TGF-β1-SMAD2 Axis Regulates Hematopoiesis and β-Globin Gene Expression Via Super-Enhancer Associated Chromatin Reorganization

Introduction: Transforming growth factor beta 1 (TGF-β1) is an important regulator of hematopoiesis. This cytokine has been shown to be associated with bone marrow failure and ineffective hematopoiesis, even though the chromatin-mediated effects that regulate these effects have not been elucidated. TGF-β1 has also been associated with accelerated erythroid differentiation, and the paradoxical anemia seen in TGF-β1 overexpression models has not been fully explained. We used a variety of in vitro and in vivo models, coupled with chromatin conformation studies, to determine the molecular changes affected by TGF-β1 in hematopoiesis.

Results: TGF-β1 can exist as three isoforms, and analysis of a large cohort of primary MDS samples (N = 183) demonstrated that TGF-β1 is the predominant isoform that is overexpressed in MDS marrow stem and progenitors cells when compared to healthy controls. We also analyzed the transcriptomic profiles of purified bone marrow mesenchymal stem cells (MSCs) and observed that these cells were also a source of TGF-β1 overproduction in MDS. We determined that in primary MDS samples, downstream SMAD2 signaling is overactive and is associated with worse anemia in large cohorts.

We utilized a transgenic mouse model that overexpresses TGF-β1 using an albumin promoter and develops anemia. Single-cell RNA-seq reveals that overexpression of TGF-β1 in our mouse model reduces the number of erythroid progenitors and mature red cells. To determine the downstream genes affected by downstream SMAD2 activation, ChIP-seq analysis of primary human erythroid progenitors at the BFU-E stage of maturation was conducted. TGF-β1-stimulated SMAD2 was bound to regulatory regions of hemoglobin and heme metabolic pathway genes, as well as the locus control region (LCR) super enhancer. We then validated that TGF-β1 stimulates the LCR enhancer, and this results in early erythroid differentiation, fewer cell divisions, and higher apoptosis during human erythropoiesis. Myc levels were found to decrease upon TGF-β1 stimulation, and chromatin capture experiments revealed loss of enhancer-promoter connections around the Myc super enhancer locus upon TGF-β1 stimulation.

Finally, we determined that exposure to plasma derived from MDS patients results in reduced erythroid output from healthy stem cells and progenitors in clonogenic assays. The inhibitory effects of TGF-β1-containing MDS patient serum on erythropoiesis can be reversed by Vactosertib, a small-molecule inhibitor of ALK5 that suppresses SMAD2 phosphorylation in various hematopoietic cell lines.

Conclusion: Our studies demonstrate TGF-β1-stimulated SMAD2 binds and regulates the LCR super enhancer and leads to increased erythroid differentiation. TGF-β1 also leads to decreased myc expression by decreasing chromatic associations with its superehnacer, and this leads to decreased cell divisions during erythropoiesis. Taken together, faster erythroid differentiation and reduced cell divisions lead to anemia phenotypes. These can be rescued with a novel, clinically useful ALK5 inhibitor, Vactosertib.