bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3031: Impact of pyruvate kinase isoforms on cysteine metabolism in pancreatic cancer
Authors: Ensink Elliot , Lunt Sophia Y.
Published: June 2022

Pancreatic cancer cells undergo metabolic alterations to survive within a nutrient-depleted tumor microenvironment. Cysteine metabolism in particular is critical for supporting pancreatic cancer cell survival through its role in redox homeostasis. Targeting cysteine metabolism presents an underexplored opportunity for therapeutic exploitation, but much remains unknown about how cysteine metabolism is influenced by microenvironmental conditions and the overall metabolic state of the cell. One critical metabolic shift in pancreatic cancer cells occurs through altered isoform expression of the glycolytic enzyme, pyruvate kinase (PKM). Pancreatic cancer cells preferentially switch from the constitutively active M1 isoform to the allosterically regulated M2 isoform of PKM. Overexpression of PKM2 in pancreatic cancer cells produces a profound reprogramming of many metabolic pathways; however, the precise relationship between PKM isoform expression and cysteine metabolism is not well understood. We hypothesize that PKM isoform selection enables pancreatic cancer cell survival within a low cystine tumor microenvironment by reprogramming metabolic activity to increase cystine import and glutathione production and modulate glucose and glutamine metabolism to resist oxidative stress. We used lentiviral CRISPR-Cas9 vectors to genetically modify two human pancreatic ductal adenocarcinoma cell lines, AsPC-1 and Panc-1, to exclusively express either PKM1 or PKM2. We evaluated the effects of PKM isoform selection on tolerance of low cystine environments by assessing viability under a range of cystine concentrations (0 – 50 μM) reflective of physiologic and cystine starvation conditions. We observe altered viability and proliferation in PKM1 vs. PKM2 expressing cells under normoxic (21%) and hypoxic (1% O2) conditions. Targeted metabolomics using stable isotope labeling tracers further reveal impacts of PKM isoform expression in central carbon metabolism when cystine is limited. Additional experiments to elucidate the precise mechanism by which PKM isoform expression impacts pancreatic cancer cells are currently under investigation. This study provides valuable insight into the complexities of metabolic reprogramming within the tumor microenvironment and enhances our understanding of how cysteine metabolism could be targeted for the treatment of pancreatic cancer.

Citation Format: Elliot Ensink, Sophia Y. Lunt. Impact of pyruvate kinase isoforms on cysteine metabolism in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3031.
Abstract 3054: Pan-cancer characterization of the tumor mycobiome and its clinical effects
Authors: Haziza Lian Narunsky , Sepich-Poore Gregory D. , Livyatan Ilana , Asraf Omer , Martino Cameron , Nejman Deborah , Gavert Nancy , Stajich Jason E. , Amit Guy , González Antonio , Wandro Stephen , Perry Gili , Ariel Ruthie , Meltser Arnon , Shaffer Justin P. , Zhu Qiyun , Balint-Lahat Nora , Barshack Iris , Dadian Maya , Gal-Yam Einav N. , Pate Sandip P. , Bashan Amir , Swafford Austin D. , Pilpel Yitzhak , Knight Rob , Straussman Ravid
Published: June 2022

While the study of the tumor microbiome and its effects on cancer biology has expanded considerably over the last few years, most of this research focused on bacteria and viruses, leaving behind the fungal kingdom. Recently, a few studies have demonstrated that specific fungi may promote tumor progression, stressing the importance of comprehensively studying the tumor mycobiome and its effects. To address this, we have characterized the mycobiome in 1,183 human tumors and their adjacent tissues, originating from eight major solid tumor types. Staining and imaging demonstrated the presence of fungi in both cancer and immune cells, with tumor-type specific distribution patterns. Quantitative PCR of the fungal 5.8s rDNA revealed the presence of fungal DNA in all tumor types. To characterize the tumor mycobiome and address potential contamination during tissue handling and processing, we subjected all samples, as well as 295 negative controls of different types, to sequencing of the ITS2 region that is situated between fungal rRNA genes. We found cancer-type specific mycobial signatures with relatively high similarity between tumors and their adjacent tissues. While the fungal mycobiome had a lower species richness as compared to the bacterial microbiome of the same tumors, fungi showed significant co-occurrences with specific bacteria, suggesting the existence of ecological niches within the tumors. We also found significant correlations with clinical parameters such as patient’s age, tumor stage, progression-free survival, overall survival, and response to immune checkpoint blockade therapy. Characterization of the tumor mycobiome may add a biologically relevant, previously overlooked, component to be considered in the study of cancer, including its effects on tumor initiation, progression, diagnosis, and response to therapy.

