INFLAMMATORY PATHWAYS IN THE BONE MARROW: ROLE OF TIMP-1 IN HEMATOPOIETIC STEM/PROGENITOR CELLS AND LEUKEMIA

Introduction. Inflammation is a protective response that contains tissue injury and preserves homeostasis. However, a prolonged inflammation hinders tissue functionality and may play a role in cancer. Hematopoietic Stem and Progenitors Cells (HSPCs) reside within the Bone Marrow, a nurturing environment shielding HSPCs from external insults. However, recent findings showed that danger signals and inflammatory cytokines actively affect HSPCs. The Tissue Inhibitor of MetalloProteinases-1 (TIMP-1) is a member of the inflammatory network: first described as an inhibitor of MMPs, TIMP-1 also displays cytokine-like functions. We recently found that TIMP-1-/- mice have decreased BM cellularity and impaired engraftment capabilities due to cell-cycle defects. Aim of this study is to investigate TIMP-1’s role in normal and leukemic hematopoiesis. Methods. Human CD34+ HSPCs were isolated from cord blood (CB) units, while leukemic cells were collected from AML patients at diagnosis. Cell proliferation was assessed by colony forming unit (CFU) assays, Long-Term Cultures (LTCs), cell cycle analyses, and CFSE staining. TIMP-1’s contribution to cell survival was evaluated by AnnexinV/PI staining. Primitive HSC potential was assessed by transplantation of TIMP-1-treated CD34+ cells into immonodeficient mice (NOD/Shi-scid/IL-2Rγnull mice). Finally, the expression of the tetraspannin receptor CD63 (TIMP-1’s putative receptor) was assessed by flow cytometry, whereas its functional role was tested by nucleofection of CD63-specific siRNAs in HSPCs. Downstream molecular targets of TIMP-1 (such as pAkt, CycD1, p27) were also evaluated by qPCR and flow cytometry. Results. rhTIMP-1 promotes CD34+ cell survival and stimulates HSPC expansion, without compromising the engraftment potential when transplanted into immunodeficient mice. The dissection of TIMP-1 signaling pathway indicated that the tetraspanin CD63 receptor is required for TIMP-1’s cytokine functions in HSPCs. CD63 activation leads to PI3K recruitment and phopshorylation of AKT, key modulators of survival/proliferation pathways in HSCs. Downstream targets of pAKT are also modulated, including the proliferation marker CycD1, which levels are increased upon exposure to TIMP-1. In AML patients, preliminary findings indicate that, at diagnosis, TIMP-1 levels are increased in both the peripheral blood and the BM. Of note, TIMP-1 promotes AML cell maintenance in vitro, possibly due to anti-apoptotic effects. Similarly to CD34+ HSPCs, the colony-forming potential of primary human AML cells is also increased upon exposure to TIMP-1, suggesting a role of TIMP-1 in AML proliferation. Conclusions. Our study suggests that TIMP-1 may play a key role in the BM microenvironment, modulating both normal HSPCs and AML cell proliferation. Our findings have the potential to provide a novel therapeutic target for therapies targeting leukemogenic mechanisms sprouting from inflammatory microenvironments.