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

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.

Extended data Figure 1

a) The Nras^G12D 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; Nras^G12D/+ 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 Nras^G12D 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; Nras^G12D/+ 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.

Extended data Figure 2. HSC competitiveness is increased in Vav1-Cre; Nras^G12D/+ mice

a) Frequencies of CD150⁺CD48⁻LSK HSCs, CD150⁻CD48⁻LSK MPPs, and LSK cells in the bone marrow (BM, top row) and spleen (sp, bottom row) of Vav1-cre; Nras^G12D/+ (G12D/+) or littermate control (con) mice (n=4) at 6-10 weeks of age. b) 5×10⁵ donor bone marrow cells from Vav1-cre; Nras^G12D/+ (G12D/+) or littermate control (con) mice at 6-10 weeks of age were transplanted into irradiated recipient mice along with 5×10⁵ recipient bone marrow cells (3 donors/genotype were each transplanted into 4 recipients/donor). c) Secondary transplantation of 3×10⁶ bone marrow cells from primary recipient mice in Extended data Figure 2b at 20 weeks after transplantation (2 primary recipients/genotype were each transplanted into 4 secondary recipients/primary recipient). Data represent mean±s.d.. Two-tailed student's t-tests were used to assess statistical significance. *P<0.05, **P<0.01, ***P<0.001.

Extended data Figure 3. HSCs from Mx1-cre; Nras^G12D/+ mice were not immortalized

A fifth round of serial transplantation of 3×10⁶ bone marrow cells from the quaternary recipients of Nras^G12D/+ (G12D/+) bone marrow cells shown in Figure 2c showed that the Nras^G12D/+ HSCs eventually exhausted all of their HSCs and MPPs and were able to only give low levels of lymphoid reconstitution. Four donor mice from Figure 2c were transplanted 20 weeks after the fourth round of transplantation into 4 recipients per quaternary donor. The data represent mean±s.d. for donor blood cells in the myeloid (Gr-1⁺ or Mac-1⁺ cells), B (B220⁺), and T (CD3⁺) cell lineages.

Extended data Figure 4. Nras^G12D (G12D/+) expression increased the reconstituting potential of CD150⁻CD48⁺LSK MPPs but did not affect the reconstituting potential of CD150⁺CD48⁺LSK, or CD150⁻CD48⁺LSK progenitors in irradiated mice

a) 10 donor MPPs, b) 25 CD150⁺CD48⁺LSK progenitors, or c) 100 CD150⁻CD48⁺LSK progenitors from Mx1-cre; Nras^G12D/+ (G12D/+) or littermate control (con) mice at 2 weeks after pIpC treatment were transplanted into irradiated recipient mice along with 3×10⁵ recipient bone marrow cells. Data represent mean±s.d. for donor blood cells in the myeloid (Gr-1⁺ or Mac-1⁺ cells), B (B220⁺), and T (CD3⁺) cell lineages. Two-tailed student's t-tests were used to assess statistical significance. None of the time points were significantly different between treatments. The data represent two independent experiments with 4 recipient mice per donor

Extended data Figure 5. Nras^G12D-induced changes in HSC function were not associated with the development of leukemia

White blood counts (WBC), hemoglobulin (Hb) levels, platelet counts, and spleen masses for recipient mice from primary transplants (a; from Figure 1d), secondary transplants (b; from Figure 2a), tertiary transplants (c; from Figure 2b) and quaternary transplants (d; from Figure 2c). In all cases, these blood cell counts were collected from mice after the analysis of blood cell reconstitution was complete (at least 20 weeks after transplantation). The transplanted mice were observed for a median time of 260 (162–315) days for primary recipient mice, 194 (122–264) days for secondary recipient mice, 224 (176–336) days for tertiary recipient mice, and 280 (279–280) days for quaternary recipient mice. We never observed evidence of leukemia or MPN by histology in these mice. Across all of the experiments, only two recipients of Nras^G12D/+ cells and two recipients of control cells died spontaneously. Data represent mean±s.d.. Two-tailed student's t-tests were used to assess statistical significance and none of the comparisons showed significant difference.

