Stable Cell Lines Research Articles

Abstract LB364: Generation and Application of a Robust Humanized Mouse Tumor Model System for Evaluation of NK-Targeting Therapeutics

Abstract With the new waves of anti-tumor immunotherapy development, novel immunotherapies based on NK cells have emerged and advanced rapidly. Considering the inherent mechanisms of NK cells, various strategies are employed in tumor immunotherapy to maximize the efficacy of NK cells, including the following: 1) immune checkpoint inhibitors to relieve NK cell inhibition signals; 2) activation of NK cell activity; 3) targeted antibody therapy for tumor cell killing via antibody-dependent cellular cytotoxicity (ADCC), and 4) cell therapy, such as CAR-NK. Although numerous NK-targeting immune therapeutics or various NK-modulating modalities have been developed or advanced toward clinical trials, there is a lack of clinically relevant preclinical animal models that can be widely accepted for the evaluation of NK-modulating agents, particularly for models that involve functional human NK cells in the test system. This study generated a humanized mouse tumor model system which involves specific reconstitution of functional and durable human NK cells in a murine system, providing a clinically relevant tumor microenvironment. We first engineered a stable monoclonal feeder cell line in vitro to expand NK cells isolated from human peripheral blood mononuclear cells (hPBMCs) using magnetic bead separation. Using this approach, the human primary NK cells were robustly and consistently amplified (up to hundreds of fold) while maintaining their purity and functionality. When compared with primary NK cells using in vitro immune assays including ADCC and FACs, the expanded NK cells showed comparable results as the parental cells. In vivo experimental results indicated that a durable and functional NK reconstitution can be established in tumor-bearing severely immune deficient mice expressing hIL-15. Furthermore, this NK-humanized tumor model system was proven to respond to standard of care (SOC) NK-targeting drugs. In summary, this novel NK humanized in vivo model may represent an effective preclinical tool for assessing NK-modulating drugs, with the hope of facilitating the development process of novel immune therapeutics employing both adaptive and innate immunity. Citation Format: Feng Lu, Weiqun Ding, Guangliang Yang, Yaoqing Jia, Zhaoxiang Wang, Xusheng Yuan, Ming Yin, Zhijian Yang, WenQing Yang. Generation and Application of a Robust Humanized Mouse Tumor Model System for Evaluation of NK-Targeting Therapeutics [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 LB364.

Abstract LB274: Nuclear transporter HIKESHI is a novel independent poor overall survival for pancreatic cancer; targeting can inhibit pancreatic cancer growth via HSP70 suppression

Abstract HIKESHI, under heat stress conditions, has been reported to transport molecular chaperone HSP70 into the nucleus to prevent cancer cell death from heat stress. The tumor-supporting roles of molecular chaperones in various solid tumors are explored extensively. Yet, the sole targeting of the molecular chaperones could not significantly impact anticancer treatment. Thus, we hypothesized that the specific transporter protein HIKESHI might improve this outcome. However, understanding of the clinical relevance of HIKESHI in pancreatic cancer remained unclear, and few studies addressed the HIKESHI role in cancer and whether targeting can inhibit pancreatic cancer cell viability and the other HSPs. Here, we first investigated the expression pattern of the HIKESHI protein in surgically resected pancreatic cancer samples (n=142) and analyzed the clinical relevance of the HIKESHI. We found that the HIKESHI protein was overexpressed in cancer compared to the adjacent normal tissue, and positive expression of cancer HIKESHI was related to poor overall survival. In addition, HIKESHI-positive expression in cancer was identified as an independent prognostic marker for overall survival. Next, using shRNA, we established the stable HIKESHI-suppressed pancreatic cancer cell line (PANC-1). HIKESHI suppression inhibited cell growth and colony formation ability in vitro. The expression and nuclear translocation of HSP70 and HSP110/105 were suppressed in HIKESHI-suppressed cells compared to the control shRNA cells despite no heat stress conditions. Furthermore, we found that the tumor growth, HIKESHI, and Ki67 expression in HIKESHI-suppressed tumors were inhibited compared to the control shRNA tumors. Next, we performed Cap Analysis of Gene Expression and IP-MS analysis to discover the potential downstream and novel interactive partner of the HIKESHI protein. The extensive mechanism of these analyses is under investigation. Our findings suggest that targeting HIKESHI may be a promising therapeutic strategy against refractory pancreatic cancer. Citation Format: Bilguun Erkhem-Ochir, Takehiko Yokobori, Gendensuren Dorjkhorloo, Haruka Okami, Takaomi Seki, Norifumi Harimoto, Ryo Muranushi, Kouki Hoshino, Kei Hagiwara, Norihiro Ishii, Mariko Tsukagoshi, Kenichiro Araki, Hiroshi Saeki, Ken Shirabe. Nuclear transporter HIKESHI is a novel independent poor overall survival for pancreatic cancer; targeting can inhibit pancreatic cancer growth via HSP70 suppression [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 LB274.

