Redundancy and Specificity of the Metalloprotease System Mediating Oncogenic NOTCH1 Activation in T-ALL.
Authors: Luisa Sulis Maria , Saftig Paul , Ferrando Adolfo A. 
Published: October 2019

Abstract
Abstract 988
Poster Board I-10
Aberrant NOTCH signaling plays a critical role in the development of over 50% of human T-cell acute lymphoblastic leukemias (T-ALL) harboring mutations of NOTCH1. The NOTCH1 receptor functions as a ligand activated transcription factor which requires a double proteolytic processing for activation. NOTCH1 is first processed by an ADAM metalloprotease in the so called S2 site located in the extracellular portion of the receptor, and subsequently by the presenilin γ-secretase complex, which cleaves at the S3 site located in the transmembrane domain. Notably, small molecule inhibitors of the γ-secretase complex effectively block NOTCH1 S3 processing and are under development as anti-NOTCH1 therapies in T-ALL. Moreover, selective inhibitors of ADAM10 and ADAM17, two ADAM proteases implicated in the S2 processing of NOTCH1, have been developed as inhibitors of ERBB signaling for the treatment of solid tumors; suggesting that inhibition of NOTCH1 S2 processing could be exploited as an anti-NOTCH1 therapy in T-ALL. However, activating mutations in NOTCH1 found in T-ALL frequently alter the structure of the HD-LNR domains that normally control the access of ADAM proteases to the S2 site, raising the possibility that S2 cleavage may be processed by alternative proteases in the context of T-ALL. To address the role of ADAM proteases in the shedding and activation of oncogenic forms of NOTCH1, we tested the effects of metalloprotease inhibitors in NOTCH1 processing and activation using a luciferase reporter assay. In these experiments we transiently transfected HeLa cells with expression vectors encoding the a NOTCH1 HD class I mutant (NOTCH1 L1601P), a NOTCH1 HD class II allele (P12 ICHIKAWA) and a NOTCH1 JME construct (Jurkat JME17), which are characterized by increased and aberrant NOTCH1 S2 processing, to test the effect of Timp1, Timp2 and Timp3 in NOTCH1 signaling. Notably Timp3, a metalloprotease inhibitor that blocks soluble, membrane and ADAM metalloproteases, decreased the activity of all three NOTCH1 mutant alleles. In contrast, inhibition of soluble metalloproteases with Timp1 had no effect on NOTCH1 activation and blocking soluble and membrane metalloproteases with Timp2 showed only a mild inhibitory activity towards the L1601P mutant allele. These results suggest that, as in the case of wild type NOTCH1, ADAM proteases are responsible for the processing of oncogenic NOTCH1 at the S2 site. Consistent with these results, inhibition of Adam10 or Adam17 in mouse embryonic fibroblasts resulted in marked decrease in the activity of all three oncogenic forms of NOTCH1. Moreover, knocking down Adam17 in Adam10 deficient cells and vice versa, knocking down Adam10 in Adam17 knockout MEFs, showed that simultaneous inactivation of Adam10 and Adam17 resulted in further decreases in the activity of oncogenic NOTCH1. These results suggest that both Adam10 and Adam17 can be competent in NOTCH1 processing. However, recent data from Adam10 conditional knockout mice has shown that Adam10 may play a more prominent role in the processing and activation of Notch1 signaling during thymocyte development. To address the specific requirement of ADAM10 for NOTCH1 processing and activation in T-ALL we performed shRNA knock down of this protease in T-ALL cell lines harboring a NOTCH1 HD class I mutation (KOPTK1), a NOTCH1 HD class II mutation (P12 ICHIKAWA) and a NOTCH1 JME mutation (Jurkat). These experiments showed that ADAM10 inactivation results in a marked decrease in the levels of activated intracellular NOTCH1 (ICN1) protein detected by Western blot and diminished expression of the NOTCH1 target Deltex1. Moreover, inhibition of ADAM10 by shRNA knock down in KOPTK1 cells, a T-ALL cell line dependent on NOTCH1 signaling for growth and proliferation, caused growth inhibition and sensitized these cells to low concentrations (10nM) of Compound E, a highly active γ-secretase inhibitor. Overall these results provide further insight on the mechanisms that control the activation of oncogenic NOTCH1 mutants and identify ADAM10 as a potential therapeutic target for the inhibition of oncogenic NOTCH1 in T-ALL.

Disclosures:
Ferrando: Merck, Pfizer: Research Funding.