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.

Spliceosome-targeted therapies trigger an antiviral immune response in triple-negative breast cancer.

Many oncogenic insults deregulate RNA splicing, often leading to hypersensitivity of tumors to spliceosome-targeted therapies (STTs). However, the mechanisms by which STTs selectively kill cancers remain largely unknown. Herein, we discover that mis-spliced RNA itself is a molecular trigger for tumor killing through viral mimicry. In MYC-driven triple-negative breast cancer, STTs cause widespread cytoplasmic accumulation of mis-spliced mRNAs, many of which form double-stranded structures. Double-stranded RNA (dsRNA)-binding proteins recognize these endogenous dsRNAs, triggering antiviral signaling and extrinsic apoptosis. In immune-competent models of breast cancer, STTs cause tumor cell-intrinsic antiviral signaling, downstream adaptive immune signaling, and tumor cell death. Furthermore, RNA mis-splicing in human breast cancers correlates with innate and adaptive immune signatures, especially in MYC-amplified tumors that are typically immune cold. These findings indicate that dsRNA-sensing pathways respond to global aberrations of RNA splicing in cancer and provoke the hypothesis that STTs may provide unexplored strategies to activate anti-tumor immune pathways.

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.

Along the Central Dogma-Controlling Gene Expression with Small Molecules.

The central dogma of molecular biology, that DNA is transcribed into RNA and RNA translated into protein, was coined in the early days of modern biology. Back in the 1950s and 1960s, bacterial genetics first opened the way toward understanding life as the genetically encoded interaction of macromolecules. As molecular biology progressed and our knowledge of gene control deepened, it became increasingly clear that expression relied on many more levels of regulation. In the process of dissecting mechanisms of gene expression, specific small-molecule inhibitors played an important role and became valuable tools of investigation. Small molecules offer significant advantages over genetic tools, as they allow inhibiting a process at any desired time point, whereas mutating or altering the gene of an important regulator would likely result in a dead organism. With the advent of modern sequencing technology, it has become possible to monitor global cellular effects of small-molecule treatment and thereby overcome the limitations of classical biochemistry, which usually looks at a biological system in isolation. This review focuses on several molecules, especially natural products, that have played an important role in dissecting gene expression and have opened up new fields of investigation as well as clinical venues for disease treatment.

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.

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.

Splice modulating therapies for human disease.

Dysregulation of splicing and alternative splicing underlies many genetic and acquired diseases. We present an overview of recent strategies and successes in modulating splicing therapeutically in clinical and preclinical contexts. Effective approaches include restoring open reading frames, influencing alternative splicing, and inducing exon inclusion to generate beneficial proteins and remove deleterious ones.

Unconventional viral gene expression mechanisms as therapeutic targets.

Unlike the human genome that comprises mostly noncoding and regulatory sequences, viruses have evolved under the constraints of maintaining a small genome size while expanding the efficiency of their coding and regulatory sequences. As a result, viruses use strategies of transcription and translation in which one or more of the steps in the conventional gene-protein production line are altered. These alternative strategies of viral gene expression (also known as gene recoding) can be uniquely brought about by dedicated viral enzymes or by co-opting host factors (known as host dependencies). Targeting these unique enzymatic activities and host factors exposes vulnerabilities of a virus and provides a paradigm for the design of novel antiviral therapies. In this Review, we describe the types and mechanisms of unconventional gene and protein expression in viruses, and provide a perspective on how future basic mechanistic work could inform translational efforts that are aimed at viral eradication.

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.

Structural Basis of Splicing Modulation by Antitumor Macrolide Compounds.

SF3B is a multi-protein complex essential for branch site (BS) recognition and selection during pre-mRNA splicing. Several splicing modulators with antitumor activity bind SF3B and thereby modulate splicing. Here we report the crystal structure of a human SF3B core in complex with pladienolide B (PB), a macrocyclic splicing modulator and potent inhibitor of tumor cell proliferation. PB stalls SF3B in an open conformation by acting like a wedge within a hinge, modulating SF3B's transition to the closed conformation needed to form the BS adenosine-binding pocket and stably accommodate the BS/U2 duplex. This work explains the structural basis for the splicing modulation activity of PB and related compounds, and reveals key interactions between SF3B and a common pharmacophore, providing a framework for future structure-based drug design.

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.

MicroRNA biogenesis is broadly disrupted by inhibition of the splicing factor SF3B1.

