UCN-01 (7-hydroxy-staurosporine) is a potent inhibitor of Ca2+ and phospholipid-dependent protein kinase C (PKC), an important regulator of signal transduction. However, the inhibitory effects of staurosporine are nonspecific and have similar potencies for other protein kinases. Additional screening revealed that the 7-hydroxy analog of staurosporine, UCN-01 was a potent inhibitor of PKC (Fig. 1), and, importantly, its inhibitory effects were more selective (25), (Table 1). Whether additional protein kinases are targets for the drug is a matter of ongoing research, with recent studies indicating that the drug can inhibit cell cycle progression in conjunction with inhibition or altered activation of CDKs. More recent studies in leukemic T-cell lines have demonstrated irreversible inhibition of cell growth after 24 h of exposure, as well as evidence of internucleosomal DNA fragmentation consistent with induction of apoptosis after as little as 312 h of exposure. These observations correlated with apparent activation of cdk-1 and -2, and suggest that targets in addition to PKC must be considered. UCN-01 has demonstrated cytotoxic effects in various cell lines and antitumor activity against murine tumors and human tumor xenografts in vivo. Other molecules targeted by UCN-01 include various cdks, leading to abrogation of G2 and S phases of cell cycle (23). Kitada et al. (26) showed that UCN-01 enhances apoptosis in vitro by inhibition of the anti-apoptotic molecules Mcl-1 and X-linked inactivator of apoptosis (XIAP). Other groups have shown inhibitory effects on the DNA repair mechanisms in normal and CLL lymphocytes exposed to ultraviolet (UV) light or 4-hydroxy-cyclophospha-mide in vitro (27). UCN-01 is currently in clinical development, primarily as a chemotherapy-sensitizing agent. Of note, as a single agent, UCN-01 produced a durable complete remission in a patient with a refractory anaplastic T-cell lymphoma and induced a complete remission with chemotherapy in a refractory large B-cell lymphoma. Combination studies of UCN-01 with fludarabine and other agents are ongoing.
Bryostatin is a natural product isolated from the marine bryozoan Bugula neritina that targets PKC, initially stimulating PKC activity in the cell membrane, followed by inhibitory effects. Of potential clinical interest is the in vitro observation that it can induce differentiation of refractory B-CLL cells to a hairy cell phenotype, rendering them susceptible to 2-chlorodeoxyadenosine (28) (Table 1). A phase I study in humans identified a maximum tolerated dose of 120 ^g/kg over 72 h, with a dose-limiting toxicity of generalized myalgia, headache, and fatigue as other common side effects. An in vitro assay for total PKC evaluation in patient peripheral blood mononuclear cells demonstrated activation within the first 2 h followed by downregulation, which was maintained for the duration of the infusion (29).
A phase II trial of bryostatin 1 in patients with relapsed low-grade lymphomas and CLL observed one complete and two partial remissions in 25 patients. Patients who progressed while receiving bryostatin 1 alone participated in a feasibility study by receiving vincristine administered immediately after infusion of bryostatin. Nine patients received sequential treatment with bryostatin 1 and vincristine. Phenotypic analysis by flow cytometry of the peripheral blood revealed a hairy cell phenotype in two of four CLL patients (28).
Depsipeptide, a bicyclic peptide produced by Chromobacterium violaceum, was first identified by Ueda et al. (30) through screening natural products for antitumor activity in myc expressing tumor cells. Mechanistically, depsipeptide inhibits cell cycle progression at the G0-G1 interface, (Fig. 2) and blocks p21 protein signal transduction, although it is not known whether if these actions explain its cytotoxic activity. Cell cycle arrest possibly occurs through inhibition of the ras-signal transduction pathway, and in vitro depsipeptide induces reversion of ras-trans-formed tumor cells to a normal morphology and regulates c-myc mRNA. One recent report also suggested that this agent might act via inhibition of the DNA histone deacetylase enzyme (31) (Table 1). In vitro, CLL cells demonstrate sensitivity to depsipeptide, through apoptosis, indicating it should be tested in this disease. Initial phase I trial pharmacokinetics demonstrated that plasma concentrations of this agent exceed those that cause both in vitro histone acetylation and favorable alterations in the bcl-2/bax ratio and also induce apoptosis in human CLL cells. Phase II studies in lymphoid malignancies are currently under way.
Proteasome inhibitors, such as PS-341 are attractive targets for therapeutic intervention because of the importance of the ubiquitin proteasome pathways in the degradation and regulation of protein action (Table 1). The proteasome has numerous protein targets, such as p53, p21, and p27, and plays a key role in a broad array of cellular processes including cell cycle regulation, cell death, gene expression, and NF-kB activation (32) (Fig. 1). Dipeptide boronic acid analogs have been developed that inhibit the chymotryptic activity of the proteasome, with effects on tumor cell growth and apoptosis. PS-341 represents a class of novel proteasome inhibitors, which selectively inhibit the proteasome with a K of 6 nM and have a wide range of activity against multiple tumor cell lines. Phase I trials indicate that the drug is relatively nontoxic when administered iv twice weekly for 4 out of 6 wk at doses ranging from 0.5 to 1.25 mg/m2. Various toxicities have been observed, including fatigue, nausea, vomiting, fever, and thrombocytopenia. Investigators have noted a direct correlation between the PS-341 dose and percent proteasome inhibition, and the biologic activity is reliably measured by inhibition of 20S proteasome activity in peripheral mononuclear cells. There is increasing evidence that proteasome inhibitors may have a potential role in the therapy of patients with CLL. Proteasome inhibition has been shown to induce apoptosis of CLL lymphocytes without affecting normal lymphocytes and can sensitize chemo- and radioresistant CLL cells to apoptosis.
Interest in arsenic trioxide (As2O3); (Table 1) has increased because of its activity in acute promyelocytic leukemia (APL), in which it has induced a high proportion of complete remissions in relapsed patients. Biologically, arsenic has been shown to inhibit tumor proliferation and induce apoptosis and to decrease or inhibit BCL-2 expression. Kapahi et al. (33) showed that arsenic is a potent inhibitor of NF-kB and IKBB activation. The limited clinical experience in B-CLL suggests that there may be some activity worth further study.
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