The Role of Small Non-Coding RNAs in Normal and Malignant Hematopoiesis

Small non-coding RNAs (sncRNAs) have emerged as important regulators of multiple cellular processes, with a growing body of evidence highlighting their dysregulation and possible pathogenic role in many malignancies, including acute myeloid leukemia (AML). We utilized various high throughput genomic approaches to identify dysregulated sncRNAs in primary samples from AML patients. While somatic point mutations and copy number alterations involving microRNA (miRNA) genes are uncommon in AML, we identified the hemizygous loss of the MIR223 gene in a male patient with therapy-related AML. Studies collectively show that MIR223 is commonly silenced in leukemia, but the contribution of MIR223 loss to disease pathogenesis is unclear. Using a mouse model of Mir223 deletion we found that loss of this gene results in a modest expansion of myeloid progenitors without significant effects on hematopoietic stem cell (HSC) function or development of malignancy with age. These results suggest that suppression of miRNA-223 is likely not an initiating event in AML but could potentially cooperate with other oncogenes in disease pathogenesis. Additionally, next generation sequencing of 200 cases of de novo AML revealed the recurring point mutation of only one miRNA gene , MIR142. Our data suggest that these MIR142 mutations likely lead to a complete loss of normal miRNA-142-3p function in addition to decreased processing or stability of miRNA-142-5p. To model this in vivo, we characterized the hematopoietic compartment of mice lacking the Mir142 gene. We observed that loss of Mir142 drastically decreased HSC repopulating activity while conferring a myeloid bias. Additionally, all AML200 cohort patients with MIR142 point mutations had coincident isocitrate dehydrogenase 1 or 2 (IDH1/2) neomorphic mutations. Transplant studies showed that deletion of Mir142 together with over-expression of mutant IDH2 resulted in anemia and neutrophilia in recipient mice, suggesting these mutations cooperate in vivo to induce aberrant hematopoiesis. Finally, previous high throughput sncRNA transcriptome sequencing studies have focused on the 15-30 nucleotide fraction that primarily consists of miRNAs, thus excluding many potentially important sncRNA species. To address this sequencing gap, we developed a transcriptome sequencing approach to assess RNA species 15-75 nucleotides in length and applied this to a cohort of 28 de novo AMLs. The most striking example of dysregulation was observed in acute promyelocytic leukemia (M3 AML). The highly conserved, imprinted DLK1-DIO3 locus at 14q32.2 contains 41 small nucleolar RNA (snoRNA) genes belonging to the SNORD112-114 family, in addition to a large number of miRNAs. Compared with healthy donor CD34+ cells or non-M3 AML, the snoRNAs and miRNAs in this locus were massively upregulated in M3 AML samples. The paternally-derived protein coding genes in this locus (DLK1, RTL1 and DIO3) showed no dysregulation in M3-AML in contrast to the aberrantly expressed, maternally-derived sncRNAs. Targeted bisulfite sequencing and expressed germline single nucleotide polymorphisms revealed that imprinting of the DKL1-DIO3 locus is not disrupted in M3-AML samples and that dysregulation of the sncRNAs in this region occur through an imprinting-independent mechanism. Collectively these data further support the importance of sncRNAs in the maintenance of the hematopoietic system and reveal how their dysregulation can lead to aberrant hematopoiesis. Furthermore, these studies highlight the importance of expanding investigation of previously less well-characterized and often un-explored larger sncRNA species