HCC Cells Research Articles

Abstract LB005: Identification of novel targets for MYC-driven hepatocellular carcinoma via transomics

Abstract Hepatocellular carcinoma (HCC) is an aggressive tumor, and treatment options for patients with unresectable HCC are limited to targeted therapies and immunotherapy. The activation and dysregulation of MYC have been implicated in multiple cancers, including HCC, where approximately 30% of human HCC samples show MYC gene amplification. However, it is challenging to target MYC directly. Therefore, we applied a novel approach to circumvent targeting MYC directly by leveraging Pepper’s proprietary transomic analysis platform, COMPASS, to identify novel targets that are predicted to mimic the effect of turning MYC off. Specifically, COMPASS unlocks functional drivers of disease to identify novel drug targets. To study the role of MYC in tumor biology, we utilize an HCC cell line with tunable MYC expression. We generate genomic, transcriptomic, proteomic, and phosphoproteomic data from samples with MYC-on and MYC-off and compare via COMPASS to identify novel targets that mimic turning MYC off. The MYC-conditional HCC cell line (EC4) allows the regulation of MYC expression via the tetracycline regulatory (Tet-Off) system. Four omic datasets were collected from each sample: genomics (next-generation sequencing, NGS), transcriptomics (NGS), proteomics (mass spectrometry), and phosphoproteomics (mass spectrometry). We employed the COMPASS target prioritization algorithm to identify and rank novel targets that mimic turning MYC off. Targets were then filtered to select those with an available pharmacological tool compound (PTC). The PTCs were used to evaluate the targets in the Huh-7 xenograft model of HCC. All targets were kinases or related to kinase activity and pathways. All PTCs inhibited tumor growth from 18.1-78.3%. One multi-tyrosine kinase inhibitor resulted in maximum tumor growth inhibition, resulting in stasis of tumor growth and increased survival compared with control vehicle-treated mice. We have previously shown how the COMPASS transomics analysis approach can identify novel drug targets for drug resistant EGFRm NSCLC. The data presented here on MYC-driven HCC further validates this approach for novel target identification. Ongoing work will investigate novel targets using gene silencing, as PTCs were unavailable for many of these novel high-ranked targets identified in this study. Further validation studies are ongoing against other difficult-to-treat MYC-driven cancers to identify novel clinical targets and drug candidates to circumvent the MYC pathway. Citation Format: Simon P. Fricker, Christopher J. Nicholson, Samuel J. Roth, Arudhir Singh, Caitlin Brown, Jon Hu, Petronela Buiga, Vishnu P. Kanakaveti, Anja Deutzmann, Dean Felsher, Samantha D. Strasser. Identification of novel targets for MYC-driven hepatocellular carcinoma via transomics [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 LB005.

Cyclovirobuxine D inhibits hepatocellular carcinoma growth by inducing ferroptosis of hepatocellular carcinoma cells

ObjectiveHepatocellular carcinoma (HCC) is one cancer with high death rates. Nowadays, there are no effective drugs to treat it. Cyclovirobuxine D (CVB-D) is the primary ingredient of the traditional Chinese medicine (TCM) Buxus microphylla. Here, we try to explore the impacts of CVB-D on human HCC cells and explain the potential mechanisms.MethodsHepG2 and Huh-7 cells were used for our experiments. The cell viability and half inhibitory concentration (IC50) were detected by MTT assays. The apoptosis ratio was examined by Annexin V-FITC/7AAD staining and flow cytometry (FCM). The Fe2+ content was examined by ferrous ion content assays. The malondialdehyde (MDA) content was evaluated by lipid peroxidation MDA assays. The reactive oxygen species (ROS) level was examined by the DCFH-DA probe. The expression of apoptotic markers (Bax and Bcl-2) and ferroptosis-related proteins (GPX4 and FSP1) was detected by western blotting. The in vivo curative effect of CVB was explored using xenograft models established in C-NKG mice.ResultsThe cell viability could be inhibited by CVB-D in HepG2 and Huh-7 cells. The IC50 value of CVB-D on HepG2 and Huh-7 cells are 91.19 and 96.29 µM at 48 h, and 65.60 and 72.80 µM at 72 h. FCM showed that the apoptosis rate was increased by CVB-D in HepG2 and Huh-7 cells. Next, ferrous ion content assays showed that the level of Fe2+ was increased by CVB-D in HepG2 and Huh-7 cells. Then, we found the level of MDA and ROS was increased by CVB-D. And the Fe2+ promotion by CVB-D could be reversed by Fer-1. Additionally, western blotting assays showed that the expression of GPX4 and FSP1 was inhibited by CVB-D in HepG2 and Huh-7 cells. Moreover, in vivo, CVB-D displayed excellent anticancer effects in HCC tumor-bearing C-NKG mice.ConclusionCVB-D suppresses the growth in HCC cells through ferroptosis.

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.

