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IDH2/R140Q mutation confers cytokine-independent proliferation of TF-1 cells by activating constitutive STAT3/5 phosphorylation - PubMed

️Mon Jan 01 2024

IDH2/R140Q mutation confers cytokine-independent proliferation of TF-1 cells by activating constitutive STAT3/5 phosphorylation

Jie Yang et al. Cell Commun Signal. 2024.

Abstract

Background: R140Q mutation in isocitrate dehydrogenase 2 (IDH2) promotes leukemogenesis. Targeting IDH2/R140Q yields encouraging therapeutic effects in the clinical setting. However, therapeutic resistance occurs in 12% of IDH2/R140Q inhibitor treated patients. The IDH2/R140Q mutant converted TF-1 cells to proliferate in a cytokine-independent manner. This study investigated the signaling pathways involved in TF-1(R140Q) cell proliferation conversion as alternative therapeutic strategies to improve outcomes in patients with acute myeloid leukemia (AML) harboring IDH2/R140Q.

Methods: The effects of IDH2/R140Q mutation on TF-1 cell survival induced by GM-CSF withdrawal were evaluated using flow cytometry assay. The expression levels of apoptosis-related proteins, total or phosphorylated STAT3/5, ERK, and AKT in wild-type TF-1(WT) or TF-1(R140Q) cells under different conditions were evaluated using western blot analysis. Cell viability was tested using MTT assay. The mRNA expression levels of GM-CSF, IL-3, IL-6, G-CSF, leukemia inhibitory factor (LIF), oncostatin M (OSM), and IL-11 in TF-1(WT) and TF-1(R140Q) cells were quantified via RT-PCR. The secretion levels of GM-CSF, OSM, and LIF were determined using ELISA.

Results: Our results showed that STAT3 and STAT5 exhibited aberrant constitutive phosphorylation in TF-1(R140Q) cells compared with TF-1(WT) cells. Inhibition of STAT3/5 phosphorylation suppressed the cytokine-independent proliferation of TF-1(R140Q) cells. Moreover, the autocrine GM-CSF, LIF and OSM levels increased, which is consistent with constitutive STAT5/3 activation in TF-1(R140Q) cells, as compared with TF-1(WT) cells.

Conclusions: The autocrine cytokines, including GM-CSF, LIF, and OSM, contribute to constitutive STAT3/5 activation in TF-1(R140Q) cells, thereby modulating IDH2/R140Q-mediated malignant proliferation in TF-1 cells. Targeting STAT3/5 phosphorylation may be a novel strategy for the treatment of AML in patients harboring the IDH2/R140Q mutation. Video Abstract.

Keywords: Acute myeloid leukemia; Cytokine-independent proliferation; Isocitrate dehydrogenase 2; R140Q mutation; Signal transducer and activator of transcription 3/5.

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Conflict of interest statement

The authors declare no competing interests.

Figures

**Fig. 1**
TF-1(R140Q) cells were resistant to apoptosis induced by GM-CSF withdrawal. After 24 h incubation in the absence or presence of 5 ng/mL GM-CSF, TF-1(WT) and TF-1(R140Q) cells reached different densities (A) and were subjected to flow cytometry assay for apoptosis (B). C TF-1(WT) and TF-1(R140Q) cells were cultured in the absence or presence of 5 ng/mL GM-CSF for 24 h, and PARP and Bcl-xL expression levels were analyzed. GAPDH was used as a loading control (left). Normalized mean gray values of cleaved-PARP (medium) and Bcl-xL (right) in TF-1(R140Q) cells were compared with TF-1(WT) cells in the absence of 5 ng/mL GM-CSF. ***P < 0.001 compared with TF-1(WT) cells. The dose of “0” group represents the vehicle control

**Fig. 2**
Aberrant constitutive activation of STAT3 and STAT5 with induced phospho-STAT3(Tyr⁷⁰⁵ ) and phospho-STAT5(Tyr⁶⁹⁴ ) levels in TF-1(R140Q) cells. A TF-1(WT) and TF-1(R140Q) cells were cultured in the absence or presence of 5 ng/mL GM-CSF for 24 h and subjected to western blotting for detection of the indicated proteins. B, C Ratios of the mean gray values of the indicated phosphorylated proteins to the mean gray values of the indicated total proteins and rations of values in TF-1(WT) and TF-1(R140Q) cells in the absence or presence of GM-CSF were analyzed. Representative results of three independent experiments are shown. Error bars are standard deviations. *P < 0.05, ***P < 0.001 compared with TF-1(WT) in the absence of GM-CSF; ^###P < 0.001 compared with TF-1(WT) in the presence of GM-CSF; ns indicates no significance. The dose of “0” group represents the vehicle control

