bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract PO-040: The role of mTOR in epigenetic regulation in cancer
Authors: Kim HaEun , Papadopoli David , Topisirovic Ivan
Published: December 2020

Changes in gene expression, including those caused by epigenetic dysregulation, represent a hallmark of cancer. The mechanistic/mammalian Target of Rapamycin (mTOR) is a serine/threonine kinase that coordinates nutrient availability to the regulation of cell growth and metabolism, which is frequently perturbed in cancer. mTOR exists in two structurally and functionally divergent complexes, mTOR complex 1 and 2 (mTORC1 and 2). mTORC1 regulates many metabolic pathways, including the serine-glycine-one carbon network (SGOC), a pathway modulating the production of S-adenosyl methionine (SAM) and α-ketoglutarate (α-KG). The latter two metabolites constitute essential co-substrates of histone methyltransferases and demethylases, respectively. However, how mTOR signaling controls epigenetic dynamics remains largely unknown. To address this important gap in knowledge, we treated two breast cancer cell lines (MCF7 and T47D) with allosteric (rapamycin) and active-site (INK128) mTOR inhibitors to study how mTOR may influence global histone methylation. We found that mTOR inhibitors significantly increased histone 3 lysine 9 tri-methylation (H3K9me3) and histone 3 lysine 27 tri-methylation (H3K27me3) but not histone 3 lysine 4 tri-methylation (H3K4me3) suggesting this effect was selective. Metabolomic tools were employed to decipher changes of epigenetic metabolites α-KG, SAM, S-Adenosyl homocysteine (SAH), methionine, and serine upon mTOR inhibition. These findings provide bases for my future work which will focus on establishing the role of alterations in mTOR signaling on epigenetic programs which drive tumorigenesis. A better understanding of this network may help identify new therapeutic targets to improve current cancer treatments.

Citation Format: HaEun Kim, David Papadopoli, Ivan Topisirovic. The role of mTOR in epigenetic regulation in cancer [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-040.
Abstract PO-053: Investigating intra-tumour metabolic heterogeneity in triple-negative breast cancer
Authors: Fukano Marina , Deblois Geneviève , Zuo Dongmei , Martinez Constanza , Yang Yang , Ragoussis Ioannis , Kleinman Claudia , Park Morag
Published: December 2020

Intra-tumour heterogeneity is a great challenge in cancer treatment. Cancer cells presenting different metabolic profiles can mutually support each other through metabolic symbiosis mechanism, which contributes to tumour proliferation, metastasis, and therapeutic resistance. Triple-negative breast cancer (TNBC) shows poor clinical outcomes due to a high level of heterogeneity and a lack of targeted therapies. Through multiplex immunofluorescence tissue section staining of TNBC patient-derived xenografts (PDX), we identified two mutually exclusive and metabolically distinct cell populations within a TNBC tumour. One cancer cell population expresses glucose transporter 1 (GLUT1) and is located in hypoxic areas, whereas another specifically expresses glutamine synthetase (GS) and is located near vasculatures. Single-cell RNA sequencing data from a TNBC PDX with mutually exclusive GLUT1- and GS-positive cells shows that GLUT1-positive cells are enriched for hypoxic and glycolytic gene signatures, while GS-positive cells are associated with glutathione, pyrimidine and purine metabolism. Importantly, our data suggest that GLUT1- and GS-positive cells exchange glutamate and lactate metabolites as part of metabolic symbiosis and targeting this mechanism could successfully impair the growth of TNBC.

Citation Format: Marina Fukano, Geneviève Deblois, Dongmei Zuo, Constanza Martinez, Yang Yang, Ioannis Ragoussis, Claudia Kleinman, Morag Park. Investigating intra-tumour metabolic heterogeneity in triple-negative breast cancer [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-053.
Abstract PO-057: Breast cancer cell metabolism: Effect of beta-hydroxybutyrate on glucose deprived breast cancer cells
Authors: Maldonado Rylee , Talana Chloe Adrienna , Uehara Kahealani , Weichhaus Michael
Published: December 2020

Ketogenic diets have the potential to lower glucose availability to cancer cells. The effect that the resulting increase in ketone bodies has on cancer cells is not fully understood. Here we explored the effect of beta-hydroxybutyrate (BHB) on glucose deprived MCF-7 and T47D breast cancer cells. Cell proliferation was decreased with lower glucose conditions which could not be rescued by either 10 mM or 25 mM BHB supplementation. Gene expression changes were altered when cells were glucose deprived. Smaller changes were observed when gene expression changes were compared between glucose deprived cells and those treated with BHB. Pathway enrichment analysis demonstrated that glucose deprivation decreased activity of the Hippo-Yap cell signaling pathway in MCF-7 breast cancer cells, while it increased expression of genes of the NRF2-pathawy and genes regulating ferroptosis in T47D breast cancer cells. No significant pathway enrichment was found when glucose deprived cells were treated with BHB. Both cell lines expressed the enzymes (OXCT1/2, BDH1, ACAT1/2) responsible for metabolizing BHB to acetyl-CoA, yet expression of these enzymes does not change with either glucose deprivation or BHB treatment. In the publicly available Cancer Genome Atlas increased expression compared to healthy tissue of enzymes responsible for metabolizing BHB to acetyl-CoA was also observed, however only a small subset of patients showed alterations in these enzymes. This alteration was linked to a significant decrease in overall survival. In conclusion, neither MCF-7 nor T47D breast cancer cells were affected by BHB during glucose deprivation, however screening of the patient’s tumor for activation of ketone bodies metabolizing enzymes may be indicated to identify who will benefit from ketogenic diet interventions.

