Thrombotic biomarkers for risk prediction of malignant disease recurrence in patients with early stage breast cancer.

In cancer patients, hypercoagulability is a common finding. It has been associated with an increased risk of venous thromboembolism, but also to tumor proliferation and progression. In this prospective study of a large cohort of breast cancer patients, we aimed to evaluate whether pre-chemotherapy abnormalities in hemostatic biomarkers levels: (i) are associated with breast cancer-specific clinico-pathological features; and (ii) can predict for disease recurrence. D-dimer, fibrinogen, prothrombin fragment 1+2, and FVIIa/antithrombin levels were measured in 701 early-stage resected breast cancer patients candidate to adjuvant chemotherapy and prospectively enrolled in the HYPERCAN study. Significant prognostic parameters for disease recurrence were identified by Cox regression multivariate analysis and used for generating a risk assessment model. Pre-chemotherapy D-dimer, fibrinogen, and pro-thrombin fragment 1+2 levels were significantly associated with tumor size and lymph node metastasis. After 3.4 years of follow up, 71 patients experienced a recurrence. Cox multivariate analysis identified prothrombin fragment 1+2, tumor size, and Luminal B HER2-negative or triple negative molecular subtypes as independent risk factors for disease recurrence. Based on these variables, we generated a risk assessment model that significantly differentiated patients at low- and high-risk of recurrence (cumulative incidence: 6.2 vs 20.7%; Hazard Ratio=3.5; P<0.001). Our prospective clinical and laboratory data from the HYPERCAN study were crucial for generating a scoring model for assessing risk of disease recurrence in resected breast cancer patients, candidate to systemic chemotherapy. This finding stimulates future investigations addressing the role of plasma prothrombin fragment 1+2 in the management of breast cancer patients to provide the rationale for new therapeutic strategies. (The HYPERCAN study is registered at clinicaltrials.gov identifier 02622815).

FANCM c.5791C>T nonsense mutation (rs144567652) induces exon skipping, affects DNA repair activity and is a familial breast cancer risk factor.

Numerous genetic factors that influence breast cancer risk are known. However, approximately two-thirds of the overall familial risk remain unexplained. To determine whether some of the missing heritability is due to rare variants conferring high to moderate risk, we tested for an association between the c.5791C>T nonsense mutation (p.Arg1931*; rs144567652) in exon 22 of FANCM gene and breast cancer. An analysis of genotyping data from 8635 familial breast cancer cases and 6625 controls from different countries yielded an association between the c.5791C>T mutation and breast cancer risk [odds ratio (OR) = 3.93 (95% confidence interval (CI) = 1.28-12.11; P = 0.017)]. Moreover, we performed two meta-analyses of studies from countries with carriers in both cases and controls and of all available data. These analyses showed breast cancer associations with OR = 3.67 (95% CI = 1.04-12.87; P = 0.043) and OR = 3.33 (95% CI = 1.09-13.62; P = 0.032), respectively. Based on information theory-based prediction, we established that the mutation caused an out-of-frame deletion of exon 22, due to the creation of a binding site for the pre-mRNA processing protein hnRNP A1. Furthermore, genetic complementation analyses showed that the mutation influenced the DNA repair activity of the FANCM protein. In summary, we provide evidence for the first time showing that the common p.Arg1931* loss-of-function variant in FANCM is a risk factor for familial breast cancer.

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

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.

Phospholipid-dependent procoagulant activity is highly expressed by circulating microparticles in patients with essential thrombocythemia.

