Sanaz Yahyanejad

Regulated Proteolysis of NOTCH2 and NOTCH3 Receptors by ADAM10 and Presenilins

ABSTRACT In mammals, there are four NOTCH receptors and five Delta-Jagged-type ligands regulating many aspects of embryonic development and adult tissue homeostasis. NOTCH proteins are type I transmembrane receptors that interact with ligands on adjacent cells and are activated by regulated intramembrane proteolysis (RIP). The activation mechanism of NOTCH1 receptors upon ligand binding is well understood and requires cleavage by ADAM10 metalloproteases prior to intramembranous cleavage by γ-secretase. How the other human NOTCH receptor homologues are activated upon ligand binding is not known. Here, we dissect the proteolytic activation mechanism of the NOTCH2 and NOTCH3 receptors. We show that NOTCH2 and NOTCH3 signaling can be triggered by both Delta-Jagged-type ligands and requires ADAM10 and presenilin-1 or -2. Importantly, we did not find any role for the highly related ADAM17/TACE (tumor necrosis factor alpha-converting enzyme) protease in ligand-induced NOTCH2 or NOTCH3 signaling. These results demonstrate that canonical ligand-induced proteolysis of the NOTCH1, -2, and -3 receptors strictly depends on consecutive cleavage of these receptors by ADAM10 and the presenilin-containing γ-secretase complex, leading to transcriptional activation.

High NOTCH activity induces radiation resistance in non small cell lung cancer

BACKGROUND AND PURPOSE Patients with advanced NSCLC have survival rates <15%. The NOTCH pathway plays an important role during lung development and physiology but is often deregulated in lung cancer, making it a potential therapeutic target. We investigated NOTCH signaling in NSCLC and hypothesized that high NOTCH activity contributes to radiation resistance. MATERIALS AND METHODS NOTCH signaling in NSCLC patient samples was investigated using quantitative RT-PCR. H460 NSCLC cells with either high or blocked NOTCH activity were generated and their radiation sensitivity monitored using clonogenic assays. In vivo, xenograft tumors were irradiated and response assessed using growth delay. Microenvironmental parameters were analyzed by immunohistochemistry. RESULTS Patients with high NOTCH activity in tumors showed significantly worse disease-free survival. In vitro, NOTCH activity did not affect the proliferation or intrinsic radiosensitivity of NSCLC cells. In contrast, xenografts with blocked NOTCH activity grew slower than wild type tumors. Tumors with high NOTCH activity grew significantly faster, were more hypoxic and showed a radioresistant phenotype. CONCLUSIONS We demonstrate an important role for NOTCH in tumor growth and correlate high NOTCH activity with poor prognosis and radioresistance. Blocking NOTCH activity in NSCLC might be a promising intervention to improve outcome after radiotherapy.

NOTCH blockade combined with radiation therapy and temozolomide prolongs survival of orthotopic glioblastoma

Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults. The current standard of care includes surgery followed by radiotherapy (RT) and chemotherapy with temozolomide (TMZ). Treatment often fails due to the radiation resistance and intrinsic or acquired TMZ resistance of a small percentage of cells with stem cell-like behavior (CSC). The NOTCH signaling pathway is expressed and active in human glioblastoma and NOTCH inhibitors attenuate tumor growth in vivo in xenograft models. Here we show using an image guided micro-CT and precision radiotherapy platform that a combination of the clinically approved NOTCH/γ-secretase inhibitor (GSI) RO4929097 with standard of care (TMZ + RT) reduces tumor growth and prolongs survival compared to dual combinations. We show that GSI in combination with RT and TMZ attenuates proliferation, decreases 3D spheroid growth and results into a marked reduction in clonogenic survival in primary and established glioma cell lines. We found that the glioma stem cell marker CD133, SOX2 and Nestin were reduced following combination treatments and NOTCH inhibitors albeit in a different manner. These findings indicate that NOTCH inhibition combined with standard of care treatment has an anti-glioma stem cell effect which provides an improved survival benefit for GBM and encourages further translational and clinical studies.

