ALBERT

Description: Background Deregulation of ribosome biogenesis is associated with carcinogenesis and in MM two of the most common recurrent mutations (DIS3 and FAM46C) are implicated in ribosomal decay and translational control. Beside its role in the regulation of protein synthesis, the importance of ribosome biogenesis is underscored by the observation that the impairment of this process leads to a ribosomal stress response with induction of p53, inhibition of c-Myc and cell cycle arrest. These effects are mediated by the direct binding of ribosomal proteins (RPs), particularly RPL11 and RPL5 to Mdm2 and c-Myc. Immunomodulatory drugs (IMiDs) anti-MM effects require their binding to Cereblon (CRBN), an adaptor protein of the Cul4A-DDB1-ROC1 ubiquitin E3 ligase complex with an ensuing down-regulation of c-Myc and up-regulation of p21. In the present study, we delineated the mechanisms through which IMiDs mediate these effects in MM cells. Methods and Results In order to identify ubiquitylated substrates that are modified by lenalidomide (Len) treatment, we performed a ubiquitin-proteome pull-down using Tandem Ubiquitin Binding Entity (Lifesensors) coupled with quantitative mass-spectroscopy proteomics (iTRAQ) in OPM2 cells exposed to Len (10 μM). Several ribosomal proteins (RPs) - S25, S26, S20 & S28 - were increased in Len treated samples suggesting that the Cul4a-CRBN complex may regulate RPs stability and hence IMiDs binding to cereblon may trigger a ribosomal stress response. Immunoblot analysis of MM cells exposed to Len lead to a rapid decrease in c-Myc expression (within 30 min) that significantly preceded the downregulation of IRF4 consistent with an IRF4-independent mechanism for c-Myc down-regulation. Furthermore, Len treatment 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 Len treated cells MDM2 co-immunoprecipitated with RPL11 and RPL5 with p53 protein stabilization (no change in p53 mRNA). In addition, an increase in the interaction between RPL11 and c-Myc was also observed consistent with the reported role for RPL11 in the post-transcriptional regulation and suppression of c-Myc. Furthermore, treatment with Lenalidomide supressed ribosomal RNA (rRNA) transcription with inhibition of pre-rRNA (47S) processing. The examination of polysome fractions (sucrose gradients) in Len treated MM cells (OPM2 and MM1S) revealed a striking reduction of 60S and 80S as well as polysomes fractions, an effect similar to that Actinomycin D (5 nM), a known RNA PolI inhibitor and potent ribosome stress inducer. Furthermore Len treatment significant reduced RPL11 within the 60S and 80S ribosome fractions. Of note, CRBN knockdown abrogated all these effects suggesting that Len binding to CRBN is upstream of this ribosome stress response. In order to investigate whether the down-regulation of c-Myc is the primary event leading to the disruption of ribogenesis, we transiently and stably silenced RPL11 in MM cells. RPL11 silencing nearly fully protected MM cells from the effects of Len and importantly it completely reversed Len-induced Mdm2 E3 ligase inhibition with up-regulation of p53, p21 and suppression of c-Myc and IRF4. Conclusion Taken together our data indicate that treatment with lenalidomide suppresses ribogenesis and induces a ribosomal stress response downstream of Cul4a-CRBN but upstream of c-Myc suppression and p53 induction. These effects likely result from the IMiDs-induced modification of the Cul4a-CRBN ubiquitome and the regulation of RPs integration into ribosomal subunits. These findings also explain the observed clinical activity of IMiDs in other ribosomopathies like 5q- MDS and Diamond-Blackfan anemia Disclosures: No relevant conflicts of interest to declare.

