bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2707: Therapeutic effects of WT1 silencing by respiratory administration of liposomal siRNA in a melanoma murine model
Authors: Ramos-Gonzalez Martin , Vázquez-Garza Eduardo , García-Rivas Gerardo , Rodríguez-Aguayo Cristian , Chávez-Reyes Arturo
Published: April 2023

Introduction: Cancer is considered among the main causes of death worldwide and the number of cases keeps rising every year, as there is not an effective treatment to offer for most of them. The lungs represent a frequent target of primary and metastatic tumors due to his high oxygenation and large air spaces to grow. Melanoma is the most aggressive type of skin cancer showing a high affinity to metastasize into the lungs, being the overexpression of the WT1 protein associated with the development of this cancer, as well as several other types.

Methods: In this study, we evaluated the effects of siRNA silencing the WT1 protein in both, in vitro in the B16F10 melanoma cell line, and in vivo in a murine model of pulmonary metastatic melanoma implementing a novel delivery strategy of neutral DOPC liposomal-siRNA system administrated via inhalation.

Results: In vitro studies showed an effective silencing of WT1 protein (86%) in the siRNAsWT1 treated cells when compared with controls, resulting in a loss of viability (40%) and proliferation (49%) of the cells, induced G1 cell-cycle arrest (11%), a restriction of migration (87%) and invasion (100%) capacities, as well as an activation of apoptosis (12%). In vivo, we tested our delivery system obtaining an efficient biodistribution in lungs, kidney, liver and spleen. The respiratory administration of LWT1siRNA after two weeks of treatment resulted in silencing of WT1 protein (59%), achieving an important antitumor activity with a 44% reduction in tumor weight. In the survival study 80% of the L-WT1siRNA treated mice were alive after all the controls mice had died, delaying the death almost a week at the end of the study.

Conclusions: This work demonstrates the efficacy of the neutral DOPC liposomal-siRNA delivery system by respiratory administration as a novel therapy to reduce the malignancy of pulmonary tumors and to increase survivability by silencing specific cancer biomarkers as WT1.

Citation Format: Martin Ramos-Gonzalez, Eduardo Vázquez-Garza, Gerardo García-Rivas, Cristian Rodríguez-Aguayo, Arturo Chávez-Reyes. Therapeutic effects of WT1 silencing by respiratory administration of liposomal siRNA in a melanoma murine model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2707.
Abstract 2813: Pyruvate kinase M2 confers radioresistance in head and neck cancer through reprograming the tumor-immune microenvironment via ECM1
Authors: Chen Fanghui , Fan Yang , Tang Chris , Richard Qin , Wei Zheng , Teng Yong
Published: April 2023

Radiotherapy plays an essential role in cancer treatment, yet radioresistance remains a major barrier to therapeutic efficacy. A better understanding of the predominant pathways determining radiotherapy response could help develop mechanism-informed therapies to improve cancer management. PKM2, an enzyme that regulates the final rate-limiting step of glycolysis, has different functions in the occurrence, progression and metastasis of cancer. However, whether PKM2 plays a vital role in radioresistance is unclear, especially in head and neck squamous cell carcinoma (HNSCC). Here we report the upregulation of PKM2 in primary post-radiotherapy tumor tissues collected from radioresistant HNSCC patients. Using multidisciplinary approaches, we found elevated PKM2 in response to radiation, which confers radioresistance in HNSCC cells via extracellular matrix protein 1 (ECM1). Mechanistically, PKM2 activates STAT5 signaling pathway in HNSCC cells upon radiation, which enhances ECM1 expression. Moreover, ECM1 upregulation mediated by PKM2 prevents effector T cells from migrating to radioresistant head and neck tumors. While depletion of ECM1 overcomes PKM2-mediated radioresistance in orthotopic xenograft mice through altering the profile of chemo-attractants, which in turn remodels the tumor microenvironment to promote more cytotoxic T cells home to tumors. These novel and significant findings not only demonstrate a novel mechanism underpinning the interaction between tumor-derived PKM2 and immune cells in radioresistance but also provide a possible therapeutic strategy to overcome the failure of radiotherapy in HNSCC patients.

Citation Format: Fanghui Chen, Yang Fan, Chris Tang, Qin Richard, Zheng Wei, Yong Teng. Pyruvate kinase M2 confers radioresistance in head and neck cancer through reprograming the tumor-immune microenvironment via ECM1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2813.
Abstract 2844: BMP inhibition overcomes chemoresistance of prostate cancer
Authors: Hao Jijun , Xie Chen , Wang Zhijun , Bar Yong , Aguilar Jose , Kyan Austin , Zhing Li
Published: April 2023

