Drug-resistant Lung Cancer Cells Research Articles

Harnessing endocytic pH gradients for cascaded formation of intracellular condensates to inhibit autophagy: A pathway to cancer therapy

Cancer cells rely on autophagy to degrade damaged organelles and survive in the harsh environment for progression and metastasis. Autophagy inhibition can improve the efficacy of anticancer therapy because many anticancer drugs can induce protective autophagy in cancer cells. However, current clinically available autophagy inhibitors are rare and have adverse effects with limited selectivity. This work develops an intracellular biomolecular condensate for autophagy inhibition to kill cancer without apparent side effects. A modularly designed amphiphilic peptide (PNP) can self-assemble to form toxic nanofibrous condensates successively from nanoparticles with the pH changes of the endocytic pathway. Mechanistic studies by CLSM and FRAP demonstrate that the PNP finally forms solid nanofibrous condensates in the lysosome after cell endocytosis. Bio-EM and cell transfection with adenovirus expressing mCherry-GFP-LC3B fusion protein (autophagy marker) experiments indicate the autophagy inhibition by PNP nanofibrous condensates. In vitro experiments show that PNP can significantly boost the activity of the clinical drug by 150-fold against drug-resistant lung cancer cells by inducing G2/M phase arrest of the cell cycle. Furthermore, PNPs demonstrate effective tumor targeting and permeability in vivo, inhibiting tumor growth and reducing the side effects of clinical chemotherapy drugs. Overall, this work provides a simple and feasible strategy for designing intracellular condensates to inhibit autophagy for cancer therapy.

Identification of naphthalimide-derivatives as novel PBD-targeted polo-like kinase 1 inhibitors with efficacy in drug-resistant lung cancer cells

Targeting polo-box domain (PBD) small molecule for polo-like kinase 1 (PLK1) inhibition is a viable alternative to target kinase domain (KD), which could avoid pan-selectivity and dose-limiting toxicity of ATP-competitive inhibitors. However, their efficacy in these settings is still low and inaccessible to clinical requirement. Herein, we utilized a structure-based high-throughput virtual screen to find novel chemical scaffold capable of inhibiting PLK1 via targeting PBD and identified an initial hit molecule compound 1a. Based on the lead compound 1a, a structural optimization approach was carried out and several series of derivatives with naphthalimide structural motif were synthesized. Compound 4Bb was identified as a new potent PLK1 inhibitor with a KD value of 0.29 μM. 4Bb could target PLK1 PBD to inhibit PLK1 activity and subsequently suppress the interaction of PLK1 with protein regulator of cytokinesis 1 (PRC1), finally leading to mitotic catastrophe in drug-resistant lung cancer cells. Furthermore, 4Bb could undergo nucleophilic substitution with the thiol group of glutathione (GSH) to disturb the redox homeostasis through exhausting GSH. By regulating cell cycle machinery and increasing cellular oxidative stress, 4Bb exhibited potent cytotoxicity to multiple cancer cells and drug-resistant cancer cells. Subcutaneous and oral administration of 4Bb could effectively inhibit the growth of drug-resistant tumors in vivo, doubling the survival time of tumor bearing mice without side effects in normal tissues. Thus, our study offers an orally-available, structurally-novel PLK1 inhibitor for drug-resistant lung cancer therapy.

Abstract 1945: Mechanisms of acquired resistance to EGFR tyrosine kinase inhibitor osimertinib and KRAS-G12C inhibitor sotorasib in lung cancer

