pico (Diagenode) (2 cycles – 30 sec ON and 30 sec Off each). Protein concentration was 38 then determined with Nanodrop by measuring the absorbance at 280 nm. Before loading, heparin-based extracts were diluted with 2X SDS Sample Buffer (10 mM Tris-HCl, pH6.8, 5% glycerol, 1% SDS, 1 mM DTT, 0.5% β-mercaptoethanol) and immunoblotting was performed with the following antibodies: anti-Topoisomerase IIα 1:1000 (Santa Cruz sc365916, RRID: AB_2238324) and anti-histone H3 1:2500 (Cell signaling Technology #4499, RRID: AB_10544584).
**In vivo Animal studies.** The Institute of Animal Care and French APAFIS committee approved all mouse protocols used in this study (French National Agreement #28412). One million of DU145 cells and SUV4-20H knock-out cells were subcutaneously injected into intact athymic nude male mice. Treatment was initiated when tumor size reached ~100 mm3 via intraperitoneal injection (i.p) two times per week. For the combination therapy experiment with DU145 xenografts, mice were randomized into four treatment groups: Vehicle (20% DMSO/corn oil), A-196 (7.5 mg/kg), etoposide (2.5 mg/kg), and combination of the two drugs. For the experiment with wild-type and SUV4-20H knock-out DU145 xenografts, male mice were randomized into different treatment groups and treated with DMSO or with 2.5 mg/kg or 6mg/kg of etoposide every three days. Mice were weighed weekly to monitor for toxicity and tumor growth was assessed by serial caliper measurements every three days. Haematology and Biochemical analysis were performed every week using MS4 and MS-Scan-e analyzers (Melet Schloesing). Survival curves were constructed following the Kaplan-Meier method using GraphPad Prism software. In vivo experiments have been approved by ethics Committee of the French ministry of higher education research and innovation with the APAFIS number #28412-2020112514195037. Statistical significance was evaluated using GraphPad Prism software by an unpaired t-test. ns: p-value > 0.05; *: p-value < 0.05; ** p-value < 0.01; ***: p-value < 0.001.

**Data availability.** All raw data and cellular models generated in this study are available from the corresponding author upon request. Prostate TCGA data used in this study were accessed via cBioPortal. The RNA sequencing data from benign, primary and metastatic tumors are available at Gene Expression Omnibus datasets
(accession numbers GSE21032 and GSE35988). The RNA seq data of DU145 cells treated with A-196 are publicly available in the Sequence Read Archive database with accession number PRJNA1162501. The code used for the calculation of drug synergy and the indexes of combination and efficiency are available at https://github.com/eric-julien/fatima_alhourani_et_al. or via code ocean :

https://codeocean.com/capsule/6334653/tree/v1

## RESULTS

**SUV4-20H2 gene expression is a marker of aggressive prostate cancer.**

Immunohistology experiments have highlighted alterations in the levels of histone H4K20 methylation in prostate tumors [32], suggesting an involvement of histone H4K20 methyltransferases in this disease. To delve deeper into this hypothesis, we first examined the expression levels of the three main histone H4K20 methyltransferases—SET8, SUV4-20H1 and SUV4-20H2— utilizing the transcriptomic data set from the well-established prostate tumors cohort of the Cancer Genome Atlas (TCGA). This cohort encompasses 497 primary tumor samples and 52 normal samples serving as non-tumoral controls [24]. No significant differences were observed in the expression levels of SET8 and SUV4-20H1 encoding genes (Figure 1A). In contrast, primary prostate tumors exhibited significantly elevated SUV4-20H2 gene expression compared to non-tumoral samples (Figure 1A). However, contrary to previous thoughts [33], the up-regulation of SUV4-20H2 in primary prostate tumors was independent of the Gleason score that currently defines tumor grades (Supplementary Figure S1A). It was slightly more pronounced in younger patients
(Supplementary Figure S1B) and associated with specific genetic features (Supplementary Figures S1C-S1G). Although differences remain modest, heightened *SUV4-20H2* expression tended to coincide with P53 mutation (Supplementary Figure S1C), the absence of structural variants of the ERG oncogene (Supplementary Figure S1D) and neoplasm disease lymph node stage 1 (Supplementary Figure S1E). In contrast, no significant correlation was identified with TMPRSS2 structural variant and SPOP mutation (Supplementary Figure S1F and S1G). The expression of *SUV4-20H2* gene also correlated with genome instability, as indicated by the higher hypoxia score and the significant increase in the aneuploidy and the MSI mantis score in primary tumors samples with heightened *SUV4-20H2* expression (Supplementary Figure S2). In agreement with these results, *SUV4-20H2* mRNA levels were found to be associated with shorter progression free and disease-free survival (Figure 1B), suggesting that *SUV4-20H2* expression is a potential marker of aggressiveness in prostate tumors. Supporting this hypothesis, the increase in *SUV4-20H2* expression was linked to the occurrence of metastasis in two other distinct cohorts of PCa patients (Figure 1C). Furthermore, compared to all types of castration-resistant prostate cancer, high *SUV4-20H2* expression emerged as a marker of neuroendocrine prostate cancer (NEPC) (Figure 1D), which represents a highly aggressive subtype of prostate cancer that commonly emerges through a trans-differentiation process and evades conventional therapies. Altogether, these clinical data indicate that the up-regulation of *SUV4-20H2* expression is a potential new marker for prostate tumors associated with specific genetic alterations and that are prone to progression towards metastatic and resistance stages.
**Targeting SUV4-20Hs induces a higher replication fork velocity that is inherently innocuous in Prostate Cancer cell lines.** The elevated expression of SUV4-20H2 and its correlation with metastatic and potential aggressiveness of PCa tumors prompted us to investigate whether this enzyme is essential in prostate cancer cells. To address this question and since the functions of SUV4-20H2 overlap with SUV4-20H1 [17], we first utilized the selective small-molecule inhibitor A-196, which specifically targets both SUV4-20H1 and SUV4-20H2. Previous studies showed that A-196 treatment reduces the conversion of H4K20me1 to higher H4K20me states [34]. Consistent with these findings, a 72-hour treatment with 4 µM of A-196 was sufficient to induce the depletion of H4K20me2/3 and the concurrent increase in H4K20me1 levels in both metastatic LNCaP and DU145 prostate cell lines (Figure 2A). Despite these genome-wide epigenetic alterations, RNA sequencing revealed that the pharmacological inhibition of SUV4-20H enzymes led to relatively modest changes in gene expression levels, with only 268 genes upregulated and 26 genes slightly downregulated in metastatic castration-resistant prostate DU145 cells (Figure 2B). The list of upregulated and downregulated genes is indicated in supplementary Tables S1 and S2, respectively. Among them, no specific pathways or cellular processes were found to be significantly under- or over-represented.

Since H4K20 methylation and SUV4-20Hs have been involved in the regulation of DNA replication [11, 14, 35, 36], we next examined the rates of DNA replication forks by DNA fiber-labeling approaches. After 72 hours of treatment with or without A-196, DU145 cells were pulsed labelled with iodo-deoxyuridine (IdU) for 30 min followed by a second pulse of chlorodeoxyuridine (CldU) for another 30 min. Then, the labeled replicating DNA was measured by microscopy using fluorescently labeled antibodies on DNA spread onto a glass slide. As shown in Figure 2C, replication fork rates were
significantly increased by more than 16% in A-196-treated cells compared to control DMSO-treated cells. However, despite the acceleration in replication fork progression, the proliferation rate and the cell-cycle distribution of A-196-treated cells remained largely unaffected (Supplementary Figures S3A and S3B).

To ascertain further these results and examine the role of each SUV4-20H enzyme in prostate cancer cells, single and double knock-out for SUV4-20H1 and SUV4-20H2 were generated in DU145 cell line using CRISPR-Cas9 approaches. As shown in Figure 2D, immunoblots with specific antibodies against SUV4-20H1 and SUV4-20H2 confirmed the successful generation of the different SUV4-20H knockout cell lines. Compared to the parental DU145 (wild-type) cell line, the loss of SUV4-20H1 alone resulted in a partial decrease in the levels of H4K20me2 and H4K20me3, while the loss of SUV4-20H2 mainly affected H4K20me3 (Figure 2D). Conversely, the concomitant knock-out of both SUV4-20H enzymes resulted in a complete depletion of H4K20me2 and H4K20me3 states, as was observed in A-196-treated cells (compare Figures 2A and 2D). As observed with the pharmacological inhibition of these enzymes by the A-196 inhibitor, loss of either SUV4-20H1 or SUV4-20H2 enzyme led to an increase in DNA replication fork velocity, with a further enhancement observed upon simultaneous loss of both enzymes (Figure 2E). However, this increase in replication fork speed did not result in a detectable change in the doubling time of the various SUV4-20H knockout cell models (Supplementary Figure S3C), similar to what was observed in A-196 treated cells. Altogether, these results confirm the efficacy of A-196 to inhibit SUV4-20H enzymes and show that the lack of conversion of H4K20me1 to higher H4K20me3 states upon loss or inhibition of SUV4-20H enzymes leads to an increased rate of replication fork progression, which largely remains inherently non-detrimental to the proliferation and survival of prostate cancer cells.
The inhibition of SUV4-20H enzymes induces a strong lethal synergy in combination with TOP2 poisons in metastatic prostate cancer cells. Given the higher velocity of DNA replication forks upon SUV4-20H inhibition, we asked whether prostate cancer cells treated with A-196 would display enhanced sensitivity to specific inducers of replication stress, notably those commonly used in cancer treatment. Following a 72 hours A-196 treatment in DU145 cell line to induce H4K20me reprogramming, we evaluated the interaction of A-196 with six selected drugs using a concentration matrix test. This involved the assessment of increasing concentrations of each individual drug with all possible combinations of the second drug. For each combination sample, the cell viability was quantified by measuring the percentage of live and dead cells with the Celigo image cytometer. The results are shown in Figure 3A according to the combination and efficiency index calculated for each drug combination. Strikingly, a positive interaction was observed between A-196 and TOP2 poisons with the highest combination and efficiency index achieved with etoposide (Figure 3A). Remarkably, the combination of A-196 with etoposide exhibited a lethal synergy in all PCa cell lines (Supplementary Figure S4A). Long-term colony formation assays further confirmed a synthetic lethality between A-196 and sub-lethal doses of etoposide in different prostate cancer lines (Figures 3B and 3C). Remarkably, the lethal interaction between A-196 and etoposide was even more effective than this previously found with the EZH2-mediated H3K27me3-targeting inhibitor GSK126 [37], which showed predominantly additive effects in our experimental conditions (Supplementary Figures S4B). We also assessed the lethal synergy between A-196 and etoposide across a representative panel of different cancer cell lines (Supplementary Figure S5). Notably, we found that the combination exhibited cancer type-dependent variability in synergistic cytotoxicity, ranging from no observable
synergy in several breast cancer cell lines to the highest efficiency in cell lines derived
from metastatic prostate tumors. Collectively, these findings reveal a unique and
robust lethal interaction between the SUV4-20H inhibitor A-196 and TOP2 poisons,
particularly etoposide, with maximal efficacy in prostate cancer cell types.

**The synthetic lethality with etoposide requires the inactivation of both SUV4-20H1 and SUV4-20H2.** To ascertain whether the enhanced sensitivity of PCa cells to etoposide was linked to the inhibition of either SUV4-20H1 or SUV4-20H2 or both enzymes, we investigated the sensitivity of the different SUV4-20H knock-out DU145 cell lines to a sub-lethal dose (0.1 µM) of etoposide over four days using the incucyte live-cell imaging system. The results are shown in Figure 3D. As anticipated, the growth of the parental cell line upon 0.1 µM of etoposide was comparable to that of control DMSO-treated cells. In contrast, the proliferation of cells depleted for each enzyme was partially reduced and virtually abolished when both enzymes were depleted (Figure 3D). Importantly, the lethal synergy between A-196 and etoposide was absent in SUV4-20H double knock-out DU145 cells (Supplementary Figure S6), indicating indeed that the enhancement of etoposide potency by A-196 is specific and not attributable to off-target effects. Altogether, these results demonstrate that the enhanced sensitivity of DU145 cells to etoposide is specific and maximized when both SUV4-20H enzymes are inactivated.

**SUV4-20H targeting selectively enhances the chromatin accumulation of TOP2α cleavage complexes in response to etoposide.** Flow cytometry analysis revealed that combined treatment with etoposide and A-196 resulted in a greater accumulation of cells in the late S and G2 phases compared to treatment with either drug alone
(Figure 4A). This cell cycle arrest was accompanied by a dose-dependent increase in cell death (Figure 4B), consistent with the induction of deleterious replication-associated DNA damage. Etoposide is known to promote DNA double-strand breaks (DSBs), notably behind the replication forks, by stabilizing TOP2α and TOP2β cleavage complexes (TOP2ccs) in chromatin and inhibiting the DNA ligation stage mediated by TOP2 enzymes [38]. To investigate whether the A-196-induced inhibition of SUV4-20H catalytic activity enhances the formation of TOP2ccs upon etoposide, we performed ICE assays. We quantified the levels of TOP2ccs upon 3 hours of treatment with etoposide in wild type cells pre-treated or not with A-196 during 3 days. As shown in Figure 4C for TOP2αccs and in Supplementary Figure S7A (upper panel) for TOP2βccs, etoposide induced a dose-dependent increase in TOP2cc levels that was markedly amplified in A-196-treated cells compared to untreated controls. For TOP2α, these findings were further supported by immunoblot analysis after heparin extraction, which revealed increased accumulation of DNA-tightly bound TOP2α in A196-treated cells following etoposide exposure (Supplementary Figure S7B). Importantly, SUV4-20H1/H2 double-knockout (KO) cells displayed similarly enhanced TOP2αccs accumulation in response to etoposide and this TOP2αccs accumulation was not further increased by A-196 treatment (Figure 4D). However, we were unable to detect significant TOP2βccs upon etoposide in SUV4-20H knock-out cells treated or not with A-196 (Supplementary Figure S7C), suggesting that the enhanced trapping of this TOP2 isoform following etoposide treatment requires the presence of SUV4-20H enzymes, even if catalytically inactive.

Given that SUV4-20H inhibition increased replication fork velocity (Figures 2C and 2E), we also examined whether A-196 treatment affects the levels of type-I topoisomerase cleavage complexes (TOP1ccs) upon etoposide or camptothecin, a
well-established and specific TOP1 poison. We found that the treatment with etoposide did not induce TOP1ccs formation under any conditions tested (Figure S7A lower panel), while camptothecin led to comparable accumulation of TOP1ccs in untreated and A-196 treated cell without affecting TOP2ccs (Supplementary Figure S7D). This was consistent with the absence of lethal synergy between A-196 and camptothecin, as illustrated in long-term colony formation assays (Supplementary Figure S7E). Collectively, these results demonstrate that both pharmacological inhibition and genetic inactivation of SUV4-20H enzymes selectively enhances the accumulation of TOP2 cleavage complexes in response to etoposide, most notably those formed with the TOP2α isoform, thereby potentiating their cytotoxic effects.

**The enhanced etoposide-induced trapping of TOP2 complexes upon SUV4-20 inhibition triggers massive DNA double strand breaks.** To examine whether the extensive etoposide-induced accumulation of TOP2ccs upon SUV4-20H inhibition was indeed associated with higher levels of DNA breaks, the levels of double-strand breaks (DSBs) were measured by pulse-field gel electrophoresis using the same treatment protocol as in Figure 4. The results are shown in Figures 5A and 5B, with untreated and DMSO-treated cells serving as negative controls and cells treated with a high concentration of etoposide (10 µM) as a positive control for DSBs. While A-196 treatment alone did not induce DNA breaks, etoposide triggered DSBs in a dose-dependent manner in control cells as expected (Figures 5A and 5B). Upon A-196 treatment, the levels of DSBs induced by etoposide were significantly enhanced (Figure 5B). To further substantiate that the lack of SUV4-20H activity strongly increases DSB formation in response to etoposide, we examined the focal accumulation of 53BP1 and of the phosphorylated form of the histone variant H2A.X (γ-H2AX) by indirect-immunofluorescence. These markers are well established
indicators of DNA breaks with known dynamics in DNA damage responses [39]. Following a 72-hour treatment with or without A-196, a sub-lethal concentration of etoposide (0.45 µM) was added for 24 hours, and cells were subsequently stained with specific antibodies for 53BP1 and γ-H2AX. Representative images and quantification of the results are shown in Figures 5C and 5D, respectively. Consistent with the pulse-field gel electrophoresis results (Figures 5A and 5B), treatment with etoposide induced a significantly higher percentage of 53BP1 and γ-H2AX positive cells when cells were pre-treated with A-196 (Figures 5C and 5D). Furthermore, 53BP1 and γ-H2AX immunostaining appeared as larger and more intense foci in nuclei treated with the drug combination than in nuclei treated only with etoposide (Figure 5C). Similar findings were obtained in SUV4-20H1 and SUV4-20H2 KO DU145 cells (Supplementary Figure S8). Collectively, the enhanced trapping of TOP2 cleavage complexes upon etoposide when SUV4-20H enzymes are inhibited or depleted is followed by higher levels of DNA double strand breaks, resulting in S/G2 cell-cycle arrest and massive cell death.

