Baruch Bulvik

Cardiac Protection by Preconditioning Is Generated via an Iron-Signal Created by Proteasomal Degradation of Iron Proteins

Ischemia associated injury of the myocardium is caused by oxidative damage during reperfusion. Myocardial protection by ischemic preconditioning (IPC) was shown to be mediated by a transient ‘iron-signal’ that leads to the accumulation of apoferritin and sequestration of reactive iron released during the ischemia. Here we identified the source of this ‘iron signal’ and evaluated its role in the mechanisms of cardiac protection by hypoxic preconditioning. Rat hearts were retrogradely perfused and the effect of proteasomal and lysosomal protease inhibitors on ferritin levels were measured. The iron-signal was abolished, ferritin levels were not increased and cardiac protection was diminished by inhibition of the proteasome prior to IPC. Similarly, double amounts of ferritin and better recovery after ex vivo ischemia-and-reperfusion (I/R) were found in hearts from in vivo hypoxia pre-conditioned animals. IPC followed by normoxic perfusion for 30 min (‘delay’) prior to I/R caused a reduced ferritin accumulation at the end of the ischemia phase and reduced protection. Full restoration of the IPC-mediated cardiac protection was achieved by employing lysosomal inhibitors during the ‘delay’. In conclusion, proteasomal protein degradation of iron-proteins causes the generation of the ‘iron-signal’ by IPC, ensuing de-novo apoferritin synthesis and thus, sequestering reactive iron. Lysosomal proteases are involved in subsequent ferritin breakdown as revealed by the use of specific pathway inhibitors during the ‘delay’. We suggest that proteasomal iron-protein degradation is a stress response causing an expeditious cytosolic iron release thus, altering iron homeostasis to protect the myocardium during I/R, while lysosomal ferritin degradation is part of housekeeping iron homeostasis.

Iron, ferritin and proteins of the methionine-centered redox cycle in young and old rat hearts.

Progressive oxidation of cellular components constitutes a major mechanism of the aging process. An emerging paradigm of redox signaling suggests that low level oxidants activate protective pathways resulting in prolonged cell survival. This report centers on the study of cardiac muscle in young and old rats, including (i) the expression of ferritin (Ft) the major iron storage protein, and (ii) the expression of the major proteins of the methionine-centered redox cycle (MCRC), which controls the cellular methionine redox status. Total amounts of Ft (protein) and its mRNA encoding for Ft L-subunit (Ft-L) were higher in the aged hearts, indicating that the iron-binding capacity of myocardial Ft increased with age. Among the proteins of the MCRC, methionine sulfoxide reductases A and B (MsrA, MsrB) and MsrA mRNA were significantly higher in hearts of old rats with a significant decrease in MsrA activity. The observed up-regulation of the expression of Msr and Ft-L could represent a protective response to the increased oxidative stress in the aging myocardium.

Aging is an organ-specific process: changes in homeostasis of iron and redox proteins in the rat

Organ-specific changes of iron- and redox-related proteins occur with age in the rat. Ferritin, the major iron storage and detoxifying protein, as well as the proteins of the methionine-centered redox cycle (MCRC) were examined in old and young animals, and showed organ-dependent changes. In spleens and livers of aged rats, ferritin (protein) levels were greater than in young ones, and their iron saturation increased, rendering higher ferritin-bound iron (FtBI). Iron saturation of the ferritin molecule in the tongues and sternohyoids of old rats was lower but ferritin level was higher than in young rats, resulting in increased FtBI with age. Ferritin level in the esophagus of older rats was lower than in young rats but its molecular iron content higher thus the total FtBI remained the same. In the larynx, both ferritin and its iron content were the same in young and old animals. MCRC proteins were measured in livers and spleens only. With aging, methionine sulfoxide reductase A and B (MsrA and MsrB) levels in livers and spleens decreased. Thioredoxin1 (Trx) and Trx-reductase1 were elevated in old spleens, but reduced in livers. Aged spleens showed reduced Msr isozyme activity; but in the liver, its activity increased. mRNA changes with age were monitored and found to be organ specific. These organ-specific changes could reflect the different challenges and the selective pathways of each organ and its resultant capacity to cope with aging.

