Sequential Analysis of miRNA Profiling during Azacitidine and Lenalidomide Therapy in Myelodysplastic Syndromes

Background and Rationale. Inositide signalling regulated by Phospholipase C (PLC) and AKT is involved in epigenetic processes and in MDS progression to AML. miRNAs are small non-coding RNAs that regulate gene expression by acting on the epigenetic machinery and are themselves controlled by epigenetic mechanisms. The expression of miRNAs has been definitively linked to cancer development and miRNA profiles are studied as new prognostic factors or therapeutic new perspectives (Jiang X et al. Nat Commun 2016). Azacitidine (AZA) is a standard first-line therapy in high-risk MDS. Its combination with Lenalidomide (LEN) has been tested, but its molecular effect is still under investigation, although the concurrent acquisition of specific point mutations on PI3KCD, PLCG2 and AKT3 genes has recently been associated with loss of response to this combination therapy (Follo MY et al. Leukemia 2019). Here we further analyzed the effect of AZA+LEN therapy on epigenetic processes and inositide regulation, focusing on miRNA expression in MDS patients.

Patients and Methods. This study included 12 high-risk MDS patients treated with AZA (75 mg/m2/day, days 1-5, sc) and LEN (10 mg/day, days 1-21, orally) every 4 weeks. Patients showing complete remission (CR), partial remission (PR) or any hematologic improvement were considered as responders, while patients showing stable disease or disease progression were considered as non responders. miRNAs expression was assessed using an Affymetrix miRNA 4.0 array on patients' cells extracted at baseline and during the therapy, at the 4th (T4) and 8th (T8) cycle of therapy.

Results. All patients included in this study were considered evaluable for response. 2 patients never responded, while 10 patients showed a positive response within T4: 9 of them maintained it at T8, whereas the remaining patient lost response at T8. These clinical results do not mirror the expected clinical outcomes, but this is due to our small population. However, our analyses could be relevant to test the molecular effect of the therapy in a time course, as well as comparing different phases (baseline vs T4; baseline vs T8; T4 vs T8). Paired analysis within the same patients during treatment course showed 61 miRNAs up- or down-regulated (p<0.01), with 23 mature miRNAs affected. Four of them (hsa-miR-3065-5p, hsa-miR-3674, hsa-miR-124-3p and hsa-miR-136-5p) specifically target inositide-specific genes, especially PLCG2 and AKT3, whose point mutation has previously been associated with loss of response to AZA+LEN therapy (Follo MY et al. Leukemia 2019). More interestingly, miR-124-3p and miR-136-5p were specifically down-regulated during therapy, hinting at a relevant role for these miRNAs on inositide regulation during AZA+LEN response. To analyze data from another point of view, miRNA profiling was also compared on patients at baseline and T4 showed alterations on 42 miRNAs, with two of them (hsa-miR-192-5p and hsa-miR-21-5p, targeting PI3K/Akt signalling) being specifically up-regulated, while comparison of baseline vs T8 showed 15 miRNAs altered, with hsa-let-7f-2-3p (targeting GSK/PI3K signalling) being down-regulated. Finally, paired analysis of T4-T8 samples showed 24 miRNAs altered, with hsa-miR-224-5p (targeting PI3K/Akt) specifically down-regulated.

Conclusions. This preliminary study shows that AZA+LEN therapy affects the expression of specific clusters of miRNAs that target the PI3K signalling and, more specifically, three inositide genes that are mutated and associated with loss of response to this combination therapy, i.e. PI3KCD, AKT3 and PLCG2. Additional studies are warranted to confirm these data and to further analyze the role of these clusters, especially to test their pathogenetic or therapeutic relevance in MDS.