Cancer Gene Therapy

Although mutations in CTNNB1 have long been associated with cancer, their impact in patients with non-small cell lung cancer (NSCLC) is not well understood. Beyond a potential role in the acquired resistance setting of EGFR-mutant NSCLC, little is known about the clinical and molecular characteristics of NSCLC patients harboring these mutations. Here, we identify 302/15,688 (1.9%) NSCLC patients with CTNNB1 mutations. These mutations frequently co-occur with EGFR and KRAS mutations and are associated with a favorable prognosis (mOS, 45.8 months overall; 19.7 months KRASmut). Patients benefit significantly from immune-checkpoint-blockade, but non-intuitively,PD-L1 TPS [≥]50%(4/6 treated with monotherapy) survive the shortest. We show that patients with proposed CTNNB1 resistance mutations to EGFR-directed therapy have significantly shorter mOS when treated with targeted therapy, compared to non-resistant CTNNB1 mutations. This study highlights unique clinicogenomic features and the nuanced impact of CTNNB1 mutations on therapeutic outcomes.

The role of red blood cells (RBCs) in tumorigenesis is poorly understood. We previously identified RBC-microRNAs with aberrations linked to lung cancer, including miR-93-5p. Here we find that miR-93-5p levels are elevated in RBC-derived exosomes among lung cancer patients and are associated with their shorter survivals. RBC-derived miR-93-5p transfers to cancer cells primarily through the exosomal pathway. The transferred RBC-miR-93-5p can target PTEN in cancer cells, and hence increase cell proliferation, invasion, and migration. RBC-derived miR-93-5p accelerates, whereas targeting miR-93-5p diminishes tumor growth in xenograft models. These findings reveal a novel biological function of RBCs in tumorigenesis, where they facilitate cancer progression by transferring the oncomiR via exosomes, thereby offering new diagnostic and treatment strategies for lung cancer.

Our laboratory recently identified a novel long noncoding RNA termed CyKILR that has two splice variants with distinct cellular localizations and opposing roles in tumorigenesis. The cytoplasmic variant, CyKILRb (exon 3 exclusion), promotes tumorigenesis, whereas the nuclear variant, CyKILRa (exon 3 inclusion), functions as a tumor suppressor. In this study, the molecular mechanism of the tumorigenic role of CyKILRb was characterized. Specifically, deep RNA sequencing analysis revealed that CyKILRb regulated the PI3K/AKT signaling pathway to block downstream tumor suppressors. In particular, downregulation of CyKILRb induced the loss of PIK3R2, an activator of PI3K, as well as RPS6KB2 and GNB2, two implicated tumor promotors, with a concomitant increase in the tumor suppressors, CDKN1A (p21) and CDKN1B (p27). In contrast, CyKILRb ectopic expression produced the opposite effect, and suppression of either PIK3R2, PI3K or AKT attenuated CyKILRb-induced cell proliferation and clonogenic survival. CyKILRb negatively regulated CyKILRa expression, which was blocked by inhibition of either PI3K or AKT. PIK3R2 ectopic expression overcame the cellular effects of CyKILRb downregulation, but not PI3K or AKT inhibition orienting the signaling pathway from CyKILRb[->]{uparrow}PIK3R2[->]PI3K[->]AKT[->]{downarrow}CyKILRa[->]enhanced oncogenicity. These findings highlight the critical role of CyKILRb in tumorigenesis and define a novel feed-forward regulatory mechanism linked to alternative RNA splicing.

