Mouse Model Of Non-small Cell Lung Cancer Research Articles

Inhibition of ULK1/2 and KRAS G12C controls tumor growth in preclinical models of lung cancer.

Mutational activation of KRAS occurs commonly in lung carcinogenesis and, with the recent FDA approval of covalent inhibitors of KRAS G12C such as sotorasib or adagrasib, KRAS oncoproteins are important pharmacological targets in non-small cell lung cancer (NSCLC). However, not all KRAS G12C -driven NSCLCs respond to these inhibitors, and the emergence of drug resistance in those patients that do respond can be rapid and pleiotropic. Hence, based on a backbone of covalent inhibition of KRAS G12C , efforts are underway to develop effective combination therapies. Here we report that inhibition of KRAS G12C signaling increases autophagy in KRAS G12C expressing lung cancer cells. Moreover, the combination of DCC-3116, a selective ULK1/2 inhibitor, plus sotorasib displays cooperative/synergistic suppression of human KRAS G12C -driven lung cancer cell proliferation in vitro and superior tumor control in vivo . Additionally, in genetically engineered mouse models of KRAS G12C -driven NSCLC, inhibition of either KRAS G12C or ULK1/2 decreases tumor burden and increases mouse survival. Consequently, these data suggest that ULK1/2-mediated autophagy is a pharmacologically actionable cytoprotective stress response to inhibition of KRAS G12C in lung cancer.

Immunotherapeutic hydrogel for co-delivery of STAT3 siRNA liposomes and lidocaine hydrochloride for postoperative comprehensive management of NSCLC in a single application

Despite standard treatment for non-small cell lung cancer (NSCLC) being surgical resection, cancer recurrence and complications, such as induction of malignant pleural effusion (MPE) and significant postoperative pain, usually result in treatment failure. In this study, an alginate-based hybrid hydrogel (SOG) is developed that can be injected into the resection surface of the lungs during surgery. Briefly, endoplasmic reticulum-modified liposomes (MSLs) pre-loaded with the signal transducer and activator of transcription 3 (STAT3) small interfering RNA and lidocaine hydrochloride are encapsulated in SOG. Once applied, MSLs strongly downregulated STAT3 expression in the tumor microenvironment, resulting in the apoptosis of lung cancer cells and polarization of tumor-associated macrophages towards the M1-like phenotype. Meanwhile, the release of lidocaine hydrochloride (LID) was beneficial for pain relief and natural killer cell activation. Our data demonstrated MSL@LID@SOG not only efficiently inhibited tumor growth but also potently improved the quality of life, including reduced MPE volume and pain relief in orthotopic NSCLC mouse models, even with a single administration. MSL@LID@SOG shows potential for comprehensive clinical management upon tumor resection in NSCLC, and may alter the treatment paradigms for other cancers.

Cancer-associated fibroblasts-derived exosomal METTL3 promotes the proliferation, invasion, stemness and glutaminolysis in non-small cell lung cancer cells by eliciting SLC7A5 m6A modification.

Cancer-associated fibroblasts (CAFs) can promote the crosstalk between cancer cells and tumor microenvironment by exosomes. METTL3-mediated N6-methyladenine (m6A) modification has been proved to promote the progression of non-small cell lung cancer (NSCLC). Here, we focused on the impacts of CAFs-derived exosomes and METTL3-mediated m6A modification on NSCLC progression. Functional analyses were conducted using Cell Counting Kit-8, EdU, colony formation, sphere formation and transwell assays, respectively. Glutamine metabolism was evaluated by detecting glutamate consumption, and the production of intercellular glutamate and α-ketoglutarate (α-KG). qRT-PCR and western blotting analyses were utilized to measure the levels of genes and proteins. Exosomes were isolated by kits. The methylated RNA immunoprecipitation assay detected the m6A modification profile of Amino acid transporter LAT1 (SLC7A5) mRNA. The NSCLC mouse model was established to conduct in vivo experiments. We found that CAFs promoted the proliferation, invasion, stemness and glutaminolysis in NSCLC cells. METTL3 was enriched in CAFs and was packaged into exosomes. After knockdown of METTL3 in CAF exosomes, it was found the oncogenic effects of CAFs on NSCLC cells were suppressed. CAFs elevated m6A levels in NSCLC cells. Mechanistically, exosomal METTL3-induced m6A modification in SLC7A5 mRNA and stabilized its expression in NSCLC cells. Moreover, SLC7A5 overexpression abolished the inhibitory effects of exosomal METTL3-decreased CAFs on NSCLC cells. In addition, METTL3 inhibition in CAF exosomes impeded NSCLC growth in vivo. In all, CAFs-derived exosomal METTL3 promoted the proliferation, invasion, stemness and glutaminolysis in NSCLC cells by inducing SLC7A5 m6A modification.

