Abstract
Synergistic deregulation of HOXA9 and the HOX-gene cofactor MEIS1 is a commonly observed phenomenon in acute myeloid leukemia (AML). The leukemogenic potential of aberrant Hoxa9 and Meis1 expression has been shown in several AML models. However, the molecular mechanisms behind Hoxa9- and Meis1-induced leukemogenesis are still not well understood.
In order to identify functionally relevant Meis1-induced microRNAs (miRNA), we profiled the global miRNA expression using a Hoxa9-Meis1 murine AML progression model. This two-step model allowed us to quantify miRNAs at a pre-leukemic stage through the overexpression of the proto-oncogene Hoxa9 (Hoxa9/ctrl), as well as after full leukemic transformation through co-overexpression of Hoxa9 and Meis1 (Hoxa9/Meis1). The pre-leukemic stage is characterized by in vitro immortalization without in vivo engraftment, whereas the transplanted leukemic cells induce full-blown AML in vivo. MiR-155 turned out to be one of the most significant differentially expressed miRNA species and its upregulation was independently validated in Hoxa9/Meis1 cells by qRT-PCR.
Subsequent analysis of various AML subtypes (CN-AML, t(11q23), t(8;21), t(15;17), n=38) showed significantly elevated levels of miR-155 in CN-AML with NPM1mut (n=10, p<0.01) and AML with t(11q23) (n=8, p<0.05) compared to healthy donor bone marrow (MNC). These results are in line with overexpression of HOXA9 (CN-AML NPM1mut: p<0.05, t(11q23): p<0.05) and MEIS1 (CN-AML NPM1mut: p<0.01, t(11q23): p<0.05) in these AML samples compared to healthy donor bone marrow cells (MNC). Expression analysis of miR-155 in healthy murine bone marrow (mbm) cells revealed miR-155 enrichment in hematopoietic stem- and progenitor cells compared to mature myeloid cells (p<0.05), mirroring a similar expression pattern as observed for Meis1.
Therefore, to dissect the leukemic potential of miR-155 to program mbm, 5-FU-stimulated mbm cells were retrovirally transduced with miR-155, leading to significantly increased proliferation in vitro (p<0.05). This finding suggests enhancement of self-renewal on the stem-/progenitor cell level by miR-155. Furthermore, mbm cells overexpressing Hoxa9 together with miR-155 (Hoxa9/miR-155) significantly increased colony formation (p<0.05) in a methylcellulose assay. In turn, absence of miR-155 (miR-155-/- mbm) significantly reduced colony formation in conjunction with Hoxa9 (p<0.05) and MLL-AF9 (p=0.05), a known positive regulator of Hoxa9 and Meis1. These findings suggest a role for miR-155 in both proliferation and self-renewal indicating that the oncogenic program of Hoxa9/Meis1 relies on the presence of miR-155.
The leukemic potency of Hoxa9/miR-155 was further investigated in a murine transplantation model in vivo. Transplantation of mbm co-overexpressing Hoxa9/miR-155 led to significantly increased engraftment levels already after four weeks (wks) (57.8%±31.3, n=16) compared to Hoxa9/ctrl (11.7%±19.3%, p<0.0001, n=17), but less than with Hoxa9/Meis1 (74.5%±20.3%, p<0.01, n=14). In contrast to Hoxa9/ctrl (22±7 wks), mice that received Hoxa9/miR-155 mbm cells had a significantly accelerated onset of a myeloproliferative disease (MPD)-like leukemia within 11 wks (11±6 wks, p<0.0001), but still a less aggressive course of disease compared to mice transplanted with Hoxa9/Meis1 (5±1 wks, p<0.0001). This result is striking considering the aggressive nature of the Hoxa9/Meis1 AML model and given how little is known about its central mechanisms. It also highlights the relevant contribution of miR-155 to the leukemic programming induced by Hoxa9/Meis1 and provides a further rational to target miR-155 in AML.
Considering the central role of the Hoxa9/Meis1 in both myeloid and lymphoid acute leukemias, we demonstrate for the first time the leukemogenic relevance of a miRNA within this transcriptional axis.
Disclosures
No relevant conflicts of interest to declare.
Donor characteristics and intra-operative total nucleated cell count influence hematopoietic progenitor yield of healthy donor bone marrow grafts