Tumor Burden In Mice Research Articles

ICOS-expressing Regulatory T Cells Influence the Composition of Antitumor CTL Populations.

The role of ICOS in antitumor T cell responses and overall tumor progression has been controversial. In this study, we compared tumor progression in mice lacking ICOS selectively in regulatory T (Treg) cells or in all T cells. Using an experimental melanoma lung metastasis model, we found that Treg cell-specific ICOS knockout reduces the overall tumor burden compared with Cre control mice, with increased CD4+-to-Treg cell and CD8+-to-Treg cell ratios in the tumor. In contrast, there was no difference in the tumor burden in mice lacking ICOS in all of the T cell compartments. This suggests a dual role of ICOS costimulation in promoting protumor and antitumor T cell responses. Consistent with reduced tumor burden, we found that Treg cell-specific deletion of ICOS leads to an increase of CD8+ CTLs that express high levels of granzyme B and perforin. Moreover, single-cell transcriptome analysis revealed an increase of Ly108+Eomeshi CD8+ T cells at the cost of the Ly108+T-bethi subset in Treg cell-specific knockout mice. These results suggest that ICOS-expressing Treg cells suppress the CTL maturation process at the level of Eomes upregulation, a critical step known to drive perforin expression and cytotoxicity. Collectively, our data imply that cancer immunotherapies using ICOS agonist Abs may work better in Treg cell-low tumors or when they are combined with regimens that deplete tumor-infiltrating Treg cells.

Berberine Protects against Hepatocellular Carcinoma Progression by Regulating Intrahepatic T Cell Heterogeneity.

Accumulating evidence suggests that berberine (BBR) exhibits anti-cancer effects in hepatocellular carcinoma (HCC). However, the mechanisms by which BBR regulates the immunological microenvironment in HCC has not been fully elucidated. In this study, a mouse model of orthotopic HCC is established and treated with varying doses of BBR. BBR showed effectiveness in reducing tumor burden in mice with HCC. Cytometry by time-of-flight depicted the alterations in the tumor immune landscape following BBR treatment, revealing the enhancement in the T lymphocytes effector function. In particular, BBR decreased the proportion of TCRbhiPD-1hiCD69+CD27+ effector CD8+ T lymphocytes and increased the proportion of Ly6ChiTCRb+CD69+CD27+CD62L+ central memory CD8+ T lymphocytes. Single-cell RNA sequencing further elucidates the effects of BBR on transcriptional profiles of liver immune cells and confirms the phenotypical heterogeneity of T lymphocytes in HCC immune microenvironment. Additionally, it is found that BBR potentially regulated the antitumor immunity in HCC by modulating the receptor-ligand interaction among immune cells mediated by cytokines. In summary, the findings improve the understanding of BBR's impact on protecting against HCC, emphasizing BBR's role in regulating intrahepatic T cell heterogeneity. BBR has the potential to be a promising therapeutic strategy to hinder the advancement of HCC.

Human-specific elimination of epithelial Siglec-XII suppresses the risk of inflammation-driven colorectal cancers.

Carcinomas are common in humans but rare among closely related "great apes." Plausible explanations, including human-specific genomic alterations affecting the biology of sialic acids, are proposed, but causality remains unproven. Here, an integrated evolutionary genetics-phenome-transcriptome approach studied the role of SIGLEC12 gene (encoding Siglec-XII) in epithelial transformation and cancer. Exogenous expression of the protein in cell lines and genetically engineered mice recapitulated approximately 30% of the human population in whom the protein is expressed in a form that cannot bind ligand because of a fixed, homozygous, human-universal missense mutation. Siglec-XII-null cells/mice recapitulated the remaining approximately 70% of the human population in whom an additional polymorphic frameshift mutation eliminates the entire protein. Siglec-XII expression drove several pro-oncogenic phenotypes in cell lines and increased tumor burden in mice challenged with chemical carcinogen and inflammation. Transcriptomic studies yielded a 29-gene signature of Siglec-XII-positive disease and when used as a computational tool for navigating human data sets, pinpointed with surprising precision that SIGLEC12 expression (model) recapitulates a very specific type of colorectal carcinomas (disease) that is associated with mismatch-repair defects and inflammation, disproportionately affects European Americans, and carries a favorable prognosis. They revealed a hitherto-unknown evolutionary genetic mechanism for an ethnic/environmental predisposition of carcinogenesis.

Abstract 6739: Efficacy and toxicity of microbial mimetic mesoporous silica nanoparticles in a mouse model of ovarian cancer