Abstract A series of molecular targeted drugs have been developed for the treatment of non-small cell lung cancer (NSCLC) harboring driver gene alterations. Although the use of these drugs has substantially improved the clinical outcomes of patients with the genetically altered NSCLC, these patients ultimately develop resistance to the drugs. Various molecular mechanisms of resistance to the molecular target therapies have been reported, it is still a crucial challenge to overcome the drug resistance. Osimertinib, the most used third-generation EGFR tyrosine kinase inhibitor to target both common EGFR mutations and secondary T790M mutation, and sotorasib, a recently developed KRAS inhibitor to target KRAS G12C mutation, are no exception. In this study, we aimed to investigate the mechanisms of acquired resistance to osimertinib and sotorasib in NSCLC by profiling drug-resistant lung cancer cell lines, which were established from EGFR-mutant HCC827(del E756_A750) and KRAS G12C-mutant H23 through exposure to each of the molecular targeted drugs. Phospho-RTK array and western blot analyses revealed MET overexpression and phosphorylation in the osimertinib-resistant HCC827 (HCC827OR) as previous studies reported. Notably in our study, HCC827OR cells were incompletely sensitive to a selective MET inhibitor savolitinib without osimertinib, suggesting that HCC827OR partially lost its dependency on mutant EGFR and got addiction to MET signaling. When sotolasib was administered to the sotolasib-resistant H23 (H23SR) cells, KRAS-GTP, and the downstream ERK and AKT phosphorylation were suppressed. To identify novel mechanisms of the resistance, we performed epigenomic profiling of active enhancer in those cells and found a candidate transcription factor, at whose gene locus H3K27 acetylation signal was increased in H23SR cells compared to parent H23 cells. Immunoblotting showed that protein expression of the gene was indeed higher in H23SR cells than the parent cells. To confirm the involvement of this gene to the resistance, further investigation is warranted. Citation Format: Yuri Yagami, Takashi Sato, Hiroki Yamamoto, Masayuki Shirasawa, Yoshiro Nakahara, Watanabe Hideo, Naoki Katsuhiko. Mechanisms of acquired resistance to EGFR tyrosine kinase inhibitor osimertinib and KRAS-G12C inhibitor sotorasib in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1945.

USP24-i-101 targeting of USP24 activates autophagy to inhibit drug resistance acquired during cancer therapy

Drug resistance in cancer therapy is the major reason for poor prognosis. Addressing this clinically unmet issue is important and urgent. In this study, we found that targeting USP24 by the specific USP24 inhibitors, USP24-i and its analogues, dramatically activated autophagy in the interphase and mitotic periods of lung cancer cells by inhibiting E2F4 and TRAF6, respectively. USP24 functional knockout, USP24C1695A, or targeting USP24 by USP24-i-101 inhibited drug resistance and activated autophagy in gefitinib-induced drug-resistant mice with doxycycline-induced EGFRL858R lung cancer, but this effect was abolished after inhibition of autophagy, indicating that targeting USP24-mediated induction of autophagy is required for inhibition of drug resistance. Genomic instability and PD-L1 levels were increased in drug resistant lung cancer cells and were inhibited by USP24-i-101 treatment or knockdown of USP24. In addition, inhibition of autophagy by bafilomycin-A1 significantly abolished the effect of USP24-i-101 on maintaining genomic integrity, decreasing PD-L1 and inhibiting drug resistance acquired in chemotherapy or targeted therapy. In summary, an increase in the expression of USP24 in cancer cells is beneficial for the induction of drug resistance and targeting USP24 by USP24-i-101 optimized from USP24-i inhibits drug resistance acquired during cancer therapy by increasing PD-L1 protein degradation and genomic stability in an autophagy induction-dependent manner.

Rapid and automatic phenotyping of cells through their annexin-mediated enforced blebbing response

Cellular blebbing has been widely recognized as a hallmark of processes such as apoptosis and cell migration. Here, we developed a novel double-layer compression microfluidic device to trigger the enforced blebbing of cells in a programmable manner. It was found that the critical compression for inducing membrane bleb in highly invasive or drug-resistant breast and lung cancer cell lines could be several times higher than that of their non-invasive or drug-sensitive counterparts. Furthermore, we showed that knockdown of annexin-6, a protein known to be heavily involved in membrane and calcium dynamics in cells, led to a significantly reduced cellular volume, reflecting a lowered intracellular pressure, and an ∼twofold increase in the critical compressive strain for triggering blebbing. The fact that hundreds of cells can be tested and automatically analyzed in our device at the same time highlights the potential of this simple and label-free method in applications such as cell sorting and disease detection.

