Mutations in TP53 and JAK2 are independent prognostic biomarkers in B-cell precursor acute lymphoblastic leukaemia.

BACKGROUND: In B-cell precursor acute lymphoblastic leukaemia (B-ALL), the identification of additional genetic alterations associated with poor prognosis is still of importance. We determined the frequency and prognostic impact of somatic mutations in children and adult cases with B-ALL treated with Spanish PETHEMA and SEHOP protocols. METHODS: Mutational status of hotspot regions of TP53, JAK2, PAX5, LEF1, CRLF2 and IL7R genes was determined by next-generation deep sequencing in 340 B-ALL patients (211 children and 129 adults). The associations between mutation status and clinicopathological features at the time of diagnosis, treatment outcome and survival were assessed. Univariate and multivariate survival analyses were performed to identify independent prognostic factors associated with overall survival (OS), event-free survival (EFS) and relapse rate (RR). RESULTS: A mutation rate of 12.4% was identified. The frequency of adult mutations was higher (20.2% vs 7.6%, P=0.001). TP53 was the most frequently mutated gene (4.1%), followed by JAK2 (3.8%), CRLF2 (2.9%), PAX5 (2.4%), LEF1 (0.6%) and IL7R (0.3%). All mutations were observed in B-ALL without ETV6-RUNX1 (P=0.047) or BCR-ABL1 fusions (P<0.0001). In children, TP53mut was associated with lower OS (5-year OS: 50% vs 86%, P=0.002) and EFS rates (5-year EFS: 50% vs 78.3%, P=0.009) and higher RR (5-year RR: 33.3% vs 18.6% P=0.037), and was independently associated with higher RR (hazard ratio (HR)=4.5; P=0.04). In adults, TP53mut was associated with a lower OS (5-year OS: 0% vs 43.3%, P=0.019) and a higher RR (5-year RR: 100% vs 61.4%, P=0.029), whereas JAK2mut was associated with a lower EFS (5-year EFS: 0% vs 30.6%, P=0.035) and a higher RR (5-year RR: 100% vs 60.4%, P=0.002). TP53mut was an independent risk factor for shorter OS (HR=2.3; P=0.035) and, together with JAK2mut, also were independent markers of poor prognosis for RR (TP53mut: HR=5.9; P=0.027 and JAK2mut: HR=5.6; P=0.036). CONCLUSIONS: TP53mut and JAK2mut are potential biomarkers associated with poor prognosis in B-ALL patients.

Association of syntenin-1 with M-RIP polarizes Rac-1 activation during chemotaxis and immune interactions.

In this study, we describe that the PDZ protein syntenin-1 is a crucial element for the generation of signaling asymmetry during the cellular response to polarized extracellular cues. We analyze the role of syntenin-1 in the control of asymmetry in two independent models of T cell polarization--the migratory response to chemoattractants and the establishment of cognate interactions between T cells and antigen-presenting cells (APCs). A combination of mutant, biochemical and siRNA approaches demonstrate that syntenin-1 is vital for the generation of polarized actin structures such as the leading edge and the contact zone with APCs. We found that the mechanism by which syntenin-1 controls actin polymerization relies on its mandatory role for activation of the small GTPase Rac. Syntenin-1 controls Rac through a specific association with the myosin phosphatase Rho interacting protein (M-RIP), which occurs in response to phosphorylation of syntenin-1 by Src at Tyr4. Our data indicate the key role of syntenin-1 in the generation of functional asymmetry in T cells and provide a novel mechanistic link between receptor activation and actin polymerization and accumulation in response to extracellular stimulation.

EWI-2 association with α-actinin regulates T cell immune synapses and HIV viral infection.

EWI motif-containing protein 2 (EWI-2) is a member of the Ig superfamily that links tetraspanin-enriched microdomains to the actin cytoskeleton. We found that EWI-2 colocalizes with CD3 and CD81 at the central supramolecular activation cluster of the T cell immune synapse. Silencing of the endogenous expression or overexpression of a cytoplasmic truncated mutant of EWI-2 in T cells increases IL-2 secretion upon Ag stimulation. Mass spectrometry experiments of pull-downs with the C-term intracellular domain of EWI-2 revealed the specific association of EWI-2 with the actin-binding protein α-actinin; this association was regulated by PIP2. α-Actinin regulates the immune synapse formation and is required for efficient T cell activation. We extended these observations to virological synapses induced by HIV and found that silencing of either EWI-2 or α-actinin-4 increased cell infectivity. Our data suggest that the EWI-2-α-actinin complex is involved in the regulation of the actin cytoskeleton at T cell immune and virological synapses, providing a link between membrane microdomains and the formation of polarized membrane structures involved in T cell recognition.

MT1-MMP collagenolytic activity is regulated through association with tetraspanin CD151 in primary endothelial cells.

