H460 Cell Lines Research Articles

STAMBPL1 promotes the progression of lung adenocarcinoma by inhibiting DHRS2 expression

BackgroundLung cancer is responsible for the majority of cancer deaths in the world. We found a significant increase of STAMBPL1 expression in lung adenocarcinoma (LUAD) tissues and cells. However, its mechanism has not been clarified. MethodsLUAD tissues and adjacent normal tissues were collected from 62 patients treated in the First Affiliated Hospital of Wenzhou Medical University from August 2018 to August 2021. In vivo, the clinical data and STAMBPL1 expression of 62 patients with LUAD were analyzed by qPCR. In vitro, cell experiments were carried out after STAMBPL1 knockdown in A549 and H1299 cells to determine cell growth, migration rate, evasiveness, colony-forming ability, and apoptosis. Gene sequencing was used to explore the expression of various genes in A549 and H1299 cells to verify that DHRS2 was up-regulated after STAMBPL1 knockdown; cell experiments further detected the role of the DHRS2 gene after DHRS2 overexpression in A549 and H1299 cells. A rescue experiment was conducted to certify that STAMBPL1 promotes NSCLC progression by regulating DHRS2 expression. ResultsAfter STAMBPL1 knockdown by siRNA. Migration, invasion, colony formation, and proliferation of siRNA groups were suppressed than those of NC groups in A549 and H1299 cells, while the cell apoptosis rate of siRNA groups increased significantly. By using gene-sequence analysis, we found that the expression level of the DHRS2 gene was up-regulated in STAMBPL1 siRNA groups, compared with STAMBPL1 NC (negative control) groups in A549 and H1299, which was verified by qPCR and WB. Further experiments showed that the DHRS2 OE group was suppressed in cell proliferation, migration, and invasion in the A549 and H1299 cell lines compared to the DHRS2 NC group, while DHRS2 OE group was significantly enhanced in the cell apoptosis in the A549 and H1299 cell lines. According to the rescue experiment, cell proliferation, migration, and invasion of the STAMBPL1 SI+DHRS2 SI group were enhanced compared with the STAMBPL1 SI+DHRS2 NC group in A549 and H1299 cells, while the STAMBPL1 SI+DHRS2 OE group were further decreased. ConclusionsThe expression of STAMBPL1 mRNA is significantly up-regulated in LUAD, promoting the progression of LUAD by down-regulating the expression of DHRS2 and acting as a potential biomarker of LUAD.

The mitotic spindle-related seven-gene predicts the prognosis and immune microenvironment of lung adenocarcinoma

PurposeAbnormalities in the mitotic spindle have been linked to a variety of cancers. Data on their role in the onset, progression, and treatment of lung adenocarcinoma (LUAD) need to be explored.MethodsThe data were retrieved from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and Molecular Signatures Database (MSigDB), for the training cohort, external validation cohort, and the hallmark mitotic spindle gene set, respectively. Mitotic spindle genes linked to LUAD prognosis were identified and intersected with differentially expressed up-regulated genes in the training cohort. Nomogram prediction models were built based on least absolute shrinkage and selection operator (LASSO) regression, univariate cox, and multivariate cox analyses. The seven-gene immunological score was examined, as well as the correlation of immune checkpoints. The DLGAP5 and KIF15 expression in BEAS-2B, A549, H1299, H1975, and PC-9 cell lines was validated with western blot (WB).ResultsA total of 965 differentially expressed up-regulated genes in the training cohort intersected with 51 mitotic spindle genes associated with LUAD prognosis. Finally, the seven-gene risk score was determined and integrated with clinical characteristics to construct the nomogram model. Immune cell correlation analysis revealed a negative correlation between seven-gene expression with B cell, endothelial cell (excluding LMNB1), and T cell CD8 + (p < 0.05). However, the seven-gene expression was positively correlated with multiple immune checkpoints (p < 0.05). The expression of DLGAP5 and KIF15 were significantly higher in A549, H1299, H1975, and PC-9 cell lines than that in BEAS-2B cell line.ConclusionHigh expression of the seven genes is positively correlated with poor prognosis of LUAD, and these genes are promising as prospective immunotherapy targets.

Downregulating MK5 to reduce immune escape induced by thebinding of PD-1 with PD-L1 in NSCLC

Lung cancer is the leading cause of cancer death in both men and women worldwide. Previous studies have indicatedthat MK5 stabilizes Yes-associated Protein (YAP) in LATS1/2-null RPE1 cells, and YAP regulates PD-L1 expression inNSCLC. Since previous studies did not associate MK5 KO with PD-L1 expression in NSCLC, this study investigateswhether downregulating MK5 in the H1299 cell line using MK5 CRISPR will reduce the expression of PD-L1 both invitro and in vivo conditions for NSCLC. The negative control is a scrambled CRISPR targeting vector, and the positivecontrol is the depletion of MK5. Through western blot and MTT in vitro and xenograft in vivo, the paper investigateswhether or not downregulating MK5 will reduce immune escape induced by the binding of PD-1 with PD-L1 inNSCLC.

Development of bifunctional CRBN-SOS1 degraders for treatment of mutant KRAS cancers.

