1. Endogenous oncogenic Nras mutation initiates hematopoietic malignancies in a dose- and cell type-dependent manner
by Jinyong Wang, Yangang Liu, Zeyang Li, Zhongde Wang, Li Xuan Tan, Myung-Jeom Ryu, Benjamin Meline, Juan Du, Ken H. Young, Erik Ranheim, Qiang Chang, and Jing Zhang
Blood
Volume 118(2):
July 14, 2011
©2011 by American Society of Hematology

2. Construction of a conditional hypomorphic Nras G12D allele.
Construction of a conditional hypomorphic Nras G12D allele. (A) Schematic diagram of WT Nras allele, targeting vector, and constructed LSL (LoxP-STOP cassette-LoxP) Nras G12Dhypo allele. The asterisk shows the substitution of amino acid aspartic acid for glycine by mutation of GGT to GAT at the codon 12. Please see “Mice” for the details of intron 1 mutations. The positions of the probes for Southern blotting are shown. (B) Southern blot analysis of BglII digested genomic DNA isolated from different ES cell clones to confirm correct targeting at the endogenous Nras locus. The WT allele is denoted by the 10.7 KB fragment. The correctly targeted LSL allele is indicated by the 7.4 KB fragment using the 5′ internal probe and by the 5.6 Kb fragment using the 3′ external probe. (C) Direct sequencing with a reverse primer of genomic DNA isolated from WT and germ line transmitted LSL Nras G12Dhypo mice demonstrates the presence of G12D mutation at the Nras locus. Arrows indicate the WT and mutated nucleotides at the codon 12. (D) Evaluation of recombination efficiency of Mox2-Cre and expression level of Nras G12D hypo allele in E14.5 fetal liver erythoid progenitors. Because Mox2-Cre recombines the conditional LSL cassette and leads to Nras G12Dhypo expression, we refer the compound mice harboring both LSL and Mox2-Cre alleles as Nras G12Dhypo/+; Mox2-Cre/+. We further crossed Nras G12Dhypo/+; Mox2-Cre/+ mice to LSL Nras G12Dhypo/+ (LSL/+) mice and generate Nras G12Dhypo/LSL progenies inherit a recombined Nras G12Dhypo allele and a nonrecombined LSL allele from parents but does not carry Mox2-Cre allele. To simplify the genotyping results, we omit the Mox2-Cre status. Southern blot analysis of SpeI digested genomic DNA using the 3′ external probe confirmed the recombination efficiency at the endogenous Nras locus (top panel). Expression levels of total Nras were measured by Western blotting and normalized against actin using the Molecular Analyst Version 1.4 software (Bio-Rad).
Jinyong Wang et al. Blood 2011;118:
©2011 by American Society of Hematology

3. Somatic activation of Nras G12D/G12D but not Nras G12D/+ leads to acute myeloproliferative disease.
Somatic activation of Nras G12D/G12D but not Nras G12D/+ leads to acute myeloproliferative disease. Five- to 6-week-old mice were injected with pI-pC as described in “Mice.” Two days after the second pI-pC injection, different tissues were isolated and analyzed. Nras G12D/+ and Nras G12D/G12D refer to pI-pC treated compound mice expressing monoallelic and biallelic oncogenic Nras, respectively, as described in “Somatic activation of Nras G12D/G12D but not Nras G12D/+ leads to an acute myeloproliferative disease.” (A) Genotyping analysis of genomic DNA to detect WT allele, LSL allele, and recombined LSL allele (1 LoxP allele). (B) Total RNA was extracted from bone marrow cells. Direct sequencing of RT-PCR amplified Nras gene using a reverse primer to confirm the sequences at the codon 12. Arrows indicate the WT and mutated nucleotides at the codon 12. (C) Flow cytometric analysis of peripheral blood (PB), spleen and bone marrow (BM) cells isolated from control (n = 5) and Nras G12D/G12D (n = 5) mice using myeloid lineage specific markers. Debris and unlysed red blood cells (low forward scatter) and dead cells (propidium iodide positive) were excluded from analysis. Data are presented as averages + SDs. (D) Splenomegaly in Nras G12/G12D mice. Results are presented as the average of spleen weights + SD. *P < .01. (E, F) 5 × 104 bone marrow cells isolated from control, Nras G12D/+, and Nras G12D/G12D mice were plated in duplicate in semisolid medium with or without GM-CSF (E) or IL-3 (F). The data are presented as average percentages (from multiple mice of each group) of maximum number of colonies formed in culture with 0.2 ng/mL of GM-CSF or 10 ng/mL of IL-3. Student t test was performed. Error bars show SD. (E) Crosses indicate P < .01. (F) *P < .05.
Jinyong Wang et al. Blood 2011;118:
©2011 by American Society of Hematology

