An official website of the United States government
Abstract Mechanistic understanding of oncogenic variants facilitates the development and optimization of treatment strategies. We recently identified in-frame, tandem duplication ofEGFRexons 18 - 25, which causes EGFR Kinase Domain Duplication (EGFR-KDD). Here, we characterize the prevalence ofERBBfamily KDDs across multiple human cancers and evaluate the functional biochemistry of EGFR-KDD as it relates to pathogenesis and potential therapeutic intervention. We provide computational and experimental evidence that EGFR-KDD functions by forming asymmetric EGF-independent intra-molecular and EGF-dependent inter-molecular dimers. Time-resolved fluorescence microscopy and co-immunoprecipitation reveals EGFR-KDD can form ligand-dependent inter-molecular homo- and hetero-dimers/multimers. Furthermore, we show that inhibition of EGFR-KDD activity is maximally achieved by blocking both intra- and inter-molecular dimerization. Collectively, our findings define a previously unrecognized model of EGFR dimerization, providing important insights for the understanding of EGFR activation mechanisms and informing personalized treatment of patients with tumors harboring EGFR-KDD. Finally, we establishERBBKDDs as recurrent oncogenic events in multiple cancers.
more »
« less
Allele-specific activation, enzyme kinetics, and inhibitor sensitivities of EGFR exon 19 deletion mutations in lung cancer
Oncogenic mutations within the epidermal growth factor receptor (EGFR) are found in 15 to 30% of all non–small-cell lung carcinomas. The term exon 19 deletion (ex19del) is collectively used to refer to more than 20 distinct genomic alterations within exon 19 that comprise the most common EGFR mutation subtype in lung cancer. Despite this heterogeneity, clinical treatment decisions are made irrespective of which EGFR ex19del variant is present within the tumor, and there is a paucity of information regarding how individual ex19del variants influence protein structure and function. Herein, we identified allele-specific functional differences among ex19del variants attributable to recurring sequence and structure motifs. We built all-atom structural models of 60 ex19del variants identified in patients and combined molecular dynamics simulations with biochemical and biophysical experiments to analyze three ex19del mutations (E746_A750, E746_S752 > V, and L747_A750 > P). We demonstrate that sequence variation in ex19del alters oncogenic cell growth, dimerization propensity, enzyme kinetics, and tyrosine kinase inhibitor (TKI) sensitivity. We show that in contrast to E746_A750 and E746_S752 > V, the L747_A750 > P variant forms highly active ligand-independent dimers. Enzyme kinetic analysis and TKI inhibition experiments suggest that E746_S752 > V and L747_A750 > P display reduced TKI sensitivity due to decreased adenosine 5′-triphosphate K m . Through these analyses, we propose an expanded framework for interpreting ex19del variants and considerations for therapeutic intervention.
more »
« less
- PAR ID:
- 10421702
- Date Published:
- Journal Name:
- Proceedings of the National Academy of Sciences
- Volume:
- 119
- Issue:
- 30
- ISSN:
- 0027-8424
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Numerous studies have shown genetic variation at the LCORL-NCAPG locus is strongly associated with growth traits in beef cattle. However, a causative molecular variant has yet to be identified. To define all possible candidate variants, 34 Charolais-sired calves were whole-genome sequenced, including 17 homozygous for a long-range haplotype associated with increased growth (QQ) and 17 homozygous for potential ancestral haplotypes for this region (qq). The Q haplotype was refined to an 814 kb region between chr6:37,199,897–38,014,080 and contained 218 variants not found in qq individuals. These variants include an insertion in an intron of NCAPG, a previously documented mutation in NCAPG (rs109570900), two coding sequence mutations in LCORL (rs109696064 and rs384548488), and 15 variants located within ATAC peaks that were predicted to affect transcription factor binding. Notably, rs384548488 is a frameshift variant likely resulting in loss of function for long isoforms of LCORL. To test the association of the coding sequence variants of LCORL with phenotype, 405 cattle from five populations were genotyped. The two variants were in complete linkage disequilibrium. Statistical analysis of the three populations that contained QQ animals revealed significant (p < 0.05) associations with genotype and birth weight, live weight, carcass weight, hip height, and average daily gain. These findings affirm the link between this locus and growth in beef cattle and describe DNA variants that define the haplotype. However, further studies will be required to define the true causative mutation.more » « less
-
