NCI-H460 Cells Research Articles

Therapeutic potential of third-generation chimeric antigen receptor T cells targeting B cell maturation antigen for treating multiple myeloma

Multiple myeloma (MM) is an incurable hematologic malignancy characterized by the rapid proliferation of malignant plasma cells within the bone marrow. Standard therapies often fail due to patient resistance. The US FDA has approved second-generation chimeric antigen receptor (CAR) T cells targeting B-cell maturation antigen (anti-BCMA-CAR2 T cells) for MM treatment. However, achieving enduring clinical responses remains a challenge in CAR T cell therapy. This study developed third-generation T cells with an anti-BCMA CAR (anti-BCMA-CAR3). The CAR incorporated a fully human scFv specific to BCMA, linked to the CD8 hinge region. The design included the CD28 transmembrane domain, two co-stimulatory domains (CD28 and 4-1BB), and the CD3ζ signaling domain (28BBζ). Lentiviral technology generated these modified T cells, which were compared against anti-BCMA-CAR2 T cells for efficacy against cancer. Anti-BCMA-CAR3 T cells exhibited significantly higher cytotoxic activity against BCMA-expressing cells (KMS-12-PE and NCI-H929) compared to anti-BCMA-CAR2 T cells. At an effector-to-target ratio of 10:1, anti-BCMA-CAR3 T cells induced lysis in 75.5 ± 3.8% of NCI-H929 cells, whereas anti-BCMA-CAR2 T cells achieved 56.7 ± 3.4% (p = 0.0023). Notably, after twelve days of cultivation, anti-BCMA-CAR3 T cells nearly eradicated BCMA-positive cells (4.1 ± 2.1%), while anti-BCMA-CAR2 T cells allowed 36.8 ± 20.1% to survive. This study highlights the superior efficacy of anti-BCMA-CAR3 T cells against both low and high BCMA-expressing MM cells, surpassing anti-BCMA-CAR2 T cells. These findings suggest potential for advancing anti-BCMA-CAR3 T cells in chimeric antigen receptor T (CAR-T) therapy for relapsed/refractory MM.

Phenylhydrazone Alkaloids from the Deep-Sea Cold Seep Derived Fungus Talaromyces amestolkiae HDN21-0307.

Alkaloids with a phenylhydrazone architecture are rarely found in nature. Four unusual phenylhydrazone alkaloids named talarohydrazones A-D (1-4) were isolated from the deep-sea cold seep derived fungus Talaromyces amestolkiae HDN21-0307 using the one strain-many compounds (OSMAC) approach and MS/MS-based molecular networking (MN) combined with network annotation propagation (NAP) and the unsupervised substructure annotation method MS2LDA. Their structures were elucidated by spectroscopic data analysis, single-crystal X-ray diffraction, and quantum chemical calculations. Talarohydrazone A (1) possessed an unusual skeleton combining 2,4-pyridinedione and phenylhydrazone. Talarohydrazone B (2) represents the first natural phenylhydrazone-bearing azadophilone. Bioactivity evaluation revealed that compound 1 exhibited cytotoxic activity against NCI-H446 cells with an IC50 value of 4.1 μM. In addition, compound 1 displayed weak antibacterial activity toward Staphylococcus aureus with an MIC value of 32 μg/mL.

Cycloheptylprodigiosin from marine bacterium Spartinivicinus ruber MCCC 1K03745T induces a novel form of cell death characterized by Golgi disruption and enhanced secretion of cathepsin D in non-small cell lung cancer cell lines

Prodiginines have been studied extensively for their anticancer activity, however, the majority of the research has focused on prodigiosin. In this study, cycloheptylprodigiosin (S-1) is extracted from marine bacterium Spartinivicinus ruber MCCC 1K03745T, and its anticancer property was investigated. It exhibits remarkable cytotoxicity against a panel of human lung cancer cell lines, with the IC50 values ranging from 84.89 nM to 661.2 nM. After 6 h of treatment, S-1 gradually accumulates on mitochondria and lysosomes. While lower doses of S-1 induce cell cycle arrest, treatment with higher doses results in cell death in apoptotic independent manner in both NCI–H1299 and NCI–H460 cell lines. Interestingly, treatment with S-1 leads to the accumulation of LC3B-II via pathways that vary among different cell lines. In addition to its role as an autophagy inhibitor, S-1 also promotes autophagy initiation as demonstrated by the increment of EGFP fragment in the EGFP-LC3 degradation assay, however, inhibition of autophagy does not rescue cells from death induced by S-1. Mechanistically, S-1 impairs autophagic flux through disrupting acidic lysosomal pH and blocking the maturation of cathepsin D. Moreover, treatment with S-1 enhanced secretion of both pro- and mature forms of cathepsin D, coincident with disintegration of trans-Golgi network. Interestingly, S-1 does not induce ferroptosis, pyroptosis or necroptosis in NCI–H1299 cells. However, treatment of NCI–H460 cells with S-1 induces methuosis, which can be suppressed by Rac1 inhibitor EHT 1864. Our data demonstrate that S-1 is an effective anticancer agent with potential therapeutic application.

Design, Synthesis, and Bioevaluation of Transcriptional Enhanced Assocciated Domain (TEAD) PROTAC Degraders.