Exploring the enhanced stability and therapeutic efficacy of Sorafenib-Cyclodextrin inclusion complex

Sorafenib (Soraf) is a second-generation tyrosine kinase inhibitor repurposed against different cancers by working on various molecular pathways, playing a vital role in cancer cell proliferation. Sorafenib is a BCS class II drug, and its low solubility further hinders its bioavailability and results in reduced therapeutic efficacy. Therefore, in this study, we aim to explore the potential of β-cyclodextrins (β-CD) derivatives to form an inclusion complex with sorafenib to enhance sorafenib's solubility, stability, and therapeutic efficacy. Our findings suggest that HP-β-CD portrayed a higher affinity towards sorafenib than SBE-β-CD and significantly improved the solubility of sorafenib. Additionally, spectroscopic analysis and solid-state studies provided valuable information regarding the intermolecular interaction between sorafenib and HP-β-CD.Furthermore, the stability of sorafenib in biological fluids and under physiological conditions was maintained under the influence of the inclusion complex, whereas plain sorafenib degraded quickly. In addition, β-CD complexation significantly improved sorafenib's intestinal permeability, as shown in physiologically relevant EpiIntestinal® models. Lastly, the sorafenib inclusion complex demonstrated increased cytotoxicity in NSCLC and HCC cell lines. This study shows the potential of HP-β-CD to improve the solubility and therapeutic efficacy of sorafenib, thus extending its clinical application.

The β2-adrenergic receptor associates with CXCR4 multimers in human cancer cells

While the existence and functional role of class C G-protein-coupled receptors (GPCR) dimers is well established, there is still a lack of consensus regarding class A and B GPCR multimerization. This lack of consensus is largely due to the inherent challenges of demonstrating the presence of multimeric receptor complexes in a physiologically relevant cellular context. The C-X-C motif chemokine receptor 4 (CXCR4) is a class A GPCR that is a promising target of anticancer therapy. Here, we investigated the potential of CXCR4 to form multimeric complexes with other GPCRs and characterized the relative size of the complexes in a live-cell environment. Using a bimolecular fluorescence complementation (BiFC) assay, we identified the β2 adrenergic receptor (β2AR) as an interaction partner. To investigate the molecular scale details of CXCR4-β2AR interactions, we used a time-resolved fluorescence spectroscopy method called pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). PIE-FCCS can resolve membrane protein density, diffusion, and multimerization state in live cells at physiological expression levels. We probed CXCR4 and β2AR homo- and heteromultimerization in model cell lines and found that CXCR4 assembles into multimeric complexes larger than dimers in MDA-MB-231 human breast cancer cells and in HCC4006 human lung cancer cells. We also found that β2AR associates with CXCR4 multimers in MDA-MB-231 and HCC4006 cells to a higher degree than in COS-7 and CHO cells and in a ligand-dependent manner. These results suggest that CXCR4-β2AR heteromers are present in human cancer cells and that GPCR multimerization is significantly affected by the plasma membrane environment.

A Novel Inhibitor of Poly(ADP-Ribose) Polymerase-1 Inhibits Proliferation of a BRCA-Deficient Breast Cancer Cell Line via the DNA Damage-Activated cGAS-STING Pathway.

Loss-of-function mutations in the Breast Cancer Susceptibility Gene (BRCA1 and BRCA2) are often detected in patients with breast cancer. Poly(ADP-ribose) polymerase-1 (PARP1) plays a key role in the repair of DNA strand breaks, and PARP inhibitors have been shown to induce highly selective killing of BRCA1/2-deficient tumor cells, a mechanism termed synthetic lethality. In our previous study, a novel PARP1 inhibitor─(E)-2-(2,3-dibromo-4,5-dimethoxybenzylidene)-N-(4-fluorophenyl) hydrazine-1-carbothioamide (4F-DDC)─was synthesized, which significantly inhibited PARP1 activity with an IC50 value of 82 ± 9 nM. The current study aimed to explore the mechanism(s) underlying the antitumor activity of 4F-DDC under in vivo and in vitro conditions. 4F-DDC was found to selectively inhibit the proliferation of BRCA mutant cells, with highly potent effects on HCC-1937 (BRCA1-/-) cells. Furthermore, 4F-DDC was found to induce apoptosis and G2/M cell cycle arrest in HCC-1937 cells. Interestingly, immunofluorescence and Western blot results showed that 4F-DDC induced DNA double strand breaks and further activated the cGAS-STING pathway in HCC-1937 cells. In vivo analysis results revealed that 4F-DDC inhibited the growth of HCC-1937-derived tumor xenografts, possibly via the induction of DNA damage and activation of the cGAS-STING pathway. In summary, the current study provides a new perspective on the antitumor mechanism of PARP inhibitors and showcases the therapeutic potential of 4F-DDC in the treatment of breast cancer.

Targeting EGFR degradation by autophagosome degraders

Several generations of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors have been developed for the treatment of non-small cell lung cancer (NSCLC) in clinic. However, emerging drug resistance mediated by new EGFR mutations or activations by pass, leads to malignant progression of NSCLC. Proteolysis targeting chimeras (PROTACs) have been utilized to overcome the drug resistance acquired by mutant EGFR, newly potent and selective degraders are still need to be developed for clinical applications. Herein, we developed autophagosome-tethering compounds (ATTECs) in which EGFR can be anchored to microtubule-associated protein-1 light chain-3B (LC3B) on the autophagosome with the assistance of the LC3 ligand GW5074. A series of EGFR-ATTECs have been designed and synthesized. Biological evaluations showed that these compounds could degrade EGFR and exhibited moderate inhibitory effects on certain NSCLC cell lines. The ATTEC 12c potently induced the degradation of EGFR with a DC50 value of 0.98 μM and a Dmax value of 81% in HCC827 cells. Mechanistic exploration revealed that the lysosomal pathway was mainly involved in this degradation. Compound 12c also exhibited promising inhibitory activity, as well as degradation efficiency in vivo. Our study highlights that EGFR-ATTECs could be developed as a new expandable EGFR degradation tool and also reveals a novel potential therapeutic strategy to prevent drug resistance acquired EGFR mutations.