Citation Format: Lian Narunsky Haziza, Gregory D. Sepich-Poore, Ilana Livyatan, Omer Asraf, Cameron Martino, Deborah Nejman, Nancy Gavert, Jason E. Stajich, Guy Amit, Antonio González, Stephen Wandro, Gili Perry, Ruthie Ariel, Arnon Meltser, Justin P. Shaffer, Qiyun Zhu, Nora Balint-Lahat, Iris Barshack, Maya Dadian, Einav N. Gal-Yam, Sandip P. Pate, Amir Bashan, Austin D. Swafford, Yitzhak Pilpel, Rob Knight, Ravid Straussman. Pan-cancer characterization of the tumor mycobiome and its clinical effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3054.
Abstract 3060: Changes in mouse gut microbiome upon exposure to N-butyl-N-(4-hydroxybutyl) nitrosamine
Authors: Ginwala Rashida , Bukavina Laura , Franklin Jodie , Sindhani Mohit , Abbosh Philip
Published: June 2022

Background: BBN (N-butyl-N-(4-hydroxybuytl) nitrosamine), a closely related compound to the carcinogens found in tobacco smoke is an attractive and most commonly used agent to model bladder cancer in mice. BBN closely recapitulates the morphological characteristics of human bladder cancer and conceptually simulates tobacco exposure as the leading risk factor for bladder cancer. We employ the BBN model in mice to decipher the microbial changes that occur in the gut of the mice during the chemical carcinogenesis induced by BBN exposure.

Methods: Starting at 8-12 weeks of age, male (N=30) and female (N=21) C57BL/6 mice were administered 0.05% BBN in drinking water for 12 weeks then switched to regular water. Control mice drank regular water. Stool was collected pre-exposure, after 6 and 12 weeks of exposure, and at the time of tumor identification (tumors typically detectable between 14-22 weeks from start of treatment using this model). DNA extracted from stool was subjected to PCR amplification using primers for the V3-V4 regions of the bacterial 16S rRNA.

Results: Gender-based differences in the microbial composition were noted in response to BBN treatment. While gut microbial diversity in the female mice was markedly reduced (Shannon, Simpson, Inverse Simpson, and Richness) upon BBN treatment, that of male mice remained unchanged from baseline. Additionally, there was a distinct clustering of microbes based on sex (p=0.024) but not BBN exposure (p=0.86) as evidenced by principal coordinate analysis. Differences in the microbial composition were seen upon BBN exposure, with BBN mice showing an increase in Bacteroidales (53%), Lactobacillales (17.5%), Coriobacteriales (19.87%) in females and male mice exhibited an increase in Erysipelotrichales (14.76%) at the order level. On the other hand while male mice showed no significant decreases, female mice exhibited reduction in abundance of Clostridiales (log change -7.79, padj <0.004), Veeruccomicrobiales (log change -2.21, padj<0.005), and Bacillales (log -17.60, padj =0.005).

Conclusions: Pronounced gut microbial changes are induced by BBN in the murine model, with microbiome of the female mice exhibiting more significant variations compared to baseline, suggesting gender-based disparities in the effect of BBN exposure.

