Biomolecular condensates are central to subcellular compartmentalization and RNA regulation. In the multinucleate fungus Ashbya gossypii, condensates composed of Whi3 protein and CLN3 mRNA help ensure nuclear cycle asynchrony in a shared cytoplasm. Here, we investigated how Whi3 protein binding sites within CLN3 mRNA are specified and influence properties of the condensate. We found that Whi3 binds to varied RNA sequences but prefers the five-nucleotide motif UGCAU, which appears at five locations in the CLN3 transcript. Mutating individual UGCAU motifs altered the saturation concentration (Csat) and dense phase concentration of RNA and Whi3 in cell-free reconstitution experiments. These defects were partially rescued by melting and refolding the mRNA, indicating that RNA structure plays a critical role in distinguishing binding sites and determining condensate properties. Lastly, a subset of mutants showed reduced condensate numbers and dysregulation of the cell cycle in cells. These data reveal that the context of otherwise identical mRNA sequences can differentially affect condensate properties.

Abstract Homozygous deletion of CDKN2A on chromosome 9p21 is the most frequent genomic loss in cancer and often extends into the adjacent MTAP gene. This co-deletion occurs in ∼10% of tumors (>200,000 cases annually in the U.S) and is particularly enriched in aggressive malignancies such as glioblastoma (GBM), malignant peripheral nerve sheath tumors (MPNST), pancreatic ductal adenocarcinoma (PDAC), and non-small cell lung cancer (NSCLC). In these tumors, MTAP loss leads to intracellular accumulation of its substrate methylthioadenosine (MTA), which partially inhibits protein arginine methyltransferase 5 (PRMT5) by competing with its methyl donor S-adenosyl-methionine (SAM) at the active site, reducing its ability to symmetrically dimethylate arginine residues (SDMA). This creates a synthetic-lethal dependence on residual PRMT5 activity and has driven the development of MTA-cooperative PRMT5 inhibitors, which show encouraging preclinical activity and single-agent response rates of 21-29% in early clinical trials in the advanced, refractory setting. We therefore hypothesized that defining the molecular consequences of PRMT5 inhibition could guide patient stratification and inform rational combination strategies capable of enhancing therapeutic efficacy and preventing resistance. To test this, we performed multi-omics profiling, including short- and long-read transcriptomics and whole-cell proteome mass spectrometry, across MTAP-deleted NSCLC, GBM, and PDAC models, integrating these data with in-house and publicly available datasets to define PRMT5-dependent methyl events and their downstream molecular consequences. Transcriptomic and proteomic analyses revealed strong and consistent pathway-level alterations across models following PRMT5 inhibition, including activation of MAPK signaling, a compensatory program validated to synergize with PRMT5 inhibitors in vitro and in vivo. Across eight distinct PRMT5 methyl-enrichment mass spectrometry studies, we identified 180 PRMT5 substrates enriched mainly for proteins involved in RNA metabolism and pre-mRNA splicing, including multiple snRNP-associated components of the spliceosome. We therefore examined the splicing consequences of PRMT5 inhibition and observed widespread increases in intron retention and reductions in exon skipping, with enrichment of splicing alterations in transcripts involved in DNA damage repair across models. Overall, this study provides a comprehensive substrate map of PRMT5 and characterizes the transcriptional, proteomic, and splicing programs activated upon its inhibition across MTAP-deleted GBM, NSCLC, and PDAC models. These findings highlight pathway and splicing-associated vulnerabilities as targets for rational combination strategies and establish a mechanistic framework to guide biomarker development and future therapeutic exploration. Citation Format: Eliana Destefanis, Blanka Bordas, Jose C. Martinez, Samar Sayedyahossein, Samuel Moffitt, Enakireru M. Erhumuoghene, Daniel Dominguez, Kathleen M. Mulvaney. Multi-omics characterization of PRMT5 inhibition identifies vulnerabilities for combination therapy in MTAP-deleted cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4131.

