Marizomib, a proteasome inhibitor for all seasons: preclinical profile and a framework for clinical trials.

The proteasome has emerged as an important clinically relevant target for the treatment of hematologic malignancies. Since the Food and Drug Administration approved the first-in-class proteasome inhibitor bortezomib (Velcade) for the treatment of relapsed/refractory multiple myeloma (MM) and mantle cell lymphoma, it has become clear that new inhibitors are needed that have a better therapeutic ratio, can overcome inherent and acquired bortezomib resistance and exhibit broader anti-cancer activities. Marizomib (NPI-0052; salinosporamide A) is a structurally and pharmacologically unique ß-lactone-γ-lactam proteasome inhibitor that may fulfill these unmet needs. The potent and sustained inhibition of all three proteolytic activities of the proteasome by marizomib has inspired extensive preclinical evaluation in a variety of hematologic and solid tumor models, where it is efficacious as a single agent and in combination with biologics, chemotherapeutics and targeted therapeutic agents. Specifically, marizomib has been evaluated in models for multiple myeloma, mantle cell lymphoma, Waldenstrom's macroglobulinemia, chronic and acute lymphocytic leukemia, as well as glioma, colorectal and pancreatic cancer models, and has exhibited synergistic activities in tumor models in combination with bortezomib, the immunomodulatory agent lenalidomide (Revlimid), and various histone deacetylase inhibitors. These and other studies provided the framework for ongoing clinical trials in patients with MM, lymphomas, leukemias and solid tumors, including those who have failed bortezomib treatment, as well as in patients with diagnoses where other proteasome inhibitors have not demonstrated significant efficacy. This review captures the remarkable translational studies and contributions from many collaborators that have advanced marizomib from seabed to bench to bedside.

Combined radiation and cytochrome CYP4B1/4-ipomeanol gene therapy using the EGR1 promoter.

BACKGROUND: Cytochrome p450 isozyme CYP4B1 converts the inert prodrug 4-ipomeanol (4-IM) into toxic alkylating metabolites. Induction of cytotoxicity by 4-IM combined with ionizing radiation (IR) in cells transfected with a fusion protein of rabbit cytochrome CYP4B1 under control of the radiation inducible EGR1 promoter was investigated. The capability of activated 4-IM to sensitize cells to IR was also assessed. MATERIALS AND METHODS: Survival fractions of cells, determined by MTT assays, stably transfected with EGR1-CYP4B1 were compared with that of cells transfected with a control plasmid after IR followed by 4-IM. Radiosensitization was tested by comparing clonogenic survival curves of cells transfected with the CYP4B1 cassette under a CMV promoter instead of EGR-1, irradiated with or without 4-IM. RESULTS: MTT assays for cytotoxicity indicated a decrease in relative survival fractions (survival with 4-IM/survival without 4-IM) of the EGR1-CYP4B1 transfected cells with increasing radiation dosage, but not of control cells. Clonogenic assays revealed decreased survival fractions with increasing radiation doses (CYP4B1 transfected and control cells) and 4-IM concentrations (CYP4B1 transfected cells), but showed no significant differences in slope of survival curves with 4-IM. CONCLUSION: The results indicate IR potentiates the cytotoxic activity of the EGR1-CYP4B1/4-IM transgene system, but activated 4-IM does not sensitize cells to IR. Thus, the EGR1-CYP4B1/4-IM system is a viable radiation-gene therapy system that may allow for improved spatial and temporal control of cytotoxicity by therapeutic radiation fields.

Isolation, characterization, and recovery of small peptide phage display epitopes selected against viable malignant glioma cells.

