Protect Tumor Cells Research Articles

Ultrasound-triggered and glycosylation inhibition-enhanced tumor piezocatalytic immunotherapy.

Nanocatalytic immunotherapy holds excellent potential for future cancer therapy due to its rapid activation of the immune system to attack tumor cells. However, a high level of N-glycosylation can protect tumor cells, compromising the anticancer immunity of nanocatalytic immunotherapy. Here, we show a 2-deoxyglucose (2-DG) and bismuth ferrite co-loaded gel (DBG) scaffold for enhanced cancer piezocatalytic immunotherapy. After the implantation in the tumor, DBG generates both reactive oxygen species (ROS) and piezoelectric signals when excited with ultrasound irradiation, significantly promoting the activation of anticancer immunity. Meanwhile, 2-DG released from ROS-sensitive DBG disrupts the N-glycans synthesis, further overcoming the immunosuppressive microenvironment of tumors. The synergy effects of ultrasound-triggered and glycosylation inhibition enhanced tumor piezocatalytic immunotherapy are demonstrated on four mouse cancer models. A "hot" tumor-immunity niche is produced to inhibit tumor progress and lung metastasis and elicit strong immune memory effects. This work provides a promising piezocatalytic immunotherapy for malignant solid tumors featuring both low immunogenicity and high levels of N-glycosylation.

The branched N-glycan of PD-L1 predicts immunotherapy responses in patients with recurrent/metastatic HNSCC

Immunotherapy has revolutionized cancer treatment, but the lack of a reliable predictive biomarker for treatment response remains a challenge. Alpha-1,6-Mannosylglycoprotein 6-β-N-Acetylglucosaminyltransferase 5 (MGAT5) is a key regulator of complex N-glycan synthesis, and its dysregulation is associated with cancer progression. The lectin Phaseolus vulgaris leukoagglutinin (PHA-L) specifically binds to mature MGAT5 products. Previous studies have indicated elevated PHA-L staining in head and neck squamous cell carcinoma (HNSCC), which implies increased activity of MGAT5. However, the specific role of MGAT5 in HNSCC remains unclear. In this study, we found significantly higher PHA-L staining and MGAT5 expression in HNSCC tumors compared to adjacent non-tumor tissues. Using a mass spectrometry (MS)-based glycoproteomic approach, we identified 163 potential protein substrates of MGAT5. Functional analysis revealed that protein substrates of MGAT5 regulated pathways related to T cell proliferation and activation. We further discovered that PD-L1 was among the protein substrates of MGAT5, and the expression of MGAT5 protected tumor cells from cytotoxic T lymphocyte (CTL) killing. Treatment of nivolumab alleviated the protective effects of MGAT5 on CTL activity. Consistently, patients with MGAT5-positive tumors showed improved responses to immunotherapy compared to those with MGAT5-negative tumors. Using purified PD-L1 from HNSCC cells and a glycoproteomic approach, we further deciphered that the N35 and N200 sites carry the majority of complex N-glycans on PD-L1. Our findings highlight the critical role of MGAT5-mediated branched N-glycans on PD-L1 in modulating the interaction with the immune checkpoint receptor PD-1. Consequently, we propose that MGAT5 could serve as a biomarker to predict patients’ responses to anti-PD-1 therapy. Furthermore, targeting the branched N-glycans at N35 and N200 of PD-L1 may lead to the development of novel diagnostic and therapeutic approaches.

CD39 is induced by nutrient deprivation in colorectal cancer cells and contributes to the tolerance to hypoxia

Rapid tumor growth and insufficient blood supply leads to the development of a hypoxic and nutrient deprived microenvironment. To survive, tumor cells need to tolerate these adverse conditions. Here we found the expression of CD39 was enhanced in necrotic regions distant from blood vessels. We speculate that this is a strategy for tumor cells to actively adapt to the hostile environment. Further studies showed that CD39 was induced by nutrient deprivation through the AMPK signalling pathway. We next explored the significance of CD39 for tumor cells. Our results showed that CD39 reduced cellular oxygen consumption, which could be significant for tumor cells if the available oxygen is limited. Metabolomics analysis showed that overexpression of CD39 significantly altered cellular metabolism, and tricarboxylic acid (TCA) cycle was identified as the most impacted metabolic pathway. In order to explore the molecular mechanism, we performed RNA-seq analysis. The results showed that CD39 significantly up-regulated the expression of pyruvate dehydrogenase kinase isozyme 2 (PDK2), thus inhibiting the activity of pyruvate dehydrogenase (PDH) and TCA cycle. Finally, CD39 was shown to protect tumor cells from hypoxia-induced cell death and reduce intratumoral hypoxia levels. CD39 has attracted a great deal of attention as a newly discovered immune checkpoint molecule in recent years. Our results indicate that CD39 not only plays a role in immune regulation, but also enables tumor cells to tolerate hypoxia by inhibiting TCA cycle and reducing cellular oxygen consumption. This study provides evidence that targeting CD39 may be a novel strategy to prevent adaptation of tumor cells in stressed conditions.

