Resistance to therapy remains a critical challenge in cancer. This is exemplified by TGF-β pathway inhibitors which, despite broad clinical testing, have failed to deliver survival benefit. More precise SMAD3 targeting has shown preclinical promise, yet resistance mechanisms to SMAD3 inhibition remain entirely unknown. Using complementary genome-wide CRISPR activation and knockout screens, we discover that cancer cells escape SMAD3 inhibition through metabolic reprogramming. Strikingly, this escape is driven not by transcriptional bypass but by the lactate transporter MCT1, with the effect preserved across BRAF-mutated melanoma, KRAS-mutated lung adenocarcinoma, and mouse melanoma. Mechanistically, SMAD3 inhibition creates energetic stress that cancer cells exploit through MCT1. By conferring metabolic flexibility and switching toward glycolysis under drug pressure, MCT1 increases anabolic activity to drive lipid and cholesterol synthesis. Pharmacological and genetic MCT1 inhibition synergizes strongly with SMAD3 blockade to impair tumor viability in BRAF-mutated melanoma, while suppressing drug-tolerant cell emergence in BRAF inhibitor-resistant cells. Critically, this synergy extends to preclinical models, where co-inhibition of SMAD3 and MCT1 potently reduces tumor growth in vivo. Given the additional and well-established role of MCT1 in shaping the tumor immune microenvironment via lactate transport, these results position MCT1 as both a metabolic and immunological target and provide a strong rationale for the repurposing of TGF-β/SMAD3 pathway inhibitors in combination.

Abstract Small cell lung cancer (SCLC) is the most aggressive subtype of lung cancer. The recent combination of chemotherapy with immune checkpoint inhibitors shows a response in only 10 to 15% of patients. At the same time, numerous studies highlight the impact of gut microbiota composition on the response to immunotherapy. To address these current challenges, we are developing a xenograft model called "MESHCAP." This model involves grafting circulating tumor cells (CTCs) from SCLC patients onto mice that have been humanized for both their immune system and their gut microbiota. With its unique dual humanization, this model will allow us to take into account the influence of the microbiota when testing new therapeutic strategies. Based on the literature, we have identified a bacterial consortium hypothesized to be beneficial for the response to immunotherapy. This consortium is cultured in the laboratory and then inoculated into immunodeficient mice whose endogenous microbiota has been previously depleted by antibiotic treatment. We are able to detect the presence of these bacteria in the feces by 16S sequencing more than a month after inoculation. In parallel, we have humanized the immune system of immunodeficient mice by injecting human CD34+ hematopoietic stem cells. This engraftment results in chimerism of the mouse immune system. Currently, we are developing the generation of doubly humanized mice for both the microbiota and the immune system. Preliminary data suggest a cross-talk between the humanized microbiota and the human immune system. In our laboratory, we routinely isolate CTCs from patients diagnosed with CTC. We then inject them subcutaneously to generate CDXs (CTC-derived xenografts). It is these CDXs that we will implant into our doubly humanized mouse model to investigate the impact of the microbiota and immune system on therapies (e.g. immunotherapy). Citation Format: Pierre Montagne, Ulrich Jarry, Mathilde Harel, Laetitia Martinetti, Anna Le Mée, Charles Ricordel, Ahmad FAILI, Samer Kayal, Rémy Pedeux, Marwan Touati, Valentin Quiniou, Hang-Phuong Pham. MESHCAP (exogenous microbiota and humanized mice for lung cancer): Towards an innovative preclinical model for testing immunotherapies [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 3386.