Abstract Ovarian cancer is predominately left undetected and untreated until the later stages of the disease where it becomes incredibly difficult to treat. Preclinical studies demonstrate that immunotherapy can be highly effective. Our lab has been working on an immunotherapy approach to treat women with ovarian cancer that utilizes mesoporous silica nanoparticles(MSN). MSN that present pathogen-associated molecular patterns (PAMPS) are able to significantly reduce tumor burden in mouse models of serous epithelial ovarian cancer when administered by intraperitoneal injection. These microbial mimetic particles are decorated with polyethyleneimine (PEI), CpG oligonucleotide, and monophosphoryl lipid A (MPLA). When in the presence of antigen presenting cells, they bind to Toll-like receptors (TLR) present on the cell surface and within the endosome. To better understand mechanisms underlying their efficacy, we injected fluorescent MSN-PEI-CpG-MPLA (100 or 500 nm) or unmodified MSN into mice with advanced ovarian cancer. The particles, regardless of the presence of TLR-agonists, were rapidly internalized by myeloid cells, with trafficking almost exclusively to immune clusters located in tumor-burdened adipose tissues when administered by intraperitoneal injection. Conversely, intravenous injection of MSN-PEI-CpG-MPLA led to predominate accumulation in liver, with no particles found in tertiary lymphoid clusters 24 hours post injection. Furthermore, unlike intraperitoneal injection, intravenous injection of MSN-PEI-CpG-MPLA was without therapeutic effect. Currently we are studying MSN safety and the impact of administration route and dose on efficacy and cytotoxicity. In summary, MSN, with and without TLR agonists, are trafficked by myeloid cells to tumor-burdened fat-associated lymphoid clusters when administered by intraperitoneal injection. Intraperitoneal injection of TLR-agonist modified MSN effectively reduces tumor burden in mice with ovarian cancer. Citation Format: Lorel Y. Medina, Rita E. Serda, Benjamin Marwedel, Melanie S. Jun, Josh Adogola. Efficacy and toxicity of microbial mimetic mesoporous silica nanoparticles in a mouse model of ovarian 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 6739.

Abstract 6320: CAR-T cells targeting LGR5: An effective treatment for chemotherapy resistant ovarian cancer

Abstract Ovarian cancer is the second leading cause of cancer-related mortality among gynaecological cancers in the Western world. CAR-T cell therapy is an adoptive immunotherapy used to treat some blood cancers. This study has investigated whether leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a suitable target for CAR-T cell therapy for ovarian cancer. We assessed the cytotoxic effects of LGR5-targeting CAR-T cells on a range of ovarian cancer cell lines and primary serous ovarian cancer cells derived from patient ascites (at diagnosis and following relapse with chemotherapy resistant disease) in monolayer culture, 3D spheroid assays and patient-derived explant assays. OVCAR3 and a chemoresistant patient-derived xenograft (PDX) models have been used to assess whether LGR5-targeting CAR-T cells can inhibit growth and metastasis in vivo. We found that LGR5 is highly expressed in high-grade serous ovarian carcinoma (HGSOC) cancer cell lines and LGR5 expression was increased in chemotherapy resistant primary ovarian cancer cells. We demonstrated that LGR5-targeting CAR-T cells were cytotoxic and significantly inhibited the survival of ovarian cancer cell lines and chemotherapy-resistant primary ovarian cancer cells with high LGR5 expression levels in both monolayer culture and 3D spheroids compared to un-transduced CD3+ T cells. In patient-derived explant assays, we observed increased apoptosis in HGSOC tissues expressing high LGR5 following treatment with LGR5-targeting CAR-T cells. In vivo, LGR5-targeting CAR-T cell treatment decreased the tumour burden in mice with OVCAR3 xenografts and a chemotherapy-resistant PDX model with high LGR5 expression, compared to CD3+ T cells. In summary, LGR5-targeting CAR-T cells have great potential to be developed as a novel immunotherapy for ovarian cancer. Citation Format: Wanqi (Jady) Wang, Veronika Bandara, Noor A. Lokman, Zoe K K. Price, Tannith M. Noye, Silvana Napoli, Batjargal Gundsambuu, Martin K. Oehler, Simon C. Barry, Carmela Ricciardelli. CAR-T cells targeting LGR5: An effective treatment for chemotherapy resistant ovarian 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 6320.

Abstract 5279: Sequential treatment with PARPi and WEE1i minimizes T cell DNA damage and enhances its immune response

Abstract Background: DNA damage response (DDR) targeted therapy, like PARP inhibitors (PARPi) and WEE1 inhibitors (WEE1i), have demonstrated potent anti-cancer activity and are pronounced to activate the cytosolic immunity pathway. Our previous work showed that sequential inhibition of PARP and WEE1 could kill tumor cells as effective as their concurrent application, with ameliorated toxicity to normal cells, suggesting high clinical translational potential of this regimen. However, the effect of sequential treatment on T lymphocytes and anti-cancer immunity remains largely unknown. Methods and results: T cells were treated with 25 μM olaparib, or 250 nM AZD1775, or olaparib combined with AZD1775 (concurrent group) for 48h, or olaparib for 24h followed by AZD1775 for 24h (sequential group) in vitro. AZD1775 and concurrent treatment reduced the T cell viability to 19% and 14.3%, while sequential treatment maintained the viability to 58.6%, closed to olaparib monotherapy (61.6%). And the most potent proliferation capacity accompanied with the lowest apoptosis level were both recorded after sequential treatment, manifested by the increasing of Ki67 and EdU and the decreasing of cleaved caspase-3. Reverse-phase protein arrays (RPPA) analyses identified relieved DNA damage in T cells receiving sequential treatment. Alkaline comet assay and detecting of γH2AX further confirmed that the T cell DNA damage caused by PARPi and WEE1i was obviously alleviated after sequential therapy. Furthermore, sequential treatment activated cGAS-STING pathway effectively and upregulated the production of CCL5, CXCL10 and IFNβ of downstream type I interferon response compared with monotherapy and concurrent treatment in ovarian cancer cell lines (SKOV3, OVCAR8, A2780). We also evaluated an ID8 intraperitoneal syngeneic immunocompetent mouse model. T cells isolated from tumors, ascites, spleen and peripheral blood in sequential group were presented with the highest proliferation potential and the lowest level of DNA damage. And the tumor burden of mice in sequential group was obviously subsided, which was comparable to that in concurrent group. Conclusions: This study demonstrated that targeting PARP and/or WEE1 impaired the T cell viability and led to DNA damage. However, administrating PARPi and WEE1i sequentially diminished this inhibitory effect on T cells on the premise of ensuring their anti-tumor effect. These data provide strong rational for the further investigation on exploring the clinical significance of sequential treatment with PARPi and WEE1i. Citation Format: Xiaofei Jiao, Jiahao Liu, Wei Mu, Li Zhu, Xuejiao Zhao, Gordon B. Mills, Qinglei Gao, Yong Fang. Sequential treatment with PARPi and WEE1i minimizes T cell DNA damage and enhances its immune response [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 5279.

