PT - JOURNAL ARTICLE AU - Federico Gaiti AU - Paulina Chamely AU - Allegra G. Hawkins AU - Mariela Cortés-López AU - Ariel D. Swett AU - Saravanan Ganesan AU - Tarek H. Mouhieddine AU - Xiaoguang Dai AU - Lloyd Kluegel AU - Celine Chen AU - Kiran Batta AU - John Beaulaurier AU - Alexander W. Drong AU - Scott Hickey AU - Neville Dusaj AU - Gavriel Mullokandov AU - Jiayu Su AU - Ronan Chaligné AU - Sissel Juul AU - Eoghan Harrington AU - David A. Knowles AU - Daniel H. Wiseman AU - Irene M. Ghobrial AU - Justin Taylor AU - Omar Abdel-Wahab AU - Dan A. Landau TI - Single-cell multi-omics defines the cell-type specific impact of splicing aberrations in human hematopoietic clonal outgrowths AID - 10.1101/2022.06.08.495292 DP - 2022 Jan 01 TA - bioRxiv PG - 2022.06.08.495292 4099 - http://biorxiv.org/content/early/2022/06/09/2022.06.08.495292.short 4100 - http://biorxiv.org/content/early/2022/06/09/2022.06.08.495292.full AB - RNA splicing factors are recurrently affected by alteration-of-function mutations in clonal blood disorders, highlighting the importance of splicing regulation in hematopoiesis. However, our understanding of the impact of dysregulated RNA splicing has been hampered by the inability to distinguish mutant and wildtype cells in primary patient samples, the cell-type complexity of the hematopoietic system, and the sparse and biased coverage of splice junctions by short-read sequencing typically used in single-cell RNA sequencing. To overcome these limitations, we developed GoT-Splice by integrating Genotyping of Transcriptomes (GoT) with enhanced efficiency long-read single-cell transcriptome profiling, as well as proteogenomics (with CITE-seq). This allowed for the simultaneous single-cell profiling of gene expression, cell surface protein markers, somatic mutation status, and RNA splicing. We applied GoT-Splice to bone marrow progenitors from patients with myelodysplastic syndrome (MDS) affected by mutations in the most prevalent mutated RNA splicing factor – the core RNA splicing factor SF3B1. High-resolution mapping of SF3B1mut vs. SF3B1wt hematopoietic progenitors revealed a fitness advantage of SF3B1mut cells in the megakaryocytic-erythroid lineage, resulting in an expansion of SF3B1mut erythroid progenitor (EP) cells. SF3B1mut EP cells exhibited upregulation of genes involved in regulation of cell cycle and mRNA translation. Long-read single-cell transcriptomes revealed the previously reported increase of aberrant 3’ splicing site usage in SF3B1mut cells. However, the ability to profile splicing within individual cell populations uncovered distinct cryptic 3’ splice site usage across different progenitor populations, as well as stage-specific aberrant splicing during erythroid maturation. Lastly, as splice factor mutations occur in clonal hematopoiesis (CH) with increased risk of neoplastic transformation, we applied GoT-Splice to CH samples. These data revealed that the erythroid lineage bias, as well as cell-type specific cryptic 3’ splice site usage in SF3B1mut cells, precede overt MDS. Collectively, we present an expanded multi-omics single-cell toolkit to define the cell-type specific impact of somatic mutations on RNA splicing, from the earliest phases of clonal outgrowths to overt neoplasia, directly in human samples.Competing Interest StatementF.G. serves as a consultant for S2 Genomics Inc. X.D., J.B., A.W.D., S.H., S.J., E.H. are employees of Oxford Nanopore Technologies Inc and are shareholders and/or share option holders. I.M.G. serves on the advisory or consulting board of Bristol Myers Squibb, Takeda, Janssen, Sanofi, Novartis, Amgen, Celgene, Cellectar, Pfizer, Menarini Silicon Biosystems, Oncopeptides, The Binding Site, GlazoSmithKlein, AbbVie, Adaptive, and 10x Genomics. O.A.-W. has served as a consultant for H3B Biomedicine, Foundation Medicine Inc, Merck, Pfizer, and Janssen, and is on the Scientific Advisory Board of Envisagenics Inc and AIChemy; O.A.-W. has received prior research funding from H3B Biomedicine and LOXO Oncology unrelated to the current manuscript. D.A.L. has served as a consultant for Abbvie, AstraZeneca and Illumina, and is on the Scientific Advisory Board of Mission Bio, Pangea, Alethiomics, and C2i Genomics; D.A.L. has received prior research funding from BMS, 10x Genomics, Ultima Genomics, and Illumina unrelated to the current manuscript.