Interestingly, PAI-1 inhibitor treatment augmented membrane-type matrix metalloprotease-1 (MT1-MMP)-dependent motility of CML-LSC, and the anti-CML effect of PAI-1 inhibitor was extinguished by the neutralizing antibody for MT1-MMP, underlining the mechanistic importance of MT1-MMP. inhibitor treatment augmented membrane-type matrix metalloprotease-1 (MT1-MMP)-dependent motility of CML-LSC, and the anti-CML effect of PAI-1 inhibitor was extinguished by the neutralizing antibody for MT1-MMP, underlining the mechanistic importance of MT1-MMP. Our findings provide evidence of, and a rationale for, a novel therapeutic tactic, based on the blockade of PAI- 1 activity, for CML patients. Introduction Chronic myeloid leukemia (CML) was the first luekemia to be identified with a specific tumorigenic chromosomal abnormality – the Philadelphia chromosome.1 Subsequent studies identified that this Ravuconazole translocation event, occurred between t(9;22) (q34;q11), fused the breakpoint cluster region gene (BCR) with the Abelson kinase gene (oncogene.2,3 This BCR/ABL fusion protein possesses constitutive tyrosine kinase activity resulting in the development of myeloid leukemia through aberrant differentiation of hematopoietic stem/progenitor cells (HSPC) toward the myeloid lineage. Although development of tyrosine kinase inhibitors (TKI) that target the abnormal activation of tyrosine kinase, such as imatinib, Ravuconazole has dramatically improved the prognosis of CML,4,5 the disease often relapses, even after total remission achieved under TKI therapy, thus remaining a central problem in the treatment of CML. It has been hypothesized that leukemic stem cells (LSC), also identified as leukemic initiating cells, are the cells that possess the unique ability to resist the cytotoxic brokers and are responsible for the relapse of leukemia,6C8 highlighting the need for therapeutic strategies that specifically target this populace of cells. LSC are thought to possess properties much like those characterizing normal HSPC, including the capacity for self-renewal, cell cycle quiescence, and resistance to traditional chemotherapy.6,9 Studies have shown that transforming growth factor-(TGF-) signaling plays supportive roles in normal hematopoiesis and leukemogenesis. Yamazaki mice (JAX, #006209) and transgenic mice (JAX, #006202), both on an FVB/N genetic background, were purchased from your Jackson Laboratory. and Ravuconazole transgenic mice were interbred to generate double-transgenic mice. These animals were managed in cages supplied with drinking water made up of 20 mg/L doxycycline (Sigma-Aldrich). At 8-10 weeks after birth, BM cells of double-transgenic (Ly5.2; CD45.2) mice were mixed with competitor BM cells obtained from congenic (Ly5.1; CD45.1) mice and then were transplanted to lethally irradiated (9 Gy) wild-type (WT) hosts. The recipients bred in the presence of doxycycline, and BCR/ABL expression was induced by doxycycline withdrawal 2 months after transplantation. All induced recipient mice progressively developed CML-like disease associated with a severe myeloid cell growth in the BM, spleen and peripheral blood (PB), and splenomegaly. The mice became moribund ~3 weeks after induction. In order to examine the effects of the combined administration of imatinib (IM) plus PAI-1 inhibitors, experiment clearly ruled out the involvement of extracellular anti-fibrinolytic function of PAI-1 in TKI resistance and suggested direct involvement of iPAI-1 in TKI resistance. Next, we examined the effect of iPAI-1 overexpression around the sensitivity to TKI (Physique 2C). Seven days after transplantation, the presence of BCR/ABLGFP+ CML cells were confirmed in mice that received any of the genetically altered CML cells. IM was orally administered to these recipient mice for 7 consecutive days, and the percentage of GFP+CML cells in the BM was analyzed on the day after final IM administration. At day 7 of post transplantation, initial engraftment of PAI-1 KD CML cells in the BM was found to be slightly lower than Rabbit Polyclonal to MAP2K1 (phospho-Thr386) that of WT CML cells. In addition, consistent with the findings explained above, downregulation of iPAI-1 expression resulted in a significant reduction of CML cells in the BM of TKI-treated mice (Physique 2D). On the contrary, although PAI-1 OE CML cells were significantly less successful in initial engraftment compared to WT CML cells and PAI-1 KD CML cells, PAI-1 OE CML cells were resistant to TKI treatment and overgrew in the BM. These results confirmed that this intensity of iPAI-1 expression governs the susceptibility of CML cells to TKI treatment. Pharmacological inhibition of iPAI-1 activity increases the susceptibility of CML cells to TKI treatment The findings explained above prompted us to investigate whether PAI-1 inhibitor administration can increase the susceptibility of CML cells to TKI treatment. CMLbearing mice produced by transplantation of the parental doubletransgenic (Ly5.2; CD45.2) mice were transplanted to lethally irradiated (9 Gy) WT hosts, and the recipients were maintained in the presence of doxycycline at least for 2 months (Physique 4A). Within 3 weeks upon the withdrawal of doxycycline and.