SIRT3 and SIRT4 double-genes remodeled the mitochondrial network to induce hepatocellular carcinoma cell line differentiation and suppress malignant phenotypes

Tumor cells with damaged mitochondria undergo metabolic reprogramming, but gene therapy targeting mitochondria has not been comprehensively reported. In this study, plasmids targeting the normal hepatocyte cell line (L-O2) and hepatocellular carcinoma cell line were generated using three genes SIRT3, SIRT4, and SIRT5. These deacetylases play a variety of regulatory roles in cancer and are related to mitochondrial function. Compared with L-O2, SIRT3 and SIRT4 significantly ameliorated mitochondrial damage in HCCLM3, Hep3B and HepG2 cell lines and regulated mitochondrial biogenesis and mitophagy, respectively. We constructed double-gene plasmid for co-express SIRT3 and SIRT4 using the internal ribosome entry site (IRES). The results indicated that the double-gene plasmid effectively expressed SIRT3 and SIRT4, significantly improved mitochondrial quality and function, and reduced mtDNA level and oxidative stress in HCC cells. MitoTracker analysis revealed that the mitochondrial network was restored. The proliferation, migration capabilities of HCC cells were reduced, whereas their differentiation abilities were enhanced. This study demonstrated that the use of IRES-linked SIRT3 and SIRT4 double-gene vectors induced the differentiation of HCC cells and inhibited their development by ameliorating mitochondrial dysfunction. This intervention helped reverse metabolic reprogramming, and may provide a groundbreaking new framework for HCC treatment.

Regulatory mechanism of the Glabrene against non-small cell lung cancer by suppressing FGFR3.

Non-small cell lung cancer (NSCLC) is a highly malignant tumor with limited effective treatment options. This study aimed to investigate the regulatory mechanism of Glabrene on NSCLC through its interaction with FGFR3. HCC827 cells were implanted into nude mice and treated with Glabrene. Tumor volume was monitored at 0, 3, 6, and 9 days after medical treatment. Tissue analysis included Hematoxylin and Eosin (HE) and Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP Nick End Labeling (TUNEL) staining, as well as immunohistochemistry for Ki67, ERK1/2, and p-ERK1/2 expression. Cell viability was determined with the CCK8 method. We utilized immunofluorescence techniques to observe apoptosis, as well as the levels of E-cadherin and Vimentin expression. Cellular proliferation was determined via plate cloning assay and cellular mobility was determined via scratch assay. Cellular invasion ability was assessed via a transwell assay. mRNA and protein levels of FGFR3, MMP1, MMP9, vimentin, E-cadherin, ERK1/2, and p-ERK1/2 were detected via qPCR and Western blot. IGF-1, VEGF, and Estradiol (E2) levels were measured through Enzyme linked immunosorbent assay (ELISA). This study verified that Glabrene was capable of suppressing tumor growth in NSCLC mice, reversing tumor tissue's pathological morphology, attenuating the capacities of cancerous cells' proliferation, migration, and invasion, and leading to apoptosis. Besides, Glabrene could reduce the FGFR3 expression in HCC827 cells. Over-expression of FGFR3 promotes the proliferation of HCC827 cells, increase both contents of IGF-1, VEGF, and E2, and expressions of MMP1, MMP9, vimentin, and p-ERK1/2, while Glabrene inhibited FGFR3. Glabrene, and inhibition of FGFR3 expression were capable of decreasing FGFR3, MMP1, MMP9, vimentin, and p-ERK1/2 expression, as well as contents of IGF-1, VEGF, and E2 in model mice and HCC827 cells, and promoting the expression of E-cadherin. Glabrene has the potential as a therapeutic agent for NSCLC by reducing cancer invasion and migration through the inhibition of ERK1/2 phosphorylation and suppression of epithelial-mesenchymal transition (EMT).

