Dianhydrogalactitol Overcomes Multiple Temozolomide Resistance Mechanisms in Glioblastoma.

Glioblastoma (GBM) is the most frequent and aggressive primary tumor type in the central nervous system in adults. Resistance to chemotherapy remains one of the major obstacles in GBM treatment. Identifying and overcoming the mechanisms of therapy resistance is instrumental to develop novel therapeutic approaches for patients with GBM. To determine the major drivers of temozolomide (TMZ) sensitivity, we performed shRNA screenings in GBM lines with different O6-methylguanine-DNA methyl-transferase (MGMT) status. We then evaluated dianhydrogalactitol (Val-083), a small alkylating molecule that induces interstrand DNA crosslinking, as a potential treatment to bypass TMZ-resistance mechanisms. We found that loss of mismatch repair (MMR) components and MGMT expression are mutually exclusive mechanisms driving TMZ resistance in vitro Treatment of established GBM cells and tumorsphere lines with Val-083 induces DNA damage and cell-cycle arrest in G2-M phase, independently of MGMT or MMR status, thus circumventing conventional resistance mechanisms to TMZ. Combination of TMZ and Val-083 shows a synergic cytotoxic effect in tumor cells in vitro, ex vivo, and in vivo We propose this combinatorial treatment as a potential approach for patients with GBM.

Anticancer activity and mechanisms of diacetyldianhydrogalactitol on hepatoma QGY-7703 cells.

Diacetyldianhydrogalactitol (DADAG) is a member of the hexitols which shows a significant anticancer effect. Despite the fact that the antitumor effects of DADAG have been studied in a number of cell lines, the mechanism of its action remains unclear. Herein, we explored antitumor effects of DADAG and the possible mechanisms by which it inhibited the growth of human hepatocellular carcinoma cell QGY-7,703 and its derived xenograft tumors. Cell proliferation was evaluated with the sulforhodamine B assay in vitro. The results suggested that DADAG had mild antiproliferative activity on QGY-7,703 cells. The antitumor effect of DADAG was assessed in nude mice xenografted with QGY-7,703 cells. We found that DADAG significantly inhibited the tumor growth. Flow cytometry results indicated that the retarded cell proliferation is associated with increased G2/M cell cycle arrest. Further studies showed that the induced G2/M cell cycle arrest is, at least partially, attributed to an upregulation of cyclin B1, phospho-cell division cycle 2 (cdc2) (Thr), phospho-cdc2 (Thr), and cdc25c protein expression, and a decrease in cdc2 protein expression. Taken together, our data show that DADAG has mild proliferative effects on QGY-7,703 cells in vitro, but it significantly inhibits the growth of QGY-7,703 in a xenograft model in vivo. The modulation of several cell cycle progression regulation proteins responsible for G2/M phase transition may account for its antitumor effects.

Complete remissions in chemotherapy of lung cancer models induced in mice by asbestos.

Chemotherapy trials were run with mice bearing transplants of carcinomas originally induced from fetal mouse lung cells by asbestos. After treatments with a nitrosourea (PCNU), mice bearing transplants of a large-cell carcinoma (ASB XIV) had complete remissions (CR) in 13 of 20 animals on a 15 mg/kg regimen, in 6 of 20 on an 8 mg/kg regimen, and in none of 10 on a 4 mg/kg regimen. With comparable total doses, treatment was most effective when PCNU was given in a few large doses. In groups where CRs occurred, continued PCNU treatment of animals without CRs prolonged survival but yielded no additional CRs. No CRs of ASB XIV occurred in 80 mice treated with eight other anticancer agents or in 50 controls injected with 0.9% NaCl solution. In mice bearing transplants of a squamous cell carcinoma (ASB XIII), treatments with PCNU were followed by CRs in 3 of 38 animals on 15 mg/kg regimens and in 3 of 28 animals on 8 mg/kg regimens. In groups of 6 mice fed a retinoid (Ro 10-9359) and treated with PCNU, CRs of ASB XIII occurred in 3 animals in each of two trials and in none in a third trial. Ro 10-9359 inhibited growth of transplants of squamous cell carcinoma LC 12 that had been induced from fetal mouse lung cells by a polycyclic hydrocarbon. In trials of four other anticancer agents vs. ASB XIII, CRs occurred only with cyclophosphamide (CPA). There were 7 CRs among 8 mice treated with CPA 100 mg/kg x 3, no CRs in 10 after 100 mg/kg x 2, one CR in 8 after 50 mg/kg x 3, and no CRs in 6 after 50 mg/kg x 4. With the 50 mg/kg x 4 regimen of CPA and 7.5 mg/kg PCNU on the same days, there were 5 CRs in 8 mice. As a single agent, aziridinylbenzoquinone (AZQ) increased life span but gave no CRs. There were CRs of ASB XIII in all of 8 mice after toxic combined therapy with PCNU and AZQ. There were no CRs in 66 control mice bearing ASB XIII.