Citation Format: Rylee Maldonado, Chloe Adrienna Talana, Kahealani Uehara, Michael Weichhaus. Breast cancer cell metabolism: Effect of beta-hydroxybutyrate on glucose deprived breast cancer cells [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-057.
Abstract PR10: Alterations in carbon and nitrogen metabolism in lung cancer
Authors: Kim Jiyeon
Published: December 2020

Lung cancer is the leading cause of cancer death worldwide. The critical barrier in lung cancer treatment is a lack of effective therapies at later stages of the disease. Lung cancer genome sequencing supports the idea that combinations of mutations act in concert to foster malignancy and disease progression. Among these, KRAS and LKB1 mutations represent “driver” changes in tumor development. Importantly, mutations in these genes also perturb signal transduction to promote a form of metabolism conducive to tumor growth. Metabolic reprogramming is considered to be fundamental to malignant transformation; thus, identifying molecular links between these mutations and metabolism, and targeting the resulting metabolic pathways, may produce better therapies. To gain broader insight into metabolic reprogramming and potential dependencies in different KRAS/LKB1 co-mutant (KL) model systems, we analyzed metabolome and gene expression profiling of tumors derived from genetically engineered mouse models of lung cancer. These two datasets independently revealed that both mouse and human KL co-mutants are associated with elevated hexosamine biosynthesis pathway (HBP) expression, a metabolic pathway that requires the interplay between nitrogen and carbon metabolism. Upregulation of the HBP in co-mutants resulted in enhanced global N-acetyl glucosamine modification (O-GlcNAcylation) and glycosylation, modification involved in important processes occurring in cancer, such as cell signaling and communication, cell-matrix interactions, immune modulation and metastasis formation. KL cells have elevated HBP metabolites and higher hexosamine biosynthesis rates. By small scale RNAi screening and chemical inhibition of the HBP, we uncovered that KL co-mutants are dependent on Glutamine-Fructose-6-Phosphate Transaminase 2 (GFPT2), a rate-limiting enzyme in the pathway. GFPT2 inhibition selectively reduced KL cell growth in culture and xenografts. Importantly, high GFPT2 expression predicts poor prognosis in human NSCLC patients, considerably more so than low LKB1 expression alone. Our study illuminates the functional significance of the interplay between carbon and nitrogen metabolism in cancer, not only for primary cancer growth but also for tumor aggressiveness and may lead to new avenues for developing novel targeted therapeutics.

Citation Format: Jiyeon Kim. Alterations in carbon and nitrogen metabolism in lung cancer [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PR10.
Abstract A12: Expression of GPR41, GPR43, and GPR109A in breast tissue in relation to breast cancer development
Authors: Kump Annie , Herrera Daniel , Stiemsma Leah T.
Published: December 2020

Introduction: There is evidence to suggest that the human microbiome may play a key role in the development of breast cancer. There is a unique microbial signature in mammary tissue that is associated with breast cancer. Further, there is mounting evidence to support the role of the gut microbiome in promoting or preventing breast cancer. Consequently, specific compositional and/or functional shifts in either of these microbiomes may promote the growth of breast tumors. Short chain fatty acids (SCFAs) are microbial metabolites produced in the large intestine by bacterial fermentation of complex carbohydrates. These metabolites exit the gut and interact with many different cell types throughout the human body, including those in the breast, via g-protein coupled receptors; GPR41, GPR43, and GPR109A. These metabolites have been identified as a potential functional connection between the gut microbiome and breast cancer but have not been studied in breast tissue in the context of breast cancer. To assess the interaction between the microbiome and breast tumor development, we analyzed the expression of these receptors in tissue collected from women who donated healthy, prediagnostic, or cancerous tissue to the Susan G. Komen and Indiana University Simon Cancer Center Tissue Banks.

Methods: DNA and RNA (n=60, n = 15 for each tissue subset: healthy, prediagnostic, adjacent normal, and tumor tissue) was copurified from these tissues using the Qiagen AllPrep® PowerFecal® DNA/RNA Kit. The RNA was then reverse transcribed into complementary DNA. Quantitative PCR was performed on the cDNA using four validated primer sets: GPR41, GPR43, GPR109A, and ribosomal 18S (housekeeping gene).

Results/Cconclusions: We did not identify any significant differences in expression levels of these receptors between the participant groups, suggesting that cancer status is not associated with increased metabolic activity of the microbiome. We are currently conducting sensitivity analyses to determine the role of clinical factors such as age, menopausal status, BMI, etc., on the expression of these receptors in breast tissue. We are also investigating the expression of enzymes and transporters in breast tissue related to host cell metabolism of the most bioactive SCFAs (acetate, propionate, and butyrate). This work will yield valuable information regarding the interaction between the human microbiome and breast tumor development. Ultimately, it may be possible to target SCFA-producing bacteria or SCFA catabolic pathways in breast tissue to prevent or treat breast cancer.