3151 Background: KRAS, a GTPase that regulates cellular growth, is the most frequently mutated oncogene in non-small cell lung, pancreatic and colorectal cancers. Hyperactivation of KRAS in mutant alleles result in upregulated signaling within the MAPK pathway leading to aberrant cell proliferation. In addition to the pan- and mutant-specific KRAS inhibitors, its upstream GEF activator, SOS1, has emerged as an attractive therapeutic target given its ability to regulate the active, GTP-loaded state of KRAS and mitigate MAPK pathway feedback. Methods: Utilizing our PRODEGY platform, we developed bifunctional CRBN-SOS1 degraders for the treatment of patients with KRAS-mutated cancers, independent of mutation type. Results: Our degraders display CRBN- and proteasome-mediated SOS1 degradation with DC50s &lt; 15nM in high-throughput (HiBiT) and traditional Western Blot assays in multiple mutant KRAS cell lines. SOS1 degradation resulted in the reduction of active KRAS, HRAS and MRAS levels when assayed by RAF-RBD pulldowns in MIA PaCa-2 (KRAS G12C) cells. Other downstream signaling markers (pERK and pS6) were also decreased upon SOS1 degradation in KRAS G12C, G12V, G12S and G13D cell lines in 3D Western Blot assays. Notably, the observed pERK and pS6 inhibition was driven by SOS1 degradation and not inhibition as decreases in signaling was no longer observed when SOS1 degraders were outcompeted with CRBN competitors. To assess the antiproliferative effects of our SOS1 degraders, we developed 3D proliferation assays in various KRAS mutant (G12A, G12C, G12D, G12V, G12S, G13C and G13D) as well as KRAS amplified cell lines and observed IC50s ranging from 0.5 - 70nM. Treatment of CRBN KO and SOS1 KO H358 and MIA PaCa-2 cell lines with our SOS1 degraders resulted in 100- to 400-fold IC50 values, validating that the functional activity observed is dependent on its targets. Consistent with our in vitro data, treatment of SOS1 degraders in MIA PaCa-2 xenografts resulted in &gt; 90% degradation and led to significant tumor growth inhibition. Given the diversity of mutations and tumors as well as acquired drug resistance to allele-specific KRAS inhibitors, we sought to determine if coupling SOS1 degradation with other EGFR-RAS-MAPK inhibitors could produce more potent responses to maximize patient benefit. Strong synergistic antiproliferative effects were observed when SOS1 degraders were co-treated with KRAS inhibitors AMG510 (G12C) and MRTX1133 (G12D) in respective KRAS-mutant cell lines as well as with MEK inhibition and EGFR inhibition. Lastly, combination of AMG510 or Trametinib with SOS1 degraders yielded strong synergy and a greater anti-tumor response in MIA PaCa-2 xenografts relative to the individual agents themselves. Conclusions: Together, these results highlight the potential of SOS1 degraders alone and in combination as therapeutic options for a variety of KRAS-driven cancers.

CISD2 promotes lung squamous carcinoma cell migration and invasion via the TGF-β1-induced Smad2/3 signaling pathway.

Although aberrant expression of CDGSH iron sulfur domain 2 (CISD2) contributes to the tumorigenesis and progression of numerous human cancers, the biological function of CISD2 and its specific prognostic value in lung squamous cell carcinoma (LUSC) have yet to be comprehensively explored. The current study aimed to elucidate the role of CISD2 in LUSC as well as the underlying molecular mechanisms. Immunohistochemistry was conducted to detect the protein expression of CISD2 and analyze whether high expression of CISD2 affects the overall survival (OS) of LUSC patients. Cell proliferation, colony formation, wound healing and Transwell invasion assays were performed to clarify whether CISD2 contributes to LUSC cell proliferation and disease progression. Quantitative real-time reverse transcription-PCR and western blot assays were used to detect the levels of transcription factors and key epithelial-mesenchymal transition (EMT)-related markers in LUSC cells after CISD2 knockdown and overexpression to determine whether CISD2 regulates transforming growth factor-beta (TGF-β)-induced EMT in LUSC. Immunohistochemistry of human tissue microarrays containing 90 pairs of adjacent and cancerous tissues revealed that CISD2 is considerably overexpressed in LUSC and strongly linked to poor OS. Functional experiments suggested that silencing endogenous CISD2 inhibited the growth, colony formation, migration, and invasion of H2170 and H226 cell lines. Exogenous overexpression of CISD2 facilitated these phenotypes in SK-MES-1 and H2170 cells. Furthermore, CISD2 promoted EMT progression by increasing the expression of mesenchymal markers (N-cadherin, vimentin, Snail, and Slug) as well as SMAD2/3 and reducing the expression of the epithelial marker E-cadherin. Mechanistically, our studies provide the first evidence that CISD2 can promote EMT by enhancing TGF-β1-induced Smad2/3 expression in LUSC cells. In conclusion, our research illustrates that CISD2 is highly expressed in LUSC and may facilitate LUSC proliferation and metastasis. Thus, CISD2 may serve as an independent prognostic marker and possible treatment target for LUSC.

