Abstract 469: Selective targeting of splicing deregulation in pediatric acute myeloid leukemia stem cells
Authors: Van der Werf Inge , Mondala Phoebe , Diep Raymond , Balaian Larisa , Mason Cayla , Kaspers Gertjan , Cloos Jacqueline , La Clair Jim , Wentworth Peggy , Whisenant Tom , Fisch Katie , Burkart Michael , Jamieson Catriona 
Published: September 2020

Abstract
While 5 year survival rates for acute lymphoblastic leukemia (ALL) in childhood are 90% (https://www.cancer.org/cancer/leukemia-in-children/detection-diagnosis-staging/survival-rates.html), acute myeloid leukemia survival rates have lagged behind at 65 to 70%. Thus, AML is the leading cause of childhood leukemic mortality and is a heterogeneous disease characterized by diverse mutations many occurring at low frequencies. Whole exome sequencing analyses have not been sufficient to predict relapse in many pediatric patients at least in part because of transcriptomic and epitranscriptomic alterations that drive therapy resistant leukemia stem cell (LSC) propagation. Recent results reported by ourselves and others suggest that LSC in AML harbor unique mRNA splicing profiles characterized by intron retention and exon skipping. In adult AML, whole transcriptome RNA sequencing data revealed global spliceosome disruption that uniquely distinguished LSCs from normal age-matched hematopoietic stem and progenitor cells (Crews et al Cell Stem Cell 2016). However, the role of splicing deregulation in LSC propagation in human pediatric AML had not been clearly elucidated. Recently, we have developed 17S-FD-895, a small molecule compound targeting SF3B1, which modulates mRNA splicing. To date, we have evaluated the effects of this splicing modulator on both self-renewal as well as pro-survival splice variants in CD34+ cells derived from both peripheral blood as well as bone marrow of pediatric AML patients. Splice isoform specific qRT-PCR demonstrated a dose-dependent increase in SF3B1 intron retention following treatment. Furthermore, splicing modulation induced MCL1 exon 2 skipping, producing pro-apoptotic MCL1-S transcripts. Hematopoietic progenitor assays demonstrated a dose-dependent reduction in LSC clonogenicity and self-renewal. In these assays, LSC were found to be sensitive to nanomolar concentrations that spared normal hematopoietic stem and progenitor cells. To further dissect the role of pre-mRNA splicing in pediatric AML, we developed a lentiviral fluorescent splicing reporter that switches from GFP to RFP expression following an exon skipping or intron retention event. In addition, whole transcriptome RNA sequencing (RNA-seq) was performed on FACS purified stem cells (CD34+/CD38-/Lin-) as well as progenitor cells (CD34+/CD38+/Lin-) from both pediatric AML as well as aged-matched normal bone marrow samples and samples were transplanted into immunocompromised mice followed by 17S-FD-895 treatment to assess sensitivity to splicing modulation in vivo. As a result of these studies, we have demonstrated LSC splicing patterns in pediatric AML that may inform novel biomarker identification as well as development of 17S-FD-895 for pediatric AML.
Citation Format: Inge Van der Werf, Phoebe Mondala, Raymond Diep, Larisa Balaian, Cayla Mason, Gertjan Kaspers, Jacqueline Cloos, Jim La Clair, Peggy Wentworth, Tom Whisenant, Katie Fisch, Michael Burkart, Catriona Jamieson. Selective targeting of splicing deregulation in pediatric acute myeloid leukemia stem cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 469.