Hepatocellular Carcinoma Xenograft Research Articles

A novel LAG3 neutralizing antibody improves cancer immunotherapy by dual inhibition of MHC-II and FGL1 ligand binding

LAG3 is an inhibitory immune checkpoint expressed on activated T and NK cells. Blocking the interaction of LAG3 with its ligands MHC-II and FGL1 renders T cells improved cytotoxicity to cancer cells. Current study generated a panel of LAG3 monoclonal antibodies (mAbs) through immunization of mice followed by phage display. Some of them bound to the D1-D2 domain of LAG3, which is known for the engagement of its ligands FGL1 and MHC-II. Three outperformers, M208, M226, and M234, showed stronger blocking activity than Relatlimab in the FGL1 binding. Furthermore, M234 showed dual inhibition of FGL1 (IC50 of 20.6nM) and MHC-II binding (IC50 of 6.2nM) to LAG3. In vitro functional tests showed that M234 significantly stimulated IFN-γ secretion from activated PBMC cells. In vivo studies in a mouse model of hepatocellular carcinoma xenografts demonstrated that combining M234 IgG with GPC3-targeted bispecific antibodies significantly improved efficacy. In addition, GPC3-targeted CAR-T cells secreting IL-21-M234 scFv fusion protein exhibited enhanced activity in inhibiting tumor growth and greatly increased the survival rate of mice. Taken together, M234 has potential in cancer immunotherapy and warrants further clinical trial.

SOAT1 regulates cholesterol metabolism to induce EMT in hepatocellular carcinoma

Cholesterol metabolism reprogramming is one of the significant characteristics of hepatocellular carcinoma (HCC). Cholesterol increases the risk of epithelial–mesenchymal transition (EMT) in cancer. Sterol O-acyltransferases 1 (SOAT1) maintains the cholesterol homeostasis. However, the exact mechanistic contribution of SOAT1 to EMT in HCC remains unclear. Here we demonstrated that SOAT1 positively related to poor prognosis of HCC, EMT markers and promoted cell migration and invasion in vitro, which was mediated by the increased cholesterol in plasmalemma and cholesterol esters accumulation. Furthermore, we reported that SOAT1 disrupted cholesterol metabolism homeostasis to accelerate tumorigenesis and development in HCC xenograft and NAFLD-HCC. Also, we detected that nootkatone, a sesquiterpene ketone, inhibited EMT by targeting SOAT1 in vitro and in vivo. Collectively, our finding indicated that SOAT1 promotes EMT and contributes to hepatocarcinogenesis by increasing cholesterol esterification, which is suppressed efficiently by nootkatone. This study demonstrated that SOAT1 is a potential biomarker and therapeutic target in NAFLD-HCC and SOAT1-targeting inhibitors are expected to be the potential new therapeutic treatment for HCC.

Discovery of 2,6-Naphthyridine Analogues as Selective FGFR4 Inhibitors for Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer and is responsible for 90% of cases. Approximately 30% of patients diagnosed with HCC are identified as displaying an aberrant expression of fibroblast growth factor 19 (FGF19)-fibroblast growth factor receptor 4 (FGFR4) as an oncogenic-driver pathway. Therefore, the control of the FGF19-FGFR4 signaling pathway with selective FGFR4 inhibitors can be a promising therapy for the treatment of HCC. We herein disclose the design and synthesis of novel FGFR4 inhibitors containing a 2,6-naphthyridine scaffold. Compound 11 displayed a nanomolar potency against Huh7 cell lines and high selectivity over FGFR1-3 that were comparable to that of fisogatinib (8) as a reference standard. Additionally, compound 11 demonstrated remarkable antitumor efficacy in the Huh7 and Hep3B HCC xenograft mouse model. Moreover, bioluminescence imaging experiments with the orthotopic mouse model support that compound 11 can be considered a promising candidate for treating HCC.

Ficolin 3 promotes ferroptosis in HCC by downregulating IR/SREBP axis-mediated MUFA synthesis

Background Targeting ferroptosis has been identified as a promising approach for the development of cancer therapies. Monounsaturated fatty acid (MUFA) is a type of lipid that plays a crucial role in inhibiting ferroptosis. Ficolin 3 (FCN3) is a component of the complement system, serving as a recognition molecule against pathogens in the lectin pathway. Recent studies have reported that FCN3 demonstrates inhibitory effects on the progression of certain tumors. However, whether FCN3 can modulate lipid metabolism and ferroptosis remains largely unknown.MethodsCell viability, BODIPY-C11 staining, and MDA assay were carried out to detect ferroptosis. Primary hepatocellular carcinoma (HCC) and xenograft models were utilized to investigate the effect of FCN3 on the development of HCC in vivo. A metabonomic analysis was conducted to assess alterations in intracellular and HCC intrahepatic lipid levels.Results Our study elucidates a substantial decrease in the expression of FCN3, a component of the complement system, leads to MUFA accumulation in human HCC specimens and thereby significantly promotes ferroptosis resistance. Overexpression of FCN3 efficiently sensitizes HCC cells to ferroptosis, resulting in the inhibition of the oncogenesis and progression of both primary HCC and subcutaneous HCC xenograft. Mechanistically, FCN3 directly binds to the insulin receptor β (IR-β) and its pro-form (pro-IR), inhibiting pro-IR cleavage and IR-β phosphorylation, ultimately resulting in IR-β inactivation. This inactivation of IR-β suppresses the expression of sterol regulatory element binding protein-1c (SREBP1c), which subsequently suppresses the transcription of genes related to de novo lipogenesis (DNL) and lipid desaturation, and consequently downregulates intracellular MUFA levels.Conclusions These findings uncover a novel regulatory mechanism by which FCN3 enhances the sensitivity of HCC cells to ferroptosis, indicating that targeting FCN3-induced ferroptosis is a promising strategy for HCC treatment.