Extended data Figure 6. Nras^G12D/+ had a bimodal effect on HSC cycling but increased the rate at which MPPs divide

a) Flow cytometric analysis of GFP expression in whole bone marrow cells from Nras^G12D/+ or littermate control mice after 12 weeks of chase without doxycycline. b) Median GFP fluorescence intensity of H2B-GFP⁻, H2B-GFP^lo and H2B-GFP^hi HSCs from wild type and Nras^G12D/+ mice (n=8 mice/genotype). GFP levels in control HSCs were set to one for comparison to relative levels in Nras^G12D/+ HSCs. c) Nras^G12D increased the rate of division by MPPs. Flow cytometric analysis of GFP expression in CD150⁻CD48⁻LSK MPPs from Mx1-cre; Nras^G12D/+; Col1A1-H2B-GFP; Rosa26-M2-rtTA mice (G12D/+) and littermate controls (con) after 12 weeks of chase(n=8 mice/genotype). Relative to control MPPs, Nras^G12D/+ MPPs included significantly more H2B-GFP⁻ frequently cycling cells and significantly fewer H2B-GFP^lo MPPs (p<0.05 by two-way ANOVA and posthoc pairwise t-tests). d) We continuously administered BrdU to Mx1-cre; Nras^G12D/+ versus control mice for 1 to 30 days and determined the frequency of BrdU+ HSCs (1 day BrdU data are from Figure 1a). Data represent mean±s.d.. Two-tailed student's t-tests were used to assess statistical significance unless stated otherwise. *P<0.05, **P<0.01, ***P<0.001.

Extended data Figure 7. Gene expression profiling demonstrates different transcriptional responses to Nras activation in quiescent as compared to frequently dividing HSCs

a) CD150⁺CD48⁻LSK HSCs and CD150⁻CD48⁻LSK MPPs were isolated from three pairs of Mx1-cre; Nras^G12D/+ and littermate controls and gene expression profiling was performed with Affymetrix mouse genome 430 2.0 microarrays. The Venn diagram shows the number of genes that were differentially expressed between Nras^G12D/+ and controls cells within each cell population (fold change≥2). b) Venn diagram of genes that were differentially expressed between Nras^G12D/+ and control GFP⁻ HSCs and GFP^high HSCs isolated from 3 pairs of Mx1-cre; Nras^G12D/+; Col1A1-H2B-GFP; Rosa26-M2-rtTA mice and littermate controls (fold change ≥and p value ≤0.05). c) Genes that were consistently increased or decreased in expression in response to Nras activation in HSCs, MPPs, GFP⁻ HSCs, and GFP^high HSCs (fold change≥2 and p≤0.05 in each cell population). d, e, f) Gene set enrichment analysis (GSEA) of cell cycle genes d), DNA replication genes e) and RNA polymerase genes f).

Extended data Figure 8. Nras activation increases STAT5 phosphorylation

a) 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; Nras^G12D/+ (G12D/+) mice, Mx1-cre; Nras^G12D/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; Nras^G12D/+ (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; Nras^G12D/+ (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; Nras^G12D/+ (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; Nras^G12D/+ (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; Nras^G12D/+ (G12D/+) mice, Mx1-cre; Pten^fl/fl (Pten^−/−) mice, or littermate controls 2 weeks after pIpC treatment. g) Socs2 transcript levels in HSCs and MPPs from Mx1-cre; Nras^G12D/+ (G12D/+) or control mice by microarray analysis (top, n=3) and qRT-PCR (bottom, n=7). h, i) Socs2 transcript levels in GFP⁻ and GFP^high HSCs from Mx1-cre; Nras^G12D/+; 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; Nras^G12D/+ 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; Nras^G12D/+; Stat5ab^−/+ mice as compared to control and Mx1-cre; Nras^G12D/+ 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; Nras^G12D/+, or Mx1-cre; Nras^G12D/+; 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.