LAPTM4B counteracts ferroptosis via suppressing the ubiquitin-proteasome degradation of SLC7A11 in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related deaths worldwide, necessitating the identification of novel therapeutic targets. Lysosome Associated Protein Transmembrane 4B (LAPTM4B) is involved in biological processes critical to cancer progression, such as regulation of solute carrier transporter proteins and metabolic pathways, including mTORC1. However, the metabolic processes governed by LAPTM4B and its role in oncogenesis remain unknown. In this study, we conducted unbiased metabolomic screens to uncover the metabolic landscape regulated by LAPTM4B. We observed common metabolic changes in several knockout cell models suggesting of a role for LAPTM4B in suppressing ferroptosis. Through a series of cell-based assays and animal experiments, we demonstrate that LAPTM4B protects tumor cells from erastin-induced ferroptosis both in vitro and in vivo. Mechanistically, LAPTM4B suppresses ferroptosis by inhibiting NEDD4L/ZRANB1 mediated ubiquitination and subsequent proteasomal degradation of the cystine-glutamate antiporter SLC7A11. Furthermore, metabolomic profiling of cancer cells revealed that LAPTM4B knockout leads to a significant enrichment of ferroptosis and associated metabolic alterations. By integrating results from cellular assays, patient tissue samples, an animal model, and cancer databases, this study highlights the clinical relevance of the LAPTM4B-SLC7A11-ferroptosis signaling axis in NSCLC progression and identifies it as a potential target for the development of cancer therapeutics.

Human lung cancer-derived mesenchymal stem cells promote tumor growth and immunosuppression

BackgroundThe presence of mesenchymal stem cells has been confirmed in some solid tumors where they serve as important components of the tumor microenvironment; however, their role in cancer has not been fully elucidated. The aim of this study was to investigate the functions of mesenchymal stem cells isolated from tumor tissues of patients with non-small cell lung cancer.ResultsHuman lung cancer-derived mesenchymal stem cells displayed the typical morphology and immunophenotype of mesenchymal stem cells; they were nontumorigenic and capable of undergoing multipotent differentiation. These isolated cells remarkably enhanced tumor growth when incorporated into systems alongside tumor cells in vivo. Importantly, in the presence of mesenchymal stem cells, the ability of peripheral blood mononuclear cell-derived natural killer and activated T cells to mediate tumor cell destruction was significantly compromised.ConclusionCollectively, these data support the notion that human lung cancer-derived mesenchymal stem cells protect tumor cells from immune-mediated destruction by inhibiting the antitumor activities of natural killer and T cells.

Tumors cells with mismatch repair deficiency induce hyperactivation of pyroptosis resistant to cell membrane damage but are more sensitive to co-treatment of IFN-γ and TNF-α to PANoptosis

Hypermutated neoantigens in cancers with DNA mismatch repair deficiency (dMMR) are prerequisites for favorable clinical responses to immune-checkpoint blockade (ICB) therapy. However, TMB is not significantly associated with favorable prognosis from Preclinical and clinical studies. It implies that except for TMB, other mechanisms should be needed to contribute to successful cancer immunotherapy. We found that the hyperactivation of PANoptotic effective molecules in dMMR tumor cells caused cell membrane damage, induced ESCRT-mediated membrane repair, and protected tumor cells from the damage caused by Triton X-100, while DNA mismatch repair proficient (pMMR) tumor cells were sensitive to Triton X-100 mediating cell membrane damage due to the lack of ESCRT-mediated membrane repair. There was hyperactivation of GSDMD, GSDME, and p-MLKL in dMMR tumor cells. Co-treatment of IFN-γ and TNF-α induced rapid death of dMMR tumor cells by inducing PANoptosis including pyroptosis, apoptosis, and no necrosis. pMMR tumor cells had defects in the PANoptosis pathway and were resistant to co-treatment of IFN-γ and TNF-α. In conclusion, we can activate immune cells to release IFN-γ and TNF-α to overcome resistance to ICB treatment.

Staphylococcus Aureus Membrane Vesicles Kill Tumor Cells Through a Caspase-1-Dependent Pyroptosis Pathway.

