Related Experiment Video
Updated: Aug 13, 2026
Intracellular Phosphoflow Cytometry of Acute Myeloid Leukemia Patient-Derived Xenotransplants
Published on: June 6, 2025
Block of C/EBP alpha function by phosphorylation in acute myeloid leukemia with FLT3 activating mutations
Hanna S Radomska1, Daniela S Bassères, Rui Zheng
1Beth Israel Deaconess Medical Center/Harvard Medical School, Boston, MA 02115, USA.
Abstract:
Mutations constitutively activating FLT3 kinase are detected in approximately 30% of acute myelogenous leukemia (AML) patients and affect downstream pathways such as extracellular signal-regulated kinase (ERK)1/2. We found that activation of FLT3 in human AML inhibits CCAAT/enhancer binding protein alpha (C/EBPalpha) function by ERK1/2-mediated phosphorylation, which may explain the differentiation block of leukemic blasts. In MV4;11 cells, pharmacological inhibition of either FLT3 or MEK1 leads to granulocytic differentiation. Differentiation of MV4;11 cells was also observed when C/EBPalpha mutated at serine 21 to alanine (S21A) was stably expressed. In contrast, there was no effect when serine 21 was mutated to aspartate (S21D), which mimics phosphorylation of C/EBPalpha. Thus, our results suggest that therapies targeting the MEK/ERK cascade or development of protein therapies based on transduction of constitutively active C/EBPalpha may prove effective in treatment of FLT3 mutant leukemias resistant to the FLT3 inhibitor therapies.
Insights
Activating FLT3 mutations in acute myelogenous leukemia (AML) block cell differentiation via ERK1/2. Targeting MEK/ERK or using active C/EBPalpha may treat FLT3-mutant AML.
Area of Science:
- Hematology
- Molecular Biology
- Oncology
Background:
- Activating mutations in FLT3 kinase occur in ~30% of acute myelogenous leukemia (AML) cases.
- FLT3 mutations impact downstream signaling, including the extracellular signal-regulated kinase (ERK) pathway.
- This aberrant signaling contributes to the differentiation block observed in leukemic cells.
Purpose of the Study:
- To investigate the mechanism by which FLT3 activation inhibits CCAAT/enhancer binding protein alpha (C/EBPalpha) function in AML.
- To explore potential therapeutic strategies targeting the FLT3-ERK-C/EBPalpha axis.
Main Methods:
- Utilized MV4;11 AML cell line models.
- Employed pharmacological inhibition of FLT3 and MEK1.
- Investigated the role of C/EBPalpha phosphorylation through site-directed mutagenesis (S21A and S21D).
Main Results:
- FLT3 activation in AML inhibits C/EBPalpha function through ERK1/2-mediated phosphorylation.
- Pharmacological inhibition of FLT3 or MEK1 induced granulocytic differentiation in MV4;11 cells.
- Stable expression of non-phosphorylatable C/EBPalpha (S21A) promoted differentiation, while a phosphomimetic mutant (S21D) did not.
Conclusions:
- ERK1/2-mediated phosphorylation of C/EBPalpha is a key mechanism underlying the differentiation block in FLT3-mutant AML.
- Targeting the MEK/ERK pathway presents a viable therapeutic strategy.
- Protein-based therapies using constitutively active C/EBPalpha may offer an alternative treatment approach for FLT3-mutant leukemias, particularly those resistant to FLT3 inhibitors.