Figure 1. Nras^G12D/+ increased HSC proliferation and competitiveness

a) A 24-hour pulse of BrdU was administered to Mx1-cre; Nras^G12D/+ (G12D/+) and littermate control (con) mice at 2 weeks and 3 months after pIpC treatment (n=3 mice/treatment). b) BrdU incorporation by CD150⁺CD48⁻LSK HSCs from Vav1-Cre; Nras^G12D/+ mice (G12D/+) or littermate controls (con) at 6-10 weeks of age (n=3). c) The total number of CD150⁺CD48⁻LSK HSCs, CD150⁻CD48⁻LSK MPPs, and LSK cells in the bone marrow and spleens of Mx1-cre; Nras^G12D/+ (G12D/+) and littermate control (con) mice at 2 weeks after pIpC treatment (n=5 mice/treatment). d) 5×10⁵ donor bone marrow cells from Mx1-cre; Nras^G12D/+ (G12D/+) or littermate control (con) mice at 2 weeks after pIpC treatment (n=3 donors/genotype) were transplanted into irradiated recipient mice (n=15 recipients/genotype) along with 5×10⁵ recipient bone marrow cells. Donor cell reconstitution in the myeloid (Mac-1⁺ cells), B (B220⁺), and T (CD3⁺) cell lineages for 4 to 20 weeks after transplantation. e) Recipients of Mx1-cre; Nras^G12D/+ (G12D/+) bone marrow cells (n=5) had significantly (p<0.05) higher proportions of donor-derived HSCs, MPPs and LSK cells compared to recipients of control bone marrow cells (con). f) 10 donor HSCs from Mx1-cre; Nras^G12D/+ (G12D/+) or littermate control (con) mice at 2 weeks after pIpC treatment (n=3 donors/genotype) were transplanted into irradiated recipient mice (n=14 recipients/genotype) along with 3×10⁵ recipient bone marrow cells. Data represent mean±s.d.. Two-tailed student's t-tests were used to assess statistical significance. *P<0.05, **P<0.01, ***P<0.001.

Figure 2. Nras^G12D/+ increased HSC and MPP self-renewal

a) Secondary transplantation (n=19 recipients/genotype) of 3×10⁶ bone marrow cells from primary recipient mice in Figure 1c (n=4 donors/genotype). Donor cell reconstitution in the myeloid (Mac-1⁺), B (B220⁺), and T (CD3⁺) cell lineages for 4 to 20 weeks after transplantation b) Transplantation of 3×10⁶ bone marrow cells from secondary recipient mice in Figure 2a (n=3 donors/genotype) into tertiary recipient mice (n=8 recipients for control and 9 recipients for Nras^G12D/+) c) Transplantation of 3×10⁶ bone marrow cells from tertiary recipient mice (n=3 donors for control and 4 donors for Nras^G12D/+) in Figure 2b into quaternary recipient mice (n=7 recipients for control and 17 for Nras^G12D/+). Each serial transplant was performed at 20 weeks after the prior round of transplantation. Data represent mean±s.d.. Two-tailed student's t-tests were used to assess statistical significance. *P<0.05, **P<0.01, ***P<0.001.

Figure 3. Nras^G12D/+ has a bimodal effect on HSC cycling

a) GFP expression in HSCs from Mx1-cre; Nras^G12D/+; Col1A1-H2B-GFP; Rosa26-M2-rtTA mice (G12D/+) and littermate controls (control) without doxycycline treatment (n=3, left), or after 6 weeks of doxycycline treatment (n=3, right). b) GFP expression in HSCs from age and sex-matched pairs of Nras^G12D/+ and control mice after labeling followed by 12 weeks of chase without doxycycline (n=8 pairs of mice from 8 independent experiments; p<0.05 by two-way ANOVA and posthoc pairwise t-tests). Despite overlapping standard deviations, differences were statistically significant in pairwise t-tests because the frequencies of H2B-GFP⁻ HSCs and H2B-GFP^hi HSCs were always higher in the Nras^G12D/+ mice. c) GFP expression in HSCs from pairs of age and sex-matched Nras^G12D/+ and control mice after 15 weeks of chase without doxycycline (n=7 mice from 5 independent experiments). Nras^G12D/+ mice always had higher frequencies of H2B-GFP^hi HSCs (p<0.05 by pairwise t-tests). d) We transplanted 15 CD150⁺CD48⁻LSK H2B-GFP^hi HSCs, 50 H2B-GFP^lo HSCs, or 75 H2B-GFP⁻ HSCs from Nras^G12D/+ or littermate control mice after 12 weeks of chase into irradiated wild-type recipients along with 3×10⁵ recipient bone marrow cells (2 independent experiments with a total of 7 recipients/genotype). Data represent mean±s.d.. Unless otherwise stated, two-tailed student's t-tests were used to assess statistical significance. *P<0.05, **P<0.01, ***P<0.001.

Figure 4. Increased STAT5 activation mediates the effect of Nras^G12D/+ on HSCs

Western 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; Stat5ab^fl/+ (Stat5ab^−/+) mice, Mx1-cre; Nras^G12D/+ (G12D/+) mice, Mx1-cre; Nras^G12D/+; Stat5ab^fl/+ (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.