Development of the proteasome inhibitor PS-341

Over the last decade, the critical role of the proteasome in cell-cycle regulation has become increasingly apparent. The proteasome, a multicatalytic protease present in all eukaryotic cells, is the primary component of the protein degradation pathway of the cell. By degrading regulatory proteins (or their inhibitors), the proteasome serves as a central conduit for many cellular regulatory signals and, thus, is a novel target for therapeutic drugs. PS-341 is a small molecule that is a potent and selective inhibitor of the proteasome. In vitro and mouse xenograft studies of PS-341 have shown antitumor activity in a variety of tumor types, including myeloma, chronic lymphocytic leukemia, prostate cancer, pancreatic cancer, and colon cancer, among others. Although PS-341 rapidly leaves the vascular compartment, a novel pharmacodynamic assay has shown that inhibition of proteasome-the biologic target-is dose dependent and reversible. These studies provided the rationale for a twice-weekly dosing schedule employed in ongoing clinical studies. Phase I trials in a variety of tumor types have shown PS-341 to be well tolerated, and phase II trials in several hematologic malignancies and solid tumor types are now in progress. Efficacy and safety data from the most advanced of these, a phase II multicenter trial in myeloma, will be available in early 2002.     

Proteasome inhibitor PS-341, a potential therapeutic agent for adult T-cell leukemia

Nuclear factor kappaB (NF-kappaB) plays a major role in the pathogenesis of human T-cell lymphotrophic virus I-associated malignancy. Proteasome inhibitors provide a rational approach to control constitutively activated NF-kappaB in human T-cell lymphotrophic virus I-infected T cells. We report that the proteasome inhibitor PS-341 decreased NF-kappaB DNA binding activity by preventing degradation of IkappaB(alpha). In our murine model of adult T-cell leukemia, PS-341 used alone did not yield prolongation of the survival of tumor-bearing mice. However, when combined with the current clinically approved drug humanized anti-Tac, therapy with PS-341 was associated with a complete remission in a proportion of treated animals, whereas only a partial response was observed in animals treated with humanized anti-Tac alone.     

Blockade of Hsp27 overcomes Bortezomib/proteasome inhibitor PS-341 resistance in lymphoma cells

Bortezomib (PS-341), a selective inhibitor of proteasome, induces apoptosis in various tumor cells, but its mechanism of action is unclear. Treatment with PS-341 induces apoptosis in SUDHL6 (DHL6), but not SUDHL4 (DHL4), lymphoma cells. Microarray analysis shows high RNA levels of heat shock protein-27 (Hsp27) in DHL4 versus DHL6 cells, which correlates with Hsp27 protein expression. Blocking Hsp27 using an antisense strategy restores the apoptotic response to PS-341 in DHL4 cells; conversely, ectopic expression of wild-type Hsp27 renders PS-341-sensitive DHL6 cells resistant to PS-341. These findings provide the first evidence that Hsp27 confers PS-341 resistance.     

The proteasome inhibitor PS-341 in cancer therapy.

The anticancer activity of the boronic acid dipeptide proteasome inhibitor PS-341 was examined in vitro and in vivo. PS-341 was a potent cytotoxic agent toward MCF-7 human breast carcinoma cells in culture, producing an IC90 of 0.05 microM on 24 h of exposure to the drug. In the EMT-6 tumor cell survival assay, PS-341 was equally cytotoxic administered p.o. or by i.p. injection up to a dose of 2 mg/kg. PS-341 was also toxic to the bone marrow colony-forming unit-granulocyte macrophage. PS-341 increased the tumor cell killing of radiation therapy, cyclophosphamide, and cisplatin in the EMT-6/Parent tumor, but was not able to overcome the in vivo resistance of the EMT-6/CTX and EMT-6/CDDP tumors. In the tumor growth delay assay, PS-341 administered p.o. had antitumor activity against the Lewis lung carcinoma, both primary and metastatic disease. In combination, regimens with 5-fluorouracil, cisplatin, Taxol and adriamycin, PS-341 seemed to produce primarily additive tumor growth delays against the s.c. tumor and was highly effective against disease metastatic to the lungs. The proteasome is an interesting new target for cancer therapy, and the proteasome inhibitor PS-341 warrants continued investigation in cancer therapy.     

