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Management of hepatoblastoma (HB), the most frequent pediatric liver cancer, is based on surgical resection and perioperative chemotherapy regimens. In this study, we aimed to identify actionable targets in HB and assess the efficacy of molecular therapies in preclinical models of HB. Paired tumor and adjacent tissues from 31 HBs and a validation set of 50 HBs were analyzed using RNA-seq, SNP, and methylation arrays. IGF2 overexpression was identified as the top targetable HB driver, present in 71% of HBs (22/31). IGF2high tumors displayed progenitor cell features and shorter recurrence-free survival. IGF2 overexpression was associated in 91% of cases with fetal promoter hypomethylation, ICR1 deregulation, 11p15.5 loss of heterozygosity or miR483-5p overexpression. The antitumor effect of xentuzumab (a monoclonal antibody targeting IGF1/2) alone or in combination with the conventional therapeutic agent cisplatin was assessed in HB cell lines, in PDX-derived HB organoids and in a xenograft HB murine model. The combination of xentuzumab with cisplatin showed strong synergistic antitumor effects in organoids and in IGF2high cell lines. In mice (n = 55), the combination induced a significant decrease in tumor volume and improved survival compared with cisplatin alone. These results suggest that IGF2 is an HB actionable driver and that, in preclinical models of HB, the combination of IGF1/2 inhibition with cisplatin induces superior antitumor effects than cisplatin monotherapy. Overall, our study provides a rationale for testing IGF2 inhibitors in combination with cisplatin in HB patients with IGF2 overexpression.

Adeno-associated virus (AAV) integrates into host genomes at low frequency, but when integration occurs in oncogenic hotspots it can cause hepatocellular carcinoma (HCC). Given the possibility of recombinant AAV (rAAV) integration leading to HCC, common causes of liver inflammation like non-alcoholic fatty liver disease (NAFLD) may increase the risk of rAAV-induced HCC. A rAAV targeting the oncogenic mouse Rian locus was used, and as expected led to HCC in all mice infected as neonates, likely due to growth-related hepatocyte proliferation in young mice. Mice infected with rAAV as adults did not develop HCC unless they were fed a diet leading to NAFLD, with increased inflammation and hepatocyte proliferation. Female mice were less susceptible to rAAV-induced HCC, and male mice with NAFLD treated with estrogen exhibited less inflammation and immune exhaustion associated with oncogenesis compared to those without estrogen. Adult NAFLD mice infected with a non-targeted control rAAV also developed HCC, though only half as frequently as those exposed to the Rian targeted rAAV. This study shows that adult mice exposed to rAAV gene therapy in the context of chronic liver disease developed HCC at high frequency, and thus warrants further study in humans given the high prevalence of NAFLD in the population.

Purpose: Immune checkpoint inhibitors combined with antiangiogenic agents produce benefits in the treatment of advanced hepatocellular carcinoma (HCC). We investigated the efficacy and immunomodulatory activity of cabozantinib alone and combined with anti-PD1 in experimental models of HCC, and explored the potential target population that might benefit from this combination. Experimental Design: C57BL/6J mice bearing subcutaneous Hepa1-6 or Hep53.4 tumors received cabozantinib, anti-PD1, their combination, or placebo. Tumor and blood samples were analyzed by flow cytometry, IHC, transcriptome, and cytokine profiling. Cabozantinib-related effects were validated in a colorectal cancer patient-derived xenograft model. Transcriptomic data from three human HCC cohorts (cohort 1: n = 167, cohort 2: n = 57, The Cancer Genome Atlas: n = 319) were used to cluster patients according to neutrophil features, and assess their impact on survival. Results: The combination of cabozantinib and anti-PD1 showed increased antitumor efficacy compared with monotherapy and placebo (P < 0.05). Cabozantinib alone significantly increased neutrophil infiltration and reduced intratumor CD8+PD1+ T-cell proportions, while the combination with anti-PD1 further stimulated both effects and significantly decreased regulatory T cell (Treg) infiltration (all P < 0.05). In blood, cabozantinib and especially combination increased the proportions of overall T cells (P < 0.01) and memory/effector T cells (P < 0.05), while lowering the neutrophil-to-lymphocyte ratio (P < 0.001 for combination). Unsupervised clustering of human HCCs revealed that high tumor enrichment in neutrophil features observed with the treatment combination was linked to less aggressive tumors with more differentiated and less proliferative phenotypes. Conclusions: Cabozantinib in combination with anti-PD1 enhanced antitumor immunity by bringing together innate neutrophil-driven and adaptive immune responses, a mechanism of action which favors this approach for HCC treatment.

