# Identification and experimental validation of druggable epigenetic targets in hepatoblastoma

Alex Clavería-Cabello, Jose Maria Herranz, Maria Ujue Latasa, Maria

Arechederra, Iker Uriarte, Antonio Pineda-Lucena, Felipe Prosper, Pedro

Berraondo, Cristina Alonso, Bruno Sangro, et al.

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► To cite this version:

Alex Clavería-Cabello, Jose Maria Herranz, Maria Ujue Latasa, Maria Arechederra, Iker Uriarte, et al. Identification and experimental validation of druggable epigenetic targets in hepatoblastoma. Journal of Hepatology, 2023, 79 (4), pp.989-1005. 10.1016/j.jhep.2023.05.031. hal-04148080

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# Identification and experimental validation of druggable epigenetic targets in hepatoblastoma

Alex Clavería-Cabello¹,†, Jose Maria Herranz¹,²,†, Maria Ujue Latasa¹,², Maria Arechederra¹,²,³, Iker Uriarte¹,², Antonio Pineda-Lucena⁴, Felipe Prosper³,⁵, Pedro Berraondo⁶,⁷, Cristina Alonso⁸, Bruno Sangro²,³,⁹, Jose Juan García Marin²,¹⁰, Maria Luz Martinez-Chantar²,¹¹, Sergio Ciordia¹², Fernando José Corrales²,¹², Paola Francalanci¹³, Rita Alaggio¹⁴, Jessica Zucman-Rossi¹⁵, Emilie Indersie¹⁶, Stefano Cairo¹⁶,¹⁷, Montserrat Domingo-Sábat¹⁸, Laura Zanatto¹⁹, Pau Sancho-Bru²,¹⁹, Carolina Armengol²,¹⁸, Carmen Berasain¹,²,³, Maite García Fernandez-Barrena¹,²,³,#,*, Matias Antonio Avila¹,²,³,#,*

Journal of Hepatology **2023**. vol. 79 | 989–1005

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**Background & Aims:** Hepatoblastoma (HB) is the most frequent childhood liver cancer. Patients with aggressive tumors have limited therapeutic options; therefore, a better understanding of HB pathogenesis is needed to improve treatment. HBs have a very low mutational burden; however, epigenetic alterations are increasingly recognized. We aimed to identify epigenetic regulators consistently dysregulated in HB and to evaluate the therapeutic efficacy of their targeting in clinically relevant models.

**Methods:** We performed a comprehensive transcriptomic analysis of 180 epigenetic genes. Data from fetal, pediatric, adult, peritumoral (n = 72) and tumoral (n = 91) tissues were integrated. Selected epigenetic drugs were tested in HB cells. The most relevant epigenetic target identified was validated in primary HB cells, HB organoids, a patient-derived xenograft model, and a genetic mouse model. Transcriptomic, proteomic and metabolomic mechanistic analyses were performed.

**Results:** Altered expression of genes regulating DNA methylation and histone modifications was consistently observed in association with molecular and clinical features of poor prognosis. The histone methyltransferase G9a was markedly upregulated in tumors with epigenetic and transcriptomic traits of increased malignancy. Pharmacological targeting of G9a significantly inhibited growth of HB cells, organoids and patient-derived xenografts. Development of HB induced by oncogenic forms of β-catenin and YAP1 was ablated in mice with hepatocyte-specific deletion of G9a. We observed that HBs undergo significant transcriptional rewiring in genes involved in amino acid metabolism and ribosomal biogenesis. G9a inhibition counteracted these pro-tumorigenic adaptations. Mechanistically, G9a targeting potently repressed the expression of c-MYC and ATF4, master regulators of HB metabolic reprogramming.

**Conclusions:** HBs display a profound dysregulation of the epigenetic machinery. Pharmacological targeting of key epigenetic effectors exposes metabolic vulnerabilities that can be leveraged to improve the treatment of these patients.

© 2023 The Author(s). Published by Elsevier B.V. on behalf of European Association for the Study of the Liver. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

## Introduction

Hepatoblastoma (HB) is the most common pediatric liver malignancy and its incidence has tripled over the past 30 years.¹ Significant advances in the clinical management of patients with HB have recently been achieved thanks to the efforts of different international consortia.² An efficient combination of surgery and chemotherapy at early disease stages may result in a 5-year survival rate of over 80% for these patients.¹ However, those diagnosed with advanced unresectable tumors, lesions that remain inoperable after chemotherapy, or recurrent disease face a much worse prognosis.¹,² On the other hand, currently implemented chemotherapy may result in lifelong severe toxic effects. Therefore, a better understanding of the

pathobiology of HB is still needed to provide more effective treatments, particularly for the most aggressive forms.³

From a genetic perspective, HBs display the lowest rate of somatic mutations across childhood cancers.⁴ Indeed, HBs only harbor an average of 2.9 mutations per tumor, and most of these alterations consist in activating mutations or deletions of the *CTNNB1* gene encoding β-catenin, which are present in over 70% of cases in association with higher malignancy.⁵ The second most frequently mutated gene, ~10% of cases, is *NFE2L2*.⁶,⁷ Transcriptomic studies identified distinct HB subclasses corresponding to tumors representing different stages of liver development or differentiation and aggressiveness.⁸⁻¹¹ The paucity of genetic alterations in HB, together with the

Keywords: Hepatoblastoma; Epigenetics; Metabolic reprogramming; G9a; c-MYC.

Received 22 November 2022; received in revised form 25 April 2023; accepted 22 May 2023; available online 10 June 2023

* Corresponding authors. Address: CIMA Building, Avda. Pio XII, n55, 31008, Pamplona, Spain. Tel.: +34 948 194700. fax: +34 948 194717.

E-mail addresses: magarfer@unav.es (M.G. Fernandez-Barrena), maavila@unav.es (M.A. Avila).

† Both authors made an equal contribution.

‡ Both authors are senior authors.

https://doi.org/10.1016/j.jhep.2023.05.031

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Epigenetic targets in hepatoblastoma

<figure><img src="image_5.png" /><figcaption>**Fig. 1. Heatmap showing the expression of epigenetic modifiers in healthy liver tissues, peritumoral liver tissues, HB tissues and HB cells.** Expression levels (RNA-sequencing data) are relative to those in healthy pediatric livers. Epigenetic modifiers are functionally grouped as DNMTs, TETs, MBPs, PRMTs, KMTs, HDMs, HMRs, HATs, HDACs and HARs. Transcriptomic data are integrated from the indicated studies. DNMTs, DNA methyltransferases; HARs, histone acetyl-readers; HATs, histone acetyltransferases; HB, hepatoblastoma; HDACs, histone deacetylases; HDMs, histone-lysine demethylases; HMRs, histone methyl-readers; KMTs, histone-lysine methyltransferases; MBPs, DNA-methyl-binding proteins; PRMTs, protein-arginine methyltransferases; TETs, DNA demethylases.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Heatmap showing the expression of epigenetic modifiers in peritumoral liver tissues and HB tissues classified according to: (A) Genome-wide DNA methylation status in the epigenetic clusters in Epi-CA and Epi-CB.¹⁶ (B) Transcriptomic molecular subgroups C1, C2A and C2B defined in Ref. [10]. Epi-CA/B, epigenetic cluster A/B; HB, hepatoblastoma.</figcaption></figure>
Epigenetic targets in hepatoblastoma

<figure><img src="image_7.png" /><figcaption>Fig. 2. Continued.</figcaption></figure>
identification of robust transcriptomic subclasses suggest that mechanisms other than structural genetic variations likely play important roles in HB development. Dysregulation of epigenetic mechanisms is currently recognized to participate in carcinogenesis in many organs, including the liver.¹² Earlier studies in HB already identified alterations in genomic DNA methylation,

finding low levels of global DNA methylation and hyper-methylation at the promoters of putative tumor suppressor genes.¹³⁻¹⁵ Importantly, more recent works including a higher number of patients with HB have provided comprehensive DNA-methylome analyses, enabling the establishment of epigenetic clusters with histological and clinical correlations,¹⁶

<figure><img src="image_8.png" /><figcaption>**Fig. 3.** Expression of selected epigenetic modifiers in healthy liver tissues, peritumoral liver tissues, HB tissues and cultured HB cells. Expression levels in fetal (F), adult (A), peritumor (PT), tumor (T), metastatic tissues (MT), recurrent tumors (R), and primary HB cell lines (PCL) relative to those found in pediatric livers (P). Data are individual values and means ± SD. Non-parametric Kruskal-Wallis ANOVA test was used. \*p<0.05, \*\*p<0.01, \*\*\*p<0.001. DNMTs, DNA methyltransferases; HARs, histone acetyl-readers; HATs, histone acetyltransferases; HB, hepatoblastoma; HDACs, histone deacetylases; HDMs, histone-lysine demethylases; HMRs, histone methyl-readers; KMTs, histone-lysine methyltransferases; MBPs, DNA-methyl-binding proteins; PRMTs, protein-arginine methyltransferases; TETs, DNA demethylases.</figcaption></figure>
Epigenetic targets in hepatoblastoma

<figure><img src="image_9.png" /><figcaption>Fig. 3. Continued.</figcaption></figure>
<table><caption>Table 1. G150 of epigenetic inhibitors in HB cell lines.</caption><thead><tr><th></th><th>HB cell line inhibitor</th><th>HuH6 (µM)</th><th>HepT1 (µM)</th><th>HepG2 (µM)</th></tr></thead><tbody><tr><td>DNMT</td><td>DNMTi (5-Azacytidine)</td><td>8.31</td><td>11.6</td><td>15.3</td></tr><tr><td>PRMT</td><td>PRMT1i (EPZ019997)</td><td>>50</td><td>>50</td><td>>50</td></tr><tr><td rowspan="8">KMT</td><td>NSD3i (BI-9321)</td><td>>100</td><td>>100</td><td>75</td></tr><tr><td>G9ai (BIX01246)</td><td>2.67</td><td>5.71</td><td>1.7</td></tr><tr><td>G9ai (UNC0642)</td><td>4.1</td><td>4.5</td><td>4.4</td></tr><tr><td>G9ai + DNMT1i (CM272)</td><td>0.130</td><td>0.290</td><td>0.495</td></tr><tr><td>SMYD3i (EPZ031686)</td><td>>100</td><td>>100</td><td>>100</td></tr><tr><td>EZH2i (UNC1999)</td><td>5.8</td><td>9.2</td><td>7.7</td></tr><tr><td>SUV39H2i (OTS186935)</td><td>3.8</td><td>3.6</td><td>2.9</td></tr><tr><td>HDM</td><td>KDM1Ai (Ladademstat)</td><td>20.3</td><td>16.2</td><td>16.5</td></tr><tr><td>HDAC</td><td>HDAC1i (Entinostat)</td><td>9.1</td><td>6.5</td><td>5.0</td></tr><tr><td>HAR</td><td>BRD4i (JQ1)</td><td>17.8</td><td>20.68</td><td>17.8</td></tr></tbody></table>

DNMT, DNA methyltransferase; HB, hepatoblastoma; HAR, histone acetyl-reader; HDAC, histone deacetylase; HDM, histone-lysine demethylases; KMT, histone-lysine methyl-transferase; PRMT, protein-arginine methyltransferases.

that have been further validated.¹¹,¹⁷,¹⁸ Notably, these epi-genomic traits were markedly associated with the previously defined transcriptomic subclasses, supporting the significance of epigenetic dysregulation in HB pathogenesis.

The ultimate causes leading to DNA methylation abnormalities in HB are not completely understood. Recent studies described the upregulation of DNA methyltransferases 1 and 3A (DNMT1, DNMT3A) and the DNMT adaptor protein UHRF1, along with the DNA demethylases TET1 and TET2 in HB.¹⁶,¹⁹,²⁰ Besides DNA methylation, epigenetic mechanisms regulating gene expression encompass other molecular processes including chromatin remodelers, non-coding RNAs and the covalent modification of histones.¹²,²⁰,²¹ Histone modifications comprise a growing list of reversible post-translational modifications (PTMs), such as acetylation, methylation, phosphorylation and sumoylation, among others.²² Histone PTMs often work in concert with other epigenetic mechanisms like DNA methylation to control the recruitment of remodeling complexes and transcription factors.²² As occurs for DNA methylation, histone PTMs are introduced, removed and recognized by a complement of epigenetic modifiers known as epigenetic writers, erasers and readers.¹² Unraveling the intricate processes of epigenetic regulation is important not only to understand carcinogenic mechanisms, but also to elucidate novel therapeutic strategies. Indeed, a variety of “epidrugs” targeting epigenetic effectors are actively being developed with promising preclinical antitumoral results, including sensitization to chemotherapy.²¹ Regarding HB, some studies highlighted the inhibitory potential of epigenetic drugs targeting histone deacetylases or the acetylated histone reader BRD4.²⁰,²³ In this work we performed an integrative transcriptional analysis of 180 epigenetic modifiers in a broad set of tumor tissues and evaluated the antitumoral effects of the inhibition of selected targets in relevant HB models. Our findings underscore the profound dysregulation of epigenetic mechanisms in HB and their involvement in key aspects of tumor biology. We have also identified new vulnerabilities in the metabolic reprogramming of HB cells that can be tackled with epigenetic inhibitors.

# Materials and methods

## Transcriptomic data

Transcriptomic data were obtained from the following sources: GSE133039:¹⁶ 34 tumoral and 32 peritumoral tissues; GSE104766:¹⁰ 19 tumoral and 23 peritumoral tissues;

GSE81928:²⁴ 21 tumoral and 6 peritumoral tissues; GSE75271:⁹ 50 tumoral and 5 peritumoral tissues; GSE111845:²⁵ 10 fetal, 10 pediatric and 10 adult healthy liver tissues; GSE89775:²⁶ 10 tumoral and 3 healthy pediatric liver tissues. GSE151347:²⁷ 7 tumoral, 11 peritumoral and 4 metastasized primary tumors. ENCODE: 2 fetal, 1 pediatric and 1 adult healthy liver tissues.

## Human tissue samples

Written informed consent was obtained from each patient. The study was approved by the Human Ethics Committee of the Hospital Universitari Germans Trias i Pujol, Badalona, Spain (protocol PI15-057), according to the 1975 Declaration of Helsinki guidelines.

Additional information is provided in the supplementary materials.