Chemoresistance is a major therapeutic challenge to prostate cancer and its underlying molecular mechanism is poorly understood. Previously, it has been suggested that bone morphogenetic protein (BMP) signaling is down-regulated during the prostate cancer progression from the early androgen-sensitive stage to the metastatic castration-resistant stage. However, no literature reports are available for BMP signaling in the more advanced-chemoresistant prostate cancer. In this study, we found the expression levels of the BMP type I receptor members, Activin-like kinase-2 (ALK2) and Activin-like kinase-3 (ALK3), were significantly higher in the chemoresistant prostate cancer cells than those in the chemosensitive prostate cancer cells. In addition, the phospho-Smad1/5/9 proteins, the pivotal intracellular effectors of the BMP signaling, were notably elevated in the chemoresistant prostate cancer cells over the chemosensitive prostate cancer cells, indicating that BMP signaling is highly activated in the chemoresistant prostate cancer cells. We also found that BMP signaling inhibition with either DMH1 or the knockdown of ALK2/ALK3 sensitized chemoresistant prostate cancer cells to the chemotherapy drug docetaxel in a dose dependent manner. Our further study indicates that DMH1 suppressed the migration and invasion of chemoresistant prostate cancer cells in vitro, and attenuated chemoresistant prostate tumor growth in the mouse xenograft model in vivo. In addition, we showed that DMH1 disrupted the sphere formation in DU145-TxR and PC3-TxR cells, suppressed the expression of marker genes of the cancer stem cells (CSCs), and inhibited the efflux activity of multidrug resistance P-glycoprotein (P-gp1). In conclusion, our study demonstrates that BMP signaling is associated with prostate cancer chemoresistance and BMP signaling inhibition effectively overcomes the cancer chemoresistance potentially through the disruption of CSCs’ stemness and P-gp1 efflux activity.

Citation Format: Jijun Hao, Chen Xie, Zhijun Wang, Yong Bar, Jose Aguilar, Austin Kyan, Li Zhing. BMP inhibition overcomes chemoresistance of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2844.
Abstract 3064: Exosome-based therapeutic strategy for neuroendocrine prostate cancer
Authors: Sreekumar Amritha , Saini Sharanjot
Published: April 2023

Background: Therapy-induced neuroendocrine prostate cancer (NEPC) is an aggressive variant of castration-resistant prostate cancer (CRPC) that is increasing in incidence with the widespread use of second generation of androgen receptor (AR)-pathway inhibitors (APIs) such as Enzalutamide (ENZ). NEPC arises from CRPC-Adenocarcinomas (CRPC-Adeno) via a reversible trans-differentiation process, referred to as neuroendocrine differentiation (NED) wherein prostate cancer cells (PCa) undergo a lineage switch. As a result of this lineage switching, PCa cells exhibit neuroendocrine (NE) features, characterized by expression of neuronal markers such as enolase 2 (ENO2), chromogranin A (CHGA) and synaptophysin (SYP). Due to lack of AR signaling, these PCa variants are impervious to anti-androgen therapy and constitute an aggressive variant of advanced CRPC with survival times < 1 year. Clinically, NEPC manifests as the presence of visceral metastatic disease including metastasis to liver, lung, central nervous system or bone despite low serum PSA levels. Current therapeutic options for NEPC are limited to highly toxic platinum drugs. The primary objective of our study is to evaluate novel, exosome-based therapies for NEPC.

Methods: Exosomes were isolated from HEK-293T cells by ultracentrifugation. Isolated exosomes were characterized by Nanoparticle Tracking Analyses and electron microscopy. These exosomes were engineered to express biotinylated CEACAM-5 antibody on their cell surface. Exosomes were packaged with a combination of drugs (EZH2 inhibitor and AR inhibitor) by sonication. Engineered exosomes were tested in vitro using NEPC cell line NCI-H660 and in vivo using LuCaP 145.1 patient-derived xenograft model.

Results: Engineered exosomes reduced the viability of NEPC cells. CEACAM5-labelled exosomes trafficked to tumor cells as CECAM5 is a surface protein expressed by NEPC cells.

Conclusions: Engineered-exosome based therapy may provide a new avenue as a targeted therapy for NEPC.

Citation Format: Amritha Sreekumar, Sharanjot Saini. Exosome-based therapeutic strategy for neuroendocrine prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3064.
Abstract 3094: Discovery of a HeliconTM peptide inhibitor of the beta-catenin-TCF interaction with in vivo activity
Authors: White Brian H. , Si Yaguang , Cappucci Sarah , Li Zhi , Ramirez Jessica D. , Ponthier Charles M. , Visness Erica , Du Peicheng , Choi Minjung , Beerepoot Pieter C. , Ortet Paula C. , Jewett Ivan T. , Alfaro-Lopez Josue , Agarwal Sorabh , La Daniel , Fulgham Aaron , McGee John H. , Orford Keith , Hurov Jonathan B. , Tremblay Martin R. , Verdine Gregory L.
Published: April 2023

Wnt signaling pathway mutations leading to constitutive activation of the driver oncogene β-catenin occur in at least 20% of all human cancers, but β-catenin itself has remained undruggable by traditional modalities. In order to inhibit the activity of β-catenin, we have developed conformationally hyperstabilized α-helical peptides (Helicons) that are cellularly permeable and bind directly to β-catenin in order to block its interaction with TCF family of transcription factors.

Starting with a helical peptide derived from phage display, a combination of structural biology insights and medicinal chemistry optimization improved β-catenin binding of lead peptides to picomolar affinity. Cellular permeability was achieved by modification of physical properties as well as cyclization strategies to enforce helicity and manage backbone amide bonds. Helicons show excellent pharmacokinetic profiles suitable for intermittent dosing. β-catenin targeting helicons demonstrate on-target activity and potent inhibition of Wnt-driven tumor growth in vivo.