Upregulation of LHPP by saRNA inhibited hepatocellular cancer cell proliferation and xenograft tumor growth.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer worldwide and no pharmacological treatment is available that can achieve complete remission of HCC. Phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) is a recently identified HCC tumor suppressor gene which plays an important role in the development of HCC and its inactivation and reactivation has been shown to result in respectively HCC tumorigenesis and suppression. Small activating RNAs (saRNAs) have been used to achieve targeted activation of therapeutic genes for the restoration of their encoded protein through the RNAa mechanism. Here we designed and validated saRNAs that could activate LHPP expression at both the mRNA and protein levels in HCC cells. Activation of LHPP by its saRNAs led to the suppression of HCC proliferation, migration and the inhibition of Akt phosphorylation. When combined with targeted anticancer drugs (e.g., regorafenib), LHPP saRNA exhibited synergistic effect in inhibiting in vitro HCC proliferation and in vivo antitumor growth in a xenograft HCC model. Findings from this study provides further evidence for a tumor suppressor role of LHPP and potential therapeutic value of restoring the expression of LHPP by saRNA for the treatment of HCC.

Berberine increases the killing effect of pirarubicin on HCC cells by inhibiting ATG4B-autophagy pathway

Pirarubicin (THP) is a new generation of cell cycle non-specific anthracycline-based anticancer drug. In the clinic, THP and THP combination therapies have been shown to be effective in hepatocellular carcinoma (HCC) patients with transcatheter arterial chemoembolisation (TACE) without serious side effects. However, drug resistance limits its therapeutic efficacy. Berberine (BBR), an isoquinoline alkaloid, has been shown to possess antitumour properties against various malignancies. However, the synergistic effect of BBR and THP in the treatment of HCC is unknown. In the present study, we demonstrated for the first time that BBR sensitized HCC cells to THP, including enhancing THP-induced growth inhibition and apoptosis of HCC cells. Moreover, we found that BBR sensitized THP by reducing the expression of autophagy-related 4B (ATG4B). Mechanistically, the inhibition of HIF1α-mediated ATG4B transcription by BBR ultimately led to attenuation of THP-induced cytoprotective autophagy, accompanied by enhanced growth inhibition and apoptosis in THP-treated HCC cells. Tumor-bearing experiments in nude mice showed that the combination treatment with BBR and THP significantly suppressed the growth of HCC xenografts. These results reveal that BBR is able to strengthen the killing effect of THP on HCC cells by repressing the ATG4B-autophagy pathway, which may provide novel insights into the improvement of chemotherapeutic efficacy of THP, and may be conducive to the further clinical application of THP in HCC treatment.

Macrophage hitchhiking for systematic suppression in postablative multifocal hepatocellular carcinoma.

Hepatocellular carcinoma (HCC), particularly the multifocal HCC, features aggressive invasion and dismal prognosis. Locoregional treatments were often refractory to eliminate tumor tissue, resulting in residual tumor cells persisting and subsequent progression. Owing to problematic delivery to the tumor tissue, systemic therapies, such as lenvatinib (LEN) therapy, show limited clinical benefit in preventing residual tumor progression. Therefore, more advanced strategies for postablative multifocal HCC are urgently needed. Motivated by the chemotaxis in tumor penetration of macrophages, we report a strategy named microinvasive ablation-guided macrophage hitchhiking (MAMH) for the targeted therapy toward HCC. In this study, the strategy leverages the natural inflammatory gradient induced by ablation to guide LEN-loaded macrophages toward tumor targeting, which increased by ~10-fold the delivery efficiency of LEN in postablative HCC in vivo. MAMH has demonstrated significant antitumor activity in various HCC models, including the hydrodynamic tail vein injection multifocal HCC mouse model and the orthotopic xenograft HCC rabbit model, systematically inhibiting residual tumor progression after ablation and prolonging the median survival of tumor-bearing mice. The potential antitumor mechanism was explored using techniques such as flow cytometry, enzyme-linked immunosorbent assay, and immunohistochemistry. We found that the strategy significantly suppressed tumor cell proliferation and neovascularization, and such enhanced delivery of LEN stimulated systemic immune responses and induced durable immune memory. The macrophage hitchhiking strategy demonstrates exceptional therapeutic efficacy and biosafety across various species, offering promising prospects for clinical translation in controlling residual tumor progression and improving outcomes following HCC ablation.

