Abstract 4962: Stroma-induced molecules and chemotherapy resistance in acute myeloid leukemia cells.

Overall objective: Acute myeloid leukemia (AML) is a devastating malignancy with a relapse rate near 50% in children, despite very toxic chemotherapy. Once a child relapses, the survival rate is very low. Therefore new, rational therapies for AML are desperately needed. Accumulating evidence shows that the bone marrow stroma protects a subset of leukemia cells from chemotherapy, eventually leading to recurrence. Our goal is to delineate the mechanisms underlying stroma-mediated chemotherapy resistance in AML. Methods, Results & Conclusions: We used a human bone marrow stromal cell line, HS-27A and two human AML cell lines, THP-1 and NB-4. HS-27A cells provide important contact-dependent interactions with AML cells and secrete only a few soluble factors. Our HS-27A cells constitutively express mOrange, to allow discrimination from AML cells after co-culture. We performed co-culture experiments to determine the ability of stromal cells to confer resistance to chemotherapy. THP-1 and NB-4 cells were cultured alone or co-cultured with HS-27A cells, and treated with etoposide, mitoxantrone or cytarabine for 48 hours before labeling with annexin V for apoptosis assay by FACS. HS-27A cells significantly protected THP-1 and NB-4 cells from all three chemotherapy agents. To discover genes in AML cells that are induced by stromal cells and may contribute to chemotherapy resistance, oligonucleotide microarray analysis was done using total RNA extracted from THP-1 or NB-4 cells cultured alone or co-cultured with HS-27A cells. We identified over 1000 genes that were similarly differentially expressed in both AML cell lines by HS-27A co-culture. Among the significantly upregulated genes was cysteine-rich, angiogenic inducer 61 (CYR61). CYR61 is a secreted, extracellular matrix-associated signaling molecule, which has been related to chemotherapy resistance in breast cancer cells and malignant melanoma cells. To validate the microarray results, qRT-PCR was done with RNA from THP-1 cells and NB-4 cells, cultured alone or co-cultured with HS-27A cells. CYR61 expression was increased over 100-fold in those cell lines when co-cultured with HS-27A cells. Next, we examined CYR61 protein expression by immunocytochemistry, with co-staining for myeloperoxidase to confirm that CYR61-expressing cells were AML cells. This study confirmed that CYR61 was highly expressed in THP-1 cells co-cultured with HS-27A cells, but barely detectable in THP-1 cells cultured alone. Further studies are underway to determine if CYR61 contributes to stroma-induced chemotherapy resistance in AML. This study suggests that CYR61 induced by the microenvironment may promote chemotherapy resistance in AML cells. Elucidating the pathways involved in chemotherapy resistance is likely to result in promising combination therapies to reduce relapse, and thereby improve survival for children with AML. Citation Format: Xin Long, Laszlo Perlaky, Tsz-Kwong Man, Michele S. Redell. Stroma-induced molecules and chemotherapy resistance in acute myeloid leukemia cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4962. doi:10.1158/1538-7445.AM2013-4962