Inhibition of STAT3 signaling leads to apoptosis of leukemic large granular lymphocytes and decreased Mcl-1 expression
P.K. Epling-Burnette, Jin Hong Liu, Robyn Catlett-Falcone, James Turkson, Marc Oshiro, Ravi Kothapalli, Yongxiang Li, Ju-Ming Wang, Hsin-Fang Yang-Yen, James Karras, Richard Jove, Thomas P. Loughran Jr.
P.K. Epling-Burnette, Jin Hong Liu, Robyn Catlett-Falcone, James Turkson, Marc Oshiro, Ravi Kothapalli, Yongxiang Li, Ju-Ming Wang, Hsin-Fang Yang-Yen, James Karras, Richard Jove, Thomas P. Loughran Jr.
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Inhibition of STAT3 signaling leads to apoptosis of leukemic large granular lymphocytes and decreased Mcl-1 expression

Abstract

Large granular lymphocyte (LGL) leukemia is characterized by the expansion of antigen-activated cytotoxic T lymphocytes. These leukemic cells are resistant to Fas-mediated apoptosis despite expressing high levels of Fas. We found that leukemic LGL from 19 patients displayed high levels of activated STAT3. Treatment of leukemic LGL with the JAK-selective tyrosine kinase inhibitor AG-490 induced apoptosis with a corresponding decrease in STAT-DNA binding activity. Moreover, using an antisense oligonucleotide approach to diminish STAT3 expression, we found that Fas sensitivity was restored in leukemic LGL. AG-490–induced apoptosis in leukemic LGL was independent of Bcl-xL or Bcl-2 expression. However, we found that the Bcl-2–family protein Mcl-1 was significantly reduced by AG-490 treatment. Activated STAT3 was shown to bind an SIE-related element in the murine mcl-1 promoter. Using a luciferase reporter assay, we demonstrated that v-src overexpression in NIH3T3 induced STAT3-dependent transcriptional activity from the mcl-1 promoter and increased endogenous Mcl-1 protein levels. We conclude that STAT3 activation contributed to accumulation of the leukemic LGL clones. These findings suggest that investigation should focus on novel strategies targeting STAT3 in the treatment of LGL leukemia.

Authors

P.K. Epling-Burnette, Jin Hong Liu, Robyn Catlett-Falcone, James Turkson, Marc Oshiro, Ravi Kothapalli, Yongxiang Li, Ju-Ming Wang, Hsin-Fang Yang-Yen, James Karras, Richard Jove, Thomas P. Loughran Jr.

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STAT3 DNA-binding to an SIE-like element in the Mcl-1 promoter and trans...
STAT3 DNA-binding to an SIE-like element in the Mcl-1 promoter and transcription regulation. (a) EMSA was performed using nuclear extracts from U266 (lanes 1–6), leukemic LGLs (patient 10169; lanes 7–12), and v-src–transformed NIH3T3 (lanes 13–17) with the Mcl-1 SIE probe labeled with P32. Competition assays were performed by adding excess cold oligonucleotide (×100) for MclSIE (lanes 2, 8, and 14), hSIE (lanes 3, 9, and 15), and nonspecific competitor (FIRE, lanes 4, 10, and 16). Blocking or supershift analysis was performed by adding anti-STAT1 antibody (lanes 5 and 11) or anti-STAT3 antibody (lanes 6, 12, and 17), respectively. (b) EMSA analysis with mcl-1 SIE using nuclear extracts of leukemic LGLs (lanes 1–8) or U266 (lanes 9 and 10) treated with solvent control (DMSO, lanes 1, 3, 5, 7, and 9) or AG-490 (lanes 2, 4, 6, 8, and 10). Results shown are representative of two experiments. (c) Transcriptional analysis was performed by transfection of NIH3T3 with 4 μg of pGL2 mcl-1 (p-203/+10mcl-luc), pGL2 mSIE mcl-1 (p-203/+10mS), and pLucSRE-luciferase alone or in combination with 4 μg v-src, pSG5-STAT3β, and pSG5 vector control expression vectors. The values shown are normalized by cotransfection with CMV-β–galactosidase expression vectors. (d) Western blot analysis in NIH3T3 without (–) or with (+) v-src overexpression for Mcl-1, STAT3, and β-actin protein expression.