bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 6024: Developing a panel of orthotopic syngeneic tumor models for IO drug discovery
Authors: Liu Gaoxiang , Sun Quanhong , Shi Wenting , Gu Qingyang , Ji Qunsheng
Published: June 2022

Immuno-oncology (IO) therapy is the focus of drug discovery in recent years. Unlike chemotherapy or targeted agents, the pharmacological efficacy of immune-oncology therapies needs to be evaluated in animal models with functional immune system. Syngeneic tumor models are established by inoculating mouse cancer cell lines to immunocompetent mice with the same genetic background. The host mice have complete immune activity and show histocompatibility with homograft tumor tissues, which can maximize the simulation of the real tumor microenvironment. Subcutaneous syngeneic models are easily established and have been widely used; however, the subcutaneous tumors lack organ-specific stromal-tumor interactions that are critical for disease progression in patients. Orthotopic tumor models are established in organ-specific sites, facilitating metastatic spread, supporting immune and stromal component interactions, and providing a more disease-relevant tumor microenvironment for IO therapy assessment. In order to facilitate the development of IO drug, we have established a panel of orthotopic syngeneic tumor models. We generated a series of luciferase-labeled bioluminescent mouse tumor cell lines, which were then engrafted into the organ of interest to establish orthotopic models. Bioluminescent imaging (BLI) enabled non-invasive in vivo imaging of orthotopic tumor burden, the real time tumor growth, and the response to therapies, monitored by quantitative bioluminescent signals. Overall, these established orthotopic syngeneic tumor models recapitulate the clinically relevant tumor microenvironment and serve as a useful tool in the pre-clinical evaluation of immunotherapies.

Citation Format: Gaoxiang Liu, Quanhong Sun, Wenting Shi, Qingyang Gu, Qunsheng Ji. Developing a panel of orthotopic syngeneic tumor models for IO drug discovery [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 6024.
Abstract 6033: Understanding the role of Cep55 in initiation and progression of cancers
Authors: Rashidieh Behnam , Tria Simon M. , Khanna Kum Kum
Published: June 2022

CEP55 is a cancer-testis antigen, highly expressed in a wide range of solid tumors as well as chromosomally unstable cancers. CEP55 is normally silent in most adult somatic tissues except germ cells; however, its expression is induced early during cell transformation. Numerous studies using cell line xenografts have shown that CEP55 is required for the viability and survival of a diverse range of cancers in vitro and in vivo. To decipher the mechanisms by which Cep55 promotes chromosomal instability and tumorigenesis in vivo, we developed a novel knock-in transgenic mouse model that ubiquitously overexpresses Cep55 (Cep55Tg/Tg) and a conditional knockout (cKO) mouse model of Cep55 (Cep55-/-). While overexpression of Cep55 was sufficient to cause spontaneous tumorigenesis in vivo, Cep55 loss hindered tumorigenesis. When we bred adult Cep55 cKO mice with other tumor-prone models: oncogenic Kras expression (Kras G12D) and Pten tumor-suppressor loss, Cep55 loss significantly delayed tumorigenesis and prolonged survival validating it as a promising therapeutic target for diverse cancers. This reduction of tumorigenesis is concomitant with the downregulation of AKT and ERK signaling pathways, attenuation of epithelial to mesenchymal transition, and increased cytotoxic CD4/CD8 T-cell tumor infiltration. This indicates that Cep55 can impair the establishment of the adaptive immune response through modification in the tumor microenvironment and regulation of cell signaling. Mass spectrophotometry of mouse embryo fibroblasts and derived exosomes from wild-type and KO mice revealed the pathways altered by Cep55 such as extracellular matrix remodeling pathways, integrin-related signaling and cellular trafficking mediators indicating the contributions of Cep55 to cancer are beyond what have been reported so far. Overall, CEP55 is a promising target to prevent tumorigenesis and the progression of cancer through various mechanisms.

Citation Format: Behnam Rashidieh, Simon M. Tria, Kum Kum Khanna. Understanding the role of Cep55 in initiation and progression of cancers [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 6033.
Abstract 6039: Application of triple negative breast cancer patient-derived organoid and xenograft model for drug screening
Authors: Ryu Seungyeon , Kim Hoe Suk , KIM Jung Eun , Yoon So-Hyun , Lee Sangeun , Baek Moonjou , Lee Han-Byoel , Lee Dong Woo , Ku Bosung , Han Wonshik
Published: June 2022

Background: Breast cancer Patient Derived Organoids (PDOs) and patient-derived xenografts (PDXs) which recapitulate the tumor heterogeneity and molecular signatures of original tumor tissue, are useful tools to enable greater precision of both approved and experimental medicines through personalized therapeutic approaches. We focus on use of ex vivo PDOs, PDX-derived organoids (PDXOs), and vivo PDXs models for drug testing and personalized therapy. In this study, we investigated the oncogenic role and therapeutic potency of targeting WEE1 in breast cancer.

Methods: Fresh surgical specimens of primary TNBC breast tumor (N=3) were used for PDOs. PDOs and PDX-derived organoids (PDXOs) were cultured in previously reported media compositions based on ECM hydrogel. PODs and PDXOs were subjected to drug screening for 22 anticancer drugs including chemoreagents and targeted drugs in the 3D HTS system. Drug sensitivity was tested in triplicate in different concentration ranges for 7 days. The IC50 for each drug was calculated by a sigmoidal dose-response curve, using the GraphPad Prism 9 program. For drug screening in PDXs models, PDOs were injected into the fourth mammary fat pads of NOD.Cg-Prkdcscid Il2rgtm1wjl/SzJ mice. WEE1 inhibitor (AZD1775) was administered by oral gavage (AZD1775, 30 mg/kg) for 3 weeks. Immunostaining of ER, PR, Her2, CK5 and Ki67 was performed in PODs, PODXs and PDX tumor tissues.

