Biochemical analysis of EGFR exon20 insertion variants insASV and insSVD and their inhibitor sensitivity
Hanchen Zhao1,2, Tyler S Beyett1,2, Jie Jiang3,4,5
1Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215.
Abstract:
Somatic mutations in the epidermal growth factor receptor (EGFR) are a major cause of non-small cell lung cancer. Among these structurally diverse alterations, exon 20 insertions represent a unique subset that rarely respond to EGFR tyrosine kinase inhibitors (TKIs). Therefore, there is a significant need to develop inhibitors that are active against this class of activating mutations. Here, we conducted biochemical analysis of the two most frequent exon 20 insertion variants, V769_D770insASV (insASV) and D770_N771insSVD (insSVD) to better understand their drug sensitivity and resistance. From kinetic studies, we found that EGFR insASV and insSVD are similarly active, but have lower Km, ATP values compared to the L858R variant, which contributes to their lack of sensitivity to 1st-3rd generation EGFR TKIs. Biochemical, structural, and cellular studies of a diverse panel of EGFR inhibitors revealed that the more recently developed compounds BAY-568, TAS6417, and TAK-788 inhibit EGFR insASV and insSVD in a mutant-selective manner, with BAY-568 being the most potent and selective versus wild-type (WT) EGFR. Cocrystal structures with WT EGFR reveal the binding modes of each of these inhibitors and of poziotinib, a potent but not mutantselective inhibitor, and together they define interactions shared by the mutant-selective agents. Collectively, our results show that these exon20 insertion variants are not inherently inhibitor resistant, rather they differ in their drug sensitivity from WT EGFR. However, they are similar to each other, indicating that a single inhibitor should be effective for several of the diverse exon 20 insertion variants.
Insights
New epidermal growth factor receptor (EGFR) inhibitors show promise against lung cancer exon 20 insertions. These mutations, common in non-small cell lung cancer (NSCLC), are often resistant to existing therapies, but novel compounds demonstrate potent and selective activity.
Area of Science:
- Oncology
- Molecular Biology
- Drug Discovery
Background:
- Somatic mutations in the epidermal growth factor receptor (EGFR) drive non-small cell lung cancer (NSCLC) development.
- EGFR exon 20 insertions are a distinct subset of activating mutations conferring resistance to standard EGFR tyrosine kinase inhibitors (TKIs).
- Developing effective therapies for EGFR exon 20 insertion-driven NSCLC remains a critical unmet need.
Purpose of the Study:
- To biochemically characterize the drug sensitivity and resistance profiles of common EGFR exon 20 insertion variants (V769_D770insASV and D770_N771insSVD).
- To evaluate the efficacy and selectivity of novel EGFR inhibitors against these specific mutations.
- To elucidate the structural basis for the differential sensitivity of mutant versus wild-type (WT) EGFR to various inhibitors.
Main Methods:
- Biochemical kinetic analyses to determine enzyme kinetics (Km, ATP) for mutant and WT EGFR.
- In vitro biochemical, structural (cocrystal), and cellular assays using a panel of EGFR inhibitors.
- Comparative analysis of inhibitor potency and selectivity against EGFR exon 20 insertion variants and WT EGFR.
Main Results:
- EGFR exon 20 insertion variants (insASV and insSVD) exhibit similar enzymatic activity but lower Km, ATP compared to L858R, explaining resistance to 1st-3rd generation TKIs.
- Novel inhibitors BAY-568, TAS6417, and TAK-788 demonstrate mutant-selective inhibition of EGFR insASV and insSVD.
- BAY-568 exhibits the highest potency and selectivity against WT EGFR among the tested novel compounds. Cocrystal structures reveal shared binding interactions for mutant-selective inhibitors.
Conclusions:
- EGFR exon 20 insertion variants are not inherently resistant but possess distinct drug sensitivity profiles compared to WT EGFR.
- The characterized novel inhibitors, particularly BAY-568, show significant potential for treating NSCLC with EGFR exon 20 insertions.
- The similar biochemical properties of common exon 20 insertion variants suggest a single inhibitor could be effective against multiple mutation types.
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