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. 2021 Feb 2;10:e63284. doi: 10.7554/eLife.63284

A small-molecule ICMT inhibitor delays senescence of Hutchinson-Gilford progeria syndrome cells

Xue Chen 1,2, Haidong Yao 1, Muhammad Kashif 1, Gwladys Revêchon 1, Maria Eriksson 1, Jianjiang Hu 1, Ting Wang 1, Yiran Liu 1, Elin Tüksammel 1, Staffan Strömblad 1, Ian M Ahearn 3, Mark R Philips 4, Clotilde Wiel 1, Mohamed X Ibrahim 1,5,✉, Martin O Bergo 1,✉
Editors: Yousin Suh6, Jessica K Tyler7
PMCID: PMC7853716  PMID: 33526168

Abstract

A farnesylated and methylated form of prelamin A called progerin causes Hutchinson-Gilford progeria syndrome (HGPS). Inhibiting progerin methylation by inactivating the isoprenylcysteine carboxylmethyltransferase (ICMT) gene stimulates proliferation of HGPS cells and improves survival of Zmpste24-deficient mice. However, we don't know whether Icmt inactivation improves phenotypes in an authentic HGPS mouse model. Moreover, it is unknown whether pharmacologic targeting of ICMT would be tolerated by cells and produce similar cellular effects as genetic inactivation. Here, we show that knockout of Icmt improves survival of HGPS mice and restores vascular smooth muscle cell numbers in the aorta. We also synthesized a potent ICMT inhibitor called C75 and found that it delays senescence and stimulates proliferation of late-passage HGPS cells and Zmpste24-deficient mouse fibroblasts. Importantly, C75 did not influence proliferation of wild-type human cells or Zmpste24-deficient mouse cells lacking Icmt, indicating drug specificity. These results raise hopes that ICMT inhibitors could be useful for treating children with HGPS.

Research organism: Mouse

Introduction

Hutchinson-Gilford progeria syndrome (HGPS) is caused by the accumulation of progerin, a mutant form of prelamin A that is farnesylated and methylated within the nuclear envelope (De Sandre-Giovannoli et al., 2003; Eriksson et al., 2003). Farnesyltransferase inhibitors (FTIs) prevent progerin farnesylation and improve some clinical phenotypes of HGPS patients, including survival, but the effect is modest (Gordon et al., 2018; Young et al., 2005). Also, a potential limitation of this approach is that FTIs are anti-proliferative (Lee et al., 2010), and children with progeria would benefit from a therapy that supports cell proliferation. We found earlier that inhibiting the methylation of progerin by inactivating the isoprenylcysteine carboxylmethyltransferase (ICMT) gene overcomes senescence and increases proliferation of HGPS cells (Ibrahim et al., 2013). Also, a knockout of Icmt substantially improves clinical phenotypes and survival of Zmpste24-deficient mice, a model of progeria (Ibrahim et al., 2013). This result raises the possibility that inhibiting ICMT activity could be a useful therapeutic strategy. An important step in the preclinical validation of this strategy would be to determine whether knockout of Icmt improves phenotypes and survival in an authentic progerin-expressing HGPS mouse model. Another step would be to determine whether pharmacologic targeting of ICMT produces similar cellular effects as genetic inactivation. To address these issues, we defined the consequences of knocking out Icmt in progerin-knock-in mice; and we synthesized a potent cell-permeable ICMT inhibitor, compound 75 (C75) (Judd et al., 2011), and examined its effects on HGPS cells.

Results

We bred mice with a hypomorphic Icmt allele (Icmthm; with ~85% reduced ICMT activity [Ibrahim et al., 2013]) with progerin-expressing lamin A knock-in mice (LmnaG609G [Osorio et al., 2011]). As expected from previous studies, LmnaG609G/G609GIcmt+/+ mice developed alopecia, stunted growth, and weight loss, and all mice had died by 129 days of age; at that time, numbers of vascular smooth muscle cell (VSMC) nuclei in aortic arch sections were reduced by ∼75% and muscle fiber size in the quadriceps muscle were 50% smaller compared with wild-type mice (Figure 1a–f). In contrast, at 129 days of age, the LmnaG609G/G609GIcmthm/hm mice were still alive with substantially higher body weights, and when they were sacrificed, VSMC numbers were found to be normalized and skeletal muscle fiber size increased (Figure 1a–f). Although these data are statistically sound, they should be interpreted with caution as the mice were difficult to breed and we only obtained three double homozygotes. Consequently, we also analyzed LmnaG609G/+Icmt+/+ mice, which had a maximal life span of 290 days (Figure 1g) (and no aorta and skeletal muscle phenotypes); importantly, all LmnaG609G/+Icmthm/hm mice were still alive at 290 days and their overall survival were increased (Figure 1g). These results are important because progerin rather than prelamin A causes progeria in LmnaG609G mice, and because homozygous LmnaG609G/G609G mice exhibit a vascular phenotype, which are prominent in children with HGPS, but absent in Zmpste24-deficient mice used in earlier studies (Ibrahim et al., 2013)—and Icmt inactivation markedly improved this phenotype.

Figure 1. Targeting Icmt improves survival and aorta and muscle phenotypes of progerin-knock-in mice.

Figure 1.

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(a) Photograph of 15-week-old littermate female mice. (b) Kaplan-Meier plot showing survival of LmnaG609G/G609Icmthm/hm (n = 3) and LmnaG609G/G609GIcmthm/+ (n = 8) mice; the three double-homozygotes were killed for analyses when all the control mice had died of progeria. (c) Body weight curves of mice in panel B. (d) Number of vascular smooth muscle cell nuclei in medial layer of aortic arch sections. Data are mean of three mice/genotype. (e) Representative photographs of aortic arch sections from panel d. (f) Skeletal muscle fiber cross-sectional diameter. Data are mean of 50 independent muscle cells diameters in M. quadriceps extensor from three mice/genotype. (g) Kaplan-Meier plot showing survival of LmnaG609G/+Icmthm/hm (n = 5) and LmnaG609G/+Icmt+/+ (n = 14) mice. *p<0.05; **p<0.01; ***p<0.005; ****p<0.001; n.s., not significant.