This study evaluates the functional procoagulant features of plasma microparticle (MP) to explore the MP contribution to the hypercoagulable state of patients with essential thrombocythemia (ET). Platelet-free plasma samples were obtained from 73 ET patients (37 positive for the JAK2V617F mutation) and 72 control subjects. The calibrated automated thrombogram (CAT) was performed in plasma samples to determine thrombin generation of MP-associated tissue factor (TF) and procoagulant phospholipid (PPL) activity, and the STA Procoag PPL assay to measure MP-PPL activity only. Both thrombin generation and PPL procoagulant activities were found significantly elevated in ET patients compared to controls, and were associated to significantly higher levels of TF antigen and FVIIa/AT complex. Thrombin generation was significantly greater in JAK2-V617F positive compared to JAK2-V617F negative patients and normal subjects. Significant correlations were found between the PPL-assay and the different parameters of the CAT assay. No difference was seen between the thrombosis and no thrombosis group. Prospective studies are needed to test whether MP-associated thrombin generation and procoagulant activity may predict for thrombosis in these patients.

Hemostatic Profile and Serological Response of Patients with Immune Thrombotic Thrombocytopenic Purpura after Receiving BNT162b2 Vaccine: A Prospective Study.

INTRODUCTION: Coronavirus disease is a clinical challenge for patients with autoimmune conditions. Patients affected by immune thrombotic thrombocytopenic purpura (iTTP) are particularly vulnerable to SARS-CoV-2 infection. Protecting these patients with vaccination is therefore mandatory, although concerns may exist on a possible increased thrombotic risk or risk of disease relapse after vaccine exposure. So far, there is no information on serological response and hemostatic activation in iTTP patients after SARS-CoV-2 vaccination. MATERIALS AND METHODS: In this study, in April 2021, we enrolled iTTP patients in clinical remission and on regular outpatient follow-up to receive the first and second dose BNT162b2 vaccine as a part of a prospective trial aimed at monitoring for 6 months after vaccination the occurrence of subclinical laboratory signs of clotting activation, as well as overt thrombotic complications or disease relapse. The seroconversion response was monitored in parallel. The results were compared with those of control non-iTTP subjects. RESULTS: A moderate decrease of ADAMTS-13 activity was recorded at 3 and 6 months in five patients with normal values at baseline, while an ADAMTS-13 relapse occurred at 6 months in one patient. Abnormalities in the endothelium activation biomarkers postvaccination were observed in iTTP patients compared with controls. The immunological response to vaccine was overall positive. No clinical iTTP relapses or thrombotic events manifested in the 6 month-follow-up after vaccination. CONCLUSION: The results of this study are in favor of efficacy and safety of mRNA vaccines in patients with iTTP, and highlight the importance of long-term monitoring of iTTP patients.

Utility of the Khorana and the new-Vienna CATS prediction scores in cancer patients of the HYPERCAN cohort.

BACKGROUND: Risk assessment models (RAMs) are relevant approaches to identify cancer outpatients at high risk of venous thromboembolism (VTE). Among the proposed RAMs, the Khorana (KRS) and the new-Vienna CATS risk scores have been externally validated in ambulatory patients with cancer. OBJECTIVES: To test KRS and new-Vienna CATS scores in 6-month VTE prediction and mortality in a large prospective cohort of metastatic cancer outpatients during chemotherapy. PATIENTS/METHODS: Newly diagnosed patients with metastatic non-small cell lung, colorectal, gastric, or breast cancers were analyzed (n = 1286). The cumulative incidence of objectively confirmed VTE was estimated with death as a competing risk and multivariate Fine and Gray regression. RESULTS: Within 6 months, 120 VTE events (9.7%) occurred. The KRS and the new-Vienna CATS scores showed comparable c-stat. Stratification by KRS provided VTE cumulative incidences of 6.2%, 11.4%, and 11.5% in the low-, intermediate-, and high-risk categories, respectively (p = ns), and of 8.5% vs. 11.8% (p = ns) in the low- vs. high-risk group by the single 2-point cut-off value stratification. Using a pre-defined 60-point cut-off by the new-Vienna CATS score, 6.6% and 12.2% cumulative incidences were obtained in the low- and high-risk groups, respectively (p < 0.001). Furthermore, having a KRS ≥2 = or a new-Vienna CATS score >60 points was also an independent risk factor for mortality. CONCLUSION: In our cohort, the 2 RAMs showed a comparable discriminating potential; however, after the application of cut-off values, the new-Vienna CATS score provided statistically significant stratification for VTE. Both RAMs proved to be effective in identifying patients at increased risk of mortality.