Citation Format: Rashida Ginwala, Laura Bukavina, Jodie Franklin, Mohit Sindhani, Philip Abbosh. Changes in mouse gut microbiome upon exposure to N-butyl-N-(4-hydroxybutyl) nitrosamine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3060.
Abstract 3062: Modifiable factors and gut microbiome alteration among colorectal cancer patients
Authors: Hoang Tung , Kim Min Jung , Park Ji Won , Jeong Seung-Yong , Lee Jeeyoo , Shin Aesun
Published: June 2022

Background: Previous study have indicated an association of microbial dysbiosis with stages of colorectal cancer (CRC). Meanwhile, higher microbial stochasticity has been introduced as the Anna Karenina principle (AKP) effect, which originates from the opening line of Leo Tolstoy and is translated in the microbiome as “all healthy microbiomes are similar; each dysbiotic microbiome is dysbiotic in its own way”. Here, we aim to elucidate the effect of modifiable factors on microbiome stochasticity related to dysbiosis in CRC.

Methods: Fecal samples from 331 CRC patients, were collected prior to their surgery at Seoul National University Hospital. Bacteria that were enriched in early- and late-stage CRC groups were identified by linear discrimination analysis effect size (LEfSE) method, which was then used to calculate microbial dysbiosis index (MDI). The association of dietary choices with microbiome variability was addressed using the LEfSE analysis. The difference of intra-sample similarity index among lifestyle factors and metabolic diseases was tested to examine the AKP effect. A tree-based analysis of food choices was constructed. We then applied Procrustes framework to analyze the shape of dietary diversity and microbiome.

Results: The LEfSE identified Leuconostoc, Oxalobacter, Acidaminococcus, and Methanobrevibacter were enriched in early-stage CRC, whereas Bacteroides, Fusobacterium, Lachnoanaerobaculum, Clostridium, and Granulicatella were enriched in late-stage CRC patients. The MDI derived from these bacteria had 64.2% predictive ability for CRC stages, with sensitivity and specificity of 61% and 69%, respectively. The AKP effect was found for history of smoking, alcohol consumption, and diabetes, whereas obesity and hypertension showed anti-AKP effect. For dietary choices, the hierarchical tree of foods and nutrients did not shape the microbial between-sample diversity. Additionally, high MDI was related with a high intake of pizza/hamburger, poultry, and light-vegetable combination.

Conclusion: Our study contributed to current evidence of the microbiome structure and dysbiosis at different stages of CRC. Furthermore, history of smoking, alcohol consumption, and diabetes showed AKP effect in CRC patients. Non-significant association between dietary choices and microbiome diversity supported a small variation of microbiome profiles explained by dietary intake at the population level.

Citation Format: Tung Hoang, Min Jung Kim, Ji Won Park, Seung-Yong Jeong, Jeeyoo Lee, Aesun Shin. Modifiable factors and gut microbiome alteration among colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3062.
Abstract 3078: A pipeline for functional precision medicine in bone and soft tissue sarcoma organoids
Authors: Al Shihabi Ahmad , Tebon Peyton J. , Chantharasamee Jomjit , Nguyen Huyen T. , Sartini Sara , Davarifar Ardalan , Tavanaie Nasrin , Nelson Scott D. , Federman Noah , Yanagawa Jane , Soragni Alice
Published: June 2022

Bone and soft tissue sarcomas span a spectrum of over one hundred histologic subtypes. Developing clinical trials and identifying effective therapies remains a challenge given the rarity of these tumors and scarcity of available tumor models. Patient-derived tumor organoids (PDTOs) are representative of the native physiology of tumors across an array of malignancies (Phan et al, 2019; Al Shihabi et al, 2021). For rare tumors such as bone and soft tissue sarcomas, PDTOs could constitute an important tool to better understand the biology of the disease and identify treatment options. Here, we present the pipeline we have established at UCLA to systematically procure tissue, develop and characterize individualized sarcoma PDTOs, and identify tumor sensitivities using high-throughput drug screening. We have collected tumor specimens from 114 patients diagnosed with sarcomas, representing more than 30 distinct subtypes of soft tissue and bone tumors. We have established suitable conditions to generate organoids from over 100 samples collected from needle biopsies, tumor resections, and metastasectomies, including soft tissue sarcomas (liposarcoma, undifferentiated pleomorphic sarcoma, leiomyosarcoma, etc.) and bone sarcomas (chondrosarcoma, osteosarcoma, Ewing sarcoma, etc.). We take advantage of our mini-ring technology to generate PDTOs in a format compatible with high-throughput drug screening (Phan et al, 2019; Nguyen et al, 2020). Our results highlight tumor-specific drug susceptibilities to chemotherapeutics, targeted agents, and combination therapies, generating results within a week from surgery. We will show how several of the organoid response patterns we identified transcend cancer subtype and patient characteristics. Our findings support the feasibility of rapidly generating individualized organoid models from bone and soft tissue sarcomas to facilitate biological interrogations and investigate response to treatment. Direct testing of PDTOs derived from clinical specimens can provide timely, actionable information for functional precision medicine approaches with the potential to improve patient outcomes.

