Notch Pathway Members Research Articles

ATRT–SHH comprises three molecular subgroups with characteristic clinical and histopathological features and prognostic significance

Atypical teratoid/rhabdoid tumor (ATRT) is an aggressive central nervous system tumor characterized by loss of SMARCB1/INI1 protein expression and comprises three distinct molecular groups, ATRT–TYR, ATRT–MYC and ATRT–SHH. ATRT–SHH represents the largest molecular group and is heterogeneous with regard to age, tumor location and epigenetic profile. We, therefore, aimed to investigate if heterogeneity within ATRT–SHH might also have biological and clinical importance. Consensus clustering of DNA methylation profiles and confirmatory t-SNE analysis of 65 ATRT–SHH yielded three robust molecular subgroups, i.e., SHH-1A, SHH-1B and SHH-2. These subgroups differed by median age of onset (SHH-1A: 18 months, SHH-1B: 107 months, SHH-2: 13 months) and tumor location (SHH-1A: 88% supratentorial; SHH-1B: 85% supratentorial; SHH-2: 93% infratentorial, often extending to the pineal region). Subgroups showed comparable SMARCB1 mutational profiles, but pathogenic/likely pathogenic SMARCB1 germline variants were over-represented in SHH-2 (63%) as compared to SHH-1A (20%) and SHH-1B (0%). Protein expression of proneural marker ASCL1 (enriched in SHH-1B) and glial markers OLIG2 and GFAP (absent in SHH-2) as well as global mRNA expression patterns differed, but all subgroups were characterized by overexpression of SHH as well as Notch pathway members. In a Drosophila model, knockdown of Snr1 (the fly homologue of SMARCB1) in hedgehog activated cells not only altered hedgehog signaling, but also caused aberrant Notch signaling and formation of tumor-like structures. Finally, on survival analysis, molecular subgroup and age of onset (but not ASCL1 staining status) were independently associated with overall survival, older patients (> 3 years) harboring SHH-1B experiencing relatively favorable outcome. In conclusion, ATRT–SHH comprises three subgroups characterized by SHH and Notch pathway activation, but divergent molecular and clinical features. Our data suggest that molecular subgrouping of ATRT–SHH has prognostic relevance and might aid to stratify patients within future clinical trials.

Glycans that regulate Notch signaling in the intestine.

Intestinal homeostasis is key to the maintenance of good health. The small intestine plays important roles in absorption, digestion, hormonal and immune functions. Crypt base columnar (CBC) stem cells residing at the bottom of crypts are nurtured by Paneth cells, and together create the stem cell niche, the foundation of intestinal homeostasis. CBC stem cells replicate to replenish their number, or differentiate into a variety of epithelial cells with specialized functions. Notch signaling is a cell-cell signaling pathway that regulates both the proliferation and differentiation of CBC stem cells. NOTCH1 and NOTCH2 stimulated by canonical Notch ligands DLL1 and DLL4 mediate Notch signaling in the intestine that, in concert with other signaling pathways including the WNT and BMP pathways, determines cell fates. Importantly, interactions between Notch receptors and canonical Notch ligands are regulated by O-glycans linked to Ser/Thr in epidermal growth factor-like (EGF) repeats of the Notch receptor extracellular domain (NECD). The O-glycans attached to NECD are key regulators of the strength of Notch signaling. Imbalances in Notch signaling result in altered cell fate decisions and may lead to cancer in the intestine. In this review, we summarize the impacts of mutations in Notch pathway members on intestinal development and homeostasis, with a focus on the glycosyltransferases that transfer O-glycans to EGF repeats of NOTCH1, NOTCH2, DLL1 and DLL4.

The enpp4 ectonucleotidase regulates kidney patterning signalling networks in Xenopus embryos

The enpp ectonucleotidases regulate lipidic and purinergic signalling pathways by controlling the extracellular concentrations of purines and bioactive lipids. Although both pathways are key regulators of kidney physiology and linked to human renal pathologies, their roles during nephrogenesis remain poorly understood. We previously showed that the pronephros was a major site of enpp expression and now demonstrate an unsuspected role for the conserved vertebrate enpp4 protein during kidney formation in Xenopus. Enpp4 over-expression results in ectopic renal tissues and, on rare occasion, complete mini-duplication of the entire kidney. Enpp4 is required and sufficient for pronephric markers expression and regulates the expression of RA, Notch and Wnt pathway members. Enpp4 is a membrane protein that binds, without hydrolyzing, phosphatidylserine and its effects are mediated by the receptor s1pr5, although not via the generation of S1P. Finally, we propose a novel and non-catalytic mechanism by which lipidic signalling regulates nephrogenesis.

