bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3099: Biological evaluation of a novel AKR1C3 inhibitor in patient-derived prostate cancer cell line and xenograft models
Authors: Yang Joy C. , Ning Shu , Adomat Hans , Gleave Martin , Gao Allen , Evans Christopher P. , Liu Chengfei
Published: June 2022

INTRODUCTION AND OBJECTIVES: Prostate cancer is a highly heterogeneous cancer type with distinct genomic and phenotypic characteristics that drive tumorigenesis and the differential response to drug therapies. A limit number of prostate cancer cell lines and patient-derived xenograft (PDX) models hinders research to improve disease outcome. Some currently available PDX models were derived from the primary tumor samples are insufficient to recapitulate the clinical response at more advanced stages. In this study, we developed patient-derived models from patients with advanced disease and evaluated a novel AKR1C3 inhibitor in these models.

METHODS: Samples received from our Pathology Biorepository Shared Resource were divided into four groups and subjected to pathological staining, RNA extraction, xenografting in NSG mice via renal capsule and subcutaneous implantation in SCID mice and conditional reprogramed cultures (CRCs) or organoid culturing. The AKR1C3 inhibitor PB was modified from celecoxib. Androgen receptor (AR), AR-V7 and AKR1C3 expression were determined by western blot. The effects of the AKR1C3 inhibitor on enzalutamide sensitivity were characterized by growth assay and colony formation assay.

RESULTS: Eight PDX models have been developed from prostate cancer patients with high Gleason score and/or at the castration-resistant stages. Among the PDX models, one spontaneous indefinite cell line PS1172 was established. Early passage CRCs showed the epithelial morphology with AR positive expression. Through serially passaging PS1172 PDX with castration in SCID mice, the castration resistant cell line 1172CR was re-cultured from castration-resistant PS1172 PDX tumors. 1172CR cells were resistant to enzalutamide treatment and expressed high level of AKR1C3 and AR-V7. A novel AKR1C3 inhibitor (PB) which displayed superior potential to inhibit AKR1C3 activity and suppress enzalutamide resistant prostate cancer cell growth was tested in these models. At the same dose, PB significantly suppressed 1172CR cell growth and colony formation compared to indomethacin and enzalutamide. PB also significantly suppressed AR/AR-V7 protein expression compared to indomethacin in 1172CR cells.

CONCLUSION: PS1172 and castration-resistant 1172CR cells are novel models with significant characteristics such as AR-V7 and AKR1C3. These novel prostate cancer models are ideal for small molecule testing and resistant mechanism investigating.

Citation Format: Joy C. Yang, Shu Ning, Hans Adomat, Martin Gleave, Allen Gao, Christopher P. Evans, Chengfei Liu. Biological evaluation of a novel AKR1C3 inhibitor in patient-derived prostate cancer cell line and xenograft models [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 3099.
Abstract 3103: Liquid tumor orthotopic PDX models of acute leukemia
Authors: Chen Hongkui , Bi Lintao , Zhao Shizhu , Wang Shuai , Huang Wenqiang , Caggiula Josh , Van Loc , Wen Danyi
Published: June 2022

Liquid tumor orthotopic xenograft models were established by intra femoral injection on NCG mice using freshly prepared leukemia cells from peripheral blood (PB) or bone marrow (BM) of AML/ALL patient samples. The establishment of the orthotopic models were monitored by the flow cytometry analysis of peripheral blood of engrafted mice:CD45+/CD33+ for AML and CD45+/CD19+ for B-ALL. The double positive cells in the peripheral blood of the successfully transplanted model can reach 5-15%. Typically, it takes 120 -170 days to establish the P0 AML orthotopic model. It takes 45-60 days to establish the P0 ALL orthotopic model. Spleen from engrafted P0 mice was used for P1-P3 reconstitution of the orthotopic model. On these orthotopic models, we tested whether medicines such as etoposide, cytarabine, vincristine, ibrutinib and imatinib are sensitive. And anti-CD47 antibody was also tested on these models. NGS profiling was used to confirm the genotype of established orthotopic PDX.

Citation Format: Hongkui Chen, Lintao Bi, Shizhu Zhao, Shuai Wang, Wenqiang Huang, Josh Caggiula, Loc Van, Danyi Wen. Liquid tumor orthotopic PDX models of acute leukemia [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 3103.
Abstract 311: Development of cludin-3 specific human monoclonal antibody(ABN501) as therapeutic anti-tumor agents
Authors: Choi Hong-Seok , Sim Euni , Kim Na Young , Lee Yong Jin , Lee Sae Hyung , Park Hayeon , Yang Hobin , Jo Jiwon , Seo Myeung-Ryun , Park Heegeon , Lee Ji-Hye , Hong Sungyoul , Shin Young Kee , Choi Jun-Young
Published: June 2022