Citation Format: Ahmad Al Shihabi, Peyton J. Tebon, Jomjit Chantharasamee, Huyen T. Nguyen, Sara Sartini, Ardalan Davarifar, Nasrin Tavanaie, Scott D. Nelson, Noah Federman, Jane Yanagawa, Alice Soragni. A pipeline for functional precision medicine in bone and soft tissue sarcoma organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3078.
Abstract 3114: PDX tumor growth comparisons in the ultra-immunodeficient NOD.CB17-PrkdcscidIL2rgtm1/Bcgen (B-NDG) mouse model
Authors: Melton Michele , Jiang Hongmei , Hyddmark Emma M. , Rothrock Travis
Published: June 2022

The purpose of the study was to compare the growth of various PDX models in the two ultraimmunodeficient mouse models NSG™ (Jackson Laboratory) and B-NDG (Inotiv). Four breast cancer PDX models from the Washington University Human in Mouse (WHIM) collection, ER+ WHIM20 and WHIM81, Her2+ WHIM43, and triple negative WHIM5, and one melanoma PDX models generated by the Wistar institute, WM4071-2, was utilized. For models WHIM20, WHIM5, and WM4071-2 all mice were injected with tumor cells on the same day, whereas for WHIM43 BND-G mice were injected in two separate batches. The WHIM81 studies were carried out at different times for the NSG™ and B-NDG animal cohorts. Studies utilizing WHIM5, WHIM20, WHIM43 and WM4071-2, included a group of athymic nude mice as well. For all models, mice were injected with 1.5x106 cells/mouse. The cells were mixed 1:1 with PBS:Corning Matrigel GFR and the injection volume was 100 µL/mouse. Mice were housed in Innovive individually vented cages and consumed Teklad Global Rodent Diet 2019 throughout the study. Tumors were measured multiple times per week using Biopticon’s TumorImager™. For all models except WHIM81, the tumors grew well and with very similar rate in both B-NDG and NSG™ mice. WHIM20 and WHIM43 grew significantly slower in the Athymic nude mice compared to both of the ultraimmunodeficient mouse models, however, the growth of WHIM5 was comparable between all three models. The results from the WHIM81 model suggests faster growth in the B-NDG model compared to the NSG™, however, it is also possible that this is due to the different batches of tumor cells utilized at each implantation event. Thus, the B-NDG model represents a novel ultraimmunodeficient mouse model that can have great utility for tumor studies, particularly for hard to grow or slower growing tumor models.

Citation Format: Michele Melton, Hongmei Jiang, Emma M. Hyddmark, Travis Rothrock. PDX tumor growth comparisons in the ultra-immunodeficient NOD.CB17-PrkdcscidIL2rgtm1/Bcgen (B-NDG) mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3114.
Abstract 3205: Role of RIG-I in tumor endothelium
Authors: Baris Adrian M. , Fraile-Bethencourt Eugenia , Khou Sokchea , Ruhl Rebecca , Hudson Clay , Anand Sudarshan
Published: June 2022

