Molecular Cancer

Resistance to cancer treatment remains the leading cause of cancer-related deaths. In tumors with low mutational burden such as pediatric cancers, alternative transcripts, including circular RNAs (circRNAs), have been identified as involved in treatment resistance. However, their isoforms are often missed by commonly used short-read sequencing. Here, we employ long-read sequencing to identify full-length circRNA isoforms associated with resistance in ALK -driven pediatric cancers. Using cell models and a cohort of ALK -translocated anaplastic large-cell lymphoma (ALK+ ALCL) patients, two circRNAs were detected as specifically upregulated in resistant cases and associated with worse disease outcomes. Similar findings were observed in the pediatric cancer neuroblastoma. These circRNAs were also more abundant in liquid biopsies from ALKi-resistant ALK+ ALCL and neuroblastoma patients. This demonstrates that long-read sequencing allows for uncovering disease-relevant circRNA isoforms that could serve as biomarkers for resistance detection in a clinical setting.

Genomic alterations driving tumorigenesis in sinonasal malignancies remain largely unexplored. Here, we perform an in vivo loss-of-function screen using a pooled custom single-guide library delivered to the sinonasal cavity by adeno-associated virus vector to identify cancer driver genes across diverse sinonasal malignancies. This approach yielded sinonasal malignancies with diverse histologies, including sinonasal squamous cell carcinoma, adenocarcinoma, poorly differentiated sinonasal carcinoma, and sinonasal neuroendocrine tumors characteristic of olfactory neuroblastoma. Surprisingly, rather than observing distinct sgRNA profiles across sinonasal tumor subtypes, common recurrent mutations were identified in Nf1 (79%), Rasa1 (74%), and Trp53 (68%) across malignancies with distinct histologies. Utilizing an orthogonal approach, we confirmed that Nf1/Trp53 were required for sinonasal tumorigenesis. Given that loss-of-function in NF1 and RASA1 may lead to increased Ras activity and downstream MEK signaling, we tested small molecule targeting of the RAS-MAPK pathway in sinonasal malignancies. Indeed, both tumor cell lines derived from our loss-of-function approach as well as from human sinonasal malignancies displayed significant sensitivity to MEK inhibition in standard in vitro culture and organoid models. These findings demonstrate that loss of NF1 and RASA1-mediated Ras-GAP activity leads to Ras activation and downstream MEK signaling which is a potential common target throughout major sinonasal tumor subtypes.

Tumor cell heterogeneity in neuroblastoma, a pediatric cancer arising from neural crest-derived progenitor cells, poses a significant clinical challenge. In particular, unlike adrenergic (ADRN) neuroblastoma cells, mesenchymal (MES) cells are resistant to chemotherapy and retinoid therapy and thereby significantly contribute to relapses and treatment failures. Previous research suggested that overexpression or activation of miR-124, a neurogenic microRNA with tumor suppressor activity, can induce the differentiation of retinoic acid-resistant neuroblastoma cells. Leveraging our established screen for miRNA-modulatory small molecules, we validated PP121, a dual inhibitor of tyrosine and phosphoinositide kinases, as a robust inducer of miR-124. A combination of PP121 and BDNF-activating bufalin synergistically arrests proliferation, induces differentiation, and maintains the differentiated state of MES SK-N-AS cells for 8 weeks. RNA-seq and deconvolution analyses revealed a collapse of the ADRN core regulatory circuitry (CRC) and the emergence of novel CRCs associated with chromaffin cells and Schwann cell precursors. Using a similar protocol, we differentiated and maintained MES neuroblastoma GI-ME-N and SH-EP cell lines, as well as glioblastoma LN-229 and U-251 cell lines, for over 16 weeks. In conclusion, our novel protocol suggests a promising treatment for therapy-resistant cancers of the nervous system. Moreover, these long-lived, differentiated cells provide valuable models for studying mechanisms underlying differentiation, maturation, and senescence.

