bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1394: Gene expression profile in BCBL-1 cell line following activation of KSHV
Authors: Haines Samantha L. , Bishop Sabrina R. , Podlutskaya Viktorija , Podlutsky Andrej
Published: September 2020

Patients with HIV/AIDS are at a 3,400 times higher risk of developing Kaposi's sarcoma when compared to healthy populations. Our hypothesis is that the upregulation of viral associated proteins is causing an increase in DNA damage, allowing for the higher incidence rate of Kaposi's sarcoma in infected populations. The direct mechanisms leading to this increased risk is unknown, but the viral infection with Kaposi's sarcoma-associated herpesvirus (KSHV) is an apparent cause of this risk. The cell is under attack of endogenous threats including oxidative stress, causing DNA damage and leading to mutations, but the viral proteins may interfere with the repair mechanisms. In previous experiments we looked at DNA repair dynamics following X-ray exposure in the two different stages and determined there is an inefficiency in the DNA repair process in the cells during active production of viral particles. This project aimed to compare the transcriptomes of the BCBL-1 cell line during latent and acute phases of KSHV production. Our results are showing that genes involved in genome stability are differently expressed during the lytic stage. Comparison of these two transcriptomes allows for identification of which proteins are being upregulated during active viral production. This may be of importance for the HIV/AIDS cancer patients because it shows the mechanisms behind genome stability in KSHV-infected cells.

1) Research reported in this publication was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Numbers UL1GM118991, TL4GM118992, or RL5GM118990. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

2) NASA EPSCoR, NNH15ZHA003C

Citation Format: Samantha L. Haines, Sabrina R. Bishop, Viktorija Podlutskaya, Andrej Podlutsky. Gene expression profile in BCBL-1 cell line following activation of KSHV [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1394.
Abstract 142: Identification of symmetric methylation and hemimethylation patterns with optimal panel design, library preparation, and error correction
Authors: Huang Hsiao-Yun , Chakravarty Ushati Das , Lai Kevin , Barnes Timothy , Chen Tzu-Chun , Sheu Jessica , Lopez Ramses , Scott Karissa , Lewis Lynette , Potts Anastasia
Published: September 2020

DNA methylation patterns are epigenetic modifications with direct implications in gene expression and chromatin structure regulation. Altered DNA methylation has been reported in various human cancers. The methylation profile of cell-free DNA (cfDNA) from plasma can be exploited to detect and diagnose tissue pathologies and is therefore of great diagnostic interest. Due to limitations of existing NGS library preparations, we present a new workflow, that integrates Unique Molecular Identifier (UMI) based error correction and optimizes library preparation and target enrichment. This workflow incorporates an engineered mutant ligase and proprietary methylated adapters that together prevent chimeras, suppress dimer-formation, and maximize conversion. Our library preparation provides >80% unique mapping efficiency and high conversion efficiency which creates superior sensitivity and specificity for detecting epigenetic signatures from a wide range of inputs (1-250 ng) and samples, including genomic DNA, FFPE DNA and cfDNA.

This workflow is suitable for both genome-wide and targeted methylation sequencing. We implemented the target methyl-seq workflow to distinguish between fully methylation and hemimethylation on only one strand of the DNA by ligating in-line UMIs prior to target enrichment, followed by bisulfite conversion and PCR. Using a custom panel for targeted methylation sequencing, we detected >60% of CpG methylation in normal human samples and >95% of CpG methylation in in vitro methylated HCT116 samples. Employing ultra-deep sequencing to an average target depth of 10,000X followed by double-stranded consensus building analysis enables accurate low frequency methylation and hemimethylation detection. The studies presented here offer tools to advance epigenetics research.

Citation Format: Hsiao-Yun Huang, Ushati Das Chakravarty, Kevin Lai, Timothy Barnes, Tzu-Chun Chen, Jessica Sheu, Ramses Lopez, Karissa Scott, Lynette Lewis, Anastasia Potts. Identification of symmetric methylation and hemimethylation patterns with optimal panel design, library preparation, and error correction [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 142.
Abstract 1455: NANOG binds polycomb repressive complex 2 to reprogram metabolism to promote slow cycling phenotype in experimental mouse and clinical carcinoma
Authors: Yeh Da-Wei , Hernandez Juan Carlos , Machida Keigo
Published: September 2020

Pluripotency transcription factor NANOG represses mitochondrial oxidative phosphorylation (OXPHOS) genes, but activates fatty acid oxidation (FAO) to support self-renewal and drug resistance of tumor-initiating stem-like cells (TICs). Hepatoceullular carcinomas (HCCs: 39%) associated with HCV and alcohol have mutations in ARID1A, a component of the chromatin remodeling complex SWI/SNF. We hypothesized that NANOG binds polycomb repressive complex 2 (PRC2) to reprogram metabolism to promote slow cycling phenotype. Here, the Genome-scale CRISPR Knock-Out (GeCKO) screening with ARID1A mutation identified PRC2 complex genes (EZH2, SUZ12 and EED) in patient-derived TICs. NANOG together with the PRC2 complex inhibits OXPHOS to generate TICs with label-retaining ability and therapy resistance. NANOG binds PRC2 complex. ARID1A gene loss promotes sorafenib-resistance phenotype and self-renewal through PRC2 complex genes. ARID1A mutation promoted HCC development in humanized FRG mice with HCCs. The studies established a novel therapy to overcome the chemoresistance of TICs for HCC treatment.

