EMBO Molecular Medicine

Spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality. The advent of approved treatments for this devastating condition has significantly changed SMA patients life expectancy and quality of life. Nevertheless, these are not without limitations, and research efforts are underway to develop new approaches to be used alone and in combination, to ensure improved and long-lasting benefits for SMA patients. Protein arginine methyltransferases (PRMT) are emerging as druggable epigenetic targets, with several small molecule PRMT inhibitors already in clinical trial stage. From a screen of highly potent and selective next generation epigenetic small molecules, we have identified MS023, a potent and selective type I PRMT inhibitor, able to promote SMN2 exon 7 inclusion and increase SMN protein levels in preclinical SMA model, by inhibiting the binding of splicing factor hnRNPA1 to SMN2 pre-mRNA. Treatment of SMA mice with MS023 results in amelioration of the disease phenotype, with strong synergistic amplification of the positive effect when delivered in combination with the SMN2-targeting antisense oligonucleotide nusinersen. Moreover, transcriptomic analysis revealed that MS023 treatment has very minimal off-target effects and that the added benefit of the combination therapy is mainly attributable to targeting neuroinflammation. Our study warrants further clinical investigation of PRMT inhibition both as a stand-alone and add-on therapy for SMA patients.

Biallelic AAGGG expansions in Replication Factor Complex Subunit 1 (RFC1) are associated with cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) and are increasingly recognised as a common cause of adult-onset ataxia and sensory neuropathy. However, the disease-causing mechanisms remain unclear. Here we leveraged in vitro assays, post-mortem brain tissue, patient-derived cell lines and a neuronal Drosophila model to demonstrate that AAGGG expansions are associated with tissue-specific reductions in the expression of RFC1 transcript, along with impaired RFC1 function and increased sensitivity to DNA damage from platinum-based drugs. CRISPR/Cas9 excision of the AAGGG repeat and flanking AluSx3 element normalized RFC1 expression in iPSC-derived neurons and rescued the DNA damage response, providing a framework for future therapeutic strategies. We also show that these biological findings are clinically relevant in heterozygous AAGGG expansion carriers, who display an increased risk and severity of neuropathy with platinum-based chemotherapy.

Targeted protein degradation is emerging as a powerful anticancer therapy, mostly focused on eliminating oncogenic drivers. In contrast, we propose using PROTACs that exploit E3 ligase defects in cancer cells to selectively protect healthy tissues from the dose-limiting toxicity of anticancer drugs. We term this approach PROTAC-Driven Protective Therapy (PDPT). PDPT consists of a combinatorial treatment of a given anticancer compound with a PROTAC that promotes the degradation of proteins required for the drug-induced toxicity. Potential targets of protective PROTACs include drug uptake transporters, enzymes activating pro-drugs, and the actual drug target in cases that mediates the drug-induced toxicity. Notably, these protective PROTACs must be designed to recruit E3 ligases that are mutated or defective in the cancer cells while remain active in healthy tissues. As a proof of concept of our strategy, we used CRBN-recruiting and VHL-recruiting PROTACs to demonstrate that PARP1 degradation alleviates the cytotoxicity of PARP inhibitors (PARPi) in E3 ligase-proficient cells, while E3 ligase-deficient cancer cells remain fully sensitive. Remarkably, PDPT also protects primary human bone marrow progenitors from PARPi-induced toxicity, which are the most clinically relevant cells affected by PARPi-associated side effects in cancer patients, supporting the clinical relevance of this strategy. We further uncover that TP53-mutant cancers display critically low expression of the E3 ligase MDM2 and show inefficient MDM2-recruiting PROTAC activity. This tumor-intrinsic feature enables PDPT using MDM2-recruiting PROTACs in TP53-mutant cancers. PDPT opens a new direction for targeted protein degradation by improving tolerability and expanding the therapeutic window of both established and future cancer therapies.