Novel STAT3 oligonucleotide compounds suppress tumor growth and overcome the acquired resistance to sorafenib in hepatocellular carcinoma.

Signal transducer and activator of transcription 3 (STAT3) plays an important role in the occurrence and progression of tumors, leading to resistance and poor prognosis. Activation of STAT3 signaling is frequently detected in hepatocellular carcinoma (HCC), but potent and less toxic STAT3 inhibitors have not been discovered. Here, based on antisense technology, we designed a series of stabilized modified antisense oligonucleotides targeting STAT3 mRNA (STAT3 ASOs). Treatment with STAT3 ASOs decreased the STAT3 mRNA and protein levels in HCC cells. STAT3 ASOs significantly inhibited the proliferation, survival, migration, and invasion of cancer cells by specifically perturbing STAT3 signaling. Treatment with STAT3 ASOs decreased the tumor burden in an HCC xenograft model. Moreover, aberrant STAT3 signaling activation is one of multiple signaling pathways involved in sorafenib resistance in HCC. STAT3 ASOs effectively sensitized resistant HCC cell lines to sorafenib in vitro and improved the inhibitory potency of sorafenib in a resistant HCC xenograft model. The developed STAT3 ASOs enrich the tools capable of targeting STAT3 and modulating STAT3 activity, serve as a promising strategy for treating HCC and other STAT3-addicted tumors, and alleviate the acquired resistance to sorafenib in HCC patients. A series of novel STAT3 antisense oligonucleotide were designed and showed potent anti-cancer efficacy in hepatocellular carcinoma in vitro and in vivo by targeting STAT3 signaling. Moreover, the selected STAT3 ASOs enhance sorafenib sensitivity in resistant cell model and xenograft model.

CircSNX6 promotes proliferation, metastasis, and angiogenesis in hepatocellular carcinoma via miR-383-5p/VEGFA signaling pathway

The role of circular RNA (circRNAs) in hepatocellular carcinoma (HCC) has been extensively studied. Previous research has highlighted the regulatory role of circSNX6 in HCC cells and tissues. However, the precise mechanism underlying HCC progression still requires comprehensive investigation. The study initially utilized quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to assess circSNX6 expression levels in HCC cell lines and tissues. Subsequently, the stability of circRNA was evaluated through Ribonuclease R and actinomycin D treatment assays. The impact of circSNX6 knockdown on proliferation, migration, invasion, and angiogenesis abilities was determined using various assays including colony formation, Transwell culture system, tube formation assay, and cell counting kit (CCK)-8 assays. Additionally, RNA immunoprecipitation chip and dual-luciferase reporter assays were employed to investigate the interactions between circSNX6 and miR-383-5p. Finally, an HCC xenograft tumor model in mice was established to assess the in vivo expression of circSNX6 and its functional role in HCC. Our findings revealed an elevated circSNX6 expression in HCC tissues, which was correlated with poor patient prognosis. Knockdown of circSNX6 suppressed HCC cell growth, invasion, metastasis, and angiogenesis. The downregulation of miR-383-5p, a target of circSNX6, significantly attenuated the tumor-suppressive effects induced by circSNX6 knockdown. Moreover, circSNX6 was found to modulate VEGFA expression by targeting miR-383-5p. The inhibition of HCC cell proliferation by miR-383-5p could be partially reversed by overexpressing VEGFA. Silencing circSNX6 also suppressed tumor formation and the metastasis of HCC cells in a mouse model. In summary, our findings suggest that circSNX6 promotes cell proliferation, metastasis, and angiogenesis in HCC by regulating the miR-383-5p/VEGFA pathway.

Abstract LB303: Suppression of stress granule formation is a novel vulnerability imposed by mutant p53

Abstract Mutation of the tumor suppressor p53 is the most common cancer specific event. Most of these mutations are missense mutations in the DNA-binding domain of p53, causing loss of wild-type p53 (wtp53) transcriptional activity. Missense mutant p53 (mutp53) also enhances cancer malignancy in a manner independent of wtp53. Hence, mutp53 is an ideal therapeutic target due to its uniqueness and high expression levels in cancers; however, directly targeting mutp53 protein itself has been challenging. To better understand the mechanism of oncogenic function of mutp53, we performed in silico TCGA database analysis and found that patients with missense mutp53-carrying hepatocellular carcinoma (HCC), but not other types of cancer, had unexpectedly better overall survival than patients with p53-null HCC. Since sorafenib (SOR) has historically been used as a single agent chemotherapy for HCC, unlike other cancer types, we hypothesized that SOR treatment may have yet unappreciated advantages for treating mutp53-expressing tumors. Indeed, knockdown of mutp53 rendered HCC cells and xenografts more resistant to SOR. Consistent with a report showing that SOR induces ER stress-mediated pro-survival stress granule (SG) formation, mutp53-expressing HCC cells, treated with SOR or other ER stress inducers, formed significantly fewer SGs with increased apoptosis, as compared with mutp53-knockdown cells. This result was partly due to mutp53’s ability to interact with an eIF2α kinase PERK and a key SG core protein, G3BP1. Moreover, the oligomerization domain of mutp53 was required for the interaction of mutp53 with G3BP1 and subsequent suppression of ER-stress-mediated SG formation and survival. These results suggest that inhibition of SG formation is a novel vulnerability imposed by mutp53, leading to sensitization of cancer cells to ER stress. Our results also emphasize the use of ER stress inducers to treat mutp53-expressing tumors and mutp53 as a biomarker of SOR sensitivity. Citation Format: Elizabeth Thoenen, Atul Ranjan, Alejandro Parrales, Shigeto Nishikawa, Dan Dixon, Sugako Oka, Tomoo Iwakuma. Suppression of stress granule formation is a novel vulnerability imposed by mutant p53 [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 LB303.

