Transforming Growth Factor Beta 1/SMAD2 Signalling Axis Regulates Leukemia Stem Cell Marker CD123: Implications for Acute Myeloid Leukemia
Authors: Azizi Taramsary Sepideh , Woodcock Joanna , Tvorogov Denis , Lopez Angel F. , Thomas Daniel 
Published: December 2024

Introduction

A key determinant of chemoresistance in acute myeloid leukaemia (AML) is the percentage of persistent leukemic stem cells (LSCs), whichaberrantly express IL3Rα (CD123) and resemble granulocyte-macrophage progenitors (GMPs). However, the signalling mechanisms that determine CD123 over-expression are not known. During normal haematopoiesis, IL-3 signalling through the IL3Rα: βc receptor complex drives the differentiation of stem cells into various myeloid lineages, including GMPs. In addition to IL-3, cytokines associated with inflammatory responses, such as TGFβ1, can modulate GMP differentiation but the relevance of TGFβ1 in LSC biology is not known. Here we investigated the TGFβ1/SMAD2/SMAD4 signalling pathway in terms of upregulation of IL3Rα.

Method

Dose-responses, flow cytometry and Western blotting were performed in AML cell lines and CD34+ cord blood stem cells using recombinant cytokines and TβRI inhibitors. SMAD2 and SMAD4 were knocked down using lentiviral transduction. Co-Immunoprecipitation, immunofluorescent confocal microscopy and chromatin immunoprecipitation -PCR were performed for SMAD2 and SMAD4. Unpaired and paired t-test and regression statistical analysis were performed on at least 3 independent biological repeats.

Results

We first studied CD123 expression in the cytokine-responsive AML cell line TF-1. We observed striking up-regulation of IL-3Rα within 21 hours (3.5x fold) compared to control, while no change was observed with GMRα. Relative to untreated cells, we confirmed by flow cytometry an increase in IL3Rα (CD123) at the cell surface after TGFβ1 stimulation, with 4x fold increase (P = 0.022) in mean fluorescence intensity (MFI). In contrast, we observed no change in GMRα. qPCR confirmed transcriptional upregulation of IL3RA mRNA 3.5x fold at 8 hours (P &lt; 0.05) and 5.8x fold at 20 hours (P&lt;0.0005). Selectively, upregulation of IL3Rα protein was only observed after TGFβ1 or TGFβ2 stimulation, but not BMP, indicating SMAD2 but not SMAD1/5 activation is likely to contribute to this upregulation. Using Lentiviral-shRNA , we confirmed SMAD2 and SMAD4 knockdown resulted in reduced IL3Rα upregulation in TF1 cells after TGFβ1 stimulation. Chromatin immunoprecipitation of SMAD2 and SMAD4 confirmed enrichment at the proximal enhancer region, upstream of IL3Rα promoter, after TGFβ1 stimulation. Interestingly, a regression analysis for differences between TβRI inhibitor (LY2109761) and untreated sample with upregulated IL3Rα AML cell lines revealed a significant correlation between IL3Rα expression and pSMAD2 (R= 0.926 and P= 0.03).

To understand the physiological relevance of this phenomenon, we exposed healthy common myeloid progenitor cells (CMPs, CD34+CD38+CD123low) to TGFβ1 and demonstrated an increase in IL3Rα MFI (2.3x fold increase, P= 0.02). Interestingly, transient exposure to TGFβ1 resulted in increased granulocyte-macrophage colonies, at the expense of CFU-GEMM (P=0.002), which was reversed by IL3Rα blocking antibody. Finally, looking at AML/MDS patient sample, we were able to show LY2109761 treatment result in striking reduction in IL3Rα and TGFβ1 responsive gene, ID1.

Conclusion: In summary, our results highlight the critical role for the TGFβ1/SMAD2/SMAD4 signalling in skewing transcriptional machinery at the regulatory site of IL3Rα gene which prime progenitors toward inflammatory myelopoiesis. Understanding this TGFβ1 regulatory mechanism in upregulating CD123 may provide valuable insights for the future development of LSC-directed therapies, particularly in AML patients.