The immune microenvironment plays a critical role in the efficacy of cancer treatment. The manipulation of cytosolic nucleic acid sensors has become a strategy to boost the immune response in cancer therapy, but it is critical to understand how these nucleic acid sensors work in the tumor microenvironment. One aspect of the immune microenvironment is the nucleic acid sensor, which recognizes pathogenic nucleic acids and activates an immune response. Retinoic acid-inducible gene I (RIG-I) is an RNA sensor that responds to dsRNA by activating a type I interferon response. It has been found that RIG-I activation can induce an anti-tumor immune response, but its role in the tumor microenvironment is unknown. Prior studies show regulation of the innate immune response in endothelium can increase the anti-tumor response. Understanding the role of RIG-I in endothelial cells, and how this can be used to potentially reprogram the tumor microenvironment, is critical for developing more effective cancer treatments. In addition, my research on the effects of RIG-I in endothelium has wide-reaching implications beyond cancer treatment. I hypothesize RIG-I activation in endothelial cells will produce an anti-tumor immune response. My findings show that endothelial RIG-I activates a robust type I interferon response and inhibits EC function including, inducing apoptosis and inhibiting migration, which is indicative that RIG-I plays a role in the endothelial immune response. RIG-I also plays a role in the expression of the several angiogenic regulators in a tissue-specific manner. This research will provide critical knowledge about the role of RIG-I in tumor angiogenesis, and provide novel treatment avenues for solid tumors.

Citation Format: Adrian M. Baris, Eugenia Fraile-Bethencourt, Sokchea Khou, Rebecca Ruhl, Clay Hudson, Sudarshan Anand. Role of RIG-I in tumor endothelium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3205.
Abstract 3238: GBP3 promotes glioblastoma resistance to temozolomide by enhancing DNA damage repair
Authors: Li Ming , Xu Hui
Published: June 2022

Glioblastoma is the most common malignant brain cancer with dismal survival and prognosis. Temozolomide (TMZ) is a first-line chemotherapeutic agent for glioblastoma, but the emergence of drug resistance limits its anti-tumor activity. We previously discovered that the interferon inducible guanylate binding protein 3 (GBP3) is highly elevated and promotes tumorigenicity of glioblastoma. Here, we show that TMZ treatment significantly upregulates the expression of GBP3 and stimulator of interferon genes (STING), both of which increase TMZ-induced DNA damage repair and reduce cell apoptosis of glioblastoma cells. Mechanistically, relying on its N-terminal GTPase domain, GBP3 physically interacts with STING to stabilize STING protein levels, which in turn induces expression of p62 (Sequestosome 1), nuclear factor erythroid 2 like 2 (NFE2L2, NRF2), and O6-methlyguanine-DNA-methyltransferase (MGMT), leading to the resistance to TMZ treatment. Reducing GBP3 levels by RNA interference in glioblastoma cells markedly increases the sensitivity to TMZ treatment in vitro and in murine glioblastoma models. Clinically, GBP3 expression is high and positively correlated with STING, NRF2, p62, and MGMT expression in human glioblastoma tumors, and is associated with poor outcomes. These findings provide novel insight into TMZ resistance and suggest that GBP3 may represent a novel potential target for the treatment of glioblastoma.

Citation Format: Ming Li, Hui Xu. GBP3 promotes glioblastoma resistance to temozolomide by enhancing DNA damage repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3238.
Abstract 3350: TSEEN genes database
Authors: Matyunina Ekaterina , Makashov Andrei , Kozlov Andrei P.
Published: June 2022

According to the hypothesis of the evolutionary role of tumors, evolutionarily new genes should be specifically expressed in tumors [A.P. Kozlov, 2014]. Indeed, TSEEN (tumor specific evolutionarily novel) genes were described by us [Kozlov, 2016]. 1015 genes were identified by the Protein Historian program as evolutionary novel for humans, that is, originated in humans, and 1392 as evolutionarily young, that is, originated in primates. As a result of analyzing the GTEx database data, we have shown that out of 1015 evolutionarily new human genes and 1392 evolutionarily young human genes, 309 genes and 592 genes are not expressed in any of 33 normal tissues, respectively. The expression level of 101 evolutionarily new human genes originated in humans and 233 evolutionarily young human genes originated in primates is significant in most tumors, according to transcriptome data from the TCGA database. Samples of 101 evolutionarily novel human genes and 233 evolutionarily young human genes were selected, which are present only in the genomes of humans and primates, not expressed in normal tissues, and expressed in one or more tumors in 10-100% of patients.For Theria and Eutheria, the similar distribution was as follows: among 365 and 896 genes novel for these taxons, 71 and 124 genes, respectively, are not expressed in normal human tissues, respectively, but at the same time have tumor-specific expression pattern (inclusion criteria of expression - fpkm >2 in 10-100% of patients).There are also 898 genes, originated in mammalia and not expressed in normal human tissues. 119 of them are expressed in at least 1 or more tumors in 10-100% of patients.The TSEEN genes database allows [us] to select genes according to their evolutionary age and expression pattern in normal and tumor tissues. We can retrieve gene(s), which are expressed tumor-specifically and are evolutionarily young or novel.As far as TSEEN genes have many immunogenic epitopes they could be promising targets for development of universal prophylactic and therapeutic antitumor vaccines, as well as universal markers for cancer laboratory diagnostics.Created TSEEN genes database is available at: https://tseendb.org/#/

