bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3765: Epigenomics at the speed of sound: High throughput epigenomic mapping of low cell input samples using acoustic enhanced immunoprecipitation
Authors: Alija Besmira , Braunstein Stephanie , Xu Jane , O'Hare Tom , Garding Angela , Viny Aaron
Published: June 2022

Mapping the epigenetic landscape of Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML) is essential for understanding gene regulatory mechanisms during cell fate changes, disease onset, progression, as well as for optimizing therapeutic regime. In addition, these information hold immense potential for accurate identification of disease biomarkers, cell of origin, and targets for epigenetic therapy. However, existing chromatin immunoprecipitation sequencing (ChIP-Seq) workflows require significant cellular material and are very time-consuming, requiring cumbersome optimization. Moreover, cellular heterogeneity can influence the accuracy of results from the perspective of sample preparation efficiency and also population-biased effects. Both normal and malignant bone marrow are heterogeneous in cell type and stage of differentiation that leads to imprecise results with regard to epigenetic determinates.In our study, we implemented Covaris’ Adaptive Focused Acoustics® (AFA®) technology to miniaturize sample volumes for both chromatin shearing and immunoprecipitation in a 96-well format. This approach enables a standardized, robust, automatable, half-day workflow for both ChIP and Hi-ChIP applications. Additionally, we evaluated low input in vitro and in vivo samples, where we utilized a range of epitopes including major histone modifications (H3K27ac) and factors (CTCF). AFA was applied to flow-sorted stem and progenitor populations from individual mice. The low cell input capability permitted deconvolution of dynamic epigenetic changes in protein binding and chromatin structure during normal lineage specification of hematopoietic stem cells. Epigenetic remodeling of these sites were consistent with established gene programs essential for myeloid differentiation. Data were found to be in alignment with standard ChIP-seq and Hi-C datasets generated from similar cell populations.The simplified and significantly shortened (Hi)ChIP-seq processes showcased in this study will be highly beneficial for both research and clinical diagnostic applications owing to their requirements for using 96-well plate format and their ability to become integrated into automated workflows. Taken together, this technology and workflow represent a high throughput epigenetic system for small cell numbers of cells with a multitude of applications enabling laboratories focused on cell biology and translational research.

Citation Format: Besmira Alija, Stephanie Braunstein, Jane Xu, Tom O'Hare, Angela Garding, Aaron Viny. Epigenomics at the speed of sound: High throughput epigenomic mapping of low cell input samples using acoustic enhanced immunoprecipitation [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 3765.
Abstract 3767: Identification of regulators of epigenetic silencing of the p16INK4a gene
Authors: Fujita Toshitsugu , Fujii Hodaka
Published: June 2022

Epigenetic silencing of tumor suppressor genes plays critical roles in oncogenesis. The CDKN2A/p16INK4a gene is one of the most frequently silenced tumor suppressor genes. To elucidate molecular mechanisms of maintenance of the silenced status of the p16INK4a gene, we applied the insertional chromatin immunoprecipitation (iChIP) method. The promoter region of the transcriptionally active and silenced alleles of the p16INK4a gene were tagged with a bacterial DNA-binding protein LexA and its binding elements. Subsequently, the promoter regions were affinity purified with antibody against a tag fused with LexA. Proteins more or less abundantly associated with the silenced promoter were identified by stable isotope labeling using amino acids in cell culture (SILAC), one of quantitative mass spectrometry methods. Knock-down experiments of the identified enzymes revealed essential components of maintenance of epigenetic silencing. The essential enzymes can be good targets of development of drugs reactivating expression of the silenced tumor suppressor genes in cancers. We are currently performing drug discovery research to identify small molecules that bind and inhibit those candidate drug target enzymes. Our locus-specific ChIP technology consisting of iChIP and the engineered DNA-binding molecule-mediated ChIP (enChIP) method is a powerful tool to identify novel drug targets for development of drugs regulating gene expression in intractable diseases.

Citation Format: Toshitsugu Fujita, Hodaka Fujii. Identification of regulators of epigenetic silencing of the p16INK4a gene [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 3767.
Abstract 377: Evaluating delivery of TFR1-targeting therapies to the CNS in a novel humanized TFR1 mouse model
Authors: Liu Chang , Shang Chengzhang , Ma Limei , Zhou Xiaofei , Guo Chaoshe
Published: June 2022

Transferrin receptor protein 1 (TFR1), also known as CD71, is a cell surface receptor that plays a major role in cellular iron uptake via receptor-mediated endocytosis. In mammalian species, this receptor is required for erythropoiesis and neural development. The efficient cellular uptake properties of TFR1 have been exploited for site-specific delivery of anti-cancer drugs and biologics, as well as targeted delivery of therapeutic genes to malignant cells overexpressing TFR1. Such delivery is achieved by several methods, including chemical conjugation of transferrin with therapeutic agents, genetic fusion of transferrin with peptides, and anti-TFR1 antibodies. Delivery of therapeutics cross the blood-brain barrier to the central nervous system (CNS) is also greatly facilitated by targeting the TFR1. At Biocytogen, we have successfully generated a humanized B-hTFR1 mouse model for evaluating TFR1-targeted therapeutics in preclinical studies. In this model, the exons 4-19 of mouse Tfr1 gene that encode the extracellular domain of the receptor were replaced by its human counterparts. As the result of this engineering, the ectodomain of the TFR1 is humanized while intracellular domain remains unchanged, maintaining the receptor’s signaling function in mice. Surface staining of erythroid cells from B-hTFR1 mice showed high levels of expression of humanized TFR1. In addition, we were able to detect anti-human TFR1 bispecific antibodies in brain tissues of the B-hTFR1 mouse after intravenous administration of the antibodies, suggesting a robust uptake of the antibodies via the hTFR1 receptor. These results indicate that the B-hTFR1 mouse model is a powerful preclinical tool to evaluate the delivery of therapeutics to the CNS in vivo.

