Hot spot mutation in receptor tyrosine kinases elicits pro-oncogenic effects

Tyrosine kinase receptors are frequently altered both in expression and activity in cancer. We performed a pan-cancer analysis to identify novel putative cancer driver mutations or/and therapeutically actionable mutations of the kinase domain of different tyrosine kinase receptors (RTK). 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. 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 and found in FGFR1-4 , FLT3, FLT4, PFGFRA, EGFR , VEGFR2 receptors, possibly leading to constitutive activation of the RTK. These amino acid residues correspond to the well-known activating V600E mutation of the oncogene B-Raf. Our analyses demonstrate that the previously uncharacterized VEGFR2 R1051Q and FGFR1 D647N mutations lead to receptor phosphorylation and metabolic changes which might be worth comparing and investigating. For this purpose, residue-substitutions were introduced by point mutation using the Quikchange 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. 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, possibly suggesting an aggressive and metastatic cell phenotype both in vitro and in vivo invasion assay (i.e. CAM). 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. These data were confirmed in vivo for VEGFR2 R1051Q mutant. Indeed, Sk-Mel-31 expressing VEGFR2 R1051Q, injected s.c. n NOD/SCID mice, are more sensitive to the VEGFR2-targeted TKI Linifanib. Our data confirm that similar alterations in the tyrosine kinase domain modulate similar biological responses and druggability.