Abstract 3916: TNG260, a small molecule CoREST inhibitor, sensitizes STK11-mutant NSCLC to anti-PD1 immunotherapy

Abstract Loss of function (LOF) of the serine-threonine kinase STK11/LKB1, which has been reported in ~15% of non-small cell lung cancer (NSCLC), drives resistance to immune checkpoint therapies such as PD1. In vivo CRISPR screens in tumor-bearing immune competent mice identified the Corepressor of Repressor Element 1 Silencing Transcription (CoREST) complex as a synthetic lethal immune evasion target for STK11- mutant tumors. Here, we demonstrate that TNG260, a small molecule drug specifically targeting the CoREST complex, can sensitize STK11 mutant lung cancers to anti-PD1 immunotherapy. Combination treatment with TNG260 and anti-PD1 arrested KRAS/STK11 mutant NSCLC tumor growth with no major toxicity observed, as confirmed in both allograft and genetically engineered mouse models (GEMMs) of STK11 mutant NSCLC. Further transcriptional analysis via bulk RNA-seq revealed that treatment of KRAS/STK11 mutant lung tumors with TNG260 promotes genes related to inflammatory responses and suppresses genes related to cell cycle and mitotic checkpoint(s). This phenotype was not observed in STK11 wildtype KRAS/P53 mutant lung tumors. Furthermore, combinational treatment of TNG260 with PD1 antibody showed increased macrophages, neutrophils and dendritic cells signatures in vivo. Our data suggests that epigenetic reprogramming via TNG260 sensitizes STK11 mutant NSCLC tumors to anti- PD1 treatment via targeting of the CoREST complex. This work identifies new vulnerabilities in STK11 LOF cancers that can be exploited therapeutically. TNG260 is currently being investigated in combination with pembrolizumab for the treatment of STK11-mutant cancer (NCT05887492), including NSCLC. Citation Format: Ayushi Patel, Soumyadip Sahu, Ke Geng, Salman Punekar, Janaye Stephens, Jiehui Deng, Ting Chen, Leanne Ahronian, Minjie Zhang, Jannik Andersen, Brian Haines, Kwok-Kin Wong. TNG260, a small molecule CoREST inhibitor, sensitizes STK11-mutant NSCLC to anti-PD1 immunotherapy [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 3916.

IL-8 from CD248-expressing cancer-associated fibroblasts generates cisplatin resistance in non-small cell lung cancer.

Chemotherapy-resistant non-small cell lung cancer (NSCLC) presents a substantial barrier to effective care. It is still unclear how cancer-associated fibroblasts (CAFs) contribute to NSCLC resistance to chemotherapy. Here, we found that CD248+ CAFs released IL-8 in NSCLC, which, in turn, enhanced the cisplatin (CDDP) IC50 in A549 and NCI-H460 while decreasing the apoptotic percentage of A549 and NCI-H460 invitro. The CD248+ CAFs-based IL-8 secretion induced NSCLC chemoresistance by stimulating nuclear factor kappa B (NF-κB) and elevating ATP-binding cassette transporter B1 (ABCB1). We also revealed that the CD248+ CAFs-based IL-8 release enhanced cisplatin chemoresistance in NSCLC mouse models invivo. Relative to wild-type control mice, the CD248 conditional knockout mice exhibited significant reduction of IL-8 secretion, which, in turn, enhanced the therapeutic efficacy of cisplatin invivo. In summary, our study identified CD248 activates the NF-κB axis, which, consecutively induces the CAFs-based secretion of IL-8, which promotes NSCLC chemoresistance. This report highlights a potential new approach to enhancing the chemotherapeutic potential of NSCLC-treating cisplatin.