We next synthesized the ICMT inhibitor C75 as described (Judd et al., 2011), and found that its IC50 was 0.5 μM (Figure 2a—figure 2 supplement S1). Prolonged C75 incubation was well tolerated by two different human HGPS cell lines and caused prelamin A accumulation and mislocalization of the RAS oncogene —markers of reduced ICMT activity—but did not affect the nuclear shape abnormalities (Figure 2b–d—figure 2 supplement S2a). C75 did not influence the electrophoretic mobility of HDJ2 which could have been indicative of effects on FTase-mediated farnesylation (figure 2 supplement S2b). Importantly, C75 increased proliferation of late-passage HGPS cell lines as judged by 45- to 70-day population-doubling assays (Figure 2e–f). The drug had increased cell viability already at 8 days, that is, before the increase in cell proliferation was evident (figure 2 supplement S2c–d). C75 also increased proliferation of Zmpste24-deficient mouse fibroblasts with normal Icmt expression but not in cells lacking Icmt, indicating drug specificity (Figure 2g–h). The drug did not affect proliferation of wild-type human fibroblasts (Figure 2i). In contrast, the FTI lonafarnib rapidly reduced proliferation of HGPS cells and abolished the effect of C75 on cell growth (Figure 2j–k). The latter finding makes sense because protein methylation cannot occur without protein farnesylation.

Figure 2. C75 inhibits isoprenylcysteine carboxylmethyltransferase (ICMT) activity and increases proliferation and reduces senescence of HGPS cells without affecting nuclear shape.

(a) Chemical structure of compound 75 (C75) and percent ICMT activity remaining after incubation with C75. (b) Western blot showing increased amounts of prelamin A in Hutchinson-Gilford progeria syndrome (HGPS) and wild-type (WT) cells incubated with C75 for 20 days; β tubulin was the loading control. (c) Left, immunofluorescence images showing prelamin A expression in HGPS cells incubated with 5 μM C75 for 20 days and 10 μM farnesyltransferase inhibitor (FTI) for 3 days; the cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Right, quantification of prelamin A staining intensity (n = 2 cell lines and six individual images/cell line). (d) Left, representative nuclei of LAP2β-stained WT cells and HGPS cells incubated with vehicle (Ctrl), 5 μM C75 for 20 days, and 10 μM FTI for 3 days. Right, quantification of misshapen nuclei. Data are mean of ∼1000 nuclei and two independent experiments per cell line and condition. (e–h) Population doubling assays of late-passage HGPS cell lines (e, f), primary Zmpste24–/– mouse fibroblasts (g), and Zmpste24–/–Icmt–/– fibroblasts (h) incubated with vehicle (Ctrl) and C75. (i) Population doubling of WT cells incubated with C75. (j, k) Population doubling assays of a late-passage HGPS cell line incubated with vehicle (Ctrl), lonafarnib, C75, and both drugs. (l–n) Senescence-associated beta-galactosidase (β-gal) staining of WT, HGPS, and Zmpste24–/– fibroblasts incubated with vehicle (Ctrl) and C75 for 20 days. (o, p) Interleukin 6 (IL-6) (o) and CDKN2A (p) expression in cells from experiment in panel m. **p<0.01; ***p<0.001; ****p<0.0001; n.s., not significant.

Figure 2.

Figure 2—figure supplement 1. C75 synthesis and absorption, distribution, metabolism, and excretion (ADME) test.

Figure 2—figure supplement 1.

(a) Schematic of the C75 synthesis reactions. (b) Mass spectra of the final product. (c) Nuclear magnetic resonance spectra of C75. (d) Left, schematic of the apparent permeability assay through a layer of Caco-2 cells. Right, quantification of apical-to-basolateral (blue) and basolateral-to-apical (red) absorption rates across a Caco-2 cell layer.

Figure 2—figure supplement 2. C75 increases viability, proliferation, and metabolic rates of Hutchinson-Gilford progeria syndrome (HGPS) cells and reduces reactive oxygen species (ROS) levels and nuclei with high levels of DNA damage.

Figure 2—figure supplement 2.

(a) Western blots showing levels of the RAS oncogene in total lysates (T) and in cytosolic (C) and membrane (M) fractions of late-passage HGPS cells and primary Zmpste24–/– mouse fibroblasts; β-actin was the loading control for cytosolic and total fractions. (b) Western blots showing electrophoretic mobility of the CAAX-protein HDJ2; a farnesyltransferase inhibitor (3-day incubation), but not C75 (20-day incubation), could partially reduce HDJ2 mobility. (c and d) Growth curves from presto blue-based cell viability assays of two HGPS cell lines. (e) Cell cycle analysis of HGPS cells. Data are mean of six technical replicates. (f) Seahorse analyses of basal and maximal respiration, and ATP production. Data are mean of two HGPS cell lines assayed in triplicate. (g) ROS levels in two HGPS cell lines incubated with C75 for 20 days. (h) Western blots showing levels of endoplasmic reticulum stress markers in lysates of two HGPS cell lines incubated with C75 for 20 days; tunicamycin was used as positive control. (i) Western blots showing levels of DNA damage markers in lysates of two HGPS cell lines incubated with C75 for 20 days. (j) Left, quantification of the frequency of γ-H2AX-positive nuclei in wild-type cells and in HGPS cells lines incubated with 5 μM C75 for 20 days. Middle, quantification of nuclei with more than six γ-H2AX-foci HGPS cells incubated with 5 μM C75 for 20 days. Data are mean of two cell lines and triplicate analyses. Right, immunofluorescence images showing γ-H2AX-positive nuclei (red); the nuclei were counterstained with DAPI (blue). *p<0.05; **p<0.01; ***p<0.001; n.s., not significant.
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Consistent with increased proliferation, C75 increased the fraction of HGPS cells in the G1 and S/G2/M phases of the cell cycle (figure 2 supplement S2e); reduced senescence-associated β-galactosidase activity (Figure 2I–m); and normalized the expression of the senescence markers IL6 and CDKN2A (Figure 2o–p). The drug also normalized oxygen consumption rates and ATP production in HGPS cells, as judged by Seahorse analyses; and reduced the levels of oxidative stress (figure 2 supplement S2f–g). In contrast, C75 did not influence expression of endoplasmic reticulum (ER) stress markers in HGPS cells; and DNA damage signaling markers, including γ-H2AX, remained unchanged (figure 2 supplement S2h–i). C75 did however reduce the fraction of cells with nuclei harboring multiple γ-H2AX foci, examined by immunofluorescence, but not the total fraction of γ-H2AX-positive nuclei (figure 2 supplement S2j). A potential interpretation of the latter finding is that C75 increases proliferation of cells with low levels of DNA damage which outcompete cells with high levels.