Intracellular localized heterogeneous protein franking by a transmembrane domain of GP64 is sufficient to be assembled on budded virions of Bombyx mori nucleopolyhedrovirus

Baculovirus has been widely used for foreign protein expression in biomedical studies, and budded virus (BV) surface display has developed into an important research tool for heterogenous membrane protein studies. The basic strategy of surface display is to construct a recombinant virus where the target gene is fused with a complete or partial gp64 gene. In this study, we further investigate and develop this BV surface displaying strategy. We constructed stable insect cell lines to express the target protein flanking with different regions of signal peptide (SP) and GP64 transmembrane domain (TMD). Subsequently, recombinant BmNPV was used to infect the cell, and the integration of heterogeneous protein into BV was detected. The results indicated that deletion of the n-region of SP (SPΔn) decreased the incorporation rate more than that of the full-length SP. However, the incorporation rate of the protein fused with h and c-region deletion of SP (SPΔh-c) was significantly enhanced by 35–40 times compare to full-length SP. Moreover, the foreign protein without SP and TMD failed to display on the BV, while the integration of foreign proteins with GP64 TMD fusion at the c-terminal was significantly enhanced by 12–26 times compared to the control. Thus, these new strategies developed the BV surface display system further.

TLR4 TIR domain and nucleolin GAR domain synergistically mediate RSV infection and induce neuronal inflammatory damage in SH-SY5Y cells.

Previous research results of our group showed that Toll-like receptor 4 (TLR4) and nucleolin synergistically mediate respiratory syncytial virus (RSV) infection in human central neuron cells, but the specific mechanism remains unclear. Here we designed and synthesized lentiviruses with TIR (674-815 aa), TLR4 (del 674-815 aa), GAR (645-707 aa), and NCL (del 645-707 aa) domains, and obtained stable overexpression cell lines by drug screening, and subsequently infected RSV at different time points. Laser confocal microscopy and coimmunoprecipitation were used for the observation of co-localization and interaction of TIR/GAR domains. Western blot analysis was used for the detection of p-NF-κB and LC3 protein expression. Real-time PCR was used for the detection of TLR4/NCL mRNA expression. ELISA assay was used to measure IL-6, IL-1β, and TNF-α concentrations and flow cytometric analysis was used for the study of apoptosis. Our results suggest that overexpression of TIR and GAR domains can exacerbate apoptosis and autophagy, and that TIR and GAR domains can synergistically mediate RSV infection and activate the NF-κB signaling pathway, which regulates the secretion of downstream inflammatory factors, such as IL-6, IL-1β, and TNF-α, and ultimately leads to neuronal inflammatory injury.

A novel method to assess antibody-dependent cell-mediated cytotoxicity against influenza A virus M2 in immunized murine models

The matrix protein 2 (M2) is a preferred target for developing a universal vaccine against the influenza A virus (IAV). This study aimed to develop a method for assessing antibody-dependent cell-mediated cytotoxicity (ADCC) associated with M2-based immunization in mice. We first established a stable cell line derived from mouse lymphoma cells (YAC-1) expressing M2 of H3N2. This cell line, designated as YAC-1-M2, was generated using a second-generation lentiviral tricistronic plasmid system to transduce the M2 gene into YAC-1 cells. The ADCC effect induced by polyclonal antibodies targeting matrix protein 2 ectodomain (M2e) was demonstrated by YAC-1-M2 cell lysis by natural killer cells (NK) derived from mice, in the presence of anti-M2 antibodies obtained from mice immunized with an mRNA vaccine based on M2e. This ADCC effect was found to be stronger compared to the effect induced by monoclonal antibodies (14C2) against M2. Moreover, the ADCC effect was enhanced as the effector-to-target ratio of NK to YAC-1-M2 cells increased. In conclusion, we established a novel method to detect ADCC of M2 of IAV, which paves the way for the development of an M2-based universal vaccine against IAV and an in-depth analysis of its mechanism of broad-spectrum immune protection in mice.

Expression and functional characterization of chimeric recombinant bovine follicle-stimulating hormone produced in Leishmania tarentolae.

Assisted reproductive techniques are routinely used in livestock species to increase and enhance productivity. Ovarian hyperstimulation is a process that currently relies on administering pituitary-derived follicle-stimulating hormone (FSH) or equine chorionic gonadotropin in combination with other hormones to promote the maturation of multiple follicles and thereby achieve superovulation. The use of partially purified preparations of FSH extracted from natural sources is associated with suboptimal and variable results. Recombinant FSH (rFSH) has been produced in a variety of heterologous organisms. However, attaining a bioactive rFSH of high quality and at low cost for use in livestock remains challenging. Here we report the production and characterization of a single chain bovine rFSH consisting of the β- and α-subunit fused by a polypeptide linker (scbFSH) using Leishmania tarentolae as heterologous expression system. This unicellular eukaryote is non-pathogenic to mammals, can be grown in bioreactors using simple and inexpensive semisynthetic media at 26°C and does not require CO2 or bovine serum supplementation. Stable cell lines expressing scbFSH in an inducible fashion were generated and characterized for their productivity. Different culture conditions and purification procedures were evaluated, and the recombinant product was biochemically and biologically characterized, including bioassays in an animal model. The results demonstrate that L. tarentolae is a suitable host for producing a homogeneous, glycosylated and biologically active form of scbFSH with a reasonable yield.

Hepatitis E virus ORF3 protein hijacking thioredoxin domain-containing protein 5 (TXNDC5) for its stability to promote viral particle release.

Hepatitis E virus (HEV) infection is the leading cause of acute viral hepatitis worldwide. After the synthesis and modification in the cells, the mature ORF3 protein is essential for HEV release. However, the host protein involved in this process has yet to be determined. Here, we reported a novel host protein, thioredoxin domain-containing protein 5 (TXNDC5), as a chaperone, contributing to HEV release by facilitating ORF3 protein stability in the endoplasmic reticulum through interacting with non-palmitoylated ORF3 protein. However, we also found that in the knockout-TXNDC5 stable cell lines, the HEV ORF3 protein may hijack other proteins for its stabilization. For the first time, our study demonstrated the involvement of TXNDC5 in viral particle release. These findings provide some new insights into the process of the HEV life cycle, the interaction between HEV and host factors, and a new direction for antiviral design.