Citation Format: Chang Liu, Chengzhang Shang, Limei Ma, Xiaofei Zhou, Chaoshe Guo. Evaluating delivery of TFR1-targeting therapies to the CNS in a novel humanized TFR1 mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 377.
Abstract 3774: The effect of CDK5 on mitochondrial function in Glioblastoma Multiforme (GBM)
Authors: Brett-Morris Adina , Mears Jason , Rochon Kristy
Published: June 2022

Glioblastoma Multiforme (GBM) is the most common and aggressive form of brain tumors.Despite conventional therapies, average patient survival is less than two years. GBMrecurrence is in part due to self-renewing tumor initiating cells, often called glioma stemcells (GSCs), which are characterized by their proliferation and tumor formation capacity,as well as their metabolic features. Thus, mitochondrial function represents a potentiallink between cellular metabolism and differentiation state. Mitochondrial fission isindispensable for growing and dividing cells. Dynamin-related protein 1 (Drp1) is essentialfor this process. In particular, there is cumulative evidence that links Drp1 with severaltypes of cancers, including GBM. Regulation of Drp1 is mainly controlled by post-translational modifications (PTMs). Importantly, recent studies have shown CDK5-dependent phosphorylation at the activating site serine 616 of Drp1 enhancesmitochondrial fission. CDK5 plays a crucial role in the central nervous system as well as,in tumor progression. Therefore, our lab hypothesized that inhibition of CDK5 would targetmitochondrial function in order to decrease tumor growth and sensitize brain tumors toconventional therapies. Western blot analysis has shown that CDK5-dependentphosphorylation of Drp1 is present in GSCs and GBM cell lines but absent in non-stemtumor cells (NSTC) and astrocytes. Small-molecule CDK5 inhibitors or CDK5 knockdownthrough siRNA in GSCs and GBM cell lines decreased activating phosphorylation of Drp1,leading to elongation of mitochondria morphology and corresponding reductions in cellviability and cell proliferation. Additionally, CDK5 inhibition showed sensitization toionizing radiation (IR) in GSCs and GBM cells. Importantly, CDK5 inhibition or knockdowncombined with IR, showed increased levels of cleaved caspase-7 and cleaved PARPsuggesting a role for apoptosis in decreased cell proliferation and sensitization to IR.Interestingly, CDK5 inhibition also diminished mitochondrial oxidative phosphorylation.Overall, these findings highlight an important role for CDK5 in regulating mitochondrialfunction in GBM, and targeting CDK5 offers a novel strategy to sensitize tumors to chemo-and radiotherapies.

Citation Format: Adina Brett-Morris, Jason Mears, Kristy Rochon. The effect of CDK5 on mitochondrial function in Glioblastoma Multiforme (GBM) [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 3774.
Abstract 3776: Frequent alteration of mitochondria functional pathways in lung cancer
Authors: Hertweck Kate L. , Persing Brian , Prodduturvar Pranitha , Singh Seema , Singh Ajay , Dasgupta Santanu
Published: June 2022

Lung cancer killed 1.8 million people globally in 2020. In the United States, there will be over 235,760 cases of lung cancer with an estimated death of 131,880 individuals in 2021. Despite significant improvement in multimodal therapeutic approaches, overall 5-year survival rate is only 21.7%. Lung cancer appears to be mitochondria enriched cancer, however, alteration pattern of hallmark molecular pathways associated with diverse mitochondrial functions remain largely unknown in lung tumorigenesis. Utilizing high-throughput genomic profiling, we identified altered copy number of a panel of 40 mitochondria targeted molecules in primary lung cancer patients and their histologically normal appearing follow up mucosal biopsies. These altered molecules are critical components of the mitochondrial protein carrier, OXPHOS, stress response, amino acid metabolism and apoptosis pathways. This finding suggests for clonal genetic relationship between multifocal lesions and potential role of these alterations in the evolution of second primary tumors, which is an existing problem among lung cancer patients. The Human Cancer Genome Atlas database was utilized to further assess the mRNA expression pattern of these 40 molecules and determine their clinico-pathological correlations. Immunohistochemistry and digital pathology system based evaluations were performed to validate expression pattern of key altered molecules. We identified overexpression of various critical mitochondria pathway molecules including AARS2, AGMAT, SDHA, NDUFB7, LONP1, DGUOK, MRM1 and GCAT in both lung adenocarcinomas (LUAD) and squamous cell carcinomas (LUSC), predicting poor prognosis. Significant loss of expression of ACSF2, ACSS1, MTCH1, SLC25A4, ACAD8 and NAGS was evident in both adenocarcinoma and squamous cell carcinoma subtypes. Molecular characterization of both LUAD and LUSC tumor subtypes identified significant association of various key molecules including ACAD8, AGMAT, DGUOK and NDUFAB1 with proximal-inflammatory, proximal-proliferative and terminal respiratory unit pathways in LUAD; and an association of DGUOK, MDH2, MRM1 and MRPL10 with basal, classical, primitive and secretory pathways in LUSC. Significant loss of SLC25A4 protein expression was evident in lung cancer tissues, which predicted poor survival of the lung cancer patients. Capturing the compendium of hallmark mitochondrial alterations in lung tumorigenesis may open up novel avenues for therapeutic and biomarker interventions to improve the overall survival of the lung cancer patients.