Citation Format: Annie Kump, Daniel Herrera, Leah T. Stiemsma. Expression of GPR41, GPR43, and GPR109A in breast tissue in relation to breast cancer development [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr A12.
Abstract A17: Human papillomavirus type 16 and 18 viral loads as predictors associated with abnormal cervical cytology among women in Saudi Arabia
Authors: Obeid Dalia , Almatrrouk Shihana , Khayat Hadeel , Al-Muammer Turfah , Tullbah Asma , Albadawi Ismail , Alahdal Mohammed , Alhamlan Fatimah
Published: December 2020

The present study aimed to screen for and genotype HPV among Saudi women; develop sensitive quantitative polymerase chain reaction (qPCR) assays to detect the viral load for the two most common HPV types, namely 16 and 18; and assess whether HPV viral load could be used as a marker for cervical abnormality and disease progression. This study examined 733 specimens (both formalin-fixed, paraffin-embedded specimens and PAP smear samples) from women who underwent cervical screening. The specimens and samples were processed for DNA extraction and then tested for HPV DNA. Approximately 165 specimens (18%) were positive for HPV. Those specimens were genotyped using a reverse line blotting hybridization assay. The results indicated that the most common HPV types detected were a single infection with HPV 16 (51%) or with HPV 18 (28%) followed by infections with multiple HPV types (~7%). A qPCR TaqMan assay developed in-house was used to determine the viral load for HPV genotypes 16 (n=80) and 18 (n=45). Viral loads for both HPV types were significantly associated with cervical cytology grade (P < 0.05). The odds ratio (OR) for the HPV 16 viral load was high for specimens with cervical cancer (OR, 18.8; 95% CI, 4.3–82.9) or for those with high-grade squamous intraepithelial lesions (OR, 14.7; 95% Cl, 2.43–88.49). For the HPV 18 viral load, the OR was significant only for specimens with cervical cancer (OR, 11.1; 95% Cl, 2.2–54.9). Logistic regression models for HPV 16 and for HPV 18 viral load levels were significant, with higher viral load associated with cervical abnormalities. These findings indicate that viral load is a predictor significantly associated with cytology abnormality in women who are positive for high-risk HPVs and suggest integrating a viral load test into current clinical screening practices for HPV-positive women.

Citation Format: Dalia Obeid, Shihana Almatrrouk, Hadeel Khayat, Turfah Al-Muammer, Asma Tullbah, Ismail Albadawi, Mohammed Alahdal, Fatimah Alhamlan. Human papillomavirus type 16 and 18 viral loads as predictors associated with abnormal cervical cytology among women in Saudi Arabia [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr A17.
Abstract A22: A mechanism of γδ T cell-mediated antitumor immunity against brain cancer
Authors: Park Jang Hyun , Lee Heung Kyu
Published: December 2020

Glioblastoma multiforme (GBM) is one of the less survivable and most malignant brain tumors. The average survival period is less than 2 years despite multiple therapies including chemotherapy and radiation. Furthermore, GBM showed severe resistance against immunotherapies such as immune checkpoint blockade mainly targeting CD8 T cells (1). Recently, several studies showed that γδ T cells can be an alternative target of immunotherapy. Unlike conventional T cells, γδ T cells recognize stress-induced surface antigens such as NKG2D-ligands and phosphor-antigens (2). Because GBM cells highly express NKG2D-ligands, γδ T cells could be proper targets for immunotherapy. However, a basic understanding of γδ T cell-mediated antitumor immunity remains unclear. Using single-cell transcriptomic analysis, we analyzed the characteristics of GBM-infiltrating γδ T cells. γδ T cells are highly suppressed by oxygen tension of tumor microenvironment in the brain tumor. We inhibited respiration of tumor cells by metformin, which is the drug for type 2 diabetes. Inhibition of tumor cell respiration sufficiently reinvigorated γδ T cells. Reinvigorated γδ T cells highly expressed NKG2D and multiple cytokines. In conclusion, this study highlights the importance of γδ T cells and the druggable pathways through reinvigoration of γδ T cells.

Citation Format: Jang Hyun Park, Heung Kyu Lee. A mechanism of γδ T cell-mediated antitumor immunity against brain cancer [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr A22.
Abstract B21: The gastrointestinal microbiota controls cancer cell intrinsic mechanisms to promote the progression of acute lymphoblastic leukemia
Authors: Mahauad-Fernandez Wadie , Zlitni Soumaya , Bhatt Ami , Felsher Dean
Published: December 2020