The tight junctions of epithelial tissues, that most malignant tumors are derived from, are associated in cell-cell interactions. Among the tight junction proteins, claudin-3 (CLDN3) is overexpressed in many types of solid cancers, such as breast, ovarian, colorectal, and gastric cancers. Although CLDN3 can be potential therapeutic target due to the overexpression in various types of cancers, high homology with other CLDN family members is a hurdle to develop antibodies specifically targeting CLDN3. In this study, human IgG1 monoclonal antibody (ABN501) against CLDN3 was developed by scFv phage display using CLDN3-overexpressing stable cells and CLDN3-embedded lipoparticles as antigens. It was confirmed that ABN501 specifically bound to human and mouse CLDN3 without cross-reactivity to other CLDN family members. Sub-nanomolar affinity in binding kinetics of ABN501 was measured in CLDN3 expressing cell lines. We observed the antibody-dependent cytotoxicity (ADCC) activity of ABN501 with human NK cells expressing CD16a (NK-92MI-CD16a), in various cancer cell lines according to CLDN3 expression levels. We also confirmed that ABN501 specifically targeted CLDN3-expressing tumors in biodistribution assay using fluorescence-conjugated ABN501. In addition, ABN501 showed anti-tumor effects when treated with NK-92MI-CD16a in xenograft mice bearing CLDN3 expressing tumors. Taken these results together, we suggest that ABN501, specifically recognizing to CLDN3, can be used as therapeutic agents for CLDN3 positive cancer, and developed in many different forms to treat cancers with its specificity for cancer diagnosis, antibody-drug conjugates, and chimeric antigen receptor (CAR) immunotherapy.

Citation Format: Hong-Seok Choi, Euni Sim, Na Young Kim, Yong Jin Lee, Sae Hyung Lee, Hayeon Park, Hobin Yang, Jiwon Jo, Myeung-Ryun Seo, Heegeon Park, Ji-Hye Lee, Sungyoul Hong, Young Kee Shin, Jun-Young Choi. Development of cludin-3 specific human monoclonal antibody(ABN501) as therapeutic anti-tumor agents [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 311.
Abstract 3117: Next-generation characterization of patient-derived xenografts
Authors: Nakashima Jonathan , Do Long , Andrews Warren , Chien Yuan-Hung , Sperry Jantzen , Carapia Bianca , Yan Deborah , Rivera Giovanni , Federman Noah , Singh Arun , Eilber Fritz C. , Datnow Brian
Published: June 2022

Patient-derived xenografts (PDXs) have provided the research community with dynamic and robust translational model systems to study cancer biology and accelerate drug development. To advance translational oncology, we have developed a library of PDXs and provide next-generation sequencing characterization of these models. Here, we provide mutation and transcriptomic profiles of our PDX collection along with comparisons against matched normal and cancer patient profiles. Lastly, we provide a web portal providing comprehensive genomic profiles for every PDX model to identify mutation, copy number, fusions, microsatellite status, gene expression, and enriched pathways to facilitate identification of clinically relevant models.

Citation Format: Jonathan Nakashima, Long Do, Warren Andrews, Yuan-Hung Chien, Jantzen Sperry, Bianca Carapia, Deborah Yan, Giovanni Rivera, Noah Federman, Arun Singh, Fritz C. Eilber, Brian Datnow. Next-generation characterization of patient-derived xenografts [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 3117.
Abstract 3118: Functional characterization and therapeutic response differences between orthotopic and subcutaneous patient-derived xenograft models
Authors: Nakashima Jonathan , Do Long , Andrews Warren , Chien Yuan-Hung , Pedros Christophe , Sperry Jantzen , Carapia Bianca , Yan Deborah , Rivera Giovanni , Singh Arun , Eilber Fritz C. , Datnow Brian
Published: June 2022

Subcutaneous patient-derived xenografts (PDXs) have provided the research community with translational models to interrogate cancer biology and assist in drug development. Orthotopic PDXs provide an even more clinically relevant model system that recapitulates the tumor environment aspects of the human disease. Here, we compare the differences in gene expression signatures of PDX models when implanted subcutaneously or orthotopically. Furthermore, we evaluate differences in in-vivo pharmacological response between the two model systems and identify several functional characterizations that selectively enhance pharmacological response in favor of the orthotopic model.

Citation Format: Jonathan Nakashima, Long Do, Warren Andrews, Yuan-Hung Chien, Christophe Pedros, Jantzen Sperry, Bianca Carapia, Deborah Yan, Giovanni Rivera, Arun Singh, Fritz C. Eilber, Brian Datnow. Functional characterization and therapeutic response differences between orthotopic and subcutaneous patient-derived xenograft models [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 3118.
Abstract 3131: High levels of potassium impair myeloid-derived suppressor cell functions
Authors: Puttalingaiah Ramesh Thylur , Ochoa Augusto C. , Sanchez-Pino Maria Dulfary
Published: June 2022

Excess salt consumption has been associated with elevated blood pressure, cardiovascular disease, obesity, diabetes, fatty liver, chronic inflammation, cancer, and autoimmune diseases. The direct impact of salts on immune cells is poorly studied; however, a recent report showed that a higher level of both sodium and potassium in the tumor microenvironment reduce tumor growth by enhancing T cell function. Separately, it was shown that intake of high levels of sodium reduced tumor growth by impairing myeloid-derived suppressor cell (MDSC) functions. Still, the effect of high levels of potassium on MDSCs has not been examined. Here, we hypothesize that an excess of potassium in an in-vitro culture of mouse bone marrow-derived MDSC (mBM-MDSC) and purified tumor-derived MDSC impairs the cellular metabolism and their immunosuppressive function. To explore this, we evaluated the effect of potassium (40 mM) on the oxygen consumption rate (OCR) as a measurement of mitochondrial oxidative phosphorylation (OXPHOS). We also tested the immunosuppressive function of MDSCs by protein expression of arginase I (ArgI), inducible nitric oxide synthase (iNOS), and the immune checkpoint PD-L1 on mBM-MDSC, as well as directly measuring their ability to inhibit T cell proliferation. While excess potassium does not influence the proliferation or viability of mBM-MDSCs, it does affect their immunosuppressive capacity by reducing ArgI, iNOS and PD-L1 expression. Furthermore, excess potassium increases the expression levels of the autophagosome marker LC3β, enhances OCR (an indicator of mitochondrial respiration), and improves the fitness of mitochondrial bioenergetics (maximum respiratory capacity). Interestingly, using the autophagy inhibitor 3-methyladenine (3-MA) in the presence of excess of potassium significantly reduces OCR and rescues ArgI expression compared to control or potassium treatment alone. Additionally, potassium significantly reduced the production of several chemokines and anti-inflammatory cytokines, including CXCL1, RANTES and IL-10, as well as the expression levels of C/EBP-β and phosphorylated AMPK, suggesting an alteration of key signaling pathways required for MDSC function. Collectively, this study provides evidence that high levels of potassium change the cellular metabolism of MDSC, reduce their immunosuppressive capacity, impair the signaling of AMPK, C/EBP-β, and promote autophagy. This may provide novel insights for further investigation on the impact of hyperkalemia or microenvironments with increased levels of potassium, such as intratumorally, on MDSCs and their role on tumor growth.

