Abstract 208: Farnesylation-Dependent Fibrosis in the Aged Murine Heart

Mesenchymal stem cells (MSC) derived from the aged murine heart express reduced levels of the multipotency marker Nanog. These CD44 + CD45 - MSC inappropriately differentiate into fibroblasts characterized by markedly enhanced basal collagen expression. While collagen synthesis in cardiac fibroblasts derived from young mice is driven by TGF-β1, paradoxically, fibroblasts derived from the aged heart displayed reduced TGF-β1 responsiveness and decreased TGF-β receptor expression (ALK5). To resolve this apparent contradiction we looked for other pathways leading to collagen expression. We found that circulating insulin levels in aged mice were elevated ∼2.5 fold when compared to young animals. We hypothesized that increased circulating insulin levels drive MSC differentiation and caused upregulation of procollagen expression in cardiac fibroblasts. Our in vitro experiment confirmed that insulin at a pathophysiological concentration (1 nM) increased procollagen type I expression only in fibroblasts isolated from old hearts. As insulin can signal through substrate prenylation, cardiac fibroblasts isolated from aged mice had ∼2 fold greater basal activity of farnesyltransferase (FTase, enzyme that transfers the prenyl chain) than fibroblasts isolated from young mice. FTase activity was further increased when fibroblasts isolated from aged hearts were stimulated with insulin. Cells derived from 3 month old mice did not upregulate FTase activity in response to insulin. Basal expression of FTase regulatory subunit (FNTA) was also upregulated by 3 fold in aged fibroblasts and expression of catalytic subunit (FTNB) was increased with insulin stimulation. FTase inhibitor reduced procollagen synthesis in aged fibroblasts. Here we present new data linking insulin-dependent upregulation of collagen type I synthesis via increased farnesylation in the aging heart.

Phase I Trial and Pharmacokinetic Study of the Farnesyltransferase Inhibitor Tipifarnib in Children With Refractory Solid Tumors or Neurofibromatosis Type I and Plexiform Neurofibromas

Purpose This pediatric phase I trial of tipifarnib determined the maximum-tolerated dose (MTD), pharmacokinetics, and pharmacodynamics of tipifarnib in children with refractory solid tumors and neurofibromatosis type 1 (NF1) –related plexiform neurofibromas. Patients and Methods Tipifarnib was administered twice daily for 21 days, repeated every 28 days starting at 150 mg/m2/dose (n = 4), with escalations to 200 (n = 12), 275 (n = 12), and 375 (n = 6) mg/m2/dose. The MTD was also evaluated on a chronic continuous dosing schedule (n = 6). Pharmacokinetic sampling was performed for 36 hours after the first dose and peripheral-blood mononuclear cells (PBMCs) were collected at baseline and steady state for determination of farnesyl protein transferase (FTase) activity and HDJ-2 farnesylation. Results Twenty-three solid tumor and 17 NF1 patients were assessable for toxicity. The MTD was 200 mg/m2/dose, and dose-limiting toxicities on cycle 1 were myelosuppression, rash, nausea, vomiting, and diarrhea. The 200 mg/m2/dose was also tolerable on the continuous dosing schedule. Cumulative toxicity was not observed in the 17 NF1 patients who received a median of 10 cycles (range, 1 to 32 cycles). The plasma pharmacokinetics of tipifarnib were highly variable but not age dependent. At steady state on 200 mg/m2/dose, FTase activity was 30% compared with baseline, and farnesylation of HDJ-2 was inhibited in PBMCs. Conclusion Oral tipifarnib is well tolerated in children receiving the drug twice daily for 21 days and a continuous dosing schedule at 200 mg/m2/dose, which is equivalent to the MTD in adults. The pharmacokinetic profile of tipifarnib in children is similar to that in adults, and at the MTD, FTase is inhibited in PBMC in vivo.

Phase II and Pharmacodynamic Study of the Farnesyltransferase Inhibitor R115777 as Initial Therapy in Patients With Metastatic Pancreatic Adenocarcinoma

Purpose: R115777 is a selective nonpeptidomimetic inhibitor of farnesyltransferase (FTase), one of several enzymes responsible for posttranslational modification that is required for the function of p21ras and other proteins. Given that RAS mutations are nearly universal in pancreatic cancer and R115777 demonstrated preclinical activity against pancreatic cell lines and xenografts, this phase II study was undertaken to determine its clinical activity and effect on target proteins in patients with measurable metastatic pancreatic adenocarcinoma. Patients and Methods: Twenty patients who had not received prior therapy for metastatic disease were treated with 300 mg of R115777 orally every 12 hours for 21 of 28 days. Inhibition of FTase activity in peripheral-blood mononuclear cells was measured using a lamin B C-terminus peptide as substrate. Western blot analysis was performed to monitor farnesylation status of the chaperone protein HDJ-2. Results: No objective responses were seen. Median time to progression was 4.9 weeks, and median survival time was 19.7 weeks. The estimated 6-month survival rate was 25%, with no patients progression-free at 6 months. Grade 3/4 toxicities were liver enzyme elevation, anemia, neutropenia, thrombocytopenia, fatigue, nausea/vomiting, rash, and anorexia. FTase activity (mean ± SD) decreased by 49.8% ± 9.8% 4 hours after treatment on day 1 and 36.1% ± 24.8% before treatment on day 15. HDJ-2 farnesylation (mean ± SD) decreased by 33.4% ± 19.8% on day 15. Conclusion: Although treatment with R115777 resulted in partial inhibition of FTase activity in mononuclear cells, it did not exhibit single-agent antitumor activity in patients with previously untreated metastatic pancreatic cancer.

Purified yeast protein farnesyltransferase is structurally and functionally similar to its mammalian counterpart

Protein farnesyltransferase (FTase) catalyses the addition of a farnesyl group to a cysteine within the so-called ‘CAAX box’ at the C-terminus of various proteins. In the present paper we report purification of Saccharomyces cerevisiae FTase to near-homogeneity. This was accomplished by constructing a yeast strain overproducing FTase approx. 100-fold. The purified enzyme was a heterodimer of approx. 90 kDa and consisted of 43 kDa and 34 kDa subunits. The 43 kDa subunit was shown to be the product of the DPR1 gene by using antibody raised against baculovirus-produced DPR1 polypeptide. The purified enzyme required Mg2+, showed a pH optimum of 7.8 and was most active at 50 degrees C. The Km values for farnesyl pyrophosphate and GST-CIIS (glutathione S-transferase fused to the C-terminal 12 amino acids of yeast RAS2 protein), KmFpp and KmGST CIIS, were 8.1 and 5.1 microM respectively. The enzyme was capable of farnesylating GST-CIIL (the same as GST-CIIS, except that the C-terminal serine is changed to leucine), a substrate protein for the enzyme geranylgeranyltransferase, although with a higher apparent Km than for GST-CIIS. Like its mammalian counterpart, yeast FTase activity was inhibited by peptides containing the C-terminal CAAX sequence (that is, one where C = cysteine, A = aliphatic amino acid and X = any amino acid). These results provide direct evidence for the idea that the yeast and mammalian FTases are structurally and functionally very similar.