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Oncogenic Nras has bimodal effects on stem cells that sustainably increase competitiveness.

Li Q, Bohin N, Wen T, Ng V, Magee J, Chen SC, Shannon K, Morrison SJ - Nature (2013)

Bottom Line: Nras(G12D) had a bimodal effect on HSCs, increasing the frequency with which some HSCs divide and reducing the frequency with which others divide.This mirrored bimodal effects on reconstituting potential, as rarely dividing Nras(G12D) HSCs outcompeted wild-type HSCs, whereas frequently dividing Nras(G12D) HSCs did not.Nras(G12D) caused these effects by promoting STAT5 signalling, inducing different transcriptional responses in different subsets of HSCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Michigan, Ann Arbor, Michigan 48109, USA.

ABSTRACT
'Pre-leukaemic' mutations are thought to promote clonal expansion of haematopoietic stem cells (HSCs) by increasing self-renewal and competitiveness; however, mutations that increase HSC proliferation tend to reduce competitiveness and self-renewal potential, raising the question of how a mutant HSC can sustainably outcompete wild-type HSCs. Activating mutations in NRAS are prevalent in human myeloproliferative neoplasms and leukaemia. Here we show that a single allele of oncogenic Nras(G12D) increases HSC proliferation but also increases reconstituting and self-renewal potential upon serial transplantation in irradiated mice, all prior to leukaemia initiation. Nras(G12D) also confers long-term self-renewal potential to multipotent progenitors. To explore the mechanism by which Nras(G12D) promotes HSC proliferation and self-renewal, we assessed cell-cycle kinetics using H2B-GFP label retention and 5-bromodeoxyuridine (BrdU) incorporation. Nras(G12D) had a bimodal effect on HSCs, increasing the frequency with which some HSCs divide and reducing the frequency with which others divide. This mirrored bimodal effects on reconstituting potential, as rarely dividing Nras(G12D) HSCs outcompeted wild-type HSCs, whereas frequently dividing Nras(G12D) HSCs did not. Nras(G12D) caused these effects by promoting STAT5 signalling, inducing different transcriptional responses in different subsets of HSCs. One signal can therefore increase HSC proliferation, competitiveness and self-renewal through bimodal effects on HSC gene expression, cycling and reconstituting potential.

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a) The NrasG12D allele was recombined in all HSCs after 3 doses (every other day) of pIpC. Two weeks after the last dose of pIpC was administered to Mx1-cre; NrasG12D/+ mice, the mice were sacrificed and individual CD150+CD48−LSK HSCs were sorted into methylcellulose cultures in 96 well plates. The cells were cultured for 14 days then DNA was extracted from individual colonies and genotyped by PCR. The size of the recombined NrasG12D allele (G12D) was 550bp and the Nras+ allele (WT) was 500bp. Nras recombination was observed in 22 of 22 HSC colonies examined. Blot is representative of three independent experiments. b) Cell cycle analysis of HSCs by pyronin Y and DAPI staining. CD150+CD48−LSK HSCs were sorted from Mx1-cre; NrasG12D/+ mice and littermate controls into 100% ethanol and stained with pyronin Y and DAPI to identify cells in G0 (left lower quadrant), G1 (left upper quadrant) and S/G2/M (right upper and lower quadrants). Data represent mean±s.d.. Statistical analysis was performed with a two-way ANOVA (P<0.01, n=4) followed by pairwise posthoc t-tests.
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Figure 5: a) The NrasG12D allele was recombined in all HSCs after 3 doses (every other day) of pIpC. Two weeks after the last dose of pIpC was administered to Mx1-cre; NrasG12D/+ mice, the mice were sacrificed and individual CD150+CD48−LSK HSCs were sorted into methylcellulose cultures in 96 well plates. The cells were cultured for 14 days then DNA was extracted from individual colonies and genotyped by PCR. The size of the recombined NrasG12D allele (G12D) was 550bp and the Nras+ allele (WT) was 500bp. Nras recombination was observed in 22 of 22 HSC colonies examined. Blot is representative of three independent experiments. b) Cell cycle analysis of HSCs by pyronin Y and DAPI staining. CD150+CD48−LSK HSCs were sorted from Mx1-cre; NrasG12D/+ mice and littermate controls into 100% ethanol and stained with pyronin Y and DAPI to identify cells in G0 (left lower quadrant), G1 (left upper quadrant) and S/G2/M (right upper and lower quadrants). Data represent mean±s.d.. Statistical analysis was performed with a two-way ANOVA (P<0.01, n=4) followed by pairwise posthoc t-tests.

