Lung Cancer Xenograft Mouse Model Research Articles

Analysis of SARS-CoV-2 omicron mutations that emerged during long-term replication in a lung cancer xenograft mouse model.

SARS-CoV-2 Omicron has the largest number of mutations among all the known SARS-CoV-2 variants. The presence of these mutations might explain why Omicron is more infectious and vaccines have lower efficacy to Omicron than other variants, despite lower virulence of Omicron. We recently established a long-term in vivo replication model by infecting Calu-3 xenograft tumors in immunodeficient mice with parental SARS-CoV-2 and found that various mutations occurred majorly in the spike protein during extended replication. To investigate whether there are differences in the spectrum and frequency of mutations between parental SARS-CoV-2 and Omicron, we here applied this model to Omicron. At 30 days after infection, we found that the virus was present at high titers in the tumor tissues and had developed several rare sporadic mutations, mainly in ORF1ab with additional minor spike protein mutations. Many of the mutant isolates had higher replicative activity in Calu-3 cells compared with the original SARS-CoV-2 Omicron virus, suggesting that the novel mutations contributed to increased viral replication. Serial propagation of SARS-CoV-2 Omicron in cultured Calu-3 cells resulted in several rare sporadic mutations in various viral proteins with no mutations in the spike protein. Therefore, the genome of SARS-CoV-2 Omicron seems largely stable compared with that of the parental SARS-CoV-2 during extended replication in Calu-3 cells and xenograft model. The sporadic mutations and modified growth properties observed in Omicron might explain the emergence of Omicron sublineages. However, we cannot exclude the possibility of some differences in natural infection.

Intermittent hypoxia inhibits anti-tumor immune response via regulating PD-L1 expression in lung cancer cells and tumor-associated macrophages

Accumulating evidence has shown an increased tumor incidence and reduced survival rate in cancer patients with obstructive sleep apnea (OSA). Although intermittent hypoxia is known to play a crucial role, the molecular mechanism by which intermittent hypoxia accelerates lung cancer progression remains to be elucidated.A lung cancer xenograft mouse model was established by subcutaneously injecting LLC cells into C57BL/6 mice. The tumor-bearing mice were exposed to either normoxia or intermittent hypoxia and received either IgG2a, anti-programmed death ligand-1 (PD-L1), PX-478, or anti-PD-L1 + PX-478 treatment.A significant upregulation of tumor associated macrophages (TAMs) papulation and PD-L1 levels was observed in lung adenocarcinoma patients with OSA. We further confirmed that hypoxia-inducible factor-1 alpha (HIF-1α) regulates PD-L1 at transcriptional levels, mainly through binding to the hypoxia response element 4. Using a lung cancer xenograft mouse model, we observed that intermittent hypoxia exposed tumors grew faster and bigger with upregulated HIF-1α and PD-L1 expression, enhanced TAMs and Treg populations, and reduced cytotoxic T cells and cytokine secretion. Finally, we found a combination of PX-478 and anti-PD-L1 exerted an encouraging tumor inhibition effect compared to single treatment. Combination therapies based on HIF-1α and PD-L1 blockade might serve as a promising strategy to treat lung cancer patients with OSA.

Abstract 477: Discovery of RGT-018: A potent, selective and orally bioavailable SOS1 inhibitor for mutant KRAS-driven cancers

