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Background: Diffuse midline gliomas (DMG) H3K27M-mutant, including diffuse intrinsic pontine glioma (DIPG), are pediatric brain tumors associated with grim prognosis. Although GD2-CAR T-cells demonstrated significant anti-tumor activity against DMG H3K27M-mutant in vivo, a multimodal approach may be needed to more effectively treat patients. We investigated GD2 expression in DMG/DIPG and other pediatric high-grade gliomas (pHGG) and sought to identify chemical compounds that would enhance GD2-CAR T-cell anti-tumor efficacy. Methods: Immunohistochemistry in tumor tissue samples and immunofluorescence in primary patient-derived cell lines were performed to study GD2 expression. We developed a high-throughput cell-based assay to screen 42 kinase inhibitors in combination with GD2-CAR T-cells. Cell viability, western blots, flow-cytometry, real time PCR experiments, DIPG 3D culture models, and orthotopic xenograft model were applied to investigate the effect of selected compounds on DIPG cell death and CAR T-cell function. Results: GD2 was heterogeneously, but widely, expressed in the tissue tested, while its expression was homogeneous and restricted to DMG/DIPG H3K27M-mutant cell lines. We identified dual IGF1R/IR antagonists, BMS-754807 and linsitinib, able to inhibit tumor cell viability at concentrations that do not affect CAR T-cells. Linsitinib, but not BMS-754807, decreases activation/exhaustion of GD2-CAR T-cells and increases their central memory profile. The enhanced anti-tumor activity of linsitinib/GD2-CAR T-cell combination was confirmed in DIPG models in vitro, ex vivo, and in vivo. Conclusion: Our study supports the development of IGF1R/IR inhibitors to be used in combination with GD2-CAR T-cells for treating patients affected by DMG/DIPG and, potentially, by pHGG.

Background Pediatric high-grade gliomas (pHGGs) are among the most common and incurable malignant neoplasms of childhood. Despite aggressive, multimodal treatment, the outcome of children with high-grade gliomas has not significantly improved over the past decades, prompting the development of innovative approaches. Methods To develop an effective treatment, we aimed at improving the suboptimal antitumor efficacy of oncolytic adenoviruses (OAs) by testing the combination with a gene-Therapy approach using a bispecific T-cell engager (BiTE) directed towards the erythropoietin-producing human hepatocellular carcinoma A2 receptor (EphA2), conveyed by a replication-incompetent adenoviral vector (EphA2 adenovirus (EAd)). The combinatorial approach was tested in vitro, in vivo and thoroughly characterized at a molecular level. Results After confirming the relevance of EphA2 as target in pHGGs, documenting a significant correlation with worse clinical outcome of the patients, we showed that the proposed strategy provides significant EphA2-BiTE amplification and enhanced tumor cell apoptosis, on coculture with T cells. Moreover, T-cell activation through an agonistic anti-CD28 antibody further increased the activation/proliferation profiles and functional response against infected tumor cells, inducing eradication of highly resistant, primary pHGG cells. The gene-expression analysis of tumor cells and T cells, after coculture, revealed the importance of both EphA2-BiTE and costimulation in the proposed system. These in vitro observations translated into significant tumor control in vivo, in both subcutaneous and a more challenging orthotopic model. Conclusions The combination of OA and EphA2-BiTE gene therapy strongly enhances the antitumor activity of OA, inducing the eradication of highly resistant tumor cells, thus supporting the clinical translation of the approach.

Rhabdomyosarcoma (RMS) is an aggressive pediatric malignancy of the muscle, that includes Fusion Positive (FP)-RMS harboring PAX3/7-FOXO1 and Fusion Negative (FN)-RMS commonly with RAS pathway mutations. RMS express myogenic master transcription factors MYOD and MYOG yet are unable to terminally differentiate. Here, we report that SNAI2 is highly expressed in FN-RMS, is oncogenic, blocks myogenic differentiation, and promotes growth. MYOD activates SNAI2 transcription via super enhancers with striped 3D contact architecture. Genome wide chromatin binding analysis demonstrates that SNAI2 preferentially binds enhancer elements and competes with MYOD at a subset of myogenic enhancers required for terminal differentiation. SNAI2 also suppresses expression of a muscle differentiation program modulated by MYOG, MEF2, and CDKN1A. Further, RAS/MEK-signaling modulates SNAI2 levels and binding to chromatin, suggesting that the differentiation blockade by oncogenic RAS is mediated in part by SNAI2. Thus, an interplay between SNAI2, MYOD, and RAS prevents myogenic differentiation and promotes tumorigenesis.