Abstract Background3′-end processing by cleavage and polyadenylation is an important and finely tuned regulatory process during mRNA maturation. Numerous genetic variants are known to cause or contribute to human disorders by disrupting the cis-regulatory code of polyadenylation signals. Yet, due to the complexity of this code, variant interpretation remains challenging. ResultsWe introduce a residual neural network model,APARENT2, that can infer 3′-cleavage and polyadenylation from DNA sequence more accurately than any previous model. This model generalizes to the case of alternative polyadenylation (APA) for a variable number of polyadenylation signals. We demonstrate APARENT2’s performance on several variant datasets, including functional reporter data and human 3′ aQTLs from GTEx. We apply neural network interpretation methods to gain insights into disrupted or protective higher-order features of polyadenylation. We fine-tune APARENT2 on human tissue-resolved transcriptomic data to elucidate tissue-specific variant effects. By combining APARENT2 with models of mRNA stability, we extend aQTL effect size predictions to the entire 3′ untranslated region. Finally, we perform in silico saturation mutagenesis of all human polyadenylation signals and compare the predicted effects of$${>}43$$ million variants against gnomAD. While loss-of-function variants were generally selected against, we also find specific clinical conditions linked to gain-of-function mutations. For example, we detect an association between gain-of-function mutations in the 3′-end and autism spectrum disorder. To experimentally validate APARENT2’s predictions, we assayed clinically relevant variants in multiple cell lines, including microglia-derived cells. ConclusionsA sequence-to-function model based on deep residual learning enables accurate functional interpretation of genetic variants in polyadenylation signals and, when coupled with large human variation databases, elucidates the link between functional 3′-end mutations and human health.more » « less
-
While worldwide efforts for improving COVID-19 vaccines are currently considered a top priority, the role of the genetic variants responsible for virus receptor protein stability is less studied. Angiotensin-converting enzyme-2 is the primary target of the SARS-CoV-1/SARS-CoV-2 spike (S) glycoprotein, enabling entry into the human body. Here, we applied computational saturation mutagenesis approaches to determine the folding energy caused by all possible mutations in ACE2 proteins within ACE2 - SARS-CoV-1-S/ACE2 - SARS-CoV-2-S complexes. We observed ACE2 mutations at residue D350 causing the most stabilizing effects on the protein. In addition, we identified ACE2 genetic variations in African Americans (rs73635825, rs766996587, and rs780574871), Latino Americans (rs924799658), and both groups (rs4646116 and rs138390800) affecting stability in the ACE2 - SARS-CoV-2-S complex. The findings in this study may aid in targeting the design of stable neutralizing peptides for treating minority patients.more » « less
-
Two amino acid variants in soybean serine hydroxymethyltransferase 8 (SHMT8) are associated with resistance to the soybean cyst nematode (SCN), a devastating agricultural pathogen with worldwide economic impacts on soybean production. SHMT8 is a cytoplasmic enzyme that catalyzes the pyridoxal 5‐phosphate‐dependent conversion of serine and tetrahydrofolate (THF) to glycine and 5,10‐methylenetetrahydrofolate. A previous study of the P130R/N358Y double variant of SHMT8, identified in the SCN‐resistant soybean cultivar (cv.) Forrest, showed profound impairment of folate binding affinity and reduced THF‐dependent enzyme activity, relative to the highly active SHMT8 in cv. Essex, which is susceptible to SCN. Given the importance of SCN‐resistance in soybean agriculture, we report here the biochemical and structural characterization of the P130R and N358Y single variants to elucidate their individual effects on soybean SHMT8. We find that both single variants have reduced THF‐dependent catalytic activity relative to Essex SHMT8 (10‐ to 50‐fold decrease inkcat/Km) but are significantly more active than the P130R/N368Y double variant. The kinetic data also show that the single variants lack THF‐substrate inhibition as found in Essex SHMT8, an observation with implications for regulation of the folate cycle. Five crystal structures of the P130R and N358Y variants in complex with various ligands (resolutions from 1.49 to 2.30 Å) reveal distinct structural impacts of the mutations and provide new insights into allosterism. Our results support the notion that the P130R/N358Y double variant in Forrest SHMT8 produces unique and unexpected effects on the enzyme, which cannot be easily predicted from the behavior of the individual variants.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1073/pnas.2206588119