Abstract Background: KRAS is the most frequently mutated oncogene with high prevalence in non-small cell lung cancers (NSCLC), colorectal cancers (CRC), and pancreatic cancers (PAC). FDA currently approved sotorasib provides breakthrough therapy for cancer patients with KRASG12C mutation, however there is still high unmet medical need for new agents that target a broader KRAS mutated tumors. A new opportunity emerges to develop a pan KRAS inhibitor by suppressing the upstream guanine nucleotide exchange factor (GEF) protein son of sevenless 1 (SOS1). SOS1 is a key activator of KRAS and facilitates the conversion of GDP-bound KRAS (off) state to GTP-bound KRAS (on) state. Binding to its catalytic domain, small molecule SOS1 inhibitor prevents KRAS activation and suppresses cancer cell proliferation. Material and Methods: Regor’s unique Computer Accelerated Rational Design (CARD) technology platform was applied to identify potent and selective SOS1 inhibitors. Biochemical assays and cellular assays were utilized to drive the structure-activity relationship (SAR). In vitro and in vivo target engagement were confirmed. In vivo efficacy study data were generated using lung and pancreatic cancer xenograft mouse models with KRAS mutation. Results: In vitro, RGT-018 blocked the interaction of KRAS::SOS1 with high selectivity, and inhibited proliferation of a broad spectrum of mutant KRAS-driven cancer cells as a single agent. Robust anti-proliferation activity was observed when RGT-018 was combined with MEK, KRASG12C, EGFR or CDK2/4/6 inhibitors in vitro. Oral administration of RGT-018 inhibited tumor growth and suppressed KRAS signaling pathway activation in tumor xenografts. Furthermore, combination with MEK or KRASG12C inhibitors led to profound tumor regression. Conclusions: The pharmacological properties of RGT-018 represent an attractive drug candidate with oral bioavailability for combination with targeted agents to treat a broader patient population driven by mutant KRAS. Citation Format: Fei Xiao, Kailiang Wang, Xinjuan Wang, Huijuan Li, Zhilong Hu, Wei Huang, Xiaoming Ren, Teng Feng, Lili Yao, Jing Lin, Chunlai Li, Liufeng Mei, Zhuanzhuan Zhang, Yangyang Liu, Xi Chen, Xiaotian Zhu, Wenge Zhong, Zhi Xie. Discovery of RGT-018: A potent, selective and orally bioavailable SOS1 inhibitor for mutant KRAS-driven cancers [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 477.

A Novel Tri-Functional Liposome Re-Educates "Cold Tumor" and Abrogates Tumor Growth by Synergizing Autophagy Inhibition and PD-L1 Blockade.

Immunotherapy has been regarded as a breakthrough in cancer treatment and achieved great success. However, the poor response rate is still a formidable challenge of current immunotherapies, especially in solid tumors without sufficient infiltration of immune cells, also known as "cold tumor." SAR405is a highly specific VPS34inhibitor and has been suggested as a potential approach converting "cold tumor" into "hot tumor" by inhibiting autophagy. In this study, a tri-functional doxorubicin (DOX) plus SAR405liposome system is established and further modified with a novel anti-PD-L1peptide JY4for targeted delivery (DOX-SAR-JY4LIPO ). The data here demonstrate that in a lung cancer xenograft mouse model, by facilitating the tumoral enrichment of both SAR405and DOX, DOX-SAR-JY4LIPO effectively increases the infiltration of cytotoxic lymphocytes in the tumor by synergizing DOX-induced immunogenic cell death (ICD) and SAR405-mediated upregulation of chemokines including CCL5and CXCL10. As results, DOX-SAR-JY4LIPO significantly inhibits tumor growth, metastasis, and resurrection by re-educating immunosuppressive tumor microenvironment. In conclusion, this study not only proves the concept of inhibiting autophagy for better immune infiltration in the tumor but also presents a novel tri-functional liposomal system that overcomes the deficiencies of current therapies and holds great promise in cancer immunotherapy.

Identification of 1,4-Benzodiazepine-2,5-dione Derivatives as Potential Protein Synthesis Inhibitors with Highly Potent Anticancer Activity.

In this study, a random multiple human tumor cell line screening of an in-stock small-molecule chemical library was performed, and a hit compound, 1,4-benzodiazepine-2,5-dione (BZD, 11a; average 50% growth inhibitory concentration (GI50 = 0.24 μM)) to 60 tumor cell lines of nine types of human cancers, was identified. Subsequent structure-activity relationship (SAR) investigation disclosed a highly potent antitumor compound, 52b, that was shown to exert promising effects against lung cancer cells by inducing cell cycle arrest and apoptosis. Further polysome profile analysis revealed that 52b inhibited protein synthesis in cancer cells. Moreover, 52b significantly prevented tumor growth in a human non-small-cell lung cancer (NCI-H522) xenograft mouse model with no observable toxic effects. These findings are the first report of the synthetic compound 52b with a 1,4-benzodiazepine-2,5-dione skeleton that acts as a potential protein synthesis inhibitor to effectively inhibit tumor growth.