Diaphorobacter nitroreducens synergize with oxaliplatin to reduce tumor burden in mice with lung adenocarcinoma.

Oxaliplatin is widely used as an effective chemotherapeutic agent in cancer treatment, but its side effects and response rate still need to be improved. Conventional probiotics potentially benefit cancer chemotherapy by regulating gut microbiota and tumor immune infiltration. This study was novel in reporting a more significant inhibitory effect of Diaphorobacter nitroreducens on lung adenocarcinoma (LADC) cells compared with common traditional probiotics and validating its potential as an adjuvant therapy for LADC chemotherapy in mice. This study investigated the impact of D. nitroreducens combined with oxaliplatin on the gut microbiota and immune infiltration of tumors as a potential mechanism to improve anticancer effects.

Abstract A025: Autotaxin-lysolipid signaling suppresses a CCL11- eosinophil axis to promote pancreatic cancer progression

Abstract Among all cancer types, pancreatic ductal adenocarcinoma (PDAC) has one of the worst outcomes. In recent studies, metabolic and microenvironmental reprogramming in PDAC has been shown to facilitate tumor progression by promoting immune escape mechanisms, however these mechanisms are still poorly understood. Recently, our lab established that autotaxin (ATX) - lysophosphatidic acid (LPA) signaling in PDAC enhances tumorigenicity, but the immunomodulatory function of this signal pathway remains largely unexplored. We now show that ablation of the LPA-producing enzyme autotaxin (ATX) in PDAC cells can reduce tumor burden in mice and reprogram the tumor immune microenvironment by increasing tumor eosinophil infiltration. The functional role of eosinophils in the context of PDAC is a poorly understood area. The presence of eosinophils in pancreatic cancer has been reported in a few case reports: for example, prior studies identified neutrophils and eosinophils together as a group, expressing high levels of CD66b. We confirmed the presence of eosinophils in PDAC in mice as well as patients, and we found that rare patient samples with the highest intratumoral eosinophil abundance had the longest survival rates. Genetic or pharmacologic ATX inhibition increased intratumoral eosinophils, which promote tumor cell apoptosis locally and suppress tumor progression. When eosinophils were present, primary tumor growth was modestly reduced, but metastatic spread was greatly reduced. Mechanistically, ATX suppresses eosinophil accumulation via an autocrine feedback loop wherein ATX-LPA signaling negatively regulates activity of AP-1 transcription factor c-Jun, in turn suppressing expression of potent eosinophil chemoattractant CCL11 (Eotaxin-1). This is the first study that highlights the anti-tumor potential of eosinophils in pancreatic cancer. Citation Format: Sohinee Bhattacharyya, Chet Oon, Holly Sandborg, Terry Morgan, Mara H. Sherman. Autotaxin-lysolipid signaling suppresses a CCL11- eosinophil axis to promote pancreatic cancer progression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A025.

Engineered CAR-Tcells targeting the non-functional P2X purinoceptor 7 (P2X7) receptor as a novel treatment for ovarian cancer.

Recent studies have identified expression of the non-functional P2X7 (nfP2X7) receptor on various malignant cells including ovarian cancer, but not on normal cells, which makes it a promising tumour-associated antigen candidate for chimeric antigen receptor (CAR)-T-cell immunotherapies. In this study, we assessed the cytotoxic effects of nfP2X7-CAR-T cells on ovarian cancer using in vitro and in vivo models. We evaluated the effects of nfP2X7-CAR-T cells on ovarian cancer cell lines (SKOV-3, OVCAR3, OVCAR5), normal peritoneal cells (LP-9) and primary serous ovarian cancer cells derived from patient ascites invitro using monolayer and 3D spheroid assays. We also evaluated the effects of nfP2X7-CAR-T cells on patient-derived tissue explants, which recapitulate an intact tumour microenvironment. In addition, we investigated the effect of nfP2X7-CAR-T cells in vivo using the OVCAR-3 xenograft model in NOD-scid IL2Rγnull (NSG) mice. Our study found that nfP2X7-CAR-T cells were cytotoxic and significantly inhibited survival of OVCAR3, OVCAR5 and primary serous ovarian cancer cells compared with un-transduced CD3+ Tcells in vitro. However, no significant effects of nfP2X7-CAR-T cells were observed for SKOV3 or normal peritoneal cells (LP-9) cells with low P2X7 receptor expression. Treatment with nfP2X7-CAR-T cells increased apoptosis compared with un-transduced Tcells in patient-derived explants and correlated with CD3 positivity.Treatment with nfP2X7-CAR-T cells significantly reduced OVCAR3 tumour burden in mice compared with un-transduced CD3 cells for 7-8 weeks. This study demonstrates that nfP2X7-CAR-T cells have great potential to be developed as a novel immunotherapy for ovarian cancer.