Abstract LB122: JNJ-87890387, a novel ENPP3 bispecific T-cell redirector (ENPP3xCD3) with tumor selectivity through targeting apical surface antigens

Abstract Clinical success of T-cell redirection therapy for solid tumors has been limited potentially due to a lack of target specificity against cancer, leading to on-target off-tumor toxicity at sub-efficacious doses. ENPP3 (ectonucleotide pyrophosphatase/phosphodiesterase family member 3) is a transmembrane protein that was identified with the help of an artificial intelligence machine learning algorithm. ENPP3 is characterized by apically restricted expression in most normal tissues and elevated, depolarized expression in several solid tumors, thus potentially providing tumor selectivity. ENPP3 expression exhibits a high prevalence in several solid tumors including renal cell carcinoma (RCC) - clear cell (93%) and papillary (78%), lung adenocarcinoma (50%), endometrioid uterine (53%) and ovarian (47%) cancers, colorectal carcinoma (51%), and hepatocellular carcinoma (HCC, 47%). JNJ-87890387 is a fully human bispecific antibody with high affinity and specific binding to ENPP3 on tumor cells and lower affinity binding to cluster of differentiation (CD)3 on T-cells. In vitro, JNJ-87890387 demonstrated potent (pM) T-cell activation and cytotoxicity against tumor cell lines with different levels of endogenous ENPP3 membrane expression, and absence of killing against cells lacking ENPP3 expression, confirming JNJ-87890387 specificity. In vivo, JNJ-87890387 demonstrated ENPP3-expression-dependent potent anti-tumor activity in multiple cell line-derived and patient tumor-derived RCC and HCC xenograft models with complete tumor regressions, and evidence of dose-dependent CD4+ and CD8+ T cell infiltration into the tumors. Preclinical safety assessment in the cynomolgus monkey with a tool molecule using the therapeutic ENPP3 binding arm but with a cynomolgus CD3 cross-reactive arm only identified toxicities consistent with the modality. In summary, these data support JNJ-87890387’s clinical development with an ongoing first-in-human Phase I study to evaluate the safety and preliminary anti-tumor activity in advanced-stage solid tumors with a high prevalence of ENPP3 expression. Citation Format: Smruthi Vijayaraghavan, Pankaj Seth, Annmarie Winkis, Kristen Chevalier, Alexa Marthaler, Katrin Sproesser, Vince Torti, Nirav Shah, Eilyn Lacy, Anna-Lena Frisk, Heather Deutsch, Gerald Chu, Michael Sharp, Jonathan Pabalan, Cholpon Tilegenova, Katherine Thorton, Josh Lauring, Bethany Mattson, Ken Tian, Jacqueline Kinyamu-Akunda, Laurie Lenox, Wan Cheung Cheung, Sheri Moores, Sylvie Laquerre. JNJ-87890387, a novel ENPP3 bispecific T-cell redirector (ENPP3xCD3) with tumor selectivity through targeting apical surface antigens [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 LB122.

Oxyberberine sensitizes liver cancer cells to sorafenib via inhibiting NOTCH1-USP7-c-Myc pathway.

Sorafenib is the first-line therapy for patients with advanced-stage HCC, but its clinical cure rate is unsatisfactory due to adverse reactions and drug resistance. Novel alternative strategies to overcome sorafenib resistance are urgently needed. Oxyberberine (OBB), a major metabolite of berberine in vivo, exhibits potential antitumor potency in various human malignancies, including liver cancer. However, it remains unknown whether and how OBB sensitizes liver cancer cells to sorafenib. Cell viability, trypan blue staining and flow cytometry assays were employed to determine the synergistic effect of OBB and sorafenib on killing HCC cells. PCR, western blot, co-immunoprecipitation and RNA interference assays were used to decipher the mechanism by which OBB sensitizes sorafenib. HCC xenograft models and clinical HCC samples were utilized to consolidate our findings. We found for the first time that OBB sensitized liver cancer cells to sorafenib, enhancing its inhibitory effect on cell growth and induction of apoptosis in vitro. Interestingly, we observed that OBB enhanced the sensitivity of HCC cells to sorafenib by reducing ubiquitin-specific peptidase 7 (USP7) expression, a well-known tumor-promoting gene. Mechanistically, OBB inhibited notch homolog 1-mediated USP7 transcription, leading to the downregulation of V-Myc avian myelocytomatosis viral oncogene homolog (c-Myc), which synergized with sorafenib to suppress liver cancer. Furthermore, animal results showed that cotreatment with OBB and sorafenib significantly inhibited the tumor growth of liver cancer xenografts in mice. These results indicate that OBB enhances the sensitivity of liver cancer cells to sorafenib through inhibiting notch homolog 1-USP7-c-Myc signaling pathway, which potentially provides a novel therapeutic strategy for liver cancer to improve the effectiveness of sorafenib.