Results: All PDOs, PDXs, and PDXOs were ER-, PR- and HER2- negative. PDOs and PDXOs (172T, 185T, 207T) showed differential response to 22 anticancer drugs. In PDOs and PDXOs models, 172T and 207T PDOs and PDXOs are highly sensitive to AZD1775, but 185T PDOs and PDXOs less sensitive or never respond to treatment with AZD1775 (10 µM). In PDX models, treatment with AZ1775 decreased tumor growth (P<0.0079) as well as distance metastasis in 207T-PDX mice, whereas in 172T- and 185T-PDX mice, the tumor growth and metastasis were not reduced compared with the vehicle group.

Conclusion: Consequently, PDOs, PDXOs and PDXs recapitulated immunohistological signatures of original tumor tissues. The results of WEE1 inhibitor response between PDOs, PDXOs and PDXs were consistent. This study may provide a preclinical tool to screen drug responses to standard-of-care and newly identified drugs.

Citation Format: Seungyeon Ryu, Hoe Suk Kim, Jung Eun KIM, So-Hyun Yoon, Sangeun Lee, Moonjou Baek, Han-Byoel Lee, Dong Woo Lee, Bosung Ku, Wonshik Han. Application of triple negative breast cancer patient-derived organoid and xenograft model for drug screening [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 6039.
Abstract 6046: Targeting TBL1 inhibits nuclear β-catenin activity and enhances immune checkpoint inhibition efficacy in osteosarcoma
Authors: Nakahata Kengo , Nirala Bikesh K. , Shuck Ryan L. , Kurenbekova Lyazat , Yustein Jason T.
Published: June 2022

Background: Osteosarcoma (OS) is the most common bone malignancy in childhood and adolescence. Though the prognosis of OS patients has been improved by multimodal therapies, patients with metastatic disease still have a very poor prognosis. We previously reported a novel TBL1/β-catenin inhibitor, Tegavivint, suppresses OS primary tumor and lung metastasis using patient-derived cell lines. Wnt/β-catenin signaling is reported to influence intratumoral T cell infiltration and using murine syngeneic OS models we investigated the effects of Tegavivint on improving immune checkpoint inhibition and the OS tumor immune microenvironment.

Methods: We used murine OS cell lines F331 and F420 for in vitro and in vivo studies. We performed cytotoxicity studies using the CCK-8 assay and western blots to investigate downstream β-catenin signaling perturbations secondary to Tegavivint. Intratibial injections were performed into C57BL/6 mice using both murine cell lines. Tegavivint and anti-PD-1 antibody was administered as monotherapy or in combination. After treatment, we compared the tumor volume in the control, monotherapy (Tegavivint or PD-1 only) and combination therapy groups (Tegavivint and PD-1). We assessed T cell tumor infiltration using immunohistochemistry and flow cytometry.

Results: In vitro, Tegavivint suppresses the growth of F331 and F420 cell lines with IC50s of 9.52nM and 49.4nM, respectively after 72hours of treatment. In vivo, anti-PD1 monotherapy did not show any significant anti-tumor activity, but Tegavivint monotherapy significantly suppressed the growth of murine tumors (p<0.05) and the combination therapy further improved overall anti-tumor activity. IHC staining showed an increase in CD8-positive T cells in Tegavivint monotherapy and combination therapy treated tumors compared with the control tumors. Furthermore, flow cytometry validated the increase in CD8-positive T cell infiltration and also showed an increase in intratumoral NK cells in Tegavivint treated tumors.

Conclusions: These results indicate that TBL1 inhibition, via Tegavivint, inhibits nuclear β-catenin activity and alters the tumor immune microenvironment and enhances immune checkpoint blockade in OS. Future directions include more comprehensive profiling and molecular analysis of the tumor and immune microenvironment, and evaluation of additional immune modulatory approaches for the treatment of OS.

Citation Format: Kengo Nakahata, Bikesh K. Nirala, Ryan L. Shuck, Lyazat Kurenbekova, Jason T. Yustein. Targeting TBL1 inhibits nuclear β-catenin activity and enhances immune checkpoint inhibition efficacy in osteosarcoma [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 6046.
Abstract 6053: Characterizing plasticity of chemotherapy-tolerant persister cells in rhabdomyosarcoma
Authors: Tang Weiliang , Fritzke Madeline , Stinson Spencer , Chen Eleanor
Published: June 2022

