IKZF1 Regulates BRD4 Retention and Recruitement at Enhancer in Myeloma Cells: Role of BRD4 in IMiDs Resistance
Bibliographic record
Abstract
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.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.003 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".