Supplementary MaterialsSupplemental data jciinsight-3-98197-s101. lymphocytes, but no evidence of myeloproliferative disease or leukemia in mice followed for 12 months. BM HSCs from mice exhibited increased multilineage repopulating capacity in main competitive transplantation assays, but secondary competitive transplants revealed exhaustion of long-term HSCs. Following total body irradiation, mice displayed accelerated hematologic recovery and increased survival. Mechanistically, HSCs from mice exhibited 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 mice were dependent on Cdk4/6 activation. Finally, BM transplantation studies revealed that augmented Kras expression produced growth of HSCs, progenitor cells, and B cells in a hematopoietic cellCautonomous manner, independent from effects around the BM microenvironment. This study provides fundamental demonstration of codon usage in a mammal using a biological result, which may speak to the importance of codon usage in mammalian biology. preferentially encoded S49076 by A or T at wobble base pairs, whereas is usually encoded by G or C, and NRAS by a mixture of all 4 nucleotides (1, 6). Codons ending in A or T are rare in mammalian exomes and rare codons have been shown to impede the efficiency of translation elongation (1, 7). Consistent with this, the rare Akt1 codons in have been shown to impede translation of the encoded mRNA, reducing protein expression (1, 6). Current understanding of the role of RAS signaling in the hematopoietic system has been driven primarily by studies in which oncogenic mutant transgenes were overexpressed in bone marrow (BM) hematopoietic stem cells (HSCs) and progenitor cells S49076 (8C18). Diverse hematopoietic effects have been observed, depending on the mutant transgene overexpressed and the mouse model itself. MacKenzie et al. (8) showed that 60% of recipient S49076 mice injected with BM cells transduced with a retrovirus encoding oncogenic developed a variety of myeloid malignancies after prolonged latency. Mx1-Cre-LoxPCdriven induction of endogenous but oncogenic in hematopoietic cells caused the development of indolent myeloproliferative disease in mice and potentiated the development of additional hematologic cancers (12). Enforced expression of a single allele of oncogenic using the Mx1-Cre model also increased HSC proliferation and serial repopulating capacity, providing possible explanation for the clonal advantage conferred by oncogenic expression (13). Furthermore, the dosage of Ras protein has been shown to correlate with the transformative effects of oncogenes in the murine hematopoietic system (12, 14). Transplantation of main murine hematopoietic cells transduced with a vector encoding oncogenic produced lymphomas and lymphoid leukemias in mice (16). In contrast, S49076 inducible expression of an gene in BM hematopoietic cells caused a rapidly fatal myeloproliferative disease in mice (9). Subsequent studies showed that activating this mutant allele in BM ckit+linC progenitor cells caused aberrant signaling downstream, as well as increased HSC competitive repopulating fitness and the ability to initiate T-lineage leukemias following transplantation (11). Recently, expression of oncogenic in Flt-3+ multipotent progenitor cells caused a neonatal myeloid leukemia in mice with features that recapitulated human juvenile myelomonocytic leukemia (18). Importantly, pharmacologic inhibition of the downstream effectors of Ras, specifically MEK and PI3K, has been shown to abrogate oncogenic KrasCdriven myeloproliferative disease in mice, suggesting that interruption of RAS-driven signaling could ameliorate disease progression in patients with hematologic malignancies and RAS mutations (15, 19). The above findings suggest an important role for Kras in normal hematopoiesis. However, this has not been directly tested since oncogenic Ras proteins, often overexpressed, have been used to chronically drive high levels of Ras signaling. It is known that Kras is required for adult hematopoiesis (20), but these studies abolished the gene, and hence, the extent to which Kras signaling underlies normal hematopoiesis remains unclear (20). Interestingly, retrovirus-mediated overexpression of oncogenic HRAS in human cord blood linC cells induced a high level of HRAS signaling, decreased proliferation, and enhanced monocyte differentiation (17). Fine-tuning the activation levels in these cells with a farnesyltransferase inhibitor produced a smaller increase in HRAS signaling and promoted a hematopoietic blast-like cell phenotype and self-renewal (17). Consistent with this observation, overexpression of a single allele of oncogenic increased HSC repopulating capacity in mice (13). Furthermore, the BM nicheCderived paracrine factor, pleiotrophin, promotes HSC regeneration in irradiated mice via upregulation of Ras/MEK/ERK signaling (21, 22). These results suggest that moderate amplification in Ras signaling could promote the growth of HSCs and progenitor cells. In order to directly test this hypothesis, we utilized a version of the gene that generates higher levels of the wild-type Kras protein S49076 without altering.