Rhabdomyosarcoma (RMS) is a rare pediatric soft tissue sarcoma (250-300 cases a year in the US). The prognosis for patients with relapsed and metastatic disease remains poor with less than 30% overall survival rate. Current treatment regimens are ineffective against relapsed disease of RMS. There remains the urgent to need to identify more effective therapy options. While some studies have demonstrated up-regulation of multi-drug resistance genes and abnormal activity of selected signaling pathways in chemotherapy-resistant RMS tumors, the cellular and molecular mechanisms underlying adaptive cell changes leading to persistent survival of treatment-resistant RMS cells remain poorly understood. The stem cell-like tumor propagating cells (TPCs) have the capacity for self-renewal and are thought to be responsible for initiating treatment-resistant relapse and metastasis of some cancer types. The drug-tolerant persister (DTP) cell population with stem-cell like features has been shown in some cancer types to exhibit plasticity in cell state and dynamic changes in chromatin and transcriptional activity. A better understanding of how the cellular plasticity of the DTPs is regulated is critical for developing therapies to overcome treatment resistance in RMS. We have shown that initial treatment of RMS cells with standard-of-care cytotoxic agent, vincristine, selects for a DTP that is enriched with CD133+ stem-like cells. By gene expression studies, MYC and YBX1 driven-gene networks are upregulated in CD133+ cells. Targeted disruption of MYC and YBX1 by CRISPR/Cas9 reduces self-renewal capacity of RMS cells, and overexpression of MYC and YBX1 confers tolerance on RMS cells to vincristine. RMS cells treated in long-term culture with incremental increase in vincristine dose gives rise to late DTP with a distinct transcriptional profile including up-regulation of many genes regulated by the myogenic regulatory factor, MYOD1. MYC binds to the promoters of YBX1 and MYOD1. The findings suggest dynamic transcriptional changes and cell state plasticity of the DTPs in RMS. Our ongoing work includes characterizing the mechanisms by which MYC and MYOD1 regulate cell state plasticity and transcriptional adaptation of the DTP, assess inter- and intra-tumoral transcriptional heterogeneity of treatment-refractory RMS tumors and determine prognostic significance of functional network modules of MYC, MYOD and their downstream genes. Our ultimate goal is to translate our findings into more effective therapies for treatment-refractory RMS patients.

Citation Format: Weiliang Tang, Madeline Fritzke, Spencer Stinson, Eleanor Chen. Characterizing plasticity of chemotherapy-tolerant persister cells in rhabdomyosarcoma [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 6053.
Abstract 6061: Novel β-Apopicropodophyllin derivative, JNC-1043, exerts anti-cancer and radiosensitizing effects
Authors: Park Jong Kuk , Kang A-Ram , Kwon Jin-Hee , Choi Jong-Ryoo , Song Jie-Young , Hwang Sang-Gu
Published: June 2022

In this study, we reported novel anti-cancer drug candidate, JNC-1043{(5S)-5-(4-Benzyloxy-3,5-dimethoxy-phenyl)-5,9-dihydro-8H-furo[3',4':6,7] naphtho[2,3-d][1,3]dioxol-6-one}, synthesized from β-Apopicropodophyllin. Firstly, we analyzed anti-cancer effect of JNC-1043. We performed MTT assay to detect IC50 values on several cancer cells - breast, lung, pancreatic, blood, gastric, colorectal cancers. In these experiments, 0.1~1 μM IC50 values of JNC-1043 were calculated, and JNC-1043 treatments induced activation of caspase-3 and -8. Additionally, effective values of pharmacokinetic parameters in male rats and 2 weeks Dose Rate Finding (DRF) of JNC-1043 also detected. This result showed JNC-1043 is a promising anti-cancer candidate. Next, we tested whether radiosensitizing effect of JNC-1043. Each IC50 values of DLD-1 and HCT116 cell lines were calculated as 0.157, 0.1145, respectively. Cell counting assays were performed and then showed combination of JNC-1043 and γ-ionizing radiation (IR) enhanced cell death. JNC-1043 only- or IR only-treatment induced cell death by 50~60%, respectively, while combination of JNC-1043 and IR increased more 20~30% cell death than those of JNC-1043 only- or IR only-treatment in both cell lines. Clonogenic assay also performed, and then dose enhancement ratios (DER) of DLD-1 and HCT116 were calculated as 1.40, 1.53, respectively. Apoptosis also was detected with Annexin V-Propidium iodide assay. Combination of JNC-1043 and IR increased apoptosis more 30~40% than those of JNC-1043 only- or IR only-treatment. DCFDA-based assay also were performed to indicate ROS production was enhanced in combination of JNC-1043 and IR. In vivo xenograft experiment, combination of JNC-1043 and IR reduced tumor sizes less 50% and increased apoptosis more 30~40% than those of JNC-1043 only- or IR only-treatment. These results implied anti-cancer and radiosensitizing effect of JNC-1043 resulted from promotion of apoptosis via enhancement of ROS production.

Citation Format: Jong Kuk Park, A-Ram Kang, Jin-Hee Kwon, Jong-Ryoo Choi, Jie-Young Song, Sang-Gu Hwang. Novel β-Apopicropodophyllin derivative, JNC-1043, exerts anti-cancer and radiosensitizing effects [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 6061.
Abstract 6065: ASF1B potentiates stem cell traits and tumor progression in hepatocellular carcinoma via histone H3.3-dependent transcriptional reprogramming
Authors: Fang Xiaona , Liu Beilei , Liu Shan , Zhang Yu , Gong Lanqi , Yan Qian , Guan Xinyuan
Published: June 2022

Hepatocellular carcinoma (HCC) is one of the frequently diagnosed cancers worldwide and has emerged as the main cause of cancer-related death with a very poor prognosis, which is closely related to cancer stem cell (CSC), a subset of cells known to be a root of tumor recurrence, metastasis, and chemoresistance in HCC. However, the characteristics of CSCs in hepatocellular carcinoma (HCC) are still elusive. Our previous in vitro hepatocyte differentiation model revealed that the expression of anti-silencing function 1B histone chaperone (ASF1B) was remarkably upregulated in the stem cell-like stages. In HCC patients, ASF1B was highly expressed in cancer tissues compared with normal counterparts and was positively correlated with poor survival. Functional assays indicated that ASF1B could considerably promote the self-renewal, growth, and metastasis of HCC both in vitro and in vivo. Mechanistically, ASF1B could directly bind to histone H3 Variant H3.3 and enhance its incorporation into the promoter regions of certain stemness- and metastasis-associated genes including SOX9, KLF4, and TBX3, contributing to their augmented transcriptional and protein expression levels. H3.3 knock-down decreased the expression of these stemness and metastasis-associated genes, eliminating the stemness and oncogenic properties of ASF1B. Overall, our study revealed that ASF1B could facilitate HCC stemness and progression via H3.3-dependent transcriptional reprogramming. ASF1B might be a novel diagnostic and prognostic biomarker for HCC patients.