Non-small cell lung cancer (NSCLC) is the deadliest cancer worldwide. Therapeutic progress stagnate, highlighting the complexity to replicate NSCLC in preclinical models. Drug discovery studies rely mostly on cancer cells in two-dimension (2D), which poorly predict drug efficacy in patients. There is a growing interest in three-dimensional (3D) preclinical models, such as 3D spheroids, to better model tumor phenotype and improve therapeutic prediction. However, a comprehensive view of 3D culture methods impact on transcriptomes, epigenomes and pharmacological responses and their correlations to NSCLC tumors is still missing. Here, we demonstrate that NSCLC spheroids undergo time-dependent transcriptomic and epigenomic changes, which peak after 3 weeks of culture. While DNA methylome remained stable, chromatin methylation and acetylation marks gained features of advanced NSCLC in a time-dependent manner. Single-cell transcriptomic profiling of spheroids demonstrated that time of 3D culture improved the correlation to NSCLC tumors. Moreover, long-term culture of 3D spheroids increased drug screening predictability, by showing resistance to drugs that failed in NSCLC patients (such as HDAC inhibitors) while demonstrating novel pharmacological vulnerabilities and synergistic interactions (such as combination of PRMT and HDAC inhibitors). Strikingly, reverting 3D spheroids back to 2D culture rapidly reversed transcriptomic, epigenetic and pharmacological signatures acquired after 3 weeks of 3D culture, highlighting the critical impact of cell culture conditions on NSCLC phenotype. Collectively, our findings demonstrate that implementing a time-dependent maturation process into 3D spheroid culture induces chromatin and transcriptomic changes that enhance NSCLC preclinical modeling.

Neuroblastoma (NB) is a highly metastatic pediatric cancer arising from the neural crest lineage. Genetic amplification of the MYCN proto-oncogene is a defining feature of NB, present in about 20% of all cases. The let-7 tumor suppressor microRNA targets the 3 UTR of MYCN mRNA. We previously demonstrated that the 3 UTR of MYCN mRNA acquires the ability to sequester let-7 in MYCN-amplified (MA) disease, thus inhibiting its function. This work established that a noncoding element within an oncogenic mRNA can contribute independently to disease pathology and genetic patterning. To further investigate the roles of noncoding RNA elements within the MYCN mRNA, we engineered cells expressing either MYCN-ORF (MYCN open reading frame only), MYCN-GL (full-length MYCN mRNA from the intact genetic locus), and Null-GL (full-length MYCN mRNA variant where EGFP replaces MYCN protein). We observe that all constructs enhance growth compared to controls in vitro and in vivo. MYCN-GL-expressing cells displayed the most robust growth in vitro despite containing multiple regulatory RNA elements. Remarkably, the Null-GL construct induces cells to grow as fast or faster than MYCN-ORF-expressing cells. Animal studies further confirmed these observations, where the Null-GL-driven tumors had the highest incidence and lowest latency, followed by MYCN-GL and then MYCN-ORF. Further, through NGS analysis, let-7, miR-101, and miR-34a targets are enriched in both MYCN-GL and Null-GL expressing cells. Thus, the 3UTR of MYCN, which is also targeted by these microRNAs, may interact with them in MYCN-GL and Null-GL cells to deliver a protective effect for the mRNA targets of these microRNAs. We also observed more dynamic differential gene expression in the -GL constructs than in ORF and GFP-expressing cells. In addition, MYCN-GL and Null-GL expressing cells are similarly enriched in gene ontology pathways for cancer, RNA metabolism, and microRNA processing pathways as compared to ORF and GFP. Whole genome sequencing also revealed more similarities in copy number variation in MYCN-GL and Null-GL than in ORF and GFP, suggesting that these constructs may provide selective pressure to favor specific CNV patterns. These observations show that full-length MYCN mRNA containing noncoding regulatory elements are more robust drivers of cell growth and oncogenicity than MYCN protein alone and provide insights into the mechanisms of oncogenic contribution. These results open an exciting door for our understanding of NB pathology and genetic patterning and have broad implications for other oncogene-driven cancers.

