New insights into the Shwachman-Diamond Syndrome-related haematological disorder: hyper-activation of mTOR and STAT3 in leukocytes

Abstract

Shwachman-Diamond syndrome (SDS) is an inherited disease caused by mutations of a gene encoding for SBDS protein. So far little is known about SBDS exact function. SDS patients present several hematological disorders, including neutropenia and myelodysplastic syndrome (MDS), with increased risk of leukemic evolution. So far, the molecular mechanisms that underlie neutropenia, MDS and AML in SDS patients have been poorly investigated. STAT3 is a key regulator of several cellular processes including survival, differentiation and malignant transformation. Moreover, STAT3 has been reported to regulate neutrophil granulogenesis and to induce several kinds of leukemia and lymphoma. STAT3 activation is known to be regulated by mTOR, which in turn plays an important role in cellular growth and tumorigenesis. Here we show for the first time, to the best of our knowledge, that both EBV-immortalized B cells and primary leukocytes obtained from SDS patients present a constitutive hyper-activation of mTOR and STAT3 pathways. Interestingly, loss of SBDS expression is associated with this process. Importantly, rapamycin, a well-known mTOR inhibitor, is able to reduce STAT3 phosphorylation to basal levels in our experimental model. A novel therapeutic hypothesis targeting mTOR/STAT3 should represent a significant step forward into the SDS clinical practice.

Figure 1. Human Phospho-kinase array.

A pool of 450 μg (150 μg each) of LY52, LY53 and LYM cell lysates (Control) or a pool of 450 μg (150 μg each) of LY190, LY193 and LY198 cell lysates (SDS) in the presence or in the absence (UT) of IL-6 (10 ng/ml) were incubated in nitrocellulose membrane pre-spotted with 43 antibodies able to recognize 43 different phospho-kinases. A cocktail of biotinylated detection antibodies was added followed by streptavidin-HRP incubation. Chemiluminescent detection reagents were applied and a signal was produced at each capture spot corresponding to the amount of phosphorylated protein bound. (a) Scanning of the arrays in which there are highlighted: i) red boxes, representing the major activated kinases; ii) blue boxes, representing phosphorylated STATs; 1, Hck; 2, mTOR; 3, PRAS40; 4, CREB; 5, p38; 6, MSK; 7, ERK; 8, AMPK2a; 9, HSP60; 10, Wnk1; 11, RSK; (a), STAT6; (b) STAT2; (c) STAT5A/B; (d) STAT5B; (e) STAT5A; (f) STAT3 (S727); (g) STAT3 (Y705). (b–s) quantitative analysis of pixel densities on developed X-ray film of the major regulated proteins.

Figure 2. Flow cytometric analysis of mTOR S2448 phosphorylation in LCLs.

(a) Representative experiment indicating mTOR S2448 phosphorylation level (green histogram) in healthy donor derived LCLs (Control) versus SDS LCLs (SDS). Red histogram indicates isotype control. Control LCLs and SDS LCLs were pre-incubated with 350 nM rapamycin (Rapa) for 1 hour before stimulation in the presence or in the absence (UT) of IL-6 (10 ng/ml) for further 15 min. (b) Median Fluorescence Intensity (MFI) and Percent of positive cells (c) derived from five independent experiments performed in LCLs derived from five different SDS patients. Data are mean ± SEM. Student’s t-test has been calculated.

Figure 3. Flow cytometric analysis of STAT3 Y705 and S727 phosphorylation in LCLs.

Representative experiment indicating STAT3 Y705 (green histogram) and S727 (blue histogram) phosphorylation level in: (a) healthy donor derived LCLs (Control) versus SDS LCLs (SDS). Red histogram indicates isotype control. Control LCLs and SDS LCLs were pre-incubated with 350 nM rapamycin (Rapa) for 1 hour before stimulation in the presence or in the absence (UT) of IL-6 (10 ng/ml) for further 15 min. (b) Median Fluorescence Intensity (MFI) and Percent of positive cells (c) for STAT3 Y705 signal. (d) Median Fluorescence Intensity (MFI) and Percent of positive cells (e) for STAT3 S727 signal. Data are mean ± SEM of five independent experiments performed in LCLs derived from five different SDS patients. Student’s t-test has been calculated.

Figure 4. IL-6-dependent nuclear translocation of STAT3 in LCLs.

Cells have been challenged with IL-6 (10 ng/ml) for 15 min. Human Phospho STAT Family Trans-AM kit was performed using 2.5 μg nuclear extracts for each sample. Histograms represent the nuclear translocation of: (a) STAT3; (b) STAT1; (c) STAT5A; (d) STAT5B. Data are mean ± SEM of 4 experiments performed in 3 different SDS cell lines versus 3 different healthy control cell lines, in duplicate. Mann-Whitney test has been reported.

Figure 5. Effect of SBDS gene silencing in healthy donor derived cells on mTOR S2448 phosphorylation.