Improving CRISPR–Cas9 directed faithful transgene integration outcomes by reducing unwanted random DNA integration

BackgroundThe field of genome editing has been revolutionized by the development of an easily programmable editing tool, the CRISPR–Cas9. Despite its promise, off-target activity of Cas9 posed a great disadvantage for genome editing purposes by causing DNA double strand breaks at off-target locations and causing unwanted editing outcomes. Furthermore, for gene integration applications, which introduce transgene sequences, integration of transgenes to off-target sites could be harmful, hard to detect, and reduce faithful genome editing efficiency.MethodHere we report the development of a multicolour fluorescence assay for studying CRISPR–Cas9-directed gene integration at an endogenous locus in human cell lines. We examine genetic integration of reporter genes in transiently transfected cells as well as puromycin-selected stable cell lines to determine the fidelity of multiple CRISPR–Cas9 strategies.ResultWe found that there is a high occurrence of unwanted DNA integration which tarnished faithful knock-in efficiency. Integration outcomes are influenced by the type of DNA DSBs, donor design, the use of enhanced specificity Cas9 variants, with S-phase regulated Cas9 activity. Moreover, restricting Cas9 expression with a self-cleaving system greatly improves knock-in outcomes by substantially reducing the percentage of cells with unwanted DNA integration.ConclusionOur results highlight the need for a more stringent assessment of CRISPR–Cas9-mediated knock-in outcomes, and the importance of careful strategy design to maximise efficient and faithful transgene integration.

Case-inspired exploration of renin mutations in autosomal dominant tubulointerstitial kidney disease: not all paths lead to the endoplasmic reticulum.

Autosomal dominant tubulointerstitial kidney disease (ADTKD) results from mutations in various genes, including REN, UMOD, MUC1, and HNF1B. ADTKD due to REN mutations (ADTKD-REN) is often characterized as a proteinopathy that triggers the endoplasmic reticulum stress (ERS) cascade, potentially sharing similarities with ADTKD-UMOD and ADTKD-MUC1 at the cellular level. This study, inspired by a patient harboring a W17R mutation, investigates ERS activation by this mutation alongside two other renin variants, W10R and L381P. We established stable cell lines expressing both wild-type and mutated renin forms (W17R, W10R, and L381P). Using luciferase reporter assays, RT-qPCR, and confocal microscopy, we evaluated ERS activation, determined the cellular localization of the renin variants, and characterized the mitochondrial network in the W17R line. The L381P line exhibited ERS activation, including transcriptional upregulation of MANF and CRELD2. No ERS activation was observed in the W17R line, while the W10R line exhibited intermediate characteristics. Notably, the W17R variant was misrouted to the mitochondria resulting in changes of the mitochondrial network organisation. ERS activation is not a universal response to different renin mutations in ADTKD-REN. The pathogenesis of the W17R mutation may involve mitochondrial dysfunction rather than the ER pathway, albeit further research is needed to substantiate this hypothesis fully. Testing CRELD2 and MANF as targeted therapy markers for a specific subgroup of ADTKD-REN patients is recommended. Additionally, fludrocortisone treatment has shown efficacy in stabilizing the renal function of our patient over a four-year period without significant side effects.

Abstract 1563: Anoikis associated metabolic modulation by UCA1 in CRC

Abstract Colorectal Cancer (CRC) is the second most lethal cancer in estimated deaths for 2023 according to the American Cancer Society. This is partially due to the low survival rate once CRC is diagnosed at a distant location. This warrants early diagnostic markers and an understanding of the mechanisms underlying the metastatic process, anoikis resistance being one of them. Our laboratory analysis of the TCGA database comprising 519 human CRC tissues shows lncRNA UCA1 overexpressed in tumors compared to normal, and higher expression of lncRNA UCA1 is responsible for the progression, metastasis, and poor survival of CRC patients. In our anchorage-independent growth model (anoikis resistant), using isogenic (oncogenic and metastatic) cell lines, we observed a very high expression of lncRNA UCA1 along with GluT1 with increased cell survival. Cancer cells, however, appear to rapidly increase their glucose uptake and directly ferment it into lactate, even in the presence of oxygen and functional mitochondria. UCA1 has been found to play an essential role in the development of metastasis and dysregulation of glycolysis in various cancers. To verify if lncRNA UCA1 modulates anoikis resistance and glucose metabolism, we generated SW480+UCA1 overexpression (OE) and SW620+shUCA1 knockdown (KD) stable cell lines using lentiviral transduction. The OE GFP-expressing cell lines were further sorted, and grown in puromycin. The KD cell lines were selected with puromycin for the generation of stable cell lines. The stable cell lines were characterized through different phenotypic assays. UCA1 OE cell lines have a higher glucose uptake and the UCA1 KD cell lines have a lower glucose uptake. The lactate production is high in the KD and low in OE cell lines. The OE and KD cell lines along with the control cell lines were subjected to an anoikis model, and glucose and lactate analysis were performed accordingly. RT-PCR and western blot analysis were performed for the target genes responsible for cell survival, stemness, and glucose metabolism. We are analyzing the Extracellular Acidification Rate (ECAR), Oxidative Consumption Rate (OCR), ATP production rate the rate and potential max rate of glycolysis, and the mitochondrial stress of the recombinant cell lines using the “Agilent Seahorse XF”. The role of lncRNA UCA1 in anoikis resistance leading to metastasis and modulating glucose metabolism will provide new insights into the pathophysiology of CRC metastasis. This information will be beneficial for developing more unique strategies for the treatment of this metastatic disease. Citation Format: Sophia Leslie, Kyle Doxtater, Samantha Lopez, Bilal Hafeez, Tamer Oraby, Timothy Loy, Manish K. Tripathi. Anoikis associated metabolic modulation by UCA1 in CRC [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 1563.