MBD2 facilitates tumor metastasis by mitigating DDB2 expression

Despite past extensive studies, the pathoetiologies underlying tumor metastasis remain poorly understood, which renders its treatment largely unsuccessful. The methyl-CpG-binding domain 2 (MBD2), a “reader” to interpret DNA methylome-encoded information, has been noted to be involved in the development of certain types of tumors, while its exact impact on tumor metastasis remains elusive. Herein we demonstrated that patients with LUAD metastasis were highly correlated with enhanced MBD2 expression. Therefore, knockdown of MBD2 significantly attenuated the migration and invasion of LUAD cells (A549 and H1975 cell lines) coupled with attenuated epithelial–mesenchymal transition (EMT). Moreover, similar results were observed in other types of tumor cells (B16F10). Mechanistically, MBD2 selectively bound to the methylated CpG DNA within the DDB2 promoter, by which MBD2 repressed DDB2 expression to promote tumor metastasis. As a result, administration of MBD2 siRNA-loaded liposomes remarkably suppressed EMT along with attenuated tumor metastasis in the B16F10 tumor-bearing mice. Collectively, our study indicates that MBD2 could be a promising prognostic marker for tumor metastasis, while administration of MBD2 siRNA-loaded liposomes could be a viable therapeutic approach against tumor metastasis in clinical settings.

Abstract B035: Targeting transcriptional elongation kinases prevents adaptation to KRASG12C inhibitors in both MAPK-dependent and -independent models of acquired resistance

Abstract The development of covalent inhibitors targeting KRASG12C mutations offers a precision medicine approach to a large cohort of lung cancer patients previously lacking opportunities for targeted therapy. Despite promising initial responses ranging from disease control to partial response in most patients undergoing treatment, the efficacy of these inhibitors has been limited by a short duration of response. Several strategies have been advanced to delay or prevent acquired drug resistance in lung cancer patients treated with KRASG12C inhibitors. Many of these involve co-administration of other targeted therapies to prevent activation of Ras-dependent mitogen activated protein kinase (MAPK) signaling, which is a hallmark response to KRASG12C inhibitors. While this approach has proven effective in some patients, combination clinical trials and postmortem analysis of tumors that progressed on KRASG12C inhibitors demonstrate that non-MAPK-dependent mechanisms of resistance remain a major challenge. We have developed a panel of lung cancer cell lines with acquired resistance to the KRASG12C inhibitor sotorasib. While Erk phosphorylation is observed in all resistant models, treatment with a panel of Ras and MAPK pathway inhibitors demonstrates differential dependence on MAPK pathway activity, including complete MAPK independence in a resistant model derived from the H358 cell line. Mutation, transcription and phosphoproteomic analysis revealed that these resistant models exhibit metabolic reprogramming and activation of a variety of DNA damage response pathways. Based on differential phosphorylation of the transcriptional elongation kinases CDK12 and CDK13 in KRASG12C-treated cells, we hypothesized that inhibiting these kinases might prevent or revert acquired drug resistance. Our prior studies demonstrate that CDK12/13 inhibition reduces expression of a broad spectrum of DNA damage response and metabolic genes, and RNA sequencing confirms that these pathways are sensitive to the CDK12/13 inhibitor SR-4835 in both sotorasib-sensitive and -resistant cells. Resistant cells show differential responses to specific DNA damage-directed therapies and cell-line specific metabolic reprogramming, but up-front combinatorial treatment with SR-4835 prevents the development of acquired resistance to sotorasib across these models. Combined treatment with sotorasib and SR-4835 does not prevent Erk phosphorylation rebound; thus, the mechanism of SR-4835 in suppressing acquired resistance is independent of MAPK pathway reactivation. In vivo combination of sotorasib and SR-4835 potentiates drug response and prevents drug resistance in the H358 cell line, which can develop both MAPK-dependent and -independent drug resistance. These results demonstrate that targeting transcriptional elongation kinases in combination with sotorasib may benefit a broader spectrum of patients than MAPK-directed combination therapies and provide rationale for suppressing the DNA damage response and metabolic reprogramming as an approach to extend the efficacy of KRASG12C inhibitors. Citation Format: Yaakov E. Stern, Pompom Ghosh, Hitendra S. Solanki, Denis Imbody, Liznair Bridenstine, Hannah L. Walker-Mimms, John W. Mosior, Andrii Monastyrskyi, Derek R. Duckett, Eric B. Haura. Targeting transcriptional elongation kinases prevents adaptation to KRASG12C inhibitors in both MAPK-dependent and -independent models of acquired resistance [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B035.

Abstract B002: Development of bifunctional CRBN-SOS1 degraders for treatment of mutant KRAS cancers