Oncogenic KRAS mutations occur in approximately 30% of lung adenocarcinoma. Despite several decades of effort, oncogenic KRAS-driven lung cancer remains difficult to treat, and our understanding of the positive and negative regulators of RAS signaling is incomplete. To uncover the functional impact of diverse KRAS-interacting proteins on lung cancer growth in vivo, we used multiplexed somatic CRISPR/Cas9-based genome editing in genetically engineered mouse models with tumor barcoding and high-throughput barcode sequencing. Through a series of CRISPR/Cas9 screens in autochthonous lung tumors, we identified HRAS and NRAS as key suppressors of KRASG12D-driven tumor growth in vivo and confirmed these effects in oncogenic KRAS-driven human lung cancer cell lines. Mechanistically, RAS paralogs interact with oncogenic KRAS, suppress KRAS-KRAS interactions, and reduce downstream ERK signaling. HRAS mutations identified in KRAS-driven human tumors partially abolished this effect. Comparison of the tumor-suppressive effects of HRAS and NRAS in KRAS- and BRAF-driven lung cancer models confirmed that RAS paralogs are specific suppressors of oncogenic KRAS-driven lung cancer in vivo. Our study outlines a technological avenue to uncover positive and negative regulators of oncogenic KRAS-driven cancer in a multiplexed manner in vivo and highlights the role of RAS paralog imbalance in oncogenic KRAS-driven lung cancer.

ObjectiveRNA adenosine-to-inosine (A-to-I) editing, catalyzed by adenosine deaminases acting on RNA (ADARs), is a pervasive post-transcriptional mechanism that expands transcriptomic and proteomic diversity. However, the global landscape of RNA A-to-I editing, as well as its functional and clinical significance in esophageal squamous cell carcinoma (ESCC), remains largely unexplored. This study aimed to systematically characterize the RNA editome of ESCC and elucidate its biological and clinical significance. MethodsWhole-transcriptome sequencing was performed on 121 primary ESCC tumors, with or without lymph node metastasis, together with matched normal tissues, to construct a high-resolution RNA editome. ADAR1-regulated RNA editing events were identified, and their functional consequences were investigated using integrated transcriptomic, phosphoproteomic, and RNA immunoprecipitation sequencing (RIP-seq) analyses. Associations between CDK13 editing, cGAS-STING-interferon-stimulated gene (ISG) signaling, and patient survival were further evaluated. ResultsA total of 222,020 high-confidence RNA editing sites were identified, of which approximately 98% were A-to-I events, including 124,486 ESCC-specific edits predominantly enriched in introns, 3' untranslated regions, and non-coding RNAs, highlighting a pervasive post-transcriptional regulatory layer. RNA A-to-I editing was extensively remodeled and globally up-regulated in non-metastatic ESCC, whereas only minimal changes were observed during lymph node metastasis, indicating that RNA editing alterations predominantly occur during early tumorigenesis. CDK13 emerged as a recurrent ADAR1 target, with RNA editing inversely correlated with CDK13 expression. ADAR1-mediated CDK13 editing established a positive feedback loop associated with enhanced interferon-stimulated gene (ISG) signaling and poorer survival in non-metastatic ESCC. Mechanistically, partial attenuation of CDK13 induced chronic, tumor cell-intrinsic activation of the cGAS-STING-ISG pathway. Integrated multi-omics analyses further revealed that CDK13 regulates phosphorylation networks governing cytoskeleton organization, intracellular trafficking, RNA homeostasis, and immune signaling. ConclusionRNA A-to-I editing represents a dynamic regulatory mechanism driving early ESCC progression and remodeling tumor cell-intrinsic immune signaling. ADAR1-mediated editing of CDK13 provides a mechanistic link between RNA editing and cGAS-STING-ISG pathway activation, revealing potential therapeutic vulnerabilities and supporting its utility as an early prognostic biomarker in ESCC.

IGF2BP1 is an oncofoetal RNA binding protein that is expressed in many tumors. We have recently described a small molecule inhibitor of IGF2BP1, termed AVJ16, that prevents binding of the protein to its RNA targets by directly associating with the protein. Here, using a multi-omics approach, we have analyzed the effects of this inhibition on RNA binding, RNA expression, and protein expression. AVJ16 treatment downregulates RNAs encoding members of several pro-oncogenic signaling pathways, including Hedgehog, Wnt, and PI3K-Akt, and there is a strong correlation between IGF2BP1 RNA binding, RNA expression, and protein expression. AVJ16 treatment of lung adenocarcinoma (LUAD) cells in culture causes a strong reduction in proliferation, colony formation, invasion, and spheroid growth while enhancing apoptosis and cell death. All of these effects are limited to cells expressing IGF2BP1. LUAD cells treated with AVJ16 show a pronounced reduction in vital dye efflux, often correlated with enhanced chemosensitivity. In syngeneic LUAD xenografts in mice, IP injection of AVJ16 prevents tumor growth, and incubation with AVJ16 induces cell death in human organoids derived from IGF2BP1-expressing LUADs but not from healthy lung tissue. These results suggest that AVJ16 is a promising candidate for mono- and/or adjuvant therapy directed against tumors expressing IGF2BP1.

