Identifying the regions responsible for isoaspartylation and aggregation of SCLC antigen ELAVL4

Small cell lung cancer (SCLC) is the most aggressive type of lung cancer, with a 5-year survival of 9%. Despite the aggressive nature, therapy options are still limited, and there is a need for the development of new treatments. Pulmonary neuroendocrine cells (PNEs), the predominant cells of origin for SCLC, uniquely express embryonic lethal abnormal vision Drosophila-like 4 (ELAVL4) and are presented on surface of SCLC cells. Clinical research has identified that 15% of SCLC patients with naturally-occurring low titer antibodies against ELAVL4 show significantly better survival. Patients with high-titer antibodies can see complete regression but suffer from a paraneoplastic autoimmune syndrome. The characteristic expression of ELAVL4 in SCLC and the observed improved survival of patients with an immune response against ELAVL4 suggest this protein may be used as a new therapeutic target. Our previous work indicates that the anti-ELAVL4 immune response is triggered by isoaspartylation in the unstructured N-terminal region of ELAVL4 adjacent to the first RNA-recognition motif domain (RRM1). Isoaspartylation occurs when asparagine or aspartic acid side chains spontaneously form a cyclic intermediate with the peptide backbone, followed by hydrolysis, resulting in an isoaspartyl kink that can be antigenic. The N-terminal region contains 6 asparagine residues and 2 aspartic acid residues. Some are followed either by a glycine, serine, or histidine, which increases the likelihood of isoaspartylation. One characteristic of isoaspartylation-prone proteins is the formation of protein aggregates, a process that may play a role in triggering immunogenicity. Here, we aim to identify which residues undergo isoaspartylation and trigger aggregation. We have generated four deletion constructs of the amino acid 1-117 ELAVL4 region (comprising residues 1-13, 8-18, 16-29, and 28-38, fused to RRM1), each including isoaspartylation-prone sites. These constructs are incubated under isoaspartyl-inducing conditions (pH 7.4, 37ºC, 7 d, in K-HEPES). We will use Western blots and mass spectrometry to determine which constructs undergo isoaspartylation and aggregation. Understanding the sites of isoaspartylation and aggregation 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. DAV, NSL and IAO are members of CaRE 2 , the Cancer Research Education and Engagement Health Center, which is supported by NIH/NCI grants U54CA233396, U54CA233444, and U54233465.