# Results

## Landscape of epigenetic gene expression in healthy liver, peritumoral liver and HB tissues

We performed a combined analysis of three previous RNA-sequencing studies comprising a total of 72 peritumoral and 91 tumoral HB tissues, four samples of metastasized tumors, plus six samples of recurrent tumors.¹⁰,¹⁶,²⁴ For comparisons, transcriptomes from normal human fetal, pediatric and adult liver tissues, as well as 11 primary HB cell lines and three established HB cell lines (HepG2, HuH6 and HepT1), were integrated. Gene expression in healthy pediatric liver samples was used as reference. We analyzed 180 genes from three different categories: i) epigenetic writers: DNMTs, histone-

<table><caption>Table 2. G150 of CM272 in HB primary cell lines.</caption><thead><tr><th>PDX cell line</th><th>CM272 (nM)</th></tr></thead><tbody><tr><td>HB-279</td><td>1,700</td></tr><tr><td>HB-282</td><td>420</td></tr><tr><td>HB-284-M</td><td>760</td></tr><tr><td>HB-243</td><td>796</td></tr><tr><td>HB-295</td><td>400</td></tr><tr><td>HB-303</td><td>530</td></tr><tr><td>HB-214</td><td>540</td></tr><tr><td>HB-229</td><td>1,600</td></tr><tr><td>HB-233</td><td>280</td></tr><tr><td>HB-305</td><td>445</td></tr><tr><td>HB-310</td><td>375</td></tr></tbody></table>

HB, hepatoblastoma; PDX, patient-derived xenograft.
Epigenetic targets in hepatoblastoma

<figure><img src="image_10.png" /><figcaption>**Fig. 4.** Analysis of G9a, DNMT1, and UHRF1 expression in HB. (A) Spearman correlation analyses of G9a, DNMT1, and UHRF1 mRNA levels in HB tissues. The regression coefficient (R) and *p* value of each correlation are indicated. (B) G9a, DNMT1 and UHRF1 mRNA levels in peritumoral tissues (PT), HB tissues classified into the Epi-CA and Epi-CB epigenetic groups and in recurrent tumors (R). (C) G9a, DNMT1 and UHRF1 mRNA levels in peritumoral tissues (PT), HB tissues classified into the C1, C2A and C2B transcriptomic groups. \* *p* < 0.05, \*\*\* *p* < 0.001. (D) Representative immunohistochemistries of G9a, DNMT1, and UHRF1 in HB tissues. Tumoral (T) and non-tumoral (NT) areas. (E) Western blot analysis of G9a, DNMT1 and UHRF1 proteins in nine primary human HB cell lines and three established HB cell lines. Samples of pediatric non-tumoral liver tissue (NT₁ and NT₂) are included. Data are individual values and means ± SD. Non-parametric Kruskal-Wallis ANOVA test was used. Epi-CA/B, epigenetic cluster A/B; HB, hepatoblastoma. (This figure appears in color on the web.)</figcaption></figure>

lysine methyltransferases (KMTs), protein-arginine methyl-
transferases (PRMTs), histone acetyl-transferases (HATs); ii) epigenetic erasers: DNA demethylases (TETs), histone-lysine
demethylases (HDMs), histone deacetylases (HDACs); and iii) epigenetic readers: DNA-methyl-binding proteins (MBPs), his-
tone methyl-readers (HMRs) and histone acetyl-readers (HARs)
(Table S1).¹² As observed in Fig. 1, and in an additional cohort
of patients⁹ (Fig. S1), marked changes in the expression of
numerous epigenetic genes, mostly upregulation, were
observed between HB samples and peritumoral tissues. Inter-
estingly, many genes upregulated in HB were also highly
expressed in fetal liver compared to pediatric liver. Overall,
these differences were preserved in primary HB cell lines. A
recent study identified two distinct epigenetic clusters in HB
(Epi-CA and Epi-CB) according to genome-wide DNA methyl-
ation status.¹⁶ As compared to Epi-CA tumors, Epi-CB tumors

have a more profound dysregulation of DNA methylation, a
high-risk transcriptomic signature and are associated with a
worse prognosis. ¹⁶ The expression of epigenetic genes in tu-
mors classified as Epi-CA and Epi-CB was more significantly
altered in the latter group (Fig. 2A, Fig. S2A). Consistently,
overall expression of epigenetic genes was more dysregulated
in the transcriptomic subgroup C2A defined by Hooks et al.,¹⁰
which identifies highly proliferative and high-risk tumors,
compared to C1 tumors with good prognosis and C2B tumors
of intermediate risk (Fig. 2B, Fig. S2B).

Expression of genes selected according to their alteration in
HB tissues is shown in Fig. 3A,B. Although transcriptional
downregulation in tumor tissues was observed in some cases,
increased expression was prevalent. In all categories we
identified genes whose expression was high in the fetal liver,
became reduced in healthy pediatric and adult organs, and was
<figure><img src="image_11.png" /><figcaption>**Fig. 5.** Antitumoral effects of CM272 and role of G9a in genetic mouse HB model. (A) Effect of CM272 treatment (48 h) on the levels of H3K9me2 in HuH6 and HepT1 cells. (B) Effect of CM272 treatment (48 h) on the global levels of DNA CpG methylation in HuH6 and HepT1 cells. (C) Colony formation assays in HuH6 and HepT1 cells treated with CM272 at their respective $GI_{50}$. (D) Effect of CM272 on HuH6 and HepT1 cells migration. (E) Effect of CM272 on the viability of HB tumoroids and healthy pediatric hepatocyte organoids. (F) Effect of CM272 on the growth of HB-303 primary HB cells subcutaneously implanted into nude mice. Representative images of tumors and tumor weight at the end of treatments are shown. (G) Effect of constitutively active YAP1 (S127A-YAP1) and β-catenin (ΔN90-β-catenin) coexpression on HB development in WT and G9a$^{ΔhepKO}$ mice. Macroscopic images of livers from WT and G9a$^{ΔhepKO}$ mice are shown. Representative H&E, and immunohistochemical stainings for G9a and DNMT1 are shown. Hepatic expression of HB relevant genes in WT and G9a$^{ΔhepKO}$ mice (uninjected), plasmid injected non-tumor tissues (G9a$^{ΔhepKO}$) and tumor tissues (WT) are shown. Data are means ± SD. Paired two-tailed Student's t tests were used (C–F). Non-parametric Kruskal-Wallis ANOVA test was used (G). \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001. HB, hepatoblastoma; KO, knockout; WT, wild-type. (This figure appears in color on the web.)</figcaption></figure>
Epigenetic targets in hepatoblastoma

A

<figure><img src="image_12.png" /></figure>

B

Response
to nutrient levels

<figure><img src="image_13.png" /></figure>

Cellular amino acid
metabolic process

<figure><img src="image_14.png" /></figure>

Amino acid
transmembrane
transport

<figure><img src="image_15.png" /></figure>

C

<figure><img src="image_16.png" /></figure>

D

<figure><img src="image_17.png" /></figure>

E

Amino acid
transmembrane
transport

<figure><img src="image_18.png" /></figure>

Ribosome biogenesis

<figure><img src="image_19.png" /></figure>

RNA metabolic process

<figure><img src="image_20.png" /></figure>

Autophagy

<figure><img src="image_21.png" /></figure>

F

<figure><img src="image_22.png" /></figure>

Essential
amino acids

<figure><img src="image_23.png" /></figure>

**Fig. 6.** Mechanisms involved in the antitumoral effects of CM272 in HB. (A) Most relevant GO functional categories of genes undergoing changes in expression identified by RNA-sequencing in HuH6 cells treated with CM272 (GI50, 48 h). Right panel shows the effect of CM272 (GI50, 48 h) on the expression of tumor suppressor genes epigenetically downregulated in HB. \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001. (B) GSEA revealed that CM272 inhibited the expression of genes involved in nutrient, amino acid, tRNA, and rRNA metabolism in HuH6 cells. (C) Volcano plot of the proteins differentially expressed in HuH6 cells treated with CM272 (GI50, 48 h). Selected functionally relevant proteins are indicated. (D) Most relevant GO categories of proteins undergoing changes in expression in HuH6 cells treated with CM272. (E) GSEA confirming inhibition of the expression of proteins involved in amino acid and RNA metabolism, and ribosome biogenesis, and enrichment of the expression of
induced in tumors, including metastatic and recurrent tissues. We confirmed the recently described upregulation of *DNMT1*, *DNMT3A*, *DNMT3B*, *TET1*, *TET2*, *TET3*, *UHRF1*, *EZH2*, *HDAC1* and *HDAC2* in HB tissues.<sup>16,19,23,28,29</sup> We also observed the marked induction of other epigenetic genes involved in tumorigenic processes, including *MBD3*, *PRMT1*, *KMT5C*, *G9a*, *NSD3*, *SMYD3*, *SUV39H2*, *JARID2*, *KDM1A*, *KAT2A*, *HDAC11*, *SIRT7*, *BRD4* and *SMARCA4*<sup>30-36</sup> among others. Interestingly, many genes overexpressed in HB samples were already upregulated in peritumoral tissues compared to healthy pediatric livers (e.g. *ZBTB4*, *MBD6*, *PRMT1*, *KMTC5C*, *KDM6B*, *SGF19*, *ING4*, *KAT8*, *KAT2B*, *SIRT7*, *BRD7*, *BRD4*).

## Pharmacological targeting of epigenetic regulators in HB cells

We next evaluated the antiproliferative potential of selected available epigenetic drugs in HB cell lines. We tested molecules targeting different classes of epigenetic modifiers significantly upregulated in tumor tissues, such as: DNMTs, KMTs (SUV39H2, SMYD3, G9a, NSD3, EZH2), KDMs (KDM1A), HATs (KAT2A), HDACs (HDAC1/3), PRMTs (Type-I PRMTs) and HARs (BRD4). Albeit some inhibitors had limited efficacy (GI<sub>50</sub> 20-100 µM), others showed GI<sub>50</sub> values in the low micromolar range (Table 1). Most effective compounds were among KMTs inhibitors, and those targeting the KMT G9a stood out. The best response was obtained with CM272, a potent substrate-competitive inhibitor of G9a that also targets DNMT1.<sup>37</sup> In view of this, we tested CM272 in 11 well-characterized patient-derived HB cell lines,<sup>38</sup> validating a robust growth-inhibitory effect (Table 2).

## Characterization of G9a and DNMT1 as epigenetic targets in HB

Epigenetic effectors undergo extensive functional crosstalk among themselves and with other transcriptional regulators.<sup>12,39</sup> This has been well described for G9a, DNMT1, and the epigenetic scaffold UHRF1 in different tumor types.<sup>19,39-41</sup> We found a significant positive correlation between the expression of G9a, DNMT1 and UHRF1 mRNA levels in HB tissues (Fig. 4A). Moreover, the expression of these three genes was higher in tumors within the epigenetic cluster Epi-CB and the transcriptomic group C2A, which include patients with poorer prognosis<sup>10,16</sup>(Fig. 4B,C). Increased levels of G9a, DNMT1 and UHRF1 proteins, and the G9a-mediated H3K9me2 histone mark,<sup>41</sup> were also validated by immunohistochemistry in representative HB tissues vs. peritumoral parenchyma (Fig. 4D, Fig. S2C), and in HB cell lines (Fig. 4E).

These findings, together with the observed antiproliferative efficacy of G9a inhibitors, particularly CM272, on HB cells supported an oncogenic role for G9a and DNMT1. Thus, we further evaluated the effects of CM272 on HB cells and relevant *in vitro* and *in vivo* models. Consistent with its pharmacological targets, CM272 treatment decreased total levels of H3K9me2 and DNA methylation (Fig. 5A,B, Fig. S3A). CM272 markedly

inhibited HB cells' clonogenic and migratory capacities (Fig. 5C,D), without inducing apoptosis (not shown). Cisplatin-based therapy is regularly implemented in patients with HB; however, resistance frequently occurs or develops.<sup>1-3</sup> Epigenetic drugs may increase the therapeutic response to chemotherapy.<sup>21,22</sup> Thus, we tested the effect of CM272 on cisplatin efficacy in HuH6 and HepT1 cells. We found that cisplatin GI<sub>50</sub> was reduced by 59% (from 2.7 to 1.1 µg/ml) and 62% (from 3.2 to 1.2 µg/ml) in HuH6 and HepT1 cells, respectively. Moreover, CM272 had a synergistic growth-inhibitory effect when combined with cisplatin or the PARP inhibitor olaparib (Fig. S3B,C). We also evaluated the antitumoral efficacy of this molecule in human HB organoids. CM272 inhibited organoids' growth at significantly lower concentrations than cisplatin (Fig. 5E). Importantly, as opposed to cisplatin, it had no effect on healthy organoids (Fig. 5E).

The antitumoral properties of CM272 were also validated in a xenograft mouse model with a primary HB cell line from a well-characterized tumor<sup>38</sup> (Fig. 5F). Tumor growth inhibition was accompanied by a tendency to reduced mitotic figures (14–54 vs. 13–45 mitoses/20x field) and increased areas of necrosis in treated mice, while no differences were found in apoptotic cells (not shown). No signs of toxicity such as weight loss or serum parameters of liver and kidney injury were observed in treated mice (not shown). The significant antiproliferative efficacy of G9a inhibitors in HB cells suggests that this KMT plays an important role in HB. Therefore we evaluated tumor development in mice expressing constitutively active forms of β-catenin (ΔN90-β-catenin) and YAP1 (YapS127A) upon tail vein injection of hydrodynamic plasmids, a recognized model for HB,<sup>42</sup> using wild-type (WT) mice, and mice with hepatocyte-specific deletion of G9a (G9a<sup>ΔhepKO</sup>) (Fig. 5G). We observed that while WT mice had tumor-laden livers, G9a<sup>ΔhepKO</sup> animals showed very few and smaller nodules (Fig. 5G, Fig. S4A, B). Lesions in WT mice had more prominent cellular crowding and more frequent mitoses (Fig. S4C). G9a and DNMT1 proteins were readily detected in tumor nodules (Fig. 5G). Expression of genes involved in HB development and known to be induced in this mouse model, such as *Ctgf*, *Cyr61*, *Survivin1* and *c-Myc*,<sup>42</sup> was markedly induced in WT mice tumors, but not in G9a<sup>ΔhepKO</sup> liver tissues (Fig. 5G). Consistently, RNA-sequencing analyses in this genetic model indicated that G9a was a key determinant in the transcriptomic rewiring observed in HB, and other pediatric tumors, including hepatocellular dedifferentiation and the establishment of a c-MYC signature characteristic of most aggressive HBs (Fig. S5A,B).

