Barton A. Kamen

Targeting Ras signaling through inhibition of carboxyl methylation: An unexpected property of methotrexate

The antifolate methotrexate is one of the most successful drugs in cancer chemotherapy. Although its efficacy is widely attributed to a decrease in nucleotide biosynthesis (1), methotrexate is known to increase homocysteine (2), a compound associated with an elevated risk of heart disease, Alzheimers disease (3), and neural tube defects (4). A potential mechanism for the detrimental effects of homocysteine is cellular hypomethylation from an increase in S-adenosylhomocysteine (5), an inhibitor of methyltransferases including isoprenylcysteine carboxyl methyltransferase (Icmt). Among the substrates of Icmt is the monomeric G protein Ras, a critical component of many signaling pathways that regulate cell growth and differentiation. Because carboxyl methylation of Ras is important for proper plasma membrane localization and function (6), we investigated the role of Icmt in the antiproliferative effect of methotrexate. After methotrexate treatment of DKOB8 cells, Ras methylation is decreased by almost 90%. This hypomethylation is accompanied by a mislocalization of Ras to the cytosol and a 4-fold decrease in the activation of p44 mitogen-activated protein kinase and Akt. Additionally, cells lacking Icmt are highly resistant to methotrexate. Whereas cells expressing wild-type levels of Icmt are inhibited by methotrexate, stable expression of myristoylated H-Ras, which does not require carboxyl methylation for membrane attachment (7), confers resistance to methotrexate. These results suggest that inhibition of Icmt is a critical component of the antiproliferative effect of methotrexate, expanding our understanding of this widely used drug and identifying Icmt as a target for drug discovery.

Dextromethorphan is effective in the treatment of subacute methotrexate neurotoxicity

Methotrexate-induced neurotoxicity (MTX-Ntox) is a frequent complication of methotrexate (MTX) therapy for patients with both malignant and inflammatory diseases. MTX-Ntox can occur after intrathecal MTX or afterlow-, intermediate-, or high-dose systemic administration. Symptoms can present in the acute, subacute, or late setting form, and can range from affective disorders, malaise, and headaches, to somnolence, focal neurologic deficits, and seizures. While the pathogenesis of MTX-Ntox is likely multifactorial, one potential biochemical pathway leading from MTX to neurotoxicity involves the folate dependent remethylation of homocysteine (Hcy). MTX therapy is known to cause elevations of both plasma and CSF Hcy. Hcy is directly toxic to vascular endothelium and it and its metabolites are excitatory agonists of the N -methyl-D-aspartate (NMDA) receptor. Competitive or noncompetitive antagonists might afford protection from or reversal of MTX-Ntox. Using high-performance liquid chromatography (HPLC) with coulometric electrochemical detection, the authors measured CSF Hcy in sequential patients with severe subacute MTX-Ntox. CSF Hcy was higher in these patients ( n = 9, median = 0.93 w M) than in asymptomatic patients ( n = 11, median 0.2 w M, p < .01). Five patients with severe subacute MTX-Ntox (most with dysarthria and/or hemiplegia) were treated with 1-2 mg/kg oral dextromethorphan (DM), a noncompetitive antagonist of the N -methyl-D-aspartate (NMDA) receptor. All five had resolution of symptoms. These data provide additional clinical support for elevated CSF Hcy in the induction of MTX-Ntox through activation of the NMDA-receptor. These data provide support for a placebo-controlled clinical trial to examine the ability of DM to prevent or alleviate MTX-Ntox.

Characterization of folate uptake by choroid plexus epithelial cells in a rat primary culture model

J. Neurochem. (2008) 104, 1494–1503.

Use of a novel genetic mouse model to investigate the role of folate in colitis-associated colon cancer.

