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Abstract

Neuroblastoma (NB) is the most common extracranial solid tumor in infants, arising from the developing sympathetic nervous system. A subgroup of approximately 6-8% of NB patients harbors an alteration in the anaplastic lymphoma kinase ALK gene, making this receptor tyrosine kinase an important therapeutic target. Small-molecule inhibitors targeting ALK are already in clinical use as monotherapies, but an optimal integration into the standard care has yet to be established. This study aims at characterizing the inhibitory properties of the third generation ALK inhibitor lorlatinib (LOR) on proliferation of tumor cells and its potential in a combinatorial therapy approach.

In this study, treatment with LOR was investigated in a selection of ALK amplified, F1174L and R1275Q mutated, as well as wild-type NB cell lines. We found that the sensitivity to LOR correlated with expression levels of activated ALK protein. ALK addicted NB-1 cells, which expressed the highest ALK protein levels, were the most sensitive amongst all tested cell lines and were further used as model system to study ALK inhibition in vitro. In these cells, LOR treatment reduced cell viability and proliferation by inhibiting the Ras-MAPK and PI3K-AKT signaling pathways, leading to a predominant G1 cell cycle arrest. Cell death was induced only at concentrations above a clinically achievable level. Despite a high initial sensitivity to LOR, cells could overcome this arrest and gain resistance after long-term treatment. Thus, LOR may not be appropriate for sustained monotherapy. Therefore, we tested whether the treatment of NB-1 cells could benefit from combination therapy with the two first-line chemotherapeutics cisplatin (CDDP) and vincristine (VCR), both in a classical two-dimensional in vitro setting and also in multicellular tumor spheroids. Indeed, combination therapy could significantly reduce cell viability in NB-1 cells. In contrast, NB cell lines that harbor an ALK alteration but express lower amounts of active ALK protein were less sensitive or even resistant to LOR and could not benefit from combination therapies. Furthermore, basal expression of ALK signature genes did not reflect the sensitivity of the different cell lines to LOR. Therefore, RNAseq data that are frequently acquired for NB patients may not be adequate to predict therapy response.

Taken together, this study shows that not only the genomic ALK status is relevant for therapeutic success, suggesting that protein levels should be examined before the treatment with ALK inhibitors. Furthermore, this study indicates that a novel combinatorial approach of LOR with CDDP or VCR is feasible for ALK addicted NBs.