Citation Format: Kate L. Hertweck, Brian Persing, Pranitha Prodduturvar, Seema Singh, Ajay Singh, Santanu Dasgupta. Frequent alteration of mitochondria functional pathways in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3776.
Abstract 3868: High throughput tissue phenotyping and imaging of the tumor immune microenvironment using novel FlexVUE™ multiplexed immunofluorescence assays
Authors: Cajigas Yvette , Hamer Peter , Ainbinder Alina , Vasaturo Angela , Wood Douglas , Maclean Kirsteen H. , Chatterjee Gourab
Published: June 2022

Immunotherapy has transformed the treatment of metastatic and recurrent solid tumors. Advances in technology in the past few years have created unprecedented opportunities to identify biomarkers of disease processes, especially by using multi-omics technologies and datasets to derive valid and useful signatures of disease. Advances in multiplexed immunofluorescence (mIF) staining and imaging technologies that allow for accurate phenotyping of individual cells while also preserving architectural features of the sample are critical to our understanding of the interplay of tumor and immune cells in the tumor immune microenvironment (TiME).Herein we describe the utility of our new flexible mIF assays (FlexVUE™ panels) to provide the necessary relevant distribution of infiltrating immune cells in tumors coupled to the use of our new UltiStacker™ software. This hardware and software package allows a detailed spatial characterization of specific cell phenotypes defined by co- or lack of expression that may help in predicting clinical responses and mechanisms of resistance to therapy. The FlexVUE™ mIF assay was used to conduct 8-plex immunophenotyping of FFPE tumor sections. Slides were stained with a cocktail of eight primary antibodies using Leica Biosystems BOND RX autostainer and imaged on the Zeiss Axio Scan.Z1. Following the first round of imaging, the slides were decoverslipped and the signal was removed. New targets were probed on the same tissue section in a reagent incubation step on the BOND RX, termed ‘exchange.’ Slides were re-imaged on the Zeiss Axio Scan Z1 using the same dye channels. The image pairs were automatically aligned using UltiStacker™ software and exported for downstream analysis with Indica Labs HALOv3.1 software. Our approach demonstrates a streamlined off the shelf workflow requiring no assay development time that supports whole slide imaging of a flexible high plex panel. In addition, our assays enable traditional same slide H&E fusion on a single tissue slide for comprehensive tissue immunophenotyping analysis of proliferating cells, tumor cells, tumor-infiltrating lymphocytes, macrophages or tertiary lymphoid structures.

Citation Format: Yvette Cajigas, Peter Hamer, Alina Ainbinder, Angela Vasaturo, Douglas Wood, Kirsteen H. Maclean, Gourab Chatterjee. High throughput tissue phenotyping and imaging of the tumor immune microenvironment using novel FlexVUE™ multiplexed immunofluorescence assays [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 3868.
Abstract 3907: A pheno-multiomic integration analysis of primary samples of acute myeloid leukemia reveals biomarkers of cytarabine resistance
Authors: Silberberg Gilad , Vishwakarama Bandana , Walling Brandon , Riveley Chelsea , Audia Alessandra , Zipeto Marianna , Sloma Ido , Wesa Amy , Ritchie Michael
Published: June 2022

The overall survival of patients diagnosed with Acute Myeloid Leukemia (AML) remains low. While initial responses to therapy are favorable, the duration of response is short and overcoming therapeutic resistance has proven difficult. A better understanding of the tumor cell biology and resistance mechanisms may shed light onto novel therapeutic targets that improve long-term outcome. In this study, we performed an exhaustive analysis to include deep tumor phenotyping, drug sensitivity profiling and comprehensive omic characterization. These datasets were included in integrative pharmaco-phenotypic-multiomic analyses to identify targets and biomarkers associated with cellular phenotype and drug response. Our results reveal that the major cellular discriminant within the cellular phenotype is CD34 expression, which associates with a high PDK-mediated metabolic profile and cytarabine sensitivity. Tumors exhibiting cytarabine resistance associate with a CD34-negative cellular phenotype and molecular characteristics such as MYC copy number gain, and increased expression of SAMDH1, FBP1 and TYMP proteins.