Antisense oligonucleotides (ASOs) belong to promising therapeutics for the treatment of neurological, muscular, and metabolic disorders. Several ASOs have been approved so far and more than 100 clinical trials are currently underway covering a dozen therapeutic areas. Yet, the mechanisms of internalization and cell trafficking of these molecules remain poorly understood. Moreover, with only a small fraction of ASOs reaching the correct cellular compartment after systemic delivery, the majority of targeted diseases require recurrent injections of ASOs. A deeper understanding of these mechanisms would guide the improvement of their potency and, thus, reduce the amount of delivered ASOs and their potential side effects. Here, using a CRISPR screen, we investigated intracellular proteins involved in ASOs efficiency using a whole genome approach and identified several potential regulators that could significantly impact ASOs potency in melanoma cells. We validated WD repeat domain 91, a regulator of endosomal maturation, as a modulator whose depletion significantly inhibits ASO productive activity. This study provides a list of ASO modulators using a biologically relevant assay to estimate the role of these proteins. In conclusion, these data could lead to a better understanding of the mechanisms favoring productive uptake or improved endosomal escape of ASOs.

While completion of the initial phase of the human genome project redefined genetics over two decades ago, the field of epigenetics is currently redefining how chromatin structure affects the gene expression to mold the proteome and the cell phenotype. Posttranslational modification (PTM) of nucleic acids and proteins is central to chromatin organization and helps govern transcription and translation to affect specific phenotypes. Zinc fingers are a common constituent of chromatin regulators and the plant homeodomain (PHD) forms of zinc finger function as readers of the histone code. Understanding the structural mechanisms driving the recognition of PTMs by PHDs is at the core of efforts to understand how chromatin architecture is maintained and modulated. Here, we describe the roles of PHD finger proteins in chromatin remodeling through their specific binding to PTMs on histone proteins with an emphasis on the Inhibitor of Growth (ING) family. Once bound to methylated chromatin, ING1-5 family members serve as scaffolds for the docking of complexes of effector proteins that are vital to gene-specific activation and/or inactivation events that occur within chromatin. All members of the ING family recognize different methylated forms of lysine 4 near the amino terminus of core histone 3 (H3K4me1/2/3), but different members target either histone acetyltransferase (HAT) or histone deacetylase (HDAC) complexes to regulate histone acetylation and subsequently chromatin structure and gene expression. ING-mediated changes in gene expression have now been shown to profoundly affect a broad range of biological processes including development, differentiation, cellular senescence, and oncogenesis.

Drug nanocapsules coated with iron oxide nanoparticles (SPION) were elaborated by the simultaneous nanoprecipitation of the drug and the nanoparticles, through solvent shifting. We examined four drugs: sorafenib, sorafenib tosylate, alpha-tocopherol and paclitaxel, to cover the cases of molecular solids, ionic solids, and molecular liquids. We first investigated the formation of the drug core in the final mixture of solvents at different concentrations. A Surfactant-Free Micro-Emulsion domain (SFME, thermodynamically stable) was observed at low drug concentration and an Ouzo domain (metastable) at high drug concentration, except for the case of paclitaxel which crystallizes at high concentration without forming an Ouzo domain. When co-nanoprecipitated with the molecular drugs in the Ouzo domain (sorafenib or alpha-tocopherol), the SPION limited the coalescence of the drug particles to less than 100 nm, forming capsules with a drug encapsulation efficiency of ca 80 %. In contrast, larger capsules were formed from the SFME or when using the ionic form (sorafenib tosylate). Finally, the sorafenib-SPION capsules exhibit a similar chemotherapeutic effect as the free drug on the hepatocellular carcinoma in vitro.

