Bromodomain inhibitors regulate the C9ORF72 locus in ALS

Abstract

A hexanucleotide repeat expansion residing within the C9ORF72 gene represents the most common known cause of amyotrophic lateral sclerosis (ALS) and places the disease among a growing family of repeat expansion disorders. The presence of RNA foci, repeat-associated translation products, and sequestration of RNA binding proteins suggests that toxic RNA gain-of-function contributes to pathology while C9ORF72 haploinsufficiency may be an additional pathological factor. One viable therapeutic strategy for treating expansion diseases is the use of small molecule inhibitors of epigenetic modifier proteins to reactivate expanded genetic loci. Indeed, previous studies have established proof of this principle by increasing the drug-induced expression of expanded (and abnormally heterochromatinized) FMR1, FXN and C9ORF72 genes in respective patient cells. While epigenetic modifier proteins are increasingly recognized as druggable targets, there have been few screening strategies to address this avenue of drug discovery in the context of expansion diseases. Here we utilize a semi-high-throughput gene expression based screen to identify siRNAs and small molecule inhibitors of epigenetic modifier proteins that regulate C9ORF72 RNA in patient fibroblasts, lymphocytes and reprogrammed motor neurons. We found that several bromodomain small molecule inhibitors increase the expression of C9ORF72 mRNA and pre-mRNA without affecting repressive epigenetic signatures of expanded C9ORF72 alleles. These data suggest that bromodomain inhibition increases the expression of unexpanded C9ORF72 alleles and may therefore compensate for haploinsufficiency without increasing the production of toxic RNA and protein products, thereby conferring therapeutic value.

C9orf72 promoter hypermethylation is reduced while hydroxymethylation is acquired during reprogramming of ALS patient cells

“…While both cell lines derived from this patient were hypermethylated, immortalized lymphocytes generated by the standard Epstein-Barr Virus (EBV) method 36, 37 possessed a larger HRE, as determined by southern blot analysis 35 . Therefore, we used immortalized lymphocyte cells (hence-forth referred to as lymphocytes) to generate iPSCs.…”

“…We identified an individual within our patient population whose fibroblast and immortalized lymphocyte cell lines maintained stable promoter hypermethylation 35 . While both cell lines derived from this patient were hypermethylated, immortalized lymphocytes generated by the standard Epstein-Barr Virus (EBV) method 36, 37 possessed a larger HRE, as determined by southern blot analysis 35 .…”

“…To examine the acquisition of C9orf72 promoter hypermethylation during neurodevelopment, we generated neural precursor cells (NPCs) and terminally differentiated motor neurons from iPSCs in a two-stage differentiation protocol as we have previously reported 35 . Expression of the NPC cell markers Nestin and Sox2 were confirmed by immunodetection (Figure 1C) while motor neuron specification was confirmed by the expression of the cytoskeletal neuronal marker Tuj1 and Isl1, which localizes to the nucleus and perinucleus of motor neurons (Figure 1D, E).…”

“…Motor neurons were differentiated according to our previously published methods 35 . Briefly, iPSC colonies were cultured in neural stem cell medium on ultra-low attachment flasks.…”

Development of Therapeutics for C9ORF72 ALS/FTD-Related Disorders

“…Specifically, BRD3 was identified as an epigenetic regulator of C9ORF72 expression. Inhibiting BDR3 in C9ORF72-ALS patient fibroblasts resulted in increased levels of the three C9ORF72 transcriptional variants [61]. BRDs share a conserved fold or Bpocket^ that comprises a left-handed bundle of four alpha helices (αZ, αA, αB, αC) that are linked by highly variable loop regions (ZA and BC loops), which form the rim of the substrate-binding pocket and determine the degree of substrate recognition [62].…”