Search

Graphical abstract, During the past two years, the world has been ravaged by a global pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Acquired mutations in the SARS-CoV-2 genome affecting virus infectivity and/or immunogenicity have led to a number of novel strains with higher transmissibility compared to the original Wuhan strain. Mutations in the receptor binding domain (RBD) of the SARS-CoV-2 spike protein have been extensively studied in this context. However, mutations and deletions within the N-terminal domain (NTD) located adjacent to the RBD are less studied. Many of these are found within certain β sheet-linking loops, which are surprisingly long in SARS-CoV-2 in comparison to SARS-CoV and other related β coronaviruses. Here, we perform a structural and epidemiological study of novel strains carrying mutations and deletions within these loops. We identify short and long-distance interactions that stabilize the NTD loops of the spike protein and form a critical epitope that is essential for the recognition by a wide variety of neutralizing antibodies from convalescent plasma. Among the different mutations/deletions found in these loops, Ala 67 and Asp 80 mutations as well as His 69/Val 70 and Tyr 144 deletions have been identified in different fast-spreading strains. Similarly, deletions in amino acids 241-243 and 246-252 have been found to affect the network of NTD loops in strains with high transmissibility. Our structural findings provide insight regarding the role of these mutations/deletions in altering the epitope structure and thus affecting the immunoreactivity of the NTD region of spike protein.

The kinase haspin phosphorylates histone H3 at threonine-3 (H3T3ph) during mitosis. H3T3ph provides a docking site for the Chromosomal Passenger Complex at the centromere, enabling correction of erratic microtubule-chromosome contacts. Although this mechanism is operational in all dividing cells, haspin-null mice do not exhibit developmental anomalies, apart from aberrant testis architecture. Investigating this problem, we show here that mouse embryonic stem cells that lack or overexpress haspin, albeit prone to chromosome misalignment during metaphase, can still divide, expand and differentiate. RNA sequencing reveals that haspin dosage affects severely the expression levels of several genes that are involved in male gametogenesis. Consistent with a role in testis-specific expression, H3T3ph is detected not only in mitotic spermatogonia and meiotic spermatocytes, but also in non-dividing cells, such as haploid spermatids. Similarly to somatic cells, the mark is erased in the end of meiotic divisions, but re-installed during spermatid maturation, subsequent to methylation of histone H3 at lysine-4 (H3K4me3) and arginine-8 (H3R8me2). These serial modifications are particularly enriched in chromatin domains containing histone H3 trimethylated at lysine-27 (H3K27me3), but devoid of histone H3 trimethylated at lysine-9 (H3K9me3). The unique spatio-temporal pattern of histone H3 modifications implicates haspin in the epigenetic control of spermiogenesis.

Simple Summary Canonical NF-κB signalling pathway acts as a tumour promoter in several types of cancer including non-small cell lung cancer (NSCLC), but the mechanism(s) by which it contributes to NSCLC is still under investigation. We show here that NF-κB RelA/p65 is required for the tumour growth of human NSCLC cells grown in vivo as xenografts in immune-compromised mice. RNA-seq transcriptome profile analysis identified the metastasis suppressor CD82/KAI1/TSPAN27 as a canonical NF-κB target. Loss of CD82 correlated with malignancy. RelA/p65 stimulates cell migration and epithelial-to-mesenchymal cell transition (EMT), mediated, in part, by CD82/KAI1, through integrin-mediated signalling, thus, identifying a mechanism mediating NF-κB RelA/p65 lung tumour promoting function. Abstract Background: The development of non-small cell lung cancer (NSCLC) involves the progressive accumulation of genetic and epigenetic changes. These include somatic oncogenic KRAS and EGFR mutations and inactivating TP53 tumour suppressor mutations, leading to activation of canonical NF-κB. However, the mechanism(s) by which canonical NF-κB contributes to NSCLC is still under investigation. Methods: Human NSCLC cells were used to knock-down RelA/p65 (RelA/p65KD) and investigate its impact on cell growth, and its mechanism of action by employing RNA-seq analysis, qPCR, immunoblotting, immunohistochemistry, immunofluorescence and functional assays. Results: RelA/p65KD reduced the proliferation and tumour growth of human NSCLC cells grown in vivo as xenografts in immune-compromised mice. RNA-seq analysis identified canonical NF-κB targets mediating its tumour promoting function. RelA/p65KD resulted in the upregulation of the metastasis suppressor CD82/KAI1/TSPAN27 and downregulation of the proto-oncogene ROS1, and LGR6 involved in Wnt/β-catenin signalling. Immunohistochemical and bioinformatics analysis of human NSCLC samples showed that CD82 loss correlated with malignancy. RelA/p65KD suppressed cell migration and epithelial-to-mesenchymal cell transition (EMT), mediated, in part, by CD82/KAI1, through integrin-mediated signalling involving the mitogenic ERK, Akt1 and Rac1 proteins. Conclusions: Canonical NF-κB signalling promotes NSCLC, in part, by downregulating the metastasis suppressor CD82/KAI1 which inhibits cell migration, EMT and tumour growth.

Carfilzomib (Cfz), an irreversible proteasome inhibitor licensed for relapsed/refractory myeloma, is associated with cardiotoxicity in humans. We sought to establish the optimal protocol of Cfz-induced cardiac dysfunction, to investigate the underlying molecular-signaling and, based on the findings, to evaluate the cardioprotective potency of metformin (Met). Mice were randomized into protocols 1 and 2 (control and Cfz for 1 and 2 consecutive days, respectively); protocols 3 and 4 (control and alternate doses of Cfz for 6 and 14 days, respectively); protocols 5A and 5B (control and Cfz, intermittent doses on days 0, 1 [5A] and 0, 1, 7, and 8 [5B] for 13 days); protocols 6A and 6B (pharmacological intervention; control, Cfz, Cfz+Met and Met for 2 and 6 days, respectively); and protocol 7 (bortezomib). Cfz was administered at 8 mg/kg (IP) and Met at 140 mg/kg (per os). Cfz resulted in significant reduction of proteasomal activity in heart and peripheral blood mononuclear cells in all protocols except protocols 5A and 5B. Echocardiography demonstrated that Cfz led to a significant fractional shortening (FS) depression in protocols 2 and 3, a borderline dysfunction in protocols 1 and 4, and had no detrimental effect on protocols 5A and 5B. Molecular analysis revealed that Cfz inhibited AMPKα/mTORC1 pathways derived from increased PP2A activity in protocol 2, whereas it additionally inhibited phosphatidylinositol 3-kinase/Akt/endothelial nitric oxide synthase pathway in protocol 3. Coadministration of Met prevented Cfz-induced FS reduction and restored AMPKα phosphorylation and autophagic signaling. Conclusively, Cfz decreased left ventricular function through increased PP2A activity and inhibition of AMPKα and its downstream autophagic targets, whereas Met represents a novel promising intervention against Cfz-induced cardiotoxicity.

