PF536 THE EFFECT OF 5‐AZACITIDINE TREATMENT ON THE OF BONE MARROW MESENCHYMAL STEM TRANSCRIPTOME IN PATIENTS WITH MYELODYSPLASTIC SYNDROMES
Authors: Liapis I. , Batsali A. , Theodorou V. , Galanopoulos A. , Spanou A. , Talianidis I. , Papadaki H.A. 
Published: June 2019

Background:

Myelodysplastic syndromes (MDS) are clonal disorders of the hematopoietic stem cells (HSC), characterized by inefficient bone marrow (BM) haemopoiesis and increased risk for leukaemic evolution. However, MDS pathogenesis is not restricted to the HSC compartment. Emerging evidence suggests a disease permitting or even a disease promoting role for the BM microenviroment (Cogle et al,2015).Mesenchymal stem cells (MSCs) are a key component of the BM niche. MDS derived MSCs (MDS‐MSCs) have been shown to hold intrinsic functional defects and an in ability to sustain normal haemopoesis (Bulycheva et al, 2015). Moreover, recent data suggest that MDS‐MSCs harbor genetic abnormalities (Blau et al,2011) and also an altered DNA methylation status (Poloni et al,2014).5‐Azacitidine (AZA), a hypomethylating agent, is widely used for MDS treatment. Our (Liapis et al,ASH 2017) and other groups have previously shown that AZA is able to partially reverse the proliferative and clonogenic deficiencies of MDS‐MSCs, whereas it is unable to affect their intrinsic genomic instability. However, knowledge of its effect on the MDS‐MSC transcriptome is still lacking.

Aims:

We aim to provide the first comparative analysis of the MDS‐MSC transcriptome before and after long term AZA treatment.

Methods:

BM samples were collected from 12 patients with high risk MDS after informed consent. Repeat harvest, after 6 cycles of AZA therapy was possible in 7 of these patients. BM samples were also collected from 7 healthy subjects undergoing orthopedic surgery. MSCs were then isolated, ex vivo expanded and re‐seeded for a total of 5 passages (P). At P2, expanded MSCs, fulfilled the minimal criteria for MSC definition (Dominici et al, 2006) and consisted of a homogenous population. Total RNA was harvested from P2 MSCs and subjected to RNA sequencing assays. We used the QuantSeq 3’ mRNA‐Seq Library Prep Kit FWD for Illumina. It utilizes the 3′prime method of sequencing only the 3′region of the RNAs and also provides quantative measurements.

Results:

Thus far, data analysis has been completed in 5 paired MDS‐MSC samples, before and after AZA therapy. Proliferation and clonogenicity parameters showed a marked improvement upon AZA treatment.410 genes were shown to have an over 2∗log2 change in their expression. Of these genes, 66 were found to be differently regulated with statistical significance (p < 0.05), in spite of the low sample count.

Surprisingly, despite AZA's function as a transcriptional promoter, long termtreatment conferred MSC gene downregulation rather than upregulation: 228 genes exhibited transcriptional suppression. Significantly downregulated genes included members of proliferative signaling cascades, among which,effectors of PI3k‐Akt and Wnt signaling. Other affected cellular processes were those of apoptosis, extracellular matrix synthesis and stromal adherence. Notable mentions of downregulated genes are those of CCND2, encoding for cyclin D2 (‐3.317∗log2, q = 0.013), the pro‐apoptotic protein BAX (‐3.05∗log2, q = 0.013) and FRZ2 (‐2.27∗log2, q = 0.013) a mediator for Wnt signaling.

182 genes were shown to be over 2∗log2 fold upregulated. Statistically significant difference was found in just 3 of them and thus, no safe conclusion can be drawn. However, analysis of the remaining MDS‐MSC and normal MSC RNA samples is still in process.

Summary/Conclusion:

Transcriptome analysis reveals that long term AZA treatment is able to restore MSC stemness. The inhibition of both the mitotic and apoptotic processes contributes to stem cell quiescence and longevity.