Targeting serine/glycine metabolism improves radiotherapy response in non-small cell lung cancer

BackgroundLung cancer is the most lethal cancer, and 85% of cases are classified as non-small cell lung cancer (NSCLC). Metabolic rewiring is a cancer hallmark that causes treatment resistance, and lacks insights into serine/glycine pathway adaptations upon radiotherapy.MethodsWe analyzed radiotherapy responses using mass-spectrometry-based metabolomics in NSCLC patient’s plasma and cell lines. Efficacy of serine/glycine conversion inhibitor sertraline with radiotherapy was investigated by proliferation, clonogenic and spheroid assays, and in vivo using a serine/glycine dependent NSCLC mouse model by assessment of tumor growth, metabolite and cytokine levels, and immune signatures.ResultsSerine/glycine pathway metabolites were significantly consumed in response to radiotherapy in NSCLC patients and cell models. Combining sertraline with radiotherapy impaired NSCLC proliferation, clonogenicity and stem cell self-renewal capacity. In vivo, NSCLC tumor growth was reduced solely in the sertraline plus radiotherapy combination treatment group. Tumor weights linked to systemic serine/glycine pathway metabolite levels, and were inhibited in the combination therapy group. Interestingly, combination therapy reshaped the tumor microenvironment via cytokines associated with natural killer cells, supported by eradication of immune checkpoint galectin-1 and elevated granzyme B levels.ConclusionOur findings highlight that targeting serine/glycine metabolism using sertraline restricts cancer cell recovery from radiotherapy and provides tumor control through immunomodulation in NSCLC.

Targeting lncRNA16 by GalNAc-siRNA conjugates facilitates chemotherapeutic sensibilization via the HBB/NDUFAF5/ROS pathway.

Chemoresistance is a significant barrier to effective cancer treatment. Potential mechanisms for chemoresistance include reactive oxygen species (ROS) accumulation and expression of chemoresistance-promoting genes. Here, we report a novel function of lncRNA16 in the inhibition of ROS generation and the progression of chemoresistance. By analyzing the serum levels of lncRNA16 in a cohort of 35 patients with non-small cell lung cancer (NSCLC) and paired serum samples pre- and post-treatment from 10 NSCLC patients receiving neoadjuvant platinum-based chemotherapy, performing immunohistochemistry (IHC) assays on 188 NSCLC tumor samples, using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS) assays, as well as RNA immunoprecipitation (RIP) and RNA pull-down analyses, we discovered that patients with increased serum levels of lncRNA16 exhibited a poor response to platinum-based chemotherapy. The expression of hemoglobin subunit beta (HBB) and NDUFAF5 significantly increases with the development of chemoresistance. LncRNA16 binds to HBB and promotes HBB accumulation by inhibiting autophagy. LncRNA16 can also inhibit ROS generation via the HBB/NDUFAF5 axis and function as a scaffold to facilitate the colocalization of HBB and NDUFAF5 in the mitochondria. Importantly, preclinical studies in mouse models of chemo-resistant NSCLC have suggested that lncRNA16 targeting by trivalent N-acetylgalactosamine (GalNAc)-conjugated siRNA restores chemosensitivity and results in tumor growth inhibition with no detectable toxicity in vivo. Overall, lncRNA16 is a promising therapeutic target for overcoming chemoresistance, and the combination of first-line platinum-based chemotherapy with lncRNA16 intervention can substantially enhance anti-tumor efficacy.

Propionate reinforces epithelial identity and reduces aggressiveness of lung carcinoma.

The epithelial-to-mesenchymal transition (EMT) plays a central role in the development of cancer metastasis and resistance to chemotherapy. However, its pharmacological treatment remains challenging. Here, we used an EMT-focused integrative functional genomic approach and identified an inverse association between short-chain fatty acids (propionate and butanoate) and EMT in non-small cell lung cancer (NSCLC) patients. Remarkably, treatment with propionate in vitro reinforced the epithelial transcriptional program promoting cell-to-cell contact and cell adhesion, while reducing the aggressive and chemo-resistant EMT phenotype in lung cancer cell lines. Propionate treatment also decreased the metastatic potential and limited lymph node spread in both nude mice and a genetic NSCLC mouse model. Further analysis revealed that chromatin remodeling through H3K27 acetylation (mediated by p300) is the mechanism underlying the shift toward an epithelial state upon propionate treatment. The results suggest that propionate administration has therapeutic potential in reducing NSCLC aggressiveness and warrants further clinical testing.