RNA G-quadruplexes (rG4s) have been implicated as important regulators of RNA metabolism and are promising targets for RNA-targeted therapeutics. rG4s typically require a canonical (G≥2N1-7)4 motif, but the sequence features that affect rG4 stability and recognition by RNA-binding proteins (RBPs) and rG4-binding ligands are not fully understood. To interrogate sequence-level drivers of rG4 folding, we applied a reverse-transcriptase stop sequencing strategy to a library of ∼3000 synthetic rG4s with varied G-tract lengths, loop lengths, and loop compositions, permitting massively parallel quantification of rG4 stability. Our data confirm known sequence-level features and characterize novel combinatorial impacts of these features. We also assessed systematically mutagenized natural rG4s, revealing unexpected mutations that significantly affect rG4 stability, including contributions from flanking sequences outside of the rG4. We further used our strategy to assess rG4 recognition preferences of the model rG4 ligand pyridostatin, revealing a preferential stabilization of rG4s containing mixed-length G-tracts. We additionally demonstrated the potential for large-scale protein-binding assays with our library to reveal rG4 features recognized by RBPs, specifically G3BP1 and FMRP. Our approach and data provide a generalizable framework to study sequence-level drivers of rG4 stability, binding by RBPs, and ligand interactions, defining basic principles of rG4 formation and downstream biology.

The addition of CDK4/6 inhibitors to endocrine therapy has significantly improved outcomes in HR+/HER2-breast cancer. However, variable patient responses and acquired resistance remain a clinical challenge. We therefore defined the comprehensive molecular response to palbociclib, the most clinically used CDK4/6 inhibitor. Global analyses of gene expression, protein abundance, splicing, and chromatin accessibility revealed broad patterns and specific changes that result from CDK4/6-inhibition in breast cancer cells. We uncovered unexpected feedback between CDK4/6 and estrogen-response signaling, which has clear clinical implications. We also revealed a widespread alternative splicing program that partially overlapped with genes whose expression is regulated, and which is expected to impact protein function. These molecular changes nominated combination therapies that interfere with the activation of CDKs or ERα. Accordingly, co-targeting CDK7, which regulates CDK2, CDK4/6 and ERα, additively impacted cell fitness. Collectively, these data reveal a complex, multi-tiered response to CDK4/6 inhibition, with implications for therapeutic efficacy.

The subcellular localization patterns of RNAs are controlled by regulatory elements contained within them. However, for most localized RNAs, the identities of these elements remain unknown. We had previously identified several localization elements that are necessary and sufficient for robust, kinesin-dependent RNA targeting to microtubule plus ends in a variety of cell types. Yet the characteristics of these elements that are critical for function remained unclear. To address this, we systematically created tens of thousands of mutant localization elements and quantified their ability to regulate subcellular RNA localization in neuronal cells. We found that the minimally active size of these localization elements is large, approximately 200 nucleotides. These elements contain multiple important subsequences, with some being completely intolerant of any changes and others being tolerant to a shuffling of nucleotide order but not to changes in nucleotide composition. Using single molecule microscopy, we verified these findings in primary rat neurons. Together, these results demonstrate that highly active mammalian RNA localization elements are large, complex, and multipartite and lay a foundation for further mechanistic studies of their function.

The subcellular localization of many mRNAs to neuronal projections allows neurons to efficiently and rapidly react to spatially restricted external cues. However, for most of these RNAs, the mechanisms that govern their localization are unknown. Here, using subcellular fractionation and single-molecule RNA FISH, we found that loss of TDP-43 results in increased accumulation of hundreds of mRNAs in neurites. Using high-throughput functional assays in cells and high-throughput binding assays in vitro, we subsequently identified specific regions within these mRNAs that mediate their TDP-43-dependent localization and interaction with TDP-43. We found that the same regions also mediated TDP-43-dependent mRNA instability, suggesting a mechanism by which TDP-43 regulates mRNA localization. ALS-associated mutations in TDP-43 resulted in similar mRNA mislocalization phenotypes as did TDP-43 loss in mouse dorsal root ganglia and human iPS-derived motor neurons. These findings establish TDP-43 as a direct negative regulator of mRNA abundance in neurites and suggest that mislocalization of specific transcripts may occur in ALS patients.

