Abstract 3650: Integrative mass spectrometry and RNA-sequencing identifies DLK1 as a candidate immunotherapeutic target in neuroblastoma
Authors: Weiner Amber K. , Radaoui Alexander B. , Kendsersky Nathan M. , Harenza-Rokita Jo Lynne , Sidoli Simone , Conkrite Karina L. , Vaksman Zalman , Rathi Komal , Raman Pichai , Martinez Daniel , Bhatti Tricia , Tsang Matthew , Pawel Bruce , Garcia Benjamin A. , Maris John M. , Diskin Sharon J. 
Published: August 2019

BACKGROUND. Neuroblastoma (NB) is an embryonal tumor of the sympathetic nervous system that accounts for 12% of childhood cancer deaths. Despite multimodal therapy, survival probability for high-risk NB patients remains below 50% and relapsed NB is largely incurable. To date, the cell surface landscape (surfaceome) of NB remains poorly defined. An unbiased survey of these proteins will facilitate the identification of candidate immunotherapeutic targets for preclinical validation.

METHODS. To identify proteins on the cell surface of NB cells, we performed plasma membrane protein extraction utilizing a sucrose density gradient methodology followed by nano-liquid chromatography coupled to mass spectrometry (nLC-MS/MS) in NB cell lines (n=12) and patient derived xenografts (PDX; n=10). We next integrated MS data with RNA-sequencing (NB=153; Normal=7859) data to evaluate proteins with an annotated extracellular domain differentially expressed in NB compared to normal tissues. Candidate immunotherapeutic targets were validated by immunohistochemistry on NB tumor and normal tissue microarray (TMA) and in-vitrofunctional studies were performed following genetic manipulation of candidate targets to assess cell proliferation, differentiation and viability.

RESULTS. We yielded on average 66% (range:60-68%) membrane protein enrichment with high reproducibility between biological replicates (80%; range:78-84%) and identified 4826 unique membrane proteins. Our approach confirmed known cell surface proteins in development as immunotherapeutic targets in NB (ALK, GPC2, NCAM1, DLL3 and CD276). We prioritized DLK1 for further evaluation due to it being the only candidate with expression directly associated with a super enhancer element (P=6.09X10-5). Genetic depletion of DLK1 resulted in neurite outgrowth suggesting induction of terminal differentiation (P=7.26X10-5). Full proteome analysis of DLK1 knockdown and control using MS showed regulation of proteins that promote outgrowth of neurites (P=3.37X10-3) and development of neurons (P=3.76X10-3).

CONCLUSION. We have developed the first MS-based surfaceome of NB. DLK1 is an epigenetically regulated oncoprotein in a large subset of high-risk NBs. We are currently developing antibody drug conjugate therapeutics designed for NBs with proven overexpression of DLK1.

Citation Format: Amber K. Weiner, Alexander B. Radaoui, Nathan M. Kendsersky, Jo Lynne Harenza-Rokita, Simone Sidoli, Karina L. Conkrite, Zalman Vaksman, Komal Rathi, Pichai Raman, Daniel Martinez, Tricia Bhatti, Matthew Tsang, Bruce Pawel, Benjamin A. Garcia, John M. Maris, Sharon J. Diskin. Integrative mass spectrometry and RNA-sequencing identifies DLK1 as a candidate immunotherapeutic target in neuroblastoma [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 3650.