Abstract 219: Splicing inhibitors reduce human AML CD34+ cell survival and self-renewal during MDS/AML evolution in a leukemia stem cell supportive niche assay.
Authors: Balaian Larisa , Burkart Michael , Yost Shawn , Rozenhak Sophie , Ball Edward D. , Frazer Kelly , Harismendy Olivier , Jamieson Catriona 
Published: August 2014

Abstract
Introduction. Myelodysplastic syndromes (MDS) are relatively common neoplasms of hematopoietic stem cells, which commonly evolve to acute myeloid leukemia (AML). Recent results suggest that MDS evolution is controlled by mutations in splicing related genes and epigenetic modifiers of gene expression. Alternative splicing driven by these mutations has been implicated in the evolution of MDS to AML. However, little is known about the cell type and context specific functional effects of these mutations on leukemia stem cells (LSC) that promote AML therapeutic resistance. Therefore, we investigated 1) the effect of splicing inhibitors on LSC survival and self-renewal during the MSD/AML progression in a bone-marrow stromal co-cultures that recapitulates key aspects of the human LSC niche and 2) the genomic mutations in LSC and non-LSC populations during disease progression. Methods. Whole exome sequencing on CD34+ and CD34- cells before and after MDS/AML progression . Mouse bone marrow cell lines transfected to produce human SCF, IL3 and G-CSF, were used as a stromal monolayer. Human CD34+ cells were selected from MDS and AML primary samples (n=6). As a normal control, cord blood CD34+ cells (CB, n=3) were utilized. Two SF3B1-targeted splicing inhibitors: FD 895 and a FD-analog were added at the initiation of co-culture at concentrations ranging from 0.1 to10 uM. Results. Whole exome DNA sequencing analysis revealed that a loss of chr21 was observed at different frequencies in CD34- and CD34+ cells. We identified a RUNX1 missense mutation with increasing prevalence in CD34+ during progression. Notably, loss of heterozygosity and a missense mutation in the histone methyltransferase EZH2 gene, implicated in MDS progression, was detected only in CD34+ post-progression to AML. After 2 weeks of stromal co-culture, survival of the cells was investigated by colony forming assay in methylcellulose. While the splicing inhibitors demonstrated no cytotoxicity towards normal CB, MDS and AML samples showed a dose-dependent inhibition of colony formation. To analyze the effect of splicing inhibitors on LSC self-renewal, replating assays were performed. While compounds at high doses mediated only a slight decrease in colony formation in CB samples, MDS and AML samples exhibited a dose dependent inhibition of LSC survival (38.2+/-8.1% p&lt;0.001) for FD895 and (13.8+/-3.6% p&lt;0.001) for FD analog. Notably, the more potent FD-analog demonstrated considerably higher ability to eradicate LSC compared to FD895. Conclusions. These data demonstrate that molecular evolution of MDS to AML may be driven by specific mutations in epigenetic modifiers, such as EZH2, and alternative splicing in CD34+ cells which gain the capacity to survive and self-renew in LSC supportive niches. These properties can be inhibited using novel splicing inhibitors with minimal toxicity toward normal progenitors.
Citation Format: Larisa Balaian, Michael Burkart, Shawn Yost, Sophie Rozenhak, Edward D. Ball, Kelly Frazer, Olivier Harismendy, Catriona Jamieson. Splicing inhibitors reduce human AML CD34+ cell survival and self-renewal during MDS/AML evolution in a leukemia stem cell supportive niche assay. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 219. doi:10.1158/1538-7445.AM2013-219