Paola Nicoli

Massive gene amplification drives paediatric hepatocellular carcinoma caused by bile salt export pump deficiency

Hepatocellular carcinoma (HCC) is almost invariably associated with an underlying inflammatory state, whose direct contribution to the acquisition of critical genomic changes is unclear. Here we map acquired genomic alterations in human and mouse HCCs induced by defects in hepatocyte biliary transporters, which expose hepatocytes to bile salts and cause chronic inflammation that develops into cancer. In both human and mouse cancer genomes, we find few somatic point mutations with no impairment of cancer genes, but massive gene amplification and rearrangements. This genomic landscape differs from that of virus- and alcohol-associated liver cancer. Copy-number gains preferentially occur at late stages of cancer development and frequently target the MAPK signalling pathway, and in particular direct regulators of JNK. The pharmacological inhibition of JNK retards cancer progression in the mouse. Our study demonstrates that intrahepatic cholestasis leading to hepatocyte exposure to bile acids and inflammation promotes cancer through genomic modifications that can be distinguished from those determined by other aetiological factors.

Dissection of transcriptional and cis-regulatory control of differentiation in human pancreatic cancer.

The histological grade of carcinomas describes the ability of tumor cells to organize in differentiated epithelial structures and has prognostic and therapeutic impact. Here, we show that differential usage of the genomic repertoire of transcriptional enhancers leads to grade‐specific gene expression programs in human pancreatic ductal adenocarcinoma (PDAC). By integrating gene expression profiling, epigenomic footprinting, and loss‐of‐function experiments in PDAC cell lines of different grade, we identified the repertoires of enhancers specific to high‐ and low‐grade PDACs and the cognate set of transcription factors acting to maintain their activity. Among the candidate regulators of PDAC differentiation, KLF5 was selectively expressed in pre‐neoplastic lesions and low‐grade primary PDACs and cell lines, where it maintained the acetylation of grade‐specific enhancers, the expression of epithelial genes such as keratins and mucins, and the ability to organize glandular epithelia in xenografts. The identification of the transcription factors controlling differentiation in PDACs will help clarify the molecular bases of its heterogeneity and progression.

Identification of MYC-Dependent Transcriptional Programs in Oncogene-Addicted Liver Tumors

Tumors driven by activation of the transcription factor MYC generally show oncogene addiction. However, the gene expression programs that depend upon sustained MYC activity remain unknown. In this study, we employed a mouse model of liver carcinoma driven by a reversible tet-MYC transgene, combined with chromatin immunoprecipitation and gene expression profiling to identify MYC-dependent regulatory events. As previously reported, MYC-expressing mice exhibited hepatoblastoma- and hepatocellular carcinoma-like tumors, which regressed when MYC expression was suppressed. We further show that cellular transformation, and thus initiation of liver tumorigenesis, were impaired in mice harboring a MYC mutant unable to associate with the corepressor protein MIZ1 (ZBTB17). Notably, switching off the oncogene in advanced carcinomas revealed that MYC was required for the continuous activation and repression of distinct sets of genes, constituting no more than half of all genes deregulated during tumor progression and an even smaller subset of all MYC-bound genes. Altogether, our data provide the first detailed analysis of a MYC-dependent transcriptional program in a fully developed carcinoma and offer a guide to identifying the critical effectors contributing to MYC-driven tumor maintenance. Cancer Res; 76(12); 3463-72. ©2016 AACR.

The mitochondrial translation machinery as a therapeutic target in Myc-driven lymphomas

The oncogenic transcription factor Myc is required for the progression and maintenance of diverse tumors. This has led to the concept that Myc itself, Myc-activated gene products, or associated biological processes might constitute prime targets for cancer therapy. Here, we present an in vivo reverse-genetic screen targeting a set of 241 Myc-activated mRNAs in mouse B-cell lymphomas, unraveling a critical role for the mitochondrial ribosomal protein (MRP) Ptcd3 in tumor maintenance. Other MRP-coding genes were also up regulated in Myc-induced lymphoma, pointing to a coordinate activation of the mitochondrial translation machinery. Inhibition of mitochondrial translation with the antibiotic Tigecycline was synthetic-lethal with Myc activation, impaired respiratory activity and tumor cell survival in vitro, and significantly extended lifespan in lymphoma-bearing mice. We have thus identified a novel Myc-induced metabolic dependency that can be targeted by common antibiotics, opening new therapeutic perspectives in Myc-overexpressing tumors.