A New Risk Prediction Model for Venous Thromboembolism and Death in Ambulatory Lung Cancer Patients.

(1) Background: Venous thromboembolism (VTE) is a frequent complication in ambulatory lung cancer patients during chemotherapy and is associated with increased mortality. (2) Methods: We analyzed 568 newly diagnosed metastatic lung cancer patients prospectively enrolled in the HYPERCAN study. Blood samples collected before chemotherapy were tested for thrombin generation (TG) and a panel of hemostatic biomarkers. The Khorana risk score (KRS), new-Vienna CATS, PROTECHT, and CONKO risk assessment models (RAMs) were applied. (3) Results: Within 6 months, the cumulative incidences of VTE and mortality were 12% and 29%, respectively. Patients with VTE showed significantly increased levels of D-dimer, FVIII, prothrombin fragment 1 + 2, and TG. D-dimer and ECOG performance status were identified as independent risk factors for VTE and mortality by multivariable analysis and utilized to generate a risk score that provided a cumulative incidence of VTE of 6% vs. 25%, death of 19% vs. 55%, and in the low- vs. high-risk group, respectively (p < 0.001). While all published RAMs significantly stratified patients for risk of death, only the CATS and CONKO were able to stratify patients for VTE. (4) Conclusions: A new prediction model was generated to stratify lung cancer patients for VTE and mortality risk, where other published RAMs failed.

Thrombin Generation and D-Dimer for Prediction of Disease Progression and Mortality in Patients with Metastatic Gastrointestinal Cancer.

Background: the tight and reciprocal interaction between cancer and hemostasis has stimulated investigations on the possible role of hemostatic biomarkers in predicting specific cancer outcomes, such as disease progression (DP) and overall survival (OS). In a prospective cohort of newly diagnosed metastatic gastrointestinal (GI) cancer patients from the HYPERCAN study, we aimed to assess whether the hemostatic biomarker levels measured before starting any anticancer therapy may specifically predict for 6-months DP (6m-DP) and for 1-year OS (1y OS). Methods: plasma samples were collected and tested for thrombin generation (TG) as global hemostatic assay, and for D-dimer, fibrinogen, and prothrombin fragment 1 + 2 as hypercoagulation biomarkers. DP and mortality were monitored during follow-up. Results: A prospective cohort of 462 colorectal and 164 gastric cancer patients was available for analysis. After 6 months, DP occurred in 148 patients, providing a cumulative incidence of 24.8% (21.4−28.4). D-dimer and TG endogenous thrombin potential (ETP) were identified as independent risk factors for 6m-DP by multivariate Fine−Gray proportional hazard regression model corrected for age, cancer site, and >1 metastatic site. After 1 year, we observed an OS of 75.7% (71.9−79.0). Multivariate Cox regression analysis corrected for age, site of cancer, and performance status identified D-dimer and ETP as independent risk factors for 1y OS. Patients with one or both hemostatic parameters above the dichotomizing threshold were at higher risk for both 6m-DP and 1-year mortality. Conclusion.: in newly diagnosed metastatic GI cancer patients, pretreatment ETP and D-dimer appear promising candidate biomarkers for predicting 6m-DP and 1y OS. In this setting, for the first time, the role of TG as a prognostic biomarker emerges in a large prospective cohort.

Validation of the Role of Thrombin Generation Potential by a Fully Automated System in the Identification of Breast Cancer Patients at High Risk of Disease Recurrence.

