bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 249: A novel suite of enzyme mixes enable robust hybrid capture sequencing from low quality FFPE DNA
Authors: Heider Margaret R. , Sun Jian , Sexton Brittany S. , Langhorst Bradley W. , Gray Andrew , Higgins Lauren , Chen Lixin , Apone Lynne , Evans Thomas C. , Nichols Nicole M. , Liu Pingfang
Published: April 2023

In cancer genomics, a common source of DNA is formalin-fixed, paraffin-embedded (FFPE) tissue from patient surgical samples, where in most cases high-quality fresh or frozen tissue samples are not available. FFPE DNA poses many notable challenges for preparing NGS libraries, including low input amounts and highly variable damage from fixation, storage, and extraction methods. Due to the high cost of sequencing and variability of coverage, regions of interest are often specifically enriched using hybrid capture-based approaches, but these methods require a high input of diverse, uniform DNA library to achieve the coverage required for somatic mutation identification in tumor samples. We developed a new DNA repair enzyme mix, enzymatic fragmentation mix, and library amplification PCR master mix, optimizing the activities of these mixes using FFPE samples ranging from DIN 1.8 to 6.8 to maximize yield, WGS library quality, and target enrichment library performance. Combining DNA damage repair and a novel enzymatic fragmentation mix upstream of library preparation reduced the false positive rate in somatic variant detection by repairing damage-derived mutations, and also improved the library yield, quality metrics (including mapping, chimeras, and properly-paired reads), complexity, coverage uniformity, and hybrid capture library quality metrics. The new PCR master mix boosts the library yield without compromising library quality in FFPE-derived samples, allowing flexibility in the PCR cycles used to accommodate high-throughput processing of FFPE samples of highly varied quality. This library prep workflow was evaluated with multiple sequencing platforms including Illumina, MGI, and Element Biosciences. This new suite of enzyme mixes allows even highly damaged FFPE samples to achieve high-quality libraries with sufficient input for hybrid capture. Increasing the useable reads and coverage enables robust detection of somatic variants as demonstrated using both reference standard DNA and patient-derived FFPE samples. Finally, the use of enzymatic fragmentation and a flexible PCR master mix make this FFPE library prep workflow compatible with high-throughput and automation-based workflows.

Citation Format: Margaret R. Heider, Jian Sun, Brittany S. Sexton, Bradley W. Langhorst, Andrew Gray, Lauren Higgins, Lixin Chen, Lynne Apone, Thomas C. Evans, Nicole M. Nichols, Pingfang Liu. A novel suite of enzyme mixes enable robust hybrid capture sequencing from low quality FFPE DNA [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 249.
Abstract 2494: Heterogeneous tumor-driven immune landscapes between lung adenocarcinomas in ever and never-smokers
Authors: Kang Xiaozheng , Wang Xin , Ji Liyan , Yan Bingfa , Gao Xuan , Xia Xuefeng , Hu Xingsheng
Published: April 2023

To depict the genomic and immune landscapes of lung adenocarcinomas (ADCs) in ever and never-smokers and guide the design of immunotherapy strategies, we applied multiple deconvolution methods that allow the identification of immune cell infiltration and immune pathway enrichment using a whole exome and transcriptome sequencing-based approach in 145 patient-matched lung ADC and non-malignant lung parenchymal tissue pairs from ever and never smokers in our in-house cohort. Results were subsequently validated by an external lung ADC cohort, including 108 never-smokers and 61 ever-smokers. Firstly, we showed that ever-smokers had an increased abundance of neutrophils and macrophage infiltration, an elevated antigen processing and presentation capacity, and activated T-cell-mediated immunity. Secondly, ever-smokers revealed elevated macrophages in acinar ADCs, decreased mast cells in lepidic ADCs, and increased neutrophils in papillary ADCs, suggesting the tumor-driven immune characteristic discrepancies among various pathologic subtypes of ADC. Thirdly, we identified mutational disparities in tumor mutation burdens, subclonal single nucleotide variants, and the predicted and expressed neoantigens between ever and never-smokers. The mutant gene KRAS preferably occurred in ever-smokers, further contributing to the infiltration of macrophages and expression of cytokines. The copy number variations of immune-related genes (NFIB, TNFAIP3) were identified as conferring to the dysregulated immune environment in ever-smokers. Moreover, a higher genomic instability index was observed in ever-smokers (p = 0.008). Fourthly, the neoantigen was significantly increased in ever-smoker lung ADCs without nodal disease. While DNA immunoediting preferably occurred in never-smokers. Notably, in lung ADC patients with nodal disease, ever-smokers exhibited slightly more homogenous mutations (p = 0.094), consistent with the evolutionary bottleneck during cancer progression. Finally, the lower immunoediting and higher microenvironment scores significantly determined the more prolonged progression-free survival in never-smokers (p < 0.050). Our multiscale genomic and immune profiling analyses thus offer valuable knowledge of tumor-driven immune disparity arising from smoking and provide a robust solution to the rational design of immune therapies.