Mentions: To conditionally activate a single allele of NrasG12D in HSCs we generated Mx-1-Cre; NrasG12D/+ mice in which the oncogenic G12D mutation was knocked into the endogenous Nras locus along with a floxed stop cassette20. To induce NrasG12D expression, mice were administered poly-inosine:poly-cytosine (pIpC) at 6-10 weeks after birth (Extended data Figure 1). At 2 weeks and 3 months after pIpC treatment, more than twice as many NrasG12D/+ CD150+CD48−Lineage−Sca-1+c-kit+ (CD150+CD48−LSK) HSCs21 incorporated a 24-hour pulse of bromo-deoxyuridine (BrdU) as compared to control HSCs (p<0.01; Figure 1a). Consistent with this, twice as many NrasG12D/+ HSCs were in G1 phase of the cell cycle as compared to control HSCs (Extended data Figure 1b). This increase in HSC proliferation did not significantly affect the number of HSCs or multipotent progenitors (MPPs) two weeks after NrasG12D activation (Figure 1c). However, Mx-1-Cre; NrasG12D/+ mice had significantly more LSK cells in the bone marrow and spleen (Figure 1c). We also observed a two-fold increase in BrdU incorporation in HSCs as well as an expansion of LSK cells in Vav1-Cre; NrasG12D/+ mice as compared to controls (Figure 1b; Extended data Figure 2a). Thus NrasG12D increased HSC division and expanded the pool of primitive hematopoietic progenitors.


Oncogenic Nras has bimodal effects on stem cells that sustainably increase competitiveness.

Li Q, Bohin N, Wen T, Ng V, Magee J, Chen SC, Shannon K, Morrison SJ - Nature (2013)

