G. Peter Beardsley

Expression of HER2/erbB-2 Correlates With Survival in Osteosarcoma

PURPOSE In osteosarcoma, prognostic factors at diagnosis other than clinical stage have not been clearly identified. The aim of this study was to determine whether human epidermal growth factor receptor 2 (HER2)/erbB-2, p-glycoprotein, or p53 expression correlated with histologic response to preoperative chemotherapy or event-free survival. PATIENTS AND METHODS We performed a retrospective immunohistochemical study on material obtained from patients treated on the Memorial Sloan-Kettering Cancer Center T12 protocol between 1986 and 1993. Paraffin-embedded tissue was identified from 53 patients (73% of patients enrolled onto protocol) and stained for HER2/erbB-2, p53, and p-glycoprotein expression using standard monoclonal antibodies and methods. RESULTS At the time of initial biopsy, 20 (42.6%) of 47 samples demonstrated high levels of HER2/erbB-2 expression. Higher frequencies of expression were observed in samples from patients with metastatic disease at presentation and at the time of relapse. Expression of HER2/erbB-2 correlated with a significantly worse histologic response (P =.03). In patients presenting with nonmetastatic disease, expression of HER2/erbB-2 at the time of initial biopsy was associated with a significantly decreased event-free survival (47% v 79% at 5 years, P =.05). p53 and p-glycoprotein expression did not correlate with histologic response or patient event-free survival. CONCLUSION The correlation of HER2/erbB-2 expression with histologic response to preoperative chemotherapy and event-free survival in this study suggests that HER2/erbB-2 should be evaluated prospectively as a prognostic indicator. The correlation also suggests that clinical trials of antibodies that target this receptor, such as recombinant humanized anti-HER2 monoclonal antibody (Herceptin; Genentech, San Francisco, CA), should be considered for the treatment of osteosarcoma.

Human 5-Aminoimidazole-4-carboxamide Ribonucleotide Transformylase/Inosine 5′-Monophosphate Cyclohydrolase A BIFUNCTIONAL PROTEIN REQUIRING DIMERIZATION FOR TRANSFORMYLASE ACTIVITY BUT NOT FOR CYCLOHYDROLASE ACTIVITY

The bifunctional enzyme aminoimidazole carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC) is responsible for catalysis of the last two steps in the de novo purine pathway. Gel filtration studies performed on human enzyme suggested that this enzyme is monomeric in solution. However, cross-linking studies performed on both yeast and avian ATIC indicated that this enzyme might be dimeric. To determine the oligomeric state of this protein in solution, we carried out sedimentation equilibrium analysis of ATIC over a broad concentration range. We find that ATIC participates in a monomer/dimer equilibrium with a dissociation constant of 240 ± 50 nm at 4 °C. To determine whether the presence of substrates affects the monomer/dimer equilibrium, further ultracentrifugation studies were performed. These showed that the equilibrium is only significantly shifted in the presence of both AICAR and a folate analog, resulting in a 10-fold reduction in the dissociation constant. The enzyme concentration dependence on each of the catalytic activities was studied in steady state kinetic experiments. These indicated that the transformylase activity requires dimerization whereas the cyclohydrolase activity only slightly prefers the dimeric form over the monomeric form.

10-formyl-5,10-dideaza-acyclic-5,6,7,8-tetrahydrofolic acid (10-formyl-DDACTHF) a potent cytotoxic agent acting by selective inhibition of human GAR Tfase and the de novo purine biosynthetic pathway

The synthesis of 10-formyl-DDACTHF (3) as a potential inhibitor of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase) is reported. Aldehyde 3, the corresponding gamma- and alpha-pentaglutamates 21 and 25 and related agents were evaluated for inhibition of folate-dependent enzymes including GAR Tfase and AICAR Tfase. The inhibitors were found to exhibit potent cytotoxic activity (CCRF-CEM IC(50) for 3=60nM) that exceeded their enzyme inhibition potency [K(i) (3)=6 and 1 microM for Escherichia coli GAR and human AICAR Tfase, respectively]. Cytotoxicity rescue by medium purines, but not pyrimidines, indicated that the potent cytotoxic activity is derived from selective purine biosynthesis inhibition and rescue by AICAR monophosphate established that the activity is derived preferentially from GAR versus AICAR Tfase inhibition. The potent cytotoxic compounds including aldehyde 3 lost activity against CCRF-CEM cell lines deficient in the reduced folate carrier (CCRF-CEM/MTX) or folylpolyglutamate synthase (CCRF-CEM/FPGS(-)) establishing that their potent activity requires both reduced folate carrier transport and polyglutamation. Unexpectedly, the pentaglutamates displayed surprisingly similar K(i)s versus E. coli GAR Tfase and only modestly enhanced K(i)s versus human AICAR Tfase. On the surface this initially suggested that the potent cytotoxic activity of 3 and related compounds might be due simply to preferential intracellular accumulation of the inhibitors derived from effective transport and polyglutamation (i.e., ca. 100-fold higher intracellular concentrations). However, a subsequent examination of the inhibitors against recombinant human GAR Tfase revealed they and the corresponding gamma-pentaglutamates were unexpectedly much more potent against the human versus E. coli enzyme (K(i) for 3, 14nM against rhGAR Tfase versus 6 microM against E. coli GAR Tfase) which also accounts for their exceptional cytotoxic potency.

