[A phase II pharmacological study of leuprolide acetate 6-month depot, TAP-144-SR (6M), in treatment-Nazve patients with prostatic cancer who received a single subcutaneous or intramuscular injection].

The aim of this phase II study was to evaluate the pharmacokinetics, pharmacodynamics, efficacy, and safety of a 6- month depot formulation of a luteinizing hormone-releasing hormone (LH-RH) agonist, TAP-144-SR (6M), in Japanese treatment-naÏve patients with prostatic cancer. Each subject received a single subcutaneous or intramuscular injection of TAP- 144-SR (6M) and was monitored for 24 weeks. The primary endpoint was the change in serum testosterone levels. The serum testosterone level in six subjects who received 22.5 mg of TAP-144 (SR) subcutaneously decreased below the castrate level after 4 weeks and remained suppressed during the 24 weeks of follow-up. With regard to safety, TAP-144-SR (6M)was not associated with any additional concerns compared to those reported for the approved 1-month and 3-month depot formulations of TAP-144-SR. In addition, 30 mg of TAP-144-SR (6M) was administered subcutaneously to six subjects, and, on the basis of the results, the optimal clinical dosage of TAP-144-SR (6M) in Japan was considered to be 22.5 mg. Outcomes with 22.5mg TAP-144-SR (6M) administered intramuscularly were similar to those with TAP-144-SR (6M) administered subcutaneously.

FLI1 monoallelic expression combined with its hemizygous loss underlies Paris-Trousseau/Jacobsen thrombopenia.

Paris-Trousseau syndrome (PTS; also known as Jacobsen syndrome) is characterized by several congenital anomalies including a dysmegakaryopoiesis with two morphologically distinct populations of megakaryocytes (MKs). PTS patients harbor deletions on the long arm of chromosome 11, including the FLI1 gene, which encodes a transcription factor essential for megakaryopoiesis. We show here that lentivirus-mediated overexpression of FLI1 in patient CD34(+) cells restores the megakaryopoiesis in vitro, indicating that FLI1 hemizygous deletion contributes to the PTS hematopoietic defects. FISH analysis on pre-mRNA and single-cell RT-PCR revealed that FLI1 expression is mainly monoallelic in CD41(+)CD42(-) progenitors, while it is predominantly biallelic in the other stages of megakaryopoiesis. In PTS cells, the hemizygous deletion of FLI1 generates a subpopulation of CD41(+)CD42(-) cells completely lacking FLI1 transcription. We propose that the absence of FLI1 expression in these CD41(+)CD42(-) cells might prevent their differentiation, which could explain the segregation of the PTS MKs into two subpopulations: one normal and one composed of small immature MKs undergoing a massive lysis, presumably originating from either FLI1(+) or FLI1(-) CD41(+)CD42(-) cells, respectively. Thus, we point to the role of transient monoallelic expression of a gene essential for differentiation in the genesis of human haploinsufficiency-associated disease and suggest that such a mechanism may be involved in the pathogenesis of other congenital or acquired genetic diseases.

Role for the nuclear factor kappaB pathway in transforming growth factor-beta1 production in idiopathic myelofibrosis: possible relationship with FK506 binding protein 51 overexpression.

The release of transforming growth factor-beta1 (TGF-beta1) in the bone marrow microenvironment is one of the main mechanisms leading to myelofibrosis in murine models and probably in the human idiopathic myelofibrosis (IMF). The regulation of TGF-beta1 synthesis is poorly known but seems regulated by nuclear factor kappaB (NF-kappaB). We previously described the overexpression of an immunophilin, FK506 binding protein 51 (FKBP51), in IMF megakaryocytes. Gel shift and gene assays show that FKBP51's overexpression in a factor-dependent hematopoietic cell line, induces a sustained NF-kappaB activation after cytokine deprivation. This activation correlates with a low level of IkappaBalpha. A spontaneous activation of NF-kappaB was also detected in proliferating megakaryocytes and in circulating CD34(+) patient cells. In normal cells, NF-kappaB activation was only detected after cytokine treatment. The expression of an NF-kappaB superrepressor in FKBP51 overexpressing cells and in derived megakaryocytes from CD34(+) of IMF patients revealed that NF-kappaB activation was not involved in the resistance to apoptosis after cytokine deprivation of these cells but in TGF-beta1 secretion. These results highlight the importance of NF-kappaB's activation in the fibrosis development of this disease. They also suggest that FKBP51's overexpression in IMF cells could play an important role in the pathogenesis of this myeloproliferative disorder.

Spontaneous STAT5 activation induces growth factor independence in idiopathic myelofibrosis: possible relationship with FKBP51 overexpression.

Spontaneous growth of megakaryocyte progenitors is one of the biologic hallmarks of idiopathic myelofibrosis (IMF). The molecular mechanisms underlying this hypersensitivity to cytokines are poorly understood. Using a differential display approach, we previously observed FK506 binding protein 51 (FKBP51) overexpression in pathologic megakaryocytes from IMF. Using an FKBP51-overexpressing cell line, we found sustained STAT5 activation associated with JAK2 phosphorylation. We subsequently tested whether this transcription factor was activated in patient samples. We detected a STAT5 nuclear translocation and activation in spontaneously grown megakaryocytes and in circulating CD34(+) cells from the majority of patients studied. The biologic role of this JAK/STAT pathway activation was demonstrated by inhibiting both the anti-apoptotic phenotype mediated by FKBP51 overexpression in UT7 cells and the spontaneous megakaryocytic growth by addition in culture of the JAK2 inhibitor AG490 or overexpression of a STAT5b dominant negative or SOCS-1. These results demonstrate that a constitutive STAT5 activation in IMF is indispensable for spontaneous growth of megakaryocytes. They also suggest that FKBP51 overexpression could be involved in STAT5 activation in IMF cells and in subsequent abnormal growth.

