Immunomodulatory drugs (IMiDs) are highly active in the treatment of multiple myeloma (MM). Blockade of the Wnt-GSK3β-beta catenin axis and suppression of eIF4E-C/EBPβ-dependent IRF4 expression were previously shown to mediate some of the IMiDs cytotoxic effects. In particular, cereblon (CRBN), an adaptor protein of the Cul4A-DDB1-ROC1 ubiquitin E3 ligase complex, was recently identified as an essential requirement for IMiDs anti-MM activity. In the present study, we examined how lenalidomide treatment modifies MM cells ubiquitome downstream of the Cul4A-DDB1-CRBN E3 ligase complex with the intent to identify novel molecular targets mediating IMiDs anti-MM effects.
Ubiquitin-proteome pull-down using Tandem Ubiquitin Binding Entity (TUBE, Lifesensors) coupled with quantitative mass-spectroscopy based proteomics (iTRAQ) was performed to identify modification to OPM2 cells ubiquitin-proteome induced by lenalidomide (10 μM for 24 hours) versus vehicle control treatment. Among the differentially modified ubiquitilated targets, histone family members H4, H2A.Z, H2B (known substrates of the Cullin-Ring Ub E3 ligases) and the 40S ribosomal proteins RPS25 and RPS26 were more than 2 fold increased in lenalidomide treated samples. Ribosomal protein RPS25 is required for internal ribosomal entry site (IRES) and 5′-cap-independent proteosynthesis. To examine the function of RPS25 in MM, siRNA-KD experiments were carried out in 2 human MM cell lines (OPM2 and MM1S). RPS25 silencing significantly (30–40%) reduced MM cells viability (MTT assay) when compared to non-targeting siRNA controls. Of interest, MM cells surviving RPS25 silencing were more resistant to IMIDs with approximately 20 to 30% reduction in lenalidomide induced cells death (Annexin V staining and MTT assay). Since, the 5′ UTR of c-myc is known to contain an IRES segement and c-myc translation can therefore be initiated by internal ribosome entry (RPS25 mediated) as well as by cap-dependent mechanisms, we next examined whether lenalidomide may downregulate c-myc through an IRF4-independent and rather RPS25-IRES dependent mechanism. Following 4 hours exposure to lenalidomide, C-MYC protein was significantly downregulated (western blot analysis) in OPM2 and MM1S cells, with no changes to IRF4 (protein or mRNA) or c-myc mRNA (qRT-PCR) at this early time point. No changes were observed in IRF4 until 24–48 hours of treatment with lenalidomide, findings consistent with an IRF4-independent C-MYC downregulation. Using the bicistronic (pRMF) luciferase reporter plasmid in which the upstream cistron encodes a Renilla luciferase translated in a cap-dependent manner, and the downstream cistron encoding a firefly luciferase translated in a cap-independent manner because of c-myc IRES insertion between the two cistrons, we examined whether treatment with lenalidomide modifies c-myc IRES ribosomal entry and translation. Following lenalidomide treatment, MM1S-pRMF transfected cells had a significant reduction in c-myc cap-independent translation as indicated by the reduction in the firely/renilla luminescence ratio. These findings were further supported by the use of the m-TOR inhibitor Rapamycin, an inhibitor of cap-dependent protein translation. Co-treatment of MM1S and OPM2 cells for 4 hours with the combination of Rapamycin (10 nM) and lenalidomide (10 μM) resulted in further reduction in C-MYC protein levels compared to either drugs alone. These results are consistent with an independent and additive (5′-cap-dependent with Rapamycin and independent with lenalidomide) rather than epistatic (both 5′-cap dependent) effects on c-myc translation.
Taken together our data indicate that treatment with lenalidomide suppresses c-myc translation in an IRF4/5′-cap-translation independent mechanism (early time point), an effect possibly resulting from a Cul4a-CRBN regulation of RPS25, a key protein-mediating ribosomal entry.
Neri:Johnson ans Johnson: Research Funding. Bahlis:Johnson and Johnson: Honoraria, Research Funding; Celgene: Honoraria.
Asterisk with author names denotes non-ASH members.