Time dependent inhibition of Morinda citrifolia essential oils against multi drug resistant bacteria and A549 lung cancer cells

Recent years, multi-drug resistant bacteria (MDRs) and lung cancer cells are threatening to human worldwide due to failure of sufficient drugs. It was suggested that the new sources needed to discover new varieties of drugs, antibiotics and other format to eradicate these infections. As like, the medicinal plant of Morinda citrifolia (M. citrifolia) was used in this study to inhibit both the bacterial infection and cancer cells. First, the LC-MS measurement was evidently reported the available essential oils from medicinal plant of M. citrifolia. It has excellent anti-bacterial properties against P. mirabilis, E. coli, P. aeruginosa and S. aureus with 24, 22, 26 and 18 zone of inhibition respectively by agar well diffusion. Subsequently, the liquid interference assay was exhibited 97 %, 96 %, 94 % and 90 % inhibition at 1000 µg/mL concentration for all the tested pathogens. In addition, M. citrifolia EOs have excellent anti-bacterial activity, and it arrested the bacterial growth completely at 1000 µg/mL concentration for 24 h after compared with other time intervals. Subsequently, the intracellular membranes and more death cells with condensed chromosome like structure were effectively observed by confocal laser scanning electron microscope. Then, invitro cytotoxicity result of M. citrifolia EOs treated A549 lung cancer cells was clearly observed. The cytotoxicity result confirmed that the 500 µg/mL of M. citrifolia EOs against A549 lung cancer cells as fixed as IC50 dose. Hence, current study conclude, M. citrifolia is the excellent medicinal plant to inactivate bacterial growth and cancer cells efficiently.

A fluorescent prodrug to fight drug-resistant lung cancer cells via autophagy-driven ferroptosis

Lung cancer is the second most common malignant tumor worldwide. The problem of drug resistance in lung cancer leads to treatment failure and recurrence in more than 90% of cancer patients. Thus, developing chemical probes that can observe drug-resistant lung cancer cells in real-time and eliminate them is of great interest. However, such tools that can simultaneously probe drug-resistant lung cancer cells and therapeutic modes of action have not yet been developed. In this work, we reported a fluorescent prodrug that can be activated in cancer cell lysosomes to release amonafide (ANF) and aniline mustard, where the fluorescent signal of ANF (λex/λem = 405/570 −620 nm) can be tracked by super-resolution imaging in live cancer cells. With imaging studies, we found that this new fluorescent prodrug uniquely displays an autophagy-driven ferroptosis-inducing effect, accompanied by the accumulation of intracellular ferrous ions and lipid peroxidation. More importantly, the fluorescent prodrug exhibits potential anti-proliferative activity, especially against drug-resistant A549R lung cancer cells (IC50, ∼ 3 μM), better than the positive control drug cisplatin (IC50, ∼ 27 μM). This work indicates that the development of fluorescent prodrugs as autophagy-driven ferroptosis inducers may represent a viable approach for targeting drug-resistant cancer cells.

Synthesis and Conformational Analysis of FR901464-Based RNA Splicing Modulators and Their Synergism in Drug-Resistant Cancers.

FR901464 is a cytotoxic natural product that binds splicing factor 3B subunit 1 (SF3B1) and PHD finger protein 5A (PHF5A), the components of the human spliceosome. The amide-containing tetrahydropyran ring binds SF3B1, and it remains unclear how the substituents on the ring contribute to the binding. Here, we synthesized meayamycin D, an analogue of FR901464, and three additional analogues to probe the conformation through methyl scanning. We discovered that the amide-containing tetrahydropyran ring assumes only one of the two possible chair conformations and that methylation of the nitrogen distorts the chair form, dramatically reducing cytotoxicity. Meayamycin D induced alternative splicing of MCL-1, showed strong synergism with venetoclax in drug-resistant lung cancer cells, and was cancer-specific over normal cells. Meayamycin D incorporates an alkyl ether and shows a long half-life in mouse plasma. The characteristics of meayamycin D may provide an approach to designing other bioactive L-shaped molecules.

Amphonal, a new polyene aldehyde from a deep-sea-derived Streptomyces amphotericinicus

A new polyene aldehyde, named amphonal (1), and two known (2 and 3) polyketides were isolated from the deep-sea-derived Streptomyces amphotericinicus OUCT16-38 strain. The structure of 1 was determined by extensive MS and NMR spectroscopic analysis. In the cytotoxicity evaluation, compound 2 showed significant growth inhibition against the drug-resistant human lung cancer cell line A549-Taxol with IC50 value of 0.44 μM, which was more potent than the positive control doxorubicin. Meanwhile, 2 showed considerable cytotoxic effect towards H1975, H1299 and HEL cell lines (IC50 = 0.93-4.73 μM) as well.

