The phosphatase inhibitor LB-100 creates neoantigens in colon cancer cells through perturbation of mRNA splicing.

Perturbation of protein phosphorylation represents an attractive approach to cancer treatment. Besides kinase inhibitors, protein phosphatase inhibitors have been shown to have anti-cancer activity. A prime example is the small molecule LB-100, an inhibitor of protein phosphatases 2A/5 (PP2A/PP5), enzymes that affect cellular physiology. LB-100 has proven effective in pre-clinical models in combination with immunotherapy, but the molecular underpinnings of this synergy remain understood poorly. We report here a sensitivity of the mRNA splicing machinery to phosphorylation changes in response to LB-100 in colorectal adenocarcinoma. We observe enrichment for differentially phosphorylated sites within cancer-critical splicing nodes of U2 snRNP, SRSF and hnRNP proteins. Altered phosphorylation endows LB-100-treated colorectal adenocarcinoma cells with differential splicing patterns. In PP2A-inhibited cells, over 1000 events of exon skipping and intron retention affect regulators of genomic integrity. Finally, we show that LB-100-evoked alternative splicing leads to neoantigens that are presented by MHC class 1 at the cell surface. Our findings provide a potential explanation for the pre-clinical and clinical observations that LB-100 sensitizes cancer cells to immune checkpoint blockade.

The diversity of splicing modifiers acting on A-1 bulged 5'-splice sites reveals rules for rational drug design.

Pharmacological modulation of RNA splicing by small molecules is an emerging facet of drug discovery. In this context, the SMN2 splicing modifier SMN-C5 was used as a prototype to understand the mode of action of small molecule splicing modifiers and propose the concept of 5'-splice site bulge repair. In this study, we combined in vitro binding assays and structure determination by NMR spectroscopy to identify the binding modes of four other small molecule splicing modifiers that switch the splicing of either the SMN2 or the HTT gene. Here, we determined the solution structures of risdiplam, branaplam, SMN-CX and SMN-CY bound to the intermolecular RNA helix epitope containing an unpaired adenine within the G-2A-1G+1U+2 motif of the 5'-splice site. Despite notable differences in their scaffolds, risdiplam, SMN-CX, SMN-CY and branaplam contact the RNA epitope similarly to SMN-C5, suggesting that the 5'-splice site bulge repair mechanism can be generalised. These findings not only deepen our understanding of the chemical diversity of splicing modifiers that target A-1 bulged 5'-splice sites, but also identify common pharmacophores required for modulating 5'-splice site selection with small molecules.

It is not just about transcription: involvement of brain RNA splicing in substance use disorders.

Alternative splicing is a co-transcriptional process that significantly contributes to the molecular landscape of the cell. It plays a multifaceted role in shaping gene transcription, protein diversity, and functional adaptability in response to environmental cues. Recent studies demonstrate that drugs of abuse have a profound impact on alternative splicing patterns within different brain regions. Drugs like alcohol and cocaine modify the expression of genes responsible for encoding splicing factors, thereby influencing alternative splicing of crucial genes involved in neurotransmission, neurogenesis, and neuroinflammation. Notable examples of these alterations include alcohol-induced changes in splicing factors such as HSPA6 and PCBP1, as well as cocaine's impact on PTBP1 and SRSF11. Beyond the immediate effects of drug exposure, recent research has shed light on the role of alternative splicing in contributing to the risk of substance use disorders (SUDs). This is exemplified by exon skipping events in key genes like ELOVL7, which can elevate the risk of alcohol use disorder. Lastly, drugs of abuse can induce splicing alterations through epigenetic modifications. For example, cocaine exposure leads to alterations in levels of trimethylated lysine 36 of histone H3, which exhibits a robust association with alternative splicing and serves as a reliable predictor for exon exclusion. In summary, alternative splicing has emerged as a critical player in the complex interplay between drugs of abuse and the brain, offering insights into the molecular underpinnings of SUDs.

QR-1011 restores defective ABCA4 splicing caused by multiple severe ABCA4 variants underlying Stargardt disease.

