Wild-type Kras Expands And Exhausts Hematopoietic Stem Cells

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Abstract

Oncogenic Kras expression specifically in hematopoietic stem cells (HSCs) induces a rapidly fatal myeloproliferative neoplasm in mice, suggesting that Kras signaling plays a dominant role in normal hematopoiesis. However, such a conclusion is based on expression of an oncogenic version of Kras. Hence, we sought to determine the effect of simply increasing the amount of endogenous wild-type Kras on HSC fate. To this end, we utilized a codon-optimized version of the murine Kras gene (Krasex3op) that we developed, in which silent mutations in exon 3 render the encoded mRNA more efficiently translated, leading to increased protein expression without disruption to the normal gene architecture. We found that Kras protein levels were significantly increased in bone marrow (BM) HSCs in Krasex3op/ex3op mice, demonstrating that the translation of Kras in HSCs is normally constrained by rare codons. Krasex3op/ex3op mice displayed expansion of BM HSCs, progenitor cells, and B lymphocytes, but no evidence of myeloproliferative disease or leukemia in mice followed for 12 months. BM HSCs from Krasex3op/ex3op mice demonstrated increased multilineage repopulating capacity in primary competitive transplantation assays, but secondary competitive transplants revealed exhaustion of long-term HSCs. Following total body irradiation, Krasex3op/ex3op mice displayed accelerated hematologic recovery and increased survival. Mechanistically, HSCs from Krasex3op/ex3op mice demonstrated increased proliferation at baseline, with a corresponding increase in Erk1/2 phosphorylation and cyclin-dependent kinase 4 and 6 (Cdk4/6) activation. Furthermore, both the enhanced colony-forming capacity and in vivo repopulating capacity of HSCs from Krasex3op/ex3op mice were dependent on Cdk4/6 activation. Finally, BM transplantation studies revealed that augmented Kras expression produced expansion of HSCs, progenitor cells, and B cells in a hematopoietic cell-autonomous manner, independent from effects on the BM microenvironment. This study provides fundamental demonstration of codon usage in a mammal having a biological consequence, which may speak to the importance of codon usage in mammalian biology.

Keywords: Hematology; Hematopoietic stem cells; Stem cells.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1

Figure 1. Kras translation is constrained by…

Figure 1. Kras translation is constrained by rare codons and increased Kras expands B lymphocytes.

Figure 1. Kras translation is constrained by rare codons and increased Kras expands B lymphocytes. (A) At left, representative histograms of Kras protein levels in BM KSL cells and HSCs in 8- to 12-week-old Krasex3op/ex3op mice (ex3op) and Krasnat/nat mice (nat). Isotype control is shown in black. Gate represents Krashi cells. At right, mean percentages of Krashi cells are shown within each mouse group (n = 9/group). ***P < 0.001. (B) Scatter plots show mean PB white blood cells (WBCs), neutrophils (Neut), and lymphocytes (Lymph) at baseline in 8- to 12-week-old Krasex3op/ex3op and Krasnat/nat mice (n = 20/group). ****P < 0.0001. (C and D) Scatter plots show the numbers of PB CD19+ B cells, CD3+ T cells, CD4+ T cells, CD8+ T cells, NK cells, naive T cells, and effector memory T cells (n = 8/group). ***P < 0.001. Two-tailed Student’s t test was used for each comparison. ns, not significant. Data are presented as mean ± SEM.
Figure 2

Figure 2. Kras ex3op/ex3op mice display increased…

Figure 2. Kras ex3op/ex3op mice display increased BM HSCs and progenitor cells.