Citation Format: Ramesh Thylur Puttalingaiah, Augusto C. Ochoa, Maria Dulfary Sanchez-Pino. High levels of potassium impair myeloid-derived suppressor cell functions [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 3131.
Abstract 3137: Elucidating the role of RNA-binding proteins in pancreatic cancer extracellular vesicle crosstalk
Authors: Finan Jennifer M. , Di Niro Roberto , Armstrong Randall , Brody Jonathan R.
Published: June 2022

Many solid tumors, including pancreatic ductal adenocarcinoma (PDAC) tumors, rely on pro-tumorigenic intercellular paracrine signaling during tumor progression. Extensive work has been done to understand paracrine signaling via cytokines, chemokines, and metabolites secreted from tumor cells, but only recent studies have begun to investigate the role of extracellular vesicles (EVs) and their cargoes in the tumor microenvironment. Studies have illustrated the ability of PDAC-derived EVs to activate and recruit pancreatic stellate cells in the tumor microenvironment; however, no work has been done in vivo to determine whether this EV uptake occurs within tumors. Additionally, little mechanistic work has been done to understand the functional consequences of EV cargo within recipient cells.

Based on transcriptomic and proteomic analysis of PDAC patient-derived EVs, mRNAs and RNA-binding proteins (RBPs) are enriched within EVs. Our lab focuses on the role of RBPs in PDAC, and thus, we will utilize enhanced crosslinking immunoprecipitation to identify the RBPs within PDAC EVs and evaluate their impact on the EV transcriptome. We have successfully isolated and characterized EVs via size exclusion chromatography isolation paired with western blotting for classical EV markers, fluorescent nanoparticle tracking analysis, and transmission electron microscopy. We have also demonstrated that we can identify mRNA cargoes that are bound and unbound by RBPs. Additionally, we have optimized PKH67 labeling and detection of PDAC EVs to assess specific and preferential uptake in vitro. Further, we are establishing the validated PalmGRET bioluminescent reporter in our patient derived cell lines to track PDAC EVs in a pancreatic orthotopic mouse model. Utilizing this method, we intend to identify cell types in the microenvironment that are importing PDAC EVs and perform functional studies to understand how these cells are impacted by EV signaling.

These studies will elucidate which cells in the PDAC tumor microenvironment import PDAC EVs, as well as interrogate the mechanistic role of PDAC EV crosstalk. This work will further characterize the role of RBPs in PDAC and begin to evaluate their role in a cell extrinsic manner.

Citation Format: Jennifer M. Finan, Roberto Di Niro, Randall Armstrong, Jonathan R. Brody. Elucidating the role of RNA-binding proteins in pancreatic cancer extracellular vesicle crosstalk [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 3137.
Abstract 3141: Toll-like receptor 4 knockout promotes cutaneous tumorigenesis in mice lack of Ikka heterozygous loss in keratinocytes
Authors: Singh Amit Kumar
Published: June 2022

Role of recognizing pathogen-associated molecular patterns in skin carcinogenesis has not been extensively studied. Toll-like receptor 4 (TLR4) recognizes gram-negative lipopolysaccharide. To explore the impact of interplay between TLR4-regulated microbiota and host epithelial cells on tumorigenesis, we adopted a mouse model that expresses hemizygous Ikkα gene induced by a keratin 5 Cre (K5.Cre, IkkααKC/+). IkkααKC mice die within 3 weeks after birth. Inducible Ikkα ablation mediated by K15.CrePR1 or K5.CreER results in spontaneous skin tumors. We found an increased incidence of spontaneous skin tumors in Tlr4-/-;IkkααKC/+ mice compared to Tlr4-/- or IkkααKC/+ mice. We detected markedly elevated bacteria numbers on the surface of skin tumors compared to the adjacent skin. Interestingly, the oral cavity of Tlr4-/-;IkkααKC/+ mice showed an elevated proportion of gram-negative bacteria compared to Tlr4-/- or IkkααKC/+ mice. To further determine the effect of bone marrow (BM)-derived hemopoietic cells on tumorigenesis, we performed BM transplant experiments. IkkααKC/+ mice with Tlr4-/- BM developed more cutaneous skin tumors than IkkααKC/+ mice with WT BM. Based on these findings, we hypothesized that Tlr4-/- hemopoietic cells regulate microbiota, which further results in altered skin carcinogenesis. We are revealing the detailed mechanism of how Tlr4-/- hemopoietic cells contribute to microbiota changes and tumorigenic promotion.

