Disruption of NOTCH signaling by a small molecule inhibitor of the transcription factor RBPJ

Abstract

NOTCH plays a pivotal role during normal development and in congenital disorders and cancer. γ-secretase inhibitors are commonly used to probe NOTCH function, but also block processing of numerous other proteins. We discovered a new class of small molecule inhibitor that disrupts the interaction between NOTCH and RBPJ, which is the main transcriptional effector of NOTCH signaling. RBPJ Inhibitor-1 (RIN1) also blocked the functional interaction of RBPJ with SHARP, a scaffold protein that forms a transcriptional repressor complex with RBPJ in the absence of NOTCH signaling. RIN1 induced changes in gene expression that resembled siRNA silencing of RBPJ rather than inhibition at the level of NOTCH itself. Consistent with disruption of NOTCH signaling, RIN1 inhibited the proliferation of hematologic cancer cell lines and promoted skeletal muscle differentiation from C2C12 myoblasts. Thus, RIN1 inhibits RBPJ in its repressing and activating contexts, and can be exploited for chemical biology and therapeutic applications.

Figure 1

Identification of small molecule RBPJ inhibitor, RIN1. (a) Schematic of the primary and counter screens. The screen was a cell-based two hybrid assay in which an active compound (stars) would disrupt the SHARP:RBPJ interaction and decrease activity of the Luciferase reporter. A minimal RBPJ-interacting domain of SHARP and a DNA-binding mutant of RBPJ were used (see Methods). (b) Assay validation using RBPJ siRNA transfection and a small molecule Luciferase inhibitor, Data is mean ± standard deviation, n = 5 wells. Z’ is a metric of dynamic range. (c) Workflow schematic. (d) Screen flowchart and structure of RIN1. (e,f) Inhibition of NOTCH2 ICD (e) and RBPJ-VP16myc fusion protein (f) activity on the Hes1-Luciferase reporter in transient transfections, n = 4 wells. (g) Effect on cell viability, n = 4 wells. (h) Effect on CMV promoter activity, n = 4 wells. Data in b-h are presented as mean ± standard deviation; experiments were repeated > 3 times. (i–k) Effect of Cycloheximide on RIN1 inhibition of RBPJ-VP16. AD-293 cells were transfected with RBPJ-VP16myc and 48 hours later were treated ± RIN1 (2 µM), ± Cycloheximide (CHX, 10 µg/ml) for an additional 17 hours and then assayed for Hes1-Luciferase activity (i) (n = 10 wells), Luciferase mRNA (j) and endogenous HES5 mRNA (k), (n = 3 samples). Western blot of RBPJ-VP16myc fusion protein under identical conditions (l) and its quantification (m), n = 3 wells. Data are presented as mean ± standard deviation. Incrementing number of symbols (* and #) denote P < 0.05, P < 0.01, P < 0.001 and P < 0.0001 respectively, using two-tailed unpaired Student’s T-test relative to empty vector (*) or to RBPJ-VP16myc + DMSO vehicle control (#) conditions. Experiments were repeated twice.

Figure 2

Comparative effects of RIN1, DAPT and CB-103 on hematologic tumor cell proliferation. (a–c) Acute T cell leukemia cell lines Jurkat (a) and KOPT-K1 (b) and non-Hodgkin’s mantle cell lymphoma Rec-1 line (c) were treated with small molecule NOTCH inhibitors during their logarithmic growth phase as indicated for 96 hours. n = 7, assay repeated twice. (d) Western blot showing levels of RBPJ in the tumor cell lines.

Figure 3

Effect of RIN1 on C2C12 myoblast differentiation. Structured illumination photomicrographs of C2C12 cells at 4 days under permissive differentiation conditions and drug treatment as indicated. Upper panels: Cells were stained for myosin heavy chain with the MF20 antibody (green) and labeled with DAPI to identify nuclei (blue) (upper panels). Lower panels: Cell body and nuclei image masks for quantification. (b,c) Image analysis, n = 3 wells, quantified the number of cell mask (green) objects (b) and the ratio of nuclei per cell mask object (c). Assay repeated 3 times.

Figure 4

Gene expression changes induced by RIN1. Jurkat cells were treated with either 8 hours with small molecules or 48 hours with transfected RBPJ siRNA and DMSO-vehicle or control (inert sequence) siRNA, respectively. The heatmap represents changes in the levels of transcript (fold change > 2, P < 0.05) that were induced by either the small molecules or RBPJ siRNA relative to their respective controls (DMSO vehicle or control siRNA) and varied < 40% between techniques (DMSO-vehicle vs. control siRNA. (b–e) qRT-PCR analysis of RBPJ target gene expression. Data are presented as mean ± standard deviation. Incrementing symbols (* and †) denote P < 0.05, P < 0.01, P < 0.001 and P < 0.0001 respectively, using two-way ANOVA with Dunnett’s post-test. *between groups indicated; ^†relative to respective control treatments (DMSO or control siRNA). ns, not significant. (f) Schematic showing NOTCH pathway inhibitors (red text) in relationship to signaling and summary of RIN1 effects on downstream gene expression.