Simple Summary It is widely thought that tumors are composed of different subpopulations of cancer cells carrying genetic alterations with some of them being common among all cells while others are unique for each subpopulation. This variable genetic profile of tumor cells is a component of what is collectively described as intratumor heterogeneity (ITH). Surviving the immune system and therapies, and establishing metastases are forces of natural selection that act upon ITH and drive tumor evolution and, eventually, the clinical presentation of patients. The aim of this prospective study was to investigate ITH in early-stage operable non-small cell lung cancer. We directly grafted human tumors in immunosuppressed mice and compared the genetic profile of the tumors grown in mice with that of the original human tumors. We identified clinical factors that affected the ability of human tumors to grow as mouse xenografts. Abstract Recent advances in sequencing technologies have allowed the in-depth molecular study of tumors, even at the single cell level. Sequencing efforts have uncovered a previously unappreciated heterogeneity among tumor cells, which has been postulated to be the driving force of tumor evolution and to facilitate recurrence, metastasis, and drug resistance. In the current study, focused on early-stage operable non-small cell lung cancer, we used tumor growth in patient-derived xenograft (PDX) models in mice as a fast-forward tumor evolution process to investigate the molecular characteristics of tumor cells that grow in mice, as well as the parameters that affect the grafting efficiency. We found that squamous cell carcinomas grafted significantly more efficiently compared with adenocarcinomas. Advanced stage, patient age and primary tumor size were positively correlated with grafting. Additionally, we isolated and characterized circulating tumor cells (CTC) from patients’ peripheral blood and found that the presence of CTCs expressing epithelial-to-mesenchymal (EMT) markers correlated with the grafting potential. Interestingly, exome sequencing of the PDX tumor identified genetic alterations in DNA repair and genome integrity genes that were under-represented in the human primary counterpart. In conclusion, through the generation of a PDX biobank of NSCLC, we identified the clinical and molecular properties of tumors that affected growth in mice.

The structure of oncogenic LMTK3 determines its role and functions allowing drug inhibition as a new therapeutic strategy., Elucidating signaling driven by lemur tyrosine kinase 3 (LMTK3) could help drug development. Here, we solve the crystal structure of LMTK3 kinase domain to 2.1Å resolution, determine its consensus motif and phosphoproteome, unveiling in vitro and in vivo LMTK3 substrates. Via high-throughput homogeneous time-resolved fluorescence screen coupled with biochemical, cellular, and biophysical assays, we identify a potent LMTK3 small-molecule inhibitor (C28). Functional and mechanistic studies reveal LMTK3 is a heat shock protein 90 (HSP90) client protein, requiring HSP90 for folding and stability, while C28 promotes proteasome-mediated degradation of LMTK3. Pharmacologic inhibition of LMTK3 decreases proliferation of cancer cell lines in the NCI-60 panel, with a concomitant increase in apoptosis in breast cancer cells, recapitulating effects of LMTK3 gene silencing. Furthermore, LMTK3 inhibition reduces growth of xenograft and transgenic breast cancer mouse models without displaying systemic toxicity at effective doses. Our data reinforce LMTK3 as a druggable target for cancer therapy.

We directly compared two different approaches used for Circulating Tumor Cell (CTC) isolation, a size-dependent microfluidic system versus an EpCAM-dependent positive selection for downstream molecular characterization of CTC both at the gene expression and DNA methylation level in Head and Neck Squamous Cell Carcinoma (HNSCC). A size-dependent microfluidic device (Parsortix, ANGLE) and an EpCAM-dependent positive immune-magnetic isolation procedure were applied in parallel, using 10 mL PB from 50 HNSCC patients and 18 healthy donors. Total RNA was isolated from enriched CTCs and RT-qPCR was used to study the expression levels of CK-19, PD-L1, EGFR, TWIST1, CDH2 and B2M (reference gene). Real time methylation specific PCR (MSP) was used to study the methylation status of RASSF1A and MLL3 genes. In identical blood draws, the label-free size-dependent CTC-isolation system was superior in terms of sensitivity when compared to the EpCAM-dependent CTC enrichment, since a significantly higher percentage of identical PB samples was found positive at the gene expression and DNA methylation level, while the specificity was not affected. Our results indicate that future studies focused on the evaluation of clinical utility of CTC molecular characterization in HNSCC should be based on size-dependent enrichment approaches.

The Notch signaling pathway has been implicated in homeostasis and disease, including cancer, in various tissues. Moreover, it has been involved both in stem cell maintenance and differentiation, in a context-dependent manner. Stem/progenitor cells, on the other hand, have long been suspected to be the cells of origin in various malignancies. In order to gain insight in the role of the Notch ligand Dll1 in mouse development, we generated a knock-in line expressing an inducible Cre recombinase. We have employed in vivo approaches in mice to genetically mark rare subpopulations of cells expressing Dll1 in various adult tissues. Moreover, we conditionally expressed a constitutively active Ras oncoprotein in these cells and showed that within days, mice develop squamous neoplasias in the skin, as well as in the stomach.

Using an efficient gene targeting approach, we developed a novel mouse line that expresses the tetracycline-controlled transactivator (tTA) from the constitutively active Eef1a1 locus in a Cre recombinase-inducible manner. The temporally and spatially controlled expression of the EF1-LSL-tTA knockin and activation of tTA-driven responder transgenes was tested using four transgenic lines that express Cre under tissue-specific promoters of the pancreas, mammary gland and other secretory tissues, as well as an interferon-inducible promoter. In all models, the endogenous Eef1a1 promoter facilitated a cell-type-specific activation of target genes at high levels without exogenous enhancer elements. The applicability of the EF1-LSL-tTA strain for biological experiments was tested in two studies related to mammary gland development and tumorigenesis. First, we validated the crucial role of active STAT5 as a survival factor for functionally differentiated epithelial cells by expressing a hyperactive STAT5 mutant in the mammary gland during postlactational remodeling. In a second experiment, we assessed the ability of the EF1-tTA to initiate tumor formation through upregulation of mutant KRAS. The collective results show that the EF1-LSL-tTA knockin line is a versatile genetic tool that can be applied to constitutively express transgenes in specific cell types to examine their biological functions at defined developmental stages.

The kinase Haspin phosphorylates histone H3 at threonine-3 (H3T3ph), creating a docking site for the Chromosomal Passenger Complex (CPC). CPC plays a pivotal role in preventing chromosome misalignment. Here, we have examined the effects of 5-Iodotubercidin (5-ITu), a commonly used Haspin inhibitor, on self-renewal and differentiation of mouse embryonic stem cells (ESCs). Treatment with low concentrations of 5-ITu eliminates the H3T3ph mark during mitosis, but does not affect the mode or the outcome of self-renewal divisions. Interestingly, 5-ITu causes sustained accumulation of p53, increases markedly the expression of histone genes and results in reversible upregulation of the pluripotency factor Klf4. However, the properties of 5-ITu treated cells are distinct from those observed in Haspin-knockout cells generated by CRISPR/Cas9 genome editing, suggesting “off-target” effects. Continuous exposure to 5-ITu allows modest expansion of the ESC population and growth of embryoid bodies, but release from the drug after an initial treatment aborts embryoid body or teratoma formation. The data reveal an unusual robustness of ESCs against mitotic perturbants and suggest that the lack of H3T3ph and the “off-target” effects of 5-ITu can be partially compensated by changes in expression program or accumulation of suppressor mutations.