Citation Format: Amit Kumar Singh. Toll-like receptor 4 knockout promotes cutaneous tumorigenesis in mice lack of Ikka heterozygous loss in keratinocytes [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 3141.
Abstract 3143: RIG-I agonism promotes immune activation in syngeneic tumor model
Authors: Zielstorff Mark , Chen Yiping , Presland Jeremy , Byford Alan , Doty Amy , Munsell Erik , Parish Craig , Baltus Gretchen , Moy Lily , Addona George , Zhang-Hoover Jie
Published: June 2022

Activation of retinoic acid-inducible gene I (RIG-I) increases pro-inflammatory cytokine production, enhances antigen presentation, and promotes tumor cell apoptosis. Here we assessed the pharmacological activity of a RIG-I agonist encapsulated in lipid nanoparticle in the B16F10 mouse syngeneic tumor model. To probe whether additional pathways may complement RIG-I agonism, we tested our compound in combination with anti-PD-1 and anti-CTLA-4 immune checkpoint inhibitors. The RIG-I agonist was administered intratumorally (IT) in B16F10 tumors. In one arm, tumors were collected four hours post treatment for cytokine analysis. In a separate arm, tumor size was measured twice a week for two weeks to assess efficacy. To understand the mechanism of action of the RIG-I agonist, high dimensional phenotyping of dissociated tumors collected at various time points was evaluated using CyTOF. Tumor-specific memory T cell response was evaluated by tumor re-challenge in vivo. Intra-tumoral dosing of the RIG-I agonist generated robust and dose-dependent production of type I IFN in the tumor that led to tumor efficacy in B16F10 model. Within 24 hours of the RIG-I agonist administration tumor infiltrating myeloid cells, NK cells, and T cells showed upregulation of activation markers such as CD69, CCR7, and Ki-67, and MHC I and PD-L1 were upregulated on CD45- cells. On day 10 of the treatment, the number of CD8+ T cells and NK cells in the tumor and their expression of granzyme B increased substantially. The RIG-I agonist dosed in combination with anti-PD-1 and anti-CTLA-4 monoclonal antibodies enhanced B16F10 tumor killing, including complete tumor regressions. No tumor growth was observed when these mice were re-inoculated with B16F10 cells. In conclusion, the RIG-I agonist encapsulated in lipid nanoparticle activates tumor infiltrating immune cells, promotes tumor killing, and combines favorably with anti-PD-1 and anti-CTLA-4 antibodies.

Citation Format: Mark Zielstorff, Yiping Chen, Jeremy Presland, Alan Byford, Amy Doty, Erik Munsell, Craig Parish, Gretchen Baltus, Lily Moy, George Addona, Jie Zhang-Hoover. RIG-I agonism promotes immune activation in syngeneic tumor 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 3143.
Abstract 3144: Pleiotrophin drives a pro-metastatic immune niche within breast tumor microenvironment
Authors: Ganguly Debolina , Ngo Tuong V. , Coleman Morgan , Sorrelle Noah , Dominguez Adrian , Toombs Jason , Schmidt Marcel , Mora Fa V. , Ortega David G. , Wellstein Anton , Brekken Rolf A.
Published: June 2022

Immune cells in the tumor microenvironment (TME) can impact cancer cell invasion, seeding and colonization of secondary metastatic sites. Our findings show that Pleiotrophin (PTN), a unique and previously under-studied heparin binding cytokine, contributes to inflammation within the TME. PTN appears to be particularly efficient in recruiting neutrophils, which have been reported to cause immune suppression. Consequently, inhibition of PTN pharmacologically or genetically results in lower breast cancer metastasis to lungs and better survival in multiple mouse models of breast cancer. Corroborating our mouse studies, we have found that higher PTN expression in breast cancer patient plasma correlates with poor prognosis. Concurrently, stage IV breast cancer patients that have lower PTN expression have a highly significant survival advantage (median survival 78.27 months) over patients expressing high levels of PTN (median survival 27.5 months). Additionally, we have also found PTN positive cancer cells are enriched in metastatic lesions suggesting that PTN-high cancer cells are more successful at colonizing and surviving at the secondary site. Uncovering the function of PTN in these unique cancer cells might have a major impact in the clinical setting. Overall, our data suggests that PTN is important in driving a pro-metastatic immune niche within the TME that promotes tumor cell escape from the primary tumor and survival of metastatic cancer cells in secondary sites. Additionally, our studies highlight that inhibition of PTN has potential to reduce metastatic burden in breast cancer and suggest that future studies testing the combination of PTN inhibition with standard chemotherapy or immune therapy are warranted.

Citation Format: Debolina Ganguly, Tuong V. Ngo, Morgan Coleman, Noah Sorrelle, Adrian Dominguez, Jason Toombs, Marcel Schmidt, Fa V. Mora, David G. Ortega, Anton Wellstein, Rolf A. Brekken. Pleiotrophin drives a pro-metastatic immune niche within breast tumor microenvironment [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 3144.
Abstract 3159: CUX1 dosage-dependent regulation of hematopoietic stem and progenitor cells
Authors: Liu Weihan , Konecki Stephanie , Martinez Tanner , McNerney Megan
Published: June 2022

