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T. Yahata et al.
all recipient mice died within 30 days. In contrast, more than 70% of mice that received cells from the donor mice that received the combined treatment survived until the end of the observation period (Figure 6D). In line with our in vitro findings, the effect of PAI-1 inhibitor on CML-LCS was completely abolished by the administration of a neu- tralizing antibody specific for MT1-MMP, indicating a critical role of this molecule in TKI sensitivity of CML- LSC (Figure 6B-D). These data confirmed that iPAI-1 blockade in combination with TKI effectively eliminates CML-LSC.
Discussion
An effective strategy to eliminate cancer stem cells is critical in securing a complete cure of patients with can- cers such as CML. The evidence that the TGF-β signaling pathway plays an essential role in the maintenance of immature CML-LSC12,13 led us to investigate the involve- ment of iPAI-1 in the function of CML-LSC and their sus- ceptibility to TKI. Our data unambiguously demonstrate that the forced expression of iPAI-1 in a CML cell line endows tumor cells with resistance to TKI treatment both in vitro and in vivo, which likely explains why iPAI-1- expressing immature CML-LSC are refractory to TKI treatment in the CML-bearing mice. This intimate associ- ation of TGF-β−iPAI-1 with CML has led to suggest that TGF-β−iPAI-1 axis itself contributes directly to malignant behavior, and that the inhibition of the TGF-β−iPAI-1 axis is beneficial in treating CML. A study has shown that iPAI-1 is able to directly bind to caspase 3,35 thus influen- cing activation of the apoptotic pathways. Indeed, our in vitro analysis demonstrated that the level of iPAI-1 expression is inversely correlated with the cellular suscep- tibility to TKI treatment, indicating that iPAI-1 contributes to TKI resistance by negatively regulating apoptosis in leukemic cells. Consistent with this, it has recently been reported that PAI-1 inhibitors induce apoptosis in vitro in a variety of tumor cell lines through the activation of the intrinsic apoptotic pathway,36,37 suggesting that PAI-1 inhibitors possess intrinsic anti-cancer activity. The com- bined administration of imatinib plus a PAI-1 inhibitor markedly improved the therapeutic outcome of TKI as evidenced by a reduction in the number of CML cells in the BM, reduced spleen size, and prolonged survival in a mouse model of CML. Furthermore, pharmacological blockade of iPAI-1 in combination with TKI effectively eliminated CML-LSC in the BM, which in turn prevented the recurrence of CML-like disease in recipients of serial transplantation experiments.
HSPC are localized in the niche where local factors that keep them in cell-cycle dormancy, such as TGF-β, are abundantly present. LSC co-opt the HSPC BM niche to gain survival benefits by hijacking the molecular physio- logical mechanisms utilized by HSPC.15,28,29 Therefore, dis- ruption of LSC localization has been proposed to be an effective means to eradicate LSC. Recently, we have shown that functional inhibition of iPAI-1 increased MT1- MMP-dependent cellular motility and caused a detach- ment of HSPC from the niche. In this report, we demon- strated that the therapeutic effect of a PAI-1 inhibitor appears to depend on the activity of MT1-MMP. Although the involvement of MT1-MMP has been implicated in physiology and pathophysiology of many types of cells,
its exact role(s) has not been determined. One of the main functions of MT1-MMP is to break down pericellular matrix proteins. It also activates pro-MMP-2, which then activates other proteases such as MMP-9 and MMP-13. The interplay of these multiple protease controls motility of both normal and malignant cells. In addition, MT1- MMP can promote cellular motility by breaking down membrane-bound adhesion molecules necessary for niche-anchoring, such as CD44, which then triggers the induction of molecules involved in hematopoietic cell traf- ficking, such as VLA4.17,18 CD44 and VLA-4 have been shown to play key roles in homing and engraftment of LSC.14,30,32–34 Consistent with this, the present study indi- cates that CML cells overexpressing iPAI-1 (where the expression of MT1-MMP is consequently suppressed17), manifest poor migration activity in the BM environment. Furthermore, analogous to our HSPC study, treatment of CML mice with a PAI-1 inhibitor increased MT1-MMP activity and altered the surface expression of CD44 and VLA-4 on CML-LSC, which resulted in enhanced motility of CML-LSC. This altered expression of adhesion/de- adhesion molecules appear to disrupt the interaction between CML-LSC and their protective environment, which renders CML-LSC susceptible to TKI therapy. In fact, it has been reported that blockade of CD44 and VLA- 4 markedly reduced LSC engraftment and prolonged the survival of LSC-transplanted mice.14,30,33,34,38 Thus, MT1- MMP activity appears to determine the therapeutic sensi- tivity of LSC. Taken together, these findings suggest that, besides its well-known role in cell cycle regulation, TGF-β signaling, in which iPAI-1-MT1-MMP axis plays an inte- gral part of its coordinated circuitry, controls the motility of both normal and malignant stem cells, thereby protec- ting them from environmental stimuli. In line with our findings, several studies have proposed mobilization of CML-LSC from the niche as a measure to counteract TKI resistance, using granulocyte-colony stimulating factor (G-GSF)39,40 or inhibitors for C-X-C motif chemokine receptor (CXCR) 4,41,42 E-selectin,43 or TGF-β,13 further exploring the therapeutic potential of this approach. The present study also demonstrates that selective upregula- tion of the iPAI-1-MT1-MMP axis in CML-LSC provides a newly recognized mechanism of drug-resistance and that modulation of this signaling pathway overcomes TKI-resistance in CML mouse. The evidence we provide in this study has led us to suggest that iPAI-1 signaling contributes directly to pathology of cancers beyond the scope of this study, and that inhibition of the TGF-β−iPAI- 1 axis can become an effective approach to treat a wide variety of cancers.
We have developed a low molecular weight synthetic inhibitor of PAI-1, TM5275 and a series of analogues with improved pharmacological and toxicological properties, including TM5509 and TM5614.44,45 A number of preclini- cal studies have demonstrated that this series of com- pounds is capable of affecting a number of metabolic, fibrotic, aging-related disorders, as well as hematopoietic regeneration.46–50 In this study, we provide the first evi- dence that inhibition of iPAI-1 can be of therapeutic benefit in the treatment of leukemia. iPAI-1 has almost identical roles in retaining normal HSC and LSC in the niche. In that sense, when CML patients are treated with a PAI-1 inhibitor, both HSC and LSC are supposed to be released from the niche. In a physiological setting, HSC are believed to be in a cycle of being released from and
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haematologica | 2021; 106(2)