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

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

Regulated Proteolysis of NOTCH2 and NOTCH3 Receptors by ADAM10 and Presenilins

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

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

Complementary use of bioluminescence imaging and contrast-enhanced micro-computed tomography in an orthotopic brain tumor model

AbstractSmall 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

The anti-malarial drug chloroquine sensitizes oncogenic NOTCH1 driven human T-ALL to gamma-secretase inhibition

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer arising from T-cell progenitors. Although current treatments, including chemotherapy and glucocorticoids, have significantly improved survival, T-ALL remains a fatal disease and new treatment options are needed. Since more than 60% of T-ALL cases bear oncogenic NOTCH1 mutations, small molecule inhibitors of NOTCH1 signalling; gamma-secretase inhibitors (GSI), are being actively investigated for the treatment of T-ALL. Unfortunately, GSI have shown limited clinical efficacy and dose-limiting toxicities. We hypothesized that by combining known drugs, blocking NOTCH activity through another mechanism, may synergize with GSI enabling equal efficacy at a lower concentration. Here, we show that the clinically used anti-malarial drug chloroquine (CQ), an inhibitor of lysosomal function and autophagy, decreases T-ALL cell viability and proliferation. This effect of CQ was not observed in GSI-resistant T-ALL cell lines. Mechanistically, CQ impairs the redox balance, induces ds DNA breaks and activates the DNA damage response. CQ also interferes with intracellular trafficking and processing of oncogenic NOTCH1. Interestingly, we show for the first time that the addition of CQ to gamma-secretase inhibition has a synergistic therapeutic effect on T-ALL and reduces the concentration of GSI required to obtain a reduction in cell viability and a block of proliferation. Overall, our results suggest that CQ may be a promising repurposed drug in the treatment of T-ALL, as a single treatment or in combination with GSI, increasing the therapeutic ratio.</p