Oncomir miR-125b regulates hematopoiesis by targeting the gene Lin28A Aadel A. Chaudhuria,1, Alex Yick-Lun Soa,1, Arnav Mehtaa, Aarathi Minisandrama, Nikita Sinhaa, Vanessa D. Jonssonb, Dinesh S. Raoc, Ryan M. O’Connelld, and David Baltimorea,2 Departments of aBiology and bComputing and Mathematical Sciences, California Institute of Technology, Pasadena, CA 91125; cDepartment of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles, CA 90095; and dDepartment of Pathology, University of Utah, Salt Lake City, UT 84112 Contributed by David Baltimore, January 23, 2012 (sent for review October 29, 2011) MicroRNA-125b (miR-125b) is up-regulated in patients with leukemia. regulation of miR-125b has profound effects on normal hema- Overexpression of miR-125b alone in mice causes a very aggressive, topoiesis, and Lin28A overexpression mimics those effects. transplantable myeloid leukemia. Before leukemia, these mice do not display elevation of white blood cells in the spleen or bone marrow; Results rather, the hematopoietic compartment shows lineage-skewing, with miR-125b Ectopic Expression Favors Myeloid Differentiation and myeloid cell numbers dramatically increased and B-cell numbers Causes a Highly Invasive Myeloid Leukemia. Previously, we showed severely diminished. miR-125b exerts this effect by up-regulating the that overexpression of miR-125b in bone marrow-transplanted number of common myeloid progenitors while inhibiting develop- recipient mice causes a myeloid leukemia 4–6 mo after bone ment of pre-B cells. We applied a miR-125b sponge loss of function marrow reconstitution (5). Here, we found that the neoplastic system in vivo to show that miR-125b physiologically regulates myeloid cells infiltrate nonhematopoietic organs, including the hematopoietic development. Investigating the mechanism by which brain, and overwhelm the periphery (Fig. 1 and Figs. S1 and S2). miR-125b regulates hematopoiesis, we found that, among a panel of Mice overexpressing miR-125a also developed an aggressive candidate targets, the mRNA for Lin28A, an induced pluripotent stem leukemia that was shown by enlarged spleen and cancer in- cell gene, was most repressed by miR-125b in mouse hematopoietic filtration into the liver and kidneys (Fig. S3). These mice died by stem and progenitor cells. Overexpressing Lin28A in the mouse 6 mo postreconstitution. Thus, dysregulated expression of miR- IMMUNOLOGY hematopoietic system mimicked the phenotype observed on inhibit- 125b or its paralogue leads to an aggressive leukemia that effi- ing miR-125b function, leading to a decrease in hematopoietic output. ciently invades nonhematopoietic organs. Relevant to the miR-125b overexpression phenotype, we also found To understand the initial events that precede the frank leu- that knockdown of Lin28A led to hematopoietic lineage-skewing, kemia, we examined the spleen and bone marrow at 7 wk after with increased myeloid and decreased B-cell numbers. Thus, the miR- reconstitution, a time well before the onset of cancer as indicated 125b target Lin28A is an important regulator of hematopoiesis and by similar white blood cell counts in the spleen (∼100 million a primary target of miR-125b in the hematopoietic system. cells for both MG and 125b spleens) and bone marrow of control MG (murine stem cell virus-GFP retro-vector) and MG-125b cancer | myelogenous | lymphocyte animals (Fig. 2A and Fig. S4 A and B). At this time, MG-125b animals showed dramatic increases in all myeloid lineages, in- icroRNA-125b (miR-125b) is up-regulated in a range of cluding granulocytes, macrophages, and dendritic cells, with T- Mhuman leukemias, including acute myeloid leukemia (1, 2), cell numbers being similar to those in the MG mice (Fig. 2A and chronic myeloid leukemia (2), acute megakaryocytic leukemia (3), Fig. S4 C–E). However, B-cell numbers were significantly de- childhood acute lymphoblastic leukemia with the ETV6/Runx1 creased in these miR-125b–overexpressing animals (Fig. 2A and fusion protein (4), and Philadelphia chromosome-positive B-cell Fig. S4F). Whether miR-125b physiologically regulates B-cell precursor childhood acute lymphoblastic leukemia (2). Indeed, development will require additional studies. Thus, miR-125b overexpression of miR-125b alone in the bone marrow of mice is overexpression at the precancerous stage causes a lineage- sufficient to induce leukemia (2, 5, 6). Recent in vitro work has also skewing of the hematopoietic compartment with myeloid cell uncovered a role for miR-125b in the development of plasma cells numbers elevated and B cells diminished. To investigate the developmental stage at which miR-125b (7) and effector T cells (8), suggesting that miR-125b regulates overexpression promotes myelopoiesis and