Citation Format: Da-Wei Yeh, Juan Carlos Hernandez, Keigo Machida. NANOG binds polycomb repressive complex 2 to reprogram metabolism to promote slow cycling phenotype in experimental mouse and clinical carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1455.
Abstract 1457: Akt phosphorylation of mitochondrial Lonp1 protease enables oxidative metabolism and advanced tumor traits
Authors: ghosh Jagadish C. , Seo Jae Ho , Agarwal Ekta , Wang Yuan , Kossenkov Andrew V. , Tang Hsin-Yao , Speicher David W. , Altieri Dario C.
Published: September 2020

Introduction: The control of protein homeostasis, or proteostasis is essential for cellular adaptation to disparate environmental cues and relies on protein (re)folding or proteolytic removal of aggregated or misfolded proteins. Defective proteostasis can irreversibly impair organelle functions and activate cell death through an unfolded protein response. Tumor mitochondria have heightened protein folding quality control, but the regulators of this process and how they impact cancer traits are not completely understood. In this study, we investigated the mechanisms of stress-regulated LonP1 (ATP-dependent protease) in tumor mitochondria and their implications for advanced cancer traits.

Experimental procedures: We monitored kinase activity in immunoprecipitation, protein expression by immunoblotting, mitochondrial ROS quantification in live cells, mitochondrial electron transport chain (ETC) activity, mitochondria time-lapse video microscopy, protein folding, proteomics analysis, mitochondrial bioenergetics, mitochondrial trafficking and tumor cell motility, and tumor growth and metastasis. A xenograft prostate cancer model was used to study antitumor activity in vivo.

Results: We showed that the ATP-directed mitochondrial protease, LonP1 is upregulated by stress conditions, including hypoxia, in tumor, but not normal cells. In mitochondria, LonP1 is phosphorylated by Akt on Ser173 and Ser181, enhancing its protease activity. Interference with this pathway induces accumulation of misfolded subunits of electron transport chain complex II and complex V, resulting in impaired oxidative bioenergetics and heightened ROS production. Functionally, this suppresses mitochondrial trafficking to the cortical cytoskeleton, shuts off tumor cell migration and invasion, and inhibits primary and metastatic tumor growth, in vivo.

Conclusion: These data identify LonP1 as a key effector of mitochondrial reprogramming in cancer and potential therapeutic target.

Citation Format: Jagadish C. ghosh, Jae Ho Seo, Ekta Agarwal, Yuan Wang, Andrew V. Kossenkov, Hsin-Yao Tang, David W. Speicher, Dario C. Altieri. Akt phosphorylation of mitochondrial Lonp1 protease enables oxidative metabolism and advanced tumor traits [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1457.
Abstract 1475: Blockade of IL17-GCSF-Bv8 inflammation axis ameliorate resistant to anti-angiogenic therapy for colon cancer
Authors: Itatani Yoshiro , Yamamoto Takamasa , Ferrara Napoleone
Published: September 2020

Anti-angiogenic therapy in combination with chemotherapy is one of the standard strategies for metastatic/unresectable colon cancers. Therefore, resistance to anti-angiogenic therapy causes a critical problem for the treatment of colon cancer patients. While some resistant mechanisms of anti-angiogenic therapy were proposed, it is not yet completely unveiled. The aim of this study was to investigate the resistant mechanism against anti-angiogenic therapy using spontaneous colon cancer mouse models. We established a mouse model of anti-VEGF resistant colon cancer. Genetically-engineered Apc/Smad4 knockout mice developed several intestinal tumors that are sensitive to anti-VEGF neutralizing antibody. When Apc/Smad4 mice were treated with dextran sulfate sodium (DSS) (DSS-Apc/Smad4 mice), they developed colon tumors that were resistant to anti-VEGF antibody. Sera from DSS-Apc/Smad4 mice showed significant upregulation of granulocyte-colony stimulating factor (GCSF) compared to water-drinking Apc/Smad4 mice. Colon tumors from DSS-Apc/Smad4 mice also showed GCSF upregulation. It also showed upregulation of IL17, one of inflammatory cytokines upstream of GCSF, and Bv8, one of angiogenic factors downstream of GCSF, suggesting that IL17-GCSF-Bv8 inflammation axis contributes to obtaining anti-VEGF resistant. Immunostaining of the tumors from DSS-Apc/Smad4 mice revealed significant accumulation of neutrophils in the tumors. Neutrophils from tumor-bearing mice showed upregulation of Bv8 expression when stimulated with GCSF. Immunostaining confirmed that neutrophils in the tumors were positive for Bv8. When DSS-Apc/Smad4 mice were treated with either anti-IL17, anti-GCSF, or anti-BV8 neutralizing antibody in combination with anti-VEGF antibody, colonic tumor formation was significantly suppressed. We also performed ELISA analysis using human serum samples from subcohort of AVF2107 trial in which metastatic colon cancer patients were treated with chemotherapy (IFL with or without bevacizumab). Higher Bv8 serum levels by ELISA were significantly correlated with poor overall survival in human colon cancer patients. Collectively, inflammatory neutrophils in the tumor microenvironment of colon cancer provide anti-VEGF resistant through IL17-GCSF-Bv8 axis, and blockade of this pathway ameliorate resistance to anti-angiogenic therapy of colon cancer mouse models.

