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. 2011 Mar 30;3(76):76ra27.
doi: 10.1126/scitranslmed.3001069.

A MEK inhibitor abrogates myeloproliferative disease in Kras mutant mice

Natalya Lyubynska  1 , Matthew F Gorman, Jennifer O Lauchle, Wan Xing Hong, Jon K Akutagawa, Kevin Shannon, Benjamin S Braun
Affiliations

A MEK inhibitor abrogates myeloproliferative disease in Kras mutant mice

Natalya Lyubynska et al. Sci Transl Med. .

Abstract

Chronic and juvenile myelomonocytic leukemias (CMML and JMML) are aggressive myeloproliferative neoplasms that are incurable with conventional chemotherapy. Mutations that deregulate Ras signaling play a central pathogenic role in both disorders, and Mx1-Cre, Kras(LSL-G12D) mice that express the Kras oncogene develop a fatal disease that closely mimics these two leukemias in humans. Activated Ras controls multiple downstream effectors, but the specific pathways that mediate the leukemogenic effects of hyperactive Ras are unknown. We used PD0325901, a highly selective pharmacological inhibitor of mitogen-activated or extracellular signal-regulated protein kinase kinase (MEK), a downstream component of the Ras signaling network, to address how deregulated Raf/MEK/ERK (extracellular signal-regulated kinase) signaling drives neoplasia in Mx1-Cre, Kras(LSL-G12D) mice. PD0325901 treatment induced a rapid and sustained reduction in leukocyte counts, enhanced erythropoiesis, prolonged mouse survival, and corrected the aberrant proliferation and differentiation of bone marrow progenitor cells. These responses were due to direct effects of PD0325901 on Kras mutant cells rather than to stimulation of normal hematopoietic cell proliferation. Consistent with the in vivo response, inhibition of MEK reversed the cytokine hypersensitivity characteristic of Kras(G12D) hematopoietic progenitor cells in vitro. Our data demonstrate that deregulated Raf/MEK/ERK signaling is integral to the growth of Kras-mediated myeloproliferative neoplasms and further suggest that MEK inhibition could be a useful way to ameliorate functional hematologic abnormalities in patients with CMML and JMML.

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Conflict of interest statement

Competing interests: None of the authors have competing interests. J.O.L. was not affiliated with Genentech while contributing to this work.