Effect of radiation fractionation on IDO1 via the NF-κB/COX2 axis in non-small cell lung cancer

Radiotherapy (RT) is the mainstay treatment modality for lung cancer. We recently reported that conventionally fractionated radiotherapy (CRT) with daily fractionation of 2Gy significantly increased the activity of indoleamine 2,3-dioxygenase (IDO1), a known immune checkpoint, which predicted poorer long-term survival in patients with non-small cell lung cancer (NSCLC), while stereotactic body radiotherapy (SBRT) using fractionation size of 10Gy did not increase IDO1 activity and had better survival. Here we hypothesized that the hypofractionated SBRT kind of dose fraction stimulates host antitumor immunity via downregulating IDO1 in which CRT could not. We tested this hypothesis in vitro and in vivo using 10Gyx1 and 2Gyx8 fractionations in the laboratory. The results demonstrated that, although there was an initial downregulation after RT, the expression of IDO1 was ultimately upregulated by both fractionation regimens. The 10Gyx1 regimen had minimum upregulation, while the 2Gyx8 regimen significantly increased in IDO1 expression which was positively correlated with the elevated expressions of p-NF-κB and COX2. Pharmacological inhibition of COX2 abolished RT-induced IDO1 expression. Furthermore, the IDO1 inhibitor, D-1-methyl-tryptophan (D-1MT), exerted RT-related tumor-killing effects in the NSCLC cell lines and mouse models. These findings suggest that, in addition to being an immune suppressor, IDO1 may serve as an adaptive resistance factor in RT. Furthermore, an unappreciated mechanism may exist, where a larger fraction size might be superior to conventional sizes in cancer treatment. This study may provide a rationale for future research in using IDO1 as a biomarker to personalize RT dose fractionation and COX2 inhibitor to decrease radiation immune suppression from CRT.

Blocking the functional domain of TIP1 by antibodies sensitizes cancer to radiation therapy

Non-small-cell lung cancer (NSCLC) and glioblastoma (GB) have poor prognoses. Discovery of new molecular targets is needed to improve therapy. Tax interacting protein 1 (TIP1), which plays a role in cancer progression, is overexpressed and radiation-inducible in NSCLC and GB. We evaluated the effect of an anti-TIP1 antibody alone and in combination with ionizing radiation (XRT) on NSCLC and GB in vitro and in vivo. NSCLC and GB cells were treated with anti-TIP1 antibodies and evaluated for proliferation, colony formation, endocytosis, and cell death. The efficacy of anti-TIP1 antibodies in combination with XRT on tumor growth was measured in mouse models of NSCLC and GB. mRNA sequencing was performed to understand the molecular mechanisms involved in the action of anti-TIP1 antibodies. We found that targeting the functional domain of TIP1 leads to endocytosis of the anti-TIP1 antibody followed by reduced proliferation and increased apoptosis-mediated cell death. Anti-TIP1 antibodies bound specifically (with high affinity) to cancer cells and synergized with XRT to significantly increase cytotoxicity in vitro and reduce tumor growth in mouse models of NSCLC and GB. Importantly, downregulation of cancer survival signaling pathways was found in vitro and in vivo following treatment with anti-TIP1 antibodies. TIP1 is a new therapeutic target for cancer treatment. Antibodies targeting the functional domain of TIP1 exhibited antitumor activity and enhanced the efficacy of radiation both in vitro and in vivo. Anti-TIP1 antibodies interrupt TIP1 function and are effective cancer therapy alone or in combination with XRT in mouse models of human cancer.

Inhalable siRNA Nanoparticles for Enhanced Tumor-Targeting Treatment of KRAS-Mutant Non-Small-Cell Lung Cancer.

Kirsten rat sarcoma (KRAS) is the most commonly mutated oncogene in lung cancers. Gene therapy is emerging as a promising cancer treatment modality; however, the systemic administration of gene therapy has been limited by inefficient delivery to the lungs and systemic toxicity. Herein, we report a noninvasive aerosol inhalation nanoparticle (NP) system, termed "siKRAS@GCLPP NPs," to treat KRAS-mutant non-small-cell lung cancer (NSCLC). The self-assembled siKRAS@GCLPP NPs are capable of maintaining structural integrity during nebulization, with preferential distribution within the tumor-bearing lung. Inhalable siKRAS@GCLPP NPs show not only significant tumor-targeting capability but also enhanced antitumor activity in an orthotopic mouse model of human KRAS-mutant NSCLC. The nebulized delivery of siKRAS@GCLPP NPs demonstrates potent knockdown of mutated KRAS in tumor-bearing lungs without causing any observable adverse effects, exhibiting a better biosafety profile than the systemic delivery approach. The results present a promising inhaled gene therapy approach for the treatment of KRAS-mutant NSCLC and other respiratory diseases.