Abstract 407: RNA:DNA hybrid/R-loop determinants variation in distinct basal-like breast cancer cells

Abstract Introduction: Despite progress in treatment methods and diagnosis, breast cancer remains one of the world's leading cancers causes of death. Among the various subtypes, basal-like breast cancer (BLBC) is highly diverse, low differentiated, most aggressive form with poor prognosis. Co-translational R-loops are created by combining G-rich RNA with complementary C-rich DNA (RNA: DNA hybrid), which creates a three-stranded nucleic acid structure. R-loops are often associated with genome instability and aggressiveness of cancers. However, their role in the diversity of BLBC remains controversial and poorly understood. This study aims to characterize RNA: DNA hybrid variations in BLBC from a cellular, genetic, and biochemical perspective. Methods: This study investigates the variation of RNA: DNA hybrid formation in stable BLBC cells. We utilize a panel of stable basal-like cell lines (MDAMB231, MDAMB436, SUM149PT) and proliferative non-cancer cells (MCF10A). They represent different genetic and cell lineages in BLBC development which allows us to study R-loop distribution in different basal lineage cells. Luminal breast cancer cells (MCF7) were used in comparative analysis. To detect RNA: DNA hybrids, we optimized Immunofluorescence (IF) specificity using RNase III and H enzymes. Predictions from Quantitative Model of R-loop Forming Sequences (QmRLFS) identify RLFS in specific gene regulatory regions. This design allows us to define R-loop position and boundaries, and optimize primers for the DRIP-qPCR which quantifies RNA: DNA hybrid enrichment in transcription regulatory sites for several breast cancer-associated genes such as BRCA1, TP53, PTEN, and AURKA. The expression of these genes was detected using RTqPCR. Results: Our IF and computational image analyses demonstrated that RNA: DNA hybrids are present not only luminal cells (as found by others), but also in the studied BLBC cells. The non-cancer proliferative cells (MCF10A) show low RNA: DNA hybrid signals bolstering our experimental technique. Variation of the RNA: DNA hybrid signals were distinct in various BLBC cells which was supported by the DRIP-qPCR and RTqPCR findings. The R-loop formation data variations was referred to the cell line contexts and the studied genes responsible for tumor suppression, DNA damage/repair signaling and impair apoptosis in breast cancer. Conclusion: Our study suggests that RNA: DNA hybrids are formed and distributed heterogeneously in the genomes and gene regions of stable BLBC cell lines, while in basal proliferative (non-cancer) cells hybrid signals are weak. A subset of the RNA: DNA hybrids might be particularly significant at certain gene loci contributing to tumor progression, indicating their significance in BLBC pathogenesis. This study provides insights into the cellular and molecular basis of the BLBC heterogeneity and implicates the RNA:DNA hybrid as a potential diagnostic and therapeutic markers. Citation Format: Ojashpi Singh Kapali, Vladimir Kuznetsov. RNA:DNA hybrid/R-loop determinants variation in distinct basal-like breast 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 407.

Abstract 3263: VEGFA mediates traditional EGFR-TKI resistance in non-small cell lung cancer with EGFR exon 20 insertions

Abstract Background: Epidermal growth factor receptor (EGFR) exon 20 insertions (Ex20ins) accounted for 4%-12% of EGFR mutations and showed primary resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs) which is an urgent issue to be solved. In our previous studies, we found that antiangiogenetic therapy had certain advantages for patients with Ex20ins in the real-world practice. Previous research showed the tumor microenvironment has been associated with resistance to targeted therapy in sensitive mutations. However, the mechanism of resistance of EGFR-TKIs and the advantage of antiangiogenetic therapy in patients with EGFR Ex20ins were still unclear. Methods: In this study, patients with EGFR exon 19 deletion (19del), exon 21 p.L858R (21L858R), and Ex20ins were collected to identify the efficacy of EGFR-TKIs. Stable cell lines expressing were established and used to compare signaling pathway activation. Matrigel angiogenesis assay was conducted in different cell lines. Next, we evaluated the efficacy of various treatments combined with antiangiogenetic agents or not in mouse models. Results: We collected 23 patients with EGFR sensitive mutations and Ex20ins, respectively, and found Ex20ins presented primary resistance to TKIs and poor survival outcomes. Based on the RNA sequence of stable cell lines, it was found that Ex20ins had a higher level of VEGFA expression and enriched in MAPK pathway compared to EGFR 19del and 21L858R. Immunohistochemical studies also revealed that a higher level of VEGFA expression was observed in a cohort of NSCLC patients with Ex20ins mutation compared with 19del or L858R. Matrigel angiogenesis assay has shown consistent result that Ex20ins promoted angiogenesis in vivo. Further experiments in mouse models identified that in the group of Osimertinib, mobocertinib alone, chemotherapy alone, mobocertinib or chemotherapy combination with bevacizumab, Osimertinib achieved the worst efficacy and additional utility of antiangiogenetic agents had a more favorable efficacy. Conclusion: Our study highlights the potential of VEGFA as a mechanism of resistance to EGFR-TKIs and induces immunosuppression microenvironment in NSCLC with Ex20ins. Furthermore, the findings suggested that antiangiogenetic therapy could be a promising therapeutic approach. Keywords: VEGFA, targeted therapy, resistance, EGFR exon 20 insertions, non-small cell lung cancer Citation Format: Yaning Yang, Chengming Liu, Guangjian Yang, Haiyan Xu, Siyu Lei, Yan Wang. VEGFA mediates traditional EGFR-TKI resistance in non-small cell lung cancer with EGFR exon 20 insertions [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 3263.