Citation Format: Xiaona Fang, Beilei Liu, Shan Liu, Yu Zhang, Lanqi Gong, Qian Yan, Xinyuan Guan. ASF1B potentiates stem cell traits and tumor progression in hepatocellular carcinoma via histone H3.3-dependent transcriptional reprogramming [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 6065.
Abstract 6068: Restoring the balance between WNT and retinoic acid signaling to promote differentiation of cancer stem cells in treatment of colorectal cancer
Authors: Facey Caroline O. , Hunsu Victoria O. , Osmond Brian T. , Opdenaker Lynn M. , Boman Bruce M.
Published: June 2022

Given that the stem cell (SC) marker ALDH is a key component of the retinoic acid (RA) signaling pathway and since APC mutation leads to increased WNT signaling and overpopulation of ALDH+ SCs in colorectal cancer (CRC), we hypothesized that human CRC evolves due to an imbalance between dysregulated RA and WNT signaling. Herein, we investigated how to target the link between these two pathways to restore proper WNT and RA signaling, and promote differentiation of CSCs along the neuroendocrine cell (NEC) lineage. Accordingly, we conducted in vitro experiments to determine if restoring wt-APC in APC-mutant CRC cells reduces Wnt/β-catenin signaling, increases sensitivity to retinoids, reduces cell proliferation, reduces expression of stem cell markers, reduces the number of ALDH+ cells, and enhances NEC maturation. We utilized Nanostring profiling, TCF reporter assay, western blot, and fluorescence activated cell sorting analyses to ascertain the effects of restoring WNT in HT29 CRC cells. We found that induction of wt-APC expression decreased WNT/β-catenin signaling and reduced protein expression of WNT-target genes. Notably, inducing wt-APC decreased ATRA-induced expression of the WNT target gene CYP26A1 (by 50%), which is predicted to increase RA signaling by lowering the degradation of RA. Indeed, wt-APC increased sensitivity of CRC cells to ATRA-induced inhibition of cell proliferation in a dose-dependent manner. Expression of ALDH1A1 decreased 3-fold by ATRA treatment and inducing wt-APC furthered the decrease by an additional 2-fold. Furthermore, we found an increase in the protein expression of several NEC markers including CHGA, GLP2R, NSE (ENO2), and SSTR1, and an increased number of GLP2R+ NECs upon inducing wt-APC. Thus, the link between WNT and RA signaling at the level of CYP26A1, provides a mechanism that can explain how mutant APC leads to decreased maturation of ALDH+ SCs along the NEC lineage and an increased number of ALDH+ SCs. Therefore, discovering ways to increase differentiation of SCs along the NEC lineage in vivo might lead to new more effective treatment strategies involving retinoids for CRC patients.

Citation Format: Caroline O. Facey, Victoria O. Hunsu, Brian T. Osmond, Lynn M. Opdenaker, Bruce M. Boman. Restoring the balance between WNT and retinoic acid signaling to promote differentiation of cancer stem cells in treatment of colorectal cancer [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 6068.
Abstract 6073: First-in-class Doublecortin-like kinase 1 (DCLK1) inhibitor for targeting cancer stem cell in hepatocellular carcinoma
Authors: Yoon Daseul , Jung Seung Hee , Jang Ju Young , Ahn Ji-Young , Jo Seung Jin , Ko Donghyun , Kim Dong-kyu , Yu Su Jong
Published: June 2022

Cancer stem cells (CSCs) have been considered promising therapeutic targets for cancer therapy. These cells have self-renewal capacity and differentiation potential and contribute to multiple tumor malignancies, such as recurrence, metastasis, heterogeneity, multidrug resistance, and radiation resistance. Doublecortin-like kinase 1 (DCLK1) was recently identified as CSC marker is upregulated in several solid tumors, which distinguishes between cancer and normal stem cell. However, the role of DCLK1 in hepatocellular carcinoma (HCC) is unclear. According to the open database like TCGA, overexpression of DCLK1 is reported to be associated with poor prognosis in HCC patients. It was also observed that DCLK1 expression was significantly increased in the advanced stage of HCC. In addition, as a result of measuring the serum DCLK1 levels obtained from patients with liver cirrhosis or HCC treated at Seoul National University Hospital, it was confirmed that the expression of DCLK1 was significantly increased in patients with HCC compared to those with liver cirrhosis. It was also confirmed that DCLK1 was expressed in human HCC cell lines and that the expression levels of DCLK1 mRNA and protein were further increased in CSC rich human HCC cell lines. Moreover, cell death was increased at the cellular level when DCLK1 was blocked via inhibiting expression of CSC markers such as CD133 and EpCAM. After evaluating the effect of DCLK1 inhibition in CSC rich human HCC cells lines, it was found that spheroid formation decreased by suppressing CSCs markers including LGR5 and CD133. In addition, it was confirmed that cell migration decreased when DCLK1 was blocked. Even in the murine HCC model, it was found that the antitumor effect was showed when administered with DCLK1 inhibitor, DCLK1-IN-1, in xenograft model with CSC rich HCC cell lines. Our results support that DCLK1 can be used as a biomarker for the detection of HCC and may be a candidate for developing targeted therapeutics to eradicate HCC.