( A ) Representative…

Figure 2. Krasex3op/ex3op mice display increased BM HSCs and progenitor cells. (A) Representative flow cytometric analysis of BM ckit+sca1+lin– (KSL) cells and CD150+CD48–KSL HSCs in ex3op mice and nat mice at baseline. Numbers represent the percentages of KSL cells and CD150+CD48–KSL cells shown within the gates. (B) Mean percentages of BM KSL cells and numbers of KSL cells in ex3op mice and nat mice at baseline (n = 9/group). *P < 0.05, **P < 0.01. (C) Mean percentages of BM HSCs and numbers of HSCs in ex3op mice and nat mice (n = 9/group). **P < 0.01, ***P < 0.001. (D) Mean numbers of CFCs in ex3op mice and nat mice (n = 10/group). **P < 0.01. (E) Poisson statistical analysis of a limiting-dilution assay revealed BM LTC-IC frequencies of 1 in 8,940 in ex3op mice and 1 in 19,235 in nat mice at 8–12 weeks. The plot shows the percentage of negative colonies detected at 3 different BM cell doses. The horizontal line represents the cell dose at which 37% of the plates show no colonies (n = 6/group). P = 0.03. Two-tailed Student’s t test performed throughout. Data are presented as mean ± SEM.
Figure 3

Figure 3. Increased wild-type Kras expands HSCs…

Figure 3. Increased wild-type Kras expands HSCs and exhausts long-term repopulating HSCs.

( A )…

Figure 3. Increased wild-type Kras expands HSCs and exhausts long-term repopulating HSCs. (A) Representative flow cytometric analysis of donor CD45.2+ hematopoietic cell engraftment in the PB of recipient CD45.1+ mice at 20 weeks following transplantation of 2 × 105 BM cells from ex3op mice or nat mice, along with 2 × 105 competitor CD45.1+ BM cells. Numbers represent percentages in each gate. (B) Panels show the mean percentages (± SD) of total donor CD45.2+ cells and donor CD45.2+ cells within the CD11b+ myeloid population, B220+ B cells, and CD3+ T cells in the PB of CD45.1+ recipient mice over time following competitive transplantation of BM cells from ex3op mice (CD45.2+) or nat mice (CD45.2+) (n = 12/group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (C) Mean percentages (± SD) of total donor CD45.2+ cells and donor CD45.2+ cells within the BM CD11b+ myeloid population, B220+ B cells, CD3+ T cells, and KSL population in CD45.1+ recipient mice at 20 weeks following competitive transplantation of BM cells from ex3op mice or nat mice (n = 12/group). *P < 0.05, ***P < 0.001. (D) Panels show the percentages of donor (DsRed+) total cell engraftment, as well as donor cell engraftment within CD11b+ myeloid cells, B220+ B cells, CD3+ T cells, and KSL cells in the BM of secondary recipient (Ds-Red negative) mice at 16 weeks following competitive transplantation of 1 × 106 BM cells collected from primary recipient mice. Primary recipient (Ds-Red negative) mice were transplanted with 2 × 105 BM cells from Krasex3op/ex3op (Ds-Red positive) mice or Krasnat/nat (Ds-Red positive) mice, along with 2 × 105 competitor (DsRed-negative) BM cells (n = 7/group). *P < 0.05, **P < 0.01, ****P < 0.0001. Data are presented as mean ± SD. Two-tailed Student’s t test was used for all analyses.
Figure 4

Figure 4. Increased wild-type Kras promotes hematopoietic…

Figure 4. Increased wild-type Kras promotes hematopoietic regeneration following myelosuppression.

( A ) Mean PB…

Figure 4. Increased wild-type Kras promotes hematopoietic regeneration following myelosuppression. (A) Mean PB WBCs, neutrophils, lymphocytes, and platelet (Plt) counts are shown in ex3op mice and nat mice at day +14 following 750 cGy TBI (n = 10/group). *P < 0.05, **P < 0.01, ***P < 0.001. ns, non-significant. (B) Mean BM cell counts, percentage of KSL cells, and numbers of KSL cells at day +14 following 750 cGy (n = 10/group). (C) Mean numbers of BM CFCs at day +14 (n = 10/group). ***P < 0.001. (D) The percentage survival of ex3op mice and nat mice is shown through day +30 following 750 cGy TBI (n = 12/group). *P = 0.03 by log-rank test for survival analysis. For all other analyses, 2-tailed Student’s t tests were performed. Data are presented as mean ± SEM.
Figure 5

Figure 5. Wild-type Kras augments hematopoietic repopulating…

Figure 5. Wild-type Kras augments hematopoietic repopulating capacity in a Cdk4/6–dependent manner.