The signaling molecule AKT binds progerin and farnesyl-prelamin A (in HGPS and Zmpste24-deficient cells, respectively) and exhibits only low levels of phosphorylation (Ibrahim et al., 2013). C75 reduced the progerin–AKT interactions and increased AKT phosphorylation (Figure 3a–d), but C75 did not influence phospho-AKT levels in wild-type cells (Figure 3b). Although these data don’t reveal whether AKT is functionally involved in the improved phenotypes upon C75 administration, we showed previously that pharmacologic AKT inhibition prevents the increased proliferation following Icmt knockout in mouse cells (Ibrahim et al., 2013). Despite improvements in multiple cellular phenotypes, C75 increased the absolute levels of progerin in HGPS cells, a consequence of reduced progerin turnover (Figure 3e–g). Moreover, C75 mislocalized some progerin and farnesyl-prelamin A away from the nuclear membrane into the nucleoplasm (Figure 3h–i).

Figure 3. The isoprenylcysteine carboxylmethyltransferase inhibitor C75 disrupts progerin-AKT interactions, increases AKT activation, and mislocalizes progerin from the nuclear membrane to the nucleoplasm.

Figure 3.

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(a) Western blot showing amounts of phosphorylated and total AKT (a.k.a protein kinase B) in whole-cell lysates of human wild-type (WT) and Hutchinson-Gilford progeria syndrome (HGPS) cells and mouse Zmpste24-deficient fibroblasts. (b, c) Left panels, western blots (WB) showing amounts of phosphorylated and total AKT in WT and HGPS cells (b) and Zmpste24-deficient (c) fibroblasts incubated with C75 for 20–30 days; β tubulin was the loading control. Right panels, quantification of the ratio of phosphorylated and total AKT from densitometry analyses of protein bands. Data are mean of two independent cell lines, each analyzed in duplicate. (d) Immunoprecipitation (IP) and WB analyses showing that C75 disrupts the association between AKT and progerin. The lysates were also used directly for WB with total AKT antibodies (input). (e) WB showing amounts of progerin in HGPS cells incubated with C75 for 20 days. (f) WB showing amounts of progerin remaining in HGPS cells incubated with vehicle and 5 μM C75 for 20 days and then with cycloheximide to stop protein synthesis. (g) Quantification of progerin amounts from the experiment in panel f and two others like it. (h, i) Left panels, WB showing amounts of progerin and prelamin A in nuclear membrane and nucleoplasm fractions of HGPS (h) and Zmpste24-deficient (i) fibroblasts, respectively, incubated with vehicle and 5 μM C75 for 20 days. Lamin B2 and nuclear P84 were loading controls for nuclear membrane and nucleoplasm fractions, respectively. Right panels, quantification of nucleoplasmic progerin/prelamin A from densitometry analyses. *p<0.05; **p<0.01; ***p<0.001.

Discussion

We conclude that genetic Icmt inactivation improves survival and unique phenotypes of an authentic HGPS mouse model. We further conclude that pharmacologic inhibition of ICMT delays senescence, restores respiration rates and ATP production, and stimulates proliferation of HGPS and Zmpste24-deficient cells, most of which is consistent with findings in Icmt-deficient cells (Ibrahim et al., 2013). Blocking methylation partially mislocalizes progerin to the nucleoplasm, disrupts its interaction with AKT, and increases AKT signaling. These positive phenotypes of blocking methylation with C75 outweighed any potential adverse effects from the modest amount of progerin accumulation. In wild-type cells, prelamin A is fully processed to mature lamin A and the cells grow and proliferate normally. In HGPS and Zmpste24-deficient cells, farnesylated and methylated progerin/prelamin A accumulates and causes senescence. Our data suggest that progerin/prelamin A methylation contributes to the toxicity of these proteins and their ability to induce senescence, and we propose that blocking progerin/prelamin A methylation mislocalizes the proteins into the nucleoplasm and thereby reduces their ability to induce DNA damage, metabolic alterations, and senescence.

A limitation of C75 is that despite good apparent permeability (Figure 2 — supplement 1d) it is predicted to have poor bioavailability (i.e., very hydrophobic and high first-passage metabolism in in silico ADME analyses). Thus, new compounds will be required for in vivo studies in mice. Nonetheless, our study takes two important steps in the preclinical validation of ICMT as a potential drug target, and thereby raises hopes that ICMT inhibition could be an effective strategy for treating children with HGPS and progeroid disorders resulting from ZMPSTE24 deficiency (Michaelis and Hrycyna, 2013).

Materials and methods

Mice

Icmthm/hm mice (Bergo et al., 2004; Wahlstrom et al., 2008) were bred with LmnaG609G/G609G mice (Osorio et al., 2011) to produce LmnaG609G/G609GIcmthm/hm mice. Controls were littermate LmnaG609G/G609GIcmthm/+ mice and LmnaG609G/G609GIcmt+/+ mice, which were indistinguishable in phenotype and collectively designated LmnaG609G/G609GIcmt+/+. We also used LmnaG609G/+Icmthm/hm mice and the controls LmnaG609G/+Icmthm/+ and LmnaG609G/+Icmt+/+ mice, which were collectively designated LmnaG609G/+Icmt+/+. Genotyping was performed by polymerase chain reaction (PCR) on genomic DNA from ear or tail biopsies. Mice were monitored daily and weighed weekly. The aortic arch and quadriceps muscle were harvested and fixed in 10% formalin for 24 hr and then stained with hematoxylin and eosin. VSMC nuclei in the aortic media and skeletal muscle fiber size were quantified as described (Greising et al., 2012; Kim et al., 2018). Mouse experiments were approved by the Animal Research Ethics Committees in Gothenburg and Linköping, Sweden.

Drug synthesis, ICMT activity assay, and apparent permeability assay

C75 was synthesized by Recipharm AB as described (Judd et al., 2011). ICMT activity was carried out as described (Choy and Philips, 2000; Zhou et al., 2016). Apparent permeability assay was carried out by analyzing the apical-to-basolateral (and vice versa) transport of C75 using Caco-2 cell monolayers as described (Hubatsch et al., 2007).

Cell culture and cell proliferation

Human HGPS cell lines (GM01972D and AG03513E) and control cell lines from unaffected parents (AG03258 and AG03512) were from the Coriell Institute. Primary mouse fibroblasts (MEFs) isolated from E13.5–E14.5 Zmpste24-deficient embryos (Bergo et al., 2002) were cultured in low-glucose Dulbecco's modified eagle medium DMEM (21885025, ThermoFisher) supplemented with 10% fetal bovine serum (26140079, ThermoFisher), 1% penicillin/streptomycin (15070063, ThermoFisher), and 1% MEM non-essential amino acid (11140068, ThermoFisher). Cell viability proliferation assays were carried out by plating 1 × 103 cells in 96-well plates. Cell viability was determined every 3 days with PrestoBlue Cell Viability Reagent (A13262, ThermoFisher); absorbance at 570 and 600 nm was measured with the Multi-mode reader (BioTek). Population doubling assays were carried out by seeding 3 × 105 cells on 10 cm plates. The cells were trypsinized, counted, and re-seeded every 3 days (MEFs) or 4–8 days (human cells). Lonafarnib (2 and 10 µM, SML1457; Sigma-Aldrich) was used in some population doubling assays. All cell lines tested negative for mycoplasma.