Dysregulation of the Hippo pathway has been observed in various cancers. The transcription factor TEAD, together with its coactivators YAP/TAZ, plays a crucial role in regulating the transcriptional output of the Hippo pathway. Recently, extensive research has focused on small molecule inhibitors targeting TEAD, but studies on TEAD degraders are comparatively rare. In this study, we designed and synthesized a series of TEAD PROTACs by connecting a pan-TEAD inhibitor with the CRBN ligand thalidomide. A representative compound, 27, exhibited potent antiproliferative activity against NF2-deficient NCI-H226 cells. It dose-dependently induced TEAD degradation dependent on CRBN and proteasome system and decreased key YAP target genes CYR61 and CTGF expressions in NCI-H226 cells. Further degradation selectivity studies revealed that 27 exhibited more potent activity against TEAD2 compared to those of the other three family members in Flag-TEADs transfected 293T cells. Therefore, 27 may serve as a valuable tool for advancing biological studies related to TEAD2.

Abstract LB070: In vitro modeling of antigen-driven exhaustion in human CAR T cells

Abstract A major hurdle for chimeric antigen receptor (CAR) T cells to function in solid tumors is chronic antigen exposure leading to terminal lymphocyte exhaustion and impaired anti-tumor activity. Therefore, it is necessary to establish improved in vitro models of antigen-driven CAR T cell exhaustion to evaluate new CAR design candidates prior to validation with costly in vivo studies. Using the human tumor antigen mesothelin (MSLN) and MSLN-targeting CAR T cells as a model, we developed two in vitro assays that drive an exhausted phenotype in human CAR T cells and can be used to predict anti-tumor responses in vivo: 1) a chronic stimulation assay with plate-bound recombinant antigen (MSLN), ICAM-1, and CD58 and 2) a high antigen-expressing 3D tumor spheroid co-culture assay. In the chronic antigen stimulation assay, repetitive exposure to plate-bound MSLN (alongside co-stimulation signals provided by recombinant ICAM-1 and CD58) reprogrammed the transcriptomic profile of MSLN CAR T cells leading to increased surface expression of inhibitory checkpoint molecules and reduced cytokine production. Moreover, chronically stimulated MSLN CAR T cells demonstrated a drastically impaired ability to kill MSLN+ tumor cells compared to their unstimulated or acutely stimulated counterparts. A similar exhausted phenotype was observed in the high-antigen tumor spheroid model: when MSLN CAR T cells were co-cultured with MSLN-high human NCI-H226 spheroids for 7 days in a 96-well plate, expression of inhibitory checkpoint molecules markedly increased on the surface of CAR T cells. This increased expression of exhaustion markers coincided with impairment of NCI-H226 spheroid clearance. By contrast, other tumor spheroid models expressing low to intermediate levels of MSLN (NCI-H1650, NCI-H2052, NCI-H292, and SKOV-3 tumor cells) did not substantially induce expression of inhibitory checkpoint molecules and the spheroids were successfully cleared by MSLN CAR T cells. Importantly, the ability (or inability) of a CAR construct to clear NCI-H226 high-antigen tumor spheroids in vitro accurately predicted CAR T cell performance in in vivo solid tumor studies: the CAR construct that showed effective killing in the in vitro spheroid model was the only condition to provide durable regression of NCI-H1650 tumors in a NSG xenograft mouse model. Additionally, the rank order of CAR efficacy for a series of MSLN CAR binders obtained using the tumor spheroid model mirrored in vivo efficacy. As the CAR T cell field develops novel strategies to combat antigen-driven exhaustion for use in solid tumors, these new antigen-dependent in vitro models of exhaustion can enable more cost-efficient and effective evaluation of those strategies than using in vivo models alone. Citation Format: Maria Steele, Tina Tan, Jason Yokoyama, Allan Wang, Ty Crowl, Jared Hammer, Jerry Chen, Kevin Haworth, Howell Moffett, Aaron Foster, Marc Lajoie, Scott Boyken, Rupesh Amin. In vitro modeling of antigen-driven exhaustion in human CAR T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB070.

Abstract LB129: Preclinical development of a bispecific antibody-trap selectively targeting CD38 and CD47 for treating hematologic malignancies

Abstract CD38 and CD47 are co-expressed in various hematologic malignancies including multiple myeloma (MM), B-cell non-Hodgkin lymphoma (NHL), B-cell acute lymphoblastic leukemia (ALL), T-cell ALL and B-cell chronic lymphocytic leukemia (CLL). We present, herein, several CD38 antibody - CD47 ligand trap bispecific antibodies (BsAbs) in an ADCC-enhanced IgG1 form that harbor high CD38 binding affinity, low CD47 binding affinity, along with negligible affinity to red blood cells (RBCs) and no induction of RBCs agglutination. Using flow cytometry and Biolayer interferometry (BLI), we confirmed that our BsAbs can simultaneously bind to both CD38 and CD47, and that binding to either antigen first does not prevent binding to the other antigen subsequently. On CD47+/CD38low Jurkat (T leukemic) cells, our BsAbs reveal less blocking effect on the CD47/SIRPα interaction than a monospecific CD47 ligand trap molecule based on which our BsAbs were designed; conversely, on CD38/CD47 double-positive Reh (pro-B ALL) cells, our BsAbs reveal 200-500-fold greater blocking effect on the CD47/SIRPα interaction than the same monospecific CD47 ligand trap molecule, indicating that the inhibition of CD47/SIRPα by our BsAbs is depended on the engagement of CD38 on tumor cells. On CD47+/CD38+ Daudi (B NHL) lymphoma cells and L-1236 (B Hodgkin) lymphoma cells, four of our BsAbs inhibited CD38 enzymatic activity, which is responsible for immune-suppressive adenosine production in tumor microenvironment. Our BsAbs presented potent dose-dependent antibody-dependent cellular cytotoxicity (ADCC) against CD47+/CD38+ Raji (B NHL) and NCI-H929 (B plasmacytoma myeloma) cells, while revealing negligible ADCC activity against CD47+/CD38− cells, indicating low off-tumor toxicity. Likewise, our BsAbs also harbor potent antibody-dependent cellular phagocytosis (ADCP) activity against Raji NHL cells, varied degree of complement-dependent cytotoxicity (CDC) activity and direct apoptosis induction against Daudi NHL cells. In a mouse xenograft model harboring NCI-H929 multiple myeloma, our BsAbs produced more potent tumor growth inhibition, than monospecific anti-CD47 ligand trap, anti-CD38 (isatuximab) or combination of isatuximab and anti-CD47 ligand trap, at the same molecular dose per body weight. Notably, clone IMM5605-12C10 eradicated all tumors and achieved 100% complete response rate in a group of six mice. In summary, simultaneous targeting CD38 and CD47 using our CD38 antibody - CD47 ligand trap bispecific design in an ADCC-enhanced IgG1 form presents a novel multimodal approach for treating hematologic malignancies that are resistant to anti-CD38 antibodies. Citation Format: Wenzhi Tian, Song Li, Dianze Chen, Yanan Yang, Huiqin Guo, Dandan Liu, Ruliang Zhang, Fan Zhang. Preclinical development of a bispecific antibody-trap selectively targeting CD38 and CD47 for treating hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB129.