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer and the second most common acute leukemia in adults. Despite best existing therapy, older children and elderly ALL patients have poor prognoses. Thus, there is a need to identify alternative therapeutics for these patients. There has been controversy about the function of the gut microbiota in cancer, with some reports showing that it supports tumor growth and others showing tumor growth inhibition. Recently, Dr. Melvyn Greaves proposed that ALL development may have a microbial trigger; however, gut microbiota studies in ALL are scarce. Little is known about the role of the gut microbiota in ALL progression and in reshaping cancer cell-intrinsic pathways that are as important as immune responses in the outcome of ALL. Recently, we found that gut microbiota depletion in immunocompromised mice reduces ALL growth by inducing apoptosis and inhibiting proliferation. We show that in immunocompromised microbiome-depleted mice, the rate of ALL tumor growth is reduced compared to microbiome-competent mice. Molecularly, we found that microbiome ablation results in changes in cancer cell-intrinsic pathways of apoptosis and proliferation in these tumors. To further understand how the gut microbiota affects these cancer cell-intrinsic pathways, we have performed RNA sequencing on tumors from microbiome-competent and microbiome-depleted mice accompanied by 16s rRNA sequencing from stool samples and mass spectrometry of blood serum to identify metabolites secreted by the gut microbiota that reach the tumor site, affecting cancer cell growth. Our goal is to define bacterial species and metabolites involved in ALL progression and to mechanistically determine how these bacterial species and metabolites regulate ALL growth via regulation of cancer cell-intrinsic pathways. These data suggest that the gut microbiota may regulate ALL progression in an immune-independent manner via transcriptional changes of cancer cell-autonomous pathways. We envision that the gut microbiota can be reshaped in ALL patients who are elderly, chemo-refractive, and/or immunocompromised to alter cancer cell-intrinsic pathways to treat ALL. The clinical impact of these findings is significant given that ALL patients who are immunocompromised could still benefit from therapeutics aiming at modulating the gut microbiota.

Citation Format: Wadie Mahauad-Fernandez, Soumaya Zlitni, Ami Bhatt, Dean Felsher. The gastrointestinal microbiota controls cancer cell intrinsic mechanisms to promote the progression of acute lymphoblastic leukemia [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr B21.
Abstract B22: The human skin bacteria Staphylococcus epidermidis ameliorates UVB-induced free radicals through reduction of labile iron
Authors: Balasubramaniam Arun , Keshari Sunita , Adi Prakaso , Huang Chun-Ming
Published: December 2020

UVB-induced skin damage results in various inflammatory disorders through the induced generation of reactive oxygen species (ROS) that quickly inundate tissue antioxidants and chronic disorders; in severe cases it can lead to skin cancer. We investigated efficacies of human skin commensal bacteria S. epidermidis (ATCC12228) with glycerol, which on fermentation produces electrons. In vivo affirmation on ICR mice has confirmed the antioxidative role of topically applied S. epidermidis with glycerol against UVB irradiation and maintained sufficient expression of 4-hydroxynonenal (4-HNE) and cyclobutane pyrimidine dimer (CPD), a major biomarker for lipid peroxidation and DNA damage. Upon UVB irradiation in keratinocyte cell lines treated with glycerol mediated S. epidermidis fermentation product show the reduced intracellular oxidative stress. S. epidermidis or glycerol alone in In vivo topical application in mice skin and in vitro fermentation product treatment in keratinocytes does not influence the level of oxidative stress. Further electrochemical behavior of glycerol mediated S. epidermidis fermented medium found to produce electron transfer; this result suggests the electrogenic and antioxidant property of S. epidermidis. The electrons produced by S. epidermidis fermentation product initiate reduction of free radicals by converting toxic Fe3+ (ferric ion) back to nontoxic Fe2+ (ferrous ion); thereby it terminates Fenton’s reaction and maintains iron hemostasis. The novel pathway linking electrons produced by probiotic skin bacteria and iron metabolism has been further analyzed.

Note: This abstract was not presented at the conference.

Citation Format: Arun Balasubramaniam, Sunita Keshari, Prakaso Adi, Chun-Ming Huang. The human skin bacteria Staphylococcus epidermidis ameliorates UVB-induced free radicals through reduction of labile iron [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr B22.
Abstract B30: Intratumoral microbes correlate with tumor-infiltrating lymphocytes in lung cancer RNAseq
Authors: Spakowicz Daniel , Hoyd Rebecca , Liu YunZhou , Sahasrabudhe Janhavi , Singh Malvenderjit J. , Arefi Isaac , Denney Andrew , Carbone David , Mo Xiaokui
Published: December 2020

Nonhuman sequences have been found in many tumors, but their effect on outcomes remains poorly understood. We hypothesize that intratumoral microbes affect the recruitment of immune cells through local immunostimulatory effects including activation nucleic acid sensing pathways. We obtained RNA-seq data from 480 tumor biopsies including melanoma (16), bladder (104), colorectal (20), renal cell carcinoma (20), sarcoma (118), and lung (202) from patients treated at The Ohio State University Comprehensive Cancer Center as part of the Oncology Research Information Exchange Network (ORIEN). Reads were aligned to human and exogenous genomes using TopHat2 and Kraken2/Bracken, respectively. Human gene expression was deconvolved to absolute abundances of immune cells using CIBERSORT. An average of 99.87% of reads aligned to the human reference genome across all samples. Inferred counts of tumor-infiltrating lymphocytes (TILs), and particularly the immune cell types CD8+ T-cells and M1 macrophages, were significantly enriched in lung cancer relative to the other tested cancers (p-values <0.0001). TILs significantly correlated with both RIG-I and cGAS expression (p-values <0.0001). Exogenous reads aligned to 1328 genomes of bacteria, archaea, viruses, and fungi after filtering. The most abundant phyla within exogenous counts were the Proteobacteria (39.5%), Firmicutes (20.1%), and Actinobacteria (5.16%). Lung cancer samples showed positive correlations between Acinetobacter junii and CD8+ T-cells (p-value=0.035) and between Acidipropionibacterium virtanenii and activated NK cells (p-value=0.035). This suggests a mechanism by which intratumoral microbes may affect immune cell infiltration and cancer outcomes.

