Abstract 7243: Mapping the isoaspartylation site(s) in the N-terminal region of ELAVL4 by using deletion constructs and testing against SCLC patient sera

Abstract Small cell lung cancer (SCLC) is the most aggressive type of lung cancer. Standard of care consists of a platin-based therapy with etoposide, combined with immune checkpoint inhibition. While 2/3 of patients respond, the cancer usually recurs, and patients have a 5-year survival rate of only 8%. New therapies are urgently needed. About 15% of SCLC patients spontaneously develop a low-titer immune response against Embryonic Lethal, Abnormal Vision Drosophila-like 4 (ELAVL4). This protein is expressed throughout the nervous system but in the lung is unique to pulmonary neuroendocrine cells (the main cells of origin of SCLC) and is present on the surface of SCLC cells. Patients with low titer anti-ELAVL4 antibodies show improved response to chemotherapy and significantly improved survival. Our previous work indicates that the anti-ELAVL4 immune response is triggered by isoaspartylation of the unstructured N-terminal ELAVL4 region; asparagine or aspartic residues spontaneously form a succinimide intermediate, followed by hydrolysis resulting in an isoaspartyl link. This creates an antigenic kink in the protein, an appears to favor dimerization/aggregation. The unstructured N-terminal region of ELAVL4 contains 6 asparagine residues and 2 aspartic acid residues. Here we aim to understand which asparagine/aspartic acid residues in the N-terminal region undergo isoaspartylation and contribute to the anti-ELAVL4 immune response. To this end, we have constructed four deletion clones with different regions of the ELAVL4 N-terminal region carrying one or two possible sites of isoaspartylation (residues 1-13, 8-18, 16-29, and 28-38). Recombinant proteins generated from these constructs will be incubated under isoaspartyl-inducing conditions (neutral pH, 37ºC) and will use Western Blot and ELISA assays todetermine reactivity of SCLC patient sera as well as sera from mice immunized with Elavl4 (1-117). Understanding the sites of isoaspartylation in ELAVL4 will help identify the optimal target for the development of anti-isoaspartylated ELAVL4 monoclonal antibodies, which may provide a novel, targeted immunotherapy for SCLC patients. Supported by the Robert E. and May R. Wright Foundation Transformative Cancer Research Grant, USC’s Undergraduate Research Associates Program, and the Norris Comprehensive Cancer Center core grant, award number P30CA014089 from the NIH/NCI. Citation Format: Shiori Fukutome, Diego A. Velarde, Davin Pan, Chunli Yan, Pamela R. Duarte, Provati Devi, Ite A. Offringa. Mapping the isoaspartylation site(s) in the N-terminal region of ELAVL4 by using deletion constructs and testing against SCLC patient sera [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7243.