Breast, lung, and colorectal cancer resistance to molecular targeted therapy is a major challenge and unfavorably impacts clinical outcomes, leading to hundreds of thousands of deaths yearly. In ERBB2+ cancers regardless of the tissue of origin, ERBB2 is the driver oncogene of resistance. We discovered that the ERBB2+ cancers are enriched with poly U sequences on their 3UTR AU rich elements which are mRNA stabilizing sequences. We developed a novel technology, in which we engineered these ERBB2 mRNA stabilizing sequences to unstable forms and specifically controlled and degraded ERBB2 transcript and protein across multiple cancer types both in the wildtype and drug resistance settings in vitro and in vivo, offering a unique novel modality to control ERBB2 and other pervasive oncogenic signals where other therapies fail. One-Sentence SummaryEngineered destabilized 3UTR ARE of ERBB2 degrades ERBB2 in many cancer types and controlled resistance. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=163 SRC="FIGDIR/small/503914v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@139d8dborg.highwire.dtl.DTLVardef@cc1fd3org.highwire.dtl.DTLVardef@13d9086org.highwire.dtl.DTLVardef@db69bf_HPS_FORMAT_FIGEXP M_FIG A. Depiction represents multiple ERBB2 expressing cancer cells with stable 3UTR ARE and the signaling cascade known to cause chemo resistance. B. Depiction of the engineered destabilized 3UTR ARE of ERBB2 and the destabilization and degradation of the ERBB2 transcript, protein and kinases involved in mediation of drug resistance C_FIG

BackgroundButyrophilins (BTNs) are immunoglobulin superfamily proteins involved in immune regulation. Among them, BTNL9 has unique structural features, including a bZIP-like domain, suggesting a potential transcriptional role. While BTNL9 is known to suppress T cell activation, its function in cancer remains largely unexplored. Recent studies suggest it may inhibit tumor progression and correlate with improved prognosis in multiple cancers. However, its molecular mechanisms and regulatory impact on lung cancer remain unclear. This study investigates the role of BTNL9 as a transcription factor and its implications for tumor progression and therapy. MethodsChIP-seq identified BTNL9-binding sites, followed by RNA-seq to assess transcriptomic profiles and validated by western blot. Drug sensitivity was evaluated through cytotoxicity assays. A xenograft model was applied to assess the effect of BTNL9 on tumor growth. TCGA data analysis examined correlations with survival, cell cycle regulators, and immune infiltration. ResultsChIP-seq identified 26,610 BTNL9 binding peaks, mapping to 9,707 genes near transcription start sites. RNA-seq and western blotting showed BTNL9 regulates cell cycle (E2F1, CDKN1A, CDK1, CDC25C, FOXM1), DNA replication (MCM2/3/7, ORC6), and p53-related transcription (BBC3, GADD45A). Integrative analysis found that 74.8% of differentially expressed genes were directly regulated by BTNL9. Functionally, BTNL9 overexpression induced cell cycle arrest, reduced proliferation, and suppressed tumor growth in vivo. BTNL9 enhanced bortezomib sensitivity in both A549 and NCI-H460 cells, with etoposide effects being more pronounced in A549. Higher BTNL9 levels strongly suppressed the expression of FOXM1, CDC25C, CDK1, CDK2, CCNA2 and CCNB1 and negatively correlated with these markers in LUAD TCGA data. Elevated BTNL9 expression was associated with improved survival, complete remission, and increased immune infiltration, including macrophages, CD8+ T cells, NK cells, and B cells in cancer tissues. ConclusionsBTNL9 functions as a transcription factor, suppresses tumor growth, and enhances drug sensitivity. Its correlation with survival and immune infiltration suggests potential role as a tumor suppressor and predictive biomarker for chemotherapy response.

TMEM147, an ER membrane protein, is linked to lung adenocarcinoma (LUAD) but its role remains unclear. This study combines bioinformatics and experiments to explore TMEM147s function in LUAD progression. TMEM147 expression was analyzed using TCGA/GEO data and validated in LUAD cells. Survival analysis assessed its prognostic value. GO/KEGG and ssGSEA revealed functional pathways and immune microenvironment interactions. Transcription factor binding predictions and in vitro assays (migration, invasion, proliferation) evaluated TMEM147s role. TMEM147 was upregulated in LUAD and correlated with poor outcomes. FLI1 was predicted as a transcriptional regulator. TMEM147 influenced immune cell infiltration and was associated with ribonucleoprotein biogenesis and oxidative phosphorylation (OXPHOS) . Silencing TMEM147 reduced cancer cell migration, invasion, and proliferation, suggesting its potential as a biomarker and therapeutic target.

