The translocation t(11;14) occurs in 20% of multiple myeloma (MM) patients and results in the upregulation of CCND1. Nearly two-thirds of t(11;14) MM cells are BCL2 primed and highly responsive to the oral BCL2 inhibitor venetoclax. While it is evident that this unique sensitivity to venetoclax depends on the BH3-proapoptotic protein priming of BCL2, the biology underlying t(11;14) MM dependency on BCL2 is poorly defined. Importantly, the epigenetic regulation of t(11;14) transcriptomes and its impact on gene regulation and clinical response to venetoclax remains elusive. In this study, by integrating ATACseq and RNAseq at the single-cell level in primary MM samples, we have defined the epigenetic regulome and transcriptome associated with t(11;14) MM. A "B cell-like" epigenetic signature was enriched in t(11;14) MM, confirming its phylogeny link to B cell rather than plasma cell biology. Of note, a loss of a "B cell-like" epigenetic signature with a gain of canonical plasma cell transcription factors was observed at the time of resistance to venetoclax. In addition, MCL1 and BCL2L1 copy number gains and structural rearrangements were linked to venetoclax resistance in t(11;14) MM patients. To date, this is the first study in which both scATAC-seq and scRNA-seq analysis are integrated into primary MM cells to obtain a deeper resolution of the epigenetic regulome and transcriptome associated with t(11;14) MM biology and venetoclax resistance.
Overall design: Single-cell mRNA sequencing (scRNA-seq):
Frozen cells were thawed at 37 °C, resuspended in RPMI 1640 medium (Gibco, Waltham, MA, USA) and washed twice with cells being collected by centrifugation at 2000 rpm for 5 min. Viable CD138+ cells were counted and resuspended in a resuspension buffer at 1,000 cells per ul. Single-cell capture, reverse transcription, and library preparation were carried out on the Chromium platform (10x Genomics, Pleasanton, CA, USA) using the Single Cell 3ʹ reagent kits v3 (10x Genomics, CG000183) according to the manufacturer’s protocol. Four thousand cells were used as input per channel. Quality control and quantification were performed using a KAPA Library Quantification qPCR kit (Roche, Basel, Switzerland) on a BioRad qPCR instrument before preparing a single pool containing equal-molar amounts of each library. This pool was then subjected to on-board cluster formation and sequencing on an Illumina NextSeq 500 sequencer with a high-output v2.5 150-cycle sequencing kit per the standard Illumina protocols. After sequencing, bcl data were converted to fastq data files using the Illumina BCL2FASTQ utility. Samples were processed with CellRanger suite v3.0.2, and downstream analyses were performed as indicated below.
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