Phage display techniques rely on nearly random oligonucleotide sequences inserted into the protein III filament binding protein of an Escherichia coli filamentous phage M13 to generate a library of phage that express more than 10(7) different peptides. Phage that expresses a sequence having high affinity for a specific molecule, cell, or tissue can then be isolated through selective binding and recovery. Selected phage cannot only be used as gene transfer vectors in themselves, but the small peptide epitopes can be sequenced and potentially recombined into the attachment proteins of viral vectors, or used by themselves to target other therapeutic agents and diagnostic imaging radiolabels. Most phage display selections are carried out against purified and/or fixed protein targets, raising concerns as to the relevance of the selected epitopes. We have selected phage from the CMTI library against viable U87-MG human malignant glioma cells using a derivation of biopanning. The library, which initially contained phage expressing 2x10(7) different epitope sequences, collapsed after four rounds of selection such that 42% of recovered clones expressed a consensus sequence. Selective binding to viable adherent U87-MG cells was subsequently demonstrated under physiologic conditions at 167% (+/-27%) unselected phage using a novel, viable enzyme-linked immunosorbent assay technique. In comparison, there was no difference in binding to control 9L rat gliosarcoma, PANC-1 human pancreatic adenocarcinoma, T98-MG human malignant glioma, or AST-4 human malignant glioma cells of selected compared to unselected phage. Using polymerase chain reaction, the epitope was recovered with flanking unique restriction sites for recombination into a herpes simplex virus type-1 vector. This study demonstrates and discusses optimized methodologies for using phage display to target viable cells.

Cytotoxicity, apoptosis, and viral replication in tumor cells treated with oncolytic ribonucleotide reductase-defective herpes simplex type 1 virus (hrR3) combined with ionizing radiation.

The viral ribonucleotide reductase (rR)-defective herpes simplex type-1 (HSV-1) virus (hrR3) has been shown previously to preferentially replicate in and kill tumor cells. This selectivity is associated with tumor cell up-regulation of mammalian rR. Ionizing radiation (IR) is currently used in the therapy of many malignancies, including glioblastoma, cervical carcinoma, and pancreatic carcinoma. IR has been shown to up-regulate mammalian rR in tumor cells and appears to increase the efficacy of at least one non-rR-deleted HSV-1 strain in an in vivo tumor model. Here, we test the hypothesis that a single therapeutic radiation fraction will increase the replication and toxicity of hrR3 for malignant cell lines in vitro. PANC-1 pancreatic carcinoma, U-87 glioblastoma, and CaSki cervical carcinoma cell lines were treated with varying doses of IR and subsequently infected with hrR3 or KOS (wild-type HSV-1 strain). Cell survival was then measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and trypan blue exclusion cytometry. At 72 hours posttreatment, irradiation with 2 Gy reduced survival from 100% to 76% in noninfected cells, from 61% to 48% in KOS-infected cells, and from 39% to 27% in hrR3-infected PANC-1 cells. As such, analysis of variance indicated that the toxicity of the two modalities was additive. Similar additivity was seen in U-87 MG and CaSki cells. Absolute survival of hrR3-infected or KOS-infected PANC-1 cells decreased as a function of time after treatment (24-72 hours) and multiplicity of infection (MOI) (0.05-5.0). However, the relative decrease in survival with the addition of IR to hrR3 or KOS in PANC-1 cells was not markedly affected by altering MOI (0.05-5.0), time (24-72 hours), radiation dose (2-20 Gy), or cell culture conditions (confluent/growth arrested). We used fluorescence-activated cell sorter analysis with the cationic lipophilic dye DiOC6 to quantify a reduction in mitochondrial membrane potential that'is associated with apoptosis. Fluorescence-activated cell sorter analysis indicated increased apoptosis in both hrR3- and IR-treated cells at 48-72 hours, with hrR3 alone producing the most induction. Viral yields from PANC-1 cells after irradiation and infection were examined. No significant differences were seen between irradiated and nonirradiated cells in viral replication, with hrR3 producing single-step titers of 3.1 +/- 0.9 x 10(5) and 4.0 +/- 1.2 x 10(5) plaque-forming units/mL in nonirradiated and irradiated cells. Thus, complementary toxicity was seen between IR and hrR3 or KOS, regardless of cell type, time, MOI, IR dose, or culture conditions, without evidence of augmented apoptosis or viral replication.

Efficient DNA subcloning through selective restriction endonuclease digestion.

Described here is a selective restriction endonuclease digestion method that eliminates the electrophoresis step that is usually used during the subcloning of new DNA sequences into typical E. coli-based plasmids. The method increases yield while decreasing laboratory resource and time utilization. By using donor and acceptor sequences that contain unique restriction sites found only outside of the intended recombination sequences, the initial digestion products can be directly combined without electrophoresis if the ligation step is followed by a selective digestion using the unique restriction enzymes before transformation. This system is based on the several order of magnitude decrease in transformation efficiency of linearized compared to circular plasmids. As an example, this method was used to obtain recombinants between a 3.6 kb acceptor plasmid and 3.0 kb insert following one ligation reaction after the failure of nine standard reactions using similar amounts of input DNA. It is particularly applicable to situations in which low subcloning efficiencies are expected. The technique can be extended to a large percentage of planned recombinations by using nonidentical compatible cohesive or blunt-ended fragments, or site-directed mutagenesis.

