Mice with Myelodysplastic Syndrome Respond to Treatment with Azanucleosides: A Preclinical Model for MDS Therapies
Authors: Gough Sheryl M , Chung Yang Jo , Shinji Maegawa , Jelinek Jaroslav , Issa Jean-Pierre , Aplan Peter 
Published: October 2019

Abstract
Abstract 3829
Therapies for the treatment of myelodysplastic syndromes (MDS) are limited. The only known curative therapy for MDS is allogeneic hematopoietic stem cell transplantation. MDS is frequently associated with epigenetic gene silencing via methylation of cytosine residues in gene regulatory regions. Clinical trials with DNA methyl-transferase inhibitors 5'azacytidine and 5-aza-2'-deoxycytidine (Decitabine, DAC) have shown hematologic responses and a survival benefit. Both azanucleosides have recently been approved for use in the clinic. However, responses are typically not durable, thus further characterization of response to treatment is required, as is the identification of new drugs to better treat MDS.
Using bone marrow cells from NUP98-HOXD13 (NHD13) transgenic mice, which have previously been shown to faithfully recapitulate key features of MDS, we co-transplanted MDS and wild type (WT) bone marrow cells into WT irradiated recipient mice. The chimaeric bone marrow transplant (BMT) produces a recipient that mimics the human condition, with patient bone marrow comprised of hematopoietic cells derived from both the MDS clone as well as normal hematopoietic precursors. WT and MDS cells in the mice can be distinguished by differential CD45 alleles (CD45.1 and CD45.2, respectively), which enables analysis and purification of the MDS and normal cells; this feat is not easily achieved with human MDS patient samples, which lack cell surface antigens specific for the MDS clone. We treated mice with 0.018mg DAC (or saline) daily for 5 days, given every 4–5 weeks, to approximate MDS patient dosing schedules. Three trial groups were divided into saline (total n=19) and DAC (total n=24) treatment cohorts. Successful treatment of the chimeric MDS mice resulted in increasing levels of WT cells in the peripheral blood, with a concomitant reduction or eradication of the MDS cells. For instance, in trial #1, DAC treated chimaeric WT/MDS mice showed less severe anemia (2.0 g/dL higher Hgb), normalized neutrophil counts (57– 85% of mice vs 20–60%) and a significant survival benefit (median 27 vs 20 weeks, p=0.004) compared to saline treated mice. The response to treatment varied within individual mice and between trials, analogous to the variability in response seen in human MDS patients.
We purified WT and MDS cells from treated mice and evaluated CpG island (CGI) DNA methylation in Chd13, a gene aberrantly methylated in MDS patients. Chd13 was hypermethylated in the murine MDS cells compared to WT cells (42–59% vs 10–16%, respectively). Of note, there was considerable variability between mice in the response to DAC treatment; half of the DAC treated mice showed a normalization of Chd13 methylation (14–20%) and half had a modest decrease in Chd13 methylation (38–40%). We next assayed global CGI methylation using a deep sequencing technique (DREAM, Digital Restriction Enzyme Analysis of Methylation). 8.2% of all CGI sites assayed were hypermethylated in MDS cells compared to WT; this increase in methylation was reduced to only 2.0% following DAC treatment of the MDS cells. This genome wide approach documented extensive hypermethylation in the NHD13 mice, similar to findings in a subset of MDS patients.
Chimaeric WT/MDS mice demonstrated variable hematologic outcomes and cytosine demethylation in response to DAC treatment, as seen in human MDS patients. Successful treatment resulted in depletion of MDS cells allowing reconstitution of the bone marrow with WT cells as would happen in the successful treatment of patients. Durable responses were seen in several recipients, including one long-term survivor with no evidence of MDS cells in the bone marrow at 24 months of age (16 months post treatment). The chimaeric WT/MDS mice therefore represent a useful pre-clinical model of human MDS, which can be used to better characterize current treatments and test novel therapies.

Disclosures:
Issa: Johnson & Johnson: Consultancy; Astex: Consultancy. Aplan:NIH Office of Technology Transfer: Patents & Royalties.