T-cell acute lymphoblastic leukemia (ALL) accounts for 15% of pediatric ALL cases and is associated with early relapse and inferior outcome. The poorer prognosis of T-ALL compared to B-precursor ALL may in part reflect the lack of unique features on which to base therapy. NOTCH1 mutations are of particular interest since these were reported in 37-71% of T-ALLs. The prognostic value of NOTCH1 mutations remains controversial as both favorable and unfavorable associations were reported, whereas in other studies there were no associations between NOTCH1 mutations and treatment outcome. NOTCH1 is a potentially attractive therapeutic target for T-ALL since constitutively activating effects of mutant NOTCH1 can be abolished with gamma secretase inhibitors (GSIs). Because of possible effects of GSIs on other cellular targets in addition to NOTCH1, we explored shRNA knockdown of NOTCH1 to identify novel NOTCH1-regulated genes that may serve as prognostic indicators or therapeutic targets in T-ALL. NOTCH1 expression was knocked down in Jurkat T-ALL cells using lentivirus expressing shRNAs for NOTCH1 or a non-targeted control (NTC) sequence. NOTCH1 knockdown was verified using western blots to measure activated NOTCH1 (ICN1) protein levels, and real-time RT-PCR (qPCR) to measure transcript levels of known NOTCH1 targets (e.g., HES1). Two clonal sublines (J.2-4 and J.2-7) were identified with significantly decreased expression of NOTCH1 compared to NTC. The J.2-4 and J.2-7 sublines showed minimal changes in cell growth, cell cycle progression and apoptosis. To characterize genotypic changes accompanying NOTCH1 knockdown, we performed microarray analysis with Agilent Whole Genome oligonucleotide microarrays and microRNA (miR) HumanV2 arrays. The miR array identified 20 miRs in J.2-4 and J.2-7 cells with altered expression compared to NTC greater than 1.5-fold (p<0.05) and ranging from 3-to10-fold. miRs hsa-Let-7e, hsa-miR-125a-5p and hsa-miR-99b, reportedly derived from a polycistronic transcript, were decreased 10-fold accompanying NOTCH1 knockdown. Using miR qPCR, we confirmed decreased levels of hsa-miR-125a-5p and hsa-miR-99b in the J.2-4 and J.2-7 sublines. From predictive algorithms, Mcl-1 is a predicted target of hsa-miR-125a-5p. On westerns, clones J.2-4 and J.2-7 showed significant increases in levels of Mcl-1 protein over NTC. In conclusion, we have developed novel T-ALL cell line models to study the impact of decreased NOTCH1 levels and activity independent of GSI treatment. Our results implicate NOTCH1 in regulating levels of hsa-miR-125a-5p with possible downstream effects on the anti-apoptotic protein, Mcl-1. Our findings suggest caution may be warranted in targeting NOTCH1 with GSIs in the therapy of T-ALL, reflecting the potential downstream impact on Mcl-1 levels resulting in decreased apoptosis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4360.