EV-mirnas predict outcomes in pediatric AML: A pilot study

Abstract Introduction: In recent years, extracellular vesicles (EVs) have gained significant interest in AML biology and prognostication. EVs are small circulating nano-sized particles implicated in cell-to-cell communication and transfer of miRNAs, proteins, metabolites/ligands across cells. In context of miRNAs, they have been shown to play a significant role in RNA silencing and gene regulation. Additionally, EV-encapsulated miRNAs profile is dependent on the cellular status. Furthermore, given the stability of miRNAs, the EV miRNA profiling is a highly attractive as biomarkers making them a promising area in oncology. With respect to AML, the circulating EVs have been implicated in bone-marrow microenvironment, immune system and contribute to drug resistance and hematopoiesis suppression. However, of the handful of studies published on exosomes/EVs in AML, the majority utilized AML cell lines and of the few on specimens from AML patients and none included pediatric AML patients. Thus, this is one of the first studies focused on investigating diagnostic and prognostic relevance of EV-derived miRNAs in pediatric AML. Methods Serum samples collected at diagnosis from 34 pediatric patients enrolled in AML02 clinical trial were included in this study. EVs were isolated using Nano pom-poms, which employs antibody-functionalized nanographene magnetic particles for specific capture of EVs. Total RNA, including small RNAs, was extracted and miRNA libraries were prepared using the NEBNext® kit. After PAGE gel-based size selection and Bioanalyzer QC the libraries were sequenced using Illumina-based next-generation sequencing (NGS), and FASTQ files were processed using the nf-core/smrnaseq pipeline. This included adapter trimming, alignment, and annotation of mature miRNAs using miRBase. Raw miRNA counts were normalized to counts per million (CPM), using edgeR package. After excluding miRNA with missing values in 2 samples, Cox proportional hazard model was used for association of miRNAs with event free survival (EFS), overall survival (OS) and measurable residual disease 1 (MRD1). Results Of the 77 miRNAs meeting the minimum expression threshold, 11 were significantly associated with EFS, 3 with OS, and 2 with MRD1. High levels of EV-miR-222-3p were predictive of better EFS (HR= 0.81, 95% CI 0.67-0.97; p= 0.03 and OS (HR= 0.63, 95% CI 0.47-0.84; p = 0.002). Several members of let-7 family, including let-7d-5p, let-7f-5p, let-7g-5p were associated with OS and EFS. EV-let-7s have previously been shown to have a tumor suppressor role and in non-small-cell lung cancer have been shown to inhibit metastatic activity by inhibiting SUV39H2/LSD1/CDH1 axis. Additionally, miR-9-5p was associated with detrimental EFS (HR-1.25, p<0.02), and a previous report has implicated it in leukemogenesis. miR-191, miR-30d, miR-24, miR27b, miR-451a and miR-486 were predictive of better EFS (at p<0.05). Notably, miR-451a levels was also predictive of MRD1 negativity, while miR-10a-5p was associated with positive MRD1 status. Conclusion This proof-of-concept pilot study provides the first evidence of EV-derived miRNAs from diagnostic specimens having the potential to serve as predictive biomarkers for clinical outcomes in pediatric AML. Several EV-miRs, including miR-9-5p, miR-486-5p, miR-222-3p and the members of let-7 family, were significantly associated with EFS, OS, or MRD1, highlighting their prognostic value. These findings support the hypothesis that EV-miRNAs contribute to leukemic progression and treatment resistance. Our ongoing studies in larger pediatric AML cohorts aim to further validate the EV-miR profiles and elucidate their mechanistic roles in disease progression and drug resistance.