Determining the Mechanism of 5-Azacytidine Resistance in Myeloid Malignancies Using Single-cell DNA Methylation Sequencing Paired With Flow Cytometry

Identification: O'Neill, K.


Description

Determining the Mechanism of 5-Azacytidine Resistance in Myeloid Malignancies Using Single-cell DNA Methylation Sequencing Paired With Flow Cytometry

K O'Neill1,4, T Hui2, D Deng4, J Parker4, M Moksa3, M Hirst3,4, A Karsan1,4

1Pathology Department, 2Genome Science and Technology Program, 3Department of Microbiology & Immunology, University of British Columbia, 4Michael Smith Genome Sciences Centre, BC Cancer Agency, Vancouver, Canada

Myeloid malignancies, especially myelodysplastic syndromes (MDS) are often treated with the DNA methyltransferase inhibitor, 5-azacytidine (5-Aza). But only half of those patients respond, and resistance typically develops within two years, followed by rapid disease progression and death [1] [2]. Recent evidence suggests 5-Aza resistance originates in rare MDS propagating cells (MDS-PC) within hematopoeitic progenitors [3]. However, the exact phenotype of these cells is unclear [3] [4], and there has been limited research into their role in resistance.

We have index-sorted and sequenced 192 CD34+CD38-Lin- single cells each from bone marrow samples before and after 5-Aza treatment from a patient with relapse after initial response, and performed whole-genome bisulfite sequencing (SC-WGBS) of each cell. We observed that while pre-treated cells have similar global methylation (mean=76.5%, SD=1.2%), 30% of the post-treated cells were globally hypomethylated. This provides the first SC-WGBS study of 5-Aza response in a clinical context, and contrasts with the much more consistent 5-Aza hypomethylation response seen in vitro in SC-WGBS studies of K562 [5].

We were also able to resolve single cell copy number alterations to 5Mbp resolution, subclonally detecting a trisomy 9 only present in the post-treatment sample, and previously reported from clinical metaphase spreads.

Neither the trisomy 9 nor the clustering of cells into hypo- and normally methylated could have been detected in bulk data. This work validates the use of single-cell DNA methylation analysis for investigating response to 5-Aza therapy. Future work includes sequencing more patients pre and post 5-Aza treatment, and more in-depth analysis of differential methylation, intercellular heterogeneity, and the role (if any) of subclonal copy number alterations.

1. Bejar, Steensma (2014) Blood 124:2793–2803.

2. Zeidan et al. (2014) British Journal of Haematology 166:352–359.

3. Woll et al. (2014) Cancer Cell 25:794–808.

4. Corces-Zimmerman, Majeti (2014) Leukemia 28:2276–82.

5. Farlik et al. (2015) Cell Reports 10:1386–1397.

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Credits: None available.

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