Targeting elevated ribosome biogenesis in cancer cells has been becoming a new treatment approach for oncogene-driven cancers. We developed a “first-in-class” selective inhibitor of ribosome biogenesis, CX-5461, which targets RNA polymerase I transcription and thereby inhibits ribosomal RNA production. CX-5461 causes cancer cell apoptosis, cell cycle arrest and senescence by inducing a p53-dependent nucleolar stress response or a p53-independent DNA damage response, and causing defects in mRNA translation and energy metabolism. We have demonstrated the remarkable potency of CX-5461 in mouse models of solid and blood cancers1–3. Importantly, CX-5461 showed safety and single-agent efficacy in phase 1 clinical trial on 17 patients with relapsed or refractory multiple myeloma and diffuse large B-cell lymphoma4. However, diseases still progressed after varying periods of time on treatment. Therefore, overcoming drug resistance is a critical step in improving the clinical efficacy of this novel therapy. We have generated CX-5461 resistant human acute myeloid leukemia (AML) single-cell clones by exposing human AML cell lines to escalating concentrations of CX-5461, dosing with CX-5461 at IC90 for 3 days, followed by single-cell sorting. We have confirmed the drug resistance of these cells by measuring cell death/proliferation and cell cycle progression upon CX-5461 treatment compared to the sensitive cells. Our preliminary results showed that p53 and DDR signalling pathways were impaired in the resistant clones. We will further investigate how tumour cells develop resistance to ribosome targeting therapy by performing polysome profiling, proteomics and metabolomics analyses to define the changes in signalling networks, mRNA translation and metabolism associated with CX-5461 resistance. We propose that this integrative analysis will help us identify therapeutic vulnerabilities and provide new strategies to combine CX-5461 with other drugs. Ultimately, we aim to improve the clinical efficacy of this new cancer therapy in patients with blood cancers.