Cyclin D1 Induces Global Epigenetic and Transcriptional Changes in Multiple Myeloma with t(11;14)(q13;q32)

Introduction: Cyclin D1, encoded by the CCND1 gene, is primarily known as a major regulator of cell cycle progression. Emerging studies have demonstrated a role for cyclin D1 in regulating gene transcription. In mantle cell lymphoma with t(11;14), cyclin D1 strongly binds to the promoters of highly transcribed genes and its overexpression led to genome-wide transcriptional downregulation. However, the transcriptional role for cyclin D1 in t(11;14) MM remains undefined. This study aims to elucidate the global chromatin and transcriptional changes induced by cyclin D1 in t(11;14) MM.

Methods: Knockout (KO) of CCND1 in U266B1, a MM cell line carrying t(11;14), was performed by the Synthego (Redwood City, CA) using CRISPR/Cas9, which generated a frameshift insertion of base pair (bp) “A” after 69,641,478 bp within exon 1. Two independent KO clones were used for downstream analysis. ChIP-seq and ATAC-seq were performed for KO (n=2) and wild-type clones (WT, n=2). ChIP-seq used antibodies against H3K4me3 (promoter) and H3K4me1 and H3K27ac (enhancer). Libraries were sequenced to 2x51 bp on the Illumina NextSeq 2000 platform. Total RNA libraries (in triplicate) were sequenced to 2x150 bp. ChIP-seq and ATAC-seq reads were mapped to hg38 with BWA, and peaks were identified by MACS2 at FDR 0.01. RNA-seq reads were mapped to hg38 with STAR and gene raw counts were estimated using featureCounts. Differential peaks and differentially expressed genes between KO and WT were identified using edgeR at FDR 0.05 and fold-change 2. Only peaks present in at least 2 samples and genes having reads-per-million value 0.1 in at least 1 sample were included, excluding those from chrY. Pathway analysis was performed with the Enrichr package.

Results: Differential expression analysis identified 2-fold more genes that were down-regulated than up-regulated in KO compared to WT (467 vs. 233). Pathway analysis revealed an enrichment of TNF-alpha signaling via NF-kappaB, inflammatory response, and apoptosis in association with down-regulated genes, while up-regulated genes are enriched in myogenesis, KRAS signaling, and epithelial mesenchymal transition pathway. In parallel, differential chromatin analysis identified 2.5% (1,161) of the peaks from H3K27ac and 0.5-1% (359-610) of the peaks for the other 3 marks that showed significant changes of signal levels between KO and WT. For the promoter mark H3K4me3, there are largely a comparable number of peaks showing increased (191 peaks) and decreased (168 peaks) signals, respectively, in KO compared to WT. However, for ATAC-seq and H3K4me1, about 70% of the differential peaks showed an increased signal in KO, in contrast with H3K27ac for which 60% of differential peaks showed decreased signal in KO. The preferential loss of H3K27ac occupancy is consistent with the observation of a higher proportion of genes being down-regulated in KO. These results indicated that knockout of CCND1 led to a global increase of chromatin accessibility, but a reduction of H3K27ac and gene expression. Further annotation revealed that, for H3K27ac and H3K4me1/ATAC-seq, 92% and 97% of the differential peaks were located >2kb away from the transcription start sites (TSSs) of expressed genes. Even for H3K4me3, 88% of the differential peaks are >2kb away from TSSs. The results suggest that the changes of chromatin upon CCND1 knockout predominantly occurred at distal regulatory regions. For up- and down-regulated genes, we estimated ChIP-seq and ATAC-seq signals in the TSS 2kb regions, revealing that up-regulated genes were overall associated with increased signals. Further analysis found that, for ATAC-seq and H3K4me3, 44% and 59% of the differential peaks located in the promoter regions were associated with differentially expressed genes, which dropped to ~20% for the two enhancer marks. These data indicate that changes in chromatin state impact the expression of the associated genes.

Conclusion: Knockout of CCND1 impacts local chromatin state, particularly at enhancer regions, and the transcriptional program. In promoter-proximal regulatory regions, the changes of chromatin accessibility and H3K4me3 are strongly correlated with the expression changes of nearby genes. Further studies will identify the genes targeted by the differential enhancers and their possible roles in MM pathogenesis mediated by cyclin D1 following the t(11;14) event.

Fonseca:Antengene: Membership on an entity's Board of Directors or advisory committees; Celgene, Bristol Myers Squibb, Bayer, Amgen, Janssen, Kite, a Gilead company, Merck Sharp & Dohme, Juno Therapeutics, Takeda, AbbVie, Aduro Biotech, Sanofi, OncoTracker: Honoraria; AbbVie, Adaptive, Amgen, Apple, Bayer, BMS/Celgene, Gilead, GSK, Janssen, Kite, Karyopharm, Merck Sharp & Dohme, Juno Therapeutics, Takeda, Arduro Biotech, Oncotracker, Oncopeptides, Pharmacyclics, Pfizer, RA Capital, Regeneron, Sanofi: Consultancy; Patent for FISH in MM - ~$2000/year: Patents & Royalties: Patent for FISH in MM - ~$2000/year. Kumar:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding; Merck: Research Funding; MedImmune/AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Research Funding; KITE: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Other: Independent review committee participation; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding. Baughn:Genentech: Consultancy.