Silencing TRIM29 Sensitizes Non-small Cell Lung Cancer Cells to Anlotinib by Promoting Apoptosis via Binding RAD50.

Previous studies have proposed that the transcriptional regulatory factor tripartite motif containing 29 (TRIM29) is involved in carcinogenesis via binding with nucleic acid. TRIM29 is confirmed to be highly expressed when the cancer cells acquire therapy-resistant properties. We noticed that TRIM29 levels were significantly increased in anlotinib-resistant NCIH1975 (NCI-H1975/AR) cells via mining data information from gene expression omnibus (GEO) gene microarray (GSE142031; log2 fold change > 1, p < 0.05). Our study aimed to investigate the function of TRIM29 on the resistance to anlotinib in non-small cell lung cancer (NSCLC) cells, including NCI-H1975 and A549 cells. Real-time RT-PCR and western blot were used to detect TRIM29 expression in anlotinib- resistant NSCLC (NSCLC/AR) cells. Apoptosis were determined through flow cytometry, acridine orange/ethidium bromide staining as well as western blot. ELISA was used to measure the content of C-X3-C motif chemokine ligand 1. Co-Immunoprecipitation assay was performed to verify the interaction between TRIM29 and RAD50 double-strand break repair protein (RAD50). TRIM29 expression was shown to be elevated in the cytoplasm and nucleus of NSCLC/ AR cells compared to normal NSCLC cells. Next, we demonstrated that TRIM29 knockdown facilitated apoptosis and enhanced the sensitivity to anlotinib in NSCLC/AR cells. Based on the refined results citing from the database BioGRID, it was proved that TRIM29 interacted with RAD50. Herein, RAD50 overexpression diminished the pro-apoptotic effect induced by silencing TRIM29 in anlotinib-resistant A549 (A549/AR) cells. Finally, we concluded that the increased sensitivity to anlotinib in NSCLC/AR cells was achieved by knocking down TRIM29, besides, the positive effects of TRIM29 knockdown were attributed to the promotion of apoptosis via binding to RAD50 in NSCLC/AR cell nucleus. Therefore, TRIM29 might become a potential target for overcoming anlotinib resistance in NSCLC treatment.

In vitro and in silico antiproliferative potential of isolated flavonoids constitutes from Pistacia integerrima.

Cancer is one of the most demanding domains for innovative, effective, safe, and affordable therapeutically active chemicals. The main aim of this study is to research new phytochemicals with anticancer activity. The current experiment identified and analyzed six compounds for anti-cancer potential supported by molecular simulation studies. The defatted methanolic extract underwent column chromatography, resulting in the isolation of six flavonoids. These include 3,5,7,4'-tetrahydroxy-flavanone (1), naringenin (2), 3,5,4'-trihydroxy-7-methoxy-flavanone (3), sakuranetin (4), spinacetin (5), and patuletin (6). The isolated compounds (1-6) were assessed for invitro anti-cancer activity against various cell lines such as HepG2 (hepatoma G2), A498 (kidney), NCI-H226 (lungs), and MDR2780AD (human ovarian). The maximum antiproliferative effect was against HepG2 and MDR2780AD. When compounds 6, 5, and 1 were compared to a standard anti-cancer medicine (paclitaxel) with an IC50 of 7.32, it was shown that compounds 6, 5, and 1 exhibited significant activity against HepG2 with IC50 values of 14.65, 20.87, and 27.09 µM, respectively. All tested compounds showed an IC50 of less than 1 µM and had notable effects against MDR2780 AD cell lines. Compound 6 exhibited notable potency against the HepG2, A498, and MDR2780AD cell lines, among the six compounds that were evaluated. In contrast, compound 3 demonstrated the most pronounced impact on the NCI-H226 cell line. Docking investigations were performed using tubulin as the specific target concerning PDB ID 4O2B. The six compounds under investigation interact hydrophobically and hydrophilically with tubulin-binding site amino acid residues.

Silencing RNA-Mediated Knockdown of IFITM3 Enhances Senecavirus A Replication.

Senecavirus A (SVA) is a non-enveloped, positive sense, single-stranded RNA virus that causes vesicular diseases in pigs. Interferon-induced transmembrane 3 (IFITM3) is an interferon-stimulated gene (ISG) that exhibits broad antiviral activity. We investigated the role of IFITM3 in SVA replication. Both viral protein expression and supernatant virus titer were significantly increased when endogenous IFITM3 was knocked down by approximately 80% in human non-smallcell lung carcinoma cell line (NCI-H1299) compared to silencing RNA control. Interestingly, overexpression of exogenous IFITM3 in NCI-H1299 cells also significantly enhanced viral protein expression and virus titer compared to vector control, which was positively correlated with induction of autophagy mediated by IFITM3 overexpression. Overall, our results indicate an antiviral role of endogenous IFITM3 against SVA. The exact molecular mechanisms by which endogenous IFITM3 limits SVA replication remain to be determined in future studies.