Non-small cell lung cancer (NSCLC) accounts for 80-85% cases of lung cancer cases. Diagnosis at advanced stages is common, after which therapy-refractory disease progression frequently occurs. Therefore, a better understanding of the molecular mechanisms that control NSCLC progression is necessary to develop new therapies. Overexpression of I{kappa}B kinase (IKK) in NSCLC correlates with poor patient survival. IKK is an NF-{kappa}B-activating kinase that is important in cell survival and differentiation, but its regulation of oncogenic signaling is not well understood. We recently demonstrated that IKK promotes NSCLC cell migration by physically interacting with dopamine- and cyclic AMP-regulated phosphoprotein, Mr 32000 (DARPP-32), and its truncated splice variant, t-DARPP. Here, we show that IKK phosphorylates DARPP-32 at threonine 34, resulting in DARPP-32-mediated inhibition of protein phosphatase 1 (PP1), subsequent PP1-mediated dephosphorylation of ERK, and activation of ERK signaling to promote lung oncogenesis. Correspondingly, DARPP-32 ablation in human lung adenocarcinoma cells reduced their anchorage-independent growth in soft agar. Mice challenged with IKK-ablated HCC827 cells exhibited less lung tumor growth than mice orthotopically administered control HCC827 cells. Our findings suggest that IKK drives NSCLC growth through activation of ERK signaling via DARPP-32-mediated inhibition of PP1 activity.

Tyrosine Kinase inhibitors (TKIs) are widely used as effective chemotherapeutic agents for treating patients with EGFR-mutated NSCLC. Unfortunately, after treatment, patients eventually develop resistance to TKI therapy. The most common resistance mechanism for the TKI Osimertinib is the overexpression of the MET Proto-Oncogene, Receptor Tyrosine Kinase (MET). We previously demonstrated that miR-411-5p A-to-I edited at position 5 (miR-411ed) can directly target MET in A549 and H1299 cells. MiR-411ed in combination with Osimertinib reduced cell proliferation in two TKI resistant EGFR-mutated cell lines: HCC827R and PC9R. MiR-411ed did not downregulate MET expression in HCC827R, suggesting an alternative mechanism for TKI response. In this study, we aim to identify the mechanism of miR-411ed TKI response using a multi-omics approach of RNAseq and protein mass spectrometry. In our cellular model, we identified miR-411ed affected genes independent of MET activity, resulting in 211 genes (RNAseq) and 36 proteins (proteomics). Pathway analysis identified an increase in interferon signaling for RNAseq and combined omics, and a decrease in ERK/MAPK signaling in proteomics. Using the IsoTar target prediction tool, we identified STAT3 as a key regulator and confirmed STAT3 protein downregulation upon transfection with miR-411ed. We further investigated the effect of miR-411ed in vivo, observing a reduction in tumor size with miR-411ed in combination with Osimertinib but not with miR-411ed or Osimertinib treatment alone, confirming the effectiveness of miR-411ed in TKI response.

Lung cancer remains the leading cause of cancer-related deaths worldwide, with tumor recurrence a major contributor to its high mortality. The genetic and molecular mechanisms of recurrence remain poorly understood. Using whole-exome sequencing of 155 primary non-small cell lung cancers, we studied the mutational landscape and driver alterations associated with recurrence. Primary tumors that developed recurrence had higher mutational burden, including hypermutated tumors explained by mutations in DNA polymerase or mismatch repair pathways. Mutational signatures of reactive oxygen species were associated with recurrence. Combined mutations in TP53 and CDKN2A were enriched in non-recurrent tumors, while ATRNL1 mutations were enriched in recurrent tumors. Pathway analyses implicated DNA repair and cilium organisation processes with tumor recurrence and highlighted 50 additional candidate genes including BRCA2. Recurrence-associated genes showed essentiality in lung cancer cell lines and included known therapeutic markers, indicating their functional and translational relevance. This analysis provides insights into the molecular basis of lung cancer recurrence and informs experiments to develop diagnostic and therapeutic strategies.

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.

