Targeting solid tumors must overcome several major obstacles, in particular, the identification of elusive tumor-specific antigens. Here, we devise a strategy to help identify tumor-specific epitopes. Glypican 2 (GPC2) is overexpressed in neuroblastoma. Using RNA sequencing (RNA-seq) analysis, we show that exon 3 and exons 7-10 of GPC2 are expressed in cancer but are minimally expressed in normal tissues. Accordingly, we discover a monoclonal antibody (CT3) that binds exons 3 and 10 and visualize the complex structure of CT3 and GPC2 by electron microscopy. The potential of this approach is exemplified by designing CT3derived chimeric antigen receptor (CAR) T cells that regress neuroblastoma in mice. Genomic sequencing of T cells recovered from mice reveals the CAR integration sites that may contribute to CAR T cell proliferation and persistence. These studies demonstrate how RNA-seq data can be exploited to help identify tumorassociated exons that can be targeted by CAR T cell therapies.

Chromosomal passenger complex (CPC) has been demonstrated to be a potential target of cancer therapy by inhibiting Aurora B or survivin in different types of cancer including neuroblastoma. However, chemical inhibition of either Aurora B or survivin does not target CPC specifically due to off-target effects or CPC-independent activities of these two components. In a previous chromatin-focused siRNA screen, we found that neuroblastoma cells were particularly vulnerable to loss of INCENP, a gene encoding a key scaffolding component of the CPC. In this study, INCENP was highly expressed by neuroblastoma cells, and its expression decreased following retinoic acidinduced neuroblastoma differentiation. Elevated levels of INCENP were significantly associated with poor prognosis in primary tumors of neuroblastoma patients with high-risk disease. Genetic silencing of INCENP reduced the growth of both MYCN-wild-type and MYCN-amplified neuroblasto-ma cell lines in vitro and decreased the growth of neuroblastoma xenografts in vivo, with significant increases in murine survival. Mechanistically, INCENP depletion suppressed neuroblastoma cell growth by inducing polyploidization, apoptosis, and senescence. In most neuroblastoma cell lines tested in vitro, apoptosis was the primary cell fate after INCENP silencing due to induction of DNA damage response and activation of the p53-p21 axis. These results confirm that CPC is a therapeutic target in neuroblastoma, and targeting INCENP is a novel way to disrupt the activity of CPC and inhibit tumor progression in neuroblastoma.Significance: Dysregulation of INCENP contributes to neuroblastoma tumorigenesis and targeting INCENP presents a novel strategy to disrupt the activity of chromosomal passenger complex and inhibit neuroblastoma progression. INCENP encodes the inner centromere protein (INCENP), which is the structural and regulatory component of the chromosomal passenger complex (CPC) comprised of INCENP, survivin, Borealin, and the Aurora B kinase (9). CPC is responsible for proper chromosomal alignment, segregation, and cytokinesis during the mitosis (9). In the CPC, INCENP plays two critical roles: firstly, it functions as a scaffold protein coordinating assembly of this complex by interacting with all the other three components, and secondarily, the interaction between INCENP and Aurora B is necessary for activation of the Aurora B kinase, the catalytic subunit of this complex (9). Thus, disruption of INCENP expression leads to dissociation of the whole complex and limits Aurora B kinase activity (10, 11). Targeting the CPC has been led by strategies aimed at targeting survivin or Aurora B kinase in neuroblastoma because inhibition of either of them significantly blocks neuroblastoma tumor cell growth in vitro and xenograft growth in vivo (12-15). Recent genome-wide meta-analyses