Breast cancer is the leading cause of cancer death in the female population, despite advances in diagnosis and treatment. The highly heterogeneous nature of the disease represents a major obstacle to successful therapy and results in a significant number of patients developing drug resistance and, eventually, suffering from tumor relapse. Cancer stem cells (CSCs) are a small subset of tumor cells characterized by self-renewal, increased tumor-initiation capacity, and resistance to conventional therapies. As such, they have been implicated in the etiology of tumor recurrence and have emerged as promising targets for the development of novel therapies. Here, we show that the histone demethylase lysine-specific demethylase 1 (LSD1) plays an important role in the chemoresistance of breast cancer cells. Our data, from a series of in vitro and in vivo assays, advocate for LSD1 being critical in maintaining a pool of tumor-initiating cells that may contribute to the development of drug resistance. Combinatory administration of LSD1 inhibitors and anti-cancer drugs is more efficacious than monotherapy alone in eliminating all tumor cells in a 3D spheroid system. In conclusion, we provide compelling evidence that LSD1 is a key regulator of breast cancer stemness and a potential target for the design of future combination therapies.

The Notch signaling pathway mediates cell-cell communication regulating cell differentiation and proliferation and cell fate decisions in various tissues. In the urinary bladder, Notch acts as a tumor suppressor in mice, while mutations in Notch pathway components have been identified in human bladder cancer as well. Here we report that the genetic inactivation of Notch in mice leads to downregulation of cell-cell and cell-ECM interaction components, including proteins previously implicated in interstitial cystitis/bladder pain syndrome (IC/BPS), structural defects and mucosal sloughing, inflammation, and leaky urine-blood barrier. Molecular profiling of ailing mouse bladders showed similarities with IC/BPS patient tissue, which also presented low Notch pathway activity as indicated by reduced expression of canonical Notch targets. Urothelial integrity was reconstituted upon exogenous reactivation of the Notch pathway, implying a direct involvement of Notch. Despite damage and inflammation, urothelial cells failed to proliferate, uncovering a possible role for α(4) integrin in urothelial homeostasis. Our data uncover a broad role for Notch in bladder homeostasis involving urothelial cell crosstalk with the microenvironment.

Aims Levosimendan (LEVO) a clinically-used inodilator, exerts multifaceted cardioprotective effects. Case-studies indicate protection against doxorubicin (DXR)-induced cardiotoxicity, but this effect remains obscure. We investigated the effect and mechanism of different regimens of levosimendan on sub-chronic and chronic doxorubicin cardiotoxicity. Methods and results Based on preliminary in vivo experiments, rats serving as a sub-chronic model of doxorubicin-cardiotoxicity and were divided into: Control (N/S-0.9%), DXR (18 mg/kg-cumulative), DXR+LEVO (LEVO, 24 μg/kg-cumulative), and DXR+LEVO (acute) (LEVO, 24 μg/kg-bolus) for 14 days. Protein kinase-B (Akt), endothelial nitric oxide synthase (eNOS), and protein kinase-A and G (PKA/PKG) pathways emerged as contributors to the cardioprotection, converging onto phospholamban (PLN). To verify the contribution of PLN, phospholamban knockout (PLN−/−) mice were assigned to PLN−/−/Control (N/S-0.9%), PLN−/−/DXR (18 mg/kg), and PLN−/−/DXR+LEVO (ac) for 14 days. Furthermore, female breast cancer-bearing (BC) mice were divided into: Control (normal saline 0.9%, N/S 0.9%), DXR (18 mg/kg), LEVO, and DXR+LEVO (LEVO, 24 μg/kg-bolus) for 28 days. Echocardiography was performed in all protocols. To elucidate levosimendan’s cardioprotective mechanism, primary cardiomyocytes were treated with doxorubicin or/and levosimendan and with N omega-nitro-L-arginine methyl ester (L-NAME), DT-2, and H-89 (eNOS, PKG, and PKA inhibitors, respectively); cardiomyocyte-toxicity was assessed. Single bolus administration of levosimendan abrogated DXR-induced cardiotoxicity and activated Akt/eNOS and cAMP-PKA/cGMP-PKG/PLN pathways but failed to exert cardioprotection in PLN−/− mice. Levosimendan’s cardioprotection was also evident in the BC model. Finally, in vitro PKA inhibition abrogated levosimendan-mediated cardioprotection, indicating that its cardioprotection is cAMP-PKA dependent, while levosimendan preponderated over milrinone and dobutamine, by ameliorating calcium overload. Conclusion Single dose levosimendan prevented doxorubicin cardiotoxicity through a cAMP-PKA-PLN pathway, highlighting the role of inotropy in doxorubicin cardiotoxicity.

A dicyanomethylene-4H-pyran based tumor-targeting and biothiol activatable fluorescent and colorimetric probe, named Biotin-Yellow, has been developed with unprecedented turn-on near-infrared fluorescence properties. Biotin-Yellow is non-toxic and can effectively recognize and discriminate human lung adenocarcinoma cells and urothelial (bladder) carcinoma cells from embryonic kidney cells and embryonic fibroblasts. Biotin-Yellow can also be employed for real-time bioimaging tracking of upregulated biothiol activity in cancer cells. The efficacy and biothiol responsiveness of Biotin-Yellow was further validated in vivo in zebrafish and mice xenografts.

Genome‐wide studies in tumor cells have indicated that chromatin‐modifying proteins are commonly mutated in human cancers. The lysine‐specific methyltransferase 2C (KMT2C/MLL3) is a putative tumor suppressor in several epithelia and in myeloid cells. Here, we show that downregulation of KMT2C in bladder cancer cells leads to extensive changes in the epigenetic status and the expression of DNA damage response and DNA repair genes. More specifically, cells with low KMT2C activity are deficient in homologous recombination‐mediated double‐strand break DNA repair. Consequently, these cells suffer from substantially higher endogenous DNA damage and genomic instability. Finally, these cells seem to rely heavily on PARP1/2 for DNA repair, and treatment with the PARP1/2 inhibitor olaparib leads to synthetic lethality, suggesting that cancer cells with low KMT2C expression are attractive targets for therapies with PARP1/2 inhibitors.

Herein, we describe a modified protocol for the generation of human induced pluripotent stem cells (hiPS) and expansion under defined, serum free and feeder free conditions. These cells exhibit a high level of plasticity towards various differentiation pathways both in vitro and in vivo. Ultimately, hiPS-derived lines achieved high standards of three dimensional differentiations on biomaterial scaffolds and promoted in vivo regeneration of complex organs, such as Anterior Cruciate Ligament (in swine ACL-rupture models) and other tissues as well.

