Cell lines were grown and STR validated as described (18–20). Cell lines were tested for mycoplasma when thawed and only grown for 20 passages following thaw. SHEP-2 MYCN-ER, and SK-N-AS MYCN-ER cells were obtained from the laboratory of Dr. Michael Hogarty at the Children's Hospital of Philadelphia. Cells were treated with 1 uM tamoxifen (Sigma h7904) to induce MYCN-ER nuclear translocation.

Lentiviral preparation was carried out as described (21). Briefly, using the clone TRCN0000020695 to deplete MYCN (Sigma), plasmids encoding shRNA along with the envelope encoding plasmid pMD2.G and packaging plasmid psPAX2 were transfected into 293T cells with Fugene 6 (Roche). Supernatant was collected 48 and 72 h later, filtered and added to IMR-05 cells in the presence of 8 ug/ml polybrene (Sigma). Puromycin (Sigma) was used to select for infected cells.

Total RNA was isolated from neuroblastoma cells utilizing RNeasy mini spin kits (Qiagen) and mRNAs were converted to cDNA using SuperScript II First Strand Synthesis kits (Life Technologies). CAMKV expression was detected using a Taqman probe (Hs01062060_g1, ThermoFisher) and MYCN was detected using (Hs00232074_m1, ThermoFisher), according to the methods previously described (19, 21).

Chromatin immunoprecipitation was performed as previously described (22) using anti-MYCN (Santa Cruz Biotechnology, Inc., clone B8.4B, sc-53993), anti-MAX (Santa Cruz Biotechnology, Inc., clone H-2, sc-8011) and anti-mouse IgG (Santa Cruz Biotechnology, Inc., sc-2025). Primer sequences are as follows: CAMKV TSS Forward: 5′-GGGCAGAATCCGCTCCGA-3′;

CAMKV TSS Reverse: 5′-GCGATGCTGGAGGTTCGCTA-3′;

CAMKV 5′ Forward: 5′-CAAAGTCTCCTATCCCACCCC-3′;

CAMKV 5′ Reverse: 5′-TTTGGGAAAGACTCTGGGCTT-3′.

Cells were washed with cold PBS and fixed with a 50% Acetone/ 50% Methanol mixture for 5 min at −20°C. Cells were blocked with 0.3% v/v Triton X-100, 2% v/v goat serum in PBS for 5 min and then incubated with primary antibody in TBS-BGT (25 mM Tris, pH 8.0, 137 mM NaCl, 3 mM KCL, 1.5 mM MgCl₂, 5 mg/ml BSA, 1 mg/ml Glycine, 0.05% v/v Tween 20, 0.02% v/v sodium azide) for 90 min at room temperature. Primary antibodies included m906IgG (32) and anti-CAMKV (Santa Cruz Biotechnology, Inc., clone S-17, sc-102408). Cells were then washed with TBS-BGT and incubated with secondary antibody in TBS-BGT. Cells were washed again in TBS-BGT and mounted to slides with prolong gold with DAPI (ThermoFisher Scientific, P36931).

10–20 × 10⁶ live neuroblastoma cells were suspended in 50 uL of medium. A 1% w/v agarose in PBS solution was boiled and cooled to 50°C in a water bath. The cell suspension was mixed with 0.5 mL of the agarose and transferred into an inverted capped Eppendorf tube with the conical bottom cut off, on ice. Once solidified, the plug was removed, cut in half, and fixed in 10% v/v buffered formalin (ThermoFisher Scientific).

CAMKV staining was performed using the CAMKV S-17 antibody (Santa Cruz Biotechnology, Inc.). Each sample was scored by the same pathologist and was designated as “1” when <10% of cells stained positively, “2” when 10–90% of tumors stained positively and “3” when >90% of cells stained positively. Only cells of targeted tissue type were evaluated for NCAM staining.

Cells were lysed and western blotting was performed as previously described (18) using the following antibodies: anti-CAMKV (Santa Cruz Biotechnology, Inc., sc-102408) was used at a 1:250 dilution, anti-ACTIN (Cell Signaling, 4967s) was used at a 1:5,000 dilution, anti-ADAM12 (abcam, ab28225) was used at 1:1,000 and anti-BCL-2 (Cell Signaling, 2870S) was used at a 1:2,000 dilution.

Membrane fractionation was performed using the Proteoextract Native Membrane Protein Extraction Kit (Millipore, 444810) according to the manufacturer's instructions.

RNA sequencing data from 150 primary neuroblastoma tumors (136 high risk) was generated by the Therapeutically Applicable Research to Generate Effective Treatments project (TARGET data matrix, http://ocg.programs/target/data-matrix). RNA sequencing data were obtained from The Cancer Genome Atlas Program (TCGA), the Children's Brain Tumor Tissue Consortium (CBTTC), the Pacific Pediatric Neuro-Oncology Consortium (PNOC), the GEO database (GSE60052), and the Genotype-Tissue Expression (GTEx) project. Gene level data was generated using STAR alignment and RSEM normalization using hg 38 as a reference genome and GENCODE V23 gene annotation (33). The voom procedure was used to normalize the RSEM generated expected counts followed by differential expression testing using the R package limma to obtain adjusted p-values and Log-fold changes (LogFCs). Tumors were stratified by MYCN expression and sequencing data was queried for genes that had a LogFC >1 in the MYCN amplified tumors compared to non-amplified tumors with an adjusted p-value of 0.05 by the Benhanini-Hochberg procedure.