Immunofluorescence and nuclear shape

Wild-type and HGPS cell lines were cultured in Glass Bottom Microwell Dishes (MatTek) for 24 hr, fixed in 4% paraformaldehyde, permeabilized with 0.4% Triton X-100, and blocked with PBS containing 2% bovine serum albumin. The cells were incubated overnight with antibodies to LAP2β (1:100, 611000, BD Biosciences), Prelamin A (1:400, MABT345, Millipore), and phospho-γH2AX (1:800, 05–636, Millipore), followed by incubation for 1 hr with secondary antibodies (1:1000, Alexa Fluor Plus 488 goat anti-mouse IgG, SA243833; 1:1000, Alexa Fluor 594 donkey anti-rat IgG, 1870948, Life Technologies) and staining with 4′,6-diamidino-2-phenylindole (DAPI) (1:500, 62248, ThermoFisher) for 15 min. Prelamin A staining intensity and the frequency of misshapen nuclei were quantified as described (Ibrahim et al., 2013). γH2AX foci were counted manually. FTI-276 (2 µM, F9553; Sigma-Aldrich) was used as a positive control for prelamin A accumulation and for restoring nuclear shape abnormalities.

Immunoblots and immunoprecipitation

Cells were lysed in buffer containing 9 M urea (U0631, Sigma-Aldrich) and complete protease inhibitor cocktail (78430, ThermoFisher), sonicated, and cleared by centrifugation (14,000 × g for 10 min). The lysates were size-fractionated on 10% Mini-PROTEAN TGX Stain-Free gels (456–8036, Bio-Rad) and transferred to nitrocellulose membranes (0.2 μm, 1704158, Bio-Rad). The membranes were incubated with primary antibodies overnight at 4°C and with secondary antibodies for 1 hr at room temperature. Primary antibodies were prelamin A (1:1000, MABT345, Millipore), progerin (1:500, 05–1231, Millipore), phospho-AKTSer473 (1:1000, 4060, Cell Signaling), total AKT (1:1000, 9272, Cell Signaling), pan-RAS (1:1000, ab69747, Abcam), lamin B2 (1:500, 33–2100, ThermoFisher), nuclear matrix protein p84 (1:1000, GTX70220, GeneTex), p21 (1:1000, sc-397, Santa Cruz Biotechnology), p53 (1:1000, sc-126, Santa Cruz Biotechnology), phospho-γH2AX (1:500, 05–636, Millipore), phospho-p53Ser15 (1:1000, 700439, ThermoFisher), antibodies in the ER stress sample kit (9956T, Cell Signaling), and β-tubulin (1:1000, T2200, Sigma-Aldrich). Secondary antibodies were anti-mouse (1:6000, 115-035-003, Jackson ImmunoResearch), anti-rabbit (1:6000, 111-035-003, Jackson ImmunoResearch), and anti-rat (1:6000, A9542, Sigma-Aldrich). Protein bands were detected and quantified on a ChemiDoc Touch Imaging System with Image lab (version 5.2.1). Cytosolic and membrane fractions were isolated with Qproteome Cell Compartment Kit (37502, QIAGEN). Immunoprecipitation (IP) was performed with the Dynabeads Protein G IP kit (100-07D, Life Technologies). To quantify progerin turnover rate, cells were incubated with cycloheximide (20 µg/ml, C4859, Sigma-Aldrich) to stop protein synthesis; lysates were prepared as above.

Senescence-associated β-galactosidase assay

Senescence-associated β-galactosidase (SA‐β Gal) staining on primary MEFs and human HGPS cell lines was performed using the Senescence Detection kit (9860, Cell Signaling). Cells were incubated with SA‐β Gal solution for 24 hr (mouse) and 4 hr (human), separately, at 37°C. Results are reported as percent of blue cells.

Quantitative PCR

RNA was isolated with the RNeasy Plus Mini kit (74136, QIAGEN) and cDNA was synthesized with the iScript cDNA synthesis kit (170–889, Bio-Rad). IL6 and CDKN2a expression was analyzed by reverse transcription quantitative PCR on a CFX384 Real-Time System (Bio-Rad) using Taqman human probe sets for IL6 (Hs00174131_m1, ThermoFisher) and CDKN2a (Hs01059210_m1, ThermoFisher). β-Tubulin (Hs00801390, ThermoFisher) was the reference gene.

Isolation of nuclear membrane and nucleoplasm fractions

Nuclear membrane and nucleoplasm separation was performed on MEFs and human fibroblasts using Minute Nuclear Envelop Protein Extraction Kit (NE-013, Invent Biotechnology), and Minute Detergent-Free Nucleoplasm Isolation Kit (NI-024, Invent Biotechnology).

Mitochondrial function assay

Mitochondrial function parameters were measured with the Cell Mito Stress Test kit using the Seahorse XFe96 Analyzer (Agilent). Cells were seeded in microplates (15,000 cells/well) (101085–004, Agilent) and cultured overnight at 37°C in a CO2 incubator. Freshly prepared DMEM-base medium supplemented with glucose, pyruvate, and glutamine, and adjusted to pH 7.4 were added to the cells and they were incubated for 45 min at 37°C in a non-CO2 incubator and then analyzed at 37°C in the XFe96 Analyzer. Basal and maximal respiration and ATP production data were normalized to viable cell numbers obtained from identically treated additional wells using the Presto Blue Cell Viability assay (A13262, ThermoFisher).

ROS measurements

HGPS cells were incubated with C75 for 20 days and then seeded in white 96-well plates (5 × 103 cells/well). ROS measurements were performed using the H2DCFDA (H2-DCF, DCF) kit (D399, Thermofisher) in FluoroBrite medium (A18967-01, Life Technologies). Fluorescence (Excitation and Emission: 492–495/517–527) was recorded with a Synergy multimode reader (BioTek).