High-dose melphalan with 6-hydroxydopamine-purged autologous bone marrow transplantation for poor-risk neuroblastoma.

Long-term results are presented of 28 patients who were diagnosed with neuroblastoma at more than 12 months of age and who received melphalan 180 mg/m2 (n = 6) or 240 mg/m2 (n = 22) to consolidate remissions of Stage IV disease or to control refractory disease. Twenty-four patients also received dianhydrogalactitol 180 to 240 mg/m2, and 11 received total body irradiation 450 to 600 cGy. Autologous bone marrow transplantation (ABMT) was performed with marrow that was unpurged (n = 2) or purged ex vivo (n = 26) with 6-hydroxydopamine (6-OHDA) 20 micrograms/ml plus ascorbate 200 micrograms/ml. The median time to an absolute neutrophil count of 500/microliters was 21 days and to self-sustaining platelet counts more than 20,000/microliters, 28 days. One patient required infusion of unpurged reserve marrow. Two groups of patients underwent ABMT: (1) 17 patients (Group I) who were in first remission a median of 7 months after diagnosis; and (2) 11 patients (Group II) who had refractory disease or were in second remission. For Group I, event-free survival was 29% at 12 months and 6% at 24 months post-ABMT. All Group II patients died of disease or ABMT-related toxicity. Overall, of the 28 patients, one is a long-term relapse-free survivor; five died of ABMT-related toxicity; ten patients with tumors present at ABMT had progressive disease within 6 months of ABMT; and 12 patients with no measurable disease at ABMT relapsed 4 to 32 months (median, 12) post-ABMT. Among the latter, six relapses involved the primary site, and six were restricted to distant sites. These results--in accord with the long-term outcome in other series--suggest that for neuroblastoma high-dose melphalan cannot be relied on to ablate residual disease or to salvage patients with refractory tumors. In addition, the pattern of relapse in several patients could be explained by infusion of incompletely purged autografts; this would support recent laboratory evidence that 6-OHDA/ascorbate is a suboptimal purging method.

Combined treatment of anaplastic astrocytoma (grade 3-4) with diacetyl-dianhydro-galactitol (DADAG).

Autoradiographic studies of labeled diacetyldianhydro-galactitol (DADAG) with tumor bearing animals revealed that the CNS accumulates high amounts of DADAG-derived radioactivity and the elimination from the brain seems to be relatively slow. This observation and the activity of DADAG against murine ependymoblastoma classified the drug as a promising agent for the treatment of malignant brain tumors. In a series of 30 evaluable consecutive patients who were operated on for anaplastic astrocytomas, DADAG has been applied during and subsequent to postoperative radiotherapy. No severe toxicity occurred. Survivals were compared with a group of patients who got irradiation alone. Statistical analysis did not show significantly better survivals in the DADAG treated group: median value was 46.5 weeks, p = 0.232.

Absence of cross-resistance between two alkylating agents: BCNU vs bifunctional galactitol.

