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PAI-1 blockade eliminates leukemia stem cells
HSPC.17,20 Therefore, we hypothesized that the TGF-β−iPAI-1 axis is similarly operational in CML-LSC. In order to investigate the activation of TGF-β−signaling in CML cells in vivo, we generated a CML-like myeloprolife- rative disease mouse model. Normal immature LSK cells obtained from fetal liver (FL) or adult BM were transduced with retrovirus carrying a human BCR/ABL-ires-GFP vec- tor and transplanted into irradiated (9 Gy) recipient mice (Figure 1A). Consistent with previous reports,12,13 we found that BCR/ABL-transduced LSK cells efficiently induced CML-like disease in recipient mice by 12-20-day post transplantation (Figure 1B). In line with our previous reports, the highest levels of TGF-β signal activation was confined to LSK cells, the majority of which are consi- dered to be CML-LSC, as demonstrated by flow cytomet- ric analysis for the phosphorylation of Smad-3 protein, a downstream signaling molecule of TGF-β which forms a complex with Smad4 in the PAI-1 promoter and enhances transcriptional activation of PAI-1 gene (Figure 1C). A gradual increase in TGF β activity in the CML-LSC frac- tion correlated with an increase in iPAI-1 expression with- in the same fraction (Figure 1C). The higher expression of iPAI-1 in CML-LSC was abolished by administration of TGF-β inhibitor (LY364947, 10 mg/kg) (Figure 1D), indi- cating that the TGF-β−iPAI-1 axis is indeed activated in CML-LSC.
iPAI-1 protects CML cells from TKI treatment
HSPC are known to be remarkably resilient (in part) because they are kept in cell cycle dormancy through acti- vation of TGF-β signaling where iPAI-1 plays a pivotal role in retaining HSPC in their protective niche.17,20 Since we found that the TGF-β−iPAI-1 axis is similarly activat- ed in CML-LSC (Figure 1), we hypothesized that func- tional activation of the TGF-β−iPAI-1 axis contributes to TKI resistance of CML-LSC by facilitating the supportive interaction within the niche. In order to test this hypoth- esis, we systemically investigated the influence of iPAI-1 on sensitivity of CML cells to TKI by using established CML-like cell lines (32D cells transduced with retrovirus carrying the human BCR/ABL-ires-GFP vector) that are genetically modified to enhance (PAI-1 overexpression [OE]) or nullify (PAI-1 knockout [KD]) the expression of iPAI-1 (Figure 2A). Twenty hours after being treated with IM in vitro, where extracellular fibrinolytic factors, such as tissue plasminogen activator, a PAI-1 target, did not exist, the apoptotic response of these cells was assessed by annexin V/PI staining. We found that a significantly high- er percentage of PAI-1 OE cells survived IM treatment than parental 32D cells while a majority of PAI-1 KD cells underwent apoptosis (Figure 2B). This in vitro experiment clearly ruled out the involvement of extracellular anti-fib- rinolytic function of PAI-1 in TKI resistance and suggest- ed direct involvement of iPAI-1 in TKI resistance.
Next, we examined the effect of iPAI-1 overexpression on the sensitivity to TKI in vivo (Figure 2C). Seven days after transplantation, the presence of BCR/ABL- GFP+CML cells were confirmed in mice that received any of the genetically modified 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 that of WT CML cells. In addition, consistent with the in
vitro findings described above, downregulation of iPAI-1 expression resulted in a significant reduction of CML cells in the BM of TKI-treated mice (Figure 2D). On the con- trary, 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 the 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 described above prompted us to investi- gate whether PAI-1 inhibitor administration can increase the susceptibility of CML cells to TKI treatment. CML- bearing mice created by transplantation of the parental BCR/ABL-ires-GFP-transduced 32D cells to non-irradiated mice were treated with either IM alone, a PAI-1 inhibitor alone or IM in combination with a PAI-1 inhibitor for 7 consecutive days and were observed for the following 40 days (Figure 3A). In this study, three different orally- active, selective PAI-1 inhibitors, namely TM5275, TM5509 and TM5614 were used. The effect of the PAI-1 inhibitors was evaluated by assessing the percentage of BCR/ABL-GFP+CML cells in the BM and the overall sur- vival of CML-bearing mice. Seven days after transplanta- tion, all recipient mice developed a CML-like disease characterized by myeloid cell expansion in the BM, spleen and PB, accompanied by splenomegaly (Figure 3B- C). Treatment with any one of the PAI-1 inhibitors alone did not markedly extend the survival of CML-bearing mice compared to the vehicle-treated CML mice (Figure 3D). Although treatment with IM by itself delayed dis- ease onset, all IM-treated CML-bearing mice experienced relapse and died before the end of the observation period (Figure 3D). Interestingly, the combined treatment of IM plus any one of the PAI-1 inhibitors significantly inhibited the persistence of CML cells in the BM, reduced spleen size, and prolonged the survival of CML-bearing mice (Figure 3B-D). Furthermore, PAI-1 blockade combined with TKI was found to be effective in overcoming the TKI-resistance in CML-mice transplanted with PAI-1 OE CML cells (Figure 3E-F).
In order to further evaluate the potential therapeutic benefit of PAI-1 inhibitor treatment, we examined the effects of the PAI-1 inhibitor using a CML-induction mouse model, a model that closely resembles CML-development in human. In this previously described tetracycline (tet)–inducible CML mouse model,26,27 BM cells of Scl/Tal1-tTAxTRE-BCR/ABL double- transgenic (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 (Figure 4A). Within 3 weeks upon the with- drawal of doxycycline and induction of BCR/ABL expres- sion, mice progressively developed CML-like disease. At the time of BCR/ABL induction, mice were treated with either IM alone, a PAI-1 inhibitor alone, or the combina- tion of IM and a PAI-1 inhibitor for 14 consecutive days. On day 15, the expression of BCR/ABL in CML cells in the BM was determined by quantitative real-time PCR. IM treatment alone reduced the level of BCR/ABL–expression in the BM LSK fraction of CML cells to approximately 1/4 of saline treated mice, whereas combined treatment of IM plus the PAI-1 inhibitor reduced the expression of
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