compromises B-cell immune cell development in addition to promoting leukemia. development, we analyzed hematopoietic stem and progenitor Lin28A is an important regulator of early embryogenesis in cell (HSPC) numbers in the bone marrow at 7 wk after bone mice and humans (9). Levels of Lin28A are enriched in ES cells marrow transplantation. In these mice, the hematopoietic stem and decrease as these cells differentiate (9). Ectopic expression of cell (HSC) numbers were similar in MG-125b animals compared Lin28A along with three other genes (Oct4, Sox2, and Nanog) with MG controls (Fig. S4 G and H). Progenitors directly causes dedifferentiation of mature human cells into induced downstream of HSCs, however, including multipotent progeni- pluripotent stem cells (10). This shows the power of Lin28A to tors (MPPs), common myeloid progenitors and granulocyte endow cells with pluripotent qualities. Still, it remains to be seen whether Lin28A also plays developmental roles in biological systems arising from adult stem cells, such as hematopoiesis. Author contributions: A.A.C., A.Y.-L.S., R.M.O., and D.B. designed research; A.A.C., A.Y.-L.S., In this study, we examine the role of miR-125b in inducing A. Mehta, A. Minisandram, N.S., V.D.J., and D.S.R. performed research; A.A.C., A.Y.-L.S., and cancer and show that overexpression of this microRNA induces R.M.O. contributed new reagents/analytic tools; A.A.C., A.Y.-L.S., V.D.J., D.S.R., and D.B. analyzed data; and A.A.C., A.Y.-L.S., and D.B. wrote the paper. a preleukemic state before aggressive frank leukemia is evident. Conflict of interest statement: D.B. is a Director of Regulus, a company devoted to com- This preleukemic state is characterized by overproduction of mercialization of antimicroRNA therapies. myeloid cells and their precursors as well as inhibition of B-cell 1A.A.C. and A.Y.-L.S. contributed equally to this work. development. We then show that the most affected target gene of 2To whom correspondence should be addressed. E-mail: [email protected]. miR-125b is Lin28A and that Lin28A down-regulation can mimic This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the preleukemic state induced by miR-125b. Furthermore, down- 1073/pnas.1200677109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1200677109 PNAS | March 13, 2012 | vol. 109 | no. 11 | 4233–4238 Downloaded by guest on September 30, 2021 Fig. 1. miR-125b overexpression causes an aggressive in- vasive myeloid leukemia. (A)Infiltration of GFP+ CD45+ and GFP+ CD11b+ cells into the brain. (Upper) Representative flow cytometric plots are shown. (Lower) Average percent GFP+ CD45+ and GFP+ CD11b+ in the brain are shown from three MG and two MG-125b mice. (B)Leukemiccellinfiltration into nonhematopoietic organs. Sections from the kidney, lung, and liver were stained with H&E. The normal structures of the MG-125b mouse kidney, lung, and liver are effaced by a dramatic infiltrate of leukemic cells. A representative im- age for each tissue is shown. The brain, kidney, lung, and liver were harvested from animals 5 mo after bone marrow reconstitution. During the time of harvest, the average per- cent GFP+ cells in the spleens of MG and MG-125b mice were 49 ± 9.7% and 91 ± 2.9%, respectively. All plots shown depict the mean with SEM. All data are representative of two independent experiments. Inf, infiltrate; G, glomerulus; A, alveolar space; CV, central vein. macrophage progenitors (CMPs/GMPs), myeloid–erythroid pro- (15–17) to isolate putative miR-125b targets with PConserved Targeting genitors (MEPs), and common lymphoid progenitors (CLPs), > 0.8 (P > 0.8 that the putative microRNA site is evolutionarily were all significantly increased (Fig. 2B). Of these progenitors, maintained because of selective microRNA targeting rather than CMPs/GMPs were the most drastically augmented, being ele- chance) (17). Application of this screen yielded 192 genes. Gene vated 8.4-fold in MG-125b animals compared with controls (Fig. ontology analyses indicate that these genes are functionally en- 2B). Notably, pre–B-cell numbers were severely decreased (Fig. riched for biological processes that include transcriptional regu- 2B), providing a developmental basis for the decreased B-cell lation, vasculature development, proteolysis, and apoptosis (Da- fi numbers that we observed in MG-125b animals. This nding also vid Functional Annotation Bioinformatics, P < 0.05). We focused indicates that miR-125b overexpression causes a developmental – on the processes that were proapoptotic or involved with stem cell block between the CLP and pre B-cell stages. In summary, we regulation, because these processes have been correlated with show here that, before the onset of leukemic disease, miR-125b leukemic