Abstract 5004: Oncolytic Jurona-driven systemic and intratumoral immunotherapy combined with immune checkpoint blockade boost immune response and survival in hepatocellular carcinoma models

Abstract The members of the vesiculovirus genus have recently become attractive immunotherapeutic agents due to their natural selectivity for tumors and lack of toxicity in humans. Here, the research shows Intratumoral (IT) or Intraperitoneal (IP) injection of JURV was found to induce dynamic tumor regression in human hepatocellular carcinoma (HCC) xenografts and syngeneic models. Furthermore, IT injections of JURV were also shown to recruit and activate cytotoxic T lymphocytes and decrease tumor-associated macrophage infiltration, resulting in the delay of tumor growth in both local and distant murine HCC tumors established in a syngeneic model. Additionally, the concurrent use of JURV and anti-PD-1 antibodies was found to synergistically modulate the tumor microenvironment (TME) via increased tumor-infiltrating CD4+ T cells and depleted CD8+ PD-1+ and NK cells. Furthermore, using RIL-175, an aggressive metastatic HCC tumor mouse model, we show that intraperitoneal (IP) (systemic) administration of JURV and anti-PD1 significantly improved long-term survival (90%) compared to the (20%) JURV or anti-PD1 alone. Through proteogenomic analysis, the activation of different effectors of the immune system was observed to cooperate to alter the TME to a favorable anti-tumor immune state. These findings further support the development of JURV as an immunovirotherapy platform for HCC, with the potential to induce abscopal effects via the activation of several tumor suppressor genes and the mechanism regulating the T helper cell responses. Citation Format: Mulu Z. Tesfay, Khandoker U. Ferdous, Aleksandra Cios, Randal S. Shelton, Camila C. Simoes, Steven R. Post, Thomas J. Kelly, Martin J. Cannon, Alexei Basnakian, Bolni M. Nagalo. Oncolytic Jurona-driven systemic and intratumoral immunotherapy combined with immune checkpoint blockade boost immune response and survival in hepatocellular carcinoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5004.

Abstract 3599: Recombinant chimeric antibody-based gamma-delta TCR targeting GPC3 induces potent antitumor response against hepatocellular carcinoma

Abstract Background and Significance: Hepatocellular carcinoma (HCC), a major type of primary liver cancer, is the third leading cause of cancer-related death worldwide. While Glypican-3 (GPC3) is highly upregulated in HCC, it is minimally expressed in normal tissues. The research in our laboratory has been focused on the development of GPC3 as an immunotherapeutic target for liver cancer. Despite many clinical trials, there has not been any approved therapy targeting GPC3 largely due to the combination of antigen heterogeneity, poor tumor penetration, and the inability to induce a durable, potent immune response in the tumor microenvironment (TME). Here, we made a recombinant chimeric antibody-based T cell receptors (named AbTCR) targeting GPC3 and explored its anti-tumor efficacy in xenograft mouse models. Methods: The AbTCR constructs were built by fusing an antigen recognition domain (hYP7 scFv or HN3 VH) specific for GPC3 to the gamma-delta TCR constant region along with a secondary T cell signaling activation motif. The NFAT-tdTomato reporter cell line was established and used to measure the AbTCR-triggered T cell signaling upon stimulation with tumor cells. Anti-tumor efficacy of AbTCR-T cells was determined via luciferase-based cell killing assay as well as multiple HCC xenograft mouse models. Results: The hYP7 GPC3 AbTCR-T cell not only enhances antigen-binding capacity and T cell activity, but also induces infiltration and expansion of T cells in the TME, thereby causing robust liver tumor regression in mice. Conclusion: Our data show that new hYP7-directed GPC3 AbTCR is a promising strategy for treating liver cancer. This work provides a rationale for the use of hYP7 GPC3 AbTCR in clinical studies for treating HCC and other GPC3-positive solid tumors. Citation Format: Dan Li, Tianyuzhou Liang, Zhijian Duan, Hongbing Zhang, Cheng Liu, Mitchell Ho. Recombinant chimeric antibody-based gamma-delta TCR targeting GPC3 induces potent antitumor response against hepatocellular carcinoma [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 3599.