Low-flow vascular malformations are congenital overgrowths composed by abnormal blood vessels potentially causing pain, bleeding, and obstruction of different organs. These diseases are caused by oncogenic mutations in the endothelium which result in overactivation of the PI3K/AKT pathway. Lack of robust in vivo preclinical data has prevented the development and translation into clinical trials of specific molecular therapies for these diseases. Here, we describe a new reproducible preclinical in vivo model of PI3K-driven vascular malformations using the postnatal mouse retina. This model reproduces human disease with Pik3ca activating mutations expressed in a mosaic pattern and vascular malformations formed in veins and capillaries. We show that active angiogenesis is required for the pathogenesis of vascular malformations caused by activating Pik3ca mutations. Using this model, we demonstrate that low doses of the AKT inhibitor miransertib both prevents and induces the regression of PI3K-driven vascular malformations. We confirmed miransertib efficacy in isolated human endothelial cells with genotypes spanning most of human low-flow vascular malformations. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=69 SRC="FIGDIR/small/452617v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@15b29b6org.highwire.dtl.DTLVardef@fa9f8org.highwire.dtl.DTLVardef@6001d8org.highwire.dtl.DTLVardef@11926c5_HPS_FORMAT_FIGEXP M_FIG C_FIG Low-flow vascular malformations are caused by PI3K signalling overactivation in endothelial cells. We have generated an optimised and robust preclinical system of PI3K-driven vascular malformations by inducing the mosaic expression of Pik3caH1047R in the retinal angiogenic endothelium. This preclinical model displays traits constituting the main hallmarks of the pathogenesis of low-flow blood vascular malformations: overactivation of PI3K signalling (high phospho-S6), vascular compartment specificity, loss of pericyte coverage, and endothelial cell hyperproliferation. Using this preclinical model we report that low dose AKT inhibitor miransertib prevents and regress PI3K-driven vascular malformations.

Biallelic variants in the SPG11 gene account for the most common form of autosomal recessive hereditary spastic paraplegia characterized by motor and cognitive impairment, with currently no therapeutic option. We previously observed in a Spg11 knockout mouse that neurodegeneration is associated with accumulation of gangliosides in lysosomes. To test whether a substrate reduction therapy could be a therapeutic option, we downregulated the key enzyme involved in ganglioside biosynthesis using an AAV-PHP.eB viral vector expressing a miRNA targeting St3gal5. Downregulation of St3gal5 in Spg11 knockout mice prevented the accumulation of gangliosides, delayed the onset of motor and cognitive symptoms, and prevented the upregulation of serum levels of neurofilament light chain, a biomarker widely used in neurodegenerative diseases. Importantly, similar results were observed upon treatment of Spg11 knockout mice with venglustat, a pharmacological inhibitor of glucosylceramide synthase expected to decrease ganglioside synthesis. Downregulation of St3gal5 or venglustat treatment of Spg11 knockout mice strongly decreased the formation of axonal spheroids, previously associated with impaired trafficking. Venglustat had similar effect on cultured human SPG11 neurons. In conclusion, this work identifies the first disease-modifying therapeutic strategy in SPG11, and provides data supporting its relevance for therapeutic testing in SPG11 patients.

Tumor recurrence is often attributed to drug-tolerant cancer stem cells. We previously demonstrated that down regulation of the Pregnane X Receptor (PXR, NR1I2) decreases chemoresistance of cancer stem cells and prevents colorectal cancer recurrence in xenograft mouse models. These is a lack of PXR antagonists that are appropriate for clinical use. In this study, we report the design and synthesis of a novel PXR agonist-based PROTAC (JMV7048) that induces polyubiquitination and degradation of human PXR protein in an E3 CRBN ubiquitin ligase- and the 26S proteasome-dependent manner. This molecule specifically degrades PXR in colon carcinoma, hepatoma, and pancreatic cancer cell lines, but not in primary cultures of human hepatocytes. Crucially, JMV7048 decreased PXR protein expression in colon cancer stem cells and sensitized them to chemotherapy significantly delaying cancer relapse in vivo. PROTACs targeting PXR protein could thus become novel therapeutic agents to enhance cancer cell sensitivity to chemotherapy.