Mechanisms of Herba Epimedii in Lung Cancer Treatment: A Combination of Molecular Docking and Network Pharmacology Analyses

Introduction Lung cancer (LC) ranks among the most prevalent malignant neoplasms globally. As a Traditional Chinese medicine (TCM), Herba Epimedii has been commonly used for treating LC. However, the specific mechanism of LC remains to be elucidated. Objectives This research aimed to explore the mechanisms of Herba Epimedii in treating LC according to molecular docking and network pharmacology. Materials and Methods The active components (ACs) and targets of Herba Epimedii were screened through the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database. LC targets were retrieved from GeneCards and DisGeNET databases. Cytoscape v3.9.0 was employed to construct a “drug-component-disease-target” network. The STRING database and Cytoscape 3.9.0 software were utilized to establish a PPI network. The DAVID database was used for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The AutoDock software was employed for molecular docking verification, and the Pymol software was applied for visual processing. Results Herba Epimedii acted on 108 LC treatment targets through 22 ACs. Five core components were screened, including quercetin, luteolin, kaempferol, anhydroicaritin, and 8-isopentenyl-kaempferol, while 12 core targets were screened, including TP53, AKT1, MYC, CASP3, ESR1, JUN, HIF1A, VEGFA, TNF, CCND1, EGFR, and IL-6. GO enrichment analysis mainly involved positive regulation of gene expression, positive regulation of the apoptotic process, enzyme binding, identical protein binding, macromolecular complex, nucleoplasm, etc. KEGG enrichment analysis mainly involved p53, interleukin-17, PI3K-Akt, and other signaling pathways. Five core components all demonstrated excellent binding activities with the core targets. Conclusion The mechanisms of Herba Epimedii in LC treatment may involve multiple components, targets, and pathways, inducing apoptosis, and inhibiting proliferation, invasion, metastasis, and drug resistance of LC cells.

3-Deoxysappanchalcone Inhibits Cell Growth of Gefitinib-Resistant Lung Cancer Cells by Simultaneous Targeting of EGFR and MET Kinases.

The mechanistic functions of 3-deoxysappanchalcone (3-DSC), a chalcone compound known to have many pharmacological effects on lung cancer, have not yet been elucidated. In this study, we identified the comprehensive anti-cancer mechanism of 3-DSC, which targets EGFR and MET kinase in drug-resistant lung cancer cells. 3-DSC directly targets both EGFR and MET, thereby inhibiting the growth of drug-resistant lung cancer cells. Mechanistically, 3-DSC induced cell cycle arrest by modulating cell cycle regulatory proteins, including cyclin B1, cdc2, and p27. In addition, concomitant EGFR downstream signaling proteins such as MET, AKT, and ERK were affected by 3-DSC and contributed to the inhibition of cancer cell growth. Furthermore, our results show that 3-DSC increased redox homeostasis disruption, ER stress, mitochondrial depolarization, and caspase activation in gefitinib-resistant lung cancer cells, thereby abrogating cancer cell growth. 3-DSC induced apoptotic cell death which is regulated by Mcl-1, Bax, Apaf-1, and PARP in gefitinib-resistant lung cancer cells. 3-DSC also initiated the activation of caspases, and the pan-caspase inhibitor, Z-VAD-FMK, abrogated 3-DSC induced-apoptosis in lung cancer cells. These data imply that 3-DSC mainly increased mitochondria-associated intrinsic apoptosis in lung cancer cells to reduce lung cancer cell growth. Overall, 3-DSC inhibited the growth of drug-resistant lung cancer cells by simultaneously targeting EGFR and MET, which exerted anti-cancer effects through cell cycle arrest, mitochondrial homeostasis collapse, and increased ROS generation, eventually triggering anticancer mechanisms. 3-DSC could potentially be used as an effective anti-cancer strategy to overcome EGFR and MET target drug-resistant lung cancer.

