VEGFR2(R1051Q) and FGFR1(D647N) correspondent mutations exhibit pro-oncogenic effects both in vitro and in vivo

Background Tyrosine kinase receptors(RTK) are frequently altered both in expression and activity in cancer. We performed a pan-cancer analysis to identify novel putative cancer driver or/and therapeutically actionable mutations of the kinase domain of different RTK(1). To pinpoint those mutations that may be clinically relevant, we exploited the recurrence of alterations on analogous amino acid residues within the kinase domain (PK_Tyr_Ser-Thr) across different kinases as a predictor of functional impact(2). By exploiting MutationAligner and LowMACA bioinformatics resources, we highlighted novel uncharacterized mutations in position 256 of the consensus sequences of KD. This alteration is located in the A-loop of TKD of FGFR1-4, FLT3, FLT4, PDGFRA, EGFR, VEGFR2 receptors, possibly leading to constitutive activation of the RTK(3). Methods Our previous results demonstrate that the substitution R1051Q in VEGFR2 leads to an increase of kinase activity and phosphorylation of receptor and supports metabolic changes. In order to evaluate if similar alterations in the tyrosine kinase domain modulate similar biological responses and druggability, we introduced the R1051Q correspondent substitution in FGFR1 (FGFR1 D647N) using the Quickchange lightning site-directed mutagenesis kit. Receptors and their mutants were expressed in HEK293T cells and assessed for ATP affinity using an ADP-glo kinase assay and phosphorylation rate through WB analysis. Cell migratory capacity and metastatic cell phenotype were assessed with both in vitro and in vivo invasion assay (i.e., CAM). Results The results confirm a similar altered ATP affinity as well as a great phosphorylation for both mutant receptors. When expressed in tumoral cells both mutants lead to a strong increase in the cell migratory capacity, compared to WT receptor. Finally, it was evaluated the activity of tyrosine kinase inhibitors (TKi) including Erdafitinib, Lenvatinib, Sunitinib and Linifanib, following receptor phosphorylation by WB. Both mutated receptors exhibit higher sensitivity to TKi. Conclusions Our data confirm our previous hypotheses concerning the biological effect of these mutations. Future studies will allow to extend this knowledge to other significant mutations in the same hotspot position. This concept is particularly important for therapeutically actionable mutations. Indeed, PD-based analyses have the potential to accelerate the choice of patient-specific targeted drugs.