Liquid-liquid phase transitions govern a wide range of protein-protein and protein-RNA interactions. Although the importance of multivalency and protein disorder in driving these transitions is clear, there is limited knowledge concerning the structural basis of phase transitions or the conformational changes that accompany this process. In this work, we found that a small human protein, SERF2, is important for the formation of stress granules. We determined the solution NMR structure ensemble of SERF2. We show that SERF2 specifically interacts with non-canonical tetrahelical RNA structures called G-quadruplexes, structures linked to stress granule formation. The biophysical amenability of both SERF2 and RNA G4 quadruplexes have allowed us to characterize the multivalent protein-RNA interactions involved in liquid-liquid phase transitions, the role that protein disorder plays in these transitions, identify the specific contacts involved, and describe how these interactions impact the structural dynamics of the components enabling a detailed understanding of the structural transitions involved in early stages of ribonucleoprotein condensate formation.

The reprogramming of alternative splicing networks during development is a hallmark of tissue maturation and identity. Alternative splicing of microexons (small, genomic regions ≤ 51 nucleotides) functionally regulate protein-protein interactions in the brain and are mis-spliced in neuronal diseases. However, little is known about the regulation and function of alternatively spliced microexons in striated muscle. Here, we investigated alternative splicing of a microexon in the synaptosome-associated protein 23 (Snap23) encoded gene. We found that inclusion of this microexon is developmentally regulated and tissue-specific, as it occurs exclusively in adult heart and skeletal muscle. The alternative region is highly conserved in mammalian species and encodes an in-frame sequence of 11 amino acids. Furthermore, we showed that alternative splicing of this microexon is mis-regulated in mouse models of heart and skeletal muscle diseases. We identified the RNA-binding proteins (RBPs) quaking (QKI) and RNA binding fox-1 homolog 2 (RBFOX2) as the primary splicing regulators of the Snap23 microexon. We found that QKI and RBFOX2 bind downstream of the Snap23 microexon to promote its inclusion, and this regulation can be escaped when the weak splice donor is mutated to the consensus 5’ splice site. Finally, we uncovered the interplay between QKI and muscleblind-like splicing regulator (MBNL) as an additional, but minor layer of Snap23 microexon splicing control. Our results are one of the few reports detailing microexon alternative splicing regulation during mammalian striated muscle development.

Genes involved in the regulation of chromatin structure are frequently disrupted in cancer, contributing to an aberrant transcriptome and phenotypic plasticity. Yet, therapeutics targeting mutant forms of chromatin-modifying enzymes have yielded only modest clinical utility, underscoring the difficulty of targeting the epigenomic underpinnings of aberrant gene regulatory networks. Here, we sought to identify novel epigenetic vulnerabilities in diffuse large B-cell lymphoma (DLBCL). Through phenotypic screens and biochemical analysis, we demonstrated that inhibition of the H3K9 demethylases KDM4A and KDM4C elicits potent, subtype-agnostic cytotoxicity by antagonizing transcriptional networks associated with B-cell identity and epigenetically rewiring heterochromatin. KDM4 demethylases associated with the KRAB zinc finger ZNF587, and their enzymatic inhibition led to DNA replication stress and DNA damage-einduced cGAS-STING activation. Broad surveys of transcriptional data from patients also revealed KDM4 family dysregulation in several other cancer types. To explore this potential therapeutic avenue, we performed high-throughput small molecule screens with H3K9me3 nucleosome substrates and identified novel KDM4 demethylase inhibitors. AI-guided protein-ligand binding predictions suggested diverse modes of action for various small molecule hits. Our findings underscore the relevance of targeting fundamental transcriptional and epigenetic mechanisms for anti-cancer therapy.

