Direct Targeting of MYCN Gene Amplification by CRISPR/Cas9 in Neuroblastoma Cells (CHP-134)

Amplification of MYCN plays a pivotal role in multiple types of tumors and correlates with poor prognosis in high-risk neuroblastoma. Despite recent advances in the treatment of neuroblastoma, no approaches directly target the master oncogene MYCN. Difficulties in targeting the MYCN protein have inspired us to develop a new gene level inhibitory strategy using a sequence-specific gene regulator. Here we generated a MYCN-targeting pyrrole-imidazole (PI) polyamide, MYCN-A3, which directly binds to and alkylates DNA at homing motifs within MYCN transcript. Pharmacological suppression of MYCN inhibited the proliferation of cancer cells harboring MYCN amplification compared with MYCN non-amplified cancer cells. In neuroblastoma xenograft mouse models, MYCN-A3 specifically downregulated MYCN expression and suppressed tumor progression with no detectable adverse effects and resulted in prolonged overall survival. Moreover, we observed the copy number reduction of MYCN in neuroblastoma cells with MYCN amplification upon treatment with MYCN-A3 but not MYCN non-targeting PI polyamide. Expression microarray experiments were also performed to compare the genome-wide effect of MYCN-A3 with CRISPR/Cas9 silencing of MYCN (MYCNcr-a). CHP-134 cells were transfected with MYCNcr-a. After 6 hours, RNA was prepared for qPCR and microarray with 2x2 replicates per condition. Samples at 150 ng were labeled with RNA Spike-In Kit and analyzed on SurePrint G3 Human GE 8x60K V2 microarrays following Agilent Technologies’ recommendations. Arrays with sample were scanned on an Agilent SureScan microarray scanner. Expression analyses were performed by limma. Pairwise fold changes in expression (FC) are defined by differences between MYCNcr-a and CRISPR/Cas9 control. Gene symbols were additionally annotated by matching associated RefSeq mRNA and ncRNA as well as Ensemble accession codes in the array definitions.