## Mechanisms involved in the antitumoral effects of CM272 in HB

In view of the marked inhibition of tumorigenesis triggered by ΔN90-β-catenin and YapS127A expression in G9a<sup>ΔhepKO</sup> mouse livers, we first evaluated if CM272 had a direct effect on the activity of these pathways, or if it affected the expression of β-catenin and YAP inhibitors in HB cells. However, CM272 treatment did not affect β-catenin nor YAP nuclear

<footnote>autophagy-related proteins in CM272-treated HuH6 cells. (F) Metabolomic analysis of HuH6 cells treated with CM272 (GI<sub>50</sub>, 48 h). Levels of amino acids, GSH and GSSG relative to untreated controls are shown. \*p < 0.05. Data are means ± SD. Paired two-tailed Student's *t* tests were used (A). NES and significance are shown (B, E). Data are individual values with means ± SD. Paired two-tailed Student's *t* tests were used (F). GO, gene ontology; GSH, reduced glutathione; GSSG, oxidized glutathione; HB, hepatoblastoma; NES, normalized enrichment score. (This figure appears in color on the web.)</footnote>
Epigenetic targets in hepatoblastoma

<figure><img src="image_24.png" /><figcaption>**Fig. 7.** CM272 inactivates the metabolic rewiring of HB cells that supports tumor growth. (A) Diagram representing key cellular pathways and mechanisms activated in cancer counteracted by CM272. Right panels show the effects of CM272 (GI₅₀, 48 h) in HuH6 cells on the expression of relevant metabolic genes commonly upregulated in tumors, and p62. (B) GSEA revealed that CM272 inhibits c-MYC transcriptional activity in HuH6 cells. (C) Effects of CM272 (GI₅₀, 48 h) on c-MYC mRNA and protein levels in HuH6 cells. (D) Effect of CM272 on c-MYC protein stability in HuH6 cells. (E) GSEA of the effect of CM272 (GI₅₀, 48 h) on ATF4-driven gene expression, and ATF4 mRNA and protein levels in HuH6 cells. (F) CM272 pretreatment (GI₅₀, 24 h) blunts the upregulation of *ATF4* expression in response to</figcaption></figure>
translocation, and with the exception of SFRP1 (see below) it did not increase the expression of negative modulators of these pathways (data not shown). To explore the antitumor mechanisms of CM272, we examined the transcriptional responses of HuH6 and HepT1 cells. In agreement with the observed anti-proliferative and anticlonogenic activities, gene ontology (GO)-based functional classification of differentially expressed genes identified general categories related to cell growth, differentiation and interaction with the cellular microenvironment (Fig. 6A). According to the pharmacological activity of CM272, the expression of RASSF1A, HHIP, SFRP1, IGFBP3, MAT1A and FBP1, tumor suppressors and key metabolic genes epigenetically repressed in HB¹⁹,²⁰ were induced (Fig. 6A, Fig. S6A), and this response was accompanied by a decrease in the levels of the G9a-mediated H3K9me2 repressive mark in their proximal promoters (Fig. S6B). Perhaps more interestingly, and aligned with categories identified by GO, gene set-enrichment analysis (GSEA) revealed significant changes in the expression of genes involved in the response to nutrient levels, amino acid transport and metabolism, mRNA translation and rRNA processing (Fig. 6B). These transcriptional effects were also captured by proteomic studies in HuH6 cells. We identified 504 differentially expressed proteins, of which 264 were upregulated and 240 downregulated. Downregulated proteins included membrane transporters (SLC1A5, SLC39A7, SLC39A14), enzymes involved in amino acid metabolism (PHGDH, PYCR1, PYCR2, ASNS), and other metabolic pathways (TALDO1, HDLPB, PCK2, MTHFD2), ribosomal proteins (RPL13, RPL21, RPL14, RPL8) and additional proteins commonly upregulated in cancer (POLD1, CBX3, YARS1) (Fig. 6C). Among the upregulated proteins, we found enzymes (AHCY, HPGD) and negative regulators of tumor growth (FTH1, DDX28, YAF2) frequently repressed in cancer. Interestingly, we also detected increased levels of key autophagy proteins (TOMM7, SQSTM1, ATG3) (Fig. 6C). GO functional analysis of differentially expressed proteins overlapped to a great extent with transcriptomic data (Fig. 6D). Similarly, GSEA revealed categories related to amino acid transmembrane transport, RNA metabolism, ribosome biogenesis and autophagy (Fig. 6E). To evaluate if the effects of CM272 on the expression of genes involved in amino acid transport and metabolism were functionally translated we performed targeted metabolomic analyses. We observed significant reductions in the intracellular levels of several essential and non-essential amino acids, as well as in total glutathione upon CM272 treatment (Fig. 6F, Fig. S7).

## CM272 impairs the adaptive response of HB cells to high metabolic demand

To sustain rapid proliferation and survival, tumor cells reprogram their metabolism to meet exacerbated bioenergetic and biosynthetic demands. According to our multiomic analyses, interference with this biosynthetic rewiring may underlie CM272 antitumoral activity. Consistently, we found that CM272 downregulated genes frequently induced in tumors such as the

amino acid transporters SLC7A11, SLC7A5, and SLC1A5; the proline biosynthetic enzyme PYCR1;⁴³ ASNS (asparagine synthetase); the enzymes involved in serine and one carbon/folate metabolism PHGDH and MTHFD2; and BCAT1 (branched-chain aminotransferase 1)⁴³⁻⁴⁵ (Fig. 7A, Fig. S8A). SLC7A11, involved in cysteine uptake and commonly induced in tumors contributing to malignancy,⁴⁵ was among the most potently downregulated genes. To test the functional significance of SLC7A11 inhibition on the antitumoral effects of CM272 we measured its efficacy in the presence of N-ace-tylcysteine, a cell membrane-permeable cysteine precursor. We observed that by replenishing intracellular cysteine levels the GI₅₀ of CM272 increased by threefold (Fig. S8B). Notably, we also observed that CM272 reduced the expression of RPS6 (ribosomal protein S6), upregulated in cancer with pro-tumorigenic consequences.⁴⁶ Importantly, the expression of most of these genes was significantly induced in HB tissues and in correlation with that of G9a (Fig S9A,B). Interestingly, we also validated the accumulation of the autophagy adaptor protein p62 (sequestosome/SQSTM1) (Fig. 7A), indicative of impaired autophagic flux.⁴⁷

Cancer metabolic rewiring is frequently orchestrated by oncogenes, among which c-MYC, with a central role in HB,⁵,⁸ stands out.⁴⁴,⁴⁵,⁴⁸ Cairo *et al.* distinguished two transcriptomic subclasses of HB, C1 and C2, with C2 tumors being less differentiated and more aggressive.⁸ C2 tumors showed a strong enrichment in the expression of bona fide MYC target genes. We performed GSEA using this gene set, and the “Hallmark MYC-targets V1” gene set, on the transcriptomic data from HuH6 cells treated with CM272. CM272 induced a significant negative enrichment in the expression of c-MYC target genes (Fig. 7B). Consistently, c-MYC expression was downregulated by CM272 in HuH6 and HepT1 cells (Fig. 7C, Fig. S10A). Interestingly, CM272 not only downregulated c-MYC mRNA levels, but it also reduced c-MYC protein half-life (Fig. 7D, Fig. S10B). The effects of CM272 on c-MYC expression were reproduced with the G9a inhibitor UNC0642 (Fig. S10C). The involvement of c-MYC inhibition in the antitumoral effects of CM272 was tested using HB cells over-expressing the oncogene. We observed that the growth-inhibitory activity of CM272, as well as its effects on the expression of key metabolic genes, were attenuated upon c-MYC overexpression (Fig. S10D,E).

Activating transcription factor 4 (ATF4) is also a critical mediator of metabolic changes supporting cancer cell survival, often in cooperation with c-MYC.⁴⁵,⁴⁹,⁵⁰ We found that ATF4 expression is upregulated in HB in correlation with that of G9a (Fig. S11A, B). Moreover, the expression of the lysine demethylase KDM4C, recently reported to contribute to ATF4 transcriptional induction in cooperation with G9a,⁵¹ was also upregulated in HB tissues (Fig. S11C). Notably, GSEA of a well-characterized ATF4 target gene set⁵² indicated that CM272 reduces ATF4 transcriptional activity (Fig. 7E). Consistently, CM272 lowered ATF4 mRNA and protein levels in HB cells (Fig. 7E, Fig. S10F). ATF4 is essential in the adaptive cellular

<footnote>amino acid deprivation in HepT1 cells. (G) Effect of CM272 pretreatment (GI₅₀, 24 h) on the response of the indicated genes to amino acid deprivation in HepT1 cells. *p* <0.05, **p** <0.01, ***p** <0.001. Data are means ± SD. Paired two-tailed Student's *t* tests were used (B-E). NES and significance are shown (B, E). Non-parametric Kruskal-Wallis ANOVA test was used (F, G). GSEA, gene set-enrichment analysis; HB, hepatoblastoma; NES, normalized enrichment score. (This figure appears in color on the web.)</footnote>
response to reduced amino acids levels, triggering the expression of genes involved in amino acid synthesis and uptake.⁴⁵ We observed that CM272, and UNC0642, markedly blunted ATF4 upregulation induced by amino acid restriction in HB cells (Fig. 7F, Fig. S10G). Consistently, the expression of ATF4 target genes, including membrane amino acid transporters and metabolic enzymes was markedly abated (Fig. 7G). In agreement with these observations, we also found that the expression of *Atf4*, and that of the ATF4 and c-MYC target genes involved in amino acid transport, metabolism and ribosome biogenesis discussed above, was significantly reduced in G9a<sup>AhepKO</sup> vs. WT mice in our HB model (Fig. S12A). Moreover, overexpression of G9a in HB cells enhanced the expression of c-MYC and ATF4, and regulated the levels of downstream metabolic genes like *SLC1A5* and *FBP1* (Fig. S12B).

Taken together, all these findings suggested that CM272 can reprogram the transcriptome of HB towards a less malignant and more differentiated phenotype. To further confirm this tenet, we evaluated the effects of CM272 on the expression of a gene set that defines the transcriptome of adult healthy hepatocytes.⁵³ To this end we selected HepT1 cells, which according to the expression of this gene set are significantly less differentiated than HuH6 cells (Fig. S12C). Interestingly, CM272 treatment induced a significant positive enrichment in the expression of this complement of genes in HepT1 cells (Fig. S12C). These transcriptomic effects had a functional translation, as CM272 treatment promoted a shift in the relative production of ATP from glycolysis to mitochondrial respiration, thus counteracting the aerobic glycolysis characteristic of tumoral cells (Fig. S12D).

Discussion

Accumulating evidence indicates that epigenetic alterations can play a role in HB development and response to therapy. Dysregulation of DNA methylation has been recognized for almost 20 years, and is currently considered a hallmark of HB, enabling the identification of epigenetic subtypes with differential biological characteristics and prognosis.¹⁶,¹⁸,²⁹ Yet, epigenetic mechanisms are very complex and intertwined, extending well beyond the control of DNA methylation. Mutational burden is low in HB, even in its most aggressive forms,⁶,⁵⁴ and to our knowledge mutations in epigenetic modifiers have not been reported. Conversely, the altered expression/activity of epigenetic modifiers has increasingly been recognized as contributing to malignancy in multiple cancer types.¹²,²² In this study, we performed what we believe is the most comprehensive analysis of the expression of epigenetic modifiers in HB tissues. We observed significant and consistent alterations, mostly upregulation, in the expression of genes belonging to every category of epigenetic gene. Interestingly, the overall pattern of epigenetic gene expression in HB was reminiscent of that in the fetal liver, and is aligned with the global DNA hypomethylation reported in fetal as well as HB tissues.¹⁶ This finding, which is consistent with the global transcriptomic reprogramming of HB towards prenatal phases,⁵ further underscores the involvement of epigenetic mechanisms in HB development. In fact, we observed that the upregulation of epigenetic genes was more pronounced in the Epi-CB and C2A molecular subgroups, corresponding to aggressive tumors resembling early developmental stages.¹⁰,¹⁶ Notably, the expression of a significant number of epigenetic

manifiers in peritumoral tissues was already elevated compared to healthy pediatric livers. This might indicate that epigenetic alterations predisposing liver tissue to neoplastic conversion may already occur before tumor development. This "field cancerization" effect is well recognized in hepatocellular carcinoma development,⁵⁵ as these tumors normally arise on a background of chronic injury and inflammation, while in HB, underlying liver disease is generally absent at diagnosis. However, recent observations indicate that field cancerization may also be involved in HB,⁵⁶ and our findings suggest that dysregulated expression of epigenetic modifiers may be part of it.

The reversible nature of epigenetic PTMs makes targeting these mechanisms an attractive strategy for cancer therapy.²¹ Our transcriptomic screening for relevant epigenetic genes in HB identified the consistent upregulation of the KMT G9a, particularly in tumors with more aggressive clinical and molecular profiles. Pharmacological interference with G9a methyltransferase activity markedly inhibited the *in vitro* and *in vivo* growth of primary HB cells, as well as that of HB tumoroids. The involvement of G9a in HB development suggested by our molecular tool CM272 was further substantiated *in vivo*. Tumor development upon hepatic delivery of oncogenic forms of β-catenin and YAP1, a genetic model of HB,⁴²,⁴⁸ was blunted in mice lacking G9a expression in hepatocytes. As shown by ourselves and others, elevated G9a expression contributes to malignant traits in solid tumors.³²,⁴¹,⁵⁷ Notably, the pro-tumorigenic activities of G9a, including the repression of tumor suppressor genes (TSGs), are frequently mediated in association with the DNMT1/UHRF1 complex.³²,⁵⁷ We found a strong correlation between G9a, DNMT1 and UHRF1 expression in HB tissues and, most interestingly, the dual G9a/DNMT1 inhibitor CM272 reactivated the expression of key TSGs known to be repressed by the DNMT1/UHRF1 complex in HB cells in association with increased H3K9 methylation, the outcome of G9a activity.¹⁹

To elucidate the antitumor mechanisms of CM272 in HB in an unbiased manner and beyond the reactivation of TSGs, we performed a series of multiomic analyses. Cancer cells reprogram their metabolism to boost biosynthesis of cellular building blocks and fuel bioenergetic demands to sustain uncontrolled proliferation and growth.⁴⁴,⁴⁵ These critical responses encompass activation of the uptake and synthesis of amino acids, including non-essential amino acids,⁴³,⁴⁵ ribosome biogenesis, protein synthesis, and salvage autophagy, among other reactions supporting macromolecule synthesis.⁴⁴,⁴⁹,⁵² We observed that CM272 markedly reduced the expression of amino acid transporters (SLC1A5, SLC7A5, SLC7A11), metabolic enzymes (BCAT1, PYCR1, ASNS), as well as genes involved in ribosome biogenesis, RNA processing and translation, such as RPS6, which are often upregulated in cancer cells.⁴³,⁴⁴,⁴⁶ These genes, which we also found overexpressed in HB tissues, are known to be important for malignant cell growth.⁴⁴⁻⁴⁶ Moreover, the downregulation of SLC7A11 expression and the concomitant reduction in intracellular reduced glutathione levels may underlie the increased efficacy of cisplatin in CM272-treated HB cells.⁵⁸

Taken together, our findings indicate that c-MYC antagonism could be a major determinant of the antitumor effects of CM272. Indeed, G9a inhibition markedly antagonized the c-MYC-driven transcriptome in HB cells. This oncogene, a well-
known target of the Wnt/β-catenin pathway, is frequently overexpressed in HB.⁵,⁸,⁵⁶ Furthermore, c-MYC is essential for the development of experimental HB triggered by oncogenic β-catenin and YAP1 expression in mouse livers.⁴²,⁴⁸ Notably, when this same HB model was implemented in G9aΔ*hep*KO mice, c-MYC expression was blunted and tumor development was drastically reduced, phenocopying the response of c-MYC-KO animals.⁴⁸ These observations may have broader significance, as they support an important role for G9a in c-MYC-driven tumorigenesis in vivo.⁵⁹ c-MYC is a master regulator of the transcriptional metabolic reprogramming occurring in cancer, and this reprogramming is central to c-MYC-induced tumor growth.⁴⁴,⁵⁰ Direct targets of c-MYC include the aforementioned amino acid transporters, enzymes, and ribosomal proteins downregulated by CM272 in HB cells. We found that G9a inhibition reduced c-MYC mRNA levels, which is consistent with recent findings in multiple myeloma cells.⁶⁰ Additionally, we also observed that G9a targeting significantly decreased c-MYC protein half-life. Although the underlying mechanisms merit further investigation, our current observations, together with the notion that c-MYC can in turn trigger G9a expression in breast cancer cells,⁵⁹ emphasize the strong interaction between these two genes in cancer.