MiR-145 as a Potential Biomarker and Therapeutic Target in Patients with Non-Small Cell Lung Cancer.

Our previous study found that miR-145 was downregulated in non-small cell lung cancer (NSCLC) tissues and that it could inhibit the cell proliferation in transfected NSCLC cells. In this study, we found that miR-145 was downregulated in NSCLC plasma samples compared to healthy controls. A receiver operating characteristic curve analysis indicated that plasma miR-145 expression was correlated with NSCLC in patient samples. We further revealed that the transfection of miR-145 inhibited the proliferation, migration, and invasion of NSCLC cells. Most importantly, miR-145 significantly delayed the tumor growth in a mouse model of NSCLC. We further identified GOLM1 and RTKN as the direct targets of miR-145. A cohort of paired tumors and adjacent non-malignant lung tissues from NSCLC patients was used to confirm the downregulated expression and diagnostic value of miR-145. The results were highly consistent between our plasma and tissue cohorts, confirming the clinical value of miR-145 in different sample groups. In addition, we also validated the expressions of miR-145, GOLM1, and RTKN using the TCGA database. Our findings suggested that miR-145 is a regulator of NSCLC and it plays an important role in NSCLC progression. This microRNA and its gene targets may serve as potential biomarkers and novel molecular therapeutic targets in NSCLC patients.

A novel 89Zr-a-PD-1 immuno-PET-CT may improve pseudoprogression detection in a lung cancer murine model receiving immunotherapy.

e15116 Background: Immune checkpoint inhibitors (ICIs) have revolutionized lung cancer treatment improving survival outcomes of non-small cell lung cancer (NSCLC) patients. Response assessment of patients on ICIs treatment represents a challenge since an increase in tumor size or the appearance of new lesions might not reflect true disease progression (P) but pseudoprogression (PP), which has been reported in a range of 3-6% in NSCLC. Conventional 18F-FDG-PET scans do not accurately discriminate P from PP. We have recently reported the efficacy of a combined blockade of PD-1 and ID1 in NSCLC mouse model. We aimed to evaluate a novel 89Zr-labelled radiotracer for micro mPET-CT to detect PP in a NSCLC murine model. Methods: Syngeneic subcutaneous tumors were generated using LLC cells (with constitutive expression of ID1 or ID1-silenced) in C57BL6J and in ID1-deficient mice, treated with PBS or with a commercially available monoclonal antibody against PD-1 (a-PD-1) (RPM1-14, 300 mg, IP, on days 7, 10 and 14). Tumor growth and response to ID1-PD-1 blockade, was measured by 18F-FDG uptake. Additionally, PP was measured labelling the anti-PD-1 with 89Zr (89Zr-a-PD-1) and analyzing 89Zr uptake at day 15, 17 and 20. Tumor microenvironment was analyzed with immunohistochemistry, multiplex immunofluorescence and quantification of relative expression of interleukins by RT-PCR. Results: 18F-FDG uptake did not show significant differences between groups, underestimating the real metabolic response induced by the treatment. However, 89Zr-mPET-CT showed a significantly higher 89Zr uptake when ID1 was inhibited in both, tumor cells and tumor micro-environment and mice were treated with a-PD-1 (p = 0.0075). The analysis of the tumor tissues in those animals by immunohistochemistry and multiplex immunofluorescence disclosed an increase in immune T CD8+ cells infiltration, being responsible for the antitumor response observed and correlating with 89Zr signal. Moreover, the analysis of interleukins expression showed that tumors with ID1 inhibition in the tumor micro-environment presented an upregulation of pro-inflammatory interleukins, favoring T- cell infiltration. Conclusions: ID1 inhibition in tumor cells and in tumor micro-environment combined with PD-1 blockade enhanced immune cell infiltration through pro-inflammatory interleukins upregulation. 89Zr-a-PD-1 immuno-PET-CT may improve PP detection in a NSCLC murine model receiving immunotherapy.

