Targeting constitutively active STAT3 in chronic lymphocytic leukemia: A clinical trial of the STAT3 inhibitor pyrimethamine with pharmacodynamic analyses.
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ABSTRACT:
SUBMITTER: Brown JR
PROVIDER: S-EPMC7954938 | biostudies-literature | 2021 Apr
REPOSITORIES: biostudies-literature
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Targeting constitutively active STAT3 in chronic lymphocytic leukemia: A clinical trial of the STAT3 inhibitor pyrimethamine with pharmacodynamic analyses.
Brown Jennifer R JR Walker Sarah R SR Heppler Lisa N LN Tyekucheva Svitlana S Nelson Erik A EA Klitgaard Josephine J Nicolais Maria M Kroll Yasmin Y Xiang Michael M Yeh Jennifer E JE Chaudhury Mousumi M Giaccone Zachary T ZT Fernandes Stacey M SM Jacobsen Eric D ED Fisher David C DC Freedman Arnold S AS Davids Matthew S MS Supko Jeffrey G JG Wu Catherine C Frank David A DA
American journal of hematology 20210111 4
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Project description:Signal transducer and activator of transcription (STAT)-3 might be phosphorylated or acetylated. Unlike the phosphorylation of STAT3, little is known about the acetylation of STAT3 in chronic lymphocytic leukemia (CLL) cells. Because acetylation activates STAT3 transcription, we sought to study the acetylation status of STAT3 in CLL cells. Using Western immunoblotting, immunoprecipitation, and flow cytometry we found that, apart from its constitutive serine phosphorylation, STAT3 is constitutively acetylated on lysine 685 residues. Because the acetyltransferase p300 was found to acetylate STAT3 on lysine 685 residues, we wondered whether p300 acetylates STAT3 in CLL cells. Using Western immunoblotting we detected high levels of p300 protein in CLL but not normal B cells. Transfection of CLL cells with p300 small-interfering (si) RNA downregulated p300 transcripts as well as p300 and acetyl-STAT3 protein levels. In addition, p300 siRNA attenuated STAT3-DNA binding and downregulated mRNA levels of STAT3-regulated genes. Furthermore, transfection of CLL cells with p300-siRNA induced a 3-fold increase in the rate of spontaneous apoptosis. Taken together, our data suggest that in CLL cells STAT3 p300 induces constitutive acetylation and activation of STAT3. Whether inhibition of STAT3 acetylation might provide clinical benefit in patients with CLL remains to be determined.
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Project description:In chronic lymphocytic leukemia (CLL), the increment in PBLs is slower than the expected increment calculated from the cells' proliferation rate, suggesting that cellular proliferation and apoptosis are concurrent. Exploring this phenomenon, we found overexpression of caspase-3, higher cleaved poly (ADP-ribose) polymerase levels (p < 0.007), and a higher apoptosis rate in cells from patients with high counts compared with cells from patients with low counts. Although we previously found that STAT3 protects CLL cells from apoptosis, STAT3 levels were significantly higher in cells from patients with high counts than in cells from patients with low counts. Furthermore, overexpression of STAT3 did not protect the cells. Rather, it upregulated caspase-3 and induced apoptosis. Remarkably, putative STAT3 binding sites were identified in the caspase-3 promoter, and a luciferase assay, chromatin immunoprecipitation, and an EMSA revealed that STAT3 activated caspase-3 However, caspase-3 levels increased only when STAT3 levels were sufficiently high. Using chromatin immunoprecipitation and EMSA, we found that STAT3 binds with low affinity to the caspase-3 promoter, suggesting that at high levels, STAT3 activates proapoptotic mechanisms and induces apoptosis in CLL cells.
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Project description:BackgroundT-cell large granular lymphocytic leukemia is a rare lymphoproliferative disorder characterized by the expansion of clonal CD3+CD8+ cytotoxic T lymphocytes (CTLs) and often associated with autoimmune disorders and immune-mediated cytopenias.MethodsWe used next-generation exome sequencing to identify somatic mutations in CTLs from an index patient with large granular lymphocytic leukemia. Targeted resequencing was performed in a well-characterized cohort of 76 patients with this disorder, characterized by clonal T-cell-receptor rearrangements and increased numbers of large granular lymphocytes.ResultsMutations in the signal transducer and activator of transcription 3 gene (STAT3) were found in 31 of 77 patients (40%) with large granular lymphocytic leukemia. Among these 31 patients, recurrent mutational hot spots included Y640F in 13 (17%), D661V in 7 (9%), D661Y in 7 (9%), and N647I in 3 (4%). All mutations were located in exon 21, encoding the Src homology 2 (SH2) domain, which mediates the dimerization and activation of STAT protein. The amino acid changes resulted in a more hydrophobic protein surface and were associated with phosphorylation of STAT3 and its localization in the nucleus. In vitro functional studies showed that the Y640F and D661V mutations increased the transcriptional activity of STAT3. In the affected patients, downstream target genes of the STAT3 pathway (IFNGR2, BCL2L1, and JAK2) were up-regulated. Patients with STAT3 mutations presented more often with neutropenia and rheumatoid arthritis than did patients without these mutations.ConclusionsThe SH2 dimerization and activation domain of STAT3 is frequently mutated in patients with large granular lymphocytic leukemia; these findings suggest that aberrant STAT3 signaling underlies the pathogenesis of this disease. (Funded by the Academy of Finland and others.).
