The PI3K/AKT/mTOR signalling pathway plays a critical role in regulating ribosome biogenesis, a fundamental cell growth process, which is invariably dysregulated in cancer. Specifically, AKT signalling promotes transcription of the ribosomal RNA (rRNA) genes by RNA Polymerase I (Pol I), which is a rate-limiting step in ribosome biogenesis . We used the Eμ-MYC model of B-cell lymphoma to demonstrate that inhibitors of the PI3K/AKT/mTOR pathway potently suppressed transcription of the rRNA genes and induced apoptosis to equivalent levels as the specific Pol I transcription inhibitor CX-5461. Remarkably, suppression of Pol I transcription by targeting the upstream signalling with PI3K/AKT/mTOR inhibitors resulted in apoptosis that was not associated with nucleolar stress nor acute activation of p53. This is in striking contrast to CX-5461, which targets Pol I machinery directly, and was previously shown to trigger nucleolar stress and to rely on intact p53 function for its anti-tumor activity in Eμ-MYC B-cell lymphoma in vivo . Furthermore,treatment with PI3K/AKT/mTOR pathway inhibitors, but not CX-5461, up-regulated the expression of the pro-apoptotic BH3-only protein BMF. Collectively these results provide evidence that differential inhibition of Pol I transcription by PI3K/AKT/mTOR pathway inhibitors (upstream) and CX-5461 (direct) induces apoptosis via distinct mechanisms and could therefore have enhanced therapeutic potential in combination. Indeed, combined treatment of Eμ-MYC cells in vitro with AKTi-1/2 and CX-5461 resulted in enhanced induction of apoptosis, providing a rational to combine these inhibitors in the clinic for the treatment of cancer.