Citation Format: Gilad Silberberg, Bandana Vishwakarama, Brandon Walling, Chelsea Riveley, Alessandra Audia, Marianna Zipeto, Ido Sloma, Amy Wesa, Michael Ritchie. A pheno-multiomic integration analysis of primary samples of acute myeloid leukemia reveals biomarkers of cytarabine resistance [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 3907.
Abstract 393: Targeting MCT inhibition to overcome enzalutamide resistance in prostate cancer
Authors: Bhattacharjee Sayani , Doan Jonathan P. , Wynn Rebecca , Nadiminty Nagalakshmi
Published: June 2022

Prostate cancers (PCa) exhibit a unique metabolic profile with reliance on different forms of glucose metabolism at different stages of disease progression. Early stage PCa cells use the more efficient TCA cycle, while metastatic PCa cells switch to glycolysis (Warburg effect), leading to the accumulation of lactate. Lactate is exported out of the cells by monocarboxylate transporters (MCTs) to maintain redox balance. Such metabolic reprogramming can lead to gain-of-function mutations and affect drug sensitivity. Enzalutamide is a second-generation antiandrogen used for the treatment of metastatic castration-resistant PCa. However, resistance to Enzalutamide develops in most patients within 9-15 months. Since Warburg effect is a hallmark of metastatic PCa, we sought to understand whether lactate transport has any effect on Enzalutamide-resistance in PCa. We measured the expression of MCTs in parental and Enzalutamide-resistant C4-2B and 22RV1 cells and found that they are overexpressed in the Enzalutamide resistant subtypes. Based on this observation, we hypothesized that targeting lactate transport might be a potential strategy to overcome Enzalutamide-resistance in PCa cells. Using cell survival and cell proliferation assays, we found that MCT antagonists resensitized Enzalutamide resistant C4-2B and 22Rv1 cells to treatment with Enzalutamide. Using the Seahorse based glycolytic rate assay we found that these combination treatments significantly reduced the extracellular acidification rate by reducing the level of glycolysis in the Enzalutamide-resistant PCa cells. We also found that treatment with these antagonists either singly, or in combination with Enzalutamide suppressed xenograft growth in SCID mice bearing subcutaneously injected parental or Enzalutamide-resistant C4-2B cells. These findings led to the conclusion that targeting MCTs could be an attractive strategy to overcome Enzalutamide resistance in prostate cancer.

Citation Format: Sayani Bhattacharjee, Jonathan P. Doan, Rebecca Wynn, Nagalakshmi Nadiminty. Targeting MCT inhibition to overcome enzalutamide resistance in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 393.
Abstract 396: Therapeutic targeting of eukaryotic translation initiation factor 4 gamma 1 (EIF4G1) in enzalutamide resistant (ENZR) prostate cancer
Authors: Jaiswal Praveen K. , Koul Sweaty , Sankaralingam Saikolappan , Koul Hari K.
Published: June 2022

Objective: Deregulation of the translation initiation machinery is one of the critical steps for oncogenic mRNA translation in tumor cells. Eukaryotic translation initiation factor 4 gamma 1 (EIF4G1) is a crucial scaffold of translation initiation complex (EIF4F). Our recent studies suggest a role for EIF4G1 in multiple tumor types including prostate cancer progression. In the present study, we tested the unknown function of EIF4G1 in Enzalutamide resistant prostate cancer cells.

Methods: We used enzalutamide resistant C4-2B (C4-2BENZR) and parental C4-2B cells for the current study. For the functional studies, we used shRNA targeting the EIF4G1 and pharmacological EIF4G-EIF4E complex inhibitor (4EGI-1). The functional assays such as clonogenic, cell proliferation, cell viability, trans-well migration/invasion, and prostasphere formation were performed. To further expand in vitro findings we employed, in-vivo tumor xenograft model derived from enzalutamide resistance C4-2B cells.

Results: We found a higher level of EIF4G1 protein in ENZ resistance (C4-2B ENZR) cell lines as compared to parental C4-2B cell lines. Furthermore, knockdown of EIF4G1 by shRNA or treatment with 4EGI-1 inhibitor impaired the clonogenic potential, prostasphere formation, cell viability/proliferation, and cell migration/invasion in C4-2B ENZR cell line, suggesting a critical role of EIF4G1 in modulating enzalutamide resistance in CRPC cells. C4-2BENZR subcutaneous tumor xenograft treated with combination of 4EGI-1 and ENZ were significantly reduced the tumor volume. Moreover 4EGI-1 sensitized ENZ Resistance C4-2B cells to Enzalutamide treatment in tumor xenograft model.

Conclusions: Overall, the findings suggest that EIF4G1 could serve as a novel therapeutic target in overcoming therapeutic resistance to current treatments in PCa.

Citation Format: Praveen K. Jaiswal, Sweaty Koul, Saikolappan Sankaralingam, Hari K. Koul. Therapeutic targeting of eukaryotic translation initiation factor 4 gamma 1 (EIF4G1) in enzalutamide resistant (ENZR) prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 396.
Abstract 3985: TGF-B/KLF10 signaling regulates muscle wasting in pancreatic cancer cachexia
Authors: Gibbard Daniel F. , Dasgupta Aneesha , Schmitt Rebecca E. , Ducharme Alexandra M. , Arneson Paige C. , Bruinsma Elizabeth S. , Hawse John R. , Doles Jason D.
Published: June 2022

