Fusion proteins deriving from translocations involving ALK gene, have a clear oncogenic potential: the aberrant constitutively activation of the tyrosine kinase domain enhances cell proliferation and survival leading also to cytoskeletal rearrangements and changes in cell shape. ALK activates different well-characterized pathways through the interaction with downstream molecules that trigger intracellular signalling cascades. In particular, the Ras-ERK (Fig 10) pathway is essential mostly for ALCL proliferation, whereas the JAK3-STAT3 pathway (Fig 11) and the PI3K-Akt (Fig 12) pathway have been shown to be vital primarily for cell survival and phenotypic changes [136]. Most of the knowledge we have about ALK signalling came from studies on NPM-ALK fusion protein, but it is conceivable to think that all the other variants could act on the same downstream pathways and with the same mode of action assuming that dimerization occurs.
The increased growth of ALCL is mainly attributed to the activation of Ras-ERK pathway.
In ALCL cells the chimera functions as a docking molecule for several downstream adaptors or scaffolding molecules such as IRS-1, SHC and SRC, which bind to the fusion protein at level of specific tyrosine residues of ALK (Fig 10). These mediators are able to activate Ras, which in turn phosphorylates ERK1 and ERK2. The SHP2/GRB2 complex interacts with ALK and SHC to enhance phosphorylation of ERK family members through SRC. Activation of phosholipase C-γ (PLCγ) is also thought to contribute to the fusion protein-mediated transformation. PLC-γ is directly bound and activated by ALK. This association leads to the formation of diacylglycerol (DAG) and inositol triphosphate (IP3), which, in turn, mobilize calcium stores from the endoplasmic reticulum and activate protein kinase C (PKC). Activated ERK signalling pathway and phosphorilated JUN N-terminal kinase (JNK) with its downstream targets (JUN and AP1), result in uncontrolled cell-cycle progression and cell growth due to the downregulation of p21 and concomitant upregulation of cyclin D3 and cyclin A. As reported in Fig 10, ERK signalling pathway is also able in turn to activate mammalian target of rapamycin (mTOR) which leads to the phosphorylation of the mTOR targets with the final end to promote cell-cycle progression [136].
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Figure 10. ALK and Ras-ERK pathway. NPM-ALK is depicted as example for an oncogenic fusion protein involving ALK.
Survival in ALCL is promoted by the activity of the key player Signal Transducer and Activator of Transcription 3 (STAT3) (Fig 11). STAT3 can be directly phosphorylated and so activated by the fusion protein or, the fusion transcript can activate JAK3, which in turn can contribute to STAT3 activation as shown in Fig 11. SHP1 is reported to be a negative regulator by dephosphorilating ALK, but in ALCL it is lost owing to gene methylation, resulting in the enhancement of STAT3 activation. In these cells, STAT3 allows the enhanced transcription of both anti-apoptotic factors and cell cycle regulators such as BCL2, BCL-XL, C/EBPβ, Survivin, MCL1 and different cyclins. The transforming property of the chimeras seems to be mediated also by STAT5B, although the mechanism of activation in this case requires JAK2. On the contrary, STAT5A has been reported to act as a tumor suppressor in ALCL, where it is epigenetically silenced and, if re-expressed, in turn an inhibit fusion proteins expression [136].
TK domain
Introduction
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Figure 11. ALK and JAK3-STAT3 pathway. NPM-ALK is depicted as example for an oncogenic fusion protein involving ALK.
NPM-ALK or another variant translocation binds to and activates PI3K through the regulatory p85 subunit of PI3K in ALCL cells, leading to the phosphorylation of its downstream effectors AKT1 and AKT2, which in turn enhance the survival of cells by blocking the function of pro-apoptotic proteins, such as BAD (Fig 12). AKT1 and AKT2 are able to phosphorylate FOXO3A, thus blocking FOXO3A-mediated transcription of target genes that promote apoptosis, cell-cycle arrest (p27 and Cyclin D2) and metabolic processes (BIM), forcing in this way G1 phase cell-cycle block. mTOR can sinergistically act promoting the survival pathway, leading to the transcription of its target genes such as p70S6K, S6RP and EIF4EBP1 (Fig 12) [136].
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Figure 12. ALK and PI3K-Akt pathway. NPM-ALK is depicted as example for an oncogenic fusion protein involving ALK.
It has been reported that NPM-ALK could also activate c-Src kinase, a tyrosine kinase receptor that plays a relevant role in cell migration, as well as in cell proliferation and growth. The chimera following its association with a specific tyrosine residue in position 418 activates c-Src. Src family kinases may also contribute to the sustained activation state of Cdc42 in ALCL cells, responsible of the regulation of the shape and migration of ALCL cells [151].
Many of the members involved in the activated pathways could be potential therapeutic targets: treatment of ALCL cells with specific inhibitors (Mek, ERK, Ras, mTOR, STAT3, JAK3, Akt) or RNA interference leads to a cell-cycle arrest, increase of apoptotic cells rate and proliferation arrest. It is plausible to think the clinical use of a combination of these drugs together with specific anti-ALK small molecules in order to obtain a synergistic effect or to use them in case of acquired resistance to ALK inhibitors.
Introduction