Background The measurement of thrombin generation (TG) potential by the calibrated automated thrombogram (CAT) assay provides a strong contribution in identifying patients at high risk of early disease recurrence (E-DR). However, CAT assay still needs standardization and clinical validation. Objective In this study, we aimed to validate the role of TG for E-DR prediction by means of the fully automated ST Genesia system. Methods A prospective cohort of 522 patients from the HYPERCAN study with newly diagnosed resected high-risk breast cancer was included. Fifty-two healthy women acted as controls. Plasma samples were tested for protein C, free-protein S, and TG by ST Genesia by using the STG-ThromboScreen reagent with and without thrombomodulin (TM). Results In the absence of TM, patients showed significantly higher peak and ETP compared with controls. In the presence of TM, significantly lower inhibition of ETP and Peak were observed in patients compared with controls. E-DR occurred in 28 patients; these patients had significantly higher peak and endogenous thrombin potential (ETP) in the absence of TM compared with disease-free patients. Multivariable analysis identified mastectomy, luminal B HER2-neg, triple negative subtypes, and ETP as independent risk factors for E-DR. These variables were combined to generate a risk assessment score, able to stratify patients in three-risk categories. The E-DR rates were 0, 4.7, and 13.5% in the low-, intermediate-, and high-risk categories (hazard ratio = 8.7; p < 0.05, low vs. high risk). Conclusion Our data validate the ETP parameter with a fully automated standardized system and confirm its significant contribution in identifying high-risk early breast cancer at risk for E-DR during chemotherapy.

Thrombin generation predicts early recurrence in breast cancer patients.

BACKGROUND: Cancer patients present with a hypercoagulable state often associated with poor disease prognosis. OBJECTIVES: This study aims to evaluate whether thrombin generation (TG), a global coagulation test, may be a useful tool to improve the identification of patients at high risk of early disease recurrence (ie, E-DR within 2 years) after breast cancer surgery. PATIENTS/METHODS: A cohort of 522 newly diagnosed patients with surgically resected high-risk breast cancer were enrolled in the ongoing prospective HYPERCAN study. TG potential was measured in plasma samples collected before starting systemic chemotherapy. Significant predictive hemostatic and clinic-pathological parameters were identified in the derivation cohort by Cox regression analysis. A risk prognostic score for E-DR was generated in the derivation and tested in the validation cohort. RESULTS: After a median observation period of 3.4 years, DR occurred in 51 patients, 28 of whom were E-DR. E-DR subjects presented with the highest TG values as compared to both late-DR (from 2 to 5 years) and no relapse subjects (P < .01). Multivariate analysis in the derivation cohort identified TG, mastectomy, triple negative and Luminal B HER2-neg molecular subtypes as significant independent predictors for E-DR, which were utilized to generate a risk assessment score. In the derivation and validation cohorts, E-DR rates were 2.3% and 0% in the low-risk, 10.1% and 6.3% in the intermediate-risk, and 18.2% and 16.7%, in the high-risk categories, respectively. CONCLUSIONS: Inclusion of TG in a risk-assessment model for E-DR significantly helps the identification of operated breast cancer patients at high risk of very early relapse.

Platelet haemostatic properties in ß-thalassaemia: the effect of blood transfusion.

