KRAS-mutant Lung Cancer Cells Research Articles

Small-molecule inhibition of MAP2K4 is synergistic with RAS inhibitors in KRAS-mutant cancers

The Kirsten rat sarcoma viral oncogene homologue KRAS is among the most commonly mutated oncogenes in human cancers, thus representing an attractive target for precision oncology. The approval for clinical use of the first selective inhibitors of G12C mutant KRAS therefore holds great promise for cancer treatment. However, despite initial encouraging clinical results, the overall survival benefit that patients experience following treatment with these inhibitors has been disappointing to date, pointing toward the need to develop more powerful combination therapies. Here, we show that responsiveness to KRASG12C and pan-RAS inhibitors in KRAS-mutant lung and colon cancer cells is limited by feedback activation of the parallel MAP2K4-JNK-JUN pathway. Activation of this pathway leads to elevated expression of receptor tyrosine kinases that reactivate KRAS and its downstream effectors in the presence of drug. We find that the combination of sotorasib, a drug targeting KRASG12C, and the MAP2K4 inhibitor HRX-0233 prevents this feedback activation and is highly synergistic in a panel of KRASG12C-mutant lung and colon cancer cells. Moreover, combining HRX-0233 and sotorasib is well-tolerated and resulted in durable tumor shrinkage in mouse xenografts of human lung cancer cells, suggesting a therapeutic strategy for KRAS-driven cancers.

Ganoderma microsporum immunomodulatory protein combined with KRASG12C inhibitor impedes intracellular AKT/ERK network to suppress lung cancer cells with KRAS mutation

KRAS mutations are tightly associated with lung cancer progression. Despite the unprecedented clinical success of KRASG12C inhibitors, recurrent mechanisms of resistance and other KRAS mutations require further therapeutic approaches. GMI, a protein from the medicinal mushroom Ganoderma microsporum, possesses antitumor activity; whereas, the biological function of GMI on regulating KRAS mutant lung cancer cells remains unknown. Herein, RNA-sequencing and bioinformatics showed that GMI may regulate KRAS-modulated MAPK and PI3K-AKT pathways in A549 (KRASG12S) cells. Further experiments demonstrated that GMI inhibited KRAS activation and suppressed ERK1/2 and AKT signaling in A549 cells. Intriguingly, GMI inhibited AKT signaling but increased phosphorylation of ERK in H358 (KRASG12C) cells. GMI significantly suppressed tumor growth in LLC1 cells-allograft and H358 cells-xenograft mice. GMI showed a synergistic effect with KRASG12C inhibitors in inhibiting cell growth, KRAS activation and KRAS-mediated downstream signaling, leading to apoptosis in H358 cells. Combination of GMI and KRASG12C inhibitor, AMG 510, resulted in more durable inhibition of tumor growth and KRAS activity in H358 cells-xenograft mice. This study highlights the potential of GMI, a dietary fungal protein, as a viable therapeutic avenue for KRAS-mutant lung cancer in combination with KRASG12C inhibitors.

Spermine-Based Poly(β-amino ester)s for siRNA Delivery against Mutated KRAS in Lung Cancer.

Polyethylenimine (PEI) is a highly efficient cationic polymer for nucleic acid delivery, and although it is commonly used in preclinical studies, its clinical application is limited because of concerns regarding its cytotoxicity. Poly(β-amino ester)s are a new group of biodegradable and biocompatible cationic polymers that can be used for siRNA delivery. In this study, we synthesized Boc-protected and deprotected poly(β-amino ester)s, P(BSpBAE) and P(SpBAE), respectively, based on spermine and 1,4-butanediol diacrylate to deliver siRNA. The polymers were synthesized by Michael addition in a step-growth polymerization and characterized via 1H NMR spectroscopy and size-exclusion chromatography (SEC). The polymers can encapsulate siRNA as determined by SYBR gold assays. Both polymers and polyplexes were biocompatible in vitro. Furthermore, the cellular uptake of P(BSpBAE) and P(SpBAE) polyplexes was more efficient than for branched PEI (25 kDa) polyplexes at the same N/P ratios. P(BSpBAE) polyplexes achieved 60% eGFP knockdown in vitro, which indicates that the Boc-protection can improve the siRNA delivery and gene silencing efficiency of PBAEs. P(BSpBAE) polyplexes and P(SpBAE) polyplexes showed different cellular uptake mechanisms, and P(BSpBAE) polyplexes demonstrated decreased endosomal entrapment, which could explain why P(BSpBAE) polyplexes more efficiently mediated gene silencing than P(SpBAE) polyplexes. Furthermore, transfection of an siRNA against mutated KRAS in KRAS-mutated lung cancer cells led to around 35% (P(BspBAE)) to 45% (P(SpBAE)) inhibition of KRAS expression and around 33% (P(SpBAE)) to 55% (P(BspBAE)) decreased motility in a migration assay. These results suggest that the newly developed spermine-based poly(β-amino ester)s are promising materials for therapeutic siRNA delivery.