4. Oncogenic Nras signaling engages myeloid progenitors into cell cycle and leads to their expansion in a dose-dependent manner.
Oncogenic Nras signaling engages myeloid progenitors into cell cycle and leads to their expansion in a dose-dependent manner. Different tissues were isolated and analyzed 2 days after pI-pC injections. (A) Quantitative analysis of myeloid progenitor (MP) compartment in bone marrow and spleen of control, Nras G12D/+, and Nras G12D/G12D mice. CMP indicates common myeloid progenitor; GMP, granulocyte-monocyte progenitor; and MEP, megakaryocyte-erythroid progenitor. Results are presented as averages + SDs. Student t test was performed: *P < .05, and **P < .01. (B) Cell cycle analysis of MPs in bone marrow of control, Nras G12D/+, and Nras G12D/G12D mice. Cell-cycle phases are defined as G0 (Ki67−, DAPIlo), G1 (Ki67+, DAPIlo), and S/G2/M (Ki67+, DAPIhi). The percentages of MPs in individual cell-cycle phases are indicated on the density plots. Average values + SDs are shown in the right graph. Student t test was performed: *P < .05. (C,D) Phospho-flow analysis of p-ERK1/2 in Lin−/low c-Kit+ bone marrow cells of control, Nras G12D/+, and Nras G12D/G12D mice 2 days after the second pI-pC injection. (C) Total bone marrow cells were freshly isolated and stimulated with or without 2ng/mL of GM-CSF at 37°C for 10 minutes. Basal condition is defined as without GM-CSF stimulation. U0126 was mixed with cells for 30 minutes before fixation or GM-CSF stimulation. Levels of p-ERK1/2 were measured using phospho-specific flow cytometry. Nonneutrophil Lin−/low c-Kit+ cells were gated for data analysis. Results obtained from one representative experiment are shown (left panel). Quantification of 6 independent experiments is shown as average values + SDs (middle panel). Solid lines indicate the median intensity of p-ERK1/2 in control cells without GM-CSF stimulation (right panels). (D) Total bone marrow cells were serum- and cytokine-starved for 1 hour and stimulated with various concentrations of GM-CSF (0, 0.16 and 2 ng/mL) at 37°C for 10 minutes. Gating strategy and plots of p-ERK1/2 are representative of 4 independent experiments. To quantify the activation of ERK1/2, median intensities of p-ERK1/2 at different GM-CSF concentrations in different animals are compared with control cells at 0 ng/mL, which is arbitrarily set at 1. Average values ± SDs are shown in the right graph. Student t test was performed: *P < .05.
Jinyong Wang et al. Blood 2011;118:
©2011 by American Society of Hematology

5. All the Nras G12D/G12D mice die with a severe myeloproliferative disease.
All the Nras G12D/G12D mice die with a severe myeloproliferative disease. After pI-pC injections, control and Nras G12D/G12D mice were kept for an extended period of time until Nras G12D/G12D mice reached a moribund stage. (A) Kaplan-Meier comparative survival analysis of control and Nras G12D/G12D mice. Cumulative survival was plotted against days after the first pI-pC injection. (B) Splenomegaly in Nras G12D/G12D mice. Results are presented as averages of spleen weights + SDs. (C) Representative histologic H&E sections from spleen show an extensive infiltration of myelomonocytic cells and extramedullary hematopoiesis in Nras G12D/G12D mice. (D) Complete blood count was performed on peripheral blood samples drawn from control and Nras G12D/G12D mice. *P < .05. (E-F) Quantitative analysis of myeloid progenitor (MP) compartment in bone marrow (E) or spleen (F) of control and moribund Nras G12D/G12D mice as described in Figure 3. Results are presented as averages + SDs. *P < .05.
Jinyong Wang et al. Blood 2011;118:
©2011 by American Society of Hematology

6. Oncogenic Nras initiates TALL in a dose-dependent manner.
Oncogenic Nras initiates TALL in a dose-dependent manner. Lethally irradiated mice (CD45.1+) were transplanted with 2.5 × 105 total bone marrow cells of control, Nras G12D/+, or Nras G12D/G12D mice along with same number of competitor cells. (A) Kaplan-Meier survival curves of reconstituted mice. Cumulative survival was plotted against days after transplantation. (B) Disease distribution patterns in recipient mice transplanted with Nras G12D/+ or Nras G12D/G12D cells. (C) Hepatosplenomegaly and enlarged thymi in TALL-het and -homo mice. (Top) Enlarged thymus in a representative recipient mouse that developed a TALL disease. (Bottom) Results are presented as averages of spleen, liver, or thymus weights + SDs. Student t test was performed: *P < (D) Representative histologic H&E sections of thymus from control, TALL-het, and TALL-homo mice. (E) Flow cytometric analysis of total thymocytes of representative control, TALL-het, and TALL-homo mice.
Jinyong Wang et al. Blood 2011;118:
©2011 by American Society of Hematology

7. Oncogenic Nras-initiated TALL tumors contain clonal Notch1 mutations.
Oncogenic Nras-initiated TALL tumors contain clonal Notch1 mutations. (A) Southern blot analysis of genomic DNA obtained from TALL-het and TALL-homo tumors. The blot was hybridized with a probe to the Vβ region of T-cell receptor β. (B) Sequence analysis of the exon 34 of Notch1 in control, TALL-het, and TALL-homo thymocytes.
Jinyong Wang et al. Blood 2011;118:
©2011 by American Society of Hematology

8. TALL-het tumors are generated through a distinct genetic mechanism from TALL-homo tumors.
TALL-het tumors are generated through a distinct genetic mechanism from TALL-homo tumors. (A) Flow cytomytric analysis of TALL-het and -homo tumor cells with T-cell markers expressed at different developmental stages. Two representative tumors of 5 for each tumor type are shown. (B) Western blot analysis of TdT expression levels in control cells, oncogenic Nras mutation-initiated TALL-het and -homo tumors (top panels), and oncogenic Kras mutation-initiated TALL-KrasG12D tumors (bottom panels). (C) Western blot analysis of total Nras expression levels in control, TALL-het, and -homo thymocytes. (D) Direct sequencing of RT-PCR amplified products in TALL-het and -homo tumors at the Nras G12 codon. Results are representative of 5 animals for each tumor type.
Jinyong Wang et al. Blood 2011;118:
©2011 by American Society of Hematology