Rocco Palermo

Notch3 and pre-TCR interaction unveils distinct NF-κB pathways in T-cell development and leukemia

Notch signaling plays a critical role in T‐cell differentiation and leukemogenesis. We previously demonstrated that, while pre‐TCR is required for thymocytes proliferation and leukemogenesis, it is dispensable for thymocyte differentiation in Notch3‐transgenic mice. Notch3‐transgenic premalignant thymocytes and T lymphoma cells overexpress pTα/pre‐TCR and display constitutive activation of NF‐κB, providing survival signals for immature thymocytes. We provide genetic and biochemical evidence that Notch3 triggers multiple NF‐κB activation pathways. A pre‐TCR‐dependent pathway preferentially activates NF‐κB via IKKβ/IKKα/NIK complex, resulting in p50/p65 heterodimer nuclear entry and recruitment onto promoters of Cyclin D1, Bcl2‐A1 and IL7‐receptor‐α genes. In contrast, upon pTα deletion, Notch3 binds IKKα and maintains NF‐κB activation through an alternative pathway, depending on an NIK‐independent IKKα homodimer activity. The consequent NF‐κB2/p100 processing allows nuclear translocation of p52/RelB heterodimers, which only trigger transcription from Bcl2‐A1 and IL7‐receptor‐α genes. Our data suggest that a finely tuned interplay between Notch3 and pre‐TCR pathways converges on regulation of NF‐κB activity, leading to differential NF‐κB subunit dimerization that regulates distinct gene clusters involved in either cell differentiation or proliferation/leukemogenesis.

Notch and NF-kB signaling pathways regulate miR-223/FBXW7 axis in T-cell acute lymphoblastic leukemia.

Notch signaling deregulation is linked to the onset of several tumors including T-cell acute lymphoblastic leukemia (T-ALL). Deregulated microRNA (miRNA) expression is also associated with several cancers, including leukemias. However, the transcriptional regulators of miRNAs, as well as the relationships between Notch signaling and miRNA deregulation, are poorly understood. To identify miRNAs regulated by Notch pathway, we performed microarray-based miRNA profiling of several Notch-expressing T-ALL models. Among seven miRNAs, consistently regulated by overexpressing or silencing Notch3, we focused our attention on miR-223, whose putative promoter analysis revealed a conserved RBPjk binding site, which was nested to an NF-kB consensus. Luciferase and chromatin immunoprecipitation assays on the promoter region of miR-223 show that both Notch and NF-kB are novel coregulatory signals of miR-223 expression, being able to activate cooperatively the transcriptional activity of miR-223 promoter. Notably, the Notch-mediated activation of miR-223 represses the tumor suppressor FBXW7 in T-ALL cell lines. Moreover, we observed the inverse correlation of miR-223 and FBXW7 expression in a panel of T-ALL patient-derived xenografts. Finally, we show that miR-223 inhibition prevents T-ALL resistance to γ-secretase inhibitor (GSI) treatment, suggesting that miR-223 could be involved in GSI sensitivity and its inhibition may be exploited in target therapy protocols.

PKC theta mediates pre-TCR signaling and contributes to Notch3-induced T-cell leukemia.

Protein kinase (PK)Cθ is a critical regulator of mature T-cell activation and proliferation, being implicated in TCR-triggered nuclear factor (NF)-κB activation and providing important survival signals to leukemic T cells. We previously showed that overexpression of pTα/pre-TCR and constitutive activation of NF-κB characterize the T-cell leukemia/lymphoma developing in Notch3-IC transgenic mice. We report here that PKCθ is a downstream target of Notch3 signaling and that its activation and membrane translocation require a functional pre-TCR in order to trigger NF-κB activation in thymocytes and lymphoma cells of transgenic mice. Furthermore, deletion of PKCθ in Notch3-IC transgenic mice reduces the incidence of leukemia, correlating with decreased NF-κB activation. This paper therefore suggests that PKCθ mediates the activation of NF-κB by pre-TCR in immature thymocytes and contributes to the development of Notch3-dependent T-cell lymphoma.