Citation Format: Yoshiro Itatani, Takamasa Yamamoto, Napoleone Ferrara. Blockade of IL17-GCSF-Bv8 inflammation axis ameliorate resistant to anti-angiogenic therapy for colon cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1475.
Abstract 1495: A transcriptional lineage tracing approach to unravel crucial gene expression patterns in breast cancer progression
Authors: Roda Niccolò , Hillje Roman , Gambino Valentina , Cheloni Stefano , Falvo Paolo , Tirelli Andrea , Bertolini Francesco , Pelicci Pier Giuseppe
Published: September 2020

Breast cancer represents a major clinical hurdle, with intra-tumor heterogeneity and metastatic outcome accounting both for poor prognosis and therapeutic failure. Traditionally, its poly-clonal structure has been investigated through the genetic barcoding of cells followed by the tracing of the offspring. However, this analysis does not allow a solid investigation of the transcriptional profile associated to clones throughout tumor progression. In this work, we exploited a recently published library of expressed barcodes to perform both clonal and trancriptional tracing in breast cancer by single-cell RNA sequencing. To this aim, human breast cancer cell line MDA-MB-231 was transduced with the above mentioned library and orthotopically transplanted in immune-deficient mice. To analyze our data we generated a pipeline aimed at identifying the barcodes at single-cell level and subsequently sequenced more than 20000 cells.Our clonal distribution analysis strongly suggested that clones within the same tumor possess different proliferative potential. Additionally, we found that breast tumors show transcriptional intra-clonal heterogeneity, since cells within the same clone display multiple transcriptional patterns. Notably, clones similar in terms of proliferative potential show profound differences in the transcriptomic profile and in the upregulated pathways. All these results represent a launching pad for further experiments aimed at identifying the transcriptional determinants involved in the spreading of breast cancer clones and in the metastatic progression.

Citation Format: Niccolò Roda, Roman Hillje, Valentina Gambino, Stefano Cheloni, Paolo Falvo, Andrea Tirelli, Francesco Bertolini, Pier Giuseppe Pelicci. A transcriptional lineage tracing approach to unravel crucial gene expression patterns in breast cancer progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1495.
Abstract 152: SOX2-mediated 5hmC dysregulation in GBM stem cells
Authors: Lopez-Bertoni Hernando M.
Published: September 2020

Primary brain tumors are among the most devastating forms of cancer and glioblastoma (GBM) represents the most aggressive and lethal form of the disease. We now know that GBM contain small subsets of cells that display tumor-propagating stem-like phenotypes (i.e. glioma stem cells or GSCs) that act as critical determinants of GBM resistance to current treatments and tumor recurrence for which there is no proven therapy. Altered patterns of DNA methylation are widely reported in human GBM. However, substantial knowledge gaps remain in our understanding of the molecular mechanisms responsible for this epigenetic dysregulation, its downstream consequences and role in the GBM tumor propagating phenotype. Understanding and ultimately targeting the epigenetic mechanisms that induce and maintain these tumor-propagating cell subsets is critical to improving GBM therapy and patient outcomes.

DNA methylation generally occurs in cytosine-guanine (CpG) sequences and is established by DNMTs, which catalyze the conversion of cytosine to 5-methylcytosine (5mC). DNA methylation is a reversible process and is partially mediated by the ten-eleven translocation (TET) family of enzymes which function as deoxygenases to catalyze the conversion of 5mC to 5-hydroxymethylcytosine (5hmC). Multiple studies found negative correlations between 5hmC levels and glioma grade and loss of 5hmC correlates with poor prognosis of GBM patients. TET enzymes and regulation of 5mC and 5hmC have the capacity to function on a global scale to regulate tumor suppressing mechanisms.

We have found that SOX2 represses the TET2 demethylase and decreases 5hmC, the enzymatic product catalyzed by TET proteins, in GSCs. TET2 repression using 2 independent shRNA hairpins efficiently decreases 5hmc levels and significantly enhances self-renewal capacity and tumor growth capacity of low-passage GSC isolates. We also show that SOX2 induces expression of miR-10b-5p, a known onco-miR predicted to target TET2, and inhibiting this miRNA rescued the reduction in 5hmC expression observed in GSCs, thus identifying miR-10b-5p as a critical mediator of SOX2-induced onco-methylation, GSC induction and glioma malignancy. Our results identify a novel targetable pathway by which SOX2 regulates the epigenetic landscape of GBM-propagating cells. This novel mechanism combined with our recent reports that SOX2 regulates DNMTs and the HMGA1 chromatin remodeling protein identified a multi-dimensional epigenetic mechanism by which oncogenic signals control GBM cell stemness and tumor propagation.

Citation Format: Hernando M. Lopez-Bertoni. SOX2-mediated 5hmC dysregulation in GBM stem cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 152.
Abstract 1545: IGF2BP2 over-expression induces epithelial mesenchymal transition & promotes tumor progression in ESCC cells
Authors: Li Pei , HU Xuan-Yu , Liu Xing-Ge , Sha Bei-Bei , DU Ying , CHEN Ping
Published: September 2020