Prominent role of TGF-beta 1 in thrombopoietin-induced myelofibrosis in mice.

Several studies suggest an implication of transforming growth factor-beta1 (TGF-beta1) in the promotion of myelofibrosis associated with hematopoietic malignancies, but the involvement of this cytokine is not fully investigated. To test directly the impact of TGF-beta1 in the pathogenesis of myelofibrosis, bone marrow stem cells from homozygous TGF-beta1 null (TGF-beta1(-/-)) and wild-type (WT) littermates were infected with a retrovirus encoding the murine thrombopoietin (TPO) protein and engrafted into lethally irradiated wild-type hosts for long-term reconstitution. Over the 4 months of follow-up, TPO levels in plasma were markedly elevated in both groups of mice, and animals typically developed a myeloproliferative syndrome characterized by thrombocytosis, leukocytosis, splenomegaly, increased numbers of progenitors in blood, and extramedullary hematopoiesis. Severe fibrosis was observed in spleen and marrow from all the mice engrafted with WT cells. In contrast, none of the mice repopulated with TGF-beta1(-/-) cells (chimerism > 70%) showed deposition of reticulin fibers at any time during the follow-up. In accordance with the development of fibrosis, latent TGF-beta1 levels in plasma and extracellular fluid of the spleen from mice engrafted with WT cells were increased 6-fold and 4-fold, respectively, over levels found in normal hosts, whereas no increase over baseline levels could be demonstrated in animals undergoing transplantation with TGF-beta1(-/-) cells. These data provide evidence that TGF-beta1 produced by hematopoietic cells is pivotal for the pathogenesis of myelofibrosis that develops in mice with TPO overexpression.

Stimulation of osteoprotegerin production is responsible for osteosclerosis in mice overexpressing TPO.

Myelofibrosis and osteosclerosis are prominent features arising in mice overexpressing thrombopoietin (TPO). The pivotal role of transforming growth factor beta 1 (TGF-beta 1) in the pathogenesis of myelofibrosis has been documented, but the mechanisms mediating osteosclerosis remain unclear. Here, we used mice deficient in osteoprotegerin (OPG), a secreted inhibitor of bone resorption, to determine whether osteosclerosis occurs through a deregulation of osteoclastogenesis. Marrow cells from opg-deficient mice (opg(-/-)) or wild-type (WT) littermates were infected with a retrovirus encoding TPO and engrafted into an opg(-/-) or WT background for long-term reconstitution. The 4 combinations of graft/host (WT/WT, opg(-/-)/opg(-/-), opg(-/-)/WT, and WT/opg(-/-)) were studied. Elevation of TPO and TGF-beta 1 levels in plasma was similar in the 4 experimental groups and all the mice developed a similar myeloproliferative syndrome associated with severe myelofibrosis. Osteosclerosis developed in WT hosts engrafted with WT or opg(-/-) hematopoietic cells and was associated with increased OPG levels in plasma and decreased osteoclastogenesis. In contrast, opg(-/-) hosts exhibited an osteoporotic phenotype and a growth of bone trabeculae was rarely seen. These findings suggest that osteosclerosis in mice with TPO overexpression occurs predominantly via an up-regulation of OPG in host stromal cells leading to disruption of osteoclastogenesis.

Overexpression of FKBP51 in idiopathic myelofibrosis regulates the growth factor independence of megakaryocyte progenitors.

Idiopathic myelofibrosis (IMF) is a chronic myeloproliferative disorder characterized by megakaryocyte hyperplasia and bone marrow fibrosis. Biologically, an autonomous megakaryocyte growth and differentiation is noticed, which contributes to the megakaryocyte accumulation. To better understand the molecular mechanisms involved in this spontaneous growth, we searched for genes differentially expressed between normal megakaryocytes requiring cytokines to grow and IMF spontaneously proliferating megakaryocytes. Using a differential display technique, we found that the immunophilin FKBP51 was 2 to 8 times overexpressed in megakaryocytes derived from patients' CD34(+) cells in comparison to normal megakaryocytes. Overexpression was moderate and confirmed in 8 of 10 patients, both at the mRNA and protein levels. Overexpression of FKBP51 in a UT-7/Mpl cell line and in normal CD34(+) cells induced a resistance to apoptosis mediated by cytokine deprivation with no effect on proliferation. FKBP51 interacts with both calcineurin and heat shock protein (HSP)70/HSP90. However, a mutant FKBP51 deleted in the HSP70/HSP90 binding site kept the antiapoptotic effect, suggesting that the calcineurin pathway was responsible for the FKBP51 effect. Overexpression of FKBP51 in UT-7/Mpl cells induced a marked inhibition of calcineurin activity. Pharmacologic inhibition of calcineurin by cyclosporin A mimicked the effect of FKBP51. The data support the conclusion that FKBP51 inhibits apoptosis through a calcineurin-dependent pathway. In conclusion, FKBP51 is overexpressed in IMF megakaryocytes and this overexpression could be, in part, responsible for the megakaryocytic accumulation observed in this disorder by regulating their apoptotic program.