Despite advancements in personalized cancer therapies, platinum-based chemotherapy remains the cornerstone for treating solid tumors, including Non-Small Cell Lung Cancer (NSCLC). The integration of novel immunotherapies with platinum compounds has shown promising outcomes for the treatment of advanced disease. However, a significant proportion of patients experience therapeutic failure due to innate or acquired resistance. Thus, identifying molecular profiles and biomarkers to monitor patient progress and treatment response is crucial for tailoring therapeutic strategies. Small extracellular vesicle (sEV)-based liquid biopsy emerges as a promising non-invasive method for cancer management. sEVs play a critical role in cell communication and provide molecular insights into the tumor environment. In this study, we characterized the microRNome content of sEVs from cisplatin-resistant and -sensitive cancer cells using small-RNA sequencing. We identified and validated three miRNAs in two cohorts of 78 and 49 patients treated with either chemotherapy alone or chemo-immunotherapy, respectively, analyzed via liquid biopsy, differentiating NSCLC patients based on progression and overall survival. Notably, miR-451a emerged as a prognostic marker for chemo and chemo-immunotherapy, while miR-142-3p was identified for the first time as a potential prognostic marker specifically for stage IV patients, irrespective of the treatment. The combination of miR-451, miR-142-3p, and miR-55745, a novel miRNA identified from our miRNome screening, serves as a valuable biomarker for both cisplatin and chemo-immunotherapy treatment responses. This study underscores the role of sEVs in acquired cisplatin resistance and introduces novel miRNA-sEV biomarkers for managing NSCLC progression.

Long noncoding RNAs (lncRNAs) are a class of regulatory RNAs with critical roles in cellular homeostasis and disease pathogenesis, including cancer. Dysregulated lncRNA expression in lung adenocarcinoma (LUAD) contributes to oncogenic mechanisms through immune and inflammatory signaling networks, including the arachidonic acid (AA) signaling pathway. Here, we investigate the functional significance of the lncRNA PACER (PTGS2 Antisense NF-{kappa}B1 Complex-Mediated Expression Regulator), which modulates cyclooxygenase-2 (COX-2), a key enzyme in the AA pathway. Using stable shRNA-mediated knockdown in A549 LUAD cells, we show that PACER silencing reduces COX-2 expression and impairs cellular proliferation, migration, and invasion. Bioinformatic analysis using LncLOOM revealed conserved motifs across primates and mice, including predicted binding sites for miR-18a-5p, miR-196-5p, miR-1306-5p, and miR-423-3p. To define PACERs structural organization, we performed selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP), generating the first secondary structure determination of PACER. SHAPE-MaP and SuperFold analyses revealed a compact, stabilized 5' domain and a flexible central and 3' region. {Delta}SHAPE analysis identified multiple sites of differential protection, and Rsample clustering resolved two dominant in vivo conformations, suggesting that PACER adopts a modular architecture with defined structural domains. These structured regions coincide with conserved motifs associated with NF-{kappa}B p50 and predicted miRNA binding sites, indicating that PACER function may depend on conformational switching that modulates protein and miRNA accessibility. Together, these findings establish PACER as a regulator of LUAD proliferation and invasion via COX-2 signaling and highlight its potential as a biomarker and therapeutic target in inflammatory cancers.