Mutual epithelium-macrophage dependency in liver carcinogenesis mediated by ST18

The ST18 gene has been proposed to act either as a tumor suppressor or as an oncogene in different human cancers, but direct evidence for its role in tumorigenesis has been lacking thus far. Here, we demonstrate that ST18 is critical for tumor progression and maintenance in a mouse model of liver cancer, based on oncogenic transformation and adoptive transfer of primary precursor cells (hepatoblasts). ST18 messenger RNA (mRNA) and protein were detectable neither in normal liver nor in cultured hepatoblasts, but were readily expressed after subcutaneous engraftment and tumor growth. ST18 expression in liver cells was induced by inflammatory cues, including acute or chronic inflammation in vivo, as well as coculture with macrophages in vitro. Knocking down the ST18 mRNA in transplanted hepatoblasts delayed tumor progression. Induction of ST18 knockdown in pre‐established tumors caused rapid tumor involution associated with pervasive morphological changes, proliferative arrest, and apoptosis in tumor cells, as well as depletion of tumor‐associated macrophages, vascular ectasia, and hemorrhage. Reciprocally, systemic depletion of macrophages in recipient animals had very similar phenotypic consequences, impairing either tumor development or maintenance, and suppressing ST18 expression in hepatoblasts. Finally, RNA sequencing of ST18‐depleted tumors before involution revealed down‐regulation of inflammatory response genes, pointing to the suppression of nuclear factor kappa B–dependent transcription. Conclusion: ST18 expression in epithelial cells is induced by tumor‐associated macrophages, contributing to the reciprocal feed‐forward loop between both cell types in liver tumorigenesis. Our findings warrant the exploration of means to interfere with ST18‐dependent epithelium–macrophage interactions in a therapeutic setting. (Hepatology 2017;65:1708‐1719).

Smyd2 is a Myc-regulated gene critical for MLL-AF9 induced leukemogenesis

The Smyd2 protein (Set- and Mynd domain containing protein 2) is a methyl-transferase that can modify both histones and cytoplasmic proteins. Smyd2 is over-expressed in several cancer types and was shown to be limiting for tumor development in the pancreas. However, genetic evidence for a role of Smyd2 in other cancers or in mouse development was missing to date. Using germ line-deleted mouse strains, we now show that Smyd2 and the related protein Smyd3 are dispensable for normal development. Ablation of Smyd2 did not affect hematopoiesis, but retarded the development of leukemia promoted by MLL-AF9, a fusion oncogene associated with acute myeloid leukemia (AML) in humans. Smyd2-deleted leukemic cells showed a competitive disadvantage relative to wild-type cells, either in vitro or in vivo. The Smyd2 gene was directly activated by the oncogenic transcription factor Myc in either MLL9-AF9-induced leukemias, Myc-induced lymphomas, or fibroblasts. However, unlike leukemias, the development of lymphomas was not dependent upon Smyd2. Our data indicate that Smyd2 has a critical role downstream of Myc in AML.

Therapeutic synergy between tigecycline and venetoclax in a preclinical model of MYC/BCL2 double-hit B cell lymphoma

The antibiotic tigecycline and the BCL2 inhibitor venetoclax cooperate to treat MYC/BCL2 double-hit lymphomas in xenografted mice. Doubling up against double-hit lymphoma Double-hit lymphomas, a type of B cell lymphomas with concurrent activation of the MYC and BCL2 oncogenes, are aggressive and difficult-to-treat tumors. In light of evidence showing that tigecycline, an antibiotic, may be effective against MYC-driven lymphomas, Ravà et al. combined it with venetoclax, a BCL2 inhibitor, to target the double-hit tumors. The authors found that the pair of clinically approved drugs showed synergy against the cancer and was also effective in combination with rituximab, a mainstay of current treatment for lymphoma. High-grade B cell lymphomas with concurrent activation of the MYC and BCL2 oncogenes, also known as double-hit lymphomas (DHL), show dismal prognosis with current therapies. MYC activation sensitizes cells to inhibition of mitochondrial translation by the antibiotic tigecycline, and treatment with this compound provides a therapeutic window in a mouse model of MYC-driven lymphoma. We now addressed the utility of this antibiotic for treatment of DHL. BCL2 activation in mouse Eμ-myc lymphomas antagonized tigecycline-induced cell death, which was specifically restored by combined treatment with the BCL2 inhibitor venetoclax. In line with these findings, tigecycline and two related antibiotics, tetracycline and doxycycline, synergized with venetoclax in killing human MYC/BCL2 DHL cells. Treatment of mice engrafted with either DHL cell lines or a patient-derived xenograft revealed strong antitumoral effects of the tigecycline/venetoclax combination, including long-term tumor eradication with one of the cell lines. This drug combination also had the potential to cooperate with rituximab, a component of current front-line regimens. Venetoclax and tigecycline are currently in the clinic with distinct indications: Our preclinical results warrant the repurposing of these drugs for combinatorial treatment of DHL.