Flow cytometry analysis

HGPS cells were incubated with 250 μl fixation/permeabilization solution (554714, BD) for 30 min on ice, in the dark; washed twice; incubated with antibodies to Ki67 (5 μl/sample; 561277, BD) at room temperature for 1 hr and 45 min; washed with PBS + 10% FCS; stained with 7AAD (5 μg/sample; A9400-1MG, Sigma) at room temperature for 20 min; resuspended and filtered into flow tubes; and analyzed using a BD LSRFortessa X-20.

Statistics

Data are presented as mean ± SEM. For statistical analyses, we used Graphpad Prism software v.7; the log-rank test was used for survival, two-way ANOVA for cell-growth curves, one-way ANOVA with Bonferroni’s post-hoc test when comparing three or more groups, and Student’s t test when comparing two groups only. Experiments were repeated 2–4 times unless stated otherwise; n indicates biological replicates.

Acknowledgements

We thank Dr. C López-Otín for the LmnaG609G mice; Dr. X Xu for technical assistance; and Dr. S Young for helpful discussions. Microscopy was performed at the LCI facility/Nikon Center of Excellence, Karolinska Institutet, supported by grants from the Knut and Alice Wallenberg Foundation, Swedish Research Council, KI infrastructure, Centre for Innovative Medicine, and Jonasson Center at the Royal Institute of Technology. The study was supported by grants from the Progeria Research Foundation, Center for Innovative Medicine (CIMED), and the Swedish Research Council (to MOB).

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Contributor Information

Mohamed X Ibrahim, Email: mohamed.ibrahim@gu.se.

Martin O Bergo, Email: martin.bergo@ki.se.

Yousin Suh, Columbia University, United States.

Jessica K Tyler, Weill Cornell Medicine, United States.

Funding Information

This paper was supported by the following grants:

  • Progeria Research Foundation to Martin O Bergo.

  • Vetenskapsrådet to Martin O Bergo.

  • Center for innovative medicine (CIMED), Karolinska Institutet, Huddinge, Sweden to Martin O Bergo.

Additional information

Competing interests

No competing interests declared.

Author contributions

Data curation, Formal analysis, Investigation, Methodology, Writing - original draft, Project administration, Writing - review and editing.

Data curation, Formal analysis, Supervision, Investigation, Writing - original draft, Project administration.

Software, Formal analysis, Supervision, Writing - original draft, Project administration.

Project administration.

Supervision, Writing - review and editing.

Software, Project administration.

Project administration.

Project administration.

Methodology, Project administration.

Supervision, Methodology, Writing - review and editing.

Investigation, Methodology.

Supervision, Project administration, Writing - review and editing.

Data curation, Formal analysis, Supervision, Methodology, Writing - original draft, Writing - review and editing.

Conceptualization, Data curation, Formal analysis, Supervision, Investigation, Methodology, Writing - original draft, Writing - review and editing.

Conceptualization, Data curation, Supervision, Funding acquisition, Methodology, Writing - original draft, Writing - review and editing.

Ethics

Animal experimentation: Mouse experiments were in strict accordance with EU and Swedish law and were approved by the research animal ethics committee in Linköping. The approval number is ID 1278-18.

Additional files

Source data 1. Exact P values.
Transparent reporting form

Data availability

Data generated in this study are presented in the manuscript and supporting files. A source file for exact P values is also included.

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Decision letter

Editor: Yousin Suh1
Reviewed by: Susan Michaelis2

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

The manuscript by Bergo and colleagues seeks to test the effects of genetic and pharmacologic inhibition of isoprenylcysteine carboxylmethyltransferase (ICMT) on rescuing phenotypes of Hutchinson Gilford Progeria Syndrome (HGPS) in culture and in vivo in mice. Using progerin-knock-in mice, the authors show that a hypomorphic Icmt allele improves survival, restores vascular smooth muscle cell numbers in the aorta, and increases skeletal muscle fibre size. In addition, using a synthetic ICMT inhibitor referred to as C75, the author show its ability to rescue classical cellular and biochemical progeria hallmarks in HGPS patient fibroblasts and Zmpste24-deficient mouse fibroblasts, including premature senescence. The study presents an extension and confirmation of the authors' previous work showing that hypomorphic Icmt improves survival in Zmpste24-deficient mice, demonstrating a potential for ICMT inhibitors as new therapeutics against HGPS and other progeroid syndromes.

Decision letter after peer review:

Thank you for submitting your article "A small-molecule ICMT inhibitor delays senescence of Hutchinson-Gilford progeria syndrome cells" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Jessica Tyler as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Susan Michaelis (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.

Summary:

The manuscript by Bergo and colleagues seeks to test the effects of genetic and pharmacologic inhibition of isoprenylcysteine carboxylmethyltransferase (ICMT) on rescuing phenotypes of Hutchinson Gilford Progeria Syndrome (HGPS) in culture and in vivo in mice. Using progerin-knock-in mice, the authors show that a hypomorphic Icmt allele improves survival, restores vascular smooth muscle cell numbers in the aorta, and increases skeletal muscle fibre size. In addition, using a synthetic ICMT inhibitor referred to as C75, the author show its ability to rescue classical cellular and biochemical progeria hallmarks in HGPS patient fibroblasts and Zmpste24-deficient mouse fibroblasts, including premature senescence. The study presents an extension and confirmation of the authors' previous work showing that hypomorphic Icmt improves survival in Zmpste24-deficient mice, demonstrating a potential for ICMT inhibitors as new therapeutics against HGPS and other progeroid syndromes. The reviewers agreed that the study provides a significant finding in establishing ICMT as a druggable target for HGPS and should be of broad interest to the community of researchers studying the biology of aging and HGPS. However, as detailed below, a number of concerns were also raised, relating in large part to insufficient clarity in the current version with respect to the authors' methods and the limitations of their approach and data.

Essential revisions:

1) This work is a continuation of their previous study in Zmpste24 mice which slightly different from HGPS model used in the current study. The rescue on the HGPS mice is not surprising. However, the number of the mice presented in this study is far from sufficient in particular given that the generation of the LAKIG609G/G609GIcmthm/hm compound mutant mice seemed to be problematic, suggesting potential genetic variation due to modified gene. This caveat should be discussed in the text. Detailed answers to the following questions must be available.

a) What is the control body weight in Figure 1C? How the initial growth of the LAKIG609G/G609GIcmthm/hm compound mutant mice is affected during the first 2 months?

b) What exactly is the breeding problem resulting in difficulty in the production of LAKIG609G/G609GIcmthm/hm mice? If the LAKIG609G/G609GIcmthm/hm compound mutants are greatly rescued phenotypically, are they fertile?

c) Figure 1B, how many breeding has been made to obtain 3 LAKIG609G/G609GIcmthm/hm compound mutants? The number of the mice used in this analysis is too little. How long can the compound mutant mice survive? What is the average lifespan?

