Impairment of AML Engraftment to the Bone Marrow Niche Following Inhibition of TGF-Beta Signaling

The cell fate of the HSC to either self-renew or differentiate is controlled by a complex interplay between cell-intrinsic and -extrinsic regulatory signals generated by the surrounding bone marrow microenvironment called the HSC niche. A balance exists within the “cross talk” between HSCs and the niche, which allows HSC dormancy, activation and differentiation. Any alterations of this balance may lead to uncontrolled cellular proliferation and ultimately the promotion of leukemia. However, it remains to be determined exactly how the hematopoietic microenvironment contributes to the deregulation of normal hematopoiesis and/or promotes the maintenance of leukemia cells as a “leukemic niche”.

To investigate this, we have now performed micro-array analysis of MS5 stromal cells that were co-cultured with a panel of leukemic cell lines and acute myeloid leukemia (AML) patient samples. The most significantly up-regulated pathways as compared to MS5 cells cultured alone involved cytoskeleton remodeling, cell cycle, cell adhesion and development through cytokine signaling. Since transcript and protein levels of number of effectors of the TGF-beta (TGF-β) signaling pathway were up-regulated in the stroma co-cultured with leukemic cells, we next investigated inhibition of this pathway using a specific inhibitor of TGF-β receptor kinase, SB-431542 (10µM). Treatment with the inhibitor significantly reduced the cell number and increased the levels of apoptosis in the AML cells co-cultured on stromal cells, whilst having mininal effect on normal cells. Treatment with SB-431542 (10mg/kg), also significantly reduced the level of AML cell engraftment on treatment in vivo (n=3) (untreated- 68.65±6.95; 56.15±22.85; 84.35±5.75 and SB-treated- 45.5±11.6; 30.5±19.6; 54.1±4.9). In order to inhibit TGF-β signaling more specifically within the stromal compartment, we next used shRNA against TGF-β Receptor II (TGFBR2) in MS5 stromal cells and co-cultured them with AML cells within 3D scaffold models (n=4), which were implanted in vivo. A significant reduction in engraftment was observed as compared to controls (shRNA control- 64.65±32.65; 87.7±7.2; 23.55±4.35; 49.65±33.65 and TGFBR2 knockdown- 20.2±3; 62.95±4.05; 15.7±1.5; 20.385±17.415). The co-culturing of normal cord blood CD34⁺ or mononuclear cells on the TGFBR2 knockdown stroma had no significant effect both in vitro and in vivo (n=3). To investigate whether TGF-β inhibition had an effect on the interaction of AML cells to the niche, we used intravital microscopy to track the cells live in vivo. HL60 (AML cell line) cells were labeled with 2µM CFSE and pretreated ±SB-431542 (10µM) on stroma, before being sorted and transplanted into immunodeficient mice. Distance to the calvaria was measured at 16 hours and we observed that the SB-431542-treated cells were positioned significantly further away from the bone surface as compared to untreated control (p=0.0001). Since the TGF-β inhibited cells appeared to have impaired ability to adhere to the bone marrow, we next investigated the relationship between extracellular matrix molecules and TGF-β signaling. We saw that stromal cells that were co-cultured with AML cells had a significantly increased expression of laminins A1, A5, B1 and G1. This effect could be recapitulated by treatment of naïve stromal cells with TGF-β2 and 3. We also observed a reciprocal decrease in expression of laminins following both treatment of AML-stromal co-cultures with SB-431542 and within TGFBR2 knockdown stroma. Furthermore, we saw an increase in the laminin receptor, integrin alpha-6 (CD49f), in AML cells treated with TGF-β 1, 2 and 3 and a reciprocal decrease following treatment with SB-431542, thereby, indicating that the abrogation of this signaling axis may be, at least, partially responsible for the impaired engraftment of AML cells to their niche following inhibition of the TGF-β pathway.

These data thus highlight the potential for the development of therapies directed at modifying the bone marrow microenvironment.