Selective Targeting of Alternative Splicing Deregulation in Pediatric Acute Myeloid Stem and Progenitor Cells

• Published in: Blood 2020-11, Vol.136 (Supplement 1), p.8-8
• Main Authors: Van Der Werf, Inge, Mondala, Phoebe, Diep, Raymond, Balaian, Larissa, Mason, Cayla, Cloos, Jacqueline, Kaspers, Gertjan J.L., La Clair, Jim, Wentworth, Peggy, Crews, Leslie A, Whisenant, Thomas, Fisch, Kathleen, Burkart, Michael, Jamieson, Catriona
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2020-136596

INTRODUCTION Because acute myeloid leukemia (AML) is the leading cause of pediatric leukemia relapse-related mortality, there is a desperate need for developing new therapeutics and providing mechanistic insights into the cell type and molecular context specific causes of relapse. Myeloid malignancies, including AML, have a propensity to disrupt the spliceosome either through acquisition of splicing factor gene mutations or epigenetic spliceosome disruption in leukemia stem cells (reviewed in Chua, Van Der Werf, Jamieson, Signer. Cancer Cell 2020;26:138-159). While leukemia stem cells (LSCs) promote relapse in adult AML as a result of their inherent capacity to become dormant and resist therapies that target dividing cells, the role of alternative splicing (AS) deregulation in relapse and therapeutic vulnerability of LSCs has not been thoroughly studied in pediatric AML. METHODS Recently, we completed pre-IND enabling studies with a selective splicing modulator, 17S-FD-895. Previously, we showed that 17S-FD-895 selectively eliminated adult AML LSC (Crews...Burkart, Jamieson. Cell Stem Cell 2016;19:599-612). As a stable pladienolide-derived small molecule splicing modulator, 17S-FD-895 targets a key component of the spliceosome, SF3B1, thereby modulating mRNA splicing. To investigate the role of splicing deregulation in pediatric AML LSC maintenance, we developed a sensitive in vitro and in vivo lentiviral splicing reporter assay, whole transcriptome RNA sequencing (RNA-seq) stem and progenitor cell splicing analysis pipelines, qRT-PCR splice isoform specific biomarkers of response, as well as stromal co-culture, hematopoietic progenitor colony survival and replating assays to assay LSC eradication. RESULTS Because splicing regulation is cell type and context dependent, we generated a comprehensive transcriptome expression map of FACS-purified hematopoietic stem cells (HSCs; CD34+CD38-Lin-) and hematopoietic progenitor cells (HPCs;CD34+CD38+Lin). By utilizing a splice variant-specific alignment algorithm, we evaluated genome wide alternative splicing events and uncovered widespread exon skipping in pediatric AML compared to non-leukemic donors. More than 2000 exon skipping events were identified in pediatric AML HSCs and HPCs. In addition, both pediatric AML HSC and HPC demonstrated a downregulation of the splicing regulator RNA-Binding fox 2 (RBFOX2), which has been linked to embryonic stem cell splice variant signatures that are vital for leukemia cell su