RNA binding proteins (RBPs) interact with and tightly regulate the fate of messenger RNAs but how RNA targets are recognized remains a challenging question. RBPs often contain multiple domains known to directly bind RNA, such as RNA recognition motifs (RRMs), as well as domains whose RNA binding capacity remains incompletely understood, e.g ., low complexity domains (LCDs). Here, we dissect HNRNPR, an RBP with three RRMs and an arginine-glycine rich (RG-rich) LCD. We apply unbiased high-throughput biochemical approaches and identify critical RNA binding domains that confer specificity. We show that not all RRMs contribute equally to binding and find that RRM3, along with a downstream C-terminal charged region, are required for RNA binding. We find that HNRNPR also binds RNA G-quadruplexes (rG4s) and map multiple rG4 binding sites including RRM3 with the C-terminal charged region and RG-rich regions within the LCD. We dissect rG4 specificity for the full length HNRNPR and LCD using a newly created RNA pool focused on rG4s and reveal that binding is dependent on RNA folding and find specific rG4 features that enhance HNRNPR-rG4 interactions. Our work highlights the complexity of RBP-RNA interactions and motivates the study of disordered regions as RNA binding domains.

30% of PDACs frequently overexpress RNA-binding proteins (RBPs), including splicing factors; however, the functional impact of aberrant splicing in PDAC progression and therapy resistance remains poorly understood. Here, we identify Survival Motor Neuron Domain Containing 1 (SMNDC1) as a key regulator of alternative splicing that sustains oncogenic MAPK signaling through exon retention in KRAS-driven pancreatic ductal adenocarcinoma (PDAC). Transcriptomic analyses revealed that SMNDC1 amplification promotes retention of cassette exons, notably exon 4 (E4) of MAPK3 (ERK1), which encodes the full activation loop, including the ERK1 activating phosphosites (Thr202/Tyr204). E4 inclusion is lower in benign, non-malignant cells in contrast to PDAC cells and tumors with elevated SMNDC1. Forced exclusion of E4 using antisense oligonucleotides (ASOs) reduced ERK1 phosphorylation, downregulated ERK target genes, and inhibited tumor initiation. Notably, SMNDC1 expression and MAPK3 E4 inclusion were elevated in PDAC cells with acquired resistance to KRAS inhibitors (KRASi: RMC-6236, adagrasib, MRTX1133, and sotorasib) and MEK inhibitors (selumetinib, trametinib). We found that PDAC cells resistant to KRAS inhibitors were resensitize to these compounds upon forcing the exclusion of E4 with ASOs or by knocking down SMNDC1. To further interrogate E4’s function in therapy resistance and oncogenic signaling, we generated isogenic PDAC lines stably expressing only either ERK1+E4 or ERK1∆E4 isoforms. ERK1+E4 cells demonstrated enhanced proliferation, MAPK signaling and resistance to KRASi. Contrary, ERK1∆E4 cells exhibited reduced oncogenic signaling, compensatory ERK2 activation, impaired proliferation, and did not re-sensitized cells to KRASi. Mechanistically, we found that ERK1∆E4 protein is rapidly degraded in cells thus acting as a dominant negative. Lastly, we hypothesized that targeting SMNDC1 in PDACs in mice could be a therapeutic approach to target KRASi-resistant tumors. Using a new SMNDC1 inhibitor (SMNDCi) we found that MRTX1133-resistant tumors decreased tumor growth in xenografts supporting the translational relevance of this approach. Together, our findings identify a previously unrecognized splicing-based mechanism of MAPK activation and therapy resistance. SMNDC1-mediated MAPK3 E4 inclusion constitutes a tunable switch for ERK1 stabilization and oncogenic output, offering a novel therapeutic vulnerability in KRAS-driven PDACs. Md Afjalus Siraj, Deanne Yugawa, Yushan Zhang, Gilbert Giri, Prabir Chakraborty, Grant Goda, Muaz faruque, Geet Rao, Daniel Dominguez, Stefan Kubicek, Resham Bhattacharya, Luisa Escobar-Hoyos, Priyabrata Mukherjee. A Splicing Switch in ERK1 Controlled by SMNDC1 Drives MAPK Pathway Reactivation and Resistance to KRAS inhibitors in PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_3):Abstract nr A003.