Citation Format: Brian H. White, Yaguang Si, Sarah Cappucci, Zhi Li, Jessica D. Ramirez, Charles M. Ponthier, Erica Visness, Peicheng Du, Minjung Choi, Pieter C. Beerepoot, Paula C. Ortet, Ivan T. Jewett, Josue Alfaro-Lopez, Sorabh Agarwal, Daniel La, Aaron Fulgham, John H. McGee, Keith Orford, Jonathan B. Hurov, Martin R. Tremblay, Gregory L. Verdine. Discovery of a HeliconTM peptide inhibitor of the beta-catenin-TCF interaction with in vivo activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3094.
Abstract 3121: Exercise and tumor genomic landscapes in 2,879 patients with cancer
Authors: Tsai Brandon L. , Liu Lydia Y. , Eng Stefan E. , Underwood Whitney P. , Lee Catherine P. , Bliss Joshua W. , Boutros Paul C. , Jones Lee W.
Published: April 2023

Approximately two-thirds of cancer diagnoses globally are attributed to modifiable lifestyle factors such as smoking, diet and inactivity. Conversely, regular exercise is linked to decreased risk of multiple cancers. However, the underlying molecular mechanisms are not fully elucidated. Specifically, how exercise impacts tumor genomic landscapes has not been considered. To address this gap, we integrated clinical annotation of exercise exposure with tumor mutational profiling of 2,879 patients with cancer. Exercise exposure was evaluated by validated questionnaire and tumor genomic profiling of ≥ 505 commonly mutated cancer genes was performed using the Memorial Sloan Kettering Cancer Center Integrated Mutation Profiling of Actional Cancer Targets (MSK-IMPACT) assay. Among the most represented cancer types were breast (n = 623), lung (n = 524), endometrial (n = 260), colorectal (n = 217) and prostate (n = 176). We found that exercise influences the genomic landscapes of tumors in a cancer-type specific manner. For instance, breast, colorectal and lung cancers from exercising patients had significantly lower tumor mutation burden (TMB) compared with non-exercisers. TMB of melanoma was significantly higher in exercisers versus non-exercisers. A pan-cancer analysis showed that mutations in SMAD3 were more frequent among exercisers, while mutations in STK11, KEAP1 and RBM10 were more frequent among non-exercisers. Overall, this study shows that exercise regulates tumor genomic landscapes and does so in a cancer-site specific manner.

Citation Format: Brandon L. Tsai, Lydia Y. Liu, Stefan E. Eng, Whitney P. Underwood, Catherine P. Lee, Joshua W. Bliss, Paul C. Boutros, Lee W. Jones. Exercise and tumor genomic landscapes in 2,879 patients with cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3121.
Abstract 3192: Antitumor activity of ex vivo expanded tumor antigen priming cytotoxic T cells against malignant melanoma
Authors: Choi Jonggwon
Published: April 2023

Background: Dendritic cells have a unique ability to pick up antigen from tissues and migrate to the lymph nodes where they can cross-present cell-associated antigens with CD8+ T cells by MHC class I. Cytotoxic T lymphocytes remove malignant cells through recognition by T cell receptors of specific antigenic peptides that appear on the surface of cancer cells by the major histocompatibility complex class I/beta-2-microglobulin complex. In this study, we show that naive CD8+ T cells against the melan A antigens cells primed by DCs loaded with melan A antigens are able to kill malignant melanoma cell line (SK-MEL-28).

Methods: Peripheral blood mononuclear cells and Co-culture dendritic cells-T cells were seeded and MART-1 (100 ng/ml) reacted for 20 hours. ELISPOT ASSAY analyzes the degree of secretion of IFN-γ induced by Melan-A/MART-1 peptide stimulation in DC-T (1st week). In addition, the subtype analysis of T cells through FACS and the cytotoxicity activity of T cells was measured by tumor cell impedance with AXION.

Results: In ELISPOT ASSAY, it was observed that the number of positive spots increased significantly over time compared to 1st week. In FACS result analysis, CD4+ and CD8+ T cells increased, CD4+ T cell was observed as a dominant T cell subtype. As a result of cell impedance assay using Axion, it was observed that the number of tumor cells (SK-MEL-28) decreased according to the CTL:T ratio. In particular, cytotoxicity was observed immediately after co-culture and gradually showed cytotoxicity up to 72 hours.

Conclusion: Our study demonstrates that DCs loaded with killed allogeneic melanoma cells can cross-prime cytotoxic T cell which is specific for melan A antigen. In the future, additional T-cell studies targeting neoantigen and clinical studies applied to solid cancer patients will be conducted.

Keywords: Dendritic cell, CD4+/CD8+ T cell, Melan A antigen, Malignant melanoma

Citation Format: Jonggwon Choi. Antitumor activity of ex vivo expanded tumor antigen priming cytotoxic T cells against malignant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3192.
Abstract 3197: Adoptive T cell therapy using IL-7 and IL-15 expanded HER2-specific CD4 T cells for metastatic breast cancer
Authors: Gautam Namrata , Kodumudi Krithika , Snyder Colin , Beyer Amber , Costa Ricardo , Han Heather , Czerniecki Brian
Published: April 2023

BACKGROUND: Adoptive cell therapy (ACT) has had limited success due to low T cell infiltration in breast cancer (BC) patients. We have shown that administration of class II HER2 peptide pulsed type I polarized dendritic cell (DC1) vaccines leads to increase in CD4 T cells in the periphery of BC patients. In this study, we investigated the feasibility of expanding CD4 T cells from peripheral blood mononuclear cells (PBMC) of HER2-DC1 vaccinated BC patients.