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) initiates IRE1α, ATF6, and PERK cascades, leading to a transcriptional/translational response known as unfolded protein response (UPR). Here we show that VEGF activates UPR mediators through a PLCγ-mediated crosstalk with the mTORC1 complex without accumulation of unfolded proteins in the ER. Activation of ATF6 and PERK contributes to the survival effect of VEGF on endothelial cells (ECs) by positively regulating mTORC2-mediated phosphorylation of AKT on Ser473, which is required for full activity of AKT. Low levels of CHOP allow ECs to evade the proapoptotic effect of this UPR product. Depletion of PLCγ, ATF6, or eIF2α dramatically inhibited VEGF-induced vascularization in mouse Matrigel plugs, suggesting that the ER and the UPR machinery constitute components of the VEGF signaling circuit that regulates EC survival and angiogenesis, extending their role beyond adaptation to ER stress.

SummaryTumor-propagating cells in acute leukemia maintain a stem/progenitor-like immature phenotype and proliferative capacity. Acute myeloid leukemia (AML) and acute T-lymphoblastic leukemia (T-ALL) originate from different lineages through distinct oncogenic events such as MLL fusions and Notch signaling, respectively. We found that Zfx, a transcription factor that controls hematopoietic stem cell self-renewal, controls the initiation and maintenance of AML caused by MLL-AF9 fusion and of T-ALL caused by Notch1 activation. In both leukemia types, Zfx prevents differentiation and activates gene sets characteristic of immature cells of the respective lineages. In addition, endogenous Zfx contributes to gene induction and transformation by Myc overexpression in myeloid progenitors. Key Zfx target genes include the mitochondrial enzymes Ptpmt1 and Idh2, whose overexpression partially rescues the propagation of Zfx-deficient AML. These results show that distinct leukemia types maintain their undifferentiated phenotype and self-renewal by exploiting a common stem-cell-related genetic regulator.

The DNA damage response (DDR) pathway and ARF function as barriers to cancer development. Although commonly regarded as operating independently of each other, some studies proposed that ARF is positively regulated by the DDR. Contrary to either scenario, we found that in human oncogene-transformed and cancer cells ATM suppressed, in a transcription-independent manner, ARF protein levels and activity. Mechanistically, ATM activated protein phosphatase 1, which antagonized Nek2-dependent phosphorylation of nucleophosmin (NPM), thereby liberating ARF from NPM and rendering it susceptible to degradation by the ULF E3-ubiquitin ligase. In human clinical samples, loss of ATM expression correlated with increased ARF levels and in xenograft and tissue culture models, inhibition of ATM stimulated the tumour-suppressive effects of ARF. These results provide insights into the functional interplay between the DDR and ARF anti-cancer barriers, with implications for tumorigenesis and treatment of advanced tumours.

The urothelium is a specialized epithelium with the single role of sealing the luminal side of the urinary tract, forming the urine-blood barrier. It is a slowly cycling epithelium, which however, ...

The urothelium is a specialized epithelium that lines the urinary tract. It consists of three different cell types, namely, basal, intermediate and superficial cells arranged in relatively distinct cell layers. Normally, quiescent, it regenerates fast upon injury, but the regeneration process is not fully understood. Although several reports have indicated the existence of progenitors, their identity and exact topology, as well as their role in key processes such as tissue regeneration and carcinogenesis have not been clarified. Here we show that a minor subpopulation of basal cells, characterized by the expression of keratin 14, possesses self-renewal capacity and also gives rise to all cell types of the urothelium during natural and injury-induced regeneration. Moreover, these cells represent cells of origin of urothelial cancer. Our findings support the hypothesis of basally located progenitors with profound roles in urothelial homoeostasis., It is unclear whether there is a progenitor/stem cell in the basal layer of the urothelium in the bladder. Here, the authors identify Keratin14 positive cells that can regenerate the bladder in both a natural and injury-induced manner, and following neoplastic transformation, can give rise to tumours.

The adult kidney contains a population of low-cycling cells that resides in the papilla. These cells retain for long periods S-phase markers given as a short pulse early in life; i.e., they are label-retaining cells (LRC). In previous studies in adult rat and mice, we found that shortly after acute kidney injury many of the quiescent papillary LRC started proliferating (Oliver JA, Klinakis A, Cheema FH, Friedlander J, Sampogna RV, Martens TP, Liu C, Efstratiadis A, Al-Awqati Q. J Am Soc Nephrol 20: 2315–2327, 2009; Oliver JA, Maarouf O, Cheema FH, Martens TP, Al-Awqati Q. J Clin Invest 114: 795–804, 2004) and, with cell-tracking experiments, we found upward migration of some papillary cells including LRC (Oliver JA, Klinakis A, Cheema FH, Friedlander J, Sampogna RV, Martens TP, Liu C, Efstratiadis A, Al-Awqati Q. J Am Soc Nephrol 20: 2315–2327, 2009). To identify molecular cues involved in the activation (i.e., proliferation and/or migration) of the papillary LRC that follows injury, we isolated these cells from the H2B-GFP mice and found that they migrated and proliferated in response to the cytokine stromal cell-derived factor-1 (SDF-1). Moreover, in a papillary organ culture assay, the cell growth out of the upper papilla was dependent on the interaction of SDF-1 with its receptor Cxcr4. Interestingly, location of these two proteins in the kidney revealed a complementary location, with SDF-1 being preferentially expressed in the medulla and Cxcr4 more abundant in the papilla. Blockade of Cxcr4 in vivo prevented mobilization of papillary LRC after transient kidney ischemic injury and worsened its functional consequences. The data indicate that the SDF-1/Cxcr4 axis is a critical regulator of papillary LRC activation following transient kidney injury and during organ repair.

Blocking Dll4–Notch signaling can reverse established diabetes via Flt3-independent induction of immature thymic DCs that enhance Treg cell generation in mice., Delta-like ligand 4 (Dll4)–Notch signaling is essential for T cell development and alternative thymic lineage decisions. How Dll4–Notch signaling affects pro-T cell fate and thymic dendritic cell (tDC) development is unknown. We found that Dll4 pharmacological blockade induces accumulation of tDCs and CD4+CD25+FoxP3+ regulatory T cells (Treg cells) in the thymic cortex. Both genetic inactivation models and anti-Dll4 antibody (Ab) treatment promote de novo natural Treg cell expansion by a DC-dependent mechanism that requires major histocompatibility complex II expression on DCs. Anti-Dll4 treatment converts CD4−CD8−c-kit+CD44+CD25− (DN1) T cell progenitors to immature DCs that induce ex vivo differentiation of naive CD4+ T cells into Treg cells. Induction of these tolerogenic DN1-derived tDCs and the ensuing expansion of Treg cells are Fms-like tyrosine kinase 3 (Flt3) independent, occur in the context of transcriptional up-regulation of PU.1, Irf-4, Irf-8, and CSF-1, genes critical for DC differentiation, and are abrogated in thymectomized mice. Anti-Dll4 treatment fully prevents type 1 diabetes (T1D) via a Treg cell–mediated mechanism and inhibits CD8+ T cell pancreatic islet infiltration. Furthermore, a single injection of anti-Dll4 Ab reverses established T1D. Disease remission and recurrence are correlated with increased Treg cell numbers in the pancreas-draining lymph nodes. These results identify Dll4–Notch as a novel Flt3-alternative pathway important for regulating tDC-mediated Treg cell homeostasis and autoimmunity.