Mammalian development, adult tissue homeostasis and the avoidance of severe diseases including cancer require a properly orchestrated cell cycle, as well as error-free genome maintenance. The key cell-fate decision to replicate the genome is controlled by two major signalling pathways that act in parallel—the MYC pathway and the cyclin D–cyclin-dependent kinase (CDK)–retinoblastoma protein (RB) pathway1,2. Both MYC and the cyclin D–CDK–RB axis are commonly deregulated in cancer, and this is associated with increased genomic instability. The autophagic tumour-suppressor protein AMBRA1 has been linked to the control of cell proliferation, but the underlying molecular mechanisms remain poorly understood. Here we show that AMBRA1 is an upstream master regulator of the transition from G1 to S phase and thereby prevents replication stress. Using a combination of cell and molecular approaches and in vivo models, we reveal that AMBRA1 regulates the abundance of D-type cyclins by mediating their degradation. Furthermore, by controlling the transition from G1 to S phase, AMBRA1 helps to maintain genomic integrity during DNA replication, which counteracts developmental abnormalities and tumour growth. Finally, we identify the CHK1 kinase as a potential therapeutic target in AMBRA1-deficient tumours. These results advance our understanding of the control of replication-phase entry and genomic integrity, and identify the AMBRA1–cyclin D pathway as a crucial cell-cycle-regulatory mechanism that is deeply interconnected with genomic stability in embryonic development and tumorigenesis.

Bone marrow (BM) is the major target organ for neuroblastoma (NB) metastasis and its involvement is associated with poor outcome. Yet, the mechanism by which NB cells invade BM is largely unknown. Tumour microenvironment represents a key element in tumour progression and mesenchymal stromal cells (MSCs) have been recognized as a fundamental part of the associated tumour stroma. Here, we show that BM-MSCs isolated from NB patients with BM involvement exhibit a greater osteogenic potential than MSCs from non-infiltrated BM. We show that BM metastasis-derived NB-cell lines secrete higher levels of exosomal miR-375, which promotes osteogenic differentiation in MSCs. Of note, clinical data demonstrate that high level of miR-375 correlates with BM metastasis in NB patients. Our findings suggest, indeed, a potential role for exosomal miR-375 in determining a favourable microenvironment in BM to promote metastatic progression. MiR-375 may, thus, represent a novel biomarker and a potential target for NB patients with BM involvement.

Background & Aim: Non-alcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease in adults and children. Along with obesity, diabetes and insulin resistance, genetic factors strongly impact on NAFLD development and progression. Dysregulated bile acid metabolism and the fibroblast growth factor 19 (FGF19) pathway play a pivotal role in NAFLD pathogenesis. However, the mechanism through which the FGF19 receptor system is associated with liver damage in NAFLD remains to be defined.Methods: We evaluated the impact of the rs17618244 G>A beta-Klotho (KLB) variant on liver damage in 249 pediatric patients with biopsy-proven NAFLD and the association of this variant with the expression of hepatic and soluble KLB. In vitro models were established to investigate the role of the KLB mutant.Results: The KLB rs17618244 variant was associated with an increased risk of ballooning and lobular inflammation. KLB plasma levels were lower in carriers of the rs17618244 minor A allele and were associated with lobular inflammation, ballooning and fibrosis. In HepG2 and Huh7 hepatoma cell lines, exposure to free fatty acids caused a severe reduction of intracellular and secreted KLB. Finally, KLB downregulation obtained by the expression of a KLB mutant in HepG2 and Huh7 cells induced intracellular lipid accumulation and upregulation of p62, ACOX1, ACSL1, IL-1 beta and TNF-alpha gene expression.Conclusion: In conclusion, we showed an association between the rsl 7618244 KLB variant, which leads to reduced KLB expression, and the severity of NAFLD in pediatric patients. We can speculate that the KLB protein may exert a protective role against lipotoxicity and inflammation in hepatocytes.Lay summary: Genetic and environmental factors strongly impact on the pathogenesis and progression of non-alcoholic fatty liver disease (NAFLD). The FGF19/FGFR4/KLB pathway plays a pivotal role in the pathogenesis of NAFLD. The aim of the study was to investigate the impact of