METHODS: PBMCs from BC patients receiving HER2-DC1 vaccines were used for CD4 T cell expansion. PBMCs were co-cultured with HER2-DC1 at 10:1 ratio for initial activation followed by expansion with cytokines- IL-2 and IL-7 or IL-15. Expanded T cells were assessed for immune marker phenotyping by flow cytometry and TCRvb analysis. For TCRvB analysis, pre, post DC1, IL-7 and IL-15 expanded CD4 T cells were pelleted for DNA isolation and sequenced using Immunoseq Analyzer platform. In addition, we investigated the in vivo efficacy of adoptively transferred mouse IL-7/IL-15 expanded Her2 specific CD4 T cells in HER2+ TUBO mouse model.

RESULTS: Co-culturing of patient PBMCs with HER2-DC1 in the presence of cytokines IL-2, IL-7 and IL-15 expanded CD4 T cells ranging from 8-43 fold expansion in different samples with 92-98% CD4 phenotype. CD4 T cells expanded in IL-7 had stem like memory phenotype while IL-15 skewed to terminally differentiated CD4 T cells. IL-7 expanded T cells had significantly higher Tim3 and Ox40 expression, and effector memory proportion as compared to the IL-15 expanded T cells. Restimulation of expanded T cells with HER2 pulsed DCs showed HER2 specificity as measured by increased interferon-gamma production We identified top five clones in PBMCs from DC1-HER2 vaccinated BC patients compared to baseline. We observed differential abundance of TCR clones under IL-7 and IL-15 conditions and identified unique clones for IL-7 and IL-15. ACT using a combination of IL-7 and IL-15 expanded mouse CD4 T cells led to 50% tumor regression in HER2+ model.

CONCLUSION: These studies demonstrate that HER2 specific CD4 T cells can be successfully expanded from HER2-DC1 vaccinated patients. Post HER2 DC1 vaccine can generate a pool of antigen specific CD4 T cells and may offer a promising ACT for HER2 BC. Further studies are warranted to demonstrate the efficacy in the clinical setting.

Citation Format: Namrata Gautam, Krithika Kodumudi, Colin Snyder, Amber Beyer, Ricardo Costa, Heather Han, Brian Czerniecki. Adoptive T cell therapy using IL-7 and IL-15 expanded HER2-specific CD4 T cells for metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3197.
Abstract 3199: Development of a flow cytometry-based assay for measuring specific CAR expression on LGR5-targeting CAR-T cells
Authors: Tyllis Timona , Abbott Caitlin , McPeake Dylan , Foeng Jade , Bandara Veronika , Gundsambuu Batjargal , Napoli Silvana , Mills Stuart , Thompson Emma , Tan Lih , Cowin Allison , Bonder Claudine , Sadlon Timothy , Barry Simon , McColl Shaun
Published: April 2023

The measurement of chimeric antigen receptor (CAR) expression is critical for the development, qualification and quantitation of CAR-T cells for clinical applications. There is a comprehensive range of tools commercially available for specifically measuring expression of the CAR on CD19-targeting CAR-T cells. However, with the rapidly expanding repertoire of CAR-T cells in development targeting a diverse range of tumour antigens, there is a paucity of readily implementable assays that specifically measure CAR expression, which is essential for translation into the clinic. We have developed several CARs targeting the cancer stem cell marker LGR5, one of which (CNA3103) is the lead candidate in an upcoming clinical trial in advanced metastatic colorectal cancer. Previously, the level of CNA3103 expression in this system has been determined indirectly by measuring expression of tEGFR on the CAR-T cell surface, taking advantage of co-expression of the CAR and tEGFR upon transduction with our lentiviral construct. tEGFR expression was found over many CAR-T cell manufacturing batches to report transduction efficiency reproducibly with high sensitivity. However, to specifically quantify expression of the CNA3103, an assay utilising an Fc-tagged recombinant human LGR5 protein, in combination with an anti-human IgG1 Fc-specific secondary antibody was developed. A number of optimisation steps including titration of multiple batches of rhLGR5-Fc, different anti-human IgG1 Fc-specific antibodies and a range of incubation parameters were tested. This resulted in development of an assay that yielded very similar reproducibility and sensitivity to that previously observed with the indirect tEGFR surrogate assay. Overall, the findings highlight the utility of using CAR-targeting recombinant proteins for evaluating CAR expression on CAR-T cells, providing a general flow cytometry-based staining strategy that may be adapted for assessing a diverse repertoire of CARs.

Citation Format: Timona Tyllis, Caitlin Abbott, Dylan McPeake, Jade Foeng, Veronika Bandara, Batjargal Gundsambuu, Silvana Napoli, Stuart Mills, Emma Thompson, Lih Tan, Allison Cowin, Claudine Bonder, Timothy Sadlon, Simon Barry, Shaun McColl. Development of a flow cytometry-based assay for measuring specific CAR expression on LGR5-targeting CAR-T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3199.
Abstract 3235: Protein kinase C is an upstream regulator of IL13Ra2 and small molecular activator enhances CAR T mediated killing
Authors: Subham Siddharth , Jeppson John D. , Schatmeyer Bryan , Forman Stephen J. , Brown Christine E. , Akhavan David
Published: April 2023

