In order to identify functionally relevant microRNAs (miRNA) in AML, we profiled global miRNA expression in a murine AML progression model based on Hoxa9 and Meis1 overexpression. We found miR-155 ...and miR-708 as the most significantly upregulated miRNA species in leukemic Hoxa9/Meis1 cells compared to the preleukemic Hoxa9/ctrl cells (both p<0.01). 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 and miR-708 in all tested samples (n=5-10) compared to total bone marrow from healthy donors, indicating potential oncogenic roles for these miRNAs.
We further investigated, in vivo, the role of miR-155 and miR-708 in AML by retroviral overexpression with Hoxa9 in murine bone marrow (mbm) cells followed by syngeneic transplantation. Overexpression of miR-155 in conjunction with Hoxa9 (Hoxa9/miR-155) caused a significantly accelerated onset of a myelomonocytic leukemia within 93 days (survival 93±23 days, p<0.0001), but still a less aggressive course of disease compared to mice transplanted with Hoxa9/Meis1 (survival 36±4 days, p<0.0001).
We then hypothesized that the combination of miR-155 and miR-708 could further replace the oncogenic potential of Meis1. Therefore, mbm cells were retrovirally transduced with Hoxa9, miR-155 and miR-708 (Hoxa9/155/708) or Hoxa9 and miR-708 (Hoxa9/miR-708) and functionally analyzed in vitro and in vivo. To our surprise, miR-708 abrogated the leukemogenic effect of Hoxa9, alone or in combination with Hoxa9/miR-155 in vivo (p=0.0117, p<0.0001, the mice were sacrificed after 197 days), with little or no engraftment.
To understand why miR-708, a potent tumor suppressor miRNA, is upregulated in the highly aggressive Hoxa9/Meis1 AML cells, we first showed in vivo that miR-708 does not impair the development of AML by Hoxa9/Meis1 cells. In order to explore the role of miR-708 in leukemia initiating cells (LIC), we FACS-sorted subpopulations enriched for LIC based on c-kit, Mac-1 and Gr-1 expression within the Hoxa9/Meis1 cells. Of the three subpopulations (c-kit+ Gr-1- Mac-1-, c-kit+ Gr-1+ Mac-1+, c-kit- Gr-1+ Mac-1+) the transplanted c-kit+ Gr-1- Mac-1- cells caused a significantly shorter survival (survival 36 days, range: 32-37d) compared to the other sorted subpopulations (p=0.0072 and p=0.0021, respectively), whereas the c-kit- Gr-1+ Mac-1+ subpopulation exhibited the longest survival (survival 44 days, range: 42-51d). Based on these findings, we measured miR-708 and miR-155 levels in the sorted Hoxa9/Meis1 subpopulations by qRT-PCR. Strikingly, no difference compared to bulk cells was detected for miR-155, highlighting its role as an oncogenic driver in all subpopulations. In contrast, miR-708 showed a distinct expression pattern with lower expression in the LIC-enriched c-kit+ Gr-1- Mac-1- subpopulation compared to bulk (p=0.032), allowing discernment of the LIC enriched population and highlighting its role as an orchestrator of the leukemic hierarchy. Adapted from these findings, we developed an AML 4-step model (Hoxa9/miR-708, Hoxa9/ctrl, Hoxa9/miR-155, Hoxa9/Meis1) based on four immortalized Hoxa9 based cell lines with completely different in vivo properties ranging from no engraftment to induction of highly aggressive AML.
Considering the central role of Hoxa9/Meis1 in AML, we demonstrate, for the first time, the concept of a tumor suppressor miRNA that stratifies the leukemic hierarchy driven by an oncogenic miRNA. Moreover, we present a new AML 4-step model as a novel tool that will allow detailed investigation of the impact of new oncogenes at every step of leukemogenesis.
Buske:CELLTRION, Inc.: Consultancy, Honoraria.
Bone-marrow transplantation (BMT) offers curative potential for patients with high-risk hematologic malignancies, but the post-transplantation period is characterized by profound immunodeficiency. ...Delayed immune reconstitution is an important contributor to transplant-related morbidity and mortality (Maury et al., Br J Haematol, 2001; Small et al., Blood, 1999). Thus, understanding the mechanisms by which the immune system reconstitutes and how environmental exposures might influence this process is an important objective in improving outcomes after BMT.