Nanosized outer membrane vesicles (OMVs) from Gram-negative bacteria have attracted increasing interest because of their antitumor activity. However, the antitumor effects of MVs isolated from Gram-positive bacteria have rarely been investigated. MVs of Staphylococcus aureus USA300 were prepared and their antitumor efficacy was evaluated using tumor-bearing mouse models. A gene knock-in assay was performed to generate luciferase Antares2-MVs for bioluminescent detection. Cell counting kit-8 and lactic dehydrogenase release assays were used to detect the toxicity of the MVs against tumor cells in vitro. Active caspase-1 and gasdermin D (GSDMD) levels were determined using Western blot, and the tumor inhibition ability of MVs was determined in B16F10 cells treated with a caspase-1 inhibitor. The vesicular particles of S. aureus USA300 MVs were 55.23 ± 8.17 nm in diameter, and 5 μg of MVs remarkably inhibited the growth of B16F10 melanoma in C57BL/6 mice and CT26 colon adenocarcinoma in BALB/c mice. The bioluminescent signals correlated well with the concentrations of the engineered Antares2-MVs (R2 = 0.999), and the sensitivity for bioluminescence imaging was 4 × 10-3 μg. Antares2-MVs can directly target tumor tissues in vivo, and 20 μg/mL Antares2-MVs considerably reduced the growth of B16F10 and CT26 tumor cells, but not non-carcinomatous bEnd.3 cells. MV treatment substantially increased the level of active caspase-1, which processes GSDMD to trigger pyroptosis in tumor cells. Blocking caspase-1 activation with VX-765 significantly protected tumor cells from MV killing in vitro and in vivo. S. aureus MVs can kill tumor cells by activating the pyroptosis pathway, and the induction of pyroptosis in tumor cells is a promising strategy for cancer treatment.

Discovery of Urea Derivatives of Celastrol as Selective Peroxiredoxin 1 Inhibitors against Colorectal Cancer Cells.

Peroxiredoxin (PRDX1) is a tumor-overexpressed antioxidant enzyme for eliminating excessive reactive oxygen species (ROS) to protect tumor cells from oxidative damage. Herein, a series of celastrol urea derivatives were developed based on its cocrystal structure with PRDX1, with the aim of pursuing a PRDX1-specific inhibitor. Among them, derivative 15 displayed potent anti-PRDX1 activity (IC50 = 0.35 μM) and antiproliferative potency against colon cancer cells. It covalently bound to Cys-173 of PRDX1 (KD = 0.37 μM), which was secured by the cocrystal structure of PRDX1 with an analogue of 15 while exhibiting weak inhibitory effects on PRDX2-PRDX6 (IC50 > 50 μM), indicating excellent PRDX1 selectivity. Treatment with 15 dose-dependently decreased the mitochondria membrane potential of SW620 cells, probably due to ROS induced by PRDX1 inhibition, leading to cell apoptosis. In colorectal cancer cell xenograft model, it displayed potent antitumor efficacy with superior safety to celastrol. Collectively, 15 represents a promising PRDX1 selective inhibitor for the development of anticolorectal cancer agents.

Mesenchymal Stromal Cells Increase the Natural Killer Resistance of Circulating Tumor Cells via Intercellular Signaling of cGAS-STING-IFNβ-HLA.

Circulating tumor cells (CTCs) shed from primary tumors must overcome the cytotoxicity of immune cells, particularly natural killer (NK) cells, to cause metastasis. The tumor microenvironment (TME) protects tumor cells from the cytotoxicity of immune cells, which is partially executed by cancer-associated mesenchymal stromal cells (MSCs). However, the mechanisms by which MSCs influence the NK resistance of CTCs remain poorly understood. This study demonstrates that MSCs enhance the NK resistance of cancer cells in a gap junction-dependent manner, thereby promoting the survival and metastatic seeding of CTCs in immunocompromised mice. Tumor cells crosstalk with MSCs through an intercellular cGAS-cGAMP-STING signaling loop, leading to increased production of interferon-β (IFNβ) by MSCs. IFNβ reversely enhances the type I IFN (IFN-I) signaling in tumor cells and hence the expression of human leukocyte antigen class I (HLA-I) on the cell surface, protecting the tumor cells from NK cytotoxicity. Disruption of this loop reverses NK sensitivity in tumor cells and decreases tumor metastasis. Moreover, there are positive correlations between IFN-I signaling, HLA-I expression, and NK tolerance in human tumor samples. Thus, the NK-resistant signaling loop between tumor cells and MSCs may serve as a novel therapeutic target.

Abstract LB257: Possible involvement of PDL1 intrinsic signaling to acquired resistance to immunotherapy for renal cell carcinoma (RCC)