Glioblastoma multiforme (GBM) is the most aggressive primary malignant brain cancer with a median survival of 16-20 months. Maximal safe resection and adjuvant chemotherapy improve survival but there is no cure and hence improved therapies are urgently needed. CAR T cell therapy has been FDA approved for hematologic malignancies but there are some barriers to their application for solid tumors in the clinic such as heterogenous tumor antigen expression. Chimeric Antigen Receptor (CAR) T Cells combine the cytolytic potency of T cells with the tumor specificity of an antibody. The interleukin 13 receptor alpha 2 (IL13Rα2) is an important target of CAR T cell therapy in ongoing GBM clinical trials. However, regulation of IL13Ra2 expression in GBM is unclear. Identifying upstream regulators of IL13Rα2 will not only help delineate IL13Rα2 carcinogenesis but also may help improve CAR T cell therapy. Utilizing knockout and overexpression constructs, we have identified protein kinase C family as an upstream regulator of IL13Rα2 transcription and translation in GBM cell lines. We have also identified a small molecule regulator of IL13Rα2, Ingenol-3-Angelate (I3A), that inhibits U87 tumor cell proliferation. Furthermore, I3A enhances IL13Rα2 re-directed CAR T cell mediated tumor cytolysis in vitro. Importantly, I3A treatment increases interferon gamma secretion by T cells. Taken together, our data implicates PKC isoforms as an upstream regulator of IL13Rα2. I3A treatment may serve as a therapeutic strategy to enhance CAR T mediated killing.

Citation Format: Siddharth Subham, John D. Jeppson, Bryan Schatmeyer, Stephen J. Forman, Christine E. Brown, David Akhavan. Protein kinase C is an upstream regulator of IL13Ra2 and small molecular activator enhances CAR T mediated killing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3235.
Abstract 3237: Dovitinib restricts EMT and promotes T cell recruitment to immune-desert gastric cancers via tumor-intrinsic IFN-γ signaling
Authors: Lu Heng , Cao Longlong , Soutto Mohammed , Bhat Nadeem , Chen Zheng , Peng Dunfa , Gomaa Ahmed , Nomura Sachiyo , Datta Jashodeep , Zaika Alexander , El-Rifai Wael
Published: April 2023

Background: Gastric cancer (GC) ranks fifth in incidence and fourth for mortality worldwide. The response to immune checkpoint blockade (ICB) therapy in GC is heterogeneous due to tumor-intrinsic and acquired immunotherapy resistance.

Methods: A novel immunophenotype-based subtyping algorithm was established to analyze tumor patient-derived bulk transcriptomic data. Gene set enrichment assays (GSEA), in vitro cell models, multiple syngeneic murine tumor models, and immune-checkpoint blockade (ICB) therapy were utilized.

Results: Our algorithm stratified human GC and showed that immune desert- and excluded-type tumors are ICB-resistant compared with immune-inflamed GC. Moreover, epithelial-mesenchymal transition (EMT) signaling is highly enriched in immune desert-type GC, and syngeneic murine tumors exhibiting mesenchymal-like, compared with epithelial-like, properties are T cell-excluded and resistant to CTLA4 blockade. Our analysis further identifies a panel of receptor tyrosine kinases (RTKs) as potential druggable targets in the immune desert-type GC. Dovitinib, an inhibitor of multiple RTKs, strikingly repressed EMT programming in mesenchymal-like immune desert syngeneic GC models. Dovitinib activates the tumor-intrinsic SNAI1/2-IFN-γ signaling axis and impedes the EMT program converting immune desert-type tumors to immune inflamed-type tumors, sensitizing these mesenchymal-like ‘cold’ tumors to CTLA4 blockade.

Conclusion: We develop a novel immunophenotype-based algorithm to reclassify GC into immune inflamed, excluded, and desert subtypes. Our analysis identified major signaling pathways and potential druggable targets relevant to patient groups, especially for refractory immune desert-type/‘cold’ tumors. Dovitinib, an RTK inhibitor, sensitizes desert-type immune-cold GCs to CTLA4 blockade and possibly in a broad spectrum of other solid tumors.

Citation Format: Heng Lu, Longlong Cao, Mohammed Soutto, Nadeem Bhat, Zheng Chen, Dunfa Peng, Ahmed Gomaa, Sachiyo Nomura, Jashodeep Datta, Alexander Zaika, Wael El-Rifai. Dovitinib restricts EMT and promotes T cell recruitment to immune-desert gastric cancers via tumor-intrinsic IFN-γ signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3237.
Abstract 342: Differential CD45 isoform expression and function in AML versus healthy leukocytes
Authors: Ales Evan , Sackstein Robert
Published: April 2023

