Targeting the LIN28B/let-7 axis by small molecules in Neuroblastoma

Neuroblastoma (NB) is a very heterogeneous tumour derived from undifferentiated cells of the neural crest. It is the most common extracranial solid tumour in children, characterised by a large variability of the clinical outcome. The clinically aggressive form of the disease, the high-risk neuroblastoma, affects about 50% of patients, which, unfortunately, despite the intensity of therapies, have a survival rate of less than 50%. Although the introduction of two new therapeutic solutions based on the use of 13-cis-retinoic acid and the immunotherapy with a chimeric monoclonal antibody against the GD2 ganglioside, 20% of patients affected by the high-risk disease are still entirely refractory to treatments, and 60% will relapse. In this panorama, identifying new strategies specific for critical NB targets is endowed with great potential to improve the survival rate and long-term quality of life and reduce the elevated toxicity of current treatments. LIN28B is an RNA binding protein extensively overexpressed in NB. Its exogenous expression in mouse sympatho-adrenergic lineage is able to reproduce the human disease, underlying the importance of LIN28B in NB pathogenesis. LIN28B prevents the maturation of let-7 miRNA family members, an important group of tumour suppressors that induce differentiation and, at the same time, decrease cell proliferation. We hypothesised that interfering with the LIN28B/let-7 miRNA interaction could lead to an increase in let-7 miRNA levels and, consequently, to a decrease in cell proliferation and an induction of cell differentiation, ultimately reducing NB aggressiveness. First, we created NB cell lines with stable LIN28B down-regulation, and we observed an expected increase in let-7 miRNA levels. We then verified if the rise in the let-7 miRNAs could induce the cells' differentiation by analysing a panel of stemness and differentiation markers such as SOX2, SOX9 and β-III-tubulin, detecting a decrease in the stemness markers and an increase in the differentiation-related markers. Following a high-throughput screening, performed and validated with two orthogonal biochemical techniques, the Alpha screen and the REMSA, we identified molecule A as the molecule with the best inhibitory activity on the LIN28B/let-7 miRNA interaction. After the biochemical validation, we proceeded to assess molecule A activity in vitro on NB cell lines. Molecule A resulted to be very unstable in cell culture conditions, therefore we decided to include the molecule in PLGA-PEG nanoparticles to preserve its stability in solution and improve its activity. Upon encapsulation, we observed a substantial increase in molecule A effects, leading to a strong increment in mature let-7 miRNAs and a consequent inhibition of cellular growth. Finally, we tested if the let-7 miRNAs increment caused by molecule A treatment was sufficient to induce NB cells differentiation, as observed with stable LIN28B-downregulation. We detected an increase in differentiation marker levels suggesting that the treatment with Molecule A nanoparticles is able to lead to the induction of neuronal differentiation processes in NB cells. Although these last results need to be confirmed with further experiments, they clearly show that the LIN28B/let-7 miRNA axis represents a good therapeutic target and that molecule A and/or other molecules able to interfere with this interaction deserve further preclinical and clinical evaluation.