Notch Signaling Pathway in Cancer—Review with Bioinformatic Analysis

Simple SummaryThe Notch signaling pathway, which controls multiple cell differentiation processes during the embryonic stage and adult life, is associated with carcinogenesis and disease progression. The aim of the present study was to highlight cancer heterogeneity with respect to the Notch pathway. Our analysis concerns the effects of the Notch signaling at different levels, including core components and downstream target genes. We also demonstrate overall and disease-free survival results, pointing out the characteristics of particular Notch components. Depending on tissue context, Notch members can be either oncogenic or suppressive. We observed different expression profile core components and target genes that could be associated with distinct survival of patients. Advances in our understanding of the Notch signaling in cancer are very promising for the development of new treatment strategies for the benefit of patients.Notch signaling is an evolutionarily conserved pathway regulating normal embryonic development and homeostasis in a wide variety of tissues. It is also critically involved in carcinogenesis, as well as cancer progression. Activation of the Notch pathway members can be either oncogenic or suppressive, depending on tissue context. The present study is a comprehensive overview, extended with a bioinformatics analysis of TCGA cohorts, including breast, bladder, cervical, colon, kidney, lung, ovary, prostate and rectum carcinomas. We performed global expression profiling of the Notch pathway core components and downstream targets. For this purpose, we implemented the Uniform Manifold Approximation and Projection algorithm to reduce the dimensions. Furthermore, we determined the optimal cutpoint using Evaluate Cutpoint software to established disease-free and overall survival with respect to particular Notch members. Our results demonstrated separation between tumors and their corresponding normal tissue, as well as between tumors in general. The differentiation of the Notch pathway, at its various stages, in terms of expression and survival resulted in distinct profiles of biological processes such as proliferation, adhesion, apoptosis and epithelial to mesenchymal transition. In conclusion, whether oncogenic or suppressive, Notch signaling is proven to be associated with various types of malignancies, and thus may be of interest as a potential therapeutic target.

Salt inducible kinases as novel Notch interactors in the developing Drosophila retina.

Developmental processes require strict regulation of proliferation, differentiation and patterning for the generation of final organ size. Aberrations in these fundamental events are critically important in tumorigenesis and cancer progression. Salt inducible kinases (Siks) are evolutionarily conserved genes involved in diverse biological processes, including salt sensing, metabolism, muscle, cartilage and bone formation, but their role in development remains largely unknown. Recent findings implicate Siks in mitotic control, and in both tumor suppression and progression. Using a tumor model in the Drosophila eye, we show that perturbation of Sik function exacerbates tumor-like tissue overgrowth and metastasis. Furthermore, we show that both Drosophila Sik genes, Sik2 and Sik3, function in eye development processes. We propose that an important target of Siks may be the Notch signaling pathway, as we demonstrate genetic interaction between Siks and Notch pathway members. Finally, we investigate Sik expression in the developing retina and show that Sik2 is expressed in all photoreceptors, basal to cell junctions, while Sik3 appears to be expressed specifically in R3/R4 cells in the developing eye. Combined, our data suggest that Sik genes are important for eye tissue specification and growth, and that their dysregulation may contribute to tumor formation.

TMIC-47. INHIBITION OF GLIOBLASTOMA GROWTH THROUGH TUMOR-MICROENVIRONMENT CROSSTALK USING CLINICALLY SUITABLE NANOBIOCONJUGATE

Abstract Tumor environment in glioblastoma (GBM) is a dynamic interactive complex between tumor, immune and stem cells and extracellular matrix (ECM). In 226 patient glioma samples, we found a clinical correlation between expression of tumor vascular laminin-411 (α4β1γ1) and cancer stem cell (CSC) markers: Notch pathway members, CD133, Nestin, and c-Myc, with faster tumor recurrence and shorter survival of patients (Tao S, et al, Cancer Res., 2019). Novel nanotechnology approach to block trimeric ECM laminin-411 was developed for GBM treatment in experimental and preclinical models on human U87MG and LN229, and patient-derived TS543 and TS576 GBM cell lines. Nanobioconjugates (NBC) based on natural polymer, poly(β-L-malic acid), with antisense against laminin-411 α4 and β-chains, endosome escape unit (Ding H, et al, PNAS, USA, 2010), antibodies for blood-brain barrier (BBB) crossing and tumor targeting was characterized for toxicity and biodistribution according to FDA guidelines. Ex vivo depletion of laminin-411 α4 and β1 chains with CRISPR/Cas9 in human GBM cells led to reduced growth of intracranial tumors in mice, and significantly increased survival in hosts compared to mice with untreated cells. A nanobioconjugate potentially suitable for clinical use and capable of crossing BBB was designed to block laminin-411 expression. In biodistribution studies the NBC labeled with 125I on tyrosines of attached antibodies was accumulated in GBM but not in healthy brain tissue. Nanobioconjugate treatment of mice carrying intracranial GBM significantly increased animal survival and inhibited multiple CSC markers, Notch signaling system through the b1 integrin-Dll4 (Notch ligand) pathway. No toxicity revealed in 4 naïve Cynomolgus macaques after administration of three therapeutic 1X and acute 10X dosages of NBC. Conclusion: An efficient strategy of GBM treatment was developed via targeting a critical component of the tumor microenvironment, laminin-411, which is independent of heterogeneous genetic mutations in glioblastomas. Support: NIH grants U01CA151815, R01CA188743, R01CA206220, R01CA209921.