Phytochemical analysis and anticancer effect of Camellia oleifera bud ethanol extract in non-small cell lung cancer A549 cells.

Camellia oleifera is a medicine food homology plant widely cultivated in the Yangtze River Basin and southern China due to its camellia oil. Camellia oleifera bud and fruit exist simultaneously, and its bud is largely discarded as waste. However, C. oleifera bud has been used in traditional Chinese medicine to treat a variety of ailments. Thus, the purpose of this study was to identify the chemical components of C. oleifera bud ethanol extract (EE) and first evaluate its anticancer effects in non-small cell lung cancer A549 cells. Based on UHPLC-Q-Orbitrap-MS analysis, seventy components were identified. For anticancer activity, C. oleifera bud EE had remarkable cytotoxic effect on non-small cell lung cancer A549 (IC50: 57.53 ± 1.54μg/mL) and NCI-H1299 (IC50: 131.67 ± 4.32μg/mL) cells, while showed lower cytotoxicity on non-cancerous MRC-5 (IC50 > 320μg/mL) and L929 (IC50: 179.84 ± 1.08μg/mL) cells. It dramatically inhibited the proliferation of A549 cells by inducing cell cycle arrest at the G1 phase. Additionally, it induced apoptosis in A549 cells through a mitochondria-mediated pathway, which decreased mitochondrial membrane potential, upregulated Bax, activated caspase 9 and caspase 3, and resulted in PARP cleavage. Wound healing and transwell invasion assays demonstrated that C. oleifera bud EE inhibited the migration and invasion of A549 cells in a dose-dependent manner. The above findings indicated that C. oleifera bud EE revealed notable anticancer effects by inhibiting proliferation, inducing apoptosis, and suppressing migration and invasion of A549 cells. Hence, C. oleifera bud ethanol extract could serve as a new source of natural anticancer drugs.

AM-DMF-SCP: Integrated Single-Cell Proteomics Analysis on an Active Matrix Digital Microfluidic Chip.

Single-cell proteomics offers unparalleled insights into cellular diversity and molecular mechanisms, enabling a deeper understanding of complex biological processes at the individual cell level. Here, we develop an integrated sample processing on an active-matrix digital microfluidic chip for single-cell proteomics (AM-DMF-SCP). Employing the AM-DMF-SCP approach and data-independent acquisition (DIA), we identify an average of 2258 protein groups in single HeLa cells within 15 min of the liquid chromatography gradient. We performed comparative analyses of three tumor cell lines: HeLa, A549, and HepG2, and machine learning was utilized to identify the unique features of these cell lines. Applying the AM-DMF-SCP to characterize the proteomes of a third-generation EGFR inhibitor, ASK120067-resistant cells (67R) and their parental NCI-H1975 cells, we observed a potential correlation between elevated VIM expression and 67R resistance, which is consistent with the findings from bulk sample analyses. These results suggest that AM-DMF-SCP is an automated, robust, and sensitive platform for single-cell proteomics and demonstrate the potential for providing valuable insights into cellular mechanisms.

Abstract 6266: Neurotensin receptor 1 regulates dimerization of HER2-HER4 and proliferation of non-small cell lung cancer cells

Abstract The neurotensin receptor 1 (NTSR1) is a 418 amino acid type A G protein-coupled receptor which causes phosphatidylinositol turnover and proliferation of non-small cell lung cancer (NSCLC) cells (Moody et al., Peptides 2021; 137: 710480). Adding the agonist NTS to NSCLC cells increases transactivation of the receptor tyrosine kinases (RTK) EGFR, HER2 and HER3 (Moody et al., Biology 2023 12: 957). The RTK tyrosine phosphorylation and growth caused by NTS is impaired by SR48692, a small molecule NTSR1 antagonist. The effects of NTS were investigated on HER4 using NSCLC cells. By Western blot, cell lines NCI-H522 and H661 had high levels of NTSR1, HER 4 and neuregulin 1 (NRG1), a ligand for HER4 but not NTSR2, HER3 or NRG2. By RT-PCT, high levels of HER4 and its isoforms JM-a, CYT1 and CYT2 were present in NCI-H522 and H661 cells. Adding NTS (0.1 μM) or NRG1 (0.01 μg/ml) to NCI-H522 or H661 cells increased P-Tyr1284-HER4 and P-ERK 3-fold. Using immunoprecipitation techniques, adding NTS to NSCLC cells increased formation of HER4-HER2 heterodimers which leads to cancer proliferation (Lucas et al., Pharmacol Rev 2022; 74:18). The increase in PY1284-HER4 caused by NTS was impaired by SR48692, HER4 siRNA, NRG1 siRNA or ibrutinib (TKI). The NTSR1 regulation of HER4 transactivation is impaired by GM6001 (MMP inhibitor]), PP2 (Src inhibitor) or N-acetylcysteine (antioxidant). The clonal growth of NSCLC cells is increased by NTS or NRG1 but decreased by SR48692 or ibrutinib. The results indicate that NTS increases the formation of HER2/HER4 heterodimers leading to the proliferation of NSCLC cells. Citation Format: Terry W. Moody, Irene Ramos-Alvarez, Robert T. Jensen. Neurotensin receptor 1 regulates dimerization of HER2-HER4 and proliferation of non-small cell lung cancer cells [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 6266.

