Reduced SMAD7 leads to overactivation of TGF-beta signaling in MDS that can be reversed by a specific inhibitor of TGF-beta receptor I kinase

Abstract

Even though myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis, the molecular alterations that lead to marrow failure have not been well elucidated. We have previously shown that the myelosuppressive TGF-β pathway is constitutively activated in MDS progenitors. Because there is conflicting data about upregulation of extracellular TGF-β levels in MDS, we wanted to determine the molecular basis of TGF-β pathway overactivation and consequent hematopoietic suppression in this disease. We observed that SMAD7, a negative regulator of TGF-β receptor I (TBRI) kinase, is markedly decreased in a large meta-analysis of gene expression studies from MDS marrow-derived CD34(+) cells. SMAD7 protein was also found to be significantly decreased in MDS marrow progenitors when examined immunohistochemically in a bone marrow tissue microarray. Reduced expression of SMAD7 in hematopoietic cells led to increased TGF-β-mediated gene transcription and enhanced sensitivity to TGF-β-mediated suppressive effects. The increased TGF-β signaling due to SMAD7 reduction could be effectively inhibited by a novel clinically relevant TBRI (ALK5 kinase) inhibitor, LY-2157299. LY-2157299 could inhibit TGF-β-mediated SMAD2 activation and hematopoietic suppression in primary hematopoietic stem cells. Furthermore, in vivo administration of LY-2157299 ameliorated anemia in a TGF-β overexpressing transgenic mouse model of bone marrow failure. Most importantly, treatment with LY-2157199 stimulated hematopoiesis from primary MDS bone marrow specimens. These studies demonstrate that reduction in SMAD7 is a novel molecular alteration in MDS that leads to ineffective hematopoiesis by activating of TGF-β signaling in hematopoietic cells. These studies also illustrate the therapeutic potential of TBRI inhibitors in MDS.

FIGURE 1. SMAD7 is significantly decreased in MDS

Differences in SMAD7 expression were also evaluated in normalized meta-analysis of MDS CD34+ (89 cases) and normal CD34+ (61 cases) cell derived gene expression microarray studies. Box plots show SMAD7 gene expression was significantly downregulated in MDS bone marrow CD34+ cells after multiple testing with Benjamin Hochberg correction with p value < 0.01. The reduction is seen in both low (n=83) and high grade (n=6) cases and also in cases with (n=29) and without (n=60) 5q deletion. Heat map shows individual values of all samples.

Figure 2. SMAD7 protein is decreased in MDS marrow progenitors

Tissue microarray was prepared from bone marrow (BM) biopsies from patients with MDS and controls with non-MDS Bone marrow biopsy samples were stained with SMAD7 antibody and show decreased expression in 4 representative cases of MDS when compared to controls. Low grade MDS samples revealed significantly less SMAD7 staining when compared to controls (Propotions test,p<0.05)

Figure 3. Reduction in SMAD7 can lead to increased sensitivity to suppressive effects of TGF-β on hematopoiesis

GFP expressing Lentiviral based shRNA against SMAD7 (A) was used to knockdown SMAD7 in hematopoeitic cells. qPCR shows decrease in SMAD7 mRNA expression after lentiviral shRNA-TBRI infection in bone marrow stromal (HS-5) cells when compared to scrambled control (B). Primary CD34+ progenitors were electroporated with GFP coexpressing anti-SMAD7 shRNA construct and sorted after 48 hours. GFP positive cells were grown in methylcellulose with cytokines and erythroid colonies were counted after 14 days. SMAD7 shRNA transfected progenitors were significantly inhibited at lower concentrations of TGF-b when compared to cells transfected with scrambled control shRNA. Expressed as Means +/- s.e.m of 3 independent experiments. (p<0.05, Two Tailed T test) (C). Bone marrow stroma derived cells (HS-5) with stable expression of anti-SMAD7 and control shRNAs were generated. These cells were transfected with plasmids expressing smad binding 3TP-Luciferase and beta-galactisidose (transfection control) and stimulated with TGF-β1 in the presence and absence of LY-215 (dose .5uM). Tgf-β1 induced control normalized Luciferase activity was significantly increased after SMAD7 knockdown. This increase was potently inhibited by LY-2157299.(D).

Figure 4. LY-2157299 is an effective and functionally active inhibitor of TGF-β signaling in hematopoietic cells

Leukemic cells (K562) and marrow stromal cells (HS-5) were treated with TGF-β1 (20ng/ml) in the presence and absence of LY-2157299 (.5uM) and assessed for SMAD2 phosphorylation by immunoblotting. LY-215 pretreament (1 hr) led to attenuation of activation/phosphorylation of SMAD2 (A,B). Healthy CD34+ progenitors were plated in methylcellulose and cytokines in the presence and absence of TGF-b and TBRI inhibitor LY-2157299 (200nM and 500nM). Colonies were scored at Day 14 and results were expressed as means + S.E.M of 3 independent experiments. TGF-b exposure led to smaller (C) and lesser numbers (D) of colonies. These effects were reversed in the presence of LY215 treatment.

Figure 5. LY-2157299 can improve anemia in a murine model of TGF-β1 driven bone marrow failure

Mice transgenic for alb/TGF-β were treated with either LY-2157299 (100 mg/kg/d) or vehicle (Placebo) daily by gastric lavage for 14 days. Blood counts were done on the 14^th day and revealed a significant rise in hematocrit after LY-2157299 treatment. (A) The mice were sacrificed and bone marrow biopsies were immunostained with antibody against phosphor-SMAD2. Representative sample shows inhibition of SMAD2 activation after LY-2157299 treatment. (B)

FIGURE 6. TBRI inhibition stimulates hematopoiesis in MDS

MDS Bone marrow derived MNCs from 10 patients were plated in methylcellulose and cytokines in the presence and absence of TBRI inhibitor LY-2157299 (200nM and 500nM). Colonies were scored at Day 14 and results were expressed as means + S.E.M of 10 independent experiments.