Combined HMG-COA reductase and prenylation inhibition in treatment of CCM

Abstract

Cerebral cavernous malformations (CCMs) are common vascular anomalies that develop in the central nervous system and, more rarely, the retina. The lesions can cause headache, seizures, focal neurological deficits, and hemorrhagic stroke. Symptomatic lesions are treated according to their presentation; however, targeted pharmacological therapies that improve the outcome of CCM disease are currently lacking. We performed a high-throughput screen to identify Food and Drug Administration-approved drugs or other bioactive compounds that could effectively suppress hyperproliferation of mouse brain primary astrocytes deficient for CCM3. We demonstrate that fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase and the N-bisphosphonate zoledronic acid monohydrate, an inhibitor of protein prenylation, act synergistically to reverse outcomes of CCM3 loss in cultured mouse primary astrocytes and in Drosophila glial cells in vivo. Further, the two drugs effectively attenuate neural and vascular deficits in chronic and acute mouse models of CCM3 loss in vivo, significantly reducing lesion burden and extending longevity. Sustained inhibition of the mevalonate pathway represents a potential pharmacological treatment option and suggests advantages of combination therapy for CCM disease.cerebral cavernous malformations | fluvastatin | zoledronic acid | mevalonate pathway | high-throughput screen C erebral cavernous malformations (CCMs) are common vascular malformations that develop in the brain, spinal cord, and, more rarely, the retina (1, 2). The lesions consist of dilated sinusoidal channels lined with a single layer of endothelium devoid of vessel wall elements. CCM disease is often asymptomatic, but on occasion the lesions rupture leading to hemorrhagic stroke. Other common symptoms are headache, seizures, and focal neurological deficits. Treatment options include observation of asymptomatic lesions, antiepileptic medication, and surgical excision of lesions in patients with symptomatic or repetitive hemorrhages or intractable seizures; however pharmacological therapies that improve outcome are lacking. CCM disease is usually sporadic, although 20% of patients carry lossof-function mutations in one of three genes: CCM1 (also known as "KRIT1"), CCM2, and CCM3 (also known as "PDCD10") (3), which encode structurally unrelated cytoplasmic proteins with critical roles in endothelial cells (4) and, uniquely for CCM3, in neurons and astrocytes as well (5, 6). CCM3 loss in neural progenitors results in hyperproliferation/activation of brain astrocytes and enhanced activity of RhoA in vivo and increased proliferation and survival of primary astrocytes in vitro (5, 6). We took advantage of these well-defined cellular phenotypes of CCM3 loss to explore potential pharmacological treatment options for CCM. High-throughput screening of available drugs identified fluvastatin, a 3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase inhibitor as a top candidate. In combination with the N-bisphosphonate zoledronic acid monohyd...

Systematic pharmacological screens uncover novel pathways involved in cerebral cavernous malformations

“…Based on 1,472 of 1,600 compounds with annotated targets in zebrafish and 4,170 of 4,748 in C. elegans , DePick predicted 47 and 134 human proteins as statistically significant targets of the compounds identified in the zebrafish and in the C. elegans screens, respectively (; ). Several of the targets identified in the C. elegans screen had previously been implicated in CCM; these included TLR4 (Tang et al , ), metalloproteinases (MMP2, MMP7, MMP13, MMP14; Zhou et al , ), and HMGCR (Nishimura et al , ), which is a strong validation of the DePick method ().…”

“…Repurposed drug screens identify compounds that suppress CCM mutant phenotypes in zebrafish and C. elegans Most screens in the past have been primarily based on simplified in vitro models that had only a limited ability to recreate the complexity of the cardiovascular system or of the complex whole organismal interactions that may be affected in the CCM pathology (Gibson et al, 2015;Nishimura et al, 2017). To identify compounds for a pharmacological suppression of CCM phenotypes, we employed diverse assays on multiple organisms that can help to discriminate distinct effects on the cardiovascular system or cell biology upon loss of CCM proteins ( Fig 1A).…”

“…Our approach to use pharmacological suppression to restore the tissue architecture that is disturbed in CCM mutants involved organismal and cell‐based assays. This provided a highly integrated platform that exceeded the complexity of previous purely cell‐based high‐throughput screens (Gibson et al , ; Nishimura et al , ) and, hence, provided a more comprehensive overview of relevant pathways affected in the disease. While these screens identified a number of compounds that had not previously been implicated in CCM, little overlap with other screens or known suppressing compounds was reported.…”

“…Our approach to use pharmacological suppression to restore the tissue architecture that is disturbed in CCM mutants involved organismal and cell-based assays. This provided a highly integrated platform that exceeded the complexity of previous purely cell-based high-throughput screens (Gibson et al, 2015;Nishimura et al, 2017) and, hence, provided a more comprehensive overview of relevant pathways A-D Shown are representative pictures of brains at P8 from iCCM2 (A, B) or iCCM3 (C, D) mice treated with vehicle (A, C) or with IR3mo (B, D). Mice were treated daily between P2 and P7 with vehicle or with IR3mo.…”

Cerebrovascular disorders associated with genetic lesions

“…A handful of candidate drugs with variable effects have been identified in studies involving cell culture and preclinical models: a) Fasudil, a ROCK inhibitor (approved in Japan and China but not in the US or Europe), which reduces vascular leakiness in Ccm1 and Ccm2 heterozygous mice and lesion burden in a chronic, sensitized model of CCM1, but not CCM2 disease [112, 114, 118]; b) simvastatin, which stabilizes the endothelium of Ccm2 heterozygous mice [75], yet does not reduce lesion burden in acute (inducible) and chronic models of CCM1 and CCM2 [118, 119]; c) cholecalciferol (vitamin D3) and tempol (scavenger of superoxide), two repurposed drugs, which decrease lesion burden in an acute model of CCM2 [119]; and d) the anti-inflammatory drugs sulindac sulfide and sulindac sulfone, which decrease lesion burden in an acute model of CCM3 [120]. More recent findings suggested that sustained inhibition of the mevalonate pathway by inhibition of HMG-CoA reductase and prenylation (respectively, by fluvastatin and the N-bisphosphonate zoledronic acid) is effective in chronic and acute models of CCM3 [83]. It should be noted, however, that these candidate drugs have yet to be tested across all three CCM models, and thus, whether they are broadly effective, even in model organisms, remains an open question.…”