Sara Rohban

In pre-cancerous lesions the overexpression of oncogenes such as Myc not only drives aberrant cellular proliferation, but also triggers a strong DNA damage response (DDR) that is in part due to DNA damage accumulating at the level of stalled replication forks. This oncogene-induced DDR is an effective barrier to cancer development and represents a relevant tumor suppressive mechanism. Conversely, at later stages of malignancy DDR signaling may function in favor of cancer progression. Such tumor promoting role of DDR may be needed for cancer cells to avoid accumulation of cytotoxic DNA damage under high level of oncogene-induced replication stress.
In an effort to identify the modulators of Myc-induced replicative stress, we carried out a high-throughput RNAi screen based on immunofluorescence detection of ϒH2AX, a DNA damage marker. Quantification of the number of cells and the percentage of ϒH2AX-positive cells, identified hits that exhibited differential cell viability and/or enhanced ϒH2AX signal in Myc-overexpressing cells compared to normal cells. Validated hits encompass a variety of pathways and biological processes and have different molecular functions.
For further mechanistic investigations we selected Rad21, a component of the cohesin complex. We provide evidence that while Rad21 is necessary for proper and efficient DNA synthesis, replication reinforcement imposed by Myc in Rad21-depleted cells results in replicative stress. In addition, we observed that Myc, as a transcription factor, could partially rescue transcriptional alterations due to Rad21 depletion. The conflicts between DNA replication and transcription in Rad21-depleted cells upon Myc activation may be the source of increased R-loops detected in these cells.
In summary, by means of a genetic loss of function screen we identified several candidates that may be involved in protecting Myc-overexpressing cells against ample replicative stress, thus revealing targets for potential therapeutic intervention in Myc-driven cancers.