Contributions
Abstract: PB1687
Type: Publication Only
Background
Acute myeloid leukemia (AML) is a disorder arising from developmental arrest of cells of the myeloid lineage. To understand how regulators of transcription mediate this block, we previously analyzed the nuclear proteome of AML blasts against developmentally matched HSPCs and found nuclear mislocalization and differential expression of S100A4. Cytosolic overexpression of this calcium binding protein has been implicated in solid tumours and is associated with poor clinical outcomes; however, little is known about the role of aberrant nuclear expression of S100A4 in AML.
Aims
(i) To validate the overexpression of S100A4 in AML patients and cell lines
(ii) To determine the effects of overexpressed S100A4 on haematopoietic growth and development using normal human CD34+ cells
(iii) To determine whether AML cell lines are dependent on S100A4 overexpression
Methods
We analysed AML patient samples (n= 24) for S100A4 expression and subcellular localisation by western blot using normal CD34+ cells as controls. Lentiviral vectors containing Nuclear Localisation Signals (NLS) were used to overexpress S100A4 into the nucleus of normal HPSCs and AML cell lines. shRNA was used to knock down S100A4 in normal HPSCs and in AML cell lines. Effects on growth and haematopoietic development were analysed by multiparameter flow cytometry.
Results
To confirm our initial studies, we analyzed a second cohort of AML patient blasts and identified S100A4 to be mislocalized to the nucleus in ~83 % of AML cases (20/24). Interestingly, S100A4 protein was not expressed in the nucleus of normal HSPC or differentiated precursors in myeloid lineage (monocytes, erythrocytes, and granulocytes) throughout development. To determine the effect of nuclear-targeted S100A4 over-expression, we infected CD34+ cells with lentivirus encoding nuclear-targeted S100A4. Whilst we could over-express S100A4 in the nucleus of some leukemic cell lines, we could not stably express it in HSPCs. We found that nuclear expression of S100A4 increased cell proliferation in TF-1 compared to uninfected controls. Conversely, knockdown of S100A4 in cell lines with elevated S100A4 expression using shRNA, induced cell death in THP-1, OCI-2 and TF-1.
Conclusion
We found that S100A4 is over-expressed and mislocalized to the nucleus in AML blasts. Whilst normal HSPCs express this protein in the cytosol of some myeloid differentiated lineages, they do not tolerate over-expression of this protein in the nucleus. In the context of transformed cells, S100A4 promotes proliferation and is essential for AML cell survival. These data suggest that S100A4 could be a target for therapy for AML.
Session topic: 3. Acute myeloid leukemia - Biology & Translational Research
Keyword(s): Acute Myeloid Leukemia
Abstract: PB1687
Type: Publication Only
Background
Acute myeloid leukemia (AML) is a disorder arising from developmental arrest of cells of the myeloid lineage. To understand how regulators of transcription mediate this block, we previously analyzed the nuclear proteome of AML blasts against developmentally matched HSPCs and found nuclear mislocalization and differential expression of S100A4. Cytosolic overexpression of this calcium binding protein has been implicated in solid tumours and is associated with poor clinical outcomes; however, little is known about the role of aberrant nuclear expression of S100A4 in AML.
Aims
(i) To validate the overexpression of S100A4 in AML patients and cell lines
(ii) To determine the effects of overexpressed S100A4 on haematopoietic growth and development using normal human CD34+ cells
(iii) To determine whether AML cell lines are dependent on S100A4 overexpression
Methods
We analysed AML patient samples (n= 24) for S100A4 expression and subcellular localisation by western blot using normal CD34+ cells as controls. Lentiviral vectors containing Nuclear Localisation Signals (NLS) were used to overexpress S100A4 into the nucleus of normal HPSCs and AML cell lines. shRNA was used to knock down S100A4 in normal HPSCs and in AML cell lines. Effects on growth and haematopoietic development were analysed by multiparameter flow cytometry.
Results
To confirm our initial studies, we analyzed a second cohort of AML patient blasts and identified S100A4 to be mislocalized to the nucleus in ~83 % of AML cases (20/24). Interestingly, S100A4 protein was not expressed in the nucleus of normal HSPC or differentiated precursors in myeloid lineage (monocytes, erythrocytes, and granulocytes) throughout development. To determine the effect of nuclear-targeted S100A4 over-expression, we infected CD34+ cells with lentivirus encoding nuclear-targeted S100A4. Whilst we could over-express S100A4 in the nucleus of some leukemic cell lines, we could not stably express it in HSPCs. We found that nuclear expression of S100A4 increased cell proliferation in TF-1 compared to uninfected controls. Conversely, knockdown of S100A4 in cell lines with elevated S100A4 expression using shRNA, induced cell death in THP-1, OCI-2 and TF-1.
Conclusion
We found that S100A4 is over-expressed and mislocalized to the nucleus in AML blasts. Whilst normal HSPCs express this protein in the cytosol of some myeloid differentiated lineages, they do not tolerate over-expression of this protein in the nucleus. In the context of transformed cells, S100A4 promotes proliferation and is essential for AML cell survival. These data suggest that S100A4 could be a target for therapy for AML.
Session topic: 3. Acute myeloid leukemia - Biology & Translational Research
Keyword(s): Acute Myeloid Leukemia