Abstract
Solid tumors that show high levels of glycolysis are often refractory to therapies such as arsenic trioxide (ATO), which mediate their anti-tumor effect via increased mitochondrial free radical formation. We have previously shown that purely glycolytic, mitochondrial gene knock-out HL60r0 cells were significantly more resistant to apoptosis induced by combined ATRA+ATO treatment than non-glycolytic HL60 cells [Herst et al. Leuk Res 2008]. Here, we investigate whether the degree of glycolytic metabolism of AML blasts isolated from diagnostic bone marrow samples reflects in vitro drug sensitivity and duration of remission from AML. Following Ethics Committee approval, AML blasts from 22 patient bone marrow (BM) samples were isolated from bone marrow aspirates previously stored in the Peter MacCallum Cancer Centre tissue bank. On each sample of AML blasts we determined the level of glycolytic metabolism by % FCCP-inhibition of reduction of the water-soluble tetrazolium dye, WST-1/PMS at the cell surface [Herst, Biochim Biophys Acta, 2007] and compared results to those measured for several cell lines and 8 primary bone marrow samples of acute lymphoblastic leukemia (ALL). In samples where sufficient (>105) cells were available, we separately assessed the degree of blast apoptosis, via annexin V/propidium iodide staining and flow cytometric analysis, induced by a 72 hour culture in either 1mM all-trans retinoic acid (ATRA), 2mM ATO or combined 1mM ATRA+2 mM ATO. Analysis of glycolysis revealed that AML blast samples distributed into two non-overlapping groups (p=0.0001) of moderate (n=13) and high levels (n=9) of glycolytic metabolism. In contrast, the level of glycolytic metabolism in ALL blasts, normal donor peripheral blood T cells and several cancer cell lines (HL60, Hela, HeLaS3w, BW1100., EL4, A20) varied extensively (Figure 1A). Paired samples of both diagnosis and subsequent relapse BM were available from 3 patients with >80% blasts in both samples. In these paired samples the level of glycolytic metabolism did not alter (all moderately glycolytic at both time points) from diagnosis to relapse, suggesting that this is a stable metabolic feature of AML that is not modified, or selected by, exposure to prior chemotherapy. Highly glycolytic AML blasts were relatively resistant to combined ATRA and ATO treatment than moderately glycolytic blasts (p=0.025) but not to ATRA or ATO treatment alone (Figure 1B). Survival from the date of bone marrow sampling was also assessed and compared between high and moderate glycolytic cohorts. At the time of analysis, with a median follow up of 4 years, 6 out of 9 patients with highly glycolytic AML blasts remain alive. Conversely, all 13 patients with moderately glycolytic AML blasts have died of progressive disease, with median survival of 64 days (p=0.005 by Gehan- Breslow-Wilcoxon test). Our results suggest that the extent of glycolytic metabolism, as measured by % FCCP-inhibition of dye reduction may be used to identify chemo-refractory and chemo-sensitive subgroups of AML and may be potentially applicable in identifying patients who may benefit from treatment intensification or novel therapies.
Figure 1: The effect of the extent of glycolytic metabolism on drug sensitivity of leukemic blasts. A. The extent of glycolytic metabolism in different cell types as determined by the % FCCP-inhibition of PMET. Values for individual BM samples, and averages of at least 3 separate experiments for resting T cells: 10, activated T cells: 63, normal BM: 62, HL60r0: 100, HeLaS3 r0: 99, HeLa r0: 98, BW1199: 74, EL4: 48, A20: 45, Molt-4: 30, HeLaS3: 37, U226: 6, HL60L 2, HeLa: 1, RPMI8226: 0. * p=0.0001 between highly glycolytic (n= 9) and moderately glycolytic (n= 13) AML blasts. B. Sensitivity of AML blasts to ATRA, ATO and combined ATRA+ATO, measured as [% viable blast after treatment]/[% viable blasts in controls]. Results are presented as average ± SEM of 4 highly glycolytic AML blasts (black bars) and 5 moderately glycolytic AML blasts (grey bars). * p=0.025 Figure 1:. The effect of the extent of glycolytic metabolism on drug sensitivity of leukemic blasts. A. The extent of glycolytic metabolism in different cell types as determined by the % FCCP-inhibition of PMET. Values for individual BM samples, and averages of at least 3 separate experiments for resting T cells: 10, activated T cells: 63, normal BM: 62, HL60r0: 100, HeLaS3 r0: 99, HeLa r0: 98, BW1199: 74, EL4: 48, A20: 45, Molt-4: 30, HeLaS3: 37, U226: 6, HL60L 2, HeLa: 1, RPMI8226: 0. * p=0.0001 between highly glycolytic (n= 9) and moderately glycolytic (n= 13) AML blasts. B. Sensitivity of AML blasts to ATRA, ATO and combined ATRA+ATO, measured as [% viable blast after treatment]/[% viable blasts in controls]. Results are presented as average ± SEM of 4 highly glycolytic AML blasts (black bars) and 5 moderately glycolytic AML blasts (grey bars). * p=0.025
Clonal deletion of self-reactive T cells in irradiation bone marrow chimeras and neonatally tolerant mice. Evidence for intercellular transfer of Mlsa.