LEUKEMIC CELL/MICROENVIRONMENT INTERACTIONS IN CHRONIC LYMPHOCYTIC LEUKEMIA: ROLE OF JAK2/STAT3 AXIS IN THE SURVIVAL OF NEOPLASTIC CLONE
(Abstract release date: 05/19/16)
EHA Library. Severin F. 06/09/16; 132570; E1021
Dr. Filippo Severin
Contributions
Contributions
Abstract
Abstract: E1021
Type: Eposter Presentation
Background
Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of mature clonal CD19+/CD5+/CD23+ B lymphocytes in peripheral blood, bone marrow, and lymphoid tissues. Despite their in vivo prolonged lifespan due to intrinsic defects, CLL leukemic cells rapidly undergo spontaneous apoptosis in vitro, highlighting the need of extrinsic signals delivered by the microenvironment. Several molecules, including those released by mesenchymal stromal cells (MSCs), signal through JAK (Janus Kinases)/STAT (Signal Transducers and Activators of Transcription) pathways. Constitutive activation of JAKs and STATs occurs at very high frequency in various hematopoietic malignancies and solid tumors. STAT3 tyrosine (Tyr) phosphorylation at residue 705 is known to be critical for the activation of JAK2/STAT3 pro-survival signaling, while the role of STAT3 Serine (Ser) phosphorylation at residue 727 has not yet been well described. In CLL, STAT3 is constitutively phosphorylated at Ser727 with respect to normal B lymphocytes, but the phosphorylation status at Tyr705 still remains unresolved.
Aims
We are aimed to evaluate JAK2/STAT3 pathway involvement in leukemic B cell survival and to study the cross-talk between JAK/STAT and BCR/Lyn axes. With the identification of new targets implicated in this cross-talk, and through the analysis of leukemic cell resistance to drug-induced apoptosis, we plan to counteract the favorable pro-leukemic cell stimuli.
Methods
B cells were collected from 34 controls and 76 CLL patients. Purified cells (2x106 cells/ml) were cultured, with/without mesenchymal stromal cells (MSCs), and treated with AG490 (10, 50 and 100μM) and Stattic (5, 7.5, and 10μM), specific inhibitors of JAK2 and STAT3 respectively, for 24, 48 and 72h. STAT3 expression and phosphorylation were evaluated by Western Blotting (WB) and Flow Cytometry (FC), and its localization was analyzed by confocal microscopy and subcellular fractionation. CLL and normal B cell viability was tested by FC with Annexin V/PI test. Lyn and SHP-1 phosphorylation was assessed by WB.
Results
We demonstrated that STAT3 is overexpressed in malignant B cells (Student’s t test, p<0.001) and the protein is higher phosphorylated at Tyr705 with respect to the normal counterpart (Student’s t test, p<0.05), thus showing its constitutive activation in CLL. We also found that the in vitro incubation of leukemic B cells with AG490 and Stattic, specific inhibitors of JAK2 and STAT3, respectively, induces a dose-dependent apoptosis of CLL B cells. We demonstrated that these inhibitors are able to induce apoptosis in CLL B cells reverting the resistance to cytotoxic agents induced by the MSCs, bypassing the provided pro-survival stimuli. In addition to JAK2/STAT3 inhibition, we showed that AG490 treatment on CLL cells can mediate the activation of SHP-1, decreasing its phosphorylation at Ser591, thus leading to inactivation of Lyn protein, by de-phosphorylation in its active site at Tyr396. Lyn, a Tyr-kinase, and SHP-1, a Tyr-phosphatase, are both involved in the prolonged lifespan of neoplastic CLL cells.
Conclusion
Bypassing the pro-survival stimuli provided by the tumor microenvironment, the ability of AG490 and Stattic to induce apoptosis in leukemic B cells, represents a starting point for the development of new therapeutic strategies in CLL. These findings also provide new insights on the cross-talk between JAK/STAT and BCR/Lyn axes in CLL.
Session topic: E-poster
Keyword(s): Chronic lymphocytic leukemia, Inhibitor, Signaling molecules, STAT3
Type: Eposter Presentation
Background
Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of mature clonal CD19+/CD5+/CD23+ B lymphocytes in peripheral blood, bone marrow, and lymphoid tissues. Despite their in vivo prolonged lifespan due to intrinsic defects, CLL leukemic cells rapidly undergo spontaneous apoptosis in vitro, highlighting the need of extrinsic signals delivered by the microenvironment. Several molecules, including those released by mesenchymal stromal cells (MSCs), signal through JAK (Janus Kinases)/STAT (Signal Transducers and Activators of Transcription) pathways. Constitutive activation of JAKs and STATs occurs at very high frequency in various hematopoietic malignancies and solid tumors. STAT3 tyrosine (Tyr) phosphorylation at residue 705 is known to be critical for the activation of JAK2/STAT3 pro-survival signaling, while the role of STAT3 Serine (Ser) phosphorylation at residue 727 has not yet been well described. In CLL, STAT3 is constitutively phosphorylated at Ser727 with respect to normal B lymphocytes, but the phosphorylation status at Tyr705 still remains unresolved.