Citation Format: Daseul Yoon, Seung Hee Jung, Ju Young Jang, Ji-Young Ahn, Seung Jin Jo, Donghyun Ko, Dong-kyu Kim, Su Jong Yu. First-in-class Doublecortin-like kinase 1 (DCLK1) inhibitor for targeting cancer stem cell in hepatocellular carcinoma [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 6073.
Abstract 6099: Differential gene expression related to TMPRSS2-ERG fusion, CDK12, RB1, TP53, AR and ZEB1 based on transcriptomic analysis of the TCGA PRAD cohort
Authors: Lautert-Dutra William , Melo Camila Morais , Chaves Luiz Paulo , Caliari André Luiz , Silva Francisco Cesar Souza , Squire Jeremy Andrew
Published: June 2022

Prostate Cancer (PCa) is considered an immunologically excluded tumor, unable to generate an effective T-cell response against cancer cells. Inactivating mutations of the PTEN tumor suppressor are one of the most common events in human malignancies that contribute to tumorigenesis by affecting many cancer Hallmark pathways. The CDK12, RB1, TP53, AR and ZEB1 genes have been variously implicated in pathways of PCa progression and immune evasion. Here, we present a comprehensive analysis of The Cancer Genome Atlas derived PCa cohort (n=488, 96 homozygous and 64 hemizygous PTEN loss). Differential gene expression (DGE) and pathway enrichment analysis were performed for all five genes using the DESeq2 tool from the Bioconductor package for R. All comparisons were designed to compensate for gene expression changes caused by PTEN loss (Table 1).

Functional pathway analysis showed that fusion positive samples presented several downregulated pathways, including leukocyte mediated cytotoxicity, IFN type I response and IFN-γ production. AR high presented upregulation of pathways such as DNA replication and chromatin remodeling, while AR low presented upregulation of pathways such as T cell activation and lymphocyte proliferation. ZEB1 high presented a wide range of enriched pathways, including upregulation of mesenchymal cell differentiation and T cell activation. CDK12-Hom group exhibited upregulation of T cell activation, T cell differentiation and IFN-γ production. TP53 mutation revealed upregulation of various pathways including DNA replication and cell division. RB1 loss showed enrichment of pathways such as T cell activation and lymphocyte activation. Our analysis showed that variation in these five genes can promote gene expression changes associated with clinically relevant pathways independently of PTEN and that AR, CDK12, TMPRSS2-ERG fusion, ZEB1 and RB1 status are potential biomarkers for immunotherapy response.

Table 1. Differential genes expression results summary Comparison # of upregulated (LFC>0) # of downregulated (LFC<0) Fusion positive vs. Fusion negative 2997 5418 AR low vs. AR medium 4133 4167 AR high vs. AR medium 4365 2589 CDK12-Hom vs. CDK12 WT 2416 713 CDK12-Hem vs. CDK12 WT 704 387 ZEB1 high vs. ZEB1 normal 6439 6792 RB1 mut vs. RB1 WT 78 335 TP53 mut vs. TP43 WT 859 1193

Citation Format: William Lautert-Dutra, Camila Morais Melo, Luiz Paulo Chaves, André Luiz Caliari, Francisco Cesar Souza Silva, Jeremy Andrew Squire. Differential gene expression related to TMPRSS2-ERG fusion, CDK12, RB1, TP53, AR and ZEB1 based on transcriptomic analysis of the TCGA PRAD cohort [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 6099.
Abstract 6105: Single high-dose irradiation induced iRhom2 up-regulation promotes macrophage anti-tumor activity through cGAS/STING signaling
Authors: Zhao Xiaomei , Du Shisuo , Wang Biao , Zeng Zhaochong
Published: June 2022

Clinical application of Stereotactic body radiotherapy (SBRT) allows high dose of radiation to be delivered to extracranial target within the body safely, however, high dose per fraction and with fewer fractions(hypofractionation) opened up the radiation oncology field to new questions on variety of dose-fractionation schedules, especially, the immunomodulatory effects of radiotherapy, which changes following the variety of dose-fractionation schedules. In the present study, we excluded the direct antitumor effect of radiation by comparing three different fractionation schedules –8*2Gy, 3*4.5Gy, 1*10Gy, which have the same biologically effective dose (BED). We found the fractionation schedules 1*10Gy had a significant higher antitumor effect, suggesting that a single high-dose induced a more enhanced anti-tumor immunity compared to the conventionally fractionation(8*2Gy) and moderately fractionation (3*4.5Gy). However, in STING deficiency mice, we failed to find a different tumor control effect among the three dose-fractionation schedules, suggesting cGAS/STING signaling was involved in regulating immunomodulatory effects that leading to different anti-tumor immunity. Further mechanism investigation revealed that single high-dose irradiation induced up-regulation of iRhom2 in infiltrated macrophage, and iRhom2 promoted cGAS/STING signaling via maintaining stability of STING and facilitating STING trafficking from ER to perinuclear microsomes. Meanwhile, a positive feedback loop between iRhom2 and TNFα sharpened the signaling responses, and amplified antitumor immunity induced by cGAS-STING-IFN pathway after single high-dose irradiation. Taken together, these results suggest that single high-dose irradiation induced iRhom2 up-regulation promotes macrophage anti-tumor activity through cGAS/STING signaling.