Citation Format: Daniel Spakowicz, Rebecca Hoyd, YunZhou Liu, Janhavi Sahasrabudhe, Malvenderjit J. Singh, Isaac Arefi, Andrew Denney, David Carbone, Xiaokui Mo. Intratumoral microbes correlate with tumor-infiltrating lymphocytes in lung cancer RNAseq [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr B30.
Abstract B31: Microbiota composition in bilateral healthy breast tissue and breast tumors
Authors: Klann Emily M. , Williamson Jessica M. , Tagliamonte Massimiliano S. , Ukhanova Maria , Asirvatham Jaya Ruth , Chim Harvey , Yaghjyan Lusine , Mai Volker
Published: December 2020

Purpose: The purpose of this pilot study was to assess the differences in breast tissue microbiota composition by breast side (left versus right) within an individual woman and compare the microbiota of healthy and breast tumor tissue between women. We further aimed to determine whether certain bacterial taxa may be associated with breast tumors.

Methods: Bilateral healthy breast tissue samples (n=36) were collected from ten women who received routine mammoplasty procedures at the University of Florida Department of Surgery. Archived breast tumor samples (n=10) were obtained from an established biorepository. Bacterial DNA was extracted from tissues, amplified via polymerase chain reaction (PCR), and sequenced on the Illumina MiSeq platform. Microbiota data were analyzed using QIIME and RStudio.

Results: The most abundant phyla in both tumor and healthy tissues were Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria. A total of 38 operational taxonomic units (OTUs) were found to be significantly different in terms of differential abundance between tumor and healthy tissues (absolute effect size range: 0.761-3.98). The OTUs with the largest absolute effect size associated with higher relative abundance in breast tumors were of Flavobacterium species (R2=3.98), Acinetobacter species (R2=3.64), [Mogibacteriaceae] family (R2=3.34), and Clostridiales order (R2=3.21). Alpha diversity (Shannon Diversity Index) was similar in healthy and tumor tissue (4.98 vs. 4.84; p=0.350). Based on unweighted UniFrac measures, breast tumor samples clustered distinctly from healthy samples (R2=0.10; p=0.001). Microbiota composition in healthy samples clustered within women (R2=0.20; p=0.012) and by breast side (left or right) within a woman (R2=0.36; p=0.001).

Conclusion: We identified significant differences in microbiota composition between women and between breasts of the same woman. These results warrant further investigation to elucidate the potential relationship between breast tissue microbiota and breast cancer.

Citation Format: Emily M. Klann, Jessica M. Williamson, Massimiliano S. Tagliamonte, Maria Ukhanova, Jaya Ruth Asirvatham, Harvey Chim, Lusine Yaghjyan, Volker Mai. Microbiota composition in bilateral healthy breast tissue and breast tumors [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr B31.
Abstract IA10: Epigenetic remodeling of host metabolic pathways by Epstein-Barr virus (EBV) immortalization
Authors: Lamontagne Jason , Wiedmer Andreas , Lieberman Paul M.
Published: December 2020

Epstein-Barr virus (EBV) reprograms host metabolism and gene expression during B-cell immortalization through a highly orchestrated process. The regulatory mechanisms coordinating this reprogramming are not fully elucidated but reflect important events in viral oncogenesis. One clue to this coordinate regulation is provided by the viral-encoded tegument protein BNRF1 that functions in viral chromatin assembly during primary infection and shares extensive structural similarity to a purine biosynthetic enzyme FGARAT (also called PFAS). FGARAT shows strong evolutionary conservation from bacteria to human. Orthologues of BRNF1 are found in all gamma herpesviruses, including KSHV ORF75, and share the common function of disarming components of the PML-nuclear body (PML-NB) and its antiviral functions. We have previously shown that the BNRF1 interacts with the histone H3.3 chaperone DAXX and displaces its interaction with ATRX. ATRX is thought to target Daxx-H3.3 to GC-rich repetitive DNA to repress viral and host telomeric transcription. We found that the BNRF1-DAXX complex changes these functions to facilitate viral gene expression during primary infection, enabling the establishment of EBV latency. However, it is not yet known how the viral FGARAT homology is linked to cellular metabolism and purine biosynthesis. Using metabolomics mass spectrometry, we provide new data indicating that the metabolic pathways are among the most significantly perturbed by EBV during B-cell immortalization. Integrating gene expression (RNA-Seq), chromatin accessibility (ATAC-Seq), and EBV transcription factor binding (ChIP-Seq) we identified several cellular metabolic genes, including adenine deaminase (ADA) and arginosuccinate synthase (ASS1), as a direct target of EBV transcriptional regulation during EBV immortalization. We find that EBV nuclear antigen EBNA1 binds directly to the ADA transcriptional regulatory regions and induces epigenetic changes at this locus. Failure to activate ADA compromises B-cell immortalization. The relationship between BNRF1, EBNA1, and nucleotide metabolism during EBV immortalization will be discussed. These findings suggest that EBV captured a highly conserved purine biosynthetic enzyme to coordinate epigenetic reprogramming with nucleotide metabolism during EBV induced B-cell immortalization.