Abstract 5866: TESC mediates resistance to lenvatinib in hepatocellular carcinoma by regulating cell autophagy

Abstract Objective: To investigate the mechanism by which TESC (Tescalcin) induces lenvatinib resistance in hepatocellular carcinoma (HCC) through autophagy mediation. Methods:1) Utilized established liver cancer organoids from over 70 patients for lenvatinib drug sensitivity screening and transcriptome sequencing to identify lenvatinib-resistant genes. 2) Constructed stable TESC overexpression and downregulation liver cancer cell lines. Employed CCK8 assays to plot IC50 drug sensitivity curves, elucidating changes in lenvatinib sensitivity in liver cancer cells after TESC overexpression or downregulation. 3) Used TESC-stable cells to establish subcutaneous tumor models and intrahepatic tumor models in nude mice. After tumor formation, administered lenvatinib orally to observe whether TESC affects liver cancer sensitivity to lenvatinib in vivo. 4) Conducted Western blot (WB) analysis to assess the expression of autophagy-related molecules (LC3B II/I, P62, and Beclin1 proteins) in TESC downregulated or overexpressed stable cell lines and their control cell lines. Utilized the LC3-GFP-mRFP fluorescence dual-labeling lentivirus system and laser confocal microscopy to examine autophagic flux. 5)Treated TESC downregulated cells with the autophagy inhibitor 3-MA. Conducted WB to assess the expression levels of autophagy-related proteins and plotted drug sensitivity curves using IC50 experiments to evaluate the impact of 3-MA on reversing liver cancer cell sensitivity to lenvatinib. Results: Compared to the control group, HCC cells overexpressing TESC showed an increased IC50 value and reduced sensitivity to lenvatinib, while TESC downregulation resulted in a decreased IC50 value and decreased sensitivity to lenvatinib. Subcutaneous tumor experiments in nude mice indicated that, compared to the control group, tumors with reduced TESC expression were more sensitive to lenvatinib. Additionally, in HCC cells overexpressing TESC, protein expression levels of LC3B II/I and Beclin1 significantly decreased, while P62 expression increased. Conversely, TESC downregulation significantly increased protein expression levels of LC3B II/I and Beclin1, while P62 expression decreased. Furthermore, results from the LC3-GFP-mRFP autophagy dual-labeling lentivirus system showed a significant reduction in fluorescence spots in the TESC overexpression group compared to the control group, indicating weakened autophagic flux. Treatment of TESC downregulated liver cancer cells with 3-MA resulted in an increased IC50 value compared to the control group. Conclusion: TESC expression levels are associated with the sensitivity of liver cancer cells to lenvatinib, possibly by inhibiting autophagy and affecting the sensitivity of liver cancer cells to lenvatinib. Citation Format: Hao Zhuang, Tingting Zhang, Jiaqi Chen. TESC mediates resistance to lenvatinib in hepatocellular carcinoma by regulating cell autophagy [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 5866.

Abstract 5942: Discovery of WRN inhibitors as targeted therapy in the treatment of microsatellite unstable (MSI-H) tumors

Abstract The synthetic lethality resulting from the inhibition of the WRN helicase protein has been observed in tumors characterized by high levels of microsatellite instability (MSI-H). This instability stems from a deficiency in the DNA mismatch repair (MMR) mechanisms, leading to the accumulation of DNA damage. This phenomenon is notably prevalent in 10-30% of colorectal, gastric, endometrial, and ovarian cancers. Specifically, the inhibition of WRN helicase activity induces DNA damage leading to cell cycle arrest and apoptosis exclusively in MSI-H cell lines. This selectivity underscores the therapeutic potential of WRN inhibitors, as they demonstrate efficacy against MSI-H cells while remaining non-toxic to microsatellite stable (MSS) cell lines. Ryvu is developing a chemical series of potent, selective WRN helicase inhibitors. Structure-based optimization facilitated the rapid expansion and delivery of a compound library with novel intellectual property (IP), demonstrating target engagement in cells and selective potency over other RecQ family members. The correlation between the cellular effect and selectivity upon treatment with these ligands was confirmed in the clonogenic assay, specifically in responder, microsatellite instable cell lines only, thereby leaving a substantial safety margin (>100x) relative to non-responder, microsatellite stable lines. Target engagement has been confirmed by dose dependent DNA damage pathway activation concomitant with WRN degradation in in vitro assays. The pharmacokinetic properties of these compounds are favorable for progressing to in vivo studies. The correlation between compound exposure and on-target effect was confirmed in PK/PD and efficacy studies in xenograft MSI-H cancer models. These data provide pharmacological proof-of-concept for the synthetic lethal effect of our inhibitors and support WRN inhibition as a new oncological therapy. Citation Format: Wojciech Schönemann, Agnieszka Ludwig-Słomczyńska, Łukasz Dudek, Marcin Król, Marco Farinone, Urszula Kulesza, Wojciech Jasnosz, Izabela Mac, Karol Zuchowicz, Katarzyna Wierzbicka, Aleksandra Buchcic-Szychowska, Andrzej Gondela, Anna Zagórska, Oliwia Matuszewska, Marta Sowińska, Marcin Nowogródzki, Marianna Girardi, Szymon Paluch, Svitlana Sukhomlinova, Agata Chłopek, Iana Levenets, Kamila Kozłowska-Tomczyk, Katarzyna Łagosz-Ćwik, Mateusz Stoszko, Szymon Woroszyło, Maciej Mikulski, Justyna Jabłońska, Magdalena Wiśniewska, Katarzyna Palus-Chramiec, Paulina Podkalicka-Gołda, Anita Janiga, Sylwia Sudoł-Tałaj, Magdalena Sieprawska-Lupa, Magdalena Miodek, Jacek Faber, Róża Starczak, Bozena Winnik, Agnieszka Świrska, Anna Kowal-Chwast, Dawid Gogola, Sanja Novak Ratajczak, Agata Dudek, Nilesh Gaud, Grzegorz Satała, Agata Stachowicz, Martin Swarbrick, Krzysztof Brzózka, Magdalena I. Zawadzka. Discovery of WRN inhibitors as targeted therapy in the treatment of microsatellite unstable (MSI-H) tumors [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 5942.