Abstract Small cell lung cancer (SCLC) represents the most aggressive subtype of lung cancer, comprising 15% of all cases. Clinical advancements in SCLC have been hindered by slow progress, attributed to significant tumor heterogeneity and early metastasis. The majority of patients experience relapse within a few months post-treatment, with 20% exhibiting chemorefractory characteristics. Recent identification of molecular subtypes in SCLC, dependent on predominant transcription factors ASCL1, NEUROD1, and POU2F3, has opened avenues for potential therapeutic targets. Preliminary studies on these transcriptomic subtypes suggest distinct therapeutic susceptibilities, and intriguingly, chemotherapy may induce alterations in transcriptomic subtypes.In the context of SCLC, diagnosis reveals a substantial presence of circulating tumor cells (CTCs) in the patient's bloodstream, which can be efficiently isolated. A previously published method allowed us to isolate CD56+ CTCs with high specificity (Ricordel et al., 2023). Our overarching hypothesis posits a connection between chemosensitivity and the heterogeneity of CTCs, leading us to seek bioclinical markers predictive of chemotherapy response. The clinical study, named CTC-CPC, is a monocentric prospective non-interventional investigation involving treatment-naïve SCLC patients. Blood samples were collected at the time of diagnosis for subsequent analysis. In our study, whole exome sequencing data from isolated CD56+ CTCs of 22 SCLC patients were analyzed. Various methods were employed to characterize inter-tumoral heterogeneity, including the identification of genetic alterations (SSMs and Indels), mutational signatures, and disrupted pathways. Prominently affected pathways included ECM organization, axon guidance, Rho GTPases, and Wnt signaling. Additionally, we unveiled substantial intra-tumoral heterogeneity using PhyloWGS, a technique for reconstructing subclonal composition and evolution from tumor sample sequencing. To further explore the impact of treatment, we aimed to clarify the expression of ASCL1, NEUROD1, and POU2F3 transcription markers in xenografts generated from patient CTCs at the time of diagnosis. Immunohistochemical studies were performed to identify potential changes in molecular subtypes at the protein level. The molecular subtypes of the four CDX we generated were characterized, revealing heterogeneity in the intratumoral expression of transcription markers for some xenografts. Subsequently, CTC-derived xenografts were subjected to chemotherapy, with observations indicating a change in the number of cells positive for ASCL1 and NEUROD1 after treatment, particularly in the CEM51.01 line treated with carboplatin, suggesting a potential modification in the tumor subtype induced by the treatment Citation Format: Laëtitia Martinetti, Pierre Montagne, Marlène Davilma, Ulrich Jarry, Aristotelis Chatziioannou, Marc Aubry, Charles Ricordel, Rémy Pedeux. Heterogeneity of CD56+ circulating tumor cells and derived xenografts and response to treatment in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2822.

The catalytically inactive caspase-8-homologous protein, c-FLIP, is a potent antiapoptotic protein highly expressed in various types of cancers. c-FLIP competes with caspase-8 for binding to the adaptor protein FADD (Fas-Associated Death Domain) following death receptors' (DRs) activation via the ligands of the TNF-R family. As a consequence, the extrinsic apoptotic signaling pathway involving DRs is inhibited. The inhibition of c-FLIP activity in tumor cells might enhance DR-mediated apoptosis and overcome immune and anticancer drug resistance. Based on an in silico approach, the aim of this work was to identify new small inhibitory molecules able to bind selectively to c-FLIP and block its anti-apoptotic activity. Using a homology 3D model of c-FLIP, an in silico screening of 1880 compounds from the NCI database (National Cancer Institute) was performed. Nine molecules were selected for in vitro assays, based on their binding affinity to c-FLIP and their high selectivity compared to caspase-8. These molecules selectively bind to the Death Effector Domain 2 (DED2) of c-FLIP. We have tested in vitro the inhibitory effect of these nine molecules using the human lung cancer cell line H1703, overexpressing c-FLIP. Our results showed that six of these newly identified compounds efficiently prevent FADD/c-FLIP interactions in a molecular pull-down assay, as well as in a DISC immunoprecipitation assay. The overexpression of c-FLIP in H1703 prevents TRAIL-mediated apoptosis; however, a combination of TRAIL with these selected molecules significantly restored TRAIL-induced cell death by rescuing caspase cleavage and activation. Altogether, our findings indicate that new inhibitory chemical molecules efficiently prevent c-FLIP recruitment into the DISC complex, thus restoring the caspase-8-dependent apoptotic cascade. These results pave the way to design new c-FLIP inhibitory molecules that may serve as anticancer agents in tumors overexpressing c-FLIP.