Eosinophilia associated with adult T-cell leukemia/lymphoma

Eosinophilia is associated with a number of disorders including malignancies. A patient is described who had eosinophilia associated with adult T-cell leukemia/lymphoma (ATL) induced by human T-lymphotropic virus type I (HTLV-I). Both tissue and peripheral blood eosinophilia and high titers of HTLV-I antibody were present. The eosinophilia was most likely caused by the malignant cells producing one or more lymphokines. The patient has achieved a durable complete remission from combination chemotherapy. Because durable remissions in ATL are rare with any known therapy and eosinophilia has not previously been associated with ATL, it is possible that the tumor in this patient was derived from a T-cell subset not usually transformed by HTLV-I. ATL is another malignancy now known to cause eosinophilia.     

Vitamin C inactivates the proteasome inhibitor PS-341 in human cancer cells.

PURPOSE: PS-341 (bortezomib, Velcade), the first proteasome inhibitor approved by the Food and Drug Administration for the treatment of patients with relapsed multiple myeloma, induces apoptosis in human cancer cell lines. Vitamin C (ascorbic acid) is an essential water-soluble vitamin required for many normal physiologic functions and has to be obtained through diet or supplemental tablets in humans. Here we studied the potential effect of vitamin C on the anticancer activity of PS-341 in human cancer cell lines. EXPERIMENTAL DESIGN: The effects of vitamin C on apoptosis induction by PS-341 alone and by PS-341 combined with tumor necrosis factor-related apoptosis-inducing ligand were studied. In addition, the effects of vitamin C and other antioxidants on PS-341-mediated proteasome inhibition were also examined. Finally, the direct chemical interaction between vitamin C and PS-341 was determined. RESULTS: Vitamin C abrogated the ability of PS-341 to induce apoptosis in various human cancer cell lines, to induce G(2)-M arrest, and to augment apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand. Moreover, vitamin C suppressed PS-341-mediated inhibition of proteasome activity. PS-341 itself did not induce generation of intracellular reactive oxygen species whereas other antioxidants failed to abrogate its biological activity. Importantly, we detected a direct chemical interaction between vitamin C and PS-341. CONCLUSION: Vitamin C directly binds to PS-431, thus inactivating PS-341 independent of its antioxidant activity. Our findings suggest that vitamin C may have a negative effect on PS-341-mediated anticancer activity.     

The proteasome inhibitor bortezomib (PS-341) inhibits growth and induces apoptosis in primary effusion lymphoma cells

The ubiquitin-proteasome pathway is responsible for degrading many critical regulatory proteins involved in immune and inflammatory responses, control of cell growth and apoptosis. Recently, proteasome inhibitors have emerged as promising new therapeutic agents in hematological malignancies. Here we show that Bortezomib (PS-341), a proteasome-inhibitor, inhibits cellular proliferation and induces apoptosis in cell lines derived from Primary Effusion Lymphoma (PEL), a subtype of non-Hodgkin's lymphoma associated with infection by human herpes virus 8 (HHV-8). Bortezomib demonstrated more cytotoxicity against PEL cells than against cell lines derived from multiple myeloma, a disease for which is in current clinical use. Apoptosis induced by Bortezomib was associated with inhibition of the classical and alternative NF-kappaB pathways, upregulation of p53, p21 and p27 and activation of caspase cascade. Finally, treatment of PEL cells with Bortezomib exerted a synergistic or additive cytotoxic effect in combination with chemotherapeutic drugs or TRAIL. Taken together, these findings suggest that Bortezomib represents a promising agent for the treatment of PEL.     