BTApep-TAT peptide inhibits ADP-ribosylation of BORIS to induce DNA damage in cancer

BackgroundBrother of regulator of imprinted sites (BORIS) is expressed in most cancers and often associated with short survival and poor prognosis in patients. BORIS inhibits apoptosis and promotes proliferation of cancer cells. However, its mechanism of action has not been elucidated, and there is no known inhibitor of BORIS. MethodsA phage display library was used to find the BORIS inhibitory peptides and BTApep-TAT was identified. The RNA sequencing profile of BTApep-TAT-treated H1299 cells was compared with that of BORIS-knockdown cells. Antitumor activity of BTApep-TAT was evaluated in a non-small cell lung cancer (NSCLC) xenograft mouse model. BTApep-TAT was also used to investigate the post-translational modification (PTM) of BORIS and the role of BORIS in DNA damage repair. Site-directed mutants of BORIS were constructed and used for investigating PTM and the function of BORIS.ResultsBTApep-TAT induced DNA damage in cancer cells and suppressed NSCLC xenograft tumor progression. Investigation of the mechanism of action of BTApep-TAT demonstrated that BORIS underwent ADP ribosylation upon double- or single-strand DNA damage. Substitution of five conserved glutamic acid (E) residues with alanine residues (A) between amino acids (AAs) 198 and 228 of BORIS reduced its ADP ribosylation. Inhibition of ADP ribosylation of BORIS by a site-specific mutation or by BTApep-TAT treatment blocked its interaction with Ku70 and impaired the function of BORIS in DNA damage repair.ConclusionsThe present study identified an inhibitor of BORIS, highlighted the importance of ADP ribosylation of BORIS, and revealed a novel function of BORIS in DNA damage repair. The present work provides a practical method for the future screening or optimization of drugs targeting BORIS.

Abstract 5458: Pharmacodynamics of WX-0593 combined with bevacizumab biosimilar on human lung cancer xenograft mouse models

Abstract Objective: To evaluate the antitumor activity of WX-0593 (anaplastic lymphoma kinase/ROS proto-oncogene 1 receptor tyrosine kinase [ALK/ROS1] tyrosine kinase inhibitor) combined with bevacizumab biosimilar in vivo. Methods: Cell line-derived xenograft (CDX) model: NCI-H3122 cell suspension was subcutaneously inoculated into the right axilla of BALB/c-nu mice. When the average tumor volume reached 100-300 mm3, the mice were randomly divided into six groups (eight mice/group) and administered with solvent, WX-0593 1.5 or 5 mg/kg, bevacizumab biosimilar 1 mg/kg, or WX-0593 1.5 or 5 mg/kg + bevacizumab biosimilar 1 mg/kg, respectively. Patient-derived xenografts (PDX) model: LU-01-1443 tumor tissue blocks of 20-30 mm3 were subcutaneously inoculated into the right axilla of BALB/c-nu mice. When the average tumor volume reached 115 mm3, the mice were randomly divided into four groups (eight mice/group) and administered with solvent, WX-0593 2.5 mg/kg, bevacizumab biosimilar 1.5 mg/kg or their combination. Tumor diameter was measured twice a week. The tolerability was also assessed. Results: CDX tumor model: There were significant differences between each group and the control group (all P <0.01). The combination of WX-0593 5 mg/kg and bevacizumab biosimilar 1 mg/kg was significantly more effective than WX-0593 5 mg/kg alone or bevacizumab biosimilar 1 mg/kg alone (both P <0.01). PDX tumor model: Compared with the control group, single-agent WX-0593 2.5 mg/kg and bevacizumab biosimilar 1.5 mg/kg delayed tumor growth. The tumor suppressive effect of WX-0593 combined with bevacizumab biosimilar was impressive (treated/control ratio =11.81%; tumor growth inhibition =92.19%) and significantly better than WX-0593 2.5 mg/kg or bevacizumab biosimilar 1.5 mg/kg alone (P =0.004, 0.003, respectively). Toxicity: There were no abnormalities in appearance and mental state in each group during the experiment, and the weight of the mice remained stable. Conclusions: The combination of WX-0593 and bevacizumab biosimilar had obvious synergistic effect and showed potent tumor suppressive activity, which was significantly better than WX-0593 or bevacizumab biosimilar alone at the same dose. There was no drug-related toxicity in all groups, and no increase in toxicity after combined administration. Citation Format: Shansong Zheng, Qingmei Zheng, Xin Dong, Xinmei Wang, Dongmei Chen, Huicheng Duan, Cuicui Ma, Chenwei Zhang. Pharmacodynamics of WX-0593 combined with bevacizumab biosimilar on human lung cancer xenograft mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5458.