Abstract Although oncogenic Kirsten rat sarcoma virus (KRAS) was once deemed undruggable, recent advances have revealed promising anti-tumor effects with pan-KRAS and KRAS-mutant specific inhibitors in both preclinical and clinical settings. Despite the success of these inhibitors, the diversity of tumors as well as tumor sensitivity results in acquired drug resistance suggesting that combinational therapeutic approaches would be necessary to avoid this problem. An attractive combination strategy would be to couple KRAS inhibition with a blockade of its guanine exchange factor, Son of Sevenless (SOS1). This would keep KRAS in its inactive state as well as mitigate upstream MAPK pathway reactivation notoriously associated with inhibition in this pathway. Utilizing our PRODEGY platform, we developed bifunctional SOS1 degraders for the treatment of patients with KRAS mutant cancers. These degraders have shown rapid and potent CRBN-mediated degradation which exhibit DC50s &amp;lt;10nM in a H358 SOS1-HiBiT cell line. Our high-throughput HiBiT data translated into effective SOS1 degradation in KRAS mutant cells harboring G12C, G12D, G12V, G12S, or G13D mutations. SOS1 degradation was validated to be dependent on the proteasome and CRBN as cotreatment with proteasomal inhibitors (MG132 or MLN4924) or treatment in a CRBN KO cells abolished degradation. Treatment of our SOS1 degraders resulted in the suppression of multiple signaling markers downstream of SOS1. Given its role in RAS activation, we observed a reduction in active KRAS, HRAS and MRAS protein upon treatment of SOS1 degraders in KRAS G12C MIA PaCa-2 cells. Furthermore, pERK and pS6 levels were decreased in a more potent or comparable manner compared to the SOS1 inhibitor, BI3406, in multiple KRAS mutant cell lines. Out competing SOS1 degraders with other CRBN binders highlighted that inhibition of pERK and pS6 was due to CRBN-mediated degradation. To determine functional activity of our SOS1 degraders, we developed and optimized 3D proliferation assays in multiple KRAS mutant (G12C, G12V, G12S, G13D) cell lines which yielded IC50s ranging from 0.5 – 70nM. Similarly, 100- to 300-fold IC50 value shifts were observed in SOS1 KO and CRBN KO H358 cell lines validating that the anti-proliferative activity of SOS1 degraders is specific to its targets. Treatment of SOS1 degraders in KRAS-mutant xenograft models resulted in &amp;gt;90% degradation of SOS1 in tumors and subsequently led to significant tumor growth inhibition as a single agent. Combination treatment of the KRAS G12C inhibitor AMG510 and SOS1 degraders produced strong synergistic antiproliferative effects in H358 and MIA PaCa-2 cell lines. Additionally, synergy was observed with SOS1 degraders and the EGFR inhibitor, Erlotinib, as well as the MEK inhibitor, Trametinib, in KRAS G13D mutant LoVo cells. The ability of our SOS1 degraders to have potency alone as well as in combination with multiple types of different RTK/RAS/MAPK pathway inhibitor highlights the value of SOS1 degraders as a potential therapeutic option for a range of KRAS mutant cancers. Citation Format: Kyle Begovich, Angela Schoolmeesters, Navin Rajapakse, Maneesh Kumar, Elena Martinez-Terroba, Arvind Shakya, Brandon Whitefield, Nataraj Pagadala, Aparajita Chourasia, Leah Fung. Development of bifunctional CRBN-SOS1 degraders for treatment of mutant KRAS cancers [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B002.

Epigenetic regulation of SMAD3 by histone methyltransferase SMYD2 promotes lung cancer metastasis

Epigenetic alterations, especially histone methylation, are key factors in cell migration and invasion in cancer metastasis. However, in lung cancer metastasis, the mechanism by which histone methylation regulates metastasis has not been fully elucidated. Here, we found that the histone methyltransferase SMYD2 is overexpressed in lung cancer and that knockdown of SMYD2 could reduce the rates of cell migration and invasion in lung cancer cell lines via direct downregulation of SMAD3 via SMYD2-mediated epigenetic regulation. Furthermore, using an in vitro epithelial-mesenchymal transition (EMT) system with a Transwell system, we generated highly invasive H1299 (In-H1299) cell lines and observed the suppression of metastatic features by SMYD2 knockdown. Finally, two types of in vivo studies revealed that the formation of metastatic tumors by shSMYD2 was significantly suppressed. Thus, we suggest that SMYD2 is a potential metastasis regulator and that the development of SMYD2-specific inhibitors may help to increase the efficacy of lung cancer treatment.

Toxicological assessment of Phormidium sp. derived copper oxide nanoparticles for its biomedical and environmental applications

Driven by the need to biosynthesized alternate biomedical agents to prevent and treat infection, copper oxide nanoparticles (CuONPs) have surfaced as a promising avenue. Cyanobacteria-derived synthesis of CuONPs is of substantive interest as it offers an eco-friendly, cost-effective, and biocompatible route. In the present study biosynthesized CuONPs were characterized and investigated regarding their toxicity. Morphological analysis using TEM, SEM and AFM showed the spherical particle size of 20.7 nm with 96% copper that confirmed the purity of CuONPs. Biogenic CuONPs with IC50 value of 64.6 µg ml−1 showed 90% scavenging of free radicals in superoxide radical scavenging assay. CuONPs showed enhanced anti-inflammatory activity by 86% of protein denaturation with IC50 value of 89.9 µg ml−1. Biogenic CuONPs exhibited significant toxicity against bacterial strains with lowest MIC value of 62.5 µg ml−1 for B. cereus and fungal strain with a MIC value of 125 µg ml−1 for C. albicans. In addition CuONPs demonstrated a high degree of synergistic interaction when combined with standard drugs. CuONPs exhibited significant cytotoxicity against non-small cell lung cancer with an IC50 value of 100.8 µg ml−1 for A549 and 88.3 µg ml−1 for the H1299 cell line with apoptotic activities. Furthermore, biogenic CuONPs was evaluated for their photocatalytic degradation potential against methylene blue dye and were able to removed 94% dye in 90 min. Free radical scavenging analysis suggested that CuONPs assisted dye degradation was mainly induced by hydroxide radicals. Biogenic CuONPs appears as an eco-friendly and cost effective photocatalyst for the treatment of wastewater contaminated with synthetic dyes that poses threat to aquatic biota and human health. The present study highlighted the blend of biomedical and photocatalytic potential of Phormidium derived CuONPs as an attractive approach for future applications in nanomedicine and bioremediation.