CD45 (the leukocyte common antigen) is a phosphatase present on all malignant and non-malignant leukocytes and hematopoietic progenitors and is the most abundant glycoprotein displayed by these cells. The human CD45 gene is alternatively spliced by reassortment of just three exons, exon 4 (“A”), exon 5 (“B”), and exon 6 (“C"), to create five distinct “R” isoforms (RO (no splice exons), RABC, RAB, RBC, and RB), with inclusion of A, B, or C exons enriching for expression of glycans (carbohydrates). Despite decades of investigation, the expression and phosphatase function of these isoforms on human AML cells remains unknown. To address this question, flow cytometry and western blot analysis was performed on AML primary cells (n=3) and KG1a and U937 cell lines versus healthy monocytes (n=3) utilizing exon-specific CD45-targetting mAbs. We then compared the contribution of CD45 phosphatase activity in KG1a and U937 cells versus healthy monocytes by employing a highly specific CD45 phosphatase inhibitor in cell culture for 24hrs, assessing induction of cell death after exposure to the inhibitor (n=3). Our results confirm reports of others indicating that "CD45RA- positivity" is a characteristic phenotype of AML cells, yet, distinctly, our data indicate that the most prevalent isoform identified on AML cells as "CD45RA-positive" is, actually, CD45RABC, the largest and most glycosylated isoform of CD45. This finding is in striking contrast to healthy monocytes, which do not express CD45RABC. Additionally, culturing of human AML cells with a CD45 phosphatase inhibitor induces rapid cell death within 24 hours but does not affect healthy monocytes. Thus, the conspicuous expression of the CD45 isoform CD45RABC among AML cells is associated with a survival advantage. Current studies in AML are directed to elaborating the prevalence of CD45RABC expression and uncovering the downstream effectors of its enzymatic activity to elucidate how such pathways sustain AML growth/survival.

Citation Format: Evan Ales, Robert Sackstein. Differential CD45 isoform expression and function in AML versus healthy leukocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 342.
Abstract 345: Cutaneous T-cell lymphoma: ectopic expression of gametocyte specific factor 1 (GTSF1) stimulates the Th phenotype
Authors: Villarreal Amelia Martínez , Litvinov Ivan
Published: April 2023

Diagnosis of Cutaneous T-Cell Lymphomas (CTCL) can take up to 6 years, increasing the risk of disease progression in these patients. Transcriptomic analyses have helped identify several diagnosis biomarkers, such as TOX, KIRD3DL2, GTSF1 and others. Although not officially a Cancer-Testis (CT) antigen, ectopic expression of Gametocyte Specific Factor 1 (GTSF1), presents the same advantages than CT antigens. Its germline-cell restricted expression potentially makes this gene easily identifiable by the immune system and a therapeutic target with less side effects. The role of GTSF1 has been elucidated in mice, where it controls retrotransposon expression during germ-cell development. Here, we investigate the molecular function of GTSF1 in CTCL. Using shRNA-mediated GTSF1 knockdown in CTCL cell lines, our current work demonstrates an unexpected role of GTSF1 regulating the Th phenotype of CTCL cells. With multiple approaches (qRT-PCR, Western blotting and a dual-luciferase based retrotransposition assay) we concluded that GTSF1 knockdown does not lead to retrotransposon reactivation. Transcriptome analysis with RNA-Seq, followed by differential gene expression analysis with DESeq2 and pathway enrichment analysis with GO, KEGG, Reactome and WikiPathways, suggested an involvement of GTSF1 in the Th phenotype of CTCL cells. Differential gene expression analysis revealed 12,496 differentially expressed genes (p adjusted < 0.05). Pathway enrichment analysis demonstrated an enrichment of several pathways including response to stimulus, immune response, response to cytokine and inflammatory response. More specifically, GTSF1 knockdown leads to an upregulation of the key cytokines associated with a Th1 phenotype, IFNG and TNF. Disease progression of CTCL is associated with a decrease in Th1 cytokine production and an enhanced Th2 cytokine production by malignant T cells. These findings shed light on the molecular function of GTSF1 as a regulator of the Th phenotype in CTCL and paves the way for the development of a valuable treatment target for CTCL patients.

Citation Format: Amelia Martínez Villarreal, Ivan Litvinov. Cutaneous T-cell lymphoma: ectopic expression of gametocyte specific factor 1 (GTSF1) stimulates the Th phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 345.
Abstract 3533: Zebrafish her3 knockout impacts developmental and rhabdomyosarcoma-related gene signatures
Authors: Kent Matthew R. , Calderon Delia , Silvius Katherine M. , Kucinski Jack P. , LaVigne Collette A. , Cannon Matthew V. , Kendall Genevieve C.
Published: April 2023

HES3 is a basic helix-loop-helix transcription factor that regulates neural stem cell renewal during development. HES3 overexpression is predictive of reduced overall survival in patients with fusion-positive rhabdomyosarcoma, a pediatric cancer that resembles immature and undifferentiated skeletal muscle and is most commonly driven by the fusion-oncogene PAX3-FOXO1. However, the mechanisms of HES3 cooperation in PAX3-FOXO1 driven rhabdomyosarcoma are unclear and are likely related to her3/HES3’s role in neurogenesis. To investigate HES3’s function during development, we generated a zebrafish CRISPR/Cas9 null mutation of her3, the zebrafish ortholog of HES3. Phenotypic characterization revealed that her3 null mutation is not embryonic lethal as they are present at expected Mendelian ratios. We observed a temporary growth delay her3 zebrafish null mutants and, rarely, eye defects in adults. Transcriptomic analysis of her3 null mutant embryos showed early dysregulation of a known downstream target, neurog1 and downregulation of genes involved in organ development, such as pctp and grinab, while genes pointing toward a terminal differentiation state, such as tmod, are upregulated. Differentially expressed genes in her3 null mutant embryos are enriched for HOX and SOX10 motifs, suggesting these may be key genes involved during HES3 dysregulation. Several cancer-related gene pathways are impacted, including the inhibition of matrix metalloproteinases and the tumor microenvironment pathway. To complement our zebrafish model, we are developing a double-inducible cell line model consisting of a tetracycline-inducible HES3 and a cumate-inducible PAX3-FOXO1 to further investigate the hypothesized cooperation between her3/HES3 and PAX3-FOXO1 in fusion-positive rhabdomyosarcoma. These two systems will allow us to elucidate conserved mechanisms of cooperation between HES3 and PAX3-FOXO1, and identify new therapeutic opportunities for children with fusion-driven rhabdomyosarcoma.

