Principal Investigator Harvey Lodish
Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are frequently associated with chromosomal translocations; most involve oncogenes or transcription factors that are up regulated or that form part of chimeric genes. The t(2;11)(p21;q23) translocationis observed in cases of MDS and AML and in her previous laboratory in Toulouse Marina Bousquet showed that this translocation triggers upregulation of miR-125b. This was the first description of microRNA deregulation by a chromosomal translocation, and implied that AML and MDS carrying the t(2;11) translocation represent a new clinico-pathological entity. Cell culture experiments demonstrate that miR-125b per se is able to block the myeloid differentiation of human cell lines under various stimulations. Since lin-4, the miR-125b ortholog in Caenorhaditis elegans, isimplicated in several developmental process, she hypothesized that deregulation of miR-125b expression would impair human and mouse haematopoiesis.
To check if the overexpression of miR-125b blocks myeloid differentiation and/or causes leukemias in vivo, Marina used a retroviral construct encoding miR-125b to infect enriched hematopoietic stem/ progenitor cell populations. These cells were then injected into lethally irradiated recipient mice. At 16 weeks all mice transplanted with fetal liver cells ectopically expressing miR-125b showed an increase in white blood cell count, in particular neutrophils and monocytes, associated with a macrocytic anemia, suggesting an important role for miR-125b early in hematopoiesis. Among these mice, half died within 12 to 29 weeks post-transplantation of B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, or a myeloproliferative neoplasm. Furthermore, co-expression of miR-125b and the BCR-ABL fusion oncogene in transplanted cells accelerated the development of leukemia, compared to control mice expressing only BCR-ABL, suggesting that miR-125b confers a proliferative advantage to the leukemic cells. Thus, she showed that overexpression of miR-125b was sufficient both to shorten the latency of BCR-ABL–induced leukemia and to independently induce leukemia in a mouse model.
To better understand the role of miR-125b in hematopoiesis Marina is trying to identify miR-125b targets using two experimental in vitro models: NB4 (human promyelocytic cell line) and 32Dcl3 (mouse promyelocytic cell line). NB4 and 32Dcl3 can be induced to differentiate to granulocytic cells with retinoic acid and G-CSF respectively. She showed that the overexpression of miR-125b in these cell lines blocks granulocytic differentiation, reduces apoptosis, and induces proliferation. To identify miR-125b targets involved in these processes, she is analyzing the total cellular gene expression pattern by both RQ-PCR and mRNA-seq, comparing cell lines expressing or not miR-125b. During the last two years, Diu Nguyen (a visiting MS student), Lauren Shield (UROP student) and Cynthia Chen (UROP student) focused on the identification of miR-125b’s targets by using mRNA-seq on NB4 and 32Dcl3 cell lines expressing or not miR-125b. By using a computational approach, they selected genes downregulated by miR-125b overexpression and containing a potential binding site for miR-125b in their 3’UTRs. By using reporter assay, they validated some putative miR-125b target segments in these mRNAs. Western blot and in vitro assays should allow us to validate new miR-125b targets.
As noted above, recent work from our lab indicates that mir-125b is a novel bona fide negative regulator of p53 in human and zebra fish. One hypothesis is that miR-125b downregulation of p53 in some specific hematopoietic cell facilitates development of leukemic cells. However, p53 is not a conserved target among all vertebrate species and in particular the binding site for miR-125b is not conserved in mouse p53 mRNA. We hypothesize that even if the miR-125b binding site is not conserved in mouse p53, the p53 pathway is regulated by miR-125b in both human and mouse.
Dr. Marina Bousquet is continuing this work in the Cancer Research Center of Toulouse, France, focusing on how overexpression of miR-125b causes a macrocytic anemia, promotes proliferation of myeloid cells, and blocks granulocyte and monocyte differentiation.