MT1-MMP plays a key role in endothelial function, as underscored by the angiogenic defects found in MT1-MMP deficient mice. We have studied the molecular interactions that underlie the functional regulation of MT1-MMP. At lateral endothelial cell junctions, MT1-MMP colocalizes with tetraspanin CD151 (Tspan 24) and its associated partner alpha3beta1 integrin. Biochemical and FRET analyses show that MT1-MMP, through its hemopexin domain, associates tightly with CD151, thus forming alpha3beta1 integrin/CD151/MT1-MMP ternary complexes. siRNA knockdown of HUVEC CD151 expression enhanced MT1-MMP-mediated activation of MMP2, and the same activation was seen in ex vivo lung endothelial cells isolated from CD151-deficient mice. However, analysis of collagen degradation in these experimental models revealed a diminished MT1-MMP enzymatic activity in confined areas around the cell periphery. CD151 knockdown affected both MT1-MMP subcellular localization and its inclusion into detergent-resistant membrane domains, and prevented biochemical association of the metalloproteinase with the integrin alpha3beta1. These data provide evidence for a novel regulatory role of tetraspanin microdomains on the collagenolytic activity of MT1-MMP and indicate that CD151 is a key regulator of MT1-MMP in endothelial homeostasis.

Multi-factorial modulation of colorectal carcinoma cells motility - partial coordination by the tetraspanin Co-029/tspan8.

Colorectal carcinoma cells Isreco1 display an ability to migrate controlled by a complex set of signals issued from the membrane. By comparing cells infected by mycoplasmas and mycoplasmas free cells, we have established that basal 2D migration is dependent on a double signal mediated by the collagen receptors integrins alpha1/2 and the Toll-Like receptor TLR2. The signal issued from mycoplasmas can be replaced by a TLR2 ligand and the functional effect is neutralized by silencing of MyD88. Following previous observation that downregulation of E-cadherin/p120 catenin increases cell motility, we now report that EGFR or CD44 inhibition have a similar effect on cell motility that is restricted to tetraspanin Co-029/tspan8 transduced IsrecoI cells (Is1-Co029). The modulation of cell migration linked to EGFR or CD44 can be neutralized by antagonizing Co-029 with the mAb Ts29.1 or by RNA interference. Altogether these data point to a crucial role of Co-029 in the modulation of colon cancer cell motility which could be related to the protumoral effect reported for this tetraspanin. Among surface molecules able to mediate Co-029 function, E-cadherin, EGFR and CD44 appear as likely candidates.

Chemokine receptors that mediate B cell homing to secondary lymphoid tissues are highly expressed in B cell chronic lymphocytic leukemia and non-Hodgkin lymphomas with widespread nodular dissemination.

B cell neoplasms present heterogeneous patterns of lymphoid organ involvement, which may be a result of the differential expression of chemokine receptors. We found that chemokine receptor (CCR)7, CXC chemokine receptor (CXCR)4, or CXCR5, the main chemokine receptors that mediate B cell entry into secondary lymphoid tissues and their homing to T cell and B cell zones therein, were highly expressed in B malignancies with widespread involvement of lymph nodes. Conversely, those pathologies with little or no nodular dissemination showed no expression to very low levels of CCR7 and CXCR5 and low to moderate levels of CXCR4. These findings provide evidence for the role of CCR7, CXCR4, and CXCR5 in determining the pattern of lymphoid organ involvement of B tumors. Functional studies were performed on B malignancies expressing different levels of CCR7, CXCR5, and CXCR4. Multiple myeloma (MM) cells did not express CCR7 nor CXCR5 and did not migrate in response to their ligands; a moderate expression of CXCR4 on MM cells was accompanied by a migratory response to its ligand, CXCL12. By contrast, cells from B cell chronic lymphocytic leukemia (B-CLL) expressed the highest levels of these chemokine receptors and efficiently migrated in response to all ligands of CCR7, CXCR4, and CXCR5. In addition, the migration index of B-CLL cells in response to both of the CCR7 ligands correlated with the presence of clinical lymphadenopathy, thus indicating that the high expression of functional chemokine receptors justifies the widespread character of B-CLL, representing a clinical target for the control of tumor cell dissemination.

Effect of an anti-human Co-029/tspan8 mouse monoclonal antibody on tumor growth in a nude mouse model.

New therapeutic agents are needed in digestive tract tumors. Co-029/tspan8 is a tetraspanin frequently expressed on human colorectal tumors, In this work, we report the effects of the monoclonal antibody Ts29.2, targeting Co-029/tspan8, on colorectal tumor cells in vitro and after implantation in nude mice. HT29, Isreco1 and SW480 colorectal tumor cell lines were used for this study. HT29 has a strong endogenous expression of Co-029/tspan8, whereas Isreco1 cells don't express Co-029/tspan8 and SW480 has only a weak expression. Isreco1 and SW480 were transduced to express Co-029/tspan8 at the same level as HT29. In order to check the specificity of the effect of monoclonal antibody Ts29.2, low Co-029/tspan8 expressing SW480 cells were injected simultaneously with transduced cells in the back, on the left and right sides of the mice. With an early treatment, Ts29.2 mAb inhibited growth of tumors expressing Co-029/tspan8 up to 70%, whereas a delayed treatment was less efficient. No effect of the antibody on cell proliferation or apoptosis induction was detected in vitro. No increase of activated caspase 3 labeling was observed in vivo and areas occupied by vessels were not significantly different between treated mice and controls. This suggests that the action of Ts29.2 is linked neither to cellular toxicity nor to the inhibition of the previously reported angiogenic properties of Co-029/tspan8. An inhibition of cell proliferation in vivo is demonstrated by a reduction of the mitotic index in HT29 tumors of Ts29.2 treated mice. The discrepancy between in vitro and in vivo data on cell proliferation suggests that the binding of Ts29.2 to tumor cells may modify their response to signals issued from the microenvironment. Given the restricted pattern of tissue expression of the tetraspanin Co-029/tspan8, these preliminary results put forth for consideration the antibody targeting of this tetraspanin in further investigations for therapeutic applications.