Citation Format: Matthew R. Kent, Delia Calderon, Katherine M. Silvius, Jack P. Kucinski, Collette A. LaVigne, Matthew V. Cannon, Genevieve C. Kendall. Zebrafish her3 knockout impacts developmental and rhabdomyosarcoma-related gene signatures. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3533.
Abstract 3555: Proteomic characterization of rhabdomyosarcoma-derived extracellular vesicles reveals a fusion-positive protein signature
Authors: Quaglietta Paula R. , Kissoondoyal Ashby , Malkin Ethan , Malkin David , Baertschiger Reto M.
Published: April 2023

Rhabdomyosarcoma is the most common pediatric soft tissue sarcoma. Current diagnostic methods involve imaging and tissue biopsy for staging, histology and fusion status assessment. There are presently no serum biomarkers for rhabdomyosarcoma diagnosis or surveillance. Novel approaches and identification of biomarkers are warranted for minimally invasive diagnostic/surveillance techniques. Extracellular vesicles (EVs) are recognized as critical mediators of intercellular communication and the pathophysiology of carcinogenesis and metastasis. This study aimed to characterize the proteome of rhabdomyosarcoma-derived EVs and identify proteomic signatures that correlate with clinical characteristics such as histological subtype, cancer stage, age and metastasis, using patient-derived cell lines. EVs were isolated from cell culture conditioned media by differential ultracentrifugation from four rhabdomyosarcoma cell lines (RH4, RH18, RH30, RD), each in triplicate. Liquid chromatography-tandem mass spectrometry identified EV protein cargo. Proteins identified in our samples were compared to all available EV data in the Vesiclepedia database, using the FunRich software. We identified 1,527 total proteins in our rhabdomyosarcoma-derived EVs. When comparing to Vesiclepedia, 96 unique proteins were identified from our EVs that were not previously annotated in EVs within the database. Expression of these 96 proteins was further evaluated based on clinical characteristics of the tumors the cell lines were derived from. We found a four-protein signature in the EV secretome of rhabdomyosarcoma cell lines correlated with FOXO1 fusion-positive status. This signature includes proteins involved in regulating extracellular matrix organization, regulating gene expression, protein biosynthesis and translation. Rhabdomyosarcoma cells secrete EVs with unique protein cargo based on clinical characteristics of the parent tumor such as histological subtype and FOXO1 fusion status. Further validation of these secreted EV proteins in biological fluids could prove an effective liquid biopsy technique for novel diagnostic approaches or surveillance in pediatric rhabdomyosarcoma.

Citation Format: Paula R. Quaglietta, Ashby Kissoondoyal, Ethan Malkin, David Malkin, Reto M. Baertschiger. Proteomic characterization of rhabdomyosarcoma-derived extracellular vesicles reveals a fusion-positive protein signature. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3555.
Abstract 3614: 3D model to study migration and invasion of lung cancer
Authors: Hardt Melina , Zatloukal Kurt , Popper Helmut H.
Published: April 2023

Background: In 2D cell cultures, migration of tumor cells is characterized by epithelial-to-mesenchymal transition (EMT), whereby tumor cells loose epithelial and express mesenchymal markers and very often change into a spindle cell phenotype. In contrast to 2D cultures, in 3D cultures tumor cells do not lose their differentiation. Small cell lung cancer usually moves as single cells or in small clusters, squamous cell and adenocarcinomas preferentially move in large clusters of cells. In tissue specimen, all of these carcinomas migrate in a hybrid EMT, not loosing their cytokeratin and E-cadherin expression. In AC and SCC also polarity and specification do occur, as some tumor cells act as leaders, providing orientation for the followers. However, 3D culture system might better depict real migration and come closer to the in vivo system as 2D cell culture systems.

Material and Method: A 3D bioprinter (TissueLabsR) was used to spot an alveolar-mimicking lung-specific matrix on cell culture wells. Microscopic channels out of pluronicsR are created within the matrix. Pluronics is removed by cooling, leaving empty channels within the protein matrix. These channels can be filled with different interleukins, such as IL23, which will direct migration of the tumor cells. Cultured cell lines from an adenocarcinoma (A549) and small cell carcinoma (NCI-H82) are layered on top of the matrix and migration into the matrix is studied using different time points.

Results and Discussion: A matrix simulating an alveolar structure was created with MatrigelR. The A549 cells were seeded on the matrix and grown for 2,5, 10, and 14 days. The cells formed cell clusters on the matrix and after one week started to invade the matrix in cell complexes. By immunohistochemistry we could show, that the A549 cells retained their epithelial differentiation. The H82 cells showed a different behavior. They did not form large clusters, but started to invade in small groups. The use of the bioprinter enabled us not only to create a 3D tumor culture system to simulate the in vivo conditions but also to create matrix structures for studying invasion and migration and to stimulate carcinoma cells with migration-activating substances.