Co-optation of Tandem DNA Repeats for the Maintenance of Mesenchymal Identity

Tandem repeats (TRs) are generated by DNA replication errors and retain a high level of instability, which in principle would make them unsuitable for integration into gene regulatory networks. However, the appearance of DNA sequence motifs recognized by transcription factors may turn TRs into functional cis-regulatory elements, thus favoring their stabilization in genomes. Here, we show that, in human cells, the transcriptional repressor ZEB1, which promotes the maintenance of mesenchymal features largely by suppressing epithelial genes and microRNAs, occupies TRs harboring dozens of copies of its DNA-binding motif within genomic loci relevant for maintenance of epithelial identity. The deletion of one such TR caused quasi-mesenchymal cancer cells to reacquire epithelial features, partially recapitulating the effects of ZEB1 gene deletion. These data demonstrate that the high density of identical motifs in TRs can make them suitable platforms for recruitment of transcriptional repressors, thus promoting their exaptation into pre-existing cis-regulatory networks.

Sustained activation of detoxification pathways promotes liver carcinogenesis in response to chronic bile acid-mediated damage

Chronic inflammation promotes oncogenic transformation and tumor progression. Many inflammatory agents also generate a toxic microenvironment, implying that adaptive mechanisms must be deployed for cells to survive and undergo transformation in such unfavorable contexts. A paradigmatic case is represented by cancers occurring in pediatric patients with genetic defects of hepatocyte phosphatidylcholine transporters and in the corresponding mouse model (Mdr2-/- mice), in which impaired bile salt emulsification leads to chronic hepatocyte damage and inflammation, eventually resulting in oncogenic transformation. By combining genomics and metabolomics, we found that the transition from inflammation to cancer in Mdr2-/- mice was linked to the sustained transcriptional activation of metabolic detoxification systems and transporters by the Constitutive Androstane Receptor (CAR), a hepatocyte-specific nuclear receptor. Activation of CAR-dependent gene expression programs coincided with reduced content of toxic bile acids in cancer nodules relative to inflamed livers. Treatment of Mdr2-/- mice with a CAR inhibitor blocked cancer progression and caused a partial regression of existing tumors. These results indicate that the acquisition of resistance to endo- or xeno-biotic toxicity is critical for cancers that develop in toxic microenvironments.

Abstract A27: Epigenomic and transcriptional analyses in a Tet-Myc driven mouse model of liver cancer

The c-myc proto-oncogene is frequently overexpressed in human hepatoblastoma due to gene amplification or constitutively active Wnt signaling. High Myc levels characterize an aggressive hepatoblastoma subtype, reproduced in mice with a liver-specific tetracyclin-regulated c-myc transgene (1, 2). Here, we used an analogous mouse model (tetO-MYC/Lap-tTA; ref. 3) to study the epigenomic and transcriptional mechanisms underlying tumor progression and maintenance. Tet-Myc induction in utero resulted in rapid and fully penetrant tumorigenesis by the age of six weeks. Pre-tumoral changes were detectable already at a late embryonic stage (E18.5) with increased liver size and hepatoblast fraction. As previously described (2), tumors remained dependent on the constitutive expression of the transgene. To study transcriptional changes during tumor progression and regression, we established RNA-seq profiles in control hepatoblasts, pre-tumoral Tet-Myc hepatoblasts and tumor nodules, in the latter before and after short-term inactivation of Tet-Myc. In parallel, we used ChIP-seq to profile the genome-wide distribution of Myc, RNAPII and several histone marks (H3K4me1, H3K4me3, H3K27ac), yielding comprehensive maps of Myc binding to regulatory elements (promoters and enhancers). This unique dataset is providing the basis for mechanistic studies addressing how Myc deregulates its target genes during liver tumorigenesis: comparison of differentially expressed mRNAs and Myc-binding profiles enables us to discriminate between directly and potentially indirectly regulated genes, for which we are currently investigating the underlying molecular basis. Furthermore, our genome-wide data are allowing us to unravel potentially druggable genes and pathways regulated by Myc during hepatoblastoma progression and maintenance. Our progress in characterizing this model will be presented at the meeting. References: 1. Cairo S et al. 2008, Cancer Cell, 14:471-84 2. Shachaf CM et al. 2004, Nature, 431:1112-7 3. van Riggelen J et al. 2010, Genes Dev, 24:1281-94 Citation Format: Theresia Kress, Valerio Bianchi, Luana D9Artista, Paola Nicoli, Mirko Doni, Enrico Radaelli, Bruno Amati. Epigenomic and transcriptional analyses in a Tet-Myc driven mouse model of liver cancer. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr A27.