Carmen Julia Tartari

Characterization of Some Molecular Mechanisms Governing Autoactivation of the Catalytic Domain of the Anaplastic Lymphoma Kinase

NPM/ALK is an oncogenic fusion protein expressed in ∼50% of anaplastic large cell lymphoma cases. It derives from the t(2;5)(p23;q35) chromosomal translocation that fuses the catalytic domain of the tyrosine kinase, anaplastic lymphoma kinase (ALK), with the dimerization domain of the ubiquitously expressed nucleophosmin (NPM) protein. Dimerization of the ALK kinase domain leads to its autophosphorylation and constitutive activation. Activated NPM/ALK stimulates downstream survival and proliferation signaling pathways leading to malignant transformation. Herein, we investigated the molecular mechanisms of autoactivation of the catalytic domain of ALK. Because kinases are typically regulated by autophosphorylation of their activation loops, we systematically mutated (Tyr → Phe) three potential autophosphorylation sites contained in the “YXXXYY” motif of the ALK activation loop, and determined the effect of these mutations on the catalytic activity and biological function of NPM/ALK. We observed that mutation of both the second and third tyrosine residues (YFF mutant) did not affect the kinase activity or transforming ability of NPM/ALK. In contrast, mutation of the first and second (FFY), first and third (FYF), or all three (FFF) tyrosine residues impaired both kinase activity and transforming ability of NPM/ALK. Furthermore, a DFF mutant, in which the aspartic residue introduces a negative charge similar to a phosphorylated tyrosine, possessed catalytic activity similar to the YFF mutant. Together, our findings indicate that phosphorylation of the first tyrosine of the YXXXYY motif is necessary for the autoactivation of the ALK kinase domain and the transforming activity of NPM/ALK.

Microparticles in tumor progression

Microparticles (MP) are shed from the surface of activated or apoptotic blood cells and their levels in plasma reflect a balance between cell stimulation, proliferation, and death. MP production occurs through vesiculation of cell membranes, and involves cytoskeletal changes and a shift in the normal phospholipid asymmetry. The expression on the majority of MP of the anionic phosphatidylserine (PS) is responsible for the capacity of MP to support blood coagulation activation. In some cases, PS expression is also associated, in the same MP, with the presence of active Tissue Factor, the main activator of blood coagulation. Elevation in plasma levels of MP have been described in numerous clinical conditions, most of which also associated with an increased thrombotic risk. Particularly, MP have been found to be increased in both solid and hematological malignancies, including myeloproliferative neoplasms. A role of MP in tumor progression has been suggested by both in vitro and in vivo studies. Evidence exists that MP of platelet origin are the main players in this process, being rich in pro-angiogenic factors. The utility of measuring MP as a diagnostic and prognostic marker is currently a subject of intense investigation. The possibility to inhibit MP production by pharmacological interventions represents a future challenge.

Oncogenic Fusion Tyrosine Kinases as Molecular Targets for Anti-Cancer Therapy

Deregulated activation of protein tyrosine kinases (PTKs) is a frequent event underlying malignant transformation in many types of cancer. The formation of oncogenic fusion tyrosine kinases (FTKs) resulting from genomic rearrangements, represents a common mechanism by which kinases escape the strict controls that normally regulate their expression and activation. FTKs are typically composed of an N-terminal dimerisation domain, provided by the fusion partner protein, fused to the kinase domain of receptor or non-receptor tyrosine kinases (non-RTKs). Since FTKs do not contain extracellular domains, they share many characteristics with non-RTKs in terms of their properties and approaches for therapeutic targeting. FTKs are cytoplasmic or sometimes nuclear proteins, depending on the normal distribution of their fusion partner. FTKs no longer respond to ligand and are instead constitutively activated by dimerisation induced by the fusion partner. Unlike RTKs, FTKs cannot be targeted by therapeutic antibodies, instead they require agents that can cross the cell membrane as with non-RTKs. Here we review the PTKs known to be expressed as FTKs in cancer and the strategies for molecularly targeting these FTKs in anti-cancer therapy.

The ALK Gene, An Attractive Target for Inhibitor Development

Anaplastic Lymphoma Kinase (ALK) is a receptor tyrosine kinase that belongs to the Insulin receptor subfamily involved as full length receptor in neural development. Even if the expression of ALK protein is down-regulated in the adults, the ALK full length is expressed in different types of tumors. Moreover, chromosomal rearrangements, involving the alk gene, can occur leading the formation of different ALK fusion proteins characterized by the kinase domain of ALK fused to several partners that determine cellular localization. Structural investigation and characterization of the ALK kinase domain in absence of its crystal structure constituted the basis of development of ALK small molecule inhibitors. Here, we described normal function of the ALK receptor and its role in tumors; formation of the constitutively activated ALK fusion proteins and we reported an update of developed small molecule inhibitors of the ALK kinase activity.

Long Term Low Molecular Weight Heparin Anticoagulant Therapy Modulates Thrombin Generation and D-dimer in Patients with Cancer and Venous Thromboembolism

ABSTRACT We enrolled 62 consecutive patients with advanced stage cancers and venous thromboembolism (VTE), prospectively followed until 1 year. All patients received 6 month low-molecular-weight heparin (LMWH) therapy. We evaluated thrombin generation (TG) and D-dimer levels at different time points, to determine whether they were sensitive to LMWH and explore a possible association with VTE recurrence, bleeding, and overall survival. During LMWH, levels of TG and D-dimer significantly dropped. No VTE recurrences occurred, one patient had cancer-related intestinal hemorrhage. LMWH treatment was effective in controlling patient hypercoagulation. No VTE recurrences were detected. High D-dimer concentration was an independent predictor of poor survival.