Plasminogen activator inhibitor-1 promotes immune evasion in tumors by facilitating the expression of programmed cell death-ligand 1.

Background: Increased levels of plasminogen activator inhibitor-1 (PAI-1) in tumors have been found to correlate with poor clinical outcomes in patients with cancer. Although abundant data support the involvement of PAI-1 in cancer progression, whether PAI-1 contributes to tumor immune surveillance remains unclear. The purposes of this study are to determine whether PAI-1 regulates the expression of immune checkpoint molecules to suppresses the immune response to cancer and demonstrate the potential of PAI-1 inhibition for cancer therapy. Methods: The effects of PAI-1 on the expression of the immune checkpoint molecule programmed cell death ligand 1 (PD-L1) were investigated in several human and murine tumor cell lines. In addition, we generated tumor-bearing mice and evaluated the effects of a PAI-1 inhibitor on tumor progression or on the tumor infiltration of cells involved in tumor immunity either alone or in combination with immune checkpoint inhibitors. Results: PAI-1 induces PD-L1 expression through the JAK/STAT signaling pathway in several types of tumor cells and surrounding cells. Blockade of PAI-1 impedes PD-L1 induction in tumor cells, significantly reducing the abundance of immunosuppressive cells at the tumor site and increasing cytotoxic T-cell infiltration, ultimately leading to tumor regression. The anti-tumor effect elicited by the PAI-1 inhibitor is abolished in immunodeficient mice, suggesting that PAI-1 blockade induces tumor regression by stimulating the immune system. Moreover, combining a PAI-1 inhibitor with an immune checkpoint inhibitor significantly increases tumor regression. Conclusions: PAI-1 protects tumors from immune surveillance by increasing PD-L1 expression; hence, therapeutic PAI-1 blockade may prove valuable in treating malignant tumors.

IMiDs uniquely synergize with TKIs to upregulate apoptosis of Philadelphia chromosome-positive acute lymphoblastic leukemia cells expressing a dominant-negative IKZF1 isoform.

The long-term prognosis of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph + ALL) is still unsatisfactory even after the emergence of tyrosine kinase inhibitors (TKIs) against chimeric BCR-ABL, and this is associated with the high incidence of genetic alterations of Ikaros family zinc finger 1 (IKZF1), most frequently the hemi-allelic loss of exons 4-7 expressing a dominant-negative isoform Ik6. We found that lenalidomide (LEN), a representative of immunomodulatory drugs (IMiDs), which have been long used for the treatment of multiple myeloma, specifically induced accumulation of Ik6 with the disappearance of functional isoforms within 24 h (i.e., abrupt and complete shut-down of the IKZF1 activity) in Ik6-positive Ph+ALL cells in a neddylation-dependent manner. The functional IKZF3 isoforms expression was also abruptly and markedly downregulated. The LEN treatment specifically suppressed proliferation of Ik6-positive-Ph+ALL cells by inducing cell cycle arrest via downregulation of cyclins D3 and E and CDK2, and of importance, markedly upregulated their apoptosis in synergy with the TKI imatinib (IM). Apoptosis of IM-resistant Ph+ALL cells with T315I mutation of BCR-ABL was also upregulated by LEN in the presence of the newly developed TKI ponatinib. Analyses of flow cytometry, western blot, and oligonucleotide array revealed that apoptosis was caspase-/p53-dependent and associated with upregulation of pro-apoptotic Bax/Bim, enhanced dephosphorylation of BCR-ABL/Akt, and downregulation of oncogenic helicase genes HILLS, CDC6, and MCMs4 and 8. Further, the synergism of LEN with IM was clearly documented as a significant prolongation of survival in the xenograft mice model. Because this synergism was further potentiated in vitro by dexamethasone, a key drug for ALL treatment, the strategy of repositioning IMiDs for the treatment of Ik6-positive Ph+ALL patients certainly shed new light on an outpatient-based treatment option for achieving their long-term durable remission and higher QOL, particularly for those who are not tolerable to intensified therapeutic approaches.

Plasminogen activator inhibitor type-1 is a negative regulator of hematopoietic regeneration in the adipocyte-rich bone marrow microenvironment.

Bone marrow adipocytes (BMAs) have recently been recognized as a niche component with a suppressive function. Obese individuals with abundant BMAs exhibit impaired hematopoietic regeneration after hematopoietic stem cell transplantation (HSCT). We hypothesized that plasminogen activator inhibitor type-1 (PAI-1), an adipokine that regulates the fibrinolytic system, contributes to impaired hematopoiesis in bone marrow (BM) microenvironment with abundant BMAs. We demonstrated that BMAs differentiated in vitro could secrete PAI-1 and were positive for PAI-1 in vivo. In addition, the abundance of BMAs was associated with high levels of PAI-1 expression. The BMA-rich microenvironment exhibited impaired hematopoietic regeneration after HSCT when compared with a BMA-less microenvironment. The impaired hematopoietic regeneration in BMA-rich microenvironment was significantly alleviated by PAI-1 knockout or PAI-1 inhibitor treatment. Obese mice with abundant BMAs, compared with normal-weight mice, exhibited higher bone marrow PAI-1 concentrations, increased fibrinolytic system suppression, and lower stem cell factor (SCF) concentrations after HSCT. PAI-1 inhibitor administration significantly activated the fibrinolytic system in obese mice, contributing to the higher SCF concentration. Moreover, PAI-1 inhibitor treatment significantly alleviated the impaired hematopoietic regeneration in obese mice both after 5-fluorouracil injection and HSCT. These results indicate that PAI-1 hinders hematopoietic regeneration in BMA-rich microenvironments. The blockade of PAI-1 activity could be a novel therapeutic means of facilitating hematopoietic reconstitution in BMA-rich patients.