The mechanism by which stem and progenitor cells commit to proliferate, self-renew, or differentiate towards different lineages remains unclear. Understanding this process is essential to decipher both normal tissue development and aberrant differentiation associated with malignant transformation. CUX1 is a non-clustered HOX-family transcription factor that is recurrently mutated in hematopoietic and solid tumours and regulates cell fate in multiple developmental systems. We reported that CUX1 is required for normal hematopoietic homeostasis of both murine and human hematopoietic stem and progenitor cells (HSPCs). To date, the transcriptional role of CUX1 in HSPCs has been unclear, partly because of the complexity of the Cux1 locus. Namely, Cux1 shares exons with a distinct gene, Casp, which encodes a highly expressed golgi-associated protein. As most transcriptomic studies combine Casp and Cux1 transcripts, the nuances of Cux1 expression are concealed. To identify the transcriptional mechanism by which CUX1 regulates HSPC functions, we compared scRNA-seq data of Kit+lin- HSPCs from transgenic mice expressing inducible shRNAs targeting Cux1, Cux1mid and Cux1low, to control counterparts expressing an shRNA targeting renilla luciferase. We demonstrate that separating Cux1 from Casp transcripts provides more power to detect the influence of Cux1 in scRNA-seq data analysis. While cell numbers within most HSPC sub-populations are largely unchanged after five days of Cux1 knockdown, the transcriptomes within multiple clusters are disrupted. The effect size is greatest in Cux1low cells, compared to Cux1mid, consistent with dosage dependent roles of CUX1 in HSPCs. Across sub-populations, Cux1 levels correlated with gene signatures of “stemness,” and Cux1 knockdown disrupted this gene signature in the core hematopoietic stem cell (HSC) population. Concomitantly, low levels of Cux1 led to upregulation of cellular metabolism and G2/M cell cycle transition pathways in core HSCs, indicating that CUX1 regulates stem cell quiescence at the hematopoietic apex. We are currently integrating this data with epigenetic datasets to determine the mechanism by which CUX1 regulates critical hematopoietic transcriptional programs. Overall, understanding the regulatory function of CUX1 will provide insight into how HSCs commit to different fates and may ultimately lead to novel cancer therapeutic interventions.

Citation Format: Weihan Liu, Stephanie Konecki, Tanner Martinez, Megan McNerney. CUX1 dosage-dependent regulation of hematopoietic stem and progenitor cells [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 3159.
Abstract 3167: The scaffolding protein DLG5 is necessary to maintain Sonic Hedgehog signaling in glioblastoma stem cells and promote tumor growth
Authors: Kundu Somanath , Nandhu Mohan S. , Longo Sharon L. , Longo John A. , Rai Shawn , Chin Lawrence S. , Richardson Timothy E. , Viapiano Mariano S.
Published: June 2022

Proteins complexes that maintain the apico-basal polarity of epithelial cells and organize cell-signaling domains are necessary for normal tissue organization and therefore regarded as tumor suppressors. Accordingly, the scaffolding proteins that form the Scribble polarity complex (SCRIB, LLGL1, and DLG members) are usually downregulated in cancer, in particular in highly aggressive or invasive tumors. Because gliomas arise from non-epithelial precursors that are likely motile before transformation, the expression and functions of these scaffolding proteins in malignant brain tumors has remained almost entirely unexplored. Surprisingly, we have found that one member of the DLG family, DLG5, is highly upregulated in malignant gliomas compared to other solid tumors. Indeed, DLG5 was the predominant DLG member found in all molecular subtypes of glioblastoma (GBM). Knockdown of DLG5 reduced the viability, invasiveness, and self-renewal of GBM stem cells belonging to the proneural and mesenchymal molecular subtypes. As a result, intracranial tumors defficient in DLG5 were smaller and less proliferative than controls, resulting in extended animal survival. At the molecular level, DLG5 knockdown decreased the expression of stemness-related genes (SOX2, NANOG, POU5F1) but increased the expression of LLGL1, a component of the Scribble complex that is associated with neural precursor differentiation. These changes were accompanied by downregulation of Sonic Hedgehog (SHh) signaling, both in vitro and in vivo, which drives GBM cell stemness and proliferation. Moreover, DLG5-deficient GBM stem cells became insensitive to exogenous SHh and were unable to upregulate Gli1 or increase cell invasion in presence of added SHh, compared to control cells. We further confirmed that DLG5 downregulation increased the ubiquitination of Gli1-containing complexes as well as Gli1 proteasomal degradation, providing a suitable mechanism for the loss of SHh signaling and tumor stemness. These results describe for the first time the expression of a DLG family member in malignant gliomas and reveal a novel, polarity-independent, pro-tumoral function of DLG5, which differs from its proposed tumor-suppressor role in other solid tumors.

Citation Format: Somanath Kundu, Mohan S. Nandhu, Sharon L. Longo, John A. Longo, Shawn Rai, Lawrence S. Chin, Timothy E. Richardson, Mariano S. Viapiano. The scaffolding protein DLG5 is necessary to maintain Sonic Hedgehog signaling in glioblastoma stem cells and promote tumor growth [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 3167.
Abstract 3185: Treatment with an engineered decoy receptor targeting the CLCF1-CNTFR signaling axis prevents oncogenesis in Kras mutant lung adenocarcinoma via both cell autonomous and non-cell autonomous mechanisms
Authors: Marini Kieren D. , Champion Emma C. , Cochrane Jennifer R. , Sweet-Cordero E. Alejandro
Published: June 2022