Citation Format: Xiaomei Zhao, Shisuo Du, Biao Wang, Zhaochong Zeng. Single high-dose irradiation induced iRhom2 up-regulation promotes macrophage anti-tumor activity through cGAS/STING signaling [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 6105.
Abstract 6122: Different characteristics of tumor immune microenvironment between LUSC and LUAD
Authors: Li Na , Hu Mengna , Zhang Ding , Ji Xiaoyu
Published: June 2022

Background: Patients with lung squamous cell carcinoma (LUSC) have higher tumor mutation burden (TMB) and PD-L1 expression than those with lung adenocarcinoma (LUAD). Several studies showed that tumor immune microenvironment (TIME), mainly the tumor-infiltrating immune cells, may affect the efficacy of immunotherapy. However, the comparison of tumor immune microenvironment between LUAD and LUSC is little known.

Methods: 14 patients with LUSC and 21 patients with LUAD were enrolled in the retrospective analysis of multiple immunofluorescence staining to assess the number of immune cells. Genomic profiling of DNA was performed on formalin-fixed paraffin-embedded tumor samples from 3752 LUAD and 459 LUSC patients by NGS with 733 cancer-related genes panel. TMB is defined as the total counts of single nucleotide variants and indels in the exon region.

Results: Overall, as the most abundant tumor-infiltrating immune cells in both groups, CD3+ cells are more in LUAD [median (number/mm2 in LUSC vs in LUAD): 312 vs 918, p = 0.02]. However, there were no significant differences in CD8+ T cells (median: 48 vs 36, p = 0.74), indicating higher infiltration of T lymphocytes rather than cytotoxic T lymphocytes (CTLs) in LUAD than that in LUSC. Unlike in tumor, LUAD patients had both more T lymphocytes (p < 0.001) and CTLs (p < 0.01) in stroma. Besides, LUSC was correlated with fewer M2 tumor-associated macrophages (TAMs) and PD-1+ immune cells (p < 0.001; p = 0.02), while no significant differences were found in tumor-infiltrating FOXP3+ cells (p = 0.41) and B lymphocytes (p = 0.61). In addition, natural killer cells were the fewest among the tumor-infiltrating lymphocytes detected in both groups (median: 2 vs 13). Similar to the previous studies, TMB level was higher in LUSC (median: 8.94 vs 3.36, p < 0.0001). Although LUSC patients had a larger proportion of positive PD-L1 expression (45% vs 30.5%, p < 0.0001) and strong PD-L1 expression in tumor cells (20.9% vs 10.7%, p < 0.0001), the number of CTLs and TAMs expressing PD-L1 is significantly lower than that in LUAD ( p = 0.04; p = 0.0001).

Conclusion: LUAD had more immune invasion, while LUSC was related to higher TMB and PD-L1 expression. It suggested that the mechanisms affecting the benefits of immunotherapy may be different. The immune microenvironment of tumor and stroma should be considered simultaneously to explore the mechanism of immunotherapy and the respective thresholds should be selected according to the types of immune cells.

Citation Format: Na Li, Mengna Hu, Ding Zhang, Xiaoyu Ji. Different characteristics of tumor immune microenvironment between LUSC and LUAD [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 6122.
Abstract 6133: Intratumoral budding in pretreatment biopsies can predict prognosis and neoadjuvant therapy response among the tumor microenvironmental components in colorectal adenocarcinoma
Authors: Yim Kwangil , Jang Won Mo , Chong Yosep , Shin OK Ran , Seo Kyung Jin
Published: June 2022

Purpose: Neoadjuvant therapy has been used as one of a treatment option in locally advanced rectal cancer and unresectable colorectal cancers (CRCC) with oligo-metastasis. Considering that radical specimen has been deformed due to this treatment, predicting prognosis is more complex. We evaluated several tumor microenvironmental factors in the pretreatment biopsies; those were known as prognostic factors in radical specimen.

Methods: From 2010 to 2021, 85 patients with neoadjuvant therapy and surgery for CRCC were enrolled in Uijeongbu St. Mary’s hospital. Intratumoral budding (ITB), tumor stromal ratio, presence of immature stroma and Klintruo-Makinen grade for inflammatory cells were evaluated in pretreatment biopsies by three pathologists, independently.

Results: Only high grade ITB (OS: p = 0.030, CSS: p = 0.005, DFS: p < 0.001) was poor survival factor in univariate analysis. In addition, high grade ITB was related with pathological stage (p = 0.017), lymphatic invasion (p = 0.010) and chemoresistance in non-metastatic CRCC group (p = 0.046). Presence of immature fibrosis was related with clinical stage (p = 0.045) and pathological stage (p = 0.033). In multivariate analysis, high grade ITB was independent poor survival factor (OS: p = 0.031, CSS: p = 0.009, DFS: p = 0.001).

Conclusion: Our study revealed that high grade ITB in pretreatment biopsied was poor survival factor in CRCC with neoadjuvant treatment. In our observation, we recommend that ITB result should be included in the pathology report when considering neoadjuvant treatment.

Citation Format: Kwangil Yim, Won Mo Jang, Yosep Chong, OK Ran Shin, Kyung Jin Seo. Intratumoral budding in pretreatment biopsies can predict prognosis and neoadjuvant therapy response among the tumor microenvironmental components in colorectal adenocarcinoma [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 6133.
Abstract 6139: GPR4 promotes the development of colitis-associated colorectal cancer in an experimental mouse model
Authors: Marie Mona A. , Sanderlin Edward J. , Hoffman Alexander , Yang Li V.
Published: June 2022