The intestinal microbiota shapes both mucosal and systemic immunity and contributes to steady-state hematopoiesis (Belkaid and Hand, Cell, 2014). To determine the role of the intestinal flora in hematopoietic reconstitution following BMT, we performed syngeneic BMT after lethal irradiation in mice with an intact intestinal flora (control, C/No abx) and in mice treated with two different antibiotic (abx) regimens administered in the drinking water: ampicillin + enrofloxacin (AmpEnro/AE) or vancomycin + amikacin (VancAmik/VA). While both regimens depleted the intestinal flora 1000-fold, ampicillin and enrofloxacin are both relatively well absorbed in the intestine while vancomycin and amikacin both have poor oral bioavailability with negligible systemic effects. BMT recipients treated with either of the abx regimens had lower white blood cell (WBC) counts and, in particular, lower lymphocyte counts compared to untreated BMT recipients (Fig 1A). Fecal microbiota transfer (FMT) with an abx-resistant intestinal flora (Caballero et al., Cell Host Microbe, 2017) rescued the impaired hematopoietic reconstitution in AE-treated mice (Fig 1B). These data rule out a direct inhibitory effect of abx on hematopoietic recovery. When we examined multiple compartments of the hematopoietic cascade we observed that flora depletion significantly impaired hematopoiesis specifically in the lymphoid and myeloid compartments of the bone marrow, thymus, and peripheral blood, while the hematopoietic stem and progenitor compartment and erythroid lineage were largely unaffected. These data are summarized in a radial plot (Fig 1C) in which each compartment of the hematopoietic cascade is a spoke and the distance from the center is proportional the cell count.
Treatment of mice with AmpEnro or VancAmik also reduced dietary energy uptake (Fig 1D), resulting in reduction of visceral adipose tissue (Fig 1E). The mice are normally fed a standard chow diet based on complex plant carbohydrates. To test if reduced uptake of calories in flora-depleted animals contributed to impaired hematopoiesis post-BMT, we supplemented the drinking water with 5% sucrose, a carbohydrate source that can be directly utilized by the host in the absence of microbiota metabolism. Sucrose supplementation increased peripheral WBC counts and lymphocyte counts in AE-treated mice (Fig 1F), indicating that a critical function of the intestinal microbiota in supporting hematopoiesis post-BMT is to improve dietary energy uptake.
We conclude that apart from specific and direct effects of the microbiota on immuno-hematopoiesis as previously described (Balmer et al., J Immunol, 2014; Clarke et al., Nat Med, 2010; Mazmanian et al., Cell, 2005), the intestinal flora can contribute to post-transplant hematopoietic reconstitution through improved dietary energy uptake.
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Peled:Seres: Research Funding. Jenq:Seres: Research Funding. van den Brink:Therakos Institute: Other: Speaking engagement; Jazz Pharmaceuticals: Consultancy; PureTech Health: Consultancy; Seres: Research Funding.
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.
No relevant conflicts of interest to declare.
MicroRNA-155 (miR-155) is one of the first described oncogenic miRNAs. Although multiple direct targets of miR-155 have been identified, it is not clear how it contributes to the pathogenesis of ...acute myeloid leukemia. We found miR-155 to be a direct target of Meis1 in murine Hoxa9/Meis1 induced acute myeloid leukemia. The additional overexpression of miR 155 accelerated the formation of acute myeloid leukemia in Hoxa9 as well as in Hoxa9/Meis1 cells in vivo. However, in the absence or after the removal of miR-155, leukemia onset and progression were unaffected. Although, miR-155 accelerated growth and homing as well as impaired differentiation, our data underscore the pathophysiological relevance of miR 155 as an accelerator rather than a driver of leukemogenesis. This further highlights the complexity of the oncogenic program of Meis1 to compensate for the loss of a potent oncogene such as miR-155. These findings are highly relevant to current and developing approaches for targeting miR-155 in acute myeloid leukemia.
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