CBFA2T3-GLIS2-dependent pediatric acute megakaryoblastic leukemia is driven by GLIS2 and sensitive to navitoclax.

Pediatric acute megakaryoblastic leukemia (AMKL) is an aggressive blood cancer associated with poor therapeutic response and high mortality. Here we describe the development of CBFA2T3-GLIS2-driven mouse models of AMKL that recapitulate the phenotypic and transcriptional signatures of the human disease. We show that an activating Ras mutation that occurs in human AMKL increases the penetrance and decreases the latency of CBF2AT3-GLIS2-driven AMKL. CBFA2T3-GLIS2 and GLIS2 modulate similar transcriptional networks. We identify the dominant oncogenic properties of GLIS2 that trigger AMKL in cooperation with oncogenic Ras. We find that both CBFA2T3-GLIS2 and GLIS2 alter the expression of a number of BH3-only proteins, causing AMKL cell sensitivity to the BCL2 inhibitor navitoclax both in vitro and in vivo, suggesting a potential therapeutic option for pediatric patients suffering from CBFA2T3-GLIS2-driven AMKL.

An EZH2 blocker sensitizes histone mutated diffuse midline glioma to cholesterol metabolism inhibitors through an off-target effect.

Background: Diffuse Midline Glioma, H3K27M-mutant (DMG) is a rare, highly aggressive pediatric tumor affecting the brainstem, and is one of the deadliest cancers. Currently available treatment options such as chemotherapy and radiotherapy do only modestly prolong survival. In this pathology, H3K27 mutations deregulate Polycomb Repressive Complex 2 (PRC2), including enzymatic activity of EZH2, which is therefore under investigation as a therapeutic target. Methods: We used a chemical EZH2 inhibitor, GSK126, small interfering RNAs, and a CRISPR/Cas9 knockout approaches in a series of DMG tumor cell lines to investigate metabolic treatment responses by proteomic analysis. A combination strategy was elaborated and studied in primary and established DMG cells, spheroid 3D cultures, and in vivo in a chick chorio-allantoic membrane DMG assay and an orthotopic intracranial DMG mouse model. Results: GSK126 shows significant (P < .05-.001) inhibitory effects in in vitro cell proliferation assays and induces apoptosis. Chemical targeting of EZH2 induced expression of proteins implicated in cholesterol metabolism. Low-dose GSK126 treatment together with statins revealed strong growth inhibition in combinatorial treatments, but not in single treatments, both in DMG cells in vitro, in DMG spheroid cultures, and in chick and mouse in vivo models (P < .05). All statistical tests were two-sided. Conclusions: Our results reveal an unexpected GSK126-inducible sensitivity to cholesterol biosynthesis inhibitors in highly aggressive pediatric glioma that warrants further evaluation as treatment strategy. This combinatorial therapy should have few side effects because of the low doses used to achieve significant anti-tumor activity.

HDAC inhibition induces expression of scaffolding proteins critical for tumor progression in pediatric glioma: focus on EBP50 and IRSp53.

BACKGROUND: Diffuse midline glioma (DMG) is a pediatric malignancy with poor prognosis. Most children die less than one year after diagnosis. Recently, mutations in histone H3 have been identified and are believed to be oncogenic drivers. Targeting this epigenetic abnormality using histone deacetylase (HDAC) inhibitors such as panobinostat (PS) is therefore a novel therapeutic option currently evaluated in clinical trials. METHODS: BH3 profiling revealed engagement in an irreversible apoptotic process of glioma cells exposed to PS confirmed by annexin-V/propidium iodide staining. Using proteomic analysis of 3 DMG cell lines, we identified 2 proteins deregulated after PS treatment. We investigated biological effects of their downregulation by silencing RNA but also combinatory effects with PS treatment in vitro and in vivo using a chick embryo DMG model. Electron microscopy was used to validate protein localization. RESULTS: Scaffolding proteins EBP50 and IRSp53 were upregulated by PS treatment. Reduction of these proteins in DMG cell lines leads to blockade of proliferation and migration, invasion, and an increase of apoptosis. EBP50 was found to be expressed in cytoplasm and nucleus in DMG cells, confirming known oncogenic locations of the protein. Treatment of glioma cells with PS together with genetic or chemical inhibition of EBP50 leads to more effective reduction of cell growth in vitro and in vivo. CONCLUSION: Our data reveal a specific relation between HDAC inhibitors and scaffolding protein deregulation which might have a potential for therapeutic intervention for cancer treatment.

Tracking cellular and molecular changes in a species-specific manner during experimental tumor progression in vivo.

Hepatoblastoma (HBL) is a pediatric liver cancer with defined molecular alterations driving its progression. Here, we describe an animal model for HBL on the chick chorioallantoic membrane (CAM), which recapitulates relevant features of HBL in patients. Expression of classic tumor-associated proteins such as ß-catenin, EpCAM and CK19 was maintained in acini-like organized tumors on CAM, as was synthesis of AFP, a tumor marker used for monitoring patient response. RNA sequencing revealed an unexpected molecular evolution of HBL cells on the CAM, with significant deregulation of more than 6,000 genes including more than half of all HOX genes. Bioinformatic analysis distinguish between tumor cell-expressed genes and chick genes, thereby shedding new light on the complex interactions taking place during HBL progression. Importantly, human tumor suppressive ribosomal genes were downregulated after implantation, whereas mitochondrial genes encoding for anti-apoptotic peptides were strongly induced in vivo. Meprin-1α expression was increased during evolution of CAM tumors and confirmed by immunohistochemistry. Cisplatin, a commonly used chemotherapeutic agent for HBL, showed significant anti-tumoral effects. Our results broaden the understanding of the molecular adaptation process of human cancer cells to the microenvironment and might help to elaborate novel therapeutic concepts for the treatment of this pediatric liver tumor.

Deregulation of MicroRNAs in Gastric Lymphomagenesis Induced in the d3Tx Mouse Model of Helicobacter pylori Infection.

Helicobacter pylori infection is considered as an excellent model of chronic inflammation-induced tumor development. Our project focuses on gastric MALT lymphoma (GML) related to H. pylori infection and mediated by the chronic inflammatory process initiated by the infection. Recently, microRNAs (miRNAs) have emerged as a new class of gene regulators, which play key roles in inflammation and carcinogenesis acting as oncogenes or tumor suppressors. Their precise characterization in the development of inflammation and their contribution in regulating host cells responses to infection by H. pylori have been little explored. Our goal was to analyze the changes in miRNAs in a GML mouse model using BALB/c mice thymectomized at day 3 post-birth (d3Tx model) and to clarify their implication in GML pathogenesis. PCR array followed by RT-qPCR identified five miRNAs (miR-21a, miR-135b, miR-142a, miR-150, miR-155) overexpressed in the stomachs of GML-developing d3Tx mice infected by H. pylori. The analysis of their putative targets allowed us to identify TP53INP1, an anti-proliferative and pro-apoptotic protein, as a common target of 4 of the 5 up-regulated miRNAs. We postulate that these miRNAs may act in synergy to promote the development of GML. miR-142a was also overexpressed in mouse sera samples and therefore could serve as a diagnostic marker. In situ hybridization on gastric samples with miR-142a revealed a global up-regulation of this miRNA by the tumor microenvironment at the lymphoma stage. Dysregulation of miR-21a, miR-135b, miR-142a, miR-150, miR-155 could play a critical role in the pathogenesis of GML and might offer potential applications as therapeutic targets and novel biomarkers for this disease.