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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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Increased STAT5 activation mediates the effect of NrasG12D/+ on HSCsWestern blots for pSTAT3, pp38, pS6, and ß-actin (a) and pSTAT5, total STAT5 and ß-actin (b) (two additional experiments are shown in Supplementary figure 8j). Cells were stimulated in culture with SCF and TPO for 30 minutes before protein extraction. c) The frequency of BrdU+CD150+CD48−LSK HSCs after a 24-hour pulse of BrdU to Mx1-cre; Stat5abfl/+ (Stat5ab−/+) mice, Mx1-cre; NrasG12D/+ (G12D/+) mice, Mx1-cre; NrasG12D/+; Stat5abfl/+ (G12D/+; Stat5ab−/+) compound mutant mice, or control mice (n=4). d) 5×105 donor bone marrow cells from mice of each genotype were transplanted into irradiated recipients along with 5×105 recipient bone marrow cells (2 independent experiments with a total of 8 recipients/genotype). Data represent mean±s.d.. Two-tailed student's t-tests were used to assess statistical significance.
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Figure 4: Increased STAT5 activation mediates the effect of NrasG12D/+ on HSCsWestern blots for pSTAT3, pp38, pS6, and ß-actin (a) and pSTAT5, total STAT5 and ß-actin (b) (two additional experiments are shown in Supplementary figure 8j). Cells were stimulated in culture with SCF and TPO for 30 minutes before protein extraction. c) The frequency of BrdU+CD150+CD48−LSK HSCs after a 24-hour pulse of BrdU to Mx1-cre; Stat5abfl/+ (Stat5ab−/+) mice, Mx1-cre; NrasG12D/+ (G12D/+) mice, Mx1-cre; NrasG12D/+; Stat5abfl/+ (G12D/+; Stat5ab−/+) compound mutant mice, or control mice (n=4). d) 5×105 donor bone marrow cells from mice of each genotype were transplanted into irradiated recipients along with 5×105 recipient bone marrow cells (2 independent experiments with a total of 8 recipients/genotype). Data represent mean±s.d.. Two-tailed student's t-tests were used to assess statistical significance.

Mentions: We did not detect increased Akt (Extended data Figure 8f), S6, or p38 (Figure 4a) phosphorylation in whole bone marrow cells, CD48−LSK cells (HSCs and MPPs) or CD48+LSK cells (mainly restricted progenitors22) from Mx1-cre; NrasG12D/+ mice.


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)

Increased STAT5 activation mediates the effect of NrasG12D/+ on HSCsWestern blots for pSTAT3, pp38, pS6, and ß-actin (a) and pSTAT5, total STAT5 and ß-actin (b) (two additional experiments are shown in Supplementary figure 8j). Cells were stimulated in culture with SCF and TPO for 30 minutes before protein extraction. c) The frequency of BrdU+CD150+CD48−LSK HSCs after a 24-hour pulse of BrdU to Mx1-cre; Stat5abfl/+ (Stat5ab−/+) mice, Mx1-cre; NrasG12D/+ (G12D/+) mice, Mx1-cre; NrasG12D/+; Stat5abfl/+ (G12D/+; Stat5ab−/+) compound mutant mice, or control mice (n=4). d) 5×105 donor bone marrow cells from mice of each genotype were transplanted into irradiated recipients along with 5×105 recipient bone marrow cells (2 independent experiments with a total of 8 recipients/genotype). Data represent mean±s.d.. Two-tailed student's t-tests were used to assess statistical significance.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4128640&req=5

Figure 4: Increased STAT5 activation mediates the effect of NrasG12D/+ on HSCsWestern blots for pSTAT3, pp38, pS6, and ß-actin (a) and pSTAT5, total STAT5 and ß-actin (b) (two additional experiments are shown in Supplementary figure 8j). Cells were stimulated in culture with SCF and TPO for 30 minutes before protein extraction. c) The frequency of BrdU+CD150+CD48−LSK HSCs after a 24-hour pulse of BrdU to Mx1-cre; Stat5abfl/+ (Stat5ab−/+) mice, Mx1-cre; NrasG12D/+ (G12D/+) mice, Mx1-cre; NrasG12D/+; Stat5abfl/+ (G12D/+; Stat5ab−/+) compound mutant mice, or control mice (n=4). d) 5×105 donor bone marrow cells from mice of each genotype were transplanted into irradiated recipients along with 5×105 recipient bone marrow cells (2 independent experiments with a total of 8 recipients/genotype). Data represent mean±s.d.. Two-tailed student's t-tests were used to assess statistical significance.
Mentions: We did not detect increased Akt (Extended data Figure 8f), S6, or p38 (Figure 4a) phosphorylation in whole bone marrow cells, CD48−LSK cells (HSCs and MPPs) or CD48+LSK cells (mainly restricted progenitors22) from Mx1-cre; NrasG12D/+ mice.

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