The Role of the Adenosinergic Pathway in the Resistance to Hypomethylating Agents
Authors: Vrachiolias George , Lamprianidou Eleftheria , Tasis Athanasios , Spyropoulos Theodoros , Kymparidou Chrysa , Dimitriou Despoina , Rimpa Christina Maria , Misidou Christina , Liapis Konstantinos , Papoutselis Menelaos Konstantinos , Bernard Elsa , Papaemmanuil Elli , Costeas PAUL ARISTIDES , Mitroulis Ioannis , Kotsianidis Ioannis 
Published: December 2024

Several lines of evidence suggest that hypomethylating agents (HMA), the only approved treatment for high-risk myelodysplastic syndromes (HR-MDS), may act via immune-mediated mechanisms. The adenosinergic pathway is activated within hypoxic tumors, where extracellular adenosine synthesized by the ectonucleotidases CD39 and CD73 subverts the anti-tumour immune responses. The impact of the adenosinergic pathway and the contribution of CD39 and CD73 on the antitumor immunity of MDS patients and the resistance to HMA are currently unknown.
Bone marrow mononuclear cells (BMMCs) were obtained from 41 patients with HR-MDS undergoing treatment with HMA. Expression of CD39, CD73 and PD1 on CD34+, CD4+ and CD8+ T cells and Tregs was measured using multiparametric flow cytometry, both before treatment initiation and after the sixth cycle of HMA administration. Bulk RNA-seq was performed in sorted BM-derived CD4+ T cells (accessible under accession number E-MTAB-8208) in order to identify prognostically relevant adenosine-related transcriptomic signatures.
Patients who achieved CR (responders) had significantly lower levels of CD39 on both total (p=0.041) and central memory CD4+ T cells (p=0.044), compared to patients who did not achieve CR (non-responders) after HMA treatment. Also, responders had significantly lower levels of PD1 on total CD4+ T cells (p=0.033) and lower CD73 on both naïve (p=0.042) and central memory CD4+ T cells (p=0.017) in comparison to non-responders. By contrast, responders displayed markedly increased CD39 expression on CD34+ cells (p=0.003). We further observed a significant downregulation of CD39 on total CD4+ T cells six months after the initiation of HMA only in responders, whereas non-responders displayed no changes. We then divided our patients into two groups based on their pretreatment levels of CD4+CD39+ T cells (CD39+_Low and CD39+_High). As expected the CD39+_Low group was enriched in responders (p=0.058). Patients in the CD39+_Low group had significantly lower levels of CD4+PD1+, CD8+PD1+, CD8+CD39+ and CD39+ Treg cells compared to those in the CD39+_High group. Regarding associations with the molecular profile patients with mutations on Splicing factors displayed lower pretreatment levels of CD4+CD39+ T cells (p=0.039), whereas those with mutations on genes involved in DNA methylation had significantly higher levels of CD4+CD39+ T cells (p=0.029).
In the transcriptomic analysis in purified CD4+ cells we used the pretreatment gene expression levels of ENTPD1, which encodes CD39 to separate patients in ENTPD1_High and ENTPD1_Low groups. Principal component analysis (PCA) revealed a complete separation of the two groups and a distinct adenosine signature based on the 14-gene signature of adenosine signaling which was generated by Sidders et al (Clin Cancer Res, 2020) and correlated with outcome in pan-cancer analysis. Using unsupervised clustering of gene expression, we found that ENTPD1_Low group had significantly lower levels of the 14 genes of the adenosine signature in comparison with ENTPD1_High group. Of note, in line with the postulated interferon-mediated mechanism of action of AZA, gene set enrichment analysis (GSEA) showed that the ENTPD1_Low group was characterized by higher expression levels of genes involved in IFN-γ and IFN-α signaling pathways.
Collectively, our results indicate a potential role of the adenosinergic pathway and in specific the ectonucleotidase CD39 in the immune-mediated mechanisms of response and resistance to AZA and set the scene for the testing of adenosine pathway modulators in HR-MDS.