Stargardt disease type 1 (STGD1), the most common form of hereditary macular dystrophy, can be caused by biallelic combinations of over 2200 variants in the ABCA4 gene. This leads to reduced or absent ABCA4 protein activity, resulting in toxic metabolite accumulation in the retina and damage of the retinal pigment epithelium and photoreceptors. Approximately 21% of all ABCA4 variants that contribute to disease influence ABCA4 pre-mRNA splicing. This emphasizes the need for therapies to restore disrupted ABCA4 splicing and halt STGD1 progression. Previously, QR-1011, an antisense oligonucleotide (AON), successfully corrected splicing abnormalities and restored normal ABCA4 protein translation in human retinal organoids carrying the prevalent disease-causing variant c.5461-10T>C in ABCA4. Here, we investigated whether QR-1011 could also correct splicing in four less common non-canonical splice site (NCSS) variants flanking ABCA4 exon 39: c.5461-8T>G, c.5461-6T>C, c.5584+5G>A and c.5584+6T>C. We administered QR-1011 and three other AONs to midigene-transfected cells and demonstrate that QR-1011 had the most pronounced effect on splicing compared to the others. Moreover, QR-1011 significantly increased full-length ABCA4 transcript levels for c.5461-8T>G and c.5584+6T>C. Splicing restoration could not be achieved in the other two variants, suggesting their more severe effect on splicing. Overall, QR-1011, initially developed for a single ABCA4 variant, exhibited potent splice correction capabilities for two additional severe NCSS variants nearby. This suggests the possibility of a broader therapeutic impact of QR-1011 extending beyond its original target and highlights the potential for treating a larger population of STGD1 patients affected by multiple severe ABCA4 variants with a single AON.

A framework for individualized splice-switching oligonucleotide therapy.

Splice-switching antisense oligonucleotides (ASOs) could be used to treat a subset of individuals with genetic diseases1, but the systematic identification of such individuals remains a challenge. Here we performed whole-genome sequencing analyses to characterize genetic variation in 235 individuals (from 209 families) with ataxia-telangiectasia, a severely debilitating and life-threatening recessive genetic disorder2,3, yielding a complete molecular diagnosis in almost all individuals. We developed a predictive taxonomy to assess the amenability of each individual to splice-switching ASO intervention; 9% and 6% of the individuals had variants that were 'probably' or 'possibly' amenable to ASO splice modulation, respectively. Most amenable variants were in deep intronic regions that are inaccessible to exon-targeted sequencing. We developed ASOs that successfully rescued mis-splicing and ATM cellular signalling in patient fibroblasts for two recurrent variants. In a pilot clinical study, one of these ASOs was used to treat a child who had been diagnosed with ataxia-telangiectasia soon after birth, and showed good tolerability without serious adverse events for three years. Our study provides a framework for the prospective identification of individuals with genetic diseases who might benefit from a therapeutic approach involving splice-switching ASOs.

Diverse targets of SMN2-directed splicing-modulating small molecule therapeutics for spinal muscular atrophy.

Designing an RNA-interacting molecule that displays high therapeutic efficacy while retaining specificity within a broad concentration range remains a challenging task. Risdiplam is an FDA-approved small molecule for the treatment of spinal muscular atrophy (SMA), the leading genetic cause of infant mortality. Branaplam is another small molecule which has undergone clinical trials. The therapeutic merit of both compounds is based on their ability to restore body-wide inclusion of Survival Motor Neuron 2 (SMN2) exon 7 upon oral administration. Here we compare the transcriptome-wide off-target effects of these compounds in SMA patient cells. We captured concentration-dependent compound-specific changes, including aberrant expression of genes associated with DNA replication, cell cycle, RNA metabolism, cell signaling and metabolic pathways. Both compounds triggered massive perturbations of splicing events, inducing off-target exon inclusion, exon skipping, intron retention, intron removal and alternative splice site usage. Our results of minigenes expressed in HeLa cells provide mechanistic insights into how these molecules targeted towards a single gene produce different off-target effects. We show the advantages of combined treatments with low doses of risdiplam and branaplam. Our findings are instructive for devising better dosing regimens as well as for developing the next generation of small molecule therapeutics aimed at splicing modulation.

Vorinostat Improves Myotonic Dystrophy Type 1 Splicing Abnormalities in DM1 Muscle Cell Lines and Skeletal Muscle from a DM1 Mouse Model.