Targeting tetraspanins in cancer.

INTRODUCTION: Tetraspanins are a family of small proteins that cross the membrane four times and form complexes by interacting between themselves and with a variety of transmembrane and cytosolic proteins, building a network of interactions referred to as tetraspanin web or tetraspanin enriched microdomains (TEMs). These domains provide a signaling platform involved in many important cellular functions and malignant processes. AREAS COVERED: The authors describe the methods and the rationale for targeting tetraspanins in the therapy of cancer in this review. EXPERT OPINION: Targeting tetraspanins in cancer may be a promising therapy due to the importance of tetraspanins in several steps of tumor formation, communication with the environment, dissemination, and metastasis.

Tetraspanin-enriched microdomains: a functional unit in cell plasma membranes.

Membrane lipids and proteins are non-randomly distributed and are unable to diffuse freely in the plane of the membrane. This is because of multiple constraints imposed both by the cortical cytoskeleton and by the preference of lipids and proteins to cluster into diverse and specialized membrane domains, including tetraspanin-enriched microdomains, glycosylphosphatidyl inositol-linked proteins nanodomains and caveolae, among others. Recent biophysical characterization of tetraspanin-enriched microdomains suggests that they might be specially suited for the regulation of avidity of adhesion receptors and the compartmentalization of enzymatic activities. Moreover, modulation by tetraspanins of the function of adhesion receptors involved in inflammation, lymphocyte activation, cancer and pathogen infection suggests potential as therapeutic targets. This review explores this emerging picture of tetraspanin microdomains and discusses the implications for cell adhesion, proteolysis and pathogenesis.

EWI-2 and EWI-F link the tetraspanin web to the actin cytoskeleton through their direct association with ezrin-radixin-moesin proteins.

EWI-2 and EWI-F, two members of a novel subfamily of Ig proteins, are direct partners of tetraspanins CD9 (Tspan29) and CD81 (Tspan28). These EWI proteins contain a stretch of basic charged amino acids in their cytoplasmic domains that may act as binding sites for actin-linking ezrin-radixin-moesin (ERM) proteins. Confocal microscopy analysis revealed that EWI-2 and EWI-F colocalized with ERM proteins at microspikes and microvilli of adherent cells and at the cellular uropod in polarized migrating leukocytes. Immunoprecipitation studies showed the association of EWI-2 and EWI-F with ERM proteins in vivo. Moreover, pulldown experiments and protein-protein binding assays with glutathione S-transferase fusion proteins containing the cytoplasmic domains of EWI proteins corroborated the strong and direct interaction between ERMs and these proteins. The active role of ERMs was further confirmed by double transfections with the N-terminal domain of moesin, which acts as a dominant negative form of ERMs, and was able to delocalize EWIs from the uropod of polarized leukocytes. In addition, direct association of EWI partner CD81 C-terminal domain with ERMs was also demonstrated. Functionally, silencing of endogenous EWI-2 expression by short interfering RNA in lymphoid CEM cells augmented cell migration, cellular polarity, and increased phosphorylation of ERMs. Hence, EWI proteins, through their direct interaction with ERM proteins, act as linkers to connect tetraspanin-associated microdomains to actin cytoskeleton regulating cell motility and polarity.

Endothelial tetraspanin microdomains regulate leukocyte firm adhesion during extravasation.

Tetraspanins associate with several transmembrane proteins forming microdomains involved in intercellular adhesion and migration. Here, we show that endothelial tetraspanins relocalize to the contact site with transmigrating leukocytes and associate laterally with both intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Alteration of endothelial tetraspanin microdomains by CD9-large extracellular loop (LEL)-glutathione S-transferase (GST) peptides or CD9/CD151 siRNA oligonucleotides interfered with ICAM-1 and VCAM-1 function, preventing lymphocyte transendothelial migration and increasing lymphocyte detachment under shear flow. Heterotypic intercellular adhesion mediated by VCAM-1 or ICAM-1 was augmented when expressed exogenously in the appropriate tetraspanin environment. Therefore, tetraspanin microdomains have a crucial role in the proper adhesive function of ICAM-1 and VCAM-1 during leukocyte adhesion and transendothelial migration.