Targeting Notch to overcome radiation resistance

Radiotherapy represents an important therapeutic strategy in the treatment of cancer cells. However, it often fails to eliminate all tumor cells because of the intrinsic or acquired treatment resistance, which is the most common cause of tumor recurrence. Emerging evidences suggest that the Notch signaling pathway is an important pathway mediating radiation resistance in tumor cells. Successful targeting of Notch signaling requires a thorough understanding of Notch regulation and the context-dependent interactions between Notch and other therapeutically relevant pathways. Understanding these interactions will increase our ability to design rational combination regimens that are more likely to be safe and effective. Here we summarize the role of Notch in mediating resistance to radiotherapy, the different strategies to block Notch in cancer cells and how treatment scheduling can improve tumor response. Finally, we discuss a need for reliable Notch related biomarkers in specific tumors to measure pathway activity and to allow identification of a subset of patients who are likely to benefit from Notch targeted therapies.

Human NOTCH2 Is Resistant to Ligand-independent Activation by Metalloprotease Adam17 *

Background: Mutations in the NOTCH1 HD domain cause NRR perturbation and ligand-independent activation and are frequent in leukemia. Results: Human NOTCH2 but not mouse Notch2 is resistant to NRR perturbation and ligand-independent activation by Adam17. Conclusion: NOTCH homologs and paralogs differ in their sensitivity to ligand-independent activation. Significance: Our data explain why activating NOTCH2 HD domain mutations are infrequent in diseases such as cancer. Cell surface receptors of the NOTCH family of proteins are activated by ligand induced intramembrane proteolysis. Unfolding of the extracellular negative regulatory region (NRR), enabling successive proteolysis by the enzymes Adam10 and γ-secretase, is rate-limiting in NOTCH activation. Mutations in the NOTCH1 NRR are associated with ligand-independent activation and frequently found in human T-cell malignancies. In mammals four NOTCH receptors and five Delta/Jagged ligands exist, but mutations in the NRR are only rarely reported for receptors other than NOTCH1. Using biochemical and functional assays, we compared the molecular mechanisms of ligand-independent signaling in NOTCH1 and the highly related NOTCH2 receptor. Both murine Notch1 and Notch2 require the metalloprotease protease Adam17, but not Adam10 during ligand-independent activation. Interestingly, the human NOTCH2 receptor is resistant to ligand-independent activation compared with its human homologs or murine orthologs. Taken together, our data reveal subtle but functionally important differences for the NRR among NOTCH paralogs and homologs.

Complementary Use of Bioluminescence Imaging and Contrast-Enhanced Micro-Computed Tomography in an Orthotopic Brain Tumor Model

Small animal models are crucial to link molecular discoveries and implementation of clinically relevant therapeutics in oncology. Using these models requires noninvasive imaging techniques to monitor disease progression and therapy response. Micro–computed tomography (CT) is less studied for the in vivo monitoring of murine intracranial tumors and traditionally suffers from poor soft tissue contrast, whereas bioluminescence imaging (BLI) is known for its sensitivity but is not frequently employed for quantifying tumor volume. A widely used orthotopic glioblastoma multiforme (GBM) tumor model was applied in nude mice, and tumor growth was evaluated by BLI and contrast-enhanced microCT imaging. A strong correlation was observed between CT volume and BLI-integrated intensity (Pearson coefficient (r) = .85, p = .0002). Repeated contouring of contrast-enhanced microCT-delineated tumor volumes achieved an intraobserver average pairwise overlap ratio of 0.84 and an average tumor volume coefficient of variance of 0.11. MicroCT-delineated tumor size was found to correlate with tumor size obtained via histologic analysis (Pearson coefficient (r) = .88, p = .005). We conclude that BLI intensity can be used to derive tumor volume but that the use of both contrast-enhanced microCT and BLI provides complementary tumor growth information, which is particularly useful for modern small animal irradiation devices that make use of microCT and BLI for treatment planning, targeting, and monitoring.Small animal models are crucial to link molecular discoveries and implementation of clinically relevant therapeutics in oncology. Using these models requires noninvasive imaging techniques to monitor disease progression and therapy response. Micro-computed tomography (CT) is less studied for the in vivo monitoring of murine intracranial tumors and traditionally suffers from poor soft tissue contrast, whereas bioluminescence imaging (BLI) is known for its sensitivity but is not frequently employed for quantifying tumor volume. A widely used orthotopic glioblastoma multiforme (GBM) tumor model was applied in nude mice, and tumor growth was evaluated by BLI and contrast-enhanced microCT imaging. A strong correlation was observed between CT volume and BLI-integrated intensity (Pearson coefficient (r)  =  .85, p  =  .0002). Repeated contouring of contrast-enhanced microCT-delineated tumor volumes achieved an intraobserver average pairwise overlap ratio of 0.84 and an average tumor volume coefficient of variance of 0.11. MicroCT-delineated tumor size was found to correlate with tumor size obtained via histologic analysis (Pearson coefficient (r)  =  .88, p  =  .005). We conclude that BLI intensity can be used to derive tumor volume but that the use of both contrast-enhanced microCT and BLI provides complementary tumor growth information, which is particularly useful for modern small animal irradiation devices that make use of microCT and BLI for treatment planning, targeting, and monitoring.

