New Gain-of-Function Mutations Prioritize Mechanisms of HER2 Activation

Abstract ERBB2 (HER2) is a well-studied oncogene with several driver mutations apart from the well-known amplification defect in some breast cancers. We used the GigaAssay to test the functional effect of HER2 missense mutations on its receptor tyrosine kinase function. The GigaAssay is a modular high-throughput one-pot assay system for simultaneously measuring molecular function of thousands of genetic variants at very high accuracy. The activities of 5,886 mutations were classified, significantly more than mutants previously reported. These variants include 112 new in vitro, 10 known, and 9 new in vivo gain-of-function (GOF) mutations. Many of the GOFs spatially cluster in sequence and structure, supporting the activation mechanisms of heterodimerization with EGFR and release of kinase inhibition by the juxtamembrane domain. Retrospective analysis of patient outcomes from the Genomic Data Commons predicts increased survival with the newly identified HER2 GOF variants. Author statement Christopher J. Giacoletto: Computational methodology, Software, Validation, Formal analysis, Investigation, Writing - Original Draft, Writing – Review and Editing, Visualization; Liz Valente: Validation, Formal analysis, Investigation, Writing - Original Draft, Writing – Review and Editing, Visualization; Lancer Brown: Validation, Formal analysis, Investigation, Writing - Original Draft, Writing – Review and Editing, Visualization; Sara Patterson; Writing - Review and Editing; Rewatee Gokhale; Writing – Review and Editing; Susan Mockus: Writing – Review and Editing; Wayne Grody Writing – Review and Editing; Hong Wen Deng: Writing – Review and Editing; Writing – Review and Editing; Jerome I. Rotter: Writing – Review and Editing; Martin R. Schiller: Conceptualization, Methodology, Validation, Formal analysis, Resources, Data Curation, Writing - Original Draft, Writing – Review and Editing, Visualization, Supervision, Project Administration, Funding acquisition. The experimental and bioinformatic investigation was performed at Heligenics. Competing Interest Statement We disclose multiple potential conflicts of interest: Liz Valente, Lancer Brown, Christopher Giacoletto, and Martin Schiller are employees of Heligenics Inc, which produces and sells MEGA-Maps produced with the GigaAssay. Susan Mockus and Wayne Grody serve on the Heligenics Board of Advisors and Jerome Rotter is a Director of Heligenics. Heligenics has filed patents (USPTO 63/375369, USPTO 63/370837, PCT US2023071962) on the GigaAssay Technology and on the data in this manuscript. Commercial use of the data must have an agreement with Heligenics. Sara Patterson,, Rewatee Gokhale, and Susan Mockus were employees of The Jackson Laboratory, who previously licensed the HER2 variant data from Heligenics as part of its commercial Clinical Knowledgebase. The results shown here are in whole or part based upon data generated by the TCGA Research Network. Funding Statement . We acknowledge partial funding of this work by a grant award from the National Human Genome Research Institute, Grant number: R43HG012537. We thank Heligenics and Jackson Laboratories for partial funding this work. Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Public tumor mutant and outcome data was obtained from individual publications, NIH GCD, ClinVar, COSMIC, and The Cancer Genome Atlas I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data Availability All data produced in the present work are contained in the manuscript.