Cancer cachexia, and its associated complications, represent a large and currently untreatable roadblock to better cancer therapy. Many potential therapies have been suggested and tested - including appetite stimulants, targeted cytokine blockers, and nutritional supplementation, but effective therapies have still not found a place in the clinic. Thus, a new approach to treating cancer cachexia is warranted. Members of the Kruppel-like factor family play wide-ranging and important roles in the development, maintenance, and metabolism of muscle. Upon review both our own and multiple publically available datasets, we identified KLF10 upregulation in a multitude of cachectic muscles - including in pancreatic, lung, and colon cancer mouse models and in human cachexia patients. Thus, in this study, we interrogated loss-of-function of KLF10 in the context of pancreatic cancer associated muscle wasting. Ex vivo experiments on satellite cells isolated from KLF10 Knockout (KO) mice showed resistance to the effect of KPC conditioned media on atrophy. In vivo studies using a KLF10 KO mouse model showed that upon orthotopic implantation of T4-KPC cells, the mice demonstrated a significant preservation of lean mass and significant alterations in the expression of muscle-specific ubiquitin ligases Trim63 and Fbxo32, as well as other genes implicated in atrophy, calcium signaling, and autophagy. Pathway analysis of RNA-seq data from these mice identified TGF-B, a known inducer of KLF10, as an important upstream regulator of the response. Further in vivo experimentation showed KLF10 KO mice to be resistant to the atrophic effects of TGF-B injections. In conclusion, we report a novel role of KLF10 in the manifestation of cachexia in pancreatic cancer.

Citation Format: Daniel F. Gibbard, Aneesha Dasgupta, Rebecca E. Schmitt, Alexandra M. Ducharme, Paige C. Arneson, Elizabeth S. Bruinsma, John R. Hawse, Jason D. Doles. TGF-B/KLF10 signaling regulates muscle wasting in pancreatic cancer cachexia [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 3985.
Abstract 4060: Identification of anti-neoplastic compounds on a triple negative breast cancer cell line from T. violacea
Authors: Dlamini Zodwa , Penny Clement , Hull Rodney , Alouna Mohammed
Published: June 2022

Background: The incidence of Invasive triple negative breast cancer (TNBC) is higher than average in South Africa, with 90% of patients being Black South Africans. Current treatments for breast cancer are associated with severe side effects. Additionally, TNBC is resistant to hormone therapy commonly used to treat other forms of breast cancer. Tulbaghia violacea, a plant native to Southern Africa, is a medicinal herb used in traditional medicine. This study aims to establish the impact of water soluble extracts from T. violacea on TNBC cancer cell lines and identify any anti-cancer compounds present in this extract.

Methods: Following the determination of the IC50 value for this extract. The anti-proliferative and anti-metastatic activities of the extract on the TNBC cell line were determined using scratch assays and cell invasion and migration assays, performed using real time cell analysis. The pro-apoptotic action of the extract on the TNBC cell line was determined using flow cytometry cell death assays. Once the anti-cancer activity of the extract was confirmed, the composition of the extract was determined in order to establish what known or possible anti-cancer compounds are present within it. This was performed using gas chromatography-mass spectroscopy (GC-MS). The structure of these identified compounds was then confirmed using Nuclear Magnetic Resonance (NMR).

Results: The IC50 value for the T. violacea water soluble extract against MDA MB 231 cells was determined to be ~350 µg/mL. The extract was also shown to have anti migration and invasion as well as pro-apoptotic effects on these cells. Fourteen known anti-cancer compounds were identified in this water soluble extract. In addition to this multiple other compounds were shown to also be present in the extract.

Discussion: The water soluble T. violacea leaf extract has cytotoxic effects and the ability to inhibit proliferation and metastasis in a TNBC cell line. The identification of fourteen compounds with known anti-cancer activity may explain the effects this extract has on these TNBC cells, however, the contribution made by any of the other compounds to this activity remains to be determined.

Conclusion: These findings suggest that one or more of the compounds present in T. violacea may serve as lead compounds for the development of new treatments for TNBC.

Citation Format: Zodwa Dlamini, Clement Penny, Rodney Hull, Mohammed Alouna. Identification of anti-neoplastic compounds on a triple negative breast cancer cell line from T. violacea [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 4060.
Abstract 408: Development of a first-in-class notch transcription factor inhibitor CB-103 to overcome resistance to chemotherapy and targeted therapies
Authors: Vigolo Michele , Urech Charlotte , Lamy Sebastien , Majumder Samarpan , Miele Lucio , Lehal Rajwinder
Published: June 2022

Human cancers possess striking ability to develop resistance to Standard of Care (SoC) designed to target specific hallmarks of cancer. This capability to develop resistance is in part due to cancer cell’s ability to activate alternative pathways and modulate tumor microenvironment to enhance cell survival, evade cell death and immunosurveillance. One such mechanism of developing resistance to SoC is the activation of the NOTCH pathway. NOTCH signaling is a developmental pathway which is known to be activated in several human cancers as a key oncogenic hit. In addition to its role as an oncogenic driver, activation of NOTCH is also known to induce resistance to several anti-cancer therapeutics in breast cancer and colorectal cancer. NOTCH signaling is known to cooperate with parallel oncogenic signals to impede activity of several drugs as a single agent. Previous agents targeting the NOTCH pathway upstream of the transcription complex were halted early in clinical development due to dose-limiting toxicities.Here we report activity of a novel first-in-class clinical stage CSL-NICD inhibitor in a panel of cell lines, xenograft and PDX models of human cancers in combination with several chemo and targeted therapies. Guided by in vitro and in vivo studies pre-clinical studies, CB-103 is currently being investigated in clinical trials as a single agent and in combination with SoC. Preliminary pre-clinical and clinical data shows desirable safety and efficacy profile of CB-103 enabling clinical development in combination with several therapeutic agents.

