RNA splicing modulator for Huntington's disease treatment induces peripheral neuropathy

Abstract

RNA splicing modulators, a new class of small molecules with the potential to modify the protein expression levels, have quickly been translated into clinical trials. These compounds hold promise for treating neurodegenerative disorders, including branaplam for lowering huntingtin levels in Huntington's disease. However, the VIBRANT-HD trial was terminated due to the emergence of peripheral neuropathy. Here, we describe the complex mechanism whereby branaplam activates p53, induces nucleolar stress in human induced pluripotent stem cell (iPSC)-derived motor neurons (iPSC-MN), and thereby enhanced expression of the neurotoxic p53-target gene BBC3. On the cellular level, branaplam disrupts neurite integrity, reflected by elevated neurofilament light chain levels. These findings illustrate the complex pharmacology of RNA splicing modulators with a small therapeutic window between lowering huntingtin levels and the clinically relevant off-target effect of neuropathy. Comprehensive toxicological screening in human stem cell models can complement pre-clinical testing before advancing RNA-targeting drugs to clinical trials.

Keywords: Biological sciences; Cell biology; Cellular neuroscience; Natural sciences; Neuroscience; Pharmacology.

Graphical abstract

Figure 1

Branaplam leads to axonal degeneration and NfL increase in human iPSC-MN (A) Paradigm illustrating experimental approach. (B) Representative pictures of beta-III-Tubulin in DMSO and 1,000 nM-treated conditions. All shown images are from UKERiff2-X-18. Scale bar: 50 μm. (C) Bar plot showing quantification of neurite disintegration index (= disintegrated area (betaIII-Tubulin intensity above mean + 3× standard deviation of DMSO)/total area (beta-III-Tubulin area). Dots depict individual values per cell line (n = 5), graph as median with interquartile range. Statistics: Friedman test: p value: 0.0167. Dunn’s multiple comparison test: ∗ < 0.05; ∗∗ < 0.01; ∗∗∗ < 0.001. Only 1,000 nM passed the significance threshold. (D) Bar plot showing quantification of neurofilament light chain (NfL) levels in media supernatant of iPSC-derived neurons after 5 days of treatment with DMSO, 100 nM, or 1,000 nM branaplam. Dots depict individual values per cell line (n = 5), graph as median with interquartile range. Statistics: Friedman test: p value: 0.0008. Dunn’s multiple comparison test: ∗ < 0.05; ∗∗ < 0.01; ∗∗∗ < 0.001. Only 1,000 nM passed the significance threshold. (E) Bar plot quantification of neurofilament light chain (NfL) levels in media supernatant of iPSC-derived neurons in Ctrl (greens, n = 4) and HD (purples, n = 4) treated for 5 days with 10 nM, 100 nM, or 1,000 nM branaplam (opaques). Dots depict individual values, graph as median with interquartile range. Statistics: paired two-way ANOVA: p value (treatment): < 0.0001; p value (disease): 0.8352; p value (interaction): 0.5045. Šídák’s multiple comparisons test: ∗ < 0.05; ∗∗ < 0.01; ∗∗∗ < 0.001.

