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 chromatin accessibility (scATAC-seq):
Frozen cells were thawed at 37 °C, resuspended in RPMI 1640 medium (Gibco, Waltham, MA, USA) and washed twice by centrifugation at 2000 rpm for 5 min. The desired number of nuclei [2000-8000] was targeted and processed according to the Nuclei Isolation for Single Cell ATAC sequencing (CG000169, 10x Genomics, Pleasanton, CA, USA). Nuclei isolation was performed as indicated in the nuclei isolation protocol for Single Cell ATAC Sequencing (10x Genomics). Based on the starting number of cells and desired final nuclei concentration, primary MM cells were lysed and resuspended in an appropriate volume of chilled Diluted Nuclei Buffer. The resulting nuclei were then immediately used to generate scATAC-seq libraries using the Chromium Single Cell ATAC reagent kit (CG000168, 10x Genomics) according to the manufacturer’s protocol. Quality control and quantification were then performed using the 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 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 standard Illumina protocol (Illumina, San Diego, CA, USA). 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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