Antimicrobial, Antioxidant, Cytotoxic, DNA Protective Activities, and Molecular Docking Studies of the Methanolic Extract of Salvia siirtica Kahraman, Celep &amp; Dogan sp. Nov. (Lamiaceae)

Background: Medicinal plants have long been utilized in traditional medicine in Turkey and worldwide. The flora of Turkey is so rich it needs to be discovered, documented, and explored. This plant species is the origin of many valuable medicinal chemicals. Salvia species was used in folk medicine for many years in Turkey. One of the new Salvia species is Salvia siirtica. Methods: The antimicrobial, cytotoxic, antioxidant, DNA restriction inhibition, and DNA protective activities of methanolic extract of S. siirtica were studied. Results: The extract has better activity than the standard antibiotics against P. aeruginosa, meanwhile, showed high DPPH scavenging activity with an IC50 value of 28.72 ± 8.02 µg/ml. The extract exhibited good cytotoxic potential with IC50 value 0.31 ± 0.07 mg/ml towards H1299 cell line. The restriction enzyme inhibition in presence of plant extract proves that extract can interact with restriction endonuclease enzymes. Apigenin and Luteolin 7- glycoside are two high components of S. siirtica were docked against potential intracellular targets to evaluate their inhibition capability. Luteolin its derivative luteolin-7-glycoside showed the highest potential in molecular docking studies. Conclusions: Determining the bioactivity of these components by isolating them from the plant may pave the way for their use as supportive agents in the fight against many diseases.

Abstract 138: Establishment of monoclonal cell subpopulations as model systems for functionally exploring resistance mechanisms in EGFR mutant non-small cell cancers

Abstract Background: Coexisting subgroups of cells within a tumor are always evolving. Understanding the molecular events that drive this evolution remains a high priority for understanding dynamic response to treatment. However, models that accurately map individual clones’ activity and sensitivity to treatment remain a challenge. To fill this gap, we established monoclonal cell populations (MCP) from a cell line and used this model system to dissect coexisting cellular and molecular events within an individual tumor and modulated response to anti-EGFR treatment in Non-Small Cell Lung Cancers (NSCLC). Materials/Methods: MCPs were established from the commercially available H1975 EGFR-mutant NSCLC cell line. Parental cells were resuspended at a density of 10 cells per mL and 100 μL of cell suspension was added to each well of a 96-well plate. Eight plates were used, and each well was inspected 24 hours post-seeding by two or more independent scientists. Wells with individual cells were monitored every 3-4 days and followed throughout the expansion process. Once MCPs were established, morphological comparison was performed using the opensource QPath software and clones were classified as circular, branched, or mixed. Selected clones were then treated with 0.7 μM of Osimertinib for 72 hours, in line with the IC50 value of the parental line and treatment responses were compared. Results: Of the 120 single cells detected, 25 expanded successfully and 16 were treated with Osimertinib. Rates of clonal expansion varied from 19 to 49 days and the most prominent morphology was branched (68.75%), followed by mixed (18.75%) and circular (12.5%). No direct correlation was observed between rate of expansion and cell morphology. Response to Osimertinib varied significantly across the clones compared to the parental line from which they were established. Cell viability after 72 hours of treatment with Osimertinib was similar between the parental line and 5 of the 25 clones. For the remaining clones, cell viability was significantly lower in the monoclonal cell subpopulations compared to the parental line. Specifically, cell viability decreased by 40% compared to the parental cell in 5 clones. In the most sensitive clones, cell viability was reduced by 63% and 75% compared to the parental. Conclusion: 25 monoclonal cell subpopulations were successfully established and expanded from the H1975 cell line. MCPs showed significant variations in their growth rate and sensitivity to Osimertinib suggesting that this approach may help identify coexisting mechanisms of response to treatment within an individual tumor. Defining molecular profiles of coexisting MCPs may lead to the identification of biomarkers that may assist physicians in predicting response to treatment or design combination regimens that concomitantly target subclones within the tumor microecology. Citation Format: Claire Blanchard, Kyle Avery, Chelsea Ward, Abduljalil M. Alsubaie, Elisa Baldelli, Mariaelena Pierobon. Establishment of monoclonal cell subpopulations as model systems for functionally exploring resistance mechanisms in EGFR mutant non-small cell cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 138.

Abstract 2009: Gemcitabine-loaded EGFR aptamer for targeted therapy of lung cancer

