Background: The BRD4 inhibitor JQ1 inhibits growth and induces apoptosis in a number of MYC-overexpressing cancer types. This study aims to assess the efficacy of JQ1 in neuroblastoma cell lines and to identify the genetic background making neuroblastoma tumors sensitive to JQ1-treatment.
Methods: A panel of 24 neuroblastoma cell lines was screened for JQ1 sensitivity by using Alamar blue viability assays, soft agar assays and FACS live/dead and cell cycle staining. MYCN mRNA and protein levels were determined by quantitative PCR and western blotting. Reverse Phase Protein Analysis (RPPA) was performed with 15 cell lines. sh/siRNA-mediated knock down of MYCN or BRD4 was used to investigate the relationship between MYCN and BRD4. Global transcript expression was profiled by RNA-seq. ChIP-seq for 6 histone modifications was used to define epigenetic changes upon JQ1 treatment.
Results: Viability screens showed that MYCN-amplified neuroblastoma cell lines are either JQ1 sensitive or resistant, whereas most non-amplified cells show intermediate response to JQ1. Reduced viability was mirrored by the absence of anchorage-independent growth and an increase of G1 and sub G1 fraction after JQ1 treatment. MYCN protein levels were reduced in 6/10 MYCN-amplified cell lines, however, this did not correlate with sensitivity. BRD4 knockdown in IMR5/75 cells did not affect MYCN levels but reduced Cyclin D1 expression. Global expression analysis of IMR5/75 cells emphasizes the importance of TP53-related signaling pathways and cell cycle-regulating genes in the response to JQ1 treatment. This was confirmed by RPPA analysis. Additionally, DNA damage repair genes were up-regulated after 6 and 12 hours JQ1 treatment. RNA expression profiles of JQ1-treated cells and xenograft tumors showed a high correlation.
Conclusions: JQ1 treatment promotes a G1 arrest and cell death in neuroblastoma cell lines. MYCN-downregulation is not the central mechanism of action. Cell cycle-related genes are affected by JQ1 treatment.