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Updated: Jun 4, 2026
Intracellular Phosphoflow Cytometry of Acute Myeloid Leukemia Patient-Derived Xenotransplants
Published on: June 6, 2025
Functional phosphoproteomic analysis reveals cold-shock domain protein A to be a Bcr-Abl effector-regulating
1Cell Survival Signalling Laboratory, Centre for Molecular Oncology and Imaging, Institute of Cancer, Barts and the London School of Medicine, Queen Mary University of London, UK.
Abstract:
One proposed strategy to suppress the proliferation of imatinib-resistant cells in chronic myeloid leukemia (CML) is to inhibit key proteins downstream of Bcr-Abl. The PI3K/Akt pathway is activated by Bcr-Abl and is specifically required for the growth of CML cells. To identify targets of this pathway, we undertook a proteomic screen and identified several proteins that differentially bind 14-3-3, dependent on Bcr-Abl kinase activity. An siRNA screen of candidates selected by bioinformatics analysis reveals cold-shock domain protein A (CSDA), shown previously to regulate cell cycle progression in epithelial cells, to be a positive regulator of proliferation in a CML cell line. We show that Akt can phosphorylate the serine 134 residue of CSDA but, downstream of Bcr-Abl activity, this modification is mediated through the activation of MEK/p90 ribosomal S6 kinase (RSK) signaling. Inhibition of RSK, similarly to treatment with imatinib, blocked proliferation specifically in Bcr-Abl-positive leukemia cell lines, as well as cells from CML patients. Furthermore, these primary CML cells showed an increase in CSDA phosphorylation. Expression of a CSDA phospho-deficient mutant resulted in the decrease of Bcr-Abl-dependent transformation in Rat1 cells. Our results support a model whereby phosphorylation of CSDA downstream of Bcr-Abl enhances proliferation in CML cells to drive leukemogenesis.
Insights
In chronic myeloid leukemia (CML), inhibiting cold-shock domain protein A (CSDA) phosphorylation downstream of Bcr-Abl blocks cancer cell proliferation. This finding offers a new therapeutic strategy for imatinib-resistant CML.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Imatinib resistance in chronic myeloid leukemia (CML) necessitates targeting proteins downstream of Bcr-Abl.
- The PI3K/Akt pathway is crucial for CML cell growth, activated by Bcr-Abl.
- Identifying novel targets within this pathway is key for therapeutic development.
Purpose of the Study:
- To identify novel targets of the PI3K/Akt pathway in CML.
- To investigate the role of cold-shock domain protein A (CSDA) in CML cell proliferation.
- To elucidate the mechanism of CSDA phosphorylation and its impact on Bcr-Abl-driven leukemogenesis.
Main Methods:
- Proteomic screening to identify 14-3-3 binding proteins.
- Bioinformatic analysis and siRNA screening to assess protein function.
- In vitro kinase assays and Western blotting to analyze CSDA phosphorylation.
- Cell proliferation assays and transformation studies using cell lines and primary CML patient cells.
Main Results:
- CSDA was identified as a positive regulator of proliferation in CML cells.
- Akt phosphorylates CSDA at serine 134, a process mediated by MEK/p90 ribosomal S6 kinase (RSK) signaling downstream of Bcr-Abl.
- Inhibition of RSK or imatinib treatment blocked proliferation in Bcr-Abl-positive leukemia cells and primary CML cells.
- Primary CML cells exhibited increased CSDA phosphorylation, and a phospho-deficient CSDA mutant reduced Bcr-Abl-dependent transformation.
Conclusions:
- CSDA phosphorylation, regulated by the Bcr-Abl-MEK/RSK pathway, enhances proliferation in CML cells.
- Targeting CSDA phosphorylation represents a potential therapeutic strategy for imatinib-resistant CML.
- This study elucidates a novel mechanism driving leukemogenesis in CML.
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