**SUV4-20Hs inhibition interferes the repair of etoposide-induced DNA breaks.** We observed that DSBs induced by the combination of A-196 and etoposide were accompanied by a pronounced activation of the DNA-damage response kinases ATM and DNA-PKcs, and to a lesser extent of ATR, as evidenced by increased levels of the phosphorylation status of these three kinases (Figure 6A). Strikingly, this activation was accompanied by a significant phosphorylation of the RPA32 subunit (Figure 6A) and the nuclear accumulation of the RPA70 subunit (Figure 6B), thereby indicating an activation of the Homologous Recombination (HR) pathway and the formation of single-strand DNA at DNA damage sites. The focal accumulation of RPA normally
involves its rapid replacement with RAD51 to form a nucleofilament competent for HR [40]. However, we observed lower levels of RAD51 nuclear foci in A-196 treated cells compared to control cells following etoposide treatment (Figure 6C). This suggests that the switch from RPA to RAD51 at DNA breaks might be somehow compromised by the inhibition of SUV4-20H activity, potentially resulting in inefficient repair of TOP2-mediated DSBs. To explore this further, DU145 cell were pre-treated for 72 hours with A-196 in order to reprogram the levels of H4K20 methylation states. Subsequently, cells were exposed to 0.45 µM of etoposide for 24 hours to induce quantitative levels of DSBs. After washing, control and A-196 treated cells were cultured with etoposide-free medium and the ability of cells to repair their DNA was evaluated by monitoring the levels of RPA, RAD51, 53BP1 and γH2A.X foci after 6 hours, 24 hours and 48 hours. In control cells treated with etoposide alone, the transition from RPA to RAD51 foci led to a rapid decrease of RPA and RAD51 foci to background levels that was observed 24 hours after etoposide removal (Figures 6D and 6E). Consistently, 53BP1 and γH2A.X focal accumulation followed similar kinetics, indicating an effective repair of DSBs (Figures 6F and 6G). In contrast, A-196 treated DU145 cells displayed defects in RAD51 focal accumulation that peaked only 6 hours after etoposide removal (Figure 6E). This coincided with a persistence of high levels of RPA and DNA PKCS phosphorylation (supplementary Figure S9). Accordingly, the levels of RPA, 53BP1 and γH2A.X focal accumulation remained elevated and showed only a slow reduction over 48 hours compared to control cells (Figures 6D, 6F and 6G). Notably, similar results were obtained in the prostate castration-resistant 22RV1 cell line, indicating that they were not specific to a particular prostate cell line (Supplementary Figure S10A-C). Moreover, FACS analysis revealed that a 24-hours pulse of sub-lethal concentration of etoposide in A-196-treated cells was sufficient to induce a S/G2 cell
cycle arrest followed by cell death as shown by the appearance of a sub-G1 fraction (Supplementary Figure S11A-B). Thus, the inhibition of SUV4-20H enhances the potency of etoposide not only by increasing TOP2ccs trapping but also by impairing the switch from RPA to RAD51, which disrupts the repair of TOP2-induced DNA breaks.

**SUV4-20H Inhibition compromises the repair of DNA double-strand breaks by disrupting H4K20me0-dependent recruitment of the BRCA1-BARD1 Complex.**

The defects in the RPA-to-RAD51 transition associated with a pronounced accumulation of 53BP1 foci suggested that the inhibition of SUV4-20H-mediated conversion of H4K20me1 to higher methylation states could impair DNA double-strand breaks (DSB) repair via homologous recombination (HR). To investigate this further and dissect the underlying mechanisms, we first examined the impact of A-196 on both non-homologous end joining (NHEJ) and HR using the well-established I-Scel GFP-reporter assays in U2OS-derived GCS5 (NHEJ) and RG37 (HR) cell lines. A previous study using similar transgenic models reported that a short-term (24-hour) treatment with 1 µM A-196 reduced NHEJ efficiency without affecting HR [34]. However, such brief exposure likely did not allow sufficient time for changes in H4K20 methylation, which typically require multiple cell divisions to become evident. Therefore, to better reflect the biological context in which H4K20 methylation states are significantly altered, reporter cells were treated with 4 µM of A-196 for 72 hours prior to DSB induction by I-Scel expression. After this treatment, immunoblot analysis confirmed consistent I-Scel expression and effective modulation of H4K20 methylation across all experimental conditions (Supplementary Figure S12). Following I-Scel expression and continued A-196 exposure, DNA repair efficiency was then assessed
36 hours later by quantifying GFP-positive cells via flow cytometry. As shown in Figure 7A, prolonged A-196 treatment and consequent inhibition of H4K20me1-to-H4K20me2/3 conversion significantly impaired HR, whereas NHEJ remained unaffected.

The activation of HR pathway critically depends on the interaction of the BRCA1-BARD1 heterodimer with unmethylated H4K20 (H4K20me0), a modification specifically recognized by the ankyrin repeat domain of BARD1 [41,42]. In a typical cellular context, DNA damage-induced reduction in H4K20me1 contributes to modulate the chromatin recruitment of 53BP1 [43,44], while BRCA1-BARD1 binding to H4K20me0 facilitates 53BP1 displacement and the subsequent loading of RAD51, a key step in HR-mediated repair [41]. We hypothesized that inhibiting SUV4-20H enzymes and altering H4K20 methylation could disrupt this process, thereby explaining the defects in the repair of DNA double-strand breaks induced by the trapping of TOP2ccs by etoposide. Indeed, compared to control cells, immunoblot analysis revealed that prostate DU145 cells treated with the SUV4-20H inhibitor A-196 or depleted of SUV4-20H enzymes exhibited a decrease in H4K20me0 rather than in H4K20me1, following etoposide treatment (Figure 7B and Supplementary figure S13A). Consistent with these results, biochemical fractionation following etoposide treatment showed a marked reduction of H4K20me0-associated proteins BRCA1 and BARD1 in the chromatin-enriched fraction with a corresponding appearance in the soluble protein fraction of cells treated with A-196 (Figure 7C) or depleted for SUV4-20H enzymes (Supplementary Figure S13B). Additionally, we noticed that BRCA1 protein levels were diminished upon etoposide treatment in the absence of SUV4-20H activity, suggesting a role for these enzymes in regulating BRCA1 expression or stability during genotoxic stress (Figure 7C and Supplementary Figure S13B). Finally,
indirect immunofluorescence revealed a significant reduction in the focal nuclear accumulation of BRCA1 in response to etoposide in A-196-treated or SUV4-20H-deficient cells (Figure 7D, Supplementary Figure S13C), underscoring the failure of BRCA1-BARD1 recruitment to sites of DNA damage. Altogether, these findings establish SUV4-20H methyltransferases as critical regulators of homologous recombination, demonstrating that loss of their catalytic activity and the resulting changes in H4K20 methylation degree impair BRCA1-BARD1 chromatin recruitment and RAD51 loading in response to etoposide-induced TOP2ccs, thereby functionally compromising HR-mediated DNA repair.

**TOP2 poisoning combined with SUV4-20H inhibition leads to enhanced anti-tumor activity in vivo.** The results described above suggest that targeting SUV4-20H enzymes is a potentially efficient strategy to increase the efficiency of etoposide and eventually reduce its effective dosage in the clinic to lower side effects. To validate this hypothesis and demonstrate *in vivo* the enhanced anti-tumor activity of etoposide when SUV4-20H enzymes are inhibited, mice bearing 100 mm³ DU145 prostate tumors were treated twice a week with vehicle (20% DMSO + corn oil), etoposide (2.5 mg per kg), A-196 (7.5 mg per kg), or the combination of the two drugs, as illustrated in Figure 8A. Importantly, toxicity or failure in liver and kidney functions were not detected in all animal groups as shown by hematological analysis (Supplementary Figure S14A and S14E). Moreover, monotherapy with A-196 or etoposide did not significantly reduce tumor growth, indicating that these two drugs are innocuous *in vivo* at these concentrations (Figures 8B and 8C). In contrast, the combination slightly but significantly delayed tumor growth and progression (Figures 8B, 8C, and 8D). Importantly, these effects were observed without apparent toxicity for the animals as
indicated by the absence of significant alterations in blood formulation and animal
weight during treatment (Supplementary Figure S14F). To further support these
results, mice were xenografted with wild-type or SUV4-20H1 and SUV4-20H2 double
knockout DU145 cells. Once tumors reached 100 mm³, they were treated with the
vehicle (DMSO) or etoposide (2.5 mg or 6 mg per kg) twice a week. Of note, without
any treatment, tumors derived from wild-type DU145 grew slightly slower than those
derived from DU145 cells lacking SUV4-20H enzymes (Figure 8E). Upon etoposide
treatment, whereas no significant difference was observed for wild-type tumors, the
progression of double SUV4-20H knockout tumors was strongly delayed and their
growth clearly impaired in a dose dependent manner (Figures 8E-8G). Taken together,
our results demonstrate that targeting the two SUV4-20H enzymes and
reprogramming H4K20 methylation signals can strongly improve the in vivo response
to etoposide while reducing its effective dose in malignant tumors and without
apparent toxicity for animal health.

DISCUSSION

Despite intensive multimodal therapy, Prostate Cancer (PCa) remains a major cause
of death due to the largely incurable nature of its metastatic and castration-resistant
stages [45]. It is therefore crucial to identify new markers for patients at high risk for
developing aggressive disease and to propose new therapeutic options. In this study,
we provide evidence that the elevated expression of the histone H4K20
methyltransferase SUV4-20H2 is a marker of aggressive prostate tumors independent
of Gleason score (Figure 1). Consistent with this, elevated levels of SUV4-20H2 are
associated with specific genetic alterations and poor prognosis and its gradual
increase during disease progression correlates with the risk of metastasis (Figure 1).
Using genetic and pharmacological approaches, we also demonstrate that targeting
SUV4-20H2 and its paralog SUV4-20H1 leads to a global reprogramming of H4-K20
methylation states and increases replication fork rates (Figure 2). Remarkably, these
epigenetic and replication alterations do not induce overt toxicity in prostate cancer
cell lines, but create conditions for synthetic lethality when combined with sub-lethal
concentrations of topoisomerase-II (TOP2) poisons, such as etoposide (Figure 3).
Specifically, we show that inhibiting SUV4-20H enzymes significantly enhances the
potency of etoposide by increasing the trapping of TOP2 cleavage complexes on
chromatin, thereby inducing higher levels of DNA double-strand breaks, and reducing
the capacity of cells to efficiently repair these breaks through BRCA-1-mediated
homologous recombination (HR) (Figures 4-7). Finally, in xenografted mouse models,
SUV4-20H inhibition or deletion boosts antitumor activity of etoposide at reduced non-
toxic doses in animals (Figure 8). Altogether, this represents a potential new strategy
for improving the anti-cancer effectiveness of etoposide notably in advanced prostate
cancer stages.

The progressive increase in SUV4-20H2 levels as the disease advances is not
unique to prostate cancer. This has been also reported in hepatocellular carcinoma,
clear renal cell carcinoma and pancreatic cancer [23,46,47], indicating it may be a
specific epigenetic feature of certain solid tumors. The mechanisms leading to SUV4-
20H2 upregulation in cancer cells remain unclear. In pancreatic cancer, this could be
partially due to gene amplification [46]. There is no such evidence in prostate tumors,
suggesting that is the consequence of a broader biological mechanism. It also remains
uncertain whether SUV4-20H2 upregulation impacts prostate cell behavior and
whether it is associated with specific changes in H4K20 methylation states.
Paradoxically, immunohistochemistry experiments have suggested that prostate
tumors often exhibit low levels in H4K20me1 and H4K20me2 while the levels of
H4K20me3, primarily mediated by SUV4-20H2, remains unchanged [32]. However,
further analysis with next-generation antibodies specific to each H4K20me state are
needed to confirm these initial results and to determine whether H4K20me alterations
in prostate tumors are related to SUV4-20H2 upregulation.

The lack of toxicity upon SUV4-20H inhibition aligns with previous findings in
various human cell lines [20,48]. By generating single and double knock-outs for
SUV4-20H2 and SUV4-20H1 in DU145 cells, we confirmed that the loss of SUV4-20H
enzymes is apparently innocuous for proliferation and survival in vitro. We did observe,
however, increased replication fork speed when SUV4-20H enzymes were inhibited
or inactivated. This acceleration could reflect altered chromatin structure resulting from
the increase in H4K20me1 and its lack of conversion to higher H4K20 methylation
states, as previously reported [15,36,49]. Alternatively, increased fork velocity may
also stem from higher availability of dNTPs due to a reduced number of active
replication origins [50,51]. Indeed, both H4K20me2 and H4K20me3 are known to
promote the licensing of replication origins by serving as binding sites for ORC
complexes and the loss of these methylation marks clearly impairs the number of
active origins during S-phase [11,12,52,53]. However, because origin activity and fork
speed influence each other [54], it remains challenging to determine whether altered
origin licensing or chromatin accessibility is the primary driver of fork acceleration upon
SUV4-20H loss. Importantly, while increased fork speed can contribute to replication
stress and genomic instability [55,56], it is unlikely that this phenotype fully accounts
for the marked sensitivity of SUV4-20H-deficient cells to etoposide. Indeed, other
replication stress-inducing agents, such as camptothecin, do not exhibit lethal synergy
in combination with SUV4-20H inhibition, arguing against a generalized replication
stress model. Instead, the heightened sensitivity to etoposide likely results from a
convergence of complementary defects in chromatin regulation, DNA replication
dynamics, and HR-mediated repair, all downstream of impaired SUV4-20H catalytic
activities and that specifically impact on TOP2 regulation and functions.

In hepatocellular carcinoma, A-196 sensitizes cells to the PARP inhibitor Olaparib [23]. Our results did not confirm this synergy in prostate cancer cells. Instead, our *in vitro* and *in vivo* results show that SUV4-20H inhibition or deletion confers a higher sensitivity of prostate cancer cells to the TOP2-poison etoposide. Mechanistically, we demonstrated that the inhibition of the catalytic activity of SUV4-20Hs enhances the etoposide-mediated accumulation of trapped TOP2α and TOP2β cleavage complexes. This may result from greater accessibility of TOP2 complexes to etoposide poisoning in the absence of SUV4-20H activity on chromatin and the subsequent accumulation of H4K20me1. Indeed, it has been recently shown that the presence of H4K20me1 facilitates chromatin openness and accessibility by disrupting chromatin folding throughout the cell cycle [57]. Noted of, A-196 treatment increased accumulation of TOP2β cleavage complexes in response to etoposide, but this effect was not observed in cells genetically depleted of SUV4-20H1 and SUV4-20H2, whether untreated or treated with A-196 (Supplementary Figure S7). While we cannot exclude the possibility that A-196 could exert some SUV4-20H-independent effects, we believe the absence of SUV4-20H enzymes may disrupt chromatin protein interactions necessary for TOP2β recruitment or activity, highlighting the need for further studies on the potential non-catalytic functions of SUV4-20Hs in TOP2β regulation. Nevertheless, this does not undermine the main conclusion that SUV4-20H
inactivation can synergize with etoposide in prostate cancer cells, particularly through
enhanced TOP2αccs accumulation.

Like SUV4-20H enzymes, treatment with inhibitors of histone deacetylases (HDACs) induces chromatin decompaction and accumulation of TOP2αccs complexes following treatment with TOP2 poisons [4,58]. However, unlike HDAC inhibitors that cause massive transcriptional alterations, A-196 treatment has weak effects on gene expression. Thus, SUV4-20H inhibition loosens chromatin without major gene regulation disruption, possibly acting in gene-poor regions. compared to clinically used HDAC inhibitors, this A-196 selectivity may reduce off-target effects and represents an advantage for combination therapy with etoposide. This is critical, as the dosage reduction of etoposide is clinically important due to its toxicity and side-effects, particularly in elderly patients with advanced prostate cancer.

Beyond enhancing TOP2ccs formation and DNA damage upon etoposide, A-196 treatment also impairs the repair of TOP2-mediated DNA breaks by disrupting BRCA1 recruitment and delaying the switch from RPA to RAD51 on damaged chromatin. This is unexpected since SUV4-20H enzymes have been previously associated with a positive role in NHEJ-mediated repair [44,48]. Yet, we did not confirm these previous results and instead found that SUV4-20H contributes to HR-mediated repair, at least when their inhibition triggers global changes in H4K20 methylation levels. Supporting a direct role in homologous recombination mechanisms, the catalytic domain of SUV4-20H2 was recently shown to interact with RAD51 [23]. We believe that HR defects stem from altered H4K20 methylation levels, since the impairment in the repair of etoposide-induced DNA breaks occurs only when changes in H4K20 methylation, triggered by SUV4-20H inhibition, precede etoposide treatment and is associated with decreased unmethylated H4K20 (H4K20me0) rather
than its mono-methylated form (H4K20me1). This imbalance between H4K20me0 and H4K20me1 upon DNA breaks may explain the recruitment of 53BP1 over the BRCA1-BARD1 complex in cells depleted for SUV4-20H enzymes. While H4K20me0 serves as binding sites for BARD1, high levels of H4K20me1 are sufficient to recruit 53BP1 even in the absence of H4K20me2 [59], thereby limiting HR by antagonizing BRCA1-mediated end resection and RAD51 loading. Consistently, we show that A-196 treatment does not affect, and even increases, 53BP1 recruitment after etoposide treatment. The cause of H4K20me0 loss is unclear but likely involves the H4K20 mono-methyltransferase SET8, which could lead to the inappropriate conversion of H4K20me0 to H4K20me1 in the vicinity of DNA damage sites. Further studies are needed to clarify how SUV4-20H enzymes preserves H4K20me0 levels from SET8 activity and supports HR in response to DSBs.

