Systematic pharmacological screens uncover novel pathways involved in cerebral cavernous malformations

Cerebral cavernous malformations (CCM) are a group of capillary-venous cavernoma pathologies that mainly affect lowly-perfused venous vessel beds and can cause bleeding within the central nervous system. CCM lesions may be deeply situated within the brain or spinal cord and thus pose major therapeutic challenges. The more common form of CCM occurs sporadically with a prevalence of ~0.5% in the general population while familial forms are much rarer. These latter forms are autosomal dominant diseases caused by the loss of CCM1/KRIT1 (Krev Interaction Trapped Protein 1), CCM2/Malcavernin, or CCM3/PDCD10 (Programmed Cell Death 10). We and others described a novel pathophysiological pathway involving the strong activation of Krüppel-like transcription factor KLF2/4 signaling within CCM-deficient endothelial cells (Renz et al., Developmental Cell 2015).

As partners of a larger EU-funded consortium, we used C. elegans, zebrafish embryos, and human umbilical vein endothelial cells (HUVECs) deficient in CCM proteins to screen 5,268 compounds from the LOPAC/Selleck, Spectrum, and GlaxoSmithKline protein kinase inhibitor libraries (Otten et al., EMBO Molecular Medicine 2018). This led to the identification of 32 compounds that suppressed CCM phenotypes in both C. elegans and zebrafish, five of which prevented actin stress fiber formation in HUVECs depleted of CCM1. These compounds have been predicted to affect diverse biological processes including angiogenesis (ENMD-2076), phospholipid metabolism (DL-erythro-dihydrosphingosine), PI3K/Akt/mTor signaling (ridaforolimus), acetylcholine signaling (DL-homatropine hydrobromide), and hormone signaling (13-cis-retinoic acid). Indirubin-3-monoxime had suppressive effects in zebrafish and HUVECs and reduced lesion number and size in CCM2 and CCM3 in mice. Molecular studies demonstrated that indirubin-3-monoxime suppressed the elevated expression levels of klf2a mRNA in zebrafish ccm2 mutants. Analyses of known and predicted targets of Indirubin-3-monoxime (and other compounds) have revealed multiple cellular targets. Curiously, a number of drugs identified in both C. elegans and zebrafish have anti-hypertensive and anti-angiogenic effects. Since C. elegans lacks a circulatory system, the drugs appear to exert effects that go beyond the well-established physiological functions of these molecular pathways in the cardiovascular system of vertebrates.

 

 

Figure 4: Cerebral cavernous malformations are mulberry-like vascular lesions (indicated by the red berry) in the central nervous system causing strokes and seizures. The authors performed an unbiased small-molecule suppression screen in zebrafish, among other model systems, with the aim of reverting or preventing the detrimental effects of this pathology. Indirubin-3-monoxime was found to alleviate the severity of lesion burden in murine preclinical models of the pathology. Shown is a carpet of herbal plants from which Indirubin can be extracted (Artwork by Kat Menschik).