LCLs derived from healthy donors were transiently transfected with 2 different specific siRNA sequences (siRNA 1 and siRNA 2) for SBDS, or with PE-conjugated siRNA sequence, or with scrambled sequence as control in the presence of cationic liposomal vector for 24 hours and stimulated with IL-6 (10 ng/ml) for further 15 min. (a) Check of efficiency rate of transfection measured by flow cytometry using a PE-conjugated siRNA. Results indicate up to 65% of transfection efficiency in our cell model. (b) Effect of SBDS gene silencing on SBDS protein expression in Control LCLs as measured by western blot analysis (SDS UT are SDS LCLs untreated as internal control). (c) Effect of SBDS gene silencing on mTOR S2448, STAT3 Y705 and STAT3 S727 phosphorylation measured byFC. (d–f) Median Fluorescence Intensity of phospho-mTOR and phosphor-STAT3 signals.

Figure 6. Flow cytometric analysis of mTOR S2448 phosphorylation in primary leukocytes.

Primary leukocytes were incubated in the absence (UT) or in the presence of IL-6 stimulation (10 ng/ml) for 15 min and analyzed by flow cytometry. (a) MFI of mTOR S2448 phosphorylation measured in primary B cells. (b) MFI of mTOR S2448 phosphorylation measured in primary PMNs. (c) MFI of mTOR S2448 phosphorylation measured in primary monocytes. Data are mean ± SEM of five independent experiments performed in LCLs obtained from five different SDS patients and compared to five different healthy donors. Student’s t-test has been reported.

Figure 7. Flow cytometric analysis of STAT3 Y705 and S727 phosphorylation in primary leukocytes.

Primary leukocytes were incubated in the absence (UT) or in the presence of IL-6 stimulation (10 ng/ml) for 15 min and analyzed by FC. (a,b) MFI of STAT3 Y705 and S727 phosphorylation (respectively) measured in primary B cells. (c,d) MFI of STAT3 Y705 and S727 phosphorylation (respectively) measured in primary PMNs. (e,f) MFI of STAT3 Y705 and S727 phosphorylation (respectively) measured in primary monocytes. Data are mean ± SEM of five independent experiments performed in LCLs obtained from five different SDS patients and compared to five different healthy donors. Student’s t-test has been reported.

Figure 8. Effect of rapamycin and STATTIC on cell proliferation and apoptosis in LCLs.

(a,b) LCLs derived from both healthy donors (a) and SDS patients (b) were incubated in the presence or in the absence of 350 nM mTOR inhibitor rapamycin, or 20 μM STAT3 inhibitor STATTIC and stimulated with increasing doses (0.01–10 ng/ml) of IL-6 for 48 hours. Cell proliferation was measured by XTT Cell Proliferation Kit II. Data are mean ± SEM of five independent experiments performed in duplicate. Student’s t-test has been calculated. (c,d) LCLs derived from both healthy donors (c) and SDS patients (d) were incubated in the presence (black bars) or in the absence (white bars) of 350 nM mTOR inhibitor rapamycin for 24 hours. Apoptosis was analyzed using the Muse Annexin V & Dead Cell Kit. Data are mean ± SEM of four independent experiments performed in duplicate. (e,f) LCLs derived from both healthy donors (e) and SDS patients (f) were incubated in the presence (black bars) or in the absence (white bars) of 20 μM STAT3 inhibitor STATTIC for 24 hours. Apoptosis was analyzed using the Muse Annexin V & Dead Cell Kit. Data are mean ± SEM of four independent experiments performed in duplicate. Student’s t-test has been calculated.

Figure 9. Model of dysregulated mTOR/STAT3 signal transduction pathways observed in leukocytes obtained from SDS patients.

Normally, IL-6 trigger a JAK1/2 activation which in turn leads to STAT3 phosphorylation, mainly at Y705 residue, causing STAT3 dimerization and translocation into the nucleus, where STAT3 is able to regulate gene expression orchestrating several cellular processes like inflammation and cell proliferation. In SDS patients, leukocytes show ERK1/2, mTOR and STAT3 hyper-activation. ERK1/2 is known to promote mTOR phosphorylation in S2448 residue, which in turn leads to mTORC1 complex activation. mTORC1 is known to regulate different cell processes, including translation, autophagy, cell growth and ribosome biogenesis, which are impaired in SDS pathology. Notably, mTORC1 is also known to induce strong phosphorylation of STAT3 both in Y705 and S727 residues in different cellular models. Here we report that mTOR inhibitor rapamycin is able to reduce STAT3 hyper-activation observed in SDS patients, restoring phosphorylation level of both Y705 and S727. Moreover, in this issue we show how loss of SBDS protein can lead to mTOR S2448 hyper-activation in LCLs obtained from healthy donors. Finally, we show that pre-incubating ERK1/2 inhibitor U0126 in SDS EBV-transformed B cells we significantly reduce IL-6 induced mTOR S2448 phosphorylation.