The Down syndrome critical region protein TTC3 inhibits neuronal differentiation via RhoA and Citron kinase.

The Down syndrome critical region (DSCR) on Chromosome 21 contains many genes whose duplication may lead to the major phenotypic features of Down syndrome and especially the associated mental retardation. However, the functions of DSCR genes are mostly unknown and their possible involvement in key brain developmental events still largely unexplored. In this report we show that the protein TTC3, encoded by one of the main DSCR candidate genes, physically interacts with Citron kinase (CIT-K) and Citron N (CIT-N), two effectors of the RhoA small GTPase that have previously been involved in neuronal proliferation and differentiation. More importantly, we found that TTC3 levels can strongly affect the NGF-induced differentiation of PC12 cells, by a CIT-K-dependent mechanism. Indeed, TTC3 overexpression leads to strong inhibition of neurite extension, which can be reverted by CIT-K RNAi. Conversely, TTC3 knockdown stimulates neurite extension in the same cells. Finally, we find that Rho, but not Rho kinase, is required for TTC3 differentiation-inhibiting activity. Our results suggest that the TTC3–RhoA–CIT-K pathway could be a crucial determinant of in vivo neuronal development, whose hyperactivity may result in detrimental effects on the normal differentiation program

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

The lymphoma-associated NPM-ALK oncogene elicits a p16INKa/pRb-dependent tumour-suppressive pathway

Oncogene induced senescence (OIS) is a barrier for tumour development. Oncogene-dependent DNA damage and activation of the ARF/p53 pathway play a central role in OIS and, accordingly, ARF and p53 are frequently mutated in human cancer. A number of leukemia/lymphoma-initiating oncogenes, however, inhibit ARF/p53 and only infrequently select for ARF or p53 mutations, suggesting the involvement of other tumour-suppressive pathways. We report that NPM-ALK, the initiating oncogene of Anaplastic Large Cell Lymphomas (ALCLs), induces DNA-damage and irreversibly arrests the cell cycle of primary fibroblasts and hematopoietic progenitors. This effect is associated with inhibition of p53 and is due to activation of the p16INK4a/pRb tumour-suppressive pathway. Analysis of NPM-ALK lymphomagenesis in transgenic mice showed p16INK4a-dependent accumulation of senescent cells in pre-malignant lesions and decreased tumour latency in the absence of p16INK4a. Accordingly, human ALCLs showed no expression of either p16INK4a or pRb. Up-regulation of the histone-demethylase Jmjd3 and de-methylation at the p16INK4a promoter contributed to the effect of NPM-ALK on p16INK4a, which was transcriptionally regulated. These data demonstrate that p16INK4a/pRb may function as an alternative pathway of oncogene-induced senescence, and suggest that the reactivation of p16INK4a expression might be a novel strategy to restore the senescence program in some tumours

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

Comprehensive population-based genome sequencing provides insight into hematopoietic regulatory mechanisms

Genetic variants affecting hematopoiesis can influence commonly measured blood cell traits. To identify factors that affect hematopoiesis, we performed association studies for blood cell traits in the population-based Estonian Biobank using high-coverage whole-genome sequencing (WGS) in 2,284 samples and SNP genotyping in an additional 14,904 samples. Using up to 7,134 samples with available phenotype data, our analyses identified 17 associations across 14 blood cell traits. Integration of WGS-based fine-mapping and complementary epigenomic datasets provided evidence for causal mechanisms at several loci, including at a previously undiscovered basophil count-associated locus near the master hematopoietic transcription factor CEBPA. The fine-mapped variant at this basophil count association near CEBPA overlapped an enhancer active in common myeloid progenitors and influenced its activity. In situ perturbation of this enhancer by CRISPR/Cas9 mutagenesis in hematopoietic stem and progenitor cells demonstrated that it is necessary for and specifically regulates CEBPA expression during basophil differentiation. We additionally identified basophil count-associated variation at another more pleiotropic myeloid enhancer near GATA2, highlighting regulatory mechanisms for ordered expression of master hematopoietic regulators during lineage specification. Our study illustrates how population-based genetic studies can provide key insights into poorly understood cell differentiation processes of considerable physiologic relevance.Peer reviewe

Anaplastic lymphoma kinase (ALK) inhibitor response in neuroblastoma is highly correlated with ALK mutation status, ALK mRNA and protein levels

Background In pediatric neuroblastoma (NBL), high anaplastic lymphoma kinase (ALK) levels appear to be correlated with an unfavorable prognosis, regardless of ALK mutation status. This suggests a therapeutic role for ALK inhibitors in NBL patients. We examined the correlation between levels of ALK, phosphorylated ALK (pALK) and downstream signaling proteins and response to ALK inhibition in a large panel of both ALK mutated and wild type (WT) NBL cell lines. Methods We measured protein levels by western blot and ALK inhibitor sensitivity (TAE684) by viability assays in 19 NBL cell lines of which 6 had a point mutation and 4 an amplification of the ALK gene. Results ALK 220 kDa (p=0.01) and ALK 140 kDa (p= 0.03) protein levels were higher in ALK mutant than WT cell lines. Response to ALK inhibition was significantly correlated with ALK protein levels (p<0.01). ALK mutant cell lines (n=4) were 14,9 fold (p<0,01) more sensitive to ALK inhibition than eight WT cell lines. Conclusion NBL cell lines often express ALK at high levels and are responsive to ALK inhibitors. Mutated cell lines express ALK at higher levels, which may define their superior response to ALK inhibition