Targeting CDK1 promotes FLT3-activated acute myeloid leukemia differentiation through C/EBPα
Hanna S. Radomska, … , Ruud Delwel, Daniel G. Tenen
Hanna S. Radomska, … , Ruud Delwel, Daniel G. Tenen
Published July 17, 2012
Citation Information: J Clin Invest. 2012;122(8):2955-2966. https://doi.org/10.1172/JCI43354.
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Research Article Oncology

Targeting CDK1 promotes FLT3-activated acute myeloid leukemia differentiation through C/EBPα

Abstract

Mutations that activate the fms-like tyrosine kinase 3 (FLT3) receptor are among the most prevalent mutations in acute myeloid leukemias. The oncogenic role of FLT3 mutants has been attributed to the abnormal activation of several downstream signaling pathways, such as STAT3, STAT5, ERK1/2, and AKT. Here, we discovered that the cyclin-dependent kinase 1 (CDK1) pathway is also affected by internal tandem duplication mutations in FLT3. Moreover, we also identified C/EBPα, a granulopoiesis-promoting transcription factor, as a substrate for CDK1. We further demonstrated that CDK1 phosphorylates C/EBPα on serine 21, which inhibits its differentiation-inducing function. Importantly, we found that inhibition of CDK1 activity relieves the differentiation block in cell lines with mutated FLT3 as well as in primary patient–derived peripheral blood samples. Clinical trials with CDK1 inhibitors are currently under way for various malignancies. Our data strongly suggest that targeting the CDK1 pathway might be applied in the treatment of FLT3ITD mutant leukemias, especially those resistant to FLT3 inhibitor therapies.

Authors

Hanna S. Radomska, Meritxell Alberich-Jordà, Britta Will, David Gonzalez, Ruud Delwel, Daniel G. Tenen

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Figure 2

C/EBPα phosphorylation on serine 21 by CDK1 in vitro and in vivo.

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C/EBPα phosphorylation on serine 21 by CDK1 in vitro and in vivo. (A) In...
(A) In vitro kinase assay. Active CDK1 enzyme was incubated with GST (lane 1) or GST-C/EBPα fusion proteins (lanes 2–5) in the presence of 32P. Lanes 2 and 3 contain WT C/EBPα-GST as a substrate. Lane 4 contains Ser21Ala mutant, and lane 5 contains Phe31Ala mutant. Proteins were resolved on acrylamide gel and blotted to nitrocellulose membrane. Upper panel: autoradiograph of the membrane. Lower panel: staining with the N-terminal C/EBPα antibody. (B) Effect of CDK1 overexpression or mitotic arrest on C/EBPα phosphorylation. 293T cells transfected with empty vector, or HA-tagged CDK1 expression vector (lanes 1 and 2). Cells expressing empty vector (lane 3) or a dominant negative form of CDK1 (lane 4; DN CDK1) were arrested at mitosis by nocodazole treatment. All samples were analyzed on the same gel, and the Western blot was stained with the antibodies indicated. (C) CDK1 silencing results in decreased C/EBPα phosphorylation on serine 21. MOLM-14 cells were transduced with a CDK1 shRNA, sorted for EGFP expression, and cultured for 2 days (total of 4 days of viral transduction). Whole-cell extracts were analyzed by Western blotting with the indicated antibodies. Signals were quantified and graphed. Black bars indicate the relative expression levels of CDK1 and C/EBPα proteins or ERK1/2 activity following the knockdown (compared with white bars for negative controls; NC). Gray bar shows the level of phospho–serine 21 C/EBPα relative to the total C/EBPα protein in cells with downregulated CDK1 expression. sh, short hairpin.