GPR4 is a proton-sensing G protein-coupled receptor expressed in endothelial cells as well as other cell types. Endothelial cell adhesion, blood vessel permeability, leukocyte infiltration, and angiogenesis are regulated by the proinflammatory functions of GPR4. Inhibition of GPR4 ameliorates inflammatory bowel disease (IBD) in mouse models. GPR4 expression is upregulated in the intestinal tissues of IBD and colorectal cancer patients and is associated with a worse prognosis. Herein, we studied the role of GPR4 in colitis-associated colorectal cancer (CAC) by inducing CAC in GPR4 knockout (KO) and wild-type (WT) mice using azoxymethane (AOM)/dextran sulfate sodium (DSS). The severity of CAC development was higher in WT mice than GPR4 KO mice when treated with AOM/DSS. Blood vessel density was significantly higher within the tumors of WT mouse colons compared to GPR4 KO mice. GPR4 KO tumors showed a significant increase in tumor necrosis in comparison to WT tumors. Fecal blood/diarrhea score, colon shortening, and mesenteric lymph node size were aggravated in WT mice compared to GPR4 KO mice treated with AOM/DSS. Our data demonstrate that GPR4 increases intestinal inflammation and angiogenesis, enhancing tumor development in the CAC mouse model, suggesting that GPR4 inhibition may reduce the risk of developing colitis-associated colorectal cancer.

Citation Format: Mona A. Marie, Edward J. Sanderlin, Alexander Hoffman, Li V. Yang. GPR4 promotes the development of colitis-associated colorectal cancer in an experimental mouse model [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 6139.
Abstract 6158: Rewiring cancer associated fibroblasts using nanomedicines improves immunotherapy efficacy
Authors: Elzoghby Ahmed , Freag May , Mohammed Mostafa , Maremanda Krishna , Jang Hae Lin , Sengupta Shiladitya
Published: June 2022

The failure of large number of patients to respond to cancer immunotherapy remains a formidable challenge. The immunosuppressive tumor microenvironment (TME) can hinder the capability of cytotoxic T cells to fight cancer. In this regard, cancer associated fibroblasts (CAFs) are among the most abundant stromal cells in TME responsible for immunosuppression. Therefore, reversal of the immunosuppressive phenotype of CAFs and is a major therapeutic goal. Cancer cells secrete exosomes and other factors to reprogram fibroblasts and induce their differentiation into pro-tumorigenic CAFs. Also, cancer cells maintain the activated fibroblast phenotype via triggering major signaling pathways e.g., TGFβ1, FGFR, or β-catenin pathways in the fibroblasts. Here, our goal was to maintain a normalized quiescent phenotype of fibroblasts to enhance the efficacy of immune checkpoint blockade. We demonstrated that a tumor-targeted EpCAM antibody modified shikonin/GW4869 combination liposomes synergistically reduces the biogenesis and release of exosomes from A549 lung cancer cells which significantly inhibits differentiation of MRC5 lung fibroblasts into CAFs. In parallel, we showed the efficacy of TAS-120/ICG-001 FGFR-Wnt/β-catenin inhibitor combination liposomes decorated with GPR77 and CD10 targeting antibodies to switch the acquired activated CAFs phenotype to a quiescent one. Co-administration of both liposomes in low drug doses significantly enhanced the infiltration of cytotoxic CD8+ T cells and improved the antitumor efficacy of αPD-L1 antibody in LLC syngeneic mice model of lung cancer. In conclusion, simultaneous blockage of the tumoral exosome-mediated activation of fibroblasts and FGFR-Wnt/β-catenin signaling may constitute an outstanding immunotherapeutic modality.

Citation Format: Ahmed Elzoghby, May Freag, Mostafa Mohammed, Krishna Maremanda, Hae Lin Jang, Shiladitya Sengupta. Rewiring cancer associated fibroblasts using nanomedicines improves immunotherapy efficacy [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 6158.
Abstract 617: MEK inhibitor rescued the efficacy of PD-1 blocker in STK11/LKB1 mutated colorectal cancer model
Authors: Liu Demi X. , Zhou Jun , Nie Chenpan , An Annie X. , Li Henry Q.X. , Wang Jingjing
Published: June 2022

Introduction: The mechanism of action of primary resistance to PD-1 blockade is largely unknown, however STK11/LKB1 alterations have been reported to have a significant role in primary resistance to PD-1/PD-L1 axis inhibitors in KRAS-mutant lung adenocarcinoma. It is importsnt to identify treatments which can rescue the efficacy of PD-1 blocker in STK11/LKB1-mutant patients. We have developed a STK11 knockout (KO) model using the CT26 colorectal cancer syngeneic line which harbors a KRAS mutation and evaluated the response to checkpoint inhibitor alone and in combination with MEK inhibitors.

Method: CT26-STK11KOwas developed by CRISPR/Cas9 technology and STK11/LTB1 deletion was verified by Western Blot. To test the response of the cell line to anti-PD-1 treatment (10mg/kg), we engrafted CT26-WT and CT26-STK11KO tumor lines into BALB/c mice. Tumor growth inhibition (TGI) was assessed as TGI%=(1-Ti/Vi) x100%; Ti as the mean tumor volume of the treatment group on the measurement day; Vi as the mean tumor volume of control group at the measurement day. At termination FACS analysis of different major types of immune cells was performed. Combination with MEK inhibitors, including Trametinib (1mg/kg) was also evaluated.

Results: In comparison to CT26-WT tumor growth, PD-1 blocker could not inhibit CT26-STK11KO tumor progression. However, Combinational treatment of Trametinib and anti-PD-1 antibody delayed CT26-STK11KO tumor growth. In CT26-WT tumor bearing mice, anti-PD-1 monotherapy increased total CD45+ immune cells, suggesting an ongoing immune response; significantly increased the ratio between M1 and M2, which might be an indicator for enhanced type I T cell response. CD8+ T cell infiltration in CT26-STK11KO tumors was dramatically decreased associated with increased percentage of G-MDSC which may have contributed to the loss of response to anti-PD-1 treatment. MET inhibitor, Trametinib, decreased G-MDSC and restored CD8+ T cell infiltration and PD-1 blocker efficacy in CT26-STK11KO tumor.