Gene expression data generated using Human Exon arrays (Affymetrix) from 250 primary tumors were obtained from the TARGET data matrix. These data were processed with Robust Mutlichip Average (RMA) normalization analysis implemented in the Affymetrix Power Tools (Affymetrix, Inc.). Samples were stratified by MYCN expression and the top and bottom 15% from this stratification were then used to query for the most differentially overexpressed genes in the MYCN-high subset of tumors.

The COMPARTMENTS database (http://compartments.jensenlab.org) was used to determine association with the plasma membrane (34). We designated only those genes to be potentially associated with the plasma membrane where the max confidence score across all GO categories was associated with Plasma Membrane or Cell Surface and was ≥3.

To determine the subset of genes which were differentially overexpressed in MYCN amplified neuroblastoma, we first used RNA-sequencing data from 150 primary tumors (136 of which were high-risk) and stratified them by MYCN expression (Figure 1A). We then filtered for genes that had a log fold change over 1 in the MYCN amplified tumors compared to non-amplified tumors with an adjusted p-value of 0.05. We similarly analyzed microarray gene expression data from 250 primary neuroblastomas stratified by MYCN expression (Figure 1B). The top and bottom 15% from this stratification were then used to query for the most differentially overexpressed genes in the MYCN-high subset. We intersected these data with a list of genes predicted to encode proteins located in the plasma membrane by the COMPARTMENTS database (34) and found 14 to be consistently differentially overexpressed in the MYCN amplified tumors (Figure 1C, Table 1, Table S1).

To prioritize these 14 candidate immunotherapeutic targets, we first surveyed for transcription start site (TSS)-proximal E-box motifs, and showed that these were present in 11 (Table 1). To determine which of these genes are transcriptionally dependent on MYCN being bound to their promoters, we performed ChIP-seq in the MYCN amplified cell lines NB-1643, NGP, and Kelly (discovery cohort). We later performed additional MYCN ChIP-seq in the MYCN amplified cell lines COG-N-415, LA-N-5, and repeated NB-1643, as well as the non-amplified cell line NB-69 (validation cohort). Ten of these 11 differentially overexpressed gene loci were occupied by MYCN, 8 at transcription start site-proximal E-boxes (Figure 1D, Figure S1, Table 1). We next looked at the normal expression of these genes and eliminated any with widespread normal tissue expression. For this analysis, we included genes that had normal tissue expression limited to the central nervous system, considering larger biologics such as antibodies would not cross the blood-brain barrier (35), reducing our gene list to 3 (Table 1). To ensure that the protein products of these genes were not only expressed at the membrane, but were membrane bound and able to be targeted with a therapeutic, we eliminated genes with secreted products. Of the remaining two genes, NME1 has been described for its prognostic value in neuroblastoma and mutations in NME1 correlate with an aggressive phenotype (36, 37). While NME1 is somewhat differentially overexpressed in MYCN amplified neuroblastoma (logFC = 1, adjusted p-value = 1.17 × 10⁻⁶, Table S1) the protein product of NME1, nm23-H1, has diffuse sub-cellular localization that does not always include the plasma membrane (38) and mostly consists of the cytosol and endoplasmic reticulum (39), suggesting it would not be an ideal candidate for targeted immunotherapy. Calmodulin kinase-like vesicle associated (CAMKV) is more significantly differentially overexpressed in MYCN amplified tumors than NME1 (logFC = 1.73, adjusted p-value = 2.02 × 10⁻⁶). CAMKV has limited normal tissue expression in the brain (Figure S2) and has a TSS-proximal E-box that is occupied by MYCN protein. While we had intended to nominate candidate immunotherapy targets for MYCN amplified neuroblastoma that lacked normal tissue expression, the analysis did not yield any targets. Thus, we expanded our analysis to include normal expression on CNS tissues with the goal of using an antibody-based therapeutic that wouldn't cross the blood-brain barrier. From this analysis, we nominated the unstudied protein CAMKV as a putative immunotherapeutic target for MYCN amplified neuroblastoma.

To further explore the association of CAMKV and MYCN co-expression, we stratified RNA-sequencing and gene expression data from primary tumors by MYCN amplification status. We performed correlation analysis in primary tumors between MYCN and CAMKV and found them to be significantly correlated (Figures 2A,C). Finally, we examined if CAMKV protein expression is selectively expressed in MYCN amplified neuroblastoma cell lines. Whole cell lysate from 7 MYCN amplified neuroblastoma cell lines was compared to two non-amplified neuroblastoma cell lines, immortalized retinal pigment epithelial cells, and three non neuroblastoma, non-neural crest origin cancer cell lines and found CAMKV protein to be exclusively expressed in the MYCN-amplified neuroblastoma cell lines (Figure 2B). Together, these data suggest that MYCN amplification is a predictive biomarker of high CAMKV mRNA and protein expression.