Citation Format: Xiaozheng Kang, Xin Wang, Liyan Ji, Bingfa Yan, Xuan Gao, Xuefeng Xia, Xingsheng Hu. Heterogeneous tumor-driven immune landscapes between lung adenocarcinomas in ever and never-smokers [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 2494.
Abstract 2508: Clinical significance of PARP7 (TIPARP) gene copy number alterations in human non-small cell and head & neck carcinomas
Authors: Ahumada Viviana A. , McEachern Kristen , Kuplast-Barr Kristy , Schalper Kurt Alex
Published: April 2023

Introduction: PARP7, encoded by the TIPARP gene, is a monoART involved in cellular stress responses and with immunomodulatory functions in cancer. The PARP7 gene is amplified in a subset of squamous cell carcinomas and ongoing clinical studies are assessing its role as anti-cancer therapeutic target (NCT04053673, NCT05127590) Here, we analyzed the frequency of PARP7 copy number alterations and its association with tumor immune microenvironment (TIME) features and outcomes in non-small cell lung cancer (NSCLC) and head & neck squamous-cell carcinoma (HNSCC) cohorts.

Methods: The PARP7 gene copy number and amplification was analyzed using a dual probe fluorescence in situ hybridization (FISH) assay in two retrospective cohorts of NSCLC (Cohort #1, n=124) and HNSCC (Cohort #2, n=83) represented in tissue microarray format. In a subset of cases, the PARP7 copy number was also studied using whole exome DNA sequencing. The TIME was assessed on consecutive tumor sections using a multiplexed quantitative immunofluorescence panel including the markers DAPI, cytokeratin, CD4, CD8 and PD-L1 coupled to computational pathology analysis. The association between the markers, with clinicopathologic variables and survival was studied.

Results: Increased PARP7 copy number (>2 copies/cell) was identified in 55% of NSCLCs (85% of them of squamous-cell histology) and 76% of HNSCCs. A high copy number (>3 copies/cell) was identified in 22% cases from both tumor type cohorts. After adjustment of the PARP7 gene copy number by the chromosome 3 centromeric probe (CEN3) signal, PARP7 amplification (>1.5 PARP7/CEN3 signal) was detected in 22% of NSCLC cases and 34% of HNSCC. A higher number of cases with PARP7 copy number gains were identified using FISH than with whole exome DNA sequencing. No significant associations between the PARP7 gains/losses and major clinicopathologic variables were found. However, higher PARP7 copy number was associated with reduced CD8+ or CD4+ tumor infiltrating lymphocytes (TILs) and a numerically lower tumor PD-L1 protein levels in HNSCC. Elevated PARP7 copy number was associated with shorter overall survival in both studied tumor types.

Conclusion: Elevated PARP7 gene copy number identifies a subset of lung and head & neck squamous-cell carcinomas with unfavorable TIME features and adverse prognosis. Detection of PARP7 gene abnormalities using FISH in tumor biopsy samples is sensitive and has potential as a predictive biomarker.

Citation Format: Viviana A. Ahumada, Kristen McEachern, Kristy Kuplast-Barr, Kurt Alex Schalper. Clinical significance of PARP7 (TIPARP) gene copy number alterations in human non-small cell and head & neck carcinomas [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 2508.
Abstract 2516: Analysis of CLL Celllular Environment and Response (ACCER) is a novel method to understand the microenvironment in CLL
Authors: Gibson Spencer , Choquette Tricia , Henson Elizabeth S. , Yang Xioyan , Johnston James B.
Published: April 2023

Microenvironments such as lymph nodes and bone marrow allow chronic lymphocytic leukemia (CLL) cells to survive after drug treatments. There are limited methods to study the to study the contribution of the microenvironment. We have adapted a solid tumour microenvironment cell culture system that provides elements of the CLL microenvironment called Analysis of CLL Cellular Environment and Response (ACCER). We optimized the cell number for patient’s primary CLL cells and HS-5 human bone marrow stromal cell line that will give sufficient cell number and viability with the ACCER. We then determined the amount of collagen type 1 to give the best extracellular matrix to seed CLL cells to the membrane. Finally, we determined that ACCER provide CLL cell protection against cell death following treatment with fludarabine and ibrutinib compared to co-culture conditions. This describes novel microenvironment model to investigate factors that promote drug resistance and cell survival in CLL. In the future, this will further validate new treatment strategies to overcome microenvironmental CLL cell survival signals.

Citation Format: Spencer Gibson, Tricia Choquette, Elizabeth S. Henson, Xioyan Yang, James B. Johnston. Analysis of CLL Celllular Environment and Response (ACCER) is a novel method to understand the microenvironment in CLL [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 2516.
Abstract 2536: Intracellular Ctla4 overexpression promotes melanoma progression and metastasis
Authors: Kazmi Hasan Raza , Mo Xuan , Zhou Bo , Alp Sarah Preston , Gross Scott , Wajeeh Hassaan , Merali Carmen , Elrod John , Soboloff Jonathan , Merali Salim , Zaidi M. Raza
Published: April 2023