Abstract 7056: The role of aggressive prostate cancer risk gene ANO7 in prostate metabolism

Abstract Prostate cancer (PCa), the most common male cancer worldwide, causes about 10% of cancer related deaths in Europe. It has a wide spectrum of clinical behavior that ranges from decades of indolence to rapid metastatic progression and lethality. However, the molecular mechanisms involved in aggressive PCa progression are still poorly understood. In PCa the expression of anoctamin 7 (ANO7) has been shown to diminish as the cancer progresses. We have previously linked single-nucleotide polymorphisms in the ANO7 gene to the risk of aggressive prostate cancer and shown that in homozygous carriers of rs77559646, the variant leads to a total loss of ANO7 protein. ANO7 is a prostate-specific gene and is highly expressed in the luminal cells of the human prostate. ANO7 belongs to a family of calcium-activated chloride channels and select members of the anoctamin family have phospholipid scramblase activity. To uncover the cellular functions of ANO7 has proven challenging because ANO7 is not expressed in commercially available cell lines. To study the cellular functions of ANO7, we have generated stable prostate cell lines overexpressing ANO7 using lentiviral transduction. To gain information into what pathways ANO7 is affecting, we performed RNA-sequencing and gene set enrichment analysis. Interestingly, we found enrichment and downregulation of mitochondrial genes participating in oxidative phosphorylation in ANO7 cells. We assessed the mitochondrial function by measuring the oxidative phosphorylation capability of the cells and showed that the basal and maximal respiratory capacity of ANO7 overexpressing cells is indeed reduced. We also analyzed which of the three major mitochondrial fuels (glucose, glutamine and fatty acids) are not used as efficiently for oxidative phosphorylation in ANO7 overexpressing cells. The result proves that ANO7 cells are not utilizing glucose as effectively as control cells. Furthermore, the glycolysis stress assay showed that glycolytic capacity is increased in ANO7 overexpressing cells. In addition, a targeted metabolite screening revealed that aspartate is decreased in ANO7 cells, which again points towards perturbed mitochondrial function as aspartate is produced from a tricarboxylic acid cycle intermediate. By inhibiting the uptake of extracellular aspartate, we measured slower proliferation of ANO7 expressing cells, while inhibiting aspartate had no effect on control cells. Interestingly, preliminary results also suggest ANO7's involvement in regulation of phospholipid acyl chain length and saturation, offering insights into altered signaling observed in ANO7 cells. Taken together, this study shows for the first time that ANO7 rewires prostate mitochondrial functions and thus cellular metabolism, which could explain why it is beneficial for cancer cells to lose ANO7 expression. Citation Format: Christoffer Löf, Nasrin Sultana, Neha Goel, Gudrun Wahlström, Johanna Schleutker. The role of aggressive prostate cancer risk gene ANO7 in prostate metabolism [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 7056.

Abstract 2065: Development of a genetically validated, cell-based reporter assay for ADAR1 editing activity

Abstract Adenosine Deaminase Acting on RNA (ADAR) enzymes perform adenosine to inosine base editing in RNA, particularly targeting adenosines located within a specific double-stranded stem-loop motif. The ADAR1 gene encodes both a universally expressed isoform (p110) and an interferon-inducible isoform (p150), which plays a role in innate immunity by mediating interferon signaling. In the context of healthy, uninfected cells, ADAR1 performs A-to-I editing on endogenous double-stranded RNA to prevent it from activating downstream dsRNA sensors RIG-I and MDA5, which in-turn activate a pro-inflammatory response. Loss of function mutations in ADAR1 result in aberrant activation of the dsRNA sensors and are involved in autoimmune disorders. ADAR1 dysfunction also impacts cancer cell growth, proliferation, and response to immunotherapy. ADAR1 expression is increased in several types of cancer and ADAR1 knock-out has been demonstrated to improve the response to certain immunotherapies such PD-1/PD-L1 blockade and to circumvent tumor immunotherapy resistance mechanisms, making ADAR1 an attractive target for therapeutic development. We have developed and validated a high-throughput, cell-based assay for measuring ADAR1 editing activity. Several ADAR editing reporter constructs were designed and evaluated for their relative response to ADAR1 over-expression. They all feature an ADAR1 hairpin target with a stop codon (UAG) which is susceptible to ADAR1-mediated editing to a tryptophan (UUG), located upstream of a firefly luciferase gene. We show that increasing transient expression of ADAR1 led to proportional increased activity of the downstream firefly luciferase, indicating dose-dependent transcript editing by ADAR1. Selected reporters were used in conjunction with ADAR2 overexpression to identify an ADAR1-biased reporter. Thus, a reporter with low luciferase activity in response to ADAR2 expression was chosen to establish stable HEK293 cell lines (which express low levels of ADAR1) with and without constitutively expressed ADAR1 protein. ADAR1-overexpressing HEK293 cells displayed high luciferase activity, in accordance with high ADAR1-mediated editing of the reporter transcript, which was decreased in response to siRNA-mediated knockdown of ADAR1 expression or pharmacological inhibition of ADAR1 activity. Sequencing of the reporter RNA confirmed that changes in reporter activity were correlated with editing of the RNA transcript. Finally, these validated ADAR1 editing reporter cell lines were further engineered to constitutively express Renilla luciferase under the control of a CMV promoter, serving as an internal control for the toxicity of ADAR1 inhibitors. In conclusion, our robust cell-based assay provides a well-validated resource to identify modulators of ADAR1 editing activity in a cellular context. Citation Format: Valerie Sapp, Jim Bilakovics, Ava Burr, Fernando Martins, Pavel Shashkin, Veronique Baron, Henry Zhu, Junguk Park. Development of a genetically validated, cell-based reporter assay for ADAR1 editing activity [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 2065.

