The Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) Regulates the Function and Migration of Leukemic Blasts through CD63/PI3K/AKT/P21 Axis

Introduction. The Tissue Inhibitor of MetalloProteinases-1 (TIMP-1) is a member of the inflammatory network that dramatically increases in response to inflammation. First described as an inhibitor of MMPs, TIMP-1 exerts pleiotropic effects in the hematopoietic microenviroment. Along with the finding that hematopoietic stem progenitor cells (HSPCs) directly respond to inflammation, a persistent inflammatory stimulation may hinder HSPC functions and eventually lead to hematological malignancies. Specifically, the inflammatory pathways activated by the bone marrow (BM) microenvironment influences the transformation and progression of leukemia. In hematological malignancies, TIMP-1 promotes proliferation in lymphoma cells and increased TIMP-1 serum levels are associated with poor prognosis in a variety of cancers. However, recent studies have revealed the dichotomy of TIMP-1 in cancer progression.

Here, in the attempt to provide further evidence for the critical role of inflammation in leukemic microenvironment, we investigated the role of TIMP-1 in leukemic blasts from patients with Acute Myeloid Leukemia (AML) and elucidated the downstream pathway ignited by rhTIMP-1.

Methods. Human CD34⁺ HSPCs were isolated from cord blood (CB) units as control samples, while leukemic cells were collected from AML patients at diagnosis (percentage of blasts >90%; n=36). Cell proliferation was assessed by cell cycle analysis, and CFSE staining. In addition, we performed colony-forming unit assays. TIMP-1's contribution to cell survival was evaluated by AnnexinV/PI staining. The expression of the tetraspannin receptor CD63 (TIMP-1's putative receptor) was assessed by flow cytometry. Leukemic blasts were assayed towards a CXCL12 gradient after exposure to rhTIMP-1 or pre-treated with LY294002 (PI3K inhibitor); migrated cells were counted and characterized for CD63 expression. Leukemic blasts were sorted in two separate fractions, CD63^- and CD63⁺, and their capability to respond to rhTIMP-1 was assessed. Downstream molecular targets of TIMP-1 (such as PI3K, pAkt, p21) were also confirmed by flow cytometry. RT-PCR was employed to detect the expression of hypoxia-inducible factor-1 (HIF-1α). Finally, co-culture system was performed with CFSE-positive leukemic blasts and mesenchymal stromal cells (MSCs) from normal or AML patients in the presence of rhTIMP-1.

Results. We firstly found that TIMP-1 levels are increased in the plasma of BM of AML patients at diagnosis. Similarly to CD34⁺ HSPCs from CB, the clonogenic potential of primary human AML blasts is increased upon exposure to rhTIMP-1. Moreover, rhTIMP-1 promotes in vitro AML blast survival, possibly due to anti-apoptotic effects and a slight but significant increase in the proportion of leukemic cells entering S-phase of the cell cycle. Such effect is related to the downregulation of cyclin-dependent kinase inhibitor p21. As for the dissection of TIMP-1 signalling pathway, our data revealed that the tetraspannin CD63 receptor is required for TIMP-1's cytokine functions. Indeed, about half of leukemic blasts expresses CD63, whose activation leads to PI3K recruitment and Akt phopshorylation, key modulators of survival/proliferation pathways. Exposure of AML blasts to rhTIMP-1 resulted in the induction of HIF-1α, which is known to play an important role in survival of leukemic stem cells. Moreover, rhTIMP-1 increased the SDF1-driven migration of leukemic blasts through the activation of PI3K. Interestingly, migrating AML blasts are highly enriched in CD63⁺ cells, whereas the CD63-negative pre-sorted fraction of leukemic cells showed reduced response to migration and pAkt expression after exposure to rhTIMP-1.Finally, in the co-culture system between AML cells and normal versus leukemic MSCs, we defined the particular role of TIMP-1 in normal and leukemic microenviroment. In particular, our results suggest that a defective capacity of AML-MSCs in sustaining the proliferation and migration of AML blasts may be restored by rhTIMP-1.

Conclusions. In our study TIMP-1 emerges as an important factor in the leukemic BM microenvironment, modulating leukemic blasts survival, migration and function. Specifically, our work suggests that TIMP-1 promotes survival and migration via CD63/PI3K/AKT/p21 signalling. Taken together, our findings indicates TIMP-1 pathway as a potential novel therapeutic target in AML.