Previously considered "housekeeping" genes, small nucleolar RNAs (snoRNAs) are increasingly understood to have wide-ranging functions in cancer, yet their role in metastasis has been less well studied. Here, we identify the snoRNA Snord67 as a regulator of lymph node (LN) metastasis in breast cancer. Snord67 expression is enriched in LN metastases in an immune-competent mouse model of female breast cancer. In an orthotopic breast cancer model, loss of Snord67 decreases LN metastasis. In a model of lymphatic metastasis, Snord67 loss decreases LN tumor growth and distant metastases. In breast cancer cell lines, Snord67 knockout results in loss of targeted 2'-O-methylation on U6 small nuclear RNA, as well as widespread changes in splicing. Together, these results demonstrate that Snord67 regulates splicing and promotes the growth of LN metastases and subsequent spread to distant metastases. SnoRNA-guided modifications of the spliceosome and regulation of splicing may represent a potentially targetable pathway in cancer.

Background: Normal cellular functions rely on iron, making dietary intake crucial for maintaining iron levels. Iron deficiency (ID) from low intake, blood loss, or pregnancy causes symptoms like fatigue, brain fog, and anemia. Intracellular iron homeostasis primarily involves post-transcriptional control by RNA-binding proteins, iron regulatory protein (IRP)-1 and IRP2, binding mRNAs of genes responsible for regulating iron levels. Emerging studies have highlighted the involvement of additional RNA-binding proteins, such as ZFP36 and SRSF7, that exhibit translational and alternative splicing control in response to low iron availability. Further, we recently uncovered iron-sensitive alternative splicing by PCBP1 and PCBP2—known intracellular iron chaperones. This budding body of evidence indicates ID triggers a broader post-transcriptional response than previously understood. Aims: We aim to study how iron deficiency impacts the transcriptome and proteome in key iron-sensitive cell types. By conducting a comparative analysis across cell lines, we seek to highlight conserved and novel mechanisms underlying the transcriptomic and proteomic changes associated with ID stress, thereby advancing our understanding of ID at a molecular level. Methods: Two well-established cell lines, HepG2 and K562, were treated with either 10 micromolar iron chelator, 21H7, or 100% DMSO overnight (n=3), and isolated RNA was polyA-selected and prepared for Illumina sequencing (mRNAseq). We characterized changes in overall gene expression and alternative splicing and evaluated effected pathways with gene ontology analysis. Parallel cell lysate samples were used for label-free whole cell mass spectrometry. Hypergeometric tests evaluated the significance of overlapping datasets, considering only co-detected genes or events as background. Spearman's rank correlation test was employed for correlation analysis. Chi-square tests were used to ascertain significant discrepancies between observed and expected results. Results: At the mRNA level, 1294 genes were upregulated and 706 downregulated for HepG2, while K562 showed 1899 genes up- and 586 downregulated. Both cell transcriptomes demonstrated more significant changes than expected by chance (p<2e-16). Comparing HepG2 and K562 revealed 407 genes (87%, p<8e-167), sharing a similar regulation profile, with a positive correlation (R=0.43, p<2e-16). Gene ontology (GO) analysis of differentially expressed genes in both cell lines identified pathways including leukocyte differentiation, nephron development, and connective tissue development, highlighting the broad role of iron in pathways for growth and development. In HepG2, we detected about 1950 alternative splicing events, with 66% of cassette exons being more excluded upon 21H7 treatment (p<2e-16). In K562, we identified nearly 5,000 alternative splicing events, indicating an unprecedented impact on RNA splicing. Unlike HepG2, only 46% of cassette exons were more excluded (p=4e-10). Between both datasets, 303 cassette exon events were found to overlap (p=7e-78). Close to 300 proteins were differentially expressed in K562, vs 137 in HepG2, leading to 43 proteins overlapping (p=3e-21). Common GO terms for differential proteins in both cell lines included response to oxygen levels and hypoxia. However, K562 GO terms uniquely included 'anchoring junction’ while HepG2 unique GO terms included muscle cell proliferation, highlighting the different responses between cell types. Conclusion: Our results show that posttranscriptional gene regulation is a major step in the cellular response to ID. Many genes were found to change in their gene expression and alternative splicing, showing wide sensitivity to iron level changes. Given the diverse metabolic demands across tissues, sensitivity to ID may vary significantly. Additionally, many alternative splicing is cell-type and tissue-specific. Thus, identifying a subset of cassette exons with significant overlap between cell types suggesting a conserved response to iron deficiency via alternative splicing. Our findings extend the role iron plays in gene regulation, especially alternative splicing.