Machine learning framework develops neutrophil extracellular traps model for clinical outcome and immunotherapy response in lung adenocarcinoma.

Neutrophil extracellular traps (NETs) are novel inflammatory cell death in neutrophils. Emerging studies demonstrated NETs contributed to cancer progression and metastases in multiple ways. This study intends to provide a prognostic NETs signature and therapeutic target for lung adenocarcinoma (LUAD) patients. Consensus cluster analysis performed by 38 reported NET-related genes in TCGA-LUAD cohorts. Then, WGCNA network was conducted to investigate characteristics genes in clusters. Seven machine learning algorithms were assessed for training of the model, the optimal model was picked by C-index and 1-, 3-, 5-year ROC value. Then, we constructed a NETs signature to predict the overall survival of LUAD patients. Moreover, multi-omics validation was performed based on NETs signature. Finally, we constructed stable knockdown critical gene LUAD cell lines to verify biological functions of Phospholipid Scramblase 1 (PLSCR1) in vitro and in vivo. Two NETs-related clusters were identified in LUAD patients. Among them, C2 cluster was provided as "hot" tumor phenotype and exhibited a better prognosis. Then, WGCNA network identified 643 characteristic genes in C2 cluster. Then, Coxboost algorithm proved its optimal performance and provided a prognostic NETs signature. Multi-omics revealed that NETs signature was involved in an immunosuppressive microenvironment and predicted immunotherapy efficacy. In vitro and in vivo experiments demonstrated that knockdown of PLSCR1 inhibited tumor growth and EMT ability. Besides, cocultural assay indicated that the knockdown of PLSCR1 impaired the ability of neutrophils to generate NETs. Finally, tissue microarray (TMA) for LUAD patients verified the prognostic value of PLSCR1 expression. In this study, we focus on emerging hot topic NETs in LUAD. We provide a prognostic NETs signature and identify PLSCR1 with multiple roles in LUAD. This work can contribute to risk stratification and screen novel therapeutic targets for LUAD patients.

Abstract 3358: Tribbles 1 (TRIB1) pseudokinase forms a complex with and activates Akt in GBM cells

Abstract Glioblastoma (GBM; WHO grade 4) is the most aggressive form of glioma with a dismal 5-year survival rate. The current therapies are largely ineffective and therapy resistance is commonly encountered. We have recently shown that TRIB1, a Ser/Thr pseudokinase contributes to therapeutic resistance in GBM cells by activating both ERK and Akt pathways. Here we show that, increased expression of TRIB1 contributed to RT/TMZ resistance by activating pro-survival Akt signaling resulting in increased viability of GBM cells. High level of Akt phosphorylation/activation is correlated with poor prognosis of GBM patients. Many pan Akt inhibitors have been developed which are currently in various phases of clinical trials. The three Akt isoforms (1, 2 and 3) are known to play a role in cancer during proliferation, transformation and metastasis. A previous report has shown that TRIB1 interacts with Akt1 in Drosophila and blocks its activation. In this study we show that Akt binds and forms a complex with TRIB1 in GBM cells and this interaction could be isoform and cell line specific. We utilized patient derived xenograft (PDX) GBM cell lines and established GBM cell lines in this study. Stable TRIB1 overexpressing cell lines were generated by antibiotic selection. Co-immunoprecipitation (Co-IP) was performed utilizing Anti-FLAG or anti-c-Myc magnetic agarose beads to evaluate protein-protein interactions. Western blotting was done to detect protein levels. The FLAG tagged TRIB1 deletion mutants were a generous gift from Dr. Takuro Nakamura (Division of Carcinogenesis, The Cancer Institute, Tokyo, Japan). Cells were transfected using Lipofectamine 2000 reagent. Co-IP studies showed that TRIB1 formed a complex with Akt in PDX GBM cell lines. We then transiently transfected respective TRIB1 deletion mutants lacking either N and C terminal residues in HEK 293 cells and performed Co-IP with Anti-FLAG magnetic agarose beads. We observed that all TRIB1 deletion mutants except TRIB1-ΔN2 (lacking first 160 amino acids) were able to pull down Akt. The western blot results showed that each Akt isoform had a variable protein expression across different cell lines. Co-IP studies in the PDX cell lines showed that TRIB1 interacted with all three Akt isoforms in T08-387 cells whereas TRIB1 consistently interacted with only Akt3 but not Akt1/2 in GBM30 cells that lack wild-type p53 expression. Our data suggest that TRIB1 forms a complex with Akt in PDX GBM cell lines. The potential Akt binding site on TRIB1 lies between 90-160 amino acids. TRIB1 can interact with different Akt isoforms in a cell type dependent manner thereby playing a role in their activation and consequent tumorigenic processes. Therefore, targeting TRIB1 mediated Akt activation could be considered a better therapeutic option over targeting Akt directly, which may decrease Akt’s oncogenic activities without risking the toxicity associated with available Akt- specific inhibitors. Citation Format: Karnika Singh, Chunhua Han, Heather Manring, Saikh Jaharul Haque, Arnab Chakravarti. Tribbles 1 (TRIB1) pseudokinase forms a complex with and activates Akt in GBM 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 3358.

