Carbenoxolone Decreases the Microenvironment-Induced Chemoresistance of Acute Myeloid Leukemia Cells

Introduction

Bone marrow niche of acute myeloid leukemia (AML) is a highly specialized microenvironment that regulates leukemic progression by favoring leukemic cell quiescence and chemoresistance. Niche components, especially mesenchymal stromal/stem cells (MSCs), have pro-survival effects on leukemia cells by protecting them from chemotherapy-induced apoptosis. The protection mechanisms of AML cells by the microenvironment are extensively studied to identify pharmacological targets to prevent AML relapse after chemotherapy. Direct intercellular communications between hematopoietic stem cells and MSCs involve connexins, such as connexin 43 (Taniguchi Ishikawa E et al. Connexin-43 prevents hematopoietic stem cell senescence through transfer of reactive oxygen species to bone marrow stromal cells. Proc Natl Acad Sci U S A. 2012,109:9071-6). The connexins are key components of gap junctions and we postulated in this study that blocking their assembly could modify cell-cell interactions in the leukemic niche and consequently the chemoresistance. To this end we evaluated the effects of carbenoxolone (CBX), a glycyrrhetinic acid derivative known to block gap junctions (Rozental R et al. How to close a gap junction channel. Efficacies and potencies of uncoupling agents. Methods Mol Biol 2001;154:447-76), already evaluated in the treatment of human diseases.

Methods

Primary AML blasts isolated from bone marrow patients and 8 AML cell lines (KG1-a, KG1, HL-60, THP-1, MOLM-13, MV4-11, ML2, NB4) were exposed to CBX to assess cell viability and proliferation (Trypan blue exclusion and MTT assays). To study the combined drug effects of CBX and cytarabine (Ara-C), isobolograms were determined by co-treatment of AML cell lines with various doses of these two molecules. The metabolic effects of CBX on AML live cells were investigated with Seahorse^® analyzer to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) and high-throughput metabolic profiling was performed by OmniLog^® technology. Direct cell-cell interactions were studied in a co-culture system of AML cells and bone marrow primary MSCs combining or not CBX (150 µM) and Ara-C (1 µM). After 48h (37°C, 5% CO₂), AML cells adherent to MSCs were collected and the two populations were discriminated by flow cytometry studies (AML cells: CD45⁺CD90^- / MSCs: CD45^-CD90⁺) to quantify cell number apoptosis/necrosis (AnnexinV/7AAD assay). Gap junction inhibition by CBX was checked by calcein transfer (dye transferred mainly by gap junction channels) from labeled MSCs to AML cells in the co-culture system. Statistical analyses were performed with Mann-Whitney-Wilcoxon test.

Results

Treatment of AML cell lines with CBX reduced cell growth and viability in a time- and dose-dependent manner and the CBX IC₅₀ was around 150 µM. Moreover, the exposure for 48h of primary AML blast to this dose of CBX induced a two time decrease in the number of viable cells. Interestingly, the isobolograms of the 8 AML cells lines identified three different profiles of resistance to chemotherapy and a synergistic effect between CBX and Ara-C. Regarding the energy metabolism, the analysis of all AML cell lines showed a major reduction of OCR and ECAR after treatment with CBX (150 µM, 48h) regardless their chemoresistance to Ara-C. Moreover, treated AML cells lacked their metabolic capacities for utilization of numerous substrates. In the co-culture experiments, contact with MSCs induced resistance to Ara-C; the apoptosis/necrosis rate observed after Ara-C exposure was 4.3- and 6.4-times decreased by MSC-contact for KG1a cells (n=5, p=0.0115) and primary blasts (n=5, p=0.0001), respectively. Co-treatment of leukemic cells with CBX and Ara-C reversed these deleterious effects of MSC-contact; the apoptosis/necrosis rate observed in MSC-contact leukemic cells was 5- and 2-times increased by CBX co-treatment for KG1a cells (n=5, p=0.007) and primary blasts (n=5, p=0.042), respectively. Finally, CBX induced a 48%-decrease in calcein transfer between leukemic cells and MSCs, highlighting its role as a gap junction inhibitor.

Conclusion

Niche-induced chemoresistance is associated with the mechanism of AML relapse after initial well-conducted chemotherapy. Combined to chemotherapy, CBX could be of interest to reduce the deleterious effects of leukemic niche by targeting gap junctions.