Abstract 3172: Significance of PELP1 signaling in liver cancer progression

Abstract Background: Hepatocellular carcinoma (HCC) is the fastest-rising cause of cancer-related mortality in the United States. Furthermore, advanced HCC has a very poor prognosis, with a median survival time of only about ten months. Effective new targeted therapies are urgently needed for the treatment of HCC. Proline, glutamic acid-, and leucine-rich protein 1 (PELP1) is a protooncogene. PELP1 signaling has been implicated in the development of several cancers, however, its role in the progression of HCC remains unclear. The goal of this project is to investigate the significance and therapeutic potential of targeting PELP1 in HCC. Methods: In this investigation, we have used six HCC cell lines (HUH7, HEP3B, SNU398, SNU475, SNU423, and SNU449). Using immunohistochemistry (IHC), the expression of PELP1 in HCC tissue microarrays was examined. PELP1shRNA lentiviral particles were used to create PELP1 knockdown (KD) cells. Using well-established in vitro tests for cell proliferation, colony formation, and apoptosis, the effects of PELP1 KD or PELP1 inhibitor (SMIP34) were investigated. RNA-seq, RT-qPCR, Western blotting, IHC, reporter gene assays, and signaling pathway analysis were used in mechanistic investigations. Preclinical evaluations were conducted using mice xenograft models. Results: TNM plot analysis showed that HCC samples had increased PELP1 expression relative to normal tissue. Immunohistochemistry analysis of tissue microarray confirmed that HCC specimens overexpressed PELP1 in comparison to normal specimens. PELP1 knockdown with shRNA dramatically decreased HCC cell invasion, clonogenicity, and proliferation. Similarly, PELP1 inhibitor SMIP34 treatment significantly reduced cell viability, invasiveness, colony forming capacity, and induced apoptosis of HCC cells. RNA-seq analyses using PELP1 KD cells confirmed that PELP1 modulates the ribosome and the eukaryotic translation elongation pathways. Mechanistic studies confirmed that SMIP34 treatment contributed to the disruption of the Rix complex, which is crucial for ribosomal biogenesis. Puromycin labelling studies confirmed that PELP1 KD or SMIP34 treatment contributes to the reduction of ribosomal biogenesis and new protein synthesis. Furthermore, PELP1 KD or treatment with SMIP34 dramatically slowed the progression of HCC tumors in xenograft models. IHC analysis revealed reduced levels of the proliferation marker Ki67 in PELP1 KD/SMIP34 treated HCC xenograft tumors compared to controls. Conclusions: Overall, the results of our study point to PELP1 as a potential therapeutic target for HCC intervention. Citation Format: Uday Pratap, Khaled Mohamed Nassar, Xue Yang, Adriana Baker, Rahul Gopalam, William C. Arnold, Timilehin Adeniran, Marian Helena Hernandez Fernandez, Gangadhara R. Sareddy, Suryavathi Viswanadhapalli, Luzhe Sun, Ratna K. Vadlamudi. Significance of PELP1 signaling in liver cancer progression [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 3172.

Prognostic PET [11C]-acetate uptake is associated with hypoxia gene expression in patients with late-stage hepatocellular carcinoma – a bench to bed study

BackgroundPositron Emission Tomography (PET) with combined [18F]-FDG and [11C]-acetate (dual-tracer) is used for the management of hepatocellular carcinoma (HCC) patients, although its prognostic value and underlying molecular mechanism remain poorly understood. We hypothesized that radiotracer uptake might be associated with tumor hypoxia and validated our findings in public and local human HCC cohorts.MethodsTwelve orthotopic HCC xenografts were established using MHCC97L cells in female nude mice, with 5 having undergone hepatic artery ligation (HAL) to create tumor hypoxia in vivo. Tumors in both Control and HAL-treated xenografts were imaged with [11C]-acetate and [18F]-FDG PET-MR and RNA sequencing was performed on the resected tumors. Semiquantitative analysis of PET findings was then performed, and the findings were then validated on the Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) cohort and patients from our institution.ResultsHAL-treated mice showed lower [11C]-acetate (HAL-treated vs. Control, tumor-to-liver SUV ratio (SUVTLR): 2.14[2.05–2.21] vs 3.11[2.75–5.43], p = 0.02) but not [18F]-FDG (HAL-treated vs. Control, SUVTLR: 3.73[3.12–4.35] vs 3.86[3.7–5.29], p = 0.83) tumor uptakes. Gene expression analysis showed the PET phenotype is associated with upregulation of hallmark hypoxia signature. The prognostic value of the hypoxia gene signature was tested on the TCGA-LIHC cohort with upregulation of hypoxia gene signature associated with poorer overall survival (OS) in late-stage (stage III and IV) HCC patients (n = 66, OS 2.05 vs 1.67 years, p = 0.046). Using a local cohort of late-stage HCC patients who underwent dual-tracer PET-CT, tumors without [11C]-acetate uptake are associated with poorer prognosis (n = 51, OS 0.25 versus 1.21 years, p < 0.0001) and multivariable analyses showed [11C]-acetate tumor uptake as an independent predictor of OS (HR 0.17 95%C 0.06–0.42, p < 0.0001).Conclusions[11C]-acetate uptake is associated with alteration of tumor hypoxia gene expression and poorer prognosis in patients with advanced HCC.

