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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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Nras activation increases STAT5 phosphorylationa) Western blot for phosphorylated ERK (pERK) in LSK stem/progenitor cells, Lin−c-kit+Sca1− progenitor cells, or whole bone marrow (WBM) cells from Mx1-cre; NrasG12D/+ (G12D/+) mice, Mx1-cre; NrasG12D/G12D (G12D/G12D) mice, or littermate controls 2 weeks after pIpC treatment b) Western blot of pERK and total ERK in 106 uncultured splenocytes from Mx1-cre; NrasG12D/+ (G12D/+) or control mice after 8 days treatment with PD0325901 MEK inhibitor or vehicle (blot is representative of four independent experiments). c) The frequency of BrdU+ CD150+CD48−LSK HSCs after a 24-hour pulse of BrdU to Mx1-cre; NrasG12D/+ (G12D/+) or control mice after 7 days of PD0325901 MEK inhibitor or vehicle (mean±s.d. from four experiments). d) Western blot of pERK and total ERK in 106 uncultured bone marrow cells from Mx1-cre; NrasG12D/+ (G12D/+) or control mice after 8 days of AZD6244 MEK inhibitor or vehicle (blot is representative of four independent experiments). e) The frequency of BrdU+ CD150+CD48−LSK HSCs after a 24-hour pulse of BrdU to Mx1-cre; NrasG12D/+ (G12D/+) or control mice after 7 days of AZD6244 MEK inhibitor or vehicle (mean±s.d. from four experiments). f) Western blot for phosphorylated Akt (pAkt) in CD48−LSK HSCs/MPPs, CD48+LSK progenitors, or WBM cells from Mx1-cre; NrasG12D/+ (G12D/+) mice, Mx1-cre; Ptenfl/fl (Pten−/−) mice, or littermate controls 2 weeks after pIpC treatment. g) Socs2 transcript levels in HSCs and MPPs from Mx1-cre; NrasG12D/+ (G12D/+) or control mice by microarray analysis (top, n=3) and qRT-PCR (bottom, n=7). h, i) Socs2 transcript levels in GFP− and GFPhigh HSCs from Mx1-cre; NrasG12D/+; Col1A1-H2B-GFP; Rosa26-M2-rtTA mice and littermate controls by microarray (h, n=3) and qRT-PCR (, n=3). j) Western blotting showed that pSTAT5 levels were significantly increased in CD48−LSK HSCs/MPPs from Mx1-cre; NrasG12D/+ mice as compared to control mice. Left panel shows western blots of pSTAT5 and total STAT5 from two independent experiments. Right panel shows quantification of pSTAT5 levels from western blots from three independent experiments (signals were quantitated using NIH ImageJ software). Blot 1 was shown in Figure 4e. k) Western blot showed that STAT5 levels were reduced in CD48−LSK HSCs/MPPs from Mx1-cre; Stat5ab−/+ or Mx1-cre; NrasG12D/+; Stat5ab−/+ mice as compared to control and Mx1-cre; NrasG12D/+ mice (blot is representative of four independent experiments). l) BrdU incorporation into common myeloid progenitors (CMPs; Lin−Sca1−ckit+CD34+CD16/32−), granulocyte macrophage progenitors (GMPs; Lin−Sca1−ckit+CD34+CD16/32+), and megakaryocyte erythroid progenitors (MEPs; Lin−Sca1−ckit+CD34−CD16/32−) from control, Mx1-cre; Stat5ab−/+, Mx1-cre; NrasG12D/+, or Mx1-cre; NrasG12D/+; Stat5ab−/+ mice after a 2.5 hour pulse of BrdU (n=4 mice/treatment). Data represent mean±s.d.. Two-tailed student's t-tests were used to assess statistical significance.