Identifying Targeted Therapies for CBFA2T3- GLIS2 Acute Myeloid Leukemia

CBFA2T3- GLIS2 fusion positive pediatric acute myeloid leukemia (AML) remains one of the worst prognostic AML subgroups. Children with this subtype of AML frequently experience upfront chemoresistance with a high cumulative incidence of relapse (42-83%) and a dismal overall survival (~20%) even with intensive therapy. New treatment approaches are urgently needed. By interrogating the Broad Institute's Cancer Dependency Map (DepMap), a data set composed of genome-scale CRISPR-Cas9 screens in over 1000 cancer cell lines, we discovered that the CBFA2T3- GLIS2 AML model M-07e was highly dependent on the kinase JAK2. Indeed, M-07e was one of the most JAK2 dependent models in the data set. Using the same genome-scale library, we next performed CRISPR-Cas9 screens in WSU-AML and CMS, two additional AML cell lines harboring this fusion, and revealed JAK2 as a strong dependency shared by the 3 cell lines. Using a doxycycline-inducible knockout (KO) system, we validated JAK2 KO by western blot in three AML cell lines and cells from a patient-derived xenograft (PDX) harboring the fusion. Using two different sgRNAs targeting JAK2 (sgJAK2) and a non-targeting (sgNT) control, we validated JAK2 dependency in all 4 AML models in competitive growth assays. We also observed impaired proliferation and induced apoptosis upon JAK2 KO. To evaluate a role for JAK2 dependency in vivo, we intravenously injected NSGS mice with WSU-AML either transduced with doxycycline-inducible sgJAK2 or sgNT. After confirmation of engraftment by bone marrow evaluation, doxycycline chow was initiated. After 2 weeks of doxycycline treatment, we observed a significantly decreased tumor burden in mice with JAK2 KO AML compared to sgNT as measured by a decrement in human CD45% cells: peripheral blood (2.8% versus 12.9%), bone marrow (37.4% versus 56.3%,) and spleen (19% versus 79.5%). We next assessed chemical inhibitors of JAK2. Both the type I JAK2 inhibitor ruxolitinib and the type II JAK2 inhibitor CHZ868 exhibited strong activity in vitro in CBFA2T3- GLIS2 cell lines and PDX cells, many resistant to cytotoxic chemotherapy. Because ruxolitinib is FDA-approved for other indications, we evaluated this drug in a cell line and PDX model of CBFA2T3- GLIS2 AML in vivo. As previously reported in myeloproliferative neoplasms, JAK2 target inhibition with ruxolitinib treatment was incomplete, and accordingly, in vivo response was modest with leukemia ultimately progressing. Evaluation of CHZ868 in vivo is ongoing. Because single agent targeted therapy is typically insufficient for durable response in the treatment of most cancers, we utilized CRISPR-Cas9 anchor screening to identify candidate resistance mechanisms and potential synergistic combinations with JAK2 inhibitors in CMS and WSU-AML cells. We compared the changes in abundance of sgRNAs in the cells either treated with ruxolitinib (IC 50) or vehicle for 14 days. The correlation of hits between the two cell lines was strong, and we found that sgRNAs targeting multiple negative regulators of the MAPK pathway were significantly enriched in the ruxolitinib-treated arm. In parallel, we generated resistant CMS sublines by chronic exposure to ruxolitinib. Ruxolitinib-resistant CMS cells were found to have a pathogenic NRAS missense mutation and an associated increase in levels of phosphorylated MEK. Accordingly, they were responsive to the MEK inhibitor trametinib. Both approaches converge on activation of the MAPK pathway as a resistance mechanism to JAK2 inhibitors in CBFA2T3- GLIS2 AML. We next hypothesized that targeting the MAPK pathway could overcome resistance to JAK2 inhibitors in this disease context. Indeed, treatment with ruxolitinib and the MEK inhibitor trametinib exhibited a synergistic effect when used in combination in cell lines and PDX cells harboring the fusion in vitro with in vivo testing as a next step. In summary, we validated JAK2 as a strong dependency in CBFA2T3- GLIS2 fusion positive pediatric AML. We demonstrated that JAK2 KO or small molecule inhibitors impaired cell viability and induced apoptosis in vitro, and JAK2 KO was highly efficacious in decreasing leukemia burden in vivo. Activation of the MAPK pathway renders resistance to JAK2 inhibitors and the combination of a JAK2 with MEK inhibitor is highly synergistic.

Dopamine Receptor 1 Impedes ILC2-Mediated Antitumor Immunity.

Ever-growing evidence has revealed that group 2 innate lymphoid cells (ILC2s) exhibit pleiotropic effects in antihelminth immunity, allergy, tissue protection, and cancer. Currently, the role of ILC2s in cancer is highly controversial regarding the intricate tumor microenvironment (TME), and the tumor-promoting or antitumor immunological mechanisms of ILC2s remain largely unknown. In this study, we report that dopamine receptor 1 (DRD1) restrains ILC2 activity in the TME. DRD1 deficiency promotes ILC2 activation, which irritates eosinophil recruitment and cytotoxic CD8+ T cell expansion during ongoing malignancy. Consequently, DRD1-deficient mice exhibit delayed tumor growth and reduced tumor progression. Furthermore, fenoldopam, a selective DRD1 agonist, restrains the ILC2 response in the TME and aggravates tumor burden in mice. Taken together, our data elaborate that the DRD1 signal acts as an excitatory rheostat in regulating ILC2-dependent antitumor immunity.