Malignant lymphomas in Japan: epidemiological analysis on adult T-cell leukemia/lymphoma

According to the Vital Statistics Japan Series in 1950-1981, the age-adjusted death rate for malignant lymphomas (ICDs, 200-202) in Japan has increased in recent years. A geographical clustering of malignant lymphomas in the Kyushu districts was observed both in the period 1969-1971 and more recently in 1979-1981. The excessive rate of malignant lymphomas in Kyushu was due to the high incidence of adult T-cell leukemia/lymphoma (ATLL). The distribution of healthy carriers of ATLV, a human retrovirus which is almost identical to HTLV, was closely related to that of patients with ATLL in Japan. Epidemiological features of the infectious mode of ATLV suggested two main routes of natural transmission, one from mother to child and the other from husband to wife. ATLV is considered to be a main causative agent of ATLL from virological and epidemiological evidence, but infection by this virus alone may not result in ATLL because the incidence of ATLL among ATLV carriers was apparently not very high even in endemic areas of ATLL. Thus, some risk factors other than ATLV, such as environmental and genetic factors, may contribute to the development of ATLL.     

Presence of HTLV-I proviral DNA in patients with adult T-cell leukemia/lymphoma in Taiwan

Human T-lymphotropic virus-I (HTLV-I) proviral DNA was demonstrated in the leukemic cells of two newly identified cases of adult T-cell leukemia/lymphoma (ATL) in Taiwan by the Southern blot hybridization method. Therefore, the ATL cases diagnosed clinicopathologically in Taiwan were, for the first time, documented to be definitely related to HTLV-I.     

Adult T-cell leukemia/lymphoma and cluster of HTLV-I associated diseases in Brazilian settings.

We studied the transmission routes of human T-cell lymphotropic virus type I (HTLV-I) within families of 82 Brazilian patients diagnosed with adult T-cell leukaemia/lymphoma (ATL). Diagnosis of ATL in 43 male and 39 female patients was based on clinical and laboratory criteria of T-cell malignancy and detection of HTLV-I monoclonal integration. Samples were tested for HTLV antibodies and infection was confirmed as HTLV-I by Western Blot and/or polymerase chain reaction (PCR) assays. Overall 26/37 (70%) of mothers, 24/37 (65%) of wives, 8/22 (36%) of husbands, 34/112 (30%) of siblings and 10/82 (12%) offspring were HTLV-I infected. In 11 ATL patients, mothers were repeatedly HTLV-I seronegative, but HTLV-I pol or tax sequences were detected in 2 out of 6 cases tested by PCR. ATL patients with seronegative mothers related the following risk factors for HTLV-I infection: 6 were breast-fed by surrogate mothers with unknown HTLV-I status, 4 had a sexually promiscuous behaviour and 1 had multiple blood transfusions at a young age. Familial aggregation of ATL and other HTLV-I associated diseases such as HTLV-I myelopathy (HAM/TSP) and or uveitis, ATL in sibling pairs or in multiple generations was seen in 9 families. There were 6 families with ATL and TSP sibling pairs. In 3 families at least one parent had died with lymphoma or presenting neurological diseases and 2 offspring with ATL. Further to the extent of vertical and horizontal transmission of HTLV-I infection within ATL families, our results demonstrate that mothers who provide surrogate breast-milk appear to be an important source of HTLV-I transmission and ATL development in Brazil.     

Clustering and clinical diversity of adult T-cell leukemia/lymphoma associated with HTLV-I in a remote black population of French Guiana

An epidemiological study was performed in French Guiana (population 115,000) to determine the prevalence and incidence of adult T-cell leukemia/lymphoma (ATL) associated with human T-cell leukemia/lymphoma virus type I (HTLV-I). From January 1990 to December 1993, all suspected cases of ATL were enrolled in this study, and their clinical, epidemiological and immunovirological features were analyzed. Out of the 19 suspected cases, 18 were considered as ATL associated with HTLV-I (8 acute forms, 8 lymphoma types and 2 smoldering cases). Before this study, only 2 ATL cases had been reported in French Guiana over a 10-year period. This demonstrates that the number of ATL cases is greatly underestimated in most tropical HTLV-I endemic areas unless a specific disease search is performed. The mean age of the patients was 41 years. While HTLV-I antibodies were present in all cases, molecular studies demonstrated a clonal integration of HTLV-I in the tumoral cells in 7 cases out of the 9 tested. Fifteen patients died within a year of diagnosis. The crude incidence rate of ATL in French Guiana is around 3.5/100,000/year, a situation similar to that found in the Caribbean and in HTLV-I-endemic regions of Japan. However it reaches around 30/100,000/year (highest incidence ever described) in a small remote ethnic group of African origin (around 6200 inhabitants). Possible causes of ATL clustering in this ethnic group are presented. © 1995 Wiley-Liss, Inc.     