Abstract 2749: DHX36 may promote the growth and metastasis of lung cancer

Abstract Introduction: Helicase is a kind of protein molecular motor that widely exists in eukaryotic cells, viruses and bacterial cells, and participates in DNA replication, transcription and other life activities. DHX36, a kind of helicase, is a new homologous gene of human DNA/RNA helicase cloned by our laboratory by homologous molecular cloning which used the sequence of the maleless gene (mle) of Drosophila. Studies have shown that DHX36 upregulates the expression of proto oncogene and promotes the occurrence of cancer. At present, there are few studies on DHX36 in lung cancer. This study aims to clarify the relationship between DHX36 and lung cancer and provide a new molecular target for the diagnosis, treatment and prognosis of lung cancer. Methods: Twenty-eight pairs of clinical tumor samples from lung adenocarcinoma and adjacent tissues were collected, and the protein expression of DHX36 in different tissues was detected by western blot. Correlation between DHX36 expression and survival prognosis of lung cancer patients was analyzed by online database. Over-expression of DHX36 in A549 and H460 cells and knockdown of DHX36 in A549 and H1975 cell lines, then the migration, invasion and growth index of cells were analyzed by real-time cell analyzer. After DHX36 stable knock-down cell line was established in H1975 cells, they were injected into nude mice by subcutaneous injection and tail vein injection respectively at the same time as the control cells, and the growth of subcutaneous tumors and the metastasis of lung tissues in nude mice were recorded and collected respectively. The tissues were analyzed by histology, immunohistochemistry, and western blot. Results: In 28 pairs of clinical lung cancer tumor samples, we found that DHX36 was higher in cancer tissues than in adjacent tissues in 23 pairs, which indicated that DHX36 might be a kind of proto oncogene to promote the occurrence and development of lung cancer. Bioinformatics analysis revealed that the high expression of DHX36 was related to the low survival of lung cancer patients. The results of RTCA showed that the abilities of growth, invasion and migration of cells in the over-expressed DHX36 groups were higher than those in the control groups. After DHX36 was knocked down, the growth, invasion and invasion of lung cancer cells were inhibited. Conclusion: DHX36 is expected to be a new molecular target of lung cancer. Next, we will knock out DHX36 in lung cancer cells to observe the tumor growth and lung metastasis in mice. Citation Format: Ting Li, Na Luo, Jiewen Fu, Chunli Wei, Junjiang Fu. DHX36 may promote the growth and metastasis of 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 2749.

Abstract 4466: Omega-3 fatty acid-induced ferroptosis in 4T1 breast cancer through CD36/FABP5 axis

Abstract Mammalian cells utilize energy from many different sources, and fatty acids are one of these sources. Dietary fats contain various structures of fatty acids, each impacting cell lipid metabolism in unique ways. Omega-3 fatty acids are known to induce breast cancer cell death through ferroptosis. However, the specific molecular mechanisms by which omega-3 fatty acids trigger ferroptosis have yet to be fully elucidated. Fatty acid binding proteins (FABPs) constitute a family of evolutionarily conserved lipid chaperones, facilitating the transport and utilization of fatty acids within cells. Notably, FABP5 is highly expressed in breast cancer, especially in the most aggressive form, triple-negative breast cancer. We hypothesized that FABP5 acts as a lipid sensor, mediating the ferroptosis induced by omega-3 fatty acids in triple-negative breast cancer cells. To test this, we created Fabp5 stable knockout cell lines in 4T1 and MDA-MB-231, two commonly used triple-negative breast cancer cell lines, using CRISPR/Cas9 technology. When treated with docosapentaenoic acid (DPA), these Fabp5-knockout cells exhibited reduced ferroptosis compared to their wild-type counterparts. Interestingly, blocking the surface protein CD36, either with inhibitors or antibodies, also reduced DPA-induced ferroptosis. Furthermore, mutations in Fabp5 decreased maximal mitochondrial respiration in both 4T1 and MDA-MB-231 cells, suggesting a crucial role for FABP5 in mediating oxygen consumption and reactive oxygen species (ROS) production during DPA-induced ferroptosis. Additionally, mice fed a high-fat diet rich in omega-3 fatty acids showed reduced tumor size and lung metastasis. Collectively, our data indicate that FABP5 is a key molecular sensor mediating oxygen consumption and ROS production, thus contributing to ferroptosis induced by omega-3 fatty acids in triple-negative breast cancer. Citation Format: Anthony Avellino, Jiaqing Hao, Matthew Yorek, Jian yu, Bing Li. Omega-3 fatty acid-induced ferroptosis in 4T1 breast cancer through CD36/FABP5 axis [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 4466.