Inhibition of Rac controls NPM–ALK-dependent lymphoma development and dissemination

Nucleophosmin-anaplastic lymphoma kinase (NPM–ALK) is a tyrosine kinase oncogene responsible for the pathogenesis of the majority of human ALK-positive lymphomas. We recently reported that it activated the Rac1 GTPase in anaplastic large-cell lymphoma (ALCL), leading to Rac-dependent formation of active invadopodia required for invasiveness. Herein, we went further into the study of this pathway and used the inhibitor of Rac, NSC23766, to validate its potential as a molecular target in ALCL in vitro and in vivo in a xenograft model and in a conditional model of NPM–ALK transgenic mice. Our data demonstrate that Rac regulates important effectors of NPM–ALK-induced transformation such as Erk1/2, p38 and Akt. Moreover, inhibition of Rac signaling abrogates NPM–ALK-elicited disease progression and metastasis in mice, highlighting the potential of small GTPases and their regulators as additional therapic targets in lymphomas

Regulation of CD45 phosphatase by oncogenic ALK in anaplastic large cell lymphoma

Anaplastic Large Cell Lymphoma (ALCL) is a subtype of non-Hodgkin lymphoma frequently driven by the chimeric tyrosine kinase NPM-ALK, generated by the t (2,5)(p23;q35) translocation. While ALK+ ALCL belongs to mature T cell lymphomas, loss of T cell identity is observed in the majority of ALCL secondary to a transcriptional and epigenetic repressive program induced by oncogenic NPM-ALK. While inhibiting the expression of T cell molecules, NPM-ALK activates surrogate TCR signaling by directly inducing pathways downstream the TCR. CD45 is a tyrosine phosphatase that plays a central role in T cell activation by controlling the TCR signaling and regulating the cytokine responses through the JAK/STAT pathway and exists in different isoforms depending on the stage of T-cell maturation, activation and differentiation. ALK+ ALCL cells mainly express the isoform CD45RO in keeping with their mature/memory T cell phenotype. Because of its regulatory effect on the JAK/STAT pathway that is essential for ALK+ ALCL, we investigated whether CD45 expression was affected by oncogenic ALK. We found that most ALK+ ALCL cell lines express the CD45RO isoform with modest CD45RA expression and that NPM-ALK regulated the expression of these CD45 isoforms. Regulation of CD45 expression was dependent on ALK kinase activity as CD45RO expression was increased when NPM-ALK kinase activity was inhibited by treatment with ALK tyrosine kinase inhibitors (TKIs). Silencing ALK expression through shRNA or degradation of ALK by the PROTAC TL13-112 caused upregulation of CD45RO both at mRNA and protein levels with minimal changes on CD45RA, overall indicating that oncogenic ALK downregulates the expression of CD45. CD45 repression was mediated by STAT3 as demonstrated by ChIP-seq data on ALCL cells treated with the ALK-TKI crizotinib or cells treated with a STAT3 degrader. Next, we found that knocking-out CD45 with the CRISPR/Cas9 system resulted in increased resistance to ALK TKI treatment and CD45 was down-regulated in ALCL cells that developed resistance in vitro to ALK TKIs. Overall, these data suggest that CD45 expression is regulated by ALK via STAT3 and acts as a rheostat of ALK oncogenic signaling and resistance to TKI treatment in ALCL

DIFFERENT AQUAPORIN-4 EXPRESSION IN GLIOBLASTOMA MULTIFORME PATIENTS WITH AND WITHOUT SEIZURES

Aquaporin-4 (AQP-4), the most important water channel in the brain, is expressed by astrocyte endfeet abutting microvessels. Altered expression levels of AQP-4 and redistribution of the protein throughout the membranes of cells found in glioblastoma multiforme (GBM) lead to development of the oedema often found surrounding the tumour mass. Dysregulation of AQP-4 also occurs in hippocampal sclerosis and cortical dysplasia in patients with refractory partial epilepsy. This work reports on analysis of the relationship between AQP-4 expression and the incidence of epileptic seizures in patients with GBM. Immunohistochemical and PCR techniques were used to evaluate AQP-4 in biopsy specimens from 19 patients with GBM, 10 of whom had a history of seizures prior to surgery. AQP-4 mRNA levels were identical in the two groups of patients, but AQP-4 expression was more frequently detected on the GBM membranes from specimens of patients with seizures than from those without (10 vs. 2,