Inflammatory bowel disease (IBD) patients are at high risk for developing folate deficiency and colon cancer. Since it is difficult to study the subtle global and gene-specific epigenetic mechanisms involved in folate-mediated tumor initiation and promotion, we have generated genetically modified mouse models by targeting the reduced folate carrier (RFC1) and folate-binding protein (Folbp1) genes. The transgenic mice were fed semi-purified diets for 8 weeks containing either normal (2 mg) or deficient (0.1 mg folate/kg diet) levels of folate. Compound heterozygous mice (Folbp1(+/-); RFC1(+/-)) fed an adequate folate diet exhibited a reduction in plasma folate concentrations compared to heterozygous (Folbp1(+/-)) and littermate wild-type mice (P<.05). In contrast, no differences were observed in colonic mucosa. Consumption of a low folate diet significantly reduced (three- to fourfold) plasma and tissue folate levels in all animal models, although plasma homocysteine levels were not altered. In order to elucidate the relationship between folate status and inflammation-associated colon cancer, animals were injected with azoxymethane followed by dextran sodium sulphate treatment in the drinking water. Mice were fed a normal folate diet and were terminated 5 weeks after carcinogen injection. The number of high multiplicity aberrant crypt foci per centimeter of colon was significantly elevated (P<.05) in compound Folbp1(+/-); RFC1(+/-) (3.5+/-0.4) mice as compared to Folbp1(+/-) (1.9+/-0.3) and wild-type control mice (1.1+/-0.1). These data demonstrate that the ablation of two receptor/carrier-mediated pathways for folate transport increases the risk for developing inflammation-associated colon cancer.

Phase 2B trial of aminopterin in multiagent therapy for children with newly diagnosed acute lymphoblastic leukemia

PurposeAminopterin offers advantages over the related antifolate, methotrexate, including greater potency, complete bioavailability, and more consistent accumulation and metabolism by patients’ blasts. This current trial was done to document the toxicity of the aminopterin within a multiagent therapeutic regimen for children with newly diagnosed ALL.Experimental DesignPatients at high risk of relapse were non-randomly assigned to therapy including oral aminopterin 4 mg/m2, in two doses 12 h apart, in place of methotrexate 100 mg/m2 in four divided doses.ResultsThirty-two patients, 22 with pre-B ALL and ten with T-lineage ALL, have been treated with aminopterin, with median follow up of 40 months. Hematologic, mucosal and hepatic toxicity has been tolerable and reversible. There have been no toxic deaths among patients in remission. During weekly AMT therapy, higher mean neutrophil counts were observed among patients who were wild type for polymorphisms in methylene tetrahydrofolate reductase and methionine synthase reductase.ConclusionsAminopterin can be safely incorporated in multiagent therapy for patients with ALL, in place of systemic methotrexate, without causing excessive toxicity. These results support a larger trial comparing the efficacy and toxicity of aminopterin and methotrexate in therapy for patients with ALL.

High time for low‐dose prospective clinical trials

The article by Glode et al. in this issue of Cancer reports on the empiric success, as defined by a decreased prostate specific antigen (PSA) level and lack of toxicity, of metronomically delivered low-dose chemotherapy for patients with hormone-refractory prostate carcinoma (HRPC). The data were collected as a retrospective evaluation of the medical records of patients treated with cyclophosphamide and dexamethasone regimen. Subjects were unable to participate in Phase II drug trials or had failed previous chemotherapy regimens. The content of this study (i.e., metronomic therapy), the process of collecting and presenting these data, and the question of how this therapeutic approach might be integrated into front-line or salvage therapy for men with prostate carcinoma are the subjects of this editorial. Although it might appear a bit superficial, the statement that the patients studied were not eligible for a Phase II trial is a major concern. Were they not eligible because the serum protein was too low? Was the bilirubin too high? Was the serum creatinine out of range? Was their performance status poor? Or were the hurdles to write and perform a trial so time consuming, because the drugs are decades old, that it was just deemed not worth the effort? For whatever reason, the lack of data leaves the reader with many questions. In this cohort only 13 of the 34 patients had ever received chemotherapy, so prior drug exposure and attendant toxicity should not have been an issue. Except for a short comment regarding bone marrow toxicity, no data are presented concerning clinical chemistry or hematology reported; either as an entry requirement or toxicity assessment. Because 25% of patients had a dose adjustment, longitudinal toxicity data may have offered insight into patient tolerability. Given the results and the usual assumption regarding eligibility requirements in traditional Phase II trials, the ineligibility of the patients could imply 1559