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. 2014 Oct 2;124(14):2252-61.
doi: 10.1182/blood-2013-02-484196. Epub 2014 Aug 22.

STAT3 supports experimental K-RasG12D-induced murine myeloproliferative neoplasms dependent on serine phosphorylation

Daniel J Gough  1 , Isabelle J Marié  1 , Camille Lobry  1 , Iannis Aifantis  1 , David E Levy  1
Affiliations

STAT3 supports experimental K-RasG12D-induced murine myeloproliferative neoplasms dependent on serine phosphorylation

Daniel J Gough et al. Blood. .

Abstract

Juvenile myelomonocytic leukemia, acute myeloid leukemia (AML), and other myeloproliferative neoplasms (MPNs) are genetically heterogeneous but frequently display activating mutations in Ras GTPases and activation of signal transducer and activator of transcription 3 (STAT3). Altered STAT3 activity is observed in up to 50% of AML correlating with poor prognosis. Activated STAT proteins, classically associated with tyrosine phosphorylation, support tumor development as transcription factors, but alternative STAT functions independent of tyrosine phosphorylation have been documented, including roles for serine-phosphorylated STAT3 in mitochondria supporting transformation by oncogenic Ras. We examined requirements for STAT3 in experimental murine K-Ras-dependent hematopoietic neoplasia. We show that STAT3 is phosphorylated on S727 but not Y705 in diseased animals. Moreover, a mouse with a point mutation abrogating STAT3 S727 phosphorylation displayed delayed onset and decreased disease severity with significantly extended survival. Activated K-Ras required STAT3 for cytokine-independent growth of myeloid progenitors in vitro, and mitochondrially restricted STAT3 and STAT3-Y705F, both transcriptionally inert mutants, supported factor-independent growth. STAT3 was dispensable for growth of normal or K-Ras-mutant myeloid progenitors in response to cytokines. However, abrogation of STAT3-S727 phosphorylation impaired factor-independent malignant growth. These data document that serine-phosphorylated mitochondrial STAT3 supports neoplastic hematopoietic cell growth induced by K-Ras.