Citation Format: Michele Vigolo, Charlotte Urech, Sebastien Lamy, Samarpan Majumder, Lucio Miele, Rajwinder Lehal. Development of a first-in-class notch transcription factor inhibitor CB-103 to overcome resistance to chemotherapy and targeted therapies [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 408.
Abstract 4082: Expression of chimeric antigen receptor therapy targets in subpopulations detected by single-cell sequencing of normal cells may contribute to off-tumor toxicity
Authors: Jing Ying , Liu Yuan , Li Qiang , Han Leng
Published: June 2022

Chimeric antigen receptor (CAR)-engineered cell therapy, which leverages genetically modified immune cells to generate a tailored immune response, has revolutionized cancer treatment (Dougan et al., 2021; Hong et al., 2020). The number, type and indications of clinical trials for CAR-engineered cell therapies have increased rapidly in recent years (MacKay et al., 2020). However, serious and potentially life-threaten toxicities, such as on-target, off-tumor effects, remain a major limitation of CAR-engineered cell therapies (Dougan et al., 2021; MacKay et al., 2020). To better understand the effects of CAR-directed immunotherapy, a systematic single-cell-level dissection of the expression of divergent CAR targets in various cell types across different normal tissues is urgently required. We analyzed the expression of 591 CAR targets, including 71 that were employed in clinical trials up to January 2021 and 520 that were predicted to have favorable safety profiles (MacKay et al., 2020), in two independent single-cell cohorts, including 342,755 adult cell scRNA-seq data from human cell landscape (HCL) based on Microwell-seq (Han et al., 2020), and 84,363 adult cell scRNA-seq data from adult human cell atlas (AHCA) based on 10× Genomics (He et al., 2020). We used a stringent cutoff by defining a CAR target as a potentially risky gene (PRG) if it was measurable in more than 100 non-immune cells and more than 2% of the total cells in at least three normal tissues. We identified 92 and 88 PRGs from HCL and AHCA by employing this criterion, with 57 PRGs shared in both HCL and AHCA datasets (hypergeometric test p = 7.17×10-29), suggesting the robustness of our analysis. To aid the research community in the design of CAR targets, we developed a user-friendly data portal, CAR target gene toxicity at single-cell level (CARTSC) (https://hanlab.tamhsc.edu/CARTSC/), to allow for browsing and searching the single target and logical ‘and’ design switch expressions at the single-cell level.

Citation Format: Ying Jing, Yuan Liu, Qiang Li, Leng Han. Expression of chimeric antigen receptor therapy targets in subpopulations detected by single-cell sequencing of normal cells may contribute to off-tumor 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 4082.
Abstract 409: Reversal of ABCG2-mediated multidrug resistance by tepotinib in vitro and in vivo
Authors: Wu Zhuoxun , Teng Qiu-Xu , Yang Yuqi , Acharekar Nikita , Wang Jing-Quan , He Min , Yoganathan Sabesan , Lin Jun , Wang Jian , Chen Zhe-Sheng
Published: June 2022

ABCG2/BCRP transporter-mediated Multidrug resistance (MDR) in cancers has been considered as one of the obstacles to the cancer chemotherapy. Our recent study characterized the MET inhibitor tepotinib as a potent ABCB1 inhibitor. In the present study, we demonstrated the MET inhibitor tepotinib effectively antagonized ABCG2-mediated MDR in vitro and in vivo. In addition, the ABCB1/ABCG2 double-transfected cell model demonstrated that tepotinib can simultaneously inhibit the two MDR transporters. Mechanistically, tepotinib stimulated ABCG2 ATPase activity without altering the protein expression level of ABCG2, MET, or p-MET. Moreover, the MDR reversal effect may be attributed to its reversible inhibition of ABCG2 substrate efflux function, thereby sensitizing the drug-resistant cancer cells to substrate drugs. The molecular docking analysis suggested that tepotinib may directly bind to the drug-binding site of ABCG2, which subsequently block the binding of ABCG2 substrates. Animal study showed that tepotinib significantly increased the intratumor accumulation of ABCG2 substrate drug topotecan and enhanced its antitumor effect in ABCG2-overexpressing tumors. In conclusion, our study provides a new strategy of repositioning tepotinib as an MDR modulator to antagonize ABCG2-mediated MDR.