Cytotoxic T Lymphocyte Antigen 4 (CTLA4) expressed on the surface of T cells is an important immunotherapeutic target in many cancers, including melanoma. However, we previously found highly upregulated expression of CTLA4 in BRAF/NRAS-mutant human melanoma cell lines and tissues. Although CTLA4 is almost entirely expressed intracellularly in melanoma cells, its intracellular functions have remained elusive. Ectopic expression of Ctla4 (Ctla4-ee) in mouse melanoma cell lines significantly promoted lung colonization in syngeneic mice as well as in immunocompromised mice, suggesting that Ctla4 had pro-tumorigenic effects independent of its extracellular immune checkpoint function. Proteomics analysis identified apoptosis to be a major affected pathway and we found that Ctla4 ectopic-expressing mouse melanoma cells (B2905-Ctla4-ee) were significantly resistant to doxorubicin-induced apoptosis as compared to empty vector (EV) controls. In a reciprocal experiment, knocking out CTLA4 in the Hs936T human melanoma cells, that exhibit high endogenous CTLA4 expression, showed significantly increased apoptosis than the parental cells. Moreover, significant upregulation of anti-apoptotic proteins (Bnip3, Birc6, Pak1, Mcl-1, Survivin, Rac1/Cdc42, Bcl-2, and Bnip3l) and downregulation of pro-apoptotic proteins (p53 and Bad) were observed in CTLA4-ee cells as compared to EV controls. CTLA4-ee cells also showed significantly higher invasion in Matrigel-coated Transwell assay. Interestingly, we also found that CTLA4 translocated to mitochondria, where it downregulated oxidative phosphorylation and increased glycolysis. These findings lead us to hypothesize that CTLA4 plays a significant role in regulating apoptosis and mitochondrial metabolic functions, leading to the promotion of melanoma progression and metastasis.

Citation Format: Hasan Raza Kazmi, Xuan Mo, Bo Zhou, Sarah Preston Alp, Scott Gross, Hassaan Wajeeh, Carmen Merali, John Elrod, Jonathan Soboloff, Salim Merali, M. Raza Zaidi. Intracellular Ctla4 overexpression promotes melanoma progression and metastasis [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 2536.
Abstract 2541: Heterogeneity of VDAC2-Bak/Bax mediated mitochondrial apoptosis can be exploited to develop effective and selective treatment against metastatic uveal melanoma
Authors: Mishra Piyush
Published: April 2023

Apoptosis, a form of programmed cell death, is an evolutionarily conserved and genetically regulated phenomenon that is pivotal in maintaining tissue homeostasis and is an important player in regulating carcinogenesis. Mitochondria along with its resident proteins are the central player in regulating apoptosis. In recent years, VDAC2, an Outer Mitochondrial Membrane (OMM) protein has been shown to be the key recruitment factor for Bak, a pro-apoptotic protein to the OMM and, therefore is crucial for tBid-induced OMM permeabilization. We have previously found that unexpectedly, hepatocellular carcinoma (HCC) cells are vastly more sensitive to tBid induced apoptosis compared to normal hepatocytes and this difference is owed to high abundance of VDAC2-Bak in HCC. These findings are in line with multiple datasets posted in Oncomine, and protein atlas websites and may have clinical relevance. Combination of S63845, a selective Mcl-1 inhibitor, with TRAIL was effective at reducing tumor growth in vivo, but only in tumors expressing VDAC2. We hypothesized that the liver metastases of some extrahepatic primary tumors might also show enhanced sensitivity to VDAC2-Bak mediated apoptosis as compared to the normal liver tissue. Testing this hypothesis is important for uveal melanoma (UM) that primarily metastasizes in the liver, and these metastases lack an effective treatment. Interestingly, on testing several UM cell lines and patient samples we observed that VDAC2-Bak are upregulated in UM liver metastasis as well as compared to normal liver tissue. As expected, high VDAC2 expressing cell lines were also more sensitive to tBid induced OMM permeabilization. We propose that these differences can be exploited to design an effective therapy for metastasized UM.

Citation Format: Piyush Mishra. Heterogeneity of VDAC2-Bak/Bax mediated mitochondrial apoptosis can be exploited to develop effective and selective treatment against metastatic uveal 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 2541.
Abstract 255: Isolation of unaltered sub-populations of immune cells using magnetic levitation
Authors: Travers Kevin , Wilson Nathan , Kale Abhijit
Published: April 2023

The analysis and characterization of subsets of immune cells from heterogeneous samples, such as peripheral blood mononuclear cells (PBMCs), requires isolation and enrichment. Common methods for this enrichment step include fluorescence activated cell sorting and positive or negative selection with magnetic beads. However, these methods alter native cell phenotypes during the enrichment process by the application of pressure, binding of antibodies, extensive manipulation and centrifugation, among other perturbations. This results in altered gene expression, generating molecular profiles that are not representative of true biological significance. These alterations are prevented by using magnetic levitation cell isolation and enrichment. The LeviCellTM platform performs hands-free cell enrichment by utilizing magnetic fields to levitate viable, healthy cells away from dead cells and debris. Unlike other single cell separation methods, the enriched target cells are not subject to mechanical force, labeling, or staining to achieve viability enrichment. This results in a single cell suspension that is ready for immediate downstream analysis, producing molecular profiles free of isolation-induced gene expression artifacts.

