Atypical activation of signaling downstream of inactivated Bcr-Abl mediates chemoresistance in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) epitomises successful targeted therapy, where inhibition of tyrosine kinase activity of oncoprotein Bcr-Abl1 by imatinib, induces remission in 86% patients in initial chronic phase (CP). However, in acute phase of blast crisis, 80% patients show resistance, 40% among them despite inhibition of Bcr-Abl1 activity. This implies activation of either Bcr-Abl1- independent signalling pathways or restoration of signalling downstream of inactive Bcr-Abl1. In the present study, mass spectrometry and subsequent in silico pathway analysis of differentiators in resistant CML-CP cells identified key differentiators, 14-3-3ε and p38 MAPK, which belong to Bcr-Abl1 pathway. Their levels and activity respectively, indicated active Bcr-Abl1 pathway in CML-BC resistant cells, though Bcr-Abl1 is inhibited by imatinib. Further, contribution of these components to resistance was demonstrated by inhibition of Bcr-Abl1 down-stream signalling by knocking-out of 14-3-3ε and inhibition of p38 MAPK activity. The observations merit clinical validation to explore their translational potential.

Disruption of desmosome function leads to increased centrosome clustering in 14-3-3γ-knockout cells with supernumerary centrosomes.

14-3-3 proteins are conserved, dimeric, acidic proteins that regulate multiple cellular pathways. Loss of either 14-3-3ε or 14-3-3γ leads to centrosome amplification. However, we find that while the knockout of 14-3-3ε leads to multipolar mitoses, the knockout of 14-3-3γ results in centrosome clustering and pseudo-bipolar mitoses. 14-3-3γ knockouts demonstrate compromised desmosome function and a decrease in keratin levels, leading to decreased cell stiffness and an increase in centrosome clustering. Restoration of desmosome function increased multipolar mitoses, whereas knockdown of either plakoglobin or keratin 5 led to decreased cell stiffness and increased pseudo-bipolar mitoses. These results suggest that the ability of the desmosome to anchor keratin filaments maintains cell stiffness, thus inhibiting centrosome clustering, and that phenotypes observed upon 14-3-3 loss reflect the dysregulation of multiple pathways.

Plakoglobin localization to the cell border restores desmosome function in cells lacking 14-3-3γ.

Desmosomes are cell-cell adhesion junctions that anchor intermediate filaments. Loss of 14-3-3γ in HCT116 cells led to defects in desmosome assembly due to a decrease in the transport of Plakoglobin (PG) to the cell border thus disrupting desmosome formation. Desmosome formation in cells lacking 14-3-3γ was restored by artificially localizing PG to the cell border by fusing it to EGFP-f (PG-EGFP-f). These results suggest that a major role of 14-3-3γ in desmosome assembly is to transport PG to the cell border leading to the initiation of desmosome formation.