A modifier in the 129S2/SvPasCrl genome is responsible for the viability of Notch1[12f/12f] mice

BackgroundMouse NOTCH1 carries a highly conserved O-fucose glycan at Thr466 in epidermal growth factor-like repeat 12 (EGF12) of the extracellular domain. O-Fucose at this site has been shown by X-ray crystallography to be recognized by both DLL4 and JAG1 Notch ligands. We previously showed that a Notch1 Thr466Ala mutant exhibits very little ligand-induced NOTCH1 signaling in a reporter assay, whereas a Thr466Ser mutation enables the transfer of O-fucose and reverts the NOTCH1 signaling defect. We subsequently generated a mutant mouse with the Thr466Ala mutation termed Notch1[12f](Notch1tm2Pst). Surprisingly, homozygous Notch1[12f/12f] mutants on a mixed background were viable and fertile.ResultsWe now report that after backcrossing to C57BL/6 J mice for 11–15 generations, few homozygous Notch1[12f/12f] embryos were born. Timed mating showed that embryonic lethality occurred by embryonic day (E) ~E11.5, somewhat delayed compared to mice lacking Notch1 or Pofut1 (the O-fucosyltransferase that adds O-fucose to Notch receptors), which die at ~E9.5. The phenotype of C57BL/6 J Notch1[12f/12f] embryos was milder than mutants affected by loss of a canonical Notch pathway member, but disorganized vasculogenesis in the yolk sac, delayed somitogenesis and development were characteristic. In situ hybridization of Notch target genes Uncx4.1 and Dll3 or western blot analysis of NOTCH1 cleavage did not reveal significant differences at E9.5. However, qRT-PCR of head cDNA showed increased expression of Dll3, Uncx4.1 and Notch1 in E9.5 Notch1[12f/12f] embryos. Sequencing of cDNA from Notch1[12f/12f] embryo heads and Southern analysis showed that the Notch1[12f] locus was intact following backcrossing. We therefore looked for evidence of modifying gene(s) by crossing C57BL/6 J Notch1 [12f/+] mice to 129S2/SvPasCrl mice. Intercrosses of the F1 progeny gave viable F2 Notch1[12f/12f] mice.ConclusionWe conclude that the 129S2/SvPasCrl genome contains a dominant modifying gene that rescues the functions of NOTCH1[12f] in signaling. Identification of the modifying gene has the potential to illuminate novel factor(s) that promote Notch signaling when an O-fucose glycan is absent from EGF12 of NOTCH1.

Blockade of a Laminin-411-Notch Axis with CRISPR/Cas9 or a Nanobioconjugate Inhibits Glioblastoma Growth through Tumor-Microenvironment Cross-talk.

There is an unmet need for the treatment of glioblastoma multiforme (GBM). The extracellular matrix, including laminins, in the tumor microenvironment is important for tumor invasion and progression. In a panel of 226 patient brain glioma samples, we found a clinical correlation between the expression of tumor vascular laminin-411 (α4β1γ1) with higher tumor grade and with expression of cancer stem cell (CSC) markers, including Notch pathway members, CD133, Nestin, and c-Myc. Laminin-411 overexpression also correlated with higher recurrence rate and shorter survival of GBM patients. We also showed that depletion of laminin-411 α4 and β1 chains with CRISPR/Cas9 in human GBM cells led to reduced growth of resultant intracranial tumors in mice and significantly increased survival of host animals compared with mice with untreated cells. Inhibition of laminin-411 suppressed Notch pathway in normal and malignant human brain cell types. A nanobioconjugate potentially suitable for clinical use and capable of crossing blood-brain barrier was designed to block laminin-411 expression. Nanobioconjugate treatment of mice carrying intracranial GBM significantly increased animal survival and inhibited multiple CSC markers, including the Notch axis. This study describes an efficient strategy for GBM treatment via targeting a critical component of the tumor microenvironment largely independent of heterogeneous genetic mutations in glioblastoma.Significance: Laminin-411 expression in the glioma microenvironment correlates with Notch and other cancer stem cell markers and can be targeted by a novel, clinically translatable nanobioconjugate to inhibit glioma growth.

Mismatch repair deficiency and aberrations in the Notch and Hedgehog pathways are of prognostic value in patients with endometrial cancer.

