Polypyrimidine Tract Binding Protein 1 (PTBP1): a splicing factor at the cross-road between metabolism, proliferation and survival in acute myeloid leukemia
Résumé
Background. Acute myeloid leukemia (AML) is one of the most common and lethal hematological cancers in adults with a high rate of disease relapse upon therapy and a 5-year overall survival rate of only 20 to 40%1,2. Alteration of RNA splicing, an essential process involved in RNA maturation, has been shown to support AML initiation and progression and to be associated with therapy resistance3-9. However, our understanding of how changes in RNA splicing may impact AML biology and clinical outcome is currently very limited. Polypyrimidine Tract Binding Protein 1 (PTBP1) is a multi-functional RNA-binding protein that plays a key role in the regulation of RNA splicing, translation, stability and localization. Besides being one of the best described splicing factors in developmental biology, PTBP1 is known to regulate the metabolism of glucose by controlling the alternative splicing of the key glycolytic enzyme pyruvate kinase M (PKM). Over-expression of PTBP1 has been linked to tumorigenesis and associated with adverse prognosis in multiple cancer types10-12. In AML, PTBP1 was identified as part of a network of splicing factors associated with poor clinical outcome13. However, how PTBP1 expression may affect AML biology, metabolism and response to therapy is currently unclear. Aims. The aim of this study is to unravel the function of the splicing factor PTBP1 in the context of AML biology and metabolism. Methods. PTBP1 knock-down was achieved by using a RNA interference approach. The functional effects of PTBP1 silencing were studied combining work on human AML cell lines in vitro and AML xenografts in immunodeficient mice. PTBP1 target genes and pathways were identified combining PTBP1-directed iCLIP experiments with RNA-seq analysis of AML cells silenced or not for PTBP1. Results. Here we show that PTBP1 silencing results in rapid negative selection of human AML cells in vitro independently of their genetic background. In vivo PTBP1 depletion has a strong anti-leukemic effect associated with induction of apoptosis. PTBP1 knock-down results in an increase of PKM1/PKM2 isoform ratio and changes in the rate of glycolysis, mitochondrial oxidative phosphorylation and redox homeostasis. However, these metabolic changes are heterogeneous among cell lines and are uncoupled from the anti-leukemic effect observed in vivo. RNA-sequencing combined with iCLIP analysis indicate that PTBP1 binds preferentially to intronic regions and its depletion leads to widespread changes in alternative RNA splicing. Pathways enrichment analysis showed that PTBP1 regulates the expression and splicing of genes involved in proliferation, DNA damage repair as well as in various metabolic pathways, extending beyond glycolysis to amino acids, carbohydrates, lipids, and pyrimidines metabolism. Our preliminary results suggest that, beyond controlling the catabolism of glucose through glycolysis, PTBP1 may be a central regulator of AML metabolism orchestrating multiple metabolic pathways to support the energy needs, proliferation and redox homeostasis of AML cells. Conclusions. Overall, our work shows that the splicing factor PTBP1 is essential for the growth and survival of human AML cells. Our results suggest that PTBP1 may be a key regulator of post-transcriptional gene expression in AML cells linking energy metabolism, cell proliferation and survival and, as such, a promising new molecular vulnerability in this disease. References: 1.Kantarjian HM et al. Clin Lymphoma Myeloma Leuk. 2021;21(9):580-597. 2.van Gils N et al. Front Oncol. 2021;11:659253. 3. Papaemmanuil E et al. N Engl J Med. 2016;374(23):2209-2221. 4. Adamia S et al. Clin Cancer Res. 2014;20(5):1135-1145. 5. Wang E et al. Cancer Cell. 2019;35(3):369-384.e7. 6. Wu B et al. Plos Biol. 2023;21(5):e3002088. 7. de Necochea-Campion R et al. J Hematol Oncol. 2016;9(1):85. 8. Wang E et al. Cancer Cell. 2023;41(1):164-180.e8. 9. Morales ML et al. Leukemia. 2023;37(8):1649-1659. 10. Gonzalez E et al. Curr Mol Med. 2023 Oct 24. On-line ahead of print. 11. Chen C et al. Front Mol Biosci. 2022 ;9:968458. 12. Biamonti G et al. Front Oncol. 2018;8:408. 13. Anande G et al. Clin Cancer Res. 2020;26(14):3597-3607.