Biology subjects

Emanuelli, G.

Publications and source records attributed to Emanuelli, G..

4 recordsLinked to original sources

bioRxiv2026

A COJEC-chemotherapy resistant model of Th-ALK(F1174L)/MYCN neuroblastoma offers insights into tumour immune evasion and development of the bone marrow metastatic niche

Multi-agent COJEC chemotherapy is the main-stay of induction treatment for patients diagnosed with high-risk neuroblastoma. However, at least 10% of patients will be primary refractory to chemotherapy and only 50% achieve 5-year overall survival. The bone marrow is the most frequent site of metastasis in these patients. Novel approaches are required to improve response rates but the inter- and intra- patient tumour heterogeneity and dynamics of the neuroblastoma immune microenvironment makes anticipation of resistance phenotypes incredibly challenging. We present here a novel immunocompetent C57 Bl/6 model of Th-ALK(F1174L)/MYCN neuroblastoma, in which spontaneous abdominal tumours are driven by expression of mutant Anaplastic Lymphoma Kinase and over-expression of Mycn in the neural crest. We have used this model to generate a personalised dosing schedule inducing COJEC-chemotherapy resistance, in which individual mice receive chemotherapy cycles dependent upon the progression of their neuroblastoma tumours. Using both single cell RNA sequencing and spatial immunophenotyping gave us extraordinary precision in our comprehensive analysis of the tumour intrinsic and microenvironmental factors associated with COJEC resistance. We found that the resistance phenotype was driven by Cdk8 upregulation in adrenergic and mesenchymal tumour cells. Infiltration of immunosuppressive myeloid-derived immune cells and remodeling of the tumour-associated stroma further contributed to COJEC resistance. In the bone marrow we observed expansion of neutrophils and evidence of NETosis associated with micro-metastatic disease. Our results further endorse the development of CDK8-targeting therapeutics for neuroblastoma patients which might boost the anti-tumour immune response. Additional studies will be required to define the roles of neutrophils and neutrophil NETosis in neuroblastoma progression and metastasis. Our C57 Bl/6 model will be pivotal in future preclinical studies of immune-modulating therapeutics.

cancer biology↗

bioRxiv2025

A GCN1-independent activator of the kinase GCN2

Mutations of EIF2AK4, which encodes the eIF2 kinase GCN2, cause a severe inherited form of pulmonary hypertension called pulmonary veno-occlusive disease (PVOD). Some pathogenic variants of GCN2 are amenable to pharmacological reactivation by low concentrations of ATP-pocket binding inhibitors. Kinase inhibition at modestly elevated concentrations limits the clinical utility of these drugs against PVOD. We therefore performed an in cellulo chemical screen for GCN2 activators and identified three structurally distinct compounds with low micromolar stimulatory activities. Unlike previously described GCN2 activators, one of these molecules activated GCN2 independently of GCN1. Modelling supported by structure activity screens suggested it binds within the ATP-pocket of GCN2, but unlike existing ligands does not protrude inward into the allosteric pocket or outward into the solvent. This overcomes a key requirement of other GCN2 activators.

cell biology↗

bioRxiv2024

Functional validation of EIF2AK4 (GCN2) missense variants associated with pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a disorder with a large genetic component. Biallelic mutations of EIF2AK4, which encodes the kinase GCN2, are causal in two ultra-rare subtypes of PAH, pulmonary veno-occlusive disease and pulmonary capillary haemangiomatosis. EIF2AK4 variants of unknown significance have also been identified in patients with classical PAH, though their relationship to disease remains unclear. To provide patients with diagnostic information and enable family testing, the functional consequences of such rare variants must be determined, but existing computational methods are imperfect. We applied a suite of bioinformatic and experimental approaches to sixteen EIF2AK4 variants that had been identified in patients. By experimentally testing the functional integrity of the integrated stress response (ISR) downstream of GCN2, we determined that existing computational tools have insufficient sensitivity to reliably predict impaired kinase function. We determined experimentally that several EIF2AK4 variants identified in patients with classical PAH had preserved function and are therefore likely to be non-pathogenic. The dysfunctional variants of GCN2 that we identified could be subclassified into three groups: misfolded, kinase-dead, and hypomorphic. Intriguingly, members of the hypomorphic group were amenable to paradoxical activation by a type-1.5 GCN2 kinase inhibitor. This experiment approach may aid in the clinical stratification of EIF2AK4 variants and potentially identify hypomorophic alleles receptive to pharmacological activation.

cell biology↗

bioRxiv2020

A roadmap for the characterization of energy metabolism in human cardiomyocytes derived from induced pluripotent stem cells

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are an increasingly employed model in cardiac research and drug discovery. A significant limitation with respect to clinical translation is their immature structural and functional phenotype. Cellular metabolism plays an integral role in determining phenotype but the metabolic profile of hiPSC-CM during maturation is poorly characterized. In this study we employ a combination of methods including extracellular flux and 13C-glucose flux analyses to study the metabolic profile of hiPSC-CM over a 12 week maturation period. Results show a progressive remodeling of pathways involved in energy metabolism and substrate utilization. The oxidative capacity of hiPSC-CM and particularly their ability to utilize fatty acids increased with time. In parallel, relative glucose oxidation was reduced while glutamine oxidation was maintained at similar levels. Anaerobic glycolysis as assessed by lactate production was maintained throughout the 12 week period but with significant alterations in proximal glycolytic enzymes such as hexokinase and phosphofructokinase. We also observed a progressive maturation of mitochondrial oxidative capacity at comparable levels of mitochondrial content between timepoints. The time-dependent restructuring of the hiPSC-CM metabolic profile indicates their potential to overcome the lack of full maturation previously reported and enhance their applicability for pharmacological studies and cardiac disease modeling.

cell biology↗