Defining the role of CAAX protein proteolysis and methylation in the pathogenesis and treatment of progeria

Hutchinson-Gilford progeria syndrome (HGPS) is a rare childhood disease characterized by failure to thrive, bone abnormalities, hair loss, and a shortened life span due to cardiovascular disease, and nuclear shape abnormalities in cultured cells. HGPS is caused by de-novo mutations in LMNA, the gene encoding prelamin A. Lamin A is produced from prelamin A following three modifications at a carboxyl-terminal CAAX motif: farnesylation of the cysteine by farnesyltransferase (FTase); release of the –AAX by RAS-converting enzyme 1 (RCE1); and methylation of the farnesylcysteine residue by isoprenylcysteine carboxyl methyltransferase (ICMT); mature lamin A is subsequently produced by proteolytic removal of the last 15 amino acids (including the farnesylcysteine residue) by Zinc metalloproteinase Ste24 homologue (ZMPSTE24). The HGPS mutation produces a truncated prelamin A—progerin—which lacks the ZMPSTE24 cleavage site; thus, farnesylated and methylated progerin accumulates at the nuclear lamina in HGPS patient’s cells and causes all disease phenotypes. Other rare forms of progeria result from inactivating mutations in ZMPSTE24 which leads to accumulation of full-length prelamin A.

Targeting FTase with small-molecule drugs prevents prelamin A and progerin farnesylation and corrects the nuclear shape abnormalities observed in cultured progeria cells and reduces symptoms in progeria mouse models. Today, FTase inhibitors (FTIs) constitute the only approved therapy for HGPS. However, FTIs have anti-proliferative properties and their effects in HGPS patients including the survival benefit are modest. Thus, new therapeutic strategies are needed. In this thesis, I have explored the biochemical and medical consequences of targeting ICMT and RCE1 on the function of prelamin A and progerin and the therapeutic benefits of these interventions.

In paper I, we followed up on our group’s earlier genetic studies showing that targeting Icmt overcomes senescence of Zmpste24-deficient cells and human HGPS cells and increases survival of Zmpste24-deficient mice with progeria. We found that knockout of Icmt improves survival of progerin knock-in mice—a more relevant model of HGPS. Moreover, we synthesized a small-molecule ICMT inhibitor, C75, and showed that it inhibits ICMT, stimulates AKT signaling, reduces senescence, and increases proliferation of cultured progeria cells but has no effect on cells lacking the Icmt gene, indicating drug specificity.

In paper II, we tested the hypothesis that targeting RCE1-mediated cleavage of prelamin A would reduce its toxicity and be useful for therapeutic purposes. It was already known that in the absence of RCE1, ZMPSTE24 can perform the same –AAX cleavage; thus, this strategy would only work on progeria caused by ZMPSTE24 deficiency. We found that knockout of Rce1 in Zmpste24-deficient mice prevented –AAX proteolysis, reduced progeria phenotypes, and improved survival, but to a lesser extent than targeting Icmt. Inhibiting RCE1 expression in cells from a ZMPSTE24-deficient patient stimulated AKT signaling, reduced senescence, and improved proliferation.

We conclude that targeting RCE1 and ICMT reduces the toxicity of prelamin A and progerin and that an ICMT inhibitor can overcome senescence and stimulate proliferation of HGPS cells. The results suggest RCE1 could be a potential drug target in future therapy of ZMPSTE24 deficiency and that ICMT inhibitors should be further developed for in-depth preclinical evaluation in HGPS therapy.

List of scientific papers

I. A small-molecule ICMT inhibitor delays senescence of Hutchinson-Gilford progeria syndrome cells. Xue Chen, Haidong Yao, Muhammad Kashif, Gwladys Revêchon, Maria Eriksson, Jianjiang Hu, Ting Wang, Yiran Liu, Elin Tüksammel, Staffan Strömblad, Ian M Ahearn, Mark R Philips, Clotilde Wiel, Mohamed X Ibrahim*, Martin O Bergo*. eLife. 2021; 10: e63284. *Authors contributed equally.
https://doi.org/10.7554/eLife.63284

II. Targeting RAS‐converting enzyme 1 overcomes senescence and improves progeria‐like phenotypes of ZMPSTE24 deficiency. Haidong Yao*, Xue Chen*, Muhammad Kashif, Ting Wang, Mohamed X. Ibrahim, Elin Tüksammel, Gwladys Revêchon, Maria Eriksson, Clotilde Wiel*, Martin O. Bergo*. Aging Cell. 2020; e13200. *Authors contributed equally.
https://doi.org/10.1111/acel.13200