Coordination of Ru(II)-Arene Fragments to Dipyridophenazine Ligands Leads to the Modulation of Their In Vitro and In Vivo Anticancer Activity

Despite extensive research on the anticancer properties of Ru complexes with dipyrido[3,2-a:2',3'-c]phenazine (dppz) ligands, their in vivo efficacy is rarely investigated. Aiming to understand whether the coordination of certain half-sandwich Ru(II)-arene fragments might improve the therapeutic potential of dppz ligands, we prepared a series of Ru(II)-arene complexes with the general formula [(η6-arene)Ru(dppz-R)Cl]PF6, where the arene fragment was benzene, toluene, or p-cymene and R was -NO2, -Me, or -COOMe. All compounds were fully characterized by 1H and 13C NMR spectroscopy and high-resolution ESI mass-spectrometry, and their purity was verified by elemental analysis. The electrochemical activity was investigated using cyclic voltammetry. The anticancer activity of dppz ligands and their respective Ru complexes was assessed against several cancer cell lines, and their selectivity toward cancer cells was assessed using healthy MRC5 lung fibroblasts. The substitution of benzene with a p-cymene fragment resulted in a more than 17-fold increase of anticancer activity and selectivity of Ru complexes and significantly enhanced DNA degradation in HCT116 cells. All Ru complexes were electrochemically active in the biologically accessible redox window and were shown to markedly induce the production of ROS in mitochondria. The lead Ru-dppz complex significantly reduced tumor burden in mice with colorectal cancers without inducing liver and kidney toxicity.

The metabolite derived from high-salt diet-induced gut microbiota modulates brain tumor environment

Abstract The Westernized diet refers to consuming high amounts of saturated fat, sugar, and salt, and low amounts of dietary fiber. Such a diet is currently thought to contribute to non-communicable diseases such as autoimmune diseases, heart disease, and cancer. A high-salt diet (HSD) is known to affect various organs, including the cardiovascular, renal, and central nervous systems. Recently, several studies reported the effect of HSD on cancer due to the enhanced osmolarity of tumor microenvironment. However, it has been known that the concentration of sodium in brain is tightly regulated. That is, there may be another effect of HSD to tumor environment in brain. In addition, the effects of HSD on brain tumor have not been elucidated. In this study, we observed an increased tumor burden in mice who received the high-salt diet. Mice on a high-salt diet also showed a change in their gut microbiota. The specific bacteria significantly increased in HSD group compared to normal-salt diet (NSD) group. This bacteria is known to produce several metabolites, and we measured these metabolites in cecum of NSD and HSD group. Among them, the one metabolite increased in HSD group and we supplemented this metabolite to mice with brain tumor. Then, we observed the exacerbation of brain tumor post the supplement compared to control group. Therefore, we speculated that the HSD exacerbated brain tumor via the increased production of metabolite by altered gut microbiota.

Abstract A003: Identification of a potential chemoprevention agent for BRCA1- mutated cancers

Abstract BRCA1 mutation carriers are predisposed to developing aggressive breast, ovarian, and other cancers at a relatively early age. Unfortunately, these cancers lack an effective chemoprevention option. We have found that cells containing mutant BRCA1 harbor defective repair of oxidative DNA damage, an early trigger to tumorigenesis. Therefore, we sought to target this defect and discover a novel chemoprevention agent for these cancers. High-throughput screening of a chemical library identified a novel class of small-molecules that enhance repair of oxidative DNA damage, which we termed DNA repair-activating agents. One of these agents, benserazide, increased base-excision repair of oxidative DNA damage utilizing a cell-based DNA repair assay, decreased levels of 8-oxo-g (i.e. most common oxidized lesion in human genome) using flow cytometric analysis, and decreased levels of oxidative DNA damage as determined by alkaline comet assay modified for detection of oxidized DNA lesions. These results were observed in mutant but not wild-type BRCA1 breast cancer cells. Benserazide, but not tamoxifen (current FDA-approved breast cancer chemoprevention agent), also decreased in vitro tumorigenesis of mutant BRCA1 or BRCA1-depleted breast cancer cells according to the soft-agar colony formation assay, and delayed tumor formation in vivo using a xenograft mouse model. Lastly, benserazide led to elevated levels of 8-oxo-dG (a by-product of base-excision repair) in conditioned media of mutant BRCA1 breast cancer cells, and concurrently increased serum levels of 8oxodG and decreased tumor burden in mice. Taken together, benserazide is a DNA-repair activating agent that enhances repair of oxidative DNA damage and decreases tumorigenesis in vitro and in vivo, and thus may serve as a potential chemoprevention agent for BRCA1-mutated cancers. Further, circulating levels of 8oxoG/8oxodG may function as a predictive biomarker for the efficacy of benserazide, which would prove useful in the clinical evaluation of benserazide for cancer chemoprevention. Citation Format: Sujeeth Shanmugam, Eboni Hosch, Dipasri Konar, James M. Ford, Elizabeth Alli. Identification of a potential chemoprevention agent for BRCA1- mutated cancers [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr A003.