Citation Format: Zhuoxun Wu, Qiu-Xu Teng, Yuqi Yang, Nikita Acharekar, Jing-Quan Wang, Min He, Sabesan Yoganathan, Jun Lin, Jian Wang, Zhe-Sheng Chen. Reversal of ABCG2-mediated multidrug resistance by tepotinib in vitro and in vivo [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 409.
Abstract 4122: Occurrence of BRAF class II and III alterations is common across solid tumors and is associated with inferior clinical outcomes in NSCLC and melanoma
Authors: Severson Paul L. , Donderici Elifnur Yay , Zhang Nicole , Franovic Aleksandra , Miller Nichol , Martin Eric , Murphy Eric , Williams Richard
Published: June 2022

Background: Oncogenic BRAF alterations can be categorized by their distinct structural and signaling properties which lead to activation of the MAPK pathway: class I - kinase active signaling of BRAF mutant monomers; class II - kinase active signaling of BRAF mutant dimers; and class III - kinase impaired BRAF that signals via RAS-dependent, heterodimers containing wild-type RAF. There are currently no approved BRAF targeted therapies for patients whose tumors bear BRAF class II or III alterations. This research utilizes the GuardantINFORM࣪ clinical-genomic database to assess the real-world occurrence and outcomes of patients with oncogenic BRAF alterations by distinct classes across solid tumors.​

Methods: This observational study utilized a database containing clinical and genomic data from ~160,000 cancer patients with ctDNA profiling by the Guardant360 assay. Patients were categorized using a broad list of known and putative class I, II or III BRAF alterations compiled from published sources. Patients were stratified by BRAF mutant class for real-world overall survival analyses.

Results: The analysis identified a cohort of over 5,890 patients with oncogenic BRAF alterations. Class II and III alterations were present in more than 2% of all ctDNA positive patients and made up approximately 55% of all the oncogenic BRAF alterations. Class II and III accounted for 65%, 30% and 20% of oncogenic BRAF alterations in NSCLC, CRC and melanoma respectively. NSCLC and melanoma patients with class II or III BRAF alterations experienced shorter overall survival relative to patients with class I alterations.

Conclusions: The analysis of this large real-world dataset identified a substantial number of cancer patients with BRAF class II and III alterations. NSCLC and melanoma patients with BRAF class II and III alterations experienced inferior clinical outcomes and represent a population that could benefit from novel targeted therapies.

Overall survival in NSCLC and melanoma patients with BRAF class I, II or III alterations Cohort Median OS Months (%95 CI) Unadjusted P-value Class I Class II Class III I vs II I vs III II vs III NSCLC (n=938) 44.8 (37.5,52.8) 34.4 (27.5,41.5) 32.3 (28.4,40.9) 0.006 0.0126 0.6611 Melanoma (n=333) 46.5 (42.3,65.5) 28.5 (8.6,30.3) 30.5 (12.5,73.8) 0.0008 0.0165 0.6529

Citation Format: Paul L. Severson, Elifnur Yay Donderici, Nicole Zhang, Aleksandra Franovic, Nichol Miller, Eric Martin, Eric Murphy, Richard Williams. Occurrence of BRAF class II and III alterations is common across solid tumors and is associated with inferior clinical outcomes in NSCLC and melanoma [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 4122.
Abstract 4202: Dual chlorotoxin and methylguanine-DNA methyltransferase (MGMT) γδ-T cells for drug resistant immunotherapy
Authors: Ding Lei , Li Yanjie , Klumpp Evan , Lamb Lawrence
Published: June 2022

High-grade gliomas are the most aggressive and refractory of brain tumors. Historical approaches to treating glioblastoma (GBM) using chimeric antigen receptor modified T cells (CAR-T) have been challenging, without a lasting benefit to patients, often due to tumor heterogeneity and a lack of tumor specific antigens. We report preliminary in vitro design and optimization of a γδ T cell-based CAR-T using Chlorotoxin (CLTX-CAR), a peptide isolated from the venom of the Israeli Deathstalker Scorpion that efficiently binds to GBM and other tumor cells with limited cross reactivity to normal tissues. This CLTX-CAR (CTLX-CAR-noZ) omits a CD3z signaling domain to maintain the natural innate targeting and tumor recognition properties of the γδ T cell through its endogenous T cell and NKG2D receptors, and CD16-based antibody-dependent cellular cytotoxicity (ADCC). This construct also includes our Drug Resistant Immunotherapy (DRI) platform that incorporates an additional p140K-MGMT encoding gene into the construct. This confers resistance to alkylating agents such as temozolomide (TMZ) that allows our DRI γδ T cells to remain viable and functional when combined with chemotherapy to force high-expression of natural killer group D ligands (NKG2DL) on the surface of tumor cells. As expected, the non-signaling, CLTX-CAR-noZ modified Jurkat cells did not result in T cell activation, as compared to the highly activated CD69 corresponding to signaling CLTX-CAR-CD3z transduced Jurkat cells. Conversely, CTLX-CAR-noZ transduced γδ T cells demonstrated higher cytotoxicity against U87MG and U251MG GBM cell lines over unmodified control γδ T cells. Dual-CLTX-CAR constructs lacking CD3z and using 2x CLTX peptides as the targeting domain (dCLTX-CAR-noZ) exhibited up to 90% lentiviral transduction efficiency into γδ T cells. When co-cultured in suspension with GBM cells in vitro, dCLTX-CAR-noZ γδ T cells demonstrated an average cytotoxicity of 62.1% against U87MG-GFP cells at an E:T ratio of 4:1 in comparison to 35.4% with non-transduced γδ T control cells. Time-lapsed microscopic recording over a period of 15hrs also verified killing of U251MG-GFP cells by dCLTX-CAR-noZ γδ T cells when cocultured with GBM cells at an E:T ratio of 2:1. Serial-killing of tumor cells by γδ T cells was documented. We are advancing this approach to xenograft animal models and as a clinical development candidate (INB-300) for both GBM and other extracranial tumors.