We have developed reagents to extend the benefits of this technology to the immuno-oncology research market. These new reagents are used to target the enrichment of all T cells, the CD4+ or CD8+ subsets of T cells, or all B cells, with other target populations in development. In all cases, unwanted cells are captured within the levitation chamber, depleting them from the sample. These reagents leverage the magnetic field across the levitation chamber. Specifically-tagged, unwanted cells are pulled to the edges of the levitation chamber while the target cells undergo viable cell enrichment. The end result is a cell population that has been enriched and at higher viability. We have seen consistent depletion rates of >98%, resulting in a final targeted population with 90% or higher purity in most cases. Here we demonstrate the performance of the LeviCell platform in the enrichment of targeted cell populations relevant to the immuno-oncology research community.

Citation Format: Kevin Travers, Nathan Wilson, Abhijit Kale. Isolation of unaltered sub-populations of immune cells using magnetic levitation [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 255.
Abstract 2557: Targeting the IGF1R signaling pathway inhibits cell growth and dissemination in BCOR mutant retinoblastoma
Authors: Lee Su-Chan , Alaali Lujain , Eberhart Charles G.
Published: April 2023

Retinoblastoma is the most common intraocular cancer of childhood, with approximately 8,000 new cases each year worldwide. While survival rates are relatively high in developed countries, death can occur in up to half of children in some regions. In addition, blindness is common in patients who survive, and improved therapies are clearly needed. While loss of RB gene function is essential for the initiation of almost all retinoblastoma, additional molecular changes driving their growth and dissemination are poorly understood. Improved understanding of the molecular pathways driving retinoblastoma growth and dissemination may allow us to develop more effective and less toxic therapies. Alterations leading to BCL-6 corepressor (BCOR) loss of function are the second most common mutations in retinoblastoma after changes affecting the RB locus, and are found in more aggressive tumors. BCOR mutations are also associated with metastasis and poor prognosis in other types of pediatric cancer, suggesting that targeting it will represent a new therapeutic approach for aggressive retinoblastoma. Here, we found that BCOR knockdown or knockout in two retinoblastoma cell lines with high baseline levels of protein caused significantly increased invasion/migration, proliferation and clonogenicity. In contrast, overexpression of BCOR in retinoblastoma cells with low/no baseline expression showed opposite effects. In orthotopic xenograft models, BCOR knockout dramatically increased the size and spread of retinoblastoma xenografts as compared to parental controls. In order to determine the molecular pathways modulated by BCOR, we used RNA sequencing and found that loss of function induces IGF1 transcription, resulting in activation of IGF1R and the ERK signaling pathway. Pharmacological inhibition of IGF1/IGF1R signaling using Lincitinib or AEW541 inhibited proliferation and migration in BCOR knockout retinoblastoma cells in vitro, and Lincitinib dramatically slowed xenograft growth in vivo, suggesting that induction of IGF1 represents an important downstream effector following BCOR loss. Our studies support a functional role for BCOR mutations in aggressive retinoblastoma, and suggest that this is mediated at least in part by increased IGF1R signaling, which represents a new therapeutic target for these malignant childhood tumors of the eye.

Citation Format: Su-Chan Lee, Lujain Alaali, Charles G. Eberhart. Targeting the IGF1R signaling pathway inhibits cell growth and dissemination in BCOR mutant retinoblastoma [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 2557.
Abstract 2589: TROP2 gene copy number variations are a hallmark of glioblastoma and MEN-2A cancers
Authors: Alberti Saverio , Trerotola Marco , Moschella Antonino , Guerra Emanuela
Published: April 2023

TROP2 mutations are not required to trigger activation of tumor growth and metastasis. However, expression of Trop-2 is affected by DNA methylation in choriocarcinomas, i.e. trophoblast-derived malignancies, and in lung adenocarcinomas, suggesting that loss of TROP2 expression may provide a selective advantage in specific tumor types. Intriguingly, amplification of the TROP1/EPCAM and TROP2/TACSTD2 genes was found to be driven by alterations of DNA methylation, with parallel LOH in sister cell clones. Consistent, mutations of TP53 were shown to induce amplification of TROP genes through loss of DNA methylation. Hence, we investigated whether LOH/DNA methylation of the TROP2 gene and gene amplification are selectively associated with specific tumor types. Our findings indicate that TROP2 amplification occurs in a subgroup of aggressive glioblastomas (15.7%, n=3/19), suggesting impact on malignant progression. On the other hand, TROP2 LOH was shown to occur in the majority of low-grade gliomas (80%, n=4/5). Investigation on the TROP2 gene in MEN-2A revealed that LOH of TROP2 was somatically acquired in 25% of phaechromocytomas and medullary thyroid carcinomas (n=6/24), but did not appear as a heritable trait in the germline. TROP2 gene copy number variations were rarely detected in other cancer types, suggesting tumor subtype-specific impact.