Description: Background: IMiDs are now recognized to promote the proteasomal degradation of IKZF1/3 and the transcriptional repression of MYC and IRF4. MYC locus rearrangement is a recurrent somatic event in MM and results in MYC repositioning near superenhancers (such as IGH@, IGL@ and others). The mechanisms of IMiDs-mediated downregulation of MYC in MM cells is not well understood, in particular it is unclear how IMiDs alter the activity of the super-enhancers driving MYC transcription. Methods and results: Transcriptome analysis (RNAseq) of MM cells (OPM2 and MM1S) treated with lenalidomide or upon the silencing of IKZF3 (lentiviral delivery of dox-inducible IKZF3 shRNA) confirmed the downregulation of MYC, IRF4 and among others the upregulation of other genes of interest such as CD38, DKK1, PDL1, FOXO3 and a host of genes involved in interferon response. Gene set enrichment analysis (GSEA) confirmed the enrichment of genes signatures associated with MYC as well as the interferon response. Notably, in t(4;14) MM cell lines we observed a significant downregulation of FGFR3 while the expression of WHSC1 was not affected. This finding was confirmed in a library of t(4;14) MM cell lines by qRT-PCR analysis. As FGFR3 is driven by the 3' IGH enhancer while WHSC1 is under the control of the intronic Em enhancer in t(4;14) MM cells, and in view of the known role of IKZF1/3 in class switch recombination, we postulated that IKZF1/3 regulate the 3' IGH enhancer activity and MYC expression in cells harbouring a MYC-IGH rearrangement. To validate this hypothesis we established the genome-wide distribution of IKZF1 in OPM2 and MM1S cells (both cell lines harbour an IGH-MYC rearrangement) by ChIP-Seq. Ikaros bound to 17660 loci of which 43% were associated with gene targets. Ikaros-binding motifs and other motifs such as STAT1, E2A, RUNX1 were also identified (MEME Tomtom and Jasper motif analysis) in the vicinity of Ikaros enrichment peaks. Importantly IKZF1 peaks also mapped to the IGH 3' enhancer loci and these results were confirmed by ChIP-PCR analysis. Of interest while no Ikaros peaks mapped to MYC (within 5 kb of TSS) by ChIP-seq, modest enrichment of Ikaros at MYC promoter was identified by ChIP-PCR in MM1S and KMS11 cells, and this enrichment was significantly attenuated by lenalidomide treatment. Since inhibition of MYC occurs as a consequence of depletion of the acetylated chromatin reader BRD4 at enhancers that drive MYC expression (Delmore et al., 2011), we examined the interaction between BRD4, IKZF1 and IMiDs treatment. Chip-Seq analysis revealed that BRD4 and IKZF1 are associated with most active enhancers and promoters in MM1S tumor cells. In particular, BRD4 and IKZF1 were enriched at the IGH 3'α2 enhancer locus. Importantly, treatment with lenalidomide (10μM, 4 hours), dramatically depleted IKZF1 and BRD4 enrichment at the 3' IGH enhancers (ChIP-PCR analysis). In contrast, treatment with the bromodomain inhibitor JQ1 depleted BRD4, but not IKZF1 from the IGH 3' enhancer locus. These findings suggest that IKZF1 regulates BRD4 binding to the 3' IGH enhancers and explain the selective effect of IMiDs on MYC transcription in transformed cells harboring a MYC rearrangement. In addition, these results suggest that IMiDs will affect MYC expression only in cells were MYC is driven by an IKZF1/IKZF3 responsive enhancer. Lastly we sought to explain how IKZF1 may regulate BRD4 enrichment at H3K27Ac marks in super-enhancer loci. Since Ikaros is a known integral component of the NuRD (nucleosome remodelling and histone deacetylase) complex, we examined whether lenalidomide treatment and the ensuing Ikaros depletion lead to local gain of NuRD at MYC TSS and depletion of H3K27 acetylation. CHD4 (helicase subunit of the NURD complex) and H3K27Ac ChIP-PCR analysis revealed significant accumulation of the NuRD complex at MYC TSS as well as reduced H3K27Ac at 3'IGH enhancers after IMiDs exposure in MM1S and OPM2 cells. Importantly, CHD4 silencing abrogated lenalidomide induced MYC transcriptional repression and cell death. Conclusion: In summary, our studies suggest that Ikaros drives MYC expression in MM cells harbouring an IGH-MYC rearrangement by regulating the accumulation of the acetylated chromatin reader BRD4 at the IGH 3' enhancer and preventing the NURD complex mediated depletion of H3K27Ac marks at these enhancers. Our findings also explain the IKZF1/3 dependent mechanism of IMiDs-mediated MYC transcriptional repression. Disclosures Neri: Celgene: Research Funding. Duggan:Jansen: Honoraria; Celgene: Honoraria. Keats:Translational Genomic Research Institute: Employment. Bahlis:Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Johnson & Johnson: Consultancy; Johnson & Johnson: Speakers Bureau; Johnson & Johnson: Research Funding; Amgen: Consultancy.