Targeting of plasminogen activator inhibitor-1 activity promotes elimination of chronic myeloid leukemia stem cells.

Therapeutic strategies that target leukemic stem cells (LSCs) provide potential advantages in the treatment of chronic myeloid leukemia (CML). Here, we show that selective blockade of plasminogen activator inhibitor-1 (PAI-1) enhances the susceptibility of CML-LSCs to tyrosine kinase inhibitor (TKI), which facilitates the eradication of CML-LSCs and leads to sustained remission of the disease. We demonstrated for the first time that TGF-ß-PAI-1 axis was selectively augmented in CML-LSCs in the bone marrow (BM), whereby protecting CML-LSCs from TKI treatment. Furthermore, the combined administration of TKI plus a PAI-1 inhibitor, in a mouse model of CML, significantly enhanced the eradication of CML cells in the BM and prolonged the survival of CML mice. The combined therapy of imatinib and a PAI-1 inhibitor prevented the recurrence of CML-like disease in serially transplanted recipients, indicating the elimination of CML-LSCs. Interestingly, PAI-1 inhibitor treatment augmented membrane-type matrix metalloprotease-1 (MT1-MMP)-dependent motility of CML-LSCs, 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.

Inhibition of plasminogen activator inhibitor-1 attenuates against intestinal fibrosis in mice.

BACKGROUND/AIMS: Intestinal fibrosis is a major complication of Crohn's disease (CD). The profibrotic protein transforming growth factor-ß (TGF-ß) has been considered to be critical for the induction of the fibrotic program. TGF-ß has the ability to induce not only the expression of extracellular matrix (ECM) including collagen, but also the production of plasminogen activator inhibitor-1 (PAI-1) that prevents enzymatic degradation of the ECM during the onset of fibrotic diseases. However, the significance of PAI-1 in the developing intestinal fibrosis has not been fully understood. In the present study, we examined the actual expression of PAI-1 in fibrotic legion of intestinal inflammation and its correlation with the abnormal ECM deposition. METHODS: Chronic intestinal inflammation was induced in BALB/c mice using 8 repeated intrarectal injections of 2,4,6-trinitrobenzene sulfonic acid (TNBS). TM5275, a PAI-1 inhibitor, was orally administered as a carboxymethyl cellulose suspension each day for 2 weeks after the sixth TNBS injection. RESULTS: Using a publicly available dataset (accession number, GSE75214) and TNBS-treated mice, we observed increases in PAI-1 transcripts at active fibrotic lesions in both patients with CD and mice with chronic intestinal inflammation. Oral administration of TM5275 immediately after the onset of intestinal fibrosis upregulated MMP-9 (matrix metalloproteinase 9) and decreased collagen accumulation, resulting in attenuation of the fibrogenesis in TNBS-treated mice. CONCLUSIONS: PAI-1-mediated fibrinolytic system facilitates collagen degradation suppression. Hence, PAI-1 inhibitor could be applied as an anti-fibrotic drug in CD treatment.

Blockade of plasminogen activator inhibitor-1 empties bone marrow niche sufficient for donor hematopoietic stem cell engraftment without myeloablative conditioning.

Upon hematopoietic stem cell transplantation (HSCT), the availability of recipients' niches in the bone marrow (BM) is one of the factors that influence donor HSC engraftment and hematopoietic reconstitution. Therefore, myeloablative conditioning, such as irradiation and/or chemotherapy, which creates empty niches in the recipients' BM, is required for the success of HSCT. However, the conventional myeloablation causes extensive damages to the patients' BM, which results in the treatment-induced severe complications and even mortality. Thus, alternative and mild conditioning could fulfill the need for safer HSCT-based therapies for hematological and nonhematological disorders. Recently, we have demonstrated that pharmacological inhibition of plasminogen activator inhibitor-1 (PAI-1) activity increases cellular motility and cause detachment of HSCs from the niches. In this study, we performed HSCT using a PAI-1 inhibitor without any myeloablative conditioning. Donor HSCs were transplanted to recipient mice that were pretreated with saline or a PAI-1 inhibitor. Saline pretreated nonmyeloablative recipients showed no engraftment. In contrast, donor cell engraftment was detected in the PAI-1 inhibitor pretreated recipients. Multilineage differentiation, including lymphoid and myeloid cells, was observed in the PAI-1 inhibitor pretreated recipients. Donor-derived cells that exhibited multilineage reconstitution as well as the existence of stem/progenitor cells were detected in the secondary recipients, confirming the maintenance of donor HSCs in the BM of PAI-1 inhibitor pretreated primary recipients. The results indicate that the PAI-1 blockade vacates functional niches in the recipients' BM, which allows the engraftment of long-term multilineage HSCs without myeloablative conditioning.