Nicotine-induced miR-21-3p promotes chemoresistance in lung cancer by negatively regulating FOXO3a.

Lung cancer is the leading cause of cancer-related mortality worldwide and cigarette smoking is reported to contribute to the lung cancer-related mortality. The present study aimed to investigate the molecular mechanism underlying nicotine-induced chemoresistance in lung cancer. The expression of microRNA (miR)-21-3p and its predicted target FOXO3a in lung cancer cells was detected via reverse transcription-quantitative PCR, in the presence or absence of nicotine. The regulatory effect of miR-21-3p and FOXO3a on lung cancer cell proliferation and apoptosis induced by docetaxel or cisplatin treatment was evaluated by performing Cell Counting Kit-8 and Annexin V/PI staining assays, respectively. The interaction between miR-21-3p and FOXO3a was analyzed by performing luciferase reporter assays and western blotting. FOXO3a overexpression rescue experiments were conducted in vitro and in vivo using a xenograft mouse model to assess the function of miR-21-3p/FOXO3a in lung cancer. Nicotine induced miR-21-3p expression in lung cancer cells in a dose-dependent manner. miR-21-3p downregulated FOXO3a expression by directly binding to the 3′-untranslated region of FOXO3a. Moreover, miR-21-3p knockdown sensitized lung cancer cells to docetaxel or cisplatin treatment. Mechanistically, FOXO3a was predicted as a direct target of miR-21-3p. FOXO3a overexpression promoted the chemosensitivity of lung cancer cells to docetaxel or cisplatin treatment. Furthermore, FOXO3a overexpression antagonized the regulatory function of miR-21-3p on docetaxel- or cisplatin-treated lung cancer cells. In the docetaxel- or cisplatin-treated lung cancer xenograft mouse model, miR-21-3p promoted chemoresistance via negatively regulating FOXO3a. Therefore, the present study demonstrated that nicotine-induced miR-21-3p promoted chemoresistance to docetaxel or cisplatin treatment via negatively regulating FOXO3a, which may serve as a novel therapeutic strategy for the treatment of patients with chemoresistant lung cancer.

Treatment of malignant pleural effusion in non-small cell lung cancer with VEGF-directed therapy

Background Vascular endothelial growth factor (VEGF) is a critical regulator of malignant pleural effusion (MPE) in non-small-cell lung cancer (NSCLC). Bevacizumab (BEV) and apatinib (APA) are novel VEGF blockers that inhibit lung cancer cell proliferation and the development of pleural effusion. Methods In this study, we established Lewis lung cancer (LLC) xenograft mouse models to compare the therapeutic effect of APA and BEV in combination with cisplatin (CDDP) against MPE. The anti-tumour and anti-angiogenic effects of this combination therapy were evaluated by 18F-FDG PET/CT imaging, TUNEL assay and Immunohistochemistry. Results The triple drug combination significantly prolonged the overall survival of the tumour-bearing mice by reducing MPE and glucose metabolism and was more effective in lowering VEGF/soluble VEGFR-2 levels in the serum and pleural exudates compared to either of the monotherapies. Furthermore, CDDP + APA + BEV promoted in vivo apoptosis and decreased microvessel density. Conclusions Mechanistically, LLC-induced MPE was inhibited by targeting the VEGF-MEK/ERK pathways. Further studies are needed to establish the synergistic therapeutic effect of these drugs in NSCLC patients with MPE. KEY MESSAGES Combined treatment of MPE with apatinib, bevacizumab and cisplatin can prolong the survival time of mice, reduce the content of MPE, decrease the SUVmax of thoracic tumour tissue, down-regulate the content of VEGF and sVEGFR-2 in serum and pleural fluid, and promote the apoptosis of tumour cells. Angiogenesis and MPE formation can be inhibited by down-regulation of HIF-1α, VEGF, VEGFR-2, MEK1 and MMP-2 molecular signalling pathway proteins.