a) The NrasG12D allele was recombined in all HSCs after 3 doses (every other day) of pIpC. Two weeks after the last dose of pIpC was administered to Mx1-cre; NrasG12D/+ mice, the mice were sacrificed and individual CD150+CD48−LSK HSCs were sorted into methylcellulose cultures in 96 well plates. The cells were cultured for 14 days then DNA was extracted from individual colonies and genotyped by PCR. The size of the recombined NrasG12D allele (G12D) was 550bp and the Nras+ allele (WT) was 500bp. Nras recombination was observed in 22 of 22 HSC colonies examined. Blot is representative of three independent experiments. b) Cell cycle analysis of HSCs by pyronin Y and DAPI staining. CD150+CD48−LSK HSCs were sorted from Mx1-cre; NrasG12D/+ mice and littermate controls into 100% ethanol and stained with pyronin Y and DAPI to identify cells in G0 (left lower quadrant), G1 (left upper quadrant) and S/G2/M (right upper and lower quadrants). Data represent mean±s.d.. Statistical analysis was performed with a two-way ANOVA (P<0.01, n=4) followed by pairwise posthoc t-tests.
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Figure 5: a) The NrasG12D allele was recombined in all HSCs after 3 doses (every other day) of pIpC. Two weeks after the last dose of pIpC was administered to Mx1-cre; NrasG12D/+ mice, the mice were sacrificed and individual CD150+CD48−LSK HSCs were sorted into methylcellulose cultures in 96 well plates. The cells were cultured for 14 days then DNA was extracted from individual colonies and genotyped by PCR. The size of the recombined NrasG12D allele (G12D) was 550bp and the Nras+ allele (WT) was 500bp. Nras recombination was observed in 22 of 22 HSC colonies examined. Blot is representative of three independent experiments. b) Cell cycle analysis of HSCs by pyronin Y and DAPI staining. CD150+CD48−LSK HSCs were sorted from Mx1-cre; NrasG12D/+ mice and littermate controls into 100% ethanol and stained with pyronin Y and DAPI to identify cells in G0 (left lower quadrant), G1 (left upper quadrant) and S/G2/M (right upper and lower quadrants). Data represent mean±s.d.. Statistical analysis was performed with a two-way ANOVA (P<0.01, n=4) followed by pairwise posthoc t-tests.
Mentions: To conditionally activate a single allele of NrasG12D in HSCs we generated Mx-1-Cre; NrasG12D/+ mice in which the oncogenic G12D mutation was knocked into the endogenous Nras locus along with a floxed stop cassette20. To induce NrasG12D expression, mice were administered poly-inosine:poly-cytosine (pIpC) at 6-10 weeks after birth (Extended data Figure 1). At 2 weeks and 3 months after pIpC treatment, more than twice as many NrasG12D/+ CD150+CD48−Lineage−Sca-1+c-kit+ (CD150+CD48−LSK) HSCs21 incorporated a 24-hour pulse of bromo-deoxyuridine (BrdU) as compared to control HSCs (p<0.01; Figure 1a). Consistent with this, twice as many NrasG12D/+ HSCs were in G1 phase of the cell cycle as compared to control HSCs (Extended data Figure 1b). This increase in HSC proliferation did not significantly affect the number of HSCs or multipotent progenitors (MPPs) two weeks after NrasG12D activation (Figure 1c). However, Mx-1-Cre; NrasG12D/+ mice had significantly more LSK cells in the bone marrow and spleen (Figure 1c). We also observed a two-fold increase in BrdU incorporation in HSCs as well as an expansion of LSK cells in Vav1-Cre; NrasG12D/+ mice as compared to controls (Figure 1b; Extended data Figure 2a). Thus NrasG12D increased HSC division and expanded the pool of primitive hematopoietic progenitors.

Bottom Line: Nras(G12D) had a bimodal effect on HSCs, increasing the frequency with which some HSCs divide and reducing the frequency with which others divide.This mirrored bimodal effects on reconstituting potential, as rarely dividing Nras(G12D) HSCs outcompeted wild-type HSCs, whereas frequently dividing Nras(G12D) HSCs did not.Nras(G12D) caused these effects by promoting STAT5 signalling, inducing different transcriptional responses in different subsets of HSCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Michigan, Ann Arbor, Michigan 48109, USA.

ABSTRACT
'Pre-leukaemic' mutations are thought to promote clonal expansion of haematopoietic stem cells (HSCs) by increasing self-renewal and competitiveness; however, mutations that increase HSC proliferation tend to reduce competitiveness and self-renewal potential, raising the question of how a mutant HSC can sustainably outcompete wild-type HSCs. Activating mutations in NRAS are prevalent in human myeloproliferative neoplasms and leukaemia. Here we show that a single allele of oncogenic Nras(G12D) increases HSC proliferation but also increases reconstituting and self-renewal potential upon serial transplantation in irradiated mice, all prior to leukaemia initiation. Nras(G12D) also confers long-term self-renewal potential to multipotent progenitors. To explore the mechanism by which Nras(G12D) promotes HSC proliferation and self-renewal, we assessed cell-cycle kinetics using H2B-GFP label retention and 5-bromodeoxyuridine (BrdU) incorporation. Nras(G12D) had a bimodal effect on HSCs, increasing the frequency with which some HSCs divide and reducing the frequency with which others divide. This mirrored bimodal effects on reconstituting potential, as rarely dividing Nras(G12D) HSCs outcompeted wild-type HSCs, whereas frequently dividing Nras(G12D) HSCs did not. Nras(G12D) caused these effects by promoting STAT5 signalling, inducing different transcriptional responses in different subsets of HSCs. One signal can therefore increase HSC proliferation, competitiveness and self-renewal through bimodal effects on HSC gene expression, cycling and reconstituting potential.

Show MeSH
Related in: MedlinePlus