An image guided small animal radiation therapy platform (SmART) to monitor glioblastoma progression and therapy response

BACKGROUND AND PURPOSE Glioblastoma multiforme is the most common malignant brain tumor. Standard treatment including surgery, radiotherapy and chemotherapy with temozolomide is not curative. There is a great need for in vitro and in vivo models closely mimicking clinical practice to ensure better translation of novel preclinical findings. METHODS AND MATERIALS A 3D spheroid model was established using the U87MG cell line. The efficacy of temozolomide, RT and combinations was assessed using growth delay assays. Orthotopic glioblastoma tumors were established, different radiation doses delivered based on micro-CT based treatment planning (SmART-plan) and dose volume histograms (DVH) were determined. Tumor growth was monitored using bioluminescent imaging. RESULTS 3D spheroid cultures showed a dose-dependent growth delay upon single and combination treatments. Precise uniform radiation was achieved in all in vivo treatment groups at all doses tested, and DVHs showed accurate dose coverage in the planning target volume which resulted in tumor growth delay. CONCLUSION We demonstrate that 3D spheroid technology can be reliably used for treatment efficacy evaluation and that mimicking a clinical setting is also possible in small animals. Both these in vitro and in vivo techniques can be combined for clinically relevant testing of novel drugs combined with radiation.

OC-0237: Adding Notch inhibition increases efficacy of standard of care treatment in glioblastoma

Purpose or Objective: The novel combretastatin analogue, OXi4503, is a vascular disrupting agent (VDA) that has recently been shown to significantly enhance a stereotactic radiation treatment. This was achieved using an OXi4503 dose of 10 mg/kg combined with a stereotactic treatment of 3 x 15 Gy. The current study was undertaken to determine the OXi4503 dose dependency when using different stereotactic radiation dose schedules.

SmART-ER imaging and treatment of glioblastoma.