The aim of this study was to investigate the prognostic value of the Hedgehog (Gli, Patched-1, Shh, Smo) and Notch (Jag1, Notch2, Notch3) pathway members, in comparison to a panel of proteins (ER, PgR, HER2/neu, Ki67, p53, p16, PTEN and MMR) previously suggested to be involved in the pathogenesis of endometrial cancer, in association with clinical outcome and standard clinicopathological characteristics. A total of 204 patients with histological diagnosis of endometrial cancer treated from 2004 to 2013 were included. The evaluation of protein expression was assessed by immunohistochemistry. Univariate analysis showed that higher Ki67 labeling, expression of PTEN, p16, Notch2 and Notch3 proteins, as well as MMR proficiency were associated with increased relapse and mortality rate. Additionally, Patched-1 protein expression was associated with worse DFS, while p53 overexpression was associated with worse OS. In multivariate analyses, patients with MMR proficient tumors had more than double risk for death than patients with MMR deficient (MMRd) tumors (adjusted HR = 2.19, 95% CI 1.05–4.58, p = 0.036). Jag1 positivity conferred reduced mortality risk (HR = 0.48, 95% CI 0.23–0.97, p = 0.042). However, as shown by hierarchical clustering, patients fared better when their tumors expressed high Jag1 protein in the absence of Notch2 and Notch3, while they fared worse when all three proteins were highly expressed. Patched-1 positivity conferred higher risk for relapse (HR = 2.04, 95% CI 1.05–3.96, p = 0.036).Aberrant expression of key components of the Notch and Hedgehog signaling pathways, as well as MMRd may serve as independent prognostic factors for recurrence and survival in patients with endometrial cancer.

Aberrant Regulation of Notch3 Signaling Pathway in Polycystic Kidney Disease

Polycystic kidney disease (PKD) is a genetic disorder characterized by fluid-filled cysts in the kidney and liver that ultimately leads to end-stage renal disease. Currently there is no globally approved therapy for PKD. The Notch signaling pathway regulates cellular processes such as proliferation and de-differentiation, which are cellular hallmarks of PKD. Thus we hypothesized that the Notch pathway plays a critical role in PKD. Evaluation of protein expression of Notch signaling components in kidneys of Autosomal Recessive PKD (ARPKD) and Autosomal Dominant PKD (ADPKD) mouse models and of ADPKD patients revealed that Notch pathway members, particularly Notch3, were consistently upregulated or activated in cyst-lining epithelial cells. Notch3 expression correlated with rapidly growing cysts and co-localized with the proliferation marker, PCNA. Importantly, Notch inhibition significantly decreased forskolin-induced Notch3 activation and proliferation of primary human ADPKD cells, and significantly reduced cyst formation and growth of human ADPKD cells cultured in collagen gels. Thus our data indicate that Notch3 is aberrantly activated and facilitates epithelial cell proliferation in PKD, and that inhibition of Notch signaling may prevent cyst formation and growth.

Effect of berberine on the renal tubular epithelial-to-mesenchymal transition by inhibition of the Notch/snail pathway in diabetic nephropathy model KKAy mice.

Renal tubular epithelial-to-mesenchymal transition (EMT) and renal tubular interstitial fibrosis are the main pathological changes of diabetic nephropathy (DN), which is a common cause of end-stage renal disease. Previous studies have suggested that berberine (BBR) has antifibrotic effects in the kidney and can reduce apoptosis and inhibit the EMT of podocytes in DN. However, the effect of BBR on the renal tubular EMT in DN and its mechanisms of action are unknown. This study was performed to explore the effects of BBR on the renal tubular EMT and the molecular mechanisms of BBR in DN model KKAy mice and on the high glucose (HG)-induced EMT in mouse renal tubular epithelial cells. Our results showed that, relative to the model mice, the mice in the treatment group had an improved general state and reduced blood glucose and 24-h urinary protein levels. Degradation of renal function was ameliorated by BBR. We also observed the protective effects of BBR on renal structural changes, including normalization of an index of renal interstitial fibrosis and kidney weight/body weight. Moreover, BBR suppressed the activation of the Notch/snail pathway and upregulated the α-SMA and E-cadherin levels in DN model KKAy mice. BBR was further found to prevent HG-induced EMT events and to inhibit the HG-induced expression of Notch pathway members and snail1 in mouse renal tubular epithelial cells. Our findings indicate that BBR has a therapeutic effect on DN, including its inhibition of the renal tubular EMT and renal interstitial fibrosis. Furthermore, the BBR-mediated EMT inhibition occurs through Notch/snail pathway regulation.

