Abstract 2024: c-Myc driven B cell lymphomas: role of the RPL5/RPL11/5S rRNA-MDM2 inhibitory complex (L5/L11/5S-MIC)

We recently demonstrated that as a consequence of impaired ribosome biogenesis, the L5/L11/5S pre-ribosomal complex is redirected to inhibit Mdm2, leading to p53 stabilization (Donati et al., 2013). In seminal studies, Eμ-Myc mice were crossed into a hypomorphic ribosomal protein (RP) L24 mutant background, which brought global translation rates to normal and extended their disease-free survival, independent of p53 (Barna et. al. 2008). However, when this mouse model harbors a single point mutation of Mdm2, which cannot bind the L5/L11/5S-MIC, mice succumb to lymphomagenesis much more rapidly than wild type Mdm2 mice (Macias et al, 2010). Moreover, inhibition of Pol I, which selectively transcribes rRNA genes, suppresses Myc driven tumors, potentially through the same L5/L11/5S-MIC (Bywater, et al, 2012). To address the role of the L5/L11/5S-MIC versus that of global translation in the Eμ-Myc B cell lymphocytes, we partially depleted mRNAs for RPL11, which in other cell types has no effect on p53 levels, or RPL7a, which is known to induce p53 stabilization in a RPL11 dependent manner. We generated stable cell lines of inducible shRNAs against RPL11 or RPL7a using a tetracycline (Tet)-regulated miR30-shRNA system, TRMPVIR retroviral vector. By titrating doxycycline, we achieved ∼50% reduction of RPL11 or RPL7a transcript levels, with Renilla shRNA (Ren) as a control. A 50% depletion of RPL11 or RPL7a mRNAs led to an equal reduction in protein synthesis and ribosome biogenesis, however, RPL11 depletion did not alter p53 levels, whereas RPL7a mRNA depletion induced p53 stabilization and Caspase-3 dependent apoptosis. RPL7A or RPL11 depletion reduced cell proliferation to half that of control cells, with more cell death observed in RPL7a depleted cells versus RPL11 depleted cells. Treatment of RPL7a depleted cells with the caspase inhibitor ZVAD, or depletion of RPL7a in a p53 negative background suppressed cell death. Expression levels of the anti-apoptotic protein Mcl-1 were elevated in Eμ-Myc cells and unchanged in RPL11 depleted cells, but reduced in RPL7a depleted cells in a p53-dependent manner. This suggests that in RPL7a depleted cells, p53 regulates Mcl-1 levels to promote cell death and tumor suppression. Finally, immunoprecipitation of Mdm2 revealed the presence of L5/L11/5S-MIC in RPL7a depleted cells, but not in RPL11 depleted cells. Although p53 was not induced in RPL11 depleted cells, reduction of RPL11 to ∼95% led to acute cell death in both p5