Interestingly, the transcription factor ATF4 cooperates with c-MYC in regulating multiple metabolic genes, with ATF4 being critical for c-MYC-dependent tumorigenesis in certain cell types.⁴⁵,⁵⁰ ATF4 expression is mainly regulated at the translational level; however, increased ATF4 transcription has been found in certain tumors.⁴⁹,⁵² We showed that ATF4 mRNA levels are elevated in HB tissues in correlation with those of G9a. Activation of ATF4 is essential for the adaptation of cancer cells to nutrient limitation, prominently to the lack of amino acids, as

occurs inside a growing tumor mass.⁴⁹ Concomitant with the reduction in amino acid levels detected in CM272-treated HB cells, we observed a significant downregulation in *ATF4* gene expression. Furthermore, the potent induction of *ATF4* expression elicited by amino acid restriction was abated upon G9a inhibition. It has been shown that *ATF4* transcription is activated by the histone demethylase KDM4C, which reduces the repressive H3K9me2 and H3K9me3 marks on the *ATF4* promoter.⁵¹ As we observed increased KDM4C expression in HB tissues, it is tempting to speculate that the concomitant upregulation of KDM4C and G9a could result in increased levels of the activating H3K9me1 mark on the *ATF4* promoter. Nevertheless, the interplay between these two epigenetic effectors in *ATF4* regulation in HB requires further elucidation.

Altogether, we have shown a remarkable alteration in the landscape of epigenetic effector expression in HB tissues. Among them, we identified the KMT G9a as a strong candidate involved in HB growth. Our mechanistic studies also led us to expose the extensive metabolic reprogramming taking place in HB. As observed in other solid tumors, c-MYC, in cooperation with ATF4, may play a central role in this metabolic rewiring. However, pharmacological inhibition of c-MYC has not been achieved yet, and thus alternative pathways to antagonize c-MYC function need to be defined. We demonstrated that G9a inhibition impaired c-MYC expression, inducing a state of metabolic anergy and leading to growth suppression in HB cells. These effects were accompanied by the induction of genes characteristic of the adult differentiated hepatocyte and the consistent reprogramming of ATP metabolism. This study suggests that epigenetic drugs may hold promise for the treatment of patients with HB, particularly those with more aggressive forms of the disease.

## Affiliations

## Abbreviations

ATF4, activating transcription factor 4; DNMTs, DNA methyltransferases; Epi-CA/B, epigenetic cluster A/B; GO, gene ontology; GSEA, gene set-enrichment analyses; HARs, histone acetyl-readers; HATs, histone acetyltransferases; HB, hepatoblastoma; HDACs, histone deacetylases; HDMs, histone-lysine demethylases; HMRs, histone methyl-readers; KMTs, histone-lysine methyltransferases; KO, knockout; MBPs, DNA-methyl-binding proteins; PDX, patient-derived xenograft; PRMTs, protein-arginine methyltransferases; PTMs, post-translational modifications; TETs, DNA demethylases; TSG, tumor suppressor gene; WT, wild-type.

## Financial support

This work was supported by grants from the Scientific Foundation of the Spanish Association Against Cancer (AECC) LABAE20011GARC (MGFB), PROYE19044-SANC (PSB), and Innova-INNOV211822SANJ (FP). Grants from Ministerio de Ciencia, Innovación y Universidades MICINN-Agencia Estatal de Investigación integrado en el Plan Estatal de Investigación Científica y Técnica y Innovación, cofinanciado con Fondos FEDER PID2019-104878RB-100/AEI/10.13039/501100011033 (MAA), PID2020-117116RB-I00 (MGFB), PID2021-127496NB-

100 (FJC), PID2020-115875RB-100/AEI/10.13039/501100011033 (APL), and PID2020-117116RB-I00 (MLMC). Intramural CSIC PIE/COVID-19 projects 202020E079 and 202020E108; Comunidad de Madrid Grant B2017/BMD-3817; Severo Ochoa Project SEV 2017-0712; European Commission-NextGenerationEU (Regulation EU 2020/2094), through CSIC's Global Health Platform (PTI Salud Global) (FJC). Instituto de Salud Carlos III (ISCIII) CIBERONC CB16/12/00489 (FP). Navarra Government Department of Health (44/2021) (also co-financed by FEDER) and Department of Economic Development AGATA (0011-1411-2020-000011 and 0011-1411-2020-000010) (FP). Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III, Spain (PI19/00819 and PI22/00526 co-funded by European Regional Development Fund/European Social Fund, "Investing in your future"); Junta de Castilla y León (SA074P20) and Fundació Marato TV3 (Ref. 201916/31) (JJGM). European Union's Horizon 2020 research and innovation program under grant agreement N° 668596 (ChiLTERN) and N° 826121 (IPC) and AGAUR (2017-SGR-490) (CA). Ligue contre le cancer (équipe labellisée), SFCE (Société Française de Lutte Contre les Cancers et les Leucémies de l'Enfant), the SIRIC CARPEM, PeLiCan.Resist InCa (Pediatric Liver CANcer database to combat RESISTance to treatment, Institut National du Cancer, France Génomique, association Etoile de Martin, Fédération Enfants et Santé, association Hubert Gouin "Enfance et Cancer," INSERM Plan Cancer

<footnote n="1">¹Hepatology Program, CIMA, CCUN, University of Navarra, Pamplona, Spain; ²CIBERehd, Instituto de Salud Carlos III, Madrid, Spain; ³Instituto de Investigaciones Sanitarias de Navarra IdiSNA, Pamplona, Spain; ⁴Molecular Therapeutics Program, CIMA, CCUN, University of Navarra, Pamplona, Spain; ⁵Oncohematology Program, CIMA, CCUN, University of Navarra, Pamplona, Spain; ⁶Immunology and Immunotherapy Program, CIMA, University of Navarra, Pamplona, Spain; ⁷CIBERonc, Madrid, Spain; ⁸OWL Metabolomics, Derio, Spain; ⁹Hepatology Unit, CCUN, Navarra University Clinic, Pamplona, Spain; ¹⁰Experimental Hepatology and Drug Targeting (HEVEFARM), University of Salamanca, IBSAL, Salamanca, Spain; ¹¹Liver Disease Laboratory, Center for Cooperative Research in Biosciences (CiCBioGUNE), Basque Research and Technology Alliance (BRTA), Derio, Spain; ¹²Functional Proteomics Laboratory, CNB-CSIC, Madrid, Spain; ¹³Pathology Unit, Children's Hospital Bambino Gesù, IRCCS, Rome, Italy; ¹⁴Pathology Unit, Children's Hospital Bambino Gesù, IRCCS, and Sapienza University, Rome, Italy; ¹⁵Centre de Recherche des Cordeliers, Sorbonne Université, Université de Paris, INSERM, and Hôpital Européen Georges Pompidou, Paris, France; ¹⁶XenTech, Evry-Courcouronnes, France; ¹⁷Champions Oncology, Rockville, MD, USA; ¹⁸Childhood Liver Oncology Group, Program of Predictive and Personalized Medicine of Cancer (PMPCC), Germans Trias i Pujol Research Institute (IGTP), Badalona, Spain; ¹⁹Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Barcelona, Spain.</footnote>
# Epigenetic targets in hepatoblastoma

(JZR). FIMA pre-doctoral fellowship to ACC, AECC post-doctoral fellowship POSTD18014AREC to MA and Ramón y Cajal Program contracts RYC-2010-07249 (CA) and RYC2018-024475-1 (MGFB) from the Spanish Ministry of Science and Innovation. This work was supported by a grant (PRYCO223102ARME) from the Scientific Foundation of the AECC (CA, MAA, MLMC, PSB, JJGM), grant EDH22PI01 from CIBERehd (CA, MAA, JJGM, PB). Grants from Fundación Eugenio Rodríguez Pascual; Fundación Échébano; Fundación Mario Losantos, Fundación M Torres (MAA, CB, MGFB).

## Conflict of interest

C. Alonso is employed by OWL Metabolomics; the other authors declare no conflict of interest.

Please refer to the accompanying ICMJE disclosure forms for further details.

## Authors' contributions

Experiments and procedures: ACC, JMH, MUL, MA, IU, APL, FP, PF, RA, PB, CA, BS, JJGM, MLMC, SC, FJC, JZR, SC, MDS, LZ, PSB, CA, CB; concept and design: ACC, JMH, MGFB, MAA; supervision: MGFB, MAA; writing of article: MAA.

## Data availability statement

RNA-sequencing data reported in this study have been deposited in the Sequence Read Archive (SRA) of NCBI, ref. PRJNA900056.

## Acknowledgements

We thank Prof. Diego Calvisi, (School of Medicine, Regensburg University, Germany) for sharing the ΔN90-β-catenin and YAP1 YapS127A plasmids. We thank Ms Laura Alvarez and Mr Roberto Barbero for their excellent technical support. We also thank the patients whose samples were used in this study. We thank Mr. Eduardo Avila for his generous support.

## Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jhep.2023.05.031.

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Supplemental information

Identification and experimental validation of druggable epigenetic targets in hepatoblastoma

Alex Clavería-Cabello, Jose Maria Herranz, Maria Ujue Latasa, Maria Arechederra, Iker Uriarte, Antonio Pineda-Lucena, Felipe Prosper, Pedro Berraondo, Cristina Alonso, Bruno Sangro, Jose Juan García Marin, Maria Luz Martinez-Chantar, Sergio Ciordia, Fernando José Corrales, Paola Francalanci, Rita Alaggio, Jessica Zucman-Rossi, Emilie Indersie, Stefano Cairo, Montserrat Domingo-Sàbat, Laura Zanatto, Pau Sancho-Bru, Carolina Armengol, Carmen Berasain, Maite García Fernandez-Barrena, and Matias Antonio Avila
# Identification and experimental validation of druggable epigenetic targets in hepatoblastoma

Alex Clavería-Cabello, Jose Maria Herranz, Maria Ujue Latasa, Maria Arechederra, Iker Uriarte, Antonio Pineda-Lucena, Felipe Prosper, Pedro Berraondo, Cristina Alonso, Bruno Sangro, Jose Juan García Marin, Maria Luz Martinez-Chantar, Sergio Ciordia, Fernando José Corrales, Paola Francalanci, Rita Alaggio, Jessica Zucman-Rossi, Emilie Indersie, Stefano Cairo, Montserrat Domingo-Sàbat, Laura Zanatto, Pau Sancho-Bru, Carolina Armengol, Carmen Berasain, Maite García Fernandez-Barrena, Matias Antonio Avila

## Table of contents

<table><tr><td>Supplementary materials and methods</td><td>2</td></tr><tr><td>Supplementary figures</td><td>13</td></tr><tr><td>Supplementary figure legends</td><td>25</td></tr><tr><td>Table S1</td><td>28</td></tr><tr><td>Supplementary references</td><td>34</td></tr></table>
# Supplementary materials and methods

## Transcriptomic data analyses

**Data retrieval from SRA:** A custom bash script was used to ensure the correct and complete download of all RNAseq datasets from SRA (SRP075759, SRP119676, SRP135703, SRP093259, SRP265069, and SRP201920) with SRAtoolkit (2.9.6-1-centos_linux64) from NCBI. ENCODE data was downloaded from the project's portal.

**Data processing:** Trimgalore version 0.6.0 with Cutadapt version 1.18¹ was used for read trimming, and quality filtering, all reads below 20nt were filtered. The mapping step was carried out using STAR version 020201² over genome version hg38 (https://www.ncbi.nlm.nih.gov/assembly/GCF_000001405.40). Read counting was performed using HTseq version 0.11.0³ and normalized using EdgeR version 3.28.1⁴ in R version 3.6.3 (https://www.R-project.org/). Trimmed mean of M-values (TMM) was selected as method for normalization in addition to low expression gene filtering for differential gene expression analysis. Gene Ontology (GO) analysis⁵ and Gene set enrichment analysis (GSEA)⁶ was done using clusterProfiler version 3.14.3⁷ with differentially expressed genes without considering the effect size in the case of GO analysis and with an absolute log fold change (LFG) greater than 1 in the case of GSEA. QIAGEN Ingenuity pathway analysis (IPA)⁸ was carried out with the same parameters. For correlation analysis Pearson correlation was used in R. Boxplots were generated using GraphPad Prism version 9.0.2.

**Data integration:** Batch effect was demonstrated to be absent in epigenetic genes expression using correlation analysis and three different unsupervised techniques (PCA, UMAP, and t-SNE) using R package M3C version 1.18.0⁹. During the process, visual inspection revealed that 3 of the pediatric livers from GSE81928, were very different from other pediatric livers and very similar to the hepatoblastoma tumors and peritumor regions, although the separate analysis of the databases, including GSE81928, render the same results and conclusions. No additional information was found in the publication. An artificial intelligence classification algorithm was built using nnet package version 7.3-13¹⁰ to test if these livers can be excluded. Data were transformed to a range of 1 to 0 to avoid the batch effect, and the algorithm was trained using GSE133039 data to classify either in peritumor or tumor. There are very few samples of normal pediatric liver, so we want to maintain them unseen by the algorithm to perform the testing, for that reason the classifier only classifies samples in peritumour (closer to the healthy) and tumour (closer to the pathology). Once trained, the outlier samples were predicted to be in the tumour group and the rest of the healthy liver samples in the peritumour group. For that reason, were excluded from the integrated dataset. Heatmaps were generated using ComplexHeatmap package version 2.2.0, and values were calculated as follows:

$$ Value_i = log \left( \frac{X_i}{\bar{C}} \right) $$
Where $X_i$ is the expression of a particular gene in the i-th sample and $\bar{C}$ the mean value for the reference group. To avoid overshadowing differences, all values over 10 times the IQR were substituted by the value of 10 times the IQR.

## Ethics statements

Regarding the use of human samples in this study: (1) written informed consent was obtained from each patient included in the study and (2) the study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the institution's human research committee.

All tissue samples were obtained from surgical specimens. Formalin-fixed paraffin-embedded tumor and paired non-tumor samples were obtained from 3 patients with HB. We also included in the study 3 adjacent non-tumor snap-frozen tissue samples obtained from 3 additional patients with HB as control samples for immunoblot analyses.

## Cell culture and in vitro pharmacological studies.

Eleven primary HB cell lines established from patients' derived xenografts as previously described were used¹¹. These cell lines were obtained from XenTech (Evry, France), and were cultured in Advanced DMEM supplemented with 10% FBS, 2 mM glutamine and antibiotics, all from Gibco Thermo-Fisher (Waltham, MA, USA), plus 20 µM Y-27632 (Rock kinase inhibitor) from Selleckchem (Houston, TX, USA). HepG2 cells were from the ATCC (Manassas, VA, USA), HuH6 cells were from RIKEN BRC (Ibaraki, Japan), and HepT1 cells were provided by Dr. Diego Calvisi (Regensburg University, Germany). These cells were cultured in DMEM supplemented with 10% FBS, 2 mM glutamine and antibiotics ¹². For amino acid deprivation studies cells were cultured as reported¹³. All cell cultures were maintained in a humidified incubator under 5% CO₂ in air at 37 °C. Times and doses of in vitro treatments are indicated throughout the manuscript, and controls received the same concentrations of DMSO (always <0.1% of final volume).

