Decreased Embryonic Splicing Repressor Expression Enhances Splicing Modulator Sensitivity in Pediatric Acute Myeloid Leukemia Stem Cells

Adults with myeloid malignancies frequently harbor mutations in splicing regulatory genes, such as SRSF2, U2AF1, and SF3B1. In pediatric AML (pAML), splicing factor mutations are relatively infrequent in bulk populations of cells. However, by performing whole exome sequencing, single cell proteogenomics (Tapestri®) as well as whole transcriptome RNA sequencing (RNA-seq) analyses of FACS-purified hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs), we discovered an increased frequency of exon skipping, typical of embryonic splicing patterns, in pAML HSCs and HPCs. By employing single cell proteogenomics (Tapestri®) analysis, we discovered SF3B1 splicing factor mutations at the single cell in pAML CD34+ cells that were not readily detectable by whole exome sequencing in bulk populations thereby suggesting that clonal expansion of minor clones may fuel leukemic stem cell (LSC) generation.

Further RNA-seq analysis demonstrated that both pAML HSCs and HPCs harbored decreased expression of a repressor of embryonic splicing, RNA-binding Fox protein 2, RBFOX2(also known as RBM9). By employing, rMATS, an algorithm that detects splicing events, we discovered differential splicing of genes involved in cell cycle regulation, DNA repair and chromatin organization in pAML more than in non-leukemic (NL) samples. In addition, differentially spliced RBFOX2 target genes were found to be involved in mRNA processing. In pAML HSCs, we observed differential splicing of THOC7, which regulates mRNA 3' end processing while in pAML HPCs, we uncovered a cluster of splicing regulators, such as SRSF7, SRSF11 and U2AF2. Thus, pAML is typified by widespread splicing deregulation in the setting of RBFOX2 downregulation.

Previous reports suggest that splicing deregulation represents a therapeutic vulnerability in a broad array of malignancies. Thus, we examined the response of pAML cell lines in vitro and in lentiviral splicing reporter humanized AML mouse models to Rebecsinib (17S-FD-895; Crews et al, Cell Stem Cell, 2016), a small molecule splicing modulator that is currently undergoing IND enabling studies. In these studies, Rebecsinib treatment reduced pAML cell survival at doses that altered splicing reporter activity and spared normal hematopoietic stem and progenitor cells (HSPCs). Notably, shRNA knockdown of RBFOX2 in cord blood CD34+ HSPCs phenocopied aspects of pAML in that it significantly increased clonogenic capacity and sensitivity to Rebecsinib in replating assays. In summary, decreased RBFOX2 expression and reversion to an embryonic splicing program typified by differential exon usage in pAML HSPCs was associated with increased sensitivity to selective splicing modulation and may represent a new therapeutic paradigm for pediatric patients with relapsed or refractory AML.

Cloos:DC-One: Other: MRD assessments, Research Funding; Takeda: Research Funding; Merus: Other: MRD assessments, Research Funding; Helsinn: Other: MRD assessments; Navigate: Patents & Royalties: Royalties for MRD analyses; Genentech: Research Funding; Janssen: Research Funding; Novartis: Consultancy, Other: MRD assessments, Research Funding; Astellas: Speakers Bureau. Burkart:Aspera Biomedicines: Consultancy, Other: co-founder of Aspera Biomedicines. Jamieson:Aspera Biomedicines, Impact Biomedicines,: Consultancy, Other: co-founder of Aspera Biomedicines and Impact Biomedicines and has received royalties for intellectual property licensed by Forty Seven Inc, Patents & Royalties: Forty Seven Inc.