Abstract 543: Stroma-mediated chemotherapy resistance in acute myeloid leukemia cells

Abstract

Abstract Overall objective: Acute myeloid leukemia (AML) is a devastating malignancy with a relapse rate near 50% in children, despite very toxic chemotherapy. Accumulating evidence shows that the bone marrow stromal environment protects a subset of leukemia cells and allows them to survive chemotherapy, eventually leading to recurrence. Furthermore, activation of signal transducer and activator of transcription 3 (Stat3) contributes to chemotherapy resistance. Indeed, Stat3 is activated in AML cells by cytokines and adhesion pathways in the stromal environment, leading to increased expression of pro-survival genes. Our goal is to determine the effect of stromal cytokines and adhesion pathways on stroma-mediated chemotherapy resistance in AML cells, which could lead to potential new therapies for AML. Methods, Results & Conclusions: We used two human bone marrow stromal cell lines, HS-5 and HS-27, for our experiments. First, we examined the ability of stroma-conditioned medium to activate Stat3 in AML cells. Kasumi-1 AML cells were treated with HS-5 or HS-27 conditioned medium, or control medium, for 15 minutes, then the level of phosphorylated Stat3 was measured by flow cytometry. Conditioned medium from HS-5, but not HS-27 cells, strongly induced phosphorylation of Stat3 in AML cells at a level comparable to that induced by G-CSF (100 ng/mL). These results suggest that HS-5 cells secrete factors that induce Stat3 activation, but HS-27 cells do not. Next, we performed co-culture experiments to determine the ability of each stromal cell line to confer resistance to chemotherapy. Human AML cell lines (NB-4 and THP-1) were cultured alone or co-cultured with PKH-26-labeled HS-5 or HS-27 cells, then treated with etoposide (30 nM – 10 uM) or mitoxantrone (3 nM – 300 nM) for 48 hours. Cells were then harvested and labeled with annexin V-FITC. The percentage of apoptotic AML cells (FITC positive and PKH-26 negative) was determined by FACS. Both HS-5 (p<0.001) and HS-27 (p<0.05) cells protected NB-4 and THP-1 cells from etoposide-induced apoptosis (apoptosis rate at the 1 uM dose: 69.4±3.0% THP-1 alone vs 48.2±1.1% HS-5 co-culture vs 59.3±3.4% HS-27 co-culture). HS-5 cells also provided protection for AML cells against mitoxantrone-induced apoptosis (apoptosis rate at the 300 nM dose: 85.1±0.8% THP-1 alone vs 57.7±5.6% HS-5 co-culture; p<0.05). In contrast, co-culture with HS-27 cells did not reduce mitoxantrone-induced apoptosis (apoptosis rate: 77.7±2.4%). The results with NB-4 were similar to THP-1 cells. These findings suggest that 1) HS-5 and HS-27 stromal cells confer resistance to various chemoagents by different mechanisms, which are consistent among human AML cell lines; and 2) resistance to individual chemotherapy agents may depend on different mechanisms, such as secreted factors vs adhesion pathways. Understanding these mechanisms will be very valuable in targeting therapies to overcome stroma-mediated chemotherapy resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 543. doi:10.1158/1538-7445.AM2011-543