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. Recently it has been shown that targeting regulators of replication checkpoint such as ATR or Chk1 in Myc-overexpressing cells caused apoptosis and prevented tumor formation, suggesting a crucial role for this pathway in ensuring cancer cell viability and offering the chance of developing new targeted therapies against cancer cells. 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 ?H2AX, a DNA damage marker. Quantification of the number of cells and the percentage of ?H2AX-positive cells, identified hits that exhibited differential cell viability (synthetic lethal hits) and/or enhanced ?H2AX signal (DDR-up hits) in Myc-overexpressing cells compared to normal cells. Validated hits encompass a variety of pathways and biological processes and have different molecular functions. As a proof of principal, we selected SRSF3 and Cdk12 and confirmed the synergistic effect of Myc overexpression and depletion of SRSF3 or Cdk12 on accumulation of cytotoxic DNA damage response as marked by H2AX phosphorylation. For further mechanistic investigations, we selected Rad21, a component of the cohesin complex, which was also reported as a Myc-synthetic lethal candidate previously. Using small inhibitory RNAs against Rad21, we confirmed that depletion of Rad21, increased ?H2AX level and subsequently led to cell death, selectively in Myc-overexpressing cells. We provide evidence that while Rad21 is necessary for proper and efficient DNA synthesis, replication reinforcement imposed by Myc overexpression 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.