Human AML cell lines with activating mutations in FLT3 express C/EBPα mRNA. (A) Cell lines (top) were not treated (0) or treated with 1 μM MLN518 for 8 or 24 h (indicated above the lanes). (top) Hybridization of the Northern blot to a human C/EBPα probe. (bottom) A control hybridization to 18S ribosomal RNA. RNA from KG1a and U937 cells served as negative and positive controls, respectively. (B) The same RNA samples were analyzed in triplicate by TaqMan Real-Time PCR and normalized to 18S RNA.

Inhibition of FLT3 induces granulocytic differentiation of MV4;11 cells. (A) MV4;11 cells were treated with 5 μM MLN518 for 3 d. Samples were withdrawn daily and phosphorylation of C/EBPα on S21 was analyzed by Western blot with phospho-specific (top) and C/EBPα (bottom) antibodies. Quantification of signals with respect to total cellular protein levels (as determined by Ponceau S staining) is shown (bottom). (B) MV4;11 cells were cultured in the presence of 5 μM MLN518 or vehicle control (0.05% DMSO). Cell aliquots were examined daily for morphological changes (top) and for NBT reduction activity (bottom). Differential counts are summarized in the chart. (C) Inhibition of FLT3 leads to down-regulation of c-myc. (top) FLT3 mutant AML cell lines were serum starved for a total of 8 h in the absence or presence of 1 μM MLN518 for the indicated number of hours (at the end of starvation period). Western blot was stained with c-myc or β-tubulin antibodies. (bottom) FLT3 mutant lines, MV4;11, MOLM-13, MOLM-14, MonoMac1 (MM1), and MonoMac6 (MM6) were grown in the absence (0) or presence of 1 μM MLN518 for the indicated times (hours). Total RNA was examined by Northern blot with probes specific for c-myc and GAPDH (to control for RNA integrity). (D) Up-regulation of granulocyte-specific gene mRNA in MLN518-treated cells. Same RNA samples as in C, were examined by Real Time-PCR with primer sets specific for the elastase, lysozyme, myeloperoxidase, and lactoferrin genes. Data were normalized for GAPDH RNA and presented as fold change.

The dephosphorylated form of C/EBPα is sufficient to mediate granulocytic differentiation of MV4;11 cells. (A) MV4;11 cells were stably transfected with inducible C/EBPα expression vectors. Independent clones were analyzed by Western blot. Parental MV4;11 cells expressing only the endogenous C/EBPα protein are shown (left). Clone nos. 7, 25, and 29 express an ectopic C/EBPα-ER fusion protein in which S21 of C/EBPα was mutated to alanine (S21A). Clones C, D, and N express C/EBPα-ER protein with S21 mutated to aspartate (S21D) to mimic phosphorylation. (B) MV4;11 cells with induced expression of S21A-C/EBPα protein undergo granulocytic differentiation. Granulocyte-specific morphological changes in Wright-Giemsa stained cytospins (indicated by red arrows) were seen (top) as well as an increase in NBT reduction activity (bottom). (C) Induction of S21D-C/EBPα did not induce granulocytic maturation as shown by morphological examinations (top) or NBT reduction assay (bottom). The inductions shown are for 24 h; however, no changes were observed after longer treatments with β-estradiol (not depicted). (D) Parental MV4;11 cells and C/EBP-ER–expressing stable lines (S21A, and S21D) were left untreated (0) or treated with 1 μM β-estradiol for 4, 8, and 24 h. RNA was collected and analyzed by Northern blot with probes specific for c-myc (top) and GAPDH (bottom). For positive controls, K562 and U937 cells were used. The U937 stable line with Zn-inducible expression of ectopic C/EBPα (reference 24) was included as negative control, as they were shown to down-regulate c-myc expression upon induction of C/EBPα expression (reference 51).

Activation of FLT3 in human AML cells leads to C/EBPα protein phosphorylation on S21 by the ERK1/2 pathway. (A) COS7 cells were untreated or treated with UV-C (60 J/m2; UV) or 100 nM PMA and incubated for 60 min. Endogenous c-Jun NH₂-terminal protein kinase (JNK), p38, and ERK were immunoprecipitated and used for in vitro kinase assays. Control tests showed that ERK phosphorylated Elk1, JNK-phosphorylated c-Jun, and p38 MAP kinase phosphorylated ATF2. (B) THP-1 cells expressing wild-type FLT3 were serum starved for 6 h (0), or starved and stimulated with 100 ng/ml FLT3 ligand (FL) for 5 and 10 min before harvest. Western blot of whole cell extracts was sequentially stained with pS21-C/EBPα, C/EBPα, pThr202/Tyr204-ERK1/2, and β-tubulin antibodies. (C) Human AML cell lines with mutant FLT3 have increased phosphorylation of C/EBPα on S21. All cell lines were serum starved for 7 h in the absence (−) or presence (+) of 1 μM MLN518. Western blot was analyzed as in B. (D) MV4;11 cells were serum starved in the absence (−) or presence of the FLT3 inhibitors AG1296 and MLN518 at concentrations marked above the lanes, or with vehicle control (DMSO). Western blot analyses were done as in B. (E) Phosphorylation status of C/EBPα in MV4;11 and MOLM-13 cells as a dose response to MLN518. Cells were serum starved for 7 h in the presence of MLN518 at concentrations indicated above the lanes. Western blot was stained as in B. (F) Time course of FLT3 inhibition and its effect on C/EBPα phosphorylation. At each time point, MV4;11, MOLM-13, or MOLM-14 cells were serum starved for a total period of 8 h. MLN518 (1 μM) was added for the final hours (indicated above the lanes) of the treatment. For example, “1 hr” means that the cells were starved for 7 h in the absence of the inhibitor, and for 1 final hour in its presence. Western blot staining was as described previously. (G) Decrease of pS21-C/EBPα by inhibition of FLT3 activity in AML patient samples. Leukemic blasts from bone marrow (patient no. 592), or peripheral blood (patient no. 667) were cultured in the absence (0), or presence (1) of MLN518 for 6 h. Western blot of whole cell extracts was analyzed with antibodies recognizing activated FLT3 (P-FLT3), phosphorylated C/EBPα (P-Ser21-C/EBPα), or total C/EBPα protein. MV4;11 cells were included for control. Quantification of C/EBPα phosphorylation normalized to the total C/EBPα protein is shown (bottom).

MV4;11 cells differentiate upon inhibition of the ERK1/2 pathway. (A) Western blot showing decreased pS21-C/EBPα (top) in response to continuous treatment with 100 μM of the MEK1 inhibitor PD98059 or vehicle control (DMSO). For DMSO-treated cells, a 72-h incubation is shown. (bottom) The same blot stained with C/EBPα antibody. (B) (top) MV4;11 cell differentiation was monitored by morphological examination of Wright-Giemsa stained cytospin preparations. Red arrows point to cells with granulocytic morphology. (bottom) Respiratory burst activity as assessed by the NBT assay.