A Molecular Test for Quantifying Functional Notch Signaling Pathway Activity in Human Cancer

Abstract

Background: The Notch signal transduction pathway is pivotal for various physiological processes, including immune responses, and has been implicated in the pathogenesis of many diseases. The effectiveness of various targeted Notch pathway inhibitors may vary due to variabilities in Notch pathway activity among individual patients. The quantitative measurement of Notch pathway activity is therefore essential to identify patients who could benefit from targeted treatment. Methods: We here describe a new assay that infers a quantitative Notch pathway activity score from the mRNA levels of generally conserved direct NOTCH target genes. Following the calibration and biological validation of our Notch pathway activity model over a wide spectrum of human cancer types, we assessed Notch pathway activity in a cohort of T-ALL patient samples and related it to biological and clinical parameters, including outcome. Results: We developed an assay using 18 select direct target genes and high-grade serous ovarian cancer for calibration. For validation, seven independent human datasets (mostly cancer series) were used to quantify Notch activity in agreement with expectations. For T-ALL, the median Notch pathway activity was highest for samples with strong NOTCH1-activating mutations, and T-ALL patients of the TLX subtype generally had the highest levels of Notch pathway activity. We observed a significant relationship between ICN1 levels and the absence/presence of NOTCH1-activating mutations with Notch pathway activity scores. Patients with the lowest Notch activity scores had the shortest event-free survival compared to other patients. Conclusions: High Notch pathway activity was not limited to T-ALL samples harboring strong NOTCH1 mutations, including juxtamembrane domain mutations or hetero-dimerization combined with PEST-domain or FBXW7 mutations, indicating that additional mechanisms may activate Notch signaling. The measured Notch pathway activity was related to intracellular NOTCH levels, indicating that the pathway activity score more accurately reflects Notch pathway activity than when it is predicted on the basis of NOTCH1 mutations. Importantly, patients with low Notch pathway activity had a significantly shorter event-free survival compared to patients showing higher activity.

RNA Based Approaches to Profile Oncogenic Pathways From Low Quantity Samples to Drive Precision Oncology Strategies

“…(G) Quantitative differences over the course of time (days 1, 2, 3, 7) without (first four groups) and with TGFβ stimulation (last four groups); microarray data from GSE84500. Creemers et al, 2018;van de Stolpe et al, 2018;van Ooijen et al, 2018;Bouwman et al, 2020;Canté-Barrett et al, 2020;Inda et al, 2020). Some validation data for Wnt, ER, FOXO, and TGFβ is shown in Figures 1B-G, illustrating that the tests accurately indicate pathway activity known from disease etiology (1B) or mutation information (1C), and represent changes in activity due to treatment (1D and 1E).…”

“…Notch pathway activity was associated with improved prognosis in T cell-acute lymphoblastic lymphoma (Canté-Barrett et al, 2020). Activity of the Hedgehog pathway was found in primary metastasized breast cancer and was associated with worse prognosis in luminal breast cancer patients who developed metastatic disease, and pathway activity was increased in higher compared to lower grade ovarian cancer (Beachy et al, 2018;Sieuwerts et al, 2020;van Lieshout et al, 2020).…”

Characterization of immunoactive and immunotolerant CD4+ T cells in breast cancer by measuring activity of signaling pathways that determine immune cell function

“…Tests to quantitatively measure functional activity of PI3K, NFκB, TGFβ, JAK-STAT, and Notch signal transduction pathways on Affymetrix Human Genome U133 Plus 2.0 expression microarrays data have been described before (13,14,16,18,19). In brief, the pathway assays are based on the concept of a Bayesian network computational model which calculates from mRNA levels of a selected set, usually between 20 and 30, target genes of the pathway-associated transcription factor a probability score for pathway activity, which is translated to a log2 value of the transcription factor odds, i.e.…”

Measurement of activity of developmental signal transduction pathways to quantify stem cell pluripotency and phenotypically characterize differentiated cells

“…In order to define a signaling pathway activity profile for stem cell pluripotency, in pluripotent hES and iPS cells and differentiated derivatives, as well as in organ-derived multipotent stem cells, activity was measured of signal transduction pathways known be important for self-renewal and differentiation. A previously described, validated set of signal transduction pathway assays was used to calculate pathway activity scores for the AR, ER, PI3K, Wnt, TGFβ, HH, Notch, and NFκB pathways using RNA expression data [16],[35],[37],[36]. To enable a more complete analysis of signaling pathway activity, for the MAPK-AP1 pathway an additional assay was developed, biologically validated on a number of different cell types, and added to the existing signaling pathway assay platform.…”

“…Assessment of combined activity of these signaling pathways was expected to provide relevant information on (pluri)potency or differentiation state of cells. We previously described a novel assay platform to quantitatively measure the functional activity of a number of signal transduction pathways (STP) in individual cell and tissue samples, based on the inference of pathway activity from measurements of mRNA levels of target genes of the transcription factor associated with the signaling pathway [16],[36],[37],, [36]. To define a pluripotency-associated pathway activity profile, and to address the potential of the STP assays to increase experimental reproducibility, we determined quantitative signal transduction pathway activity profiles of different types of hES and iPS cell lines and differentiated derivatives across different labs, as well as primary organ-derived stem cells.…”