Myotonic dystrophy type 1 (DM1), the most common form of adult muscular dystrophy, is caused by an abnormal expansion of CTG repeats in the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. The expanded repeats of the DMPK mRNA form hairpin structures in vitro, which cause misregulation and/or sequestration of proteins including the splicing regulator muscleblind-like 1 (MBNL1). In turn, misregulation and sequestration of such proteins result in the aberrant alternative splicing of diverse mRNAs and underlie, at least in part, DM1 pathogenesis. It has been previously shown that disaggregating RNA foci repletes free MBNL1, rescues DM1 spliceopathy, and alleviates associated symptoms such as myotonia. Using an FDA-approved drug library, we have screened for a reduction of CUG foci in patient muscle cells and identified the HDAC inhibitor, vorinostat, as an inhibitor of foci formation; SERCA1 (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) spliceopathy was also improved by vorinostat treatment. Vorinostat treatment in a mouse model of DM1 (human skeletal actin-long repeat; HSALR) improved several spliceopathies, reduced muscle central nucleation, and restored chloride channel levels at the sarcolemma. Our in vitro and in vivo evidence showing amelioration of several DM1 disease markers marks vorinostat as a promising novel DM1 therapy.

CDK11 regulates pre-mRNA splicing by phosphorylation of SF3B1.

RNA splicing, the process of intron removal from pre-mRNA, is essential for the regulation of gene expression. It is controlled by the spliceosome, a megadalton RNA-protein complex that assembles de novo on each pre-mRNA intron through an ordered assembly of intermediate complexes1,2. Spliceosome activation is a major control step that requires substantial protein and RNA rearrangements leading to a catalytically active complex1-5. Splicing factor 3B subunit 1 (SF3B1) protein-a subunit of the U2 small nuclear ribonucleoprotein6-is phosphorylated during spliceosome activation7-10, but the kinase that is responsible has not been identified. Here we show that cyclin-dependent kinase 11 (CDK11) associates with SF3B1 and phosphorylates threonine residues at its N terminus during spliceosome activation. The phosphorylation is important for the association between SF3B1 and U5 and U6 snRNAs in the activated spliceosome, termed the Bact complex, and the phosphorylation can be blocked by OTS964, a potent and selective inhibitor of CDK11. Inhibition of CDK11 prevents spliceosomal transition from the precatalytic complex B to the activated complex Bact and leads to widespread intron retention and accumulation of non-functional spliceosomes on pre-mRNAs and chromatin. We demonstrate a central role of CDK11 in spliceosome assembly and splicing regulation and characterize OTS964 as a highly selective CDK11 inhibitor that suppresses spliceosome activation and splicing.

Control of backbone chemistry and chirality boost oligonucleotide splice switching activity.

Although recent regulatory approval of splice-switching oligonucleotides (SSOs) for the treatment of neuromuscular disease such as Duchenne muscular dystrophy has been an advance for the splice-switching field, current SSO chemistries have shown limited clinical benefit due to poor pharmacology. To overcome limitations of existing technologies, we engineered chimeric stereopure oligonucleotides with phosphorothioate (PS) and phosphoryl guanidine-containing (PN) backbones. We demonstrate that these chimeric stereopure oligonucleotides have markedly improved pharmacology and efficacy compared with PS-modified oligonucleotides, preventing premature death and improving median survival from 49 days to at least 280 days in a dystrophic mouse model with an aggressive phenotype. These data demonstrate that chemical optimization alone can profoundly impact oligonucleotide pharmacology and highlight the potential for continued innovation around the oligonucleotide backbone. More specifically, we conclude that chimeric stereopure oligonucleotides are a promising splice-switching modality with potential for the treatment of neuromuscular and other genetic diseases impacting difficult to reach tissues such as the skeletal muscle and heart.

Addressing Today's Absorption, Distribution, Metabolism, and Excretion (ADME) Challenges in the Translation of In Vitro ADME Characteristics to Humans: A Case Study of the SMN2 mRNA Splicing Modifier Risdiplam.