Heterogeneous nuclear ribonucleoprotein K promotes the progression of lung cancer by inhibiting the p53-dependent signaling pathway.

BackgroundHeterogeneous nuclear ribonucleoprotein K (hnRNPK) is a nucleic acid‐binding protein. Reportedly, hnRNPK is overexpressed in many human tumors, and such overexpression is associated with poor prognosis, implicating the role of hnRNPK as an oncogene during tumorigenesis. In this study, hnRNPK expression in lung cancer tissues was investigated.MethodsBriefly, hnRNPK was knocked down in lung cancer cell lines, and effects of knockdown on the cell proliferation, migration, and cell cycle were assessed using a cell counting kit‐8 (CCK‐8) assay, colony formation assay, transwell assay and flow cytometry. The effects of hnRNPK knockdown on the p53‐dependent signaling pathway were examined using western blotting. Finally, the effect of hnRNPK knockdown on tumor growth was verified in vivo using a lung cancer xenograft mouse model.ResultshnRNPK knockdown inhibited the cell proliferation, migration and cell cycle. In addition to phenotypic changes, hnRNPK knockdown upregulated expressions of pCHK1, pCHK2, and p53，p21，cyclin D1, thereby mediating the DNA damage response (DDR). The regulatory function of hnRNPK during p53/p21/cyclin D1 signaling in hnRNPK‐knockdown A549 cells was confirmed by suppressed the protein expression of associated signaling pathways, which inhibited DDR.ConclusionhnRNPK plays a crucial role in the progression of lung cancer, ultimately affecting survival rate. Inhibition of progression of lung cancer cells induced by hnRNPK‐knockdown is dependent on activation of p53 by the p53/p21/cyclin D1 pathway.

Administration of Gapmer-type Antisense Oligonucleotides Targeting γ-Glutamylcyclotransferase Suppresses the Growth of A549 Lung Cancer Xenografts.

γ-Glutamyl cyclotransferase (GGCT) is up-regulated in various cancer types, including lung cancer. In this study, we evaluated efficacy of gapmer-type antisense oligonucleotides (ASOs) targeting GGCT in an A549 lung cancer xenograft mouse model and studied their mechanisms of action. GGCT was inhibited using GGCT-ASOs and cell proliferation was evaluated by dye exclusion test. Western blot analysis was conducted to measure expression of GGCT, p21, p16 and p27, phosphorylation of AMP-activated protein kinase, and caspase activation in A549 cells. Induction of apoptosis and up-regulation of reactive oxygen species were assessed by flow cytometry using annexin V staining and 2',7'-dichlorodihydrofluorescein diacetate dye, respectively. GGCT-ASOs suppressed GGCT expression in A549 cells, inhibited proliferation, and induced apoptosis with activation of caspases. GGCT-ASOs also increased expression of cell-cycle regulating proteins, phospho-AMPK and ROS levels. Systemic administration of GGCT-ASOs to animals bearing A549 lung cancer xenografts showed significant antitumor effects without evident toxicity. GGCT-ASOs appear to be promising as novel cancer therapeutic agents.

Human Amniotic Epithelial Cells and Their Derived Exosomes Protect Against Cisplatin-Induced Acute Kidney Injury Without Compromising Its Antitumor Activity in Mice.

Background: Cisplatin is a widely used chemotherapeutic drug, whereas the clinical application is greatly limited by its nephrotoxic side effect. Currently, there has been no effective treatment to prevent cisplatin-induced acute kidney injury (cisplatin-AKI). Human amniotic epithelial cells (hAECs) and their derived exosomes (EXOs) have been proven to effectively protect against ischemia reperfusion-induced AKI, yet their roles in cisplatin-AKI are still unknown. Methods: C57BL/6J mice were given two doses of cisplatin at 20 or 15 mg/kg of body weight to induce AKI with or without mortality. hAECs or EXOs were injected via tail vein 1 day after cisplatin administration. Serum and kidney tissues were collected on the fourth day after 15 mg/kg cisplatin treatment to explore the nephro-protective effects of hAECs and EXOs on cisplatin-AKI. Lung cancer xenograft model was built by subcutaneous injection of A549 cells into BALB/c nude mice to evaluate the effect of hAECs or EXOs on cisplatin chemotherapy. Results: Cisplatin nephrotoxicity was significantly attenuated by hAECs and EXOs as evidenced by reduced mortality rate and decreased serum creatinine (sCr) and reduced tubular injury score. hAECs or EXOs exerted the nephro-protective effects via suppression of TNF-α/MAPK and caspase signaling pathways. In the A549 lung cancer xenograft mouse model, administration of hAECs or EXOs did not promote tumor growth or compromise the therapeutic effects of cisplatin on tumors. Conclusion: This study is the first to demonstrate that hAECs and their derived exosomes have nephro-protective effects in cisplatin-AKI in vivo. Importantly, neither hAECs nor EXOs compromise the antitumor activity of cisplatin. These results potentially support the use of hAECs and their derived EXOs as nephro-protectors against cisplatin-induced nephrotoxicity clinically.

