Contribution of an Aged Microenvironment to Aging-Associated Myeloproliferative Disease

The molecular and cellular mechanisms of the age-associated increase in the incidence of acute myeloid leukemia (AML) remain poorly understood. Multiple studies support that the bone marrow (BM) microenvironment has an important influence on leukemia progression. Given that the BM niche itself undergoes extensive functional changes during lifetime, we hypothesized that one mechanism for the age-associated increase in leukemia incidence might be that an aged niche promotes leukemia progression. The most frequent genetic alteration in AML is the t(8;21) translocation, resulting in the expression of the AML1-ETO fusion protein. Expression of the fusion protein in hematopoietic cells results in mice in a myeloproliferative disorder. Testing the role of the age of the niche on leukemia progression, we performed both transplantation and in vitro co-culture experiments. Aged animals transplanted with AML1-ETO positive HSCs presented with a significant increase in the frequency of AML-ETO positive early progenitor cells in BM as well as an increased immature myeloid cell load in blood compared to young recipients. These findings suggest that an aged BM microenvironment allows a relative better expansion of pre-leukemic stem and immature myeloid cells and thus imply that the aged microenvironment plays a role in the elevated incidence of age-associated leukemia

Aging of the Microenvironment Influences Clonality in Hematopoiesis

<div><p>The mechanisms of the age-associated exponential increase in the incidence of leukemia are not known in detail. Leukemia as well as aging are initiated and regulated in multi-factorial fashion by cell-intrinsic and extrinsic factors. The role of aging of the microenvironment for leukemia initiation/progression has not been investigated in great detail so far. Clonality in hematopoiesis is tightly linked to the initiation of leukemia. Based on a retroviral-insertion mutagenesis approach to generate primitive hematopoietic cells with an intrinsic potential for clonal expansion, we determined clonality of transduced hematopoietic progenitor cells (HPCs) exposed to a young or aged microenvironment <em>in vivo.</em> While HPCs displayed primarily oligo-clonality within a young microenvironment, aged animals transplanted with identical pool of cells displayed reduced clonality within transduced HPCs. Our data show that an aged niche exerts a distinct selection pressure on dominant HPC-clones thus facilitating the transition to mono-clonality, which might be one underlying cause for the increased age-associated incidence of leukemia.</p> </div

The clonal composition of the hematopoiesis is different in old compared to young microenvironment. (A)

<p>Schematic representation of the gammaretroviral SF91/IRES-eGFP vector used for insertional mutagenesis and the experimental setup (LTR: long terminal repeat with strong enhancer element, wPRE: woodchuck hepatitis virus posttranscriptional regulatory element). Lineage depleted (lin-) BM cells from young C57BL/6 mice were pre-stimulated with a cytokine cocktail and transduced. Cells were subsequently transplanted in equal amounts into young and aged recipient mice. (The graft contained 51.6%, 30.7%, 32.4% GFP+ cells in the 3 transduction/transplantation.) 24–26 week post-transplantation recipients were sacrificed and PB, spleen and BM analyzed by flow cytometry for lineage differentiation markers as well as the number of primitive L-S+K+ cells. At the same time CFC-assays on BM cells were performed and DNA from GFP+ colonies (representative pictures) isolated for LM-PCR. GFP expression among BM cells of young <b>(B)</b> and aged <b>(C)</b> recipient mice. (young mice n = 6, aged mice n = 5, from 3 independent experiments).</p

Insertion sites in CFCs isolated from young and aged recipient mice.

<p>RISs isolated from CFC mice with a chimerism higher than 15% (also displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042080#pone-0042080-g002" target="_blank">Figure 2</a>/D, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042080#pone.0042080.s003" target="_blank">Material and Methods S1</a>) are listed. Gene categories were defined by the DAVID gene ontology (GO)²¹, using high classification stringency option by clustering. For Endocytosis GO group, the Fisher Exact test gave a P-value of 4.0E−2 and for Regulation of transcription GO group P-value = 2.9E−1.</p>*<p> = same RISs were found in 2 different young recipient mice (transplanted with two different pools of transduced cells), all the other RISs were unique within the experiments.</p

Model for the influence of an aged microenvironment on leukemia progression.

<p>(<b>A</b>) The standard model describes leukemia development as the clonal evolution of an aberrant clone: a founder cell mutates through multiple subsequent steps, frequently via a chronic phase (CML) to ultimately result in acute myeloid leukemia (AML). The velocity of expansion of the aberrant clone increase along the three phases of leukemia (pre-leukemia, chronic leukemia, acute leukemia). The transitions between the phases are not well described in both cellular and molecular terms, but might be caused by intrinsic/genetic and/or extrinsic changes (figure adapted from ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031523#pone.0031523-Nowell1" target="_blank">[46]</a>). (<b>B</b>) An aged microenvironment increases the velocity of the expansion of an aberrant pre-leukemic clone. The faster expansion of myeloproliferation-initiating stem cells in aged BM thus promoting the leukemic process. In a larger aberrant cell population, the probability of generating additional hits is increased, resulting in a more likely and thus earlier transition to the next phase of leukemia.</p

<i>In vitro</i> culture of AML1-ETO positive stem/progenitor cells on aged and young endosteal cells.

<p>(<b>A</b>) Experimental set-up for isolation of a cell fraction close to the endosteum ( =  endosteal cells) and representative phase contrast images (×10) from adherent endosteal cells from young and aged mice, bar represent 50 µm (<b>B</b>) Experimental set-up for co-culture experiments and FACS analysis for analysis of the Lin−,Sca-1+, c-Kit+ (LSK) cell compartment. (<b>C</b>) Frequency of GFP+ cells among all cells, (<b>D</b>) frequency of GFP+ cells in the LSK population (<b>E</b>) and the total number GFP+LSK cells. n = 3, * = p<0.05. Bars represent the mean ± SEM.</p

Coexistence of balanced HSC populations is characteristic for young microenvironment whereas an aged microenvironment favors the expansion of single dominant hematopoietic clone.

<p>(<b>A</b>) Schematic representation of the retroviral insertional mutagenesis screen. DNA was extracted from individual CFCs (average: 7.3±1.8 GFP+CFCs/mouse were isolated) and digested with four-cutter enzymes. After LM-PCR, the recovered bands were isolated and sequenced. The sequences were aligned to the mouse genome with NCBI/BLAST program. The RISs were identified and the closest genes (within a ±100 kb window) were listed. Based on the LM-PCR band-pattern and the identified RISs, clonal analyses were performed. (<b>B</b>) Representative agarose gel analysis of LM-PCR performed on methylcellulose colonies isolated from BM of one representative young transplanted mouse. Clonality was identified based on the band pattern and DNA sequence information on individual bands. Distinct colors represent distinct clones. (<b>C</b>) Insertion sites recovered by LM-PCR from CFCs of one representative aged recipient mouse. The red arrows depict distinct GFP peaks. i.c.: internal control bands represent PCR products amplified from the viral genome. (<b>D</b>) Distribution of clonality within the GFP+ CFC population of young and aged recipient mice determined by the LM-PCR pattern and sequence information. (<b>E</b>) Flow cytometric analyses of PB of young and aged recipient mice (same mice were used in clonal analysis and RISs identification). Myeloid cells were identified based on Gr1 and Mac1 expression and B220 was used as B cell marker. (young mice n = 6, aged mice n = 5, from 3 independent experiments), * = p<0.05 (<b>F</b>) Proposed model for clonality of hematopoiesis in young and aged microenvironment.</p