High throughput droplet single-cell Genotyping of Transcriptomes (GoT) reveals the cell identity dependency of the impact of somatic mutations

Abstract

Defining the transcriptomic identity of clonally related malignant cells is challenging in the absence of cell surface markers that distinguish cancer clones from one another or from admixed non-neoplastic cells. While single-cell methods have been devised to capture both the transcriptome and genotype, these methods are not compatible with droplet-based single-cell transcriptomics, limiting their throughput. To overcome this limitation, we present single-cell Genotyping of Transcriptomes (GoT), which integrates cDNA genotyping with high-throughput droplet-based single-cell RNA-seq. We further demonstrate that multiplexed GoT can interrogate multiple genotypes for distinguishing subclonal transcriptomic identity. We apply GoT to 26,039 CD34 + cells across six patients with myeloid neoplasms, in which the complex process of hematopoiesis is corrupted by CALR-mutated stem and progenitor cells. We define highresolution maps of malignant versus normal hematopoietic progenitors, and show that while mutant cells are comingled with wildtype cells throughout the hematopoietic progenitor landscape, their frequency increases with differentiation. We identify the unfolded protein response as a predominant outcome of CALR mutations, with significant cell identity dependency. Furthermore, we identify that CALR mutations lead to NF-kB pathway upregulation specifically in uncommitted early stem cells. Collectively, GoT provides highthroughput linkage of single-cell genotypes with transcriptomes and reveals that the transcriptional output of somatic mutations is heavily dependent on the native cell identity.Somatic mutations underlie the development of clonal outgrowth and malignant transformation 1-5 . Ongoing clonal evolution through acquisition of further genetic alterations often result in multiclonal cancer populations 6-10 . Transcriptional read-outs are critical for the study of the molecular basis of these processes. However, clonally-derived populations often lack cell surface markers that distinguish them from normal cells or that can help distinguish subclones, limiting the effectiveness of bulk RNA sequencing for investigating the clonal architecture of malignant cell populations. For example, myeloproliferative neoplasms (MPNs) have been shown to result from recurrent somatic mutations in JAK2, CALR and MPL, which are present across multiple progenitor classes, including CD34 + , CD38hematopoietic stem progenitor cells (HSPC) and downstream progenitor cells including megakaryocytic-erythroid progenitors (MEP) 11,12 . These malignant clones often represent a subset of the bone marrow progenitor population without distinctive cell surface markers to distinguish them from non-neoplastic hematopoietic cells.Furthermore, MPNs are known to undergo clonal diversification with subclones harboring driver gene mutations 11,13,14 , without a tractable avenue for isolation of subclones based on cell surface markers. Therefore, our current understanding of the impact of the somatic mutations in MPN has relied primarily on cell l...

The Expression of Myeloproliferative Neoplasm-Associated Calreticulin Variants Depends on the Functionality of ER-Associated Degradation

“…We showed that expression of CALR mutant proteins associated with MPN does not trigger an ER stress. These results were unexpected given that it was recently deduced from transcriptomic data that ER homeostasis was perturbed in CALR variant-expressing MPN [28,29]. However, our data suggest that this UPR pathway activation is not a direct consequence of the expression of CALR mutant proteins.…”

“…However, our data suggest that this UPR pathway activation is not a direct consequence of the expression of CALR mutant proteins. On the contrary, it might result from the modification of cell properties, such as an increased proliferation, as suggested by a more predominant activation of the UPR in committed progenitors compared to hematopoietic stem cells (HSC) [29]. Salati et al recently reported a decreased sensitivity toward ER-stress induced apoptosis in cells expressing CALR variants [30].…”

Expanding the CITE-seq tool-kit: Detection of proteins, transcriptomes, clonotypes and CRISPR perturbations with multiplexing, in a single assay

“…The recovery of clonotype information together with surface protein marker expression allowed fine separation of specific cell populations of interest, enabling careful determination of molecular phenotypes. Analogous to the use of TCR clonotype information in this study, we have recently used expressed mutations to define and further characterize clonal populations in scRNA-seq data-sets (Genotyping of Transcriptomes, GoT 18 ), an approach that could readily be combined with ECCITE-seq. The method we describe is inherently customizable and we envisage additional oligo-tagged ligands, such as peptide-loaded MHC complexes for detecting specific TCRs, labeled antigens for detection of antigen specific B cells, or antibodies directed against intracellular proteins being added to future iterations of this system.…”

Identification of leukemic and pre-leukemic stem cells by clonal tracking from single-cell transcriptomics

“…MutaSeq has distinct advantages and disadvantages compared to other methods for mutational profiling during single-cell RNA-seq currently proposed on preprint servers. GoT-Seq (Nam et al , biorxiv) performs targeted amplifications from cDNA libraries obtained from droplet-based singlecell RNA-seq, and therefore is biased towards mutations near the cDNA 3’ or 5’ end. Moreover, it provides a decreased molecular sensitivity compared to MutaSeq (median 4420 genes detected per CD34+ bone marrow cell, compared to ~2300 in GoT-Seq).…”