Esophageal cancer (EC) is a common form of malignant tumor with high morbidity & mortality worldwide. IGF2BP2/ IMP2 (insulin-like growth factor 2 mRNA binding protein 2) belongs to a large family of RBPs (RNA binding proteins) that coordinates the export, trafficking, precise localization and translation of RNA in cells. Analysis of TCGA data (results from GEPIA, http://gepia.cancer-pku.cn/) showed that IGF2BP2 over-expressed in ESCA and many other types of human cancers. However, whether and how IGF2BP2 affects EC progression is still unclear. In this study, the expression of IGF2BP2 in esophageal squamous cancer cell lines and normal esophageal epithelial cells(Het) were investigated to explore the relationship between IGF2BP2 and epithelial-mesenchymal transition(EMT)in the ESCC. Methods: IGF2BP2 was knocked down in the Kyse150 cells by siRNA while upregulated by infection lentivirus with IGF2BP2 cDNA in the Eca109 cells. Then Western blotting was used to detect the protein expression of IGF2BP2, p38, P-p38, ERK and EMT markers in these cells. Results: In this study, the results showed that the expression of the epithelial marker, E-cadherin protein increased and the mesenchymal markers, N-cadherin, Snail, and Slug protein decreased after IGF2BP2 knockdown in Kyse150 cells. Meanwhile, the EMT features were promoted in the Eca109 cell after IGF2BP2 upregulation, shown as a reduction in epithelial marker E-cadherin with an increase in mesenchymal markers: N-cadherin, Snail, and Slug. Results from Western blotting showed that IGF2BP2 protein expression was independent of p38 and ERK protein levels. Conclusion: These data indicated that IGF2BP2 could promote the process of EMT in ESCC cells and might serve as a new therapeutic target for the treatment of esophageal cancer. Keywords: IGF2BP2; esophageal cancer; EMT

Citation Format: Pei Li, Xuan-Yu HU, Xing-Ge Liu, Bei-Bei Sha, Ying DU, Ping CHEN. IGF2BP2 over-expression induces epithelial mesenchymal transition & promotes tumor progression in ESCC cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1545.
Abstract 1587: Stress keratin 17 overexpression downregulates CD8+ T cell infiltration in papillomavirus-associated disease
Authors: Wang Wei , Gronski Ellery , Lambert Paul
Published: September 2020

High-risk human papillomaviruses (HPVs) cause 5% of human cancers. Despite the availability of HPV vaccines, there remains a strong urgency to find ways to treat persistent HPV infections, as current HPV vaccines are not therapeutic for individuals already infected. It has not well understood how papillomaviruses evade host immune surveillance to establish long-term infections, which can lead to neoplastic disease including cancer. Using a mouse papillomavirus (MmuPV1) infection model in immunocompetent FVB/N mice, we found that stress keratins are upregulated in mouse papillomavirus-induced papillomas. In mice deficient for stress keratin K17 (K17KO), we observed early regression of papillomas dependent on T-cells. Expression of cellular genes involved in immune response were significantly upregulated in the papillomas arising on the K17KO mice correlating with increased numbers of infiltrating CD8+T cells and upregulation of IFNγ-related genes, including CXCL9 and CXCL10, prior to complete regression. This T cell-dependent regression also required ligand accessibility to CXCR3, a receptor for CXCL9 and CXCL10. By analyzing public available TCGA data, we found an inverse correlation between K17 mRNA expression and CD8A, GZMB and CXCL9 mRNA expression in a cohort of head and neck cancer patients. We validated these observations by antibody immunofluorescence staining of a tissue microarray (TMA) composed of 380 tumor tissues from head and neck cancer patients (HPV+ n=265, HPV- n=49, HPV unknown n=66). We stained TMA with CD8 and K17. Out of 380 cancer specimens, there are n=46 that had either high percentage of CD8+ cells in tumor (Z score>=1.64, n=14) or a high mean fluorescence intensity of K17 (Z score>=1.64, n=33). The heatmap of these 46 samples showed that the high K17 stained tissue and high CD8+ T cell infiltrated tissues are mutually exclusive, consistent with our prior analysis from TCGA mRNA data from head and neck cancer patients. In the process of exploring the mechanism of K17-mediated immunosuppression, we found that mouse papillomas arising from K17 knockout mice lacked nuclear staining for YAP1 whereas a significant amount of nuclear staining for YAP1 was observed in WT FVB papillomas, suggesting K17 may regulate Yap1 activation to mediate downstream immunosuppressive effect.

Citation Format: Wei Wang, Ellery Gronski, Paul Lambert. Stress keratin 17 overexpression downregulates CD8+ T cell infiltration in papillomavirus-associated disease [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1587.
Abstract 1596: Profiling immune cell populations in the tumor microenvironment with complementary capillary-based and single-cell western assays
Authors: Haitjema Charles , Grant Rainer , Wentz Lindsay , Luhowskyj Stefan , Cooper Jeff , Boge Annegret , Brumbaugh Kathy , Gardner Kelly , Heger Christopher
Published: September 2020