Proinflammatory cytokines in the tumor microenvironment can promote tumor growth, yet their value as therapeutic targets remain underexploited. Previously, we validated the functional significance of the cardiotrophin-like cytokine factor 1 (CLCF1)-ciliary neurotrophic factor receptor (CNTFR) signaling axis in lung adenocarcinoma (LUAD) and generated a high-affinity soluble receptor (eCNTFR-Fc) that sequesters CLCF1, thereby inhibiting its oncogenic effects. eCNTFR-Fc inhibits tumor growth in multiple xenograft models and in an autochthonous, highly aggressive genetically engineered mouse model of LUAD, driven by activation of oncogenic Kras and loss of Trp53. Abrogation of CLCF1 through eCNTFR-Fc appears most effective in tumors driven by oncogenic KRAS. From our genetically engineered mouse and our syngeneic modelling of LUAD we have observed that eCNTFR seems to work via two mechanisms, firstly in a tumor intrinsic fashion where it may modulate altered guanosine triphosphate loading, as we see differential effectiveness of eCNTFR-Fc, depending on the presence of KRAS mutations that retain the intrinsic capacity to hydrolyze guanosine triphosphate. Alternatively, eCNTFR also effects the tumor microenvironment and alters the composition of the tumor immune infiltrate, where using single cell genomics we see vast changes to the myeloid compartment, and alteration of the CAF populations, which we have previously shown to be the primary location of CLCF1 expression in mouse LUAD. To further enhance the efficacy of eCNTFR-Fc we have identified potential targets for combination therapy from both the autonomous and non-cell autonomous mechanisms of eCNTFR-Fc. We conclude that eCNTFR-Fc acts through two distinct mechanisms to be a highly effective treatment for Kras mutant LUAD, and combining eCNTER with targets, identified by a rational combination approach, could further enhance its therapeutic potential.

Citation Format: Kieren D. Marini, Emma C. Champion, Jennifer R. Cochrane, E. Alejandro Sweet-Cordero. Treatment with an engineered decoy receptor targeting the CLCF1-CNTFR signaling axis prevents oncogenesis in Kras mutant lung adenocarcinoma via both cell autonomous and non-cell autonomous mechanisms [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 3185.
Abstract 3204: Differential expression of angiogenesis-associated genes in smoker and non-smoker lung adenocarcinoma patients
Authors: Singh Shubhangi , Dasgupta Santanu , Turbat-Herrera Elba A. , Singh Seema , Singh Ajay P.
Published: June 2022

Tobacco smoke (TS) is one of the most prominent risk factors for lung and several other malignancies. Toxic chemicals present in TS induce DNA damage leading to carcinogenic mutations. Moreover, TS can also cause epigenetic and gene expression changes in tumor and other cells of the tumor microenvironment, especially through its addictive component nicotine. We and others have reported that nicotine promotes tumor angiogenesis via its direct impact on the endothelial cells or by altering the cancer cell secretome. In this study, we wanted to explore the association of smoking habits with the differential expression of angiogenesis-related genes in lung adenocarcinoma (LUAD) patients. Gene Set Enrichment Analysis (GSEA) identified a set of 48 angiogenesis-related genes that were surveyed for differential expression in smoker versus non-smoker patients in The Cancer Genomic Atlas (TCGA) database using an interactive web application, UALCAN. Gender and age of the LUAD patients were considered as confounding variables since patients of different age groups and gender can have biological and physiological differences, which could influence gene expression regardless of the smoking habits. We identified a total of fourteen differentially-expressed genes, of which four (HTATIP2, NCL, SPHK1, and VEGFA) were overexpressed and ten (AGGF1, AMOT, C1GALT1, ERAP1, NPR1, PML, RNH1, SHH, SPINL5, and TNFSF12) were downregulated in LUAD of smokers than those of non-smokers. Five of the differentially-expressed genes (HTATIP2, NCL NPR1, PML, and TNFSF12) exhibited significantly different transcript levels in males versus females and three (SPHK1, AGGF1, and SPINK5) showed varying pattern of expression in LUAD of different age group patients. Literature survey found prior published evidence for an association of two of the fourteen differentially expressed genes (NCL and NPR) with nicotine exposure. Together, our findings identify smoking-associated angiogenic gene signatures that could be potential effectors of nicotine signaling that promotes cancer angiogenesis and should be validated further by conducting laboratory investigations.

Citation Format: Shubhangi Singh, Santanu Dasgupta, Elba A. Turbat-Herrera, Seema Singh, Ajay P. Singh. Differential expression of angiogenesis-associated genes in smoker and non-smoker lung adenocarcinoma patients [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 3204.
Abstract 3207: Notch decoys as potential anti-angiogenic biotherapeutics
Authors: Sargis Timothy , Youn Seock-Won , Lee Hyun , Naiche L.A. , Kitajewski Jan
Published: June 2022

Notch signaling is activated by ligands Delta-like 4 (Dll4) and Jagged1 in the tumor microenvironment to promote tumor angiogenesis and perfusion. Notch activation is associated with poor outcomes in several cancers, particularly triple negative breast cancer (TNBC), and affects both tumor angiogenesis and metastasis. The development of therapeutics targeting angiogenesis, such as the Notch pathway, has again attracted attention. Previous approaches to globally inhibit the Notch pathway or block Dll4/Notch1 activation, such as γ-secretase inhibitors (GSI), have raised safety concerns due to gastrointestinal toxicity due to accumulation of secretory goblet cells in the intestine. Similarly, anti-Dll4 therapy resulted in pathological changes in the liver as well as severe vascular neoplasms when evaluated using preclinical animal models. Development of new approaches for targeting the Notch pathway remains a critical clinical problem currently unaddressed. Our lab has previously developed ligand-specific inhibitors of Notch signaling, called Notch decoys, which are comprised of Fc fusions to specific EGF-like repeats of the Notch1 extracellular domain. These Notch decoys bind ligand non-productively and interfere with ligand function. Jagged-specific Notch1 decoys inhibit angiogenesis in vitro and significantly impair tumor growth, tumor angiogenesis, and perfusion without apparent toxicity in mouse models of TNBC. However, this previous work utilized Notch decoys produced via viral expression vectors, which preclude dosage control and limit clinical applicability. We have developed a new generation of Notch “mini” decoys that contain fewer EGF-like repeats. These mini decoys show improved secretion characteristics and can be purified as active proteins in clinically relevant quantities. Using a variety of binding assays, we observed that these Notch mini decoys demonstrate strong but distinct binding to Notch ligands Dll4 and Jagged1 and block Notch signaling when evaluated in cultured cells. Our newly generated Notch decoys can now be sufficiently purified for use in a dose-dependent manner to test the therapeutic role of Notch inhibition on tumor angiogenesis using preclinical animal models.