Potential applications of clickable probes in EGFR activity visualization and prediction of EGFR-TKI therapy response for NSCLC patients

The epithelial growth factor receptor (EGFR) is abnormally overexpressed on the cell surface of cancer cells and is strongly associated with cancer cell proliferation, migration, differentiation, apoptosis, and angiogenesis. Tools enabling the visualization of EGFR in a structure-function approach are highly desirable to predict EGFR mutations and guide EGFR tyrosine kinase inhibitor (TKI) treatment making. Here, we describe the design, synthesis, and application of new, potent and selective clickable probes 13 (HX03), 20 (HX04) and 24 (HX05) by introducing an alkyne ligation handle to visualize EGFR activity in living cancer cells and tissue slices. These clickable probes are versatile chemical tools based on the key pharmacophore (4-anilinoquinazoline) of EGFR-TKIs (e.g., canertinib, dacomitinib and afatinib) and are able to irreversibly target the kinase domain of EGFR. Among them, 13 exhibits the highest reactivity towards EGFR kinase, particularly to EGFR kinase with primary mutations. Using activity-based protein profiling strategy, 13 showed high sensitivity and selectivity in labeling of endogenous EGFR in a native cellular context. Moreover, 13 was applied to visualize EGFR mutant activity in tumour tissues from non-small-cell lung cancer (NSCLC) xenograft mouse models, and patients with NSCLC for the prediction of EGFR-TKI sensitivity. These results demonstrate that strategically designed EGFR-TKI-based probes allow discriminating EGFR mutations in human tissues and hold promise as useful diagnostic tools in predicting EGFR-TKI therapy response.

Targeting AKR1B1 inhibits glutathione de novo synthesis to overcome acquired resistance to EGFR-targeted therapy in lung cancer.

Acquired resistance represents a bottleneck to molecularly targeted therapies such as epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) treatment in lung cancer. A deeper understanding of resistance mechanisms can provide insights into this phenomenon and help to develop additional therapeutic strategies to overcome or delay resistance. Here, we identified a pharmacologically targetable metabolic mechanism that drives resistance to EGFR TKIs in lung cancer cell lines and patient-derived xenograft mice. We demonstrated that aldo-keto reductase family 1 member B1 (AKR1B1) interacts with and activates signal transducer and activator of transcription 3 (STAT3) to up-regulate the cystine transporter solute carrier family 7 member 11 (SLC7A11). This leads to enhanced cystine uptake and flux to glutathione de novo synthesis, reactive oxygen species (ROS) scavenging, protection from cell death, and EGFR TKI drug resistance in lung cancer cell lines and xenograft mouse models. Suppression of AKR1B1 with selective inhibitors, including the clinically approved antidiabetic drug epalrestat, restored the sensitivity of resistant cell lines to EGFR TKIs and delayed resistance in lung cancer patient-derived xenograft mice. Our findings suggest a metabolic mechanism for resistance to a molecularly targeted therapy and provide a potential therapeutic target for overcoming resistance to EGFR TKIs, including the third-generation inhibitor osimertinib.

Single-cell lineages reveal the rates, routes, and drivers of metastasis in cancer xenografts.

Detailed phylogenies of tumor populations can recount the history and chronology of critical events during cancer progression, such as metastatic dissemination. We applied a Cas9-based, single-cell lineage tracer to study the rates, routes, and drivers of metastasis in a lung cancer xenograft mouse model. We report deeply resolved phylogenies for tens of thousands of cancer cells traced over months of growth and dissemination. This revealed stark heterogeneity in metastatic capacity, arising from preexisting and heritable differences in gene expression. We demonstrate that these identified genes can drive invasiveness and uncovered an unanticipated suppressive role for KRT17 We also show that metastases disseminated via multidirectional tissue routes and complex seeding topologies. Overall, we demonstrate the power of tracing cancer progression at subclonal resolution and vast scale.

