Lenalidomide Induced Degradation of Aiolos Disrupts RNA Polimerase I Activity and Ribosomal Biogenesis in Myeloma Cells

Background and rational: The B cell specific zinc finger transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) were recently identified as ubiquitylation substrates of the IMiDs bound CLR4-CRBN E3 ligase complex resulting in their proteasomal degradation. This downregulation of Ikaros and Aiolos represses the transcription of IRF4 and MYC and is believed to trigger MM cells death. It is clear however that Aiolos and Ikaros and in addition to IRF4 and MYC do activate or repress the transcription of other genes that contribute to the IMiDs effect in myeloma cells, in particular since overexpression of IRF4 was not sufficient to rescue MM cells from lenalidomide cytotoxic effects. Our group (Slaby J et al. Blood ASH 2013) has recently demonstrated that lenalidomide induces a ribosomal stress response with induction of TP53, downregulation of c-Myc and cell cycle arrest. This ribosomal stress response is mediated by the direct binding of ribosomal proteins (RPs), particularly RPL11 and RPL5 to Mdm2 and c-Myc. In the current study, and since IMiDs effects are Aiolos-dependent, we investigated how Aiolos contributes to ribosomal biogenesis and IMiDs induced stress response. Methods and Results: In order to investigate the impact of IKZF3 inhibition on ribosome biogenesis we stably transduced OPM2 myeloma cell lines with lentiviral plasmids carrying a doxycycline inducible shRNA targeting IKZF3. As expected, Aiolos silencing in these cells resulted in IRF4 and MYC downregulation. In addition, and as we previously observed with lenalidomide treatment, Aiolos silencing transiently stabilised and subsequently down-regulated MDM2 expression with up-regulation of p53 and its down-stream targets (p21, PUMA). Under ribosomal stress conditions, polysome-free RPs are released into the nucleoplasm where they bind MDM2 and suppress its E3 ligase activity. Consistent with this effect, in Aiolos silenced cells MDM2 co-immunoprecipitated with RPL11 and RPL5 with p53 protein stabilization (no change in TP53 mRNA). Furthermore Aiolos resulted in a rapid and robust decrease in the level of transcription of rDNA by RNA polymerase I as determined by qRT-PCR quantification of pre-rRNA (47S). The downregulation of 47S in lenalidomide treated cells is observed within 2 hours of treatment and mimics the kinetics of Aiolos downregulation. In addition ChiP assay of lenalidomide treated cells revealed decreased binding of RNA polymerase 1A to the 18S ribosomal DNA promoter region. Of note silencing of CRBN in OPM2 cells, blocks lenalidomide dowregulation of Aiolos and fully abrogated the supression of rDNA transcription. Overall these findings are consitent with an Aiolos-dependent suppression of RNA polymerase I activity. Lastly, and examining the effects of lenalidomide of the RNA polymerase I complex, we observed a significant transcriptional downregulation of the TAF-1A subunit and Aiolos ChiP studies revealed strong binding at TAF-1A TSS. Conclusions: Our studies indicate that IMiDs downregulation of Aiolos results in the suppression of RNA-polymerase I activity with the disruption of ribogenesis and induction of a ribosomal stress response.