Citation Format: Saverio Alberti, Marco Trerotola, Antonino Moschella, Emanuela Guerra. TROP2 gene copy number variations are a hallmark of glioblastoma and MEN-2A cancers [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 2589.
Abstract 26: BaF3 RAS cell panel, a powerful cell model for Ras inhibitor discovery and development
Authors: YAO PENG , Wang Yu , Liu Chang , Wang Na , Meng Wei , Ning Jinying , Hao Feng
Published: April 2023

RAS, abbreviated from Rat sarcoma virus, is composed of three small GTPase proteins, HRAS, KRAR and NRAS. Over-activation of these genes due to different mutation were found in up to 20% of all human cancers and about 90% in pancreatic cancer. The identification of RAS mutation, especially KRAS-G12S or G12D mutation, as cancer diver genes has recently led to rapid development of anti-tumor therapeutics agents such as sotorasib (AMG 510), adagrasib (MRTX-849) and MRTX1133, but there are still no good drugs for the cancer patients with other RAS mutations. Ba/F3 line is a popular cell model for studying both kinases and their inhibitors, because some kinases can render the growth of Ba/F3 cells to be depended on the over-activation of the kinases instead of exogenous IL-3 supplement, while their inhibitors can antagonize these effects. Our group has generated a Ba/F3 RAS cell panel with 29 KRAS, 11 HRAS and 20 NRAS different single or double mutants for RAS inhibitor screening and development, all of these lines were well validated with PCR sequencing, Western Blot and KRAS G12C and G12D inhibitors. In addition to use the cell lines for in vitro assays, the transformed Ba/F3 lines can grow well in immune-deficient mice as in vivo models. Our data indicate that the cell line panel of Ba/F3 is a powerful system for new RAS inhibitor discovery and development.

Citation Format: PENG YAO, Yu Wang, Chang Liu, Na Wang, Wei Meng, Jinying Ning, Feng Hao. BaF3 RAS cell panel, a powerful cell model for Ras inhibitor discovery and development [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 26.
Abstract 260: Long RNA and DNA sequencing reveals HEK293 diversity between commercial sources
Authors: Blum Laura , Sexton Brittany , Sanford Jonathan C. , Janzen Evan , Sun Luo , Koetsier Paul A. , Krishnan Keerthana , Apone Lynne , Langhorst Bradley W.
Published: April 2023

HEK293 cells are used extensively in cancer research, with more than 4800 articles between 2017 and 2022 (PubMed search “cancer[MeSH Major Topic] and hek293”). Studies involving transfection to overexpress, knock down, or modify proteins in these cells are common. While HEK293 cells are relatively easy to grow and maintain, early use of high throughput sequencing Yao-Cheng et. al. identified genomic variability between different sources of cell lines and revealed unexpected details about their developmental lineage (likely adrenal, not kidney)[1]. Genomic or transcriptomic reorganization may complicate interpretation of results from targeted intervention experiments. Here, we evaluate HEK293 cells from various commercial sources using long read sequencing technology. Oxford Nanopore direct cDNA profiling was used to compare isoform usage and examine gene fusions. Long DNA libraries were prepared using a high molecular weight isolation method, sequenced on Oxford Nanopore, and assembled to produce long contiguous fragments. Genome assemblies generated from long read sequencing support the genomic diversity reported by Yao-Cheng et.al, though the specific copy number variants are distinct from those previously reported. Assembled genomes and transcriptomes for these specific lots of HEK293 cells will be available at NCBI. Given the plastic nature of HEK293 cells, we recommend confirmation of genomic and transcriptomic structure of the specific cells in use before and after intervention.

[1] Lin, Yao-Cheng, Morgane Boone, Leander Meuris, Irma Lemmens, Nadine Van Roy, Arne Soete, Joke Reumers, et al. “Genome Dynamics of the Human Embryonic Kidney 293 Lineage in Response to Cell Biology Manipulations.” Nature Communications 5, no. 1 (September 3, 2014): 4767. https://doi.org/10.1038/ncomms5767.

Citation Format: Laura Blum, Brittany Sexton, Jonathan C. Sanford, Evan Janzen, Luo Sun, Paul A. Koetsier, Keerthana Krishnan, Lynne Apone, Bradley W. Langhorst. Long RNA and DNA sequencing reveals HEK293 diversity between commercial sources [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 260.
Abstract 2604: Functional analysis of p53 codon 72 polymorphism with cysteine substitution in cancer cells
Authors: Zhang Shengliang , El-Deiry Wafik S.
Published: April 2023

The p53 codon p72 polymorphism with proline (p53P72)/arginine (p53R72) substitution is associated with cancer incidence. Cysteine(C) substitution at p53 codon 72 polymorphism (p53C72) is rarely studied in cancer patients. A patient who developed AML and who carries p53C72 was described (El-Deiry, 2022 WIN Symposium). The patient’s family has high rates of cancer susceptibility in the women some of whom carry FANCC alterations and other alleles. To examine the functional characteristics of different p53 alterations at codon 72, we generated plasmids expressing p53C72, p53 R72 or p53P72. The variants of p53 were overexpressed in p53-null cancer cells by transient transfection of the plasmids. We tested functions of the p53 codon 72 polymorphisms (p53C72, p53R72 and p53P72) in two cancer cell lines (Soas-2 and HCT116 p53 null) with different expression levels of the p53 variants at different time points. To examine the effect of codon 73 variants on p53 transactivation, we performed a PG13-Luciferase reporter assay and examined the endogenous p53 targets at the protein level. The luciferase-reporter assay showed that all the three variants of the p53 codon p72 variants increased PG-13 luciferase expression at early time points. The Western blot assay showed that the p53C72, the p53R73 and the p53P72 increased endogenous p21, DR5, puma and MDM2 at the protein levels at different time points. The p53C72 increased the p53 targets and PG13-driven luciferase expression much higher than the p53R72 and the p53P72. These results suggest that the p53 codon 72 variants (C72, R72 and P72) retain wild-type activity for p53-mediated transcription, and p53C72 has the highest potency among them. We examined the effect of the p53 variants on cell growth and death. PARP cleavage (a marker of cell apoptosis) was induced in cells overexpressing all the three p53 codon 72 variants. We observed a lesser potency of p53C72 to induce PARP cleavage compared to p53R72 and p53P72. Colony formation assay showed a suppressive effect of p53C72, p53R72 and p53P72 on cancer cell growth. Our studies demonstrate some differences of in wild-type functions of p53 p53C72, p53R72 and p53P72 alleles.