Abstract Immune checkpoint inhibitor (ICI) therapy has shown unprecedented results in RCC treatment, but durable responses have been hampered by acquired resistance (AR, initially respond but eventually develop cancer progression). Mechanisms for AR remain ambiguous. Previously, we reported elevated blood GPNMB to strongly associate with AR development. To study molecular mechanisms for AR-linked GPNMB elevation, we created an ICI-resistant RenCa (R-RenCa) mouse model by repeated selection of largest tumors in mice treated with PD1 Ab ICI. Parental cells (Gpnmblow) respond to ICI (S-RenCa), while R-RenCa is completely unresponsive to ICI and acquired Gpnmb overexpression (but PDL1 remained unchanged), recapitulating strong association of GPNMB with AR development. R-RenCa tumors displayed more immunosuppressive and angiogenic phenotypes and enhanced response to anti-Gpnmb Ab. In probing for genetic alterations unique to R-RenCa, whole exome seq analysis showed R-RenCa to bear a greater tumor mutation burden (184.9 and 174.6 mut/Mb for R- and S-RenCa, respectively) but no somatic mutations in beta-2 microglobulin and IFN-signal molecule genes known to be involved in AR. RNA-seq analysis showed; more transcriptionally active genes in R-RenCa tumors (heatmap analysis); more responsive genes to ICI (Volcano analysis); and dysregulation in microphthalmia (Mitf) family gene expression. Mitf RNA was upregulated 3-fold, but Tfe3 and other members downregulated 2-3-fold. RNA start sites of Mitf transcripts are highly heterogenous in R-RenCa compared to S-RenCa (Sashimi plot), indicating generation of more isoforms. Since Gpnmb promoter has E-boxes (DNA binding sites) for Mitf factor, we examined whether Mitf is responsible for Gpnmb expression using promoter-Luc reporter assays and found 4-fold higher in R-RenCa, which was lost completely in E-box-mutated promoter. In seeking transcriptional activators for Mitf gene, we found Sox10 factor to be upregulated in R-RenCa. The molecular link of Sox10-Mitf-Gpnmb was shown by the data of gene-knock down experiments that Sox10-siRNA diminished Mitf and Gpnmb expression completely, and Mitf-siRNA had no effect on Sox10 expression but diminished Gpnmb expression. We then studied mechanisms for Sox10 activation and found PDL1 signaling (triggered by Ab-crosslinking) to induce Sox10 RNA expression in tumor cells, which was 80% abrogated by deleting the RMLDVEKC cytoplasmic motif known to protect tumor cells from IFN cytotoxicity. For human relevance of Sox10-Mitf axis activation, we employed nested qRT-PCR of cell-free RNA (cfRNA) isolated from plasma of RCC patients (n=3) immediately before and after AR. SOX10 and MITF cfRNAs were increased by 2.3 ± 0.85 and 1.77 ± 0.05-fold, respectively, between these two time points. Thus, PDL1 signaling is involved in AR by dysregulating the SOX10-MITF-GPNMB cascade, indicating the potential of the cascade-targeting to break AR hindrance for ICI therapy. Citation Format: Jin-Sung Chung, Lei Guo, Vinita Popat, Ponciano D. Cruz, Lin Xu, Hans Hammers, Kiyoshi Ariizumi. Possible involvement of PDL1 intrinsic signaling to acquired resistance to immunotherapy for renal cell carcinoma (RCC) [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 LB257.

E2F1-EP300 co-activator complex potentiates immune escape in nasopharyngeal carcinoma through the mediation of MELK.

Nasopharyngeal carcinoma (NPC) is characterized by a highly suppressive microenvironment that protects tumor cells against immune attack and facilitates tumor progression. MELK is upregulated in various tumors, whereas its function in the immune escape remains largely unknown. In this study, we investigated the role of MELK during immune escape in NPC. Differentially expressed genes were filtered using GEO datasets and PPI network analysis. NPC cell colony formation and motility were examined, and the impact of CD8⁺ T cells on NPC cells was evaluated. A xenograft model was constructed to detect the growth of tumor cells and the T-cell phenotype of tumor infiltration. ChIP-qPCR and dual-luciferase assays were used to verify the transcriptional regulation of MELK by EP300/E2F1. MELK was overexpressed in NPC, and sh-MELK suppressed the clonogenic ability, migration, and invasion of NPC cells and promoted the killing effects of CD8⁺ T cells. These in vitro findings were reproduced in vivo. EP300 synergized E2F1 to regulate the transcription of MELK in NPC cells. Loss of EP300 or E2F1 reverted the malignant phenotype of NPC cells and promoted the immune effect of CD8⁺ T cells. MELK further suppressed the immune effect of CD8⁺ T cells in the presence of sh-E2F1. EP300 coordinated with E2F1 to promote the transcription of MELK which promoted the growth of NPC cells and repressed the killing effect of CD8⁺ T cells. Blockage of MELK may be a potential way to suppress the immune escape of NPC cells.