Histone deacetylase inhibitor belinostat regulates metabolic reprogramming in killing KRAS-mutant human lung cancer cells.

Kirsten rat sarcoma virus (KRAS) oncogene, found in 20%-25% of lung cancer patients, potentially regulates metabolic reprogramming and redox status during tumorigenesis. Histone deacetylase (HDAC) inhibitors have been investigated for treating KRAS-mutant lung cancer. In the current study, we investigate the effect of HDAC inhibitor (HDACi) belinostat at clinically relevant concentration on nuclear factor erythroid 2-related factor 2 (NRF2) and mitochondrial metabolism for the treatment of KRAS-mutant human lung cancer. LC-MS metabolomic study of belinostat on mitochondrial metabolism was performed in G12C KRAS-mutant H358 non-small cell lung cancer cells. Furthermore, l-methionine (methyl-13 C) isotope tracer was used to explore the effect of belinostat on one-carbon metabolism. Bioinformatic analyses of metabolomic data were performed to identify the pattern of significantly regulated metabolites. To study the effect of belinostat on redox signaling ARE-NRF2 pathway, luciferase reporter activity assay was done in stably transfected HepG2-C8 cells (containing pARE-TI-luciferase construct), followed by qPCR analysis of NRF2 and its target gene in H358 cells, which was further confirmed in G12S KRAS-mutant A549 cells. Metabolomic study reveals significantly altered metabolites related to redox homeostasis, including tricarboxylic acid (TCA) cycle metabolites (citrate, aconitate, fumarate, malate, and α-ketoglutarate); urea cycle metabolites (Arginine, ornithine, argino-succinate, aspartate, and fumarate); and antioxidative glutathione metabolism pathway (GSH/GSSG and NAD/NADH ratio) after belinostat treatment. 13 C stable isotope labeling data indicates potential role of belinostat in creatine biosynthesis via methylation of guanidinoacetate. Moreover, belinostat downregulated the expression of NRF2 and its target gene NAD(P)H:quinone oxidoreductase 1 (NQO1), indicating anticancer effect of belinostat is mediated, potentially via Nrf2-regulated glutathione pathway. Another HDACi panobinostat also showed potential anticancer effect in both H358 and A549 cells via Nrf2 pathway. In summary, belinostat is effective in killing KRAS-mutant human lung cancer cells by regulating mitochondrial metabolism which could be used as biomarkers for preclinical and clinical studies.

Abstract 1078: Sotorasib and metformin combination enhances cytotoxicity and apoptosis in KRAS mutant lung cancer cell lines

Abstract KRAS is the most frequently mutated oncogene in lung adenocarcinoma (50%) and it is associated with a poor response to antineoplastic treatments, as well as poorer survival rates. KRAS activates the MAPK pathway, promoting cell survival and increasing protein synthesis. Novel inhibitors of KRAS G12C mutation (sotorasib) have demonstrated short-lasting responses due to resistance mediated by the AKT-mTOR-P70S6K pathway. In this context, metformin is a promising candidate to avoid this inconvenience by inhibiting mTOR and P70S6K. Therefore, this project aimed to explore the effects of the combination of sotorasib and metformin on cytotoxicity, apoptosis, and the activity of the mTOR pathway in lung cancer cell lines with or without KRAS mutations. Methods: We performed dose-effect curves to determine the IC50 concentration of sotorasib, and IC10 of metformin. Then, these doses were used to treat lung cancer cell lines A549 (KRAS G12S), H522 (without KRAS), and H23 (KRAS G12C). Cellular cytotoxicity was evaluated by MTT assay, apoptosis induction through flow cytometry with Annexin V and 7AAD, and the activity of P70S6K and AKT proteins by western blot. Main outcomes and measures: cell survival, apoptosis, expression, and phosphorylation of intermediaries belonging to the mTOR pathway. Results: The combined treatment showed a synergic effect on cytotoxicity, increased apoptosis induction, and notable inhibition of AKT and P70S6K1 proteins, compared to sotorasib alone or to control groups in all the cell lines, but predominantly in KRAS-mutated cells (H23 and A549). Conclusions: The combination of metformin with sotorasib showed synergic effects on cytotoxicity, increased apoptosis induction, and a remarkable inhibition of downstream proteins involved in the signaling of growth factor receptors in all tested cells. Moreover, these results suggest a sensitizing effect of metformin to sotorasib treatment in cells without KRAS mutations, such as H522. Citation Format: Norma Yanet Hernandez-Pedro, Pedro Barrios-Bernal, José Lucio-Lozada, Maritza Ramos-Ramirez, Oscar Arrieta. Sotorasib and metformin combination enhances cytotoxicity and apoptosis in KRAS mutant lung cancer cell lines [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 1078.