**Fig. 3**
Effects of AGI-6780 on STAT3 and STAT5 activation in TF-1(R140Q) cells. A TF-1(R140Q) cells were treated with AGI-6780 for 2 days and subjected to western blotting for detection of the indicated proteins. GAPDH was used as a loading control. Ratios of the mean gray value of phospho-STAT3 (Tyr⁷⁰⁵) to the mean gray value of STAT3 (B), ratio of the mean gray value of pSTAT5 (Tyr⁶⁹⁴) to the mean gray value of STAT5 (C) and ratios in the treated group to the 0 µM of AGI-6780 group were analyzed. ***P < 0.001 compared with 0 µM of AGI-6780. D TF-1(R140Q) cells were treated with AGI-6780 for 7 days in the absence of GM-CSF, and cell viability was assayed by the MTT method. The inhibition rate was evaluated. The results are repetitive of three independent experiments with similar results, and error bars indicates standard deviations. ***P < 0.001 compared with 0 µM of AGI-6780 for 7 days. The dose of “0” group represents the vehicle control

**Fig. 4**
STAT3 and STAT5 inhibitors suppressed cytokine-independent TF-1(R140Q) cell proliferation. A TF-1(R140Q) cells were treated with C188-9 or NSC74859 for 24 h and subjected to western blotting for detection of the indicated proteins. β-Actin was used as a loading control. B The ratios of the mean gray value of phospho-STAT3(Tyr⁷⁰⁵) to the mean gray value of STAT3, (C) the ratios of the mean gray value of phospho-STAT5(Tyr⁶⁹⁴) to the mean gray value of STAT5 were calculated and the ratios of the treated groups to the control group were analyzed. The results represent three independent experiments with similar results, and the error bars indicate standard deviations. ***P < 0.001 compared with the control (0 µM C188-9). ^###P < 0.001 compared with the control (0 µM NSC74859). TF-1(R140Q) cells were treated with C188-9 (D), NSC74859 (E) or STAT5-IN-1 (F) for 3 days in the absence of GM-CSF, and cell viability was evaluated using MTT assays. The inhibition rate of three independent experiments with similar results, and error bars mark indicate standard deviations. **P < 0.01, ***P < 0.001 compared with the vehicle control. G TF-1(R140Q) cells were induced with 50 ng/mL EPO to differentiate for 7 days in the presence of AGI-6780 (0.2, 1µM), C188-9 (2.5, 5, 7.5 µM) or NSC74859 (50, 100, 200 µM), respectively. Afterwards, the cells were collected, and color change was photographed. The dose of “0” and/or “-” group represents the vehicle control

**Fig. 5**
Autocrine GM-CSF, OSM, and LIF in TF-1(R140Q) cells determined the cytokine-independent transformation of TF-1(R140Q) cells. A mRNA expression levels of GM-CSF, IL-3, IL-6, G-CSF, LIF, OSM, and IL-11 in TF-1(WT) and TF-1(R140Q) cells in the absence of GM-CSF for 24 h. ***P < 0.001, compared with TF-1(WT) cells. B Secretion of GM-CSF, OSM, and LIF from TF-1(WT) and TF-1(R140Q) cells in the absence of GM-CSF for 24 h. ***P < 0.001, compared with TF-1(WT) cells. C Effects of AGI-6780 treatment on the mRNA expression of GM-CSF, OSM, and, LIF in TF-1(R140Q) cells in the absence of GM-CSF for 24 h. ***P < 0.001 compared with the control (0 µM AGI-6780). D Effects of AGI-6780 on GM-CSF, OSM, and LIF protein secretion in TF-1(R140Q) cells in the absence of GM-CSF for 24 h. ***P < 0.001 compared with the control (0 µM of AGI-6780). The dose of “0” group represents the vehicle control

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IDH2/R140Q mutation confers cytokine-independent proliferation of TF-1 cells by activating constitutive STAT3/5 phosphorylation - PubMed