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Figures

Figure 1
Figure 1
STAT3 is phosphorylated on S727, not Y705, in K-RasG12D-driven MPNs in vivo. STAT3-Flox mice of the following genotypes, Mx:STAT3F/+ (M:F/+), Mx:K-RasG12DLSL:F/+ (M:K:F/+), or K-RasG12DLSL:STAT3F/+ (K:F/+), were injected with a single 100-µg dose of poly(I:C). Mice were allowed to recover for 2 weeks before peripheral blood was collected, red blood cells lysed, and total leukocytes assessed for STAT3 phosphorylation on S727 (pS STAT3) or Y705 (pY STAT3) by immunoblotting (A). Antibody against total STAT3 served as a loading control. As a positive control for STAT3 phosphorylation, wild-type mice were injected with 30 µg of LPS 4 hours prior to harvesting peripheral blood leukocytes (LPS). (B) After 2 weeks of recovery following poly(I:C) injection, bone marrow was harvested from Mx:F/+ (Control) or Mx:K-Ras:F/+ mice (M:K) and expression of (i) pS727 STAT3, (ii) pY705 STAT3, (iv) pSTAT5, and (v) pERK was determined in lineage-negative, c-Kit+ cell populations by flow cytometery using phospho-specific antibodies. As a positive control for STAT3 Y705 phosphorylation (iii), mice were injected with 30 µg of LPS 4 hours prior to harvesting bone marrow. Pooled data from 3 animals for each genotype are plotted in the histogram showing the MFI ± standard deviation. Statistical significance was calculated using the Student t test. *P < .05, ***P < .001. MFI, mean fluorescence intensity.
Figure 2
Figure 2
Blocking STAT3 S727 phosphorylation ameliorates K-RasG12D–driven MPNs. Control animals (M:F/+) lacking K-RasG12D, Mx:STAT3F/SA (M:F/SA), Mx:K-RasG12DLSL:STAT3F/+ (M:K:F/+), or MxK-RasG12DLSL:STAT3F/SA (M:K:F/SA) mice were injected with poly(I:C) and peripheral white blood cells enumerated every 2 weeks (A). Each point represents the mean of cohorts of 5 animals, with error bars representing 1 standard deviation from the mean. *P < .05 and ****P < .0001 for comparisons between the M:K:F/+ and M:K:F/SA cohorts (calculated by 2-way ANOVA with the Tukey multiple comparison test). On the day animals were sacrificed due to morbidity (week 14 for Mx:K-Ras:F/+) or at the end of the experiment in the case of all other cohorts (week 20), peripheral white blood cells (B) or granulocytes (C) were counted and plotted. Each point represents cell numbers from an independent animal. **P < .01 and ***P < .001 for comparisons between the M:K:F/+ and M:K:F/SA cohorts (1-way ANOVA with the Tukey multiple comparison test). (D) Animals were sacrificed and tissues collected for histological analysis (20 weeks post poly(I:C) injection for Mx:F/+, Mx:F/SA, Mx:K-Ras:F/SA; 14 weeks for Mx:K-Ras:F/+). The peripheral blood (subpanels a-d) and bone marrow smears (subpanels e-h) were prepared with Wright-Giemsa stain and imaged with a 60× objective (inset: 2× digital enlargement). Spleen (subpanels h-o) and liver sections (subpanels p-w) were stained with hematoxylin and eosin and imaged with a 10× or 40× objective, as indicated. ANOVA, analysis of variance.
Figure 3
Figure 3
Blocking STAT3 S727 phosphorylation significantly increases the life of K-RasG12D animals. Kaplan-Meier survival curves of animals of the indicated genotypes. Control animals (Mx:F/+) and Mx:STAT3F/SA (Mx:F/SA) animals survived until the experiment was terminated at over 300 days. Mx:K-Ras:F/+ (Mx:K:F/+) mice died rapidly from MPNs with a median survival of 83 days. Mx:K-Ras:F/SA (Mx:K:F/SA) animals survived significantly longer (***P < .001 log-rank test) with a median survival of 115 days. Cohorts included at least 10 animals, except Mx:F/SA (5 animals).
Figure 4
Figure 4
Activated K-Ras-induced increase in splenic hematopoietic progenitor cells requires STAT3 S727. Animals reconstituted with bone marrow of the indicated genotypes were injected with poly(I:C) and allowed to develop early (cohort 1, 1.5-fold increase in peripheral white blood cell counts) and later signs of MPNs (cohort 2, more than twofold increase). Bone marrow (A,C) and splenic leukocytes (B,D) were harvested and the proportions and total numbers of MPs, LSK, CMPs (Lin−, cKit+, FCγR−, CD34+), GMPs (Lin−, cKit+, FCγR+, CD34+), and MEPs (Lin−, cKit+, FCγR−, CD34lo) were assessed by flow cytometry. Representative flow cytometry dot plots are shown in panels A and B. Proportion and total numbers of cells per organ are shown for the early (1) and later cohorts (2) in panels C and D. Number of animals per cohort were: 2 Mx:F/+ (cohort 1); 2 Mx:F/+ (cohort 2); 6 Mx:K-Ras:F/+ (cohort 1), 4 Mx:K-Ras:F/+ (cohort 2); 5 Mx:K-Ras:F/SA (cohort 1); 4 Mx:K-Ras:F/SA (cohort 2). Statistical significance was calculated using the Student t test. *P < .05.
Figure 5
Figure 5
Nontranscriptional, mitochondrial STAT3 supports factor-independent colony formation. Control, Mx:K-Ras:F/+, Mx:K-Ras:F/F, or Mx:K-Ras:F/SA mice were injected with poly(I:C) and allowed to recover for 1 week. Bone marrow was harvested and lineage depleted. Cells (103) were plated in methylcellulose supplemented with 50 ng/mL SCF, 10 ng/mL IL-6, 10 ng/mL IL-3, 3 units/mL EPO, and 20 ng/mL GM-CSF (A) or without growth factors (B). Colonies were counted and identified after 10 days in culture. (C-D) Mx:K-Ras:F/F mice were injected with poly(I:C) and allowed to recover for 1 week. Bone marrow was harvested and transduced with pMIG retroviruses expressing WT, YF, MTS, or EV. Lineage-negative, GFP-positive cells were sorted and 103 cells per plate were cultured in methylcellulose supplemented with 50 ng/mL SCF, 10 ng/mL IL-6, 10 ng/mL IL-3, 3 units/mL EPO, and 20 ng/mL GM-CSF (C) or without growth factors (D). Colony numbers and cell types were enumerated after 10 days. Histograms represent the mean of triplicate experiments and error bars are 1 standard deviation from the mean of the total number of colonies. *P < .05 and **P < .001 of differences between groups calculated by the 1-way ANOVA and the Tukey multiple comparison test. EV, empty vector control; MTS, mitochondrially restricted STAT3 mutant; NS, not significant; WT, wild-type STAT3; YF, STAT3 Y705F mutant.
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