Background: Transforming growth factor β (TGFβ) has a dual role in cancer, including hepatocellular carcinoma (HCC). It acts as a tumor suppressor in early stages of hepatocarcinogenesis, but promotes epithelial-to-mesenchymal transition, angiogenesis and immunosuppression in advanced stages. PMEPA1 (prostate transmembrane protein androgen induced 1) negatively regulates TGFβ signaling by interacting with SMAD proteins, and has been shown to promote TGFβ oncogenic effects in other cancers. Thus, we aimed to explore the role of PMEPA1 in HCC pathogenesis. Methods: We analyzed transcriptomic, genomic, epigenomic and clinicopathological data of a discovery cohort comprising 228 HCCs and a validation cohort of 361 HCCs. PMEPA1 levels were quantified by qPCR in the discovery cohort. PMEPA1 overexpression was validated in 6 independent publicly available cohorts (n = 916) with either microarray or RNAseq data. We also evaluated PMEPA1 expression at the single-cell level using a publicly available single-cell RNA sequencing (scRNAseq) dataset comprising 22 HCC samples. Genetically engineered mouse models were generated by performing hydrodynamic tail-vein injection of equal amounts of plasmids overexpressing MYC and MYC+PMEPA1. Molecular and histopathological analyses of the murine tumors are currently ongoing. Results: PMEPA1 was overexpressed in 18% of human HCCs [FC > 2; n = 203/1144], a feature associated with TGFβ signaling (p < 0.05). Overexpression of PMEPA1 was associated with gene body hypermethylation (p < 0.0001). HCCs displaying both TGFβ signaling and high PMEPA1 levels (11% of cases) were significantly enriched in signatures of immune exhaustion, late TGFβ activation, tumor microenvironment (TME) response to TGFβ and active stroma (p < 0.05) when compared with HCCs with TGFβ signalling alone (8% of cases) or PMEPA1 overexpression alone (11% of cases). Analyses of PMEPA1 expression at the single-cell level revealed that this gene is not only expressed by tumoral cells but also by the TME cell populations, including endothelial cells, pericytes, fibroblasts, and dendritic cells. In vivo, overexpression of MYC+PMEPA1 led to HCC development in 9/16 mice and was associated with a significantly reduced survival when compared to mice overexpressing MYC alone (p = 0.014), which did not develop tumors. Compared to healthy liver controls, MYC+PMEPA1 tumors presented upregulation of AKT. Conclusion: PMEPA1 upregulation is linked to TGF-β activation and an aggressive phenotype in human HCC. Overexpression of PMEPA1 in combination with MYC in vivo led to HCC development, showing for the first time the oncogenic role of PMEPA1 in this cancer type. Citation Format: Marta Piqué-Gili, Carmen Andreu-Oller, Roser Pinyol, Roger Esteban, Marina Bárcena-Varela, Judit Peix, Jordi Abril-Fornaguera, Katherine E. Lindblad, Miguel Torres-Martin, Júlia Huguet-Pradell, Daniela Sia, Amaia Lujambio, Josep M Llovet. PMEPA1 has an oncogenic role in the context of TGF-β signaling in hepatocellular carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PR02.