Abstract Lung cancer is the leading cause of cancer-related death in the world and non-small cell lung cancer (NSCLC) is the most common types of the lung cancer. Epidermal growth factor receptor (EGFR) is an attractive therapeutic target due to its high expression in NSCLC. Gemcitabine (Gem) has shown antitumor activity against many kinds of solid tumors, including NSCLC. Aptamers emerged as promising drug delivery vehicles because of their high binding affinity for the target molecules. The objective of this study was to develop the aptamer-drug conjugates using the EGFR-specific aptamer. EGFR aptamers selected by the SELEX process and gemcitabine was internally incorporated into the EGFR aptamer. In vitro binding affinity of EGFR and Gem-loaded EGFR aptamer was evaluated on EGFR-positive A549, H460, H1299 cell lines, as well as EGFR-negative H522 cells using confocal microscopy. To determine whether the Gem-loaded EGFR aptamer was effectively internalized, Z-stack images were acquired after 1 hr incubation of Cy5-conjugated EGFR aptamer at 37°C. The ex vivo biodistribution of Cy7-conjugated EGFR aptamer was investigated in NSCLC xenograft nude mice. To confirm the antiproliferative effects of Gem-loaded EGFR aptamer, the MTT cell proliferation assay was carried out in NSCLC cells. In the confocal image of EGFR-positive cells, Gem-loaded EGFR aptamers were selectively bound to the cell surface, while no aptamer binding was observed in H522 cells. Z-stack imaging showed that internalized Gem-loaded EGFR aptamer accumulated in EGFR-positive cells. The ex vivo biodistribution data showed that the Gem-loaded EGFR aptamer exhibited higher binding in EGFR-positive A549 tumors than scramble EGFR aptamer. A549 cells showed significant reduction in proliferation following treatment with Gem-loaded EGFR aptamer. Our results indicated that Gem-loaded EGFR aptamer has a potential therapeutic agent in EGFR-positive NSCLC therapy. Citation Format: Jun Young Park, Ye Lim Cho, Ju Ri Chae, Won Jun Kang. Gemcitabine-loaded EGFR aptamer for targeted therapy of 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 2009.

Abstract 428: The role of sphingosine kinase 2 in overcoming EGFR-tyrosine kinase inhibitor resistance, EMT and NSCLC tumor progression

Abstract Background: Tyrosine Kinase Inhibitors (TKIs) such as Erlotinib (ER) and Osimertinib (OR) are being used to treat lung cancer patients with EGFR mutations; however, patients develop resistance to these drugs within 10-18 months. EGFR-TKI resistance may be mediated by epithelial-mesenchymal transition (EMT), which causes a motile and invasive phenotype similar to mesenchymal cells. Sphingosine kinase 2 (SphK2), which is upregulated in Non-Small Cell Lung Cancer (NSCLC), phosphorylates sphingosine to sphingosine-1-phosphate (S1P) and increases cell proliferation, tumor survival and cell migration. The sphingolipid rheostat controls the cell fate by maintaining a balance between pro-survival and pro-apoptotic sphingolipids, which is regulated by SphK2. However, the role of SphK2 in TKI resistance and EMT is not well understood. Hypothesis: We hypothesize that the inhibition/downregulation of SphK2 will reverse EMT, reduce tumorigenicity and overcome ER and OR resistance. Study design and Results: We studied expression of SphK2 in both drug resistant (OR and ER) and drug sensitive (parental) H358, H2170, H3255 and PC9 NSCLC cell lines. In drug resistant cells, qPCR and immunoblotting studies showed that SphK2 was upregulated by 1.5-1.7 and 1.5-3.3 fold in ER and OR resistant cell lines respectively compared to parental cells. 48 hours after SphK2 knockdown with siRNA treatment, SphK2 was downregulated by 4.6-fold and EMT mediator E-cadherin expression was upregulated by 2.9-fold. EMT proteins Vimentin and N-cadherin were downregulated by 3.8 and 2.2 fold respectively (p≤0.01). Using an MTT assay, differences in percentage viability of cells with different treatments were calculated with respect to their viability in the presence of OR. We found that 81.43% of H358OR and 76.35% of the H3255OR cells were inhibited by a combination of Opaganib, a Sphk2 inhibitor, and OR, this is 27.74% and 12.28% higher compared to the additive effect of drug and inhibitor in H358OR and H3255 cells respectively. Furthermore, transwell migration assays, wound healing assays and spheroid assays were performed in OR resistant cells and the results indicated inhibition of the above assays by 94.97%, 82.36%, 88.67% respectively when treated with a combination of Opaganib and OR as compared to the diluent. This indicates the potential of SphK2 inhibition to overcome TKI resistance and EMT. To analyze the role of SphK2 expression in cancer progression, IHC was performed on 63 early and 48 late-stage lung cancer patient sections. We observed upregulation of SphK2 in late-stage lung cancer (p≤0.05), indicating its role in cancer progression. Conclusion: In this study, SphK2 was shown to decrease tumor survival, cancer cell proliferation, and expression of EMT markers. Thus, SphK2 may be a promising clinical target for overcoming EGFR-TKI resistance and NSCLC tumor progression. Citation Format: S M Nasifuzzaman, Namrata Dube, Katie Lam, Robert J. Hillwig, Neelu Puri. The role of sphingosine kinase 2 in overcoming EGFR-tyrosine kinase inhibitor resistance, EMT and NSCLC tumor progression [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 428.