Figures

Fig. 1
Fig. 1. PD0325901 inhibits MEK in vivo
(A) Flow cytometry was used to measure ERK and STAT5 phosphorylation in primary myeloid progenitor cells from the bone marrow of Mx1-Cre, KrasG12D and wild-type mice in the presence or absence of 10 ng/mL GM-CSF. After gating for surface-marker expression, phosphoprotein staining was analyzed as a histogram (open) in comparison with an unstimulated control samples (gray). (B) Mx1-Cre, KrasG12D mice were treated with a single dose of the MEK inhibitor PD0325901 (PD) or vehicle, and bone marrow and spleens were harvested 12 h, 18 h, and 24 h later. Bone marrow and spleen cells were stimulated with GM-CSF and analyzed for phosphorylated ERK (pERK). (C) Bone marrow and spleen cells were analyzed for phosphorylation of STAT5 (pSTAT5), 12 h after treatment with PD0325901. The phosphorylation of STAT5 in cells that do not phosphorylate ERK indicates that some signals induced by GM-CSF are intact after exposure to PD0325901, demonstrating specific inhibition of the Raf/MEK/ERK pathway.
Fig. 2
Fig. 2. PD0325901 controls myeloproliferative neoplasia in Mx1-Cre, KrasG12D mice
(A) Total leukocyte (WBC) count, (B) hemoglobin (Hb) concentration, and (C) reticulocyte frequency in peripheral blood of Mx1-Cre, KrasG12D (red) or wild-type control (blue) mice treated with PD0325901 (PD; solid line) or vehicle (dashed line). Error bars show s.e.m. (n=5–7 per group). Significance by Student t test comparing PD0325901 treated vs. vehicle treated Mx1-Cre, KrasG12D mice at the 4 week time point: *p<0.05; **p< 0.01; ***p< 0.001. (D) Mouse spleen weight after 5 weeks of therapy. (E) Kaplan-Meier survival estimates indicating a survival benefit to PD0325901 treatment in Mx1-Cre, KrasG12D mice with significance of p<0.0001 by the log rank test (n=6 and 7, respectively, for wild-type mice treated with PD0325901 and vehicle; n=8 and 9 for Mx1-Cre, KrasG12D mice treated with PD0325901 and vehicle). (F) Genotypes of individual granulocyte/macrophage colonies (lanes 1–5) derived from the bone marrow of a representative Mx1-Cre, KrasG12D mouse treated daily with PD0325901 for five weeks. Each colony was derived from a single myeloid progenitor, indicating a predominance of cells with a heterozygous KrasG12D/+ genotype in the progenitor population. Lanes (+) and (−) show controls for KrasG12D/+ and wild-type genotypes, respectively. A total of 44 colonies grown from three independent KrasG12D mice that received PD0325901 were analyzed, and all demonstrated Cre-mediated activation of the KrasG12D allele.
Fig. 3
Fig. 3. PD0325901 improves myeloid and erythroid differentiation in vivo
Frequencies of precursor populations were determined by flow cytometry(A) Typical distributions of myeloid progenitors in bone marrow of wild-type and Mx1-Cre, KrasG12D mice treated with vehicle or PD0325901 (PD) for five weeks. Cells gated for lineage negative/low (Lin−/lo), c-kit+, Sca1− are shown. GMP: granulocyte/macrophage progenitors, MEP: megakaryocyte/erythroid progenitors, CMP: common myeloid progenitors that populate all four myeloid lineages (B) Ratios of GMP to CMP in bone marrow of wild-type control (white bars) or Mx1-Cre, KrasG12D (black bars) mice treated with vehicle (solid bar) or PD0325901 (striped bar) for 3 days or 5 weeks, as indicated. All error bars show s.e.m. (n=3–9 per group). In Mx1-Cre, KrasG12D mice, differences in treatment with vehicle vs. PD are significant according to Welch’s corrected t test at 3 days (*p<0.05) and 5 weeks (**p<0.01). (C) Peripheral blood concentrations of leukocytes (WBC) and hemoglobin (Hb) in Mx1-Cre, KrasG12D mice after 3 days of PD0325901 treatment. Differences are not statistically significant (n=4–6 per group). (D) Bone marrow and (E) spleen populations of monocytes (M; Mac1+ Ly6C+), immature granulocytes (G1; Mac1+ Ly6Cmed Gr1med), intermediate granulocytes (G2; Mac1med Ly6Cmed Gr1high), and mature granulocytes (G3; Mac1high Gr1high). Differences from the vehicle-treated wild-type cohort are indicated (*p<0.05, **p< 0.01, ***p< 0.001), as determined by Bonferroni testing after 2-way ANOVA (n=3–4 mice per group). (F) Bone marrow and (G) spleen distributions of erythroid populations, with erythroid precursors at sequential stages of maturation labeled as E1 (c-kit+ CD105+), E2 (CD71high TER119+), or E3 (CD71med TER119+). Statistical significance is indicated as in Fig. 3D.
Fig. 4
Fig. 4. PD0325901 improves function of Mx1-Cre, KrasG12D myeloid progenitors in vitro
(A) Bone marrow was harvested from wild-type and Mx1-Cre, KrasG12D mice after 5 weeks of treatment with PD0325901 (PD) or vehicle and plated in methylcellulose with a range of GM-CSF concentrations. CFU-GM from Mx1-Cre, KrasG12D mice treated with PD0325901 remain hypersensitive to GM-CSF. Error bars for all graphs show s.e.m., which is smaller than the plotting symbol for some data points. (B) Bone marrow cells from pIpC-treated wild-type or Mx1-Cre, KrasG12D mice were plated in methylcellulose medium containing a high concentration of GM-CSF (10 ng/mL) and varying doses of PD0325901. The DMSO concentration was constant. Colonies were enumerated after 8 days, and counts were normalized to the number arising without PD0325901. CFU-GM from wild-type and Mx1-Cre, KrasG12D mice have similar sensitivities to PD0325901 at saturating concentrations of GM-CSF. (C) A low concentration of PD0325901 (0.1 μM) causes a modest reduction in the number of colonies derived in the presence of 10 ng/mL GM-CSF. A DMSO control is shown for comparison. To account for variation, the number of colonies for each sample was normalized by dividing colony number by the average number of colonies arising from a control sample in (wild-type cells in DMSO) in the same experiment. **p<0.01 vs. wild-type DMSO control by Bonferroni testing after 2-way ANOVA. (D) Bone marrow cells from pIpC-treated wild-type or Mx1-Cre, KrasG12D mice were plated with a range of GM-CSF concentrations and a fixed concentration of PD0325901 (0.1 μM) or DMSO control. (E) CFU-GM from pIpC-treated wild-type or Mx1-Cre, KrasG12D mice were harvested from methylcellulose culture after 7 days with 10 ng/mL GM-CSF and either 0.1 μM PD0325901 or DMSO. Average colony size was calculated as total cell number per plate divided by colony number (n = 3–5). Statistical significance is denoted as in Fig. 4C. (F) Harvested cells were stained for myeloid differentiation antigens F4/80 and Gr1. All colony assays were performed in triplicate from independent mice.
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