Background and Aims: Management of hepatoblastoma (HB), the most frequent pediatric liver cancer, is based on surgical resection and perioperative chemotherapy regimens, commonly cisplatin. Here, we aimed to identify actionable targets in HB and assess the efficacy of molecular therapies in preclinical models of HB.Methods: Paired tumor and adjacent tissues from 31 resected HBs and a validation set of 50 HBs were analyzed at the transcriptomic, genomic and epigenomic level using RNAseq, SNP and methylation arrays, respectively. The main targetable driver in HB was identified by gene co-expression network analysis (GCN) and its overexpression was confirmed by qRT-PCR. The anti-tumor effect of driver inhibition with molecular therapies alone or in combination with cisplatin was assessed in cell lines, patient-derived HB organoids and in a HB xenograft murine model.Results: Seven network modules were significantly deregulated in tumors compared to non-tumoral samples, including IGF2 signaling pathway, cell cycle and survival and immune response. IGF2 overexpression (FC> 4 vs adjacent tissue) was identified as the top targetable HB driver (study cohort: 71%, 22/31; independent validation cohorts: 78% and 76%). IGF2high tumors displayed progenitor cell features and were significantly enriched in molecular classes with aggressive phenotypes and CTNNB1 mutations, while IGF2low tumors were enriched in inflammatory and TGF-β signaling. IGF2high tumors correlated with shorter recurrence-free survival after resection (median 34 months vs not reached for IGF2low; p = 0.02). IGF2 overexpression correlated in most cases (86%) with fetal promoter hypomethylation (50%), 11p15.5 loss of heterozygosity (LOH, 57%) or overexpression of miR483 (55%). Xentuzumab (anti-IGF1/2 mAb) alone or combined with cisplatin reduced proliferation and clonogenic capacity in IGF2high cell lines. The combination of xentuzumab and cisplatin exhibited synergistic effects in terms of cell viability in organoids derived from IGF2high human HBs. The combination treatment induced apoptosis and reduced IGF2 pathway activation in vitro. In mice (n = 13-14 per arm), this combination induced a significant decrease in the viable tumor volume (p < 0.01), extended survival compared to cisplatin alone (p < 0.05) and inhibited tumor angiogenesis (p < 0.05).Conclusion: IGF2 is an actionable driver in HB and its overexpression was associated with fetal promoter hypomethylation, LOH or miR483 overexpression. The combination of a mAb against IGF1/2 (xentuzumab) with cisplatin led to remarkable anti-tumoral effects in pre-clinical models, providing the rationale for exploring this regimen in IGF2high HB patients. Citation Format: Jordi Abril-Fornaguera, Laura Torrens, Juan Carrillo-Reixach, Alex Rialdi, Ugne Balaseviciute, Júlia Huguet-Pradell, Carla Montironi, Philipp Haber, Álvaro Del Río-Álvarez, Montserrat Domingo-Sàbat, Laura Royo, Nicholas Akers, Catherine E Willoughby, Judit Peix, Miguel Torres-Martin, Marc Puigvehi, Roser Pinyol, Stefano Cairo, Margaret Childs, Rudolf Maibach, Rita Alaggio, Piotr Czauderna, Bruce Morland, Bojan Losic, Vincenzo Mazzaferro, Ernesto Guccione, Daniela Sia, Carolina Armengol, Josep M Llovet. Identification of IGF2 as genomic driver and actionable therapeutic target in hepatoblastoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO009.

Background and aims: Lenvatinib is an effective drug in advanced HCC. Its combination with the anti-PD1 (programmed cell death protein 1) immune checkpoint inhibitor, pembrolizumab, has generated encouraging results in phase Ib and is currently being tested in phase III trials. Here, we aimed to explore the molecular and immunomodulatory effects of lenvatinib alone or in combination with anti-PD1. Approach and results: We generated three syngeneic models of HCC in C57BL/6J mice (subcutaneous and orthotopic) and randomized animals to receive placebo, lenvatinib, anti-PD1, or combination treatment. Flow cytometry, transcriptomic, and immunohistochemistry analyses were performed in tumor and blood samples. A gene signature, capturing molecular features associated with the combination therapy, was used to identify a subset of candidates in a cohort of 228 HCC patients who might respond beyond what is expected for monotherapies. In mice, the combination treatment resulted in tumor regression and shorter time to response compared to monotherapies (P < 0.001). Single-agent anti-PD1 induced dendritic and T-cell infiltrates, and lenvatinib reduced the regulatory T cell (Treg) proportion. However, only the combination treatment significantly inhibited immune suppressive signaling, which was associated with the TGFß pathway and induced an immune-active microenvironment (P < 0.05 vs. other therapies). Based on immune-related genomic profiles in human HCC, 22% of patients were identified as potential responders beyond single-agent therapies, with tumors characterized by Treg cell infiltrates, low inflammatory signaling, and VEGFR pathway activation. Conclusions: Lenvatinib plus anti-PD1 exerted unique immunomodulatory effects through activation of immune pathways, reduction of Treg cell infiltrate, and inhibition of TGFß signaling. A gene signature enabled the identification of ~20% of human HCCs that, although nonresponding to single agents, could benefit from the proposed combination.

