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NPM1 mutations were more sensitive to dexamethasone- induced apoptosis than samples without NPM1 mutations (Figure 4C).
Overlap between mutant NPM1 up-regulated target genes and a dexamethasone-associated gene expression signature
In line with these results, interrogation of a transcrip- tomic data set from a series of AML patients with NPM1 mutations and a data mining algorithm revealed that the NPM1 mutation gene signature was highly enriched in
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genes responsive to several small molecules, including dexamethasone as well as all-trans retinoic acid and dactinomycin, which have previously demonstrated ther- apeutic activity in this subgroup of AML (Figure 4D and E, and Online Supplementary Table S7).23 To address the genet- ic heterogeneity of AML, further transcriptomic analyses from two independent data sets revealed more complex molecular interactions between dexamethasone and some AML subgroups, such as those with RUNX1 mutations or the CBF-MYH11 rearrangement (Online Supplementary Figure S4A).23,24 Overall, enrichment in the dexamethasone
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Figure 4. Activity of dexamethasone against acute myeloid leukemia (AML). (A) Anti-leukemic effect of dexamethasone on AML cell lines cultured in minimum essential medium alpha whose cytogenetic and molecular characteristics are shown in Online Supplementary Table S4 (two-way ANOVA test, P<0.001). OCI-AML3 and KG1a were significantly more sensitive compared to the other AML cell lines (Tukey mul- tiple comparison test, P<0.001). (B) Kaplan–Meier curves for survival in a xenotransplantation mouse model of AML using the OCI-AML3 cell line (NPM1 and DNMT3A-R882C mutated) and NSG mice treat- ed with vehicle, dexamethasone (10 mg/kg/day, 5 days), cytarabine (30 mg/kg/day, 5 days), or dexam- ethasone plus cytarabine. P-values of the log-rank test are vehicle versus cytarabine (P=0.0105), vehi- cle versus dexamethasone (P=0.0014), vehicle versus dexamethasone plus cytarabine (P<0.0001), cytarabine versus dexamethasone (P=0.1474), cytarabine versus dexamethasone plus cytarabine (P=0.0001), and dexamethasone versus dexamethasone plus cytarabine (P=0.0943). (C) Apoptosis induction by dexamethasone in primary AML samples (#1 to 37, Online Supplementary Table S8) according to NPM1 mutational status (16 samples with NPM1 mutations and 18 samples that were wild-type for NPM1). AML samples were incubated with or without 100 and 300 nM of dexamethasone for 24 hours and then processed for apoptosis studies using annexin-V/propidium iodide staining. Results are presented as mean percentages of annexin V-positive cells in treated versus untreated cells (P=0.049 and P=0.04 for 100 and 300 nM when comparing NPM1 mutated versus NPM1 wild- type samples, respectively). (D) Gene ontology enrichment analysis using the data set of Verhaak et al. in AML with NPM1 mutations.23 (E) Gene-gene associations between mutant NPM1 up-regulated target genes and small molecule gene signatures and a data-mining algorithm (Genomatix). Gene expression analyses revealed that the NPM1 mutation gene signature was highly enriched in genes responsive to treatment using small molecules, such as all trans-retinoic acid (ATRA), retinoic acid (RA), dexametha- sone, and to a lesser extent dactinomycin (Online Supplementary Table S7).
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