P-345: Connecting 1q amplification and PHF19 expression in high-risk myeloma clones

Introduction: Myeloma is a plasma cell malignancy in which a high-risk set of patients can be defined by genomic markers including cytogenetic groups such as loss of 1p, gain or amplification (gain/amp) 1q, and TP53 abnormalities. Increasingly, other markers are also being identified that are associated with progression such as PHF19.

Methods: Bone marrow aspirates from smoldering multiple myeloma (SMM; n=10), newly diagnosed multiple myeloma (NDMM; n=22), and relapsed/refractory multiple myeloma (RRMM; n=17) patients were collected. Samples underwent CD138+ sorting and single cell multiomic sequencing (RNA-seq and ATAC-seq; 10X Genomics). The multiomic single cell data were integrated using Seurat via weighted nearest neighbors (WNN). Copy number variants (CNVs) were generated using InferCNV. The transcription factor (TF) databases, hTFtarget and TF2DNA were used to identify TFs regulating genes of interest. Previously published paired ChIP-seq and RNA-seq from myeloma cell lines were used to determine TF binding sites. Finally, differences in TF, cell cycle inhibitors, and epigenetic regulator chromatin accessibility and expression in subclones within patient samples with high-risk CNVs were evaluated.

Results: We previously reported a subset of myeloma cells present primarily in RRMM patients that we denoted relapse/refractory plasma cells (RRPC) that express PHF19 (P < 0.0001) and are enriched for amp(1q) (P < 0.0001). To determine how PHF19 expression is controlled we examined TF binding sites and identified 92 TFs predicted to bind to the promoter region. Given the association of the RRPCs with amp(1q) we identified seven TF loci encoded on 1q including ZNF648, ATF3, KDM5B, PBX1, RBBP5, RFX5, and USF1 of which PBX1 (log2FC=0.78, P < 0.0001), RFX5 (log2FC=0.81, P< 0.0001), and RBBP5 (log2FC=0.93, P< 0.0001) were significantly up-regulated in RRPCs. ChIP-seq and RNA-seq data were available for PBX1 in MM1S and U266 cell lines, and indicated PBX1 binding at the promoter of PHF19 which was in the top 4% of MM1S ChIP-seq peaks and the top 8% of U266 ChIP-seq peaks. Silencing PBX1 via shRNA significantly decreased PHF19 expression in both cell lines. In our single cell data PBX1 (log2FC=0.78, P < 0.0001), PHF19 (log2FC=2.43 P< 0.0001), and FOXM1, a previously identified target of PBX1 in myeloma, (log2FC=3.64, P< 0.0001) were upregulated in RRPCs. PHF19 expression was detectable in 47% of RRPCs compared to only 9% of cells in other clusters (5.22 fold increase) and PBX1 expression was detectable in 22% of RRPCs compared to only 10% of cells in other clusters (2.44 fold increase).

Conclusions: Here we show a link between amp(1q), increased expression of the transcription factor PBX1 which leads to increased expression of the high-risk marker PHF19. This link gives further strength and biological meaning to the impact of amp(1q) on high-risk disease.