2 research outputs found
Illuminating the Molecular Mechanisms of Tyrosine Kinase Inhibitor Resistance for the FGFR1 Gatekeeper Mutation: The Achilles’ Heel of Targeted Therapy
Human
fibroblast growth factor receptors (FGFRs) 1–4 are
a family of receptor tyrosine kinases that can serve as drivers of
tumorigenesis. In particular, <i>FGFR1</i> gene amplification
has been implicated in squamous cell lung and breast cancers. Tyrosine
kinase inhibitors (TKIs) targeting FGFR1, including AZD4547 and E3810
(Lucitanib), are currently in early phase clinical trials. Unfortunately,
drug resistance limits the long-term success of TKIs, with mutations
at the “gatekeeper” residue leading to tumor progression.
Here we show the first structural and kinetic characterization of
the FGFR1 gatekeeper mutation, V561M FGFR1. The V561M mutation confers
a 38-fold increase in autophosphorylation achieved at least in part
by a network of interacting residues forming a hydrophobic spine to
stabilize the active conformation. Moreover, kinetic assays established
that the V561M mutation confers significant resistance to E3810, while
retaining affinity for AZD4547. Structural analyses of these TKIs
with wild type (WT) and gatekeeper mutant forms of FGFR1 offer clues
to developing inhibitors that maintain potency against gatekeeper
mutations. We show that AZD4547 affinity is preserved by V561M FGFR1
due to a flexible linker that allows multiple inhibitor binding modes.
This is the first example of a TKI binding in distinct conformations
to WT and gatekeeper mutant forms of FGFR, highlighting adaptable
regions in both the inhibitor and binding pocket crucial for drug
design. Exploiting inhibitor flexibility to overcome drug resistance
has been a successful strategy for combatting diseases such as AIDS
and may be an important approach for designing inhibitors effective
against kinase gatekeeper mutations
Temporal Resolution of Autophosphorylation for Normal and Oncogenic Forms of EGFR and Differential Effects of Gefitinib
Epidermal growth factor receptor (EGFR) is a member of
the ErbB family of receptor tyrosine kinases (RTK). EGFR overexpression
or mutation in many different forms of cancers has highlighted its
role as an important therapeutic target. Gefitinib, the first small
molecule inhibitor of EGFR kinase function to be approved for the
treatment of nonsmall cell lung cancer (NSCLC) by the FDA, demonstrates
clinical activity primarily in patients with tumors that harbor somatic
kinase domain mutations in EGFR. Here, we compare wild-type EGFR autophosphorylation
kinetics to the L834R (also called L858R) EGFR form, one of the most
common mutations in lung cancer patients. Using rapid chemical quench,
time-resolved electrospray mass spectrometry (ESI-MS), and Western
blot analyses, we examined the order of autophosphorylation in wild-type
(WT) and L834R EGFR and the effect of gefitinib (Iressa) on the phosphorylation
of individual tyrosines. These studies establish that there is a temporal
order of autophosphorylation of key tyrosines involved in downstream
signaling for WT EGFR and a loss of order for the oncogenic L834R
mutant. These studies also reveal unique signature patterns of drug
sensitivity for inhibition of tyrosine autophosphorylation by gefitinib:
distinct for WT and oncogenic L834R mutant forms of EGFR. Fluorescence
studies show that for WT EGFR the binding affinity for gefitinib is
weaker for the phosphorylated protein while for the oncogenic mutant,
L834R EGFR, the binding affinity of gefitinib is substantially enhanced
and likely contributes to the efficacy observed clinically. This mechanistic
information is important in understanding the molecular details underpinning
clinical observations as well as to aid in the design of more potent
and selective EGFR inhibitors