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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 3

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

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Increased wild-type Kras expands HSCs and exhausts long-term repopulatin...
(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.

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