Small molecules that present complex absorption, distribution, metabolism, and elimination (ADME) properties can be challenging to investigate as potential therapeutics. Acquiring data through standard methods can yield results that are insufficient to describe the in vivo situation, which can affect downstream development decisions. Implementing in vitro-in vivo-in silico strategies throughout the drug development process is effective in identifying and mitigating risks while speeding up their development. Risdiplam (Evrysdi)-an orally bioavailable, small molecule approved by the US Food and Drug Administration and more recently by the European Medicines Agency for the treatment of patients ≥2 months of age with spinal muscular atrophy-is presented here as a case study. Risdiplam is a low-turnover compound whose metabolism is mediated through a non-cytochrome P450 enzymatic pathway. Four main challenges of risdiplam are discussed: predicting in vivo hepatic clearance, determining in vitro metabolites with regard to metabolites in safety testing guidelines, elucidating enzymes responsible for clearance, and estimating potential drug-drug interactions. A combination of in vitro and in vivo results was successfully extrapolated and used to develop a robust physiologically based pharmacokinetic model of risdiplam. These results were verified through early clinical studies, further strengthening the understanding of the ADME properties of risdiplam in humans. These approaches can be applied to other compounds with similar ADME profiles, which may be difficult to investigate using standard methods. SIGNIFICANCE STATEMENT: Risdiplam is the first approved, small-molecule, survival of motor neuron 2 mRNA splicing modifier for the treatment of spinal muscular atrophy. The approach taken to characterize the absorption, distribution, metabolism, and excretion (ADME) properties of risdiplam during clinical development incorporated in vitro-in vivo-in silico techniques, which may be applicable to other small molecules with challenging ADME. These strategies may be useful in improving the speed at which future drug molecules can be developed.

Identification of a small molecule splicing inhibitor targeting UHM domains.

Splicing factor mutations are frequent in myeloid neoplasms, blood cancers, and solid tumors. Cancer cells harboring these mutations present a particular vulnerability to drugs that target splicing factors such as SF3b155 or CAPERα. Still, the arsenal of chemical probes that target the spliceosome is very limited. U2AF homology motifs (UHMs) are common protein interaction domains among splicing factors. They present a hydrophobic pocket ideally suited to anchor small molecules with the aim to inhibit protein-protein interaction. Here, we combined a virtual screening of a small molecules database and an in vitro competition assay and identified a small molecule, we named UHMCP1 that prevents the SF3b155/U2AF65 interaction. NMR analyses and molecular dynamics simulations confirmed the binding of this molecule in the hydrophobic pocket of the U2AF65 UHM domain. We further provide evidence that UHMCP1 impacts RNA splicing and cell viability and is therefore an interesting novel compound targeting an UHM domain with potential anticancer properties.

Splicing modulators elicit global translational repression by condensate-prone proteins translated from introns.

Chemical splicing modulators that bind to the spliceosome have provided an attractive avenue for cancer treatment. Splicing modulators induce accumulation and subsequent translation of a subset of intron-retained mRNAs. However, the biological effect of proteins containing translated intron sequences remains unclear. Here, we identify a number of truncated proteins generated upon treatment with the splicing modulator spliceostatin A (SSA) via genome-wide ribosome profiling and bio-orthogonal noncanonical amino acid tagging (BONCAT) mass spectrometry. A subset of these truncated proteins has intrinsically disordered regions, forms insoluble cellular condensates, and triggers the proteotoxic stress response through c-Jun N-terminal kinase (JNK) phosphorylation, thereby inhibiting the mTORC1 pathway. In turn, this reduces global translation. These findings indicate that creating an overburden of condensate-prone proteins derived from introns represses translation and prevents further production of harmful truncated proteins. This mechanism appears to contribute to the antiproliferative and proapoptotic activity of splicing modulators.

Aberrant RNA splicing and therapeutic opportunities in cancers.