A study of toxicity and comparative therapeutic efficacy of vindesine-prednisone vs. vincristine-prednisone in children with acute lymphoblastic leukemia in relapse. A Pediatric Oncology Group study.

SummaryVindesine (des-acetyl Vinblastine) is a synthetic derivative of vinblastine, and was produced with the hope that it would have less neurotoxicity and hematopoietic toxicity than other vinca alkaloids.Phase I and II studies also demonstrated significant activity in lymphoid malignancies, especially Acute Lymphoblastic Leukemia (ALL). The present study was designed to compare therapeutic effectiveness of twice weekly vindesine (2 mg/M2/dose) plus Prednisone (60 mg/M2/dose) (Treatment 1) to weekly Vincristine (2 mg/M2/dose) plus Prednisone (60 mg/M2/day) (Treatment 2). All patients were less than 21 years of age, and had documented bone marrow relapse (blast count>25%). In 39 patients presumed sensitive to vincristine, there were 11 complete responses out of 20 patients (55%) randomized to receive vindesine/ prednisone and 7 complete responses out of 19 patients (37%) treated with Vincristine/Prednisone. In 37 patients resistant to vincristine, there were 7 complete responses (19%). Vindesine was more toxic than Vincristine. Major toxicities of vindesine included paraesthesias, peripheral neuropathy and ileus. Vindesine hematological toxicity appeared greater, but such toxicity is hard to assess in patients with bone marrow disease. In this study, vindesine and vincristine had similar efficacy, but vindesine use was associated with more toxicity.

Induction of HL-60 leukemia cell differentiation by tetrahydrofolate inhibitors of de novo purine nucleotide biosynthesis.

Summary5,10-Dideazatetrahydrofolic acid (DDATHF) is a folate antimetabolite that shows activity against glycinamide ribonucleotide (GAR) transformylase, a folate-requiring enzyme in the de novo purine nucleotide biosynthetic pathway. Previous studies from our laboratory have shown that DDATHF is an effective inducer of the maturation of HL-60 promyelocytic leukemia. In solution, DDATHF is a mixture of two diastereomers due to an asymmetric configuration at carbon 6. Incubation of HL-60 cells with 1 μM of each diastereomer resulted in an inhibition of cellular proliferation after 48 h that preceded an increase in the number of differentiated myeloid cells, as determined by the ability of cells to reduce nitroblue tetrazolium (NBT) and by the binding of the myeloid-specific antibody Mo 1. Several analogs of DDATHF were also tested as inducers of the differentiation of HL-60 cells. With the exception of the 10-acetyl analog of 5-deazatetrahydrofolic acid, all compounds displayed similar activities as inducers of maturation. The finding that both stereoisomers of DDATHF, as well as the analogs tested, could selectively reduce intracellular purine nucleotide levels suggested that these compounds inhibited purine nucleotide biosynthesis de novo. This possibility was confirmed by the finding that hypoxanthine completely prevented the reduction of intracellular purine nucleotide levels, as well as the induction of differentiation and the inhibition of cellular growth, by these folate analogs. The results suggest that GAR transformylase is a target for a series of compounds whose structures resemble that of tetrahydrofolate and indicate that the inhibition of GAR transformylase by these compounds is sufficient to induce the maturation of HL-60 leukemia cells.

Mechanism of ganciclovir-induced chain termination revealed by resistant viral polymerase mutants with reduced exonuclease activity

Significance Nucleoside analogues include important drugs that target DNA polymerases and cause chain termination. However, how ganciclovir, the first line of treatment for human cytomegalovirus infections, does this is unknown. Ganciclovir resistance is a serious clinical problem. Many ganciclovir-resistant isolates contain substitutions in the 3′–5′ exonuclease domain of the catalytic subunit of viral DNA polymerase. How these mutations confer resistance is a long-standing question. This study shows that both wild-type and exonuclease mutant polymerases incorporate ganciclovir into DNA and continue synthesis, but whereas the wild-type enzyme excises nucleotides two positions downstream of incorporated ganciclovir, the mutant enzymes do not, permitting chain extension. These results show how a therapeutically important drug causes chain termination and explain an unusual mechanism of drug resistance. Many antiviral and anticancer drugs are nucleoside analogs that target polymerases and cause DNA chain termination. Interestingly, ganciclovir (GCV), the first line of therapy for human cytomegalovirus (HCMV) infections, induces chain termination despite containing the equivalent of a 3′-hydroxyl group. Certain HCMV GCV resistance (GCVr) mutations, including ones associated with treatment failures, result in substitutions in the 3′–5′ exonuclease (Exo) domain of the catalytic subunit of the viral DNA polymerase (Pol). To investigate how these mutations confer resistance, we overexpressed and purified wild-type (WT) HCMV Pol and three GCVr Exo mutants. Kinetic studies provided little support for resistance being due to effects on Pol binding or incorporation of GCV-triphosphate. The mutants were defective for Exo activity on all primer templates tested, including those with primers terminating with GCV, arguing against the mutations increasing excision of the incorporated drug. However, although the WT enzyme terminated DNA synthesis after incorporation of GCV-triphosphate and an additional nucleotide (N+1), the Exo mutants could efficiently synthesize DNA to the end of such primer templates. Notably, the Exo activity of WT Pol rapidly and efficiently degraded N+2 primer templates to N+1 products that were not further degraded. On N+1 primer templates, WT Pol, much more than the Exo mutants, converted the incoming deoxynucleoside triphosphate to its monophosphate, indicative of rapid addition and removal of incorporated nucleotides (“idling”). These results explain how GCV induces chain termination and elucidate a previously unidentified mechanism of antiviral drug resistance.