Conclusions: We successfully generated a CT26-STK11KO cell line which was valuable for evaluation of PD-1 blocker and MEK inhibitor in STK11/LTB1 deficient tumor, in which Trametinib could rescue the efficacy of PD-1 treatment in CT26-STK11KO tumor via inhibiting G-MDSC. Taken together, our data suggest that altered immune cell infiltration, particularly increased G-MDSCs, in tumor microenvironment might be the reason for poor PD-1 blocker efficacy in CT26-STK11KO tumor.

Citation Format: Demi X. Liu, Jun Zhou, Chenpan Nie, Annie X. An, Henry Q.X. Li, Jingjing Wang. MEK inhibitor rescued the efficacy of PD-1 blocker in STK11/LKB1 mutated colorectal cancer model [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 617.
Abstract 6171: A single-cell solution for solid tumors to detect mutations and quantify copy number variations
Authors: Parikh Saurabh , Patel Khushali , Li Alex , Parikh Anup
Published: June 2022

Cancer is a highly heterogeneous disease characterized by the presence of chromosomal structural copy number variation (CNV) and driver gene mutation events, which appear somatically at the early stages of oncogenesis and can drive tumor progression. Previously, we have developed a high throughput single cell DNA analysis platform that leverages droplet microfluidics and a multiplex-PCR based targeted DNA sequencing approach. It demonstrates high sensitivity detection of SNVs and indels in the same cells and generation of high resolution maps of clonal architecture based on mutational profiling. Here we expand upon the same approach and create an algorithm to accurately quantify CNVs along with genetic mutations from the same single cells. To estimate the CNV the amplicon read counts were corrected for artefacts related to cell to cell variability due sequencing efficiencies along with amplicon to amplicon variability that occur due to amplification efficiency differences. We used a spiked in diploid cell line as a reference to normalize all the cells. The spiked cell line was identified using its mutational profile. We validated the system using cell lines with known CNVs and found that we could at least accurately estimate copy numbers ranging from 0 through 5. Some of the regions were also validated orthogonally using droplet digital PCR. Another set of validation was performed using well characterized cell lines from NIST. We enable multiple visualizations of the copy number estimates in karyotype plots and line plots projected on SNV clones. With improved biochemistry, panel design and novel data analysis algorithms we develop a complete solution to detect amplification or loss of function in oncogenes and tumor suppressors reliably. Integration of CNVs and SNVs facilitates more accurate reconstruction of tumor evolution to better understand cancer progression mechanisms as well for quality control of gene edited cells, to further advance cancer research and therapy.

Citation Format: Saurabh Parikh, Khushali Patel, Alex Li, Anup Parikh. A single-cell solution for solid tumors to detect mutations and quantify copy number variations [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 6171.
Abstract 6175: Developing antibody-drug conjugates against cholangiocarcinoma using a novel molecular target
Authors: Zhu Bing , Yao Shi-Li , Wang Xin-Yan , Xu Rui , Guo Peng , Ying Jie-Er
Published: June 2022

As a rare malignancy, cholangiocarcinoma (CCA) is a lethal disease with a 5-year overall survival rate of merely 5%. Highly invasive and asymptomatic characteristics of CCA cause most patients diagnosed at advanced stages, severely deteriorating their clinical outcomes. The drug treatment options for CCA are limited and surgical resection of CCA frequently suffers from a high recurrence rate of 66%. Albeit Pemigatinib, a FGFR2 inhibitor, has recently been approved by FDA as the first CCA-targeted therapeutic, only less than 10% of patients with CCA carry FGFR2 fusion mutations that can benefit from this drug. Ivosidenib, another targeted drug against IDH1 mutation, only works for 13% CCA patients carrying this mutation. Therefore, discovering new molecular targets and developing associated targeted drugs remain a significant and unmet medical need in CCA therapy. In this study, we first screened potential molecular targets for CCA by comparing the expression levels of a panel of cancer-related cell membrane antigens between human CCA cells and normal human intrahepatic biliary epithelial cells through flow cytometry. CD54 was identified as a remarkable molecular target of CCA with strong binding force to its antibodies and high endocytosis efficiency by flow cytometry and confocal fluorescent microscopy. Meanwhile, we confirmed that CD54 was absent in most normal human tissues but was highly upregulated in CCA tumor tissues through immunohistochemical (IHC) staining. Based on this newly discovered CCA target, we constructed a series of CD54 antibody-drug conjugates (ADCs) using different chemical linkers and payloads. We next evaluated and compared the half maximum inhibitory concentrations (IC50s) of each ADC in ablating human CCA cells in vitro in order to identify an optimized ADC formulation. Furthermore, we validated its anti-tumor efficacy against primary and peritoneal metastatic CCA tumors in vivo in comparison with conventional chemotherapy. Eventually, we determined the biodistribution and safety profile of this CD54 ADC in order to ensure that it is well-tolerated under the effective dose. In general, we identified CD54 as a novel molecular target for CCA through antibody library screening, and explored its potential in the development of CCA-targeted ADCs, providing a promising targeted therapeutic for clinical treatment of CCA.

Citation Format: Bing Zhu, Shi-Li Yao, Xin-Yan Wang, Rui Xu, Peng Guo, Jie-Er Ying. Developing antibody-drug conjugates against cholangiocarcinoma using a novel molecular target [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 6175.