Dendritic cells (DCs) in tissues and lymphoid organs comprise distinct functional subsets that differentiate in situ from circulating progenitors. Tissue-specific signals that regulate DC subset differentiation are poorly understood. We report that DC-specific deletion of the Notch2 receptor caused a reduction of DC populations in the spleen. Within the splenic CD11b(+) DC subset, Notch signaling blockade ablated a distinct population marked by high expression of the adhesion molecule Esam. The Notch-dependent Esam(hi) DC subset required lymphotoxin beta receptor signaling, proliferated in situ, and facilitated CD4(+) T cell priming. The Notch-independent Esam(lo) DCs expressed monocyte-related genes and showed superior cytokine responses. In addition, Notch2 deletion led to the loss of CD11b(+)CD103(+) DCs in the intestinal lamina propria and to a corresponding decrease of IL-17-producing CD4(+) T cells in the intestine. Thus, Notch2 is a common differentiation signal for T cell-priming CD11b(+) DC subsets in the spleen and intestine.

Notch signalling is a central regulator of differentiation in a variety of organisms and tissue types. Its activity is controlled by the multi-subunit γ-secretase (γSE) complex. Although Notch signalling can play both oncogenic and tumour-suppressor roles in solid tumours, in the haematopoietic system it is exclusively oncogenic, notably in T-cell acute lymphoblastic leukaemia, a disease characterized by Notch1-activating mutations. Here we identify novel somatic-inactivating Notch pathway mutations in a fraction of patients with chronic myelomonocytic leukaemia (CMML). Inactivation of Notch signalling in mouse haematopoietic stem cells (HSCs) results in an aberrant accumulation of granulocyte/monocyte progenitors (GMPs), extramedullary haematopoieisis and the induction of CMML-like disease. Transcriptome analysis revealed that Notch signalling regulates an extensive myelomonocytic-specific gene signature, through the direct suppression of gene transcription by the Notch target Hes1. Our studies identify a novel role for Notch signalling during early haematopoietic stem cell differentiation and suggest that the Notch pathway can play both tumour-promoting and -suppressive roles within the same tissue.

The kidney papilla contains a population of cells with several characteristics of adult stem cells, including the retention of proliferation markers during long chase periods (i.e., they are label-retaining cells [LRCs]). To determine whether the papillary LRCs generate new cells in the normal adult kidney, we examined cell proliferation throughout the kidney and found that the upper papilla is a site of enhanced cell cycling. Using genetically modified mice that conditionally expressed green fluorescence protein fused to histone 2B, we observed that the LRCs of the papilla proliferated only in its upper part, where they associate with “chains” of cycling cells. The papillary LRCs decreased in number with age, suggesting that the cells migrated to the upper papilla before entering the cell cycle. To test this directly, we marked papillary cells with vital dyes in vivo and found that some cells in the kidney papilla, including LRCs, migrated toward other parts of the kidney. Acute kidney injury enhanced both cell migration and proliferation. These results suggest that during normal homeostasis, LRCs of the kidney papilla (or their immediate progeny) migrate to the upper papilla and form a compartment of rapidly proliferating cells, which may play a role in repair after ischemic injury.

T-cell acute lymphoblastic leukaemia (T-ALL) is a blood malignancy afflicting mainly children and adolescents1. T-ALL patients present at diagnosis with increased white cell counts and hepatosplenomegaly, and are at an increased risk of central nervous system (CNS) relapse2,3. For that reason, T-ALL patients usually receive cranial irradiation in addition to intensified intrathecal chemotherapy. The marked increase in survival is thought to be worth the considerable side-effects associated with this therapy. Such complications include secondary tumours, neurocognitive deficits, endocrine disorders and growth impairment3. Little is known about the mechanism of leukaemic cell infiltration of the CNS, despite its clinical importance4. Here we show, using T-ALL animal modelling and gene-expression profiling, that the chemokine receptor CCR7 (ref. 5) is the essential adhesion signal required for the targeting of leukaemic T-cells into the CNS. Ccr7 gene expression is controlled by the activity of the T-ALL oncogene Notch1 and is expressed in human tumours carrying Notch1-activating mutations. Silencing of either CCR7 or its chemokine ligand CCL19 (ref. 6) in an animal model of T-ALL specifically inhibits CNS infiltration. Furthermore, murine CNS-targeting by human T-ALL cells depends on their ability to express CCR7. These studies identify a single chemokine–receptor interaction as a CNS ‘entry’ signal, and open the way for future pharmacological targeting. Targeted inhibition of CNS involvement in T-ALL could potentially decrease the intensity of CNS-targeted therapy, thus reducing its associated short- and long-term complications.

Considering the strong association between dysregulated insulin-like growth factor (IGF) signaling and various human cancers, we have used an expedient combination of genetic analysis and pharmacological treatment to evaluate the potential of the type 1 IGF receptor (Igf1r) for targeted anticancer therapy in a mouse model of mammary tumorigenesis. In this particular strain of genetically modified animals, histopathologically heterogeneous invasive carcinomas exhibiting up-regulation of the Igf1r gene developed extremely rapidly by mammary gland-specific overexpression of constitutively active oncogenic Kras* (mutant Kras G12D ). Immunophenotyping data and expression profiling analyses showed that, except for a minor luminal component, these mouse tumors resembled basal-like human breast cancers. This is a group of aggressive tumors of poor prognosis for which there is no targeted therapy currently available, and it includes a subtype correlating with KRAS locus amplification. Conditional ablation of Igf1r in the mouse mammary epithelium increased the latency of Kras*-induced tumors very significantly (≈11-fold in comparison with the intact model), whereas treatment of tumor-bearing animals by administration of picropodophyllin (PPP), a specific Igf1r inhibitor, resulted in a dramatic decrease in tumor mass of the main forms of basal-like carcinomas. PPP also was effective against xenografts of the human basal-like cancer cell line MDA-MB-231, which carries a KRAS G13D mutation.