Lung cancer is one of the most common and deadliest cancers. Preclinical models are essential to study new therapies and combinations taking tumor genetics into account. We have established cell lines expressing the luciferase gene from lines with varied genetic backgrounds, commonly encountered in patients with pulmonary adenocarcinoma. We have characterized these lines by testing their response to multiple drugs. Thus, we have developed orthotopic preclinical mouse models of NSCLC with very high engraftment efficiency. These models allow the easy monitoring of tumor growth, particularly in response to treatment, and of tumor cells dissemination in the body. We show that concomitant treatment with osimertinib (3rd generation tyrosine kinase inhibitor targeting mutated EGFR) and bevacizumab (anti-angiogenic targeting VEGF) can have a beneficial therapeutic effect on EGFR-mutated tumors. We also show that the addition of afatinib to osimertinib-treated tumors in escape leads to tumor growth inhibition. No such effect is observed with selumetinib or simvastatin. These preclinical mouse models therefore make it possible to test innovative therapeutic combinations and are also a tool of choice for studying resistance mechanisms.

Supplementary Figure 5 from WNT16B Is a New Marker of Cellular Senescence That Regulates p53 Activity and the Phosphoinositide 3-Kinase/AKT Pathway

Supplementary Movie 1 from Inhibitor of Growth 4 Suppresses Cell Spreading and Cell Migration by Interacting with a Novel Binding Partner, Liprin α1

Circulating tumor cells (CTC) have been studied in various solid tumors but clinical utility of CTC in small cell lung cancer (SCLC) remains unclear. The aim of the CTC-CPC study was to develop an EpCAM-independent CTC isolation method allowing isolation of a broader range of living CTC from SCLC and decipher their genomic and biological characteristics. CTC-CPC is a monocentric prospective non-interventional study including treatment-naïve newly diagnosed SCLC. CD56+ CTC were isolated from whole blood samples, at diagnosis and relapse after first-line treatment and submitted to whole-exome-sequencing (WES). Phenotypic study confirms tumor lineage and tumorigenic properties of isolated cells for the 4 patients analyzed with WES. WES of CD56+ CTC and matched tumor biopsy reveal genomic alteration frequently impaired in SCLC. At diagnosis CD56+ CTC were characterized by a high mutation load, a distinct mutational profile and a unique genomic signature, compared to match tumors biopsies. In addition to classical pathways altered in SCLC, we found new biological processes specifically affected in CD56+ CTC at diagnosis. High numeration of CD56+ CTC (> 7/ml) at diagnosis was associated with ES-SCLC. Comparing CD56+ CTC isolated at diagnosis and relapse, we identify differentially altered oncogenic pathways (e.g. DLL3 or MAPK pathway). We report a versatile method of CD56+ CTC detection in SCLC. Numeration of CD56+ CTC at diagnosis is correlated with disease extension. Isolated CD56+ CTC are tumorigenic and show a distinct mutational profile. We report a minimal gene set as a unique signature of CD56+ CTC and identify new affected biological pathways enriched in EpCAM-independent isolated CTC in SCLC.

The INhibitor of Growth (ING) proteins (ING1, ING2, ING3, ING4 and ING5) are a family of epigenetic regulators. Their decreased expression in numerous cancers led to identifying the ING proteins as gatekeeper tumor suppressors as they regulate cell cycle progression, apoptosis and senescence. Subsequently, they were also described as caretaker tumor suppressors through their involvement in DNA replication and the DNA damage response (DDR). Recent studies have identified new interactions of the ING proteins with proteins or pathways implicated in cell proliferation, the maintenance of stem cells pluripotency or the DDR. Furthermore, the ING proteins have been identified as regulators of ribosomal RNA synthesis and of mRNA stability and as regulators of mitochondrial DNA transcription resulting in the regulation of metabolism. These new findings highlight new antitumorigenic activities of the ING proteins that are potential targets for cancer treatment.

