Abstract 49: Synergistic antileukemic therapies in NOTCH1-induced T-ALL
Authors: Sanchez-Martin Marta , Ambesi-Impiombato Alberto , Qin Yue , Herranz Daniel , Bansal Mukesh , Girardi Tiziana , Paietta Elisabeth , Tallman Martin S. , Rowe Jacob M. , Keersmaecker Kim De , Califano Andrea , Ferrando Adolfo 
Published: August 2018

Over 60% of patients with T-cell acute lymphoblastic leukemia (T-ALL) harbor activating mutations in Notch1 gene, making this receptor a promising therapeutic target for T-ALL. Υ-secretase inhibitors (GSIs), which block a proteolytic cleavage required for NOTCH1 activation, have been clinically explored for the treatment of T-ALL. However, lack of primary responses and relapses have prevented further development of GSIs in the clinic, highlighting the need to identify novel therapies synergistic with NOTCH1 inhibition for the treatment of highly aggressive NOTCH1-mutant T-ALL.

To identify new antileukemic drugs for T-ALL we designed an expression-based drug screen utilizing drug profiles contained in the Connectivity Map (cMAP) to search for positive connections with gene signatures driven by NOTCH1 inhibition in this disease. This analyses uncovered 17 compound candidates for synergistic activity with NOTCH1 inhibition, including drugs with known mechanism of action: inhibitors of the mTOR/PI3K/AKT pathway and histone deacetylase inhibitors; as well as compounds with unrelated mechanisms, pyrvinium pamoate (antihelmintic) and withaferin A (antiangiogenic). Pair-wise gene set enrichment analyses on the expression profile driven by each of these drugs in T-ALL cells revealed compounds with transcriptional programs partially overlapping those driven by PI3K/AKT inhibitors, and drugs with distinctive expression profiles, such as withaferin A, suggestive of a unique mode of action.

We next characterized the activity of each of the cMAP hits alone and in combination with the GSI DBZ in vitro using a panel of 5 NOTCH1-mutant T-ALL cell lines (CUTLL1, DND41, HPBALL, RPMI and CCRF-CEM). In this analysis, 5 drugs scored as highly active (IC50&lt;0.5) and synergistic (combination index, CI &lt;0.4) in combination with the GSI DBZ: withaferin A, parthenolide, rapamycin, wortmannin, and vorinostat. In addition, 3 out of the 5 drug candidates displayed proapoptotic activity by flow cytometry in cells treated with each of the drugs alone and in combination with DBZ. Among them, withaferin A stood up as the most cytotoxic GSI-synergistic drug candidate. In addition, evaluation of withaferin A activity as single agent and in combination with DBZ in genetic mouse models and primary-derived xenografts of NOTCH1-mutant T-ALL in vivo demonstrated strong antileukemic effects for the combination of GSI and withaferin A.

Functional annotation and DeMAND algorithm analyses on the expression profile of withaferin A revealed downregulation of genes and pathways involved in protein translation and identified 21 components of the ribosome and the translation machinery as potential effectors of the antitumoral activity of the drug in T-ALL, including two subunits of the eIF2A translation complex (eIF2S1 and eIF2S2) which controls translation initiation via phosphorylation of the eIF2S1 subunit under conditions of cellular stress. Polysome profiling, nascent protein, and Western blot analyses of cells treated with withaferin A confirmed an inhibitory effect of the drug in protein translation mediated by eIF2S1 phosphorylation. Consistently, expression of a non-phosphorylable mutant form of eIF2S1 in leukemia cell lines rescued the effects of withaferin A in vitro.

Our results illustrate the power of our expression-based and in vivo validation approach toward the identification of antitumor drug combinations for the treatment of human cancer.

Citation Format: Marta Sanchez-Martin, Alberto Ambesi-Impiombato, Yue Qin, Daniel Herranz, Mukesh Bansal, Tiziana Girardi, Elisabeth Paietta, Martin S. Tallman, Jacob M. Rowe, Kim De Keersmaecker, Andrea Califano, Adolfo Ferrando. Synergistic antileukemic therapies in NOTCH1-induced T-ALL [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 49.