Citation Format: Shengliang Zhang, Wafik S. El-Deiry. Functional analysis of p53 codon 72 polymorphism with cysteine substitution in cancer 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 2604.
Abstract 2625: KRAS activity-mediated mechanism of castration-resistant prostate cancer progression
Authors: Kamijima Taiki , Izumi Kouji , Kadono Yoshifumi , Mizokami Atsushi
Published: April 2023

Purpose: Prostate cancer is highly androgen-dependent, and androgen receptor (AR) signaling is suppressed by treatment. However, there are cases in which disease progresses rapidly at the end of treatment even after AR suppression, suggesting that the disease progresses by mechanisms different from AR. The aim of this study is to investigate the KRAS signaling pathway as one of the mechanisms.

Methods: We used the hormone-sensitive prostate cancer (HSPC) cell line LNCaP and the castration-resistant prostate cancer (CRPC) cell line DU145. We performed KRAS knockdown by si-RNA and activation of KRAS signaling by EGF stimulation. To examine KRAS signaling, PCR and Western blotting were performed. The changes in proliferation and migration due to inhibition and activation of KRAS signaling were confirmed.

Results: RT-PCR showed a fusion gene of ubiquitin-conjugating enzyme UBE2L3 and KRAS (UBE2L3-KRAS) in DU145. Western blotting showed increased phosphorylation of ERK and p38 downstream of KRAS in DU145. KRAS knockdown inhibited proliferation and migration in DU145 but not in LNCaP. In EGF-stimulated activation of KRAS signaling, Western blotting showed activation of downstream KRAS signaling in LNCaP, but no change in proliferation and migration in fetal bovine serum (FBS)-added medium. In contrast, EGF stimulation of DU145 further activated downstream KRAS signaling and promoted proliferation and migration. The AR-dependent LNCaP showed almost no effect of KRAS signaling activation on cancer progression, whereas DU145 showed that KRAS signaling is involved in cancer progression.

Conclusion: While HSPC is highly AR-dependent and does not show any effect of KRAS on cancer progression, KRAS may be involved in cancer progression in some CRPCs.

Citation Format: Taiki Kamijima, Kouji Izumi, Yoshifumi Kadono, Atsushi Mizokami. KRAS activity-mediated mechanism of castration-resistant prostate cancer progression [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 2625.
Abstract 263: Automating next generation sequencing workflows for custom hyb panels: high quality library preparation and target enrichment on the Biomek NGeniuS System
Authors: Roseman Nicole , Tucker Laura , Criscuolo Francesco , Lewis Lynette , Liu Li , Buit Tyler , Mandair Jasmeen , Smith Zachary , Cortes Calvin
Published: April 2023

This study investigates a custom 2Mb xGen Hyb panel designed against mutated gene targets implicated in several cancers using an end-to-end assay workflow on the Biomek NGeniuS platform. Automation in laboratories is advantageous to generate reproducible next generation sequencing (NGS) sample libraries, however, assay automation differs from instrument vendor to vendor but also from lab to lab with different deck layouts of the same instrument. These differences require a significant amount of optimization to design the workflow and begin generating NGS libraries. In addition to this optimization, some research, e.g., cancer research utilizes low input and/or degraded samples such as cfDNA from plasma or FFPE biopsies. Integrated DNA Technologies (IDT) and Beckman Coulter - Life Sciences together provide a walk away NGS automation solution for these research samples directly upon instrument installation with no optimization required by combining Beckman Coulter’s Biomek NGeniuS with IDT’s xGen cfDNA & FFPE DNA Library Prep kit and xGen Hybridization Capture. This trio provides a solution for laboratories to minimize technician hands-on time, error reduction, and rework while improving flexibility. IDT’s xGen cfDNA & FFPE DNA Library Prep kit utilizes novel chemistry to maximize sample input conversion, suppress adapter-dimer formation, and facilitate consensus analysis. IDT’s xGen Hybridization Capture products maintain high library diversity, obtain high on-target, and provide consistent and uniform sequencing coverage regardless of panel size. We demonstrate performance of this complete workflow with a custom 2Mb xGen Hyb panel yielding high library complexity, high on-target coverage, and capture uniformity. The combination of IDT’s xGen library preparation solutions on the NGeniuS Biomek platform provides a reliable and consistent solution that can be instantly downloaded from the portal straight to analysis of precious low input and degraded samples.