Abstract 2417: Detection and quantification of site-specific DNA methylation from liquid biopsies as a pharmacodynamic biomarker of OTX-2002, a novel MYC-targeting epigenomic mRNA therapeutic

Abstract Omega Therapeutics has developed a novel platform of programmable epigenomic mRNA medicines capable of modifying chromatin state to specifically tune gene expression at the pre-transcriptional level. Epigenomic controllers (ECs) unlock targets that have been historically considered “undruggable,” with one of the most elusive being the MYC oncogene. A direct MYC-targeting anti-cancer agent has previously remained intangible, largely due to the absence of a drug binding pocket and tight autoregulation. A clinical trial is underway (MYCHELANGELO, NCT05497453) to investigate pre-transcriptional inhibition of MYC with OTX-2002 in patients with hepatocellular carcinoma (HCC). OTX-2002, a first-in-class mRNA therapeutic delivered via lipid nanoparticles (LNP), encodes two proteins that durably modify chromatin, in part, through CpG DNA methylation at the MYC locus. Using both liquid and solid biopsy sampling from in vivo studies, we investigated whether target engagement for OTX-2002 could be assessed by MYC methylation. Detection of DNA methylation has long held promise as an oncology biomarker given its functional roles in various cancer types and the potential signal afforded by methylated CpGs. Indeed, DNA methylation has been shown to serve as a robust analyte in liquid biopsy-derived multi-cancer early detection (MCED) and minimal residual disease (MRD) tests that have recently been utilized in the clinic. Many of these platforms are founded on complex models that leverage methylation signals across &amp;gt;1 million CpGs that can span tens of megabases of genomic space. We faced a distinct challenge when compared to these MCED/MRD tests in developing a pharmacodynamic methylation assay for OTX-2002 as the target region consists of just a few kilobases. With a tissue specific LNP delivery system, ultra-high sensitivity was required to identify rare ctDNA events from the larger cfDNA population. To this end, we designed a minimal hybridization/capture panel that targeted ~50 kb of genomic space, effectively allowing for ultra-deep methylation sequencing of MYC. When this technique was paired with enzymatic (EM) conversion for methylation detection and supported by an analysis pipeline focused on epiallele identification, this assay was able to detect methylation down to the theoretical limit in a dilution series of control genomic DNA, 1 in 104 copies of MYC. This degree of sensitivity translated to successful preclinical detection of on-target methylation by OTX-2002 from DNA extracted from plasma samples collected from mice-bearing human HCC xenografts. Overall, we present a non-invasive and exquisitely sensitive method of assessing target engagement and site-specific pharmacodynamic activity of a novel epigenomic medicine that can be directly translated to the clinical setting. Citation Format: Justin Chen, William Senapedis, Stephen Siecinski, Elmer Figueroa, Adam Katz, Samuel Mildrum, Yaoyu E. Wang, Houda Belaghzal, Kayleigh Gallagher, Graeme Hodgson, Charles W. O'Donnell, Thomas G. McCauley. Detection and quantification of site-specific DNA methylation from liquid biopsies as a pharmacodynamic biomarker of OTX-2002, a novel MYC-targeting epigenomic mRNA therapeutic [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 2417.

Abstract 4553: DXC004A, a novel EGFR antibody-tubulysin analog conjugate demonstrated potential to broaden therapeutic opportunities for non-small cell lung cancer

Abstract EGFR is a member of the epidermal growth factor receptor (HER) family, and the over-expression of EGFR plays an important role in the growth and progression of various tumors, including non-small cell lung cancer. Monoclonal EGFR antibodies, such as cetuximab and panitumumab, have proven efficacy in various types of cancer. However, treatment with the anti-EGFR agents can be associated with toxicities of the skin, nails, hair, and eyes. Nimotuzumab (Nimo) is the first humanized monoclonal antibody targeting EGFR and has been granted approval for use in squamous cell carcinoma of head and neck (SCCHN), glioma and nasopharyngeal cancer in different countries. In contrast to cetuximab, Nimotuzumab is distinguished by achieving higher or similar complete remission rate (CRR) or overall remission rate (ORR) of the primary tumor in clinical applications but much less toxicity, resulting in a better safety profile, which has been attributed to its about 10-fold lower affinity. Paclitaxel combined with cisplatin is the first-line chemotherapy regimen for non-small cell lung cancer. A phase 2 clinical trial has confirmed that Nimotuzumab combined with concurrent chemoradiation therapy (radiation concurrent with docetaxel and cisplatin, CCRT) was well tolerated for locally advanced squamous cell lung cancer. However, the combination strategy of Nimo-CCRT has only demonstrated similar OS and PFS to those in the CCRT group. DXC004A is an ADC drug targeting EGFR, in which a Nimotuzumab derivative and a Tubulysin B analog is conjugated by a functional linker. DXC004A is in phase I clinical trial and has demonstrated clinical activity in advanced non-small cell lung cancers and good tolerability. In vitro and in vivo results had confirmed that DXC004A monotherapy had better anti-tumor activity than the combination of Nimotuzumab and Tubulysin analog in HCC827 cell lines with high EGFR expression. Meanwhile, a single injection of low-dose DXC004 (2.5 mg/kg) in HCC827 xenograft model showed very good durable anti-tumor effect. Moreover, the combination of DXC004A with Cisplatin exhibited significantly better activities than that of Nimo-CCRT combination, DXC004A or cisplatin alone, in vitro and in vivo, which might solve the predicament of poor efficacy of Nimo-CCRT combination therapy. This synergy results suggested that DXC004A plus cisplatin would possibly be a new adjuvant therapy for non-small cell lung cancer in further clinical studies. Citation Format: Xiaobo Cai, Min Cao, Huihui Guo, Qingliang Yang, Yongxiang Chen, Xiangfei Kong, Yong Du, Zhicang Ye, Zhixiang Guo, Lingli Zhang, Lu Bai, Junxiang Jia, Yunxia Zheng, Wei Zheng, Jun Zheng, Wenjun Li, Yuanyuan Huang, Zhongliang Fan, Binbin Chen, Meng Dai, Robert Y. Zhao. DXC004A, a novel EGFR antibody-tubulysin analog conjugate demonstrated potential to broaden therapeutic opportunities for non-small cell 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 4553.

