Deregulation of Splicing in Pediatric Acute Myeloid Stem and Progenitor Cells
Authors: Mondala Phoebe , Van Der Werf Inge , Balaian Larisa , Steel Kathleen , Ladel Luisa , Mason Cayla , Diep Raymond , Cloos Jacqueline , Kaspers Gertjan , van Roosmalen Markus J , Van Boxtel Ruben , Chan Warren , Mark Adam , Donohoe Mary , La Clair James , Wentworth Peggy , Fisch Kathleen , Crews Leslie A , Whisenant Thomas , Burkart Michael , Jamieson Catriona 
Published: November 2021

Abstract
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Currently, the limited capacity of pediatric acute myeloid leukemia (AML) therapies to prevent recurrence has contributed to high mortality rates. While dormant self-renewing leukemia stem cells (LSCs) contribute to adult AML relapse, their role in pediatric AML therapeutic resistance has not been clearly elucidated and thus was investigated in the context of this study.
Through whole transcriptome sequencing (RNA-seq) analyses of FACS-purified human hematopoietic stem cells (HSCs; CD34 +CD38 -Lineage -) and progenitor cells (HPCs; CD34 +CD38 + Lineage -) from pediatric AML (n=10) compared with adult de novo (n=5) and secondary AML (n=6) as well as non-leukemic pediatric bone marrow samples (n=6), we identified widespread splicing alterations in pediatric AML compared to non-leukemic donors, indicative of a disruption in splicing regulation. In this study, we identified 2,000 exon skipping events in pediatric AML HSCs and HPCs. Moreover, we detected increased exon skipping and intron retention in stem cell self-renewal and survival transcripts in pediatric AML stem and progenitor cells. Specifically, the pro-survival isoform of MCL1, MCL1 long, was significantly increased in comparison to its pro-apoptotic counterpart, MCL1 short. In addition, self-renewal, RNA editing and splice isoform altering adenosine deaminase RNA specific 1 (ADAR1) p150 isoform levels were significantly (p=0.05) upregulated in pediatric AML progenitors suggesting that splicing and RNA editing deregulation could fuel pediatric AML stem and progenitor cell propagation.
After successful completion of pre-IND development of a pharmacologically stable, potent, and selective small molecule splicing modulator, Rebecsinib (17S-FD-895) (Crews, Balain et al Cell Stem Cell 2016; Chan et al Cell Reports 2020), we developed a dual fluorescence lentiviral splicing reporter that assays the on target anti-leukemic efficacy of Rebcsinib and to assess the therapeutic index between LSCs and normal hematopoietic stem and progenitor cells. In hematopoietic progenitor assays, we observed a dose-dependent reduction in clonogenicity and replating of CD34 + cells isolated from pediatric AML samples following treatment with Rebecsinib. While pediatric AML samples were more sensitive to splicing modulation than adult de novo or adult secondary AML samples, normal cord blood progenitor samples were unaffected by splicing modulator treatment. In addition, we identified dose-dependent alterations in lentiviral splicing reporter activity in pediatric leukemia cells engrafted in a humanized AML mouse xenograft model following intravenous treatment with one dose of 10mg/kg and 20mg/kg of Rebecsinib. Finally, we observed a reduction in ADAR1 p150 transcripts by RNA-seq analysis of hematopoietic tissues in serially transplanted patient derived AML xenografts after Rebecsinib treatment suggesting that inhibition of ADAR1 splicing prevents LSC self-renewal.
Cumulatively, these data demonstrate that stem and progenitor cell specific deregulation of pre-mRNA splicing and ADAR1 activation represent a therapeutic vulnerability to splicing modulation, which provides a strong rationale for developing Rebecsinib for preventing pediatric AML recurrence.

Disclosures
Cloos: Astellas: Speakers Bureau; DC-One: Other, Research Funding; Genentech: Research Funding; Helsinn: Other; Janssen: Research Funding; Merus: Other, Research Funding; Navigate: Patents & Royalties; Novartis: Consultancy, Other, Research Funding; Takeda: Research Funding. Crews: Ionis Pharmaceuticals: Research Funding. Burkart: Algenesis: Other: Co-founder. Jamieson: Forty Seven Inc.: Patents & Royalties.