Molecular and cellular consequences of mevalonate kinase deficiency.

Mevalonate kinase deficiency (MKD) is an autosomal recessive metabolic disorder associated with recurrent autoinflammatory episodes. The disorder is caused by bi-allelic loss-of-function variants in the MVK gene, which encodes mevalonate kinase (MK), an early enzyme in the isoprenoid biosynthesis pathway. To identify molecular and cellular consequences of MKD, we studied primary fibroblasts from severely affected patients with mevalonic aciduria (MKD-MA) and more mildly affected patients with hyper IgD and periodic fever syndrome (MKD-HIDS). As previous findings indicated that the deficient MK activity in MKD impacts protein prenylation in a temperature-sensitive manner, we compared the subcellular localization and activation of the small Rho GTPases RhoA, Rac1 and Cdc42 in control, MKD-HIDS and MKD-MA fibroblasts cultured at physiological and elevated temperatures. This revealed a temperature-induced altered subcellular localization and activation in the MKD cells. To study if and how the temperature-induced ectopic activation of these signalling proteins affects cellular processes, we performed comparative transcriptome analysis of control and MKD-MA fibroblasts cultured at 37 °C or 40 °C. This identified cell cycle and actin cytoskeleton organization as respectively most down- and upregulated gene clusters. Further studies confirmed that these processes were affected in fibroblasts from both patients with MKD-MA and MKD-HIDS. Finally, we found that, similar to immune cells, the MK deficiency causes metabolic reprogramming in MKD fibroblasts resulting in increased expression of genes involved in glycolysis and the PI3K/Akt/mTOR pathway. We postulate that the ectopic activation of small GTPases causes inappropriate signalling contributing to the molecular and cellular aberrations observed in MKD.

Mevalonate kinase-deficient THP-1 cells show a disease-characteristic pro-inflammatory phenotype.

Objective: Bi-allelic pathogenic variants in the MVK gene, which encodes mevalonate kinase (MK), an essential enzyme in isoprenoid biosynthesis, cause the autoinflammatory metabolic disorder mevalonate kinase deficiency (MKD). We generated and characterized MK-deficient monocytic THP-1 cells to identify molecular and cellular mechanisms that contribute to the pro-inflammatory phenotype of MKD. Methods: Using CRISPR/Cas9 genome editing, we generated THP-1 cells with different MK deficiencies mimicking the severe (MKD-MA) and mild end (MKD-HIDS) of the MKD disease spectrum. Following confirmation of previously established disease-specific biochemical hallmarks, we studied the consequences of the different MK deficiencies on LPS-stimulated cytokine release, glycolysis versus oxidative phosphorylation rates, cellular chemotaxis and protein kinase activity. Results: Similar to MKD patients' cells, MK deficiency in the THP-1 cells caused a pro-inflammatory phenotype with a severity correlating with the residual MK protein levels. In the MKD-MA THP-1 cells, MK protein levels were barely detectable, which affected protein prenylation and was accompanied by a profound pro-inflammatory phenotype. This included a markedly increased LPS-stimulated release of pro-inflammatory cytokines and a metabolic switch from oxidative phosphorylation towards glycolysis. We also observed increased activity of protein kinases that are involved in cell migration and proliferation, and in innate and adaptive immune responses. The MKD-HIDS THP-1 cells had approximately 20% residual MK activity and showed a milder phenotype, which manifested mainly upon LPS stimulation or exposure to elevated temperatures. Conclusion: MK-deficient THP-1 cells show the biochemical and pro-inflammatory phenotype of MKD and are a good model to study underlying disease mechanisms and therapeutic options of this autoinflammatory disorder.

Identification of FDA-approved drugs that increase mevalonate kinase in hyper IgD syndrome.

Mevalonate kinase deficiency (MKD) is an autoinflammatory metabolic disorder caused by bi-allelic loss-of-function variants in the MVK gene, resulting in decreased activity of the encoded mevalonate kinase (MK). Clinical presentation ranges from the severe early-lethal mevalonic aciduria to the milder hyper-IgD syndrome (MKD-HIDS), and is in the majority of patients associated with recurrent inflammatory episodes with often unclear cause. Previous studies with MKD-HIDS patient cells indicated that increased temperature, as caused by fever during an inflammatory episode, lowers the residual MK activity, which causes a temporary shortage of non-sterol isoprenoids that promotes the further development of inflammation. Because an increase of the residual MK activity is expected to make MKD-HIDS patients less sensitive to developing inflammatory episodes, we established a cell-based screen that can be used to identify compounds and/or therapeutic targets that promote this increase. Using a reporter HeLa cell line that stably expresses the most common MKD-HIDS variant, MK-V377I, C-terminally tagged with bioluminescent NanoLuc luciferase (nLuc), we screened the Prestwick Chemical Library®, which includes 1280 FDA-approved compounds. Multiple compounds increased MK-V377I-nLuc bioluminescence, including steroids (i.e., glucocorticoids, estrogens, and progestogens), statins and antineoplastic drugs. The glucocorticoids increased MK-V377I-nLuc bioluminescence through glucocorticoid receptor signaling. Subsequent studies in MKD-HIDS patient cells showed that the potent glucocorticoid clobetasol propionate increases gene transcription of MVK and other genes regulated by the transcription factor sterol regulatory element-binding protein 2 (SREBP-2). Our results suggest that increasing the flux through the isoprenoid biosynthesis pathway by targeting the glucocorticoid receptor or SREBP-2 could be a potential therapeutic strategy in MKD-HIDS.

Compromised Protein Prenylation as Pathogenic Mechanism in Mevalonate Kinase Deficiency.

Mevalonate kinase deficiency (MKD) is an autoinflammatory metabolic disorder characterized by life-long recurring episodes of fever and inflammation, often without clear cause. MKD is caused by bi-allelic pathogenic variants in the MVK gene, resulting in a decreased activity of the encoded enzyme mevalonate kinase (MK). MK is an essential enzyme in the isoprenoid biosynthesis pathway, which generates both non-sterol and sterol isoprenoids. The inflammatory symptoms of patients with MKD point to a major role for isoprenoids in the regulation of the innate immune system. In particular a temporary shortage of the non-sterol isoprenoid geranylgeranyl pyrophosphate (GGPP) is increasingly linked with inflammation in MKD. The shortage of GGPP compromises protein prenylation, which is thought to be one of the main causes leading to the inflammatory episodes in MKD. In this review, we discuss current views and the state of knowledge of the pathogenetic mechanisms in MKD, with particular focus on the role of compromised protein prenylation.