Abstract 6141: Mechanisms by which Mito-lonidamine inhibits EGFR mutant lung cancer

Abstract Lung cancer remains the leading cause of cancer mortality. Efficacious targeted therapy options are urgently needed for lung cancer prevention and treatment. Epidermal growth factor receptor (EGFR) mutant lung cancer represents about 15% of lung cancer cases generally occurring in patients with minimal to no smoking history and with higher prevalence in females. Linkages to environmental exposures have been postulated. Our group synthesized Mito-lonidamine (Mito-LND), a mitochondrial targeted analog of lonidamine which inhibits aerobic glycolysis in cancer cells. We previously identified that Mito-LND induces mitophagy in KRAS mutant lung cells and inhibits lung tumorigenesis and brain metastasis in vivo. Yet, the cancer inhibitory capacity of Mito-LND in EGFR mutant lung cancer has not been investigated. Viability staining assessed Mito-LND induced cell death in EGFR mutant cell lines PC9 (parent) and PC9BrM3 (brain metastatic). Transcriptomic profiling was conducted following Mito-LND treatment and results analyzed in Metacore to assess molecular changes. Deconvolution analysis interrogated alterations in immune cell populations. To discern cell death mechanisms, we evaluated FDA-approved inhibitors for autophagy (chloroquine, CQ), mitophagy/apoptosis (cyclosporin A, CsA) and necrosis (Ponatinib) in EGFR mutant cell lines using viability assays with Mito-LND treatment. In PC9 cells, the IC50 for Mito-LND at 24h and 48h was 1.5µM and 0.5µM, respectively and in PC9BrM3 cells at 24h and 48h was 1.5µM and 0.7µM, respectively. These data support that EGFR mutant cell lines are >2-fold more sensitive to Mito-LND induced cell death compared to KRAS mutant lung cell lines H2030 and H2030BrM3. Transcriptomic profiling results show that Mito-LND treatment upregulated pathway maps linked to heat shock protein and HIF-1A signaling in both PC9 and PC9BrM3 cells. Process networks linked to unfolded protein response, response to hypoxia and oxidative stress, apoptosis and the immune response were upregulated with Mito-LND treatment. Deconvolution analysis revealed monocytes and macrophages were upregulated in Mito-LND treated EGFR mutant cell lines, with CD8+ T cells increased in PC9BrM3 cells and regulatory T cells increased in PC9 cells. Last, CsA significantly inhibited Mito-LND induced cell death in both cell lines, while CQ had no effect suggesting blocking apoptosis, but not late autophagy inhibits cell death induction in EGFR mutant cell lines. Further, necrosis inhibition with Ponatinib synergistically increased Mito-LND induced cell death in EGFR mutant cell lines. These results begin to define the cancer inhibitory mechanisms of Mito-LND in EGFR mutant lung cancer cells which appear to differ from those observed in KRAS mutant lung cancer cells. Future directions include dissecting apoptotic and necrosis driven Mito-LND induced cell death using additional pharmacological inhibitors and genetic approaches. Citation Format: Katherine M. Weh, Connor Howard, Yun Zhang, Jean-Jack Riethoven, Jennifer Clarke, Gang Cheng, Balaraman Kalyanaraman, Ming You, Laura Kresty. Mechanisms by which Mito-lonidamine inhibits EGFR mutant lung cancer [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 6141.

Abstract 412: Drug combination screen identifies pemigatinib, an FGFR inhibitor, as a mechanism to overcome KRASG12C inhibitor resistance in lung cancer