Abstract 1578: Novel Cereblon mediated bifunctional degraders of SOS1 for treatment of pan-KRAS mutant tumors

Abstract KRAS, a GTPase that regulates cellular growth, is the most frequently mutated oncogene with implications in non-small cell lung, pancreatic and colorectal cancers. Hyperactivation of KRAS in mutant alleles favors KRAS in its active GTP-bound state over its inactive GDP-bound state. Once deemed undruggable, recent advances have revealed promising anti-tumor effects with pan-KRAS and KRAS-mutant specific inhibitors in both preclinical and clinical settings. Despite the success of these inhibitors, the diversity of tumors as well as tumor sensitivity results in acquired drug resistance suggesting that combinational therapeutic approaches would be necessary to avoid this problem. An attractive dual approach would be to couple KRAS inhibition with a blockade of its guanine exchange factor (GEF), SOS1, to keep KRAS in its inactive state as well as prevent upstream pathway reactivation. Utilizing our PRODEGY platform, we developed bifunctional SOS1 degraders for the treatment of patients with KRAS mutant cancers. These degraders have shown rapid and potent Cereblon (CRBN)-mediated degradation which exhibit DC50s &amp;lt;10nM in a H358 SOS1-HiBiT cell line. Our high- throughput HiBiT data translated into effective SOS1 degradation in KRAS mutant cells harboring G12C, G12D, G12V, G12S, or G13D mutations. SOS1 degradation was validated to be dependent on the proteasome and CRBN as cotreatment with proteasomal inhibitors (MG132 or MLN4924) or treatment in a CRBN KO cells abolished degradation. Additionally, treatment of SOS1 degraders resulted in suppression of downstream signaling markers, pERK and pS6, in both 3D immunoblots and high-throughput assays with pERK IC50s &amp;lt;10nM. Chemical competitor assays also revealed that inhibition of pERK and pS6 was dependent on CRBN-mediated competition. To determine functional activity of our SOS1 degraders, we developed and optimized 3D proliferation assays in multiple KRAS mutant (G12C, G12D, G12V, G12S, G13D) cell lines which yielded IC50s ranging from 0.5 - 70nM. Similarly, 100- to 300-fold IC50 value shifts were observed in SOS1 KO and CRBN KO H358 cell lines validating that the anti-proliferative activity of SOS1 degraders is specific to its targets. Treatment of SOS1 degraders in KRAS-mutant xenograft models resulted in &amp;gt;75% degradation of SOS1 in tumors and subsequently led to significant tumor growth inhibition as a single agent. SOS1 degradation also provided synergistic anti-proliferation effects with other RAS/MAPK/AKT inhibition in in vitro studies. For example, combination treatment of AMG510 and SOS1 degraders resulted in strong synergistic effects in KRAS G12C H358 cells. Together, our data SOS1 degradation could be a valuable potential treatment option for a range of KRAS mutant cancers. Citation Format: Kyle Begovich, Angela Schoolmeesters, Navin Rajapakse, Maneesh Kumar, Arvind Shakya, Brandon Whitefield, Elena Martinez-Terroba, Nataraj Pagadala, Shenlin Huang, Aparajita H. Chourasia, Leah Fung. Novel Cereblon mediated bifunctional degraders of SOS1 for treatment of pan-KRAS mutant tumors [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 1578.

Discovery of Novel EGFR Inhibitor Targeting Wild-Type and Mutant Forms of EGFR: In Silico and In Vitro Study.

Targeting L858R/T790M and L858R/T790M/C797S mutant EGFR is a critical challenge in developing EGFR tyrosine kinase inhibitors to overcome drug resistance in non-small cell lung cancer (NSCLC). The discovery of next-generation EGFR tyrosine kinase inhibitors (TKIs) is therefore necessary. To this end, a series of furopyridine derivatives were evaluated for their EGFR-based inhibition and antiproliferative activities using computational and biological approaches. We found that several compounds derived from virtual screening based on a molecular docking and solvated interaction energy (SIE) method showed the potential to suppress wild-type and mutant EGFR. The most promising PD13 displayed strong inhibitory activity against wild-type (IC50 of 11.64 ± 1.30 nM), L858R/T790M (IC50 of 10.51 ± 0.71 nM), which are more significant than known drugs. In addition, PD13 revealed a potent cytotoxic effect on A549 and H1975 cell lines with IC50 values of 18.09 ± 1.57 and 33.87 ± 0.86 µM, respectively. The 500-ns MD simulations indicated that PD13 formed a hydrogen bond with Met793 at the hinge region, thus creating excellent EGFR inhibitory activity. Moreover, the binding of PD13 in the hinge region of EGFR was the major determining factor in stabilizing the interactions via hydrogen bonds and van der Waals (vdW). Altogether, PD13 is a promising novel EGFR inhibitor that could be further clinically developed as fourth-generation EGFR-TKIs.

Steroidal alkaloids from the roots of Veratrum stenophyllum

Three new steroidal alkaloids, veratrasines A − C (1–3), along with ten known analogues (4–13) were isolated from the roots of Veratrum stenophyllum. Their structures were elucidated by NMR and HRESIMS data and comparison with the reported data in the literatures. A plausible biosynthetic pathway for 1 and 2 were proposed. Compounds 1, 3, and 8 showed moderate cytotoxic activity against MHCC97H and H1299 cell lines.