Aims
We are aimed to evaluate JAK2/STAT3 pathway involvement in leukemic B cell survival and to study the cross-talk between JAK/STAT and BCR/Lyn axes. With the identification of new targets implicated in this cross-talk, and through the analysis of leukemic cell resistance to drug-induced apoptosis, we plan to counteract the favorable pro-leukemic cell stimuli.
Methods
B cells were collected from 34 controls and 76 CLL patients. Purified cells (2x106 cells/ml) were cultured, with/without mesenchymal stromal cells (MSCs), and treated with AG490 (10, 50 and 100μM) and Stattic (5, 7.5, and 10μM), specific inhibitors of JAK2 and STAT3 respectively, for 24, 48 and 72h. STAT3 expression and phosphorylation were evaluated by Western Blotting (WB) and Flow Cytometry (FC), and its localization was analyzed by confocal microscopy and subcellular fractionation. CLL and normal B cell viability was tested by FC with Annexin V/PI test. Lyn and SHP-1 phosphorylation was assessed by WB.
Results
We demonstrated that STAT3 is overexpressed in malignant B cells (Student’s t test, p<0.001) and the protein is higher phosphorylated at Tyr705 with respect to the normal counterpart (Student’s t test, p<0.05), thus showing its constitutive activation in CLL. We also found that the in vitro incubation of leukemic B cells with AG490 and Stattic, specific inhibitors of JAK2 and STAT3, respectively, induces a dose-dependent apoptosis of CLL B cells. We demonstrated that these inhibitors are able to induce apoptosis in CLL B cells reverting the resistance to cytotoxic agents induced by the MSCs, bypassing the provided pro-survival stimuli. In addition to JAK2/STAT3 inhibition, we showed that AG490 treatment on CLL cells can mediate the activation of SHP-1, decreasing its phosphorylation at Ser591, thus leading to inactivation of Lyn protein, by de-phosphorylation in its active site at Tyr396. Lyn, a Tyr-kinase, and SHP-1, a Tyr-phosphatase, are both involved in the prolonged lifespan of neoplastic CLL cells.
Conclusion
Bypassing the pro-survival stimuli provided by the tumor microenvironment, the ability of AG490 and Stattic to induce apoptosis in leukemic B cells, represents a starting point for the development of new therapeutic strategies in CLL. These findings also provide new insights on the cross-talk between JAK/STAT and BCR/Lyn axes in CLL.
Session topic: E-poster
Keyword(s): Chronic lymphocytic leukemia, Inhibitor, Signaling molecules, STAT3
Abstract: E1021
Type: Eposter Presentation
Background
Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of mature clonal CD19+/CD5+/CD23+ B lymphocytes in peripheral blood, bone marrow, and lymphoid tissues. Despite their in vivo prolonged lifespan due to intrinsic defects, CLL leukemic cells rapidly undergo spontaneous apoptosis in vitro, highlighting the need of extrinsic signals delivered by the microenvironment. Several molecules, including those released by mesenchymal stromal cells (MSCs), signal through JAK (Janus Kinases)/STAT (Signal Transducers and Activators of Transcription) pathways. Constitutive activation of JAKs and STATs occurs at very high frequency in various hematopoietic malignancies and solid tumors. STAT3 tyrosine (Tyr) phosphorylation at residue 705 is known to be critical for the activation of JAK2/STAT3 pro-survival signaling, while the role of STAT3 Serine (Ser) phosphorylation at residue 727 has not yet been well described. In CLL, STAT3 is constitutively phosphorylated at Ser727 with respect to normal B lymphocytes, but the phosphorylation status at Tyr705 still remains unresolved.
Aims
We are aimed to evaluate JAK2/STAT3 pathway involvement in leukemic B cell survival and to study the cross-talk between JAK/STAT and BCR/Lyn axes. With the identification of new targets implicated in this cross-talk, and through the analysis of leukemic cell resistance to drug-induced apoptosis, we plan to counteract the favorable pro-leukemic cell stimuli.
Methods
B cells were collected from 34 controls and 76 CLL patients. Purified cells (2x106 cells/ml) were cultured, with/without mesenchymal stromal cells (MSCs), and treated with AG490 (10, 50 and 100μM) and Stattic (5, 7.5, and 10μM), specific inhibitors of JAK2 and STAT3 respectively, for 24, 48 and 72h. STAT3 expression and phosphorylation were evaluated by Western Blotting (WB) and Flow Cytometry (FC), and its localization was analyzed by confocal microscopy and subcellular fractionation. CLL and normal B cell viability was tested by FC with Annexin V/PI test. Lyn and SHP-1 phosphorylation was assessed by WB.
