Abstract

Abstract 3524

Background:

Although Notch signaling contributes to T cell leukemogenesis, the role of Notch in human AML is unclear. We and others have found that activation of Notch signaling inhibits AML growth and survival, e.g. a tumor suppressor like effect. However it is not known what the consequences of activating or inhibiting Notch signaling are in human AML in vivo.

Approach:

To determine whether Notch signaling would have growth inhibiting effects in vivo, we stably-transduced ML1 human AML cells with the constitutively-active forms of Notch1 and Notch2 (ICN1, ICN2), the common Notch target gene Hairy/Enhancer of Split 1 (HES1) or the pan-Notch inhibitor dominant-negative Mastermind-like (dnMAML) and performed in vivo competitive proliferation assays. Briefly, following transduction, each vector type were sorted to 50% GFP+ (containing the gene of interest) and GFP- (untransduced control cells). Groups of NSG mice were injected with these 50:50 mixtures of ML1 cells, and peripheral blood levels of GFP- and GFP+ cells were measured with flow cytometry for anti-human CD45 and GFP.

Results:

Peripheral blood engraftment of human CD45+ ML1 cells by week 5 was similar (2–5%) for ICN1, ICN2 and HES1 injected mice, but was significantly higher (23%) in dnMAML injected mice (Panel A). Similar to in vitro competitive proliferation assays, ICN1, ICN2 and HES1 all led to decreased relative numbers of GFP%+ cells, with 1%, 4% and 16% respectively (Panel B). Importantly, dnMAML had little effect on AML proliferation in vitro, however led to dramatic increases in GFP% as well as early morbidity and mortality due to increased leukemia burden, with 93% GFP+ (Panel B), demonstrating a selective advantage for dnMAML-expressing ML1 in vivo. When GFP+ (transduced) ML1 peripheral engraftment was directly compared to GFP- (parental CD45+) engraftment, the GFP- control cells had similar engraftment rates (2–5%) across groups of mice, while the GFP+ engraftment rates were significantly lower in ICN1, ICN2, and HES1 groups, but significantly higher in the dnMAML group (Panel C), demonstrating enhanced engraftment/proliferation in dnMAML-expressing cells.

Conclusions:

This suggests a previously unreported concept, namely that endogenous Notch ligands can inhibit human AML growth in vivo. This data supports the hypothesis that Notch behaves as a tumor suppressor in AML, and suggests the potential use of Notch agonists in human AML.

Disclosures:

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.