Delta-like 4/Notch signaling promotes ApcMin/+ tumor initiation through angiogenic and non-angiogenic related mechanisms

BackgroundDelta like 4 (Dll4)/Notch signaling is a key regulator of tumor angiogenesis. Additionally, the role of Dll4 has been studied on tumor stem cells. However, as these cells are implicated in tumor angiogenesis, it is conceivable that the effect of Dll4 on these cells may be a consequence of its angiogenic function. Our aim was to evaluate the expression and dissect the functions of Dll4 in the ApcMin/+ model of colorectal cancer.MethodsWe evaluated the protein expression pattern of Dll4 and other Notch members in the ApcMin/+ tumors relatively to the normal gut and compared endothelial-specific with ubiquitous Dll4 knockout mice on an ApcMin/+ background.ResultsAll Notch pathway members were present in the normal small and large intestine and in the adenomas of the same regions. Dll4, all Notch receptors and Hes1 expression seemed upregulated in the tumors, with some regional differences. The same members and Hes5, instead of Hes1, presented ectopic expression in the tumor parenchyma. Dll4 expression was most pronounced in the tumor cells but it was also present in the tumor blood vessels and in other stromal cells. Ubiquitous and endothelial-specific Dll4 deletion led to an equivalent reduction of tumor growth because of a similarly marked tumoral angiogenic phenotype promoting non-productive vasculature and consequently hypoxia and apoptosis. The ubiquitous Dll4 inhibition led to a stronger decrease of tumor multiplicity than the endothelial-specific deletion by further reducing tumor proliferation and tumor stem cell density through upregulation of the cyclin-dependent kinase inhibitors 1C and 1B and downregulation of Myc, Cyclin D1 and D2 independently of β-catenin activation. This phenotype was associated to the observed increased epithelial differentiation deviated towards the secretory lineages by Atoh1 and Klf4 upregulation only in the ubiquitous Dll4 mutants.ConclusionsDll4 seems to promote ApcMin/+ tumorigenesis through both angiogenic and non-angiogenic related mechanisms.

Predictive values of Notch signalling in renal carcinoma.

IntroductionNotch signalling, an evolutionarily conserved mechanism of cellular differentiation and tissue remodelling, is frequently deregulated in several human malignancies, including renal cell carcinoma (RCC). However, the prognostic value of individual Notch pathway members in RC subtypes remains indefinable. The present study investigates whether the differential expression of Notch members has a contrary effect on disease-free survival (DFS) in clear cell renal cell carcinoma (KIRC), papillary cell renal cell carcinoma (KIRP) and chromophobe renal cell carcinoma (KICH) patients.Material and methodsThe predictive value of 19 Notch members was evaluated in KICH, KIRC and KIRP patient cohorts from The Cancer Genome Atlas (TCGA). Results in the form of Kaplan-Meier survival plots with the p-value calculated (log-rank test, p < 0.05) enabled the patients to be split into favourable/unfavourable prognosis groups regarding expression of Notch members.ResultsMore specifically, lowered expression of ADAM17 correlated with good prognosis in KICH, KIRC and KIRP (HR = 7.79, p = 0.03; HR = 3.98, p = 0.051; HR = 11.24, p < 0.001, respectively). Additionally, HES4 differentiated KICH and KIRC, as its higher expression correlated with good prognosis in KICH and favourable lowered expression in KIRC (HR = 0.11, p = 0.015; HR = 2.42, p < 0.001, respectively).ConclusionsOur analysis could be valuable for better understanding of the molecular mechanism of renal carcinoma. The expression of Notch pathway members could be a useful biomarker for predicting favourable/unfavourable prognosis in patients with RCC.

Leukemia-Induced Dysregulation of Bone Marrow Skeletal Stem Cells (SSC) Subpopulations and Their Hematopoietic Supportive Function