Dianhydrogalactitol (DAG) increased the life span of both BCNU-sensitive and -resistant L1210 tumor-bearing mice. However, the BCNU-resistant strain showed slightly lower sensitivity against DAG, which could be overcome by an increase in drug dose of ca. 20%. The somewhat lower sensitivity was proportional to a slightly reduced DNA cross-linking formation induced by DAG in BCNU-resistant cells. The amount of DNA cross-links was determined by measurement of the 1,6-di(guaninyl)-galactitol content of DNA. The slight reduction in cross-links is not attributable to DNA repair but rather to other factors that seem to prevent the formation of DNA-drug adducts. The absence of cross-resistance is explained by different kinds of DNA damage caused by the two alkylating agents and the presumably different defense mechanisms developed by cells against these lesions.

Development and some characteristics of a P388 leukemia strain resistant to 1, 2:5, 6-dianhydrogalactitol.

Repeated intraperitoneal treatment of standard P388 mouse leukemia with dianhydrogalactitol (DAG) resulted in the development of a P388/DAG experimental mouse tumor which was resistant to the drug. Resistance was stable without DAG treatment throughout 80 passages. P388/DAG shows cross-resistance to alkylating agents such as nitrogen-mustard, cyclophosphamide and diacetyl-DAG but not to selected antimetabolites and tubulin binders and exhibits reduced sensitivity to nitrosoureas. Resistance to DAG could not be overcome by the administration of maximally tolerated dose of DAG to tumor bearing mice. The resistant tumor is one chromosome short and shows a 13-fold increase of cells possessing a submetacentric marker chromosome.

Chemotherapy of human non-Hodgkin lymphoma (NHL) xenografts.

Three human NHL (B-cell type) were established successfully as serially transplantable xenografts in artificially immunesuppressed CBA mice. All of them preserved the phenotypic characteristics of the original tumour even after several passages. The transplanted tumours were highly sensitive to cyclophosphamide and methotrexate, reflecting the results obtained in clinical practice, and relatively sensitive to dianhydrogalactitol.

Phase II trials of hexamethylmelamine, dianhydrogalactitol, razoxane, and beta-2'-deoxythioguanosine as single agents against advanced measurable tumors of the pancreas. Gastrointestinal Tumor Study Group.

Phase II trials of several single agents demonstrated only minimal objective response rates in patients with pancreatic carcinoma and measurable tumors: hexamethylmelamine (7%; four responses among 55 patients); dianhydrogalactitol (2.5%; one response among 40 patients); razoxane (7%; two responses among 29 patients); and beta-2'-deoxythioguanosine (6%; two responses among 32 patients). Among patients with a good performance status (0-2) and no prior chemotherapy, response rates were 8% for hexamethylmelamine (two responses among 26 patients); 8% for dianhydrogalactitol (one response among 13 patients); 8% for razoxane (one response among 12 patients); and 10% for beta-2'-deoxythioguanosine (two responses among 20 patients). None of these agents given by the methods of this study offers substantive benefit to the patient with advanced pancreatic cancer.

Characteristics and chemotherapeutic sensitivity of a human testicular cancer grown in artificially immunosuppressed mice.

Seven human testicular tumors were transplanted into artificially immunosuppressed mice. Two of them grew progressively (TT2 and TT6) and a serially transplantable line was developed from TT2. The xenografts maintained only the embryonal carcinoma components of originally mixed (embryonal cell carcinoma and choriocarcinoma) donor tumor. Although the histology did not change remarkably with passages, the xenografts lost their capacity to express human choriogonadotropin and alpha-fetoprotein. The latency period shortened, the growth rate remained similar with subsequent transplantations. The tumor cells of the TT2 line presented the human character according to chromosome analysis and were built up of two subsets of cells with a different DNA index estimated by flow cytometry. The embryonal cell carcinoma line was highly sensitive to CY and cisDDP. PVB combination was also effective, although the tumor growth inhibition proved to be only temporary.

Damages of DNA synthesis in normal and tumor cells with sugar alcohol derivatives.