Results
We demonstrated that STAT3 is overexpressed in malignant B cells (Student’s t test, p<0.001) and the protein is higher phosphorylated at Tyr705 with respect to the normal counterpart (Student’s t test, p<0.05), thus showing its constitutive activation in CLL. We also found that the in vitro incubation of leukemic B cells with AG490 and Stattic, specific inhibitors of JAK2 and STAT3, respectively, induces a dose-dependent apoptosis of CLL B cells. We demonstrated that these inhibitors are able to induce apoptosis in CLL B cells reverting the resistance to cytotoxic agents induced by the MSCs, bypassing the provided pro-survival stimuli. In addition to JAK2/STAT3 inhibition, we showed that AG490 treatment on CLL cells can mediate the activation of SHP-1, decreasing its phosphorylation at Ser591, thus leading to inactivation of Lyn protein, by de-phosphorylation in its active site at Tyr396. Lyn, a Tyr-kinase, and SHP-1, a Tyr-phosphatase, are both involved in the prolonged lifespan of neoplastic CLL cells.
Conclusion
Bypassing the pro-survival stimuli provided by the tumor microenvironment, the ability of AG490 and Stattic to induce apoptosis in leukemic B cells, represents a starting point for the development of new therapeutic strategies in CLL. These findings also provide new insights on the cross-talk between JAK/STAT and BCR/Lyn axes in CLL.
Session topic: E-poster
Keyword(s): Chronic lymphocytic leukemia, Inhibitor, Signaling molecules, STAT3
Type: Eposter Presentation
Background
Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of mature clonal CD19+/CD5+/CD23+ B lymphocytes in peripheral blood, bone marrow, and lymphoid tissues. Despite their in vivo prolonged lifespan due to intrinsic defects, CLL leukemic cells rapidly undergo spontaneous apoptosis in vitro, highlighting the need of extrinsic signals delivered by the microenvironment. Several molecules, including those released by mesenchymal stromal cells (MSCs), signal through JAK (Janus Kinases)/STAT (Signal Transducers and Activators of Transcription) pathways. Constitutive activation of JAKs and STATs occurs at very high frequency in various hematopoietic malignancies and solid tumors. STAT3 tyrosine (Tyr) phosphorylation at residue 705 is known to be critical for the activation of JAK2/STAT3 pro-survival signaling, while the role of STAT3 Serine (Ser) phosphorylation at residue 727 has not yet been well described. In CLL, STAT3 is constitutively phosphorylated at Ser727 with respect to normal B lymphocytes, but the phosphorylation status at Tyr705 still remains unresolved.
Aims
We are aimed to evaluate JAK2/STAT3 pathway involvement in leukemic B cell survival and to study the cross-talk between JAK/STAT and BCR/Lyn axes. With the identification of new targets implicated in this cross-talk, and through the analysis of leukemic cell resistance to drug-induced apoptosis, we plan to counteract the favorable pro-leukemic cell stimuli.
Methods
B cells were collected from 34 controls and 76 CLL patients. Purified cells (2x106 cells/ml) were cultured, with/without mesenchymal stromal cells (MSCs), and treated with AG490 (10, 50 and 100μM) and Stattic (5, 7.5, and 10μM), specific inhibitors of JAK2 and STAT3 respectively, for 24, 48 and 72h. STAT3 expression and phosphorylation were evaluated by Western Blotting (WB) and Flow Cytometry (FC), and its localization was analyzed by confocal microscopy and subcellular fractionation. CLL and normal B cell viability was tested by FC with Annexin V/PI test. Lyn and SHP-1 phosphorylation was assessed by WB.
Results
We demonstrated that STAT3 is overexpressed in malignant B cells (Student’s t test, p<0.001) and the protein is higher phosphorylated at Tyr705 with respect to the normal counterpart (Student’s t test, p<0.05), thus showing its constitutive activation in CLL. We also found that the in vitro incubation of leukemic B cells with AG490 and Stattic, specific inhibitors of JAK2 and STAT3, respectively, induces a dose-dependent apoptosis of CLL B cells. We demonstrated that these inhibitors are able to induce apoptosis in CLL B cells reverting the resistance to cytotoxic agents induced by the MSCs, bypassing the provided pro-survival stimuli. In addition to JAK2/STAT3 inhibition, we showed that AG490 treatment on CLL cells can mediate the activation of SHP-1, decreasing its phosphorylation at Ser591, thus leading to inactivation of Lyn protein, by de-phosphorylation in its active site at Tyr396. Lyn, a Tyr-kinase, and SHP-1, a Tyr-phosphatase, are both involved in the prolonged lifespan of neoplastic CLL cells.
Conclusion
Bypassing the pro-survival stimuli provided by the tumor microenvironment, the ability of AG490 and Stattic to induce apoptosis in leukemic B cells, represents a starting point for the development of new therapeutic strategies in CLL. These findings also provide new insights on the cross-talk between JAK/STAT and BCR/Lyn axes in CLL.
Session topic: E-poster
Keyword(s): Chronic lymphocytic leukemia, Inhibitor, Signaling molecules, STAT3
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