BackgroundGroup 3 (G3) is one of the most common, aggressive and fatal subtypes of the paediatric cerebellar tumour Medulloblastoma (MB), primarily driven by the MYC oncogene. Targeting MYC has long been challenging and this, combined with our incomplete understanding of G3 MB molecular bases, has hindered the development of effective targeted therapies. Long noncoding RNAs (lncRNAs), with their extensive oncogenic roles, cancer-specific expression, and connection to MYC biology, offer opportunities for unravelling this complexity and providing new insights and therapeutic targets. MethodologyUsing genome-wide, molecular and cellular assays, we characterised the activity of the MYC-dependent, anti-apoptotic lncRNA lncMB3 in G3 MB cells. ResultsThrough transcriptomic and interactomic analyses, we clarified lncMB3 function and mode-of-action. LncMB3 controls the TGF-{beta} pathway, critically altered in G3 medulloblastomagenesis. This regulation occurs via the direct coding-noncoding RNA interaction between lncMB3 and the mRNA for the epigenetic factor HMGN5, with both sharing targets in the TGF-{beta} cascade. This axis converges on apoptosis through OTX2, another G3 MB driver gene, and photoreceptor lineage genes. Synergistic effects between lncMB3 targeting and cisplatin treatment underscores the relevance of this regulatory network in vitro. Finally, we propose novel ferritin-based nanocarriers as efficient delivery tools for antisense oligonucleotides targeting lncMB3. ConclusionsLncMB3 emerges as a central node linking MYC amplification to apoptosis inhibition through a circuit involving RNA-based mechanisms, G3 MB key drivers and underexplored factors. This integrated framework deepens our understanding of G3 MB molecular underpinnings and lay the foundation for translating lncRNA research into potential applications.

High-risk neuroblastoma is characterized by MYCN amplification and high MYCN or MYC gene expression. These patients have a poor prognosis and there is an urgent need for more effective drugs. While strategies to develop inhibitors that directly target the MYC proteins have remained largely unsuccessful, recent preclinical studies have identified ATR, a key protein of the DNA damage response, as a promising alternative therapeutic target. Here we identified a strong RUVBL1 and RUVBL2 signature in transcriptomics data derived from different MYCN-driven mice tumors treated with ATR inhibitors. The RUVBL proteins form a complex with ATPase activity that has broad cellular functions and we demonstrate that pharmacological inhibition of this protein complex results in a strong reduction of MYC signaling, cell cycle arrest, DNA damage and apoptosis. We confirmed the association with MYCN and identified the RUVBL genes as independent prognosticators in human primary neuroblastoma data.

Neuroblastoma is a heterogeneous paediatric cancer arising from developmentally arrested neuronal precursors, where restoring differentiation offers therapeutic promise. ASCL1, a pro-neural transcription factor, is widely expressed in neuroblastoma and can drive either proliferation or differentiation depending on the cellular context. Here, we show that distinct MYCN-amplified neuroblastoma cell lines exhibit differing cell cycle and differentiation responses to ASCL1 overexpression. By comparing genome-wide ASCL1 chromatin binding, transcriptional changes, and protein-protein interactions, we found that ASCL1 binds more extensively to neuronal proteins in a cell line that is more susceptible to ASCL1-driven differentiation, but associates with cell cycle regulators in less responsive cells. We show that CDK2-Cyclin A2 bind ASCL1 in less responsive cells, with CDK-mediated phosphorylation of ASCL1 limiting the ability of ASCL1 to drive differentiation. Our study reveals that context-dependent interactions of ASCL1 with protein partners on the chromatin control its ability to re-engage a differentiation program in neuroblastoma.

H19, a lnc-pri-miRNA that encodes miR-675, is dysregulated in numerous cancers. However, the specific mechanisms underlying H19 processing, particularly miR-675 formation, remain unclear. Our study reveals that H19 is highly expressed and m6A modified in a METTL3-dependent manner in glioblastoma (GBM) and glioma stem cells (GSCs). Silencing METTL3 reduced both H19 and miR-675 levels, whereas overexpressing METTL3 promoted miR-675 processing without affecting H19 levels. Further, miR-675 derived from exogenously expressed H19 was affected considerably more in METTL3 silenced glioma cells compared to H19 levels, suggesting differential requirements in the processing of m6A modified H19 transcript. We demonstrate that H19 interacts with m6A reader proteins, IGF2BP2 and HNRNPA2B1, and silencing either reduced H19 and miR-675 levels. However, a high level of miR-675 seen in METTL3 overexpressing cells is severely affected in HNRNPA2B1-silenced compared to IGF2BP2-silenced glioma cells. Interestingly, IGF2BP2 silencing more significantly affected H19 stability from exogenous H19 construct, while HNRNPA2B1 silencing severely impacted miR-675 processing. Site-directed mutagenesis confirmed the presence of two m6A sites in the first exon of H19, with site #1 facilitating HNRNPA2B1 interaction to promote miR-675 processing. In contrast, the IGF2BP2 interaction is promoted by site #2, resulting in enhanced H19 stability. H19-METTL3-HNRNPA2B1-miR675 axis inhibited Calneuron 1 (CALN1), a known target of miR-675, to promote glioma cell migration. Notably, a low CALN1/high H19 predicted a poor prognosis in GBM patients and was further exacerbated by a high METTL3 or HNRNPA2B1 but not IGF2BP2 transcript levels. Thus, we found that the H19 transcript is highly expressed in GBM and m6A modified, and the m6A reader proteins, IGF2BP2 and HNRNPA2B1, regulate the H19 processing differently to promote glioma cell migration.