Abstract 5431: Identification of serine/threonine kinase 39 (STK39) as a driver for multidrug resistance in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is a common type of cancer globally and is associated with a poor prognosis due to its high recurrence rate and resistance to treatment. Solid evidence showed the existence of cancer stem cells (CSCs) that are crucial for therapeutic resistance. However, there are only a few kinase inhibitors that have been found to specifically target liver T-ICs for the HCC treatment. To understand the regulatory mechanism of liver CSCs, we employed RNA-Seq analysis to compare the gene expression profiles, with a particular focus on protein kinases, between (1) sorted CD133+ liver CSCs and CD133- differentiated cells extracted from two HCC cell lines (Huh7 and PLC8024), as well as cultured hepatospheres and adherent differentiated cells of HCC cell line PLC/PRF/5. Our analysis identified Serine/Threonine kinase 39 (STK39) as a commonly up-regulated protein kinase in both CD133+ liver CSCs and CSC-enriched hepatospheres. Based on our analysis of the TCGA data, a positive correlation has consistently been observed between STK39 mRNA expression and the expression of liver CSC markers, including CD24, CD133, and CD47. Utilizing publicly available transcriptome datasets from the NCBI Gene Expression Omnibus (GEO), including GSE14520 and GSE25097, we found that overexpression of STK39 is detected in HCC tumor tissues (p<0.001) and is significantly associated with poorer overall survival and disease-free survival. Through the use of lentiviral based overexpression and knockdown approaches, we have demonstrated the role of STK39 in regulating liver CSC properties, including self-renewal, tumorigenicity, cell invasiveness, and the expression of liver CSC markers. STK39 expression was found to be significantly elevated in in HCC cells that were resistant to both sorafenib and lenvatinib, and its inhibition resulted in increased sensitivity of HCC cells to these treatments. To identify the downstream target of STK39 for regulation of cancer stemness, tandem affinity purification coupled with mass spectrometry was employed. The analysis led to the identification of PARP1 as a novel protein binding partner of STK39. Upon suppression of STK39 in HCC cells, it was observed that the phosphorylation of PARP1 at Threonine 368 (T368) was significantly inhibited, indicating its role as a downstream effector of STK39-mediated liver CSC functions. Interestingly, STK39-mediated PARP1 phosphorylation led to development of resistance to PARP1 inhibitor, rucaparib. Additionally, STK39 plays a critical role in regulating PARP1-mediated chromatin decondensation, which enables it to open chromatin structure at the promoter regions of SOX2/OCT4. In summary, treatment of molecular targeted drugs including rucaparib and lenvatinib with STK39 inhibitor may be a potential therapeutic strategy for treatment of this deadly disease. Citation Format: Carmen Oi Ning Leung, Terence Kin-Wah Lee. Identification of serine/threonine kinase 39 (STK39) as a driver for multidrug resistance 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 5431.

Abstract 1117: To investigate the effect of fluoxetine combined with radiation therapy on hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is a highly prevalent and lethal primary liver tumor. It commonly occurs in individuals with chronic liver diseases, such as hepatitis B or C related cirrhosis. Despite conventional treatments, such as surgery and chemotherapy, the prognosis for HCC remains poor. The optimal treatment for patients with HCC is contingent on various factors, including the size and location of the tumor, the functional capacity of their liver, and their overall health status. Radiation therapy (RT) is a prevalent cancer treatment modality that targets rapidly dividing tumor cells by utilizing high-energy radiation. It is frequently administered alongside other treatments, such as surgery and chemotherapy, to improve the likelihood of successful cancer management. The timing of RT may vary, as it can be delivered before, during, or after other therapies depending on the patient's specific clinical situation. It's important to note that there are limits to the amount of radiation a patient's body can safely receive over their lifetime. For this reason, RT is often combined with other cancer treatments to enhance its effectiveness in sensitizing HCC cells. Fluoxetine is an anti-depressant drug that has also been shown to inhibit the growth of HCC. However, the potential role of fluoxetine in combination with RT for the treatment of HCC remains uncertain. In this study, we aim to identify whether fluoxetine may sensitize HCC to RT and evaluate its underlying mechanism. In this study, we employed the MTT assay and colon information to assess the cytotoxic effects of fluoxetine and radiotherapy (RT) at varying doses on HCC. Our findings, obtained through flow cytometry, revealed that the combination treatment can elicit extrinsic/intrinsic apoptosis in HCC cells. Specifically, this combination led to the loss of mitochondrial membrane potential, activation of cleaved-caspase-3, -8, -9, Fas, and FasL. Additionally, flow cytometry analysis showed an increase in the activation of PARP-1 and TUNEL, signifying DNA damage induction. Furthermore, we observed that fluoxetine not only enhances this DNA damage but also inhibits DNA repair, as shown through flow cytometry by the suppression of p-ATM and p-CHK2. Our western blotting assay confirmed that fluoxetine effectively inhibits HCC progression by activating LC3B, indicating the induction of autophagy in HCC cells. Moreover, the combined treatment, as demonstrated by transwell and wound healing assays, significantly reduced the migration and invasion capabilities of HCC cells. To conclude, the induction of apoptosis, DNA damage, and autophagy emerges as a crucial mechanism contributing to the heightened sensitivity of HCC cells to RT when combined with Fluoxetine. These results suggest that the combination of Fluoxetine with RT presents a promising novel approach for the treatment of HCC. Citation Format: Bo-Xun Chen, Tsai Lan Liao, Rong-Er Lu, Fei-Ting Hsu, Yuan Chang. To investigate the effect of fluoxetine combined with radiation therapy on 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 1117.