Citation Format: Melina Hardt, Kurt Zatloukal, Helmut H. Popper. 3D model to study migration and invasion of lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3614.
Abstract 3636: Liver endothelium secreted LRG1 promotes metastatic colorectal cancer growth through the HER3/RSK/EIF4B AXIS
Authors: Rathore Moeez Ghani , Wright Michelle , Huang Wei , Taylor Derek , Li Yamu , Winter Jordan , Wang Zhenghe , Greenwood John , Moss Stephen , Wang Rui
Published: April 2023

Background: 25% of patients diagnosed with colorectal cancer (CRC) have liver metastasis at presentation, and ~80% of all metastatic CRC are developed in the liver. We previously reported that liver endothelial cells (ECs), a key component of the liver microenvironment, secrete LRG1 to promote CRC growth via activating human epidermal growth factor receptor (ERbB3, also known as HER3). However, we found that LRG1-induced HER3 activation is distinct from the canonical neuregulin 1 (NRG1)-induced HER3 pathway. The present study further validated LRG1 as a new HER3 ligand for promoting mCRC growth and elucidated the novel downstream signaling pathway induced by LRG1-HER3.

Methods: We first measured the binding affinity between HER3 and LRG1 by Biolayer interferometry (BLI). We then used in vitro and in vivo xenograft approaches to determine the effect of LRG1 monoclonal antibody (15C4) on HER3 activation and CRC growth. To further determine the role of LRG1 in promoting CRC growth in the liver, we used murine CRC cells in a syngeneic orthotropic liver injection model to establish CRC allografts in the liver of LRG1−/− mice with systemic LRG1 knockout and wild-type siblings (LRG1+/+). We also performed unbiased phospho-MS analysis and subsequent validations to determine the downstream signaling pathway activated by LRG1-HER3.

Results: We identified that LRG1 binds to HER3 with the affinity at ~100nM. The LRG1 antibody 15C4 completely attenuated LRG1-induced HER3 activation and in vitro and xenograft growth in vivo. Moreover, LRG1−/− mice with CRC allografts in the liver had 2 times longer overall survival than tumor-bearing LRG1+/+ mice. Lastly, unbiased -omics analysis identified eIF4-protein synthesis is significantly activated by LRG1. With target-specific inhibitors, we further determined that LRG1-HER3 activates the PI3K-PDK1-RSK1/3-eIF4 axis independent of AKT.

Conclusions: We identified LRG1 as a novel HER3 ligand and demonstrated that the liver microenvironment-derived LRG1 plays a key oncogenic role in mCRC, by activating a novel RSK-eIF4 survival pathway. Our findings highlighted the potential of blocking LRG1-HER3 and involved downstream pathways for treating patients with mCRC.

Citation Format: Moeez Ghani Rathore, Michelle Wright, Wei Huang, Derek Taylor, Yamu Li, Jordan Winter, Zhenghe Wang, John Greenwood, Stephen Moss, Rui Wang. Liver endothelium secreted LRG1 promotes metastatic colorectal cancer growth through the HER3/RSK/EIF4B AXIS. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3636.
Abstract 3639: Impact of KRAS oncogenic signaling in tumorigenesis and immune evasion driven by extracellular vesicles (EVs)
Authors: Vargas Zulaida M. Soto , Arteaga David C. , Sohal Ikjot S. , Hasan Humna , Kasinski Andrea L.
Published: April 2023

Mutations in Kristen rat sarcoma homolog (KRAS) occur in about 25% of cancers, making this protein one of the most frequently altered genes in cancer. KRAS mutations are not only associated with poor patient survival, but also with increased tumor aggressiveness and metastasis. Tumor development and metastasis are impacted by intercellular communication. Key mediators of cell-to-cell communication include vesicles secreted by cells, also known as extracellular vesicles (EVs). EVs released by cancer cells can mediate malignant transformation of non-cancerous cells as well as influence pre-metastatic niche formation. Conversely, inhibiting the secretion and release of EVs by cancer cells decreases tumor growth and metastasis. However, the mechanisms through which EVs drive tumorigenesis in KRAS mutant tumors are still understudied. Our lab recently reported that EVs from lung cancer cells drive invasion of non-tumorigenic lung epithelial cells. Here, we found that KRAS oncogenic signaling in the donor cells contributes to the EV-driven migration and invasion of non-tumorigenic lung epithelial cells. Previous studies have demonstrated that KRAS signaling drives immune evasion and promotes an “immune cold” tumor microenvironment. Our preliminary studies showed that EVs derived from KrasG12C lung cancer cells (H358) inhibit T cell proliferation and that blocking KRASG12C signaling, using ARS-1620 (KrasG12C inhibitor), impairs EV-driven T cell immunosuppression. We performed a comprehensive proteomic comparison between EVs isolated from H358 treated with ARS-1620 and those isolated from cells treated with DMSO (vehicle) and identified proteins involved in migration and invasion, such as LAMB-3, to be downregulated in EVs isolated from ARS-1620 treated cells. Ongoing studies aim to pinpoint the detailed mechanisms by which cancer cells utilize mutant KRAS signaling to promote loading of oncogenic cargo into EVs, and the resulting contributions to tumorigenesis and immune evasion.

Citation Format: Zulaida M. Soto Vargas, David C. Arteaga, Ikjot S. Sohal, Humna Hasan, Andrea L. Kasinski. Impact of KRAS oncogenic signaling in tumorigenesis and immune evasion driven by extracellular vesicles (EVs). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3639.