Background and Aims: Management of hepatoblastoma (HB), the most frequent pediatric liver cancer, is based on surgical resection and perioperative platin-based regimens, commonly cisplatin. Here, we aimed to identify actionable targets in HB and assess the efficacy of molecular therapies in HB cell lines, organoids and murine models. Methods: Paired tumor and adjacent tissues from 31 resected HBs and a validation set of 50 HBs were analyzed at the transcriptomic, genomic and epigenomic level using RNAseq, SNP and methylation arrays, respectively. The main targetable driver in HB was identified by gene co-expression network analysis (GCN) and its overexpression was confirmed by qRT-PCR. The anti-tumor effect of driver inhibition with molecular therapies alone or in combination with cisplatin was assessed in cell lines, patient-derived HB organoids and in a HB xenograft murine model. Results: IGF2 overexpression (FC> 4 vs adjacent tissue) was identified as the top targetable HB driver (71%, 22/31). IGF2high tumors displayed progenitor cell features and were significantly enriched in molecular classes with aggressive phenotypes and CTNNB1 mutations, while IGF2low tumors were enriched in inflammatory and TGF-β signaling. IGF2high tumors correlated with shorter recurrence-free survival after resection (median 34 months vs not reached for IGF2low; p = 0.02). IGF2 overexpression correlated in most cases (86%) with fetal promoter hypomethylation (50%), 11p15.5 loss of heterozygosity (LOH, 57%) or overexpression of miR483 (55%). Xentuzumab (anti-IGF1/2 mAb) alone or combined with cisplatin reduced proliferation and clonogenic capacity in IGF2high cell lines. The combination of xentuzumab and cisplatin exhibited synergistic effects in terms of cell viability in organoids derived from IGF2high human HBs. In mice, this combination induced a significant decrease in the viable tumor volume and extended survival compared to cisplatin alone (n = 13-14 per arm, p = 0.04). Conclusion: IGF2 is an actionable driver in HB and its overexpression was associated with fetal promoter hypomethylation, LOH or miR483 overexpression. The combination of a mAb against IGF1/2 (xentuzumab) with cisplatin led to remarkable anti-tumoral effects in pre-clinical models, providing the rationale for exploring this regimen in IGF2high HB patients. Citation Format: Jordi Abril-Fornaguera, Laura Torrens, Juan Carrillo-Reixach, Alexander Rialdi, Ugne Balaseviciute, Carla Montironi, Philipp Haber, Álvaro Del Río-Álvarez, Montserrat Domingo-Sàbat, Laura Royo, Nicholas Akers, Catherine E. Willoughby, Judit Peix, Miguel Torres-Martin, Marc Puigvehi, Roser Pinyol, Stefano Cairo, Margaret Childs, Rudolf Maibach, Rita Alaggio, Piotr Czauderna, Bruce Morland, Bojan Losic, Vincenzo Mazzaferro, Ernesto Guccione, Daniela Sia, Carolina Armengol, Josep M. Llovet. Identification of IGF2 as genomic driver and actionable therapeutic target in hepatoblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4005.

Background: Transforming growth factor β (TGFβ) has a dual role in cancer, including hepatocellular carcinoma (HCC). It acts as a tumor suppressor in early stages of hepatocarcinogenesis, but promotes epithelial-to-mesenchymal transition, angiogenesis and immunosuppression in advanced stages. PMEPA1 (prostate transmembrane protein androgen induced 1), a direct target gene of the TGFβ pathway that negatively regulates TGFβ signaling by interacting with SMAD proteins, has been shown to promote TGFβ oncogenic effects in other cancers and we decided to explore its role in HCC pathogenesis. Methods: We analyzed transcriptomic, genomic, epigenomic and clinicopathological data of a discovery cohort of 228 HCCs and a validation cohort of 361 HCCs. PMEPA1 levels were quantified by qPCR in the discovery cohort. PMEPA1 overexpression was validated in 6 independent cohorts (n = 916) using microarray and RNAseq data. Genetically engineered mouse models were generated through hydrodynamic tail-vein injection. Equal amounts of plasmids overexpressing PMEPA1, TGFB1 and MYC were injected in mice to evaluate survival. Molecular and histopathological analyses of the murine tumors are currently ongoing. Results: PMEPA1 was overexpressed in 18% of human HCCs [FC > 2; n = 203/1144], a feature associated with TGFβ signaling (p < 0.05). Overexpression of PMEPA1 was associated with gene body hypermethylation (p < 0.0001). HCCs displaying both TGFβ signaling and high PMEPA1 levels (11% of cases) were significantly enriched in signatures of immune exhaustion, late TGFβ activation, TME response to TGFβ and active stroma (p < 0.05) compared to HCCs with TGFβ signalling alone (8% of cases) or PMEPA1 high levels alone (11% of cases). In vivo, overexpression of MYC+PMEPA1 led to HCC development in 9/16 mice and showed a lower survival when compared to the control arm, overexpression of MYC alone (p = 0.014). On the contrary, only 1/12 mice in the MYC+PMEPA1+TGFβ1 condition developed HCC and none of the MYC+TGFβ1 mice. Conclusion: PMEPA1 upregulation is linked to TGF-β activation and an aggressive phenotype in human HCC. Overexpression PMEPA1 in combination with MYC in vivo led to HCC development, showing for the first time the oncogenic role of PMEPA1 in this cancer. Citation Format: Marta Piqué-Gili, Carmen Andreu-Oller, Roser Pinyol, Marina Bárcena-Varela, Roger Esteban-Fabró, Judit Peix, Jordi Abril-Fornaguera, Katherine E. Lindblad, Miguel Torres-Martin, Daniela Sia, Amaia Lujambio, Josep M. Llovet. PMEPA1 is an oncogene linked to TGF-β signaling in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 840.