EZH2 Promotes Cholangiocarcinoma Development and Progression through Histone Methylation and microRNA-Mediated Down-Regulation of Tumor Suppressor Genes

Cholangiocarcinoma (CCA) is a highly malignant cancer of the biliary tree. Although studies have implicated enhancer of Zeste homolog 2 (EZH2) in CCA growth, the role of EZH2 in CCA development has not been investigated, and the mechanism for EZH2-regulated gene expression in CCA remains to be further defined. The current study used a mouse model of CCA induced by hydrodynamic tail vein injection of Notch1 intracellular domain and myristoylated-AKT plasmids. Mice with liver-specific EZH2 knockout displayed reduced CCA development. In a xenograft model, EZH2 knockdown significantly decreased CCA progression. Administration of the EZH2 inhibitor GSK126 decreased CCA tumor burden in mice. Accordingly, EZH2 depletion or inhibition reduced the growth and colony formation capability of CCA cells. Analysis of high-throughput data identified a set of 12 tumor-inhibiting genes as targets of EZH2 in CCA. The experimental results suggest that EZH2 may down-regulate these tumor-inhibiting genes through methylation of lysine 27 on histone H3 (H3K27) in the gene louses and through regulation of specific miRNAs. High mobility group box 1 was shown to facilitate the methyltransferase activity of EZH2, which is implicated in the regulation of CCA cell growth. The study shows that EZH2 promotes CCA development and progression through a complicated regulatory network involving tumor-inhibiting genes, miRNAs, and high mobility group box 1, which support targeting EZH2 as a potentially effective strategy for CCA treatment.

Chidamide Combined with Cytarabine Plays a Role in the Treatment of AML By Down-Regulating the MYC-RRP9 Pathway

Purpose: To explore the therapeutic effect and mechanism of Chidamide combined with Cytarabine in acute myeloid leukemia (AML), and to provide a new treatment strategy for AML patients. Methods: The effects of Chidamide and Cytarabine alone or in combination on the phenotype of AML cell lines were detected by MTT, flow cytometry and Western blot. Transcriptome sequencing and GSEA software were used to explore the mechanism of Chidamide combined with Cytarabine in the treatment of AML. RRP9 overexpression and knockdown AML cell lines were constructed by lentivirus to explore the effects of RRP9 on the proliferation, drug sensitivity, precursor ribosomal RNA (rRNA) process and protein production capacity of AML cell lines. Mature rRNA was detected by qPCR, nascent RNA was detected by EU kit, nucleolar area was observed by electron microscope, and nascent protein was detected by protein synthesis kit. Lentivirus and chromatin immunoprecipitation (CHIP) were used to explore the upstream regulatory genes of RRP9. The effects of Chidamide combined with Cytarabine on the survival rate and molecular level of primary AML cells were detected by CCK-8 and qPCR, respectively. An AML mouse model was constructed to explore the effects of Chidamide and Cytarabine on tumor burden and survival time in AML mice. Results: Chidamide combined with Cytarabine was more effective in killing AML cell lines than single drug, and the combination index was less than 1. The apoptosis rate (P<0.01) and the apoptotic protein level (P<0.05) induced by double drug were both higher than those induced by single drug. Chidamide combined with Cytarabine significantly down-regulated MYC pathway RRP9. In this pathway, RRP9 was selected for further research. An AML cell line overexpressing RRP9 was constructed. RRP9 overexpression promoted the proliferation of AML cell line (p<0.01) and induced the AML cell line to be resistant to the dual-drug combination (P<0.01). Construction of RRP9 knockdown AML cell line. The mature rRNA (P<0.01), nascent RNA (P<0.05), nucleolar area (P<0.0001) and nascent protein level (P<0.05) of cells in the RRP9 knockdown group were significantly lower than those in the control group. In addition, Chidamide combined with Cytarabine reduced the levels of mature rRNA (P<0.05), nascent RNA, nucleolar area (P<0.05), and nascent protein (P<0.05) in AML cell lines. The expression levels of RRP9 in MYC-overexpressing cells and knockdown cells were higher and lower than those in the control group, respectively (P<0.05). CHIP showed that the enrichment abundance of MYC at the RRP9 promoter site was significantly higher than that of IgG (P<0.05). The downregulation of MYC by dual-drug combination was stronger than that of single-drug(P<0.05). Chidamide combined with Cytarabine had stronger killing effect on AML primary cells than single drug, and the combination index was less than 1. Chidamide combined with Cytarabine down-regulated MYC (P<0.05) and RRP9 (P<0.01) mRNA levels in AML primary cells. The results of in vivo imaging of small animals showed that Chidamide alone could significantly reduce the tumor burden in mice (P<0.01), and the combination with Cytarabine further reduced the tumor burden (P<0.01). The survival time of mice in the Chidamide combined with Cytarabine treatment group was significantly longer than that in the control group (median survival time 28 days (28-31 days) vs 23 days (19-26 days)) (P<0.001), and the difference was still statistically significant compared with the single drug (P<0.05). Conclusion: Chidamide combined with cytarabine has a synergistic therapeutic effect on AML cell lines, primary cells and AML mice, which inhibits mature rRNA, total RNA and protein synthesis in AML cells caused AML cell death by synergistically down-regulating the MYC-RRP9 pathway.