Abstract 607: Development of a novel, selective, and potent inhibitor of RON kinase

Abstract Purpose: Receptor d’origine nantais (RON) is a highly conserved transmembrane protein expressed at low levels in healthy adult tissues of epithelial origin and at various levels in immune cells such as macrophages. Aberrant activation of RON has been described in many solid tumors, and it contributes to tumorigenesis by modulating the immune tumor microenvironment, activating numerous oncogenic signaling pathways, and protecting tumor cells under stress. The overexpression of RON protein and the subsequent generation of oncogenic variants are mainly responsible for the persistent activation of downstream signaling pathways. In breast cancer, more than 80% of primary cancer samples are positive for RON expression, with overexpression reported in approximately 50% of cases. Importantly, tumoral RON expression correlates with increased breast cancer progression, metastasis, and poor prognosis independent of molecular subtype. We hypothesize that a small molecule inhibitor of RON that can target both full-length and truncated isoforms will promote anti-tumor immunity within the tumor microenvironment and block pro-metastatic signaling pathways. Herein, we report the development of a novel, orally bioavailable, selective, and potent inhibitor of RON kinase. Methods: With structure-guided and iterative medicinal chemistry approaches, we identified a novel chemical entity that potently inhibited RON kinase activity. Further optimization resulted in the identification of ZB-60 with improved potency, selectivity, and desirable drug-like properties. In vitro kinase assays were performed using ADP-Glo RON kinase assay kit (Promega). Changes in phosphorylation of RON was demonstrated by Western blotting. Plasma pharmacokinetics (PK) was conducted in mice to measure exposures and to determine the oral bioavailability of ZB-60. In vivo efficacy of ZB-60 was evaluated in syngeneic mouse models of breast (EMT6) and colorectal (CT26) cancer. Results: ZB-60 inhibited RON kinase activity with an IC50 of 56 nM and showed greater than 40-fold selectivity against MET and other closest members of the tyrosine kinase family. In ZB-60 treated breast cancer cells, there was a dose dependent reduction of RON phosphorylation and downstream signaling. PK analysis showed a high oral bioavailability with low clearance. In vivo evaluation of ZB-60 showed inhibition of tumor growth in EMT6 and CT26 models. Ongoing studies are focused on testing ZB-60 in combination with checkpoint inhibitors. The results from these studies along with ADME-TOX and pharmacokinetics will be presented. Conclusion: ZB-60 has target specific activity and exhibits favorable drug-like properties. Citation Format: Mohan Rao Kaadige, Trason Thode, Jaime Fornetti, Alexis Weston, Serina Ng, Tithi Ghosh-Halder, Taylor Bargenquast, Brian Durbin, Srinivas Kasibhatla, Raffaella Soldi, Alana Welm, Sunil Sharma. Development of a novel, selective, and potent inhibitor of RON kinase [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 607.

Abstract 4438: Time restricted eating improves antitumor immune responses via modulation of insulin-like growth factor 1 in patients with head and neck cancer

Abstract Background- Treatment of metastatic head and neck cancer remains an area of unmet need with median survival less than a year. We tested the efficacy of a novel circadian aligned fasting intervention called “time restricted eating” (TRE), to improve ICB response in tumor bearing head and neck syngeneic mice models (MOC1), and showed significant reduction in tumor growth, which we then recapitulated in a pilot, open labeled, interventional clinical trial in 30 metastatic head and neck cancer patients. We noted significant improvement in median progression free survival (5 months vs not reached (NR), HR-0.2, p-0.0008) and overall survival (8 months vs NR, HR-0.00000045) in patients observing TRE vs those who did not. The favorable effect of TRE was mediated via modulation of gut microbiome, microbial tryptophan metabolome, and insulin signalling, specifically reduction in circulating insulin-like growth factor 1 (IGF1). We hypothesized that IGF1 mediates ICB resistance via protecting tumor cells from cytotoxic T cell activity. Methods- We performed in vitro luciferase cytotoxicity assays using HLA-A2+NY-ESO+ squamous cell cancer 4 (SCC-4) human cancer cells as targets, and HLA-A2-restricted NY-ESO-1-specific T cell receptor (TCR) -transduced T cells. We co-cultured the tumor cells and the T cells for 24 hours under different conditions of IGF-1 concentration (100 ng, 50 ng, and 10 ng) and T cell-to-tumor cell ratio (1:5, 1:10, and 1:20). We also treated the tumor cells with IGF-1 alone to assess its direct cytotoxic effect. Results We observed that IGF-1 impaired the ability of the T cells to kill the tumor cells in a dose-dependent manner, while IGF-1 had no cytotoxic effect on the tumor cells in the absence of T cells. Conclusion- While the role of IGF1 in treatment resistance has been described, its role and mechanism in antitumor immune response is largely unknown. We, for the first time demonstrate the role of IGF1 in impairing T cell mediated antitumor activity in a dose dependent fashion, thus contributing to ICB resistance. Further work will help us develop alternative strategies to modulate IGF1 and improve ICB resistance, in patients who are unable to tolerate TRE. Citation Format: Mostafa Eysha, Carmen A. Galindo, Jameel Muzaffar, Jessica Mine, Ricardo Chauriogonzalez, Mariam Baig, Luis Lopez-bailon, Muhammad Alhomsi, Mahmoud Hathout, Jiangbo Wang, Wanyu Zhang, Daliya Rizvi, Angie Huang, Kate Lee, Daniel Kye, Amit Sen, Jose Conejo-Garcia, Shahla Bari. Time restricted eating improves antitumor immune responses via modulation of insulin-like growth factor 1 in patients with head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4438.