Background & Aims: Hepatoblastoma (HB) is a rare disease. Nevertheless, it is the predominant pediatric liver cancer, with limited therapeutic options for patients with aggressive tumors. Herein, we aimed to uncover the mechanisms of HB pathobiology and to identify new biomarkers and therapeutic targets in a move towards precision medicine for patients with advanced HB. Methods: We performed a comprehensive genomic, transcriptomic and epigenomic characterization of 159 clinically annotated samples from 113 patients with HB, using high-throughput technologies. Results: We discovered a widespread epigenetic footprint of HB that includes hyperediting of the tumor suppressor BLCAP concomitant with a genome-wide dysregulation of RNA editing and the overexpression of mainly non-coding genes of the oncogenic 14q32 DLK1-DIO3 locus. By unsupervised analysis, we identified 2 epigenomic clusters (Epi-CA, Epi-CB) with distinct degrees of DNA hypomethylation and CpG island hypermethylation that are associated with the C1/C2/C2B transcriptomic subtypes. Based on these findings, we defined the first molecular risk stratification of HB (MRS-HB), which encompasses 3 main prognostic categories and improves the current clinical risk stratification approach. The MRS-3 category (28%), defined by strong 14q32 locus expression and Epi-CB methylation features, was characterized by CTNNB1 and NFE2L2 mutations, a progenitor-like phenotype and clinical aggressiveness. Finally, we identified choline kinase alpha as a promising therapeutic target for intermediate and high-risk HBs, as its inhibition in HB cell lines and patient-derived xenografts strongly abrogated tumor growth. Conclusions: These findings provide a detailed insight into the molecular features of HB and could be used to improve current clinical stratification approaches and to develop treatments for patients with HB. Lay summary: Hepatoblastoma is a rare childhood liver cancer that has been understudied. We have used cutting-edge technologies to expand our molecular knowledge of this cancer. Our biological findings can be used to improve clinical management and pave the way for the development of novel therapies for this cancer. (c) 2020 European Association for the Study of the Liver. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)., This article was possible thanks to the inputs from the Instituto de Salud Carlos III, ISCIII (PI09/00751, PI10/02082, PI13/02340). The project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 668596 (ChiLTERN) and grant agreement No 826121 (iPC). JCR is supported by the Catalan Agency for Management of University and Research Grants (AGAUR, 2019 FI_B01024). LT is supported by an Accelerator Award (CRUCK, AECC, AIRC) (HUNTER, C9380/A26813). DS is supported by the Gilead Research Scholar in Liver Disease. JML is supported by the European Union's Horizon 2020 research and innovation programme (HEPCAR, 667273-2), Institucio Catalana de Recerca i Estudis Avancats (ICREA), U.S. Department of Defense (CA150272P3), an Accelerator Award (CRUCK, AECC, AIRC) (HUNTER, C9380/A26813), National Cancer Institute, Tisch Cancer Institute (P30-CA196521), Samuel Waxman Cancer Research Foundation, Spanish National Health Institute (SAF2016-76390) and AGAUR (SGR-1358). CA and MRS were supported by Ramon y Cajal (RYC-2010-07249) and Miguel Servet (CPII14/00021) programs of the Ministry of Science and Innovation of Spain and ISCIII, respectively. CA, MRS and MS received funding from CIBERehd (CB06/04/0033) and AGAUR (2017-SGR-490). IGTP is a member of the CERCA network of institutes. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.