A Protein Farnesyltransferase Inhibitor Ameliorates Disease in a Mouse Model of Progeria

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Science  17 Mar 2006:
Vol. 311, Issue 5767, pp. 1621-1623
DOI: 10.1126/science.1124875


Progerias are rare genetic diseases characterized by premature aging. Several progeroid disorders are caused by mutations that lead to the accumulation of a lipid-modified (farnesylated) form of prelamin A, a protein that contributes to the structural scaffolding for the cell nucleus. In progeria, the accumulation of farnesyl–prelamin A disrupts this scaffolding, leading to misshapen nuclei. Previous studies have shown that farnesyltransferase inhibitors (FTIs) reverse this cellular abnormality. We tested the efficacy of an FTI (ABT-100) in Zmpste24-deficient mice, a mouse model of progeria. The FTI-treated mice exhibited improved body weight, grip strength, bone integrity, and percent survival at 20 weeks of age. These results suggest that FTIs may have beneficial effects in humans with progeria.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder characterized by the precocious development of aging-like phenotypes (1, 2). Children with HGPS exhibit retarded growth, osteoporosis, osteolytic lesions in bones, alopecia, loss of subcutaneous fat, and ultimately occlusive vascular disease, which generally leads to death by the teenage years (1, 2). HGPS is caused by a LMNA mutation that leads to the production of a mutant prelamin A that cannot undergo the endoproteolytic processing step that yields mature lamin A (3). Lamin A is a key structural protein of the nuclear lamina, an intermediate filament meshwork that acts as a scaffold for the cell nucleus (1, 2).

Prelamin A contains a CaaX motif that triggers the addition of a 15-carbon farnesyl lipid (a cholesterol biosynthetic intermediate) to a C-terminal cysteine (2, 4). In normal cells, the farnesylation of prelamin A is thought to facilitate its targeting to the inner nuclear membrane, where it is cleaved by the zinc metalloproteinase ZMPSTE24 (5). ZMPSTE24 cleaves 15 amino acids from the C terminus of prelamin A that includes the farnesylcysteine, releasing mature lamin A. In HGPS, the mutant prelamin A cannot be processed by ZMPSTE24 and therefore retains its farnesyl lipid anchor; this mutant farnesylated protein is targeted to the nuclear rim, interferes with the integrity of the lamina, and causes misshapen cell nuclei (nuclei with blebs, folds, or wrinkles) (3). A more severe progeroid disorder, restrictive dermopathy, is caused by mutations leading to loss of ZMPSTE24 (2, 6, 7); partial loss of ZMPSTE24 can lead to mandibuloacral dysplasia, a progeroid disorder similar to HGPS (8, 9). In ZMPSTE24-deficient cells, wild-type farnesyl–prelamin A accumulates along the nuclear envelope and likewise leads to misshapen cell nuclei (6, 7, 10).

When the farnesylation of prelamin A in HGPS cells and ZMPSTE24-deficient cells is blocked with a farnesyltransferase inhibitor (FTI), prelamin A does not accumulate at the nuclear rim and the percentage of cells with misshapen nuclei is reduced (2, 1115). These in vitro observations strongly suggest that the accumulation of farnesyl–prelamin A interferes with the scaffolding function of the nuclear lamina. If the disrupted nuclear scaffolding were important to the pathogenesis of progeria, then FTIs might be expected to ameliorate disease symptoms in animal models of the disorder.

To explore this hypothesis, we treated Zmpste24-deficient mice (Zmpste24–/–) with a potent FTI, ABT-100 (16, 17). Like mice harboring a targeted HGPS mutation (11), Zmpste24–/– mice exhibit osteoporosis, osteolytic lesions in the ribs near the costovertebral joints, retarded growth after weaning, and a shorter life span (4 to 8 months, versus >24 months in wild-type mice) (10, 18). The osteolytic lesions in the ribs lead to spontaneous rib fractures beginning at 3 months of age. The Zmpste24–/– mice also lose grip strength by 3 to 4 months of age, as judged by their inability to hang upside down from a grid (10, 18).

The FTI (or vehicle alone) was orally administered to groups of male and female Zmpste24–/– mice (n = 7 to 9) and male and female littermate wild-type mice (n = 6 to 9) via the drinking water, beginning at 5 weeks of age (17). The plasma concentrations of ABT-100 were in the same range as those previously shown to be effective in inhibiting the growth of human tumor xenografts (16, 17).

The FTI inhibited protein farnesylation in vivo, as judged by Western blots of HDJ-2, a bio-marker of FTI activity (17, 19, 20). About 20 to 50% of the HDJ-2 in extracts of tail biopsies was nonfarnesylated (Fig. 1A). This level of nonfarnesylated HDJ-2 is similar to the levels attained in studies investigating the anticancer activity of other FTIs in mice (2123). As expected, nonfarnesylated prelamin A was detected in the tail extracts from FTI-treated but not untreated wild-type mice (Fig. 1B) (17, 20, 24).

Fig. 1.

Administration of an FTI in the drinking water of wild-type mice inhibits protein farnesylation in vivo. (A) Western blots of tail samples developed with a monoclonal antibody to HDJ-2, showing the appearance of nonfarnesylated HDJ-2 in mice treated with an FTI. Extracts of mouse embryonic fibroblasts grown in the presence and absence of an FTI (2.5 μM) were included as controls. (B) Western blot of tail extracts of wild-type mice with a prelamin A–specific antibody (9), showing the appearance of prelamin A in the setting of FTI treatment. Actin was measured as a control for sample loading.