Genome-directed discovery of antiproliferative bafilomycins from a deepsea-derived Streptomyces samsunensis

Genomic bioinformatics analysis identified a bafilomycin biosynthetic gene cluster (named bfl) in the deepsea-derived S. samsunensis OUCT16-12, from which two new (1 and 2, named bafilomycins R and S) along with four known (3–6) bafilomycins were targetly obtained. The structure of 3 was clearly identified for the first time, thus named bafilomycin T herein. Differ from the fumarate substitution at C-21 of known bafilomycins, its location on C-23 is a unique feature of 1 and 2. The stereochemistry of the compounds was established based on NOE correlations, ketoreductase (KR)-types in PKS modules of bfl, and ECD calculations. Moreover, a detailed biosynthetic model of 1–6 in S. samsunensis OUCT16-12 was provided based on the gene function prediction and sequence identity. Compared with the positive control doxorubicin, 1–6 showed more potent antiproliferative activities against drug-resistant lung cancer cell line A549-Taxol, with IC50 values ranging from 0.07 μM to 1.79 μM, which arrested cell cycle in G0/G1 phase to hinder proliferation.

The role of USP51 in attenuating chemosensitivity of lung cancer cells to cisplatin by regulating DNA damage response.

Lung cancer is the most frequent type of cancer affecting both men and women globally, and it is associated with a high mortality rate. It is clinically treated with cisplatin, a platinum-based drug that works by generating DNA lesions, which activates DNA damage response and induces cell death. However, chemoresistance by cancer cells limits the clinical usefulness of cisplatin as an anticancer drug. Here, we uncovered a role of ubiquitin-specific protease 51 (USP51) in the chemosensitivity of lung cancer cells to cisplatin by regulating DNA damage response. USP51 was more upregulated in lung cancer tissues of chemotherapy-resistant patients than those of chemotherapy-sensitive patients with adjacent, nontumor tissues. USP51 overexpression in lung cancer cells in vitro reduced γ-H2AX formation and promoted checkpoint kinase 1 (CHK1) phosphorylation, whereas USP51 knockdown showed opposite effects, indicating that USP51 played an important role in promoting DNA damage repair. Finally, USP51 knockdown weakened cisplatin resistance in A549/DDP cells and significantly suppressed tumor growth in vivo, suggesting that a USP51 inhibitor combined with cisplatin may be considered as an effective treatment strategy to eliminate drug-resistant lung cancer cells.

Emergence of Resistance to MTI-101 Selects for a MET Genotype and Phenotype in EGFR Driven PC-9 and PTEN Deleted H446 Lung Cancer Cell Lines.

Simple SummaryMTI-101 is a first-in-class novel cyclic peptide shown to have anti-tumor activity in both multiple myeloma and castrate-resistant prostate in vivo cancer models. These data suggest the potential for a broad spectrum of anti-cancer activity for this class of compounds. To further delineate determinants of sensitivity and resistance that were not dependent on oncogenic drivers, two isogenic drug-resistant cell lines were generated with chronic exposure to MTI-101 in a non-small cell lung cancer (NSCLC) PC-9 (EGFR driven) and a small cell lung cancer (SCLC) H446 (PTEN deleted and c-MYC amplified). Our data indicate that the chronic exposure of MTI-101 selects for a stable mesenchymal-to-epithelial (MET) genotype and phenotype in both PC-9 and H446 lung cancer cell lines.MTI-101 is a first-in-class cyclic peptide that kills cells via calcium overload in a caspase-independent manner. Understanding biomarkers of response is critical for positioning a novel therapeutic toward clinical development. Isogenic MTI-101-acquired drug-resistant lung cancer cell line systems (PC-9 and H446) coupled with differential RNA-SEQ analysis indicated that downregulated genes were enriched in the hallmark gene set for epithelial-to-mesenchymal transition (EMT) in both MTI-101-acquired resistant cell lines. The RNA-SEQ results were consistent with changes in the phenotype, including a decreased invasion in Matrigel and expression changes in EMT markers (E-cadherin, vimentin and Twist) at the protein level. Furthermore, in the EGFR-driven PC-9 cell line, selection for resistance towards MTI-101 resulted in collateral sensitivity toward EGFR inhibitors. MTI-101 treatment showed synergistic activity with the standard of care agents erlotinib, osimertinib and cisplatin when used in combination in PC-9 and H446 cells, respectively. Finally, in vivo data indicate that MTI-101 treatment selects for increased E-cadherin and decreased vimentin in H446, along with a decreased incident of bone metastasis in the PC-9 in vivo model. Together, these data indicate that chronic MTI-101 treatment can lead to a change in cell state that could potentially be leveraged therapeutically to reduce metastatic disease.

