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DEX and DAS impair in vivo T-ALL propagation
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Figure 6. Dexamethasone (DEX) and dasatinib (DAS) act synergistically to induce cell death in T-cell acute lymphoblastic leukemia (T-ALL). (A) Cell viability of parental SUPT1 cells (mock), shCtrl (NTC), shLCK#1 or shLCK#3 transduced SUPT1 cells upon treatment with increasing DEX concentrations (0-1699 nM). (B) Cell viability of SUPT1 with and without DAS (left; black line, no DAS; blue line, 0.8 mM; red line, 2.0 mM) in combination with increasing concentrations of DEX (0-600 nM) as derived from the drug matrix with titration of DEX (0-600 nM) and DAS (0 - 50 uM; right). (Right) Combenefit analysis of drug matrix demonstrates drug synergy in SUPT1 cells at clinically relevant drug concentrations. (C) (Left) LK203 cells were expanded ex vivo on OP9-DL1 feeder cells for 1 week prior to treatment with and without DAS (black line, no DAS; blue line, 0.08 μM; red line, 2.0 mM; orange line, 10 mM) in combination with increasing concentrations of DEX (0-600 nM) as derived from a drug matrix with DEX (0-600 nM) and DAS (0 - 50 uM) (Online Supplementary Figure S6E). (Right) Cell death analysis in LK203 cells exposed to control (Ctrl) conditions, DAS (1 mM), DEX (100 nM) or DAS+DEX combination treatment. (D) (Left) Normalized GILZ mRNA expression in Jurkat cells after transduction with shNTC or shLCK#3 with or without DEX exposure (100 nM). (Right) Normalized GILZ mRNA expression in Jurkat cells after exposure to Ctrl conditions, DAS (2 mM), DEX (100 nM) or DAS+DEX combination treatment at the same concentrations. Student's t-test: **P<0.01, ***P<0.005, ****P<0.001.
cle as measured by hCD45 or hCD7 engraftment in peripheral blood, bone marrow, spleen and CNS (Figure 7D-F and Online Supplementary Figure S7A). Single agent DEX reduced CNS leukemia burden in 7 out of 9 samples, reflecting its proven efficacy in reducing CNS relapses,27 whilst DAS showed some reduction in CNS burden in 4 out of 8 samples (Online Supplementary Figure S7B). Combination therapy was particularly effective, with complete eradication of measurable CNS leukemia in five patient samples and evidence of an additive effect with DEX in 3 out of 4 of the remaining patient samples (Online Supplementary Figure S7B). When considering the cohort of nine patient samples overall, combination treatment sig- nificantly reduced leukemic infiltrates compared to con- trol (P=0.02). Representative histology images are shown in Figure 7G. Spleen weight was substantially reduced in mice receiving combination treatment, compared with the single or control treatment arms (Online Supplementary Figure S7C and D). DEX+DAS also significantly reduced hCD45+ or hCD7+ leukemia cell engraftment in liver tissue of all six PDX samples analyzed (Online Supplementary Figure S7E). The initial therapeutic advantage of DEX+DAS in mice derived from PDX LK080 was lost at the end of the experiment, most likely because these mice were kept alive for 3 weeks after completion of treatment. In the case of L907, however, the benefit of combination treatment was not observed until the last time point (Online Supplementary Figure S7G).
Discussion
Using a phase II-like murine trial, we demonstrate here the efficacy of the drug combination DEX+DAS in impair- ing expansion of human T-ALL samples. This effect is apparent in an unselected, biologically heterogeneous, cohort of PDX samples. This trial format recapitulates early phase human clinical trials and indicates that this drug combination could be widely applicable in the treat- ment of T-ALL. Studies by Serafin et al. first proposed a role for this drug combination.28 Our murine trial extends these initial observations with an extensive cohort consist- ing of nine different PDX demonstrating treatment advan- tage for both DEX sensitive and resistant T-ALL.
Significant superiority of DEX+DAS was demonstrated even after exclusion of mice who reached their clinical end points prematurely. These untoward events highlight the practicalities of performing murine trials.
We propose that the impaired in vivo expansion results from a combination of cell cycle arrest as well as cell death. Several mechanisms could provide plausible expla- nations for the occurrence of cell cycle arrest. DAS is a protein tyrosine kinase inhibitor which targets Abl and SFK family members. We have confirmed that DAS effec- tively inhibits activity of the SFK member LCK by pre- venting phosphorylation, leading to G0/G1 arrest. DAS has previously been shown to inhibit cyclin dependent kinase 1 (CDK1), which plays a central role in G1/S and G2/M
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