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Viability and stress protection of chronic lymphoid leukemia cells involves overactivation of mitochondrial phosphoSTAT3Ser727.

Capron C, Jondeau K, Casetti L, Jalbert V, Costa C, Verhoeyen E, Verhoyen E, Massé JM, Coppo P, Béné MC, Bourdoncle P, Cramer-Bordé E, Dusanter-Fourt I - Cell Death Dis (2014)

Bottom Line: Importantly, pSTAT3Ser(727), but neither Tyr705-phosphorylated STAT3 nor total STAT3, levels correlate with prolonged in vivo CLL B cells survival.Mt pSTAT3Ser(727) appears to be a newly identified cell-protective signal involved in CLL cells survival.Targeting pSTAT3Ser(727) could be a promising new therapeutic approach.

View Article: PubMed Central - PubMed

Affiliation: 1] Institut Cochin, Inserm U1016, Paris, France [2] Service d'Hématologie-Immunologie, Hôpital Ambroise Paré, Boulogne-Billancourt, France [3] Université Paris Descartes, Sorbonne Paris Cité, Paris, France [4] CNRS UMR8104, Paris, France [5] Université de Versailles St Quentin en Yvelines, Guyancourt, France.

ABSTRACT
Chronic lymphoid leukemia (CLL) is characterized by the accumulation of functionally defective CD5-positive B lymphocytes. The clinical course of CLL is highly variable, ranging from a long-lasting indolent disease to an unpredictable and rapidly progressing leukemia requiring treatment. It is thus important to identify novel factors that reflect disease progression or contribute to its assessment. Here, we report on a novel STAT3-mediated pathway that characterizes CLL B cells-extended viability and oxidative stress control. We observed that leukemic but not normal B cells from CLL patients exhibit constitutive activation of an atypical form of the STAT3 signaling factor, phosphorylated on serine 727 (Ser(727)) in the absence of detectable canonical tyrosine 705 (Tyr705)-dependent activation in vivo. The Ser(727)-phosphorylated STAT3 molecule (pSTAT3Ser(727)) is localized to the mitochondria and associates with complex I of the respiratory chain. This pSer(727) modification is further controlled by glutathione-dependent antioxidant pathway(s) that mediate stromal protection of the leukemic B cells and regulate their viability. Importantly, pSTAT3Ser(727), but neither Tyr705-phosphorylated STAT3 nor total STAT3, levels correlate with prolonged in vivo CLL B cells survival. Furthermore, STAT3 activity contributes to the resistance to apoptosis of CLL, but not normal B cells, in vitro. These data reveal that mitochondrial (Mt) pSTAT3Ser(727) overactivity is part of the antioxidant defense pathway of CLL B cells that regulates their viability. Mt pSTAT3Ser(727) appears to be a newly identified cell-protective signal involved in CLL cells survival. Targeting pSTAT3Ser(727) could be a promising new therapeutic approach.

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GSH metabolism regulates the phosphorylation of STAT-Ser727 of CLL-BC. (a andb) Comparison of apoptosis, ROS, pSTAT3Ser727 and total STAT3 expression of CLL-BCcultured alone for 4 days in the presence or absence of NAC (1 mM) or GSH (2 mM).Where indicated, PEITC (1–5 μM, 5 h) was added at the end of theculture. (c) Time course of ROS, pSTAT3Ser727 and total STAT3 expression of CLL-BCcultured alone in the presence of PEITC (5 μM) for the indicated time. ROS weremeasured by DHE staining of CD45/CD19+/CD5+ cells;pSTAT3Ser727 and total STAT3 levels were evaluated upon immunolabeling/FCM. Data areexpressed as relative to untreated cells. The mean±S.E.M. of four separate experiments withfour different CLL patient samples are shown (*P<0.05; **P<0.01;***P<0.001; ****P<0.0001). (d) FCM ofpMEK (right) and pSTAT3Ser727 (left) of CLL-BC upon a two-day culture in the absence(black) or presence of 10 nM (dark grey) or 100 nM (medium grey) of MEK inhibitorPD0325901. B cells were labeled with rabbit control (light grey), pMEK or pSTAT3Ser727 antibodies,as indicated. Results are expressed as mean fluorescence intensity (arbitrary unit, a.u.)
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fig6: GSH metabolism regulates the phosphorylation of STAT-Ser727 of CLL-BC. (a andb) Comparison of apoptosis, ROS, pSTAT3Ser727 and total STAT3 expression of CLL-BCcultured alone for 4 days in the presence or absence of NAC (1 mM) or GSH (2 mM).Where indicated, PEITC (1–5 μM, 5 h) was added at the end of theculture. (c) Time course of ROS, pSTAT3Ser727 and total STAT3 expression of CLL-BCcultured alone in the presence of PEITC (5 μM) for the indicated time. ROS weremeasured by DHE staining of CD45/CD19+/CD5+ cells;pSTAT3Ser727 and total STAT3 levels were evaluated upon immunolabeling/FCM. Data areexpressed as relative to untreated cells. The mean±S.E.M. of four separate experiments withfour different CLL patient samples are shown (*P<0.05; **P<0.01;***P<0.001; ****P<0.0001). (d) FCM ofpMEK (right) and pSTAT3Ser727 (left) of CLL-BC upon a two-day culture in the absence(black) or presence of 10 nM (dark grey) or 100 nM (medium grey) of MEK inhibitorPD0325901. B cells were labeled with rabbit control (light grey), pMEK or pSTAT3Ser727 antibodies,as indicated. Results are expressed as mean fluorescence intensity (arbitrary unit, a.u.)