Abstract 707: Mahanine, a carbazole alkaloid attenuates lung cancer progression

Abstract Lung cancer continues to be a major cause of cancer mortality. Emerging evidence suggests that cancer likely involves multiple mutations. Therefore, compounds targeting multiple signal transduction pathways would be ideal for the prevention and treatment of cancer. Mahanine, a carbazole alkaloid isolated from an Indian folklore medicinal plant Murraya koenigii (known as Curry Patta), widely consumed in traditional dishes, have been shown to possess anticancer activities and confound many oncogenic signaling events in breast, colon, and prostate cancers. In the current study, we determined the anticancer potential of mahanine against drug-sensitive and drug-resistant lung cancer cells and postulate the possible mechanism of action. Mahanine was isolated from the dried leaves of Murraya koenigii by hydro alcoholic extraction and fractionation with ethyl acetate, followed by preparative UPLC to isolate pure (>95%) compound and its identity was confirmed by LC-MS/MS and NMR spectroscopy. Antiproliferative activity of mahanine was determined against drug- sensitive (A549 and H1299) and taxol-resistant (A549-TR) human lung cancer cells. Mahanine showed a dose- and time-dependent inhibition of both drug-sensitive and drug-resistant lung cancer cell growth. Interestingly, the IC50 values of mahanine were essentially the same for drug sensitive (A549) (12.5 µM) and drug- resistant (12.5 µM) lung cancer cells; the IC50 value was somewhat lower (10 µM) against highly metastatic H1299 lung cancer cells. Mahanine attenuated the colony formation and reduced the wound healing of lung cancer cells. Mechanistically, mahanine dose dependently impeded the oncogenic markers such as KRAS, MET, AKT, mTOR and cMYC. Together, mahanine demonstrate ability to inhibit both drug- sensitive and drug-resistant lung cancer cells and modulate distinct oncogenic molecular targets providing potential to be developed for the treatment of drug-resistant and metastatic NSCLC (Supported from Duggan Endowment funds). Keywords: Mahanine, Lung cancer, Oncogenes, Plant therapeutics. Citation Format: Raghuram Kandimalla, Farrukh Aqil, Disha N. Moholkar, Suman K. Samanta, Ramesh C. Gupta. Mahanine, a carbazole alkaloid attenuates lung cancer progression [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 707.

Abstract 2412: Emergence of resistance to MTI-101 selects for favorable MET phenotype in lung cancer

Abstract Lung cancer remains the most-deadly of all cancers, and thus clinical evidence indicates novel treatment strategies are needed to improve patient outcomes. Our laboratory has been developing a novel cyclic peptide coined MTI-101, which induces cell death via calcium overload, a finding dependent on the TRPC1/TRPC5/TRPC4 complex. To better understand determinants of sensitivity and resistance to MTI-101, we developed drug resistant isogenic lung cancer cell lines. Two isogenic drug resistant cell lines were generated with chronic exposure to MTI-101 in PC-9 (NSCLC EGFR Driven) and H446 (SCLC PTEN Deleted and c-MYC amplified). Drug resistance was confirmed in both cell lines with over two-fold increases in IC50 values upon treatment with MTI-101 in MTT assays. Interestingly, pushing the resistant levels beyond a two-fold increase was not obtained as cultures did not sustain growth with increased selection pressure. Using the isogenic systems coupled with RNA-SEQ, the top enriched GSEA hallmark was downregulation of genes that contribute to EMT in both MTI-101 resistant lines. The RNA-SEQ data correlated with changes in the phenotype which included a significant decreased invasion in Matrigel and changes of MET markers (E-Cadherin, Vimentin) at the protein level. Furthermore, in the EGFR driven PC-9 cell line, selection for resistance towards MTI-101 resulted in collateral sensitivity to EGFR inhibitors. To determine the stability of the MET phenotype, cells were removed from drug, then tested for invasion and drug resistance every month. Our data indicated that the MET phenotype and genotype was stable for a minimum of six months. RNA-SEQ data continued to demonstrate decreased expression of EMT in cells removed from drug compared to the parental cell line. MTI-101 treatment in PC-9 and H446 cells showed synergistic activity with standard of cares Erlotinib, Osimertinib, and Cisplatin when used in combination on the wildtype cells as well. Finally, in vivo data indicates that MTI-101 treatment selects for increased E-Cadherin and decreased Vimentin. Similar to the cell line model, this finding correlated with decreased incidence of bone metastasis in the PC-9 in vivo model. Together our data indicate that MTI-101 treatment could be used as a tool to delay the emergence of resistance and potentially inhibit or delay the emergence of metastatic disease for the treatment of lung cancer. Citation Format: Clark Jones, Lori Hazlehurst. Emergence of resistance to MTI-101 selects for favorable MET phenotype in lung cancer [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 2412.