Abstract 587: Tumor treating fields induce the integrated stress response, alter the transcriptional signatures of cellular metabolism, and modulate immune-related cytokines dependent and independent of p53

Abstract Tumor treating fields (TTFields) are an FDA-approved therapy for the treatment of glioblastoma and pleural mesothelioma. TTFields employ alternating electric fields delivered by cutaneous transducer arrays to induce cytostatic and cytotoxic effects on solid tumors. Additionally, recent studies suggest that TTFields may enhance anti-tumor immunity. We performed RNA-seq and multiplexed cytokine profiling in HCT116 wild-type and HCT116 p53-/- human colorectal cancer cells to elucidate TTFields’ effects on the transcriptome and secretome. TTFields lead to profound changes in the transcriptome related to metabolism, including downregulation of transcripts coding for multiple enzymes involved in the Krebs cycle, fatty acid synthesis, and glycolysis. Furthermore, though only HCT116 p53-wild type cells strongly induced the p53 pathway, both cell types’ transcriptomes showed strong downregulation of cell cycle progression. Our cytokine data showed that key tumor-promoting cytokine IL-8 was downregulated independently of p53. We identified upregulation of immunomodulatory cytokines MIF and MICB independently of p53. While MICB promotes NK cell activation, MIF drives tumor progression. Therefore, MICB may be one way in which TTFields enhance anti-tumor immunity; targeting MIF during TTFields treatment may lead to synergy by abolishing its pro-tumor effects. We also identified a p53-dependent decrease in the TRAIL decoy receptor DcR3, which normally protects tumor cells from TRAIL-induced apoptosis. Further mechanistic work showed that TTFields induce EIF2-alpha phosphorylation, the central protein involved in activating the integrated stress response (ISR), across multiple cancer types, leading to suppression of global protein translation. TTFields treatment also resulted in increased expression of surface calreticulin, an endoplasmic reticulum stress marker, which activates the ISR. Future mechanistic work will explore the impact of identified cytokines and the ISR on anti-tumor immunity both in vitro and in vivo, as well as metabolic alterations induced by TTFields. This work identifies potential biomarkers and rationales for therapeutic combinations exploiting TTFields-induced molecular alterations. Citation Format: Praveen R. Srinivasan, Andrew George, Charissa Chou, Maximilian Pinho-Schwermann, Maryam Ghandali, Vida Tajiknia, Yaara Porat, Moshe Giladi, Andrea Armstead, Alper Uzun, Eric T. Wong, Wafik S. El-Deiry. Tumor treating fields induce the integrated stress response, alter the transcriptional signatures of cellular metabolism, and modulate immune-related cytokines dependent and independent of p53 [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 587.

Ti2C3 MXene-based nanocomposite as an intelligent nanoplatform for efficient mild hyperthermia treatment

Photothermal therapy (PTT) has attracted much attention due to its less invasive, controllable and highly effective nature. However, PTT also suffers from intrinsic cancer resistance mediated by cell survival pathways. These survival pathways are regulated by a variety of proteins, among which heat shock protein (HSP) triggers thermotolerance and protects tumor cells from hyperthermia-induced apoptosis. Confronted by this challenge, we propose and validate here a novel MXene-based HSP-inhibited mild photothermal platform, which significantly enhances the sensitivity of tumor cells to heat-induced stress and thus improves the PPT efficacy. The Ti3C2@Qu nanocomposites are constructed by utilizing the high photothermal conversion ability of Ti3C2 nanosheets in combination with quercetin (Qu) as an inhibitor of HSP70. Qu molecules are loaded onto the nanoplatform in a pH-sensitive controlled release manner. The acidic environment of the tumor causes the burst-release of Qu molecules, which deplete the level of heat shock protein 70 (HSP70) in tumor cells and leave the tumor cells out from the protection of the heat-resistant survival pathway in advance, thus sensitizing the hyperthermia efficacy. The nanostructure, photothermal properties, pH-responsive controlled release, synergistic photothermal ablation of tumor cells in vitro and in vivo, and hyperthermia effect on subcellular structures of the Ti3C2@Qu nanocomposites were systematically investigated.

Abstract 2667: Mucin 5AC inhibition via intratracheal RNAi delivery as an effective alternative strategy for prevention and treatment of Kras mutant lung cancer