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Figure 12: Nras activation increases STAT5 phosphorylationa) Western blot for phosphorylated ERK (pERK) in LSK stem/progenitor cells, Lin−c-kit+Sca1− progenitor cells, or whole bone marrow (WBM) cells from Mx1-cre; NrasG12D/+ (G12D/+) mice, Mx1-cre; NrasG12D/G12D (G12D/G12D) mice, or littermate controls 2 weeks after pIpC treatment b) Western blot of pERK and total ERK in 106 uncultured splenocytes from Mx1-cre; NrasG12D/+ (G12D/+) or control mice after 8 days treatment with PD0325901 MEK inhibitor or vehicle (blot is representative of four independent experiments). c) The frequency of BrdU+ CD150+CD48−LSK HSCs after a 24-hour pulse of BrdU to Mx1-cre; NrasG12D/+ (G12D/+) or control mice after 7 days of PD0325901 MEK inhibitor or vehicle (mean±s.d. from four experiments). d) Western blot of pERK and total ERK in 106 uncultured bone marrow cells from Mx1-cre; NrasG12D/+ (G12D/+) or control mice after 8 days of AZD6244 MEK inhibitor or vehicle (blot is representative of four independent experiments). e) The frequency of BrdU+ CD150+CD48−LSK HSCs after a 24-hour pulse of BrdU to Mx1-cre; NrasG12D/+ (G12D/+) or control mice after 7 days of AZD6244 MEK inhibitor or vehicle (mean±s.d. from four experiments). f) Western blot for phosphorylated Akt (pAkt) in CD48−LSK HSCs/MPPs, CD48+LSK progenitors, or WBM cells from Mx1-cre; NrasG12D/+ (G12D/+) mice, Mx1-cre; Ptenfl/fl (Pten−/−) mice, or littermate controls 2 weeks after pIpC treatment. g) Socs2 transcript levels in HSCs and MPPs from Mx1-cre; NrasG12D/+ (G12D/+) or control mice by microarray analysis (top, n=3) and qRT-PCR (bottom, n=7). h, i) Socs2 transcript levels in GFP− and GFPhigh HSCs from Mx1-cre; NrasG12D/+; Col1A1-H2B-GFP; Rosa26-M2-rtTA mice and littermate controls by microarray (h, n=3) and qRT-PCR (, n=3). j) Western blotting showed that pSTAT5 levels were significantly increased in CD48−LSK HSCs/MPPs from Mx1-cre; NrasG12D/+ mice as compared to control mice. Left panel shows western blots of pSTAT5 and total STAT5 from two independent experiments. Right panel shows quantification of pSTAT5 levels from western blots from three independent experiments (signals were quantitated using NIH ImageJ software). Blot 1 was shown in Figure 4e. k) Western blot showed that STAT5 levels were reduced in CD48−LSK HSCs/MPPs from Mx1-cre; Stat5ab−/+ or Mx1-cre; NrasG12D/+; Stat5ab−/+ mice as compared to control and Mx1-cre; NrasG12D/+ mice (blot is representative of four independent experiments). l) BrdU incorporation into common myeloid progenitors (CMPs; Lin−Sca1−ckit+CD34+CD16/32−), granulocyte macrophage progenitors (GMPs; Lin−Sca1−ckit+CD34+CD16/32+), and megakaryocyte erythroid progenitors (MEPs; Lin−Sca1−ckit+CD34−CD16/32−) from control, Mx1-cre; Stat5ab−/+, Mx1-cre; NrasG12D/+, or Mx1-cre; NrasG12D/+; Stat5ab−/+ mice after a 2.5 hour pulse of BrdU (n=4 mice/treatment). Data represent mean±s.d.. Two-tailed student's t-tests were used to assess statistical significance.

Mentions: We detected the activation of the canonical Ras effector, ERK, in bone marrow cells from Mx1-cre; NrasG12D/+ and Mx1-cre; NrasG12D/G12D mice but not in LSK stem/progenitor cells or Lineage−c-kit+Sca-1− myeloid progenitors (Extended data Figure 8a). We treated Mx1-cre; NrasG12D/+ or control mice with the MEK inhibitors, PD0325901 (5mg/kg/day) or AZD6244 (25mg/kg/day), and assessed the effects on BrdU incorporation in CD150+CD48−LSK HSCs. After eight days of treatment, splenocytes from PD0325901-treated mice of both genotypes showed reduced pERK levels (Extended data Figure 8b), but this did not affect the increased rate of BrdU incorporation by NrasG12D/+ HSCs (Extended data Figure 8c). In contrast, when we performed the same experiments with AZD6244, pERK activation was completely blocked in bone marrow and spleen (Extended data Figure 8d) and the increased cycling of NrasG12D/+ HSCs was abolished (Supplementary figure 8e). These data suggest that the more stringent inhibition of pERK activation by AZD6244 blocks the effect of NrasG12D/+ on HSC cycling.