Abstract 371: Exosomal paclitaxel formulation, alone and in combination with cisplatin, enhances drug’s efficacy against lung cancer

Abstract Lung cancer is the second most common cancer in both men and women. Despite several treatment options, chemotherapy remains a major standard-of-care treatment modality. Over time cancer cells acquire drug resistance and evade its effects for survival. The combination of chemotherapeutic drugs with the non-overlapping mechanism of actions are one alternative approach to treat drug-resistant cancers. Paclitaxel (PAC) and cisplatin (CisPt) are widely used to treat non-small cell-lung cancer (NSCLC). However, dose-limiting toxicity, metastasis and chemoresistance have restricted its use. Our recent findings suggested that PAC, when loaded onto folic acid (FA)-functionalized colostrum exosomes (FA-ExoPAC) and administered orally, exceeded the efficacy of i.v. PAC but matched efficacy of i.v. albumin-bound PAC nanoformulation (Abraxane [Abx]) against A549 orthotopic lung tumors. Furthermore, unlike i.v. PAC and i.v. Abx, oral FA-ExoPAC lacked immune toxicity. In order to advance the FA-ExoPAC to clinical studies, we determined if the exosomal formulation will interfere in CisPt therapy. MTT assay showed similar antiproliferative activity of ExoPAC and PAC (IC50 6.25 nM) against drug-sensitive A549 lung cancer cells. However, the antiproliferative activity of ExoPAC (IC50 38 nM) was much greater than for PAC (IC50 >>100 nM) against drug-resistant A549TR lung cancer cells. The activity was further enhanced with FA-functionalized ExoPAC. The enhanced efficacy of the drug presumably resulted from higher cell uptake of ExoPAC and FA-ExoPAC by both drug-sensitive and drug-resistant cells. To determine the effect of the combination of ExoPAC and CisPt, A549 and A549TR cells were treated with increasing concentrations of both the drugs, alone and in combination, for 72 h. MTT assay showed that CisPt (20 µM) and ExoPAC (80 nM) inhibited the growth of A549 cells by 47% and 75%, respectively, the effect was significantly increased with the combination (>95% inhibition). The combination effect was likewise enhanced against the A549TR, except the respective concentration of CisPt (40 µM) and ExoPAC (200 nM) was increased to achieve >95% growth inhibition; the inhibition was lower (50–60%) when treated individually. Together, these data suggest that targeted oral therapy with FA-ExoPAC can potentially eliminate drug resistance and inhibit metastasis with minimal or no side effects and warrants testing in the clinical settings (Supported from the NIH grant R44-CA-221487 and Agnes Brown Duggan Endowment). Citation Format: Raghuram Kandimalla, Disha N. Moholkar, Jayaprakash Jeyabalan, Wendy Spencer, Ramesh C. Gupta, Farrukh Aqil. Exosomal paclitaxel formulation, alone and in combination with cisplatin, enhances drug’s efficacy against lung cancer [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 371.

Membrane Repairing Capability of Non-Small Cell Lung Cancer Cells Is Regulated by Drug Resistance and Epithelial-Mesenchymal-Transition.

