Forodesine, an inhibitor of purine nucleoside phosphorylase, induces apoptosis in chronic lymphocytic leukemia cells

Abstract Purine nucleoside phosphorylase (PNP) deficiency in humans results in T lymphocytopenia. Forodesine, a potent inhibitor of PNP, was designed based on the transition-state structure stabilized by the enzyme. Previous studies established that forodesine in the presence of deoxyguanosine (dGuo) inhibits the proliferation of T lymphocytes. A phase 1 clinical trial of forodesine in T-cell malignancies demonstrated significant antileukemic activity with an increase in intracellular dGuo triphosphate (dGTP). High accumulation of dGTP in T cells may be dependent on the levels of deoxynucleoside kinases. Because B-cell chronic lymphocytic leukemia (B-CLL) cells have high activity of deoxycytidine kinase (dCK), we hypothesized that these lymphocytes would respond to forodesine. This postulate was tested in primary lymphocytes during in vitro investigations. Lymphocytes from 12 patients with CLL were incubated with forodesine and dGuo. These CLL cells showed a wide variation in the accumulation of intracellular dGTP without any effect on other deoxynucleotides. This was associated with DNA damage-induced p53 stabilization, phosphorylation of p53 at Ser15, and activation of p21. The dGTP accumulation was related to induction of apoptosis measured by caspase activation, changes in mitochondrial membrane potential, and PARP cleavage. Based on these data, a phase 2 clinical trial of forodesine has been initiated for CLL patients.

Download Full-text

Development of Forodesine Hydrochloride (FH), an Inhibitor of Purine Nucleoside Phosphorylase, for Patients with Chronic Lymphocytic Leukemia (CLL).

Blood ◽

10.1182/blood.v106.11.2119.2119 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2119-2119 ◽

Cited By ~ 1

Author(s):

Kumudha Balakrishnan ◽

Farhad Ravandi ◽

Michael J. Keating ◽

Varsha Gandhi

Keyword(s):

Clinical Trial ◽

T Cells ◽

Cell Death ◽

Purine Nucleoside Phosphorylase ◽

Response To Therapy ◽

Lymphocytic Leukemia ◽

Purine Nucleoside ◽

High Accumulation ◽

Nucleoside Phosphorylase

Abstract Mammalian purine nucleoside phosphorylase (PNP) catalyzes the cleavage of inosine, deoxyinosine, guanosine, and deoxyguanosine (dGuo) to their corresponding base and sugar 1-phosphate by phosphorolysis. PNP deficiency in humans produces a relatively selective depletion of T cells without much effect on normal B cells. FH, a potent inhibitor of PNP, was designed based on the transition-state analog structure stabilized by the enzyme. Previous studies demonstrated that FH in the presence of dGuo inhibits the proliferation of T-lymphocytes (Kicska et al. PNAS 2001). Based on these observations, we conducted a phase I clinical trial of FH in patients with advanced T-cell malignancies. Significant antileukemic activity was correlated with an increase in plasma FH (median 5 μM) and dGuo (median 14 μM), and an accumulation of intracellular dGuo triphosphate (dGTP) (Gandhi et al, Blood, in press, 2005). High accumulation of dGTP in T-cells may be dependant on activity of deoxynucleoside kinases. Because B-CLL cells have high activity of deoxycytidine kinase, we hypothesized that they would be sensitive to FH. This postulate was tested in primary CLL lymphocytes during in vitro investigations. Lymphocytes from patients with CLL were incubated in vitro with FH (2 μM) in the presence of 10 μM dGuo. Lymphocytes from 3 of 4 patients showed an elevation in the intracellular dGTP levels to a median 30-fold at 8 hr, without any effect on other deoxynucleotides. This increase in dGTP was associated with phosphorylation of p53 at ser15, stabilization of p53, and an increase in p21 protein. The dGTP accumulation was related to induction of apoptosis measured by activation of caspase 8, 9, and 3 and cleavage of PARP. Incubation with either FH or dGuo alone did not result in dGTP accumulation or cell death suggesting that PNP inhibition by FH and phosphorylation of dGuo to dGTP are essential for CLL cell death. Based on these encouraging results, availability of oral formulation and to validate these in vitro data during clinical trial, a phase II study of FH in patients with advanced, fludarabine-refractory CLL has been initiated. FH is administered orally at a dose of 200 mg/day for 7 days each week for 4 weeks (cycle 1). Patients are evaluated after 1 full cycle of therapy and in the absence of serious side effects, or disease progression, they continue the treatment for up to 5 more cycles. Laboratory endpoints such as level of FH and dGuo in plasma, PNP activity, dGTP levels in cells, will be measured and correlated with cytoreduction. It is postulated that a high kinase/low nucleotidase activity leading to the accumulation of intracellular dGTP in target CLL cells and apoptosis (akin to what has been seen in T-cells) will result in response to therapy. This is the first trial of a PNP inhibitor for treatment of B-CLL.