Abstract 6490: Pt1a and sorafenib combination treatment exerts synergistic anti-hepatocellular carcinoma effect through RNA m6A methylation modification

Abstract Hepatocellular carcinoma (HCC) is the most common type of liver cancer and has a poor prognosis. The survival benefits of sorafenib in patients with advanced HCC are modest owing to acquired resistance. Accumulating evidence has demonstrated that cancer stemness is an important cause of drug resistance and the root cause of tumor recurrence. [PtII(C^N^N)(NHC2Bu)]PF6 (Pt1a) is a newly developed platinum-based drug containing N-heterocyclic carbene with proven anticancer properties against a range of tumor types. However, its role in targeting cancer stemness and its ability to sensitize cancer cells to overcome molecular-targeted therapy drug resistance in HCC remains elusive. Herein, we demonstrate the synergistic inhibitory effect of sorafenib and Pt1a combination treatment on HCC through RNA m6A methylation modification. In cultured HCC cells and patient-derived organoids, the drug combination synergistically reduced the cell viability and promoted cell apoptosis compared to single-drug treatment. Combination treatment also attenuated self-renewal, as evidenced by a marked decrease in tumor-initiating cancer cell frequency, as measured by limiting dilution spheroid formation assay. These findings were further validated in HCC patient-derived xenograft mouse models and a mouse HCC model induced by hydrodynamic tail vein delivery of NRAS+AKT coactivation plasmids. Notably, the combination treatment with sorafenib and Pt1a, but not a single treatment, led to a significant reduction in tumor burden. The combination treatment was also effective in decreasing tumor-initiating cell frequency, as measured by subjecting the harvested tumor cells for limiting dilution spheroid formation assay ex vivo. Mechanistically, integrative RNA-sequencing and thermal proteome profiling analyses both indicated the enrichment of m6A-related pathways in combination drug treatment. An increased global m6A level upon combination treatment was validated using m6A dot blotting, confirming that the synergistic effect may be mediated by the elevated global m6A level in HCC cells. Collectively, our study demonstrated that Pt1a and sorafenib combination treatment synergistically inhibited the viability and stemness of HCC cells by increasing global RNA m6A level. These findings supported the potential of Pt1a as a therapeutic option for HCC and overcoming sorafenib drug resistance. Ongoing studies are focused on identifying m6A regulators and downstream targets involved in combination treatment. Citation Format: Yinjia Huang, Yalu Cui, Ianto Bosheng Huang, Yanyan Wang, Yang Xuan, Pui Ki Wan, Chun Nam Lok, Chi Ming Che, Stephanie Ma. Pt1a and sorafenib combination treatment exerts synergistic anti-hepatocellular carcinoma effect through RNA m6A methylation modification [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 6490.

Abstract 2785: Beyond the role of transcobalamin 1 (TCN1) as a vitamin B12-binding protein in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is a common form of primary liver cancer and is associated with poor prognosis. Although sorafenib and lenvatinib have shown promising results in HCC treatment, their clinical benefits for patients have been limited owing to drug resistance and tumor relapse. This may be associated with the presence of tumor-initiating cells (T-ICs), which require further investigation to identify the key pathways or molecules involved in their regulation. Through in vitro enrichment of liver T-IC populations via a series of sphere passages under treatment with chemotherapeutic drugs, subsequent bulk RNA sequencing analysis identified a vitamin B-12 binding protein, transcobalamin 1 (TCN1), as the most upregulated molecule in such enriched T-IC populations. This result was consistent with the enrichment profiles of the stem cell signature in TCN1High HCC samples in the clinical dataset. TCN1 is a secretory protein expressed at high levels in salivary glands and glandular cells; however, its function beyond its native role as a vitamin B12-binding protein in the regulation of T-ICs is unclear. Using lentiviral shRNA overexpression and knockdown approaches, we identified a critical role for TCN1 in regulating the properties of T-ICs, including self-renewal, invasiveness, tumorigenicity, and drug resistance, through in vitro and in vivo assays. Clinically, we also observed a substantial increase in TCN1 mRNA signatures in HCC samples, which was associated with poor prognosis of HCC patients. By analyzing the TCGA-LIHC dataset with confirmation by immunoprecipitation, we report, for the first time, that TCN1 directly interacts with integrin subunit alpha 3 (ITGA3), thereby regulating the Notch signaling pathway. Furthermore, ELISA results revealed the presence of the secretory form of TCN1 in HCC cells, which exerts a T-IC-enhancing effect on HCC cells via autocrine regulation. In conclusion, our study provides novel insights into how TCN1 functions in HCC via a non-canonical mechanism, which goes beyond its conventional role as a vitamin B12-binding protein and uncovers a potential novel therapeutic target for HCC. Citation Format: Gregory Kenneth Muliawan, Carmen Oi Ning Leung, Terence Kin Wah Lee. Beyond the role of transcobalamin 1 (TCN1) as a vitamin B12-binding protein 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 2785.