Abstract 2889: Cartilage oligomeric matrix protein as a potential biomarker and therapeutic target of radiation resistance in NSCLC

Abstract Despite recent advances in lung cancer treatment, the survival rate of non-small cell lung cancer (NSCLC) is ~57% largely due to treatment resistance. Cartilage Oligomeric Matrix Protein (COMP) has previously been implicated in promoting metastasis and is associated with poor survival outcomes in breast, prostate and colorectal cancers. Patients with prostate cancer and osteoarthritis (a condition characterized by elevated circulating COMP) had both increased tumor COMP expression and rate of metastasis. Additionally, elevated COMP expression, among others, was observed in a study that investigated the gene expression of invasive NSCLC cell lines, though association with COMP was not further studied. Therefore, COMP could potentially increase the metastatic potential for NSCLC and thus serve as a therapeutic target to reduce tumor aggressiveness. We performed proliferation and migration assays on NSCLC cell lines, A549 and NCI-H1975, treated with 2 µg/mL COMP to identify COMP’s role in NSCLC progression. Plated cells in monolayer were treated with either COMP (2µg/mL) or PBS (control), and then within each group received either: 1] one of two different inhibitors of COMP signaling (either the αᵥ integrin antagonist cilengitide (1µM), or the Src inhibitor PP2 (1µM)), or 2] no additional treatment. Replicates from each group then received 2Gy Cs137 γ-radiation, or no irradiation. Proliferation and migration were monitored via the IncuCyte ZOOM live-cell imaging system over a 48hr interval. Radiation alone reduced both proliferation (-22%, p<0.0001) and migration (-40%, p<0.01) of A549 cells. However, exogenous COMP treatment attenuated the effects of radiation, maintaining both A549 proliferation (19% vs irradiated control, p<0.05) and migration (91% vs irradiated control, p<0.0001). While the overall pattern for NCI-H1975 cells with/without COMP treatment was similar, the magnitude of the responses was reduced vs A549 cells and non-significant. In contrast, radiation resistance with COMP treatment was abrogated by exogenous treatment of both COMP inhibitors. Cilengitide treatment resulted in lower proliferation rates of the COMP treated, irradiated A549 (-16%, p<0.05) and NCI-H1975 cell lines (-58%, p<0.0001) vs COMP treatment alone, while PP2 reduced proliferation and migration rates for both irradiated and non-irradiated COMP treated cell lines (p<0.0001). PP2 treatment alone did not reduce migration rates of either irradiated control cell lines. In conclusion, COMP serves to radioprotect NSCLC in an undetermined manner, and could serve as both a prognostic biomarker for and therapeutic target against radiation resistance in patients with NSCLC. Support: Milton Raben Foundation Citation Format: Kaitlyn E. Reno, Sun H. Park, Michael K. Farris, Ryan T. Hughes, Joseph E. Moore, Chirayu M. Patel, Jeffrey S. Willey. Cartilage oligomeric matrix protein as a potential biomarker and therapeutic target of radiation resistance in NSCLC [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 2889.

Abstract 1931: Targeting YAP1/TEAD signaling re-sensitizes MAPK/ERK pathway inhibitors in KRAS- driven cancer cells

Abstract Many TEAD small-molecule inhibitors (SMIs) have recently reported initial clinical evaluations on Hippo-mutated cancer types. Several studies have demonstrated that activation of the YAP1/TEAD transcriptional complex via a Hippo-independent manner can drive resistance to MAPK/ERK pathway inhibitors. Here, we elucidated the potential mechanism of TEAD inhibition overcoming MAPK/ERK pathway resistance with a TEAD SMI (hereafter abbreviated as TEADi). Firstly, the MoA of TEADi was validated in Hippo-mutated cells. TEADi inhibited cell growth of Hippo-mutated cancer cell lines NCI-H226 and NCI-H2052 but not that of MKN45, a YAP1-deletion cell line. Co-IP and qRT-PCR results demonstrated that TEADi disrupted the interaction between YAP1 and TEAD, and thus markedly repressed the expression of CTGF and CYR61, two downstream targets of YAP1/TEAD, in NCI-H226 cells. Secondly, we hypothesized that hyper-activation of TEAD confers resistance to KRAS mutant cancer types. To test this hypothesis, we generated two resistant cell lines. One is KARS G12C inhibitor Sotorasib-resistant NCI-H358 (NCI-H358-R). The other is MEK inhibitor Trametinib-resistant HCT116 (HCT116-R). Immunofluoresence assay showed that YAP1 nucleus translocation was enhanced in both resistant cells (NCI-H358-R and HCT116-R), but not in their parental counterparts (NCI-H358-P and HCT116-P). The enhanced YAP1 nucleus translocation resulted in increased transcription activities of TEAD in both resistant cells, as illustrated by luciferase reporter assay. Accordingly, CTGF and CYR61 were observed upregulated in both resistant cells. The above data indicate that YAP1/TEAD mediated-transactivation plays a role in MAPK pathway resistance. Indeed, TEADi treatment alone displayed substantial difference in cell growth inhibition between parental and resistant cell lines in both NCI-H358 and HCT116, further confirming that the resistant cells are more dependent on YAP1/TEAD signaling. Finally, we evaluated the combinational efficacies of TEADi and MAPK/ERK pathway inhibitors in the resistant cells. It was observed that addition of TEADi could significantly restore the response of NCI-H358-R and HCT116-R to Sotorasib and Trametinib, respectively. Mechanistically, TEADi efficiently suppressed TEAD transcriptional activities and subsequently the expression of CTGF and CYR61 in both resistant cells. Moreover, TEADi had almost no impact on ERK phosphorylation in either of the resistant cells, suggesting that re-sensitization of MAPK/ERK pathway inhibitors by TEADi is independent of primary onco-genetic signaling pathway. Taken together, our study demonstrates that inhibition of YAP1/TEAD signaling would be an efficient approach to overcome resistance to MAPK/ERK pathway inhibitors in the patients carrying KRAS mutations, and provides the scientific basis for development of combination therapy strategies. Citation Format: Xianwen Yang, Guiping Li, Peiling Ren, Xiaoyun Shi, Ying Yu, Baoyu Hao, Pan Wang, Min Cheng, Guangxiu Dai. Targeting YAP1/TEAD signaling re-sensitizes MAPK/ERK pathway inhibitors in KRAS- driven cancer cells [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 1931.