2) The lack of important controls in several figures presented, especially the data from WT cells, is a concern. Detailed answers to the following questions must be available.

a) The WT doubling should be included in Figure 2E, F and G, as an important control.

b) Figure 2I lack an important control to compare the growth of WT under C75 treatments with that in LAKIG609G/G609GIcmthm/hm. If C75 inhibits prelamin A processing, it will produce unprocessed yet farnesylated prelamin A.

c) In Figure 3B. Authors should show what happen to WT cells in AKT phosphorylation in response to C75 treatment.

3) Why does Figure 2J, K showed inhibitory effects of FTI lonafarnib? If the FTI lonafarnib and C75 both inhibit the nuclear membrane localization of progerin, why does C75 promote HGPS proliferation whereas FTI lonafarnib does not? Figure 2K does not necessarily mean that C75 failed rescue HGPS cells in the absence of methylation. It can simply mean the inhibitory effect of FTI. What is the impact of C75 on WT cells in terms of nuclear shape and lamin A processing? Text and data presentation should be clarified to address these.

4) The treatment of WT and Zmpste24 cells by C75 should have exactly the same effect given that RCE1 is involved in the first cleavage. Can authors explain why their data showed different response of WT and Zmpste24/HGPS cells? Text and data presentation should be clarified to address this point.

5) In Figure 3, authors showed that C75 treatment stabilized progerin therefore increasing the accumulation of progerin, specifically it nucleoplasm. It is very confusing as we know that it is the membrane progerin or prelamin A that gives rise to the increased DNA damage and senescence phenotype. Is the nuclear membrane-bound progerin/prelamin A or the increased nucleoplasmic prelamin A/progerin (farnesylate yet unmethylated) that results in reduced senescence? How? Text and data presentation should be clarified to address these points.

6) The only issue that detracts slightly from complete enthusiasm for this compelling study is the lack of thorough characterization of the new drug C75 as strictly a methylation inhibitor. Unanticipated effects of a drug on more than one target enzyme are not unprecedented, especially for lamin A processing enzymes. For instance, the HIV aspartyl protease inhibitor lopinavir unexpectedly is a zinc metalloprotease inhibitor for Zmpste24 (Coffinier et al., 2007; PMID: 17652517). Likewise, a GGTI inhibitor was unexpectedly shown to block Zmpste24 activity (Chang et al., 2012; PMID: 22448028). In the present study, C75 treatment causes accumulation of prelamin A (Figure 2B) and release of some RAS from the membrane fraction (Figure 2—figure supplement 2B), which are both expected outcomes of FTase inhibition. Is it possible that C75 could be inhibiting (albeit to a lesser degree than it inhibits ICMT) the farnesyltransferase complex? Perhaps the authors could look at another farnesylated substrate, such as HDJ-2, to show C75 has no effect on its mobility by SDS-PAGE? The mobility shift of HDJ-2 is often used as a test for farnesyltransferase inhibitors (FTIs), and would be a useful control for C75 treatment. Alternatively, or in addition, it could be helpful to test if C75 has any FTI activity in an in vitro assay. Text and data presentation should be clarified to address this point.

7) Importantly, the authors do make a significant effort to address the issue of C75 specificity to some extent, in that they show that proliferation of the Zmpste24-/- cells is improved by C75 (Figure 2G) but proliferation of the Zmpste24-/-Ictm∆/∆ double mutant cells is unchanged upon C75 treatment (Figure 2H), suggesting genetically or pharmacologically blocking ICMT have same effect. Likewise, HGPS cells co-treated with C75 and FTI's (Figure 2K) abolishes the population doubling increase observed with C75 alone, (expected for FTIs hitting a step upstream of that inhibited by C75) strengthening the likelihood that C75 acts by inhibiting mainly ICMT in vivo. These two important figures lack error bars, suggesting the experiments should be repeated. Text and data presentation should be clarified to address this point.

8) If C75 were found to have some modest FTI activity in addition to inhibiting methylation, this would be an important piece of information to establish for this new drug. In any case, the main conclusion of this work – that C75 improves HGPS phenotypes – is clear and well supported. Text and data presentation should be clarified to address this point.

9) Mechanistically, there is little new information provided compared with their early study (Ibrahim et al., 2013). AKT phosphorylation was shown to be relevant to the rescue but no direct evidence to show blocking AKT phosphorylation attenuates C75 effect. Text and data presentation should be clarified to address this point.

10) No in vivo data were presented to show C75 could rescue the premature aging in HGPS mice. Text should be clarified to address this point.

eLife. 2021 Feb 2;10:e63284. doi: 10.7554/eLife.63284.sa2

Author response

Essential revisions:

1) This work is a continuation of their previous study in Zmpste24 mice which slightly different from HGPS model used in the current study. The rescue on the HGPS mice is not surprising. However, the number of the mice presented in this study is far from sufficient in particular given that the generation of the LAKIG609G/G609G Icmthm/hm compound mutant mice seemed to be problematic, suggesting potential genetic variation due to modified gene. This caveat should be discussed in the text. Detailed answers to the following questions must be available.

We agree with this important point (although survival extension was statistically significant with an n of 3 these numbers are lower than what we would have liked. Indeed, this was the reason we included the following text: “Although these data are statistically sound, they should be interpreted with caution as the mice were difficult to breed and we only obtained three double homozygotes.” It was also the reason we also analyzed the impact of Icmt deficiency on heterozygous LAKI mice. We are happy to provide more information as requested in your points below.

a) What is the control body weight in Figure 1C? How the initial growth of the LAKIG609G/G609G Icmthm/hm compound mutant mice is affected during the first 2 months?

Because there was only one male mouse, we combined the body weight curves and showed change in body weight instead of actual body weight in Figure 1C. Author response image 1 shows graphs for the actual body weights of females (A) and males (B) from this graph. Specifically, the body weight of control females was 15–17 g at week 9 (63 days; peak) after which they continually lost weight (see Author response image 1A). Body weight for male control mice was 18–23g at the peak (week 8–9) with subsequent weight loss. Importantly, after the control mice peaked in weight, the LAKIG609G/G609G Icmthm/hmmice remained at the peak weight and even continued to gain some weight.

Author response image 1.

Author response image 1.

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b) What exactly is the breeding problem resulting in difficulty in the production of LAKIG609G/G609G Icmthm/hm mice? If the LAKIG609G/G609G Icmthm/hm compound mutants are greatly rescued phenotypically, are they fertile?