The archaeal eIF2 homologue: functional properties of an ancient translation initiation factor

The eukaryotic translation initiation factor 2 (eIF2) is pivotal for delivery of the initiator tRNA (tRNAi) to the ribosome. Here, we report the functional characterization of the archaeal homologue, a/eIF2. We have cloned the genes encoding the three subunits of a/eIF2 from the thermophilic archaeon Sulfolobus solfataricus, and have assayed the activities of the purified recombinant proteins in vitro. We demonstrate that the trimeric factor reconstituted from the recombinant polypeptides has properties similar to those of its eukaryal homologue: it interacts with GTP and Met-tRNAi, and stimulates binding of the latter to the small ribosomal subunit. However, the archaeal protein differs in some functional aspects from its eukaryal counterpart. In contrast to eIF2, a/eIF2 has similar affinities for GDP and GTP, and the β-subunit does not contribute to tRNAi binding. The detailed analysis of the complete trimer and of its isolated subunits is discussed in light of the evolutionary history of the eIF2-like proteins.

Acetylation controls Notch3 stability and function in T-cell leukemia

Post-translational modifications of Notch3 and their functional role with respect to Notch3 overexpression in T-cell leukemia are still poorly understood. We identify here a specific novel property of Notch3 that is acetylated and deacetylated at lysines 1692 and 1731 by p300 and HDAC1, respectively, a balance impaired by HDAC inhibitors (HDACi) that favor hyperacetylation. By using HDACi and a non-acetylatable Notch3 mutant carrying K/R1692−1731 mutations in the intracellular domain, we show that Notch3 acetylation primes ubiquitination and proteasomal-mediated degradation of the protein. As a consequence, Notch3 protein expression and its transcriptional activity are decreased both in vitro and in vivo in Notch3 transgenic (tg) mice, thus impairing downstream signaling upon target genes. Consistently, Notch3-induced T-cell proliferation is inhibited by HDACi, whereas it is enhanced by the non-acetylatable Notch3-K/R1692−1731 mutant. Finally, HDACi-induced Notch3 hyperacetylation prevents in vivo growth of T-cell leukemia/lymphoma in Notch3 tg mice. Together, our findings suggest a novel level of Notch signaling control in which Notch3 acetylation/deacetylation process represents a key regulatory switch, thus representing a suitable druggable target for Notch3-sustained T-cell acute lymphoblastic leukemia therapy.

NF-kB/NOS cross-talk induced by mitochondrial complex II inhibition : Implications for Huntington's disease

Nuclear factor-kB (NF-kB) is a family of DNA-binding proteins that are important regulators involved in immune and inflammatory responses, as well as in cell survival and apoptosis. In the nervous system NF-kB is activated under physiological and pathological conditions including learning and memory mechanisms and neurodegenerative diseases. NF-kB is activated in neurons in response to excitotoxic, metabolic and oxidative stress and there is a body of evidence to suggest that glutamate induces NF-kB by the main ionotropic glutamate receptors. In the present study, 3 nitroproprionic acid (3NP), an irreversible inhibitor of succinate dehydrogenase (SD, complex II) has been employed to provide an experimental model of Huntingtons disease (HD). Specifically, we described 3NP-induced activation of NF-kB and of iNOS and nNOS genes in striatal treated slices. To aim to better understand the relationship between these identified dysregulated genes and mitochondrial dysfunction, we investigated in SK-N-MC human neuroblastoma cells following 3NP treatment, whether NF-kB nuclear translocation and activation might be involved in the mechanisms by which 3NP leads to transcriptional activation of NOS genes. These results are relevant to more precisely define the role of NF-kB in neuronal cells and better understand its putative involvement in neurodegeneration.

Protective effect of pioglitazone, a PPARγ ligand, in a 3 nitropropionic acid model of Huntington's disease

The peroxisome proliferator-activated receptor γ (PPARγ) is a member of the PPAR family. PPARγ is the target of insulin-sensitising thiazolidinediones (TZDs), drugs used for the treatment of non-insulin-dependent diabetes. Recently, several studies have shown that PPARγ activators can also prevent or attenuate neurodegeneration. The PPARγ agonist pioglitazone provides neuroprotection to dopaminergic neurons in lipopolysaccharide (LPS) and MPTP-induced Parkinsons disease experimental models. Here, we investigated whether PPARγ activation by pioglitazone protected striatal cells from mitochondrial dysfunction and oxidative stress in a 3 nitropropionic acid (3NP)-induced experimental model of Huntingtons disease (HD). Our results suggested that pioglitazone has beneficial effects on mitochondrial dysfunction by interfering with the NF-κB signalling pathway, which has been implicated in the pathogenesis of HD. Additionally, we demonstrated that the nuclear translocation of HDAC3 is regulated by 3NP via IκBα and that treatment with pioglitazone prevented these effects. These results suggested that IκBα-dependent nuclear translocation is responsible for PPARγ inhibition by 3NP and pointed to histone modifications as a novel approach for treating HD.