Abstract 3347: KIFC1 inhibition elicits antineoplastic activity in small cell lung cancer by inducing anaphase catastrophe

Abstract Cancer cells often have more than two centrosomes (supernumerary centrosomes) and this is one of the hallmarks of cancer. We previously demonstrated that CDK2 antagonism inhibits clustering of supernumerary centrosomes at mitosis leading to multipolar cell division and apoptotic death in non-small cell lung cancer. This antineoplastic process was designated anaphase catastrophe. Anaphase catastrophe can preferentially affect cancer cells sparing normal cells with two centrosomes. On the other hand, as CDK2 functions in normal cell cycle progression, its inhibition can also affect normal and malignant cells. Identification of a novel drug target that would preferentially cause anaphase catastrophe is needed. The motor protein KIFC1 affects centrosome clustering. Small cell lung cancer (SCLC), known for its poor prognosis with limited therapeutic options, displays disruptions in the cell cycle checkpoint mechanism due to inactivation of p53 and RB. We hypothesized SCLC was susceptible to mitotic abnormalities, including anaphase catastrophe. In this study, we explored the possibility of KIFC1 as a novel therapeutic target that triggers anaphase catastrophe in SCLC. In-silico GEO database analysis revealed elevated levels of KIFC1 and PLK4 in SCLCs compared with other cancers and normal tissues. PLK4 plays a key role in centrosome amplification. Our in-silico analysis results imply that centrosome amplification and their clustering frequently occur in SCLC. We performed in-vitro functional analysis of KIFC1 inhibition in SCLC cell lines (NCI-H146, SHP77, NCI-H209, and NCI-H524 cells), using siRNAs and CRISPR-Cas9 system for genetic inhibition and AZ82, a specific KIFC1 inhibitor, for pharmacologic inhibition of KIFC1. Cell proliferation was measured by the CellTiter-Glo Luminescent Assay and apoptosis was scored by annexin V and PI staining followed by flow cytometry analysis. Cell growth was reduced and apoptosis was statistically significantly increased after genetic inhibition of KIFC1 by siRNAs and by use of the CRISPR-Cas9 system in SCLC cell lines. These antineoplastic effects also occurred after AZ82 treatments, in a dose-dependent manner in these SCLC cell lines. To examine the mitotic status of SCLC cells after KIFC1 inhibition, SCLC cells were stained with α-tubulin antibody and DAPI before analysis with a fluorescence microscope. Besides normal bipolar mitotic cells, multipolar mitotic cells were observed, and their population was increased after KIFC1 knockdown with siRNAs. This indicated that clustering of supernumerary centrosomes was inhibited by targeting KIFC1 in SCLC cells. Taken together, KIFC1 is a potential therapeutic target to induce anaphase catastrophe in cancer cells. Targeting KIFC1 is a novel therapeutic strategy to consider for SCLC that currently has limited treatment options. Citation Format: Natsuki Nakagawa, Masakatsu Tokunaga, Mirei Ka, Yuriko Sugiura, Takahiro Iida, Takahiro Ando, Kousuke Watanabe, Xi Liu, Ethan Dmitrovsky, Hidenori Kage, Masanori Kawakami. KIFC1 inhibition elicits antineoplastic activity in small cell lung cancer by inducing anaphase catastrophe [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 3347.

Abstract 6513: Discovery and characterization of a KRASG12C/D/V degrader with potent anti-tumor activity in KRASmut-driven preclinical models

Abstract KRAS mutations are one of the most common oncogenic drivers for multiple cancers. KRASG12X mutations are identified in approximately 25% of lung cancer, 24% of colorectal (CRC), and 79% of pancreatic ductal adenocarcinoma (PDAC). Although the approval of Sotorasib in 2021 by the FDA partially addressed the needs of patients with KRASG12C mutant, other mutations, such as G12D and G12V, still lack effective target therapies. Here, we report RD0255359, a proteolysis targeting chimera (PROTAC) KRASG12C/D/V degrader with highly potent cell viability and KRASG12C/D/V degradation ability. In vitro, RD0255359 exhibited a remarkable KRAS degradation property in NCI-H358 (G12C), AGS (G12D), and SW620 (G12V) cell lines at concentrations below 10 nM. RD0255359 suppressed the proliferation of these cells with nanomolar IC50 without affecting the viability of KRASWT or KRAS-independent cell lines. Meanwhile, using protease inhibitor MG132 or corresponding ligand pretreatment and VHLKD-AGS cell experiment confirmed that RD0255359-induced KRAS degradation was VHL and UPS dependent. In vivo, weekly IV injection of 1-10 mg/kg RD0255359 led to tumor growth suppression or tumor regression in PK-59 PDAC xenograft model. The in vivo PK/PD demonstrated that durable KRAS degradation could be achieved with once- weekly IV injection. Notably, after injection, the drug can distribute into tumor tissue rapidly and abundantly, and maintaining a high concentration till the next administration. Taken together, RD0255359 is a highly potent and selective KRASG12C/D/V degrader with favorable PK properties. It exhibits a promising therapeutic index in preclinical studies. Citation Format: Zhengqing Li, Renqi Xu, Xiaofeng Yang, Zhengyao Zou, Xiaoqiang Kong, Haibo Chen, Yongqing Sun, Ying Zhao, Jing Guo, Cheng Yang, Han Han, Xiaoyun Liu, Hong Lan, Lieming Ding, Quan Zhou, Hao Wu, Jiabing Wang. Discovery and characterization of a KRASG12C/D/V degrader with potent anti-tumor activity in KRASmut-driven preclinical models [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 6513.