Animal models are essential for assessing the preclinical efficacy of candidate drugs, but animal data often fails to replicate in human clinical trials. This translational gulf is due in part to the use of models that do not accurately replicate human disease processes and phenotyping strategies that do not capture sensitive, disease-relevant measures. To address these challenges with the aim of validating candidate neuroprotective drugs, we combined a mouse prion (RML scrapie) model that recapitulates the key common features of human neurodegenerative disease including bona fide neuronal loss, with unbiased and machine learning-assisted behavioural phenotyping. We found that this approach measured subtle, stereotyped, and progressive changes in motor behaviour over the disease time course that correlated with the earliest detectable histopathological changes in the mouse brain. To validate the utility of this model system, we tested whether the anti-diabetic drug pioglitazone could slow prion disease progression. Pioglitazone crosses the blood-brain-barrier and has been shown to reduce neurodegenerative disease severity in other mouse models. We found that in addition to significantly slowing the emergence of early-stage clinical signs of neurodegeneration, pioglitazone significantly improved motor coordination throughout the disease time course and reduced neuronal endoplasmic reticulum stress. Together, these findings suggest that pioglitazone could have neuroprotective properties in humans, confirm the utility of the scrapie mouse model of neurodegeneration, and provide generalisable experimental and analysis methods for the generation of data-rich behavioural data to accelerate and improve preclinical validation.

Mutant Ras oncoproteins, particularly KRAS, are among the most prevalent drivers of cancer. Small-molecule inhibitors of KRAS have been developed, bearing high potential for cancer therapy but considerable risk of resistance development. To avoid cancer cell adaptation, effective combinatorial partners for increasing immediate efficacy remain to be explored. Here, we demonstrate that combining the KRAS inhibitor Sotorasib with the CDK4/6 inhibitor Palbociclib synergistically eliminates pancreatic ductal adenocarcinoma (PDAC) cells and organoids harboring KRAS G12C mutations. This synergy was particularly pronounced after drug washout, indicating a durable impact. Similar effects were observed in non-small-cell lung cancer (NSCLC) cells. Additionally, MRTX1133, a KRAS G12D inhibitor, synergized with Palbociclib to suppress KRAS G12D-mutant PDAC-derived cells. Mechanistically, these combinations induced sustained cell cycle arrest through reduced RB phosphorylation, decreased E2F1 levels, and increased CDKN1B/p27 expression. Deletion of CDKN1B largely rescued tumor cell proliferation, underscoring its critical role in mediating the observed synergy. These findings support the therapeutic potential of combining KRAS and CDK4/6 inhibitors for treating PDAC and other Ras-driven cancers.

SARS-CoV-2 remains an acute threat to human health, endangering hospital capacities worldwide. Many studies have aimed at informing pathophysiologic understanding and identification of disease indicators for risk assessment, monitoring, and therapeutic guidance. While findings start to emerge in the general population, observations in high-risk patients with complex pre-existing conditions are limited. To this end, we biomedically characterized quantitative proteomics in a hospitalized cohort of COVID-19 patients with mild to severe symptoms suffering from different (co)-morbidities in comparison to both healthy individuals and patients with non-COVID related inflammation. Deep clinical phenotyping enabled the identification of individual disease trajectories in COVID-19 patients. By the use of this specific disease phase assignment, proteome analysis revealed a severity dependent general type-2 centered host response side-by-side with a disease specific antiviral immune reaction in early disease. The identification of phenomena such as neutrophil extracellular trap (NET) formation and a pro-coagulatory response together with the regulation of proteins related to SARS-CoV-2-specific symptoms by unbiased proteome screening both confirms results from targeted approaches and provides novel information for biomarker and therapy development. Graphical AbstractSars-CoV-2 remains a challenging threat to our health care system with many pathophysiological mechanisms not fully understood, especially in high-risk patients. Therefore, we characterized a cohort of hospitalized COVID-19 patients with multiple comorbidities by quantitative plasma proteomics and deep clinical phenotyping. The individual patients disease progression was determined and the subsequently assigned proteome profiles compared with a healthy and a chronically inflamed control cohort. The identified disease phase and severity specific protein profiles revealed an antiviral immune response together with coagulation activation indicating the formation of NETosis side-by-side with tissue remodeling related to the inflammatory signature. O_FIG O_LINKSMALLFIG WIDTH=197 HEIGHT=200 SRC="FIGDIR/small/22271106v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1e791faorg.highwire.dtl.DTLVardef@20d3d6org.highwire.dtl.DTLVardef@1339e42org.highwire.dtl.DTLVardef@1db3710_HPS_FORMAT_FIGEXP M_FIG C_FIG