Lung cancer, the most lethal malignancy globally, urgently requires effective early detection methods. Current non-invasive approaches based on plasma cell-free DNA (cfDNA) fragmentomics are often constrained by limited sensitivity in early-stage patients due to low tumor DNA fraction. To overcome this, we introduce a novel computational feature--First-Order Transition Probability (FOTP)--to decode nucleotide sequential dependencies within cfDNA fragments. Through systematic analysis of 1,036 participants and low-pass whole-genome sequencing, we demonstrate that the first 10 bp at the 5' end harbor the most discriminative information for cancer detection. An SVM model leveraging FOTP achieved an AUC of 0.942, with 73.9% sensitivity for stage I and 81.8% for stage II lung cancer at 95% specificity, significantly outperforming existing fragmentomic features. Furthermore, the method generalized robustly across independent and multi-cancer validation sets, including HCC, CRC, and HNSCC, and exhibited potential for tissue-of-origin identification. These findings are supported by nucleotide frequency stability and entropy patterns beyond the initial 10 bp, reflecting underlying nuclease cleavage biases and chromatin features. This work establishes FOTP as a biologically interpretable and highly efficient feature for pan-cancer early detection, offering a scalable pathway toward population-wide screening programs.

Neuroblastoma (NB) can be a highly aggressive malignancy in children. However, the precise mechanisms driving NB tumorigenesis remain elusive. This study revealed the critical role of CREB phosphorylation in NB cell proliferation. By employing a CRISPR-Cas9 knockout screen targeting calcium/calmodulin-dependent protein kinase (CaMK) family members, we identified the CaM kinase-like vesicle-associated (CAMKV) protein as a kinase that mediates direct phosphorylation of CREB to promote NB cell proliferation. CAMKV was found to be a transcriptional target of MYCN/MYC in NB cells. CAMKV knockout and knockdown effectively suppressed NB cell proliferation and tumor growth both in vitro and in vivo. Bioinformatic analysis revealed that high CAMKV expression is significantly correlated with poor patient survival. High-risk NB frequently had high CAMKV protein levels by Immunohistochemical staining. Integrated transcriptomic and proteomic analyses of CAMKV knockdown cells unveiled downstream targets involved in CAMKV-regulated phosphorylation and signaling pathways, many of which are linked to neural development and cancer progression. We identified small molecule inhibitors targeting CAMKV and further demonstrated the efficacy of one inhibitor in suppressing NB tumor growth and prolonging the survival of mice bearing xenografted tumors. These findings reveal a critical role for CAMKV kinase signaling in NB growth and identified CAMKV kinase as a potential therapeutic target and prognostic marker for patients with NB.

MYCN activates canonical MYC targets involved in ribosome biogenesis, protein synthesis and represses neuronal differentiation genes to drive oncogenesis in neuroblastoma (NB). How MYCN orchestrates global gene expression remains incompletely understood. Our study finds that MYCN binds promoters to up-regulate canonical MYC targets but binds to both enhancers and promoters to repress differentiation genes. MYCN-binding also increases H3K4me3 and H3K27ac on canonical MYC target promoters and decreases H3K27ac on neuronal differentiation gene enhancers and promoters. WDR5 is needed to facilitate MYCN promoter binding to activate canonical MYC target genes, whereas MYCN recruits G9a to enhancers to repress neuronal differentiation genes. Targeting both MYCNs active and repressive transcriptional activities using both WDR5 and G9a inhibitors synergistically suppresses NB growth. We demonstrate that MYCN cooperates with WDR5 and G9a to orchestrate global gene transcription. The targeting of both these cofactors is a novel therapeutic strategy to indirectly target the oncogenic activity of MYCN.

