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C. Di Genua et al.
gest that AML1-ETO is acquired in pre-leukemic HSC. First, AML1-ETO mRNA could still be detected in AML patients who had been in clinical remission for up to 150 months.14 Secondly, AML1-ETO remains stable in patients who relapse, while additional mutations were highly dynamic with mutations both gained and lost at relapse.15 Finally, evidence from mouse models support the concept that pre-leukemic mutations confer a competitive advan- tage to cells within the phenotypic HSC compartment, without causing transformation of downstream progeni- tor cells.16,17 In particular, Aml1-ETO knock-in mice did not develop leukemia, but Aml1-ETO-expressing cells had an enhanced in vitro replating ability, indicating greater self- renewal capacity.16
In contrast, signaling transduction mutations of genes such as FLT3, KIT or KRAS occur as late events that are detected in the transformed leukemic progenitors but rarely detected in the pre-leukemic HSC compartment.11,12 RAS mutations also frequently co-occur with t(8;21) (NRAS = 12.9%, KRAS = 4.3%).15 In AML patients who achieve remission, RAS mutations are unstable and often lost at subsequent relapse, with gain of a novel signaling transduction mutation (e.g. FLT3-ITD), while the initiating translocation remains. This is consistent with RAS muta- tions occurring as a late event during leukemic transforma- tion.15 Finally, the KrasG12D/+;Mx1-Cretg/+ mouse model devel- ops a fatal myeloproliferative neoplasm (MPN); however, these mice do not develop AML.18,19 Collectively, these studies provide evidence that RAS mutations are second- ary events in AML development and are not present with- in pre-leukemic HSC. Mouse models in which activating signaling pathway mutations were introduced into wild- type (WT) HSC have revealed both cell-intrinsic and cell- extrinsic effects on the HSC compartment, usually result- ing in a depletion of HSC.20-24 However, the impact of sig- naling transduction mutations on pre-leukemic HSC remains unclear. This is of considerable importance for understanding why signaling mutations are absent from the pre-leukemic HSC compartment.
We hypothesized that the absence of signaling muta- tions in the HSC may reflect a detrimental impact of such mutations on pre-leukemic HSC. To address this question, we used conditional mouse genetics to introduce Aml1- ETO and K-RasG12D separately or in combination, both expressed from their endogenous loci, into WT HSC, to determine the effect of K-Ras activation on a well-defined pre-leukemic HSC population. While Aml1-ETO expres- sion enhanced the long-term repopulating ability of HSC, expression of K-RasG12D in Aml1-ETO-expressing HSC led to loss of quiescence and self-renewal-associated gene expression, and was detrimental to their function. Such functional impairment would limit clonal expansion of pre-malignant HSC co-expressing AML1-ETO and activat- ed RAS, providing a molecular and cellular basis for the observed absence of activating RAS mutations in pre- leukemic HSC.
Methods
Animals
All mouse lines were maintained on a C57Bl/6J genetic back- ground. Conditional knock-in mice expressing Aml1-ETO (Aml1ETO/+)16 and K-Ras (KrasG12D/+),25 either individually or com- bined (Aml1ETO/+;KrasG12D/+), were crossed to the Mx1-Cre mouse
line.26 All mice were bred and maintained in accordance with UK Home Office regulations. Experiments were conducted following approval by the University of Oxford Animal Welfare and Ethical Review Body (project license n. 30/3103).
Competitive transplantation
Competitive transplants were performed as previously described.27 See Online Supplementary Appendix for further details.
Serial transplantations were performed by co-transplanting 1.25x105 CD45.2 fetal liver (FL) cells with 5x106 CD45.1 WT bone marrow (BM) competitor cells into lethally irradiated recipients (2x500rads). Bulk secondary and tertiary transplants were per- formed by transplanting 3x106 BM cells from primary and second- ary recipients respectively into lethally irradiated recipients at eight weeks post-poly(I:C) for secondary transplants or 12 weeks post-transplantation for tertiary transplants. Tertiary transplanted mice were analyzed 12 weeks post-transplantation.
Flow cytometry and fluorescence-activated cell sorting
Details of antibodies and viability dyes are shown in Online Supplementary Table S1. All antibodies were used at pre-deter- mined optimal concentrations. Hematopoietic stem and progeni- tor cells were analyzed as previously described.28 Cell acquisition and analysis were performed on a BD LSRFortessa (BD Biosciences, San Jose, CA, USA) using BD FACSDivaTM software (BD Biosciences). Cell sorting was performed on a BD FACSAriaII cell sorter (BD Biosciences). Cells used in cell sorting experiments were c-Kit-enriched (MACS Miltenyi Biotec, Bergisch Gladbach, Germany) and were stained with specific antibodies following ini- tial Fc-block incubation. Gates were set using a combination of fluorescence minus one controls and populations known to be negative for the antigen.
For HSC cell cycle staining, BM cells were c-Kit-enriched and stained following initial Fc-block incubation. Stained cells were then fixed and permeabilized using BD cytofix/cytoperm fixation and permeabilization solution (BD Biosciences). Cells were stained with Ki-67 PE (BD Biosciences) overnight. Cells were then stained with 4',6-diamidino-2-phenylindole (DAPI) (0.5 mg/mL) (ThermoFisher Scientific, Waltham, MA, USA) for one hour before analysis.
In vitro serial replating assay
Serial replating was performed as previously described.5 Briefly,
100 CD45.2 Lineage–Sca1+cKit+ (LSK) BM cells were sorted from mice transplanted with 2.5x105 FL cells and 1x106 CD45.1 WT BM competitor cells eight weeks post-poly(I:C). Cells were seeded into 1 mL of methylcellulose medium (Methocult, M3434, STEM- CELL Technologies, Vancouver, BC, Canada) and incubated in 37°C, in 5% CO2, with ≥95% humidity. Colonies (≥30 cells) were counted after eight days. Cells were re-suspended and re-plated at 1x104 cells per 1 mL of methylcellulose medium. Cells were then counted and re-plated after 6-7days.
RNA-sequencing
Statistical analysis
Unless otherwise indicated, statistical significance of differences
Fifty cells per biological replicate were sorted into 4 mL of lysis buffer containing; 0.2% Triton X-100 (Sigma-Aldrich, St Louis, MO, US), 2.5 mM OligodT (Biomers, Ulm, Germany), 2.5 mM dNTPs (ThermoFisher Scientific), RNase Inhibitor 20 U (Takara Bio USA Inc., Mountain View, CA, USA) and ERCC spike-in 1:4x106 (ThermoFisher Scientific). For details on cDNA synthesis, library preparation and data analysis see Online Supplementary Appendix.
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haematologica | 2019; 104(11)