Efficacy and Imaging-Enabled Pharmacodynamic Profiling of KRAS G12C Inhibitors in Xenograft and Genetically Engineered Mouse Models of Cancer.

KRAS is one of the most commonly mutated oncogenes in lung, colorectal, and pancreatic cancers. Recent clinical trials directly targeting KRAS G12C presented encouraging results for a large population of non-small cell lung cancer (NSCLC), but resistance to treatment is a concern. Continued exploration of new inhibitors and preclinical models is needed to address resistance mechanisms and improve duration of patient responses. To further enable the development of KRAS G12C inhibitors, we present a preclinical framework involving translational, non-invasive imaging modalities (CT and PET) and histopathology in a conventional xenograft model and a novel KRAS G12C knock-in mouse model of NSCLC. We utilized an in-house developed KRAS G12C inhibitor (Compound A) as a tool to demonstrate the value of this framework in studying in vivo pharmacokinetic/pharmacodynamic (PK/PD) relationship and anti-tumor efficacy. We characterized the Kras G12C-driven genetically engineered mouse model (GEMM) and identify tumor growth and signaling differences compared to its Kras G12D-driven counterpart. We also find that Compound A has comparable efficacy to sotorasib in the Kras G12C-driven lung tumors arising in the GEMM, but like observations in the clinic, some tumors inevitably progress on treatment. These findings establish a foundation for evaluating future KRAS G12C inhibitors that is not limited to xenograft studies and can be applied in a translationally relevant mouse model that mirrors human disease progression and resistance.

Dipeptidyl peptidase 4 inhibitor reduces tumor-associated macrophages and enhances anti-PD-L1-mediated tumor suppression in non-small cell lung cancer

PurposeThe efficacy of immune checkpoint inhibitors such as programmed cell death ligand 1 (PD-L1) antibodies in non-small cell lung cancer (NSCLC) is limited, and combined use with other therapies is recommended. Dipeptidyl peptidase 4 (DPP4) inhibitors, a class of small molecule inhibitors, are highly effective for treating type 2 diabetes. Emerging evidence implicates DPP4 inhibitors as immunomodulators that modify aspects of innate and adaptive immunity. We evaluated the combination of a DPP4 inhibitor (anagliptin) and PD-L1 blockade in an NSCLC mouse model.MethodsThe effect of the combination of anti-PD-L1 and anagliptin was evaluated in subcutaneous mouse models of NSCLC. Tumor-infiltrating immune cells were analyzed by flow cytometry. Bone marrow-derived monocytes of C57BL/6 mice were isolated in vitro to examine the underlying mechanism of anagliptin on the differentiation and polarization of macrophage.ResultsAnagliptin dramatically improved the efficacy of PD-L1 antibody monotherapy by inhibiting macrophage formation and M2 polarization in the tumor microenvironment. Mechanistically, anagliptin suppressed the production of reactive oxygen species in bone marrow monocytes by inhibiting NOX1 and NOX2 expression induced by macrophage colony-stimulating factor, reduced late ERK signaling pathway activation, and inhibited monocyte-macrophage differentiation. However, the inhibitory effect was reactivated by lipopolysaccharide and interferon-gamma interacting with corresponding receptors during M1 macrophage polarization, but not M2.ConclusionsAnagliptin can enhance PD-L1 blockade efficacy in NSCLC by inhibiting macrophage differentiation and M2 macrophage polarization, and combination therapy may be a promising strategy for treating PD-L1 blockade therapy-resistant patients with NSCLC.

Combinational study with network pharmacology, molecular docking and preliminary experiments on exploring common mechanisms underlying the effects of weijing decoction on various pulmonary diseases