Citation Format: Lei Ding, Yanjie Li, Evan Klumpp, Lawrence Lamb. Dual chlorotoxin and methylguanine-DNA methyltransferase (MGMT) γδ-T cells for drug resistant immunotherapy [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 4202.
Abstract 4210: Gene therapy approach to suppress metastasis in a mouse melanoma model
Authors: Heller Richard , Scott Megan , Mangiamele Cathryn , Synowiec Jody , Shi Guilan
Published: June 2022

Immune checkpoint inhibitors (ICIs) have been demonstrated to have significant clinical benefits. Effectiveness of ICIs have been limited by both primary and acquired resistance. In addition, absence of T cell infiltration within the tumor site is one of the major obstacles limiting ICIs efficacy against solid tumors. Interleukin-12 (IL-12) is a potent immune stimulator that has been shown to stimulate growth and survival of T cells as well as NK cells. Utilizing a B16.F10 melanoma model in C57Bl/6 mice, we observed a significant reduction in T regulatory cells and myeloid derived suppressor cells while T effector cells were significantly increased within the tumor microenvironment (TME) following intratumor delivery of a plasmid encoding IL-12 using gene electrotransfer. Monotherapy with plasmid IL-12 delivered with gene electrotransfer (pIL-12 GET) resulted in prolonged disease-free survival as well as long term immune memory. To determine the robustness of this therapeutic approach and to evaluate the responsiveness in combination with anti-PD1, we used a two-tumor model consisting of a subcutaneous B16F10 tumor and B16F10 cells expressing luciferase injected via the intraperitoneal route in a C57Bl/6 mouse. Intratumor delivery of pIL-12 GET as a monotherapy resulted in reduction or elimination of the subcutaneous tumor. However, the monotherapy approach was only successful in reducing the peritoneal spread in about 50% of the mice. When pIL-12 GET was combined with anti-PD1 administered via an intraperitoneal injection not only was there an elimination of the subcutaneous tumor, but it resulted in the elimination of intraperitoneal metastatic growth. The elimination of peritoneal spread was confirmed utilizing an In Vivo Imaging System. Observations on day 60 revealed background levels of luminescence in 90% of mice treated with the combination therapy thus confirming long-term disease-free survival of these mice. This level of response was not seen in mice treated with anti-PD-1 alone or with simple injection of pIL-12. In addition, the combination therapy resulted in an enhanced memory response which protected against growth of new tumors following challenge. In addition, exposure of tumor cells to IL-12 resulted in upregulation of MHC class 1 and PDL1 expression. The results have this study suggest that the combination of pIL-12 GET combined with ICIs could be utilized as an effective combination therapy for melanoma.

Citation Format: Richard Heller, Megan Scott, Cathryn Mangiamele, Jody Synowiec, Guilan Shi. Gene therapy approach to suppress metastasis in a mouse melanoma 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 4210.
Abstract 4227: NL-201, a de novo agonist of IL-2 and IL-15 receptors, demonstrates antitumor activity in preclinical B cell lymphoma models
Authors: Huard Justin , Mortales Christie , Chen Alex , Swanson Ryan , Walkey Carl , Arthur Bill
Published: June 2022

NL-201 is a computationally-designed IL-2/IL-15 cytokine mimetic under investigation in a phase 1 clinical trial for patients with solid tumors. This de novo protein signals through the IL-2Rβ/ɣ complex in an IL-2Rα-independent fashion, supporting immune function of both T cells and natural killer (NK) cells. In syngeneic tumor models of solid tumors, NL-201 demonstrates marked antitumor activity through multiple mechanisms of action, including upregulation of granzyme B in cytotoxic CD8+ T cells and NK cells. Here, we further explored the therapeutic potential of NL-201 in preclinical models of B cell malignancies. In cell-based studies, NL-201 augmented antibody-dependent cellular cytotoxicity (ADCC) of Toledo DLBCL (diffuse large B cell lymphoma) cells incubated with human NK cells and CD20 antibodies. Importantly, direct activation by NL-201 was specific to NK cells and did not impact STAT5 phosphorylation nor proliferation of the tested malignant cells. The administration of NL-201 monotherapy in mice bearing A20 B cell lymphoma syngeneic tumors yielded robust inhibition of tumor growth. Tumor xenografts from implanted Pfeiffer cells have also been evaluated to understand the activity of NL-201 in a model closely resembling DLBCL physiology. In summary, NL-201 is active as a single agent and in combination with CD20-targeted therapies in vitro, as well as demonstrating monotherapy activity in in vivo preclinical models of B cell malignancies as well as in combination with checkpoint inhibitors. The NL-201 enhancement of cytotoxicity by CD8+ T cells and NK cells supports additional exploration aimed at optimizing combinatorial approaches. These findings will inform the design of planned clinical studies with NL-201 in patients with B-cell lymphomas and other hematological malignancies.

Citation Format: Justin Huard, Christie Mortales, Alex Chen, Ryan Swanson, Carl Walkey, Bill Arthur. NL-201, a de novo agonist of IL-2 and IL-15 receptors, demonstrates antitumor activity in preclinical B cell lymphoma 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 4227.