The combination of A-196 with etoposide was previously tested in U2OS cells where only a mild sensitivity was reported [48], suggesting that the lethal synergy between these drugs varies across cancer cell types. Our findings support this hypothesis, showing the strongest synergy in cell lines derived from metastatic prostate tumors. This differential sensitivity may be influenced by the expression levels of SUV4-20H2, which are highest within metastatic stages. Additionally, the pronounced effectiveness of the drug combination of A-196 and etoposide may also be driven by the elevated expression of TOP2α, which has been associated with prostate tumor progression and resistance to anti-androgen therapies [37,60]. The elevated levels of TOP2α have suggested a potential benefit of targeting it in advanced stages of prostate tumors. Thus, the TOP2-poisons mitoxantrone and etoposide have already been included as standard-of-care treatment for metastatic and castration-resistant prostate cancer [6]. However, given the inconvenient risk-benefit ratio, these
drugs are currently limited to combination with platinum agents to treat the most
aggressive forms of neuroendocrine prostate cancer [6,60]. As an alternative strategy,
combining an inhibitor of SUV4-20Hs with etoposide could be of utmost interest in
clinic for managing advanced and metastatic prostate cancer. This will however
require a deeper understanding of SUV4-20H functions in normal and pathological
context. It will be essential notably to map SUV4-20H chromatin binding sites, to
identify their potential substrates beyond histone H4K20, and to clarify the connections
with topoisomerases activity before and after DNA damage. Finally, the development
of more potent, selective SUV4-20H inhibitors and tumor-targeted delivery will be
essential for advancing this epigenetic drug to clinical trials.

ACKNOWLEDGMENTS

This work was supported by several grants from SIRIC Montpellier Cancer (INCa-DGOS-INSERM-ITMO Cancer-18004), Ligue Contre le Cancer Languedoc-Rousillon, MUSE Innovation program of Montpellier University (project CAPRE) and Institut National du Cancer (INCa_19385) and Lebanese University Rectorat (Beirut, Lebanon). MGX acknowledges financial support from France Génomique National infrastructure and Agence Nationale pour la Recherche (ANR-10-INBS-09). Institutionnel support was provided by the Institut National de la Santé et de la Recherche Médicale (INSERM), Montpellier University and by the Centre National de la Recherche Scientifique (CNRS). F.A. was supported by a PhD fellowship provided by Montpellier University, CNRS-Liban and by the Ligue Nationale Contre le Cancer (LNCC). The authors acknowledge the use of artificial intelligence (AI)-based assistance for language correction and editorial improvements of the manuscript.
## AUTHORS CONTRIBUTION

EJ designed the project. VB, RAM and EJ supervised the research. FA, MA, HD, CR, AG, EJ and JP performed the in vitro experiments. FA, MT, PD, JP and AT performed the in vivo experiments. EJ, MT, SG and XM performed RNA sequencing and analysis. FA, CR, PP, VB, RAM and EJ analyzed the other sets of experimental data. BM, FA, and EJ contributed to the analysis of TCGA and other public datasets. PP, CR, CG, FC, MD and DT provided conceptual advices. FA, VB and EJ wrote the paper. FA, VB, CR, PP, CG, RAM and EJ contributed to manuscript editing.

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FIGURE LEGENDS
**Figure 1: SUV4-20H2 up-regulation is a poor prognosis factor in prostate cancer (A) Violin plots showing SET8, SUV4-20H1, and SUV4-20H2 mRNA levels in normal (n=52) and tumor (n=497) prostate samples (TCGA). Dashed: median; dotted: quartiles; ns: non-significant; ***p < 0.0001 (Mann-Whitney). (B) Kaplan-Meier curves for progression-free (left) and disease-free (right) intervals based on SUV4-20H2 expression (TCGA) (C). SUV4-20H2 expression in benign, primary and metastatic tumors from GSE35988(left) and GSE21032 (right) cohorts. Median: dashed line, quartile: dotted line, ns: non-significant, **p < 0.001, ***p < 0.0001 (Mann-Whitney U test). (D) Violin plot showing SUV4-20H2 mRNA levels in CRPC (n=31) and NEPC (n=15) samples from GSE21032 and GSE35988 datasets. **p < 0.001; ***p < 0.0001. (Mann-Whitney U test).**

**Figure 2: SUV4-20H inactivation induces higher replication fork rates in metastatic prostate cancer cells (A) Immunoblot of histone H4 and H4K20 methylation levels in LNCaP and DU145 cells treated or not with A-196 for 72 hours as indicated. (B) Volcano plot of RNA-seq data from DU145 cells treated or not with A-196 (5 µM, 10 days). Blue and red dots represent the genes that are significantly upregulated and downregulated respectively (FDR=0.01, log2FC ≥ 0.69). (C) Replication assay design with IdU (green) and CldU (red) incubation and example of DNA fiber results. Dot plot shows IdU (green) and CldU (red) track lengths in DU145 cells treated or not with A-196 for 72 hours, n=3, 100 replication tracks per condition, ***p < 0.0001 (unpaired t test) (D). Immunoblots of histone H4, tubulin and H4K20 methylation in wild-type and SUV4-20H knockout DU145 cells (n=3) (E) Dot plot showing IdU and CldU track lengths in wild-type and SUV4-20H knockout cells, n=3;**
100 replication tracks per condition, *p < 0.01; **p < 0.001; ***p < 0.0001, (unpaired t test).

**Figure 3: targeting SUV4-20H enzymes induces a lethal synergy specifically in combination with etoposide.** (A) Scatter plot showing combination index (y-axis) and efficacy index (x-axis) for A-196 combined with genotoxic and replication stress-inducing agents in DU145 cells. Each dot represents one experiment. The dotted line marks the synergy threshold. (B) Clonogenic assays with DU145, 22RV1, and PC3 cells treated with A-196 (4 mM) and etoposide (0.2, 0.3 and 0.6 µM, respectively) for 72 hours. (C) Quantification of clonogenic assay in DU145 cells (A-196 4 µM + etoposide 0.2 µM, 72 hours), n=3; ***p < 0.0001. (D) Proliferation curve (mean ± s.d.) of wild-type and SUV4-20H knockout DU145 cells treated with etoposide, n=4.

**Figure 4: the inhibition of SUV4-20Hs enhances the accumulation of TOP2αccs complexes on chromatin upon etoposide (A) FACS analysis of 7AAD and BrdU staining in DU145 cells treated with DMSO or A-196 for 72h and then for 24h with etoposide as indicated, n=3. (B) Quantification of propidium iodide staining after A-196 and etoposide. Data = mean ± s.d; n=3, ** p < 0.01, *** p < 0.001 (unpaired t test) (C) Immunoblots (upper panel) and quantification (lower panel) of TOP2αccs in identical and increasing concentrations of nuclear extracts of DU145 cells treated or not with A196 for 4 days prior to etoposide treatment. Data = mean ± s.d., n=4. *** p < 0.001 (unpaired t test) (D) Immunoblots (upper panel) and quantification (lower panel) of TOP2αccs in wild-type and SUV4-20H knockout cells treated as above. Data = mean ± s.d., n=3. *** p < 0.001 (unpaired t test).**
**Figure 5:** The enhanced toxicity of etoposide in A-196 treated cells is related to higher DSB formation. (A) Pulse-field gel electrophoresis showing uncut DNA (upper band) and broken DNA fragments (lower band) in DU145 cells treated with DMSO or A-196 (4µM) for 72 hours before treatment for 3 hours with etoposide as indicated. The star indicates a non-specific DNA fragmentation found in all conditions. (B) Quantification of broken DNA fragments in DU145 cells treated as above, n=4, data = mean ± s.d, \*p<0.01, \*\*p <0.001 (unpaired t test). (C) Representative images of 53BP1 and γ-H2AX staining in DU145 cells treated with DMSO or A-196 (4µM) for 72 hours prior etoposide treatment (0.45 µM) for 24 hours. Scale bar = 10µm. (D) Quantification of 53BP1 and γ-H2AX foci per nucleus of DU145 cells treated as above, n=3 (200 cells per condition), data = mean ± s.d, ns: non-significant, \*p < 0.01, \*\*p < 0.001, \*\*\*p < 0.0001 (unpaired t test).

**Figure 6:** A-196 treatment compromises the ability of DU145 cells to repair TOP2-induced DNA breaks. (A) Immunoblot analysis of DNA repair proteins and H4K20me states in DU145 cells treated with DMSO or A-196 (4µM) for 72 hours and then exposed to etoposide (0.45µM) for 24 hours. n=3. (B) Representative images of RPA70 staining in DU145 cells treated as above. Scale bar = 10µm. (C) RAD51 staining in DU145 cells treated as above. Scale bar = 10µm. (D) Quantification of RPA70 foci per nucleus treated with DMSO or with A-196 (4µM) for 72h prior to etoposide treatment (0.45µM) for 24h and then collected at different times after etoposide removal as indicated, n=3, Data = mean ± s.d, (>200 cells per condition), ns: non-significant, \*p < 0.01, \*\* p < 0.001, \*\*\* p < 0.0001 (unpaired t test) (E) Quantification of RAD51 foci per nucleus treated as above. n=3, Data = mean ± s.d., (>200 cells per condition), ns: non-significant, \*p < 0.01, \*\* p < 0.001, \*\*\* p < 0.0001
(unpaired t test) (F) Quantification of R53BP1 foci per nucleus treated as above. n=3, mean ± SD, (>200 cells per condition), ns: non-significant, \*p-value < 0.01, \*\* p-value < 0.001, \*\*\* p-value < 0.0001. (G) Quantification of γ-H2AX foci per nucleus treated as above. n=3; data = mean ± s.d., (>200 cells per condition), ns: non-significant, \*p < 0.01, \*\* p < 0.001, \*\*\* p < 0.0001 (unpaired t test).

**Figure 7: SUV4-20H inhibition or depletion impairs BRCA1-mediated homologous recombination. (A) Flow cytometry analysis of Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ) efficiency in U2OS-derived GCS5 (NHEJ) and RG37 (HR) cell lines treated as indicated. (> 20 000 cells per sample), data = mean ± s.d., n = 3. \* p<0.01 (unpaired t-test). (B) Immunoblots analysis of H4K20me0, H4K20me1 and total histone H4 levels in DU145 cells untreated or treated with A-196 for 4 days prior to etoposide treatment for 24 hours as indicated. (C) Immunoblot analysis of BRCA1, BARD1, MEK1 and histone H4 levels in chromatin-enriched and soluble protein fractions of DU145 cells treated as above. MEK1 and H4 are controls for the soluble and chromatin fraction respectively. (D) Representative images of BRCA1 and RAD5 staining and quantification of BRCA1 foci per nucleus treated as above, n=3, data=mean ± s.d., (>200 cells per condition), ns: non-significant, \*p < 0.01 (unpaired t-test).**

**Figure 8: The depletion of SUV4-20H enzymes improves in vivo therapeutic response to etoposide. (A) Experimental and treatment timeline of DU145 xenograft mice. (B) Tumor growth curves of the DU145 xenograft tumors untreated or treated as indicated. (n=4 mice per group). (C) Final tumor volume DU145 xenograft tumors treated during 2 weeks as indicated. ns: non-significant, \*\* p< 0.001 (unpaired t test).**
(D) Graphical representation of non-progression percentage of DU145-derived xenograft tumors treated as indicated. (E) Growth curves of wild-type (WT) tumors (upper panel) and SUV4-20H knock-out tumors (lower panel) treated with DMSO or etoposide as indicated; (n=4 mice per group). (F) Final tumor volume of wild-type and SUV4-20H knockout tumors treated during four weeks. Mean ± s.d., ns: > 0.05, **p < 0.001 (unpaired t test) (G) Graphical representation of non-progression percentage of SUV4-20H knockout tumors treated as indicated.
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<figure><img src="image_42.png" /><figcaption>Supplementary Figure S1</figcaption></figure>
**Figure S1: Clinical features associated with SUV4-20H2 expression in prostate tumors. (A) Bar plot representing the mRNA expression of SUV4-20H2 according to the Gleason Score (GS). Error bars represent the minimum and maximum level of expression, ***p < 0.0001. Mann-Whitney U test. (B) Dot plot presenting SUV4-20H2 mRNA expression in two age categories of prostate cancer patients. Patients from 41-60 years (red dots) and patients from 61-80 years (orange dots). Mean values (black lines). **, p < 0.001. Mann-Whitney U test. (C-G) Dot plot representing SUV4-20H2 mRNA expression in prostate cancer patients (C) with or without TP53 mutations, (D) with or without ERG structural variant (E) in the two stages of Neoplasm Disease Lymph Node, (F) with or without SPOP mutations and (G) with or without TMPRSS2 structural variants. Mean values (black lines) and inter-quartiles (whiskers). ns: non-significant, * p < 0.01, ** p<0.001 Mann-Whitney U test.**
<figure><img src="image_43.png" /><figcaption>**Figure S2: SUV4-20H2 upregulation correlates with genetic instability in the cohort of prostate tumors in TCGA database.** Dot plot showing the fraction Genome Altered (top left), the MSI MANTIS Score (top middle), the Aneuploidy Score (top right), the Buffa Hypoxia Score (bottom left), and the Winter Hypoxia Score (bottom right) in patients with low SUV4-20H2 expression (red dots) and high SUV4-20H2 expression (blue dots) levels from the prostate TCGA database. Mean values (black lines). **, p < 0.001; ***, p < 0.0001. Mann-Whitney U test.</figcaption></figure>
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**Figure S3:** The inhibition or the loss of SUV4-20H enzymes is inherently non-detrimental to the proliferation and survival of prostate DU145 cells. (A) cell-cycle analysis by FACS of DU145 cells treated with A-196 (4µM) or not (DMSO) for 72h. Total DNA was stained with 7-AAD, and replicating cells was labelled with BrdU, n=3. (B) Graph showing the proliferation curve of DU145 cells treated with increasing concentration of A-196 as indicated during 4 days. The number of cells was quantified as phase objective count using Incucyte live cell imaging assay, n=2. (C) Graph representing the doubling time of wild-type DU145 (WT) and of DU145 cells knock-out (KO) for SUV4-20H1 or SUV4-20H2 or both enzymes (SUV4-20H1/H2 KO). Cells were counted as phase objective count using Incucyte live cell imaging assay, n=3, ns: non-significant p-value. (unpaired t-test).
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<figure><img src="image_50.png" /><figcaption>Figure S4</figcaption></figure>

Alhourani F et al.
<caption>Figure S4 : Features of the lethal synergy between the SUV4-20Hs inhibitor A-196 and etoposide in metastatic prostate cancer cell lines.</caption>

(A) Scatter plot representing the combination index (y) and the efficiency index (x) of combination treatment with increasing concentrations of A-196 and etoposide for 72h in 22RV1, DU145, PC3, and LNCaP cell lines. Each dot represents one experiment. (B) Representative viability matrix (top) and synergy scores (bottom) for DU145 and PC3 cell lines after 72h of treatment with increasing doses of A-196 and etoposide or with increasing doses of EZH2i (GSK126) and etoposide as indicated. Synergy occurs when score values are greater than 20 (Red), additivity occurs when score values are between -20 and 20 (Black), and antagonism occurs when score values are less than -20 (Green). Score and color intensity are dependent on the degree of synergy, antagonism or additivity.
<figure><img src="image_51.png" /><figcaption>**Figure S5 :** Synergistic cytotoxicity of A-196 and etoposide across different cancer cell types. Scatter plot representing the combination index (y) and the efficiency index (x) of combination treatment with increasing concentrations of A-196 and etoposide for 72h in various kidney, colon, lung, breast and metastatic prostate cancer cell lines as indicated. Each small dot represents one drug synergy experiment. Dotted circles group dots coming from the same cancer cell type.</figcaption></figure>
SUV4-20H1/H2 KO DU145 cells

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**Figure S6:** The lethal synergy between the inhibitor A-196 and etoposide is largely abolished in SUV-420H1/H2 KO DU145 cells. Representative viability matrix (top) and synergy scores (bottom) for SUV4-20H1/H2 KO DU145 cell line after 72h of treatment with increasing doses of A-196 and etoposide as indicated. Synergy occurs when score values are greater than 20 (Red), additivity occurs when score values are between -20 and 20 (Black), and antagonism occurs when score values are less than -20 (Green).
<figure><img src="image_53.png" /><figcaption>B</figcaption></figure>