Abstract Background: Lung cancer is one of the most common tumors, accounting for approximately 1.8 million deaths worldwide in 2020. KRAS is a frequently mutated oncogene, with mutations reported in roughly 20-25% of non-small cell lung cancer cases. Specifically, KRAS mutations at amino acid 12, resulting in a glycine to cysteine (G12C) substitution, occur in approximately 13% and 3% of lung and colon cancers, respectively, and less frequently in other solid tumors. Recently, the development of covalent KRASG12C inhibitors has shown meaningful anticancer activity in patients. However, many patients with KRASG12C do not respond and/or develop resistance to single-agent treatment. Here we investigate combination therapies that may overcome resistance and broaden patient response to KRASG12C inhibitors. Methods: In vitro preclinical KRASG12C models including lung and colon cancer cell lines were treated with a panel of small molecule and covalent KRASG12C inhibitors. The activity of single-agent versus combination treatments was measured in cell viability assays, and screen hits were validated by in vitro mechanism of action studies. Results: A curated set of 152 compounds was used in combination with screen monitoring cell-viability assays across 12 KRAS mutant lung and colon cancer cell lines. Compounds tested included kinase inhibitors targeting oncogenic signaling pathways, epigenetics modifiers, regulators of apoptosis, chemotherapeutics, and other anticancer agents. The screen identified synergy between covalent KRASG12C and several other inhibitors including those for FGFR, EGFR, and SHP2. Notably, pemigatinib, a potent and selective FGFR1-3 inhibitor, had a significantly high Bliss synergy score. Selectivity and siRNA knockdown experiments were performed to identify the specific FGFR isoform involved in synergistic anticancer activity. Inhibition of FGFR1 activity was shown to be essential, whereas an FGFR2-3 specific inhibitor demonstrated only modest activity in combination with KRASG12C inhibitors. Additionally, FGFR1 knockdown combined with KRASG12C inhibition resulted in increased drug sensitivity; in contrast, knockdown of other FGFR family members did not demonstrate similar increase in sensitivity to KRASG12C inhibition. Conclusions: Cell-viability combination screening identified strong synergy between KRASG12C covalent inhibitors and the FGFR1-3 inhibitor pemigatinib in KRASG12C cancer cell lines. The synergistic effect of pemigatinib and KRASG12C inhibition was notably strongest in mesenchymal cancer cell lines. This effect was further validated in in vitro selectivity and siRNA studies. Together, our results support pemigatinib as a promising agent for combination therapy with KRAS inhibitors in lung cancer. Citation Format: Angela Abdollahi, Margaret Favata, Darlise DiMatteo, Sean Schuette, Erika Boarder, Mark Rupar, Ricardo Macarron, Aidan Gilmartin, Hui Wang, Alejandro Amador-Arjona. Drug combination screen identifies pemigatinib, an FGFR inhibitor, as a mechanism to overcome KRASG12C inhibitor resistance in lung cancer [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 412.

Abstract 364: Multi-omics approaches revealed the involvement of immune cells in skeletal muscles of lung cancer cachexia mouse model

Abstract Cancer-associated cachexia (CAC) is a multifactorial syndrome characterized by loss of body weight and skeletal muscle mass that cannot be fully recovered by nutritional support. It accounts for approximately 20 % of cancer-related deaths. However, there are no approved treatments for CAC yet. In order to better understand CAC, we wanted to apply the multi-omics approaches to simultaneously assess tumors and skeletal muscles in CAC mouse models. First, we established a lung cancer cachexia mouse model by orthotopic transplantation of KRAS-mutant lung cancer cells. Lung tumor-bearing mice developed cachexia at 7 weeks post injection, showing over 20 % of body weight loss and wasting of skeletal muscles and fats. The histological and immunoblotting analyses revealed degradation of gastrocnemius (GA) and tibialis anterior (TA) muscles in CAC group. Then, we performed the bulk RNA sequencing to assess transcriptomic changes in lung tumors and GA muscles of CAC mice. In lung tumors, genes associated to ATP metabolic process was upregulated. However, skeletal muscles of CAC mice showed upregulation of genes involved in protein degradation and immune responses, while genes related to energy metabolism were downregulated. Notably, the immunohistochemical staining revealed the increased numbers of infiltrating macrophages and CD8+ T cells in skeletal muscles of CAC mice. These data suggest the involvement of immune cells in lung cancer-associated cachexia. To further identify the distinct immune cell populations involved in CAC, the single cell RNA sequencing was conducted using CD45+ immune cells infiltrated into GA muscles of CAC mice. The enrichment of natural killer cells, dendritic cells and T cells was detected in GA of CAC mice. Moreover, we confirmed the roles of the infiltrating immune cells by using the neutralizing antibodies in CAC mice. Taken together, these data suggest the involvement of the immune cells in the skeletal muscle wasting of CAC mice. These multi-omics approaches will shed light on development of therapeutics for cancer-induced cachexia. Citation Format: Se-Young Park, Bomi Kim, Myunggyo Lee, Haeseung Lee, Na-Young Song. Multi-omics approaches revealed the involvement of immune cells in skeletal muscles of lung cancer cachexia mouse model [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 364.

Natural product manoalide promotes EGFR-TKI sensitivity of lung cancer cells by KRAS-ERK pathway and mitochondrial Ca2+ overload-induced ferroptosis.