Synergistic efficacy in human ovarian cancer cells by histone deacetylase inhibitor TSA and proteasome inhibitor PS-341

Histone deacetylase inhibitors and proteasome inhibitor are all emerging as new classes of anticancer agents. We chose TSA and PS-341 to identify whether they have a synergistic efficacy on human ovarian cancer cells. After incubated with 500 nM TSA or/and 40 nM PS-341, we found that combined groups resulted in a striking increase of apoptosis and G2/M blocking rates, no matter in A2780, cisplatin-sensitive ovarian cancer cell line OV2008 or its resistant variant C13*. This demonstrated that TSA interacted synergistically with PS-341, which raised the possibility that combined the two drugs may represent a novel strategy in ovarian cancer.     

Phase I trial of the proteasome inhibitor PS-341 in patients with refractory hematologic malignancies.

PURPOSE: To determine the maximum-tolerated dose (MTD), dose-limiting toxicity (DLT), and pharmacodynamics (PD) of the proteasome inhibitor bortezomib (previously known as PS-341) in patients with refractory hematologic malignancies. PATIENTS AND METHODS: Patients received PS-341 twice weekly for 4 weeks at either 0.40, 1.04, 1.20, or 1.38 mg/m(2), followed by a 2-week rest. The PD of PS-341 was evaluated by measurement of whole blood 20S proteasome activity. RESULTS: Twenty-seven patients received 293 doses of PS-341, including 24 complete cycles. DLTs at doses above the 1.04-mg/m(2) MTD attributed to PS-341 included thrombocytopenia, hyponatremia, hypokalemia, fatigue, and malaise. In three of 10 patients receiving additional therapy, serious reversible adverse events appeared during cycle 2, including one episode of postural hypotension, one systemic hypersensitivity reaction, and grade 4 transaminitis in a patient with hepatitis C and a substantial acetaminophen ingestion. PD studies revealed PS-341 induced 20S proteasome inhibition in a time-dependent manner, and this inhibition was also related to both the dose in milligrams per meter squared, and the absolute dose of PS-341. Among nine fully assessable patients with heavily pretreated plasma cell dyscrasias completing one cycle of therapy, there was one complete response and a reduction in paraprotein levels and/or marrow plasmacytosis in eight others. In addition, one patient with mantle cell lymphoma and another with follicular lymphoma had shrinkage of nodal disease. CONCLUSION: PS-341 was well tolerated at 1.04 mg/m(2) on this dose-intensive schedule, although patients need to be monitored for electrolyte abnormalities and late toxicities. Additional studies are indicated to determine whether incorporation of dose/body surface area yields a superior PD model to dosing without normalization. PS-341 showed activity against refractory multiple myeloma and possibly non-Hodgkin's lymphoma in this study, and merits further investigation in these populations.     

Malignant lymphomas in Japan: Epidemiological analysis of adult T-cell leukemia/lymphoma (ATL)

The incidence of malignant lymphomas in Japan is relatively low compared to that in western European countries and the United States. However, in limited areas in Japan a specific type of lymphoid malignancy called adult T-cell leukemia/lymphoma (ATL), which is caused by human T-cell leukemia virus type I (HTLV-I), is highly prevalent, and there are also many healthy carriers of HTLV-I in the same areas. A cross-sectional seroepidemiological study of HTLV-I showed that the age-specific proportion of healthy HTLV-I carriers in these ATL-endemic areas increased with age, especially over 40, and was higher in females than in males. Three main routes of HTLV-I transmission are recognized: 1) vertical transmission from mother to child mainly through breast milk; 2) horizontal transmission from man to woman through semen, and; 3) parenteral transmission from carrier donor to non-carrier recipient. The annual incidence rate of ATL among HTLV-I carriers is estimated at 2.0 in males and 0.5 in females, and the cumulative risk for ATL in HTLV-I carriers during a 70-year life span is 1%–5%. Possible risk factors for ATL in addition to HTLV-I infection were considered, i.e. genetic factors, environmental factors, nutritional condition, thymus involution etc., but none of these were found to be clearly associated with ATL. To determine whether there exist particularly susceptible hosts for ATL in the ATL endemic areas, HLA types were examined, but no conclusive results on the positive relationships between HLA types and ATL manifestation or HTLV-I infection were obtained. From follow-up studies on the age-specific distribution of HTLV-I carriers in Japan, it is now speculated that the HTLV-I infection rate might have decreased naturally in the more recent generational cohort groups, even in the ATL-endemic areas. However, ATL in Japan is an important subject for study in the field of cancer epidemiology, and several trial intervention programs for the prevention of ATL, such as controls of vertical transmission from mother to child through breast milk, are now ongoing in the ATL-endemic areas of Japan.     