Epidermal growth factor receptor (EGFR) signaling is frequently dysregulated in various cancers. The ubiquitin ligase Cbl (Casitas B-lineage lymphoma proto-oncogene) regulates degradation of activated EGFR through ubiquitination and acts as an adaptor to recruit proteins required for trafficking. We used Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) mass spectrometry (MS) to compare Cbl complexes with or without epidermal growth factor (EGF) stimulation. We identified over a hundred novel Cbl interactors, and a secondary siRNA screen found that knockdown of Flotillin-2 (FLOT2) led to increased phosphorylation and degradation of EGFR upon EGF stimulation in HeLa cells. In PC9 and H441 cells, FLOT2 knockdown increased EGF-stimulated EGFR phosphorylation, ubiquitination, and downstream signaling, reversible by the EGFR inhibitor erlotinib. CRISPR knockout (KO) of FLOT2 in HeLa cells confirmed EGFR downregulation, increased signaling, and increased dimerization and trafficking to the early endosome. FLOT2 interacted with both Cbl and EGFR. EGFR downregulation upon FLOT2 loss was Cbl-dependent, as co-knockdown of Cbl and Cbl-b restored EGFR levels. Overexpression of FLOT2 decreased EGFR sjgnaling and growth. Overexpression of wild type (WT) FLOT2, but not the soluble G2A FLOT2 mutant, inhibited EGFR phosphorylation upon EGF stimulation in HEK293T cells. FLOT2 loss induced EGFR-dependent proliferation and anchorage-independent growth. Lastly, FLOT2 KO increased tumor formation and tumor volume in nude mice and NSG mice, respectively. These data demonstrated that FLOT2 negatively regulated EGFR activation and dimerization, as well as its subsequent ubiquitination, endosomal trafficking, and degradation, leading to reduced proliferation in vitro and in vivo.

Clinical responses to EGFR tyrosine kinase inhibitors are restricted only to tumors harboring specific activating mutations and even then, not all tyrosine kinase inhibitors provide clinical benefit. We here show that the addition of EGFR-TKIs results in a strong and rapid intracellular accumulation of the protein. However, this accumulation was observed only in the context of a combination of a TKI-sensitive mutation with a clinically effective TKI: TKI-insensitive mutations did not show this accumulation nor did clinically ineffective TKIs induce accumulation. All TKIs effectively inhibited EGFR phosphorylation and downstream pathway activation, irrespective of the mutation present in EGFR. The discrepancy between molecular activity of TKIs and their efficacy in patients therefore is mimicked by the mutation- and TKI-specificity of intracellular accumulation. Using this intracellular accumulation as assay, we were able to predict response to gefitinib in a panel of cell-lines (harboring different EGFR mutations) and predicted clinical benefit to EGFR TKIs on a cohort of unselected pulmonary adenocarcinoma patients (hazard ratio 0.21, P=0.0004). Even in patients harboring rare mutations with unknown TKI-sensitivity, intracellular accumulation was predictive of the clinical response. The intracellular accumulation depended on a continued presence of TKI indicating that TKIs exert a continued effect on the protein even after its dephosphorylation. It is therefore possible that accumulation is caused by conformational changes induced by both the mutation and the TKI and this change induces a block in intracellular trafficking. Interestingly, intracellular accumulation was observed independent of the genetic background of the cell, indicating that accumulation is almost entirely dictated by the combination of mutation and TKI. Our results therefore suggest that TKI-sensitivity is tumor-type independent.

We identified a germline TP53 c.758C>T (p.T253I) mutation in the TP53 tumor suppressor gene in a pediatric adrenocortical carcinoma (ACC) patient. Characteristic to pathogenic p53 mutations, we observed upregulation of total p53 protein levels in the patients ACC and concurrent suppression of the wild-type (WT) TP53 allele. As ACC can be associated with Li-Fraumeni Syndrome (LFS) and the mutation has not yet been linked to LFS, we sought to characterize the functionality of the T253I mutation. We acquired p53-/- HEK293 cells and stably transduced them with GFP-tagged wild type (T253) or T253I p53 as well as two established pathogenic p53 mutants (C176Y and R213X). Compared to p53 WT, levels of T253I p53 increased while MDM2 levels decreased, suggesting a loss of MDM2-mediated regulation of T253I p53. Additionally, T253I showed a reduction in DNA damage responsive events, diminished DNA binding capabilities, and blunted transactivation capacity. These experimental data lead us to conclude that T253I represents a pathologic variant in TP53 that may predispose to LFS-associated tumors.