Tolerance of autologous and allogeneic bone grafts to therapeutic radiation in humans.

PURPOSE: To examine the effect of perioperative irradiation on bone graft healing and functional integrity. METHODS AND MATERIALS: Fifty-five bone grafts (10 autologous and 45 allogeneic) performed between 1978 and 1995 were evaluated retrospectively. Sixteen received preoperative radiation, 11 received postoperative, and 13 were treated with a combination of pre- and postoperative radiation. Fifteen nonirradiated grafts were randomly selected to serve as controls. Twenty-three of the grafts were placed in patients who received chemotherapy in the perioperative period. Functional graft survival and radiographic healing quality were evaluated. RESULTS: Overall rates of graft survival at 1 year were 89% for autografts and 79% for allografts. Graft survival rates were 86% and 68% at 1 and 5 years for the irradiated group, and 67% and 58% for the control group. No significant difference was seen in the Kaplan-Meier graft survival curves of the two groups. There was a nonsignificant trend toward improved radiographic healing quality in the control group. No significant differences in outcome based on treatment chronology were found with survival rates of 88% for preoperative treatment and 100% for postoperative treatment. No relation between outcome and bone dose (preoperative + postoperative dose), graft dose (postoperative dose), or mean dose/day was found. There was a trend (p = 0.0525) toward worse outcome seen in the Kaplan-Meier curves of patients who received chemotherapy. This difference, however, was not seen in the 1-year survival rates or healing quality. Tobacco use tended toward predicting failure, with 63% graft survival compared to 85% in nonsmokers (p = 0.09). Healing quality was significantly lower in the smoking group. CONCLUSION: The low failure rate of grafts in irradiated sites, overall and compared to controls from this study and relevant literature, as well as the lack of dose and time effects, does not support significant deviation from the indicated treatment regimen for patients who have received or are expected to receive a graft. The trend toward decreased quality of radiographic bone healing, and data published in relevant literature indicating improved healing when radiation is withheld until 3-4 weeks postoperatively suggest this delay should be attempted when not expected to otherwise compromise patient outcome. A nonsignificant trend only for the effect of chemotherapy on bone grafts was seen, thus we do not recommend changes in its use as appropriate for disease management other than a preference against use during the immediate perioperative period.

Gene therapy of gliomas: receptor and transcriptional targeting.

Through incremental increases in the overall therapeutic ratio of combined modality regimens, each addition of unique selective toxicity to a tumor moves one step closer to a cure. The primary advantage of adding gene therapy strategies to current oncologic regimens is the ability to design multiple levels of unique biologic selectivity into vectors using recombinant technology. This article presents an overview of current and potential methods for designing vectors targeted to high grade gliomas through selective cell entry or transcriptional regulation. Cell entry based methodologies are founded on increasing relative uptake of the vector through the chemical or recombinant addition of epitopes which bind to receptors selectively expressed on target cells. Transcriptional targeting utilizes promoter and enhancer systems which have potential for selectively activating transcription for transgene expression or vector propagation in target cells.

Targeting gene therapy vectors to CNS malignancies.

Gene therapy offers significant advantages to the field of oncology with the addition of specifically and uniquely engineered mechanisms of halting malignant proliferation through cytotoxicity or reproductive arrest. To confer a true benefit to the therapeutic ratio (the relative toxicity to tumor compared to normal tissue) a vector or the transgene it carries must selectively affect or access tumor cells. Beyond the selective toxicities of many transgene products, which frequently parallel that of contemporary chemotherapeutic agents, lies the potential utility of targeting the vector. This review presents an overview of current and potential methods for designing vectors targeted to CNS malignancies through selective delivery, cell entry, transport or transcriptional regulation. The topic of delivery encompasses physical and pharmaceutic means of increasing the relative exposure of tumors to vector. Cell entry based methodologies are founded on increasing relative uptake of vector through the chemical or recombinant addition of ligand and antibody domains which selectively bind receptors expressed on target cells. Targeted transport involves the potential for using cells to selectively carry vectors or transgenes into tumors. Finally, promoter and enhancer systems are discussed which have potential for selectivity activating transcription to produce targeted transgene expression or vector propagation.