Enhance anti-lung tumor efficacy of chimeric antigen receptor-T cells by ectopic expression of C–C motif chemokine receptor 6

Chimeric antigen receptor-T (CAR-T) cells have limited therapeutic efficacy against solid tumors, partially due to their limited ability to reach and invade into the neoplastic foci. By gene expression profiling interactive analysis, we identified that the C–C motif chemokine ligand (CCL) 20 is highly expressed in lung and other most incidence and/or mortality cancers such as colon, rectum, stomach, and liver cancers. Forced expression of C–C motif chemokine receptor 6 (CCR6), the biunique receptor of CCL20, results in robust trafficking of CAR-T cells toward CCL20-secreting tumor cells. In a lung cancer xenograft mouse model, CCR6-expressing CAR-T cells efficiently migrate to and infiltrate into solid tumors upon infusion, leading to effective tumor clearance and significantly prolonged survival of tumor-bearing mice. In addition, culturing CCR6-CAR-T cells with interleukin (IL)-7 and IL-15 further improved their anti-lung cancer activity. Our findings provide supporting evidence for the clinical development of chemokine receptor-engineered CAR-T cells for solid tumor immunotherapy.

Abstract 5956: New molecular mechanisms of small cell lung cancer growth

Abstract Lung cancer is the leading cause of cancer death among both men and women in the United States. Small cell lung cancer (SCLC) results in over 30,000 deaths of Americans annually and comprises 15-17% incidences of lung cancer. SCLC is the most aggressive form of lung cancer due to its rapid doubling time and early widespread metastasis. Most SCLC patients present with advanced disease, respond to initial systemic chemotherapy, and then treatment refractory progression usually occurs within one year due to acquired drug resistance. Consequently, the median survival time of SCLC patients is only 9 to 20 months and merely 7% of SCLC patients survive beyond five years. A better molecular understanding of SCLC is necessary to develop much needed new treatment strategies. For the first time, we demonstrate that dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32) and its splice variant t-DARPP stimulate tumors of neuroendocrine origin, and we show DARPP-32 proteins are aberrantly overexpressed in human SCLC. Upregulation of t-DARPP protein in a subset of SCLC patients positively correlates with overexpression of genes associated with Notch signaling, including achaete-scute homologue 1 (ASCL1). We reveal that DARPP-32 isoforms are transcriptionally activated by ASCL1 in human SCLC cells. To determine whether DARPP-32 drives SCLC growth in vivo, we utilized an orthotopic lung cancer xenograft mouse model. Mice challenged with DARPP-32-ablated DMS-53 cells show a substantial decrease in lung tumor growth relative to controls. Correspondingly, we demonstrate increased tumor growth in mice harboring lung tumors formed by administration of DMS-53 or H1048 cells overexpressing DARPP-32 and t-DARPP proteins. Mechanistically, we describe how DARPP-32 isoforms promote SCLC growth through increased proliferation, Akt/Erk-mediated survival, and anti-apoptotic signaling. Taken together, we define new regulatory mechanisms of SCLC oncogenesis that suggest DARPP-32 isoforms may represent a negative prognostic indicator for SCLC and serve as a potential target for the development of new therapies. Citation Format: Sk. Kayum Alam, Li Wang, Yanan Ren, Christina E. Hernandez, Farhad Kosari, Anja C. Roden, Rendong Yang, Luke H. Hoeppner. New molecular mechanisms of small cell lung cancer growth [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5956.

