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T. Yahata et al.
LSC are thought to possess properties similar to those characterizing normal HSPC, including the capacity for self-renewal, cell cycle quiescence, and resistance to tradi- tional chemotherapy.6,9 Studies have shown that trans- forming growth factor-β (TGF-β) signaling plays suppor- tive roles in normal hematopoiesis and leukemogenesis. Yamazaki et al.10,11 reported that TGF-β signaling is essen- tial for the maintenance of HSPC within the bone marrow (BM) microenvironmental niche and promotes Smad- dependent HSPC hibernation. In the same vein, Naka et al.12,13 showed that TGF-β signaling is also essential for the maintenance of CML-LSC. Thus, suppression of TGF-β signaling deserves investigation for the purpose of eliminating CML-LSC.
Like their normal hematopoietic counterparts, CML- LSC are presumed to reside in specific niches in the BM microenvironment, which likely contribute to relapse after chemotherapy.14–16 We have recently uncovered a pathway by which TGF-β signaling regulates HSPC retention in the niche.17 TGF-β induces the expression of plasminogen activator inhibitor-1 (PAI-1), a major physio- logic serine protease inhibitor (serpin) of the fibrinolytic system. In HSPC, PAI-1, known conventionally as an extracellular fibrinolysis-related serpin, functions intracel- lularly (subsequently referred to as iPAI-1) and inhibits the proteolytic activity of proprotein convertase, furin, which consequently diminishes membrane type-1 metallopro- tease (MT1-MMP) activity.17 MT1-MMP promotes HSPC motility by breaking down pericellular matrix proteins and adhesion molecules necessary for anchoring HSPC to the niche.18,19 Genetic or pharmacological inhibition of TGF-β-iPAI-1 signaling increases MT1-MMP-dependent cellular motility, causing detachment of HSPC from the niche, making them receptive to extraneous stimuli.17,20 In line with our report, a number of studies have demon- strated that both TGF-β and PAI-1 play important roles in numerous physiological and pathological conditions, including wound healing, obesity, cardiovascular disease and cancer.21–23 Therefore, we hypothesize that blockade of iPAI-1 activity facilitates the dislocation of CML-LSC in the BM, which in turn renders CML-LSC susceptible to TKI therapy, resulting in the eradication of CML-LSC and sustained disease remission.
Methods
Additional methods are presented in the Online Supplementary Methods, available on the Haematologica web site.
Pharmacological properties of inhibitors
Three different PAI-1 inhibitory compounds (TM5275, TM5509 and TM5614) were used.24 All three selectively and effectively inhibited PAI-1 activity with a half-maximal inhibi- tion (IC50) value <6.95 mM in a tPA-dependent hydrolysis assay. The PAI-1 inhibitors (up to 100 mM) did not interfere with other serpin/serine protease systems such as 1-antitrypsin/trypsin and α2-antiplasmin/plasmin. The TGF-β inhibitor, LY364947, is a potent and selective inhibitor of the TGF- β receptor I, inhibiting phosphorylation of Smad3 by TGF-β receptor I kinase (IC50=59 nM).
CML mouse model
Several different mouse models of CML-like disease were uti- lized in this study. First, we used a BCR/ABL1 transduction/trans-
plantation-based CML model (BCR/ABL1-CML mice) as previ- ously described.12,25 Briefly, normal LSK cells (4–5×104 cells per recipient mouse) isolated from mononuclear cells of fetal liver (embryonic day E14.5) or adult BM (8-12 weeks old) were trans- duced with the human BCR/ABL1-ires green fluorescent protein (GFP) retrovirus and transplanted into irradiated (9 Gy) recipient C57BL/6J mice purchased from CLEA Japan (Tokyo). Another set of experiments, gene-modified 32D cell lines were also transplan- ted into non-irradiated C3H/HeNCrl mice (CLEA Japan). CML- like disease developed in these recipient mice by 12–20 days post transplantation.
A tetracycline (tet)-inducible CML mouse model26,27 was also used in this study. Tal1-tTA mice (JAX, #006209) and TRE- BCR/ABL1 transgenic mice (JAX, #006202), both on an FVB/N genetic background, were purchased from the Jackson Laboratory. Tal1-tTA and TRE-BCR/ABL1 transgenic mice were interbred to generate Tal1-tTAxTRE-BCR/ABL1 double-transgenic mice. These animals were maintained in cages supplied with drinking water containing 20 mg/L doxycycline (Sigma-Aldrich). At 8-10 weeks after birth, BM cells of Scl/Tal1-tTAxTRE-BCR/ABL double-transgenic (Ly5.2; CD45.2) mice were mixed with com- petitor 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 with- drawal 2 months after transplantation. All induced recipient mice progressively developed CML-like disease associated with a severe myeloid cell expansion in the BM, spleen and peripheral blood (PB), and splenomegaly. The mice became moribund ~3 weeks after induction.
In order to examine the in vivo effects of the combined admin- istration of imatinib (IM) plus PAI-1 inhibitors, BCR/ABL1-CML- affected mice received vehicle alone, or IM (Gleevec; 150-400 mg/kg/day; Novartis) and/or PAI-1 inhibitor (TM5257, 100 mg/kg/day; TM5009 or TM5614, 10 mg/kg/day) in vehicle, and, at the same time, mice were intraperitoneally injected with 1 mg/kg anti-MT1-MMP antibody (Merck Millipore) or non- immune species- and isotype-matched control antibody for 5 consecutive days. Treatment was delivered by oral gavage on days 8–30 post transplantation. Three to 4 months after trans- plantation, PB, spleen, and BM samples were collected from indi- vidual recipients and subjected to analyses and secondary trans- fer experiments.
Protocols concerning animal experiments and recombinant gene experiments were approved by the Animal Care Committee and the Gene Recombination Experiment Safety Committee of Tokai University.
Results
TGF-β-iPAI-1 axis is activated in CML-LSC
LSC are analogous to HSPC in so many aspects; both
cell types are characteristically slow cell-cycling and have a high tendency to localize in particular sites, so called niche dependency.14,15,28–30 In addition, the surface markers of CML-LSC and normal HSPC are shown to be almost identical both in murine models and in human samples. For example, transplantation studies of BCR/ABL-induced murine CML models demonstrated that BCR/ABL- expressing LSC activity is confined to the fraction of Lineage (Lin)–Sca1+c-Kit+ (LSK) cells that contains murine HSPC population.12,31 We have recently shown that TGF-β signaling regulates the motility of HSPC in the niche through selective upregulation of iPAI-1 expression in
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haematologica | 2021; 106(2)