Citation Format: Jason Lamontagne, Andreas Wiedmer, Paul M. Lieberman. Epigenetic remodeling of host metabolic pathways by Epstein-Barr virus (EBV) immortalization [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr IA10.
Abstract IA24: Engineered Salmonella for drug delivery to solid tumors
Authors: Forbes Neil S.
Published: December 2020

Engineered bacteria have the potential to overcome the limitations that cause cancer therapies to fail. We are engineering bacteria to deliver therapeutic payloads and quantifying the mechanisms that control bacterial therapy. We have shown that bacteria preferentially target tumors and actively penetrate tissue. Bacterial motility has a linear relationship with colonization density. Manipulating chemoreceptors in the membrane can direct bacteria to drug-resistant tumor regions, and inducing inflammation can promote colonization by modifying tumor vasculature. When engineered to express α-hemolysin from Staphylococcus aureus, bacteria kill 99% of culture cells in 5 minutes and reduce tumor volume in mice. Engineering Salmonella with quorum-sensing provides a density-dependent switch that induces protein expression only after bacteria have colonized tumor tissue. This technique prevents system toxicity after delivery of therapeutic molecules. We have also developed bacteria that selectively invade cancer cells and release molecules that modulate epigenetic targets, e.g., EZH2 and PP1. Salmonella were engineered with a genetic cassette that induces lysis specifically after invasion into mammalian cells. Lysis releases the bacterial content into the cytoplasm of cells. In culture, Salmonella lyse inside cancer cells and release GFP, which diffuses throughout the cellular cytoplasm. Releasing a peptide that interferes with the binding of the phosphatase, PP1, with its regulator, NIPP1, induces cell death after bacterial lysis. In three-dimensional tissue culture, administration of these bacteria induces cell death. These techniques establish Salmonella as a tunable platform for cancer therapy. By understanding the mechanisms of bacterial delivery, new strategies will be developed to treat hard-to-treat cancers.

Citation Format: Neil S. Forbes. Engineered Salmonella for drug delivery to solid tumors [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr IA24.
Abstract IA01: Single cell analysis of CRISPR T cells
Authors: Stadtmauer Edward , Fraietta Joseph , Satpathy Ansuman , June Carl H.
Published: December 2020

CRISPR-Cas9 gene editing provides a powerful tool to enhance the natural ability of human T cells to fight cancer. We report a first-in-human phase 1 clinical trial to test the safety and feasibility of multiplex CRISPR-Cas9 editing to engineer T cells in three patients with refractory cancer. Two genes encoding the endogenous T cell receptor (TCR) chains, TCRα (TRAC) and TCRβ (TRBC), were deleted in T cells to reduce TCR mispairing and to enhance the expression of a synthetic, cancer-specific TCR transgene (NY-ESO-1). Removal of a third gene encoding programmed cell death protein 1 (PD-1; PDCD1), was performed to improve antitumor immunity. Adoptive transfer of engineered T cells into patients resulted in durable engraftment with edits at all three genomic loci. Although chromosomal translocations were detected, the frequency decreased over time. Modified T cells persisted for up to 9 months, suggesting that immunogenicity is minimal under these conditions and demonstrating the feasibility of CRISPR gene editing for cancer immunotherapy.

Citation Format: Edward Stadtmauer, Joseph Fraietta, Ansuman Satpathy, Carl H. June. Single cell analysis of CRISPR T cells [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr IA01.
Abstract IA02: Slide-seq: A platform for understanding cellular circuits in tissue
Authors: Chen Fei
Published: December 2020

The precise spatial localization of molecular signals within tissues richly informs the mechanisms of tissue formation and function. Here we introduce Slide-seq, a technology which enables transcriptome-wide measurements with near-single cell spatial resolution. We’ll describe recent experimental and computational advances to enable Slide-seq in biological contexts in biological contexts where high detection sensitivity is important. More broadly, we’ll discuss the promise and challenges of spatial transcriptomics for tissue genomics.