RNA G-quadruplexes (rG4s) are key regulatory elements in gene expression, yet the effects of genetic variants on rG4 formation remain underexplored. Here, we introduce G4mer, an RNA language model that predicts rG4 formation, classifies rG4 subtypes, and evaluates the effects of genetic variants across the transcriptome. G4mer significantly improves accuracy over existing methods and uncovers subtype-specific differences in mutational sensitivity and evolutionary constraint, highlighting sequence length and flanking motifs as important rG4 features. Applying G4mer to $${5}^{{\prime} }$$ 5 ′ untranslated region (UTR) variations, we identify variants in breast cancer-associated genes that alter rG4 formation and validate their impact on structure and gene expression. These results demonstrate the potential of integrating computational models with experimental approaches to study rG4 function, especially in diseases where non-coding variants are often overlooked. To support broader applications, G4mer is available as both a web tool and a downloadable model.

Oncogenic KRAS is a central driver of pancreatic ductal adenocarcinoma (PDAC), primarily through maintenance of ERK signaling. However, activation of the KRAS-MEK-ERK through a KRAS-independent manner is not fully understood. Here, we identify one alternative mechanism of ERK activation mediated by the RNA-binding protein survival motor neuron domain containing protein 1 (SMNDC1), which regulates splicing of ERK1 (MAPK3). SMNDC1 is amplified or overexpressed in ∼30% of PDACs and is associated with decreased survival in both human and murine models. Gain- and loss-of-function studies revealed that SMNDC1 promotes tumor growth in part by driving inclusion of MAPK3-E4, which encodes the kinase-activating phosphorylation sites Thr202/Tyr204. Use of an ERK kinase biosensor (ERK-KTR), which translocates to the nucleus upon ERK inactivation, confirmed that E4 inclusion is critical for maintaining downstream ERK activity. Conversely, forced exclusion of MAPK3-E4 using antisense oligonucleotides (ASOs) diminished ERK signaling, despite the continued presence of wild-type ERK2, underscoring the functional significance of the ERK1 splice isoform. To examine this regulation at the tissue and cell level, we performed BaseScope in situ hybridization on a tissue microarray comprising over 200 PDAC patients. While expression of MAPK3 transcripts with and without E4 was broadly correlated, we observed a consistent shift toward E4 inclusion in tumor samples, indicating selective pressure for the active isoform in PDAC. Mechanistically, high-throughput RNA Bind-n-Seq (RBNS) and enhanced CLIP (eCLIP) in PDAC cells revealed that SMNDC1 binds a C-rich polypyrimidine tract at the distal 3′ splice site of MAPK3-E4. Mutational analysis demonstrated that the conserved Tudor domain of SMNDC1 is required for this RNA interaction and for E4 splicing. Additionally, we identified a feedback mechanism whereby inclusion of a conserved poison exon in SMNDC1 triggers its own degradation, suggesting tight post-transcriptional control of this splicing regulator. Altogether, these findings define a sequence- and domain-specific splicing switch that enhances ERK activity through MAPK3-E4 inclusion, providing a KRAS-independent mechanism of MAPK pathway activation and a potential therapeutic vulnerability in PDAC. Deanne E. Yugawa, Md Afjalus Siraj, Muaz Faruque, Gilbert Giri, Grant Goda, Daniel Dominguez, Pablo Perez-Pinera, Resham Bhattacharya, Priyabrata Mukherjee, Luisa F. Escobar-Hoyos. Tightly regulated alternative RNA splicing of MAPK3 (ERK1) as a Kras-independent mechanism of oncogenesis in PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_3):Abstract nr A060.

