Defining the functional role and potential as an immunotherapeutic target of ALCAM in neuroblastoma

Abstract Despite intensive, multimodal therapy, only half of children diagnosed with high-risk neuroblastoma will survive five years, and survivors harbor significant short- and long-term treatment-related co-morbidities. Although monoclonal antibody therapy targeting GD2 has improved outcomes, GD2-directed immunotherapy remains one of the only FDA-approved immunotherapies for pediatric cancer, and therapy is toxic due to GD2 expression on pain fibers. Thus, there is a critical need to uncover new immunotherapy targets in neuroblastoma. ALCAM is a cell adhesion molecule that promotes tumor growth in a variety of cancers and is highly expressed in neuroblastoma. We generated three inducible CRISPRi cell lines to deplete ALCAM and elucidate its role in neuroblastoma. Depletion of ALCAM reduced cell growth, reduced Ki-67 staining, and increased cleaved PARP. To determine the mechanism of ALCAM overexpression, we used ChIP-sequencing to show MYCN oncoprotein binding at the ALCAM promoter. We generated luciferase reporters from the ALCAM promoter and a putative upstream (10kb) enhancer, which we defined using Promoter-based Capture-C. Treatment with the MYC(N)/MAX dimerization inhibitor MYCi975 reduced ALCAM expression by immunoblotting and luciferase signal from the ALCAM promoter. We validated the activity of the upstream enhancer and uncovered an AP-1 binding motif that is critical for enhancer activity. Finally, as ALCAM is expressed in several normal tissues, we investigated an ALCAM-targeted conditionally activated antibody drug conjugate (ADC), CX-2009 (praluzatamab ravtansine), which delayed tumor growth in two out of three PDX models. Together, these findings credential ALCAM as an immunotherapeutic target in neuroblastoma.