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.
Highlights
NOTCH proteins are trans-membrane receptors that transduce signals from cell-bound JAGGED (JAG) and Delta-like (DLL) families of ligands to mediate cell-cell interactions in processes as diverse as fetal development, heart disease and cancer[1,2]
To identify selective inhibitors of RBPJ, we developed a primary screen to detect inhibitors of a functional interaction between RBPJ and the scaffold protein SHARP that was followed by secondary assays to establish efficacy against NOTCH
RBPJ Inhibitor-1 (RIN1) treatment induced a profile of transcript changes that was distinct from the changes induced by DAPT, including opposing effects on bonafide NOTCH target genes HES1, HES5, HEY1 and DTX (Fig. 4)
Summary
NOTCH proteins are trans-membrane receptors that transduce signals from cell-bound JAGGED (JAG) and Delta-like (DLL) families of ligands to mediate cell-cell interactions in processes as diverse as fetal development, heart disease and cancer[1,2]. There is only one small molecule reported to selectively inhibit NOTCH signaling and none known to target RBPJ5. Newer generation GSIs exhibit a biased inhibition of APP over NOTCH9,10, there are no NOTCH-selective GSIs. Clinical use of GSIs cause numerous side effects, notably intestinal crypt cell proliferation[11], and skin rashes and tumors[12]. (b) Assay validation using RBPJ siRNA transfection and a small molecule Luciferase inhibitor, Data is mean ± standard deviation, n = 5 wells. Inhibition of NOTCH2 ICD (e) and RBPJ-VP16myc fusion protein (f) activity on the Hes1-Luciferase reporter in transient transfections, n = 4 wells. 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 assayed for Hes1-Luciferase activity (i) (n = 10 wells), Luciferase mRNA (j) and endogenous HES5 mRNA (k), (n = 3 samples).
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