Hematopoietic differentiation at single-cell resolution in NPM1-mutated AML

Recent data suggest that NPM1-mutated AMLs are heterogeneous in terms of co-mutations and expression of transcriptomic programs and surface proteins [1, 2]. Such heterogeneity may reflect a variable differentiation blockade of leukemic cells. Indeed, blasts in some NPM1-mutated AMLs may display an immature progenitor morphology, immunophenotype, and transcriptional program, or have a more mature monocytic and/or dendritic differentiation in other patients [1,2,3]. These differences can be clinically important, as immature blasts might have higher stemness capacity, a feature associated with poorer outcomes in AML [4]. Conversely, blasts with monocytic differentiation may have immunosuppressive capacities, and relative BCL-2 independence [5]. Previous reports based on bulk sequencing revealed only weak associations between NPM1/FLT3-ITD genotype and immature phenotype and between NPM1/FLT3-TKD or NPM1/RAS genotypes and monocytic/dendritic differentiation [2, 3]. Novel technologies allow simultaneous genotyping and analysis of surface protein expression at single-cell resolution and may help to resolve the interconnection between genotype and cell differentiation in leukemia [6, 7]. We used a droplet-based multi-omics single-cell platform to characterize the genetic clonal architecture in eleven NPM1-mutated AML diagnostic samples and investigate the relationship between co-mutations and phenotypic hematologic differentiation at the single-cell level.

We retrospectively included viably frozen samples from 11 patients with NPM1-mutated AMLs diagnosed in Saint Louis or Lille university hospitals banked after informed consent between May 2016 and July 2019. The project was approved by INSERM IRB (CEEI-20-274). Samples were selected if they had an NPM1 mutation and at least two additional mutations covered by the Mission Bio AML amplicons panel. Molecular information was available from routine bulk high-throughput sequencing (HTS) using previously published custom capture panels at Saint Louis (n = 8, Table S1) or Lille (n = 3, Table S2) university hospitals [8]. Cryopreserved mononucleated cells were thawed and live cells were stained using a 15 antibodies derived tags (ADT) panel (Fig. 1A and Table S3). Cells were processed according to the manufacturer protocol, using Mission Bio 20-genes AML amplicon panel (Table S4). Libraries were sequenced on a Novaseq 6000 (Illumina). Fastq files were analyzed using Mission Bio Tapestri Pipeline V2 (Fig. 1B). Filt3R was used for FLT3-ITD detection. sc-DNAseq analysis was focused on variants also detected on bulk HTS, using the TapestriR package. A genotype was considered informative if the single-cell sequencing depth (scDP) was ≥10x. An allele was retained if supported by at least 3 reads, and a single-cell variant allelic frequency (scVAF) ≥15% for an scDP between 20–100× or ≥10% for an scDP >100x, or considered non-informative otherwise. infSCITE was used to infer phylogenetic trees from mutation matrices as published [7]. Inferred clonal architectures (Fig. S1A) were used to correct raw cell genotypes. Cells with insufficient genotype information or with a genotype violating the clonal hierarchy owing to cell doublets or sequencing errors were excluded for downstream analyses (Fig. S1B). Protein data was analyzed using Seurat package V4.0 [9]. ADT counts were transformed using centered log-ratio transformation and differential expression was tested using ALDEX2 [10]. Bulk and single-cell DNA-Seq will be available at European Genome-phenome Archive (EGA) under accession code EGAS00001006565. Other data will be available upon reasonable request to the principal investigator.

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