Thank you for this question. The problem is that Mendelian inheritance of the genes does not happen in the pups that are born and that fewer pups than normal are born. The answer to why this happens is unknown but it likely begins with the fact that the heterozygous LAKI mice are quite difficult to breed (smaller than normal litters). You are probably very well aware of these problems; but we would like to mention that from 20 years of experience with breeding genetically modified mouse strains, we have seen this phenomenon at least a dozen other times – i.e., that when we combine two or more alleles that on their own produce offspring with normal inheritance, no pups with double homozygosity are born. Sometimes this phenomenon goes away when the mice are bred onto a new genetic background, but in this case it didn’t. Because we have obtained only three double homozygotes thus far, these mice became too precious to be used for breeding, and we are afraid we can’t answer the question of whether they are fertile. We are continuing to breed these mouse strains, but will not be able to produce more for this particular submission. Since the current data is statistically significant, and we see similar effects with heterozygous LAKI mice (also significant), we hope that you will agree that this could be sufficient for the scope of this study, and that the robust rescue of the vascular phenotype (which is essential for the children with progeria) along with the drug data addresses new aspects of whether targeting ICMT would be effective in HGPS therapy.

c) Figure 1B, how many breeding has been made to obtain 3 LAKIG609G/G609G Icmthm/hm compound mutants? The number of the mice used in this analysis is too little. How long can the compound mutant mice survive? What is the average lifespan?

Please see also the answers to point 1b for more details. For the LAKI allele, only heterozygotes (hets) are used in breeding and even they often produce smaller size litters than normal; for the Icmthm allele, both hets and homozygotes are used. The success rate should thus be that 1 in 4, 1 in 8, or 1 in 16 should be double homozygotes. We have bred to date 87 females and genotyped >300 pups. We can add that breeding the Zmpste24-knockout allele with Icmthm/hm was not this difficult. And again, because we only obtained three double homozygotes we killed them after all the control mice had died of progeria so that we could analyze the aortas and muscle fibers. Thus, we can’t answer the question of how long they survive. Please see Figure 1G for survival of the LAKI heterozygotes; these mice don’t show vascular or muscle phenotypes in the same way that homozygotes do, and thus we opted for survival experiments for the hets.

2) The lack of important controls in several figures presented, especially the data from WT cells, is a concern. Detailed answers to the following questions must be available.

a) The WT doubling should be included in Figure 2E, F and G, as an important control.

We agree with this comment. In our original figure drafts the effect of the drug on WT human cells was included, but the curves became “messy”, crowded, and very difficult to read. Thus, we opted to show this data separately in the figure panel Figure 2I. As you can see, the drug only minimally affect proliferation of WT cells. We hope you will agree with this strategy. Thus, the control in terms of effects on WT cells is already included. Another control is shown in Figure 2H which is Zmpste24- and Icmt- double-deficient cells where we find that the ICMT inhibitor drug has no impact in cells already lacking the Icmt gene – an indication of drug specificity.

b) Figure 2I lack an important control to compare the growth of WT under C75 treatments with that in LAKIG609G/G609G Icmthm/hm. If C75 inhibits prelamin A processing, it will produce unprocessed yet farnesylated prelamin A.

We understand the comment but would like to mention that it is very difficult to compare human and mouse cell lines as they behave differently in terms of physical growth rate and population doubling time and we would therefore prefer to keep them separate as is. The reader can still compare the shape and slope of the curves side-by-side in the two panels. We hope you will agree with this argument.

c) In Figure 3B. Authors should show what happen to WT cells in AKT phosphorylation in response to C75 treatment.

Low levels of phospho-AKT is a reproducible finding in HGPS and Zmpste24-deficient cells – and these levels are increased and sometimes even restored to WT levels upon ICMT inhibition. WT cells already have high phospho-AKT levels and we had not included effects of C75 in these cells in our previous submission. Thus, in response to this important comment we analyzed levels of phosphorylated and total AKT in WT human cells incubated with C75. The western blots show that levels of phosphorylated and total AKT do not change in WT cells incubated with C75. The new western blot and quantification is added to Figure 3B along with comments in the Results and figure legend text. Thank you for this comment.

3) Why does Figure 2J, K showed inhibitory effects of FTI lonafarnib? If the FTI lonafarnib and C75 both inhibit the nuclear membrane localization of progerin, why does C75 promote HGPS proliferation whereas FTI lonafarnib does not? Figure 2K does not necessarily mean that C75 failed rescue HGPS cells in the absence of methylation. It can simply mean the inhibitory effect of FTI. What is the impact of C75 on WT cells in terms of nuclear shape and lamin A processing? Text and data presentation should be clarified to address these.

There are two main conclusions of Figure 2J and K: First, that the recently approved and only therapy for HGPS (i.e., FTI) is a drug with potent anti-proliferative properties (verified also by genetic strategies in previous studies) a result which we believe should increase the interest in targeting ICMT. Second, the cell-killing effect of FTIs is independent of ICMT-mediated methylation. This result makes sense as protein methylation can’t occur in the absence of farnesylation. FTase and ICMT process many protein substrates, in addition to prelamin A. The cell-killing effect of FTIs likely stems from reducing the farnesyl-dependent membrane targeting or function of several other proteins such as lamin B, CENP, RAS, or RHOA etc. It is fascinating to us that targeting ICMT which renders many proteins unmethylated can increase proliferation of HGPS cells—an indication that prelamin A methylation is an underlying cause and an indication that CAAX-protein methylation is dispensable for many cell functions.

Regarding the impact of C75 on nuclear shape of WT cells; since C75 (and Icmt knockout) does not influence nuclear shape in HGPS cells, it is highly unlikely that it would affect nuclear shape in WT cells where the levels of misshapen nuclei are low.

Regarding the question on the effects of C75 on lamin A processing in WT cells. We have previously shown that knockout of Icmt blocks prelamin A methylation and reduces the efficiency of the upstream ZMPSTE24-mediated cleavage resulting in partial prelamin A accumulation. To address your highly relevant question regarding the C75 drug, we performed western blots on C75-exposed WT cells. The data confirm the previous genetic analysis that ICMT inhibition causes prelamin A accumulation. The new data are added to Figure 2B, right panel, along with text additions in the Results and figure legends. Thank you for this suggestion.

4) The treatment of WT and Zmpste24 cells by C75 should have exactly the same effect given that RCE1 is involved in the first cleavage. Can authors explain why their data showed different response of WT and Zmpste24/HGPS cells? Text and data presentation should be clarified to address this point.