Individualizing management of aggressive fibromatoses.

PURPOSE: To examine prognostic indicators in aggressive fibromatoses that may be used to optimize case-specific management strategy. METHODS AND MATERIALS: One hundred and seven fibromatoses presenting between 1971 and 1992 were analyzed. The following treatment modalities were utilized: (a) surgery alone for 51 tumors; (b) radiation alone for 15 tumors; and (c) radiation and surgery (combined modality) for 41 tumors. Outcome analysis was based on 5-year actuarial local control rates. RESULTS: Control rates among surgery, radiation therapy, and combined modality groups were 69%, 93%, and 72%. Multivariate analysis identified age < 18 years, recurrent disease, positive surgical margins, and treatment with surgery alone as predictors for failure. Patients treated with surgery alone had control rates of 50% (3 of 6) for gross residual, 56% for microscopically positive margins, and 77% for negative margins. Radiation and surgery resulted in rates of 59% for gross residual, 78% for microscopically positive margins, and 100% (6 of 6) for negative margins. For recurrent vs. primary tumors, control was achieved in 48% vs. 77%, 90% vs. 100% (5 of 5), and 67% vs. 79% in the Surgery, Radiation, and Combined modality Groups, respectively. Patients presenting with multiple disease sites tended to have aggressive disease. A radiation dose-control relation to > 60 Gy was seen in patients with unresected or gross residual disease. Of the patients, 23 with disease involving the plantar region had a control rate of 62%, with significantly worse outcomes in children. CONCLUSIONS: These results are consistent with those found in the relevant literature. They support primary resection with negative margins when feasible. Radiation is a highly effective alternative in situations where surgery would result in major functional or cosmetic defects. When negative surgical margins are not achieved in recurrent tumors, radiation is recommended. Perioperative radiation should be considered in other high-risk groups (recurrent disease, positive margins, and plantar tumors in young patients). Doses of 60-65 Gy for gross disease and 50-60 Gy for microscopic residual are recommended. Observation may be considered for primary tumors with disease remaining in situ when they are located such that progression would not cause significant morbidity. Although plantar lesions in children may represent a group at high risk for recurrence or aggressive behavior, the greater potential for radiation-induced morbidity in this group must also temper its use. Given the inconsistent nature and treatment response of this tumor, it is fundamental that treatment recommendations should be made based on the risk:benefit analysis for the individual patient, dependent on tumor characteristics and location, as well as patient characteristics and preferences.

Hemoglobin difference between black and white women with comparable iron status: justification for race-specific anemia criteria.

To determine the appropriateness of race-specific criteria for anemia, we used the sample of women of child-bearing age from the Second National Health and Examination Survey to examine the relationship between hemoglobin and iron status for blacks and whites. After adjustment for major factors known to cause hemoglobin variation, including iron nutrition status, black women overall had a significantly lower mean hemoglobin value (126 +/- 12 g/L) than white women (134 +/- 11 g/L). Comparison of the probability plots of black and white hemoglobin distributions found the difference across the distributions to not be uniform, likely because a subset of black women had lower hemoglobin values rather than because of a generalized lowering. This finding suggests that it may not be appropriate to have a separate criteria for all blacks to accommodate the subset with lower hemoglobin. However, evaluation of the screening performance of hemoglobin found that race-specific anemia criteria (10 g/L difference) yielded a comparable sensitivity and specificity in detecting iron deficiency for both races. In contrast, a fixed anemia criterion did not yield comparable screening performances for the two races. This functional evaluation supports considering race-specific anemia criteria for screening iron deficiency.

Toxicology of daily administration to mice of the radiation potentiator SR 4233 (WIN 59075).

We have investigated the feasibility of administration of an effective dose of the hypoxic cytotoxin, SR 4233, Monday-Friday daily for 6 weeks. From a thorough hematological, histopathological and clinical chemistry evaluation throughout the course and during a 3-week recovery period, we conclude that daily administration of a radiopotentiating dose of SR 4233 in mice is well tolerated and that bone marrow suppression is likely to be the dose-limiting toxicity.