Sickle cell disease (SCD) is due to a mutation in the {beta}-globin (HBB) gene causing the production of the toxic sickle hemoglobin (HbS, a2{beta}S2). Transplantation of autologous hematopoietic stem/progenitor cells (HSPCs) transduced with lentiviral vectors (LVs) expressing an anti-sickling {beta}-globin ({beta}AS) is a promising treatment; however, it is only partially effective and patients still present elevated HbS levels. Here, we developed a bifunctional LV expressing {beta}AS3-globin and an artificial microRNA (amiR) specifically downregulating {beta}S-globin expression with the aim of reducing HbS levels and favoring {beta}AS3 incorporation into Hb tetramers. Efficient transduction of SCD HSPC by the bifunctional LV led to a substantial decrease of {beta}S-globin transcripts in HSPC-derived erythroid cells, a significant reduction of HbS+ red cells and effective correction of the sickling phenotype, outperforming {beta}AS gene addition and BCL11A gene silencing strategies. The bifunctional LV showed a standard integration profile and neither the HSPC viability, engraftment and multi-lineage differentiation nor the erythroid transcriptome and miRNAome were affected by the treatment, confirming the safety of this therapeutic strategy. In conclusion, the combination of gene addition and gene silencing strategies can improve the efficacy of current LV-based therapeutic approaches without increasing the mutagenic vector load, thus representing a novel treatment for SCD.

Proteolytic cell surface release ( shedding) of the prion protein (PrP), a broadly expressed GPI-anchored glycoprotein, by the metalloprotease ADAM10 impacts on neurodegenerative and other diseases in animal and in vitro models. Recent studies employing the latter also suggest shed PrP (sPrP) to be a ligand in intercellular communication and critically involved in PrP-associated physiological tasks. Although expectedly an evolutionary conserved event, and while soluble forms of PrP are present in human tissues and body fluids, neither proteolytic PrP shedding and its cleavage site nor involvement of ADAM10 or the biological relevance of this process have been demonstrated for the human body thus far. In this study, cleavage site prediction and generation (plus detailed characterization) of sPrP-specific antibodies enabled us to identify PrP cleaved at tyrosin 226 as the physiological and strictly ADAM10-dependent shed form in humans. Using cell lines, neural stem cells and brain organoids, we show that shedding of human PrP can be stimulated by PrP-binding ligands without targeting the protease, which may open novel therapeutic perspectives. Site-specific antibodies directed against human sPrP also detect the shed form in brains of cattle, sheep and deer, hence in all most relevant species naturally affected by fatal and transmissible prion diseases. In human and animal prion diseases, but also in patients with Alzheimers disease, sPrP relocalizes from a physiological diffuse tissue pattern to intimately associate with extracellular aggregates of misfolded proteins characteristic for the respective pathological condition. Findings and research tools presented here will accelerate novel insight into the roles of PrP shedding (as a process) and sPrP (as a released factor) in neurodegeneration and beyond.

Diamond-Blackfan Anemia Syndrome (DBAS) is characterized by impaired erythropoiesis due to dysfunctional ribosome biogenesis and aberrant cellular signaling. Here, we investigate how ribosomal stress-induced activation of the NLRP1 inflammasome modulates erythroid differentiation in DBAS. We demonstrate that FDA/EMA-approved tyrosine kinase inhibitors (TKIs) effectively mitigate defective erythropoiesis in Diamond-Blackfan anemia syndrome (DBAS) by inhibiting NLRP1 inflammasome activation. Specifically, nilotinib enhances erythroid differentiation in K562 cells through suppression of the ZAK/P38/NLRP1/CASP1 axis, leading to increased GATA1 protein levels and upregulation of key erythroid genes involved in iron acquisition, hemoglobin synthesis, and erythrocyte structure. These effects were validated in human CD34+ hematopoietic stem and progenitor cells (HSPCs) and zebrafish models, where nilotinib, along with other TKIs (imatinib, dasatinib, and bosutinib), promoted erythropoiesis at the expense of myelopoiesis and reduced caspase-1 activity. Importantly, in RPS19-deficient zebrafish and human models and HSPCs from patients with DBAS, nilotinib, imatinib and dasatinib rescued defective erythroid differentiation and restored hemoglobin levels. These findings highlight the potential of TKIs to address the erythroid defects observed in ribosomopathies like DBAS. Given the limited treatment options available for DBAS and other congenital anemias, our study provides compelling evidence for repurposing TKIs as a novel therapeutic strategy to alleviate pathological NLRP1 activation and improve erythropoiesis. This work opens new avenues for managing ribosome-related disorders and advancing personalized medicine approaches for hematopoietic diseases.