Download Full-text

Purine nucleoside phosphorylase in chronic lymphocytic leukemia (CLL)

Blood ◽

10.1182/blood.v52.5.886.886 ◽

1978 ◽

Vol 52 (5) ◽

pp. 886-895 ◽

Cited By ~ 19

Author(s):

M Borgers ◽

H Verhaegen ◽

M De Brabander ◽

J De Cree ◽

W De Cock ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Healthy Subjects ◽

Purine Nucleoside Phosphorylase ◽

Peripheral Blood Lymphocytes ◽

Lymphocytic Leukemia ◽

Purine Nucleoside ◽

Salvage Pathway ◽

Purine Salvage ◽

Nucleoside Phosphorylase ◽

A Minor

Abstract Purine nucleoside phosphorylase (PNP), the enzyme schematically next to adenosine deaminase in the purine salvage pathway, has been demonstrated cytochemically in peripheral blood lymphocytes of healthy subjects and chronic lymphocytic leukemia (CLL) patients. The enzyme activity is confined to the cytosol. In healthy subjects the majority of lymphocytes are strongly reactive for PNP, whereas the rest are devoid of cytochemically demonstrable activity. The percentage of PNP- positive cells largely corresponds to the number of E rosette-forming cells and is inversely proportional to the number of Ig-bearing cells. In six of seven CLL patients studied only a minor percentage of the lymphocytes showed strong PNP activity, whereas the large majority (88%- -98%) possessed trace activity. Such patients have a high number of Ig- bearing cells and a low number of E rosette-forming cells. A different pattern of markers was found in the lymphocytes of the seventh CLL patient: 66% were strongly reactive for PNP, an important number formed E rosettes, and a minor percentage were Ig bearing. These data indicate that PNP can be useful as a “nonmembrane” marker in the differentiation of the B and T cell origin in CLL and deserves to be studied in other lymphoproliferative disorders.

Download Full-text

Combined assay of adenosine deaminase, purine nucleoside phosphorylase, and lactate dehydrogenase in the early clinical evaluation of B-chronic lymphocytic leukemia

American Journal of Hematology ◽

10.1002/ajh.2830270302 ◽

1988 ◽

Vol 27 (3) ◽

pp. 157-162 ◽

Cited By ~ 9

Author(s):

Joan Lluis Vives Corrons ◽

Ciril Rozman ◽

Maria Assumpció Pujades ◽

Dolors Colomer ◽

Encarna Perez Vila ◽

...

Keyword(s):

Lactate Dehydrogenase ◽

Chronic Lymphocytic Leukemia ◽

Adenosine Deaminase ◽

Clinical Evaluation ◽

Purine Nucleoside Phosphorylase ◽

Lymphocytic Leukemia ◽

Purine Nucleoside ◽

Nucleoside Phosphorylase

Download Full-text

Intralymphocytic Adenosine Deaminase (ADA) and Purine Nucleoside Phosphorylase (PNPase) Activity in Chronic Lymphocytic Leukemia (CLL) : Effect of Thymostimulin

Advances in Experimental Medicine and Biology - Purine Metabolism in Man-IV ◽

10.1007/978-1-4757-0390-0_62 ◽

1984 ◽

pp. 327-332

Author(s):