The plasma membrane separates the interior of the cells from the extracellular fluid and protects the cell from disruptive external factors. Therefore, the self-repairing capability of the membrane is crucial for cells to maintain homeostasis and survive in a hostile environment. Here, we found that micron-sized membrane pores induced by cylindrical atomic force microscope probe puncture resealed significantly (~1.3–1.5 times) faster in drug-resistant non-small cell lung cancer (NSCLC) cell lines than in their drug-sensitive counterparts. Interestingly, we found that such enhanced membrane repairing ability was due to the overexpression of annexin in drug-resistant NSCLC cells. In addition, a further ~50% reduction in membrane resealing time (i.e., from ~23 s to ~13 s) was observed through the epithelial-mesenchymal-transition, highlighting the superior viability and potential of highly aggressive tumor cells using membrane resealing as an indicator for assessing the drug-resistivity and pathological state of cancer.

Acetyltanshinone IIA reduces the synthesis of cell cycle-related proteins by degrading p70S6K and subsequently inhibits drug-resistant lung cancer cell growth

Targeted therapies using tyrosine kinase inhibitors (TKIs) against epidermal growth factor receptor (EGFR) have improved the outcomes of patients with non-small cell lung cancer (NSCLC). However, due to genetic mutations of EGFR or activation of other oncogenic pathways, cancer cells can develop resistance to TKIs, resulting in usually temporary and reversible therapeutic effects. Therefore, new anticancer agents are urgently needed to treat drug-resistant NSCLC. In this study, we found that acetyltanshinone IIA (ATA) displayed much stronger potency than erlotinib in inhibiting the growth of drug-resistant NSCLC cells and their-derived xenograft tumors. Our analyses revealed that ATA achieved this effect by the following mechanisms. First, ATA could bind p70S6K at its ATP-binding pocket to prevent phosphorylation, and second by increasing the ubiquitination of p70S6K to cause its degradation. Since phosphorylation of S6 ribosome protein (S6RP) by p70S6K can induce protein synthesis at the ribosome, the dramatic reduction of p70S6K after ATA treatment led to great reductions of new protein synthesis on several cell cycle-related proteins including cyclin D3, aurora kinase A, polo-like kinase, cyclin B1, survivin; and reduced the levels of EGFR and MET. In addition, ATA treatment increased the levels of p53 and p21 proteins, which blocked cell cycle progression in the G1/S phase. Taken together, as ATA can effectively block multiple signaling pathways essential for protein synthesis and cell proliferation, ATA can potentially be developed into a multi-target anti-cancer agent to treat TKI-resistant NSCLC.

Cannabidiol Inhibits Tumorigenesis in Cisplatin-Resistant Non-Small Cell Lung Cancer via TRPV2.

Simple SummaryDrug resistance is the key factor contributing to the therapeutic failure of lung cancer and the deaths related to lung cancer. Our study demonstrated that small molecular weight non-psychotropic phytochemical, cannabidiol (CBD), inhibits growth and metastasis of drug-resistant non-small cell lung cancer cells (NSCLC) cells in-vitro and in-vivo. We further discovered that CBD mediates its anti-cancer effects in part via an ion channel receptor, TRPV2, present on lung adenocarcinoma. Moreover, we showed that CBD induces apoptosis of cisplatin-resistant cells by modulating oxidative stress pathways. Overall, these studies indicate that CBD could be used as a promising therapeutic strategy in TRPV2 expressing cisplatin-resistant NSCLC.Chemotherapy forms the backbone of current treatments for many patients with advanced non-small-cell lung cancer (NSCLC). However, the survival rate is low in these patients due to the development of drug resistance, including cisplatin resistance. In this study, we developed a novel strategy to combat the growth of cisplatin-resistant (CR) NSCLC cells. We have shown that treatment with the plant-derived, non-psychotropic small molecular weight molecule, cannabidiol (CBD), significantly induced apoptosis of CR NSCLC cells. In addition, CBD treatment significantly reduced tumor progression and metastasis in a mouse xenograft model and suppressed cancer stem cell properties. Further mechanistic studies demonstrated the ability of CBD to inhibit the growth of CR cell lines by reducing NRF-2 and enhancing the generation of reactive oxygen species (ROS). Moreover, we show that CBD acts through Transient Receptor Potential Vanilloid-2 (TRPV2) to induce apoptosis, where TRPV2 is expressed on human lung adenocarcinoma tumors. High expression of TRPV2 correlates with better overall survival of lung cancer patients. Our findings identify CBD as a novel therapeutic agent targeting TRPV2 to inhibit the growth and metastasis of this aggressive cisplatin-resistant phenotype in NSCLC.