Abstract Lung cancer, including lung adenocarcinoma (LUAD), is the leading cause of cancer-related deaths in the United States. KRAS mutations are the most prevalent oncogenic driver alteration in human LUAD. We have previously shown that high expression of Mucin 5 AC (MUC5AC), a main airway secretory mucin, is significantly associated with poor prognosis. Mucins are essential for mucociliary clearance and the maintenance of airway homeostasis, however, their overproduction could negatively affect the lung immune microenvironment and has been shown to promote airway diseases such as chronic obstructive pulmonary disease (COPD) and lung cancer. Previously, we have shown that genetic deletion of MUC5AC in a murine model of K-ras mutant lung adenocarcinoma (KM-LUAD) impedes tumor development and reduces pro-tumor inflammatory and immune phenotypes. These findings indicate that MUC5AC is a potential druggable target against KM-LUAD. In this study, we sought a translational approach and inhibited MUC5AC in our K-rasG12D mutant mouse model of LUAD via RNA interference (RNAi). Six-week-old mice were given RNAi (5mg/kg) or vehicle (saline) via biweekly intratracheal administration for 8 weeks. This approach led to a significant decrease in tumor burden (40%) and a trending increase in total white blood cell counts, predominantly macrophages in the lung tumor microenvironment which was associated with phenotypic changes in macrophages characterized by reduced expression of Fizz1, and M2 pro-tumor macrophage marker. Additionally, RNAi-treated mice had reduced expression of Siglec-E, a mucin receptor and known inhibitor of the TLR4 endocytosis and promoter of NF-ĸB signaling, as well as a significant decrease in IL-6, a product of the NF-ĸB pathway and promoter of STAT3 signaling which we have previously shown to have an essential role in promotion of KM-LUAD. In summary, these findings elucidate a potential mechanism by which MUC5AC protects tumor cells from apoptosis and immune-mediated cytotoxic activity but also highlights how MUC5AC RNAi is an effective indirect preventive and therapeutic strategy in high-risk individuals (smokers with COPD) and patients with early stage KM-LUAD, respectively. Citation Format: Melvin Zarghooni, Maria T. Grimaldo, Avantika Krishna, Michael J. Clowers, Linda Phan, Yasmina H. Rezai, Arnav Gaitonde, Carlos Rodriguez Reyna, Walter Velasco Torrez, Erik Bush, Seyed Javad Moghaddam. Mucin 5AC inhibition via intratracheal RNAi delivery as an effective alternative strategy for prevention and treatment of Kras mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2667.

Abstract 2915: Targeting complement factor H renders STK11-mutant lung adenocarcinoma sensitive to anti-PD-1 therapy in mice

Abstract STK11 mutations occurs in 15-20% of lung adenocarcinoma (LUAD) and is associated with KRAS mutations, poor survival and resistance to immune checkpoint inhibitors (ICI). We observed tumor cell lines and primary human STK11-mutant LUAD upregulates of expression of C3 and complement factor H (FH). Deletion of C3 in STK11 knock-out tumor cells resulted in reduced FH expression. FH is a co-factor of Factor I, which converts C3b to proteolytically inactive iC3b thereby limiting activation of the alternative pathway (AP) C3 convertase. FH binds to proteoaminoglycans on the cell membrane and protects cells from AP complement-dependent cytotoxicity. With increased C3 generation by STK11-mutant tumor cells, we reasoned that systemic or tumor-derived FH protects tumor cells from complement attack. We hypothesized that tumor-derived C3 would promote STK11-mutant LUAD growth in immune intact mice by promoting immune evasion and would drive ICI resistance. Based on the expected tumor-protective effects of FH, we also hypothesized that anti-FH antibodies would control tumor growth and render tumor more sensitive to ICI. We evaluated growth of subcutaneously administered CMT167-STK11KO with and without C3 deletion (CMT167-STK11KO is murine KRASG12V LUAD with CRISPR/Cas9 deletion of STK11). Deletion of C3 in CMT167-STK11KO (CMT167-STK11KO/C3KO) resulted in dramatic inhibition of tumor growth and in sensitivity to anti-PD-1 therapy in vivo. In contrast, deletion of C3 in STK11-KO tumors had no effect on tumor growth in nude mice or following CD8 depletion in WT mice. These findings support a role of tumor-derived C3 in evasion of T cell anti-tumor immunity and as an obstacle to anti-PD-1 therapy. GT103 is a therapeutic antibody that binds tumor cell-attached CFH, causes complement activation, kills tumor cells, initiates a shift from a pro- to an anti-tumor immune microenvironment, and is currently being evaluated in a Phase 1b-2 clinical trial in combination with pembrolizumab (NCT05617313). We observed that single agent GT103 or anti-PD-1 has limited efficacy in CMT167-STK11KO tumor in WT mice. However, the combination of GT103 and anti-PD-1 was very effective in limiting tumor growth. Our results support a role of tumor-derived C3 in suppressing CD8 T-cell immunity and in driving ICI resistance and that the addition of GT103 renders STK11-KO tumors sensitive to anti-PD-1 in mice. These studies establish rationale for evaluating this combination in patients with STK11-mutant LUAD. Citation Format: Sora Suzuki, Anm Nazmul Khan, Thejaswini Giridharan, Catrina Ting, Ryan Bushey, Elizabeth Gottlin, Michael Campa, Edward F. Patz, Edwin H. Yau, Brahm Segal. Targeting complement factor H renders STK11-mutant lung adenocarcinoma sensitive to anti-PD-1 therapy in mice [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 2915.