Specialized bone marrow microenvironment (BMM) niches are essential for hematopoietic stem cell (HSC) maintenance and differentiation, and include perivascular cells with mesenchymal stem cells (MSC) capacity. The role of BMM niches in regulating LSC growth remains unclear. We have shown that cytokines altered in CML BMM contribute a competitive growth advantage to CML LSC over normal HSC (Zhang et al. Cancer Cell. 2012; 21(4):577). However, the effect of leukemic hematopoiesis on BM MSC and their progeny, and the effect of such alterations on the regulation of LSC versus HSC growth, is not well studied. Recently rigorous single-cell analyses and lineage tracing has been used to delineate a BM skeletal stem cell (SSC) hierarchy (Chan et al. Cell. 2015, 160(1-2):285). Eight distinct subpopulations were identified within the CD45-Ter119-Tie2-AlphaV+ BM mesenchymal cells, of which the mSSC (Thy-6C3-CD105-CD200+) subpopulation generated all of the other subpopulations. Two multipotent progenitor populations, pre-BCSP (Thy-6C3-CD105-CD200-) and BCSP (Thy-6C3-CD105+) gave rise to bone, cartilage and hematopoietic supportive stroma. The 6C3 (Thy-6C3+CD105+) and HEC (Thy-6C3+CD105-) populations gave rise to stroma, the BLSP (Thy+6C3-CD105-) and Thy (Thy+6C3-CD105+CD200-); populations formed bone, and the PCP (Thy+6C3-CD105+CD200+) population formed cartilage. To examine how these mesenchymal populations related to those targeted in the Cre-transgenic mouse lines commonly used to study BMM, we performed lineage tracing by crossing Prx-1-Cre, Sp7-Cre, BGLAP-Cre, and UBC-Cre mice with Rosa26CAG-tdTomato mice, in which tdTomato is expressed by cells targeted for Cre-mediated recombination. Interestingly, mSSC and 6C3 cells were targeted in all Cre lines, while the Thy population was only targeted in Prx1-Cre mice. We examined SSC population distribution and function following CML development using the SCL-tTA-BCR-ABL transgenic mouse model. The frequency and absolute number of bone-forming Thy (WT: 442.5±65.66; CML: 2501±705.5; p=0.012, n=14) and stroma-forming 6C3 (WT:1061±193.7; CML: 2271±358.5; p=0.009, n=14) cells were significantly increased in BM of CML mice compared to wild-type (WT) mice, while primitive mSSC, pre-BCSP and BCSP populations remained unchanged. Expression of major hematopoietic regulatory molecules, transcription factors and cell cycle regulatory genes in CML and WT BMM subsets was measured by qPCR analysis using the Fluidigm system. Integrin α4 and Tenascin levels were reduced, whereas PDGFRβ, BMP2 and Hif-1α levels were increased in CML compared to normal mSSC. Several WNT pathway members were significantly upregulated in CML compared to WT 6C3 stromal cells, whereas CXCL12, KitL, IL-3, and IGF-1 were reduced. Expression of cell cycle regulators, Hedgehog and Notch pathway members remained unchanged. In ongoing experiments to investigate whether these subsets have altered HSC supportive capacity towards normal and CML stem cells, we co-cultured FACS sorted normal or CML LTHSC with FACS sorted normal or CML mSSC, BCSP, 6C3, and Thy cells for 3 days followed by transplantation into lethally irradiated WT mice. Normal LTHSC engraftment at 4 weeks post-transplant was enhanced by coculture with normal mSSC but not with CML mSSC. In contrast, there was increased engraftment of normal LTHSC co-cultured with CML compared to normal BCSP, Thy or 6C3 cells. CML LTHSC engraftment at 4 weeks was not enhanced by either normal or CML mSSC, but was enhanced following co-culture with normal as well as CML Thy and 6C3 cells. These results indicate that SSC subpopulations from CML BM demonstrate altered capacity to support normal LTHSC compared to their normal counterparts, but that CML and WT SSC populations may have similar effects on CML LTHSC. This experiment is ongoing with results of longer-term engraftment pending. In conclusion, we have further characterized a phenotypically-defined skeletal stem cell hierarchy and identified significant alterations in distribution of SSC subpopulations within CML compared to WT BM, with significantly altered expression of important hematopoietic regulatory molecules and HSC supportive function. We hypothesize that leukemia-induced SSC alterations may confer a growth advantage to CML LSC over normal HSC, and that characterization of key regulatory mechanisms could facilitate therapeutic intervention. DisclosuresNo relevant conflicts of interest to declare.

Bruceantin inhibits multiple myeloma cancer stem cell proliferation

ABSTRACTMultiple myeloma (MM) continues to claim the lives of a majority of patients. MM cancer stem cells (CSCs) have been demonstrated to sustain tumor growth. Due to their ability to self-renew and to express detoxifying enzymes and efflux transporters, MM-CSCs are rendered highly resistant to conventional therapies. Therefore, managing MM-CSCs characteristics could have profound clinical implications. Bruceantin (BCT) is a natural product previously demonstrated to inhibit the growth of MM in RPMI 8226 cells-inoculated mouse xenograft models, and to cause regression in already established tumors. The objectives of the present study were to test the inhibitory effects of BCT on MM-CSCs growth derived from a human primary tumor, and to explore a mechanism of action underlying these effects. BCT exhibited potent antiproliferative activity in MM-CSCs starting at 25 nM. BCT induced cell cycle arrest, cell death and apoptosis in MM-CSCs as well as inhibited cell migration and angiogenesis in vitro. Using a qPCR screen, it was found that the gene expression of a number of Notch pathway members was altered. Pretreatment of MM-CSCs with the γ-secretase inhibitor RO4929097, a Notch pathway inhibitor, reversed BCT-induced effects on MM-CSCs proliferation. In this study, BCT was shown to be an effective agent in controlling the proliferation, viability and migration of MM-CSCs as well as angiogenesis in vitro. The effect on MM-CSCs proliferation may be mediated by the Notch pathway. These results warrant further investigation of BCT in a broader set of human-derived MM-CSCs and with in vivo models representative of MM.

Drosophila Cyclin G Is a Regulator of the Notch Signalling Pathway during Wing Development.