Mentions: The accumulation of Mt pSTAT3Ser727 in stroma-supported CLL-BC led us to search forstromal signals that would regulate pSTAT3Ser727 activation in these cells. CLL-BCintrinsically have high levels of reactive oxygen species (ROS) when compared with normallymphocytes.1,18 This makesCLL-BC more dependent on such cellular antioxidants as GSH. A critical metabolic interaction betweenCLL-BC and bone marrow stromal cells was recently reported to enhance GSH synthesis, and thusincrease the ability of CLL-BC to maintain the redox balance and promote cell survival.6 We tested the relationship between GSH, oxidative stress andpSTAT3Ser727 expression of CLL-BC. Dihydroethidium (DHE) provides a clear indication ofprimary ROS levels19 by binding to superoxide anions thatare produced predominantly by the respiratory chain of mitochondria in most cells except phagocytes.As shown in Figure 6a, the addition of GSH to CLL-BC culture medium, inabsence of stromal cells, indeed enhanced ROS detoxification, thereby lowering superoxide anionslevels and promoting cell survival. It further enhanced pSTAT3Ser727 expression of CLL-BCwithout changing pSTAT3Tyr705 activation (data not shown) and total STAT3 levels. Similardata were obtained upon addition of N-acetylcysteine (NAC), another antioxidant. This increasedactivation of pSTAT3Ser727 by antioxidants targeted Mt STAT3, as assessed byimmunolabeling coupled to confocal microscopy (data not shown).


Viability and stress protection of chronic lymphoid leukemia cells involves overactivation of mitochondrial phosphoSTAT3Ser727.

Capron C, Jondeau K, Casetti L, Jalbert V, Costa C, Verhoeyen E, Verhoyen E, Massé JM, Coppo P, Béné MC, Bourdoncle P, Cramer-Bordé E, Dusanter-Fourt I - Cell Death Dis (2014)

GSH metabolism regulates the phosphorylation of STAT-Ser727 of CLL-BC. (a andb) Comparison of apoptosis, ROS, pSTAT3Ser727 and total STAT3 expression of CLL-BCcultured alone for 4 days in the presence or absence of NAC (1 mM) or GSH (2 mM).Where indicated, PEITC (1–5 μM, 5 h) was added at the end of theculture. (c) Time course of ROS, pSTAT3Ser727 and total STAT3 expression of CLL-BCcultured alone in the presence of PEITC (5 μM) for the indicated time. ROS weremeasured by DHE staining of CD45/CD19+/CD5+ cells;pSTAT3Ser727 and total STAT3 levels were evaluated upon immunolabeling/FCM. Data areexpressed as relative to untreated cells. The mean±S.E.M. of four separate experiments withfour different CLL patient samples are shown (*P<0.05; **P<0.01;***P<0.001; ****P<0.0001). (d) FCM ofpMEK (right) and pSTAT3Ser727 (left) of CLL-BC upon a two-day culture in the absence(black) or presence of 10 nM (dark grey) or 100 nM (medium grey) of MEK inhibitorPD0325901. B cells were labeled with rabbit control (light grey), pMEK or pSTAT3Ser727 antibodies,as indicated. Results are expressed as mean fluorescence intensity (arbitrary unit, a.u.)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4237234&req=5

