Dysregulation of protein synthesis is acknowledged as a key driver of cancer cell growth and highly coordinated to reprogramming of metabolism that accompanies malignant transformations [1,2]. We demonstrated that combining RNA Polymerase I transcription inhibitor CX-5461, with mammalian target of rapamycin complex 1 inhibitor Everolimus robustly improved the therapeutic efficacy of these agents in a MYC-driven mouse model of haematological cancer [3]. However, the mechanisms underlying this cooperativity are still not fully understood. We hypothesise that this cooperativity is driven, at least in part, by dramatic changes in the cell’s translation and metabolic profiles. This project employs polysome profiling and metabolomics analysis to characterise the signature of responses to these inhibitors on global mRNA translation efficiency and metabolic profiles, respectively, both in vitro and in vivo using the Eμ-MYC mouse model of B-cell lymphoma. Results from polysome profiling analysis showed that the abundances of over 1200 mRNA species associated with the polysomes were altered in the mouse lymph node cells that are resistant to the combination therapy due to translational as opposed to transcriptional regulation. In vitro experiments to validate this data are currently underway. As part of the protocol optimisation, our preliminary metabolomics experiments identified perturbations in the cells’ metabolic profiles in response to acute treatment with Everolimus, which agrees with published data [4]. In contrast, acute treatment with CX-5461 did not cause any dramatic metabolic effect. Further experiments involving longer treatment durations are required to further elucidate the signature of responses to these inhibitors, both as single agents or combination, on the cell’s metabolic profile. Defining the key changes in the translatome and metabolome upon drug response will provide critical insights into the mechanisms of the aforementioned cooperativity, and identify novel molecular processes that can be targeted to improve the therapeutic efficacy of these pharmacological agents.