Abstract Recently, we identified a novel oncogenic EGFR exon 18 - 25 Kinase Domain Duplication (EGFR-KDD) in patients with lung cancer (PMID: 26286086). This novel EGFR alteration contains two tandem, in-frame EGFR TKDs. Computational modeling suggested that the linker between the two TKDs may support the formation of an asymmetric intra-molecular dimer. In this study, we sought to provide experimental support for the model that EGFR-KDD could form ligand-independent asymmetric intra-molecular dimers. We constructed point mutations (Table 1) disrupting the potential intra- (C1, N2) and inter-molecular dimer interfaces (N1 and C2) between each TKD of EGFR-KDD. We also introduced catalytically inactivating mutation into each TKD individually (Dead1 and Dead2). We stably expressed these mutants in YAMC and NR6, two cell lines lacking endogenous wild-type EGFR expression. In the absence of ligand, ‘wild-type’ EGFR-KDD was phosphorylated on known EGFR auto-phosphorylation sites, Y992, Y1068, and Y1173. However, mutations of the intra-molecular dimer interface (C1, N2) abrogated EGFR-KDD auto-phosphorylation, which is an accepted surrogate for EGFR enzymatic activity. In support of these data, expression of EGFR-KDD, but not the intra-molecular dimer interface mutants (C1, N2), supported growth in soft agar, an assay utilized in support of oncogenic phenotypes. Interestingly, phosphorylation of the dimer interface mutants (C1, N2) was rescued by ligand stimulation, suggesting the formation of EGFR-KDD multimers. Our structural modeling by Rosetta suggests that EGFR-KDD can form high order multimers with itself or with wild-type EGFR, and we observed co-precipitation of EGFR-KDD with itself and wild-type EGFR in vitro. Collectively, our findings define a previously unrecognized model of asymmetric intra-molecular dimers for EGFR, which provides important insights for the understanding of EGFR activation mechanisms and shed light on the exploration of therapeutic benefit. Table 1:Kinase domain mutations constructed for these studiesLobeMutationFunctionsN1I706QDisruption of the 1st N-lobe interfaceDead1D837NKinase dead mutation of the 1st kinase domainC1V948RDisruption of the 1st C-lobe interfaceN2I1057QDisruption of the 2nd N-lobe interfaceDead2D1188NKinase dead mutation of the 2nd kinase domainC2V1299RDisruption of the 2nd C-lobe interface Citation Format: Zhenfang Du, Jean-Nicolas Gallant, Monica Red-Brewer, Benjamin Brown, Jonathan Sheehan, Jens Meiler, Christine M. Lovly. EGFR kinase domain duplication (EGFR-KDD) is activated by asymmetric intra-molecular dimerization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 867.