Citation Format: Ekaterina Matyunina, Andrei Makashov, Andrei P. Kozlov. TSEEN genes database [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3350.
Abstract 3351: Identification of a NOTCH transcriptomic signature for basal-like breast cancer
Authors: Braune Eike-Benjamin , Geist Felix , Wienke Dirk , Seshire Anita , Lendahl Urban
Published: June 2022

Breast cancer is a heterogeneous disease, where different subtypes differ in terms of biology, treatment options and prognosis. It is an emerging view that dysregulation of major signaling pathways, including Notch signaling, contributes to breast cancer development and progression, but precisely how and in which subtypes Notch signaling is hyperactivated still largely remains to be determined. To gain further insights into dysregulation of Notch in cancer, we set out to identify and evaluate a novel Notch transcriptomic signature focusing initially on breast cancer. We first established Notch-regulated transcriptomes from six breast cancer cell of the basal subtype in response to activation of Notch (by culturing on immobilized Notch ligand) or blockade of Notch (by γ-secretase inhibitors) for 8 and 72 hours. Our data reveal that a core set of Notch-responsive genes can be identified from both time-points and we are validating the transcriptomic signature on a broader set of additional 19 breast cancer cell lines from various breast cancer subtypes. The Notch transcriptomic signature showed robust coherence scores in breast cancer patient data, and robust correlation with basal-like transcriptomes was demonstrated. Analysis of single cell transcriptomic data from breast cancer patients demonstrated that the Notch signature showed additionally good correlation with immune cells in the tumor stroma, notably tumor associated macrophages, indicating that Notch signaling is upregulated in specific cell types of the tumor stroma. Collectively, the data provide a robust transcriptional signature to read out activation of the Notch signaling pathway in basal-like breast cancer and the signature will be important to better stratify patients and to evaluate and predict outcome of future Notch-based therapies for breast cancer.

Citation Format: Eike-Benjamin Braune, Felix Geist, Dirk Wienke, Anita Seshire, Urban Lendahl. Identification of a NOTCH transcriptomic signature for basal-like breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3351.
Abstract 3364: Diagnosing Li-Fraumeni syndrome from the somatic genome
Authors: Laverty Brianne , Subasri Vallijah , Light Nicholas , Malkin David
Published: June 2022

Background: Li-Fraumeni syndrome (LFS) is a hereditary cancer predisposition syndrome caused by germline mutations in the tumor suppressor gene TP53. LFS is estimated to occur in 1:1000 - 1:5000 people and is associated with a 80% lifetime cancer risk. This syndrome is diagnosed using familial cancer history and germline TP53 sequencing; however, clinical heterogeneity and variants of unknown significance limit diagnostic precision. Accurate diagnosis is imperative to implement surveillance for secondary malignancies and familial genetic testing.

Methods: We hypothesized that LFS cancers evolve uniquely from sporadic cancers, implying that the somatic genomes of LFS patients exhibit distinct characteristics that can infer the predisposition syndrome. To investigate this, we interrogated mutational signatures, TP53 copy number, TP53 loss of heterozygosity, ploidy, and the incidence of chromothripsis in tumor compared to normal samples (blood or fibroblast) of individuals with germline TP53 mutations (n=27), somatic TP53 mutations (n=17) and WT for TP53 (n=158). We created a random forest model with 10-fold cross validation to determine if somatic features could diagnose LFS.