For proliferation studies, cells were seeded at a density of 2500-3000 cells/well in 96-well plates (triplicates). After overnight incubation, cells were treated for 48h with vehicle alone (0.1% DMSO) or with the different compounds tested in complete medium. Experiments were repeated three times for each cell line. Cell viability was measured using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA). The concentration of drug inhibiting cell growth by 50% relative to the untreated control ($G_{150}$) was calculated after curve fitting with GraphPad Prism-v5 software as described¹⁴. Calculations of the combination index (CI) for CM272 with cisplatin (cis-diamminedichloroplatinum II, CDDP) and olaparib, both from Selleckchem, were done as reported¹⁵. For chemosensitization studies, HuH6 and HepT1 cells were seeded in 96-well plates as indicated above and were pretreated, or not, with 50 or 100 nM CM272 respectively for 48h. Then, we calculated the $G_{150}$ of cisplatin in HuH6 and HepT1 cells as reported before¹². UNC0642 and cycloheximide (CHX) were from Sigma-Aldrich (St. Louis, MO, USA). The $G_{150}$ of CM272 was also calculated in HepT1 and HuH6 cells that were pretreated with 1 mM N-acetylcysteine (NAc) (Sigma-Aldrich) for 3 h and then incubated with different concentrations of CM272 for 48h also in the presence of NAc.
## Gene expression analyses

### RNA isolation and quantitative real-time RT-qPCR

Total RNA from cell lines was extracted using the automated Maxwell system from Promega (Madison, WI, USA). For retro-transcription, RNA samples were exposed for 1min at 90 °C for denaturalization followed by 1 h at 37 °C using a mix containing: 50 mM Tris-HCl pH 8.3, 75 mM KCl and 3 mM MgCl₂, 10 ng/µL of random primers, 0.5 mM of each deoxyribonucleic triphosphate (dNTP), 5 mM of dithiothreitol (DTT), 1.2 U/µL RNase inhibitors (RNase out) and 6 U/µL of M-MLV inverse transcriptase enzyme. All reagents were from Invitrogen (Carlsbad, CA, USA), except dNTPs that were from Roche Diagnostics (Mannheim, Germany). Quantitative reverse transcription PCR (qRT-PCR) was performed as reported, and gene expression was normalized relative to that of the housekeeping gene *H3F3A* as described¹⁶. Primer sequences are available upon request.

### RNA sequencing (RNAseq)

RNA was subjected to quantity and quality control using Qubit HS RNA Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) and 4200 Tapestation with High Sensitivity RNA ScreenTape (Agilent Technologies). All RNA samples were high-quality, with RIN values higher than 8. Library preparation was performed using the Illumina Stranded mRNA Prep Ligation kit (Illumina) following the manufacturer's protocol. All sequencing libraries were constructed from 100 ng of total RNA according to the manufacturer's instruction. Briefly, the protocol selects and purifies poly(A) containing RNA molecules using magnetic beads coated with poly(T) oligos. Poly(A)-RNAs are fragmented and reverse transcribed into first cDNA strand using random primers. The second cDNA strand is synthesized in the presence of dUTP to ensure strand specificity. Resulting cDNA fragments are purified with AMPure XP beads (Beckman Coulter), adenylated at 3' ends and then ligated with uniquely indexed sequencing adapters. Ligated fragments are purified, and PCR amplified to obtain the final libraries. The quality and quantity of the libraries were verified using Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and 4200 Tapestation with High Sensitivity D1000 ScreenTape (Agilent Technologies). Libraries were then sequenced using a NextSeq2000 sequencer (Illumina). 30-40 million pair-end reads (200 bp; Rd1:101; Rd2:101) were sequenced for each sample and demultiplexed using Cutadapt. RNAseq was carried out at the Genomics Unit of the Center for Applied Medical Research (CIMA, Universidad de Navarra, Pamplona). RNAseq data has been deposited in the Sequence Read Archive (SRA) of NCBI under the title: "Experimental validation of druggable epigenetic targets in hepatoblastoma", ref. PRJNA900056.

## Immunohistochemical analyses

Three µm thick consecutive sections cut from formalin-fixed paraffin-embedded liver/HB tissues were deparaffined with xylene, dehydrated with ethanol, and incubated with 3% hydrogen peroxide to block endogenous peroxidase. Antigen retrieval was
performed by heating in 10 mM Tris-EDTA buffer pH 9 before incubation with antibodies for G9a (ab185050, 1:200), DNMT1 (ab188453, 1:100), UHRF1 (ab194236, 1:100) and Myc tag (ab9106 1:100), all from Abcam. YAP1 antibody was from Cell Signaling (8418, 1:100), and H3K9me2 antibody was from Sigma-Aldrich (07-212, 1:100). HRP-conjugated Envision secondary antibody (K4003, DAKO) followed by DAB reagent (K3468, DAKO) were applied for the detection procedure. For signal quantification, images were analyzed with the QuPath software v0.3.2¹⁷.Tissue sections were counterstained with Hematoxylin (Sigma-Aldrich) and dehydrated. Negative controls were included omitting primary antibodies.

## Chromatin immunoprecipitation (ChIP)

ChIP assays were performed in HB cells treated with vehicle or CM272 (48h, 150 nM for HuH6 and 300 nM for HepT1). After treatment, cells underwent chromatin crosslinking with 1% formaldehyde for 10 min followed by quenching with 1.25 mM glycine during 5 min. Cells were then washed twice and harvested in ice-cold PBS with proteases inhibitors. After centrifugation at 1200 rpm for 5 min, cell pellet was lysed with LB1 buffer (50 mM Hepes-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100) with proteases inhibitors. After incubation for 10 min at 4 °C, cell nuclei were obtained by dounce homogenization followed by centrifugation at 3000 g for 10 min. Nuclear pellet was washed in LB2 buffer (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) with protease inhibitors, and centrifuged at 3000 g, 10 min. Then nuclei were resuspended in LB3 (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-deoxycholate, 0.5% N-Lauryl sarcosine) with protease inhibitors. The lysate was subjected to sonication to shear the chromatin into fragments of 200-1000 bp. After centrifugation at 14.000 rpm for 10min, samples were precleared with pre-washed protein G magnetic beads for 2h at 4 °C. Beads were discarded and equal amounts of DNA were incubated overnight with 5 µg of anti-H3K9me2 antibody (C15410060, Diagenode, Liege, Belgium) or normal rabbit IgG (2729S, Cell Signaling). After incubation with antibody, pre-washed protein G magnetic beads were added and incubated for an additional 4 hours at 4 °C with gentle agitation. Beads were consecutively washed with low-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8, 150 mM NaCl, 0.1 M DTT, protease inhibitors), high-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8, 500 mM NaCl, 0.1 M DTT, protease inhibitors), LiCl wash buffer (0.25 M LiCl, 1% NP-40, 1% Na-deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8, 0.1 M DTT, protease inhibitors), and twice with TE pH 8 buffer (100 mM Tris-HCl pH 8, 10 mM EDTA pH 8). Each wash was performed in rotation for 5 min at 4 °C. After the washes, beads were resuspended in 250 µL of Elution Buffer (25 mM Tris-HCl pH 7.5, 5 mM EDTA, 0.5% SDS) and incubated in rotation for 15 min at room temperature. Subsequently, 10 µL of 5 M NaCl, 10 µL of Tris-HCl pH 7, 5 µL of 0.5 M EDTA pH 8, 2 µL of proteinase K (20 µg) were added to the samples, and incubated for 1 h at 45 °C. This was followed by an incubation at 65 °C for 4 hours to reverse the crosslinking. 10 µL of RNAse A were added and incubated at 37 °C for 30 min. Finally, DNA extraction was performed using the PCR Purification Kit (Qiagen) following the manufacturer's instructions. Purified DNA was subsequently subjected to qPCR as detailed above using the primers listed in the table below.
<table><thead><tr><th>Gene</th><th>Sense</th><th>Sequence</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="2">FBP1</td><td>Fw</td><td>5'-CATAATTCAGTCACCTCCTACCAGG-3'</td><td rowspan="2">18</td></tr><tr><td>Rv</td><td>5'-AGGCATGGTAGTGTGACATGGTT-3'</td></tr><tr><td rowspan="2">SFRP1</td><td>Fw</td><td>5'-ACGCCGTGATCCATTCCC-3'</td><td rowspan="2">19</td></tr><tr><td>Rv</td><td>5'-CGGCTCAACACCCCTTAAAAA-3'</td></tr><tr><td rowspan="2">HHIP</td><td>Fw</td><td>5'-TTCCCACCTCCTACGGCC-3'</td><td rowspan="2">19</td></tr><tr><td>Rv</td><td>5'-TCCTCTCTCCTCCCGCTT-3'</td></tr><tr><td rowspan="2">IGFBP3</td><td>Fw</td><td>5'-GCTCCCTGAGACCCAAATGTAA-3'</td><td rowspan="2">19</td></tr><tr><td>Rv</td><td>5'-GCTCGGCATTCGTGTGTACC-3'</td></tr><tr><td rowspan="2">MAT1A</td><td>Fw</td><td>5'-AGGTGGGTTGTGAACTCTGG-3'</td><td rowspan="2">15</td></tr><tr><td>Rv</td><td>5'-AAAGCGAAAGTGCCTGAAAA-3'</td></tr><tr><td rowspan="2">ACTB</td><td>Fw</td><td>5'-GCCAACGCCAAAACTCTCC-3'</td><td rowspan="2">19</td></tr><tr><td>Rv</td><td>5'-CAGTCAGCATTTTTTTACCCC-3'</td></tr></tbody></table>

## Histone extraction

Histones were isolated as previously described¹². Briefly, cells were lysed in a buffer containing 10 mM Tris-HCl pH 7.4, 10 mM NaCl, and 3 mM MgCl₂. Supernatants were removed after centrifugation at 2500 rpm for 10 min at 4 °C, and pellets were lysed in the previous buffer but containing 0.5% NP40 on ice for 10 min with gentle stirring. Nuclei were pelleted by centrifugation at 2500 rpm for 10 min at 4 °C and resuspended in 5 mM MgCl2 and 0.8 M HCl. Nuclei were incubated in this buffer during 30 min at 4 °C to extract the histones. Samples were then centrifuged at 14,000 rpm for 10 min at 4 °C to pellet debris, and supernatants were transferred to a clean tube where TCA 50% was added to precipitate the histones. After washing the pellets with acetone samples were air-dried and resuspended in 100 mM Tris-HCl pH 7.5, 1 mM EDTA, and 1% SDS. The histone concentration in the extract was measured using the BCA assay (Pierce Technologies, Rockford, IL, USA) according to manufacturer's specifications.

## Immunoblot analyses

Cells and tissues were lysed in RIPA buffer as described previously¹⁶. Histone extracts, as well as homogenates from cells and tissues, were subjected to immunoblot (Western blot) analysis as reported ¹². The antibodies used are listed in the table below. Blots were probed with anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (2118S, Cell Signaling Technology) or anti-β-ACTIN (ab6276, Abcam) to demonstrate equal loading.
Representative images are shown throughout the study. Representative images are shown throughout the study.

<table><thead><tr><td>Target</td><td>Dilution</td><td>Reference</td><td>Company</td></tr></thead><tbody><tr><td>G9A</td><td>1:1000</td><td>3306</td><td>Cell Signaling Technology (Leiden, The Netherlands)</td></tr><tr><td>DNMT1</td><td>1:1000</td><td>5032</td><td>Cell Signaling Technology</td></tr><tr><td>UHRF1</td><td>1:500</td><td>Ab57083</td><td>Abcam (Cambridge, United Kingdom)</td></tr><tr><td>β-ACTIN</td><td>1:2500</td><td>Ab6276</td><td>Abcam</td></tr><tr><td>HISTONE 3</td><td>1:2500</td><td>05-928</td><td>Merck-Millipore (Darmstadt, Germany)</td></tr><tr><td>H3K9me2</td><td>1:1000</td><td>07-212</td><td>Merck-Millipore</td></tr><tr><td>ATF4</td><td>1:1000</td><td>11815</td><td>Cell Signaling Technology</td></tr><tr><td>MYC</td><td>1:1000</td><td>M4439</td><td>Sigma Aldrich (St. Louis, MO, USA)</td></tr><tr><td>ASNS</td><td>1:1000</td><td>92479</td><td>Cell Signaling Technology</td></tr><tr><td>SLC7A11</td><td>1:1000</td><td>12691</td><td>Cell Signaling Technology</td></tr><tr><td>SLC7A5</td><td>1:1000</td><td>5347</td><td>Cell Signaling Technology</td></tr><tr><td>SLC1A5</td><td>1:1000</td><td>OSS00244W</td><td>Thermo Fisher Scientific. Life Technologies (Waltham, MA, USA)</td></tr><tr><td>PYCR1</td><td>1:1000</td><td>13108-1-AP</td><td>ProteinTech (Rosemont, IL, USA)</td></tr><tr><td>BCAT1</td><td>1:1000</td><td>16431</td><td>Cell Signaling Technology</td></tr><tr><td>MTHFD2</td><td>1:1000</td><td>Ab56772</td><td>Abcam</td></tr><tr><td>PHGDH</td><td>1:1000</td><td>Ab240744</td><td>Abcam</td></tr></tbody></table>

## Colony formation and migration assays.

Colony formation assays were performed with the indicated cell lines essentially as described$^{12,20}$. Five thousand cells were seeded in complete medium in six-well plates and treated (CM272 50nM) the next day. Media was changed every two days and cultures maintained until differences between treatment conditions were noticeable (10 days). Plates were washed with PBS, fixed with 4% formaldehyde (Sigma-Aldrich) in PBS for 10 minutes, and stained with crystal violet. Representative pictures were taken. At least two biological replicates with three technical replicates were performed.

For migration assays, HuH6 and HepT1 (7x10$^5$) cells were seeded in Corning (Glendale, AZ, USA) Transwell polycarbonate membrane cell culture upper wells (8.0 µm pore) in regular culture medium supplemented with 0.5% FBS. Lower wells contained regular culture medium supplemented with 30% FBS. Once cells were attached, cultures were treated with CM272 (G150) for 24h. Membranes were washed with PBS, and cells were fixed with 4% paraformaldehyde for 24h, washed with PBS, and stained with 0.5% crystal violet in methanol at 2%. Experiments were performed in triplicates, and 3 to 5 pictures were taken from each transwell at 5X using an inverted microscope (Leica, Wetzlar, Germany). For quantification, images were analyzed using the ImageJ software$^{21}$.
## Immunofluorescence

For immunofluorescent detection of methylated CpGs, HuH6 and HepT1 cells were cultured on coverslips. After overnight incubation, cells were treated with CM272 (150 nM for HuH6 and 300 nM for HepT1). Immunofluorescence was performed as previously reported²². Briefly, after 48h treatment, cells were fixed with ice-cold methanol for 15 min at room temperature and washed twice with PBS. 50 mM NH₄Cl in PBS was used for quenching for 10 min. After washing three times with PBS, cells were permeabilized with 0,2% Triton X-100 for 5 min at 4 °C. After permeabilization, DNA was denatured incubating with 4 M HCl for 15 min followed by 100 mM Tris-HCl pH 8.5 for 10 min. After washing, coverslips were blocked with Superblocking buffer (Thermo Fisher Scientific) for 1 h at room temperature and incubated overnight at 4 °C with anti-5-methylCytosine antibody (BI-MECY 0100, Eurogentec, Seraing, Belgium) diluted in 1% BSA in PBS. After washing with 1% BSA in PBS, cells were incubated with fluorophore-conjugated secondary antibodies in 1% BSA in PBS for 1 h at room temperature, washed and stained with vectashield (Vector laboratories, Burlingame, CA, USA) containing DAPI. Images were obtained using the Zeiss Axio Imager.M1 microscope (Zeiss, Oberkochen, Germany).

