The effects of chromosomal copy number variations (CNV) on transcriptional programs at single cell resolution in multiple myeloma

Chromosome copy number variations (CNV) are a near-universal feature of cancer however their specific effects on cellular function are poorly understood. Single-cell RNA sequencing (scRNA-seq) can reveal cellular gene expression however cannot directly link this to CNVs. Here we report scRNA-seq normalization methods that improve inter-cellular expression alignment, increasing the sensitivity of scRNA-seq for CNV detection. We additionally report sciCNV, a tool for inferring CNVs from scRNA-seq data. Together, these tools enable simultaneous profiling of DNA CNV and RNA within single cells. We apply these tools to multiple myeloma and examine the roles of pan cancer CNVs +8q and +1q. Cells with +8q23-24 upregulate MYC, MYC-target genes, mRNA translation, protein synthesis capacity and DEPTOR; and show reduced transcriptome size. Cells with +1q21-44 reconfigure translation to suppress unfolded protein stress and show increased proliferation, oxidative phosphorylation and MCL1. Overall, we report scRNA-seq methods that can reveal the function of CNVs in cancer. Overall design: Single cell RNA-seq (10X Genomics) was used to profile the transcriptomes of plasma cells and B cells from the bone marrow of patients with multiple myeloma (MM). FACS was employed to enrich the profiled populations for the B cell - plasma cell lineage. Novel normalization methods (RTAM2) and single cell inferred copy number variation (sciCNV) methods were applied to the data to identify the effect of specific CNVs on transcriptional programs in MM cells. Normal plasma cells were identified by their expression of non-tumor immunoglobulin isotype and by their lack of tumor clone CNVs.

染色体拷贝数变异（Chromosome copy number variations, CNV）是癌症近乎普遍存在的特征，但其对细胞功能的具体调控效应仍有待阐明。单细胞RNA测序（Single-cell RNA sequencing, scRNA-seq）可揭示细胞基因表达谱，但无法直接将表达特征与CNV建立关联。本研究报道了可优化细胞间表达对齐的scRNA-seq标准化方法，提升了scRNA-seq用于CNV检测的灵敏度。此外，本研究还开发了sciCNV——一款可从scRNA-seq数据中推断CNV的分析工具。二者联用可实现单细胞内DNA拷贝数变异与RNA表达的同步谱分析。本研究将上述工具应用于多发性骨髓瘤样本，探究了泛癌CNV+8q与+1q的生物学功能。携带+8q23-24变异的细胞可上调MYC、MYC靶基因的表达，增强mRNA翻译与蛋白质合成能力，并提升DEPTOR的表达水平，同时伴有转录组规模的缩减；而携带+1q21-44变异的细胞则通过重构翻译过程以抑制未折叠蛋白应激，同时表现出增殖能力增强、氧化磷酸化水平提升以及MCL1表达上调的特征。综上，本研究开发的scRNA-seq相关方法可用于解析癌症中CNV的生物学功能。整体实验设计：采用单细胞RNA测序（10X Genomics平台）对多发性骨髓瘤（Multiple myeloma, MM）患者骨髓中的浆细胞与B细胞进行转录组谱分析。通过荧光激活细胞分选（Fluorescence-activated cell sorting, FACS）富集B细胞-浆细胞谱系的目标群体。将新型标准化方法RTAM2与单细胞拷贝数变异推断工具sciCNV应用于测序数据，以解析特定CNV对MM细胞转录程序的调控效应。正常浆细胞通过其表达非肿瘤性免疫球蛋白亚型，且不携带肿瘤克隆型CNV的特征进行鉴定。