Results: No signatures were significantly associated with LFS (Wilcoxon rank-sum test, Benjamin-Hochberg FDR correction). As previously reported in the literature, LFS compared to non-LFS cancers in our cohort were more likely to: be hyperdiploid (odds ratio (OR) = 11.83, FDR < 0.0001, Fisher exact test), have undergone TP53 loss of heterozygosity (OR = 23.15, FDR < 0.0001, Fisher exact test) and experience chromothripsis (OR = 7.76, FDR < 0.001, Fisher exact test).

The area under the receiver operating curve (AUROC) for our random forest model with 10-fold cross validation was 0.90, the area under the precision recall curve (AUPRC) was 0.59, the positive predictive value (PPV) was 0.70, the negative predictive value (NPV) was 0.93 and the F1-score was 0.52. This implies that the somatic genomic features are reliable indicators of this germline syndrome. We have obtained access to a future 50 LFS samples from the Pediatric Cancer Genome Project dataset, which we hope will improve our model’s performance.

Conclusion: We have developed a machine learning tool that uses somatic features to identify LFS, a germline cancer predisposition syndrome. As the importance of precision oncology becomes apparent, a tool to identify LFS patients from the somatic genome will facilitate early diagnosis. This will allow individuals to enter a surveillance program for early detection of secondary tumors, leading to improved outcomes.

Citation Format: Brianne Laverty, Vallijah Subasri, Nicholas Light, David Malkin. Diagnosing Li-Fraumeni syndrome from the somatic genome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3364.
Abstract 3384: Combined DNA methylation analysis in size based CTC fraction and matched plasma-cfDNA provides prognostic information in early stage NSCLC
Authors: Markou Athina N. , Londra Dora , Tserpeli Victoria , Kollias John , Tsaroucha Emilia , Pateras Ioannis , Potaris K , Vamvakaris Ioannis , Kotsakis Athanasios , Georgoulias Vasilis , Lianidou Evi
Published: June 2022

Background: Circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) analysis represents a liquid biopsy approach for real-time monitoring of tumor evolution. DNA methylation is considered to be an early event in the process of cancer development and progression. The aim of the present study was to evaluate whether detection of DNA methylation of selected tumour suppressor genes in size-based CTC fraction and matched ctDNA could provide prognostic information in early stage NSCLC.

Methods: The methylation status of five selected gene promoters (APC, RASSFIA1, FOXA1, SLFN11, SHOX2) was examined by highly specific and sensitive real time methylation specific PCR assays in: a) a training group of 35 primary tumours and their corresponding adjacent non-cancerous tissues of early stage NSCLC patients, b) a validation group of 22 primary FFPEs tumor tissues and 42 peripheral blood samples of early stage NSCLC patients that gDNA was isolated from FFPEs, sized-based CTCs and plasma, and c) a control group of healthy blood donors (n=12).

Results: All five gene promoters tested were highly methylated in the training group of primary tumours; methylation of SHOX2 promoter in the primary tissues was associated with unfavorable outcome. RASSFIA and APC were found methylated in plasma-cfDNA samples at 14.3% and 11.9% respectively, whereas in the corresponding size-based CTC-enriched fractions SLFN11 and APC promoters were methylated in 7.1%. The incidence of relapse was higher in patients with i) promoter methylation of APC and SLFN11 in plasma-cfDNA (P=0.037 and P=0.042 respectively) and ii) at least one gene promoter was methylated in the sized-based CTC fraction or plasma-cfDNA.

Conclusions: Although in liquid biopsy components methylation of these gene promoters was significantly lower than in the primary tumors, the combination of DNA methylation analysis in sized-based CTC fraction and plasma-cfDNA revealed clinical relevance. Additional studies are required to validate our findings in a large cohort of early stage NSCLC patients.