In animals, microRNA (miRNA) biogenesis begins with cotranscriptional cleavage of the primary (pri-)miRNA by the Microprocessor complex. Cotranscriptional splicing has been shown to influence Microprocessor cleavage when miRNAs are hosted in introns of protein-coding pri-miRNAs, but the impact of splicing on production of miRNAs hosted in long non-coding (lnc)RNAs is largely unknown. Here, we investigated the role of splicing in the biogenesis of miR-122, an lncRNA-hosted, highly expressed, medically important, liver-specific miRNA. We found that splicing inhibition by the SF3B1 inhibitor pladienolide B (PlaB) led to strong and rapid reduction in transcription of endogenous, but not plasmid-encoded, pri-miR-122, resulting in reduced production of mature miR-122. To allow detection of rapid changes in miRNA biogenesis despite the high stability of mature miRNAs, we used SLAMseq to globally quantify the effects of short-term splicing inhibition on miRNA synthesis. We observed an overall decrease in biogenesis of mature miRNAs following PlaB treatment. Surprisingly, miRNAs hosted in exons and introns were similarly affected. Together, this study provides new insights into the emerging role of splicing in transcription, demonstrating novel biological importance in promotion of miR-122 biogenesis from an lncRNA, and shows that SF3B1 is important for global miRNA biogenesis.

The cryo-EM structure of the SF3b spliceosome complex bound to a splicing modulator reveals a pre-mRNA substrate competitive mechanism of action.

Somatic mutations in spliceosome proteins lead to dysregulated RNA splicing and are observed in a variety of cancers. These genetic aberrations may offer a potential intervention point for targeted therapeutics. SF3B1, part of the U2 small nuclear RNP (snRNP), is targeted by splicing modulators, including E7107, the first to enter clinical trials, and, more recently, H3B-8800. Modulating splicing represents a first-in-class opportunity in drug discovery, and elucidating the structural basis for the mode of action opens up new possibilities for structure-based drug design. Here, we present the cryogenic electron microscopy (cryo-EM) structure of the SF3b subcomplex (SF3B1, SF3B3, PHF5A, and SF3B5) bound to E7107 at 3.95 Å. This structure shows that E7107 binds in the branch point adenosine-binding pocket, forming close contacts with key residues that confer resistance upon mutation: SF3B1R1074H and PHF5AY36C The structure suggests a model in which splicing modulators interfere with branch point adenosine recognition and supports a substrate competitive mechanism of action (MOA). Using several related chemical probes, we validate the pose of the compound and support their substrate competitive MOA by comparing their activity against both strong and weak pre-mRNA substrates. Finally, we present functional data and structure-activity relationship (SAR) on the PHF5AR38C mutation that sensitizes cells to some chemical probes but not others. Developing small molecule splicing modulators represents a promising therapeutic approach for a variety of diseases, and this work provides a significant step in enabling structure-based drug design for these elaborate natural products. Importantly, this work also demonstrates that the utilization of cryo-EM in drug discovery is coming of age.

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.

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.

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.

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.

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.

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.

G-quadruplex-forming nucleic acids interact with splicing factor 3B subunit 2 and suppress innate immune gene expression.

G-quadruplex (G4), a non-canonical higher-order structure formed by guanine-rich nucleic acid sequences, affects various genetic events in cis, including replication, transcription and translation. Whereas up-regulation of innate immune/interferon-stimulated genes (ISGs) is implicated in cancer progression, G4-forming oligonucleotides that mimic telomeric repeat-containing RNA suppress ISG induction in three-dimensional (3D) culture of cancer cells. However, it is unclear how G4 suppresses ISG expression in trans. In this study, we found that G4 binding to splicing factor 3B subunit 2 (SF3B2) down-regulated STAT1 phosphorylation and ISG expression in 3D-cultured cancer cells. Liquid chromatography-tandem mass spectrometry analysis identified SF3B2 as a G4-binding protein. Either G4-forming oligonucleotides or SF3B2 knockdown suppressed ISG induction, whereas Phen-DC3, a G4-stabilizing compound, reversed the inhibitory effect of G4-forming oligonucleotides on ISG induction. Phen-DC3 inhibited SF3B2 binding to G4 in vitro. SF3B2-mediated ISG induction appeared to occur independently of RNA splicing because SF3B2 knockdown did not affect pre-mRNA splicing under the experimental conditions, and pharmacological inhibition of splicing by pladienolide B did not repress ISG induction. These observations suggest that G4 disrupts the ability of SF3B2 to induce ISGs in cancer. We propose a new mode for gene regulation, which employs G4 as an inhibitory trans-element.