1.Non-small cell lung cancer (NSCLC) remains a leading cause of cancer-related mortality, driven by tumor heterogeneity, metastasis, and therapeutic resistance. While Rho GTPases are well-established regulators of oncogenic processes, the role of the atypical GTPases in NSCLC remains unexplored. Here, we identified RHOV as one of the commonly upregulated Rho GTPases in NSCLC. Analysis of four independent patient cohorts revealed that elevated RHOV expression serves as a robust and independent prognosticator of NSCLC patients specifically early-stage disease. Functionally, RHOV knockdown significantly inhibited cell proliferation, whereas its overexpression enhanced proliferation. Similarly, RHOV depletion suppressed cell migration by disrupting cytoskeletal dynamics, while its overexpression promoted migratory capacity. Mechanistically, we demonstrated that RHOV is a direct transcriptional target of the TGF{beta}-SMAD3 signaling pathway. RNA-seq analysis identified MYC as a critical downstream mediator of RHOV; RHOV knockdown reduced MYC expression, impairing mitochondrial oxidative phosphorylation and inducing ROS-mediated DNA damage--a phenotype rescued by MYC overexpression. Furthermore, RHOV inhibition sensitized NSCLC cells to etoposide but not doxorubicin. immunoprecipitation coupled with LC-MS revealed PEAK1 as a key interactor of RHOV. The RHOV-PEAK1 complex proved essential for NSCLC proliferation, as PEAK1 silencing abolished RHOV- driven MYC upregulation and tumor growth. This axis sustains MYC levels and activates PI3K/MAPK signaling. Intriguingly, PEAK1 depletion elevated TGF-{beta} levels, which suppressed RHOV expression, establishing a negative feedback loop wherein PEAK1 maintains RHOV by inhibiting TGF-{beta} signaling. Collectively, our findings establish RHOV as a prognostic biomarker and a driver of NSCLC progression via the RHOV-PEAK1-MYC axis, highlighting its potential as a therapeutic target. HighlightsO_LIRHOV upregulation predicts poor NSCLC survival, particularly in early-stage disease. C_LIO_LIThe RHOV-PEAK1 interaction is crucial for NSCLC growth and cell migration. C_LIO_LIRHOV inhibition sensitizes NSCLC cells to Etoposide treatment. C_LIO_LIRHOV expression is sustained via a PEAK1-TGF{beta} negative feedback loop. C_LI

Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy with high mortality rates, often exhibiting resistance to conventional treatments such as radiotherapy (RT) or a combination of chemotherapy and radiotherapy (CRT). The nerve growth factor receptor (NGFR, also known as p75NTR or CD271) is a well-established cancer stem cell marker in melanoma, where it has been linked to resistance to multiple therapies. In HNSCC, NGFR has been reported as a poor prognostic marker, with its overexpression associated with disease progression. However, its contribution to therapy resistance in HNSCC remains unknown. Here, we show in a cohort of RT/CRT-treated patients that NGFR expression identifies individuals with poor prognosis and increased risk of recurrence following standard RT/CRT. Moreover, we found that NGFR is upregulated in the human Detroit 562 cisplatin (CDDP)-resistant HNSCC cell line in vitro and in vivo. Functional studies demonstrated that genetic knock out of NGFR in these cisplatin-resistant cells restored sensitivity to CDDP in vivo. These results indicate that NGFR contributes to cisplatin resistance in HNSCC. NGFR is upregulated in tumors from patients with poorer prognosis and an increased risk of recurrence after standard radiotherapy and/or RT/CRT, as well as in cisplatin-resistant models. Altogether, our findings open the way to consider NGFR as a new potential therapeutic target to overcome or mitigate cisplatin resistance in HNSCC.