The tumor microenvironment (TME) is a complex mixture of cancerous and non-cancerous cells, including immune cells like T-cells, macrophages, and neutrophils. The TME plays a key role in tumorigenesis and metastasis, and it has recently been recognized that it can dramatically shape a response to therapy. Thus, there is a pressing need to accurately identify and quantify the variety of cell types in any given TME. However, studying the TME presents major challenges. For example, the heterogeneity of the environment requires sensitive and high-resolution techniques to parse subpopulations of different cell types. This challenge is compounded by the severely limited sample size that can be obtained from donor tissues. To address these challenges, we use an in-capillary immunoassay with small sample sizes (3 µL) to identify immune cells commonly found in the TME. We also leverage single-cell western to uncover trends in population heterogeneity. Human peripheral blood mononuclear cells (PBMCs) were differentiated into dendritic cells (DCs) and regulatory T cells (Tregs), and natural killer (NK) cells were expanded from isolated NK cells. These samples were then analyzed by in-capillary immunoassay and single-cell western. These analyses revealed the identification and characterization of cell types, at both the single-cell and population level, based on the differential expression of protein biomarkers. Specifically, in-capillary immunoassay analysis identified mature populations by CD209 and CD83 expression for DCs, a CD56+/CD3- phenotype for NK cells, and CD25 and Foxp3 expression for Tregs. Analysis of single cells provided further detail within these populations, for example, we observed FoxP3low and FoxP3high subpopulations in Tregs, and an unexpectedly large (81%) CD56-/CD3- subpopulation in undifferentiated PBMCs, suggesting the presence of other cell subtypes. We anticipate that the small sample size, automation, single-cell resolution, and multiplexing ability of these assays collectively will enable a more efficient and deeper characterization of the TME not possible with traditional immunoassays like western blot and flow cytometry.

Citation Format: Charles Haitjema, Rainer Grant, Lindsay Wentz, Stefan Luhowskyj, Jeff Cooper, Annegret Boge, Kathy Brumbaugh, Kelly Gardner, Christopher Heger. Profiling immune cell populations in the tumor microenvironment with complementary capillary-based and single-cell western assays [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1596.
Abstract 1616: Elucidating the role of PI3K lipid effectors in BRAF mutant melanoma
Authors: Parkman Gennie , Cho Joseph , Holmen Sheri , McMahon Martin
Published: September 2020

Metastatic melanoma accounts for the highest number of skin cancer deaths, and while current treatments for this disease have progressed significantly in recent years, stage IV melanoma currently has only a 15-20% five-year survival rate. This highlights the critical need for new therapeutics to treat this complex disease. The PI3K/AKT pathway and RAS-RAF-MEK-ERK pathway play a major role in melanoma initiation and progression. Mutations in BRAF are found in around 50% of all human melanomas, and although there has been significant success in the development of therapies for BRAF mutant melanomas, most patients will experience primary or secondary resistance. Furthermore, mutant BRAF has been shown to be insufficient for melanomagenesis, yet the mechanism by which mutationally activated BRAF cooperates with oncogenes and loss of tumor suppressors to promote melanoma tumorigenesis and progression is not well understood. Dysregulated PI3K/AKT signaling is frequently observed concurrently with BRAF mutant melanomas; however, pharmacological inhibitors of AKT have been largely ineffective in clinical trials as single agents. Studies in our lab have shown that there is a discordant effect between pharmacological inhibition and genetic targeting of AKT, suggesting that therapeutic inhibition of this protein kinase is insufficient to block its activity in melanoma. We have shown that genetic knockdown of all three AKT paralogs leads to complete cell lethality in multiple human melanoma cell lines, and overexpression of each of the AKT proteins is able to significantly rescue this deleterious effect, which is dependent on functional kinase activity. Based on these preliminary results, we seek to determine if the PI3K pathway depends solely on AKT signaling for its role in melanoma initiation and progression. Dysregulation of the PI3K lipid signaling cascade in melanoma is conventionally believed to occur through activation of AKT, and we have previously shown that either loss of PTEN or mutationally activated PI3K cooperates with mutant BRAF to promote melanoma formation. However, it has not been shown if constitutively active AKT alone is able to substitute for this effect. Further research will elucidate the role of AKT in the initiating stages of melanoma and provide a more thorough understanding of the PI3K/AKT signaling pathway in promoting BRAF mutant melanomagnesis.

Citation Format: Gennie Parkman, Joseph Cho, Sheri Holmen, Martin McMahon. Elucidating the role of PI3K lipid effectors in BRAF mutant melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1616.
Abstract 1622: Microsatellite instability causes colorectal cancer cell death to trigger anti-tumor immune response
Authors: Lizardo Darleny Y. , Wang Yi-Jun , Yang Liheng , Yu Jian , Zhang Lin
Published: September 2020

Colorectal cancer (CRC) is the second leading cause of cancer deaths in the western world. Approximately 15% of CRCs have defects in DNA mismatch repair (MMR) genes, which lead to frequent mutations in repetitive genomic sequences known as microsatellites. This kind of genomic instability, referred to as microsatellite instability (MSI), contributes to the emergence of highly immunogenic neoantigen peptides that can stimulate an antitumor response. As such, a subset of CRC patients with deficient MMR has a positive response to immune checkpoint blockade immunotherapy. However, the molecular basis of MSI-induced antitumor immune response is not well understood. To address this issue, we used CRISPR to genetically inactivate MutL homolog 1 (MLH1), a key MMR protein, in two microsatellite stable (MSS) murine colorectal cancer cell lines, CT26 and MC-38, which enabled us to directly compare isogenic MSI and MSS tumors in mice with an immuno-deficient or -competent microenvironment. The growth of MLH1-KO cells was comparable to the parental cells in immunocompromised mice. Upon transplantation into syngeneic, immunocompetent mice, the parental CT26 and MC-38 tumors rapidly proliferate. In contrast, the MLH1-KO tumors grew poorly under the same conditions, which was accompanied by apoptotic cell death, upregulation of inflammatory cytokines, and tumor infiltration of CD8+ T lymphocytes. Blocking apoptosis by caspase inhibition suppressed cytokine induction and T lymphocyte infiltration, suggesting a key role of apoptosis in triggering an antitumor immune response. Our current efforts are towards elucidating the mechanism of cell death and defining the associated immune signatures. Ultimately, a better understanding of the immunological consequences of MSI status in CRCs may help to develop more effective CRC therapies.