Background: Manoalide (MA), a proven natural inhibitor of PLA2 has anticancer effects, but its potential application and mechanism as an anticancer drug to promote EGFR-TKI sensitivity in lung cancer cells have not been studied. Methods: KRAS-mutated lung cancer cells and organoids, acquired osimertinib-resistant lung cancer cell lines HCC827OR, were used as EGFR-TKI-resistant models. CCK-8, clone formation, apoptosis assays, and calcein-AM staining were performed to investigate the inhibitory effects of MA in lung cancer cells and organoids. The flow cytometry or confocal microscope was used to detect lipid droplets, ROS, lipid peroxidation, mitochondria Ca2+, and iron content. The oxygen consumption rate (OCR) and fatty acid oxidation (FAO) were used to estimate the effect of MA on mitochondrial function. Results: MA inhibits the proliferation of KRAS-mutated lung cancer cells and organoids. In addition, MA induces ER stress in a ROS-dependent mechanism. The ROS induced by MA is mainly in mitochondrial and causes lipid peroxidation, thereby inhibiting mitochondrial FAO metabolism and promoting the accumulation of lipid droplets. MA also suppresses the KRAS-ERK pathway through ROS and promotes the sensitivity of KRAS-mutated lung cancer cells and organoids to osimertinib. Furthermore, MA induces ferroptosis by suppressing the NRF2-SLC7A11 axis and mitochondrial Ca2+ overload induced-FTH1 pathways to promote the sensitivity of osimertinib-resistant lung cancer cells to osimertinib. Conclusions: MA is a candidate EGFR-TKI sensitizer in KRAS-mutated and osimertinib-resistant lung cancer cells.

BCL6 is regulated by the MAPK/ELK1 axis and promotes KRAS-driven lung cancer.

Mutational activation of KRAS is a common oncogenic event in lung cancer, yet effective therapies are still lacking. Here, we identify B cell lymphoma 6 (BCL6) as a lynchpin in KRAS-driven lung cancer. BCL6 expression was increased upon KRAS activation in lung tumor tissue in mice and was positively correlated with the expression of KRAS-GTP, the active form of KRAS, in various human cancer cell lines. Moreover, BCL6 was highly expressed in human KRAS-mutant lung adenocarcinomas and was associated with poor patient survival. Mechanistically, the MAPK/ERK/ELK1 signaling axis downstream of mutant KRAS directly regulated BCL6 expression. BCL6 maintained the global expression of prereplication complex components; therefore, BCL6 inhibition induced stalling of the replication fork, leading to DNA damage and growth arrest in KRAS-mutant lung cancer cells. Importantly, BCL6-specific knockout in lungs significantly reduced the tumor burden and mortality in the LSL-KrasG12D/+ lung cancer mouse model. Likewise, pharmacological inhibition of BCL6 significantly impeded the growth of KRAS-mutant lung cancer cells both in vitro and in vivo. In summary, our findings reveal a crucial role of BCL6 in promoting KRAS-addicted lung cancer and suggest BCL6 as a therapeutic target for the treatment of this intractable disease.

Tailored modulation of stemness and drug resistance marker characteristics in K-Ras mutant lung cancer cells via PD-L1 gene suppression

AimsWe aimed to analyze the association of tumor cell-specific Programmed Cell Death Ligand 1 (PD-L1) expression with stemness markers and multi-drug resistance genes in non-small cell lung cancer. Main methodsATP Binding Cassette Subfamily G Member 2 (ABCG2), Aldehyde dehydrogenase 1 family, member A1 (ALDH1A1), CD44, Epithelial cell adhesion molecule (EPCAM), Kruppel-like factor 4 (KLF4), MYC Proto-Oncogene (MYC), Nanog Homeobox (NANOG), SRY-Box Transcription Factor 2 (SOX2) as well as ATP Binding Cassette Subfamily C Member (ABCC) 1–6, ABCC10–12 with ATP Binding Cassette Subfamily B Member (ABCB) 1 and ABCB4–9 belongs to ABC transporters family were analyzed and their correlation with PD-L1 was evaluated using Cancer Cell Line Encyclopedia and The Cancer Genome Atlas data sets. We validated potential lung cancer stemness markers harboring ABCG2, CD44, ALDH1A1 and SOX-2, which are affected as a result of siRNA-mediated suppression of PD-L1 via flow cytometry. K-Ras downstream signaling proteins as well as multidrug resistance proteins were also investigated. Key findingsPD-L1 was found to be positively correlated with CD44, whereas negatively correlated with ALDH1A1 (Pearson r = 0.44, r = −0.48; respectively) in 45 K-Ras mutated NSCLC cells based on CCLE database. While ABCC5 was dominantly decreased in K-Ras mutant lung cancer cells affected by PD-L1 gene suppression, expression changes were observed in the activation of distinct signaling pathways in a cell line-dependent manner. SignificanceThe evaluation of markers in lung adenocarcinoma with different types of K-Ras mutations in terms of both stemness and drug resistance markers will contribute to the development of personalized immunotherapy regimens.