Upon accumulation of improperly folded proteins in the Endoplasmic Reticulum (ER), the Unfolded Protein Response (UPR) is triggered to restore ER homeostasis. The induction of stress genes is a sine qua non condition for effective adaptive UPR. Although this requirement has been extensively described, the mechanisms underlying this process remain in part uncharacterized. Here, we show that p97/VCP, an AAA+ ATPase known to contribute to ER stress-induced gene expression, regulates the transcription factor GLI1, a primary effector of Hedgehog (Hh) signaling. Under basal (non-ER stress) conditions, GLI1 is repressed by a p97/VCP-HDAC1 complex while upon ER stress GLI1 is induced through a mechanism requiring both USF2 binding and increase histone acetylation at its promoter. Interestingly, the induction of GLI1 was independent of ligand-regulated Hh signaling. Further analysis showed that GLI1 cooperates with ATF6f to induce promoter activity and expression of XBP1, a key transcription factor driving UPR. Overall, our work demonstrates a novel role for GLI1 in the regulation of ER stress gene expression and defines the interplay between p97/VCP, HDAC1 and USF2 as essential players in this process.

Supplementary Methods and Materials, Figure Legends 4-5, Tables 1-3 from WNT16B Is a New Marker of Cellular Senescence That Regulates p53 Activity and the Phosphoinositide 3-Kinase/AKT Pathway

Supplementary Figures 1-4, Video Legend from Inhibitor of Growth 4 Suppresses Cell Spreading and Cell Migration by Interacting with a Novel Binding Partner, Liprin α1

Circulating Tumor Cells (CTC) have been studied in various solid tumors. However, clinical utility of CTC in Small Cell Lung Cancer (SCLC) remains poorly understood. Most published studies are based on Cellsearch® isolation methods that did not capture CTC undergoing epithelial-mesenchymal transition. The aim of the CTC-CPC study was to develop an EpCAM-independent CTC isolation method allowing isolation of living CTC from SCLC and decipher their genomic characteristics.

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) are currently recommended as first-line treatment for advanced non-small-cell lung cancer (NSCLC) with EGFR-activating mutations. Third-generation (3rd G) EGFR-TKIs, including osimertinib, offer an effective treatment option for patients with NSCLC resistant 1st and 2nd EGFR-TKIs. However, the efficacy of 3rd G EGFR-TKIs is limited by acquired resistance that has become a growing clinical challenge. Several clinical and preclinical studies are being carried out to better understand the mechanisms of resistance to 3rd G EGFR-TKIs and have revealed various genetic aberrations associated with molecular heterogeneity of cancer cells. Studies focusing on epigenetic events are limited despite several indications of their involvement in the development of resistance. Preclinical models, established in most cases in a similar manner, have shown different prevalence of resistance mechanisms from clinical samples. Clinically identified mechanisms include EGFR mutations that were not identified in preclinical models. Thus, NRAS genetic alterations were not observed in patients but have been described in cell lines resistant to 3rd G EGFR-TKI. Mainly, resistance to 3rd G EGFR-TKI in preclinical models is related to the activation of alternative signaling pathways through tyrosine kinase receptor (TKR) activation or to histological and phenotypic transformations. Yet, preclinical models have provided some insight into the complex network between dominant drivers and associated events that lead to the emergence of resistance and consequently have identified new therapeutic targets. This review provides an overview of preclinical studies developed to investigate the mechanisms of acquired resistance to 3rd G EGFR-TKIs, including osimertinib and rociletinib, across all lines of therapy. In fact, some of the models described were first generated to be resistant to first- and second-generation EGFR-TKIs and often carried the T790M mutation, while others had never been exposed to TKIs. The review further describes the therapeutic opportunities to overcome resistance, based on preclinical studies.

