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Wild-type Kras expands and exhausts hematopoietic stem cells
Joshua P. Sasine, … , Christopher M. Counter, John P. Chute
Joshua P. Sasine, … , Christopher M. Counter, John P. Chute
Published June 7, 2018
Citation Information: JCI Insight. 2018;3(11):e98197. https://doi.org/10.1172/jci.insight.98197.
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Research Article Hematology Stem cells

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.

Authors

Joshua P. Sasine, Heather A. Himburg, Christina M. Termini, Martina Roos, Evelyn Tran, Liman Zhao, Jenny Kan, Michelle Li, Yurun Zhang, Stéphanie C. de Barros, Dinesh S. Rao, Christopher M. Counter, John P. Chute

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Figure 5

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

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Wild-type Kras augments hematopoietic repopulating capacity in a Cdk4/6–...
(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.

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