A model for estimating flood damage in Italy: preliminary results

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po 130 ser235 residue drives eif6 oncogenic activity in npm alk induced t cell lymphomagenesis

Introduction Dysregulation of mRNA translational control in cancer leads to cell transformation, metabolic reprogramming and angiogenesis. eIF6 is an oncogenic translation factor, which regulates the initiation phase of translation acting on 60S availability in the cytoplasm and controlling active 80S complex formation. eIF6 activation is mTORC1-independent and driven by PKCβ mediated phosphorylation on Ser235. An increment of eIF6 expression is reported in several cancer cell lines and human tumours, due to amplification or overexpression. In mice, eIF6 haploinsufficiency blocks Myc-driven lymphomagenesis. Intriguingly, high levels of PKC and eIF6 are found in T-cell lymphomas. In particular, in Anaplastic Large Cell Lymphoma (ALCL) eIF6 is overexpressed and hyperactivated. Material and methods Here, we aimed to define the role of eIF6 phosphorylation in NPM-ALK mediated T-cell lymphomagenesis, combining multidisciplinary studies on murine and cellular models. We used a conditional eIF6 SA KI mouse model in which Ser235 is replaced by an Ala. Results and discussions First, we addressed the effect of eIF6 mutated protein expression in all tissues: homozygosity is lethal after gastrulation while heterozygous mice are viable but resistant to NPM-ALK driven lymphomagenesis. Then, we investigated the role of Ser235 phosphorylation specifically in T-cell lineage, crossing eIF6 SA KI mice with CD4-Cre mice. Physiological T-cell development and subsets composition are not affected by the eIF6 mutated protein. In cancer, eIF6 SA/SA CD4-Cre NPM-ALK mice have a significant increase in survival time, compared to wt with a delay in the appearance of lymphoma up to 6 months. Histological analysis and ex vivo cultures confirm the delay in disease development. eIF6 SA/SA CD4-Cre NPM-ALK thymocytes are smaller respect to wt counterparts and show a striking senescence-like phenotype in vitro . Similarly, in vitro generated eIF6 SA/SA MEFs show a markedly reduced proliferation and increased SA β-gal positivity. This phenotype is completely rescued by transducing eIF6 wild-type, but not by eIF6 SA . Currently, we are investigating the molecular mechanisms by which eIF6 phosphorylation affects ALK-induced malignancy and whether it may modulate premature cell senescence, thus establishing an effective barrier to T-cell lymphomagenesis. Conclusion Our work demonstrates for the first time that eIF6 phosphorylation plays an essential role in mammals development, cell homeostasis and is rate-limiting for T-cell lymphomagenesis in vivo

Inhibition of Anaplastic Lymphoma Kinase (ALK) Activity Provides a Therapeutic Approach for CLTC-ALK-Positive Human Diffuse Large B Cell Lymphomas

ALK positive diffuse large B-cell lymphomas (DLBCL) are a distinct lymphoma subtype associated with a poor outcome. Most of them feature a t(2;17) encoding a clathrin (CLTC)-ALK fusion protein. The contribution of deregulated ALK-activity in the pathogenesis and maintenance of these DLBCLs is not yet known. We established and characterized the first CLTC-ALK positive DLBCL cell line (LM1). LM1 formed tumors in NOD-SCID mice. The selective ALK inhibitor NVP-TAE684 inhibited growth of LM1 cells in vitro at nanomolar concentrations. NVP-TAE684 repressed ALK-activated signalling pathways and induced apoptosis of LM1 DLBCL cells. Inhibition of ALK-activity resulted in sustained tumor regression in the xenotransplant tumor model. These data indicate a role of CLTC-ALK in the maintenance of the malignant phenotype thereby providing a rationale therapeutic target for these otherwise refractory tumors