Blocking STAT3 by pyrvinium pamoate causes metabolic lethality in KRAS-mutant lung cancer

Signal transducer and activator of transcription 3 (STAT3) exerts a profound role in regulating mitochondrial function and cellular metabolism. Mitochondrial STAT3 supports RAS-dependent malignant transformation and tumor growth. However, whether pharmacological blockade of STAT3 leads to metabolic lethality in KRAS-mutant lung cancer remains unclear. Pyrvinium pamoate, a clinical antihelminthic drug, preferentially inhibited the growth of KRAS-mutant lung cancer cells in vitro and in vivo. Mechanistic study revealed that pyrvinium dose-dependently suppressed STAT3 phosphorylation at tyrosine 705 and serine 727. Overexpression mitochondrial STAT3 prominently weakened the therapeutic efficacy of pyrvinium. As a result of targeting STAT3, pyrvinium selectively triggered reactive oxygen species release, depolarized mitochondrial membrane potential and suppressed aerobic glycolysis in KRAS-mutant lung cancer cells. Importantly, the cytotoxic effects of pyrvinium could be significantly augmented by glucose deprivation both in vitro and in a patient-derived lung cancer xenograft mouse model in vivo. The combined efficacy significantly correlated with intratumoural STAT3 suppression. Our findings reveal that KRAS-mutant lung cancer cells are vulnerable to STAT3 inhibition exerted by pyrvinium, providing a promising direction for developing therapies targeting STAT3 and metabolic synthetic lethality for the treatment of KRAS-mutant lung cancer.

A Novel Paclitaxel Conjugate with Higher Efficiency and Lower Toxicity: A New Drug Candidate for Cancer Treatment.

Paclitaxel-lipoate (IDD-1040) is a conjugate formed by the chemical joining of the two compounds, by condensing a lipoic acid moiety to the C2′ of paclitaxel. IDD-1040 was evaluated for its anti-tumor activity and potential druggability, using an in vivo non-small-cell, lung cancer (NSCLC) xenograft mouse model. In the in vivo studies, IDD-1040 showed a maximum tolerated dose (MTD) of 250 mg/kg compared to paclitaxel (PTX), with an MTD of 20 mg/kg. Most interesting, IDD-1040 demonstrated higher anti-tumor activity, and its inhibitory activity on tumor volume (cell growth) was dose-dependent. That anti-tumor activity persisted for two weeks after cessation of IDD-1040 treatment, as opposed to PTX cessation, after which the tumor relapsed, confirming that IDD-1040 exhibits superior tumor inhibition. Similar to PTX treatment, no marked body weight decrease was observed during IDD-1040 treatment, indicating a low toxicity profile. The increase in animal body weight noted over time was due to the increasing weight of tumors, recorded in all the mouse test groups. The results also showed that mortality rate of mice was reduced by treatment with IDD-1040, more so than with PTX. Furthermore, in a preliminary study on the ex vivo distribution of IDD-1040, neutropenia was primarily concentrated in the liver 1 h after injection, and most of the drug was metabolized by the liver in 24 h. All of these results demonstrate IDD-1040’s great potential as a candidate drug for cancer treatment.

Sulforaphane inhibits epithelial–mesenchymal transition by activating extracellular signal-regulated kinase 5 in lung cancer cells

Epithelial–mesenchymal transition (EMT) contributes to the initiation, invasion, metastasis and drug resistance of cancer. The function of extracellular signal-regulated kinase 5 (ERK5) in lung cancer progression remains elusive. In this study, we investigated the effect of sulforaphane (SFN) on lung cancer EMT and the role of ERK5 in its effect. Wound healing and Transwell assays were applied to examine the migratory and invasive capacity in vitro. Quantitative real-time polymerase chain reaction and immunoblotting analysis were performed to investigate the expression of mRNA and protein levels. Small-interfering RNA was used to silence ERK5. Xenograft model was used to confirm the effect of SFN in vivo. Enhanced EMT and decreased ERK5 activation were observed in lung cancer cells in comparison with normal human bronchial epithelial cells. SFN diminished the migratory and invasive capacity of lung cancer cells. Additionally, significantly increased expression of epithelial markers (E-cadherin and ZO-1), decreased expression of mesenchymal markers (N-cadherin and Snail1) and activation of ERK5 were observed after SFN treatment. The inhibitory effect of SFN on lung cancer cell EMT was attenuated by ERK5 silencing. SFN-induced EMT suppression and ERK5 activation were further confirmed in lung cancer xenograft mouse model. The present study illustrated for the first time that ERK5 activation mediates SFN suppression of lung cancer cell EMT. These findings could provide new insights into the function of ERK5 in EMT regulation and the potential therapeutic application of SFN in cancer intervention.