Small animal imaging and radio therapy (SmART) platforms combine non-invasive imaging and precise conformal irradiation and are emerging as an important links between preclinical cancer research and clinical treatment to test novel irradiation schedules, evaluate radiosensitizers, and to monitor disease progression and response [1]. With interest we read the article by Kirschner et al. ‘‘In vivo micro-CT imaging of untreated and irradiated orthotropic glioblastoma xenografts in mice: capabilities, limitations and a comparison with bioluminescence imaging’’ in Journal of Neuro-Oncology [2]. Kirschner has used a comparable approach as in Yahyanejad et al. ‘‘Complementary Use of Bioluminescence Imaging and ContrastEnhanced Micro-Computed Tomography in an Orthotropic Brain Tumor Model’’ Mol Imaging [3]. Both studies describe the implementation of SmART for a preclinical Glioblastoma (GBM) model in mice. GBM is a deadly disease with no cure and in dire need of new treatment options. Both studies investigated the correlation between bioluminescent imaging (BLI) to contrast-enhanced micro-CT (CECT) as a tool to estimate tumor volume and monitor disease progression and response. BLI is a cheap, fast and very sensitive non-invasive based imaging approach based on the expression of the enzyme firefly luciferase in tumor cells, which emits lights when the substrate luciferin is injected. Micro-CT based follow up of brain tumor growth requires the use of exogenous contrast agents. Both studies use the U87MG GBM cell line that establishes relatively quickly (*1 month) so that therapeutic interventions can be performed in a reasonable timeframe. In Kirschner’s study, immunosuppressed NOD/SCID mice were used, while in our study CD1 nude mice were used. Similar surgical techniques were used between the two studies, except that in our study fewer cells were injected (1 vs. 2.5 9 10). In our study, all mice that were implanted (100 %, n = 33), established tumors, whereas in the other study only three quarters grew tumors (74 %, n = 27). CECT imaging was used in both studies with similar energies (80 kVp). Both studies used Iodinated contrast agents iomeprol (300 ll/iv) or Ominipaque 350 (150 ll/iv), which both appear to be well tolerated. The CECT-derived tumor volumes compared to histological determination were systematically smaller in both studies 23 and 20 %, respectively, most likely as a consequence of tissue fixation. Importantly, both studies observed strong correlation coefficients of 0.96 and 0.88 between micro-CT and histology demonstrating that CECT imaging in orthotropic GBM mouse model can be used to delineate brain tumor growth longitudinally in vivo (supplementary material). Additionally, both studies reported good interand intra-observer correspondence between lCT delineated tumor volumes. This is crucial because micro-CT could suffice for initial tumor delineation and planning and BLI imaging is accurate enough (across 59 tumor volume doublings) to measure tumor control in response to treatments. To monitor tumor growth longitudinally both studies used U87MG tumor cells expressing firefly luciferase. While the Kirschner’s study reported a moderate correlation (Spearman r = 0.543) between CT-derived tumor Electronic supplementary material The online version of this article (doi:10.1007/s11060-015-1801-6) contains supplementary material, which is available to authorized users.

Abstract 3243: Notch inhibitors and chemoradiation in an orthotopic glioblastoma model

Glioblastoma multiforme (GBM) is the most common and malignant brain tumor in adults. Aggressive multimodal treatment using surgery followed by radiotherapy and chemotherapy extends the median survival of GBM patients to approximately one year after diagnosis. Treatment is not curative because of the intrinsic and acquired radiation resistance of a subpopulation of tumor cells. Notch inhibition has been shown to impair the tumorigenic capacity of these cells as well as enhance their sensitivity towards radiation. Therefore, we investigated if a highly potent and clinically approved Notch pathway inhibitor (g-secretase inhibitor) improves tumor control when combined with radiotherapy and chemotherapy in an orthotopic GBM mouse model. To investigate the treatment efficacy of combinations treatments between standard of care treatment (radiotherapy and temozolomide) and Notch inhibitors, we used a three dimensional spheroid growth assay using both established and primary human glioma cell lines in which spheroid volume growth delay was quantitatively monitored. In addition, we assessed the expression of the putative glioma stem cell marker CD133 using flow cytometry. Furthermore, therapeutic efficacy of these combination treatments was als assessed in vivo in an orthotopic glioma tumor model (U87-luc) wherein tumor progression was evaluated using bioluminescence (BLI) and contrast-enhanced micro-computed tomography (CT) imaging. A small animal precision irradiation platform (PXI, XRAD 225Cx, CT, USA) was used for micro-CT imaging and irradiation delivery of conformal doses between 2- 10 Gy to intracranial tumors in mice receiving single or combination treatments with Notch inhibitors and chemotherapy. Results: Notch blockade alone did not affect the sphere volume compared to control, whereas combination treatment with radiation and / or temozolomide resulted in a substantial spheroid growth delay (p = 0.004). Irradiation enhanced the expression of the stem cell marker CD133, while Notch blockade reduced CD133 expression. In intracranial tumours we found a strong correlation between CT and BLI imaging of tumor growth (Pearson coefficient (r) = 0.85, p = 0.001). The potential of Notch inhibition combined with precision radiotherapy and chemotherapy in our orthotopic GBM model is currently being determined and the outcome of this study will be presented. Citation Format: Sanaz Yahyanejad, Patrick Granton, Stefan van Hoof, Lydie Barbeau, Jan Theys, Frank Verhaegen, Marc Vooijs. Notch inhibitors and chemoradiation in an orthotopic glioblastoma model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3243. doi:10.1158/1538-7445.AM2015-3243