(C) PLOS One
This story was originally published by PLOS One and is unaltered.
. . . . . . . . . .



Single-cell transcriptome analyses reveal critical roles of RNA splicing during leukemia progression [1]

['Baohong Wu', 'Department Of Hematology', 'Institute Of Hematology', 'State Key Laboratory Of Biotherapy', 'Cancer Center', 'West China Hospital', 'Sichuan University', 'Chengdu', 'Sichuan', 'Xuelan Chen']

Date: 2023-05

Leukemogenesis is proposed to be a multistep process by which normal hematopoietic stem and progenitor cells are transformed into full-blown leukemic cells, the details of which are not fully understood. Here, we performed serial single-cell transcriptome analyses of preleukemic and leukemic cells (PLCs) and constructed the cellular and molecular transformation trajectory in a Myc-driven acute myeloid leukemia (AML) model in mice, which represented the transformation course in patients. We found that the Myc targets were gradually up-regulated along the trajectory. Among them were splicing factors, which showed stage-specific prognosis for AML patients. Furthermore, we dissected the detailed gene network of a tipping point for hematopoietic stem and progenitor cells (HSPCs) to generate initiating PLCs, which was characterized by dramatically increased splicing factors and unusual RNA velocity. In the late stage, PLCs acquired explosive heterogeneity through RNA alternative splicing. Among them, the Hsp90aa1 hi subpopulation was conserved in both human and mouse AML and associated with poor prognosis. Exon 4 skipping of Tmem134 was identified in these cells. While the exon skipping product Tmem134β promoted the cell cycle, full-length Tmem134α delayed tumorigenesis. Our study emphasized the critical roles of RNA splicing in the full process of leukemogenesis.

Funding: This work was supported by the National Natural Science Foundation of China (82130007 to YL, 82170171 to CC), the Sichuan Science and Technology Program (2021YFS0027 to LC, 2020YFQ0059 to CC, 2018JZ0077 to YL), the 1.3.5. Project for Disciplines of Excellence, West China Hospital, Sichuan University (ZYJC21009 to YL and ZYGD22012 to YL), the Sichuan University Postdoctoral Interdisciplinary Innovation Fund to XC, the Post-Doctor Research Project, West China Hospital, Sichuan University (2023HXBH019 to BW and 2023HXBH033 to XC), the China Postdoctoral Science Foundation (2022M722272 to JX). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: All scRNA-seq and RNA-seq data in this study are deposited in NCBI Gene Expression Omnibus under accession number GSE142645. The analysis code can be found at GitHub ( https://github.com/pangxueyu233/Super-RNA-splicing-entropy-drives-stepwise-leukemogenesis ). All flow cytometry data for S1 Fig are deposited in the FlowRepository database under accession number FR-FCM-Z64U and FR-FCM-Z64T.

Tumorigenesis, a process to accumulate enough genetic alterations for normal cells to be transformed into malignant cells, can take decades in patients [ 1 – 5 ]. Similarly, leukemogenesis is conceptualized as the multistage process for normal hematopoietic stem and progenitor cells (HSPCs) to become full-blown leukemic cells [ 6 , 7 ]. It has been shown that largely normal clonal hematopoiesis (CH) can progress into low-risk myelodysplastic syndrome (MDS) and then high-risk MDS or myeloproliferative neoplasm (MPN), eventually leading to full-blown acute myeloid leukemia (AML) [ 7 ]. However, AML is among the human malignancies with the lowest mutation burdens [ 8 ], and there is accumulating evidence suggesting that the majority of genetic alterations exist in preleukemic conditions such as CH and MDS [ 9 – 12 ]. Therefore, we wondered whether molecular rewiring other than mutations plays a significant role during leukemogenesis. Recent advances in omics studies, especially single-cell RNA sequencing (scRNA-seq), combined with our murine AML model, have provided unique opportunities to reveal the trajectory of leukemogenesis and further molecular characterization after acquiring initiating mutations.

Results

Progressively deteriorated RNA splicing abnormality during leukemogenesis To dissect the molecular events underlying stepwise leukemogenesis, we performed gene set variation analysis (GSVA) for specific pathways of PLCs of each stage. We found that the Myc target genes gradually increased from T 0 to T 3 , along with genes involved in oxidative phosphorylation and DNA repair, and the genes of the p53 pathways gradually decreased over time, all of which were consistent with the gradually increased aggressiveness of PLCs over time (Fig 2A). PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 2. The Myc targets, not Myc, triggered a progressively deteriorating RNA splicing abnormality during leukemogenesis. (A) Heatmap showing GSVA scores of hallmark pathways at each time point during leukemogenesis. (B) The top 10 KEGG pathways of Myc_targets detached in PLCs. (C) The box plot showed the relative expression levels of SFs in PLC during leukemogenesis; p values were calculated by the Wilcoxon signed-rank test. (D) Heatmap showing the relative expression levels of 4 subtypes of MYC targets involved in high SFs (rows) during leukemogenesis (columns) in murine AML. (E) Kaplan–Meier survival curves of TARGET-AML patients with low or high SFs expression at T0, T1, T2, and T3. The p value was calculated by the log-rank test. The underlying data for Fig 2C and 2E can be found in S1 Data. AML, acute myeloid leukemia; GSVA, gene set variation analysis; PLC, preleukemic and leukemic cell; SF, splicing factor. https://doi.org/10.1371/journal.pbio.3002088.g002 Since myc was the sole driver in this AML model, we first checked the expression levels of Myc. We found that the expression of either ectopic or endogenous Myc was not constitutively increased from T 0 to T 3 (S3A Fig). However, we observed that the expression levels of Myc targets constitutively increased from T 0 to T 3 (S3B Fig). Since the expression level of Myc itself was not progressively changed in PLCs, it was unlikely that the increase in Myc targets was just a selection of cells with high levels of Myc, which further suggested that stepwise leukemogenesis was not a result of simply selecting Myc expression. Importantly, by analyzing the transcriptome of the TCGA AML cohort, we found that the signature of Myc target, but not the expression of Myc itself, was associated with the poor prognosis of AML patients (S3C Fig). Furthermore, the KEGG analysis of the Myc target genes revealed that the spliceosome pathway was the most enriched among the Myc targets in PLCs, along with the cell cycle pathway and RNA transport pathway [16] (Fig 2B). The expression level of splicing factors gradually increased from T 0 to T 3 (Fig 2C), and stage-specific splicing regulatory factors (SFs) were identified by gene expression (Figs 2D and S3D). We found that the expression of these stage-specific SFs associated with PLCs, but not those associated with normal cells, had prognostic value in human AML patients, and the prognostic value of PLC SFs progressively increased from T 1 to T 3 (Fig 2E). Taken together, these data suggested that aggressively increased expression of splicing factors, independent of the Myc expression level, might underlie the progression from preleukemic to fully transformed leukemia in our stepwise model of leukemogenesis.

[END]
---
[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002088

Published and (C) by PLOS One
Content appears here under this condition or license: Creative Commons - Attribution BY 4.0.

via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/

You are viewing proxied material from magical.fish. The copyright of proxied material belongs to its original authors. Any comments or complaints in relation to proxied material should be directed to the original authors of the content concerned. Please see the disclaimer for more details.