Metastatic melanoma patients carrying a BRAFV600 mutation can be treated with BRAF inhibitors (BRAFi), in combination with MEK inhibitors (MEKi), but innate and acquired resistance invariably occurs. Resistance can involve transcriptional- and epigenetic-based phenotypic adaptations, as yet unpredictable. Predicting patient response to targeted therapies is crucial to guide clinical decision. We describe here the development of a highly efficient patient-derived xenograft model adapted to patient melanoma biopsies, using the avian embryo as a host (AVI-PDX). In this in vivo paradigm, we depict a fast and reproducible tumor engraftment of patient samples within the embryonic skin, preserving key molecular and phenotypic features. We show that sensitivity and resistance to BRAFi/MEKi targeted therapies can be reliably modeled in these AVI-PDX, as well as synergies with other drugs, such as HDACi. We further provide proof-of-concept that the AVI-PDX models the diversity of responses of melanoma patients to BRAFi/MEKi, within days, hence positioning it as a valuable tool for the design of personalized medicine assays and for the evaluation of novel combination strategies.

Glioblastoma (GB) is a fatal disease in which most targeted therapies have clinically failed. However, pharmacological reactivation of tumor suppressors has not been thoroughly studied as yet as a GB therapeutic strategy. Tumor suppressor Protein Phosphatase 2A (PP2A), is inhibited by non-genetic mechanisms in GB, and thus it would be potentially amendable for therapeutic reactivation. Here we demonstrate, that small molecule activators of PP2A (SMAPs), NZ-8-061 and DBK-1154, effectively cross the in vitro model of blood-brain barrier (BBB), and in vivo partition to mouse brain tissue after oral dosing. In vitro, SMAPs exhibit robust cell killing activity against five established GB cell lines, and nine patient-derived primary glioma cell lines. Collectively these cell lines have heterogenous genetic background, kinase inhibitor resistance profile, and stemness properties; and they represent different clinical GB subtypes. Oral dosing of either of the SMAPs significantly reduced growth of infiltrative intracranial GB tumors. DBK-1154, with both higher degree of brain/blood distribution, and more potent in vitro activity against all tested GB cell lines, also significantly increased survival of mice bearing orthotopic GB xenografts. In summary, this report presents a proof-of-principle data for BBB-permeable tumor suppressor reactivation therapy for glioblastoma cells of heterogenous molecular background.

5,6-dimethylxanthenone-4-acetic acid (DMXAA) is a mouse-selective stimulator of interferon gene (STING) agonist exerting STING-dependent anti-tumor activity. Although DMXAA can not fully activate human STING, DMXAA reached phase III in lung cancer clinical trials. How DMXAA is effective against human lung cancer is completely unknown. Here, we show that DMXAA is a partial STING agonist interfering with agonistic STING activation, which may explain its partial anti-tumor effect observed in humans, as STING was reported to be pro-tumorigenic for lung cancer cells with low antigenicity. Furthermore, we developed a DMXAA derivative--3-hydroxy-5-(4-hydroxybenzyl)-4-methyl-9H-xhanthen-9one (HHMX)--that can potently antagonize STING-mediated immune responses both in humans and mice. Notably, HHMX suppressed aberrant responses induced by STING gain-of-function mutations causing STING-associated vasculopathy with onset in infancy (SAVI) in in vitro experiments. Furthermore, HHMX treatment suppressed aberrant STING pathway activity in peripheral blood mononuclear cells from SAVI patients. Lastly, HHMX showed a potent therapeutic effect in SAVI mouse model by mitigating disease progression. Thus, HHMX offers therapeutic potential for STING-associated autoinflammatory diseases.

Meningococcal infections remain particularly difficult to treat. Despite antibiotic therapy, the state of the patients often rapidly deteriorates. Early clinical studies suggest that meningococci acquire a form of resistance to antibiotic treatments during infections. Taking advantage of a humanized animal model of infection, we confirm that adherent bacteria become highly resistant to antibiotic treatments as early as 3-6 hours post infection, although fully sensitive in vitro. Within this time frame, meningococci adhere to the endothelium via their type IV pili, proliferate and eventually fill the vessel lumen. Using intravital imaging, we show that rapidly upon infection blood flow is dramatically decreased, thus limiting antibiotic access to infected vessels. Concomitantly, fibrin is deposited inside infected vessels in proximity to bacterial aggregates. Pharmacologically impairing thrombin generation by inhibiting Factor X activity not only improves blood flow in infected vessels, but also enhances the efficacy of the antibiotic treatment. Our results indicate that the combined administration of anticoagulants together with antibiotics might represent a therapeutic approach to treat meningococcal sepsis more efficiently.

