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Glucocorticoids and selumetinib in RAS pathway-mutated ALL
selumetinib and the drug combination at 0.25x, 0.5x, 1x, 2x and 4x their respective GI50 concentrations and viability data were evaluated by median effect analysis. The CI for all RAS pathway-mutated samples were indicative of strong synergy with a mean of 0.1 (range, 0.02-0.15) (Figure 1A,B and Online Supplementary Figure S1). Synergism was not observed in ALL primagraft cells with- out RAS pathway activation (CI >1.2). Mechanistic assess- ments were performed with the GI50 concentration of both drugs for 24 h. As expected, ALL cells treated with selume- tinib resulted in almost complete inhibition of ERK phos- phorylation and downregulation of MCL1 levels. Dexamethasone treatment also downregulated pERK lev- els. Treatment with dexamethasone or selumetinib was associated with increased levels of BIM which were fur- ther enhanced with the drug combination. A representa- tive western blot and a histogram of the combined densit- ometry values (n=4 patient-derived xenografts) are shown in Figure 1C and Online Supplementary Figure S2A. The apoptotic marker, cleaved PARP, was enhanced with the drug combination in some, but not all, patient-derived xenograft samples at this time point (Online Supplementary Figure S2B). While the loss in cell viability in non-dividing patient-derived xenograft ALL cells must be due to increased cell death, we also showed enhanced apoptosis with the drug combination for the NRAS-mutated R3F9 cell line (Online Supplementary Figure S2C,D), an effect reduced by BIM knockdown (Online Supplementary Figure S2E,F). There were similar levels of induction of the glu- cocorticoid receptor target gene, GILZ, in cells treated with both dexamethasone and with the drug combination suggesting that enhanced glucocorticoid receptor tran- scriptional activity is not a component of the synergism (Online Supplementary Figure S3). Synergism between selumetinib and other drugs for example, gemcitabine, is highly schedule dependent and sequential rather than simultaneous dosing appears optimal.25 Thus, we assessed synergism in primagraft ALL cells dosed simultaneously or with only selumetinib or dexamethasone for 24 h fol- lowed by both drugs for an additional 72 h, prior to cell viability assessments. We saw similar synergism across all experimental parameters (Figure 1D) and thus we selected simultaneous drug administration in subsequent in vivo studies.
Pharmacokinetic studies to define a clinically relevant oral dose and exclude drug-drug interactions
To determine the optimal oral dose of dexamethasone that will achieve clinically relevant serum levels, pharma- cokinetic studies were performed in CD1 mice. Mice (n=27) were dosed with 0.5, 1 and 5 mg/kg dexametha- sone by oral gavage. Blood samples were taken after 15 min,30min,1h,3h,6hand24handserumdexametha- sone levels were analyzed (Online Supplementary Figure S4A). A Tmax of 60 min was observed, with Cmax values of 48.9, 94.7 and 766.5 ng/mL following the 0.5, 1 and 5 mg/kg doses, respectively. Given the reported Cmax average of 40-90 ng/mL in recent UK and American ALL trials, 1 mg/kg was deemed the most appropriate dose level.26,27
Dexamethasone can induce cytochrome P450 forms, including CYP3A4, the principal isoform responsible for selumetinib oxidative metabolism; we therefore per- formed selumetinib pharmacokinetic analyses, following administration of the drug alone (25 mg/kg) and after co- administration with 1 mg/kg dexamethasone (Online
Supplementary Figure S4B,C). A Tmax of 60 min was observed, with Cmax values for selumetinib of 4.74 μg/mL compared to 5.49 μg/mL, respectively (P>0.05, Student t test). Other parameters were also similar (Online Supplementary Figure S4C), indicative of no drug-drug interaction (P>0.05 for all).
Selumetinib and dexamethasone show synergy in vivo and clear central nervous system disease
The drug combination was evaluated in vivo and its effects compared to those of single drugs and control vehi- cle in primagrafts derived from diagnostic ALL (NRAS Q61R and KRAS G12D) and relapse (KRAS G13D) samples. Scheduling and dosing, by oral gavage, are shown in Figure 2A-G. Given the significant weight loss (>20%) associated with dexamethasone, dosing could not be prolonged, even when the dose was lowered from 1 mg/kg bid to 0.25 mg/kg sid. There was no additional toxicity observed in mice given the drug combination. Nevertheless, at the end of the dosing period, there was a significant reduction in spleen size with selumetinib or dexamethasone alone but a statistically greater reduction in mice given the drug combi- nation, with spleen weights approaching those of healthy mice (P<0.001) (combined data are shown in Figure 2G). In addition, brains were assessed for the depth of leukemia infiltration in the leptomeninges. For mice engrafted with L897 and L779 primagraft cells, there was a significant reduction in leukemic infiltration in drug-treated mice, with a mean and standard deviation of 66.3 μ ± 100.6 for animals given the control vehicle, compared to 3.1 μ ± 12.5 for those treated with dexamethasone and 5.37 μ ± 21.475 for those treated with selumetinib (Online Supplementary Figure S5A). Mice treated with the drug combination showed no leukemic infiltration (P<0.05 for all by the Student t test). For L779, there was demonstrable CNS disease once peripheral ALL exceeded 1% i.e. pre-dosing (Online Supplementary Figure S5B). Clearance of CNS disease in mice engrafted with L829R cells was unevaluable because of minimal CNS leukemia in both control and drug-treated mice. The results of the pharmacodynamic assessment of engrafted spleens after short-term dosing were consistent with observations in vitro; inhibition of ERK phosphoryla- tion and lower MCL1 levels associated with selumetinib dosing, similar induction of GILZ with dexamethasone dos- ing, and modest enhancement of BIM levels with the drug combination (Figure 3A-D). Annexin V binding in circulat- ing ALL cells, as detected by multi-parameter flow cytome- try, increased at both 24 h and 48 h in all drug-treated mice and was highest for those given the drug combination (Figure 3E).
Discussion
Selumetinib is a potent, selective, allosteric inhibitor of MEK1/2 with demonstrated anti-tumor activity and a favorable toxicity profile. It has progressed to phase III clinical trials for several types of adult solid cancers.28-30 In the pediatric setting, selumetinib has recently undergone phase I clinic testing as a monotherapy in children with BRAF-driven recurrent/refractory pediatric low-grade glioma which defined a maximum tolerated dose of 25 mg/m2/dose bid.31 Sustained responses (1 complete, 7 par- tial) were observed in some children and selumetinib was well tolerated, with the most common toxicity being rash.
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