As in patients with HGPS (1, 2), retarded growth is a key feature of the mice with progeria (10, 11, 18). We therefore monitored the effects of the FTI on body weight. In wild-type mice, FTI treatment reduced body weight in females (P = 0.022) and tended to reduce body weight in males (P = 0.077) relative to wild-type mice receiving the vehicle alone (Fig. 2). The reason for the reduced weight gain in the FTI-treated wild-type mice is not known, but it could be due to inhibition of protein farnesylation or to a toxic effect of the FTI. In contrast, FTI-treated Zmpste24–/– mice gained more weight than did the vehicle-treated Zmpste24–/– mice. This was true for both females (P < 0.0001) and males (P < 0.0001) (Fig. 2).

Fig. 2.

Effect of FTI treatment on body weight. Wild-type (WT) and Zmpste24–/– (KO) mice were given FTI (solid symbols) or vehicle alone (open symbols) beginning at 5 weeks of age. Body weights were measured weekly; the mean change in body weight from baseline for females (upper panel) and males (lower panel) is shown. The body weight curves for the FTI-treated Zmpste24–/– mice were significantly different from those of the vehicle-treated Zmpste24–/– mice, both in males (P < 0.0001) and in females (P < 0.0001), as judged by repeated-measures analysis of variance. Numbers of mice for each group: female wild-type mice on vehicle, n = 9; female wild-type mice on ABT-100, n = 7; male wild-type mice on vehicle, n = 5; male wild-type mice on ABT-100, n = 4; female Zmpste24–/– mice on vehicle, n = 7; female Zmpste24–/– mice on ABT-100, n = 7; male Zmpste24–/– mice on vehicle, n = 7; male Zmpste24–/– mice on ABT-100, n = 6. Error bars: SEM.

Zmpste24–/– mice normally lose the ability to hang upside down from a grid by 3 to 4 months of age (10, 18). In this study, 100% of the female (7/7) and male (7/7) Zmpste24–/– mice receiving the vehicle exhibited abnormal grip strength by 16 weeks of age (Fig. 3, A and B). In contrast, only 28% of the female (2/7) and 33% of the male (2/6) mice treated with the FTI exhibited a grip abnormality (Fig. 3, A and B). The delay in the appearance of the grip abnormality was significant in both females (P = 0.0015) and males (P = 0.036). Moreover, the mean length of time that the FTI-treated mice (n = 13) could hang on to the grid was longer than for the vehicle-treated Zmpste24–/– mice (n = 14) (37 s versus 5 s, P < 0.0001) (fig. S1).

Fig. 3.

FTI treatment improves grip strength, increases survival, and reduces the number of rib fractures in Zmpste24–/– mice. (A and B) Plots of grip strength in female and male mice over time. Zmpste24–/– mice were given vehicle alone (open symbols) or the FTI (solid symbols) starting at 5 weeks of age. The number of mice with a grip abnormality (inability to hang upside down from a grid for 60 s) was expressed as a percentage of the total number of mice in each group. On the basis of a log rank test, the FTI significantly improved grip strength in female (P = 0.0015) and male (P = 0.036) Zmpste24–/– mice. None of the wild-type mice developed a grip abnormality. (C) Survival of Zmpste24–/– mice on FTI versus vehicle alone. The number of surviving male and female mice was recorded weekly and is expressed as a percentage of the total number of mice. The significance of differences was determined with a log rank test. (D) Number of rib fractures in Zmpste24–/– mice on FTI versus vehicle alone. Wild-type and Zmpste24–/– mice were given vehicle alone or the FTI, starting at 5 weeks of age. At 20 weeks of age, the surviving mice were killed and the number of rib fractures was counted. The number of fractured ribs in the FTI-treated Zmpste24–/– mice was significantly lower than in the vehicle-treated Zmpste24–/– mice (P = 0.0002), as determined by the nonparametric Kruskal-Wallis test.

Zmpste24–/– mice invariably develop osteolytic lesions and rib fractures by 3 to 4 months of age (10, 18). Our goal was to score rib fractures in vehicle- and FTI-treated mice at a uniform time point. By 20 weeks of age, 6 of 14 vehicle-treated Zmpste24–/– mice had died, whereas only 1 of 13 FTI-treated mice had died (P = 0.0439) (Fig. 3C). At that time point, all of the surviving Zmpste24–/– mice were killed. The median number of rib fractures in the FTI-treated Zmpste24–/– mice was two, compared with 14 in the vehicle-treated mice. This difference was highly significant (P = 0.0002) (Fig. 3D). An improvement in bone was also apparent by computerized tomography scanning of the thoracic spine (fig. S2).

These studies show that an FTI ameliorates disease phenotypes in a mouse model of progeria, just as the FTI reduces the frequency of misshapen nuclei in progeria fibroblasts (2, 1115). However, our results do not formally prove that the improvement in disease phenotypes is a direct consequence of the improvement in nuclear morphology. Also, the disease phenotypes in the Zmpste24–/– mice were not completely eliminated by the FTI treatment. The FTI-treated Zmpste24–/– mice still had an abnormal growth curve relative to that of the wild-type mice; some treated mice still developed abnormal grip strength and a few mice ultimately developed several rib fractures. Additional studies will be required to determine whether a higher dose of an FTI and more complete inhibition of protein farnesylation could provide greater benefits. Different methods of drug delivery should also be tested. A study of much longer duration, involving a larger number of mice, is also needed to better define the impact of the FTI on survival.

The benefits of FTI treatment on disease phenotypes in mice with progeria are likely to fuel interest in testing FTIs in children with HGPS, which is uniformly fatal and for which no therapy exists. Two orally available FTIs, lonafarnib and tipifarnib, have already been extensively tested as antitumor agents in humans and are well tolerated in short-term studies (21, 25). For treatment of children with progeria, it will be important to identify an FTI treatment program that is efficacious in blocking protein farnesylation, yet is well tolerated over the long term.

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