Targeted therapy against chemoresistant colorectal cancers: Inhibition of p38α modulates the effect of cisplatin in vitro and in vivo through the tumor suppressor FoxO3A.

Chemoresistance is a major obstacle to effective therapy against colorectal cancer (CRC) and may lead to deadly consequences. The metabolism of CRC cells depends highly on the p38 MAPK pathway, whose involvement in maintaining a chemoresistant behavior is currently being investigated. Our previous studies revealed that p38α is the main p38 isoform in CRC cells. Here we show that p38α pharmacological inhibition combined with cisplatin administration decreases colony formation and viability of cancer cells and strongly increases Bax-dependent apoptotic cell death by activating the tumor suppressor protein FoxO3A. Our results indicate that FoxO3A activation up-regulates transcription of its target genes (p21, PTEN, Bim and GADD45), which forces both chemosensitive and chemoresistant CRC cells to undergo apoptosis. Additionally, we found that FoxO3A is required for apoptotic cell death induction, as confirmed by RNA interference experiments. In animal models xenografted with chemoresistant HT29 cells, we further confirmed that the p38-targeted dual therapy strategy produced an increase in apoptosis in cancer tissue leading to tumor regression. Our study uncovers a major role for the p38-FoxO3A axis in chemoresistance, thereby suggesting a new therapeutic approach for CRC treatment; moreover, our results indicate that Bax status may be used as a predictive biomarker.

The Molecular Basis of Notch Signaling Regulation: A Complex Simplicity

The Notch receptors have attracted considerable attention for their ability to control cellular functions that regulate embryo development and tissue homeostasis. Notch receptors act by controlling the expression of a specific set of target genes. If Notch signaling system can be so simple, and yet so complex in its pleiotropic effects, then a sophisticated network of regulatory mechanisms is required to maintain the control over the initiation, activity and termination of this signaling pathway. A multitude of regulatory mechanisms has been discovered that controls the interaction of Notch receptors with their ligands, the assembling of a Notch transcriptional activation complex and the termination of Notch signals. The intracellular and extracellular domains of the Notch receptors are synthesized as single proteins, pairing with each other during their trafficking through the exocytotic route. The mechanisms operating in the phase preceding the generation of the heterodimeric signal-competent Notch receptors can be as elaborate and physiologically important as those operating downstream of Notch receptor activation. These regulatory mechanisms, which are essential to understand the role of Notch signaling in human physiology and pathology are reviewed here.

Prolyl-isomerase Pin1 controls Notch3 protein expression and regulates T-ALL progression

Deregulated Notch signaling is associated with T-cell Acute Lymphoblastic Leukemia (T-ALL) development and progression. Increasing evidence reveals that Notch pathway has an important role in the invasion ability of tumor cells, including leukemia, although the underlying molecular mechanisms remain mostly unclear. Here, we show that Notch3 is a novel target protein of the prolyl-isomerase Pin1, which is able to regulate Notch3 protein processing and to stabilize the cleaved product, leading to the increased expression of the intracellular domain (N3IC), finally enhancing Notch3-dependent invasiveness properties. We demonstrate that the combined inhibition of Notch3 and Pin1 in the Notch3-overexpressing human leukemic TALL-1 cells reduces their high invasive potential, by decreasing the expression of the matrix metalloprotease MMP9. Consistently, Pin1 depletion in a mouse model of Notch3-induced T-ALL, by reducing N3IC expression and signaling, impairs the expansion/invasiveness of CD4+CD8+ DP cells in peripheral lymphoid and non-lymphoid organs. Notably, in in silico gene expression analysis of human T-ALL samples we observed a significant correlation between Pin1 and Notch3 expression levels, which may further suggest a key role of the newly identified Notch3-Pin1 axis in T-ALL aggressiveness and progression. Thus, combined suppression of Pin1 and Notch3 proteins may be exploited as an additional target therapy for T-ALL.