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Supplementary Figure S7
**Figure S7: TOP2 and TOP1 DNA cleavage complexes are differentially affected by the inhibition or depletion of SUV4-20H enzymes in DU145 cells.** (A) Immunoblots of the ICE assays showing DNA Cleavage Complexes of TOP2β (upper panel, TOP2βccs) and TOP1 (lower panel, TOP1ccs) in increasing concentrations of nuclear extracts of untreated or A196-treated DU145 cells during four days prior to etoposide treatment as indicated. DNA inputs were normalized prior to slot blotting by agarose gel quantification. (B) Immunoblot analysis showing the total levels of TOP2α (upper panel) before heparin extraction and of the levels of chromatin-bound TOP2α (lower panel) after heparin extraction in DU145 cells treated with DMSO or A-196 (4μM) for 72h and then treated with increasing doses of etoposide for 3 hours as indicated. HSC70 is used as loading control in total cell extracts and histone H3 is used as quantitative control of chromatin-bound proteins after heparin extraction, n=3. Lower panel is a bar plot quantification of chromatin-bound TOP2α levels in control and treated cells as indicated above using ImageJ software, n=3, \* p-value ≤ 0.05, \*\* p-value < 0.01, \*\*\* p-value < 0.001. (C) Immunoblots of the ICE assays showing the absence of detectable TOP2β DNA cleavage complexes (TOP2βccs) in increasing concentrations of nuclear extracts of SUV4-20H1/H2 depleted DU145 cells treated or not with A-196 during four days prior to etoposide treatment as indicated. DNA inputs were normalized prior to slot blotting by agarose gel quantification. (D) Immunoblots of the ICE assays showing TOP2 and TOP1 DNA cleavage complexes in increasing concentrations of nuclear extracts of untreated or A196-treated DU145 cells during four days prior to three hours of camptothecin treatment as indicated. DNA inputs were normalized prior to slot blotting by agarose gel quantification. (E) Images (left panel) and quantification (bar plot) of clonogenic assays for prostate cancer cells treated with A-196 (4μM) and camptothecin (5 nM) for 72 hours. \*p-value ≤ 0.05, n=3.
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**Figure S8:** The loss of SUV4-20H proteins increases DNA damage in response to etoposide similarly to their inhibition with A-196 compound. Upper and middle panels are representative images of 53BP1 and γ-H2AX staining in DU145 WT and DU145 SUV4-20H KO cells treated with 0.45 µM of etoposide or not (DMSO) for 24hours as indicated. Scale bar = 10µm. Lower panel, Violin plot representing the number of 53BP1 (Left) and γ-H2AX (right) foci per nucleus of DU145 Wild-Type (WT) and SUV4-20H KO cells treated as indicated. Median (plain line), quartile (dashed line). n = 1, >200 cells per condition, \*p < 0.01, \*\*p < 0.001, \*\*\*p < 0.0001 (unpaired-t-test).
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**Figure S9: RPA and DNAPKcs remain phosphorylated in A-196-treated cells after etoposide removal.** DU145 cell line was treated with 4µM A-196 or not (DMSO) for 72h. Immunoblot analysis showing the maintenance of RPA and DNAPKcs phosphorylation 6, 24, and 48h after 24 hours exposure with 0.45µM of etoposide in DU145 cell line that was previously treated with 4µM A-196 or not (DMSO) during 72 hours in order to induce H4K20me reprogramming.

**Supplementary Figure S9**
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<figure><img src="image_66.png" /><figcaption>Supplementary Figure S10</figcaption></figure>
**Figure S10: The pharmacological inhibition of SUV4-20Hs enhances etoposide responses in castration-resistant 22RV1 prostate cell lines. (A)**

Representative images of γ-H2AX staining in 22RV1 cell line treated with A-196 (4μM) or not (DMSO) for 72 hours, then etoposide (0.45 μM) was added for an additional 24 hours as indicated. Scale bar = 10μm. (B) Immunoblot analysis showing the expression levels of indicated proteins in 22RV1 cell line treated as indicated above. Total DNA-PKcs, ATM, and ATR are used as a loading control, n=2. (C) Bar plot representing the number of γ-H2AX foci per nucleus in 22RV1 cell line treated with A-196 (4μM) or not (DMSO) for 72 hours, followed by etoposide treatment (0.45μM) for 24 hours. 0h corresponds to etoposide removal and after 6, 24, and 48h of etoposide removal as indicated. A-196 is maintained when indicated (+), n=2, >200 cells per condition.
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<figure><img src="image_68.png" /><figcaption>Supplementary Figure S11</figcaption></figure>
**Figure S11: A 24-hour treatment with a harmless dose of etoposide induces S/G2 arrest and cell death in A-196-treated DU145 cells. (A) Cell cycle distribution of DU145 cells 6, 24, and 48h after 24 hours treatment with etoposide (0.45µM) and treatment or not with A-196 (4µM) 72 hours before etoposide incubation. Total DNA was stained with 7-AAD, and nascent DNA was labeled with BrdU, n=3. (B) Bar plot representing the % of sub-G1 cells for DU145 cell line treated or not as indicated, n=3, data = mean ± s.d. ns: non-significant, * p < 0.01, ** p < 0.001, ***p < 0.0001 (unpaired t-test).**
<figure><img src="image_69.png" /><figcaption>**Figure S12:** Immunoblot analysis levels of H4K20 methylation and the restriction enzyme I-Scel in U2OS-derived GCS5 (NHEJ) and RG37 (HR) cell lines treated with A-196 during four days prior to I-Scel expression.</figcaption></figure>
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<figure><img src="image_71.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_72.png" /><figcaption>Supplementary Figure S13</figcaption></figure>
**Figure S13: Alterations in H4K20 methylation and BRCA1-mediated HR pathway in SUV4-20H depleted cells treated with etoposide. (A) Representative immunoblots showing the levels of total histone H4 and the levels of H4K20me0 and H4K20me1 in DU145 cells untreated or treated with A-196 and in SUV4-20H depleted cells treated or with DU145 during 4 days prior to etoposide treatment for 24 hours as indicated. (B) Representative immunoblots showing the localization of BRCA1, BARD1, MEK1 and histone H4 in chromatin-enriched fraction and in the complementary soluble proteins fraction in DU145 cells depleted for SUV4-20H enzymes and treated with etoposide for 24 hours as indicated. MEK1 serves as control for soluble fraction whereas histone H4 serves as control for the chromatin-enriched fraction (C) Bar plot representing the number of BRCA1, RPA and RAD51 foci per nucleus in DU145 wild-type and SUV4-20 depleted cells treated with etoposide for 24 hours as indicated. n=3, data = mean ± s.d., >200 cells per condition and per experiment, ns: non-significant, *p < 0.01, ** p<0.001. (unpaired t-test).**
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Blood Cell Analysis

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Liver Function

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Kidney Function
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glycemia