Abstract 5246: Therapeutic efficacy of glypican-3 peptide-linked chimeric antigen receptor-macrophages in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the third most lethal cancer and sixth most diagnosed cancer worldwide. Recently, chimeric antigen receptor-T cell (CAR-T) therapy has been demonstrated to be a promising immunotherapy for the treatment of hematologic malignancies such as leukemia. However, its application in solid tumors has proven to be challenging. To overcome this limitation, chimeric antigen receptor-macrophages (CAR-Ms) have emerged as a new immunotherapy against solid cancers, capitalizing on the unique attributes of macrophages in penetrating the tumor microenvironment, promoting antigen presentation, and priming T cells. Traditionally, CAR-M therapy involves the transfer of an edited CAR gene into macrophages via viral infection. However, this method is expensive, involves tedious procedures, and has long-term safety concerns. In the present study, we employed peptides to mimic the genetic approach for producing CAR-Ms to reduce the above uncertainties. To achieve this, we developed a novel bioconjugation method called the phthalaldehydeamine capture (PAC) reaction to produce peptidic CAR-Ms (pCAR-Ms) targeting Glypican-3 (GPC3) in HCC cells. This was achieved by linking GPC3 specific peptides to Raw264.7 murine macrophages. Using an in vitro co-culture system, we demonstrated the specificity and efficacy of pCAR-Ms against GPC3, as evidenced by a significant increase in the number of phagocytosed cells with high GPC3 expression when compared to their low-expression counterparts, by flow cytometry and confocal microscopy. The specificity of pCAR-Ms was further proven in GPC3-knockdown HCC cells, with a significant decrease in the phagocytic rate. Using an HCC xenograft model derived from MHCC97L cells, we confirmed the specific homing of pCAR-Ms to tumor sites via intravenous injection of lipopolysaccharide (LPS)-differentiated pCAR-Ms. Strikingly, pCAR-Ms suppressed HCC tumor growth when compared with untagged macrophages and PBS control. In conclusion, we provided evidence of the potency and specificity of non-viral-based pCAR-Ms in targeting HCC with simple and short production time and increased flexibility. Citation Format: Wing Hei Leung, Oi Ning Leung, Tsun Ting Wong, Kin Wah Lee. Therapeutic efficacy of glypican-3 peptide-linked chimeric antigen receptor-macrophages in hepatocellular carcinoma [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 5246.

Upregulation of CYR61 by TGF-β and YAP signaling exerts a counter-suppression of hepatocellular carcinoma

TGF-β and Hippo signaling are two critical pathways engaged in cancer progression by regulating both oncogenes and tumor suppressors, yet how the two pathways coordinately exert their functions in the development of hepatocellular carcinoma (HCC) remains elusive. In this study, we firstly conducted an integrated analysis of public liver cancer databases and our experimental TGF-β target genes, identifying CYR61 as a pivotal candidate gene relating to HCC development. The expression of CYR61 is downregulated in clinical HCC tissues and cell lines than that in the normal counterparts. Evidence revealed that CYR61 is a direct target gene of TGF-β in liver cancer cells. In addition, TGF-β-stimulated Smad2/3 and the Hippo pathway downstream effectors YAP and TEAD4 can form a protein complex on the promoter of CYR61, thereby activating the promoter activity and stimulating CYR61 gene transcription in a collaborative manner. Functionally, depletion of CYR61 enhanced TGF-β- or YAP-mediated growth and migration of liver cancer cells. Consistently, ectopic expression of CYR61 was capable of impeding TGF-β- or YAP-induced malignant transformation of HCC cells in vitro, and attenuating HCC xenograft growth in nude mice. Finally, transcriptomic analysis indicates that CYR61 can elicit an antitumor program in liver cancer cells. Together, these results add new evidence for the crosstalk between TGF-β and Hippo signaling and unveil an important tumor suppressor function of CYR61 in liver cancer.