The regulation and impact of tumor-specific isoforms in cancer is challenging to understand, with unclear links between oncogenic signaling and mRNA processing and decay. Using transcriptomic analyses and experimental validation, we demonstrate that clear cell renal cell carcinoma (ccRCC) tumors exhibit coordinated intron retention (IR) and poison exon (PE) inclusion, generating transcripts targeted by nonsense-mediated decay (NMD). ccRCC tumors segregate into two subtypes based on IR: tumors with low IR levels and those with high IR levels, the latter associated with aggressive clinical features and poorer survival. We identify mammalian target of rapamycin (mTOR) signaling as a key modulator of IR and PE inclusion, showing mTOR inhibition significantly increases NMD-targeted isoforms, linking mTOR signaling with RNA surveillance pathways. Additionally, tumors with high IR levels exhibit increased T cell infiltration signatures. Collectively, these findings reveal a mechanism by which aggressive ccRCC tumors sustain aberrant RNA isoforms, offering insights for biomarker and therapeutic development.

Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest cancers, in part because we do not sufficiently understand its genetic basis to design effective therapies. Although KRAS mutations are present in 96% of PDACs, only 60% have concurrent TP53 mutations, leaving 40% with wild-type TP53 and undefined drivers. Identifying additional mutations is challenging due to the long tail of low-frequency events in PDAC (<10%), yet these rare mutations could reveal key drivers of tumorigenesis and therapeutic vulnerabilities. To identify novel driver mutations beyond TP53, we performed a genetic interaction analysis based on mutual exclusivity, which suggests that mutations exclusive to one another function within the same pathway. Using unbiased genetic interaction analyses, we queried >37,000 PDAC cases to prioritize rare mutations mutually exclusive with TP53 mutations. Mutations in the splicing factors SF3B1 and RBM10 (∼15% combined) emerged as the most significant and mutually exclusive with mutant TP53. Thus, we hypothesized that mutations K700E in SF3B1 and truncating mutations in RBM10 drives tumorigenesis and therapy resistance. We established the first autochthonous mouse models co-expressing mutant Kras with either Sf3b1K700E or Rbm10 loss, revealing these splicing alterations can cause PDAC. To uncover how splicing-factor mutations drive PDAC, we performed deep RNA sequencing and eCLIP in lineage-traced tumor cells from mouse models with wild-type or mutant Sf3b1/Rbm10. While tumors with these mutations lack chromosomal deletions, they exhibit widespread splicing defects, particularly in exon selection. Thousands of mRNAs transcribed from chromosomes 2, 7, 11, and 17 are mis-spliced and degraded by nonsense-mediated decay (NMD), leading to post-transcriptional gene loss. These patterns are conserved in human PDAC models. Notably, TP53-mutant PDACs require chromosomal deletions of chr11 and chr7 for tumor development, suggesting that SF3B1 and RBM10 mutations mimic this effect through splicing-dependent mechanisms. To define precision therapies for splicing-mutant tumors, we found that PDACs with these mutations are up to 5000X more sensitive to Gemcitabine versus 5FU. We combined Gemcitabine with the splicing modulator H3B-8800, which synergistically decreased tumor growth and extended survival of xenograft mice. Together, our findings reveal that diverse genetic events—TP53 mutations, SF3B1 mutations, or RBM10 loss—collaborate with mutant KRAS to by converging on a shared tumorigenic mechanism: loss of gene expression from specific chromosomes. In the case of inactivating mutations in TP53, this decrease in gene expression is caused by chromosome deletions, a genetic mechanism. In contrast, mutations in SF3B1 and RBM10 result in decreased gene expression through alternative splicing and mRNA decay, a post-transcriptional mechanism. This work uncovers a novel genetic convergence in PDAC and suggests that targeting splicing-mediated vulnerabilities may offer therapeutic opportunities across molecular subtypes. Natasha Pinto Medici, Diana Martinez-Saucedo, Tianyi Chu, Saúl Rojas-Sánchez, Daniel Lee, Robert Tseng, Vincent Cannataro, Julia C.F. Quintanilha, Ryon P. Graf, Gilbert Giri, Sheila Oliveira, Amelia Lower, Isabela Fuentes, Mariana do Carmo, Lyanne Delgado-Coka, Lucia Roa-Pena, Sumedha Chowdhury, Muaz Furaque, Emma Delannoy, Nicolas Lecomte, Christine Iacobuzio-Donahue, John P. Morris IV, Katerina Politi, Pablo Perez-Piñera, Marie E. Robert, Kenneth Shroyer, Scott Lowe, Michael Cecchini, Omar Abdel-Wahab, Steven D. Leach, Jeffrey P. Townsend, Daniel Dominguez, Mathieu Quesnel-Vallières, Luisa Escobar-Hoyos. Altered RNA splicing drives pancreatic cancer by mimicking chromosome deletions [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_3):Abstract nr A004.