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Project description:Signal Transducer and Activator of Transcription 3 (STAT3) is considered as an oncogene being constitutively activated in a wide variety of primary human tumours, which often become addicted to its activity. Using primary fibroblasts derived from a knock-in mouse expressing only the constitutively active form STAT3-C as well as STAT3-dependent tumor cell lines MDA-MB468, DU145 and SKBR3, we provide evidence that STAT3 can act as a central regulator of cell metabolism. STAT3C/C MEFs are resistant to apoptosis and senescence and they show reduced mitochondrial activity but activate aerobic glycolysis, as shown by enhanced expression of master regulators of glycolysis such as PDK-1 and HIF-1α, and by increased lactate production, glucose avidity and sensitivity to glycolysis inhibitors. HIF-1α is up-regulated in the STAT3C/C MEFs and is responsible for the glycolytic phenotype. Moreover, inhibition of STAT3 activity in STAT3-addicted tumour cell lines restores mitochondrial activity and down-regulates glycolytic metabolism, preceding the induction of massive apoptosis. Thus, the essential role observed for STAT3 in so many different cancer cell types may be partly explained by its ability to act as a molecular switch of cellular metabolism triggering abnormal cell survival/proliferation. Three biological replicates of MEFs from mice with constitutively active Stat3 are compared to three biological replicates of wild-type MEFs.
2010-11-17 | E-GEOD-21507 | biostudies-arrayexpress
Project description:Signal Transducer and Activator of Transcription 3 (STAT3) is considered as an oncogene being constitutively activated in a wide variety of primary human tumours, which often become addicted to its activity. Using primary fibroblasts derived from a knock-in mouse expressing only the constitutively active form STAT3-C as well as STAT3-dependent tumor cell lines MDA-MB468, DU145 and SKBR3, we provide evidence that STAT3 can act as a central regulator of cell metabolism. STAT3C/C MEFs are resistant to apoptosis and senescence and they show reduced mitochondrial activity but activate aerobic glycolysis, as shown by enhanced expression of master regulators of glycolysis such as PDK-1 and HIF-1α, and by increased lactate production, glucose avidity and sensitivity to glycolysis inhibitors. HIF-1α is up-regulated in the STAT3C/C MEFs and is responsible for the glycolytic phenotype. Moreover, inhibition of STAT3 activity in STAT3-addicted tumour cell lines restores mitochondrial activity and down-regulates glycolytic metabolism, preceding the induction of massive apoptosis. Thus, the essential role observed for STAT3 in so many different cancer cell types may be partly explained by its ability to act as a molecular switch of cellular metabolism triggering abnormal cell survival/proliferation.
2010-11-17 | GSE21507 | GEO
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Project description:Phosphorylation of receptor tyrosine kinases (RTKs) has a key role in cellular functions contributing to the malignant phenotype of tumor cells. We and others have previously demonstrated that RTK ROR1 is overexpressed in chronic lymphocytic leukemia (CLL). Silencing siRNA downregulated ROR1 and induced apoptosis of CLL cells. In the present study we analysed ROR1 isoforms and the phosphorylation pattern in CLL cells (n=38) applying western blot and flow-cytometry using anti-ROR1 antibodies and an anti-phospho-ROR1 antibody against the TK domain. Two major ROR1 bands with the size of 105 and 130 kDa respectively were identified, presumably representing unglycosylated (immature) and glycosylated (mature) ROR1 respectively as well as a 260 kDa band which may represent dimerized ROR1. A ROR1 band of 64 kDa that may correspond to a C-terminal fragment was also noted, present only in the nucleus. The 105 kDa ROR1 isoform was more frequently expressed in non-progressive as compared to progressive CLL patients (p=0.03). The 64, 105, 130 and 260 kDa bands were constitutively phosphorylated both at tyrosine and serine residues. Phosphorylation intensity of the mature (130 kDa) isoform was significantly higher in progressive than in non-progressive disease (p<0.001). Incubation of CLL cells with a mouse anti-ROR1 KNG or an anti-ROR1 CRD mAb respectively induced dephosphorylation of ROR1 before entering apoptosis. In conclusion CLL cells expressed different isoforms of ROR1 which were constitutively phosphorylated. The mature, phosphorylated ROR1 isoform was associated with a progressive disease stage. Targeting ROR1 by mAbs induced specific dephosphorylation and leukemic cell death. ROR1 might be an interesting therapeutic target.
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