Abstract 5338: Evaluation of checkpoint inhibitor efficacy in a humanized immune system mouse model lacking murine Fc gamma receptors

Abstract Humanized immune system (HIS) mice are critical immuno-oncology tools for evaluating antibody-based therapeutic target engagement and efficacy in a human-like context. Most antibody-based therapeutics have an IgG backbone and thus engage Fc gamma receptors. Traditional HIS mice retain murine immune cells such as neutrophils and macrophages which can interact with human IgG-based therapeutics and confound preclinical results. To determine whether knockout of murine Fc gamma receptors (FcγRs) in a super-immunodeficient mouse model would alter anti-PD1 efficacy compared to the parent strain, we studied tumor growth kinetics, human reconstitution, and tumor infiltrating leukocytes (TILs) in each strain engrafted with HCC827 lung adenocarcinoma tumor cells treated with pembrolizumab or vehicle. Methods: HIS NOG (huNOG) or HIS FcγR knockout NOG mice (FcResolv™ huNOG) were created using identical protocols with CD34+ cells from three human donors shared across both strains. Baseline reconstitution was evaluated in naïve animals via flow cytometry. HCC827 cells were inoculated in remaining animals. Animals were randomized on day 7 post-tumor implantation into 12 groups. Body weight, clinical observations, and tumor growth were measured. Mice received treatment from D7, dosed twice weekly for four weeks, and were then euthanized for FACS analysis, collecting blood, spleen, and tumor samples. Results: For a given donor, no significant differences were seen between strains in human immune reconstitution and kinetics. Pembrolizumab treatment showed significant tumor growth inhibition in one donor in FcResolv huNOG, but not in donor-matched huNOG mice. Evaluation of human TILs in pembrolizumab-treated animals showed significant differences between the strains across all donors, with FcResolv huNOG mice showing increased CD8+ T cells and decreased tumor-associated macrophages compared to vehicle-treated mice, and no significant differences in huNOG. Evaluation of murine TILs revealed differences in murine macrophage populations, regardless of treatment, with Ly6Clo dominant in FcResolv huNOG and Ly6Chi dominant in huNOG. In the presence of pembrolizumab, the murine myeloid and granulocyte population within the spleen is significantly decreased in FcResolv huNOG mice whereas no change was seen in huNOG. Conclusions: Our study demonstrates when treated with anti-PD1, FcResolv huNOG mice show expected pharmacodynamic changes and donor-dependent efficacy whereas, despite identical donors and creation protocols, pembrolizumab-treated huNOG mice showed neither. These differences are due to the presence or absence of murine FcγRs and their impact on antibody IgG-based therapeutics. Citation Format: Nicholas Smith, Monika Buczek, Megan MacBride, Esther Andino, Emily Sack, Debra Freer, Michelle Vedder, Kathleen Bott, Louise Baskin, Damien France, Francis Bichat, Caroline Mignard, Janell Richardson. Evaluation of checkpoint inhibitor efficacy in a humanized immune system mouse model lacking murine Fc gamma receptors [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 5338.

Abstract 591: Discovery of ZM-2322, a highly selective, potent inhibitor of membrane-associated tyrosine- and threonine-specific cdc2-inhibitory kinase (PKMYT1)

Abstract The genome DNA is consistently threatened by a variety of intrinsic and exogenous DNA damage factors. Cell cycle checkpoint is one important mechanism in response to DNA damage and maintaining genome stability. In tumor cells, gene alterations, such as CCNE1 amplification (encoding for Cyclin E1), result in G1/S checkpoint loss and replication stress, leading to high dependency on the G2/M checkpoint. PKMYT1 inhibits crucial checkpoint protein CDK1 activity by phosphorylating CDK1 at the residues of Thr-14 and Tyr-15 in the G2/M phase. In CCNE1 amplification cells, the inhibition of PKMYT1 leads to the reduction of pCDK1 and loss of G2/M checkpoint, consequently results in premature mitosis and the accumulation of DNA damage, then finally induces cell death. Here we identify a novel small molecular ZM-2322, which displays robust intracellular binding to PKMYT1 and inhibits the specific downstream phosphorylation of CDK1 Thr-14 with IC50 values of 14 nM and 29 nM, respectively. ZM-2322 strongly inhibits the proliferation of the HCC1569 cell line with CCNE1 high-level amplification and shows a >400 × selectivity folds over a CCNE1 wildtype cell line SKOV3. ZM-2322 also shows significant inhibition of tumor growth in a dose-dependent manner in the HCC1569 xenograft model, and the efficacy correlates well with PK and target inhibition. There is evidence showing that loss-of-function mutations of FBXW7, a cyclin E E3 ligase, lead to cyclin E accumulation and are synthetic-lethal with PKMYT1 inhibition. Consistently, a potent anti-proliferation of ZM-2322 is observed in DLD-1 FBXW7 -/- cells, but not DLD-1 parental cells. These data suggest a high potency of ZM-2322 on PKMYT1 inhibition and a potential good kinase selectivity which is further confirmed by using a kinase panel biochemical assay. As expected, when compared head-to-head with another clinical PKMYT1 inhibitor RP-6306 which shows inhibitory effect on 27 of 217 human kinases with inhibition rates >90% at 1 μM, ZM-2322 is found to be a much more PKMYT1-selective inhibitor and hits only several individual kinases under the same assay condition, suggesting a potentiality in better safety profile and higher dose tolerance. Furthermore, our data illustrates that in combination with gemcitabine, ZM-2322 elicits strong synergetic anti-proliferation activities on HCC1569 cells in vitro and results in tumor regression with a minor effect on body weight in vivo. Besides, a strong synergy effect is also observed in the combo of ZM-2322 and ATR inhibitor in HCC1569 in vitro. These data indicate that ZM-2322 has the potential of application expansion and good tolerance under continuous administration. Taken together, our data suggest that ZM-2322 is a potent and highly selective PKMYT1 inhibitor. Citation Format: Feng Zhou, Lu Liu, Lei Jiang, Baoying Cheng, Zhentao Li, Dongxing Zhu, Jianbin Xue, Liting Xue, Renhong Tang. Discovery of ZM-2322, a highly selective, potent inhibitor of membrane-associated tyrosine- and threonine-specific cdc2-inhibitory kinase (PKMYT1) [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 591.