Development of 10-Hydroxycamptothecin-crizotinib conjugate based on the synergistic effect on lung cancer cells

The effect of the combination of 10-Hydroxycamptothecin (HCPT) and crizotinib (CRI) on EGFR- and KRAS-mutant lung cancer cells was investigated and the conjugates of the two drugs were synthesised. HCPT combined with CRI synergistically inhibited the cell growth and proliferation of H1975, HCC827, and H460 without aggravating adverse effect on the normal cells. The combination synergistically enhanced the cell apoptosis rate through releasing Cyto-C by activation of Bcl-2 family-mediated mitochondrial signalling, which was associate with inactivating of EGFR related downstream signalling pathways including AKT, ERK, JNK, and p38 MAPK. Based on this synergy, the conjugates of HCPT and CRI (compounds CH-1 and CH-2) with different chemical bonds were synthesised. Compound CH-1 exhibited stronger cytotoxicity than HCPT and CRI alone or in combination. The combination of HCPT and CRI might be a promising therapeutic regimen and the conjugate CH-1was a potential target drug for the treatment of lung cancer.

Synthesis and Biological Evaluation of Cardiac Glycosides for Cancer Therapy by Targeting the DNA Damage Response.

Cardiac glycosides (CGs) are bioactive compounds originally used to treat heart diseases, but recent studies have demonstrated their anticancer activity. We previously demonstrated that Antiaris toxicaria 2 (AT2) possesses anticancer activity in KRAS mutated lung cancers via impinging on the DNA damage response (DDR) pathway. Toward developing this class of molecules for cancer therapy, herein we report a multistep synthetic route utilizing k-strophanthidin as the initial building block for determination of structure-activity relationships (SARs). A systematic structural design approach was applied that included modifications of the sugar moiety, the glycoside linker, stereochemistry, and lactone ring substitutions to generate a library of O-glycosides and MeON-neoglycosides derivatives. These molecules were screened for their anticancer activities and their impact on DDR signaling in KRAS mutant lung cancer cells. These results demonstrate the ability to chemically synthesize CG derivatives and define the SARs to optimize AT2 as a cancer therapeutic.

Circadian disruption enhances HSF1 signaling and tumorigenesis in Kras-driven lung cancer.

Disrupted circadian rhythmicity is a prominent feature of modern society and has been designated as a probable carcinogen by the World Health Organization. However, the biological mechanisms that connect circadian disruption and cancer risk remain largely undefined. We demonstrate that exposure to chronic circadian disruption [chronic jetlag (CJL)] increases tumor burden in a mouse model of KRAS-driven lung cancer. Molecular characterization of tumors and tumor-bearing lung tissues revealed that CJL enhances the expression of heat shock factor 1 (HSF1) target genes. Consistently, exposure to CJL disrupted the highly rhythmic nuclear trafficking of HSF1 in the lung, resulting in an enhanced accumulation of HSF1 in the nucleus. HSF1 has been shown to promote tumorigenesis in other systems, and we find that pharmacological or genetic inhibition of HSF1 reduces the growth of KRAS-mutant human lung cancer cells. These findings implicate HSF1 as a molecular link between circadian disruption and enhanced tumorigenesis.

Accommodation of ring C expanded deoxyvasicinone in the HDAC inhibitory pharmacophore culminates into a tractable anti-lung cancer agent and pH-responsive nanocarrier

A fragment recruitment process was conducted to pinpoint a suitable fragment for installation in the HDAC inhibitory template to furnish agents endowed with the potential to treat lung cancer. Resultantly, Ring C expanded deoxyvasicinone was selected as an appropriate surface recognition part that was accommodated in the HDAC three-component model. Delightfully, fused quinazolinone 6 demonstrating a magnificent anticancer profile against KRAS and EGFR mutant lung cancer cell lines (IC50 = 0.80–0.96 μM) was identified. Results of the mechanistic studies confirmed that the cell growth inhibitory effects of compound 6 stems for HDAC6 (IC50 = 12.9 nM), HDAC1 (IC50 = 49.9 nM) and HDAC3 inhibition (IC50 = 68.5 nM), respectively. Compound 6 also suppressed the colony formation ability of A549 cells, induced apoptosis, and increased autophagic flux. Key interactions of HDAC inhibitor 6 within the active site of HDAC isoforms were figured out through molecular modeling studies. Furthermore, a pH-responsive nanocarrier (Hyaluronic acid - fused quinazolinone 6 nanoparticles) was designed and assessed using a dialysis bag approach under both normal and acidic circumstances that confirmed the pH-sensitive nature of NPs. Delightfully, the nanoparticles demonstrated selective cell viability reduction potential towards the lung cancer cell lines (A549 lung cancer cell lines) and were found to be largely devoid of cell growth inhibitory effects under normal settings (L929, mouse fibroblast cells).