Niclosamide Inhibits the Growth of Acute Lymphoblastic Leukemia Cells By Increasing Intracellular ROS and Activation of p53-PUMA/NOXA

Acute lymphoblastic leukemia (ALL) is a heterogeneous clonal malignancy originated from B- or T-cell lymphoid precursor cells. The prognosis is extremely poor in refractory or relapsed cases. Niclosamide is an FDA-approved oral anthelmintic drug with anti-cancer property. In the present study, we investigated the anti-leukemic effect of niclosamide in ALL cells in vitro and in vivo. ALL cell lines REH and NALM-6, and primary cells of patients with ALL were treated with niclosamide according to the concentration gradient, and then detected by trypan blue, MTS, and soft agar colony assays. The transcriptional and translational levels of apoptosis/anti-apoptosis-related genes were assessed by immunoblotting and quantitative real-time PCR. Flow cytometry was used to detect the effect of niclosamide on apoptosis of ALL cells, mitochondrial membrane potential, and intracellular specific reactive oxygen species (ROS) levels. For animal studies, we generated a mouse xenograft model with NOD prkdc-/-IL-2Rg-/- mice intravenously engrafted with NALM-6-luc cells, the NALM-6 cells infected with retrovirus expressing luciferase. Mice were treated with 25 mg/kg/day niclosamide or solvent twice a day intraperitoneally. The effect of niclosamide against leukemia cells in mice was dynamically evaluated by an in vivo imaging system. Flow cytometry was used to analyze the percentage of leukemia cells in spleens and bone marrows of sacrificed mice. The overall survival of mice was evaluated by Kaplan-Meier survival curve. In the cell viability assay, we found that niclosamide effectively inhibited REH, NALM-6 and primary ALL cells with IC50 values of 1.72, 3.58 and 1.36 μmol/L, respectively. Immunoblotting showed increased levels of pro-apoptotic proteins p53, PUMA, NOXA, cyto C and PARP, but decreased level of anti-apoptotic protein survivin in ALL cells treated with niclosamide in a dose- and time-dependent manner. Flow cytometry showed that niclosamide induced apoptosis and mitochondrial membrane potential in ALL cells. We also found that the level of ROS began to increase in cells treated by niclosamide for 2 hours, which reached the maximum at 8 hours. The treatment of ROS inhibitor N-acetylcysteine (NAC) attenuated niclosamide-induced apoptosis of REH and NALM-6 cells, which reduced by 18.12% and 8.02%, respectively. NAC also inhibited niclosamide-induced accumulation of intracellular p53/PUMA/NOXA proteins in these cells. In the animal study, in vivo imaging system showed significantly reduced tumor burdens in mice treated with niclosamide compared with controls, with bioluminescence signals of 15.53 and 4.04 on day 21, respectively. Mice treated with niclosamide survived longer than controls (32.5 days vs 26.5 days, p < 0.01), which had lower percentage of CD45+ CD19+ leukemia cells in their bone marrows and spleens, compared with the control group (61.10% vs 34.76% and 54.88% vs 26.40%, respectively). In the present study, we found that niclosamide potently inhibited the growth of ALL cells and induced apoptosis via increased levels of ROS and the activation of p53 signaling. Our findings suggest that niclosamide may be a promisingly potent agent for ALL therapy.

Molecular Analysis of Short- versus Long-Term Survivors of High-Grade Serous Ovarian Carcinoma.

Simple SummaryOvarian cancer is commonly associated with poor survival; patients with a diagnosis of high-grade serous ovarian carcinoma (HGSC) have an overall survival of 39% at 5 years. For reasons not well known, about 32% of ovarian cancer patients survive 10 years or more. In this study, our goal was to determine the molecular differences that drive long-term survival (LTS) in patients with HGSC. Indeed, this study shows that patients with LTS have a distinct pattern of gene expression, with TMEM62 being related to LTS. Increased TMEM62 expression led to decreased proliferation of ovarian cancer cells in vitro and decreased tumor burden in vivo.Despite having similar histologic features, patients with high-grade serous ovarian carcinoma (HGSC) often experience highly variable outcomes. The underlying determinants for long-term survival (LTS, ≥10 years) versus short-term survival (STS, <3 years) are largely unknown. The present study sought to identify molecular predictors of LTS for women with HGSC. A cohort of 24 frozen HGSC samples was collected (12 LTS and 12 STS) and analyzed at DNA, RNA, and protein levels. OVCAR5 and OVCAR8 cell lines were used for in vitro validation studies. For in vivo studies, we injected OVCAR8 cells into the peritoneal cavity of female athymic nude mice. From RNAseq analysis, 11 genes were found to be differentially expressed between the STS and LTS groups (fold change > 2; false discovery rate < 0.01). In the subsequent validation cohort, transmembrane protein 62 (TMEM62) was found to be related to LTS. CIBERSORT analysis showed that T cells (follicular helper) were found at higher levels in tumors from LTS than STS groups. In vitro data using OVCAR5 and OVCAR8 cells showed decreased proliferation with TMEM62 overexpression and positive correlation with a longevity-regulating pathway (KEGG HSA04213) at the RNA level. In vivo analysis using the OVCAR8-TMEM62-TetON model showed decreased tumor burden in mice with high- vs. low-expressing TMEM62 tumors. Our results demonstrate that restoring TMEM62 may be a novel approach for treatment of HGSC. These findings may have implications for biomarker and intervention strategies to help improve patient outcomes

DUSP1 inhibition in the treatment of high grade serous ovarian carcinoma (141.5)