Figure 2

Branaplam leads to p53 activation and cell-cycle arrest (A) Experimental analysis strategy. DMSO (n = 8) vs. branaplam (n = 8) treated fibroblast RNA-seq data was used to determine differential gene expression and analysis of enrichment of transcription factor (TF) binding sites (TFBS), followed by validation using ENCODE ChIP-seq data and determine of the cellular mode of action. (B) Volcano plot of differentially expressed genes (blue: downregulated, red: upregulated). y axis depicts negative log₁₀ of the adjusted p value from the DESeq2 output. (C) Scatterplot of significantly enriched (x axis, false discovery rate [FDR]) TFBS in genes upregulated upon branaplam treatment. y axis depicts percentage genes associated TF that are differentially upregulated upon branaplam. In red are 10 TFs with high significance and percentages. (D) Scatterplot of significantly enriched (x axis, false discovery rate [FDR]) TFBS in genes downregulated upon branaplam treatment. y axis depicts percentage genes associated TF that are differentially downregulated upon branaplam. In red are 5 TFs with high significance and percentages. (E) Cumulative distribution plot of log₂(fold changes) of branaplam vs. DMSO in Ctrls in genes with p53 ChIP-seq peak within region of 5,000 bp upstream of gene start (red, n = 865 genes) or in a randomly shuffled background of equal size (black, n = 865 genes). Significance calculated with 2 sample Kolmogorov-Smirnov (KS) test. (F) Cumulative distribution plot of log₂(fold changes) of branaplam vs. DMSO in HD in genes with p53 ChIP-seq peak within region of 5,000 bp upstream of gene start (red, n = 865 genes) or in a randomly shuffled background of equal size (black, n = 865 genes). Significance calculated with 2 sample Kolmogorov-Smirnov (KS) test. (G) Bar plot of CDKN1A RPKM values with Ctrl (greens, n = 4) and HD (purples, n = 4) fibroblast with DMSO and branaplam (opaque) treatment. Dots depict individual values, graph as median with interquartile range. Statistics: paired two-way ANOVA: p value (treatment): < 0.0001; p value (disease): 0.8851; p value (interaction): 0.3535. Šídák’s multiple comparisons test: p value (Ctrl): 0.001; p value (HD): 0.0004. (H) Bar plot of BBC3 RPKM values with Ctrl (greens, n = 4) and HD (purples, n = 4) fibroblast with DMSO and branaplam (opaque) treatment. Dots depict individual values, graph as median with interquartile range. Statistics: paired two-way ANOVA: p value (treatment): <0.0001; p value (disease): 0.1239; p value (interaction): 0.3071. Šídák’s multiple comparisons test: p value (Ctrl): 0.0006; p value (HD): 0.002. (I) Paradigm of EdU FACS cell cycle analysis and example scatterplots and gatings from DMSO, 10 nM branaplam, 1,000 nM branaplam and paclitaxel of UKERf33Q. (J) Bar plot of percentages of cells in G₀/G₁ phase in Ctrl (greens, n = 4) and HD (purples, n = 4) treated for 72h with 10 nM, 100 nM, 1,000 nM branaplam (opaques) or 24 h 10 nM paclitaxel (white/blue). Dots depict individual values, graph as median with interquartile range. Statistics: paired two-way ANOVA: p value (treatment): < 0.0001; p value (disease): 0.7840; p value (interaction): 0.0166. Šídák’s multiple comparisons test: ∗ < 0.05; ∗∗ < 0.01; ∗∗∗ < 0.001. (K) Bar plot of percentages of cells in S phase in Ctrl (greens, n = 4) and HD (purples, n = 4) treated for 72 h with 10 nM, 100 nM, 1,000 nM branaplam (opaques) or 24 h 10 nM paclitaxel (white/blue). Dots depict individual values, graph as median with interquartile range. Statistics: paired two-way ANOVA: p value (treatment): < 0.0001; p value (disease): 0.0345; p value (interaction): 0.0018. Šídák’s multiple comparisons test: ∗ < 0.05; ∗∗ < 0.01; ∗∗∗ < 0.001. (L) Bar plot of percentages of cells in G₂/M phase in Ctrl (greens, n = 4) and HD (purples, n = 4) treated for 72h with 10 nM, 100 nM, 1,000 nM branaplam (opaques) or 24 h 10 nM paclitaxel (white/blue). Dots depict individual values, graph as median with interquartile range. Statistics: paired two-way ANOVA: p value (treatment): < 0.0001; p value (disease): 0.0127; p value (interaction): <0.0001. Šídák’s multiple comparisons test: ∗ < 0.05; ∗∗ < 0.01; ∗∗∗ < 0.001.

Figure 3

Branaplam leads to activation of nucleolar stress and expression of BBC3 and axonal degeneration in human iPSC-MN (A) Paradigm illustrating experimental approach. (B) Representative pictures of betaIII-Tubulin-positive neurons (magenta) with NPM1 signal (LUT, blue = low fluorescence intensity, white = high fluorescence intensity) in the nucleus (white) of UKERiff2-X-18. Scale bar: 10 μm. (C) Bar plot quantifying fluorescence intensity ratio of NPM1 in non-nucleolar nucleoplasm/nucleolar area (n = 4 cell lines). Dots depict individual values per cell line, graph as median with interquartile range. Statistics: Friedman test: p value: 0.0062. Dunn’s multiple comparison test: ∗ < 0.05; ∗∗ < 0.01; ∗∗∗ < 0.001. Significance in post-hoc analysis only found between DMSO and 1,000 nM. (D) Western blot of BBC3 (also known as PUMA) in iPSC-MN of 5 cell lines treated for 5 days with DMSO, 100 nM, and 1,000 nM branaplam. GAPDH serves as loading control. (E) Bar plot showing densitometric quantification of BBC3 signal normalized to GAPDH. Dots depict individual values per cell line, graph as median with interquartile range. Statistics: Friedman test: p value: 0.0008. Dunn’s multiple comparison test: ∗ < 0.05; ∗∗ < 0.01; ∗∗∗ <0.001. Significance in post-hoc analysis only found between DMSO and 1,000 nM.