The mammalian insulin-like growth factor 1 (IGF1), which is a member of a major growth-promoting signaling system, is produced by many tissues and functions throughout embryonic and postnatal development in an autocrine/paracrine fashion. In addition to this local action, IGF1 secreted by the liver and circulating in the plasma presumably acts systemically as a classical hormone. However, an endocrine role of IGF1 in growth control was disputed on the basis of the results of a conditional, liver-specific Igf1 gene knockout in mice, which reduced significantly the level of serum IGF1, but did not affect average body weight. Because alternate interpretations of these negative data were tenable, we addressed genetically the question of hormonal IGF1 action by using a positive experimental strategy based on the features of the cre / loxP recombination system. Thus, we generated bitransgenic mice carrying in an Igf1 null background a dormant Igf1 cDNA placed downstream of a transcriptional “stop” DNA sequence flanked by loxP sites (floxed) and also a cre transgene driven by a liver-specific promoter. The Igf1 cDNA, which was inserted by knock-in into the mutated and inactive Igf1 locus itself to ensure proper transcriptional regulation, was conditionally expressed from cognate promoters exclusively in the liver after Cre-mediated excision of the floxed block. Our genetic study demonstrated that the endocrine IGF1 plays a very significant role in mouse growth, as its action contributes approximately30% of the adult body size and sustains postnatal development, including the reproductive functions of both mouse sexes.

The involvement of Myc in the development of human breast cancer is known for quite some time, but an analogous role of Notch signaling is only now emerging from fragmented pieces of information. Recently, a Notch/Myc relationship in oncogenesis was revealed from a mouse model, in which mammary tumors are induced by the intracellular domain of Notch1 (N1(IC)). In fact, a combination of genetic and molecular data demonstrated that Myc is a direct transcriptional target of Notch1 participating as an indispensable downstream effector in N1(IC)-induced tumorigenic action. A medically-relevant correlative observation based on immunophenotyping was the coexpression of Notch1 and Myc in a significant fraction of human breast cancer specimens. A Notch1/Myc oncogenic association was also observed in T-cell acute lymphoblastic leukemia.

BACKGROUND Pancreatic ductal adenocarcinoma (PDA) is frequently driven by oncogenic KRAS(KRAS*) mutations. We developed a mouse model of KRAS*-induced PDA and, based on genetic results demonstrating that KRAS* tumorigenicity depends on Myc activity, we evaluated the therapeutic potential of an orally administered anti-Myc drug. METHODS We tested the efficacy of Mycro3, a small-molecule inhibitor of Myc-Max dimerization, in the treatment of mouse PDA (n = 9) and also of xenografts of human pancreatic cancer cell lines (NOD/SCID mice, n = 3-12). Tumor responses to the drug were evaluated by PET/CT imaging, and histological, immunohistochemical, molecular and microarray analyses. The Student's t test was used for differences between groups. All statistical tests were two-sided. RESULTS Transgenic overexpression of KRAS* in the pancreas resulted in pancreatic intraepithelial neoplasia in two-week old mice, which developed invasive PDA a week later and became moribund at one month. However, this aggressive form of pancreatic tumorigenesis was effectively prevented by genetic ablation of Myc specifically in the pancreas. We then treated moribund, PDA-bearing mice daily with the Mycro3 Myc-inhibitor. The mice survived until killed at two months. PET/CT image analysis (n = 5) demonstrated marked shrinkage of PDA, while immunohistochemical analyses showed an increase in cancer cell apoptosis and reduction in cell proliferation (treated/untreated proliferation index ratio: 0.29, P < .001, n = 3, each group). Tumor growth was also drastically attenuated in Mycro3-treated NOD/SCID mice (n = 12) carrying orthotopic or heterotopic xenografts of human pancreatic cancer cells (eg, mean tumor weight ± SD of treated heterotopic xenografts vs vehicle-treated controls: 15.2±5.8 mg vs 230.2±43.9 mg, P < .001). CONCLUSION These results provide strong justification for eventual clinical evaluation of anti-Myc drugs as potential chemotherapeutic agents for the treatment of PDA.

The Notch signaling pathway controls cell fates through interactions between neighboring cells by positively or negatively affecting the processes of proliferation, differentiation and apoptosis in a context-dependent manner. This pathway has been implicated in human cancer as both an oncogene and a tumor suppressor. Here we report new inactivating mutations in Notch pathway components in over 40% of human bladder cancers examined. Bladder cancer is the fourth most commonly diagnosed malignancy in the male population of the United States. Thus far, driver mutations in fibroblast growth factor receptor 3 (FGFR3) and, less commonly, in RAS proteins have been identified. We show that Notch activation in bladder cancer cells suppresses proliferation both in vitro and in vivo by directly upregulating dual-specificity phosphatases (DUSPs), thus reducing the phosphorylation of ERK1 and ERK2 (ERK1/2). In mouse models, genetic inactivation of Notch signaling leads to Erk1/2 phosphorylation, resulting in tumorigenesis in the urinary tract. Collectively our findings show that loss of Notch activity is a driving event in urothelial cancer.

Transposable elements have been used widely in the past 20 years for gene transfer and insertional mutagenesis in Drosophila . Transposon-based technology for gene manipulation and genomic analysis currently is being adopted for vertebrates. We tested the ability of Minos , a DNA transposon from Drosophila hydei , to transpose in mouse tissues. Two transgenic mouse lines were crossed, one expressing Minos transposase in lymphocytes under the control of the CD2 promoter/locus control region and another carrying a nonautonomous Minos transposon. Only mice containing both transgenes show excision of the transposon and transposition into new chromosomal sites in thymus and spleen cells. In addition, expression of Minos transposase in embryonic fibroblast cell lines derived from a transposon-carrying transgenic mouse resulted in excision of the transposon. These results are a first step toward a reversible insertional mutagenesis system in the mouse, opening the way to develop powerful technologies for functional genomic analysis in mammals.

To assess the ability of the transposable element Minos to act as a vector for genetic manipulation of the silkworm Bombyx mori, an extrachromosomal transposition assay based on three plasmids was performed. The three plasmids - helper, donor and target - were co-injected into preblastoderm embryos. Low molecular weight DNA was extracted from the embryos at the stage of blastokinesis and used to transform Escherichia coli. High frequency of transposition was observed in the presence of a helper plasmid possessing an intronless Minos transposase gene, whereas transposition did not occur in the presence of a helper plasmid with the intron-bearing transposase gene. Sequence analysis of the insertion sites showed that Minos always inserts into a TA dinucleotide. Although the insertions are distributed throughout the target gene, there was a preference for certain insertion sites. However, no consensus could be identified in the sequence flanking the target site. The results strongly suggest that the transposable element Minos has the potential to be used as a vector in the silkworm and probably in other lepidopteran insects.

Summary Production of Aβ by γ-secretase is a key event in Alzheimer's disease (AD). The γ-secretase complex consists of presenilin (PS) 1 or 2, nicastrin (ncstn), Pen-2, and Aph-1 and cleaves type I transmembrane proteins, including the amyloid precursor protein (APP). Although ncstn is widely accepted as an essential component of the complex required for γ-secretase activity, recent in vitro studies have suggested that ncstn is dispensable for APP processing and Aβ production. The focus of this study was to answer this controversy and evaluate the role of ncstn in Aβ generation and the development of the amyloid-related phenotype in the mouse brain. To eliminate ncstn expression in the mouse brain, we used a ncstn conditional knockout mouse that we mated with an established AD transgenic mouse model (5XFAD) and a neuronal Cre-expressing transgenic mouse (CamKIIα-iCre), to generate AD mice (5XFAD/CamKIIα-iCre/ncstnf/f mice) where ncstn was conditionally inactivated in the brain. 5XFAD/CamKIIα-iCre/ncstnf/f mice at 10 week of age developed a neurodegenerative phenotype with a significant reduction in Aβ production and formation of Aβ aggregates and the absence of amyloid plaques. Inactivation of nctsn resulted in substantial accumulation of APP-CTFs and altered PS1 expression. These results reveal a key role for ncstn in modulating Aβ production and amyloid plaque formation in vivo and suggest ncstn as a target in AD therapeutics.