Abstract 4378: Identification of novel TXNIP-dependent antitumor pathways in breast cancer

Abstract Thioredoxin-interacting protein (TXNIP) plays a critical role in glucose metabolism and redox signaling. TXNIP has recently emerged as a potent tumor suppressor in various cancer models. In previous studies, we found TXNIP was activated by UNC0642, an inhibitor of histone methyltransferase, in MDA-MB-231 breast cancer cells. TXNIP activation was coupled with significant suppression of cell proliferation and tumor growth. In contrast, TXNIP knockdown increased MDA-MB-231 cell proliferation and in tumor growth and metastasis in vivo. TXNIP-reconstitution in TXNIP-deficient HCC1954 cells reduced cell proliferation and migration coupled with increased reactive oxygen species production. TXNIP-reconstitution also significantly decreased mitochondrial respiration, mitochondrial membrane potential and glycolysis in HCC1954 cells. To understand the mechanisms underlying TXNIP’s antitumor activity, we performed co-immunoprecipitation and proteomic analyses to uncover TXNIP-interacting proteins. We identified calpastatin (CAST) as a novel TXNIP-interacting protein in breast cancer cells. CAST is an endogenous inhibitor of calpains, a family of calcium-activated cysteine proteases. Intriguingly, CAST association with TXNIP was readily detectable in TXNIP-low, but not in TXNIP-high breast cancer cells. The disassociation of CAST from TXNIP in TXNIP-high cells might allow CAST to inhibit calpain activity to augment TXNIP’s antitumor function. Through RNAseq analysis, we identified a positive correlation between TXNIP expression and IL-24 activation in various breast cancer cells, which were also confirmed by western blotting. IL-24 has emerged as an important cancer therapy target because of its role in promoting cancer-specific apoptosis. Our data also demonstrated that TXNIP upregulated the level of IL-24 and downregulated STAT-3. Moreover, TXNIP exerts multifaceted effects on BC development through the IL24-STAT3 signaling axis, deepening our understanding of its complex role in the disease. Taken together, these studies identified novel TNXIP-dependent pathways and their potential new mechanisms in breast cancer pathogenesis with significant clinical implications. Citation Format: Jasvinder Singh, Bindeshwar Sah, Robert Clarke, Liang Liu. Identification of novel TXNIP-dependent antitumor pathways in breast 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 4378.

Abstract 5963: VGLL1-derived peptides demonstrated anticancer effect by inhibiting VGLL1-TEAD4 interaction

Abstract YAP/TAZ, cofactors of TEA domain-containing transcription factor TEADs (TEAD1-4), play a key role in the HIPPO pathway, which is involved in various function including embryonic development, organ size, cell proliferation, tissue regeneration, and the development and progression of several cancers. YAP/TAZ is therefore considered as master regulators of various cancers. However, we are interested in VGLL1, a cofactor of TEAD4, because it is mainly involved in the proliferation and metastasis of various cancers, including gastric, breast, pancreatic, and ovarian cancers. In gastric cancer, VGLL1 expression is regulated by the PI3K/AKT/β-catenin pathway. VGLL1 is then phosphorylated at S84 by TGF-β/ribosomal S6 kinase 2 (RSK2) and interacts with TEAD4 to express target genes such as MMP9, cyclin D1, IGFBP3, IGFBP5 and ITGαV. which are involved in cancer malignancy. We designed S84-containing VGLL1-derived peptides (VGLL1S84 peptides, SCVP), which consist of 7 to 15 amino acids linked to CPP, a cell-penetration peptide. We evaluated SCVP to inhibit the VGLL1-TEAD4 interaction and their therapeutic potential as anticancer agents. SCVP suppressed the growth of gastric and breast cancer cells expressing high levels of VGLL1, EGFR, and/or HER2. In the RNA-seq analysis of NUGC3 to elucidate the molecular mechanisms of SCVP induced cell death, SCVP-treated cells showed gene expression changes in metabolic pathway, RAS signaling, PI3K-AKT signaling, Wnt signaling, TNF signaling, and mTOR signaling. However, RNA-seq analysis of breast cancer cells treated with SCVP showed changes in the expression of genes involved in cell cycle, DNA replication, DNA repair, and apoptosis. Then, we performed CRISPR/Cas9 screening using SCVP and sgRNA library to explore companion biomarkers and genetic interactors of VGLL1 for targeted cancer therapy. As a result, we identified several candidate genes, including ZNF292 and PLK1, as synthetic lethal genes of VGLL1. As expected, HCC70 cells with ZNF292 mutation showed increased sensitivity to SCVP. We also found that SCVP in combination and the PLK inhibitor volasertib resulted in a synergistic effect on the growth inhibition of breast cancer cells. In conclusion, we demonstrated VGLL1 as a novel target for anticancer drug development, and elucidated the molecular mechanism and therapeutic potential of the SCVP in gastric and breast cancers. Citation Format: Sangmi Ock, Changsub Lee, Wongi Jung, Ina Whang, Kyung-Soo Kim, Jongmin Han, Sujin Yim, Bo-Kyung Kim, Hong-Sub Lee, Kwan-Young Chang, Misun Won. VGLL1-derived peptides demonstrated anticancer effect by inhibiting VGLL1-TEAD4 interaction [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 5963.