Abstract 640: In vivo rescue of p53 tumor suppressor function with ADGN-531 across Pan-p53 alterations

Abstract p53 tumor suppressor mutations result in loss of function of p53 in approximately 50% of all human cancers.TP53 missense, nonsense mutations and p53 deletion are present in about 50%, 12% and 5% of all p53 driven cancers, respectively. To date there is no effective treatment for rescuing p53 function in cancers using a pan-p53 approach. The use of ADGN-531 is a new potent strategy combining mRNA with a tumor selective peptide nanocarrier to rescue of p53 tumor suppressor function. ADGN-531 contains proprietary p53-mRNA complexed with proprietary short amphipathic peptides in the form of nanoparticles. ADGN-531 was evaluated on 20 different cancer cell lines harboring various p53 mutations. In-vivo efficacy of IV-administered ADGN-531 nanoparticles was evaluated on colorectal SW403 (p53 nonsense/KRASG12V), osteosarcoma SaOs2 (p53 null) and NSCLC NCI-H358 (p53 null/KRASG12C) mouse xenografts at a dose of 0.25-1.0 mg/kg weekly. ADGN-531/sotorasib synergy was evaluated in vitro on NCI-H358 (p53 null/KRASG12C) and Mia-PACA (p53R48W/KRASG12C) cells and in vivo on NCI-H358 mouse xenograft. ADGN-531 markedly inhibits the growth of a large panel of cancer cells harboring p53-missense or nonsense mutations or p53-deletion, by rescuing p53 transcriptional activity and subsequent expression of its target proteins. ADGN-531 induces cell cycle arrest in G1 due to p21 upregulation and apoptosis following BAX and PUMA activation. IV-administration of ADGN-531 resulted in dose responsive tumor growth inhibition in SaOs2, SW403 and H358 xenografts independent of KRAS status. We demonstrated a strong synergy in a NSCLC H358 xenograft (tumor regression 80%) when restoring P53 function with ADGN-531 (0.5mg/kg, weekly) along with inhibition of KRASG12C mutant using sotorasib (10mg/kg, daily). ADGN-531 potentiated the effect of sotorasib on sotorasib resistant cells harboring high level of KRASG12C in permanent active state. ADGN-531 treatments are well tolerated, no sign of clinical toxicity was detected after single or repeated administrations. ADGN-531 is effective in rescuing p53 function both in vitro and in vivo across a wide range of p53 alterations. Our study provides a proof-of-concept that restoration of tumor suppressor function by ADGN-531 could be used as a single agent therapy in P53 altered tumors independent of other driver mutations e.g., KRAS, or with other therapies for potent combinatorial cancer treatment. Citation Format: Gilles Divita, Audrey Grunenberger, Elodie Czuba, Veronique Josserand, Melanie Guidetti, Neil Desai. In vivo rescue of p53 tumor suppressor function with ADGN-531 across Pan-p53 alterations [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 640.

Abstract 5815: Next-generation RNAi therapeutics: siRNA-loaded exosomes targeting KRAS G12C in non-small cell lung cancer (NSCLC)

Abstract Background: NSCLC represents the predominant subtype of lung cancer, boasting an overall 5-year survival rate of 26%, a figure that fluctuates based on the disease stage. Within NSCLC, certain cancer gene mutations, notably KRAS G12C, are currently classified as 'undruggable targets,' creating hurdles in achieving effective treatment. The development of innovative anticancer drugs aimed at these challenging targets is expected to present a viable solution to these difficulties. Methods: Exosomes have garnered attention as an inventive drug delivery system for in vivo gene delivery through systemic injection. We used a new post-loading approach by actively loading KRAS G12C siRNA into exosomes using the shock waves (SWEET: shock wave exosome engineering technology ) in this study. The effect of in vitro and in vivo were tested in NCI-H358 (KRAS G12C mt NSCLC) cells and its xenograft mouse model. The tumor size and weight were measured twice a week. Results: We developed an exosome-based therapeutic using siRNA to effectively inhibit KRAS G12C, targeting NSCLC. The SWEET platform's post-loading method showed a high efficiency in loading KRAS G12C siRNA into exosomes. The KRAS G12C siRNA-loaded exosomes effectively silenced oncogenic KRAS G12C expression in cancer cells; they inhibited tumor growth when administered intravenously in a NSCLC xenograft mouse model. Remarkably, the anticancer effect of KRAS G12C-loaded exosome was similar to that of Sotorasib, a KRAS G12C mutation targeting anti-cancer drug. Conclusion: Our study showed KRAS G12C siRNA loaded exosomes were effectively delivered to NSCLC tumor tissue via intravenous (IV) administration. And its anti-cancer effects were very similar to sotorasib, showing potential as a treatment for KRAS G12C mutation-expressing cancer. The effective delivery of siRNA to tumor tissues through exosomes highlights the potential of this technology as a promising approach for developing new RNAi therapeutics for cancer. Citation Format: Hyo Kyeong Kim, Yujeong Choi, Kyoung Hwa Kim, Yeongju Byun, Tae Hee Kim, Jae Hwan Kim, Shung Hyun An, DaeHo Bae, MyeongKwan Choi, Jihwa Chung, Kihwan Kwon. Next-generation RNAi therapeutics: siRNA-loaded exosomes targeting KRAS G12C in non-small cell lung cancer (NSCLC) [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 5815.