( A )…

Figure 5. Wild-type Kras augments hematopoietic repopulating capacity in a Cdk4/6–dependent manner. (A) Increased percentage of BrdU-positive cells are shown within BM KSL cells, myeloid cells, B cells, and T cells in ex3op mice compared with nat mice at 24 hours after BrdU administration (n = 5/group). **P < 0.01. (B) At left, representative analysis of BrdU incorporation in BM CD150+CD48–KSL HSCs in nat mice and ex3op mice at day +25 following continuous BrdU administration in drinking water. At right, mean percentages of BrdU-positive HSCs are shown in each group (n = 6/group). ***P < 0.001. (C) At left, representative phospho-Erk1/2 (p-Erk1/2) expression in BM KSL cells from ex3op mice and nat mice in response to 20 ng/ml thrombopoietin (TPO) treatment for 5 minutes. At right, bar graphs show p-Erk1/2–positive KSL cells in each group (n = 5/group). *P < 0.05. (D) Relative mRNA levels of cell cycle regulatory genes in BM KSL cells from ex3op mice and nat mice at baseline (n = 8/group). **P < 0.01, ***P < 0.001. (E) Mean numbers of CFCs following 72-hour culture of BM KSL cells from ex3op mice or nat mice with media containing TPO, stem cell factor, and Flt-3 ligand (TSF), with or without the Erk1/2 inhibitor, BVD-523, or the Cdk4/6 inhibitor, palbociclib (palbo) (n = 6/group). *P < 0.05, ****P < 0.0001. (F) Mean percentages of phospho-Rb (p-Rb) in BM KSL cells in ex3op mice and nat mice at baseline (n = 4/group). *P < 0.05. (G) Adult C57BL/6 mice were irradiated with 850 cGy TBI, and then transplanted with 1 × 105 BM cells from ex3op mice or nat mice and treated on days +5 and +6 with either 85 mg/kg palbo or saline. Percentage survival of each group of mice is shown (n = 17/group). Log-rank test was used for survival analysis. *P < 0.05, ****P < 0.0001. (H) At left, representative microscopic images of femurs stained with H&E (×10 magnification) at day +14 from irradiated mice transplanted with ex3op BM cells, nat BM cells, or ex3op BM cells + palbociclib treatment. At right, mean BM cell counts (± SEM) at day +14 are shown for each group (n = 9/group). **P < 0.01. (I) Scatter plot shows numbers of BM KSL cells at day +14 in irradiated recipient mice transplanted with nat BM cells, ex3op BM cells, and ex3op BM cells + palbo treatment (n = 9). *P < 0.05. Except as noted, P values were obtained using a 2-tailed Student’s t test. Data are presented as mean ± SEM.
Figure 6

Figure 6. Kras regulates hematopoiesis in a…

Figure 6. Kras regulates hematopoiesis in a hematopoietic cell–autonomous manner.

( A ) Scatter plots…

Figure 6. Kras regulates hematopoiesis in a hematopoietic cell–autonomous manner. (A) Scatter plots show no differences in PB WBCs, lymphocytes, hemoglobin (Hgb), or platelets (Plt) between 8-week-old nat;ex3op mice and nat;nat mice. Increased PB neutrophils are detected in nat;ex3op mice (n = 8/group). **P < 0.01. (B) Mean numbers of PB CD19+ B cells and (C) BM cell counts in each group (n = 8/group). (D) Percentages of BM KSL and HSCs from each group (n = 8/group). Data are presented as mean ± SEM. Two-tailed Student’s t test was used for all analyses. ns, not significant.
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References

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