There has been accumulating evidence that RNA splicing is frequently dysregulated in a variety of cancers and that hotspot mutations affecting key splicing factors, SF3B1, SRSF2 and U2AF1, are commonly enriched across cancers, strongly suggesting that aberrant RNA splicing is a new class of hallmark that contributes to the initiation and/or maintenance of cancers. In parallel, some studies have demonstrated that cancer cells with global splicing alterations are dependent on the transcriptional products derived from wild-type spliceosome for their survival, which potentially creates a therapeutic vulnerability in cancers with a mutant spliceosome. It has been c. 10 y since the frequent mutations affecting splicing factors were reported in cancers. Based on these surprising findings, there has been a growing interest in targeting altered splicing in the treatment of cancers, which has promoted a wide variety of investigations including genetic, molecular and biological studies addressing how altered splicing promotes oncogenesis and how cancers bearing alterations in splicing can be targeted therapeutically. In this mini-review we present a concise trajectory of what has been elucidated regarding the pathogenesis of cancers with aberrant splicing, as well as the development of therapeutic strategies to target global splicing alterations in cancers.

Small molecule splicing modifiers with systemic HTT-lowering activity.

Huntington's disease (HD) is a hereditary neurodegenerative disorder caused by expansion of cytosine-adenine-guanine (CAG) trinucleotide repeats in the huntingtin (HTT) gene. Consequently, the mutant protein is ubiquitously expressed and drives pathogenesis of HD through a toxic gain-of-function mechanism. Animal models of HD have demonstrated that reducing huntingtin (HTT) protein levels alleviates motor and neuropathological abnormalities. Investigational drugs aim to reduce HTT levels by repressing HTT transcription, stability or translation. These drugs require invasive procedures to reach the central nervous system (CNS) and do not achieve broad CNS distribution. Here, we describe the identification of orally bioavailable small molecules with broad distribution throughout the CNS, which lower HTT expression consistently throughout the CNS and periphery through selective modulation of pre-messenger RNA splicing. These compounds act by promoting the inclusion of a pseudoexon containing a premature termination codon (stop-codon psiExon), leading to HTT mRNA degradation and reduction of HTT levels.

Discovery of highly reactive self-splicing group II introns within the mitochondrial genomes of human pathogenic fungi.

Fungal pathogens represent an expanding global health threat for which treatment options are limited. Self-splicing group II introns have emerged as promising drug targets, but their development has been limited by a lack of information on their distribution and architecture in pathogenic fungi. To meet this challenge, we developed a bioinformatic workflow for scanning sequence data to identify unique RNA structural signatures within group II introns. Using this approach, we discovered a set of ubiquitous introns within thermally dimorphic fungi (genera of Blastomyces, Coccidioides and Histoplasma). These introns are the most biochemically reactive group II introns ever reported, and they self-splice rapidly under near-physiological conditions without protein cofactors. Moreover, we demonstrated the small molecule targetability of these introns by showing that they can be inhibited by the FDA-approved drug mitoxantrone in vitro. Taken together, our results highlight the utility of structure-based informatic searches for identifying riboregulatory elements in pathogens, revealing a striking diversity of reactive self-splicing introns with great promise as antifungal drug targets.

Orally administered branaplam does not impact neurogenesis in juvenile mice, rats, and dogs.

Branaplam is a therapeutic agent currently in clinical development for the treatment of infants with type 1 spinal muscular atrophy (SMA). Since preclinical studies showed that branaplam had cell-cycle arrest effects, we sought to determine whether branaplam may affect postnatal cerebellar development and brain neurogenesis. Here, we describe a novel approach for developmental neurotoxicity testing (DNT) of a central nervous system (CNS) active drug. The effects of orally administered branaplam were evaluated in the SMA neonatal mouse model (SMNΔ7), and in juvenile Wistar Hannover rats and Beagle dogs. Histopathological examination and complementary immunohistochemical studies focused on areas of neurogenesis in the cerebellum (mice, rats, and dogs), and the subventricular zone of the striatum and dentate gyrus (rats and dogs) using antibodies directed against Ki67, phosphorylated histone H3, cleaved caspase-3, and glial fibrillary acidic protein. Additionally, image-analysis based quantification of calbindin-D28k and Ki67 was performed in rats and dogs. The patterns of cell proliferation and apoptosis, and neural migration and innervation in the cerebellum and other brain regions of active adult neurogenesis did not differ between branaplam- and control-treated animals. Quantitative image analysis did not reveal any changes in calbindin-D28k and Ki67 expression in rats and dogs. The data show that orally administered branaplam has no impact on neurogenesis in juvenile animals. Application of selected immunohistochemical stainings in combination with quantitative image analysis on a few critical areas of postnatal CNS development offer a reliable approach to assess DNT of CNS-active drug candidates in juvenile animal toxicity studies.

