MYCN activation in hTERT immortalized RPE cells as a model for oncogene induced senescence

The role of senescence in suppression of MYCN driven cancer initiation has not been intensively studied. In MYCN driven neuroblastoma, an aggressive pediatric tumor arising from sympathetic neuroblasts, CDK4/6 inhibition can induce senescence in these cells, amongst others through dose-dependent decreases in phosphorylated RB and FOXM1. Here, we propose a model to study the early effects of MYCN induced senescence in RPE1–MYCN-ER cells. Upon tamoxifen induced activation of MYCN, we observed proliferation and cell cycle arrest and robust induction of senescence as monitored by beta-galactosidase staining and increased expression of p21 and p16. We believe that MYCN induction renders cells susceptible to the action of DNA damaging agents. Therefore, we have established a high-content microscopy (HCM)-based compound-screening assay that provides a combined readout on DNA damage, cell morphology and replication stress response. The assay is based on multiplexed staining for nuclei (DAPI), single-stranded (pRPA32) and double stranded breaks (yH2AX) as well as replicative stress (EdU), and was validated using compounds that are known to induce DNA damage or replicative stress. We are now applying it to assess interactions between these compounds and MYCN activation. In doing so, we expect to reveal common modes of action between selected compounds and to infer putative synergies for combinatorial treatments.