Abstract P03: Splicing factor mutations induce stress granule upregulation in myeloid malignancies

Abstract

Abstract Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are aggressive blood cancers with a 5-year relative survival rate of only 37% and 28%, respectively [Sekeres MA, Taylor J. JAMA. 2022; https://www.cancer.net/cancer-types/leukemia-acute-myeloid-aml/statistics]. This highlights the need to better characterize the crucial oncogenic mechanisms sustaining cancer stem cell fitness in order to develop more effective therapies. Somatic mutations affecting splicing factor genes represent a hallmark of myeloid blood cancers [Saez B, et al. Blood. 2017]. Splicing factor mutations alter the splicing process of thousands of RNA molecules, but how they provide clonal advantage with consequent ineffective hematopoiesis remains an unanswered question. We integrated fractionated enhanced UV-crosslinking and immunoprecipitation (freCLIP-seq), bulk RNA-seq and immunofluorescence imaging demonstrating that U2AF1 mutations in myeloid malignancies alter the biology of stress granules (SGs), whose upregulation has been linked to tumorigenesis [Redding A, Grabocka E. Trends Cancer. 2023]. U2AF1 mutations, through RNA binding and splicing alterations, increase the availability of SG components leading to enhanced stress adaptation. Crucially, U2AF1-mutant cells show improved cell fitness that can be reverted by a SG inhibitor [Biancon G, et al. Mol Cell. 2022]. These observations have been recently validated at single-cell level in MDS patients (n=10) without splicing factor mutations, with U2AF1 S34F mutation, or with SRSF2 P95H mutation that carries an equally poor prognosis as U2AF1-mutant disease. We isolated CD34+ blasts from bone marrow samples and performed 10x Genomics 5' scRNA-seq. Integrating cell type annotation (https://github.com/TebaldiLab/TheCellMarkerAccordion) with mutational analysis (VarTrix v1.1.19), we clearly distinguished an enrichment of U2AF1 mutant cells and of SRSF2 mutant cells in hematopoietic multipotent progenitor cells. The enrichment is observed also in monocytic cells that are also characterized by high SG score. Of note, it has been recently showed that leukemic stem cells with monocytic phenotype are associated to relapse in AML patients treated with BCL2-inh [Pei S, et al. Cancer Discov. 2023]. We then identified the top influential SG genes in each annotated mutant population and conducted functional enrichment analysis. In hematopoietic multipotent progenitor cells, the biological processes mainly affected by the SG upregulation are regulation of translation, of microtubule, of transcription and of cell growth. Changes in SGs may represent the collective result of splicing aberrancies that drive ineffective hematopoiesis in MDS and AML, or other potential tumorigenic phenotypes. Splicing factor mutations are in fact reported in several cancer types [Stanley RF, Abdel-Wahab O. Nat Cancer. 2022] and SGs are involved in several cancer hallmarks [Boija A, et al. Cancer Cell. 2021]. Alterations in SGs provide therefore novel therapeutic opportunities in blood cancers and potentially in other cancers. Citation Format: Giulia Biancon, Emma Busarello, Matthew Cheng, Domitilla Baccon, Jennifer VanOudenhove, Fabio Ciceri, Toma Tebaldi, Stephanie Halene. Splicing factor mutations induce stress granule upregulation in myeloid malignancies [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P03.