Objective‘Homotherapy for heteropathy’ is a theory by which different diseases with similar pathogenesis can be treated with one Chinese formula. We aimed to explore the key components and core targets of Weijing decoction (WJD) in treating various lung diseases, namely, pneumonia, chronic obstructive pulmonary disease (COPD), acute lung injury (ALI), pulmonary fibrosis, pulmonary tuberculosis and non-small cell lung cancer (NSCLC), via network pharmacology, molecular docking and some experiments. SignificanceThis is the first study on the mechanism of WJD in treating various lung diseases by ‘homotherapy for heteropathy’. This study is helpful for the transformation of TCM formula and development of new drugs. MethodsActive components and therapeutic targets of WJD were obtained via TCMSP and UniProt databases. Targets of the six pulmonary diseases were harvested from the GeneCards TTD, DisGeNet, UniProt and OMIM databases. Drug-disease intersection targets, corresponding Venn diagrams, herb-component-target networks and protein–protein interaction networks were established. Furthermore, GO biological function and KEGG enrichment analysis were completed. Moreover, the binding activity between main compounds and core targets was measured through molecular docking. Finally, the xenograft NSCLC mouse model was established. Immune responses were evaluated by flow cytometry and mRNA expression levels of critical targets were measured by real-time PCR. ResultsJUN, CASP3 and PTGS2 were the most critical targets in six pulmonary diseases. The active compounds beta-sitosterol, tricin and stigmasterol stably bound to many active sites on target proteins. WJD had extensive pharmacological regulation, involving pathways related to cancer, inflammation, infection, hypoxia, immunity and so on. ConclusionsEffects of WJD against various lung diseases involve lots of compounds, targets and pathways. These findings will facilitate further research as well as clinical application of WJD.

Abstract 3878: Targeting Hippo-YAP, BRD4 and RAS-MAPK interplay in lung cancer to forestall drug resistance

Abstract Introduction: Identifying parallel survival pathways or compensatory mechanisms that limit the response to targeted therapy is critical to discover effective combinatorial strategies for the treatment of lung cancer and to prevent emergence of drug resistance Results: Using combination drug screening in a panel of cell line models of non-small cell lung cancer (NSCLC), we identified the tumor suppressor STK11 (aka LKB1) as a molecular biomarker of sensitivity to BET and RAS-MAPK pathway inhibitors combination in NSCLC. We found a small molecule inhibitor of BET (bromodomain and extra terminal) family proteins (BRD4, 3, 2), PLX51107, suppressed BRAF and KRAS mutant NSCLC cell proliferation and in combination with RAS (KRAS G12C), RAF or MEK inhibitor led to pronounced loss in cell viability with potent MYC downregulation and synergistic apoptosis induction in vitro and potent tumor regression or stasis in vivo in several KRAS mutant CDX and PDX mouse models of NSCLC. However, BRAF and KRAS mutant NSCLC cells with co-occurring mutations in STK11 were resistant to BET and RAS, RAF or MEK co-inhibition. To unravel the underlying mechanism of resistance to BET and RAS-MAPK inhibitors upon STK11 loss we modeled STK11 deficiency in BRAF and KRAS mutant NSCLC cells and tumors. We found that STK11 loss creates an unprecedented dependence on YAP/TEAD TFs. STK11 silencing in BRAF and KRAS mutant NSCLC cells resulted in nuclear transfer/activation of the HIPPO pathway effector, YAP, upregulation of the YAP target BIRC5 (aka Survivin) and rescued the synthetic lethality due to BET and RAS, RAF or MEK co-inhibition. Phenocopying STK11 loss, constitutively active YAP (YAP5SA), but not transcriptionally inactive YAP (YAPS94A), mitigated the deleterious effects of BET and RAS, RAF or MEK co-suppression in BRAF and KRAS mutant NSCLC cells with potent upregulation of the pro-survival factor BIRC5. Conversely, we found that co-silencing YAP or TEADs with BETs re-sensitized BRAF and KRAS mutant NSCLC cells with co-occurring mutations in STK11 to BET and RAS-MAPK inhibitors combination and caused synthetic lethality. Conclusion: This study uncovered a functional interaction between BET proteins, STK11, HIPPO-YAP and RAS-MAPK signaling pathways in lung cancer. These results will help identify combinatorial strategies for treating aberrant RAS-MAPK pathway driven lung cancer more effectively and forestall drug resistance. Citation Format: Nilanjana Chatterjee, Victor Olivas, Wei Wu, Ben Powell, Trever Bivona. Targeting Hippo-YAP, BRD4 and RAS-MAPK interplay in lung cancer to forestall drug resistance. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3878.

