Neri, Paola

Abstract 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.

Abstract 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.

Abstract MYC dysregulation, the most common genetic aberration in multiple myeloma, is frequently due to the translocation of super-enhancers to the MYC locus. Several drugs target proteins that are enriched at many of these enhancers including BRD4 (BET inhibitors, BETi) and Ikaros (IMiDs), although their mechanism of action remains poorly understood. Here we present a characterization of the responses to these drugs in a collection of over sixty myeloma cell lines having a diversity of MYC rearrangements. We found that the anti-proliferative effects of these drugs significantly correlated with changes in MYC protein levels, consistent with both drugs targeting MYC expression. Despite this common target, there was no statistically significant correlation between the individual BETi and IMiD responses, suggesting that they act through different mechanisms. Of those lines having extremes of sensitivity or resistance, there were two major groups (BETiS/IMiDS and BETiS/IMiDR), a smaller group of four lines resistant to both drugs individually (BETiR/IMiDR) and only one line was BETiR/IMiDS. In the BETiR/IMiDR group, resistance to BETi was mediated by a BRD4-independent mechanism as BRD4 was efficiently released from the MYC-associated enhancers. In all three of BETiR/IMiDR cell lines that we examined, treatment with BETi and IMiD together abolished proliferation and down-regulated MYC, consistent with parallel BRD4- and Ikaros-dependent pathways driving MYC expression. These resistant lines all expressed high levels of the transcription factor ETV4 and knocking out its gene sensitized a line to each drug individually. Thus ETV4 appears to be necessary for the parallel pathways driving MYC expression. There were nine lines in the BETiS/IMiDR group. Sensitivity to BETi in these lines could be explained by either low ETV4 expression or by BETi repressing Ikaros levels (which was only observed in BETiS lines, suggesting that BRD4 drives IKZF1 expression in these lines). Thus, in ETV4-containing lines, BETi sensitivity is due to the simultaneously targeting of the BRD4- and Ikaros-dependent pathways. IMiD resistance likely was due to several reasons. In one cell line, OCIMY5, IMiD had little effect on Ikaros levels, likely due to the previously reported low levels of Cereblon. Seven of the eight remaining lines expressed high levels of either ETV4, or the other potential super-enhancer binding factors IRF4 or RUNX1. In the eight BETiS/IMiDS cell lines, IMiD strongly reduced both Ikaros and Aiolos protein levels, which likely caused IMiD sensitivity. As with the BETiS lines described above, the lines in this group either lacked ETV4 or BETi repressed Ikaros levels. In conclusion, by examining drug response in a collection of genetically annotated myeloma cell lines we have been able to identify factors that contribute the broad range of responses to BETi and IMiDs in myeloma cells. Citation Format: Daniel L. Riggs, Camille Herzog, Victoria M. Garbitt, Niamh Keane, Courtney J. Hillukka, Zachary J. Hammond, Julia E. Wiedmeier, Seth J. Welsh, Shulan Tian, Huihuang Yan, Ranjan Maity, Nizar Bahlis, Paola Neri, W Michael Kuehl, Marta Chesi, P Leif Bergsagel. IMiDs and BET inhibitors target distinct pathways of MYC dysregulation by super-enhancers in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3015.