Aberrant spliceosome activity via elevated intron retention and upregulation and phosphorylation of SF3B1 in chronic lymphocytic leukemia

Manoj Kumar Kashyap; Hiren Karathia; Deepak Kumar; Roberto Vera Alvarez; Jose Vicente Forero-Forero; Eider Moreno; Juliana Velez Lujan; Carlos Ivan Amaya-Chanaga; Newton Medeiros Vidal; Zhe Yu; Emanuela M Ghia; Paula A Lengerke-Diaz; Daniel Achinko; Michael Y Choi; Laura Z Rassenti; Leonardo Mariño-Ramírez; Stephen M Mount; Sridhar Hannenhalli; Thomas J Kipps; Januario E Castro

doi:10.1016/j.omtn.2024.102202

Aberrant spliceosome activity via elevated intron retention and upregulation and phosphorylation of SF3B1 in chronic lymphocytic leukemia

Mol Ther Nucleic Acids. 2024 Apr 26;35(2):102202. doi: 10.1016/j.omtn.2024.102202. eCollection 2024 Jun 11.

Authors

Manoj Kumar Kashyap^{1

2

3}, Hiren Karathia^{4

5

6}, Deepak Kumar¹, Roberto Vera Alvarez⁷, Jose Vicente Forero-Forero⁸, Eider Moreno⁸, Juliana Velez Lujan¹, Carlos Ivan Amaya-Chanaga¹, Newton Medeiros Vidal⁷, Zhe Yu¹, Emanuela M Ghia^{1

2

9}, Paula A Lengerke-Diaz⁸, Daniel Achinko⁷, Michael Y Choi^{1

2

9}, Laura Z Rassenti^{1

2

9}, Leonardo Mariño-Ramírez⁷, Stephen M Mount¹⁰, Sridhar Hannenhalli¹¹, Thomas J Kipps^{1

2

9}, Januario E Castro^{1

8}

Affiliations

¹ Moores Cancer Center, University of California, San Diego, La Jolla, CA 92093-0820, USA.
² Division of Hematology Oncology, Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA.
³ Amity Stem Cell Institute, Amity Medical School, Amity University Haryana, Panchgaon (Manesar), Gurugram (HR) 122413, India.
⁴ Advanced Biomedical Computational Science and National Center for Advancing Translational Sciences, National Cancer Institute, National Institutes of Health, Frederick, MD, USA.
⁵ Greenwood Genetic Center, Greenwood, SC, USA.
⁶ Center for Bioinformatics and Computational Biology, University of Maryland, College Park, MD 20742, USA.
⁷ National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, USA.
⁸ Department of Internal Medicine, Division of Hematology-Oncology, Mayo Clinic, Phoenix, AZ 85054, USA.
⁹ Center for Novel Therapeutics, University of California, San Diego, La Jolla, CA 92037, USA.
¹⁰ Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20742, USA.
¹¹ Cancer Data Science Lab, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.

Abstract

Splicing factor 3b subunit 1 (SF3B1) is the largest subunit and core component of the spliceosome. Inhibition of SF3B1 was associated with an increase in broad intron retention (IR) on most transcripts, suggesting that IR can be used as a marker of spliceosome inhibition in chronic lymphocytic leukemia (CLL) cells. Furthermore, we separately analyzed exonic and intronic mapped reads on annotated RNA-sequencing transcripts obtained from B cells (n = 98 CLL patients) and healthy volunteers (n = 9). We measured intron/exon ratio to use that as a surrogate for alternative RNA splicing (ARS) and found that 66% of CLL-B cell transcripts had significant IR elevation compared with normal B cells (NBCs) and that correlated with mRNA downregulation and low expression levels. Transcripts with the highest IR levels belonged to biological pathways associated with gene expression and RNA splicing. A >2-fold increase of active pSF3B1 was observed in CLL-B cells compared with NBCs. Additionally, when the CLL-B cells were treated with macrolides (pladienolide-B), a significant decrease in pSF3B1, but not total SF3B1 protein, was observed. These findings suggest that IR/ARS is increased in CLL, which is associated with SF3B1 phosphorylation and susceptibility to SF3B1 inhibitors. These data provide additional support to the relevance of ARS in carcinogenesis and evidence of pSF3B1 participation in this process.

Keywords: CLL; E7107; MT: RNA/DNA Editing; RNA splicing; RNA-seq; SF3B1; alternative RNA splicing; intron retention; intron usage; macrolide; pladienolide-B; spliceosome.