Oncogenic stimuli trigger the DNA damage response (DDR) and induction of the alternative reading frame (ARF) tumor suppressor, both of which can activate the p53 pathway and provide intrinsic barriers to tumor progression. However, the respective timeframes and signal thresholds for ARF induction and DDR activation during tumorigenesis remain elusive. Here, these issues were addressed by analyses of mouse models of urinary bladder, colon, pancreatic and skin premalignant and malignant lesions. Consistently, ARF expression occurred at a later stage of tumor progression than activation of the DDR or p16INK4A, a tumor-suppressor gene overlapping with ARF. Analogous results were obtained in several human clinical settings, including early and progressive lesions of the urinary bladder, head and neck, skin and pancreas. Mechanistic analyses of epithelial and fibroblast cell models exposed to various oncogenes showed that the delayed upregulation of ARF reflected a requirement for a higher, transcriptionally based threshold of oncogenic stress, elicited by at least two oncogenic ‘hits’, compared with lower activation threshold for DDR. We propose that relative to DDR activation, ARF provides a complementary and delayed barrier to tumor development, responding to more robust stimuli of escalating oncogenic overload. Oncogenic stimuli trigger the DNA damage response (DDR) and induction of the alternative reading frame (ARF) tumor suppressor, both of which can activate the p53 pathway and provide intrinsic barriers to tumor progression. However, the respective timeframes and signal thresholds for ARF induction and DDR activation during tumorigenesis remain elusive. Here, these issues were addressed by analyses of mouse models of urinary bladder, colon, pancreatic and skin premalignant and malignant lesions. Consistently, ARF expression occurred at a later stage of tumor progression than activation of the DDR or p16(INK4A), a tumor-suppressor gene overlapping with ARF. Analogous results were obtained in several human clinical settings, including early and progressive lesions of the urinary bladder, head and neck, skin and pancreas. Mechanistic analyses of epithelial and fibroblast cell models exposed to various oncogenes showed that the delayed upregulation of ARF reflected a requirement for a higher, transcriptionally based threshold of oncogenic stress, elicited by at least two oncogenic 'hits', compared with lower activation threshold for DDR. We propose that relative to DDR activation, ARF provides a complementary and delayed barrier to tumor development, responding to more robust stimuli of escalating oncogenic overload.

To explore the potential involvement of aberrant Notch1 signaling in breast cancer pathogenesis, we have used a transgenic mouse model. In these animals, mouse mammary tumor virus LTR-driven expression of the constitutively active intracellular domain of the Notch1 receptor (N1 IC ) causes development of lactation-dependent mammary tumors that regress upon gland involution but progress to nonregressing, invasive adenocarcinomas in subsequent pregnancies. Up-regulation of Myc in these tumors prompted a genetic investigation of a potential Notch1/Myc functional relationship in breast carcinogenesis. Conditional ablation of Myc in the mammary epithelium prevented the induction of regressing N1 IC neoplasms and also reduced the incidence of nonregressing carcinomas, which developed with significantly increased latency. Molecular analyses revealed that both the mouse and human Myc genes are direct transcriptional targets of N1 IC acting through its downstream Cbf1 transcriptional effector. Consistent with this mechanistic link, Notch1 and Myc expression is positively correlated by immunostaining in 38% of examined human breast carcinomas.

The olive fruit fly (olive fly) Bactrocera oleae (Dacus), recently introduced in North America, is the most destructive pest of olives worldwide. The lack of an efficient gene transfer technology for olive fly has hampered molecular analysis, as well as development of genetic techniques for its control. We have developed a Minos-based transposon vector carrying a self-activating cassette which overexpresses the enhanced green fluorescent protein (EGFP). Efficient transposase-mediated integration of one to multiple copies of this vector was achieved in the germ line of B. oleae by coinjecting the vector along with in vitro synthesized Minos transposase mRNA into preblastoderm embryos. The self-activating gene construct combined with transposase mRNA present a system with potential for transgenesis of very diverse species.

The product of the imprinted gene paternally expressed gene-10 (PEG10) has been reported to support proliferation in hepatocellular carcinomas, but how this gene is regulated has been an open question. We find that MYC knockdown by RNA interference suppresses PEG10 expression in Panc1 pancreatic carcinoma and HepG2 hepatocellular carcinoma cells and that knockdown of PEG10 inhibits the proliferation of Panc1, HepG2, and Hep3B cells. Conversely, PEG10 was up-regulated by inducing c-MYC expression in a B-lymphocyte cell line. Chromatin immunoprecipitation from Panc1 cells showed c-MYC bound to an E-box-containing region in the PEG10 first intron and site-directed mutagenesis showed that the most proximal E-box is essential for promoter activity. In a mouse mammary tumor virus (MMTV)-MYC transgenic mouse model of breast cancer, most but not all of the mammary carcinomas had strongly increased Peg10 mRNA compared with normal mammary gland. By immunohistochemistry, normal human breast and prostate epithelium was negative for the major isoform [reading frame-1 (RF1)] of PEG10 protein, but this cytoplasmic protein was strongly expressed in a subset of breast carcinomas in situ and invasive ductal carcinomas (∼30%) and in a similar percentage of prostate cancers. As in the mouse model, we found positive, but not absolute, correlations between PEG10 and c-MYC in tissue arrays containing 161 human breast cancers (P < 0.002) and 30 prostate cancers (P = 0.014). Immunostaining of human placenta showed PEG10 and c-MYC proteins coexpressed in proliferating cytotrophoblast and coordinately lost in postmitotic syncytiotrophoblast. These findings link cancer genetics and epigenetics by showing that a classic proto-oncogene, MYC, acts directly upstream of a proliferation-positive imprinted gene, PEG10. (Cancer Res 2006; 66(2): 665-72)

Much of the information about the function of D. melanogaster genes has come from P-element mutagenesis. The major drawback of the P element, however, is its strong bias for insertion into some genes (hotspots) and against insertion into others (coldspots). Within genes, 5′-UTRs are preferential targets. For the successful completion of the Drosophila Genome Disruption Project, the use of transposon vectors other than P will be necessary. We examined here the suitability of the Minos element from Drosophila hydei as a tool for Drosophila genomics. Previous work has shown that Minos, a member of the Tc1/mariner family of transposable elements, is active in diverse organisms and cultured cells; it produces stable integrants in the germ line of several insect species, in the mouse, and in human cells. We generated and analyzed 96 Minos integrations into the Drosophila genome and devised an efficient “jump-starting” scheme for production of single insertions. The ratio of insertions into genes vs. intergenic DNA is consistent with a random distribution. Within genes, there is a statistically significant preference for insertion into introns rather than into exons. About 30% of all insertions were in introns and ∼55% of insertions were into or next to genes that have so far not been hit by the P element. The insertion sites exhibit, in contrast to other transposons, little sequence requirement beyond the TA dinucleotide insertion target. We further demonstrate that induced remobilization of Minos insertions can delete nearby sequences. Our results suggest that Minos is a useful tool complementing the P element for insertional mutagenesis and genomic analysis in Drosophila.

