Magnetic Resonance Imaging Institute for Biomedical Research

Modulation of splicing has become an established therapeutic strategy, with proven clinical applications and continued potential to target specific exons to influence gene expression. Recent advancements led to the identification of small molecule splicing modifiers such as Risdiplam and Branaplam. These compounds induce the inclusion of exons that are typically skipped due to their weak 5' splice site. While Risdiplam has a preference to induce exons with a NGA sequence at the 3' exon end, Branaplam has a proclivity towards introducing AGA-ending exons. However, the variables that determine the selectivity and specificity of splicing modulators are still not completely understood, as evidenced by the hundreds of unaffected NGA-ending exons present in the human genome. In this study, we delve into the molecular mechanisms governing the specificity of splicing-modifying compounds, focusing on their interactions with RNA structures at splice sites. Using biochemical assays, whole transcriptome analyses, and genetic perturbation approaches, our findings reveal contributions of primary sequence dependencies that help determine the required secondary structural conformation, thus governing responsiveness to splicing-modulator induction. Based on these learnings, we were able to reprogram the specificity of splicing modulators by genetic manipulation of the U1 snRNA component of the spliceosome. Our findings further the understanding of splicing modulators and might help to identify novel targets and to design new splicing-modifying compounds.

Iron homoeostasis is tightly regulated, with hepcidin and soluble transferrin receptor (sTfR) playing significant roles. However, the genetic determinants of these traits and the biomedical consequences of iron homoeostasis variation are unclear. In a meta-analysis of 12 cohorts involving 91,675 participants, we found 43 genomic loci associated with either hepcidin or sTfR concentration, of which 15 previously unreported. Mapping to putative genes indicated involvement in iron-trait expression, erythropoiesis, immune response and cellular trafficking. Mendelian randomisation of 292 disease outcomes in 1,492,717 participants revealed associations of iron-related loci and iron status with selected health outcomes across multiple domains. These associations were largely driven by HFE, which was associated with the largest iron variation. Our findings enhance understanding of iron homoeostasis and its biomedical consequences, suggesting that lifelong exposure to higher iron levels is likely associated with lower risk of anaemia-related disorders and higher risk of genitourinary, musculoskeletal, infectious and neoplastic diseases.

Abstract Clonal hematopoiesis (CH), a phenomenon linked to aging, correlates with inflammation and myeloid malignancies. Here, we explore the interaction of CH, with terminally differentiated lymphoid malignancy, and multiple myeloma (MM). Analysis of CH in clinical cohorts revealed a higher prevalence among MM patients and a lower deep response to proteasome inhibitors. By utilizing the bone marrow samples from MM patients with CH, single-cell transcriptome analyses indicated frequent interaction between CH and MM cells, mediated by CCR10-CCL2, resulting in the upregulation of the MAPK pathway and angiogenesis, findings corroborated by exosome RNA analysis. Conditioned media from TET2 knockdown macrophages significantly enhanced MM cell proliferation compared to that from wild-type cells, an effect reversible by a CCR10 inhibitor. Our results underscore the pivotal role of TET2 CH in driving CCR10-high myeloma progression through paracrine oncogenic effects via exosomal interactions on CCR10, suggesting its potential as a therapeutic target.

Schizophrenia is marked by poor social functioning that can have a severe impact on quality of life and independence, but the underlying neural circuity is not well understood. Here we used a translational model of subanesthetic ketamine in mice to delineate neural pathways in the brain linked to social deficits in schizophrenia. Mice treated with chronic ketamine (30 mg/kg/day for 10 days) exhibit profound social and sensorimotor deficits as previously reported. Using three- dimensional c-Fos immunolabeling and volume imaging (iDISCO), we show that ketamine treatment resulted in hypoactivation of the lateral septum (LS) in response to social stimuli. Chemogenetic activation of the LS rescued social deficits after ketamine treatment, while chemogenetic inhibition of previously active populations in the LS (i.e. social engram neurons) recapitulated social deficits in ketamine-naïve mice. We then examined the translatome of LS social engram neurons and found that ketamine treatment dysregulated genes implicated in neuronal excitability and apoptosis, which may contribute to LS hypoactivation. We also identified 38 differentially expressed genes (DEGs) in common with human schizophrenia, including those involved in mitochondrial function, apoptosis, and neuroinflammatory pathways. Chemogenetic activation of LS social engram neurons induced downstream activity in the ventral part of the basolateral amygdala, subparafascicular nucleus of the thalamus, intercalated amygdalar nucleus, olfactory areas, and dentate gyrus, and it also reduces connectivity of the LS with the piriform cortex and caudate-putamen. In sum, schizophrenia-like social deficits may emerge via changes in the intrinsic excitability of a discrete subpopulation of LS neurons that serve as a central hub to coordinate social behavior via downstream projections to reward, fear extinction, motor and sensory processing regions of the brain.