The bitter fate of the sweet heart: impairment of iron homeostasis in diabetic heart leads to failure in myocardial protection by preconditioning.

Cardiovascular dysfunction is a major complication of diabetes. Examining mechanistic aspects underlying the incapacity of the diabetic heart to respond to ischemic preconditioning (IPC), we could show that the alterations in iron homeostasis can explain this phenomenon. Correlating the hemodynamic parameters with levels of ferritin, the main iron storage and detoxifying protein, without and with inhibitors of protein degradation, substantiated this explanation. Diabetic hearts were less sensitive to ischemia-reperfusion stress, as indicated by functional parameters and histology. Mechanistically, since ferritin has been shown to provide cellular protection against insults, including ischemia-reperfusion stress and as the basal ferritin level in diabetic heart was 2-fold higher than in controls, these are in accord with the greater resistance of the diabetic heart to ischemia-reperfusion. Additionally, during ischemia-reperfusion, preceded by IPC, a rapid and extensive loss in ferritin levels, during the prolonged ischemia, in diabetic heart but not in non-diabetic controls, provide additional substantiation to the explanation for loss of respond to IPC. Current research is shedding light on the mechanism behind ferritin degradation as well, suggesting a novel explanation for diabetes-induced loss of cardioprotection.

Single Dose of the CXCR4 Antagonist BL-8040 Induces Rapid Mobilization for the Collection of Human CD34+ Cells in Healthy Volunteers

Purpose: The potential of the high-affinity CXCR4 antagonist BL-8040 as a monotherapy-mobilizing agent and its derived graft composition and quality were evaluated in a phase I clinical study in healthy volunteers (NCT02073019). Experimental Design: The first part of the study was a randomized, double-blind, placebo-controlled dose escalation phase. The second part of the study was an open-label phase, in which 8 subjects received a single injection of BL-8040 (1 mg/kg) and approximately 4 hours later underwent a standard leukapheresis procedure. The engraftment potential of the purified mobilized CD34+ cells was further evaluated by transplanting the cells into NSG immunodeficient mice. Results: BL-8040 was found safe and well tolerated at all doses tested (0.5–1 mg/kg). The main treatment-related adverse events were mild to moderate. Transient injection site and systemic reactions were mitigated by methylprednisolone, paracetamol, and promethazine pretreatment. In the first part of the study, BL-8040 triggered rapid and substantial mobilization of WBCs and CD34+ cells in all tested doses. Four hours postdose, the count rose to a mean of 8, 37, 31, and 35 cells/μL (placebo, 0.5, 0.75, and 1 mg/kg, respectively). FACS analysis revealed substantial mobilization of immature dendritic, T, B, and NK cells. In the second part, the mean CD34+ cells/kg collected were 11.6 × 106 cells/kg. The graft composition was rich in immune cells. Conclusions: The current data demonstrate that BL-8040 is a safe and effective monotherapy strategy for the collection of large amounts of CD34+ cells and immune cells in a one-day procedure for allogeneic HSPC transplantation. Clin Cancer Res; 23(22); 6790–801. ©2017 AACR.

CXCR4 promotes neuroblastoma growth and therapeutic resistance through miR-15a/16-1 mediated ERK and BCL2/cyclin D1 pathways