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.

Abstract 433: BCAT1 promotes HCC metabolic reprogramming and survival through HIF-1α stabilization

Abstract Hepatocellular carcinoma (HCC) is a highly lethal primary liver malignancy characterized by hypoxia, a common feature of solid tumors, particularly in HCC. Hypoxia-inducible factors (HIFs) play a pivotal role in mediating various cellular processes, such as metabolic adaptation, to support the survival of hypoxic cells. The cytosolic form of branched-chain amino transferase (BCAT1) is responsible for the reversible transamination reaction between branched-chain keto-amino acids (BCKAs) and branched-chain amino acids (BCAAs), facilitating the interconversion between α-ketoglutarate (α-KG) and glutamate. Interestingly, we found that in the context of HCC, BCAT1 consumes α-KG, thereby favoring the stabilization of HIF-1α through suppressing the α-KG-dependent prolyl hydroxylase (PHD). Knockdown (KD) or knockout (KO) of BCAT or BCAT inhibitor (ERG245) suppressed HIF-1 stability, as well as HIF-1 dependent transcriptomic and metabolic changes. Mass spectrometry (MS)-based isotopic carbon tracing showed that KD/KO or ERG 245 suppressed hypoxia/HIF-1-induced glycolytic flux. Interestingly, ERG245 effectively inhibited the proliferative output of hypoxic HCC cells and suppressed HCC growth. ERG245 also profoundly inhibited HCC growth in vitro and in vivo together with Sorafenib, a tyrosine kinase inhibitor (TKI) used in HCC by increasing the apoptotic cell death. Furthermore, we confirmed that the expressions of BCAT1 and the HIF-1α-targeted metabolic genes are positively correlated in human HCCs. Our findings unequivocally demonstrate that BCAT1 confers a growth advantage to HCC cells by stabilizing HIF-1α and orchestrating HIF-1α-mediated metabolic reprogramming. Consequently, targeting BCAT1 emerges as a promising therapeutic strategy for HCC patients. Funding Support: This work was supported by the Centre for Oncology and Immunology under the Health@InnoHK initiative funded by the Innovation and Technology Commission, The Government of Hong Kong SAR, China and National Natural Science Foundation of China (NSFC) (82173123, C.C.-L.W.) Citation Format: Misty Shuo Zhang, Kenneth Kin-Leung Kwan, Weixing Li, Adonia E. Papathanassiu, Irene Oi-Lin Ng, Carmen Chak-Lui Wong. BCAT1 promotes HCC metabolic reprogramming and survival through HIF-1α stabilization [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 433.

Abstract 5546: CRISPR-based kinome screen identifies Ikbkb as a regulator of phagocytic evasion in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is one of the most lethal and prevalent malignancies worldwide and is associated with poor prognosis. Recently, immune checkpoint inhibitors (ICIs) have revolutionized the oncology landscape by targeting genes critical for immune checkpoint pathways, leading to the immune evasion of cancer cells. Phagocytosis checkpoints have emerged as essential checkpoints for cancer immunotherapy by functioning as “don’t eat me” signal to suppress immune response. Given the druggable properties of protein kinases, together with recent reports showing the antitumor immunity of kinase inhibitors, we aimed to identify protein kinases critical for phagocytic evasion in HCC by transducing a lentiviral sgRNA expression plasmid containing 2,852 unique single-guide RNAs (sgRNAs) targeting the 713 mouse kinase gene in mouse HCC cells, RIL-175, and subsequently co-incubated with RAW 264.7 macrophages. Based on the deep sequencing analysis comparing the sgRNA distribution in RIL-175 with or without co-incubation, we identified the Ikbkb gene as top-sensitizing hit on this screen, with at least two-fold sgRNA reduction upon incubation with macrophages. To verify the phagocytic evasive role of Ikbkb in HCC, we have employed CRISPR/Cas9 knockout approach to delete Ikbkb in mouse HCC cell line, RIL-175 cells. The results showed that upon co-incubation with RAW 264.7 cells, the Ikbkb knockout cells exhibited increased phagocytosis, as confirmed by both flow cytometry analysis and microscopy observation. The analysis of the TCGA-LIHC cohort revealed that the expression level of Ikbkb was elevated and associated with unfavorable clinical outcomes. Notably, Ikbkb expression was found to be significantly correlated with established anti-phagocytic markers, including CD24, CD47, and CD274, further supporting their roles in the anti-phagocytic activities of HCC cells. In summary, we have uncovered IKBKB as a cancer-intrinsic regulator of phagocytosis, which has the potential to target IKBKB as a novel therapeutic strategy to enhance the efficacy of current ICI treatments for HCC patients. Citation Format: Sze Man Chan, Terence Kin Wah Lee. CRISPR-based kinome screen identifies Ikbkb as a regulator of phagocytic evasion 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 5546.