YAP is a key transcriptional co-activator of TEADs, it regulates cell growth and is frequently activated in cancer. In Malignant Pleural Mesothelioma (MPM), YAP is activated by loss-of-function mutations in upstream components of the Hippo pathway, while, in Uveal Melanoma (UM), YAP is activated in a Hippo-independent manner. To date, it is unclear if and how the different oncogenic lesions activating YAP impact its oncogenic program, which is particularly relevant for designing selective anti-cancer therapies. Here we show that, despite YAP being essential in both MPM and UM, its interaction with TEAD is unexpectedly dispensable in UM, limiting the applicability of TEAD inhibitors in this cancer type. Systematic functional interrogation of YAP regulatory elements in both cancer types reveals convergent regulation of broad oncogenic drivers in both MPM and UM, but also strikingly selective programs. Our work reveals unanticipated lineage-specific features of the YAP regulatory network that provide important insights to guide the design of tailored therapeutic strategies to inhibit YAP signaling across different cancer types.

Accurate estimation of the pKas of cysteine residues in proteins could inform targeted approaches in hit discovery. The pKa of a targetable cysteine residue in a disease-related protein is an important physiochemical parameter in covalent drug dis- covery, as it influences the fraction of nucleophilic thiolate amenable to chemical protein modification. Traditional structure-based in silico tools are limited in their predictive accuracy of cysteine pKas relative to other titratable residues. Additionally, there are limited comprehensive benchmark assessments for cysteine pKa predictive tools. This raises the need for extensive assessment and evaluation of methods for cysteine pKa prediction. Here, we report the performance of several computational pKa methods, including single structure and ensemble-based approaches, on a diverse test set of experimental cysteine pKas retrieved from the PKAD data- base. The dataset consisted of 16 wildtype and 10 mutant proteins with experimentally measured cysteine pKa values. Our results highlight that these methods are varied in their overall predictive accuracies. Among the test set of wildtype proteins evaluated, the best method yielded a mean absolute error of 2.3 pK units highlighting the need for improvement of existing pKa methods for accurate cysteine pKa estimation. Given the limited accuracy of these methods, further development is needed before these approaches can be routinely employed to drive design decisions in early drug discovery efforts.

Abstract Targeted protein degradation using heterobifunctional Proteolysis-Targeting Chimeras (PROTACs) or molecular glues has arisen as a powerful therapeutic modality for degrading disease targets. While PROTAC design is becoming more modular and straightforward, the discovery of novel molecular glue degraders has been more challenging. While several recent studies have showcased phenotypic screening and counter-screening approaches to discover new molecular glue degraders, mechanistically elucidating the ternary complex induced by the small-molecule that led to the initial phenotype—i.e. identifying the degraded target and relevant components of the ubiquitin-proteasome system—has remained cumbersome and laborious. To overcome these obstacles, we have coupled the screening of a covalent ligand library for anti-proliferative effects in leukemia cells with quantitative proteomic and chemoproteomic approaches to rapidly discover both novel covalent molecular glue degraders and their associated ternary complex components and anti-proliferative mechanisms. We have identified a cysteine-reactive covalent ligand EN450 that impairs leukemia cell viability in a NEDDylation and proteasome-dependent manner. Chemoproteomic profiling revealed covalent interaction of EN450 with an allosteric C111 in the E2 ubiquitin ligase UBE2D. Follow-up quantitative proteomic profiling revealed the proteasome-mediated degradation of the oncogenic transcription factor NFKB1 as a putative degradation target. Subsequent validation studies demonstrated that EN450 induced the ternary complex formation between UBE2D and NFKB1 and that both UBE2D and NFKB1 were important for the anti-proliferative mechanisms of EN450. Our study thus puts forth the discovery of a novel molecular glue degrader that uniquely induced the proximity of an E2 ligase with a transcription factor to induce its degradation and anti-proliferative effects in cancer cells. Taken more broadly, our study showcases a rapid and modular approach for discovering novel covalent molecular glue degraders and their respective ternary complex components in an unbiased fashion.

Ras proteins are membrane-anchored GTPases that regulate key cellular signaling networks. It has been recently shown that different anionic lipid types can affect the properties of Ras in terms of dimerization/clustering on the cell membrane. To understand the effects of anionic lipids on key spatiotemporal properties of dimeric K-Ras4B, we perform all-atom molecular dynamics simulations of the dimer K-Ras4B in the presence and absence of Raf[RBD/CRD] effectors on two model anionic lipid membranes: one containing 78% mol DOPC, 20% mol DOPS, and 2% mol PIP2 and another one with enhanced concentration of anionic lipids containing 50% mol DOPC, 40% mol DOPS, and 10% mol PIP2. Analysis of our results unveils the orientational space of dimeric K-Ras4B and shows that the stability of the dimer is enhanced on the membrane containing a high concentration of anionic lipids in the absence of Raf effectors. This enhanced stability is also observed in the presence of Raf[RBD/CRD] effectors although it is not influenced by the concentration of anionic lipids in the membrane, but rather on the ability of Raf[CRD] to anchor to the membrane. We generate dominant K-Ras4B conformations by Markov state modeling and yield the population of states according to the K-Ras4B orientation on the membrane. For the membrane containing anionic lipids, we observe correlations between the diffusion of K-Ras4B and PIP2 and anchoring of anionic lipids to the Raf[CRD] domain. We conclude that the presence of effectors with the Raf[CRD] domain anchoring on the membrane as well as the membrane composition both influence the conformational stability of the K-Ras4B dimer, enabling the preservation of crucial interface interactions.