Notch signalling regulates a multitude of differentiation processes during Drosophila development. For example, Notch activity is required for proper wing vein differentiation which is hampered in mutants of either the receptor Notch, the ligand Delta or the antagonist Hairless. Moreover, the Notch pathway is involved in several aspects of Drosophila oogenesis as well. We have identified Drosophila Cyclin G (CycG) as a molecular interaction partner of Hairless, the major antagonist in the Notch signalling pathway, in vitro and in vivo. Loss of CycG was shown before to cause female sterility and to disturb the architecture of the egg shell. Nevertheless, Notch dependent processes during oogenesis appeared largely unaffected in cycG mutant egg chambers. Loss of CycG modified the dominant wing phenotypes of Notch, Delta and Hairless mutants. Whereas the Notch loss of function phenotype was ameliorated by a loss of CycG, the phenotypes of either Notch gain of function or of Delta or Hairless loss of function were enhanced. In contrast, loss of CycG had only a minor effect on the wing vein phenotype of mutants affecting the EGFR signalling pathway emphasizing the specificity of the interaction of CycG and Notch pathway members.

Whole Exome Sequencing of Flow Purified Tumour Cells Reveals Recurrently Mutated Genes and Pathways in Adult T-Cell Lymphoma/Leukaemia (ATLL)

IntroductionATLL is an aggressive lymphoid malignancy with a very poor prognosis arising in patients with HTLV-1 infection. Asymptomatic HTLV1+ individuals have a polyclonal expansion of CD4+ T-cells driven by the products of the HTLV-1 tax and HBZ genes. In ATLL, integration site analysis shows a monoclonal outgrowth (frequently associated with downregulation of tax) which is presumed to be driven by acquisition of oncogenic somatic mutations. Genome-wide studies identifying these mutations in patients of African or African Caribbean origin have not been reported; limited data are available on a Japanese population.MethodsWe obtained nine cryopreserved peripheral blood mononuclear cell samples from seven patients of African Carribean origin. ATLL cells (CD3+CD4+CD25+CCR4+) and healthy B- and myeloid cells (CD3-CD33+ and CD3-CD20+) were sorted to high purity (>99%) by flow cytometry. B- and myeloid populations were pooled and used as a germline control. DNA was extracted from the sorted populations and whole exome sequencing libraries were prepared using Sure Select XT v5 (Agilent) and sequenced using an Illumina HiSeq2500. Mean exome coverage was 145.3x in tumour samples and 131x in control samples with 96.3% and 96% respectively covered at >20x. Variants were called using GATK, germline variants were filtered out using VarScan, and all remaining variants were manually inspected using IGV. Copy number changes were called using ExomeDepth.ResultsWe identified somatic mutations in all cases, including short insertions or deletions (InDels, median 3 per exome, range 2-12), single nucleotide variations (SNV) affecting the amino acid sequence (median 40, range 4-132) and noncoding SNV (median 35, range 12-114). Median variant allele frequency (VAF) was >20% in 8/9 tumour samples (overall median 40%, range 24-47%) indicating reliable isolation of substantially pure clonal tumour cells. We excluded one sample with a low VAF (18%) from further analysis.We identified two recurrent somatic point mutations: CCR4 Y331I (2 patients; a known activating mutation present in ~25% of ATLL cases) and NRG1 T232M (2 patients; a novel mutation). Both patients with CCR4 Y331I had large duplications encompassing the mutant CCR4 allele along with CCR1-CCR9; one of the two patients had been treated with the anti-CCR4 antibody Mogamulizumab and had been in complete remission for 20 months. We identified a further six genes harbouring somatic mutations in more than one patient: ANKRD30A, CDH7, PRKCB, RYR2, SETD5 and STAT3 (each mutated in two patients).In addition to these recurrently mutated genes, pathway analysis using ConsensusPath identified three pathways which were mutated in most or all patients. NOTCH pathway members were mutated in 6/6 cases (with 3/6 cases carrying two mutations) in one of the following genes: NOTCH1, NOTCH2, MAMLD1, DMXL2, PTCRA, NOTCH2NL, TBL1XR1, HDAC10.T-cell activation and costimulation pathway members were mutated in 5/6 patients (range 1-5 mutations). The following genes were affected: CARD11, CCR4, CD40LG, CHUK, DPP8, HLA-A, HLA-B, IFNAR2, NLRP2, P2RX7, PLCG1, RASSF5, SH2B3, ZEB1.Finally we observed multiple mutations affecting the FGFR/EGFR - PI3K signalling pathway (6/6 patients, 1-6 mutations per patient). The affected genes were ADCY1, APBB1B, FGFR2, HGF, LRRFIP1, NCAM1, NRG1, PDGFC, PI3KCD, PLCG1, PLCXD1, PRKCB, STAT3 and ZMYM2.In one patient, all 261 somatic mutations present in the blood were detected in a lymph node biopsy taken at the same time; an additional mutation (a frameshift deletion in FIP1L1) was present in the node but not detected in the blood. In another patient, a sample taken at the time of relapse five years after initial presentation was also analysed: all 97 mutations called at diagnosis were present at relapse, and 15 additional somatic mutations had been acquired.ConclusionsWhole exome sequencing identified two recurrent somatic point mutations and six recurrently mutated genes which, given their co-occurrence in this small sample, are likely to be tumour drivers. Genes encoding components of the NOTCH, T-cell activation and FGFR/EGFR-PI3K pathways were mutated in most or all ATLL samples analysed, identifying these pathways as potential therapeutic targets. We now plan to validate these candidate genes in a large and longitudinal patient cohort and compare these with results from an ongoing study in the Japanese population. DisclosuresNo relevant conflicts of interest to declare.