fig6: GSH metabolism regulates the phosphorylation of STAT-Ser727 of CLL-BC. (a andb) Comparison of apoptosis, ROS, pSTAT3Ser727 and total STAT3 expression of CLL-BCcultured alone for 4 days in the presence or absence of NAC (1 mM) or GSH (2 mM).Where indicated, PEITC (1–5 μM, 5 h) was added at the end of theculture. (c) Time course of ROS, pSTAT3Ser727 and total STAT3 expression of CLL-BCcultured alone in the presence of PEITC (5 μM) for the indicated time. ROS weremeasured by DHE staining of CD45/CD19+/CD5+ cells;pSTAT3Ser727 and total STAT3 levels were evaluated upon immunolabeling/FCM. Data areexpressed as relative to untreated cells. The mean±S.E.M. of four separate experiments withfour different CLL patient samples are shown (*P<0.05; **P<0.01;***P<0.001; ****P<0.0001). (d) FCM ofpMEK (right) and pSTAT3Ser727 (left) of CLL-BC upon a two-day culture in the absence(black) or presence of 10 nM (dark grey) or 100 nM (medium grey) of MEK inhibitorPD0325901. B cells were labeled with rabbit control (light grey), pMEK or pSTAT3Ser727 antibodies,as indicated. Results are expressed as mean fluorescence intensity (arbitrary unit, a.u.)
Mentions: The accumulation of Mt pSTAT3Ser727 in stroma-supported CLL-BC led us to search forstromal signals that would regulate pSTAT3Ser727 activation in these cells. CLL-BCintrinsically have high levels of reactive oxygen species (ROS) when compared with normallymphocytes.1,18 This makesCLL-BC more dependent on such cellular antioxidants as GSH. A critical metabolic interaction betweenCLL-BC and bone marrow stromal cells was recently reported to enhance GSH synthesis, and thusincrease the ability of CLL-BC to maintain the redox balance and promote cell survival.6 We tested the relationship between GSH, oxidative stress andpSTAT3Ser727 expression of CLL-BC. Dihydroethidium (DHE) provides a clear indication ofprimary ROS levels19 by binding to superoxide anions thatare produced predominantly by the respiratory chain of mitochondria in most cells except phagocytes.As shown in Figure 6a, the addition of GSH to CLL-BC culture medium, inabsence of stromal cells, indeed enhanced ROS detoxification, thereby lowering superoxide anionslevels and promoting cell survival. It further enhanced pSTAT3Ser727 expression of CLL-BCwithout changing pSTAT3Tyr705 activation (data not shown) and total STAT3 levels. Similardata were obtained upon addition of N-acetylcysteine (NAC), another antioxidant. This increasedactivation of pSTAT3Ser727 by antioxidants targeted Mt STAT3, as assessed byimmunolabeling coupled to confocal microscopy (data not shown).

Bottom Line: Importantly, pSTAT3Ser(727), but neither Tyr705-phosphorylated STAT3 nor total STAT3, levels correlate with prolonged in vivo CLL B cells survival.Mt pSTAT3Ser(727) appears to be a newly identified cell-protective signal involved in CLL cells survival.Targeting pSTAT3Ser(727) could be a promising new therapeutic approach.

View Article: PubMed Central - PubMed

Affiliation: 1] Institut Cochin, Inserm U1016, Paris, France [2] Service d'Hématologie-Immunologie, Hôpital Ambroise Paré, Boulogne-Billancourt, France [3] Université Paris Descartes, Sorbonne Paris Cité, Paris, France [4] CNRS UMR8104, Paris, France [5] Université de Versailles St Quentin en Yvelines, Guyancourt, France.

ABSTRACT
Chronic lymphoid leukemia (CLL) is characterized by the accumulation of functionally defective CD5-positive B lymphocytes. The clinical course of CLL is highly variable, ranging from a long-lasting indolent disease to an unpredictable and rapidly progressing leukemia requiring treatment. It is thus important to identify novel factors that reflect disease progression or contribute to its assessment. Here, we report on a novel STAT3-mediated pathway that characterizes CLL B cells-extended viability and oxidative stress control. We observed that leukemic but not normal B cells from CLL patients exhibit constitutive activation of an atypical form of the STAT3 signaling factor, phosphorylated on serine 727 (Ser(727)) in the absence of detectable canonical tyrosine 705 (Tyr705)-dependent activation in vivo. The Ser(727)-phosphorylated STAT3 molecule (pSTAT3Ser(727)) is localized to the mitochondria and associates with complex I of the respiratory chain. This pSer(727) modification is further controlled by glutathione-dependent antioxidant pathway(s) that mediate stromal protection of the leukemic B cells and regulate their viability. Importantly, pSTAT3Ser(727), but neither Tyr705-phosphorylated STAT3 nor total STAT3, levels correlate with prolonged in vivo CLL B cells survival. Furthermore, STAT3 activity contributes to the resistance to apoptosis of CLL, but not normal B cells, in vitro. These data reveal that mitochondrial (Mt) pSTAT3Ser(727) overactivity is part of the antioxidant defense pathway of CLL B cells that regulates their viability. Mt pSTAT3Ser(727) appears to be a newly identified cell-protective signal involved in CLL cells survival. Targeting pSTAT3Ser(727) could be a promising new therapeutic approach.

Show MeSH
Related in: MedlinePlus