BACKGROUND: Patients with thalassaemia may have thromboembolic events and, even without thrombosis, they have a subclinical hypercoagulable state. In this setting, several coagulation laboratory abnormalities have been described, but thus far no studies have explored the contribution of platelet adhesive and procoagulant properties to blood clotting activation. In this study, we dissected the platelet procoagulant effect and influence of blood transfusions on haemostasis and platelet function in thalassaemic patients. MATERIAL AND METHODS: Sixteen subjects with thalassaemia were studied (9 with transfusion-dependent ß-thalassaemia, 7 "trait" carriers). Splenectomised and non-splenectomised patients undergoing blood transfusion were compared. All splenectomised patients were then compared to "trait" carriers and to healthy controls (n=9). The following parameters were measured in transfusion-dependent patients before and after monthly transfusions and compared to those of controls: levels of platelet surface activation markers (P-selectin, tissue factor, and fibrinogen), whole blood platelet aggregation, tissue factor or adenosine diphosphate (ADP)-induced platelet thrombin generation (TG) potential, and D-dimer. RESULTS: Before transfusion, platelets from splenectomised patients showed significantly higher ADP-induced tissue factor expression, ADP- and collagen-induced platelet aggregation and TG potential than those from non-splenectomised patients and controls. Blood transfusion in splenectomised patients reduced platelet activation, aggregation and TG potential. DISCUSSION: Splenectomised patients with ß-thalassaemia had a prothrombotic state, characterised by enhanced platelet reactivity and function, and high platelet-induced TG potential. One hour after blood transfusions platelet and coagulation parameters improved, supporting the hypothesis that transfusion might have a protective role on platelet haemostatic status.

Structural insights into the ATP binding pocket of the anaplastic lymphoma kinase by site-directed mutagenesis, inhibitor binding analysis, and homology modeling.

Anaplastic lymphoma kinase (ALK) is a valid target for anticancer therapy; however, potent ALK inhibitors suitable for clinical use are lacking. Because the majority of described kinase inhibitors bind in the ATP pocket of the kinase domain, we have characterized this pocket in ALK using site-directed mutagenesis, inhibition studies, and molecular modeling. Mutation of the gatekeeper residue, a key structural determinant influencing inhibitor binding, rendered the fusion protein, NPM/ALK, sensitive to inhibition by SKI-606 in the nanomolar range, while PD173955 inhibited the NPM/ALK mutant at micromolar concentrations. In contrast, both wild type and mutant NPM/ALK were insensitive to imatinib. Computer modeling indicated that docking solutions obtained with a homology model representing the intermediate conformation of the ALK kinase domain reflected closely experimental data. The good agreement between experimental and virtual results indicate that the ALK molecular models described here are useful tools for the rational design of ALK selective inhibitors. In addition, 4-phenylamino-quinoline compounds may have potential as templates for ALK inhibitors.

An enzyme-linked immunosorbent assay to screen for inhibitors of the oncogenic anaplastic lymphoma kinase.

The discovery of novel anti-cancer drugs targeting anaplastic lymphoma kinase (ALK), an oncogenic tyrosine kinase, raises the need for in vitro assays suitable for screening compounds for ALK inhibition. To this aim we have developed and optimized an ALK-specific enzyme-linked immunosorbent assay that employs a novel ALK peptide substrate and purified ALK kinase domain.

ADP-induced platelet aggregation and thrombin generation are increased in Essential Thrombocythemia and Polycythemia Vera.

INTRODUCTION: Essential Thrombocythemia (ET) and Polycythemia Vera (PV) patients are characterized by an increased rate of thrombotic complications and by several abnormalities of platelets, more pronounced in JAK2V617F positive patients. The aim of this study was to characterize the platelet aggregation as well as the platelet procoagulant potential induced by several different agonists in ET and PV patients. MATERIALS AND METHODS: Venous blood samples were obtained from 65 ET and 51 PV patients. Whole blood impedance aggregometry was utilized to characterize platelet aggregation induced by collagen, ADP, thrombin receptor activating peptide and arachidonic acid, while the Calibrated Automated Thrombogram (CAT) assay was used to determine the thrombin generation (TG) potential induced by ADP in platelet-rich plasma. CAT assay was also performed in the presence of annexin V to evaluate the contribution of platelet phospholipids to TG. RESULTS AND CONCLUSIONS: ADP-induced platelet aggregation and TG were significantly increased in ET and PV patients compared to controls. The highest values were observed in JAK2V617F positive patients and in patients on aspirin. In these subjects, annexin V was less effective in inhibiting both basal and ADP-induced TG. This study demonstrates for first time that platelets from ET and PV patients are more responsive to the ADP stimulus, in terms of both increased platelet aggregation, and enhanced TG, particularly in the JAK2V617F positive patients. Our data support the hypothesis that the use of ADP receptor inhibitors, in addition to aspirin, might be considered in the prevention of thrombosis in these conditions, by allowing a more complete inhibition of platelet functions.