The molecular mechanisms of apoptosis accompanied with the epigenetic regulation of the NY-ESO-1 antigen in non-small lung cancer cells treated with decitabine (5-aza-CdR)

Dysregulated epigenetic modifications are common in lung cancer but have been reversed using demethylating agent like 5-Aza-CdR. 5-Aza-CdR induces/upregulates the NY-ESO-1 antigen in lung cancer. Therefore, we investigated the molecular mechanisms accompanied with the epigenetic regulation of NY-ESO-1 in 5-Aza-CdR-treated NCI–H1975 cell line. We showed significant induction of the NY-ESO-1 protein (**p < 0.0097) using Cellular ELISA. Bisulfite-sequencing demonstrated 45.6% demethylation efficiency at the NY-ESO-1 gene promoter region and RT-qPCR analysis confirmed the significant induction of NY-ESO-1 at mRNA level (128-fold increase, *p < 0.050). We then investigated the mechanism by which 5-Aza-CdR inhibits cell proliferation in the NCI–H1975 cell line. Upregulation of the death receptors TRAIL (2.04-fold *p < 0.011) and FAS (2.1-fold *p < 0.011) indicate activation of the extrinsic apoptotic pathway. The upregulation of Voltage-dependent anion-selective channel protein 1 (1.9-fold), Major vault protein (1.8-fold), Bax (1.16-fold), and Cytochrome C (1.39-fold) indicate the activation of the intrinsic pathway. We also observed the differential expression of protein Complement C3 (3.3-fold), Destrin (−5.1-fold), Vimentin (−1.7-fold), Peroxiredoxin 4 (−1.6-fold), Fascin (−1.8-fold), Heme oxygenase-2 (−0.67-fold**p < 0.0055), Hsp27 (−0.57-fold**p < 0.004), and Hsp70 (−0.39-fold **p < 0.001), indicating reduced cell growth, cell migration, and metastasis. The upregulation of 40S ribosomal protein S9 (3-fold), 40S ribosomal protein S15 (4.2-fold), 40S ribosomal protein S18 (2.5-fold), and 60S ribosomal protein L22 (4.4-fold) implied the induction of translation machinery. These results reiterate the decisive role of 5-Aza-CdR in lung cancer treatment since it induces the epigenetic regulation of NY-ESO-1 antigen, inhibits cell proliferation, increases apoptosis, and decreases invasiveness.

Establishment of Human Lung Adenocarcinoma Radioresistant Cell Lines and the Mechanism of Radioresistance

Radiotherapy is one of the most common treatments for lung cancer, and about 40%-50% of patients after radiotherapy will appear uncontrolled or recurrence in the case of local tumors. Radioresistance is the predominant cause of local therapeutic failure. Nevertheless, the lack of in vitro radioresistance models is an influential factor obstructing the study of its mechanism. Therefore, the establishment of radioresistant cell lines, H1975DR and H1299DR, was beneficial to explore the mechanism of radioresistance in lung adenocarcinoma. The radioresistant cell lines of H1975DR and H1299DR were obtained from H1975 and H1299 cells irradiated with equal doses of X-rays; Clonogenic assays were performed to compare the clone-forming ability of H1975 vs H1975DR cells, H1299 vs H1299DR cells, then fitting cell survival curve by linear quadratic model; The comet assay was employed to examine DNA damage repair and calculate the percentage of DNA tails; The optical microscopy, CCK-8, flow cytometry, Transwell invasion assays were used to compare biological characteristics such as cell morphology, cell proliferation, apoptosis level, cycle distribution, cell migration and invasion; Western blot was carried out to measure the protein expression of DNA damage repair factors, such as DNA-PKcs, 53BP1, RAD51, and p-ATM. After five months of continuous irradiation and stable culture, radioresistant cell lines H1975DR and H1299DR were obtained. The cell proliferation activity, clone formation ability and DNA damage repair ability of the two radioresistant cell lines were significantly improved under X-ray irradiation. The proportion of the G2/M phase was markedly decreased and the proportion of the G0/G1 phase was increased. Cell migration and invasion ability were significantly enhanced. Relative expression levels of p-DNA-PKcs (Ser2056), 53BP1 in the nonhomologous end-joining (NHEJ) repair pathway and p-ATM (Ser1981), RAD51 in the homologous recombination (HR) repair pathway were higher than those in H1975 and H1299. H1975 and H1299 cell lines can be able to differentiate into lung adenocarcinoma radioresistant cell lines H1975DR and H1299DR by equal dose fractional irradiation, which provided an in vitro cytological model for the study of radiotherapy resistance mechanism of lung cancer patients.

Guided Isolation of An Uncommon Cembranoid Orthoester, Sarcotortin A, and Three Skeletal Diverse Terpenoids from the Hainan Soft Coral Sarcophyton tortuosum Based on Molecular Networking Strategy.

Applying the emerging molecular networking strategy, an uncommon cembranoid orthoester, sarcotortin A (1), featuring a 3/14/8/5-fused scaffold, an unusual eunicellane-type diterpenoid, sarcotorolide A (2), and two new biscembranoids, ximaolides M and N (7 and 8), along with nine known terpenoids 3-6 and 9-13 were isolated from the Hainan soft coral Sarcophyton tortuosum. The structure and absolute configuration of all new compounds were established by a combination of spectroscopic data, X-ray diffraction analysis, and/or quantum chemical computational approaches. The plausible biogenetic relationship among these skeletally different terpenoids was proposed and discussed. In in vitro bioassay, new compound 7 exhibited a remarkable inhibitory activity against protein tyrosine phosphatases 1B (PTP1B) with the IC50 value of 8.06 μM. In addition, compounds 4 and 10 displayed significant inhibitory effects on lipopolysaccharide (LPS)-induced inflammatory responses in RAW264.7 macrophages cells with the IC50 values of 19.13 and 16.45 μM, respectively. Compound 9 showed interesting cytotoxicity against H1975, MDA-MB231, A549, and H1299 cancer cell lines with IC50 values of 31.59, 34.96, 43.87, and 27.93 μM, respectively.