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Supplementary Figure S14
**Figure S14: Assessment of Toxicity Induced by A-196 and Etoposide in Athymic Nude Mice.** (A) hematological analysis of white and red blood cells, of lymphocytes and of platelets in athymic nude mice after 18 days of treatment with A-196 or etoposide or the combination of two drugs. All mice were divided into four cohorts of 4 animals: Vehicle (DMSO + cornoil), A-196 (7.5 mg/kg) etoposide (6 mg/kg), and combination. Tumor sizes were measured every 3 days. (B) hematological analysis of ASA and ALAT units per ml as measure of liver function in mice treated as described above. data = mean ± SD, four animals per cohort. (C) levels of creatinin (measure of kidney function) in the blood of mice treated as described above data = mean ± SD, four animals per cohort. (D) levels of glucose in the blood of mice treated as described above data = mean ± SD, four animals per cohort. (E) Graphical representation of the body weight measurement of mice after three weeks of treatment as indicated. Intact athymic nude males were inoculated with DU145 cells by subcutaneous injection for 2 weeks. All mice were divided into four cohorts of 4 animals: Vehicle (DMSO + cornoil), A-196 (7.5 mg/kg) etoposide (2.5 mg/kg), and combination. Tumor sizes were measured every 3 days. (F) Graphical representation of the final body weight of mice wild-type and SUV4-20H depleted DU145 xenograft tumors in mice treated with etoposide every three days (IP injection at two different concentrations as indicated). Data = mean ± SD.
<table><caption>Figure S3: List of upregulated genes according to the log2 (FC) value in DU145 cells after H4K20me reprogramming induced by the chemical inhibitor A-196. (FDR 0.01, log2 (FC) > 0.69).</caption><thead><tr><th>feature</th><th>gene_ID</th><th>baseMean</th><th>Log2(FC)</th><th>lfcSE</th><th>stat</th><th>pvalue</th><th>pADJ</th><th></th></tr></thead><tbody><tr><td>ROR2</td><td></td><td>4920</td><td>18.45223854</td><td>2.623863042</td><td>0.52173683</td><td>-5.029093</td><td>4.93E-07</td><td>2.04E-05</td></tr><tr><td>GPR1</td><td></td><td>2825</td><td>11.24881559</td><td>2.552345225</td><td>0.65141651</td><td>-3.9181463</td><td>8.92E-05</td><td>0.001405156</td></tr><tr><td>SRGN</td><td></td><td>5552</td><td>16.62868286</td><td>2.33328246</td><td>0.52668872</td><td>-4.4300977</td><td>9.42E-06</td><td>0.000229313</td></tr><tr><td>MIG7</td><td></td><td>9878</td><td>63.5159673</td><td>2.046565684</td><td>0.26404106</td><td>-7.7509372</td><td>9.12E-15</td><td>2.92E-12</td></tr><tr><td>LINGO1</td><td></td><td>84894</td><td>24.12753368</td><td>1.946457058</td><td>0.4646131</td><td>-4.1894149</td><td>2.80E-05</td><td>0.000564973</td></tr><tr><td>PRSS8</td><td></td><td>5652</td><td>25.59028959</td><td>1.917053474</td><td>0.41207674</td><td>-4.6521759</td><td>3.28E-06</td><td>9.57E-05</td></tr><tr><td>KRT81</td><td></td><td>3887</td><td>648.5026397</td><td>1.896571464</td><td>0.12574093</td><td>-15.083167</td><td>2.09E-51</td><td>1.57E-47</td></tr><tr><td>PSG5</td><td></td><td>5673</td><td>34.90040043</td><td>1.849731777</td><td>0.35076787</td><td>-5.2733786</td><td>1.34E-07</td><td>6.80E-06</td></tr><tr><td>SLC2A3</td><td></td><td>6515</td><td>40.86456767</td><td>1.800377519</td><td>0.3148504</td><td>-5.7181998</td><td>1.08E-08</td><td>7.41E-07</td></tr><tr><td>TNS4</td><td></td><td>84951</td><td>106.7196892</td><td>1.788654414</td><td>0.21641945</td><td>-8.2647582</td><td>1.40E-16</td><td>7.27E-14</td></tr><tr><td>IGFBP3</td><td></td><td>3486</td><td>1442.63088</td><td>1.72902404</td><td>0.0831751</td><td>-20.78776</td><td>5.59E-96</td><td>8.42E-48</td></tr><tr><td>ALPG</td><td></td><td>251</td><td>154.5802133</td><td>1.725943396</td><td>0.2118319</td><td>-8.1477031</td><td>3.71E-16</td><td>1.80E-13</td></tr><tr><td>PEAR1</td><td></td><td>375033</td><td>61.01473851</td><td>1.720203329</td><td>0.26602441</td><td>-6.4663365</td><td>1.00E-10</td><td>1.23E-08</td></tr><tr><td>COL1A2</td><td></td><td>1278</td><td>37.48349737</td><td>1.690507785</td><td>0.32982156</td><td>-5.1255224</td><td>2.97E-07</td><td>1.30E-05</td></tr><tr><td>PRRT4</td><td></td><td>401399</td><td>82.69868077</td><td>1.685179781</td><td>0.22712222</td><td>-7.4197047</td><td>1.17E-13</td><td>3.10E-11</td></tr><tr><td>KREMEN2</td><td></td><td>79412</td><td>100.5774223</td><td>1.64405213</td><td>0.219774</td><td>-7.4806489</td><td>7.40E-14</td><td>2.14E-11</td></tr><tr><td>GKN2</td><td></td><td>200504</td><td>325.9291107</td><td>1.638989896</td><td>0.13386133</td><td>-12.243938</td><td>1.81E-34</td><td>6.82E-31</td></tr><tr><td>SCUBE2</td><td></td><td>57758</td><td>19.16037968</td><td>1.62410663</td><td>0.45637362</td><td>-3.5587215</td><td>0.00037266</td><td>0.004303488</td></tr><tr><td>TNFSF15</td><td></td><td>9966</td><td>34.82894534</td><td>1.617358718</td><td>0.34263226</td><td>-4.7203925</td><td>2.35E-06</td><td>7.34E-05</td></tr><tr><td>CCL20</td><td></td><td>6364</td><td>22.14383216</td><td>1.617211937</td><td>0.44564693</td><td>-3.6289085</td><td>0.00028462</td><td>0.003506256</td></tr><tr><td>KLHL35</td><td></td><td>283212</td><td>21.15817844</td><td>1.611841494</td><td>0.42931226</td><td>-3.7544735</td><td>0.00017371</td><td>0.002397209</td></tr><tr><td>RHCG</td><td></td><td>51458</td><td>21.04340477</td><td>1.590905387</td><td>0.44680847</td><td>-3.5605981</td><td>0.00037001</td><td>0.004279407</td></tr><tr><td>TEX48</td><td></td><td>100505478</td><td>25.16979784</td><td>1.573470802</td><td>0.39039865</td><td>-4.0304207</td><td>5.57E-05</td><td>0.000966652</td></tr><tr><td>CLDN7</td><td></td><td>1366</td><td>408.7767271</td><td>1.552425951</td><td>0.12645004</td><td>-12.276991</td><td>1.20E-34</td><td>6.05E-31</td></tr><tr><td>BAIAP2L2</td><td></td><td>80115</td><td>26.36411284</td><td>1.542749432</td><td>0.42765982</td><td>-3.607422</td><td>0.00030925</td><td>0.003731357</td></tr><tr><td>CALHM5</td><td></td><td>254228</td><td>19.76155454</td><td>1.489224697</td><td>0.45235083</td><td>-3.2921896</td><td>0.00099411</td><td>0.009541931</td></tr><tr><td>ACP7</td><td></td><td>390928</td><td>76.35662391</td><td>1.481918959</td><td>0.23754092</td><td>-6.238584</td><td>4.42E-10</td><td>4.57E-08</td></tr><tr><td>LINC01116</td><td></td><td>375295</td><td>30.82930352</td><td>1.466103627</td><td>0.35944621</td><td>-4.0787845</td><td>4.53E-05</td><td>0.000820007</td></tr><tr><td>MESP2</td><td></td><td>145873</td><td>20.07124093</td><td>1.461196886</td><td>0.44016472</td><td>-3.3196593</td><td>0.00090127</td><td>0.008865664</td></tr><tr><td>HES2</td><td></td><td>54626</td><td>36.46603671</td><td>1.448925255</td><td>0.34423062</td><td>-4.2091702</td><td>2.56E-05</td><td>0.000527677</td></tr><tr><td>INSYN1</td><td></td><td>388135</td><td>42.31556738</td><td>1.424989157</td><td>0.31758228</td><td>-4.486992</td><td>7.22E-06</td><td>0.000184821</td></tr><tr><td>KLRF1</td><td></td><td>51348</td><td>27.12628109</td><td>1.389216249</td><td>0.39839069</td><td>-3.48707</td><td>0.00048834</td><td>0.0053136</td></tr><tr><td>LIPH</td><td></td><td>200879</td><td>279.439512</td><td>1.38494443</td><td>0.13569461</td><td>-10.206333</td><td>1.86E-24</td><td>3.50E-21</td></tr><tr><td>GJA1</td><td></td><td>2697</td><td>25.91307546</td><td>1.384095067</td><td>0.38155289</td><td>-3.6275314</td><td>0.00028614</td><td>0.003520155</td></tr><tr><td>TMC4</td><td></td><td>147798</td><td>83.19146686</td><td>1.37507354</td><td>0.27343464</td><td>-5.028893</td><td>4.93E-07</td><td>2.04E-05</td></tr><tr><td>ENO4</td><td></td><td>387712</td><td>25.19348472</td><td>1.374523012</td><td>0.39833239</td><td>-3.4506935</td><td>0.00055915</td><td>0.005946621</td></tr><tr><td>FRMD4B</td><td></td><td>23150</td><td>42.1681164</td><td>1.370055634</td><td>0.3093908</td><td>-4.4282365</td><td>9.50E-06</td><td>0.000230556</td></tr><tr><td>IL1RL1</td><td></td><td>9173</td><td>55.05120475</td><td>1.363960711</td><td>0.28844589</td><td>-4.7286536</td><td>2.26E-06</td><td>7.10E-05</td></tr><tr><td>SERPINB5</td><td></td><td>5268</td><td>87.28331549</td><td>1.363911806</td><td>0.21867166</td><td>-6.2372592</td><td>4.45E-10</td><td>4.57E-08</td></tr><tr><td>SYNE4</td><td></td><td>163183</td><td>60.56675234</td><td>1.356310187</td><td>0.2883243</td><td>-4.7041134</td><td>2.55E-06</td><td>7.79E-05</td></tr><tr><td>BCL2A1</td><td></td><td>597</td><td>36.17280179</td><td>1.350262575</td><td>0.32525266</td><td>-4.1514267</td><td>3.30E-05</td><td>0.000644868</td></tr><tr><td>LAD1</td><td></td><td>3898</td><td>56.56492221</td><td>1.338251326</td><td>0.2730649</td><td>-4.9008543</td><td>9.54E-07</td><td>3.52E-05</td></tr><tr><td>SMIM24</td><td></td><td>284422</td><td>24.80006715</td><td>1.336496473</td><td>0.385496</td><td>-3.4669529</td><td>0.00052639</td><td>0.005630064</td></tr><tr><td>FGFBP1</td><td></td><td>9982</td><td>258.8525854</td><td>1.32832119</td><td>0.14789658</td><td>-8.9814193</td><td>2.67E-19</td><td>2.69E-16</td></tr><tr><td>KCNQ2</td><td></td><td>3785</td><td>47.11011029</td><td>1.325689709</td><td>0.28364837</td><td>-4.6737081</td><td>2.96E-06</td><td>8.91E-05</td></tr><tr><td>ADGRE2</td><td></td><td>30817</td><td>53.07536437</td><td>1.32539148</td><td>0.27993183</td><td>-4.7346937</td><td>2.19E-06</td><td>6.90E-05</td></tr><tr><td>C1QTNF1</td><td></td><td>114897</td><td>30.54785839</td><td>1.316786382</td><td>0.37840125</td><td>-3.479868</td><td>0.00050166</td><td>0.005427691</td></tr><tr><td>SH3TC2</td><td></td><td>79628</td><td>77.23430256</td><td>1.31462071</td><td>0.23123706</td><td>-5.6851643</td><td>1.31E-08</td><td>8.83E-07</td></tr><tr><td>PTGER1</td><td></td><td>5731</td><td>87.3473274</td><td>1.30450354</td><td>0.22316773</td><td>-5.8453951</td><td>5.05E-09</td><td>3.72E-07</td></tr><tr><td>SUSD3</td><td></td><td>203328</td><td>53.45991928</td><td>1.303690798</td><td>0.29104026</td><td>-4.4794175</td><td>7.48E-06</td><td>0.00018989</td></tr><tr><td>EPS8L1</td><td></td><td>54869</td><td>39.09838468</td><td>1.298901279</td><td>0.31366253</td><td>-4.1410789</td><td>3.46E-05</td><td>0.000666156</td></tr><tr><td>RGS11</td><td></td><td>8786</td><td>99.38296809</td><td>1.296040131</td><td>0.22719658</td><td>-5.7044878</td><td>1.17E-08</td><td>7.96E-07</td></tr></tbody></table>
<table>
  <tr>
    <td>SYT16</td>
    <td>83851</td>
    <td>24.37139607</td>
    <td>1.291526852</td>
    <td>0.39287204</td>
    <td>-3.2873982</td>
    <td>0.00101118</td>
    <td>0.009662931</td>
  </tr>
  <tr>
    <td>ADAMTS10</td>
    <td>81794</td>
    <td>66.79513446</td>
    <td>1.279209405</td>
    <td>0.26927414</td>
    <td>-4.7505839</td>
    <td>2.03E-06</td>
    <td>6.48E-05</td>
  </tr>
  <tr>
    <td>FSTL1</td>
    <td>11167</td>
    <td>273.6856945</td>
    <td>1.270633268</td>
    <td>0.13209215</td>
    <td>-9.6192942</td>
    <td>6.63E-22</td>
    <td>8.01E-19</td>
  </tr>
  <tr>
    <td>GNG2</td>
    <td>54331</td>
    <td>36.70623625</td>
    <td>1.263249169</td>
    <td>0.32212215</td>
    <td>-3.9216464</td>
    <td>8.79E-05</td>
    <td>0.00139217</td>
  </tr>
  <tr>
    <td>PRTN3</td>
    <td>5657</td>
    <td>26.73468759</td>
    <td>1.261026304</td>
    <td>0.38112933</td>
    <td>-3.3086572</td>
    <td>0.00093745</td>
    <td>0.009132064</td>
  </tr>
  <tr>
    <td>INSC</td>
    <td>387755</td>
    <td>26.12036959</td>
    <td>1.251816994</td>
    <td>0.37855586</td>
    <td>-3.3068224</td>
    <td>0.00094361</td>
    <td>0.009162474</td>
  </tr>
  <tr>
    <td>PRRG2</td>
    <td>5639</td>
    <td>52.0153423</td>
    <td>1.24521457</td>
    <td>0.27735858</td>
    <td>-4.4895476</td>
    <td>7.14E-06</td>
    <td>0.000183552</td>
  </tr>
  <tr>
    <td>TMEM92</td>
    <td>162461</td>
    <td>51.47978062</td>
    <td>1.24199206</td>
    <td>0.29807346</td>
    <td>-4.1667314</td>
    <td>3.09E-05</td>
    <td>0.000610302</td>
  </tr>
  <tr>
    <td>AATK</td>
    <td>9625</td>
    <td>105.9125476</td>
    <td>1.229507595</td>
    <td>0.20855265</td>
    <td>-5.8954302</td>
    <td>3.74E-09</td>
    <td>2.92E-07</td>
  </tr>
  <tr>
    <td>DNER</td>
    <td>92737</td>
    <td>118.711709</td>
    <td>1.229463263</td>
    <td>0.18907993</td>
    <td>-6.5023468</td>
    <td>7.91E-11</td>
    <td>1.01E-08</td>
  </tr>
  <tr>
    <td>QPCT</td>
    <td>25797</td>
    <td>33.37953613</td>
    <td>1.227254865</td>
    <td>0.33768865</td>
    <td>-3.6342793</td>
    <td>0.00027876</td>
    <td>0.003454683</td>
  </tr>
  <tr>
    <td>CARMIL3</td>
    <td>90668</td>
    <td>38.07731103</td>
    <td>1.213134083</td>
    <td>0.31868143</td>
    <td>-3.8067297</td>
    <td>0.00014082</td>
    <td>0.002029834</td>
  </tr>
  <tr>
    <td>TERT</td>
    <td>7015</td>
    <td>79.54932962</td>
    <td>1.197803605</td>
    <td>0.2279637</td>
    <td>-5.2543612</td>
    <td>1.49E-07</td>
    <td>7.36E-06</td>
  </tr>
  <tr>
    <td>ADM</td>
    <td>133</td>
    <td>230.2165564</td>
    <td>1.187228883</td>
    <td>0.14710669</td>
    <td>-8.0705295</td>
    <td>7.00E-16</td>
    <td>3.20E-13</td>
  </tr>
  <tr>
    <td>TMEM61</td>
    <td>199964</td>
    <td>70.1777136</td>
    <td>1.186339047</td>
    <td>0.24539596</td>
    <td>-4.8343869</td>
    <td>1.34E-06</td>
    <td>4.63E-05</td>
  </tr>
  <tr>
    <td>FAM83E</td>
    <td>54854</td>
    <td>47.20990941</td>
    <td>1.185858525</td>
    <td>0.29022394</td>
    <td>-4.0860121</td>
    <td>4.39E-05</td>
    <td>0.000801634</td>
  </tr>
  <tr>
    <td>FOXL1</td>
    <td>2300</td>
    <td>44.74835767</td>
    <td>1.182475765</td>
    <td>0.29450783</td>
    <td>-4.0150911</td>
    <td>5.94E-05</td>
    <td>0.001021097</td>
  </tr>
  <tr>
    <td>AZU1</td>
    <td>566</td>
    <td>52.1387685</td>
    <td>1.181461361</td>
    <td>0.30766937</td>
    <td>-3.8400357</td>
    <td>0.00012302</td>
    <td>0.001819429</td>
  </tr>
  <tr>
    <td>PKP1</td>
    <td>5317</td>
    <td>33.69265487</td>
    <td>1.180476485</td>
    <td>0.33251659</td>
    <td>-3.5501281</td>
    <td>0.00038504</td>
    <td>0.004409278</td>
  </tr>
  <tr>
    <td>MAPK15</td>
    <td>225689</td>
    <td>44.48308856</td>
    <td>1.178291887</td>
    <td>0.29682631</td>
    <td>-3.9696342</td>
    <td>7.20E-05</td>
    <td>0.001193383</td>
  </tr>
  <tr>
    <td>ABCG1</td>
    <td>9619</td>
    <td>75.99991908</td>
    <td>1.174243707</td>
    <td>0.23249065</td>
    <td>-5.0507136</td>