Glycogen synthase kinase-3 inhibitor AR-A014418 suppresses pancreatic cancer cell growth via inhibition of GSK-3-mediated Notch1 expression

BackgroundGlycogen synthase kinase-3 (GSK-3) can act as either a tumour promoter or suppressor by its inactivation depending on the cell type. There are conflicting reports on the roles of GSK-3 isoforms and their interaction with Notch1 in pancreatic cancer. It was hypothesized that GSK-3α stabilized Notch1 in pancreatic cancer cells thereby promoting cellular proliferation. MethodsThe pancreatic cancer cell lines MiaPaCa2, PANC-1 and BxPC-3, were treated with 0–20 μM of AR-A014418 (AR), a known GSK-3 inhibitor. Cell viability was determined by the MTT assay and Live-Cell Imaging. The levels of Notch pathway members (Notch1, HES-1, survivin and cyclinD1), phosphorylated GSK-3 isoforms, and apoptotic markers were determined by Western blot. Immunoprecipitation was performed to identify the binding of GSK-3 specific isoform to Notch1. ResultsAR-A014418 treatment had a significant dose-dependent growth reduction (P< 0.001) in pancreatic cancer cells compared with the control and the cytotoxic effect is as a result of apoptosis. Importantly, a reduction in GSK-3 phosphorylation lead to a reduction in Notch pathway members. Overexpression of active Notch1 in AR-A014418-treated cells resulted in the negation of growth suppression. Immunoprecipitation analysis revealed that GSK-3α binds to Notch1. ConclusionsThis study demonstrates for the first time that the growth suppressive effect of AR-A014418 on pancreatic cancer cells is mainly mediated by a reduction in phosphorylation of GSK-3α with concomitant Notch1 reduction. GSK-3α appears to stabilize Notch1 by binding and may represent a target for therapeutic development. Furthermore, downregulation of GSK-3 and Notch1 may be a viable strategy for possible chemosensitization of pancreatic cancer cells to standard therapeutics.

Notch signaling deregulation in multiple myeloma: A rational molecular target.

Despite recent therapeutic advances, multiple myeloma (MM) is still an incurable neoplasia due to intrinsic or acquired resistance to therapy. Myeloma cell localization in the bone marrow milieu allows direct interactions between tumor cells and non-tumor bone marrow cells which promote neoplastic cell growth, survival, bone disease, acquisition of drug resistance and consequent relapse. Twenty percent of MM patients are at high-risk of treatment failure as defined by tumor markers or presentation as plasma cell leukemia. Cumulative evidences indicate a key role of Notch signaling in multiple myeloma onset and progression. Unlike other Notch-related malignancies, where the majority of patients carry gain-of-function mutations in Notch pathway members, in MM cell Notch signaling is aberrantly activated due to an increased expression of Notch receptors and ligands; notably, this also results in the activation of Notch signaling in surrounding stromal cells which contributes to myeloma cell proliferation, survival and migration, as well as to bone disease and intrinsic and acquired pharmacological resistance. Here we review the last findings on the mechanisms and the effects of Notch signaling dysregulation in MM and provide a rationale for a therapeutic strategy aiming at inhibiting Notch signaling, along with a complete overview on the currently available Notch-directed approaches.

LG-04 * NOTCH SIGNALING ACTIVATION IN PEDIATRIC LOW-GRADE ASTROCYTOMA

Pilocytic astrocytoma (PA) is the most common primary brain tumor in children, and various signaling pathways have been implicated in its biology. The Notch signaling pathway has been found to play a role in development, stem cell biology and the pathogenesis of several cancers but its role in PA has not been investigated. We studied alterations in Notch signaling components in clinical PA tumor tissue, to identify much needed therapeutic targets. In this study, we found Notch pathway members to be overexpressed at the mRNA (NOTCH1, NOTCH2, HEY1, HEY2) and protein (HES1) level in PA at various anatomical sites compared to non-neoplastic brain. The changes were not associated with specific BRAF alterations. Inhibiting the Notch pathway in pediatric low grade astrocytoma-derived cell lines (Res 186 and Res 259) using either RNA interference or a gamma secretase inhibitor resulted in a significant, but variable reduction in cell growth and migration. This study suggests a role for Notch signaling in the context of pediatric low grade astrocytoma tumorigenesis, and that Notch signaling is a viable pathway to target therapeutically.