Citation Format: Darleny Y. Lizardo, Yi-Jun Wang, Liheng Yang, Jian Yu, Lin Zhang. Microsatellite instability causes colorectal cancer cell death to trigger anti-tumor immune response [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1622.
Abstract 1632: Long-term maintenance of human mature NK cells from hCD34+HSCs engraftment supported by immune humanized B-NDG hIL15 mice
Authors: Lin Qingcong , Hu Yuting , Zhou Xiaofei , Su Youhong , Shen Yuelei
Published: September 2020

Human HSC (hematopoietic stem cells) engrafted humanized mice are a powerful model that allows researchers to examine the human immune system and evaluate cellular immunotherapy in the preclinical stage. Successful reconstitution of the human immune system in B-NDG mice (a complete immunodeficient mouse model previously developed by Biocytogen) has been accomplished using CD34+ hematopoietic stem cells. However, the species barrier between human immune cells and the mouse microenvironment can obstruct human immunity acquisition, as well as innate immune function development, in the mice upon the reconstitution of immune system.

To address this issue, Biocytogen has developed and validated the B-NDG hIL15 mice that combines a B-NDG mouse background and expresses human IL15 cytokine, which plays important roles in the innate and adaptive cell homeostasis, as well as peripheral immune function. The targeting strategy stipulates the insertion of CDS region of the human IL15 gene after the 5′UTR of the mouse Il15. In this work, human CD34+ hematopoietic stem cells have been successfully grafted in B-NDG hIL15 mice, to reconstitute a functional human immune system. At 4 weeks post CD34+ cell implantation, the percentage of hCD45+ cells reached over 25%, containing human T, B, and NK cells. In summary, these CD34+ reconstituted B-NDG hIL15 mice are validated to be promising tools for the efficacy study of clinically relevant therapeutic agents given the mice's long-term maintenance of human NK cells upon the reconstitution of immune system.

Citation Format: Qingcong Lin, Yuting Hu, Xiaofei Zhou, Youhong Su, Yuelei Shen. Long-term maintenance of human mature NK cells from hCD34+HSCs engraftment supported by immune humanized B-NDG hIL15 mice [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1632.
Abstract 1637: ATP-citrate lyase transgenic mice frequently develop lymphoma/leukemia spontaneously
Authors: Kanda Hiroaki , Nomura Kimie , Iizuka Toshihiko , Fujiwara Mutsunori , Ishikawa Yuichi , Migita Toshiro
Published: September 2020

ATP-citrate lyase (ACLY) catalyzes the generation of acetyl-CoA from citrate. ACLY expression is upregulated and/or ACLY is phosphorylated in several types of cancer. We generated ACLY transgenic mice (Tg) to examine the role of ACLY in carcinogenesis.

(Materials and methods)

Using human ACLY cDNA under the ROSA26 promoter, Tg with a C57BL background was established. We observed the Tg without treatment. Statistical analyses were performed using the Chi-square and Fisher's exact test.

(Results)

Of 24 male (M) Tg, aged 24–30 months, 14 (58%), 7 (29%), and 1 (4%) spontaneously developed lymphoma/leukemia (LL), lung carcinoma (LC), and small intestinal carcinoma (SIC), respectively. Since some C57BL mouse colonies are genetically susceptible to LL, and we have previously examined human cases of carcinoma, the Tg were back-crossed with C3H mice, which are resistant to LL and susceptible to carcinoma, especially hepatocellular carcinoma (HCC). A total of 129 (M: 67, female (F): 62) Tg were observed for 18 months. Forty-nine C3H mice (M: 19, F: 30) from the same colony were used as controls. Multiple tumors were counted individually. Seventy-three Tg (57%; M: 41, 61%; F: 32, 52%) and 8 controls (16%; M: 4, 21%; F: 4, 13%) developed cancer. The Tg developed significantly more tumors (total (T), M, F: all p<0.05), including 33 HCC (26%; M: 22, 33%; F: 11, 18%), 28 LL (22%; M: 12, 18%; F: 16, 26%), 7 LC (5%; M: 6, 9%; F: 1, 2%), 4 sarcomas (Sa)(3%; M: 2, 3%; F: 2, 3%), 2 SIC (2%; M: 2, 3%; F: 0), 2 skin appendage carcinomas (SAC) (2%; M: 0; F: 2, 3%), and 1 ovarian cancer (Ov) (1%; F: 1, 2%), than the control mice, which developed 7 HCC (14%; M: 4, 21%; F: 3, 10%), 1 LC (2%; M: 1, 5%; F: 0), and 1 Ov (2%; F: 1, 3%). No LL was seen in the controls. The frequency of HCC did not differ significantly between the Tg and controls (T, M, F: all p>0.05).

(Discussion)

ACLY was reported to be related to the carcinogenesis of anaplastic large cell lymphoma (Basappa J. et al. Blood 2015 126:465). This study suggests that ACLY might contribute to LL development.

