Dexamethasone Accumulation in Dexamethasone Sensitive and Resistant Acute Lymphoblastic Leukemia

Introduction:

Glucocorticoids (GC) have been at the forefront of acute lymphoblastic leukemia (ALL) treatment for a number of decades. However, there is heterogeneity of response, both in terms of GC-related toxicity and leukemia cell sensitivity. Contributing factors include marked pharmacokinetic variability observed in children (Yang et al. JCO, 2008; Jackson et al.AACR annual meeting abstract CT115, 2016) and a several hundred fold range of GC cellular response. Cellular resistance can be caused by deletion of the glucocorticoid receptor (GR) but is more commonly downstream of the GR. One neglected upstream parameter relates to GC accumulation, which may be an important factor in ALL GC response, given the evidence for drug transporters in ALL cells. Therefore, this study aimed to determine whether variation in intracellular dexamethasone (dex) levels is a determinant of dex sensitivity in an ALL setting.

Methods:

A number of cell lines including PreB697, GC resistant PreB697 sub-lines and REH cells, along with primagraft material from 9 patients and 6 primary patient samples (5 presentation and 1 relapse) were studied. The relative sensitivity of cells to dex was assessed using Alamar Blue drug sensitivity assay. Two methods were developed to assess intracellular dex accumulation; a liquid-chromatography mass spectrometry (LC/MS) method and a flow cytometry method, using dex conjugated to the fluorochrome FITC analysed on a FACSCalibur flow cytometry machine. GR status of the cells was confirmed by western blotting.

Results:

Dex GI50 values (concentration giving 50% growth inhibition) ranged from 37nM in PreB697 cells to >1000nM in GC resistant sub-lines and REH cells. Dex GI50 values in patient ALL cells ranged from 2 to >1000nM. Dex resistant cells were defined as having a dex GI50 of >500nM. The mean GI50 of the dex sensitive cells was 3.8nM. Western blotting suggested wildtype GR status in all samples, with R3D11 and REH serving as hemizygous deleted and GR negative controls, respectively.

The mean dex accumulation was measured in cells using an LC/MS assay developed from a fully validated assay measuring plasma dex concentrations (Jackson et al. NCRI annual meeting abstract BACR9, 2014). Dex was stable in RF10 media for at least 8 hours and there was no matrix effect of RF10 media on dex chromatograms compared to dex in plasma. An incubation concentration of 500nM was chosen as this is the observed median value of dex cell exposure clinically. Dex concentrations were quantifiable in cell numbers of 1 x 10⁶after incubation with 500nM dex, allowing measurement of patient samples where limited numbers of cells are available.

Dex accumulation in cell lines after incubation with 500nM dex for 4h was 2.1 and 1.8 pmol/million cells in PreB697 and dex resistant sub-lines, respectively (range for resistant subclones 1.2 - 2.1pmol/million cells). There was greater variability in patient cells with a 40-fold range seen, but dex accumulation was not significantly different between sensitive (mean, 1.0 pmol/million; range, 0.1-2.3) and resistant cells (1.4 pmol/million; range, 0.4-4.4) (unpaired students t-test, p=0.17).

To assess intra-leukemia heterogeneity in terms of dex accumulation, a flow based assay was established using dex-FITC. Incubation conditions of 500nM dex-FITC at 37°C for 45 minutes were optimal. Dex-FITC accumulation did not differ significantly between sensitive and resistant cells; mean fluorescence intensity of 4.2 (range 1.5-5.9) versus 4.1 (range 2.0 - 9.1) in sensitive and resistant cells, respectively (p=0.97, unpaired students t test). Dex-FITC accumulation appeared uniform within the ALL samples examined.

Conclusions:

These data suggest that variations in dex accumulation are unlikely to play a role in dex resistance in ALL, at least in vitro. Advancement of the flow-based dex accumulation assay to include leukaemia-associated immunophenotype markers will allow measurement in dex-resistant MRD in vivo. Given that 35% of patients do not achieve plasma concentrations of 200nM dex (Jackson et al. AACR annual meeting abstract CT115, 2016), a combined approach incorporating pharmacokinetic assessments, drug accumulation and cellular response in ALL cells, may allow a comprehensive understanding of dex pharmacology in order to optimise its clinical utility.