Citation Format: Timothy Sargis, Seock-Won Youn, Hyun Lee, L.A. Naiche, Jan Kitajewski. Notch decoys as potential anti-angiogenic biotherapeutics [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 3207.
Abstract 3236: CDK7 Expression is a biomarker of resistance to olaparib
Authors: Silberberg Gilad , Vishwakarma Bandana , Heverly Paul , Zipeto Marianna , DeOrnellis David , Ritchie Michael
Published: June 2022

The overall survival of patients diagnosed with Ovarian Cancer remains low. The current initial treatment regimen for patients with newly diagnosed advanced ovarian cancer includes the use of paclitaxel and carboplatin in combination with surgical cytoreduction. Remission is often achieved, but 80% of patients will have a recurrence within 3 years of remission. Recent treatment advances include the use of poly(ADP)-ribose polymerase (PARP) inhibitors for recurrent tumors with associated BRCA mutations. While maintenance treatment with PARP inhibitors results in remission for a significant number of patients, acquired resistance to therapy has been observed. In this study, we evaluated biomarkers associated with resistance to the PARP inhibitor Olaparib in a cohort of platinum pretreated Ovarian Cancer tumors. We identified Cyclin H and CDK7 expression, among others, as biomarkers associated with resistance to Olaparib. Further prospective analyses confirmed CDK7 expression as a biomarker of resistance to Olaparib in NSCLC.

Citation Format: Gilad Silberberg, Bandana Vishwakarma, Paul Heverly, Marianna Zipeto, David DeOrnellis, Michael Ritchie. CDK7 Expression is a biomarker of resistance to olaparib [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 3236.
Abstract 3252: Tumor treating fields suppression of ciliogenesis enhances temozolomide toxicity
Authors: Shi Ping , Tian Jia , Ulm Brittany , Mallinger Julianne , Khoshbouei Habibeh , Deleyrolle Loic , Sarkisian Matthew
Published: June 2022

Tumor Treating Fields (TTFields) are low intensity, alternating intermediate frequency (200kHz) electrical fields that extend survival of glioblastoma patients receiving maintenance temozolomide (TMZ) chemotherapy. How TTFields exert efficacy on cancer cells over normal cells, or interact with TMZ is unclear. Primary cilia are microtubule-based organelles triggered by extracellular ligands, mechanical and electrical field stimulation, and are capable of promoting cancer growth and TMZ chemoresistance. We found in both low and high grade patient glioma cell lines that TTFields ablated cilia within 24 hours. Halting TTFields treatment led to recovered frequencies of elongated cilia. Cilia on normal primary astrocytes, neurons and multiciliated ependymal cells were less affected by TTFields. The TTFields-mediated loss of glioma cilia was partially rescued by chloroquine pretreatment, suggesting the effect is in part due to autophagy activation. We also observed death of ciliated cells during TTFields by live imaging. Notably, TMZ-induced stimulation of ciliogenesis in both adherent cells and gliomaspheres was blocked by TTFields. Moreover, the inhibitory effects of TTFields and TMZ on tumor cell recurrence correlated with the relative timing of TMZ exposure to TTFields and ARL13B positive cilia. Finally, TTFields disrupted cilia in patient tumors treated ex vivo. Our findings suggest TTFields efficacy may depend on the degree of tumor ciliogenesis and relative timing of TMZ treatment.

Citation Format: Ping Shi, Jia Tian, Brittany Ulm, Julianne Mallinger, Habibeh Khoshbouei, Loic Deleyrolle, Matthew Sarkisian. Tumor treating fields suppression of ciliogenesis enhances temozolomide toxicity [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 3252.
Abstract 3265: Discovery of a first-in-class reversible DNMT1-selective inhibitor
Authors: Pappalardi Melissa B.
Published: June 2022

DNA methylation, a key epigenetic driver of transcriptional silencing, is universally dysregulated in cancer. Reversal of DNA methylation by hypomethylating agents, such as the cytidine analogs decitabine or azacytidine, has demonstrated clinical benefit in hematologic malignancies. These nucleoside analogs are incorporated into replicating DNA where they inhibit DNA cytosine methyltransferases DNMT1, DNMT3A, and DNMT3B through irreversible covalent interactions. These agents induce significant toxicity to normal blood cells thus limiting their clinical doses. We report the discovery of a series of potent first-in-class DNMT1-selective inhibitors that compete with the active-site loop of DNMT1 for penetration into hemi-methylated DNA. These compounds induce robust loss of DNA methylation, transcriptional activation, and cancer cell growth inhibition in vitro leading to superior tumor regression and survival in mouse models of acute myeloid leukemia due to improved in vivo tolerability compared with decitabine.