Background: Iron is essential for cellular function. Homeostasis of iron is controlled post-transcriptionally via highly conserved mechanisms. We recently found broad changes in alternative splicing with reduced iron availability, consistent with numerous operant mechanisms of iron sensitive RNA regulation. Poly C binding proteins (PCBPs) are RNA-binding proteins that regulate alternative splicing, translation, and RNA stability. PCBPs have also been found to be critical iron chaperones regulating intracellular iron flux by delivering iron to target proteins, such as ferritin. We have found PCBP1 to have iron-sensitive alternative splicing activity. Specifically, we found PCBP1-mediated splicing events that change with iron chelation and loss of PCBP1 attenuates iron chelation-induced splicing. Moreover, PCBP1 has enhanced association with RNA during iron chelation and mutation of PCBP1 residues associated with iron binding also enhances RNA association. The specific molecular mechanisms by which iron modulates PCBP1 mRNA regulation is not known. Because PCBP1 is a splicing factor that participates in ribonucleoprotein complexes, we hypothesize that low iron levels affect the PCBP1 protein interactome to influence splicing outcomes. Study Aim: Determine how protein-protein interactions contribute to iron-sensitive RNA regulation by PCBP1. Methods: We performed an APEX-proximity labeling assay to assess PCBP1 protein interactors under conditions of iron chelation or iron overload. APEX-PCBP1 fusion protein was expressed in K562 cells, and cells were treated with iron chelation (21H7) or iron overload (ferric ammonium citrate, FAC). Cells were then treated with biotin-phenol and hydrogen peroxide to biotinylate proteins proximal to APEX-PCBP1. Omission of hydrogen peroxide served as background controls. LC-MS was performed on streptavidin pulldown of whole cell lysate to determine enrichment or depletion of biotinylated proteins. Results: Altering iron levels resulted in widespread changes in biotinylated proteins that were proximal to PCBP1. Notably, we found a network of pre-mRNA binding proteins that are preferentially biotinylated under iron chelation compared to iron overload, including LSM5 (Log2 fold change [L2FC]=3.48, 21H7/FAC), LSM1 (L2FC=3.45, 21H7/FAC), and PRPF39 (L2FC =2.62, 21H7/FAC). Notably, ACO1 biotinylation was dramatically increased in chelation compared to overload (L2FC =5.46, 21H7/FAC). We also found various kinases with increased biotinylation under iron chelation compared to iron overload, including MAP kinases such as MAPK14 (L2FC =3.40, 21H7/FAC) and MAP2K2 (L2FC = 1.89, 21H7/FAC). Conclusions: These data support a model wherein low iron levels promote PCBP1 association with splicing factors to enhance alternative splicing regulation. Future work will explore the role of post-translational modifications in iron-sensitive PCBP1 function.