Description: Background: MYC rearrangement is a recurrent somatic event in MM and results in super-enhancers repositioning within 3Kb (centromeric or telomeric) of the MYC locus. BRD4, a member of the bromodomain family, binds H3K27Ac and regulates enhancers activity, RNA polymerase II transcription initiation and elongation and in particular it regulates MYC transcription in MM cells. Immunomodulatory drugs (IMiDs) promote the proteasomal degradation of IKZF1/3 and the transcriptional repression of MYC and IRF4. However the exact mechanisms through which IMiDs downregulate MYC are not well understood. Methods and Results: Using a extended panel of 12 MM cell lines we have first confirmed our previous observartion that IMIDs universally degraded IKZF1/3, however unlike in IMiDs-sensitive cell lines it failed to downregulate MYC in resistant cells. Therefore, this finding suggests that MYC transcriptional regulation rather than IKF1/3 degradation is the "bottleneck" in IMiDs anti-myeloma activity or resistance. In addition, RNA-seq of paired and serially collected primary myeloma cells with acquired resistance to IMIDsconfirmed the enrichment of genes signatures associated with MYC at the time of acquired resistance. Of note, in a library of t(4;14) MM cell lines lenalidomide significantly downregulates FGFR3 while the expression of WHSC1 was not affected. As in t(4;14) cells FGFR3 is driven by the 3' IGH enhancer while WHSC1 is under the control of the intronic Em enhancer, and in view of the known role of IKZF1 in CSR, we posit that IKZF1 regulates plasma cells enhancer activity and hence MYC expression. To validate this hypothesis we first established the genome-wide distribution of IKZF1 in MM1S, RPMI8226 and OPM2 cells by ChIP-Seq. This analysis revealed the distribution of IKZF1 across the genome, predominantly to intronic an intragenic loci with only ~ 8% or reads mapping within +/- 5 kb of genes TSS. Importantly IKZF1 was also enriched at known myeloma cells enhancers or super-enhancers such as IGLL5, TXNDC5, BCL2L1, and DUSP22. We also mapped BRD4 binding to the genome in 8226 and MM1S cell lines and confirmed previously described mapping to known B-cell enhancers. Of note IKZF1 nearly universally overlapped with all BRD4 enhancers and superenhancers loci. These findings lead us to propose that IKZF1 depletion in response to IMiDs treatment will result in enhancer destabilization and displacement of BRD4 from enhancer loci. Indeed, Chip-Seq analysis of IKZF1 and BRD4 genomic distribution pre- and post- treatment with lenalidomide (10 uM for 4 or 8h) in IMiDs sensitive cell lines significantly depleted IKZF1 and BRD4 at superenhancer loci. In contrast, in IMiDs resistant cell lines (RPMI8226) and despite IKZF1 depletion, BRD4 was largely retained at these loci. These findings suggest that in IMiDs sensitive cells, IKZF1 is the major transcription factor mediating BRD4 recruitment to enhancer and superenhancer loci. In contrast, in IMiDs resistant cells and similar to BET-bromodomain inhibitor (JQ1) resistance, transcriptional plasticity or hyper-phosphorylation of BRD4 attributable to the phosphatase PP2A inactivation may also be mediating IMiDs resistance. Indeed the levels of p-BRD4 determined by western blotting were significantly higher in IMiDs resistant INA6 and RPMI8226 cells compared to MM1S cells. Furthermore, treatment of INA6 cells with the PP2A activator phenothiazine significantly sensitized INA6 cells to lenalidomide. Lastly, in order to investigate whether IMiDs resistance was associated with acquired new MYC rearrangements with an "IKZF1_resisant enhancer," we performed mate-pair sequencing of IMiDs resistance patients (18 post acquisition of IMIDs resistance and 7 pairs of pre- and post- acquisition of IMIDs resistance). MYC structural rearrangements (indels, translocations, inversions) were detected using DELLY and MANTA. Of note several MYC rearrangement were identified including MYC duplication in 1 patient, MYC structural rearrangement (±3Kb of TSS) in 9 patients (enhancer partner: TXNDC5, IGHa2, NSMCE2, IGLL5) and a newly acquired IRF4-IGH a2 rearrangement in 1 patient with pre-existing MYC-IGLL5 rearrangement. Conclusion:We have demonstrated that IMIDs alter enhancer activity in myeloma cells by depleting IKZF1 / IKZF3 and disrupting BRD4 binding to enhancers and super-enhancers, stalling of RNA polymerase II and MYC transcription. Disclosures Neri: Celgene and Jannsen: Consultancy, Honoraria. Bergsagel:Amgen, BMS, Novartis, Incyte: Consultancy; Novartis: Research Funding. Bahlis:Onyx: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: Travel Expenses, Research Funding, Speakers Bureau; BMS: Honoraria; Celgene: Consultancy, Honoraria, Other: Travel Expenses, Research Funding, Speakers Bureau.

