Leukemogenic MicroRNA-29a Causes Myeloproliferation by Inhibiting Apoptosis and Inducing Aberrant Self-Renewal in a Developmental Stage Specific Manner

Abstract 2556

We recently reported that miR-29a is highly expressed in AML as well as in hematopoietic stem cells (HSC). Overexpression of miR-29a in mouse bone marrow resulted in myeloproliferative disease characterized by a granulocyte/macrophage differentiation bias and progressive disease culminating in AML. Prior to the development of AML, committed myeloid progenitors including common myeloid progenitors (CMP) and granulocyte/macrophage progenitors (GMP) aberrantly acquired the ability to self-renew, demonstrated by long-term engraftment of FACS-purified transplanted cells. In order to elucidate the mechanisms of miR-29a induced leukemogenesis, we characterized the immature hematopoietic compartment during the myeloproliferative phase of disease. Similar to our prior studies, miR-29a primary chimeras exhibited granulocytic hyperplasia as soon as 6–8 weeks after transplantation, with increased frequencies of GFP+Gr-1+Mac-1+ granulocytes in the peripheral blood of animals transplanted with miR-29a versus empty vector transduced cells (mean 34.7% vs. 9.2%, p<0.05). miR-29a expressing primary chimeras also showed increased frequencies of LSK (Lin-Sca1+c-kit+) cells and myeloid progenitors (Lin-Sca1-c-kit+) compared with empty vector controls (2.34% vs. 0.54%, p<0.05; and 6.85% vs. 14.3%, p<0.05, respectively). miR-29a expressing LSK cells also exhibited decreased apoptosis as assessed by annexin V staining (mean 10.8% vs. 5.9%, p<0.05), as well as a marked increase in the number of cells in G0/G1 (mean 85.3% vs. 64%; p<0.05); both of these effects were significantly attenuated or absent in more mature myeloid progenitors. Evaluation of miR-29a target mRNAs by qPCR revealed down-regulation of DNMT3a and DNMT3b transcripts in miR-29a progenitor cells. Secondary transplants were performed with FACS-sorted cells including myeloid-biased (My-bi, Lin-Sca1+c-kit+CD150hiCD34-) and lymphoid-biased (Ly-bi, Lin-Sca1+c-kit+CD150lowCD34-) HSC, multipotent progenitors (MPP, Lin-Sca1+c-kit+CD150negCD34+), and myeloid progenitors. Five months after transplant, mice transplanted with miR-29a expressing MPP (4/5) or myeloid progenitors (3/7) exhibited a myeloid expansion, consistent with acquisition of aberrant self-renewal and transfer of disease-initiating cells. In contrast, GFP+ chimerism was either low or undetectable in secondary recipients transplanted with My-bi HSC (1/4) or Ly-bi HSC (0/6). Finally, we measured miR-29a expression levels in AML blasts from several mouse models of AML including MLL-AF9, AML-ETO9a, and NUP98-HOXD13 and show that miR-29a expression is increased 6–12 fold over normal c-kit+ cells, consistent with our previously published results demonstrating high levels of miR-29a in primary AML blasts compared to their normal HSC/progenitor counterparts. Together, these results indicate that miR-29a induces myeloproliferation by inducing self-renewal in hematopoietic progenitors including MPP and committed myeloid progenitors. The enhanced self-renewal and expansion of LSK cells is likely due to the anti-apoptotic effect of miR-29a, which is developmental stage specific and associated with decreased cell cycling and decreased DNMT3a/b expression. These findings indicate that myeloproliferation may be due to expansions caused by increased self-renewal of progenitors and not necessarily through enhanced HSC function.