    <td>4.40E-07</td>
    <td>1.85E-05</td>
  </tr>
  <tr>
    <td>DAW1</td>
    <td>164781</td>
    <td>704.0963443</td>
    <td>1.17040429</td>
    <td>0.10050349</td>
    <td>-11.645409</td>
    <td>2.42E-31</td>
    <td>7.30E-28</td>
  </tr>
  <tr>
    <td>MESP1</td>
    <td>55897</td>
    <td>125.9850592</td>
    <td>1.168758284</td>
    <td>0.19760234</td>
    <td>-5.9146986</td>
    <td>3.32E-09</td>
    <td>2.65E-07</td>
  </tr>
  <tr>
    <td>SLC19A3</td>
    <td>80704</td>
    <td>160.5869051</td>
    <td>1.168087804</td>
    <td>0.1629161</td>
    <td>-7.1698734</td>
    <td>7.51E-13</td>
    <td>1.66E-10</td>
  </tr>
  <tr>
    <td>PLEKHG6</td>
    <td>55200</td>
    <td>89.9020547</td>
    <td>1.165825029</td>
    <td>0.21115683</td>
    <td>-5.5211335</td>
    <td>3.37E-08</td>
    <td>2.10E-06</td>
  </tr>
  <tr>
    <td>F12</td>
    <td>2161</td>
    <td>246.6795035</td>
    <td>1.159745718</td>
    <td>0.14480005</td>
    <td>-8.009291</td>
    <td>1.15E-15</td>
    <td>4.97E-13</td>
  </tr>
  <tr>
    <td>KIAA1549L</td>
    <td>25758</td>
    <td>78.35250561</td>
    <td>1.157360534</td>
    <td>0.23754431</td>
    <td>-4.8721879</td>
    <td>1.10E-06</td>
    <td>3.95E-05</td>
  </tr>
  <tr>
    <td>NPW</td>
    <td>283869</td>
    <td>105.8936479</td>
    <td>1.154700486</td>
    <td>0.22086826</td>
    <td>-5.2280054</td>
    <td>1.71E-07</td>
    <td>8.30E-06</td>
  </tr>
  <tr>
    <td>PRPH</td>
    <td>5630</td>
    <td>60.60847336</td>
    <td>1.150684085</td>
    <td>0.25847786</td>
    <td>-4.4517705</td>
    <td>8.52E-06</td>
    <td>0.000210745</td>
  </tr>
  <tr>
    <td>WAKMAR2</td>
    <td>100130476</td>
    <td>71.63023732</td>
    <td>1.14251858</td>
    <td>0.22859593</td>
    <td>-4.997983</td>
    <td>5.79E-07</td>
    <td>2.30E-05</td>
  </tr>
  <tr>
    <td>TGFBR3L</td>
    <td>100507588</td>
    <td>148.5738217</td>
    <td>1.139567267</td>
    <td>0.17618308</td>
    <td>-6.4680859</td>
    <td>9.93E-11</td>
    <td>1.23E-08</td>
  </tr>
  <tr>
    <td>MLPH</td>
    <td>79083</td>
    <td>266.6372247</td>
    <td>1.137833538</td>
    <td>0.14459003</td>
    <td>-7.8693773</td>
    <td>3.56E-15</td>
    <td>1.25E-12</td>
  </tr>
  <tr>
    <td>MYPN</td>
    <td>84665</td>
    <td>242.4723645</td>
    <td>1.136844128</td>
    <td>0.15528838</td>
    <td>-7.3208579</td>
    <td>2.46E-13</td>
    <td>6.17E-11</td>
  </tr>
  <tr>
    <td>DENND1C</td>
    <td>79958</td>
    <td>64.11457106</td>
    <td>1.131167045</td>
    <td>0.26369848</td>
    <td>-4.289623</td>
    <td>1.79E-05</td>
    <td>0.000386812</td>
  </tr>
  <tr>
    <td>ANGPTL4</td>
    <td>51129</td>
    <td>516.9217151</td>
    <td>1.130554621</td>
    <td>0.11758255</td>
    <td>-9.6149863</td>
    <td>6.91E-22</td>
    <td>8.01E-19</td>
  </tr>
  <tr>
    <td>SPRY4</td>
    <td>81848</td>
    <td>73.5328524</td>
    <td>1.112077335</td>
    <td>0.26379917</td>
    <td>-4.2156211</td>
    <td>2.49E-05</td>
    <td>0.000514221</td>
  </tr>
  <tr>
    <td>TMEM121</td>
    <td>80757</td>
    <td>133.3329789</td>
    <td>1.1005935</td>
    <td>0.1851874</td>
    <td>-5.9431337</td>
    <td>2.80E-09</td>
    <td>2.29E-07</td>
  </tr>
  <tr>
    <td>KRT86</td>
    <td>3892</td>
    <td>191.2190306</td>
    <td>1.097548034</td>
    <td>0.16769116</td>
    <td>-6.5450559</td>
    <td>5.95E-11</td>
    <td>8.07E-09</td>
  </tr>
  <tr>
    <td>PALM3</td>
    <td>342979</td>
    <td>74.33614676</td>
    <td>1.096101271</td>
    <td>0.22704921</td>
    <td>-4.8275935</td>
    <td>1.38E-06</td>
    <td>4.73E-05</td>
  </tr>
  <tr>
    <td>PTH1R</td>
    <td>5745</td>
    <td>149.7158525</td>
    <td>1.093244644</td>
    <td>0.16870737</td>
    <td>-6.4801239</td>
    <td>9.16E-11</td>
    <td>1.15E-08</td>
  </tr>
  <tr>
    <td>WNT10B</td>
    <td>7480</td>
    <td>81.82237985</td>
    <td>1.0914269</td>
    <td>0.21813086</td>
    <td>-5.003542</td>
    <td>5.63E-07</td>
    <td>2.25E-05</td>
  </tr>
  <tr>
    <td>CCDC74B</td>
    <td>91409</td>
    <td>98.15432006</td>
    <td>1.090156528</td>
    <td>0.22300379</td>
    <td>-4.8885113</td>
    <td>1.02E-06</td>
    <td>3.72E-05</td>
  </tr>
  <tr>
    <td>PSG1</td>
    <td>5669</td>
    <td>41.08828565</td>
    <td>1.086438461</td>
    <td>0.31590135</td>
    <td>-3.4391701</td>
    <td>0.0005835</td>
    <td>0.006148779</td>
  </tr>
  <tr>
    <td>CES3</td>
    <td>23491</td>
    <td>105.524497</td>
    <td>1.084469211</td>
    <td>0.20160109</td>
    <td>-5.3792824</td>
    <td>7.48E-08</td>
    <td>4.10E-06</td>
  </tr>
  <tr>
    <td>HAAO</td>
    <td>23498</td>
    <td>52.58847493</td>
    <td>1.068841739</td>
    <td>0.27269592</td>
    <td>-3.919537</td>
    <td>8.87E-05</td>
    <td>0.001401154</td>
  </tr>
  <tr>
    <td>STAP2</td>
    <td>55620</td>
    <td>59.24706745</td>
    <td>1.057775929</td>
    <td>0.25205649</td>
    <td>-4.1965827</td>
    <td>2.71E-05</td>
    <td>0.000548128</td>
  </tr>
  <tr>
    <td>RASGRP2</td>
    <td>10235</td>
    <td>58.4144214</td>
    <td>1.054615843</td>
    <td>0.25257484</td>
    <td>-4.1754588</td>
    <td>2.97E-05</td>
    <td>0.000592022</td>
  </tr>
  <tr>
    <td>RASD2</td>
    <td>23551</td>
    <td>104.2740408</td>
    <td>1.052837372</td>
    <td>0.19916553</td>
    <td>-5.2862429</td>
    <td>1.25E-07</td>
    <td>6.42E-06</td>
  </tr>
  <tr>
    <td>STC1</td>
    <td>6781</td>
    <td>715.4362869</td>
    <td>1.051846598</td>
    <td>0.09871355</td>
    <td>-10.655544</td>
    <td>1.64E-26</td>
    <td>3.54E-23</td>
  </tr>
  <tr>
    <td>COL9A3</td>
    <td>1299</td>
    <td>86.02519414</td>
    <td>1.050265829</td>
    <td>0.21766091</td>
    <td>-4.8252386</td>
    <td>1.40E-06</td>
    <td>4.77E-05</td>
  </tr>
  <tr>
    <td>KCTD19</td>
    <td>146212</td>
    <td>133.2238846</td>
    <td>1.049612917</td>
    <td>0.1795657</td>
    <td>-5.8452861</td>
    <td>5.06E-09</td>
    <td>3.72E-07</td>
  </tr>
  <tr>
    <td>COL13A1</td>
    <td>1305</td>
    <td>333.3462086</td>
    <td>1.048441262</td>
    <td>0.13142287</td>
    <td>-7.9776164</td>
    <td>1.49E-15</td>
    <td>5.83E-13</td>
  </tr>
  <tr>
    <td>B4GALNT2</td>
    <td>124872</td>
    <td>75.82651354</td>
    <td>1.041848865</td>
    <td>0.22527115</td>
    <td>-4.6248658</td>
    <td>3.75E-06</td>
    <td>0.000106784</td>
  </tr>
  <tr>
    <td>SULT2B1</td>
    <td>6820</td>
    <td>62.90469693</td>
    <td>1.039053937</td>
    <td>0.26376156</td>
    <td>-3.9393683</td>
    <td>8.17E-05</td>
    <td>0.001315348</td>
  </tr>
</table>
<table>
  <tr>
    <td>LOXL1</td>
    <td>4016</td>
    <td>59.04851642</td>
    <td>1.031827614</td>
    <td>0.25037855</td>
    <td>-4.1210703</td>
    <td>3.77E-05</td>
    <td>0.000713067</td>
  </tr>
  <tr>
    <td>DUSP4</td>
    <td>1846</td>
    <td>337.8021977</td>
    <td>1.029430073</td>
    <td>0.12347994</td>
    <td>-8.3368204</td>
    <td>7.63E-17</td>
    <td>4.42E-14</td>
  </tr>
  <tr>
    <td>DUOX1</td>
    <td>53905</td>
    <td>55.20277185</td>
    <td>1.028411103</td>
    <td>0.2631906</td>
    <td>-3.9074766</td>
    <td>9.33E-05</td>
    <td>0.001453469</td>
  </tr>
  <tr>
    <td>RNF223</td>
    <td>401934</td>
    <td>61.6766655</td>
    <td>1.028127207</td>
    <td>0.2508176</td>
    <td>-4.0991031</td>
    <td>4.15E-05</td>
    <td>0.000765036</td>
  </tr>
  <tr>
    <td>L1CAM</td>
    <td>3897</td>
    <td>147.1292354</td>
    <td>1.026349808</td>
    <td>0.17047105</td>
    <td>-6.0206691</td>
    <td>1.74E-09</td>
    <td>1.49E-07</td>
  </tr>
  <tr>
    <td>RASAL1</td>
    <td>8437</td>
    <td>96.76487197</td>
    <td>1.025525268</td>
    <td>0.2123231</td>
    <td>-4.830022</td>
    <td>1.37E-06</td>
    <td>4.70E-05</td>
  </tr>
  <tr>
    <td>TSPAN8</td>
    <td>7103</td>
    <td>74.67486716</td>
    <td>1.013403596</td>
    <td>0.25029498</td>
    <td>-4.048837</td>
    <td>5.15E-05</td>
    <td>0.000906185</td>
  </tr>
  <tr>
    <td>APLP1</td>
    <td>333</td>
    <td>895.159106</td>
    <td>1.011559277</td>
    <td>0.08952731</td>
    <td>-11.298891</td>
    <td>1.33E-29</td>
    <td>3.34E-26</td>
  </tr>
  <tr>
    <td>CCN3</td>
    <td>4856</td>
    <td>51.32140079</td>
    <td>1.001808508</td>
    <td>0.28125864</td>
    <td>-3.5618764</td>
    <td>0.00036821</td>
    <td>0.004265166</td>
  </tr>
  <tr>
    <td>DNAJC12</td>
    <td>56521</td>
    <td>68.02304609</td>
    <td>0.998112957</td>
    <td>0.26787451</td>
    <td>-3.7260468</td>
    <td>0.00019451</td>
    <td>0.002614817</td>
  </tr>
  <tr>
    <td>FBXO6</td>
    <td>26270</td>
    <td>141.6609501</td>
    <td>0.996914984</td>
    <td>0.19183057</td>
    <td>-5.1968515</td>
    <td>2.03E-07</td>
    <td>9.61E-06</td>
  </tr>
  <tr>
    <td>TMEM74B</td>
    <td>55321</td>
    <td>83.7987934</td>
    <td>0.996606645</td>
    <td>0.22047436</td>
    <td>-4.5202837</td>
    <td>6.18E-06</td>
    <td>0.000164721</td>
  </tr>
  <tr>
    <td>WIPF3</td>
    <td>644150</td>
    <td>230.5592519</td>
    <td>0.992347567</td>
    <td>0.14056024</td>
    <td>-7.0599449</td>
    <td>1.67E-12</td>
    <td>3.35E-10</td>
  </tr>
  <tr>
    <td>MDFI</td>
    <td>4188</td>
    <td>407.501634</td>
    <td>0.987207414</td>
    <td>0.12376914</td>
    <td>-7.9761999</td>
    <td>1.51E-15</td>
    <td>5.83E-13</td>
  </tr>
  <tr>
    <td>AOX1</td>
    <td>316</td>
    <td>267.990915</td>
    <td>0.986157108</td>
    <td>0.1433555</td>
    <td>-6.879102</td>
    <td>6.02E-12</td>
    <td>1.01E-09</td>
  </tr>
  <tr>
    <td>ANXA9</td>
    <td>8416</td>
    <td>64.46002649</td>
    <td>0.984659939</td>
    <td>0.23776464</td>
    <td>-4.1413221</td>
    <td>3.45E-05</td>
    <td>0.000666156</td>
  </tr>
  <tr>
    <td>PIP5KL1</td>
    <td>138429</td>
    <td>795.0865471</td>
    <td>0.984086474</td>
    <td>0.26612169</td>
    <td>-3.6978816</td>
    <td>0.00021741</td>
    <td>0.002844021</td>
  </tr>
  <tr>
    <td>HYI</td>
    <td>81888</td>
    <td>1004.496251</td>
    <td>0.983378249</td>
    <td>0.10019549</td>
    <td>-9.8145957</td>
    <td>9.74E-23</td>
    <td>1.63E-19</td>
  </tr>
  <tr>
    <td>TMEM151A</td>
    <td>256472</td>
    <td>63.75800639</td>
    <td>0.982006016</td>
    <td>0.25093574</td>
    <td>-3.9133764</td>
    <td>9.10E-05</td>
    <td>0.001425768</td>
  </tr>
  <tr>
    <td>GOS2</td>
    <td>50486</td>
    <td>476.1092333</td>
    <td>0.980988167</td>
    <td>0.13843367</td>
    <td>-7.0863407</td>
    <td>1.38E-12</td>
    <td>2.92E-10</td>
  </tr>
  <tr>
    <td>CITED4</td>
    <td>163732</td>
    <td>45.95955322</td>
    <td>0.980709831</td>
    <td>0.28725559</td>
    <td>-3.414067</td>
    <td>0.00064001</td>
    <td>0.006656263</td>
  </tr>
  <tr>
    <td>TSPAN18</td>
    <td>90139</td>
    <td>230.3174438</td>
    <td>0.977636407</td>
    <td>0.14157438</td>
    <td>-6.9054615</td>
    <td>5.00E-12</td>
    <td>8.77E-10</td>
  </tr>
  <tr>
    <td>NKD2</td>
    <td>85409</td>
    <td>907.3636535</td>
    <td>0.97174049</td>
    <td>0.11359917</td>
    <td>-8.5541163</td>
    <td>1.19E-17</td>
    <td>8.14E-15</td>
  </tr>
  <tr>
    <td>SCARA5</td>
    <td>286133</td>
    <td>168.2033401</td>
    <td>0.9707754</td>
    <td>0.16664693</td>
    <td>-5.8253423</td>
    <td>5.70E-09</td>
    <td>4.15E-07</td>
  </tr>
  <tr>
    <td>KCNN4</td>
    <td>3783</td>
    <td>441.8956473</td>
    <td>0.970399152</td>
    <td>0.11981812</td>
    <td>-8.0898349</td>
    <td>5.54E-16</td>
    <td>2.61E-13</td>
  </tr>
  <tr>
    <td>LGALS8-AS1</td>
    <td>100287902</td>
    <td>63.75948556</td>
    <td>0.964251441</td>
    <td>0.24407532</td>
    <td>-3.9506307</td>
    <td>7.79E-05</td>
    <td>0.001271254</td>
  </tr>
  <tr>
    <td>SSPN</td>
    <td>8082</td>
    <td>46.88251539</td>
    <td>0.96058585</td>
    <td>0.28368406</td>
    <td>-3.3861114</td>
    <td>0.00070891</td>
    <td>0.007257617</td>
  </tr>
  <tr>
    <td>CHST13</td>
    <td>166012</td>
    <td>105.8485718</td>
    <td>0.955003518</td>
    <td>0.21270506</td>
    <td>-4.4898016</td>
    <td>7.13E-06</td>
    <td>0.000183552</td>
  </tr>
  <tr>
    <td>HES7</td>
    <td>84667</td>
    <td>361.1279759</td>
    <td>0.953185027</td>
    <td>0.1331864</td>
    <td>-7.1567747</td>
    <td>8.26E-13</td>
    <td>1.80E-10</td>
  </tr>
  <tr>
    <td>NEFL</td>
    <td>4747</td>
    <td>200.7455509</td>
    <td>0.953014683</td>
    <td>0.14642085</td>
    <td>-6.5087364</td>
    <td>7.58E-11</td>
    <td>9.93E-09</td>
  </tr>
  <tr>
    <td>ACE</td>
    <td>1636</td>
    <td>87.38067234</td>
    <td>0.9503404</td>
    <td>0.21568934</td>
    <td>-4.406061</td>
    <td>1.05E-05</td>
    <td>0.000251407</td>
  </tr>
  <tr>
    <td>ARTN</td>
    <td>9048</td>
    <td>74.16282496</td>
    <td>0.945514505</td>
    <td>0.22997746</td>
    <td>-4.1113356</td>
    <td>3.93E-05</td>
    <td>0.000737337</td>
  </tr>
  <tr>
    <td>LAT2</td>
    <td>7462</td>
    <td>195.4935926</td>
    <td>0.944088696</td>
    <td>0.15347302</td>
    <td>-6.1514963</td>
    <td>7.68E-10</td>
    <td>7.56E-08</td>
  </tr>
  <tr>
    <td>CAMK2N2</td>
    <td>94032</td>
    <td>81.8012444</td>
    <td>0.94112401</td>
    <td>0.22606705</td>
    <td>-4.1630304</td>
    <td>3.14E-05</td>
    <td>0.000618661</td>
  </tr>
  <tr>
    <td>ZNF296</td>
    <td>162979</td>
    <td>44.1168505</td>
    <td>0.939442905</td>
    <td>0.28454441</td>
    <td>-3.3015687</td>
    <td>0.00096146</td>
    <td>0.009287931</td>
  </tr>
  <tr>
    <td>UNC13D</td>
    <td>201294</td>
    <td>230.2962252</td>
    <td>0.937010502</td>
    <td>0.14403224</td>
    <td>-6.505561</td>
    <td>7.74E-11</td>
    <td>1.01E-08</td>
  </tr>
  <tr>
    <td>LINC02381</td>
    <td>400043</td>
    <td>54.97634785</td>
    <td>0.935778332</td>
    <td>0.25902702</td>
    <td>-3.6126669</td>
    <td>0.00030306</td>
    <td>0.003671361</td>
  </tr>
  <tr>
    <td>CA12</td>
    <td>771</td>
    <td>169.6710151</td>
    <td>0.930606053</td>
    <td>0.15759902</td>
    <td>-5.9048975</td>
    <td>3.53E-09</td>
    <td>2.78E-07</td>
  </tr>
  <tr>
    <td>CREG2</td>
    <td>200407</td>
    <td>64.41168355</td>
    <td>0.929879574</td>
    <td>0.23881498</td>
    <td>-3.8937239</td>
    <td>9.87E-05</td>
    <td>0.001519576</td>
  </tr>
  <tr>
    <td>PLEKHA6</td>
    <td>22874</td>
    <td>152.7473984</td>
    <td>0.929368766</td>
    <td>0.17681652</td>
    <td>-5.2561196</td>
    <td>1.47E-07</td>