Abstract 5988: Intra-carotid artery brain metastasis models for evaluation of lung and breast cancer drugs

Abstract Non-CNS metastatic brain cancer is about 10 times more common than CNS cancer. Lung cancer and breast cancer account for most brain metastasis. KRAS-mutant NSCLC compose a third lung adenocarcinoma, among which 17% to 55% will develop brain metastases. Likewise, more than a third HER2-positive breast cancer will develop brain metastasis. Among existing animal models, the ectopic injection in brain cannot reflect the mechanism of tumor invasion and metastasis, while tail vein and intra-cardiac injection usually produces extra-cerebral metastatic disease and the animals have to be sacrificed before brain metastases appeared. Here we developed several metastasis models injected by intra-carotid artery (ICA) to address these problems. Two KRAS-mutant lung cancer cells and two HER2-positive breast cancer cells selected by their metastasize potential were luciferase labeled for in vivo imaging and drug evaluation. The tumor pathology in brain was evaluated by H&E staining. Compared to ectopic models, these ICA models have natural dissemination and progression in the brain, which recapitulate the different pattern of metastatic lesions in the clinic. With synchronized growth rate and little variation compared to tail vein and intra-cardiac models, these tumor growth kinetics allow for more appropriate animal efficacy study. Both KRAS-sensitive and insensitive models have been developed and validated for the next generation drug development; HER-2 positive breast models with ICA method also showed a good discriminative power on anti-HER2 modalities including antibodies, small molecules and ADC. Meanwhile, ICA models with human immune cell reconstitution were also developed to support immune therapies in pre-clinical stage. In summary, we have developed a panel of intra-carotid artery brain metastatic models for drug evaluation, and demonstrated their superiority in growth kinetics, brain microenvironment, drug selectivity and immune component. These models can be a promising predictive tool for novel drug/modality discovery to target brain metastasis. Citation Format: Fuyang Wang, Yuying Yang, Haoxia Zhao, Chunlei Dai, Bingrui Han, Mengyao Zhang, Xia Xiong, Chao Zhang, Xuzhen Tang, Qunsheng Ji. Intra-carotid artery brain metastasis models for evaluation of lung and breast cancer drugs [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 5988.

Abstract 3187: Understanding the role of cancer-associated fibroblasts (CAFs) on pathogenesis of KRAS mutated cancers by defining differences in the basal state proteome and secretome

Abstract Background: KRAS is commonly mutated in solid tumors. Cancer-associated fibroblasts (CAFs) are a key tumor stroma component, which can affect cancer progression and drug response. We hypothesized that defining the differential secreted proteins between CAFs and KRAS mutant-cancer cells could reveal potential mechanisms by which CAFs influence cancer progression and drug resistance. Methods: We studied 12 cell lines: 3 KRAS mutant-colorectal cancer cell lines (H747, LIM2099 and SW620), 3 KRAS mutant-lung cancer cell lines (H1792, H2030 and H23), 3 KRAS mutant-pancreatic cancer cell lines (CAPAN1, DANG, MIAPACA2) and 3 CAF cell line models (colorectal and lung CAF and pancreatic stellate cells). We quantified proteins in cell lines and serum-free media after 24 hour incubation in cells using mass spectrometry in a multiplexed manner (12plex) in triplicates to characterize the proteome and the profile of secreted proteins (secretome), respectively. Prediction software (signalP, secretomeP, TMHMM) and databases (surfaceome and cell surface protein atlas) were used to annotate secreted or transmembrane proteins. Results: Our proteome analysis quantified 9307 proteins. T-test between the cell types (CAF vs cancer) identified 1770 differentially expressed proteins, where 712 proteins were CAF-enriched. This included known CAF markers (e.g α smooth muscle actin) but also proteins not known to be CAF markers such as heat shock protein beta-5/6 (HSPB5/6). The secretome analysis identified 2330 secreted or transmembrane proteins where 202 were differentially expressed between cancer cells and CAFs. 190 proteins were CAF-enriched with the highest ranking being syndecan-2 (SDC2), collagen α-28(I) chain (COL28A1), decorin (DCN) and wnt family member 5B (WNT5B). Interestingly, basal proteome analysis of KRAS mutant-cancer cells showed expression of receptors corresponding to the CAF-enriched ligands, such as receptor tyrosine kinase-like orphan receptor 1/2 (ROR1/2) which respond to WNT5B. This suggests that CAF-enriched ligands may activate certain downstream pathways, including non-canonical WNT signaling, in KRAS mutant-cancer cells. Conclusion: We undertook deep quantitative proteomics where we identified potential novel CAF markers HSPB5/6. We have also found proteins in the CAF secretome, such as WNT5B, which may drive drug resistance in KRAS mutant-cancer cells. Citation Format: Rachel Lau, Lu Yu, Theodoros Roumeliotis, Lisa Pickard, Adam Stewart, Jyoti Choudhary, Udai Banerji. Understanding the role of cancer-associated fibroblasts (CAFs) on pathogenesis of KRAS mutated cancers by defining differences in the basal state proteome and secretome [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 3187.

