P210 Tyrosine Kinase Regulates the Expression of 14-3-3sigma in Hematopoietic Progenitors Expressing the bcr-abl Fusion Gene.

The 14-3-3sigma is a member of a highly conserved family of acidic proteins that bind to serine/threonine-phosphorylated residues in a context specific manner analogous to the Src homology 2 (SH2)- and phospho-tyrosine- binding domains. Its central role in the subcellular distribution and transcriptional activity of partners molecules controlling cell cycle progression, survival, senescence, adhesion and metabolism made 14-3-3sigma isoform the most directly linked to cancer pathogensis and progression. Here we provide evidence for its involvement in deregulated proliferation and extended survival of Chronic Myeloid Leukemia (CML) hematopoietic progenitors. In LAMA cell line and CD34+ cells of CML patients at clinical diagnosis p210 bcr-abl dephosphorylation and enzymatic activity inhibition induced by in vitro exposure to 1 microM tyrosine kinase (TK) inhibitor Imatinib mesylate (IM) were associated with a significant reduction of 14-3-3sigma transcript and protein. 14-3-3sigma reduced expression arises from transcriptional mechanisms and involves epigenetic modifications of chromatin structure. In LAMA cell line PCR amplification of DNA from immunoprecipitated chromatin (ChIP) revealed histone H4 deacetylation at a discrete region of 14-3-3sigma promoter critical for gene transcription since 2^nd up to 24^th hour of IM treatment. Conversely, bisulfite-SSCP sequencing of PCR amplification of gene 5′ coding sequences encompassing the transcription start site did not show significant differences in CpG methylation status relative to p210 TK activity. Up-modulation and increased nuclear import of p27Kip1, downmodulation of cyclin D1 and Cdc25A levels, Bad dephosphorylation at Ser¹³⁶ and Bax translocation to mitochondria proceeding from 14-3-3sigma reduction gave rise to cell cycle arrest in late G1-/early S-phase and committment to apoptotic death. Interestingly, higher levels of 14-3-3sigma protein were associated with the development of IM resistance in vitro and in vivo. To conclude, our study supports a role of 14-3-3sigma in the pathogenesis of CML hematopoiesis as well as in the disease progression towards a drug-resistant phenotype mostly due to its regulatory control on pro-apoptotic and growth-arrest proteins downstream of forkhead transcription factor Foxo3a and c-jun N-terminal kinase (JNK).