Loss of MiR-143 and MiR-145 Inhibits Hematopoietic Stem Cell Self-Renewal through Dysregulated TGFβ Signaling
Authors: Lam Jeffrey C , Wegrzyn-Woltosz Joanna , Ibrahim Rawa , Slowski Kate , Umlandt Patricia , Fuller Megan , Karsan Aly 
Published: October 2019

Myelodysplastic syndromes (MDS) are a collection of hematopoietic malignancies in which genomic abnormalities within the hematopoietic stem cell (HSC) compartment results in dysplasia of the marrow cells and ineffective hematopoiesis. As a result, the primary cause of mortality in these patients is eventual bone marrow failure although MDS patients also have a significantly increased risk of transformation to acute myeloid leukemia (AML). The most common karyotypic change in MDS is an interstitial deletion of the long arm of chromosome 5, del(5q) MDS. Patients with an isolated interstitial deletion of chromosome 5q are referred to as having 5q- syndrome. Mapping of the commonly deleted region (CDR) within 5q- syndrome has identified a 1.5-megabase region on band 5q32. MicroRNA (miRNA) -143 and -145 are located within the CDR of del(5q) MDS and have been implicated in the pathogenesis of the disease. However, their functional role in myelodysplastic syndromes has not been well studied.

To investigate the role of miR-143 and miR-145, we utilized a gene-targeted mouse model containing deletion of miR-143 and miR-145. Here we show that mouse marrow lacking miR-143 and miR-145 have a decrease in short-term repopulating HSC and progenitors of the myeloid lineage by flow cytometry, as well as of hematopoietic progenitor activity using colony forming assays. Additionally, we performed a limiting dilution assay of miR-143-/-145-/- bone marrow and observed significantly fewer functional HSCs compared to wildtype marrow. To explore the molecular mechanism behind this defect, we performed Ingenuity Pathway Analysis of the predicted targets of miR-143 and miR-145. We identified the transforming growth factor-beta (TGFβ)-signaling pathway as a common target of these two miRNAs. Gene Set Enrichment Analysis of del(5q) using mRNA expression of MDS patient CD34+ marrow cells show an enriched TGFβ-signature compared to healthy controls. In addition, the defect in hematopoietic progenitor activity in miR-143-/-145-/- marrow can be rescued by inhibiting Smad3 using the chemical inhibitor SIS3.

We validated the TGFβ pathway adaptor protein, Disabled-2 (DAB2), as a target of miR-145 and show that TGFβ signaling is activated upon loss of miR-145 or enforced expression of DAB2. Enforced expression of DAB2 in mouse marrow is able to recapitulate many of the features of miR-143-/-145-/- mice. DAB2 overexpressing marrow formed significantly fewer colonies in progenitor assays, and in competitive transplants, vector-transduced marrow was able to out compete DAB2-overexpressing marrow in both primary transplants as well as in secondary limiting dilution assays. Interestingly, compared to wildtype mice, aged miR-143-/-145-/- mice showed decreased hemoglobin and platelet counts with elevated white blood cell counts. This phenotype was also observed in a subset of mice with enforced DAB2 expression where a proportion of mice developed a transplantable myeloproliferative disorder.

Together, our data identifies a role for miR-143 and miR-145 in the pathogenesis of del(5q) MDS where their loss results in a defect in HSC activity. We observe that the TGFβ signaling pathway is activated in patient marrow and we validate DAB2 as a direct target of miR-145. We provide evidence that the defect observed in miR-143-/-145-/- marrow is mediated in part by DAB2 where its enforced expression leads to a defect in HSC self-renewal but contributes to myeloproliferation.

Disclosures

Karsan: Celgene: Research Funding.