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

Program: Oral and Poster Abstracts
Type: Oral
Session: 616. Acute Myeloid Leukemia: Novel Therapy, excluding Transplantation: Novel promising therapies for relapsed/refractory AML
Hematology Disease Topics & Pathways:
Biological, survivorship, HSCs, Therapies, Pediatric, Biological Processes, Technology and Procedures, epigenetics, Cell Lineage, Study Population, Clinically relevant, Quality Improvement , stem cells, RNA sequencing
Saturday, December 5, 2020: 10:45 AM

Inge Van Der Werf, MSc1,2, Phoebe Mondala, BS2,3*, Raymond Diep, BS3*, Larissa Balaian, PhD4*, Cayla Mason, BS3*, Jacqueline Cloos, PhD5*, Gertjan J.L. Kaspers, Prof. MD, PhD6, Jim La Clair7*, Peggy Wentworth8*, Leslie A Crews, PhD3, Thomas Whisenant, PhD9*, Kathleen Fisch, PhD9*, Michael Burkart, PhD7* and Catriona Jamieson, MD, PhD10,11

1Amsterdam UMC, Voorschoten, Netherlands
2authors contributed equally, .
3Division of Regenerative Medicine, University of California, San Diego, La Jolla, CA
4Moores Cancer Center, UCSD, La Jolla, CA
5Cancer Center Amsterdam, Department of Hematology, VU University Medical Center, Amsterdam, Netherlands
6Princess Máxima Center for Pediatric Oncology, Utrecht, Netherlands
7Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA
8UCSD, La Jolla
9Center for Computational Biology and Bioinformatics, University of California San Diego, La Jolla, CA
10Moores Cancer Center, University of California, San Diego, La Jolla, CA
11Division of Regenerative Medicine, Department of Medicine, Moores Cancer Center, and Sanford Stem Cell Clinical Center, University of California, San Diego, La Jolla, CA


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.


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.


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 survival (Holm...Jamieson. PNAS 2015;112:15444-15449; Denichenko et al Nat Commun 2019;10:1590). Treatment with 17S-FD-895 induced a dose-dependent reduction in clonogenicity (p=0.001; Student’s t-test) and self-renewal of CD34+ cells (p=0.001; Student’s t-test) isolated from pediatric AML samples. Pediatric AML samples were significantly more sensitive to splicing modulation than adult de novo or adult secondary AML. Moreover, normal cord blood HSC and HPC samples were unaffected by splicing modulator treatment. Our lentiviral splicing reporter assays demonstrated a dose dependent increase in MAPT intron retention in in pediatric leukemia cell lines as measured by a switch from GFP to RFP. Finally, splice isoform specific RT-PCR demonstrated a dose-dependent increase in SF3B1 intron retention following treatment as well as MCL1 exon 2 skipping, producing pro-apoptotic MCL1-S transcripts.


Cumulatively, our data indicate that spliceosome modulation via 17S-FD-895-mediated targeting of SF3B1 constitutes a novel potential therapeutic strategy for pediatric patients with AML.

Disclosures: Kaspers: Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; AbbVie: Ended employment in the past 24 months; Janssen R&D: Ended employment in the past 24 months; Helsinn Healthcare: Ended employment in the past 24 months. Crews: Ionis Pharmaceuticals: Research Funding. Jamieson: Forty Seven Inc: Patents & Royalties; Bristol-Myers Squibb: Other.

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