Identification of a Circulating Serum miRNA Signature As a Predictive Biomarker and Potential Therapeutic Target in Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is an aggressive hematological malignancy. Despite the R-CHOP immunochemotherapy, ≈35% of patients are refractory/relapsing. Among the prognostic factors there are genomic alterations and gene expression profiles, which define the activated B-cell-like (ABC) and germinal centre B-cell-like (GCB) cell-of-origin (COO) subtypes. However, none of these indicators can entirely predict treatment response. Thus, the identification of new prognostic/predictive biomarkers that might complement those currently in use, is demanding.

MicroRNAs (miRNAs), small non-coding RNAs with an epigenetic function, impact on several cancer processes including drug resistance. They are deregulated both in tumour tissues and patient blood, where they circulate in a stable form and can be easily detected, thus making miRNAs interesting non-invasive biomarker candidates. Moreover, recent advances in high-throughput sequencing techniques have unveiled their prognostic/predictive utility in numerous pathologies including lymphomas. In this regard, we previously found the serum miR-22-3p as a circulating prognostic biomarker being correlated with progression-free survival (PFS), independently from other prognostic factors. However, a signature of miRNAs, rather than a single miRNA, may represent a more reliable biomarker and a better mirror of the complexity of the disease.

In this study, using a small-RNA sequencing on serum samples from 33 de novo DLBCL patients (15 refractory subjects and 18 responsive to R-CHOP), we selected circulating miRNAs differentially expressed between refractory and responding patients (area under the ROC curve-AUC>0.7; p value≤0.05). The combined expression of three selected serum miRNAs (miR-200c-3p, miR-421 and miR-324-5p) improved the predictive performance compared to the use of the single miRNAs (from AUC 0.72 to 0.8) and was also correlated to PFS (p=0.02). Interestingly, among these three miRNAs, miR-421 and miR-324-5p were also differentially expressed in DLBCL tumor tissues on the basis of the response to treatment, being significantly downregulated in refractory patients compared to the responsive ones (fold change of 0.27 and 0.37 respectively). Investigating the functional role of this two-miRNA signature in the biology of DLBCL, we also found that its overexpression induced a proliferation and viability decrease in the GCB-COO DLBCL but not in ABC subtype, thus suggesting a tumor suppressive role of this restricted signature depending on the COO context. Furthermore, we identified and validated EGLN1 and TXNRD1 as novel direct targets of miR-421 and miR-324 respectively. Interestingly, both of them encode for proteins with an impact on the oxygen metabolism and the redox homeostasis of cells, processes frequently altered in DLBCL and impacting on tumor cell survival and response to treatment. Lastly, ongoing experiments indicate that the inhibition of the identified miRNA targets impacts viability by triggering cell death programs upon their silencing. Of note, inhibitors for the enzymes encoded by the target genes constitute already approved drugs for hematological or inflammatory diseases, giving hope for the possibility of a drug repositioning approach for DLBCL treatment.

Altogether these findings, representing a significant and timely contribution to the DLBCL biology understanding, support a potential application of a two-miRNA signature and its targets for novel combined therapeutic interventions in DLBCL and may be of critical importance especially with regards to precision medicine.

No relevant conflicts of interest to declare.