Citation Format: Nicole Roseman, Laura Tucker, Francesco Criscuolo, Lynette Lewis, Li Liu, Tyler Buit, Jasmeen Mandair, Zachary Smith, Calvin Cortes. Automating next generation sequencing workflows for custom hyb panels: high quality library preparation and target enrichment on the Biomek NGeniuS System [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 263.
Abstract 2645: A high-throughput platform for targets screening by membrane protein library
Authors: Li dan , Xiong Junyi , Ning Jinying , Hao Feng
Published: April 2023

The specificity of antibodies, CAR-T cells and other biologic therapeutics to their binding targets is important to evaluate drug quality. The non-specific binding may lead serious toxic side effects. Therefore, specificity detection is an important step in the evaluation process of drug development. At present, the non-specific binding of antibody drugs is often verified only after serious adverse reactions founding in clinical trials, which may result in drug safety accidents and the abandonment of previous studies. To address the need for antibody target deconvolution and receptor identification, we developed a novel approach “AB5000”. AB5000 is an excellent solution for antibody specificity screening which enable early off-target screening and can de-risk the antibody drug development program. The AB5000 makes use of the full coverage membrane protein, representing over 5465 unique human membrane proteins including receptors, transporters, enzymes, miscellaneous and other membrane protein. Gene vector was transfected into target cells. Each membrane protein introduces a C-terminal epitope tag, allowing confirmation of protein expression and cellular location. To provide the highest level of sensitivity, we use luciferase reporter cell line system which is more rapid, simple and highly sensitive than flow cytometry or other binding assays. The AB5000 has widely application in testing the specificity of antibodies, deconvolution of orphan antibodies and ligands, detection of CAR-T proteins. The AB5000 represents a rapid, comprehensive, and highly sensitive platform for identifying the membrane protein targets of a wide variety of antibodies, protein ligands, and functional interactions. It is a promising powerful tool for biotherapeutic modalities development.

Citation Format: dan Li, Junyi Xiong, Jinying Ning, Feng Hao. A high-throughput platform for targets screening by membrane protein library [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 2645.
Abstract 268: Antitumor CD4+ T cells reprogram tumor metabolism to drive progressive tumor regression
Authors: Brandle Caitlin , Okoko Ogacheko D. , Gazi Md Yeashin , Wang Xin , Kehinde-Ige Mercy , Shi Huidong , Zhou Gang
Published: April 2023

Adoptive cell therapy has revolutionized the landscape of cancer immunotherapy, exhibiting unprecedented success in treating various types of cancer. Substantial investigative efforts have been devoted to gaining a deeper understanding of the ways in which adoptively transferred T cells eliminate tumors and to discovering strategies that enhance T cell functionality in the tumor microenvironment. We have established an experimental mouse model in which adoptive transfer of tumor-specific CD4+ T cells leads to eradication of established tumors. Using this model, we previously reported on the profound disruption of tumor metabolism induced by adoptive T cell therapy. However, the detailed mechanisms that enable tumor-reactive CD4+ T cells to alter tumor metabolism remain largely undefined. In the current study, we aimed to identify the key metabolic enzymes and pathways targeted by antitumor CD4+ T cells that lead to progressive tumor regression. Utilizing next-generation sequencing strategies, including bulk and single-cell RNA sequencing, we examined the metabolic changes cancer cells undergo in response to treatment, and the immune cell compositions in the tumor microenvironment. Additionally, we performed an in vivo CRISPR knockout screen to identify the metabolic genes that either sensitize tumor cells to adoptive cell therapy or contribute to tumor resistance. Results from our study may also illuminate potential targets in cancer metabolism that can be exploited using small-molecule inhibitors when adoptive cell therapy is not feasible.

Citation Format: Caitlin Brandle, Ogacheko D. Okoko, Md Yeashin Gazi, Xin Wang, Mercy Kehinde-Ige, Huidong Shi, Gang Zhou. Antitumor CD4+ T cells reprogram tumor metabolism to drive progressive tumor regression [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 268.
Abstract 2708: Nano-immunotherapy: efficacy of nanoconjugate QN-247 in a triple negative breast cancer (TNBC) mouse model
Authors: Arshad Tariq , Fait Stephen , Gammon Guy , Hertig Andrew , Sarno Mark J.
Published: April 2023

Introduction and Purpose: Triple-negative breast cancer (TNBC) accounts for 12% of breast cancers (BC) in the US and 15% worldwide. It lacks expression of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Compared to other types of BC, it is more aggressive and has higher risks of recurrence. In the metastatic setting, survival outcomes are much worse than other BC subtypes, with a 5-years survival rate of 8-16%. There is an urgent need to develop better treatment options for TNBC patients. We have developed a novel therapeutic, QN-247, by conjugating an anti-proliferative DNA aptamer to gold nanospheres. The aptamer-gold nanosphere construct shows increased half-life and cytotoxic effects relative to the aptamer alone. QN-247 inhibits nucleolin, a key regulatory protein overexpressed in triple-negative breast cancer (TNBC) cells, thereby reducing their proliferation.

Description of Experimental Procedures: A subcutaneous, TNBC model (MDA-MB-231 xenograft) in nude mice was used in this efficacy study. When tumor volumes reached sufficient size (85 mm3), QN-247 test compound, aptamer alone, and vehicle control was administered daily (QD) at 1 mg/kg via intraperitoneal injection for 12 days. Tumor volumes and body weights were measured regularly through day 20. Nine animals were included in each treatment group. Summary of DataA statistically significant reduction in mean tumor volumes of QN-247 treated mice compared to the vehicle control and aptamer alone was observed. Tolerability of the QN-247 treatment was excellent with no evidence of toxicity.

