Therapeutic potential of proteasome inhibitors in congenital erythropoietic porphyria.

Congenital erythropoietic porphyria (CEP) is a rare autosomal recessive disorder characterized by uroporphyrinogen III synthase (UROS) deficiency resulting in massive porphyrin accumulation in blood cells, which is responsible for hemolytic anemia and skin photosensitivity. Among the missense mutations actually described up to now in CEP patients, the C73R and the P248Q mutations lead to a profound UROS deficiency and are usually associated with a severe clinical phenotype. We previously demonstrated that the UROS(C73R) mutant protein conserves intrinsic enzymatic activity but triggers premature degradation in cellular systems that could be prevented by proteasome inhibitors. We show evidence that the reduced kinetic stability of the UROS(P248Q) mutant is also responsible for increased protein turnover in human erythroid cells. Through the analysis of EGFP-tagged versions of UROS enzyme, we demonstrate that both UROS(C73R) and UROS(P248Q) are equally destabilized in mammalian cells and targeted to the proteasomal pathway for degradation. We show that a treatment with proteasomal inhibitors, but not with lysosomal inhibitors, could rescue the expression of both EGFP-UROS mutants. Finally, in CEP mice (Uros(P248Q/P248Q)) treated with bortezomib (Velcade), a clinically approved proteasome inhibitor, we observed reduced porphyrin accumulation in circulating RBCs and urine, as well as reversion of skin photosensitivity on bortezomib treatment. These results of medical importance pave the way for pharmacologic treatment of CEP disease by preventing certain enzymatically active UROS mutants from early degradation by using proteasome inhibitors or chemical chaperones.

Metabolic correction of congenital erythropoietic porphyria with iPSCs free of reprogramming factors.

Congenital erythropoietic porphyria (CEP) is due to a deficiency in the enzymatic activity of uroporphyrinogen III synthase (UROS); such a deficiency leads to porphyrin accumulation and results in skin lesions and hemolytic anemia. CEP is a candidate for retrolentivirus-mediated gene therapy, but recent reports of insertional leukemogenesis underscore the need for safer methods. The discovery of induced pluripotent stem cells (iPSCs) has opened up new horizons in gene therapy because it might overcome the difficulty of obtaining sufficient amounts of autologous hematopoietic stem cells for transplantation and the risk of genotoxicity. In this study, we isolated keratinocytes from a CEP-affected individual and generated iPSCs with two excisable lentiviral vectors. Gene correction of CEP-derived iPSCs was obtained by lentiviral transduction of a therapeutic vector containing UROS cDNA under the control of an erythroid-specific promoter shielded by insulators. One iPSC clone, free of reprogramming genes, was obtained with a single proviral integration of the therapeutic vector in a genomic safe region. Metabolic correction of erythroblasts derived from iPSC clones was demonstrated by the disappearance of fluorocytes. This study reports the feasibility of porphyria gene therapy with the use of iPSCs.

Neonatal bone marrow transplantation prevents liver disease in a murine model of erythropoietic protoporphyria.

BACKGROUND & AIMS: Erythropoietic protoporphyria (EPP) is an inherited disorder of heme biosynthesis caused by partial ferrochelatase deficiency, resulting in protoporphyrin IX (PPIX) accumulation in erythrocytes, responsible for skin photosensitivity. In some EPP patients, the development of cholestatic liver injury due to PPIX accumulation can lead to hepatic failure. In adult EPP mice, bone marrow transplantation (BMT) leads to skin photosensitivity correction but fails to reverse liver damages, probably because of the irreversible nature of liver fibrosis. Our aim was to determine the time course of liver disease progression in EPP mice and to evaluate the protective effect of BMT into neonates. METHODS: We studied the development of liver disease from birth in EPP mice, in relation with erythroid and hepatic PPIX accumulation. To prevent the development of liver disease, BMT was performed into newborn mice using a novel busulfan-mediated preconditioning assay. RESULTS: We showed that hepatic PPIX accumulates during the first 2 weeks and correlates with the onset of a progressive liver fibrosis in 12-day-old EPP mice. Transplantation of normal congenic hematopoietic stem cells into EPP neonates led to long-term donor hematopoiesis recovery. A full correction of erythroid PPIX accumulation and skin photosensitivity was obtained. Furthermore, five months after neonatal BMT, liver damage was almost completely prevented. CONCLUSIONS: We demonstrated for the first time that BMT could be successfully used to prevent liver disease in EPP mice and suggested that BMT would be an attractive therapeutic option to prevent severe liver dysfunction in EPP patients.

Pharmacologic Manipulation of Wnt Signaling and Cancer Stem Cells.

Wnt (Wingless-related integration site)-signaling orchestrates self-renewal programs in normal somatic stem cells as well as in cancer stem cells. Aberrant Wnt signaling is associated with a wide variety of malignancies and diseases. Although our understanding has increased tremendously over the past decade, therapeutic targeting of the dysregulated Wnt pathway remains a challenge. Here we review recent preclinical and clinical therapeutic approaches to target the Wnt pathway.

Effects of CD49d-targeted antisense-oligonucleotide on α4 integrin expression and function of acute lymphoblastic leukemia cells: Results of in vitro and in vivo studies.

We recently demonstrated the effectiveness of blocking CD49d with anti-functional antibodies or small molecule inhibitors as a rational targeted approach to the treatment of acute leukemia in combination with chemotherapy. Antisense oligonucleotide promises to be no less specific than antibodies and inhibitors, but more interesting for pharmacokinetics and pharmacodynamics. We addressed this using the published CD49d antisense drug ATL1102. In vitro, we incubated/nucleofected the ALL cell line Kasumi-2 with ATL1102. In vivo, immunodeficient hosts were engrafted with primary ALL cells and treated with ATL1102. Changes in expression of CD49d mRNA and CD49d protein, and of cooperating gene products, including ß1 integrin and CXCR4, as well as survival in the mouse experiments were quantified. We observed dose-dependent down-regulation of CD49d mRNA and protein levels and its partner integrin ß1 cell surface protein level and, up-regulation of CXCR4 surface expression. The suppression was more pronounced after nucleofection than after incubation, where down-regulation was significant only at the higher doses. In vivo effects of ATL1102 were not sufficient to translate into "clinical" benefit in the leukemia model. In summary, antisense oligonucleotides are successful tools for specifically modulating gene expression but sufficient delivery to down-regulate CD49d in vivo may be difficult to achieve.

The Wnt signaling pathway in cancer.

The Wnt signaling pathway is critically involved in both the development and homeostasis of tissues via regulation of their endogenous stem cells. Aberrant Wnt signaling has been described as a key player in the initiation of and/or maintenance and development of many cancers, via affecting the behavior of Cancer Stem Cells (CSCs). CSCs are considered by most to be responsible for establishment of the tumor and also for disease relapse, as they possess inherent drug-resistance properties. The development of new therapeutic compounds targeting the Wnt signaling pathway promises new hope to eliminate CSCs and achieve cancer eradication. However, a major challenge resides in developing a strategy efficient enough to target the dysregulated Wnt pathway in CSCs, while being safe enough to not damage the normal somatic stem cell population required for tissue homeostasis and repair. Here we review recent therapeutic approaches to target the Wnt pathway and their clinical applications.