Extracellular vesicle-derived LINC00482 induces microglial M2 polarization to facilitate brain metastasis of NSCLC

Considering the crucial role of long non-coding RNAs (lncRNAs) in non-small cell lung cancer (NSCLC), we tried to analyze the role of extracellular vesicle (EV)-derived LINC00482 in the occurrence of brain metastasis in NSCLC. LINC00482 expression was quantified in EVs isolated from serum samples of NSCLC patients (serum-EVs). Ectopic expression and depletion assays were conducted in the microglial cell line HMC3 co-cultured with serum-EVs and in xenograft mouse models of NSCLC to explore the roles of EV-carried LINC00482. LINC00482 was enriched in serum-EVs and induced M2 polarization of microglial cells HMC3 in vitro. LINC00482 competitively bound to miR-142-3p and upregulated the expression of miR-142-3p target gene TGF-β1 in HMC3 cells, thus promoting microglial M2 polarization. EV-derived LINC00482-induced M2 microglia promoted the malignant properties of NSCLC cells. In vivo data demonstrated that EVs transmitted LINC00482 to regulate the miR-142-3p/TGF-β1 axis, induce microglial M2 polarization and affect the pre-metastatic niche, thus enhancing brain metastasis of NSCLC. Overall, suppression of the expression of tumor-derived LINC00482 or LINC00482-containing EVs, may serve as an effective target for contributing to the reduction of brain metastasis of NSCLC.

Tumor-intrinsic IRE1α signaling controls protective immunity in lung cancer

IRE1α-XBP1 signaling is emerging as a central orchestrator of malignant progression and immunosuppression in various cancer types. Employing a computational XBP1s detection method applied to TCGA datasets, we demonstrate that expression of the XBP1s mRNA isoform predicts poor survival in non-small cell lung cancer (NSCLC) patients. Ablation of IRE1α in malignant cells delays tumor progression and extends survival in mouse models of NSCLC. This protective effect is accompanied by alterations in intratumoral immune cell subsets eliciting durable adaptive anti-cancer immunity. Mechanistically, cancer cell-intrinsic IRE1α activation sustains mPGES-1 expression, enabling production of the immunosuppressive lipid mediator prostaglandin E2. Accordingly, restoring mPGES-1 expression in IRE1αKO cancer cells rescues normal tumor progression. We have developed an IRE1α gene signature that predicts immune cell infiltration and overall survival in human NSCLC. Our study unveils an immunoregulatory role for cancer cell-intrinsic IRE1α activation and suggests that targeting this pathway may help enhance anti-tumor immunity in NSCLC.

IL6 Mediates Suppression of T- and NK-cell Function in EMT-associated TKI-resistant EGFR-mutant NSCLC.

Patients with advanced non-small cell lung cancer (NSCLC) harboring activating EGFR mutations are initially responsive to tyrosine kinase inhibitors (TKI). However, therapeutic resistance eventually emerges, often via secondary EGFR mutations or EGFR-independent mechanisms such as epithelial-to-mesenchymal transition. Treatment options after EGFR-TKI resistance are limited as anti-PD-1/PD-L1 inhibitors typically display minimal benefit. Given that IL6 is associated with worse outcomes in patients with NSCLC, we investigate whether IL6 in part contributes to this immunosuppressed phenotype. We utilized a syngeneic genetically engineered mouse model (GEMM) of EGFR-mutant NSCLC to investigate the effects of IL6 on the tumor microenvironment and the combined efficacy of IL6 inhibition and anti-PD-1 therapy. Corresponding in vitro studies used EGFR-mutant human cell lines and clinical specimens. We identified that EGFR-mutant tumors which have oncogene-independent acquired resistance to EGFR-TKIs were more mesenchymal and had markedly enhanced IL6 secretion. In EGFR-mutant GEMMs, IL6 depletion enhanced activation of infiltrating natural killer (NK)- and T-cell subpopulations and decreased immunosuppressive regulatory T and Th17 cell populations. Inhibition of IL6 increased NK- and T cell-mediated killing of human osimertinib-resistant EGFR-mutant NSCLC tumor cells in cell culture. IL6 blockade sensitized EGFR-mutant GEMM tumors to PD-1 inhibitors through an increase in tumor-infiltrating IFNγ+ CD8+ T cells. These data indicate that IL6 is upregulated in EGFR-mutant NSCLC tumors with acquired EGFR-TKI resistance and suppressed T- and NK-cell function. IL6 blockade enhanced antitumor immunity and efficacy of anti-PD-1 therapy warranting future clinical combinatorial investigations.