A limited number of non-amplified tumors appeared to have high CAMKV expression (Figures 2A,C). A recently defined subset of high-risk neuroblastoma tumors that lack MYCN amplification overexpress the MYC protein and have a poor prognosis comparable to those with amplified MYCN (40–42). While the majority of CAMKV-expressing cell lines are MYCN amplified (Figure 3A), the highest CAMKV-expressing cell line is NB-69 which does not harbor MYCN amplification, but has the highest MYC expression (Figure 3B). To further interrogate how MYCN and/or MYC protein(s) control CAMKV expression, we performed validation CHIP-sequencing for MYCN protein in the MYCN amplified neuroblastoma cell lines: COG-N-415, LA-N-5, NB-1643, and for MYC protein in the MYCN non-amplified lines: NB-69 and SK-N-SH (Figure 3C). We additionally performed MYCN ChIP-Seq on the non-amplified NB-69 cell line as a negative control. The MYCN amplified cell lines COG-N-415, LA-N-5, and NB-1643 all show MYCN occupancy at the CAMKV TSS-proximal E-boxes, similar to KELLY and NGP from the discovery cohort (top two tracks). The NB-69 cell line does not harbor MYCN amplification and has no MYCN occupancy at the TSS-proximal E-box; NB-69 is the highest MYC- and CAMKV-expressing cell line, and indeed shows MYC occupancy at the CAMKV TSS-proximal E-box (Figure 3C). The SK-N-SH cell line has the second highest MYC expression after NB-69, the second-highest CAMKV expression among non-amplified cell lines, and also has MYC occupancy at the CAMKV TSS-proximal E-box (Figure 3C). Together, these data suggest that in some high-risk neuroblastomas that lack MYCN amplification, MYC can drive CAMKV transcription.

To investigate whether MYCN protein regulates CAMKV expression, we transiently depleted MYCN from MYCN amplified IMR-05 cells using shRNA and saw a significant reduction in CAMKV mRNA levels (Figure 4A, p < 0.005), suggesting that MYCN is required for CAMKV transcription in MYCN amplified neuroblastoma cells. Next, to determine if MYCN protein could drive CAMKV transcription, we used SHEP and SK-N-AS cells with a tamoxifen-inducible construct to translocate MYCN to the nucleus in cells that do not normally express MYCN. Upon MYCN translocation, we observed a significant increase in CAMKV mRNA expression in these cells, indicating that MYCN is also sufficient to drive CAMKV transcription (Figure 4B, p < 0.01). The MYCN ChIP-seq data revealed that the CAMKV TSS has a MYCN-occupied E-box motif. To confirm this occupancy, we designed primers to amplify the locus containing the E-box motif and a 5′ locus that did not have an E-box motif or an occupancy peak in the ChIP-seq data sets. We performed ChIP-qPCR in three MYCN amplified cell lines (KELLY, NGP, and IMR-05) and the non-amplified cell line, SK-N-AS. These data confirmed that the MYCN and MAX proteins occupy the E-box motif at the TSS in MYCN amplified cell lines and not in SK-N-AS cells and do not have significant binding to the 5′ locus (Figure 4C).

CAMKV is known to localize to neurite outgrowths in the brain and thus is predicted to be a membrane-bound protein. Furthermore, CAMKV is predicted to have a transmembrane domain (43). To confirm transmembrane localization in neuroblastoma, we first performed a membrane extraction in four MYCN amplified neuroblastoma cell lines [KELLY, CHP-134, LA-N-5, and SK-N-BE (2)c], three non-amplified neuroblastoma cell lines (NB-Ebc1, SK-N-AS, SK-N-FI) and RPE-1 cells. We used ADAM12 as a marker for the membrane fraction (44) and BCL-2 as a marker for the soluble fraction of cells (45). CAMKV protein was present in the membrane fraction of the four MYCN amplified neuroblastoma cell lines and NB-EBc1 cells [which have known low basal MYCN protein expression without locus amplification (18)] (Figure 5A). Next, we created cell plugs from both MYCN amplified and non-amplified neuroblastoma cell lines and performed immunocytochemistry for CAMKV. This revealed CAMKV protein to be expressed and membrane-bound in MYCN amplified SK-N-BE (2)c cells but not expressed in non-amplified SK-N-AS cells (Figure 5B). Finally, the membrane localization of CAMKV was confirmed in MYCN amplified NGP neuroblastoma cells by immunofluorescence (Figure 5C). We used NCAM1 (CD56), an established cell surface molecule expressed in neuroblastoma as a marker for the plasma membrane (32, 46) and found NCAM1 and CAMKV proteins co-localize on the plasma membrane of NGP cells. Together, these data show that CAMKV is a membrane-bound protein in MYCN amplified neuroblastoma with the potential to be targeted using a CAMKV-specific biologic.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.