(PA-211) Cyclin D1 Beyond the Cell Cycle: A New Role in t(11;14) MM

Cyclin D1, encoded by CCND1, is a cell cycle regulator, overexpressed in ~15% of multiple myeloma (MM) cases via CCND1-IGH super-enhancer juxtaposition through t(11;14). Cyclin D1 also plays a non-canonical role in transcriptional regulation, but its function in gene regulation in t(11;14) MM remains undefined. We investigated the global transcriptional and epigenomic consequences of cyclin D1 overexpression in t(11;14) MM.

Methods: Two CCND1 knockout (KO) t(11;14) MM U266B1 clones were generated by CRISPR/Cas9, introducing a frameshift insertion (A) after chr11:69641478 (hg38, exon 1). ChIP-seq (H3K4me3, H3K4me1, H3K27ac) was performed in KO and wild-type in duplicates, RNA-seq in triplicates. Libraries were sequenced on Illumina NextSeq 2000 (2x51 bp for ChIP/ATAC, 2x150 bp for RNA-seq). Reads were mapped to hg38 by BWA (ChIP/ATAC) or STAR (RNA-seq), peaks called by MACS2 (FDR< 0.01), gene counts by featureCounts. Differential peaks and differentially expressed genes were identified by edgeR (FDR<0.05, fold-change ≥2), including peaks in ≥2 samples and genes with transcript-per-million (TPM) ≥0.1 in ≥1 sample. Pathway analysis performed using Enrichr. To define a CCND1 signature, RNA-seq from two independent cohorts was compared (high CCND1 t(11;14) (≥300 TPM, n=271) vs. low CCND1 hyperdiploid cases ( < 30 TPM, n=233). Co-immunoprecipitation was done in t(11;14) KMS12BM cells.

Results: Differential expression comparing CCND1-high t(11;14) and CCND1-low MM cases identified 22 upregulated and 21 downregulated genes associated with CCND1 expression in 2 patient cohorts and CCND1-modified U266 cells. Enriched pathways (p < 0.05) included Notch signaling, early/late estrogen response, epithelial-mesenchymal transition and reduced mitotic spindle and myogenesis. Dysregulated genes included those related to apoptosis, ubiquitin-proteosome, extracellular matrix, adhesion, and immune response, demonstrating a broader role for cyclin D1 beyond cell cycle control. To explore the epigenomic basis of this regulation, we examined histone marks in the U266 KO model. Among differentially expressed genes, 6 (27%; ASS1, PMAIP1, MAN1C1, MYEOV, CCR7, DTX1) and 5 (24%; UACA, RASAL2, PTPRZ1, PPT2, IL23R) showed visually distinct changes in histone marks, suggesting a chromatin-mediated mechanism of gene regulation by cyclin D1. The most prominent changes involved H3K27ac, mirroring the gene expression changes. As H3K27ac is deposited by histone acetyltransferases CBP, we tested for cyclin D1-CBP interaction. Co-IP confirmed this interaction, suggesting a role for this complex in chromatin remodeling and transcriptional control in t(11;14) MM.

Conclusions:

A cyclin D1-associated gene signature was identified supporting its role in t(11;14) MM beyond the cell cycle. These findings highlight cyclin D1 as a therapeutic vulnerability and open new avenues for personalized treatment strategies.