    <td>7.32E-06</td>
  </tr>
  <tr>
    <td>CELF5</td>
    <td>60680</td>
    <td>77.10973701</td>
    <td>0.924779148</td>
    <td>0.22425403</td>
    <td>-4.1238018</td>
    <td>3.73E-05</td>
    <td>0.000708213</td>
  </tr>
  <tr>
    <td>GALNT6</td>
    <td>11226</td>
    <td>106.6789657</td>
    <td>0.924467922</td>
    <td>0.19847072</td>
    <td>-4.6579561</td>
    <td>3.19E-06</td>
    <td>9.38E-05</td>
  </tr>
  <tr>
    <td>MIR210HG</td>
    <td>100506211</td>
    <td>305.2809665</td>
    <td>0.918654646</td>
    <td>0.1306354</td>
    <td>-7.0322032</td>
    <td>2.03E-12</td>
    <td>3.98E-10</td>
  </tr>
  <tr>
    <td>BSPRY</td>
    <td>54836</td>
    <td>227.2447292</td>
    <td>0.917159098</td>
    <td>0.13770544</td>
    <td>-6.6602969</td>
    <td>2.73E-11</td>
    <td>3.81E-09</td>
  </tr>
  <tr>
    <td>HLA-DRB1</td>
    <td>3123</td>
    <td>262.5134278</td>
    <td>0.916561311</td>
    <td>0.15019547</td>
    <td>-6.1024565</td>
    <td>1.04E-09</td>
    <td>9.66E-08</td>
  </tr>
  <tr>
    <td>TMEM229B</td>
    <td>161145</td>
    <td>74.70278513</td>
    <td>0.91530222</td>
    <td>0.2299158</td>
    <td>-3.9810323</td>
    <td>6.86E-05</td>
    <td>0.00114513</td>
  </tr>
  <tr>
    <td>TNFSF10</td>
    <td>8743</td>
    <td>124.1158188</td>
    <td>0.914374165</td>
    <td>0.1814377</td>
    <td>-5.0396042</td>
    <td>4.66E-07</td>
    <td>1.96E-05</td>
  </tr>
  <tr>
    <td>C6orf132</td>
    <td>647024</td>
    <td>146.2977545</td>
    <td>0.911115912</td>
    <td>0.16643076</td>
    <td>-5.4744442</td>
    <td>4.39E-08</td>
    <td>2.59E-06</td>
  </tr>
  <tr>
    <td>NDRG1</td>
    <td>10397</td>
    <td>5511.921069</td>
    <td>0.91059877</td>
    <td>0.09463563</td>
    <td>-9.6221553</td>
    <td>6.45E-22</td>
    <td>8.01E-19</td>
  </tr>
  <tr>
    <td>ETV4</td>
    <td>2118</td>
    <td>382.2606543</td>
    <td>0.910135915</td>
    <td>0.122825</td>
    <td>-7.4100217</td>
    <td>1.26E-13</td>
    <td>3.28E-11</td>
  </tr>
  <tr>
    <td>STC2</td>
    <td>8614</td>
    <td>2312.761748</td>
    <td>0.90933215</td>
    <td>0.0936837</td>
    <td>-9.7064071</td>
    <td>2.83E-22</td>
    <td>4.27E-19</td>
  </tr>
  <tr>
    <td>ARHGAP4</td>
    <td>393</td>
    <td>132.8908935</td>
    <td>0.903255714</td>
    <td>0.19232874</td>
    <td>-4.6964157</td>
    <td>2.65E-06</td>
    <td>8.06E-05</td>
  </tr>
  <tr>
    <td>BMPER</td>
    <td>168667</td>
    <td>130.8648173</td>
    <td>0.90209066</td>
    <td>0.19304472</td>
    <td>-4.6729622</td>
    <td>2.97E-06</td>
    <td>8.91E-05</td>
  </tr>
  <tr>
    <td>CXCL8</td>
    <td>3576</td>
    <td>619.2939693</td>
    <td>0.897782059</td>
    <td>0.10487006</td>
    <td>-8.5608996</td>
    <td>1.12E-17</td>
    <td>8.04E-15</td>
  </tr>
</table>
<table>
  <tr>
    <td>EFEMP2</td>
    <td>30008</td>
    <td>176.5913295</td>
    <td>0.895842237</td>
    <td>0.17583343</td>
    <td>-5.0948345</td>
    <td>3.49E-07</td>
    <td>1.51E-05</td>
  </tr>
  <tr>
    <td>ARHGAP40</td>
    <td>343578</td>
    <td>93.09264182</td>
    <td>0.894190187</td>
    <td>0.2243495</td>
    <td>-3.9857018</td>
    <td>6.73E-05</td>
    <td>0.001126583</td>
  </tr>
  <tr>
    <td>ESRP1</td>
    <td>54845</td>
    <td>162.1343505</td>
    <td>0.893779676</td>
    <td>0.16936976</td>
    <td>-5.2770914</td>
    <td>1.31E-07</td>
    <td>6.70E-06</td>
  </tr>
  <tr>
    <td>ERFE</td>
    <td>151176</td>
    <td>92.09975946</td>
    <td>0.892687051</td>
    <td>0.22960862</td>
    <td>-3.8878639</td>
    <td>0.00010113</td>
    <td>0.001545673</td>
  </tr>
  <tr>
    <td>WNT5B</td>
    <td>81029</td>
    <td>160.0304325</td>
    <td>0.887757817</td>
    <td>0.16207502</td>
    <td>-5.47745</td>
    <td>4.31E-08</td>
    <td>2.56E-06</td>
  </tr>
  <tr>
    <td>PPP1R3G</td>
    <td>648791</td>
    <td>235.5291983</td>
    <td>0.882562288</td>
    <td>0.15178607</td>
    <td>-5.8145145</td>
    <td>6.08E-09</td>
    <td>4.36E-07</td>
  </tr>
  <tr>
    <td>NALT1</td>
    <td>101928483</td>
    <td>73.82571681</td>
    <td>0.882333458</td>
    <td>0.22415836</td>
    <td>-3.9362059</td>
    <td>8.28E-05</td>
    <td>0.001329951</td>
  </tr>
  <tr>
    <td>TNNT1</td>
    <td>7138</td>
    <td>1079.679098</td>
    <td>0.882065221</td>
    <td>0.11185542</td>
    <td>-7.885762</td>
    <td>3.13E-15</td>
    <td>1.12E-12</td>
  </tr>
  <tr>
    <td>NLRP1</td>
    <td>22861</td>
    <td>68.81433374</td>
    <td>0.877828642</td>
    <td>0.23141733</td>
    <td>-3.793271</td>
    <td>0.00014868</td>
    <td>0.00212455</td>
  </tr>
  <tr>
    <td>IRF5</td>
    <td>3663</td>
    <td>300.4880596</td>
    <td>0.874219343</td>
    <td>0.13124575</td>
    <td>-6.6609346</td>
    <td>2.72E-11</td>
    <td>3.81E-09</td>
  </tr>
  <tr>
    <td>NGEF</td>
    <td>25791</td>
    <td>134.4466052</td>
    <td>0.871866556</td>
    <td>0.17899821</td>
    <td>-4.8708116</td>
    <td>1.11E-06</td>
    <td>3.96E-05</td>
  </tr>
  <tr>
    <td>WFDC2</td>
    <td>10406</td>
    <td>229.5906195</td>
    <td>0.866056206</td>
    <td>0.16703136</td>
    <td>-5.1849918</td>
    <td>2.16E-07</td>
    <td>1.01E-05</td>
  </tr>
  <tr>
    <td>LOC101929705</td>
    <td>101929705</td>
    <td>97.4656173</td>
    <td>0.865835587</td>
    <td>0.22188554</td>
    <td>-3.9021722</td>
    <td>9.53E-05</td>
    <td>0.001479581</td>
  </tr>
  <tr>
    <td>LTBP4</td>
    <td>8425</td>
    <td>3622.859577</td>
    <td>0.865384675</td>
    <td>0.09706596</td>
    <td>-8.9154289</td>
    <td>4.86E-19</td>
    <td>4.31E-16</td>
  </tr>
  <tr>
    <td>TNFRSF14</td>
    <td>8764</td>
    <td>82.94775855</td>
    <td>0.860947661</td>
    <td>0.24328473</td>
    <td>-3.5388479</td>
    <td>0.00040188</td>
    <td>0.004563728</td>
  </tr>
  <tr>
    <td>FER1L4</td>
    <td>80307</td>
    <td>791.2926466</td>
    <td>0.86051595</td>
    <td>0.26195762</td>
    <td>-3.2849434</td>
    <td>0.00102003</td>
    <td>0.009722853</td>
  </tr>
  <tr>
    <td>MMP1</td>
    <td>4312</td>
    <td>124.2437872</td>
    <td>0.856306634</td>
    <td>0.19526006</td>
    <td>-4.3854675</td>
    <td>1.16E-05</td>
    <td>0.000271675</td>
  </tr>
  <tr>
    <td>NPAS1</td>
    <td>4861</td>
    <td>152.5696309</td>
    <td>0.856014151</td>
    <td>0.17329785</td>
    <td>-4.9395544</td>
    <td>7.83E-07</td>
    <td>2.99E-05</td>
  </tr>
  <tr>
    <td>PADI2</td>
    <td>11240</td>
    <td>192.5521913</td>
    <td>0.851369666</td>
    <td>0.14737706</td>
    <td>-5.7768127</td>
    <td>7.61E-09</td>
    <td>5.34E-07</td>
  </tr>
  <tr>
    <td>KIRREL2</td>
    <td>84063</td>
    <td>106.022171</td>
    <td>0.847762833</td>
    <td>0.21080115</td>
    <td>-4.0216233</td>
    <td>5.78E-05</td>
    <td>0.000996593</td>
  </tr>
  <tr>
    <td>LRRC34</td>
    <td>151827</td>
    <td>266.3317996</td>
    <td>0.84771187</td>
    <td>0.13317215</td>
    <td>-6.365534</td>
    <td>1.95E-10</td>
    <td>2.14E-08</td>
  </tr>
  <tr>
    <td>HK2</td>
    <td>3099</td>
    <td>721.8788965</td>
    <td>0.841619913</td>
    <td>0.09863449</td>
    <td>-8.5327146</td>
    <td>1.43E-17</td>
    <td>9.37E-15</td>
  </tr>
  <tr>
    <td>CARD11</td>
    <td>84433</td>
    <td>99.87209684</td>
    <td>0.84009749</td>
    <td>0.19674858</td>
    <td>-4.2699037</td>
    <td>1.96E-05</td>
    <td>0.00041625</td>
  </tr>
  <tr>
    <td>LYPD3</td>
    <td>27076</td>
    <td>750.5661199</td>
    <td>0.839210796</td>
    <td>0.10153214</td>
    <td>-8.2654693</td>
    <td>1.39E-16</td>
    <td>7.27E-14</td>
  </tr>
  <tr>
    <td>GGT1</td>
    <td>2678</td>
    <td>170.2338261</td>
    <td>0.838593829</td>
    <td>0.17985591</td>
    <td>-4.662587</td>
    <td>3.12E-06</td>
    <td>9.26E-05</td>
  </tr>
  <tr>
    <td>GNA15</td>
    <td>2769</td>
    <td>124.7685583</td>
    <td>0.838135526</td>
    <td>0.18643827</td>
    <td>-4.4955123</td>
    <td>6.94E-06</td>
    <td>0.000179092</td>
  </tr>
  <tr>
    <td>CEMIP</td>
    <td>57214</td>
    <td>2550.564939</td>
    <td>0.837849162</td>
    <td>0.09680853</td>
    <td>-8.654704</td>
    <td>4.94E-18</td>
    <td>3.72E-15</td>
  </tr>
  <tr>
    <td>MFSD2A</td>
    <td>84879</td>
    <td>85.05677016</td>
    <td>0.832445443</td>
    <td>0.22837874</td>
    <td>-3.6450216</td>
    <td>0.00026737</td>
    <td>0.003354724</td>
  </tr>
  <tr>
    <td>EML1</td>
    <td>2009</td>
    <td>261.5867901</td>
    <td>0.831691515</td>
    <td>0.1405623</td>
    <td>-5.9168888</td>
    <td>3.28E-09</td>
    <td>2.64E-07</td>
  </tr>
  <tr>
    <td>VTN</td>
    <td>7448</td>
    <td>161.051236</td>
    <td>0.831650907</td>
    <td>0.17712339</td>
    <td>-4.6953196</td>
    <td>2.66E-06</td>
    <td>8.09E-05</td>
  </tr>
  <tr>
    <td>LHX2</td>
    <td>9355</td>
    <td>218.2030754</td>
    <td>0.831502868</td>
    <td>0.14037207</td>
    <td>-5.9235636</td>
    <td>3.15E-09</td>
    <td>2.55E-07</td>
  </tr>
  <tr>
    <td>NUDT8</td>
    <td>254552</td>
    <td>154.9426855</td>
    <td>0.829421617</td>
    <td>0.16594117</td>
    <td>-4.998287</td>
    <td>5.78E-07</td>
    <td>2.30E-05</td>
  </tr>
  <tr>
    <td>MAPK8IP2</td>
    <td>23542</td>
    <td>589.6770752</td>
    <td>0.828204191</td>
    <td>0.11906053</td>
    <td>-6.9561606</td>
    <td>3.50E-12</td>
    <td>6.35E-10</td>
  </tr>
  <tr>
    <td>GRIN1</td>
    <td>2902</td>
    <td>92.24683377</td>
    <td>0.827697065</td>
    <td>0.21354714</td>
    <td>-3.8759455</td>
    <td>0.00010621</td>
    <td>0.00160865</td>
  </tr>
  <tr>
    <td>SPDEF</td>
    <td>25803</td>
    <td>79.69208905</td>
    <td>0.827523581</td>
    <td>0.23811556</td>
    <td>-3.4753024</td>
    <td>0.00051028</td>
    <td>0.005484941</td>
  </tr>
  <tr>
    <td>MACROD1</td>
    <td>28992</td>
    <td>302.8167764</td>
    <td>0.823241513</td>
    <td>0.16012796</td>
    <td>-5.141148</td>
    <td>2.73E-07</td>
    <td>1.23E-05</td>
  </tr>
  <tr>
    <td>C19orf81</td>
    <td>342918</td>
    <td>75.7654431</td>
    <td>0.821408689</td>
    <td>0.22330828</td>
    <td>-3.678362</td>
    <td>0.00023474</td>
    <td>0.003024431</td>
  </tr>
  <tr>
    <td>CARD6</td>
    <td>84674</td>
    <td>120.7787593</td>
    <td>0.816560102</td>
    <td>0.18727216</td>
    <td>-4.3602855</td>
    <td>1.30E-05</td>
    <td>0.000297257</td>
  </tr>
  <tr>
    <td>EVI2B</td>
    <td>2124</td>
    <td>86.27110041</td>
    <td>0.812457486</td>
    <td>0.21259582</td>
    <td>-3.8216061</td>
    <td>0.00013259</td>
    <td>0.001926771</td>
  </tr>
  <tr>
    <td>KISS1R</td>
    <td>84634</td>
    <td>81.61187624</td>
    <td>0.812337315</td>
    <td>0.22750783</td>
    <td>-3.5705906</td>
    <td>0.00035618</td>
    <td>0.004161145</td>
  </tr>
  <tr>
    <td>IGDCC4</td>
    <td>57722</td>
    <td>387.5145404</td>
    <td>0.810478647</td>
    <td>0.12033341</td>
    <td>-6.7352753</td>
    <td>1.64E-11</td>
    <td>2.44E-09</td>
  </tr>
  <tr>
    <td>DNAAF3</td>
    <td>352909</td>
    <td>124.4113965</td>
    <td>0.809810314</td>
    <td>0.18669963</td>
    <td>-4.3375036</td>
    <td>1.44E-05</td>
    <td>0.000324625</td>
  </tr>
  <tr>
    <td>LOXL4</td>
    <td>84171</td>
    <td>292.7765977</td>
    <td>0.8094291</td>
    <td>0.12631602</td>
    <td>-6.4079686</td>
    <td>1.47E-10</td>
    <td>1.70E-08</td>
  </tr>
  <tr>
    <td>PAQR5</td>
    <td>54852</td>
    <td>130.8065966</td>
    <td>0.807257329</td>
    <td>0.17634129</td>
    <td>-4.5778122</td>
    <td>4.70E-06</td>
    <td>0.000129686</td>
  </tr>
  <tr>
    <td>HSPB8</td>
    <td>26353</td>
    <td>112.9524042</td>
    <td>0.807071955</td>
    <td>0.19314033</td>
    <td>-4.1786816</td>
    <td>2.93E-05</td>
    <td>0.000587577</td>
  </tr>
  <tr>
    <td>NRARP</td>
    <td>441478</td>
    <td>152.3778337</td>
    <td>0.80559869</td>
    <td>0.16610383</td>
    <td>-4.8499705</td>
    <td>1.23E-06</td>
    <td>4.31E-05</td>
  </tr>
  <tr>
    <td>SPTBN4</td>
    <td>57731</td>
    <td>112.7296362</td>
    <td>0.803707662</td>
    <td>0.19571364</td>
    <td>-4.106549</td>
    <td>4.02E-05</td>
    <td>0.000745787</td>
  </tr>
  <tr>
    <td>TLL2</td>
    <td>7093</td>
    <td>74.61128171</td>
    <td>0.802517438</td>
    <td>0.24424284</td>
    <td>-3.2857357</td>
    <td>0.00101716</td>
    <td>0.009703555</td>
  </tr>
  <tr>
    <td>ADAMTS7</td>
    <td>11173</td>
    <td>254.4261637</td>
    <td>0.801254598</td>
    <td>0.14863247</td>
    <td>-5.3908448</td>
    <td>7.01E-08</td>
    <td>3.90E-06</td>
  </tr>
  <tr>
    <td>FAM131C</td>
    <td>348487</td>
    <td>286.1934338</td>
    <td>0.797796753</td>
    <td>0.13046101</td>
    <td>-6.115212</td>
    <td>9.64E-10</td>
    <td>9.14E-08</td>
  </tr>
  <tr>
    <td>CACNG8</td>
    <td>59283</td>
    <td>122.0639063</td>
    <td>0.793757395</td>
    <td>0.18997696</td>
    <td>-4.1781772</td>
    <td>2.94E-05</td>
    <td>0.00058881</td>
  </tr>
  <tr>
    <td>PPFIA3</td>
    <td>8541</td>
    <td>239.2503444</td>
    <td>0.793684794</td>
    <td>0.16307982</td>
    <td>-4.8668487</td>
    <td>1.13E-06</td>
    <td>4.02E-05</td>
  </tr>
  <tr>
    <td>NEIL1</td>
    <td>79661</td>
    <td>246.8892769</td>
    <td>0.786665229</td>
    <td>0.15634154</td>
    <td>-5.0317096</td>
    <td>4.86E-07</td>
    <td>2.02E-05</td>
  </tr>
  <tr>
    <td>PGF</td>
    <td>5228</td>
    <td>100.2225987</td>
    <td>0.786223869</td>
    <td>0.22314421</td>
    <td>-3.523389</td>
    <td>0.00042607</td>
    <td>0.00476321</td>
  </tr>
</table>
<table>
  <tr>
    <td>SECTM1</td>
    <td>6398</td>
    <td>500.3181305</td>
    <td>0.785272862</td>
    <td>0.11172778</td>
    <td>-7.0284475</td>
    <td>2.09E-12</td>
    <td>4.03E-10</td>
  </tr>
  <tr>
    <td>STK31</td>
    <td>56164</td>
    <td>304.5528069</td>
    <td>0.783875451</td>
    <td>0.13342086</td>
    <td>-5.875209</td>
    <td>4.22E-09</td>
    <td>3.23E-07</td>
  </tr>
  <tr>
    <td>CRIP1</td>
    <td>1396</td>
    <td>3658.828319</td>
    <td>0.783628413</td>
    <td>0.10106702</td>
    <td>-7.7535525</td>
    <td>8.94E-15</td>
    <td>2.92E-12</td>
  </tr>
  <tr>
    <td>ST8SIA6</td>
    <td>338596</td>
    <td>71.02272435</td>
    <td>0.783245793</td>
    <td>0.23388035</td>
    <td>-3.3489167</td>
    <td>0.00081128</td>
    <td>0.008139826</td>
  </tr>
  <tr>
    <td>KRT15</td>
    <td>3866</td>
    <td>71.02812729</td>
    <td>0.779300657</td>
    <td>0.23574865</td>