ObjectivePFKP (Phosphofructokinase, Platelet Type isoform), as an essential metabolic enzyme, contributes to the high glycolysis rates seen in cancers, while its role in oncogenic pathways, especially from a non-metabolic aspect, is not fully understood. MethodsHere we performed a comprehensive analysis of published RNA-seq, microarray data, and immunohistochemistry of tissue microarray to evaluate the significance of PFKP expression in non-small cell lung cancer (NSCLC). Functionally, we tested the cell proliferation, colony formation, invasion, and migration upon PFKP knockdown in lung cancer cells. Mechanistically, we performed RNA-seq, DIA-mass spectrum, western blot, and qPCR to probe the change of cell signaling pathways upon PFKP silencing. Co-immunoprecipitation and mass spectrum were used to uncover potential PFKP interacting proteins. ResultsWe found that PFKP was highly expressed in NSCLC and was related to poor patient survival. Knockdown of PFKP significantly inhibited cell proliferation, colony formation, invasion, and migration of NSCLC cells. Mechanistically, we found that PFKP can directly bind with AXL and promote its phosphorylation at Y779, thus activating the AXL signaling pathway and promoting MET phosphorylation. In addition, several glycolysis, glutaminolysis, and TCA cycle proteins were downregulated following PFKP silencing. ConclusionsThese data demonstrate that PFKP, beyond its known role in glycolysis, also has a distinct non-metabolic function in affecting lung cancer progression by directly interacting with the AXL-MET axis, thus indicating a potential therapeutic target for lung cancer.

Lung adenocarcinomas frequently harbour actionable oncogenic mutations that are vulnerable to treatment with targeted therapies. While responses to targeted therapies are often initially dramatic, relapse is almost inevitable and prevents durable responses in advanced-stage patients. Relapse is, in part, caused by drug tolerant persister cells (DTPs) which are able to survive treatment by entering a reversible, dormant state. Although long non-coding RNAs (lncRNAs) regulate processes thought to allow DTPs to survive and become stably resistant, the potential roles of lncRNAs in DTPs are largely unknown. In this study, we sought to investigate the expression of lncRNAs in in vitro DTP models of lung adenocarcinoma. We found that the lncRNAs Metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1) and Nuclear Paraspeckle Assembly Transcript 1 (NEAT1) were enriched in DTPs and that knocking down MALAT1 enhanced the effect of targeted therapies in both EGFR- and KRAS-mutant DTP models. To better understand pathways that MALAT1 might regulate in DTPs, bulk RNA-sequencing was performed and several pathways that may contribute to the actions of MALAT1 in DTPs were identified. Overall, our work describes a role for the lncRNA MALAT1 in DTPs in NSCLC and suggests that MALAT1 may be a novel target for the prevention of drug tolerance and subsequent resistance to targeted therapy in NSCLC.

BackgroundLung cancer remains the deadliest malignancy. Although PD-1/PD-L1 immune checkpoint inhibitors have improved survival, their benefit is limited by persistent immune resistance. Identifying systemic regulators of tumor immune microenvironment may enhance therapeutic efficacy. We previously showed that RBC-derived miR-93-5p promotes PTEN loss and PI3K-AKT activation. This study examined whether RBC-derived miR-93-5p is associated with immune checkpoint activity and features of T-cell exhaustion in lung cancer. MethodsRBCs, RBC-derived exosomes, and plasma were isolated from 80 lung cancer patients and 30 controls, and tumor and matched noncancerous lung tissues were collected. miR-93-5p expression was quantified by droplet digital PCR. PD-1, PD-L1, CD8+ T-cell infiltration, CD69, IFN-{gamma}, and TNF- were evaluated by immunohistochemistry. Associations with clinicopathologic features and survival were statistically analyzed. ResultsmiR-93-5p levels in RBCs, their exosomes, and tumor tissues were significantly elevated in lung cancer. Higher miR-93-5p was associated with increased PD-1 and PD-L1 expression and reduced CD69, IFN-{gamma}, and TNF-, consistent with T-cell exhaustion. Elevated miR-93-5p correlated with advanced disease and reduced survival. ConclusionsRBC-derived miR-93-5p is associated with immune checkpoint activation, T-cell exhaustion, and poor clinical outcomes, suggesting impaired antitumor immunity. Targeting this axis may improve the efficacy of immunotherapy in lung cancer.