Regulatory T-cell function of adult T-cell leukemia/lymphoma cells

Adult T-cell leukemia/lymphoma (ATLL) patients are highly immunocompromised, but the underlying mechanism responsible for this state remains obscure. Recent studies demonstrated that FOXP3, which is a master control gene of naturally occurring regulatory T (Treg) cells, is expressed in the tumor cells from a subset of patients with ATLL. Since most ATLL cells express both CD4 and CD25, these tumors might originate from CD4(+)CD25(+)FOXP3(+) Treg cells, based on their phenotypic characteristics. However, whether ATLL cells actually function as Treg cells has not yet been clearly demonstrated. Here, we show that ATLL cells from a subset of patients are not only hypo-responsive to T-cell receptor-mediated activation, but also suppress the proliferation of autologous CD4(+) non-ATLL cells. Furthermore, ATLL cells from this subset of patients secrete only small amounts of IFN-gamma, and suppress IFN-gamma production by autologous CD4(+) non-ATLL cells. These are the first data showing that ATLL cells from a subset of patients function as Treg cells in an autologous setting. The present study provides novel insights into understanding the immunopathogenesis of ATLL, i.e., how HTLV-1-infected cells can survive in the face of host immune responses. It also adds to our understanding of ATLL patients' severely immunocompromised state.     

Tumor microenvironment of adult T-cell leukemia/lymphoma

Adult T-cell leukemia/lymphoma (ATLL) is a malignancy caused by the human T-cell leukemia virus type 1. Aggressive ATLL is refractory to conventional chemotherapy and has a poor prognosis. Better therapeutic approaches, including cancer immunotherapy, are required to improve survival and prognosis. The genetic landscape of ATLL reveals frequent genetic alterations in genes associated with immune surveillance, including major histocompatibility complex (MHC) class I, CD58 antigen, and programmed cell death ligand 1. Clinicopathological investigations also revealed tumor immunity mechanisms in ATLL, including immune checkpoint molecules, MHC molecules, tumor-associated macrophages, and chemokines. However, the tumor microenvironment of ATLL remains complex because ATLL itself originates from T-cells, usually expressing regulatory T-cell markers. In this review, we discuss the recent literature describing the tumor microenvironment of ATLL.     

Pathology and immunology of adult T-cell leukemia/lymphoma

Adult T-cell leukemia/lymphoma is a disseminated malignancy of T lymphocytes infected by the human T-lymphotrophic virus type I. It is endemic in southern Japan, the Caribbean basin, the southeastern United States, and central Africa. The virus is transmitted through intimate contact or exposure to blood products; only a small proportion of those infected develop adult T-cell leukemia/lymphoma. The malignant cells are activated T-helper cells that have suppressor function and multiple cytogenetic abnormalities. The disease occurs in mid to late life, years after exposure to the human T-lymphotrophic virus type I, with hypercalcemia, skin involvement, lymphadenopathy, hepatospenomegaly, lytic bone lesions, and leukemia. Although the disease usually has a rapidly progressive clinical course, there is a spectrum of clinical and pathologic features with smoldering and subacute forms. Our knowledge of this malignancy has grown rapidly and is leading to greater understanding of the relationships between human retroviruses and oncogenes.