Activity of Targeted Molecular Therapeutics Against Primary AML Cells: Putative Role of the Bone Marrow Microenvironment.

Abstract

In hematological malignancies, there are reciprocal interactions between leukemic cells and cells of the bone marrow (BM) microenvironment such as mesenchymal stem cells (MSC). It is speculated that specific BM niches may provide a sanctuary for subpopulations of leukemic cells to evade chemotherapy-induced death and allow acquisition of a drug-resistant phenotype. In this study, we compared anti-leukemia effects of Ara-C and various signal transduction and apoptosis inhibitors in a co-culture system of primary AML and human bone marrow-derived MSC. AML blasts from 11 primary AML samples with high (>70%) blast count were co-cultured with MSC for 24 hours, after which they were exposed to the indicated concentrations of inhibitors for 48–96 hrs. Concentrations were selected on the basis of preliminary cell line studies which determined efficient inhibition of drug targets. Induction of apoptosis was analyzed by Annexin V flow cytometry after gating on the CD90 APC(−) (non-MSC) population. MSC protected leukemic blasts from spontaneous apoptosis in all 11 samples studied (mean annexinV positivity, 49.5±7.2% vs 25.3±4.8%, p<0.001) and from Ara-C-induced cytotoxicity in 6 out of 11 samples (p=0.02). No difference in the degree of protection was noted when MSC from older vs. younger donors were used (not shown). Co-culture of leukemic cells with MSC resulted in significant (p<0.03) suppression of inhibitor-induced apoptosis for all agents tested (Table 1), however PI3K/AKT inhibitors seemed to overcome MSC-mediated resistance. In addition, specific inhibitors of Bcl-2 and MDM2 induced apoptosis not only in suspension, but also in the MSC co-culture system, while Raf-1/MEK inhibitors were less effective. The AKT inhibitor A443654 caused apoptosis induction not only in leukemic cells, but also in MSC, which likely accounted for its high efficacy in stromal co-cultures (53±6% annexin V+). In a different study (Tabe et al, ASH 2005), we report that interactions of leukemic and BM stromal cells result in the activation of PI3K/ILK/AKT signaling in both, leukemic and stromal cells. We therefore propose that disruption of these interactions by specific PI3K/AKT inhibitors represents a novel therapeutic approach to eradicate leukemia in the BM microenvironment via direct effects on leukemic cells and by targeting activated BM stromal cells. Furthermore, Bcl-2 and MDM2 inhibitors appear to retain their efficacy in stroma-cocultured AML cells, while the efficacy of chemotherapy and Raf/MEK inhibitors in these conditions may be reduced. Further studies are aimed at the elucidation of the role of the BM microenvironment and its ability to activate specific signaling pathways in the pathogenesis of leukemias and on efforts to disrupt the MSC/leukemia interaction (Zeng et al, ASH 2005). Focus on this stroma-leukemia-stroma crosstalk may result in the development of strategies that enhance the efficacy of therapies in hematological malignancies and prevent the acquisition of a chemoresistant phenotype.Table 1Leukemia Cell Apoptosis in a MSC/AML Co-Culture SystemTargetBcl-2/XLMDM2PI3KAKTRaf-1MEKCompound, concentrationAra-C, 1 μMABT-737, 0.1 μMNutlin-3A, 2.5 μMLY294002, 10 μMA443654, 1 μMBAY43-9006, 2.5 μMCI1040, 3 μMAML28±769±745±753.8±13.375±735±1127±11AML + MSC16±438±628±631.2±6.953±618±815±5Apoptosis was determined as percentage of Annexin V(+)CD90(−) cells, and calculated by the formula: % specific apoptosis = (test − control) x 100 / (100 − control).