Citation Format: Fei Chen. Slide-seq: A platform for understanding cellular circuits in tissue [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr IA02.
Abstract IA08: Tracking the impact of tumor mutations in the T cell differentiation landscape of lung cancer
Authors: Quezada Sergio A.
Published: December 2020

Tumour mutational burden (TMB) predicts immunotherapy outcome in non-small cell lung cancer (NSCLC), consistent with immune recognition of tumour neoantigens. However, persistent antigen exposure is detrimental for T cell function. How TMB affects CD4 and CD8 T cell differentiation in untreated tumours and whether this affects patient outcomes is unknown. Here, we paired high-dimensional flow cytometry, exome, single-cell and bulk RNA sequencing from patients with resected, untreated NSCLC to examine these relationships. TMB was associated with compartment-wide T cell differentiation skewing, characterized by loss of TCF7-expressing progenitor-like CD4 T cells, and an increased abundance of dysfunctional CD8 and CD4 T cell subsets with strong phenotypic and transcriptional similarity to neoantigen-reactive CD8 T cells. A gene signature of redistribution from progenitor-like to dysfunctional states was associated with poor survival in lung and other cancer cohorts. Single-cell characterization of these populations informs potential strategies for therapeutic manipulation in NSCLC.

Citation Format: Sergio A. Quezada. Tracking the impact of tumor mutations in the T cell differentiation landscape of lung cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr IA08.
Abstract IA16: Cancer evolution, immune evasion and metastasis
Authors: Swanton Charles
Published: December 2020

Increasing evidence supports complex subclonal relationships in solid tumours, manifested as intratumour heterogeneity. Parallel evolution of subclones, with distinct somatic events occurring in the same gene, signal transduction pathway or protein complex, suggests constraints to tumour evolution that might be therapeutically exploitable. Data from TRACERx, a longitudinal lung cancer evolution study will be presented. Drivers of tumour heterogeneity change during the disease course and contribute to the temporally distinct origins of lung cancer driver events. APOBEC driven mutagenesis appears to be enriched in subclones in multiple tumour types. Oncogene, tumour suppressor gene and drug induced DNA replication stress are found to drive APOBEC mutagenesis. Phylogenetic tracking detects minimal residual disease and clonal evolution of disease from primary to metastatic sites, presenting opportunities for drug development. On-going chromosomal instability, manifested as Mirrored Subclonal Allelic Imbalance (MSAI) is found to be a major driver of intratumour heterogeneity across cancer types, contributing to parallel evolution and selection. The finding of subclonal driver events, evidence of ongoing selection within subclones, combined with genome instability driving cell-to-cell variation is likely to limit the efficacy of targeted monotherapies, suggesting the need for new approaches to drug development and clinical trial design and integration of cancer immunotherapeutic approaches. Multiple adaptive mechanisms to neo-antigen evolution have been found in TRACERx that emphasise the role of cancer chromosomal instability driving immune evasion and HLA/MHC loss and loss of clonal neo-antigens as well as epigenetic repression of neo-antigens. The clonal neo-antigenic architecture may act as a tumour vulnerability, targeting multiple clonal neo-antigens present in each tumour to mitigate resistance and treatment failure.

Citation Format: Charles Swanton. Cancer evolution, immune evasion and metastasis [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr IA16.
Abstract IA22: Single cell proteomics to capture human dysplasia and dysfunction
Authors: Bendall Sean
Published: December 2020

Single cell analysis, starting with the earliest low parameter fluorescent cytometry and microscopy experiments, helped define the major cell subsets of human cellular systems as we understand them today. Now, a novel combination of elemental mass spectrometry with single cell analysis (mass cytometry – CyTOF, Science 2011) and imaging (multiplexed ionbeam imaging – MIBI, Nature Med. 2014, Cell 2018) offers routine examination of >50 parameters without fluorescent agents or interference from spectral overlap using heavy metal isotopes as reporters. With these platforms, we have reached new levels of organizational understanding in human pathobiology. Here, we will review the evolution of these technologies and present our latest efforts to create assays with these platforms in order to quantify multiple levels of regulation including metabolism, proteome dynamics, and chromatin content regulating the epigenome. Further to this, we will highlight recent efforts to leverage structural characteristics of leukemia dysplasias to facilitate automated identification and classification from multiplexed single cell datasets (Tsai & Glass et al. Nature Med. 2020). Overall, these new approaches can reveal new layers of human cellular organization and structure that can be exploited to understand dysfunction in general and provide avenues for more automated and robust clinical classification.

Citation Format: Sean Bendall. Single cell proteomics to capture human dysplasia and dysfunction [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr IA22.
Abstract PO-015: Ex-vivo stimulation of chimeric antigen receptor T cell pre-infusion products predicts clinical response in pediatric B cell acute lymphoblastic leukemia
Authors: Woodhouse Steven , Bai Zhiliang , Fan Rong , Camara Pablo G. , Melenhorst J. Joseph
Published: December 2020

CAR T cell therapy has shown remarkable efficacy against refractory hematological malignancies, frequently leading to complete remission. However, some patients do not respond to the therapy, and many patients who do show an initial response eventually relapse. The factors determining these diverse patient responses remain to be understood. We sought to assess whether the intrinsic molecular characteristics of CAR T pre-infusion products could be predictive of clinical outcomes, reasoning that differences in T cell cellular states and subtype proportions determine response. To investigate the heterogeneity and temporal dynamics of CD19-targeted CAR T cells at a molecular level and the relationship to clinical variables in pediatric B cell acute lymphoblastic leukemia, we profiled by single-cell RNA-seq patient-derived pre-infusion CAR T cells before and after stimulation ex-vivo by antigen presenting cells. Computational analysis of data from 12 pediatric patients with chemotherapy-resistant or refractory disease revealed distinct populations of T cells associated with clinical response. We found that CAR T cells from patients with a favorable response had an increased potential to transition to an active, cytokine-expressing state upon stimulation with CD19, and that responding patients were particularly associated with a robust mixed CD4/CD8 Type 1 helper-like response. Conversely, we found that patients who do not did not respond to CAR T therapy had a higher proportion of non-cytokine expressing CD8 cytotoxic T cells upon stimulation. These results demonstrate the potential of single-cell gene expression profiling coupled with computational analysis to uncover the molecular factors determining response to immunotherapy.