In wild-type cells, prelamin A is fully processed to mature lamin A and the cells grow and proliferate normally (i.e., mature lamin A is not farnesylated or methylated as the C-terminus including the farnesylmethylcysteine has been cleaved off); whereas In HGPS and Zmpste24-deficient cells, farnesylated and methylated progerin/prelamin A accumulates and causes senescence. Therefore, we don’t expect that C75 would produce the same effects in WT cells as in HGPS and Zmpste24-deficient cells. Our data suggest that progerin/prelamin A methylation contributes to the toxicity of these proteins and their ability to induce senescence; and we propose that blocking progerin/prelamin A methylation mislocalizes the proteins into the nucleoplasm and thereby reduces their ability to induce DNA damage, metabolic alterations, and senescence.

We hope this explanation is sufficient. Parts of this text has been added to the Results.

5) In Figure 3, authors showed that C75 treatment stabilized progerin therefore increasing the accumulation of progerin, specifically it nucleoplasm. It is very confusing as we know that it is the membrane progerin or prelamin A that gives rise to the increased DNA damage and senescence phenotype. Is the nuclear membrane-bound progerin/prelamin A or the increased nucleoplasmic prelamin A/progerin (farnesylate yet unmethylated) that results in reduced senescence? How? Text and data presentation should be clarified to address these points.

We agree with the assessment that nuclear membrane–bound progerin/prelamin A causes DNA damage and senescence. Therefore, we think it is fairly straightforward that if progerin becomes detached from the nuclear membrane—because it is no longer methylated, it can no longer cause senescence. We hope you will agree with this conclusion. This is now discussed in the main text starting on 113.

6) The only issue that detracts slightly from complete enthusiasm for this compelling study is the lack of thorough characterization of the new drug C75 as strictly a methylation inhibitor. Unanticipated effects of a drug on more than one target enzyme are not unprecedented, especially for lamin A processing enzymes. For instance, the HIV aspartyl protease inhibitor lopinavir unexpectedly is a zinc metalloprotease inhibitor for Zmpste24 (Coffinier et al., 2007; PMID: 17652517). Likewise, a GGTI inhibitor was unexpectedly shown to block Zmpste24 activity (Chang et al., 2012; PMID: 22448028). In the present study, C75 treatment causes accumulation of prelamin A (Figure 2B) and release of some RAS from the membrane fraction (Figure 2—figure supplement 2B), which are both expected outcomes of FTase inhibition. Is it possible that C75 could be inhibiting (albeit to a lesser degree than it inhibits ICMT) the farnesyltransferase complex? Perhaps the authors could look at another farnesylated substrate, such as HDJ-2, to show C75 has no effect on its mobility by SDS-PAGE? The mobility shift of HDJ-2 is often used as a test for farnesyltransferase inhibitors (FTIs), and would be a useful control for C75 treatment. Alternatively, or in addition, it could be helpful to test if C75 has any FTI activity in an in vitro assay. Text and data presentation should be clarified to address this point.

Thank you for these relevant comments and discussion. In response to this comment, we run HDJ-2 western in HGPS cells incubated with two concentrations of C75. We found C75 did not have any effect on HDJ-2 mobility. An important reason that we tend to believe the drug is specific for ICMT (and does not affect FTase) is that the drug produces essentially identical effects as Icmt deficiency. Indeed, knockout of Icmt – where FTase is fully functional – leads to RAS mislocalization and prelamin A accumulation (Bergo et al., JBC 2000; Bergo et al., 2002). Regardless, we agree with you that an FTase activity assay would be the best option to rule out non-specific effects on FTase, but we unfortunately do not have this assay set up. We hope you agree with us that the other data (including your excellent suggestion of HDJ-2 western blots) and arguments herein (and in point 7 below) are sufficient. Please see new figure panel (Figure supplement 2—figure supplement 2B) and brief text in the Results.

7) Importantly, the authors do make a significant effort to address the issue of C75 specificity to some extent, in that they show that proliferation of the Zmpste24-/- cells is improved by C75 (Figure 2G) but proliferation of the Zmpste24-/- Ictm∆/∆ double mutant cells is unchanged upon C75 treatment (Figure 2H), suggesting genetically or pharmacologically blocking ICMT have same effect. Likewise, HGPS cells co-treated with C75 and FTI's (Figure 2K) abolishes the population doubling increase observed with C75 alone, (expected for FTIs hitting a step upstream of that inhibited by C75) strengthening the likelihood that C75 acts by inhibiting mainly ICMT in vivo. These two important figures lack error bars, suggesting the experiments should be repeated. Text and data presentation should be clarified to address this point.

Thank you for this comment and for pointing out the missing error bars mistake. The experiment with Zmpste24/Icmt double knockout cells is shown in Figure 2H and actually did contain error bars (see the last data point enlarged): but the data is very reproducible and the technical replicates have very small differences. However, the error bars were definitely missing in Figure 2K and we have now added them. The experiments in all the cell population doubling experiments have been performed multiple times and are highly reproducible.

8) If C75 were found to have some modest FTI activity in addition to inhibiting methylation, this would be an important piece of information to establish for this new drug. In any case, the main conclusion of this work- that C75 improves HGPS phenotypes- is clear and well supported. Text and data presentation should be clarified to address this point.

Thank you for your comment. Please see answers to point 6 for a discussion on this topic and added text in the Results and a figure panel to Figure 2—figure supplement 2B.

9) Mechanistically, there is little new information provided compared with their early study (Ibrahim et al., 2013). AKT phosphorylation was shown to be relevant to the rescue but no direct evidence to show blocking AKT phosphorylation attenuates C75 effect. Text and data presentation should be clarified to address this point.

We agree with this comment. The goal of this study as outlined in the Introduction was to determine whether knockout of Icmt would improve phenotypes in an authentic progerin-expressing HGPS mouse model and to synthesize and pre-clinically validate a new ICMT inhibitor. The study does provide some new evidence that blocking methylation improves HGPS phenotypes, including the vascular and muscle fiber phenotypes which was not shown before; and new data on effects of C75 on cell respiration/metabolism/ROS levels/ER stress/DNA damage. To address your comment, we have added new text in the Discussion.

10) No in vivo data were presented to show C75 could rescue the premature aging in HGPS mice. Text should be clarified to address this point.

Yes, this is the main limitation of the study and we believe it is clearly shown and discussed in the revised manuscript.

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    Source data 1. Exact P values.
    elife-63284-data1.xlsx (9KB, xlsx)
    Transparent reporting form
    elife-63284-transrepform.docx (245.7KB, docx)

    Data Availability Statement

    Data generated in this study are presented in the manuscript and supporting files. A source file for exact P values is also included.

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