The rates of incorporation of 2-14C-thymidine into DNA of melanoma B16, bone marrow, gastrointestinal mucosa, spleen and liver at various time after administration of dianhydrogalactitol (DAG), 3,4-diacetyldianhydrogalactitol (DiacDAG) and 3,4-disuccinyldianhydrogalactitol (DisuDAG) at maxima nonlethal single doses to tumor-bearing mice were studied. The sugar alcohol derivatives induced the stable inhibition in DNA synthesis of tumor cells. DNA synthesis in normal dividing cells was shown to recover more rapidly than in melanoma B16 cells after administration of all drugs. DisuDAG is characterized by stronger inhibitory effect on DNA synthesis in melanoma B16 cells at the half of the single maxima nonlethal dose compared with DAG and DiacDAG. Differing from DAG, DiacDAG and DisuDAG did not effect the incorporation of 2-14C-thymidine into DNA of liver cells. In vivo inhibition of DNA synthesis in melanoma B16 cells with DiacDAG was not due to damage of the TCA soluble fraction.

Phase II evaluation of dianhydrogalactitol in the treatment of advanced non-squamous cervical carcinoma. A Gynecologic Oncology Group study.

In an on-going Phase II evaluation, dianhydrogalactitol (NSC 132313) was administered intravenously to 28 patients with advanced or recurrent non-squamous cell carcinoma of the cervix. The initial dosage was 60 mg/m2/wk with escalation to 75 mg/m2/wk if there were no adverse effects. Twenty-seven patients were evaluable for toxicity and response. There was one complete response and one partial response. Adverse effects were not infrequent but tolerable.

Phase II trial of galactitol 1,2:5,6-dianhydro (NSC 132313) in the treatment of advanced gynecologic malignancies: a Gynecologic Oncology Group study.

Dianhydrogalactitol was administered intravenously to patients with advanced or recurrent gynecologic malignancies on a weekly schedule. The initial dosage was 60 mg/m2 with escalation to 75 mg/m2 if there were no adverse effects. Forty-two patients with ovarian epithelial adenocarcinoma (OEA) and forty-one patients with squamous carcinoma of the cervix (SCC) were entered into this study. Of these, 39 patients with OEA and 36 with SCC were evaluable for toxicity and response. Seven patients (19.4%) with SCC had an objective response, while six patients (15.4%) with OEA had an objective response. Adverse effects were frequent but tolerable. There were no drug-related deaths, and only two patients suffered life-threatening hematologic toxicity. Myelosuppression was observed more frequently among the patients with OEA. A higher percentage of OEA patients had received prior chemotherapy. The level of activity and frequency of adverse effects observed at this dose schedule warrant further studies of this drug in these two tumors.

Phase II evaluation of galactitol in head and neck cancer: a Southwest Oncology Group Study.

A phase II evaluation of DAG in head and neck cancer patients produced one partial response and one patient with prolonged stable disease from nine fully evaluable patients with squamous carcinomas. One partial response was produced in a patient with anaplastic thyroid carcinoma. No drug deaths or life-threatening toxicity were reported. Further evaluation of DAG in thyroid tumors is warranted.

Ineffectiveness of chemotherapy in patients with metastatic ependymoma of the cauda equina.

Six cases of intraspinal ependymoma metastasizing outside the central nervous system have been reported. This report documents a seventh case. This patient received multiple courses of combination and single-agent chemotherapy without evidence of tumor regression. All seven patients had four things in common: early onset of disease, numerous operations, massive local recurrence at the time distant metastases were noted, and a long time period from diagnosis to death.

Effect of phase I and II chemotherapeutic agents against human lymphomas heterotransplanted in nude mice.

Ten chemotherapeutic agents, mostly phase I and II drugs, were tested for activity against two human lymphomas heterotransplanted in nude mice. Three of these agents have been tested in phase II trials in patients with lymphoma and found to lack activity; a corresponding lack of activity was found in lymphoma-bearing nude mice. Apart from cyclophosphamide, which is known to have activity against lymphoma and was used as a positive control, only dianhydrogalactitol (DAG) had antitumor activity in the lymphoma-bearing nude mice. Tumor regressions induced by DAG in a heterotransplanted diffuse histiocytic lymphoma were determined to be significant using a statistical method designed for such studies. These data suggest there should be further study of DAG in patients with lymphoma.