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.

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.

Pathogenesis and treatment of thrombohemorrhagic diathesis in acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is a distinct subtype of myeloid leukemia characterized by t(15;17) chromosomal translocation, which involves the retinoic acid receptor-alpha (RAR-alpha). APL typically presents with a life-threatening hemorrhagic diathesis. Before the introduction of all-trans retinoic acid (ATRA) for the cure of APL, fatal hemorrhages due, at least in part, to the APL-associated coagulopathy, were a major cause of induction remission failure. The laboratory abnormalities of blood coagulation found in these patients indicate the occurrence of a hypercoagulable state. Major determinants of the coagulopathy of APL are endogenous factors expressed by the leukemic cells, including procoagulant factors, fibrinolytic proteins, and non-specific proteolytic enzymes. In addition, these cells have an increased capacity to adhere to the vascular endothelium, and to secrete inflammatory cytokines [i.e. interleukin-1beta (IL-1beta) and tumor necrosis factor (TNF-alpha)], which in turn stimulate the expression of prothrombotic activities by endothelial cells and leukocytes. ATRA can interfere with each of the principal hemostatic properties of the leukemic cell, thus reducing the APL cell procoagulant potential, in parallel to the induction of cellular differentiation. This effect occurs in vivo, in the bone marrow of APL patients receiving ATRA, and is associated with the improvement of the bleeding symptoms. Therapy with arsenic trioxide (ATO) also beneficially affects coagulation in APL. However, early deaths from bleeding still remain a major problem in APL and further research is required in this field. In this review, we will summarize our current knowledge of the pathogenesis of the APL-associated coagulopathy and will overview the therapeutic approaches for the management of this complication.

Dissection of the RET/ß-catenin interaction in the TPC1 thyroid cancer cell line.

The RET receptor tyrosine kinase is a member of the cadherin superfamily and plays a pivotal role in cell survival, differentiation and proliferation. Currently, 12 ret/ptc chimeric oncogenes, characterized by the fusion between the intracellular domain of RET and different activating genes, which can cause ligand-independent dimerization and constitutive activation, have been described. ß-catenin is usually involved in the maintenance of cell-to-cell adhesion and mediates the Wnt/ß-catenin pathway important during embryogenesis and in cellular malignant transformation. Recently, a novel mechanism of RET-mediated function through the ß-catenin pathway has been reported in multiple endocrine neoplasia type 2 and in sporadic thyroid carcinomas. Here, we investigated the effects of the ZD6474, a small molecule RET-inhibitor, on RET/ß-catenin interaction. We confirmed the ZD6474 mediated-inhibition of recombinant RET kinase and of growth of cells expressing RET/PTC. Interestingly, we firstly observed reduced cellular mobility and changed morphology of TPC1 treated cells suggesting that RET-inhibitor could affect ß-catenin cellular distribution as resulted in its co-immunoprecipitation with E-cadherin. We further investigated this hypothesis showing that TPC1 treated cells displayed predominantly ß-catenin cytosolic localization. Surprisingly, RET and ß-catenin co-immunoprecipitated in both ZD6474-treated and untreated TPC1 cells, suggesting that RET/ß-catenin interaction might not be affected by RET kinase inactivation. All together these results suggest that RET kinase activation is crucial for ß-catenin stabilization (pY654), localization and its signaling pathway activation but not for ß-catenin/RET physical interactions, in human papillary thyroid carcinomas. In conclusion, ZD6474, by inhibiting RET kinase, down-modulates ß-catenin pathway leading its recruitment to the membrane by E-cadherin.

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 approximately 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.

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.