Expression of Sialic Acid α2-3 and α2-6 in MCF Cells Upon Stimulation with Lipopolysaccharide

Protein sialylation is an important post-translational modification of glycoproteins that is also involved in tumor progression and metastasis. Increased expression of sialic acid can disrupt receptor-ligand interactions and protect tumor cells from the immune system. In this work, the expression of sialic acid α2-3 and α2-6 in MCF-7 stimulated with LPS at a concentration of 20 ng/ml cells was determined, by flow cytometry, fluorescence microscopy, and RT- qPCR. An increase in the expression of sialic acid α2-3 and α2-6 recognized by Maackia amurensis and Sambuccus nigra lectins was observed. While the percentage of cells expressing α2, 3 sialic acid recognized by the Maackia amurensis lectin remained at 20%, in the case of α2, 6 sialic acid recognized by the Sambucus nigra lectin, it was observed that 44% of the population expressed it after 2h of stimulation. The expression of ST3GAL1 and ST6GAL1 was evaluated by RT- qPCR, and the results obtained showed that Lipopolysaccharide did not produce significant changes in ST6GAL1 mRNA levels, while for ST3GAL1 it is observed that Lipopolysaccharide causes an increase in mRNA at 4 hours while at 2 and 6 hours it decreases. Our results suggest that lipopolysaccharide causes an increase in the expression of α2, 3 sialic acid at four hours after stimulation, decreasing at six hours of stimulation, while the expression of α2, 6 sialic acid increases at six hours of stimulation with LPS. Further studies are needed to evaluate the effect of protein sialylation caused by lipopolysaccharide.

Glycan-Lectin interactions between platelets and tumor cells drive hematogenous metastasis.

Glycosylation is a ubiquitous cellular or microenvironment-specific post-translational modification that occurs on the surface of normal cells and tumor cells. Tumor cell-associated glycosylation is involved in hematogenous metastasis. A wide variety of tumors undergo aberrant glycosylation to interact with platelets. As platelets have many opportunities to engage circulating tumor cells, they represent an important avenue into understanding the role glycosylation plays in tumor metastasis. Platelet involvement in tumor metastasis is evidenced by observations that platelets protect tumor cells from damaging shear forces and immune system attack, aid metastasis through the endothelium at specific sites, and facilitate tumor survival and colonization. During platelet-tumor-cell interactions, many opportunities for glycan-ligand binding emerge. This review integrates the latest information about glycans, their ligands, and how they mediate platelet-tumor interactions. We also discuss adaptive changes that tumors undergo upon glycan-lectin binding and the impact glycans have on targeted therapeutic strategies for treating tumors in clinical settings.

Aberrant MET Receptor Tyrosine Kinase Signaling in Glioblastoma: Targeted Therapy and Future Directions.

Brain tumors represent a heterogeneous group of neoplasms characterized by a high degree of aggressiveness and a poor prognosis. Despite recent therapeutic advances, the treatment of brain tumors, including glioblastoma (GBM), an aggressive primary brain tumor associated with poor prognosis and resistance to therapy, remains a significant challenge. Receptor tyrosine kinases (RTKs) are critical during development and in adulthood. Dysregulation of RTKs through activating mutations and gene amplification contributes to many human cancers and provides attractive therapeutic targets for treatment. Under physiological conditions, the Met RTK, the hepatocyte growth factor/scatter factor (HGF/SF) receptor, promotes fundamental signaling cascades that modulate epithelial-to-mesenchymal transition (EMT) involved in tissue repair and embryogenesis. In cancer, increased Met activity promotes tumor growth and metastasis by providing signals for proliferation, survival, and migration/invasion. Recent clinical genomic studies have unveiled multiple mechanisms by which MET is genetically altered in GBM, including focal amplification, chromosomal rearrangements generating gene fusions, and a splicing variant mutation (exon 14 skipping, METex14del). Notably, MET overexpression contributes to chemotherapy resistance in GBM by promoting the survival of cancer stem-like cells. This is linked to distinctive Met-induced pathways, such as the upregulation of DNA repair mechanisms, which can protect tumor cells from the cytotoxic effects of chemotherapy. The development of MET-targeted therapies represents a major step forward in the treatment of brain tumours. Preclinical studies have shown that MET-targeted therapies (monoclonal antibodies or small molecule inhibitors) can suppress growth and invasion, enhancing the efficacy of conventional therapies. Early-phase clinical trials have demonstrated promising results with MET-targeted therapies in improving overall survival for patients with recurrent GBM. However, challenges remain, including the need for patient stratification, the optimization of treatment regimens, and the identification of mechanisms of resistance. This review aims to highlight the current understanding of mechanisms underlying MET dysregulation in GBM. In addition, it will focus on the ongoing preclinical and clinical assessment of therapies targeting MET dysregulation in GBM.