CXCR4 expression in neuroblastoma tumors correlates with disease severity. In this study, we describe mechanisms by which CXCR4 signaling controls neuroblastoma tumor growth and response to therapy. We found that overexpression of CXCR4 or stimulation with CXCL12 supports neuroblastoma tumorigenesis. Moreover, CXCR4 inhibition with the high-affinity CXCR4 antagonist BL-8040 prevented tumor growth and reduced survival of tumor cells. These effects were mediated by the upregulation of miR-15a/16-1, which resulted in downregulation of their target genes BCL-2 and cyclin D1, as well as inhibition of ERK. Overexpression of miR-15a/16-1 in cells increased cell death, whereas antagomirs to miR-15a/16-1 abolished the proapoptotic effects of BL-8040. CXCR4 overexpression also increased miR-15a/16-1, shifting their oncogenic dependency from the BCL-2 to the ERK signaling pathway. Overall, our results demonstrate the therapeutic potential of CXCR4 inhibition in neuroblastoma treatment and provide a rationale to test combination therapies employing CXCR4 and BCL-2 inhibitors to increase the efficacy of these agents.Significance: These results provide a mechanistic rationale for combination therapy of CXCR4 and BCL-2 inhibitors to treat a common and commonly aggressive pediatric cancer.Cancer Res; 78(6); 1471-83. ©2017 AACR.

Abstract 3556: CXCR4 controls BCL-2 expression and function by regulating miR-15a/16-1 expression in tumor cells

Background: CXCR4 is overexpressed in the majority of tumor cells and its degree of expression often correlates with disease severity. Binding of CXCL12 to the CXCR4 receptor activates signaling pathways which are crucial for the interaction of hematopoietic cells with the microenvironment and cell survival. Signaling activated through CXCR4 was shown to be detrimental by increasing survival of tumor cells and promoting resistance to therapy in many types of cancer. CXCR4-antagonists, such as BL-8040, currently in phase II trials, were shown to selectively inhibit tumor cell growth and to induce apoptosis in vitro and in vivo. However, the molecular mechanism by which CXCR4 overexpression triggers tumor cell survival and its inhibition leads to cell death is not fully understood. Objective: To study the mechanism by which the CXCR4 pathway controls malignant cell survival and death through regulating miR-15a/16-1 expression. Method: We assessed the effect of CXCR4 overexpression, its activation and inhibition, on the expression of miR-15a/16-1 and their target genes, BCL-2, MCL-1 and cyclin D1, in a variety of tumor cells in vitro and in vivo. Results: We found that overexpression of CXCR4 in tumor cells or stimulation of cells with its ligand, CXCL12, lead to up-regulation of miR-15a/16-1, resulting in down-regulation of their target genes BCL-2, MCL-1 and cyclin D1. Furthermore, overexpression of CXCR4 in these cells increases tumorgenesis and shifts their oncogenic dependency from the BCL-2 to the CXCR4/ERK signaling pathway. Antagonists of CXCR4 such as BL-8040 were shown to induce apoptotic cell death of malignant cells. BL-8040 was found to increase the expression of miR-15a/16-1 and reduce the expression of BCL2, MCL1 and cyclin D1. Importantly, CXCR4 inhibition using BL-8040 induced apoptosis in vitro and in vivo in AML and neuroblastoma tumors. This was mediated by inhibition of survival signals by ERK and down-regulation of BCL-2 expression. In support of these results overexpression of miR-15a/16-1 in AML and neuroblastoma cells was shown to induce their apoptosis. Conclusions: Our results demonstrate, for the first time to the best of our knowledge, that CXCR4 signaling regulates the expression of miR-15a/16-1 and their target genes. Our results suggest that overexpression of CXCR4 may override the survival dependency of tumor cells on BCL-2, MCL-1, and cyclin D1 leading to resistance of tumor cells to inhibition of these pathways. Furthermore, these results indicate that ligands of CXCR4 may tip the balance toward cell death by down- regulating survival signals through miR-15a/16-1 suppression of BCL2, MCL1 and cyclin D1 expression. Citation Format: Shiri Klein Silberman, Michal Abraham, Baruch Bulvik, Hanna Wald, Orly Eizenberg, Dvorah Olam, Lola Weiss, Katia Beider, Ori Wald, Shlomo Bulvik, Abraham Avigdor, Ohad Benjamini, Eithan Galun, Arnon Nagler, Yaron Pereg, Amnon Peled. CXCR4 controls BCL-2 expression and function by regulating miR-15a/16-1 expression in tumor cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3556.