<b>Objectives:</b> Dual Specificity Phosphatase (DUSP) proteins regulate signaling cascades involved in tumor progression, but there are limited studies of DUSPs in epithelial ovarian carcinoma, especially in high-grade serous ovarian carcinoma (HGSOC). The study objective was to elucidate <i>DUSP1</i>'s contribution to HGSOC progression and examine a <i>DUSP1/6</i> inhibitor (DUSPi) in therapy-resistant HGSOC. <b>Methods:</b> Bioinformatic analyses were conducted using The Cancer Genome Atlas (TCGA) Ovarian Serous Cystadenocarcinoma Firehose Database and PanCancer Atlas. qPCR was completed to determine the mRNA expression of <i>DUSP1</i>. Fifty percent inhibitory concentration of DUSPi was determined using colony formation assays. Reverse-phase protein array (RPPA) examined the effect of DUSPi on cellular signaling. The antitumoral effect of DUSPi was examined using a patient-derived xenograft (PDX) model. Quantitative data were expressed as mean +/- standard error of the mean. RPPA relative protein levels were determined for each and designated as log2 intensities and subsequently median-centered. Analysis of variance with Tukey correction was used for multiple comparisons. The level of significance was set at 0.05. <b>Results:</b> Of 24 DUSP proteins examined in HGSOC, <i>DUSP1</i> mRNA was significantly upregulated (p<0.001, one-way ANOVA). Progression-free survival (PFS) and overall survival (OS) were significantly higher in patients with low <i>DUSP1</i> expression (PFS: 15.41 vs 22.22 months, Log-rank test, p=0.0010, HR: 1.65, 95% CI: 1.213-2.259; OS: 41.52 vs 52.40 months, Log-rank test, p=0.0183, HR: 1.462, 95% CI: 1.064-2.00). Elevated <i>DUSP1</i> protein expression was noted in our tissue microarray of 137 serous ovarian carcinoma primary and recurrent tumors. Olaparib-resistant HGSOC cells had significantly increased <i>DUSP1</i> expression compared to olaparib-sensitive HGSOC cells (unpaired t-test, p<0.0001). The RPPA analysis revealed 34 proteins with significantly (p<0.05) altered expression after treatment with DUSPi. DUSPi led to the significant downregulation of <i>WEE1</i> and <i>ATR</i>. Phosphor-AMPK (AMPK-a2_pS345), part of the mTORC pathway that can serve both tumor-suppressive and oncogenic roles, was elevated and dual phosphorylated ERK (MAPK_pT202_Y204). RPPA findings were confirmed in TCGA data and by immunoblotting. A therapy-resistant PDX model was used to examine DUSPi <i>in vivo</i> activity. DUSPi led to a decrease in total flux (photons/sec), indicating tumor regression, in the mice treated with DUSPi compared to control. At the end of the study, there was no evidence of ascites or tumor burden in mice treated with DUSPi. <b>Conclusions:</b> <i>DUSP1</i> expression is associated with poor progression-free and overall survival. After treatment with a novel <i>DUSP1/6</i> inhibitor (DUSPi), cell viability was significantly reduced. We defined the differential activation of signaling following DUSPi via RPPA. DUSP inhibition significantly resulted in altering the expression of multiple pathway proteins, including increasing AMPK phosphorylation. Finally, we identified a robust <i>in vivo</i> antitumor response with DUSPi in PDX models.

Abstract 975: Obesity-induced changes in the tumor microenvironment impact the response to chemotherapy and overall ovarian cancer metastatic success

Abstract Background The majority of women with epithelial ovarian cancer (OvCa) are diagnosed with metastatic disease, resulting in a poor 5-year survival of 31%. Obesity is a recognized epidemic that increases OvCa incidence, enhances metastatic success and reduces survival. We have previously demonstrated a link between obesity and OvCa metastatic success in a diet-induced obesity (DIO) mouse model where a significantly enhanced tumor burden was associated with a decreased M1/M2 tumor-associated macrophage (TAM) ratio (Liu Y et al. Can, Res. 2015; 75:5046-57). TAMs are the most abundant immune cell type in the ovarian tumor microenvironment and are generally categorized as M1-polarized (cytotoxic to tumor cells), or M2-polarized (growth promoting). For this study we sought to examine if a similar correlation between body mass index (BMI) and TAMs were also true for human ovarian cancer patients. Furthermore, we used the DIO mouse model to examine any potential effect of obesity on response to chemotherapy. Methods We analyzed high grade serous ovarian tumors from 6 normal weight patients (BMI 20-25), and 10 obese patients (BMI ≥35) for an in-depth analysis of immune cells using MultiOmyx™, an immunofluorescence (IF) multiplexing assay. After multiplexing FFPE sections with a custom panel of 13 immuno-oncology biomarkers, images were analyzed by applying the deep-learning based cell classification platform NeoLYTX.For the analysis of response to chemotherapy we used a DIO mouse model in which mice were fed a low-fat diet (LFD) vs high-fat diet (HFD) and then injected with ID8-Trp53-/- cells to establish tumor burden. Mice bearing equivalent tumor burden were treated with weight-adjusted standard of care chemotherapy. After sacrifice, remaining tumor burden was evaluated by quantitative fluorescence imaging. Results We found that the mean M1/M2 ratio in obese OvCa patients was nearly half (ratio=0.16) compared to patients who were normal weight (ratio=0.29), consistent with our previously published mouse data. Quantitation of residual tumor burden in the mouse model showed significantly reduced efficacy of chemotherapy (i.e., greater remaining tumor burden) in HFD mice (n=11/cohort). The M1/M2 TAM ratio in these mouse tumors is now under evaluation. Conclusions Our observations suggest that the negative impact of obesity on OvCa survival may be due in part to an increased M1/M2 TAM ratio and reduced response to chemotherapy. These data will be key to a more detailed understanding of the contribution of host obesity to ovarian tumor progression. Citation Format: Anna Juncker-Jensen, Yueying Liu, Tyvette S. Hilliard, Nicholas Stavrou, M Sharon Stack. Obesity-induced changes in the tumor microenvironment impact the response to chemotherapy and overall ovarian cancer metastatic success [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 975.