Table:Summary of developed cancersNumberTotal cancerHCCLLLCSICSaSACOvACLY12973(57%)*33(26%)28(22%)7(5%)2(2%)4(3%)2(2%)1(1%)Male6741(61%)**22(33%)12(18%)6(9%)2(3%)2(3%)0-Female6232(52%)***11(18%)16(26%)1(2%)02(3%)2(3%)1(2%)Control498(16%)*7(14%)01(2%)0001(2%)Male194(21%)**4(21%)01(5%)000-Female304(13%)***3(10%)000001(3%)*, **, ***; p<0.05

Citation Format: Hiroaki Kanda, Kimie Nomura, Toshihiko Iizuka, Mutsunori Fujiwara, Yuichi Ishikawa, Toshiro Migita. ATP-citrate lyase transgenic mice frequently develop lymphoma/leukemia spontaneously [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1637.
Abstract 1652: Tumor-specific PET imaging with an 18F-labeled tryptophan analogue in a transgenic medulloblastoma mouse model
Authors: Yue Xuyi , Xin Yangchun , Li Hua , Li Zhiqin , Cai Hancheng , Langhans Sigrid A.
Published: September 2020

In vivo positron emission tomography (PET) imaging is a key modality to evaluate disease status of brain tumors. In recent years, tremendous efforts have been made in developing PET imaging methods for pediatric brain tumors. Carbon-11 labelled tryptophan derivatives are feasible as PET imaging probes in brain tumor patients with activation of the kynurenine pathway, but the short half-life of carbon-11 limits its application. Using a transgenic mouse model for the sonic hedgehog (Shh) subgroup of medulloblastoma, here we evaluated the potential of the newly developed 1-(2-[18F]fluoroethyl)-L-tryptophan (1-L-[18F]FETrp) as a PET imaging probe for this common malignant pediatric brain tumor. 1-L-[18F]FETrp was synthesized on a PETCHEM automatic synthesizer with good chemical and radiochemical purities and enantiomeric excess values. Imaging was performed in tumor-bearing Smo/Smo medulloblastoma mice using a Perkin-Elmer G4 PET-X-Ray scanner. Medulloblastoma tumors showed significant and specific accumulation of 1-L-[18F]FETrp. 1-L-[18F]FETrp also showed significantly higher tumor uptake than its D-enantiomer 1-D-[18F]FETrp. The uptake of 1-L-[18F]FETrp in normal brain tissue was low, suggesting that 1-L-[18F]FETrp may prove a valuable PET imaging probe for the Shh subgroup of medulloblastoma and possibly other pediatric and adult brain tumors.

Citation Format: Xuyi Yue, Yangchun Xin, Hua Li, Zhiqin Li, Hancheng Cai, Sigrid A. Langhans. Tumor-specific PET imaging with an 18F-labeled tryptophan analogue in a transgenic medulloblastoma mouse model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1652.
Abstract 1655: Hyperspectral metabolic imaging with a camera
Authors: Hedde Per Niklas , Gratton Enrico
Published: September 2020

New methods to quantify metabolism in cells, organoids, and tissues are highly demanded for cancer diagnosis and treatment. We developed a light sheet-based volumetric imaging platform to rapidly quantify metabolism via fluorescence spectral signatures of intrinsically fluorescent biomolecules.

The ability to quantify metabolism in live cells and tissues can be very powerful as the metabolic activity in rapidly proliferating cells, such as cancer cells, is fundamentally different from non-proliferating cells. Most cancer cells rely on aerobic glycolysis to ferment glucose into lactate independent of the presence of oxygen whereas healthy, differentiated cells primarily rely on oxidative phosphorylation. The rapid generation of cytosolic adenosine triphosphate (ATP) during proliferation results in elevated ratios of the reduced form of nicotinamide adenine dinucleotide (NADH) to NAD+ in cancerous cells that can be detected with fluorescence microscopy of intrinsically (auto)fluorescent molecules such as NADH and flavin adenine dinucleotide (FAD) and its variants. This opens a window to quantify ongoing redox reactions as well as important enzymatic reactions via changes of their fluorescence spectral signatures reporting on the metabolic activity of cells. We developed a light sheet-based hyperspectral imaging platform leveraging custom designed sine/cosine filters and spectral phasor data analysis to directly obtain spectral information with a regular camera. The approach was verified by spectrally resolving 3D reconstructions of fluorescence images of fragments of mouse intestinal crypts. The acquisition speed of light sheet-based hyperspectral imaging is on the order of seconds for one 3D stack allowing for sequential, repeated imaging of several samples over the course of hours to days.

In summary, the overall goal of this project is to develop a modular imaging platform to overcome the limitations of current, laser scanning-based spectral imaging and to make quantification of metabolism viable in live three-dimensional cell, organoid and tissue samples, that can then be used to, for example, to study the effects of cancer chemotherapy reagents on the metabolism of patient-derived organoids.

Citation Format: Per Niklas Hedde, Enrico Gratton. Hyperspectral metabolic imaging with a camera [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1655.
Abstract 1656: Quantifying task performance with photon-processing detectors
Authors: Caucci Luca , Henscheid Nicholas P. , Barrett Harrison H.
Published: September 2020

Purpose. To estimate task performance (e.g., the ability to correctly detect a small lesion in a normal tissue) when the imaging system uses a new class of detectors, called photon-processing detectors.

Experimental Procedure. We simulated a single-photon emission computed tomography (SPECT) system in which multiple cameras image a 3D object from different angles. Each camera uses multiple measurements (e.g., photomultiplier tube outputs) to perform estimation of event parameters (or “attributes”) of each detected gamma-ray photon. Photon attributes, such as position, energy and direction of propagation, are stored at full precision. The likelihood ratio applied to photon-processing data is used to assess task performance for the detection of a signal buried in a random background. Statistical methods (including Markov chain Monte Carlo estimation) are used to calculate the performance of the likelihood ratio on this task.