Metabolic synthetic lethality by targeting NOP56 and mTOR in KRAS-mutant lung cancer

BackgroundOncogenic KRAS mutations are prevalent in human cancers, but effective treatment of KRAS-mutant malignancies remains a major challenge in the clinic. Increasing evidence suggests that aberrant metabolism plays a central role in KRAS-driven oncogenic transformation. The aim of this study is to identify selective metabolic dependency induced by mutant KRAS and to exploit it for the treatment of the disease.MethodWe performed an integrated analysis of RNAi- and CRISPR-based functional genomic datasets (n = 5) to identify novel genes selectively required for KRAS-mutant cancer. We further screened a customized library of chemical inhibitors for candidates that are synthetic lethal with NOP56 depletion. Functional studies were carried out by genetic knockdown using siRNAs and shRNAs, knockout using CRISPR/Cas9, and/or pharmacological inhibition, followed by cell viability and apoptotic assays. Protein expression was determined by Western blot. Metabolic ROS was measured by flow cytometry-based quantification.ResultsWe demonstrated that nucleolar protein 5A (NOP56), a core component of small nucleolar ribonucleoprotein complexes (snoRNPs) with an essential role in ribosome biogenesis, confers a metabolic dependency by regulating ROS homeostasis in KRAS-mutant lung cancer cells and that NOP56 depletion causes synthetic lethal susceptibility to inhibition of mTOR. Mechanistically, cancer cells with reduced NOP56 are subjected to higher levels of ROS and rely on mTOR signaling to balance oxidative stress and survive. We also discovered that IRE1α-mediated unfolded protein response (UPR) regulates this process by activating mTOR through p38 MAPK. Consequently, co-targeting of NOP56 and mTOR profoundly enhances KRAS-mutant tumor cell death in vitro and in vivo.ConclusionsOur findings reveal a previously unrecognized mechanism in which NOP56 and mTOR cooperate to play a homeostatic role in the response to oxidative stress and suggest a new rationale for the treatment of KRAS-mutant cancers.

A Tasquinomod-loaded dopamine-modified pH sensitive hydrogel is effective at inhibiting the proliferation of KRAS mutant lung cancer cells.

Hydrogels can maintain a high local drug concentration during treatments and may be useful to local targeting diseased areas. We propose a pH sensitive hydrogel consisting of poly-vinylpyrrolidone (PVP) and chitosan as a new treatment method for KRAS mutant lung cancer. Addition of dopamine improved the drug loading and release effects of this hydrogel. We demonstrate that Tasquinimod-loading of this dopamine-modified pH sensitive hydrogel is more effective than Tasquinimod alone for inhibiting the proliferation of KRAS mutant lung cancer cells. Combination of conventional drugs with hydrogels may thus provide a new treatment modality for lung cancer.

CCL5 production in lung cancer cells leads to an altered immune microenvironment and promotes tumor development

ABSTRACT Current immunotherapies for lung cancer are only effective in a subset of patients. Identifying tumor-derived factors that facilitate immunosuppression offers the opportunity to develop novel strategies to supplement and improve current therapeutics. We sought to determine whether expression of driver oncogenes in lung cancer cells affects cytokine secretion, alters the local immune environment, and influences lung tumor progression. We demonstrate that oncogenic EGFR and KRAS mutations, which are early events in lung tumourigenesis, can drive cytokine and chemokine production by cancer cells. One of the most prominent changes was in CCL5, which was rapidly induced by KRASG12V or EGFRL858R expression, through MAPK activation. Immunocompetent mice implanted with syngeneic KRAS-mutant lung cancer cells deficient in CCL5 have decreased regulatory T cells (Tregs), evidence of T cell exhaustion, and reduced lung tumor burden, indicating tumor-cell CCL5 production contributes to an immune suppressive environment in the lungs. Furthermore, high CCL5 expression correlates with poor prognosis, immunosuppressive regulatory T cells, and alteration to CD8 effector function in lung adenocarcinoma patients. Our data support targeting CCL5 or CCL5 receptors on immune suppressive cells to prevent formation of an immune suppressive tumor microenvironment that promotes lung cancer progression and immunotherapy insensitivity.