Abstract 4513: Exploration degradation of intrinsically disordered protein YAP induced by PROTACs

Abstract Yes-associated protein (YAP), a potent oncogene and a key player in the Hippo tumor suppression pathway, has long been considered challenging to target due to its partially intrinsically disordered nature. However, recent advancements in High-throughput Screening (HTS) have yielded a breakthrough, as several YAP binders have been identified, igniting new possibilities in the quest to combat YAP-driven malignancies. Building upon this progress, a novel approach utilizing Proteolysis-Targeting Chimera (PROTAC) technology was employed to design and synthesize a series of YAP degraders. Here, our degraders were created by linking NSC682769, a previously reported YAP binder, with either VHL ligand 2 or pomalidomide using various linkers of different lengths and types. Among these degraders, YZ-6 emerges as the most promising, inducing rapid and sustained YAP degradation via promoting its ubiquitination-proteasome-dependent proteolysis. This process effectively suppressed YAP/TEAD-led transcription in both YAP-dependent NCI-H226 and Huh7 cancer cell lines. In addition to its degradation capabilities, YZ-6 also exhibited antiproliferative activity in both cell lines. Importantly, YZ-6 efficiently suppressed tumor development in the Huh7 xenograft mouse model, accompanied by a remarkable decrease in YAP levels, without adverse effects on the mice. These findings highlight the potential of PROTAC-mediated degradation as a viable strategy for reducing oncogenic YAP levels and attenuating downstream signaling in cancer cells. Moreover, the development of PROTACs based on NSC672869 holds promise for treating YAP-driven malignancies, opening new avenues for cancer therapy. Citation Format: Chen Zhou, Chunbao Sun, Liya Pi, Chenglong Li. Exploration degradation of intrinsically disordered protein YAP induced by PROTACs [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 4513.

Abstract 1884: DXC006, Anti-CD56-CPT113 ADC displays favorable anti-tumor efficacy, pharmacokinetic and safety profiles in preclinical studies

Abstract CD56, also known as neural cell adhesion molecule 1 (NCAM1), is a type I plasma membrane glycoprotein involved in cell-cell and cell-matrix adhesion. It is overexpressed in almost all neuroblastomas, 95% of small cell lung cancers and 78% of multiple myeloma patients[1], whereas CD56 is less frequently or less abundantly expressed in normal tissues, making it a potential target for tumor therapy. So far, no CD56-targeting ADCs have entered late-stage clinical trials.DXC006 is an antibody-drug conjugate of a humanized anti-CD56 antibody (DXA006) linked to the topoisomerase I inhibitor CPT113 through a cleavable peptidyl linker and CROSSCONJU™ technology. The pharmacologic activity and mechanism of action of DXC006 were investigated in several human cancer cell lines and xenograft mouse models. Pharmacokinetic and safety profiles were also assessed in rats and cynomolgus monkeys.DXC006 binds specifically to CD56 and internalizes into tumor cells, after intracellular trafficking to lysosome and CPT113 releasing, induces DNA damage. In vitro cell based assays showed that DXC006 remarkably inhibited the cell growth of CD56-positive cell lines of IMR-32 (neuroblastoma), NCI-H526 (small cell lung cancer), and NCI-H929 (multiple myeloma), with IC50 of 0.0046~0.061 nmol/L. Although DXC006 also binds to CD56 expressed NK cells, we found that the inhibition of DXC006 to human NK cells was much less effective, with IC50 higher than 675 nmol/L, which is ten thousand-folds less potent than that of targeted abnormal cells. In vivo, DXC006 demonstrated good durable antitumor activities in three CD56-positive xenograft models of IMR-32, NCI-H526 and NCI-H929 cell lines, with a single dose of 2 mg/Kg, 3 mg/Kg, and 5 mg/Kg, respectively. No animal death or moribund outcome was observed in two months during preclinical safety studies in 150 mg/Kg Q2W dosed rats. For cynomolgus monkeys, the highest non-severely toxic dose (HNSTD) of DXC006 was 20 mg/Kg, in Q2W, four times of administration. PK profiles of both DXC006 the naked antibody DXA006 were comparable in both rats and monkeys. In summary, DXC006 demonstrated strong antitumor activity in various types of cancer cells both in vitro and in vivo, and good pharmacokinetic and safety profiles in rat and monkeys, a potential to be explored in clinical applications. [1] Yang Feng, Yanping Wang, Zhongyu Zhu, et al. Differential killing of CD56-expressing cells by drug-conjugated human antibodies targeting membrane-distal and membrane-proximal nonoverlapping epitopes. mAbs 2016; 799-810. Citation Format: Xiangfei Kong, Huihui Guo, Yong Du, Zhicang Ye, Yongxiang Chen, Zhixiang Guo, Lingli Zhang, Lu Bai, Junxiang Jia, Yunxia Zheng, Wei Zheng, Jun Zheng, Wenjun Li, Yuanyuan Huang, Zhongliang Fan, Mengmeng Liu, Binbin Chen, Meng Dai, Juan Wang, Fang Du, Miaomiao Chen, Qingliang Yang, Robert Y. Zhao. DXC006, Anti-CD56-CPT113 ADC displays favorable anti-tumor efficacy, pharmacokinetic and safety profiles in preclinical studies [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 1884.