Isoginkgetin leads to decreased protein synthesis and activates an ATF4-dependent transcriptional response.

Isoginkgetin (IGG) is a small molecule inhibitor of pre-mRNA splicing. Failure to accurately remove introns could lead to the production of aberrant mRNAs and proteins. The cellular responses to splicing stress are not well defined. Here, we used oligonucleotide microarrays to assess genome wide changes in gene expression associated with exposure to IGG. Two of the 3 enriched pathways identified using PANTHER analysis of differentially expressed transcripts are linked to the ATF4 transcription factor. We confirmed that ATF4 was selectively translated and upregulated in response IGG despite an almost complete block to total protein synthesis. Importantly, partial disruption of the ATF4 gene using CRISPR-mediated gene editing prevented IGG-induced changes in gene expression. Remarkably, another spliceosome inhibitor, pladienolide B, did not inhibit translation, activate ATF4 or increase ATF4-dependent gene expression. Taken together, IGG activates ATF4 and an ATF4-dependent transcriptional response but these effects are not common to all spliceosome inhibitors.

ELAVL4, splicing, and glutamatergic dysfunction precede neuron loss in MAPT mutation cerebral organoids.

Frontotemporal dementia (FTD) because of MAPT mutation causes pathological accumulation of tau and glutamatergic cortical neuronal death by unknown mechanisms. We used human induced pluripotent stem cell (iPSC)-derived cerebral organoids expressing tau-V337M and isogenic corrected controls to discover early alterations because of the mutation that precede neurodegeneration. At 2 months, mutant organoids show upregulated expression of MAPT, glutamatergic signaling pathways, and regulators, including the RNA-binding protein ELAVL4, and increased stress granules. Over the following 4 months, mutant organoids accumulate splicing changes, disruption of autophagy function, and build-up of tau and P-tau-S396. By 6 months, tau-V337M organoids show specific loss of glutamatergic neurons as seen in individuals with FTD. Mutant neurons are susceptible to glutamate toxicity, which can be rescued pharmacologically by the PIKFYVE kinase inhibitor apilimod. Our results demonstrate a sequence of events that precede neurodegeneration, revealing molecular pathways associated with glutamate signaling as potential targets for therapeutic intervention in FTD.

Pharmacologic modulation of RNA splicing enhances anti-tumor immunity.

Although mutations in DNA are the best-studied source of neoantigens that determine response to immune checkpoint blockade, alterations in RNA splicing within cancer cells could similarly result in neoepitope production. However, the endogenous antigenicity and clinical potential of such splicing-derived epitopes have not been tested. Here, we demonstrate that pharmacologic modulation of splicing via specific drug classes generates bona fide neoantigens and elicits anti-tumor immunity, augmenting checkpoint immunotherapy. Splicing modulation inhibited tumor growth and enhanced checkpoint blockade in a manner dependent on host T cells and peptides presented on tumor MHC class I. Splicing modulation induced stereotyped splicing changes across tumor types, altering the MHC I-bound immunopeptidome to yield splicing-derived neoepitopes that trigger an anti-tumor T cell response in vivo. These data definitively identify splicing modulation as an untapped source of immunogenic peptides and provide a means to enhance response to checkpoint blockade that is readily translatable to the clinic.

Systematically Studying the Effect of Small Molecules Interacting with RNA in Cellular and Preclinical Models.

The interrogation and manipulation of biological systems by small molecules is a powerful approach in chemical biology. Ideal compounds selectively engage a target and mediate a downstream phenotypic response. Although historically small molecule drug discovery has focused on proteins and enzymes, targeting RNA is an attractive therapeutic alternative, as many disease-causing or -associated RNAs have been identified through genome-wide association studies. As the field of RNA chemical biology emerges, the systematic evaluation of target validation and modulation of target-associated pathways is of paramount importance. In this Review, through an examination of case studies, we outline the experimental characterization, including methods and tools, to evaluate comprehensively the impact of small molecules that target RNA on cellular phenotype.