M. Petrini ◽

F. Bruschi ◽

F. Caracciolo ◽

G. Carulli ◽

B. Grassi ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Adenosine Deaminase ◽

Purine Nucleoside Phosphorylase ◽

Lymphocytic Leukemia ◽

Purine Nucleoside ◽

Nucleoside Phosphorylase

Download Full-text

Purine nucleoside phosphorylase in chronic lymphocytic leukemia (CLL)

Blood ◽

10.1182/blood.v52.5.886.bloodjournal525886 ◽

1978 ◽

Vol 52 (5) ◽

pp. 886-895

Author(s):

M Borgers ◽

H Verhaegen ◽

M De Brabander ◽

J De Cree ◽

W De Cock ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Healthy Subjects ◽

Purine Nucleoside Phosphorylase ◽

Peripheral Blood Lymphocytes ◽

Lymphocytic Leukemia ◽

Purine Nucleoside ◽

Salvage Pathway ◽

Purine Salvage ◽

Nucleoside Phosphorylase ◽

A Minor

Purine nucleoside phosphorylase (PNP), the enzyme schematically next to adenosine deaminase in the purine salvage pathway, has been demonstrated cytochemically in peripheral blood lymphocytes of healthy subjects and chronic lymphocytic leukemia (CLL) patients. The enzyme activity is confined to the cytosol. In healthy subjects the majority of lymphocytes are strongly reactive for PNP, whereas the rest are devoid of cytochemically demonstrable activity. The percentage of PNP- positive cells largely corresponds to the number of E rosette-forming cells and is inversely proportional to the number of Ig-bearing cells. In six of seven CLL patients studied only a minor percentage of the lymphocytes showed strong PNP activity, whereas the large majority (88%- -98%) possessed trace activity. Such patients have a high number of Ig- bearing cells and a low number of E rosette-forming cells. A different pattern of markers was found in the lymphocytes of the seventh CLL patient: 66% were strongly reactive for PNP, an important number formed E rosettes, and a minor percentage were Ig bearing. These data indicate that PNP can be useful as a “nonmembrane” marker in the differentiation of the B and T cell origin in CLL and deserves to be studied in other lymphoproliferative disorders.

Download Full-text

Involvement of CED-3/ICE Proteases in the Apoptosis of B-Chronic Lymphocytic Leukemia Cells

Blood ◽

10.1182/blood.v89.9.3378 ◽

1997 ◽

Vol 89 (9) ◽

pp. 3378-3384 ◽

Cited By ~ 71

Author(s):

Beatriz Bellosillo ◽

Mireia Dalmau ◽

Dolors Colomer ◽

Joan Gil

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Lymphocytic Leukemia ◽

Interleukin 4 ◽

Leukemia Cells ◽

Parp Cleavage ◽

Dependent Manner ◽

Kinase C ◽

Dose Dependent ◽

Pkc Activation

Abstract B-chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of long-lived B lymphocytes that express high levels of Bcl-2. We examined the involvement of CED-3/ICE-like proteases in the apoptosis of B-CLL cells. One of the substrates of these proteases is poly(ADP [adenosine 5′-diphosphate]-ribose) polymerase (PARP). The effect of different factors that induce the apoptosis of B-CLL cells on the proteolytic cleavage of PARP has been studied. Treatment of B-CLL cells with different concentrations of dexamethasone (1 to 1,000 μmol/L) induced in a dose-dependent manner the cleavage of PARP. Dexamethasone induced PARP cleavage after 12 hours of incubation, which was almost complete at 48 hours. PARP cleavage during apoptosis of B-CLL cells was studied in cells from eight patients and a correlation was found between cell viability and the degree of PARP cleavage. Incubation in vitro of B-CLL cells with fludarabine for 48 hours induced PARP cleavage in all the cases studied. Protein kinase C (PKC) activation with 100 nmol/L TPA (12-O-tetradecanoylphorbol 13-acetate) or incubation with interleukin-4 (10 ng/mL) prevented either dexamethasone- or fludarabine-induced proteolysis of PARP. Incubation of B-CLL cells with the CED-3/ICE–like protease inhibitor Z-VAD.fmk inhibited spontaneous and dexamethasone-induced PARP cleavage and DNA fragmentation in a dose-dependent manner. Furthermore, Z-VAD.fmk prevented the cytotoxic effect of dexamethasone. These results indicate that CED-3/ICE–like proteases play an important role in the apoptosis of B-CLL cells.