The central nervous system (CNS) is a common site of metastatic spread for both non-small cell and small cell lung cancer, yet the therapeutic strategies to prevent and decrease lung cancer brain metastases remain limited. Tyrosine kinase inhibitors have shown promising results in increasing the overall response in brain metastases, owing to their brain penetrance and increased effectiveness; however, their use is limited to the small group of tumors carrying specific oncogenic drivers. Among these, inhibitors with activity against neurotrophic tyrosine receptor kinases (NTRKs) are showing promising effects in reducing CNS metastases in cancers driven by gene rearrangements of these drugs targets. However, wild-type NTRKs are susceptible to activation by their canonical ligands, which are expressed throughout the brain metastatic niche and can, in a paracrine manner, activate NTRK function in cancer cells. Here we show that NTRKs are expressed in primary tumors, brain metastases, and lung cancer cells with various driver mutations expressing wild-type NTRK2 (WT-TrkB). We demonstrate that WT-TrkB activates downstream signaling and proliferation in response to exogenous BDNF and conditioned media from reactive astrocytes known to secrete BDNF in the brain niche. Importantly, the FDA-approved NTRK inhibitor entrectinib blocked BDNF and astrocyte-induced survival pathways in multiple lung cancer cell lines, decreased their proliferation in vitro, and effectively prevented brain metastatic colonization and progression in vivo without significant effects on extracranial disease. Thus, these studies suggest that brain-dependent activation of NTRK is critical for brain metastases of WT-NTRK+ lung cancers, and therefore, NTRK inhibitors can be used to target non-fusion NTRK function to prevent or decrease brain metastases. SIGNIFICANCEThese studies demonstrate that NTRK wild-type receptors are important drivers of brain metastatic colonization and progression in different subtypes of lung cancer, independent of their driver alterations. Thus, they provide rationale to expand the use of FDA-approved NTRK inhibitors with brain penetrance for the prevention of CNS metastases.

Breast cancer is a heterogeneous disease that can be molecularly classified based on the expression of hormone receptors and the overexpression of the HER2 receptor (ERBB2). Targeted therapies for HER2-positive breast cancer, including trastuzumab, antibody drug conjugates (ADCs) and tyrosine kinase inhibitors, have significantly improved patient outcomes. However, both primary and acquired resistance to these treatments pose challenges that can limit their long-term efficacy. Addressing these obstacles is vital for enhancing therapeutic strategies and patient care. Alternative splicing, a post-transcriptional mechanism that enhances transcript diversity (isoforms) within a cell, can result in isoform-encoded proteins with varied functions, cellular localizations, or binding properties. In this study, we undertook a comprehensive characterization of the alternative splicing isoforms of HER2, assessed their expression levels in primary breast tumors and cell lines, and explored their role in resistance to anti-HER2 therapies. Our results have significantly expanded the catalog of known HER2 protein-coding isoforms from 13 to 90, revealing distinct patterns of protein domains, cellular localization, and protein structures, as well as mapping their antibody-binding sites. Additionally, by profiling expression in 561 primary breast cancer samples and analyzing mass spectrometry data for translation evidence, we discovered a complex landscape of splicing isoform expression in primary tumors, revealing novel isoforms that were previously unrecognized and are not evaluated in routine clinical practice. This extends beyond the traditional profile based solely on HER2 gene expression and translation. Finally, by assessing HER2 isoform expression in cell cultures that are either sensitive or resistant to trastuzumab and ADCs (T-DM1 or T-DXd), we found that drug-resistant tumor cells shifted their expression toward splicing isoforms that lack the antibody-binding domains. Our results substantially broaden the understanding of HER2 protein-coding isoforms, revealing distinct mechanisms of potential resistance to anti-HER2 therapies, particularly ADCs, by uncovering a new dimension of splicing isoform diversity. This expanded landscape of HER2 isoforms, marked by unique domain patterns and altered antibody-binding sites, emphasizes the crucial role of alternative splicing investigations in advancing precision-targeted cancer therapies.