Pharmacologic studies on the dibutyl and γ-monobutyl esters of methotrexate in the rhesus monkey

SummaryThe pharmacokinetics and metabolism of dibutyl methotrexate (DBMTX) and γ-monobutyl methotrexate (γ-MBMTX) were studied in Rhesus monkeys. When a bolus IV dose of either [3H]DBMTX or [3H]γ-MBMTX was given, the principal species in serum for up to 1 h was the monoester, with MTX accounting for < 10% of the total radioactivity. Products other than γ-MBMTX and MTX were formed in substantial amounts with DBMTX, but not with γ-MBMTX. Total radioactivity recovered in the bile 5 h after [3H]DBMTX injection accounted for 32% of the administered dose, indicating high hepatic extraction for this lipophilic compound. Serum and CSF levels of unchanged γ-MBMTX, as well as of MTX arising via esterase cleavage, were measured by HPLC after IV infusion of γ-MBMTX (10 g/m2). Efflux of monoester from CSF was slower than disappearance from serum. However, γ-MBMTX levels in CSF were no higher than could be attained by infusing MTX itself at the same dose rate. While CSF/serum ratios were ca. 10-fold higher for γ-MBMTX than for MTX, this difference could be explained on the basis of the very different affinities of the two compounds for serum proteins. HPLC analysis of serum processed by methanol precipitation as opposed to ultrafiltration of the proteins showed γ-MBMTX to be >99% bound, whereas for MTX this value was 50% or less. When γ-MBMTX and MTX levels measured after ultrafiltration were corrected for this difference in serum protein binding the total amount of the two drugs in serum became almost equivalent.

Novel pyrrolo[2,3-d]pyrimidine antifolate TNP-351: cytotoxic effect on methotrexate-resistant CCRF-CEM cells and inhibition of transformylases of de novo purine biosynthesis

N-{4-[3-(2,4-Diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl}-l-glutamic acid (TNP-351), characterized by a pyrrolo[2,3-d]pyrimidine ring, is a novel antifolate that exhibits potent antitumor activities against mammalian solid tumors. The mechanism of action of TNP-351 was evaluated using some methotrexate-resistant CCRF-CEM human lymphoblastic leukemia cell lines as well as partially purified enzymes folylpolyglutamate synthetase (FPGS), aminoimidazolecarboxamide ribonucleotide transformylase (AICARTFase), and glycinamide ribonucleotide transformylase (GARTFase) from parent CCRF-CEM cells. TNP-351 was found to inhibit the growth of L1210 and CCRF-CEM cells in culture, with the doses effective against 50% of the cells (ED50 values) being 0.79 and 2.7 nM, respectively. The growth inhibition caused by TNP-351 was reversed by leucovorin or a combination of hypoxanthine and thymidine. The methotrexate-resistant CCRF-CEM cell line, which has an impaired methotrexate transport, showed less resistance to TNP-351 than to methotrexate. TNP-351 was also found to be an excellent substrate for FPGS with a Michaelis constant (Km) of 1.45 μM and a maximum of velocity (Vmax) of 1,925 pmol h−1 mg−1. Inhibitory activities of TNP-351-Gn (n=1–6) for AICARTFase were found to be significantly enhanced with increasing glutamyl chain length [inhibition constants (Ki): G1, 52 μM; G6, 0.07 μM]. Neither TNP-351 nor its polyglutamates were very strong inhibitors of GARTFase. These findings have significant implications regarding the mechanism of action of TNP-351.

Altered transport of folic acid and antifolates through the carrier mediated reduced folate transport system in a human leukemia cell line resistant to (6R) 5,10-dideazatetrahydrofolic acid (DDATHF).

5,10-Dideazatetrahydrofolic acid is a potent antiproliferative agent in vitro and in vivo, arresting or delaying the proliferation of tumor cells in a number of murine and human xenografts models 1,2. In contrast to classical antifolates, (dihydrofolate reductase inhibitors), DDATHF blocks de novo purine biosynthesis as the result of a potent inhibition of both glycinamide ribonucleotide (GAR) and aminoimidazole carboxamide ribonucleotide (AICAR) transformylases.