Oncogene-induced replicative stress (RS) drives tumor progression by disrupting genome stability, primarily through transcription-replication conflicts (TRCs), which promote R-loop accumulation and trigger the DNA damage response (DDR). In this study, we investigate the role of chromatin regulators in exacerbating TRCs and R-loop accumulation in cancer. We find that in breast cancer patients, the simultaneous upregulation of MYC and the H2A.Z-specific chaperone ANP32E correlates with increased genomic instability. Genome-wide analyses reveal that ANP32E-driven H2A.Z turnover alters RNA polymerase II processivity, leading to the accumulation of long R-loops at TRC sites. Furthermore, we show that ANP32E overexpression enhances TRC formation and activates an ATR-dependent DDR, predisposing cancer cells to R-loop-mediated genomic fragility. By exploiting the vulnerability of ANP32E-expressing cancer cells to ATR inhibitors, we find that tumors relied on this DDR pathway, whose inhibition halts their pro-metastatic capacity. These findings identify ANP32E as a key driver of TRC-induced genomic instability, indicating ATR inhibition as a potential therapeutic strategy for ANP32E-overexpressing tumors.