Citation Format: Melissa B. Pappalardi. Discovery of a first-in-class reversible DNMT1-selective inhibitor [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 3265.
Abstract 3266: The KDM5 inhibitor PBIT reduces proliferation of castration-resistant prostate cancers via the induction of senescence
Authors: Smith Tunde M. , Chen Zhenbang , Stewart LaMonica V.
Published: June 2022

The members of the KDM5 family of lysine demethylases are best known for their ability to demethylate di- and trimethylated histone H3 at lysine 4 (H3K4me3). However, recent studies suggest at least three family members (KDM5A, KDM5B and KDM5C) promote prostate cancer growth and progression and may serve as effective therapeutic targets for this disease. Previous work from our laboratory has shown that 2-4(4-methylphenyl)-1,2-benzisothiazol-3(2H)-one (PBIT), a compound that inhibits the enzymatic activity of multiple KDM5 isoforms, reduces the viability of castration-resistant prostate cancer cells. The goal of this study was to further characterize the anti-tumor effects of PBIT within human prostate cancers. Two models of human castration-resistant prostate cancer were used to define the effects of PBIT: the androgen receptor (AR) positive C4-2B cells and the PC3 cells, which express little to no AR. Our group initially demonstrated via quantitative RT-PCR analysis that PC3 and C4-2B cells express varying amounts of KDM5A, KDM5B, and KDM5C, the therapeutic targets of PBIT. Presto Blue assays were next performed to determine the extent to which PBIT alters cell proliferation. Micromolar concentrations of PBIT significantly reduced prostate cancer cell proliferation in a time- and concentration-dependent manner. Data from Cell Death ELISAs suggest that PBIT at a concentration of 10 μM did not significantly induce apoptosis within C4-2B or PC3 cells. However, PBIT does appear to induce cellular senescence within the PC3 cell line. The level of senescence associated beta-galactosidase staining was increased in PC3 cells following treatment with 10 μM PBIT. Furthermore, PBIT increased protein levels of a second senescence marker, lamin B1. Together, these data suggest that the induction of senescence contributes to the anti-proliferative effect of PBIT.

Citation Format: Tunde M. Smith, Zhenbang Chen, LaMonica V. Stewart. The KDM5 inhibitor PBIT reduces proliferation of castration-resistant prostate cancers via the induction of senescence [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 3266.
Abstract 328: LCB84, a TROP2-targeted ADC, for treatment of solid tumors that express TROP-2 using the hu2G10 tumor-selective anti-TROP2 monoclonal antibody, a proprietary site-directed conjugation technology and plasma-stable tumor-selective linker chemistry
Authors: Kim Hyejung , Guerra Emanuela , Baek Eunji , Jeong Yeojin , You Hyogeun , Yu Byeongjun , Jang Taeik , Saverio Alberti , Chung Chul-Woong , Park Changsik
Published: June 2022

LCB84 is a human Trop-2-targeting antibody drug conjugate (ADC) composed of monomethyl auristatin E (MMAE) as payload and the Hu2G10 (by Mediterranea Theranostic) humanized IgG1 antibody that selectively targets the ADAM10-activated Trop-2 protein selectively expressed in transformed cancer cells (1). LCB84 was prepared using ConjuAll࣪, a proprietary site-directed conjugation technology of LegoChem Biosciences, which incorporates a conjugation ‘handle’ joined by enzymatic prenylation to a specifically engineered recognition sequence (CaaX) on antibody light chains. This conjugation handle facilitates simple versatile chemical conjugation to the linker-payload. A proprietary plasma-stable cleavable linker that is recognized and cleaved by a cancer-associated lysosomal enzyme, β-glucuronidase, was used to enable efficient and traceless payload release in a cancer-specific manner. LCB84 has been evaluated for anti-tumor activity and showed superior anticancer efficacy in triple-negative breast cancer (TNBC), pancreatic ductal adenocarcinoma (PDAC), gastric cancer and non-small cell lung cancer (NSCLC) cell line-derived xenograft (CDX) models compared to the ADC competitors Trodelvy and DS-1062. The LCB84 treatments were well tolerated, with no changes in body weight compared to control animals, for all dosing groups. LCB84 has robust cross-reactivity against primate Trop-2, which allows rigorous toxicity studies in monkeys. Remarkably, preliminary toxicity studies using cynomolgus monkeys showed that LCB84 is well tolerated, with calculated therapeutic index (TI, MTD/MED) of ~30 for single dosing and ~40 for repeat dosing. In conclusion, LCB84 is highly effective against Trop-2-positive CDX models in mice at doses that are well tolerated in mice and in primate models. Use of this proprietary plasma-stable cancer-selective linker technology and the Hu2G10 anti-Trop-2 monoclonal antibody that targets cancer-activated Trop-2 has led to a greatly improved next generation ADC for the treatment of various Trop-2-positive solid cancers including TNBC, PDAC, NSCLC and gastric cancer.

Citation Format: Hyejung Kim, Emanuela Guerra, Eunji Baek, Yeojin Jeong, Hyogeun You, Byeongjun Yu, Taeik Jang, Alberti Saverio, Chul-Woong Chung, Changsik Park. LCB84, a TROP2-targeted ADC, for treatment of solid tumors that express TROP-2 using the hu2G10 tumor-selective anti-TROP2 monoclonal antibody, a proprietary site-directed conjugation technology and plasma-stable tumor-selective linker chemistry [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 328.