The development of efficient non-viral methodologies for genome-wide insertional mutagenesis and gene tagging in mammalian cells is highly desirable for functional genomic analysis. Here we describe transposon mediated mutagenesis (TRAMM), using naked DNA vectors based on the Drosophila hydei transposable element Minos. By simple transfections of plasmid Minos vectors in HeLa cells, we have achieved high frequency generation of cell lines, each containing one or more stable chromosomal integrations. The Minos-derived vectors insert in different locations in the mammalian genome. Genome-wide mutagenesis in HeLa cells was demonstrated by using a Minos transposon containing a lacZ-neo gene-trap fusion to generate a HeLa cell library of at least 10(5) transposon insertions in active genes. Multiple gene traps for six out of 12 active genes were detected in this library. Possible applications of Minos-based TRAMM in functional genomics are discussed.

Fast and reliable methods for assessing the mobility of the transposable element Minos have been developed. These methods are based on the detection of excision and insertion of Minos transposons from and into plasmids which are co-introduced into cells. Excision is detected by polymerase chain reaction (PCR) with appropriate primers. Transposition is assayed by marker rescue in Escherichia coli, using a transposon plasmid that carries a tetracycline resistance gene and a target plasmid carrying a gene that can be selected against in E. coli. Using both assays, Minos was shown to transpose in Drosophila melanogaster cells and embryos, and in cultured cells of a mosquito, Aedes aegypti, and a lepidopteran, Spodoptera frugiperda. In all cases, mobility was dependent on the presence of exogenously supplied transposase, and both excision and transposition were precise. The results indicate that Minos can transpose in heterologous insect species with comparable efficiencies and therefore has the potential to be used as a transgenesis vector for diverse species.

Introduction: Recently, whole genome sequencing studies revealed that loss of function somatic mutations in NOTCH1 receptor are present in more than 15% of human head and neck cancers making NOTCH1 signalling an attractive target for the development of new, highly specific antitumor drugs. Here, we present a novel Notch 1 receptor variant containing an in frame deletion of 231 nucleotides (named as del31 Notch) of exon 31. Materials and Methods: Total RNA from 64 frozen head and neck and 72 bladder cancer and matched normal tissue were used for cDNA synthesis by conventional reverse transcription using random primers. In a next step, the fragments corresponding to the full size Notch1 receptor were PCR amplified and subjected to Sanger sequencing analysis. We analysed the sequencing data, focusing on truncation and exonic deletion events in the Notch 1 receptor. Results: During PCR amplification of the cDNA fragment corresponding to exon 31 of Notch 1 receptor in our samples, we identified an unexpected PCR product that was slightly smaller in size than wild type exon. Sequencing of this product revealed a Notch1 truncated transcript that lacks a part of exon 31 sequence, harbouring a 231 nt in frame deletion. Further characterization of the truncated receptor revealed that its intracellular domain contains a shorter ankyrin domain (ANK) and that its transactivation efficiency is less than 65% compared to the wild type receptor. Sequencing analysis of genomic DNA in our samples revealed that this exonic deletion mutation was not present in genomic DNA, suggesting that the truncated variant is possibly generated by an abnormal splicing event. In support to this proposed mechanism, the 5’ end of the deleted sequencing is located near to 3’ acceptor slice site of the intron between exons 30 and 31. To identify the incidence of Notch1 del31 variant in cancer specimens, we designed a Notch1 del31 variant-specific PCR assay. Variant specific polymerase chain reaction showed a high incidence of Notch1 del31 variant expression in head and neck cancer (8/64), while the incidence of this variant was rare in bladder cancer (1/72). We did not observe expression of Notch1 del31 variant in tissue adjacent to tumors or in normal tissues. Discussion: We present a novel variant of Notch1 receptor which exhibits lower transactivation activity compared to wild type receptor. Loss of function mutations of Notch1 are associated with head and neck carcinogenesis.Therefore, Notch1 del 31 truncated variant may promote carcinogenesis by suppressing the Notch signalling in head and neck epithelium. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A183. Citation Format: Amanda Psyrri, Eirini Pectasides, M Avgeris, Apostolos Klinakis, Andreas Scorilas, Clarence Sasaki, George Fountzilas, Theodoros Rampias. Identification of an exon 31-deletion variant of Notch1 receptor with frequent expression in head and neck cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A183.

Intensive chemotherapy helps achieve remission in the vast majority of acute lymphoblastic leukemia (ALL) patients. A subset of these cases, T-cell ALL (T-ALL), fare significantly worse, presenting with more severe disease, lower remission rates and a greater likelihood of relapse, particularly within the central nervous system (CNS). All modern treatment protocols involve intensive prophylactic CNS chemotherapy and cranial irradiation. The dramatic increase in overall survival is thought to be worth the significant long term side effects associated with CNS treatment such as secondary tumors, neurocognitive deficits and neuroendocrine disorders. Despite its significance, very little is known about the basic mechanisms of leukemic lymphocyte infiltration into the CNS. Over 50% of T-ALL patients have activating mutations in the Notch1 protein. According to our gene expression profiling results, Notch1 activation leads to the induction of CCR7, a chemokine receptor known to play a role in normal lymphocyte homing in extramedullary tissues and CNS. To study CNS infiltration, we generated two murine models expressing activated Notch1 (N1-IC), a bone marrow transplant (WT/WTN1) and a conditional knockin. Both developed an aggressive T-ALL with immature T-cells infiltrating into secondary lymphoid tissues, but more importantly, they had CNS involvement. Numerous malignant appearing CD3 positive cells were found in the leptomeningeal space of the brain in both mouse models. When we transplanted CCR7 knock out bone marrow cells expressing N1-IC, the recipient mice (WT/CCR7KON1) developed an aggressive T-ALL as before. However, a close examination of the brain failed to show any leukemic infiltration in the host mice. In agreement, wild-type N1-IC positive cells transplanted into plt mice (plt/ WTN1) lacking the CCR7 ligands, CCL19 and CCL21, induced T-cell leukemia, but again, these cells failed to infiltrate into the CNS. In addition, WT/CCR7KON1 and plt/WTN1 mice survived longer than WT/WTN1 mice (61 and 71 vs 47 days, p