Background: GD2-targeting dinutuximab therapy set a precedent for the use of immunotherapy to treat of high-risk neuroblastoma. However, this therapy is associated with toxicity and relapses on or after therapy do occur, highlighting the need for new tumor-specific immunotherapy targets.
Methods: We developed a computation pipeline designed to used RNA sequencing (n=136 high-risk tumors) and gene expression profiling data from 250 neuroblastoma tumors to identify cell surface proteins predicted to be highly differentially expressed in MYCN amplified neuroblastomas compared to a normal human tissues. We then performed ChIP-seq in the MYCN amplified cell lines KELLY and NGP to identify gene promoters that are occupied by MYCN protein to define the intersection with the differentially expressed gene list.
Results: From this pipeline, we identified 116 putative immunotherapy targets with predicted transmembrane domains. The most significant differentially expressed cell surface molecule in high-risk neuroblastomas harboring MYCN amplification was the calmodulin kinase-like vesicle-associated gene (CAMKV, p=2x10-6), which encodes a protein that binds calmodulin in the presence of calcium, but lacks the kinase activity of other calmodulin kinase family members. We have confirmed that the CAMKV protein is selectively expressed in 7 (of 7 tested) MYCN amplified neuroblastoma cell lines and the transcription of CAMKV is directly controlled by MYCN. From membrane fractionation and immunohistochemistry, we have verified that CAMKV is membranous in MYCN-amplified neuroblastoma cell lines and xenografted tumors. Immunohistochemistry also showed that CAMKV is not expressed in human pediatric normal tissues with the expected exception of neural tissues.
Conclusions: CAMKV is a differentially expressed cell surface protein that is transcriptionally regulated by MYCN, making it a candidate for immunotherapeutic targeting. Due to the normal CNS expression of CAMKV, we are pursuing an antibody drug conjugate strategy (does not cross blood-brain barrier).