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)

Nras activation increases STAT5 phosphorylationa) Western blot for phosphorylated ERK (pERK) in LSK stem/progenitor cells, Lin−c-kit+Sca1− progenitor cells, or whole bone marrow (WBM) cells from Mx1-cre; NrasG12D/+ (G12D/+) mice, Mx1-cre; NrasG12D/G12D (G12D/G12D) mice, or littermate controls 2 weeks after pIpC treatment b) Western blot of pERK and total ERK in 106 uncultured splenocytes from Mx1-cre; NrasG12D/+ (G12D/+) or control mice after 8 days treatment with PD0325901 MEK inhibitor or vehicle (blot is representative of four independent experiments). c) The frequency of BrdU+ CD150+CD48−LSK HSCs after a 24-hour pulse of BrdU to Mx1-cre; NrasG12D/+ (G12D/+) or control mice after 7 days of PD0325901 MEK inhibitor or vehicle (mean±s.d. from four experiments). d) Western blot of pERK and total ERK in 106 uncultured bone marrow cells from Mx1-cre; NrasG12D/+ (G12D/+) or control mice after 8 days of AZD6244 MEK inhibitor or vehicle (blot is representative of four independent experiments). e) The frequency of BrdU+ CD150+CD48−LSK HSCs after a 24-hour pulse of BrdU to Mx1-cre; NrasG12D/+ (G12D/+) or control mice after 7 days of AZD6244 MEK inhibitor or vehicle (mean±s.d. from four experiments). f) Western blot for phosphorylated Akt (pAkt) in CD48−LSK HSCs/MPPs, CD48+LSK progenitors, or WBM cells from Mx1-cre; NrasG12D/+ (G12D/+) mice, Mx1-cre; Ptenfl/fl (Pten−/−) mice, or littermate controls 2 weeks after pIpC treatment. g) Socs2 transcript levels in HSCs and MPPs from Mx1-cre; NrasG12D/+ (G12D/+) or control mice by microarray analysis (top, n=3) and qRT-PCR (bottom, n=7). h, i) Socs2 transcript levels in GFP− and GFPhigh HSCs from Mx1-cre; NrasG12D/+; Col1A1-H2B-GFP; Rosa26-M2-rtTA mice and littermate controls by microarray (h, n=3) and qRT-PCR (, n=3). j) Western blotting showed that pSTAT5 levels were significantly increased in CD48−LSK HSCs/MPPs from Mx1-cre; NrasG12D/+ mice as compared to control mice. Left panel shows western blots of pSTAT5 and total STAT5 from two independent experiments. Right panel shows quantification of pSTAT5 levels from western blots from three independent experiments (signals were quantitated using NIH ImageJ software). Blot 1 was shown in Figure 4e. k) Western blot showed that STAT5 levels were reduced in CD48−LSK HSCs/MPPs from Mx1-cre; Stat5ab−/+ or Mx1-cre; NrasG12D/+; Stat5ab−/+ mice as compared to control and Mx1-cre; NrasG12D/+ mice (blot is representative of four independent experiments). l) BrdU incorporation into common myeloid progenitors (CMPs; Lin−Sca1−ckit+CD34+CD16/32−), granulocyte macrophage progenitors (GMPs; Lin−Sca1−ckit+CD34+CD16/32+), and megakaryocyte erythroid progenitors (MEPs; Lin−Sca1−ckit+CD34−CD16/32−) from control, Mx1-cre; Stat5ab−/+, Mx1-cre; NrasG12D/+, or Mx1-cre; NrasG12D/+; Stat5ab−/+ mice after a 2.5 hour pulse of BrdU (n=4 mice/treatment). Data represent mean±s.d.. Two-tailed student's t-tests were used to assess statistical significance.
