Expression profiling of snoRNAs in normal hematopoiesis and AML

Key Points A subset of snoRNAs is expressed in a developmental- and lineage-specific manner during human hematopoiesis. Neither host gene expression nor alternative splicing accounted for the observed differential expression of snoRNAs in a subset of AML.</jats:p

MicroRNA-223 regulates granulopoiesis but is not required for HSC maintenance in mice

MIR233 is genetically or epigenetically silenced in a subset of acute myeloid leukemia (AML). MIR223 is normally expressed throughout myeloid differentiation and highly expressed in hematopoietic stem cells (HSCs). However, the contribution of MIR223 loss to leukemic transformation and HSC function is largely unknown. Herein, we characterize HSC function and myeloid differentiation in Mir223 deficient mice. We show that Mir223 loss results in a modest expansion of myeloid progenitors, but is not sufficient to induce a myeloproliferative disorder. Loss of Mir223 had no discernible effect on HSC quiescence, long-term repopulating activity, or self-renewal capacity. These results suggest that MIR223 loss is likely not an initiating event in AML but may cooperate with other AML associated oncogenes to induce leukemogenesis

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

Loss of <i>Mir223</i> is not associated with alterations in HSC number or function.

<p>(A) Representative plots showing the gating strategy to identify multipotent progenitors (MPP, CD34⁺Flt3⁺), CD34^-KLS (CD34^-Flt3^-KLS), or CD150⁺CD48^-KLS cells. (B) Progenitor data for 8–12 week old wild-type (WT) or pooled male and female Mir223 deficient mice (223KO). (C) Representative plots showing cell cycle analysis of CD150⁺CD48^-KLS cells. (D) CD150⁺CD48^-KLS cell cycle data. (E) Wild-type or <i>Mir223</i> deficient bone marrow (Ly5.2) was mixed at a 1:1 ratio with wild-type (Ly5.1) competitor bone marrow and transplanted into irradiated recipients (Ly5.1/5.2). Shown is the percentage of Ly5.2 donor cells (n = 9–10 mice from two independent experiments). (F) Lineage distribution in the bone marrow 24 weeks after transplantation. (G) Serial transplants were performed with either Ly5.2 wild-type or <i>Mir223</i> deficient donor marrow. The percentage of donor Ly5.2 cells in the blood 6–8 weeks after transplantation is shown. (n = 4–5 mice per cohort at each time point). All data represent the mean ± SEM. *p<0.05, **p<0.005, ***p<0.001.</p

Loss of <i>Mir223</i> is associated with minimal perturbations of basal granulopoiesis.

<p>(A) Peripheral blood from male and female wild-type and <i>Mir223</i> deficient mice (223KO) was analyzed at 8–12 weeks of age. (B) Absolute number of neutrophils (PMN, Gr-1^HiCD115^-), monocytes (Mono, Gr-1^INTCD115⁺), B cells (B220⁺), and T cells (CD3e⁺) in peripheral blood. (C) Bone marrow mononuclear cell count (BMMNC). (D) Representative plots showing the gating strategy to identify mature neutrophils, granulocyte precursors (Pre, Gr-1^INTCD115^-) and monocytes. (E) Bone marrow lineage and granulocyte precursor data. (F) Representative plots showing the gating strategy to identify megakaryocyte-erythroid progenitors (MEP, CD34^-FcγR^-), common myeloid progenitors (CMP, CD34⁺FcγR^-) or granulocyte-macrophage progenitors (GMP, CD34⁺FcγR⁺); data are gated on lineage^-c-kit⁺sca-1^- cells. (G) Progenitor data.</p

Loss of <i>Mir223</i> is associated with an increased stress granulopoiesis response.

<p>(A) Wild-type and <i>Mir223</i> deficient mice (223KO) were treated with 150 mg/kg of 5-FU and neutrophil counts were followed for 35 days (n = 8–10 mice per cohort from two independent experiments). (B) Representative plots showing Ki-67 and DAPI staining of GMPs. Harvested on day 14 following 5-FU. (C) Shown is the percentage of GMPs in the G2/M/S phase of the cell cycle. All data represent the mean ± SEM. *p<0.05, **p<0.005, ***p<0.001.</p

Neutrophils counts are normal in aged <i>Mir223</i> deficient mice.

<p>(A and B) Wild-type and <i>Mir223</i> deficient (223KO) mice at least 6 months old were analyzed for (A) peripheral blood or (B) bone marrow neutrophil counts. The absolute number of neutrophils (PMN, Gr-1^HiCD115^-) is shown. All data represent the mean ± SEM.</p

No gender specific alterations in hematopoiesis are present in <i>Mir223</i> deficient mice.

<p>(A and B) Peripheral blood from 8–12 week old wild-type and (A) female <i>Mir223</i>^-/Y or (B) male <i>Mir223</i>^-/- mice was analyzed at 8–12 weeks of age. The absolute number of peripheral blood mononuclear cells (PBMNC), neutrophils (PMN, Gr-1^HiCD115^-), monocytes (Mono, Gr-1^INTCD115⁺), B cells (B220⁺), and T cells (CD3e⁺) are shown. All data represent the mean ± SEM. *p<0.05.</p