Statement of Conclusions: This study demonstrates that QN-247 exerts a powerful anti-tumor effect. Additional preclinical mouse xenograft tumor models will be treated with QN-247 to study its effect on other cancers.

Citation Format: Tariq Arshad, Stephen Fait, Guy Gammon, Andrew Hertig, Mark J. Sarno. Nano-immunotherapy: efficacy of nanoconjugate QN-247 in a triple negative breast cancer (TNBC) mouse 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 2708.
Abstract 2717: A potent and highly tumor-selective KRAS inactivator for tumor targeting
Authors: Liu Shihui , Zuo Zehua , Liu Jie
Published: April 2023

Cancer driver mutations in the RAS-RAF-MEK-ERK pathway occur in 46% of all human cancers. This has inspired the successful development of many small molecule inhibitors of BRAF and MEK. However, development of RAS small molecule inhibitors has not been successful in large part due to the lack of a sufficiently large and deep hydrophobic pocket in RAS proteins to allow for small molecule binding. Notably, in this regard, many bacterial pathogens have evolved potent protein toxins to disrupt the RAS-RAF-MEK-ERK pathway. Fortunately, these naturally designed potent toxins, such as anthrax toxin, Fortunately, these potent, naturally designed toxins, such as anthrax toxin and DUF5, can be structurally modified to achieve high tumor specificity. DUF5 (amino acids Glu3581-Gln4089) is a toxin effector domain of MARTX (a multifunctional-autoprocessing repeats-in-toxin) from Vibrio vulnificus. DUF5 was recently identified to be a specific and potent endopeptidase that cleaves and inactivates RAS. To address the above critical unmet medical needs, in this work, we have generated an anthrax toxin/DUF5-based, highly tumor-selective RAS inactivator and evaluated its anti-tumor activity in a variety of tumor models. This tumor-selective RAS inactivator can specifically bind to the major anthrax toxin receptor CMG2 on tumor cells and tumor stromal cells, and strictly relies on the simultaneous presence of two distinct tumor-associated proteases, MMPs and urokinase, to gain entry into tumor cells and tumor stromal cells to proteolytically inactivate RAS signaling, achieving potent selective targeting. Our data show that our tumor-selective RAS inactivator displays potent cytotoxicity to cancer cells whose survival depends on oncogenic RAS signaling. Our engineered toxin displayed potent in vivo antitumor activity to a range of tumors with oncogenic RAS mutations in syngeneic and human xenograft mouse tumor models. Therefore, we have developed a tumor-selective, safe, and highly potent RAS inactivator that has high potential for targeting tumors with oncogenic RAS mutations.

Citation Format: Shihui Liu, Zehua Zuo, Jie Liu. A potent and highly tumor-selective KRAS inactivator for tumor targeting [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 2717.
Abstract 2748: Identifying therapeutic mechanism of action and new potential patient populations using PRISM
Authors: Nguyen Ellen , Nair Shiker , Davis John , Masciotti Antonella , Mochi Connor , Finn John , Ponsi Cole , Engel Brienne , Mapa Claudine , Kocak Mustafa , Ronan Melissa , Rees Matthew G. , Roth Jennifer A.
Published: April 2023

Our multiplexed cell viability platform, PRISM (profiling relative inhibition simultaneously in mixtures), enables screening of potential cancer therapeutics at an unprecedented scale. We routinely assess the effects of perturbations against more than 900 cancer cell lines concurrently through the use of unique oligonucleotide barcodes stably transduced into individual cancer cell lines. Following barcode transduction, individual cell lines are pooled together in groups of 20-25 based on growth rate similarity, then thawed into 384-well assay-ready plates containing compounds of interest. After 5 days of growth, isolated mRNA is used to detect transcribed barcode abundance of each individual cancer cell line to measure relative viability. We leverage the baseline cellular features (e.g., gene expression, cell lineage, mutation, copy number, metabolomics, proteomics, genome-wide RNAi and CRISPR dependencies) of each cell line to interpret viability profiles, enabling identification of drivers of differential sensitivity and potential biomarkers of compound response. Critically, the scale and throughput of PRISM has enabled the generation of large, publicly available datasets and the rapid characterization of emerging therapeutic targets and classes (e.g., isoform-selective RAS inhibitors and degraders). Although the read-out of PRISM is relative cell line viability, the platform can be used to answer a multitude of scientific questions. For example, PRISM data can be used to identify potential patient populations who would most benefit from treatment with a specific compound, uncover unexpected off-target toxicities, and validate mechanism of action hypotheses on a more holistic scale. Ultimately, PRISM offers a large-scale and comprehensive platform for the testing and validation of anti-cancer compounds to aid in the search for new oncology therapeutics.

Citation Format: Ellen Nguyen, Shiker Nair, John Davis, Antonella Masciotti, Connor Mochi, John Finn, Cole Ponsi, Brienne Engel, Claudine Mapa, Mustafa Kocak, Melissa Ronan, Matthew G. Rees, Jennifer A. Roth. Identifying therapeutic mechanism of action and new potential patient populations using PRISM [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 2748.