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Figure 12: Nras activation increases STAT5 phosphorylationa) Western blot for phosphorylated ERK (pERK) in LSK stem/progenitor cells, Lin−c-kit+Sca1− progenitor cells, or whole bone marrow (WBM) cells from Mx1-cre; NrasG12D/+ (G12D/+) mice, Mx1-cre; NrasG12D/G12D (G12D/G12D) mice, or littermate controls 2 weeks after pIpC treatment b) Western blot of pERK and total ERK in 106 uncultured splenocytes from Mx1-cre; NrasG12D/+ (G12D/+) or control mice after 8 days treatment with PD0325901 MEK inhibitor or vehicle (blot is representative of four independent experiments). c) The frequency of BrdU+ CD150+CD48−LSK HSCs after a 24-hour pulse of BrdU to Mx1-cre; NrasG12D/+ (G12D/+) or control mice after 7 days of PD0325901 MEK inhibitor or vehicle (mean±s.d. from four experiments). d) Western blot of pERK and total ERK in 106 uncultured bone marrow cells from Mx1-cre; NrasG12D/+ (G12D/+) or control mice after 8 days of AZD6244 MEK inhibitor or vehicle (blot is representative of four independent experiments). e) The frequency of BrdU+ CD150+CD48−LSK HSCs after a 24-hour pulse of BrdU to Mx1-cre; NrasG12D/+ (G12D/+) or control mice after 7 days of AZD6244 MEK inhibitor or vehicle (mean±s.d. from four experiments). f) Western blot for phosphorylated Akt (pAkt) in CD48−LSK HSCs/MPPs, CD48+LSK progenitors, or WBM cells from Mx1-cre; NrasG12D/+ (G12D/+) mice, Mx1-cre; Ptenfl/fl (Pten−/−) mice, or littermate controls 2 weeks after pIpC treatment. g) Socs2 transcript levels in HSCs and MPPs from Mx1-cre; NrasG12D/+ (G12D/+) or control mice by microarray analysis (top, n=3) and qRT-PCR (bottom, n=7). h, i) Socs2 transcript levels in GFP− and GFPhigh HSCs from Mx1-cre; NrasG12D/+; Col1A1-H2B-GFP; Rosa26-M2-rtTA mice and littermate controls by microarray (h, n=3) and qRT-PCR (, n=3). j) Western blotting showed that pSTAT5 levels were significantly increased in CD48−LSK HSCs/MPPs from Mx1-cre; NrasG12D/+ mice as compared to control mice. Left panel shows western blots of pSTAT5 and total STAT5 from two independent experiments. Right panel shows quantification of pSTAT5 levels from western blots from three independent experiments (signals were quantitated using NIH ImageJ software). Blot 1 was shown in Figure 4e. k) Western blot showed that STAT5 levels were reduced in CD48−LSK HSCs/MPPs from Mx1-cre; Stat5ab−/+ or Mx1-cre; NrasG12D/+; Stat5ab−/+ mice as compared to control and Mx1-cre; NrasG12D/+ mice (blot is representative of four independent experiments). l) BrdU incorporation into common myeloid progenitors (CMPs; Lin−Sca1−ckit+CD34+CD16/32−), granulocyte macrophage progenitors (GMPs; Lin−Sca1−ckit+CD34+CD16/32+), and megakaryocyte erythroid progenitors (MEPs; Lin−Sca1−ckit+CD34−CD16/32−) from control, Mx1-cre; Stat5ab−/+, Mx1-cre; NrasG12D/+, or Mx1-cre; NrasG12D/+; Stat5ab−/+ mice after a 2.5 hour pulse of BrdU (n=4 mice/treatment). Data represent mean±s.d.. Two-tailed student's t-tests were used to assess statistical significance.
Mentions: We detected the activation of the canonical Ras effector, ERK, in bone marrow cells from Mx1-cre; NrasG12D/+ and Mx1-cre; NrasG12D/G12D mice but not in LSK stem/progenitor cells or Lineage−c-kit+Sca-1− myeloid progenitors (Extended data Figure 8a). We treated Mx1-cre; NrasG12D/+ or control mice with the MEK inhibitors, PD0325901 (5mg/kg/day) or AZD6244 (25mg/kg/day), and assessed the effects on BrdU incorporation in CD150+CD48−LSK HSCs. After eight days of treatment, splenocytes from PD0325901-treated mice of both genotypes showed reduced pERK levels (Extended data Figure 8b), but this did not affect the increased rate of BrdU incorporation by NrasG12D/+ HSCs (Extended data Figure 8c). In contrast, when we performed the same experiments with AZD6244, pERK activation was completely blocked in bone marrow and spleen (Extended data Figure 8d) and the increased cycling of NrasG12D/+ HSCs was abolished (Supplementary figure 8e). These data suggest that the more stringent inhibition of pERK activation by AZD6244 blocks the effect of NrasG12D/+ on HSC cycling.

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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