Download Full-text

Intracellular delivery of purine nucleoside phosphorylase (PNP) fused to protein transduction domain corrects PNP deficiency in vitro

Cellular Immunology ◽

10.1016/j.cellimm.2006.07.003 ◽

2006 ◽

Vol 240 (2) ◽

pp. 107-115 ◽

Cited By ~ 22

Author(s):

Ana Toro ◽

Melissa Paiva ◽

Cameron Ackerley ◽

Eyal Grunebaum

Keyword(s):

Purine Nucleoside Phosphorylase ◽

Intracellular Delivery ◽

Purine Nucleoside ◽

Protein Transduction ◽

Protein Transduction Domain ◽

Nucleoside Phosphorylase

Download Full-text

Selective in vitro inhibition of human molt-4 T lymphoblasts by the novel purine nucleoside phosphorylase inhibitor, CI-972

Biochemical and Biophysical Research Communications ◽

10.1016/0006-291x(91)91042-b ◽

1991 ◽

Vol 178 (3) ◽

pp. 1351-1358 ◽

Cited By ~ 12

Author(s):

Richard B. Gilbertsen ◽

Mi K. Dong ◽

Lynn M. Kossarek ◽

Jagadish C. Sircar ◽

Catherine R. Kostian ◽

...

Keyword(s):

Purine Nucleoside Phosphorylase ◽

Purine Nucleoside ◽

The Novel ◽

Nucleoside Phosphorylase ◽

In Vitro Inhibition ◽

T Lymphoblasts

Download Full-text

Concurrent up-regulation of BCL-XL and BCL2A1 induces approximately 1000-fold resistance to ABT-737 in chronic lymphocytic leukemia

Blood ◽

10.1182/blood-2008-08-173310 ◽

2009 ◽

Vol 113 (18) ◽

pp. 4403-4413 ◽

Cited By ~ 223

Author(s):

Meike Vogler ◽

Michael Butterworth ◽

Aneela Majid ◽

Renata J. Walewska ◽

Xiao-Ming Sun ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Lymph Nodes ◽

Peripheral Blood ◽

De Novo ◽

Phase 1 ◽

Lymphocytic Leukemia ◽

Interleukin 4 ◽

Synergistic Activity

Abstract ABT-737 and its orally active analog, ABT-263, are rationally designed inhibitors of BCL2 and BCL-XL. ABT-263 shows promising activity in early phase 1 clinical trials in B-cell malignancies, particularly chronic lymphocytic leukemia (CLL). In vitro, peripheral blood CLL cells are extremely sensitive to ABT-737 (EC50 ∼7 nM), with rapid induction of apoptosis in all 60 patients tested, independent of parameters associated with disease progression and chemotherapy resistance. In contrast to data from cell lines, ABT-737–induced apoptosis in CLL cells was largely MCL1-independent. Because CLL cells within lymph nodes are more resistant to apoptosis than those in peripheral blood, CLL cells were cultured on CD154-expressing fibroblasts in the presence of interleukin-4 (IL-4) to mimic the lymph node microenvironment. CLL cells thus cultured developed an approximately 1000-fold resistance to ABT-737 within 24 hours. Investigations of the underlying mechanism revealed that this resistance occurred upstream of mitochondrial perturbation and involved de novo synthesis of the antiapoptotic proteins BCL-XL and BCL2A1, which were responsible for resistance to low and high ABT-737 concentrations, respectively. Our data indicate that after therapy with ABT-737–related inhibitors, resistant CLL cells might develop in lymph nodes in vivo and that treatment strategies targeting multiple BCL2 antiapoptotic members simultaneously may have synergistic activity.

Download Full-text