Erlotinib resistance remains a critical barrier in treating EGFR-mutant non-small cell lung cancer (NSCLC). While distinct resistance mechanisms have been identified, the temporal evolution of transcriptional states and the role of non-coding RNAs in this process remain poorly understood. To address this, we performed a secondary single-cell RNA sequencing (scRNA-seq) analysis of PC9 cells treated with Erlotinib (GEO Accession: GSE149383). We employed pseudotime trajectory inference (Monocle3) and rigorous in silico modeling to map resistance evolution and predict miRNA-lncRNA interactions. Our trajectory analysis revealed a biphasic evolution of resistance: an early phase characterized by ribosomal stress responses (RPS5, RPL21) followed by a late proliferative phase driven by cell cycle regulators (CENPF, HMGB2). Notably, the long non-coding RNA NEAT1 showed dynamic upregulation during this transition. Computational modeling identified miR-124-3p as a high-confidence regulator of NEAT1, with structural analysis confirming a thermodynamically stable interaction ({Delta}G = - 14.8 kcal/mol). These findings suggest that Erlotinib resistance is not a static state but a dynamic process involving sequential transcriptional reprogramming. We propose the miR-124-3p/NEAT1 axis as a potential therapeutic target to disrupt the stress-adaptation phase of drug resistance.

Long non-coding RNAs (lncRNAs) are emerging as key regulators in cancer, influencing gene expression, chromatin remodeling, and signaling. Evidence from The Cancer Genome Atlas (TCGA) and other datasets supports their role in tumor progression. Although the human genome harbors thousands lncRNA genes, only a small subset has been validated in cancer. In this study, we used the LncBook catalog ([~]95,000 lncRNAs) to identify [~]12,500 lncRNAs with expression evidence across major TCGA cancer types. These were stratified by clinical annotations, including cancer stage (I-IV) and metastatic state (M0/M1). Using significant differential expression (z-score >|3|) for consecutive transitions, we identified a set of influential transitional lncRNAs (Tr-lncRNAs) that signify cancer transitions. Analyzing seven transitions revealed that over 70% of Tr-lncRNAs were cancer-type specific, while only 2-4% were shared across 17 major cancers. Each cancer type had 30-80 Tr-lncRNAs, with more than half uniquely expressed in one type. Most Tr-lncRNAs were previously uncharacterized. A pan-cancer analysis revealed 14 shared Tr-lncRNAs, including known ones such as XIST and H19. Our findings highlight distinct lncRNA expression patterns during cancer progression and provide new insights into cis-regulatory antisense mechanisms. We discuss the potential of Tr-lncRNAs as diagnostic biomarkers and therapeutic targets in cancer.

Rhabdomyosarcoma (RMS) is an aggressive pediatric tumor with poor prognosis for metastasis and recurrent disease. Large scale sequencing endeavors demonstrate that RMS tumors have limited mutations and a dearth of driver mutations that are precisely targetable. However, IGF2 signaling is known to be grossly altered in RMS. The IGF2 signalling molecule binds both its innate IGF1 receptor as well as the insulin-receptor-variant-A (IR-A) with high affinity. Mitogenic and proliferative signalling via the canonical IGF2 pathway is therefore augmented by IR-A. The insulin receptor (IR) which is a transmembrane tyrosine-kinase receptor exists in two alternatively spliced isoforms, IR-A and IR-B. In this study, we show that RMS patients express increased IR-A compared to control tissues that express predominantly the IR-B isoform. We also found that Hif1a is significantly increased in RMS tumors, portraying their hypoxic phenotype. Furthermore, the alternative-splicing of IR adapts to produce more IR-A in response to hypoxic stress. Upon examining the pre-mRNA structure of the gene, we identified a hypoxia-responsive-element, which is also the binding site for the RNA-binding protein CUG-BP1. We designed Splice-Switching-Oligonucleotides (SSO) against this binding site to decrease the levels of IR-A in RMS cell-lines and consequently rescue the IR-B expression levels. SSO treatment resulted in significant reductions in proliferation, migration and angiogenesis. Our data show promising insight into how impeding the IGF-2 pathway by reducing IR-A expression mitigates tumor growth. Our data reveal that RMS tumors use IR alternative-splicing as yet another survival strategy which can be exploited as therapeutic intervention in conjunction with already established anti-IGF-1 receptor therapies.