## Organoids culture and treatments

Tumour and adjacent non-tumour tissues from the liver of HB patients were collected and organoids were generated following the protocol described by Broutier et al.²³. Procedures were approved by the Ethics Committee of the Hospital Clinic of Barcelona, Spain (protocol number HCB-2019-0041). Cells were resuspended in 50 µL of BME (Basement Membrane Extract, Type 2, Pathclear, Bio-Techne, Minneapolis, MN, USA) and seeded in 24-well plates. Once the BME solidified, the cells were cultured in human liver organoid isolation medium as previously published²⁴. Once a week organoids were passaged by mechanical dissociation and enzymatic disruption with TrypLE Express (Gibco, Grand Island, NY, USA), and then resuspended in fresh BME.

For organoid-viability assays, organoids were mechanically dissociated by pipetting before being resuspended in 10 µl of BME and dispensed into white 96-well plates (Greiner). Media supplemented with CM272 or cisplatin (cis-diamminedichloroplatinum II, CDDP) was refreshed every 2 days and after 7 days of drug incubation. Cell viability was assessed using CellTiter-Glo (Promega).

## Plasmid transfections

Expression vectors were produced in competent E. coli bacteria grown in lysogeny broth (LB) (Sigma-Aldrich) under sterile conditions at 37 °C in agitation overnight. Plasmids were purified using the Endofree Plasmid Maxi Kit from Qiagen (Hilden, Germany), following manufacturer's instructions. DNA amount was measured in Nanodrop ND-
1000 Spectrophotometer from Thermo Fisher Scientific. Overexpression experiments were performed in HuH6 and HepT1 seeded in 6-multiwell plates with complete medium at 120.000 cells per well. After overnight incubation, for G9a overexpression cells were washed with PBS and transfections were made with 1.5 µg of pEGFP-hG9a (Ref. 33025, Addgene, Cambridge, MA) or empty vector, pEGFP-Ctrl (Ref. 86775, Addgene), using 1.5 µL/well of Lipofectamine 2000 (Invitrogen) transfection reagent in Opti-MEM Reduced Serum Medium from Gibco (Thermo Fisher Scientific) without antibiotics. Cells were harvested after 24h of transfection. RNA and proteins were extracted to confirm G9a overexpression and further gene expression analyses.

Transient transfections for c-MYC overexpression were performed with 1.5 µg of pLENTI6-V5 plasmid (Addgene) encompassing human c-MYC cDNA (kindly provided by Dr. Silvestre Vicent, CIMA, Universidad de Navarra, Pamplona, Spain) or empty vector using 1.5 µL of Lipofectamine 2000 (Invitrogen) transfection reagent in Opti-MEM Reduced Serum Medium from Gibco (Thermo Fisher Scientific) without antibiotics in cells grown in 6-wells plates. After 24h of transfection, cells were treated with CM272 at $G_{1}50$ concentrations. After 48h of incubation, RNA was extracted for gene expression analyses, and cell proliferation was assessed by crystal violet (0.5% w/v) staining and the addition of 0.2 mL of 1% acetic acid solution containing 50% ethanol to extract the dye from the cells. After 20 min at room temperature and gentle agitation on a microplate shaker for 5 min, extracts were transferred to a microplate reader equipped with a 540 nm filter to measure absorbance.

## Mouse models

All animals received humane care according to the "Guide for the care and Use of Laboratory Animals" written by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86-23 revised 1985). Protocols were approved by the Animal Care Committee of the University of Navarra and were performed following their guidelines (ethical committee approval #R-CP001-15GN). Animal experiments followed the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines (http://www.nc3rs.org.uk/arrive-guidelines), developed by the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) to improve standards and reporting of animal research.

### Mouse xenograft studies.

The subcutaneous tumor model was established injecting $10x10^{6}$ of HB-303-LEF cells, a patient's derived primary cell line¹¹, on the right flank region of male athymic nude mice (6-8 weeks)(Envigo, Valencia, Spain) as described¹². When tumors reached ~100 mm³, mice (n=4-6) were randomized into control and treatment groups. Mice received 5 mg/kg (i.p.) of CM-272 or same volume of vehicle (phosphate-buffered saline, PBS), for the indicated times. This dose was selected based on our previous studies. At the end of treatment tumors were harvested, fixed with 4% formalin for histological analyses or snap-frozen, and stored at -80°C.

### Generation of G9a<sup>ΔhepKO</sup> mice and hydrodynamic tail-vein injection model
*G9a(Ehmt2)fl/fl* animals (C57BL/6 background) were supplied by Dr. S. Aznar-Benitah (Institute for Research in Biomedicine, IRB, Barcelona, Spain)²⁵, and were originally obtained from Dr. Y. Shinkai (Saitama University, Japan). Hepatocyte-specific *G9a* knockout mice (G9aΔHep) were generated by crossing *G9afl/fl* mice with transgenic *Alb-Cre* mice (Jackson Laboratory).

For the hydrodynamic tail-vein injection model, a sterile 0.9% NaCl solution/plasmid mix was prepared containing DNA. We used 20 µg of YapS127A, 20 µg of ΔN90-CTNNB1 and 1.6 µg of SB13 transposase-encoding plasmids dissolved in 2 mL of 0.9% NaCl solution, sterile filtered, and a volume corresponding to 10% of the weight of each mouse in volume was injected into the lateral tail vein of 4 weeks old male *G9aΔhepKO* and *G9aWT* mice as described²⁶,²⁷.

## Proteomic analyses

**Lysis and protein extraction:** After treatments, HuH6 cells were scraped in PBS, pelleted (3,500 x g for 5 min at 4°C), and washed in PBS. Cells were pelleted again and pellets were snap-frozen in liquid N₂ and stored. Each lyophilized cell pellet was dissolved with lysis buffer containing 5% SDS (Sigma-Aldrich), 100 mM triethylammonium bicarbonate (ThermoFisher Scientific). Samples were homogenized by micro tip probe ultrasonication for 1 min using an UP50H ultrasonic lab homogenizer (Hielscher Ultrasonics) in the presence of 1µL of endonuclease. The homogenate was centrifuged at 20,000 × g for 10 min at 4 °C, and the supernatant was used for further analysis. Protein concentration was estimated by Pierce 660nm protein assay (ThermoFisher Scientific), and 50µg of each sample was reduced and alkylated by adding 5 mM tris(2-carboxyethyl)phosphine and 10 mM chloroacetamide for 60 minutes at 37 °C.

**S-Trap™ Digestion:** Protein digestion was performed using S-Trap filters (Protifi, Huntington, NY, USA) following the protocol described in²⁸ with slight modifications. Briefly, 50 µg of protein of each sample was diluted with 40 µL of 5% SDS. Afterwards, 12% phosphoric acid and then seven volumes of binding buffer (90% methanol; 100 mM TEAB) were added to the sample (final phosphoric acid concentration: 1.2%). The protein solution was loaded on a S-Trap filter in two consecutive steps, including an intermediate 2 min centrifugation at 3000 x g. was After washing the filter 3 times with 150 µL of binding buffer 3.3 µg of Pierce MS-grade trypsin (ThermoFisher Scientific) in 20 µL of 100 mM TEAB solution was added to each sample in a ratio 1:15 (w/w) and spun through the S-Trap. Flow-through was then reloaded to the top of the S-Trap column and allowed to digest in a wet chamber at 37 °C overnight. To elute the tryptic peptides, two step-wise buffers were applied: (1) 40 µL of 25 mM TEAB and 2) 40 µL of 80% acetonitrile and 0.2% formic acid in H₂O), separated by a 2 min centrifugation at 3000 x g in each case. Eluted peptides were pooled and dried in a SpeedVac. After cleaning in homemade C18 microcolumns, peptide concentration was estimated by Qubit™ Fluorometric Quantitation (ThermoFisher Scientific).

**Tagging with TMT 16plex™ reagent:** The equivalent of 25 micrograms of triptic peptides per sample were subsequently labelled using TMT-16plex Isobaric Mass Tagging Kit (Thermo Fisher Scientific) following the manufacturer's instructions. Labeled samples
were combined, and the concentration of the peptide mixture adjusted for LC-ESI MS/MS analysis.

**Liquid chromatography and mass spectrometry analysis (LC-ESI-MS/MS):** One µg aliquot of each fraction was subjected to 1D-nano LC ESI-MS/MS (Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric) analysis using an Ultimate 3000 nano HPLC system (Thermo Fisher Scientific) coupled online to an Orbitrap Exploris 240 mass spectrometer (Thermo Fisher Scientific). Peptides were separated in a 50 cm × 75 µm Easy-spray PepMap C18 analytical column at 45 °C at a flow rate of 300 nL/min using a 120 min gradient ranging from 2 % to 95 % mobile phase B (mobile phase A: 0.1% formic acid (FA); mobile phase B: 80 % acetonitrile (ACN) in 0.1% FA). The loading solvent was 2 % ACN) in 0.1 % FA, and injection volume was 5 µl.

Data acquisition was performed using a data-dependent top-20 method, in full scan positive mode, scanning 375 to 1200 m/z. Survey scans were acquired at a resolution of 60,000 at m/z 200, with Normalized Automatic Gain Control (AGC) target adjusted to 300 (%) and a maximum injection time (IT) selected to AUTO. The top 20 most intense ions (charges ranging from 2-5) from each MS1 scan were selected and fragmented via Higher-energy collisional dissociation (HCD). Resolution for MS2 spectra was set to 45,000 at m/z 200, with AGC target of 100 and a maximum ion injection time in AUTO. Isolation of precursors was performed with a 0.7 m/z window, dynamic exclusion was set to 45s, and the HCD collision energy was set to 32.

**Proteomics data analysis and sequence search:** Raw instrument files were processed using Proteome Discoverer (PD) version 2.4 (ThermoFisher Scientific). MS2 spectra were searched by combining four search engines (Mascot (v2.7.0), MsAmanda (v2.4.0), MsFragger (v3.1.1), and Sequest HT) and a target/decoy database containing *Homo sapiens* protein sequences downloaded from Uniprot Knowledgebase Database. Static modifications with TMT Pro reagents (+304.207 Da) on lysine and N-termini of the peptide, and carbamidomethylation on cysteine were considered, while oxidation of methionine residues (+15.9949 Da) was set as a dynamic modification. A maximum number of 2 tryptic missclevages was allowed. Peptide precursor and MS/MS mass tolerance were set to 10 ppm and 0.02 Da, respectively. The false discovery rate (FDR) for proteins, peptides, and peptide spectral matches (PSMs) peptides was set to 1%. The quantification values for proteins were calculated using the summed abundance of all peptides considered for the identification. To calculate the p-values and adjusted p-values for quantification results, the "t-test background based" statistical method was used.

## Metabolomic analyses

HuH6 and HepT1 cells were treated with CM-272 at the corresponding $GI_{50}$ concentrations for 48h. At the end of treatments, cells were washed twice with cold PBS, scraped, counted and equivalent amounts of cells were pelleted. For metabolite extraction, pellets were resuspended in cold water and vortexed, proteins were precipitated by successively adding methanol (615 µl) and chloroform (435 µl), followed
by vortexing. Samples were spiked with tryptophan-d₅ (indole-d₅)(Sigma-Aldrich) as internal standard. Samples were then incubated at -20°C for 30 minutes, vortexed and centrifuged (18,000 g for 15 minutes at 4 °C). Supernatants (1000 µl) were collected, dried under vacuum, and resuspended in methanol. After centrifugation (18,000 g for 5 minutes at 4 °C). Finally, 10 µl were transferred to microtubes and derivatized for amino acid analysis as described²⁹. For ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) analyses an ACQUITY UPLC coupled to an Aquality-SQD system (Waters Corp., Milford, MA; USA) was used. Data processing and normalization were done as described³⁰. These analyses were performed by OWL Metabolomics (Derio, Biscay, Spain).

## Metabolic flux analysis

The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of HuH6 and HepT1 cells were measured using a Seahorse XFp extracellular flux analyzer (Seahorse Bioscience, Billerica, MA, USA). In brief, cells were plated on Seahorse XFp plates at a density of 3000 cells per well and cultured in DMEM 10% medium with/without CM272 (75 or 150 nM for Huh6, and 150 or 300 nM for HepT1) during 48h. Following the treatment, the medium was replaced with Seahorse XF DMEM medium pH 7.4 (supplemented with 10 mM glucose, 2 mM glutamine and 1 mM pyruvate) with/without CM272 (at the same concentration). Cells were then incubated for 1 hour at 37 °C without CO₂ and then basal levels of OCR and ECAR were recorded. Based on these determinations, relative production of ATP from mitochondria (mitoATP) and from glycolysis (glycoATP) was estimated according to the manufacturer's instructions, using oligomycin (1.5 µM) and rotenone/antimycin A (0.5 µM). Data were normalized to protein content per well, as assessed by Bradford (Bio-Rad, Hercules, CA, USA).

## Statistical analyses

Statistical analysis was performed using GraphPad Prism-v5 software. For comparison between two groups, paired two-tailed Student's t-test, or Kruskal-Wallis ANOVA-test were used according to sample distribution. All reported *p* values were two-tailed and differences were considered significant when *p*<0.05.
**Supplementary figures**

<figure><img src="image_25.png" /><figcaption>Fig. S1.</figcaption></figure>
A

<figure><img src="image_26.png" /></figure>

B

<figure><img src="image_27.png" /></figure>

C

<figure><img src="image_28.png" /><figcaption>Fig. S2.</figcaption></figure>
<figure><img src="image_29.png" /><figcaption>A</figcaption></figure>

<figure><img src="image_30.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_31.png" /></figure>

<figure><img src="image_32.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /><figcaption>Fig. S3.</figcaption></figure>
<figure><img src="image_35.png" /><figcaption>Fig. S4.</figcaption></figure>
<figure><img src="image_36.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_37.png" /></figure>

Fig. S5.
<figure><img src="image_38.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_39.png" /><figcaption>HepT1 cells</figcaption></figure>

<figure><img src="image_40.png" /></figure>

Fig. S6.
<figure><img src="image_41.png" /><figcaption>Fig. S7.</figcaption></figure>
<figure><img src="image_42.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_43.png" /></figure>

Fig. S8.
<figure><img src="image_44.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_45.png" /></figure>

Fig. S9.
A

<figure><img src="image_46.png" /></figure>

B

<figure><img src="image_47.png" /></figure>

C

<figure><img src="image_48.png" /></figure>

D

<figure><img src="image_49.png" /></figure>

E

<figure><img src="image_50.png" /></figure>

F

<figure><img src="image_51.png" /></figure>

G

<figure><img src="image_52.png" /><figcaption>Fig. S10.</figcaption></figure>
A

<figure><img src="image_53.png" /></figure>

B

<figure><img src="image_54.png" /></figure>

C

<figure><img src="image_55.png" /></figure>

Fig. S11.
<figure><img src="image_56.png" /><figcaption>Fig. S12.</figcaption></figure>
**<u>Supplementary figure legends</u>**

**Fig. S1.** Heatmap showing the expression of epigenetic modifiers in HB tissues from the study of Sumazin et al.³¹. Expression values were normalized to those in peritumoral tissues.