    <td>-3.3056421</td>
    <td>0.00094759</td>
    <td>0.009195227</td>
  </tr>
  <tr>
    <td>CXCL5</td>
    <td>6374</td>
    <td>192.9660684</td>
    <td>0.779012539</td>
    <td>0.15693046</td>
    <td>-4.9640619</td>
    <td>6.90E-07</td>
    <td>2.66E-05</td>
  </tr>
  <tr>
    <td>APOE</td>
    <td>348</td>
    <td>609.7918727</td>
    <td>0.778919055</td>
    <td>0.1124174</td>
    <td>-6.9288121</td>
    <td>4.24E-12</td>
    <td>7.61E-10</td>
  </tr>
  <tr>
    <td>LINC00957</td>
    <td>255031</td>
    <td>153.322615</td>
    <td>0.774428044</td>
    <td>0.17971361</td>
    <td>-4.3092342</td>
    <td>1.64E-05</td>
    <td>0.000360933</td>
  </tr>
  <tr>
    <td>INAVA</td>
    <td>55765</td>
    <td>150.2835474</td>
    <td>0.768813713</td>
    <td>0.17032476</td>
    <td>-4.5138107</td>
    <td>6.37E-06</td>
    <td>0.000168343</td>
  </tr>
  <tr>
    <td>RCOR2</td>
    <td>283248</td>
    <td>86.42220791</td>
    <td>0.767655419</td>
    <td>0.21809518</td>
    <td>-3.5198183</td>
    <td>0.00043184</td>
    <td>0.004809953</td>
  </tr>
  <tr>
    <td>COL16A1</td>
    <td>1307</td>
    <td>74.73285051</td>
    <td>0.766268744</td>
    <td>0.22285204</td>
    <td>-3.4384641</td>
    <td>0.00058502</td>
    <td>0.006156644</td>
  </tr>
  <tr>
    <td>NID2</td>
    <td>22795</td>
    <td>206.6733637</td>
    <td>0.76461715</td>
    <td>0.14281526</td>
    <td>-5.3538897</td>
    <td>8.61E-08</td>
    <td>4.63E-06</td>
  </tr>
  <tr>
    <td>NFASC</td>
    <td>23114</td>
    <td>123.8104847</td>
    <td>0.763613986</td>
    <td>0.18356468</td>
    <td>-4.1599178</td>
    <td>3.18E-05</td>
    <td>0.000624703</td>
  </tr>
  <tr>
    <td>NPTX1</td>
    <td>4884</td>
    <td>107.4769931</td>
    <td>0.760881802</td>
    <td>0.21111349</td>
    <td>-3.6041363</td>
    <td>0.00031319</td>
    <td>0.003769819</td>
  </tr>
  <tr>
    <td>BDH1</td>
    <td>622</td>
    <td>269.3464489</td>
    <td>0.760550976</td>
    <td>0.12970928</td>
    <td>-5.8635046</td>
    <td>4.53E-09</td>
    <td>3.41E-07</td>
  </tr>
  <tr>
    <td>HOXC5</td>
    <td>3222</td>
    <td>126.4353462</td>
    <td>0.760474207</td>
    <td>0.18689236</td>
    <td>-4.0690492</td>
    <td>4.72E-05</td>
    <td>0.000848908</td>
  </tr>
  <tr>
    <td>ALDH1L2</td>
    <td>160428</td>
    <td>355.8860086</td>
    <td>0.757370394</td>
    <td>0.12913045</td>
    <td>-5.8651572</td>
    <td>4.49E-09</td>
    <td>3.40E-07</td>
  </tr>
  <tr>
    <td>NKX2-8</td>
    <td>26257</td>
    <td>241.1288378</td>
    <td>0.756972873</td>
    <td>0.1630436</td>
    <td>-4.6427636</td>
    <td>3.44E-06</td>
    <td>9.91E-05</td>
  </tr>
  <tr>
    <td>SLC7A5</td>
    <td>8140</td>
    <td>9021.500531</td>
    <td>0.756704409</td>
    <td>0.08474358</td>
    <td>-8.9293425</td>
    <td>4.29E-19</td>
    <td>4.04E-16</td>
  </tr>
  <tr>
    <td>SLC37A1</td>
    <td>54020</td>
    <td>294.736694</td>
    <td>0.756059665</td>
    <td>0.12666779</td>
    <td>-5.9688339</td>
    <td>2.39E-09</td>
    <td>2.00E-07</td>
  </tr>
  <tr>
    <td>GRIN3B</td>
    <td>116444</td>
    <td>196.2720497</td>
    <td>0.755211105</td>
    <td>0.16037971</td>
    <td>-4.7088943</td>
    <td>2.49E-06</td>
    <td>7.69E-05</td>
  </tr>
  <tr>
    <td>NES</td>
    <td>10763</td>
    <td>420.3418837</td>
    <td>0.754343306</td>
    <td>0.12689539</td>
    <td>-5.9446076</td>
    <td>2.77E-09</td>
    <td>2.28E-07</td>
  </tr>
  <tr>
    <td>NAGS</td>
    <td>162417</td>
    <td>517.4634759</td>
    <td>0.751533754</td>
    <td>0.11512603</td>
    <td>-6.5279223</td>
    <td>6.67E-11</td>
    <td>8.89E-09</td>
  </tr>
  <tr>
    <td>SELENOP</td>
    <td>6414</td>
    <td>177.8911917</td>
    <td>0.751386044</td>
    <td>0.1802385</td>
    <td>-4.1688433</td>
    <td>3.06E-05</td>
    <td>0.000605469</td>
  </tr>
  <tr>
    <td>CORO6</td>
    <td>84940</td>
    <td>318.5474265</td>
    <td>0.751367427</td>
    <td>0.1265908</td>
    <td>-5.935403</td>
    <td>2.93E-09</td>
    <td>2.39E-07</td>
  </tr>
  <tr>
    <td>SH2B2</td>
    <td>10603</td>
    <td>118.947673</td>
    <td>0.750363275</td>
    <td>0.18284776</td>
    <td>-4.1037597</td>
    <td>4.06E-05</td>
    <td>0.000751632</td>
  </tr>
  <tr>
    <td>PPP1R16B</td>
    <td>26051</td>
    <td>138.950063</td>
    <td>0.749104055</td>
    <td>0.18163824</td>
    <td>-4.1241539</td>
    <td>3.72E-05</td>
    <td>0.000708024</td>
  </tr>
  <tr>
    <td>TNNI3</td>
    <td>7137</td>
    <td>132.2212764</td>
    <td>0.743780309</td>
    <td>0.17812449</td>
    <td>-4.1756207</td>
    <td>2.97E-05</td>
    <td>0.000592022</td>
  </tr>
  <tr>
    <td>SPINT1</td>
    <td>6692</td>
    <td>1427.904482</td>
    <td>0.739873537</td>
    <td>0.09321818</td>
    <td>-7.9370087</td>
    <td>2.07E-15</td>
    <td>7.80E-13</td>
  </tr>
  <tr>
    <td>TMEM30B</td>
    <td>161291</td>
    <td>208.6124251</td>
    <td>0.739506893</td>
    <td>0.15533709</td>
    <td>-4.760659</td>
    <td>1.93E-06</td>
    <td>6.23E-05</td>
  </tr>
  <tr>
    <td>HOXB9</td>
    <td>3219</td>
    <td>719.7567467</td>
    <td>0.733324266</td>
    <td>0.09371731</td>
    <td>-7.8248538</td>
    <td>5.08E-15</td>
    <td>1.73E-12</td>
  </tr>
  <tr>
    <td>DGCR5</td>
    <td>26220</td>
    <td>415.2265283</td>
    <td>0.733311477</td>
    <td>0.1171613</td>
    <td>-6.2589906</td>
    <td>3.87E-10</td>
    <td>4.08E-08</td>
  </tr>
  <tr>
    <td>CYS1</td>
    <td>192668</td>
    <td>124.9665949</td>
    <td>0.729862903</td>
    <td>0.19739312</td>
    <td>-3.6975094</td>
    <td>0.00021773</td>
    <td>0.002845723</td>
  </tr>
  <tr>
    <td>MSI1</td>
    <td>4440</td>
    <td>174.0078681</td>
    <td>0.728301602</td>
    <td>0.1549027</td>
    <td>-4.7016714</td>
    <td>2.58E-06</td>
    <td>7.87E-05</td>
  </tr>
  <tr>
    <td>NOXA1</td>
    <td>10811</td>
    <td>438.5813501</td>
    <td>0.726074836</td>
    <td>0.13697035</td>
    <td>-5.3009637</td>
    <td>1.15E-07</td>
    <td>6.01E-06</td>
  </tr>
  <tr>
    <td>STEAP1B</td>
    <td>256227</td>
    <td>85.33303595</td>
    <td>0.722806195</td>
    <td>0.21503853</td>
    <td>-3.3612869</td>
    <td>0.0007758</td>
    <td>0.007851805</td>
  </tr>
  <tr>
    <td>CSGALNACT1</td>
    <td>55790</td>
    <td>492.2332612</td>
    <td>0.719983981</td>
    <td>0.10552991</td>
    <td>-6.8225584</td>
    <td>8.94E-12</td>
    <td>1.39E-09</td>
  </tr>
  <tr>
    <td>HHIPL1</td>
    <td>84439</td>
    <td>166.094212</td>
    <td>0.719110143</td>
    <td>0.16820727</td>
    <td>-4.2751431</td>
    <td>1.91E-05</td>
    <td>0.000408891</td>
  </tr>
  <tr>
    <td>LGI3</td>
    <td>203190</td>
    <td>133.9902869</td>
    <td>0.714822984</td>
    <td>0.18299302</td>
    <td>-3.9062856</td>
    <td>9.37E-05</td>
    <td>0.001459139</td>
  </tr>
  <tr>
    <td>EPOP</td>
    <td>100170841</td>
    <td>515.2525388</td>
    <td>0.713143084</td>
    <td>0.11825305</td>
    <td>-6.0306529</td>
    <td>1.63E-09</td>
    <td>1.41E-07</td>
  </tr>
  <tr>
    <td>PRR29</td>
    <td>92340</td>
    <td>113.1888916</td>
    <td>0.707842237</td>
    <td>0.20369721</td>
    <td>-3.4749727</td>
    <td>0.00051091</td>
    <td>0.005487772</td>
  </tr>
  <tr>
    <td>CPT1C</td>
    <td>126129</td>
    <td>169.2052761</td>
    <td>0.707303159</td>
    <td>0.17491735</td>
    <td>-4.0436318</td>
    <td>5.26E-05</td>
    <td>0.00092117</td>
  </tr>
  <tr>
    <td>MAP3K7CL</td>
    <td>56911</td>
    <td>173.7332349</td>
    <td>0.707210153</td>
    <td>0.15334903</td>
    <td>-4.611768</td>
    <td>3.99E-06</td>
    <td>0.000112886</td>
  </tr>
  <tr>
    <td>DDIT4</td>
    <td>54541</td>
    <td>4612.761209</td>
    <td>0.706918936</td>
    <td>0.09167448</td>
    <td>-7.7111858</td>
    <td>1.25E-14</td>
    <td>3.91E-12</td>
  </tr>
  <tr>
    <td>FBXL16</td>
    <td>146330</td>
    <td>274.4928042</td>
    <td>0.7055123</td>
    <td>0.14992743</td>
    <td>-4.7056918</td>
    <td>2.53E-06</td>
    <td>7.75E-05</td>
  </tr>
  <tr>
    <td>EGFL7</td>
    <td>51162</td>
    <td>1431.442486</td>
    <td>0.704617691</td>
    <td>0.09383432</td>
    <td>-7.5091682</td>
    <td>5.95E-14</td>
    <td>1.79E-11</td>
  </tr>
  <tr>
    <td>HAPLN3</td>
    <td>145864</td>
    <td>121.7735612</td>
    <td>0.704242028</td>
    <td>0.19496599</td>
    <td>-3.6121276</td>
    <td>0.0003037</td>
    <td>0.003674802</td>
  </tr>
  <tr>
    <td>PTPRU</td>
    <td>10076</td>
    <td>298.2059944</td>
    <td>0.704162355</td>
    <td>0.13998469</td>
    <td>-5.0302812</td>
    <td>4.90E-07</td>
    <td>2.03E-05</td>
  </tr>
  <tr>
    <td>PTK6</td>
    <td>5753</td>
    <td>280.9850334</td>
    <td>0.700013536</td>
    <td>0.13569735</td>
    <td>-5.1586383</td>
    <td>2.49E-07</td>
    <td>1.14E-05</td>
  </tr>
  <tr>
    <td>LY6K</td>
    <td>54742</td>
    <td>3321.602029</td>
    <td>0.699376851</td>
    <td>0.09726835</td>
    <td>-7.1901787</td>
    <td>6.47E-13</td>
    <td>1.46E-10</td>
  </tr>
  <tr>
    <td>LINC01588</td>
    <td>283551</td>
    <td>302.3285816</td>
    <td>0.694576518</td>
    <td>0.13260986</td>
    <td>-5.2377442</td>
    <td>1.63E-07</td>
    <td>7.90E-06</td>
  </tr>
  <tr>
    <td>GAMT</td>
    <td>2593</td>
    <td>962.5101188</td>
    <td>0.690666562</td>
    <td>0.11701248</td>
    <td>-5.9025034</td>
    <td>3.58E-09</td>
    <td>2.81E-07</td>
  </tr>
  <tr>
    <td>ARHGEF37</td>
    <td>389337</td>
    <td>377.9249812</td>
    <td>0.690610008</td>
    <td>0.11263038</td>
    <td>-6.1316492</td>
    <td>8.70E-10</td>
    <td>8.46E-08</td>
  </tr>
  <tr>
    <td>ABHD1</td>
    <td>84696</td>
    <td>124.401849</td>
    <td>0.690258138</td>
    <td>0.18061774</td>
    <td>-3.8216519</td>
    <td>0.00013256</td>
    <td>0.001926771</td>
  </tr>
</table>
<table><caption>**Figure S4:** List of downregulated genes according to the log2 (FC) value in DU145 cells after H4K20me reprogramming induced by the chemical inhibitor A-196. (FDR 0.01, log2 (FC) < -0.69).</caption><thead><tr><th>feature</th><th>GENE_ID</th><th>baseMean</th><th>log2 (FC)</th><th>lfcSE</th><th>stat</th><th>pvalue</th><th>padj</th></tr></thead><tbody><tr><td>LOC106699570</td><td></td><td>106699570</td><td>17.42473228</td><td>-1.904069604</td><td>0.50286723</td><td>3.7864261</td><td>0.00015283</td><td>0.00216664</td></tr><tr><td>FOS</td><td></td><td>2353</td><td>183.8014951</td><td>-1.342251894</td><td>0.189906621</td><td>7.06795734</td><td>1.57E-12</td><td>3.25E-10</td></tr><tr><td>DMBT1</td><td></td><td>1755</td><td>45.46205698</td><td>-1.263428103</td><td>0.330732387</td><td>3.82009187</td><td>0.0001334</td><td>0.00193305</td></tr><tr><td>ADCY10P1</td><td></td><td>221442</td><td>55.10428121</td><td>-1.174711118</td><td>0.272116831</td><td>4.31693664</td><td>1.58E-05</td><td>0.00035114</td></tr><tr><td>NPNT</td><td></td><td>255743</td><td>315.0236295</td><td>-1.067635284</td><td>0.126946054</td><td>8.41014946</td><td>4.09E-17</td><td>2.47E-14</td></tr><tr><td>SLC6A3</td><td></td><td>6531</td><td>400.2994783</td><td>-1.002281214</td><td>0.135050473</td><td>7.42153056</td><td>1.16E-13</td><td>3.10E-11</td></tr><tr><td>CDRT1</td><td></td><td>374286</td><td>89.25968942</td><td>-0.88722126</td><td>0.208786431</td><td>4.24942012</td><td>2.14E-05</td><td>0.00045037</td></tr><tr><td>PIK3R3</td><td></td><td>8503</td><td>122.177714</td><td>-0.87831471</td><td>0.179999623</td><td>4.87953638</td><td>1.06E-06</td><td>3.82E-05</td></tr><tr><td>DOK3</td><td></td><td>79930</td><td>89.73894095</td><td>-0.862781595</td><td>0.215101644</td><td>4.01104138</td><td>6.05E-05</td><td>0.00103406</td></tr><tr><td>SLC43A2</td><td></td><td>124935</td><td>1445.525001</td><td>-0.850219224</td><td>0.116646136</td><td>7.28887605</td><td>3.13E-13</td><td>7.60E-11</td></tr><tr><td>KLF9</td><td></td><td>687</td><td>561.014703</td><td>-0.831657626</td><td>0.11168078</td><td>7.44673904</td><td>9.57E-14</td><td>2.67E-11</td></tr><tr><td>SPTBN5</td><td></td><td>51332</td><td>155.2348596</td><td>-0.819106354</td><td>0.173870577</td><td>4.71101188</td><td>2.46E-06</td><td>7.64E-05</td></tr><tr><td>TRIM54</td><td></td><td>57159</td><td>79.47157666</td><td>-0.799459098</td><td>0.228800467</td><td>3.49413228</td><td>0.00047561</td><td>0.00520885</td></tr><tr><td>DOCK11</td><td></td><td>139818</td><td>2305.752769</td><td>-0.798677069</td><td>0.090460799</td><td>8.82898533</td><td>1.06E-18</td><td>8.84E-16</td></tr><tr><td>MAN1A1</td><td></td><td>4121</td><td>575.9081358</td><td>-0.795760118</td><td>0.115407457</td><td>6.8952227</td><td>5.38E-12</td><td>9.32E-10</td></tr><tr><td>KCNH1-IT1</td><td></td><td>100874296</td><td>81.42937364</td><td>-0.789676957</td><td>0.226177253</td><td>3.4914075</td><td>0.00048048</td><td>0.00525463</td></tr><tr><td>MEGF9</td><td></td><td>1955</td><td>9276.57</td><td>-0.751134724</td><td>0.094104477</td><td>7.98192333</td><td>1.44E-15</td><td>5.83E-13</td></tr><tr><td>TP53INP1</td><td></td><td>94241</td><td>1089.326995</td><td>-0.73598954</td><td>0.107057073</td><td>6.87473996</td><td>6.21E-12</td><td>1.03E-09</td></tr><tr><td>BMF</td><td></td><td>90427</td><td>585.1229806</td><td>-0.732894095</td><td>0.133468401</td><td>5.49114315</td><td>3.99E-08</td><td>2.41E-06</td></tr><tr><td>PRUNE2</td><td></td><td>158471</td><td>221.8443018</td><td>-0.725537499</td><td>0.183014094</td><td>3.96438047</td><td>7.36E-05</td><td>0.00121463</td></tr><tr><td>SLC17A8</td><td></td><td>246213</td><td>132.0300483</td><td>-0.721163324</td><td>0.171828611</td><td>4.1969921</td><td>2.70E-05</td><td>0.00054787</td></tr><tr><td>TAF9B</td><td></td><td>51616</td><td>298.0540008</td><td>-0.71872771</td><td>0.137573548</td><td>5.22431615</td><td>1.75E-07</td><td>8.44E-06</td></tr><tr><td>PER3</td><td></td><td>8863</td><td>188.2085307</td><td>-0.71398397</td><td>0.156438552</td><td>4.5639899</td><td>5.02E-06</td><td>0.00013678</td></tr><tr><td>ABCC4</td><td></td><td>10257</td><td>3193.606927</td><td>-0.700513497</td><td>0.080049118</td><td>8.75104583</td><td>2.11E-18</td><td>1.68E-15</td></tr><tr><td>TNFRSF21</td><td></td><td>27242</td><td>2824.396757</td><td>-0.699777502</td><td>0.082491467</td><td>8.4830289</td><td>2.19E-17</td><td>1.38E-14</td></tr><tr><td>ATP11C</td><td></td><td>286410</td><td>3190.79314</td><td>-0.695962027</td><td>0.08716572</td><td>7.98435474</td><td>1.41E-15</td><td>5.83E-13</td></tr></tbody></table>
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