Urine cytology is non-invasive, easy to collect, with medium sensitivity and a high specificity. It is an effective way to detect high-grade bladder cancer (BC), but it is less effective on low-grade BC because the rate of equivocal results is much higher. Recently, the fluorescent properties of plasma membranes of urothelial tumor cells (UTC) found in urine cytology have been shown to be useful in improving the early detection of BC. This phenomenon is called peri-membrane fluorescence (PMF). Based on previous studies that have identified the PMF on UTCs, the main objective was to characterize this phenomenon. For this study, a software was specially created to quantify the PMF of all tested cells and different treatments performed. PMF was not found to be a morphological and discriminating feature of UTCs, all cells in shape and not from urine show PMF. We were able to highlight the crucial role of plasma membrane integrity in the maintenance of PMF. Finally, it was found that the induction of a strong cellular stress induced a decrease in PMF, mimicking what was observed in non-tumor cells collected from urine. These results suggest that PMF is found in cells able to resist this stress, such as tumor cells.

ER stress is mediated by three sensors and the most evolutionary conserved IRE1α signals through its cytosolic kinase and endoribonuclease (RNase) activities. IRE1α RNase activity can either catalyze the initial step of XBP1 mRNA unconventional splicing or degrade a number of RNAs through regulated IRE1-dependent decay. Until now, the biochemical and biological outputs of IRE1α RNase activity have been well documented; however, the precise mechanisms controlling whether IRE1α signaling is adaptive or pro-death (terminal) remain unclear. We investigated those mechanisms and hypothesized that XBP1 mRNA splicing and regulated IRE1-dependent decay activity could be co-regulated by the IRE1α RNase regulatory network. We identified that RtcB, the tRNA ligase responsible for XBP1 mRNA splicing, is tyrosine-phosphorylated by c-Abl and dephosphorylated by PTP1B. Moreover, we show that the phosphorylation of RtcB at Y306 perturbs RtcB interaction with IRE1α, thereby attenuating XBP1 mRNA splicing. Our results demonstrate that the IRE1α RNase regulatory network is dynamically fine-tuned by tyrosine kinases and phosphatases upon various stresses and that the extent of RtcB tyrosine phosphorylation determines cell adaptive or death outputs.

Endoplasmic Reticulum (ER) stress is a hallmark of various diseases, which is dealt with through the activation of an adaptive signaling pathway named the Unfolded Protein Response (UPR). This response is mediated by three ER-resident sensors and the most evolutionary conserved, IRE1α signals through its cytosolic kinase and endoribonuclease (RNase) activities. IRE1α RNase activity can either catalyze the initial step of XBP1 mRNA unconventional splicing or degrade a number of RNAs through Regulated IRE1- Dependent Decay (RIDD). The balance between these two activities plays an instrumental role in cells’ life and death decisions upon ER stress. Until now, the biochemical and biological outputs of IRE1α RNase activity have been well documented, however, the precise mechanisms controlling whether IRE1 signaling is adaptive or pro-death (terminal) remain unclear. This prompted us to further investigate those mechanisms and we hypothesized that XBP1 mRNA splicing and RIDD activity could be co-regulated by the IRE1α RNase regulatory network. We showed that a key nexus in this pathway is the tRNA ligase RtcB which, together with IRE1α, is responsible for XBP1 mRNA splicing. We demonstrated that RtcB is tyrosine phosphorylated by c-Abl and dephosphorylated by PTP1B. Moreover, we identified RtcB Y306 as a key residue which, when phosphorylated, perturbs RtcB interaction with IRE1α, thereby attenuating XBP1 mRNA splicing and favoring RIDD. Our results demonstrate that the IRE1α RNase regulatory network is dynamically fine-tuned by tyrosine kinases and phosphatases upon various stresses and that the nature of the stress determines cell adaptive or death outputs. ### Competing Interest Statement EC and LAE are founders of Cell Stress Discoveries Ltd. The authors declare no conflicting interests.