**Fig. S2.** Violin plots show the combined expression of the fifty most upregulated epigenetic modifiers in: (A) the epigenetic clusters Epi-CA and Epi-CB of HB tissues according to Carrillo-Reixach et al.³², and: (B) the transcriptomic molecular subgroups C1, C2A and C2B defined by Hooks et al.³³. Gene expression values were normalized to those found in peritumoral tissues. Data are means ± SD (horizontal dashed lines within violin plots indicate the mean and the SD). Kruskal-Wallis ANOVA test was used. \*\*\*\*p<0.0001. (C) Immunohistochemical analysis of H3K9me2 in human HB tissues. A representative image including tumoral (T) and non-tumoral (NT) areas is shown.

**Fig. S3.** (A) Effect of CM272 treatment for the indicated times on the total levels of H3K9me2 in HuH6 and HepT1 cells. (B and C) Combination study of the growth inhibitory effects of CM272 and cisplatin (B), or CM272 and the poly-ADP-ribose polymerase (PARP) inhibitor olaparib (C), in HB cell lines. Grey columns indicate the existence of a synergistic effect, i.e. a combination index (CI) < 1, at the indicated drugs concentrations.

**Fig. S4.** (A) Immunohistochemical analyses of ΔN90-β-catenin (Myc-tag), YAP1, G9a, and DNMT1 in liver tissue samples from WT and G9a<sup>ΔhepKO</sup> mice eight weeks after injection with ΔN90-β-catenin and YAP1 expressing plasmids. Representative images are shown. (B) Quantification of immunostaining of ΔN90-β-catenin (Myc-tag) in the livers of WT and G9a<sup>ΔhepKO</sup> mice eight weeks after injection with plasmids. Images were analyzed with the QuPath software v0.3.2. Five mice were included per group, and six 5X fields were analyzed per mouse. Data are means ± SD. Paired two-tailed Student's t tests were used. \*p<0.05. (C) Representative H&E stained liver tissue sections from WT and G9a<sup>ΔhepKO</sup> mice eight weeks after injection with ΔN90-β-catenin and YAP1 expressing plasmids. Higher magnification images show more prominent cellular crowding and more frequent mitosis (arrows) found in WT mice.

**Fig. S5.** (A) Most relevant GO functional categories of genes undergoing changes in expression identified by RNASeq in liver tissue samples from WT and G9a<sup>ΔhepKO</sup> mice eight weeks after injection with ΔN90-β-catenin and YAP1 expressing plasmids. (B) GSEA of transcriptional profiles of WT and G9a<sup>ΔhepKO</sup> mice revealed that lack of G9a expression results in a significant negative enrichment in the expression of genes upregulated in human HB (CAIRO_HEPATOBLASTOMA_CLASSES-UP gene set), concomitantly with a significant positive enrichment in genes downregulated in human HB tissues (CAIRO_HEPATOBLASTOMA_CLASSES_DOWN gene set) as defined by Cairo et al.³⁴. A negative enrichment in the expression of a gene set including pediatric cancer markers (WHITEFORD_PEDRIATRIC_CANCERnalINKERS gene set) and a c-MYC oncogenic signature characteristic of the most aggressive HB tumors (BILD_MYC_ONCOGENIC restSIGNATURE) was also observed in samples from G9a<sup>ΔhepKO</sup> mice.
mice. Compared to WTs, G9a<sup>Ahep</sup>KO mice showed a strong positive enrichment in the expression of a gene set that defines the adult liver-specific/enriched transcriptome (HSIAO_LIVER_SPECIFIC-GENES gene set)³⁵. Normalized enrichment score (NES) and significance is shown.

**Fig. S6.** (A) Effect of CM272 treatment (GI₅₀, 48 h) in HepT1 cells on the expression of tumor suppressor genes epigenetically downregulated in HB as analyzed by qRT-PCR. Data are means ± SD. Paired two-tailed Student's t tests were used. *p*<0.05, **p** <0.01, ***p** <0.001. (B) H3K9me2 Q-ChIP analysis in the proximal promoter region of the indicated genes in control and CM272-treated (48h at GI₅₀ concentrations) HuH6 and HepT1 cells. The proximal promoter of the housekeeping gene β-actin (ACTB) was tested for H3K9me2 levels as control. Data are means ± SD. Paired two-tailed Student's t tests were used. *p*<0.05, **p** <0.01, ns: not significant.

**Fig. S7.** Metabolomic analysis of HepT1 cells treated with CM272 (GI₅₀, 48 h). The levels of indicated amino acids, as well as reduced (GSH) and oxidized (GSSG) glutathione, are also shown. Values are relative to those in untreated HepT1 cells. Data are individual values with means ± SD. Paired two-tailed Student's t tests were used. *p*<0.05, ns: not significant.

**Fig. S8.** (A) Effects of CM272 treatment (GI₅₀, 48 h) in HepT1 cells on the expression of relevant metabolic genes commonly upregulated in tumors at the protein and mRNA levels. Data are means ± SD. Paired two-tailed Student's t tests were used. *p*<0.05, **p** <0.01, ***p** <0.001. (B) Calculation of CM272 GI₅₀ in HuH6 and HepT1 cells treated or not with N-acetylcysteine (NAc) at 1mM.

**Fig. S9.** (A) Expression levels of the indicated metabolic genes in fetal (F), adult (A), peritumor (PT), tumor (T), metastatic tissues (MT) and recurrent tumors (R) referred to those found in pediatric livers (P). Data are individual values and means ± SD. Nonparametric Kruskal-Wallis ANOVA test was used. *p*<0.05, **p** <0.01, ***p** <0.001. (B) Spearman correlation analysis of the mRNA levels of G9a and the indicated genes in HB tissues³². The regression coefficient (R) and p-value of each correlation are indicated.

**Fig. S10.** (A) Expression of *c-MYC* in HepT1 cells treated with CM272 (GI₅₀, 48 h) analyzed at the mRNA and protein levels. (B) Representative Western blots analyzing c-MYC protein levels in HuH6 cells pretreated or not with CM272 (GI₅₀, 48 h) and then incubated with cycloheximide (CHX, 100 µg/mL) for the indicated periods of time. (C) Effect of UNC0642 treatment (2.5 µM, 48h) on the levels of c-MYC protein in HuH6 and HepT1 cells. (D) Western blot showing c-MYC protein levels in HepT1 cells transiently transfected with a control plasmid (*pControl*) or a plasmid expressing c-MYC (*p_c-MYC*). Twenty-four hours after transfections cells were treated or not with CM272 (at GI₅₀) for another day, and then cell viability was determined by crystal violet staining and colorimetric quantification. *p*<0.05. (E) qPCR analysis of the expression of the indicated genes in HuH6 cells transfected with *pControl* or *p_c-MYC* and treated or not with CM272 as described above. *p*<0.05. (F) Expression of *ATF4* in HepT1 cells treated with CM272 (GI₅₀, 48 h) analyzed at the mRNA and protein levels. (G) HuH6 and HepT1 cells were pretreated with UNC0642 (2.5 µM) for 24h and then incubated for 24h more
in media with and without amino acids (Aa). ATF4 protein levels were analyzed by Western blot. *p*<0.05, **p** <0.01. Data are means ± SD. Paired two-tailed Student's t tests were used (A, F). Nonparametric Kruskal-Wallis ANOVA test was used (D, E).

**Fig. S11.** (A) Expression of *ATF4* in fetal (F), adult (A), peritumor (PT), tumor (T), metastatic tissues (MT), recurrent tumors (R) and primary HB cell lines (PCL) are referred to those found in pediatric livers (P). *p*<0.05, **p** <0.01, ***p** <0.001. (B) Spearman correlation analysis of *G9a* and *ATF4* mRNA levels in HB tissues. (C) Expression of *KDM4C* in peritumoral and tumoral HB tissues *p*<0.05, **p** <0.01. Data are individual values and means ± SD. Nonparametric Kruskal-Wallis ANOVA test was used (A). The regression coefficient (R) and p-value of each correlation are indicated (B). Data are means ± SD. Paired two-tailed Student's t tests were used (C).

**Fig. S12.** (A) Relative expression levels of the indicated genes in the livers of WT and *G9a*ΔhepKO mice eight weeks after injection with ΔN90-β-catenin and YAP1 expressing plasmids as analyzed by RNAseq. (B) Western blot showing G9a protein levels in HuH6 cells transiently transfected with a control plasmid (pControl) or a plasmid expressing G9a (pG9a). The graph below shows the expression of the indicated genes analyzed by qPCR in these transiently transfected HuH6 cells. *p*<0.05. (C) GSEA of the HSIAO_LIVER_SPECIFIC_GENES gene set that defines the liver-specific/enriched transcriptome³⁵ performed on HuH6 and HepT1 RNAseq data indicating that HepT1 cells are less differentiated. Right panel shows a GSEA of this gene set using RNAseq data from control and CM272 treated (at G1₅₀, 48h) HepT1 cells. (D) Relative ATP production from oxidative phosphorylation (mitoATP) and glycolysis (glycoATP) in HuH6 and HepT1 cells treated with the indicated concentrations of CM272 for 48h. *p*<0.05, **p** <0.01. Data are individual means and p-value of each gene from the RNAseq analysis (A). Data are individual values with means ± SD. Paired two-tailed Student's t tests were used (B). Normalized enrichment score (NES) and significance is shown (C). Data are means ± SD. Paired two-tailed Student's t tests were used (D).
<table><caption>**Table S1.** Epigenetic modifiers included in the HB transcriptomic meta-analysis.</caption><thead><tr><th>Class</th><th>Gene symbol</th><th>Description</th><th>Family</th></tr></thead><tbody><tr><td rowspan="3">DNMTs (DNA methyltransferases)</td><td>DNMT1</td><td>DNA methyltransferase 1</td><td>Writer</td></tr><tr><td>DNMT3A</td><td>DNA methyltransferase 3 alpha</td><td>Writer</td></tr><tr><td>DNMT3B</td><td>DNA methyltransferase 3 beta</td><td>Writer</td></tr><tr><td rowspan="3">TETs (methylcytosine dioxygenases)</td><td>TET1</td><td>Tet methylcytosine dioxygenase 1</td><td>Eraser</td></tr><tr><td>TET2</td><td>Tet methylcytosine dioxygenase 2</td><td>Eraser</td></tr><tr><td>TET3</td><td>Tet methylcytosine dioxygenase 3</td><td>Eraser</td></tr><tr><td rowspan="14">MBPs (methyl binding proteins)</td><td>BAZ2A</td><td>Bromodomain adjacent to zinc finger domain 2A</td><td>Reader</td></tr><tr><td>BAZ2B</td><td>Bromodomain adjacent to zinc finger domain 2B</td><td>Reader</td></tr><tr><td>MBD1</td><td>Methyl-CpG binding domain protein 1</td><td>Reader</td></tr><tr><td>MBD2</td><td>Methyl-CpG binding domain protein 2</td><td>Reader</td></tr><tr><td>MBD3</td><td>Methyl-CpG binding domain protein 3</td><td>Reader</td></tr><tr><td>MBD4</td><td>Methyl-CpG binding domain 4, DNA glycosylase</td><td>Reader</td></tr><tr><td>MBD5</td><td>Methyl-CpG binding domain protein 5</td><td>Reader</td></tr><tr><td>MBD6</td><td>Methyl-CpG binding domain protein 6</td><td>Reader</td></tr><tr><td>MECP2</td><td>Methyl-CpG binding protein 2</td><td>Reader</td></tr><tr><td>UHRF1</td><td>Ubiquitin like with PHD and ring finger domains 1</td><td>Reader</td></tr><tr><td>ZBTB33</td><td>Zinc finger and BTB domain containing 33</td><td>Reader</td></tr><tr><td>ZBTB38</td><td>Zinc finger and BTB domain containing 38</td><td>Reader</td></tr><tr><td>ZBTB4</td><td>Zinc finger and BTB domain containing 4</td><td>Reader</td></tr><tr><td>ZFP57</td><td>ZFP57 zinc finger protein</td><td>Reader</td></tr><tr><td rowspan="9">PRMTs (arginine (R) methyltransferases)</td><td>CARM1</td><td>Coactivator associated arginine methyltransferase 1</td><td>Writer</td></tr><tr><td>PRMT1</td><td>Protein arginine methyltransferase 1</td><td>Writer</td></tr><tr><td>PRMT2</td><td>Protein arginine methyltransferase 2</td><td>Writer</td></tr><tr><td>PRMT3</td><td>Protein arginine methyltransferase 3</td><td>Writer</td></tr><tr><td>PRMT5</td><td>Protein arginine methyltransferase 5</td><td>Writer</td></tr><tr><td>PRMT6</td><td>Protein arginine methyltransferase 6</td><td>Writer</td></tr><tr><td>PRMT7</td><td>Protein arginine methyltransferase 7</td><td>Writer</td></tr><tr><td>PRMT8</td><td>Protein arginine methyltransferase 8</td><td>Writer</td></tr><tr><td>PRMT9</td><td>Protein arginine methyltransferase 9</td><td>Writer</td></tr></tbody></table>