Description: Background Chromosomal region maintenance (CRM1), also known as exportin 1 (XPO1) plays an important role in the nuclear-cytoplasmic shuttling. The nuclear export receptor, XPO1, is considered as a regulator of subcellular distribution of several proteins involved in the regulation of centrosome duplication such as nucleophosmin (NPM), breast and ovarian cancer susceptibility protein 1 (BRCA1) and many tumor suppressor proteins (p53, p21, FOXO and pRB). Furthermore, XPO1 is required for the export of assembled ribosomal subunits (60S & 40S) from the nucleolus back into the cytoplasm. Inhibition of XPO1 triggers a ribosomal stress response that may result in the death of transformed cells with stressed ribogenesis. Silencing of XPO1 is reported to be synthetically lethal in MM cells, however the mechanisms that mediate this effect are not fully elucidated. Methods and Results To determine the effect of XPO1 inhibition in MM, cells were exposed to different doses of KPT330 (Karyopharm), a selective inhibitor of nuclear-cytoplasmic transport by irreversibly binding to the XPO1 cargo recognition site. Nanomolar concentrations of KPT330 (50-150 nM) induced apoptosis (Puma up-regulation and caspase 3 cleavage) and suppressed the proliferation of myeloma cell lines MM1S, OPM2 while KMS11 cells were more resistant. Mechanistically treatment with KPT330 up-regulated the expression of p53, as well as p21, p27 and MDM2 at the protein and RNA levels and significantly decreased the expression of c-Myc and IRF4. Cognizant of the role XPO1 in cytoplasmic-nuclear shuttling of ribosomal subunits, we reasoned that c-Myc downregulation and p53 induction in MM cells exposed to KPT330 results from ribosomal biogenesis stress. Therefore we analyzed the cellular co-localization of ribosomal proteins (RPL5, RPL11), c-Myc and MDM2 in presence of KPT330. A shifting of RPL11 and RPL5 from the nucleolus to the nucleoplasm and cytosol was observed in presence of KPT330 where they accumulated in ribosome-free cellular fractions. Co-immunoprecipitation studies showed that RPL11 and RPL5 released from the nucleolus bind MDM2 and c-Myc. This binding of RPL11 and RPL5 to MDM2 and c-Myc is known to suppress their function and expression. Therefore our data explain the suppression of MDM2 E3 ligase activity with p53 stabilization and reduction of c-Myc at the post-transcriptional levels. Study of ribosome fractions with sucrose gradients showed that in the presence of KPT330, 40S and the polysomes were completely suppressed while 60S and 80S subunits were significantly downregulated in OPM2 and MM1S cell lines. Consistent with the disruption of ribosomal function and the translational machinery, c-Myc mRNA levels were significantly decreased in 40S, 60S and 80S fractions after treatment with KPT330. Confirming the role of the ribosomal stress response in KPT330-mediated MM cells' death, silencing of ribosomal proteins RPL11 or RPL5 fully protected them from KPT330 cytotoxicity. Furthermore silencing RPL11 or RPL5, suppressed the effects of KPT330 on MDM2, p53, p21 and c-MYC. Conclusion Inhibition of XPO1 induces a perturbation in the ribosome subunits transfer, disruption of ribosomal assembly and the induction of a ribosomal stress response in MM cells. Perturbation on the nucleolar-cytoplasmic shuttling by KPT330 and the targeting of the translational factory represents a novel therapeutic approach in multiple myeloma. Disclosures: No relevant conflicts of interest to declare.