Citation Format: Steven Woodhouse, Zhiliang Bai, Rong Fan, Pablo G. Camara, J. Joseph Melenhorst. Ex-vivo stimulation of chimeric antigen receptor T cell pre-infusion products predicts clinical response in pediatric B cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-015.
Abstract PO-026: GLUER: integrative analysis of multi-omics data at single-cell resolution
Authors: Peng Tao , Pourfarhangi Kamyar Esmaeili , Tan Kai
Published: December 2020

Single-cell omics assays such as RNA-Seq, ATAC-Seq and methylome sequencing have been developed to identify cell types and/or states in heterogeneous tissues. However, it is challenging to integrate these different types of single-cell omics data. Moreover, the advent of spatially resolved single-cell imaging data represent another challenge for integrative analysis with omics data. Here, we present a new algorithm, inteGrative anaLysis of mUlti-omics at single-cEll Resolution (GLUER), for integration of single-cell multi-omics data as well as imaging data. We tested GLUER using multiple datasets generated using multiomics data generated on the same single cells, which was taken as the ground truth. Our results demonstrate that GLUER has significantly improved performance in terms of the accuracy of matching cells with different data modalities, which in turn enhances downstream analyses such as clustering and trajectory inference. GLUER provides a principled analytical framework for studying the heterogeneity of cell populations using multi-omics and imaging data.

Citation Format: Tao Peng, Kamyar Esmaeili Pourfarhangi, Kai Tan. GLUER: integrative analysis of multi-omics data at single-cell resolution [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-026.
Abstract PO-050: Peripheral neurons invade oral squamous cell carcinoma microenvironment and drive tumorigenesis
Authors: Atherton Megan A. , Nilsen Marci L. , Scheff Nicole N.
Published: December 2020

The impact of peripheral neurons on carcinogenesis is an understudied process in head and neck cancer. Peripheral nerve innervation is increased in preclinical models of oral cancer; sympathectomy resulted in smaller, less invasive cancers. Local neurotransmitter release from peripheral neurons innervating cancer has been linked to cancer growth and immune suppression. We hypothesize that local neurotransmitter release from trigeminal sensory neurons (TGNs) and sympathetic postganglionic neurons promotes cancer proliferation and suppresses the immune response. We used real-time PCR to determine RNA expression in human oral cancer cell lines and a colorimetric cell proliferation assay to quantify cancer cell growth in response to the sympathetic and sensory neurotransmitters. Oral cancer cell lines, HSC-3 and SCC-4, overexpress the norepinephrine (NE) receptor, adrenergic receptor beta 2 (ADRβ2) and sensory neurotransmitter receptors, receptor activity modifying protein1 (RAMP1) and neurokinin1 receptor (NK1R) compared to the non-tumorigenic cell line, HaCaT. Treatment with sympathetic (NE) and sensory (CGRP, substance p) neurotransmitters for 48 hours significantly increased cell proliferation in HSC-3 (>2-fold) and SCC-4 (>1.5-fold) cell lines, but had no effect on HaCaT cell proliferation. Using optical clearing and immunohistochemistry, we observed increased PGP9.5+ neuronal fiber density in the microenvironment using high-resolution microscopy in a tongue oral cancer xenograft mouse model. In vitro we measured a 44.6 ± 1.14% increase in TGN sprouting after 48-hour exposure to conditioned media from the HSC-3, compared to conditioned media from HaCaT. Lastly, HSC-3 cell culture supernatant injection (50 µl) into the tongue of C57Bl/6 female mice resulted in infiltration of CD45+ immune cells (9.10 ± 1.2 % of total live cells). Temporary nerve block with 1% bupivacaine injection into the lingual nerve prior to a 50 µl injection of HSC-3 supernatant into the tongue resulted in significantly more CD45+ immune cell infiltration (21.66 ± 3.4 %) compared to HSC-3 supernatant without pharmacologic nerve block. These results together suggest neurotransmitter-induced increase in oral cancer cell proliferation and neurotransmitter-induced suppression of the cancer-evoked immune response. Understanding the nerve-cancer interaction might improve strategies to treat head ad neck cancer by targeting peripheral neurons in the cancer microenvironment.

Citation Format: Megan A. Atherton, Marci L. Nilsen, Nicole N. Scheff. Peripheral neurons invade oral squamous cell carcinoma microenvironment and drive tumorigenesis [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-050.