TGF-ß-induced intracellular PAI-1 is responsible for retaining hematopoietic stem cells in the niche.

Hematopoietic stem and progenitor cells (HSPCs) reside in the supportive stromal niche in bone marrow (BM); when needed, however, they are rapidly mobilized into the circulation, suggesting that HSPCs are intrinsically highly motile but usually stay in the niche. We questioned what determines the motility of HSPCs. Here, we show that transforming growth factor (TGF)-ß-induced intracellular plasminogen activator inhibitor (PAI)-1 activation is responsible for keeping HSPCs in the BM niche. We found that the expression of PAI-1, a downstream target of TGF-ß signaling, was selectively augmented in niche-residing HSPCs. Functional inhibition of the TGF-ß-PAI-1 signal increased MT1-MMP-dependent cellular motility, causing a detachment of HSPCs from the TGF-ß-expressing niche cells, such as megakaryocytes. Furthermore, consistently high motility in PAI-1-deficient HSPCs was demonstrated by both a transwell migration assay and reciprocal transplantation experiments, indicating that intracellular, not extracellular, PAI-1 suppresses the motility of HSPCs, thereby causing them to stay in the niche. Mechanistically, intracellular PAI-1 inhibited the proteolytic activity of proprotein convertase Furin, diminishing MT1-MMP activity. This reduced expression of MT1-MMP in turn affected the expression levels of several adhesion/deadhesion molecules for determination of HSPC localization, such as CD44, VLA-4, and CXCR4, which then promoted the retention of HSPCs in the niche. Our findings open up a new field for the study of intracellular proteolysis as a regulatory mechanism of stem cell fate, which has the potential to improve clinical HSPC mobilization and transplantation protocols.

Inhibition of plasminogen activator inhibitor type-1 activity enhances rapid and sustainable hematopoietic regeneration.

The prognosis of patients undergoing hematopoietic stem cell transplantation (HSCT) depends on the rapid recovery and sustained life-long hematopoiesis. The activation of the fibrinolytic pathway promotes hematopoietic regeneration; however, the role of plasminogen activator inhibitor-1 (PAI-1), a negative regulator of the fibrinolytic pathway, has not yet been elucidated. We herein demonstrate that bone marrow (BM) stromal cells, especially osteoblasts, produce PAI-1 in response to myeloablation, which negatively regulates the hematopoietic regeneration in the BM microenvironment. Total body irradiation in mice dramatically increased the local expression levels of fibrinolytic factors, including tissue-type plasminogen activator (tPA), plasmin, and PAI-1. Genetic disruption of the PAI-1 gene, or pharmacological inhibition of PAI-1 activity, significantly improved the myeloablation-related mortality and promoted rapid hematopoietic recovery after HSCT through the induction of hematopoiesis-promoting factors. The ability of a PAI-1 inhibitor to enhance hematopoietic regeneration was abolished when tPA-deficient mice were used as recipients, thus indicating that PAI-1 represses tPA-dependent hematopoietic regeneration. The PAI-1 inhibitor not only accelerated the expansion of the donor HSCs during the early-stage of regeneration, but also supported long-term hematopoiesis. Our results indicate that the inhibition of PAI-1 activity could be a therapeutic approach to facilitate the rapid recovery and sustained hematopoiesis after HSCT.

Accumulation of oxidative DNA damage restricts the self-renewal capacity of human hematopoietic stem cells.

Stem cells of highly regenerative organs including blood are susceptible to endogenous DNA damage caused by both intrinsic and extrinsic stress. Response mechanisms to such stress equipped in hematopoietic stem cells (HSCs) are crucial in sustaining hematopoietic homeostasis but remain largely unknown. In this study, we demonstrate that serial transplantation of human HSCs into immunodeficient mice triggers replication stress that induces incremental elevation of intracellular reactive oxygen species (ROS) levels and the accumulation of persistent DNA damage within the human HSCs. This accumulation of DNA damage is also detected in HSCs of clinical HSC transplant patients and elderly individuals. A forced increase of intracellular levels of ROS by treatment with a glutathione synthetase inhibitor aggravates the extent of DNA damage, resulting in the functional impairment of HSCs in vivo. The oxidative DNA damage activates the expression of cell-cycle inhibitors in a HSC specific manner, leading to the premature senescence among HSCs, and ultimately to the loss of stem cell function. Importantly, treatment with an antioxidant can antagonize the oxidative DNA damage and eventual HSC dysfunction. The study reveals that ROS play a causative role for DNA damage and the regulation of ROS have a major influence on human HSC aging.