<table><thead><tr><th>Class</th><th>Gene symbol</th><th>Description</th><th>Family</th></tr></thead></table>
<table><tr><td>ASH1L</td><td>ASH1 like histone lysine methyltransferase</td><td>Writer</td></tr><tr><td>ASH2L</td><td>ASH2 like, histone lysine methyltransferase complex subunit</td><td>Writer</td></tr><tr><td>DOT1L</td><td>DOT1 like histone lysine methyltransferase</td><td>Writer</td></tr><tr><td>EHMT1</td><td>Euchromatic histone lysine methyltransferase 1</td><td>Writer</td></tr><tr><td>EHMT2</td><td>Euchromatic histone lysine methyltransferase 2</td><td>Writer</td></tr><tr><td>EZH1</td><td>Enhancer of zeste 1 polycomb repressive complex 2 subunit</td><td>Writer</td></tr><tr><td>EZH2</td><td>Enhancer of zeste 2 polycomb repressive complex 2 subunit</td><td>Writer</td></tr><tr><td>FBXO10</td><td>F-box protein 10</td><td>Writer</td></tr><tr><td>KMT2A</td><td>Lysine methyltransferase 2A</td><td>Writer</td></tr><tr><td>KMT2B</td><td>Lysine methyltransferase 2B</td><td>Writer</td></tr><tr><td>KMT2C</td><td>Lysine methyltransferase 2C</td><td>Writer</td></tr><tr><td>KMT2D</td><td>Lysine methyltransferase 2D</td><td>Writer</td></tr><tr><td>KMT2E</td><td>Lysine methyltransferase 2E (inactive)</td><td>Writer</td></tr><tr><td>KMT5A</td><td>Lysine methyltransferase 5A</td><td>Writer</td></tr><tr><td>KMT5B</td><td>Lysine methyltransferase 5B</td><td>Writer</td></tr><tr><td>KMT5C</td><td>Lysine methyltransferase 5C</td><td>Writer</td></tr><tr><td>MECOM</td><td>MDS1 and EVI1 complex locus</td><td>Writer</td></tr><tr><td>NSD1</td><td>Nuclear receptor binding SET domain protein 1</td><td>Writer</td></tr><tr><td>NSD2</td><td>Nuclear receptor binding SET domain protein 2</td><td>Writer</td></tr><tr><td>NSD3</td><td>Nuclear receptor binding SET domain protein 3</td><td>Writer</td></tr><tr><td>PRDM16</td><td>PR/SET domain 16</td><td>Writer</td></tr><tr><td>PRDM2</td><td>PR/SET domain 2</td><td>Writer</td></tr><tr><td>PRDM6</td><td>PR/SET domain 6</td><td>Writer</td></tr><tr><td>PRDM8</td><td>PR/SET domain 8</td><td>Writer</td></tr><tr><td>PRDM9</td><td>PR/SET domain 9</td><td>Writer</td></tr><tr><td>SETD1A</td><td>SET domain containing 1A, histone lysine methyltransferase</td><td>Writer</td></tr><tr><td>SETD1B</td><td>SET domain containing 1B, histone lysine methyltransferase</td><td>Writer</td></tr><tr><td>SETD2</td><td>SET domain containing 2, histone lysine methyltransferase</td><td>Writer</td></tr><tr><td>SETD3</td><td>SET domain containing 3, actin histidine methyltransferase</td><td>Writer</td></tr><tr><td>SETD7</td><td>SET domain containing 7, histone lysine methyltransferase</td><td>Writer</td></tr><tr><td>SETDB1</td><td>SET domain bifurcated histone lysine methyltransferase 1</td><td>Writer</td></tr><tr><td>SETDB2</td><td>SET domain bifurcated histone lysine methyltransferase 2</td><td>Writer</td></tr><tr><td>SETMAR</td><td>SET domain and mariner transposase fusion gene</td><td>Writer</td></tr><tr><td>SMYD1</td><td>SET and MYND domain containing 1</td><td>Writer</td></tr><tr><td>SMYD2</td><td>SET and MYND domain containing 2</td><td>Writer</td></tr><tr><td>SMYD3</td><td>SET and MYND domain containing 3</td><td>Writer</td></tr><tr><td>SMYD5</td><td>SMYD family member 5</td><td>Writer</td></tr><tr><td>SUV39H1</td><td>SUV39H1 histone lysine methyltransferase</td><td>Writer</td></tr><tr><td>SUV39H2</td><td>SUV39H2 histone lysine methyltransferase</td><td>Writer</td></tr></table>
<table><thead><tr><th>Class</th><th>Gene symbol</th><th>Description</th><th>Family</th></tr></thead><tbody><tr><td rowspan="20">HDMs (histone demethylases)</td><td>HIF1AN</td><td>Hypoxia inducible factor 1 subunit alpha inhibitor</td><td>Eraser</td></tr><tr><td>JARID2</td><td>Jumonji and AT-rich interaction domain containing 2</td><td>Eraser</td></tr><tr><td>JMJD1C</td><td>Jumonji domain containing 1C</td><td>Eraser</td></tr><tr><td>JMJD6</td><td>Jumonji domain containing 6, arginine demethylase and lysine hydroxylase</td><td>Eraser</td></tr><tr><td>KDM1A</td><td>Lysine demethylase 1A</td><td>Eraser</td></tr><tr><td>KDM1B</td><td>Lysine demethylase 1B</td><td>Eraser</td></tr><tr><td>KDM2A</td><td>Lysine demethylase 2A</td><td>Eraser</td></tr><tr><td>KDM2B</td><td>Lysine demethylase 2B</td><td>Eraser</td></tr><tr><td>KDM3A</td><td>Lysine demethylase 3A</td><td>Eraser</td></tr><tr><td>KDM3B</td><td>Lysine demethylase 3B</td><td>Eraser</td></tr><tr><td>KDM4A</td><td>Lysine demethylase 4A</td><td>Eraser</td></tr><tr><td>KDM4B</td><td>Lysine demethylase 4B</td><td>Eraser</td></tr><tr><td>KDM4C</td><td>Lysine demethylase 4C</td><td>Eraser</td></tr><tr><td>KDM4D</td><td>Lysine demethylase 4D</td><td>Eraser</td></tr><tr><td>KDM4E</td><td>Lysine demethylase 4E</td><td>Eraser</td></tr><tr><td>KDM5A</td><td>Lysine demethylase 5A</td><td>Eraser</td></tr><tr><td>KDM5B</td><td>Lysine demethylase 5B</td><td>Eraser</td></tr><tr><td>KDM5C</td><td>Lysine demethylase 5C</td><td>Eraser</td></tr><tr><td>KDM5D</td><td>Lysine demethylase 5D</td><td>Eraser</td></tr><tr><td>KDM6A</td><td>Lysine demethylase 6A</td><td>Eraser</td></tr><tr><td>KDM6B</td><td>Lysine demethylase 6B</td><td>Eraser</td></tr><tr><td>KDM7A</td><td>Lysine demethylase 7A</td><td>Eraser</td></tr><tr><td>KDM8</td><td>Lysine demethylase 8</td><td>Eraser</td></tr><tr><td>PHF2</td><td>PHD finger protein 2</td><td>Eraser</td></tr><tr><td>PHF8</td><td>PHD finger protein 8</td><td>Eraser</td></tr><tr><td>RIOX1</td><td>Ribosomal oxygenase 1</td><td>Eraser</td></tr><tr><td>RIOX2</td><td>Ribosomal oxygenase 2</td><td>Eraser</td></tr><tr><td>UTY</td><td>Ubiquitously transcribed tetratricopeptide repeat containing, Y-linked</td><td>Eraser</td></tr></tbody></table>
<table><thead><tr><th>Class</th><th>Gene symbol</th><th>Description</th><th>Family</th></tr></thead><tbody><tr><td rowspan="18">HMRs (histone methyl readers)</td><td>ARID4A</td><td>AT-rich interaction domain 4A</td><td>Reader</td></tr><tr><td>ATRX</td><td>ATRX chromatin remodeler</td><td>Reader</td></tr><tr><td>BPTF</td><td>Bromodomain PHD finger transcription factor</td><td>Reader</td></tr><tr><td>CBX5</td><td>Chromobox 5</td><td>Reader</td></tr><tr><td>DNMT3L</td><td>DNA methyltransferase 3 like</td><td>Reader</td></tr><tr><td>EED</td><td>Embryonic ectoderm development</td><td>Reader</td></tr><tr><td>ING2</td><td>Inhibitor of growth family member 2</td><td>Reader</td></tr><tr><td>ING4</td><td>Inhibitor of growth family member 4</td><td>Reader</td></tr><tr><td>ING5</td><td>Inhibitor of growth family member 5</td><td>Reader</td></tr><tr><td>L3MBTL 1</td><td>L3MBTL histone methyl-lysine binding protein 1</td><td>Reader</td></tr><tr><td>MSL3</td><td>MSL complex subunit 3</td><td>Reader</td></tr><tr><td>ORC1</td><td>Origin recognition complex subunit 1</td><td>Reader</td></tr><tr><td>PHF1</td><td>PHD finger protein 1</td><td>Reader</td></tr><tr><td>PHF19</td><td>PHD finger protein 19</td><td>Reader</td></tr><tr><td>SFMBT1</td><td>Scm like with four mbt domains 1</td><td>Reader</td></tr><tr><td>SGF29</td><td>SAGA complex associated factor 29</td><td>Reader</td></tr><tr><td>SPIN1</td><td>Spindlin 1</td><td>Reader</td></tr><tr><td>TP53BP1</td><td>Tumor protein p53 binding protein 1</td><td>Reader</td></tr><tr><td>WDR5</td><td>WD repeat domain 5</td><td>Reader</td></tr><tr><td rowspan="18">HATs (histone acetyltransferases)</td><td>ATF2</td><td>Activating transcription factor 2</td><td>Writer</td></tr><tr><td>CLOCK</td><td>Clock circadian regulator</td><td>Writer</td></tr><tr><td>CREBBP</td><td>CREB binding protein</td><td>Writer</td></tr><tr><td>EP300</td><td>E1A binding protein p300</td><td>Writer</td></tr><tr><td>GTF3C4</td><td>General transcription factor IIIC subunit 4</td><td>Writer</td></tr><tr><td>HAT1</td><td>Histone acetyltransferase 1</td><td>Writer</td></tr><tr><td>KAT2A</td><td>Lysine acetyltransferase 2A</td><td>Writer</td></tr><tr><td>KAT2B</td><td>Lysine acetyltransferase 2B</td><td>Writer</td></tr><tr><td>KAT5</td><td>Lysine acetyltransferase 5</td><td>Writer</td></tr><tr><td>KAT6A</td><td>Lysine acetyltransferase 6A</td><td>Writer</td></tr><tr><td>KAT6B</td><td>Lysine acetyltransferase 6B</td><td>Writer</td></tr><tr><td>KAT7</td><td>Lysine acetyltransferase 7</td><td>Writer</td></tr><tr><td>KAT8</td><td>Lysine acetyltransferase 8</td><td>Writer</td></tr><tr><td>MEAF6</td><td>MYST/Esa1 associated factor 6</td><td>Writer</td></tr><tr><td>NCOA1</td><td>Nuclear receptor coactivator 1</td><td>Writer</td></tr><tr><td>NCOA2</td><td>Nuclear receptor coactivator 2</td><td>Writer</td></tr><tr><td>NCOA3</td><td>Nuclear receptor coactivator 3</td><td>Writer</td></tr></tbody></table>
<table><thead><tr><th>Class</th><th>Gene symbol</th><th>Description</th><th>Family</th></tr></thead><tbody><tr><td rowspan="14">HDACs (histone deacetylases)</td><td>HDAC1</td><td>Histone deacetylase 1</td><td>Eraser</td></tr><tr><td>HDAC10</td><td>Histone deacetylase 10</td><td>Eraser</td></tr><tr><td>HDAC11</td><td>Histone deacetylase 11</td><td>Eraser</td></tr><tr><td>HDAC2</td><td>Histone deacetylase 2</td><td>Eraser</td></tr><tr><td>HDAC3</td><td>Histone deacetylase 3</td><td>Eraser</td></tr><tr><td>HDAC6</td><td>Histone deacetylase 6</td><td>Eraser</td></tr><tr><td>HDAC8</td><td>Histone deacetylase 8</td><td>Eraser</td></tr><tr><td>SIRT1</td><td>Sirtuin 1</td><td>Eraser</td></tr><tr><td>SIRT2</td><td>Sirtuin 2</td><td>Eraser</td></tr><tr><td>SIRT3</td><td>Sirtuin 3</td><td>Eraser</td></tr><tr><td>SIRT4</td><td>Sirtuin 4</td><td>Eraser</td></tr><tr><td>SIRT5</td><td>Sirtuin 5</td><td>Eraser</td></tr><tr><td>SIRT6</td><td>Sirtuin 6</td><td>Eraser</td></tr><tr><td>SIRT7</td><td>Sirtuin 7</td><td>Eraser</td></tr></tbody></table>
<table><thead><tr><th>Class</th><th>Gene Symbol</th><th>Description</th><th>Family</th></tr></thead><tbody><tr><td rowspan="20">HARs (Histone Acetyl readers)</td><td>ATAD2</td><td>ATPase family AAA domain containing 2</td><td>Reader</td></tr><tr><td>ATAD2B</td><td>ATPase family AAA domain containing 2B</td><td>Reader</td></tr><tr><td>BAZ1B</td><td>Bromodomain adjacent to zinc finger domain 1B</td><td>Reader</td></tr><tr><td>BRD1</td><td>Bromodomain containing 1</td><td>Reader</td></tr><tr><td>BRD2</td><td>Bromodomain containing 2</td><td>Reader</td></tr><tr><td>BRD3</td><td>Bromodomain containing 3</td><td>Reader</td></tr><tr><td>BRD4</td><td>Bromodomain containing 4</td><td>Reader</td></tr><tr><td>BRD7</td><td>Bromodomain containing 7</td><td>Reader</td></tr><tr><td>BRD9</td><td>Bromodomain containing 9</td><td>Reader</td></tr><tr><td>BRDT</td><td>Bromodomain testis associated</td><td>Reader</td></tr><tr><td>BRPF1</td><td>Bromodomain and PHD finger containing 1</td><td>Reader</td></tr><tr><td>BRPF3</td><td>Bromodomain and PHD finger containing 3</td><td>Reader</td></tr><tr><td>BRWD1</td><td>Bromodomain and WD repeat domain containing 1</td><td>Reader</td></tr><tr><td>BRWD3</td><td>Bromodomain and WD repeat domain containing 3</td><td>Reader</td></tr><tr><td>CECR2</td><td>CECR2 histone acetyl-lysine reader</td><td>Reader</td></tr><tr><td>DPF1</td><td>Double PHD fingers 1</td><td>Reader</td></tr><tr><td>DPF2</td><td>Double PHD fingers 2</td><td>Reader</td></tr><tr><td>DPF3</td><td>Double PHD fingers 3</td><td>Reader</td></tr><tr><td>MLLT3</td><td>MLLT3 super elongation complex subunit</td><td>Reader</td></tr><tr><td>PBRM1</td><td>Polybromo 1</td><td>Reader</td></tr><tr><td>PHIP</td><td>Pleckstrin homology domain interacting protein</td><td>Reader</td></tr><tr><td>SMARCA2</td><td>SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 2</td><td>Reader</td></tr><tr><td>SMARCA4</td><td>SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4</td><td>Reader</td></tr><tr><td>SP100</td><td>SP100 nuclear antigen</td><td>Reader</td></tr><tr><td>SP110</td><td>SP110 nuclear body protein</td><td>Reader</td></tr><tr><td>SP140</td><td>SP140 nuclear body protein</td><td>Reader</td></tr><tr><td>TAF1</td><td>TATA-box binding protein associated factor 1</td><td>Reader</td></tr><tr><td>TAF1L</td><td>TATA-box binding protein associated factor 1 like</td><td>Reader</td></tr><tr><td>TRIM24</td><td>Tripartite motif containing 24</td><td>Reader</td></tr><tr><td>TRIM28</td><td>Tripartite motif containing 28</td><td>Reader</td></tr><tr><td>TRIM33</td><td>Tripartite motif containing 33</td><td>Reader</td></tr><tr><td>TRIM66</td><td>Tripartite motif containing 66</td><td>Reader</td></tr><tr><td>ZMYND11</td><td>Zinc finger MYND-type containing 11</td><td>Reader</td></tr><tr><td>ZMYND8</td><td>Zinc finger MYND-type containing 8</td><td>Reader</td></tr></tbody></table>
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