ABT-737 Targets Leukemic Stem Cells In Mouse Models of Mutant NRASD12/hBCL-2- Mediated Acute Myeloid Leukemia Progression with Increased Survival

Animal models enable us to understand disease progression and provide us with reagents to test various therapeutic strategies. We have previously developed a mouse model of myelodysplasia/acute myelogenous leukemia (MDS/AML) progression using mutant NRASD12 and overexpression of human hBCL-2 (Omidvar et al Cancer Res 67:11657-67, 2007). Expanded leukemic stem cells (LSC) were identified as Lin-/Sca1+/KIT+ (LSK) populations, with increased myeloid colony growth and were transplantable. Increased hBCL-2 and RAS-GTP complex were observed in both MDS/AML diseases. The MDS-like disease had increased apoptosis, whilst the AML-like mice had liver apoptosis patterns similar to wild type. The single NRASD12 line also had increased apoptosis. In this present study using a BCL-2 homology domain 3 (BH3) mimetic ABT-737 (Abbott), we have evaluated the effects of targeting BCL-2 in our preclinical models.

Treatment with the inhibitor shows a reduction of LSK cells, reduced progenitor numbers in colony assays and clearance of the liver infiltrations in both MDS and AML models. Gene expression profiling of the MDS mice shows regulation of 399 genes upon treatment including 58 genes expressed by the single mutant RAS mice and not expressed in the untreated AML mice. 78 genes were shared between single NRASD12 and diseased mice and not the treated mice. These studies potentially identify the contribution of NRASD12 genes to disease progression. By confocal microscopy we observed that in the MDS mice the majority of the RAS and BCL-2 co-localized to the plasma membrane, where active pro-apoptotic RAS is normally located, whereas in the AML disease RAS and BCL-2 co-localized in the mitochondria, where BCL-2 is normally found (Omidvar et al 2007). After treatment with the inhibitor the AML co-localization of RAS and BCL-2 shifted to the plasma membrane where single NRASD12 is normally localized. Furthermore, increased RAS-GTP levels was detected in both Sca1+ and Mac1+ enriched spleen cells and interestingly an increase in BCL-2 expression was observed in peripheral blood and in spleen cells after treatment; this increase in BCL-2 was associated with a decrease in the phosphorylation of serine 70 and an increase in phosphorylation of threonine 56 of BCL-2. ABT-737 treatment led to increased phosphorylated ERK resembling RAS and reduced MEK and AKT phosphorylation, changes detected by western blots and the nanoimmunoassay (NIA, NanoPro, Cell Biosciences) that might account for the increased apoptosis, measured by TUNEL and In vivo imaging by single-photon emission computed tomography (SPECT) using Tc-99m-labelled AnnexinV (SPECT). In contrast, although treated MDS mice had increased apoptosis they did not have an increase in overall expression of BCL-2 or in RAS-GTP levels. Treatment of both MDS and AML models with this inhibitor significantly extended lifespan from diagnosis with mean survival of 28 days untreated vs 80 days treated (p=0.0003) and mean survival from birth of 39 untreated vs 85 days treated (p<0.0001) respectively

Genomics, proteomics and imaging have been employed in the MDS/AML models to characterize disease progression and follow response to treatment to the BH3 mimetic ABT-737 in order to gain molecular insights in the evaluation of the efficacy. ABT-737 appears to target LSCs, induce apoptosis, regulating RAS and BCL-2 signalling pathways, which translated into significantly increased survival.

Padua:Vivavacs SAS: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Auboeuf:GenoSplice technology: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. de la Grange:GenoSplice technology: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Fenaux:Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Janssen Cilag: Honoraria, Research Funding; ROCHE: Honoraria, Research Funding; AMGEN: Honoraria, Research Funding; GSK: Honoraria, Research Funding; Merck: Honoraria, Research Funding; Cephalon: Honoraria, Research Funding. Tu:Cell Biosciences Inc;: Employment. Yang:Cell Biosciences Inc;: Employment. Weissman:Amgen, Systemix, Stem cells Inc, Cellerant: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Felsher:Cell Bioscience:. Chomienne:Vivavacs SAS: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.