Spinal muscular atrophy (SMA) is a monogenic neurodegenerative disorder caused by decreased levels of Survival of Motor Neuron (SMN) protein. If left untreated, SMA patients have a poor prognosis, marked by the degeneration of motor neurons, progressive muscle weakness and atrophy. The approval of SMN-restoring therapies that improve symptoms and lifespan in patients with SMA has created emerging, non-neuronal phenotypes and an urgent need for deepening our understanding of disease pathogenesis. Leveraging the knowledge that SMN loss drives alterations in translation, we used multiple tissues from a mouse model of SMA to uncover early translational alterations in key mRNAs and proteins, which act as contributors to pathogenesis and hallmarks of the disease. Among hundreds of differentially translated mRNAs, Col1a1 emerged as a translation-specific manifestation of early defects in the mouse model. These findings were confirmed in fibroblasts derived from patients with varying levels of disease severity. Notably, treatment with SMN-restoring therapies rescued COL1A1 protein levels, particularly in fibroblasts from patients with the most severe forms of the disease. Overall, our study identifies COL1A1 as an indicator of disease severity in SMA, which captures early molecular alterations and respond to SMN-modifying therapies.

Primary cilia (PC) are important signaling hubs in cells and their deregulation has been associated with various diseases including cancer. Here we explored the role of PC in colorectal cancer (CRC) and colitis. In the colon we found PC to be mostly present on different subtypes of fibroblasts. Colons of mice exposed to either chemically induced colitis-associated colon carcinogenesis (CAC) or dextran sodium sulfate (DSS)-induced colitis had decreased numbers of PC. We employed conditional knock-out strains for the PC essential genes, Kif3A and Ift88, to generate mice with reduced numbers of PC on colonic fibroblasts. These mice showed an increased susceptibility in the CAC model as well as in DSS-induced colitis. Colons from DSS-treated mice with PC-deficiency on fibroblasts displayed an elevated production of the pro-inflammatory cytokine IL-6 and colonic epithelial cells had diminished levels of HES-1, a key transcription factor of Notch signaling. Notably, an analysis of PC presence on biopsies of patients with ulcerative colitis as well as CRC patients revealed decreased numbers of PC on colonic fibroblasts in pathological versus surrounding normal tissue. Taken together, we provide evidence that a decrease in colonic PC numbers promotes colitis and CRC. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=177 HEIGHT=200 SRC="FIGDIR/small/871772v2_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@1f8eccdorg.highwire.dtl.DTLVardef@1829b3borg.highwire.dtl.DTLVardef@19d632aorg.highwire.dtl.DTLVardef@1d9833b_HPS_FORMAT_FIGEXP M_FIG C_FIG

RNA mis-splicing correction therapies have been developed for neurological disorders like spinal muscular atrophy and neuronal ceroid lipofuscinosis. In Huntingtons disease (HD), pathogenic mis-splicing was initially observed in genes linked to neurodegeneration, such as HTT itself, MAPT, and TAF1. Later, genome-wide analyses identified a broader mis-splicing signature in HD brains, involving additional neurodegeneration-related genes. Correcting each mis-spliced gene individually would be unfeasible, highlighting the need to target upstream splicing factors altered in HD. Our previous motif-enrichment analyses of intronic sequences flanking the exons mis-spliced in HD identified RBFOX and U2AF2 as candidate splicing factors, both of which are reduced in HD brains. In this study, we tested their pathogenic relevance generating conditional transgenic mouse models that overexpress RBFOX1 or U2AF2 in forebrain neurons and combining them with HD mice. Our results show that moderate overexpression of RBFOX1, but not U2AF2, corrects multiple HD-associated mis-splicing events and alleviates HD mice neuropathology and motor symptoms. These findings demonstrate that RBFOX1 downregulation contributes to HD pathology and underscore the therapeutic potential of strategies aimed at increasing RBFOX1 levels.

Withdrawal StatementThe authors have withdrawn their manuscript because the reported synergy of TOP2A inhibitors plus MLN4924 proved to be untrue (not reproducible). Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author (mberens@tgen.org).