Results. Our results show an improvement with respect to conventional (i.e., pixelated) detectors when photon-processing detectors are used. The area under the receiver operating characteristic (ROC) curve is used to assess task performance, and we use this figure-of-merit to compare photon-processing detectors with conventional detectors.

Conclusions. Increased performance is observed in the detection of a small lesion when photon-processing detectors are used instead of pixelated detectors. Applications that will benefit from photon-processing detectors include tumor diagnosis, drug development, therapy assessment and the study of tumor metabolism.

Citation Format: Luca Caucci, Nicholas P. Henscheid, Harrison H. Barrett. Quantifying task performance with photon-processing detectors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1656.
Abstract 169: Discovery of clinically distinct CLL subgroups by integrative mapping of large-scale genetic, epigenetic, expression and clinical data
Authors: Knisbacher Binyamin A. , Lin Ziao , Stewart Chip , Hahn Cynthia K. , Stevenson Kristen E. , Neuberg Donna S. , Martín-Subero José I. , Puente Xose S. , Campo Elias , Wu Catherine J. , Getz Gad
Published: September 2020

Chronic lymphocytic leukemia (CLL) accounts for one third of all diagnosed leukemias. It exhibits tremendous clinical variability, ranging from indolent disease kinetics to a fast progressing aggressive disease. Two large-scale studies by our groups, each analyzing more than 500 CLL subjects, have shown that a large part of this clinical variability can be explained by underlying molecular features of the cancerous B-cells, including genetic and epigenetic alterations. Despite this progress, patients do not yet fully benefit from these discoveries in their treatment plans, and prognosis is not tailored to them based on the molecular features of their cancer. To achieve precision medicine, we compile a large dataset of multi-omic and clinical data from over 1100 CLL patients. Our extensive cohort confers power to detect novel driver genes, doubling the number of known drivers (114 total) and identifying drivers specific to major CLL subtypes, such as IGHV-mutated and IGHV-unmutated. The analysis identifies recurrent copy number alterations, order-of-event timing and mutational processes acting in CLL. We integrate these with expression data from 700 CLL RNA-seqs and epigenetic data to define molecular subtypes that present differential clinical outcomes. Altogether, this work delineates the molecular landscape of CLL and sets the stage for improved classification, prognostics and treatment of CLL patients.

Citation Format: Binyamin A. Knisbacher, Ziao Lin, Chip Stewart, Cynthia K. Hahn, Kristen E. Stevenson, Donna S. Neuberg, José I. Martín-Subero, Xose S. Puente, Elias Campo, Catherine J. Wu, Gad Getz. Discovery of clinically distinct CLL subgroups by integrative mapping of large-scale genetic, epigenetic, expression and clinical data [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 169.
Abstract 1690: Blockade of antitumor milieu-induced tumor cell PIM2 expression potently synergizes with anti-PD-L1 therapy in hepatocellular carcinoma
Authors: Wang Juncheng , Lu Shi-Xun , Chen Jinbin , Chen Dongping , Chen Minshan , Zhou Zhongguo
Published: September 2020

Background & Aims: Despite being a typical oncogene, PIM kinase has been reported to suggest a favorable prognosis in some solid tumors, but no research has yet explored the underlying mechanisms. Furthermore, an inconsistent prognosis also hinders the clinical application of PIM inhibitors. We aimed to explore the clinical significance and regulatory mechanism of PIM2, as well as the potential clinical application of PIM2 inhibitor in hepatocellular carcinoma (HCC).

Methods: PIM2 expression was assessed in 464 HCC patients who underwent curative resection, and its correlations with prognosis, immune status, and tumor infiltrating leukocytes (TILs) were investigated. Moreover, the underlying regulatory mechanisms of PIM2 and the clinical application of PIM2 inhibitor were also studied.

Results: We found that PIM2 primarily displayed an oncogenic function in HCC. However, elevated PIM2 expression in tumor cells predicted a favorable prognosis. Mechanistically, high PIM2 expression in tumors was associated with an adaptive immune response elicited by TILs, and IL-1β, together with TNF-α and IFN-γ, derived from TILs induced the expression of PIM2 by activating the p38/Erk/MAPK and NF-κB signaling pathways in tumor cells. Specifically, high PIM2 expression in tumors was significantly correlated with a high density of T cells and monocytes, and activated T cell-secreting IFN-γ-polarized monocytes induced the tumor cell PIM2 expression in the tumor milieu. Moreover, blockade of antitumor milieu-induced tumor cell PIM2 expression potently augmented the therapeutic efficacy of anti-PD-L1 therapy in HCC.

Conclusions: Our study provides important insights into a possible adaptive immune resistance mechanism by which the antitumor milieu induces oncogenic PIM2 expression in tumor cells. Moreover, specific inhibition of PIM2 in tumor cells augments the therapeutic efficacy of anti-PD-L1 therapy in HCC.

Citation Format: Juncheng Wang, Shi-Xun Lu, Jinbin Chen, Dongping Chen, Minshan Chen, Zhongguo Zhou. Blockade of antitumor milieu-induced tumor cell PIM2 expression potently synergizes with anti-PD-L1 therapy in hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1690.