Citation Format: Athina N. Markou, Dora Londra, Victoria Tserpeli, John Kollias, Emilia Tsaroucha, Ioannis Pateras, K Potaris, Ioannis Vamvakaris, Athanasios Kotsakis, Vasilis Georgoulias, Evi Lianidou. Combined DNA methylation analysis in size based CTC fraction and matched plasma-cfDNA provides prognostic information in early stage NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3384.
Abstract 3392: Rapid, scalable isolation of human tumor nuclei for single cell genomics
Authors: Gibbons Michael , Puleo Alaina , Herschleb Jill , Taylor Sarah
Published: June 2022

Sample preparation remains a significant roadblock to the implementation and scaling of single cell technologies in clinical and translational research. Time consuming, custom dissociation protocols performed on fresh tissue by experts are often utilized to obtain high quality single cell data. However, these complex protocols result in low sample throughput and high variability driven by logistical challenges and user variability. Additionally, many samples that are of interest for clinical and translational studies arrive in research labs snap frozen, leaving nuclei extraction as the only viable path to generate single cell/nuclei data. To overcome these challenges, we present a scalable, easy to use, spin column-based nuclei isolation platform that generates single nuclei that are ready for input into downstream 10x Genomics single cell assays in under an hour.

We processed eight unique human tumors in parallel (skin melanoma, breast, ovary, prostate, lung, kidney, colorectal, and pancreas). Starting from snap frozen tissue, we were able to generate single nuclei suspensions for all samples, in parallel, within 90 minutes. The resulting single nuclei were run through the 10x Genomics Single Cell Gene Expression, Single Cell Immune Profiling, and Single Cell ATAC workflows.

Single cell gene expression data retained high data complexity across all tumor types tested. Expected cell type clusters, including tissue-specific tumor cells, surrounding/infiltrating immune cells and stromal cells were easily identified and profiled via gene expression analysis. Even fragile cell types often missing in single-cell data (e.g. mature adipocytes) were preserved and profiled in this experiment. Additionally, epigenetic changes as measured by Single Cell ATAC profiling were consistent with both general and tissue-specific epigenetics of each cancer type analyzed.

In summary, this platform streamlines sample preparation for single cell studies from snap frozen tissue, enabling these samples to be used much more routinely and unlock biobanked sample cohorts. The findings presented here demonstrate that this fast and easy to use nuclei isolation system provides a consistent platform for generating high quality single nuclei data at scale for clinical, translational and basic research studies alike.

Citation Format: Michael Gibbons, Alaina Puleo, Jill Herschleb, Sarah Taylor. Rapid, scalable isolation of human tumor nuclei for single cell genomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3392.
Abstract 3408: Longitudinal measurement of HPV copy number in cell free DNA predicts progression free survival in HPV positive oropharyngeal cancer
Authors: Adamowicz Martyna Joanna , Warlow Sophie J. , Thomson John P. , Carey Lara M. , Thain Helen , Wescott Robert , Cuschieri Kate , Li Lucy Q. , Conn Brendan , Hay Ashley , Nixon Iain J. , Aitman Timothy J.
Published: June 2022

Oropharyngeal squamous cell carcinoma (OPSCC) is increasing in global prevalence and is divided into two types dependent on association with human papillomavirus (HPV), with a more favorable prognosis in HPV+ve tumors. Assay of HPV copy number in plasma cell-free DNA (cfDNA) provides a minimally invasive method for detecting and monitoring tumor-derived HPV, with potential for enhancing clinical care. We have evaluated the utility of cfDNA droplet digital PCR (ddPCR) as a method for characterisation and longitudinal monitoring of patients with OPSCC in a prospectively recruited cohort of 104 OPSCC patients. ddPCR assay of cfDNA for five HPV types showed overall 95% concordance with p16 immunohistochemistry (IHC) and PCR analysis of solid tumor tissue. Longitudinal sampling in 48 HPV+ve patients, with median follow-up of 20 months, strongly predicted patient outcomes. Progression-free survival, stratified respectively by the presence or absence of detectable HPV cfDNA at a median of 13 weeks post-treatment, was 50% and 88% (p=0.001, hazard ratio 10.0 (95% CI 2.1-47.1)). In two patients, greater reliance on sequential HPV measurement would have avoided surgical intervention which ultimately did not confirm disease recurrence. The high concordance of pre-treatment plasma cfDNA-HPV analysis with p16 IHC and HPV-PCR of solid tissue, together with the predictive value and utility of sequentially measured post-treatment cfDNA-HPV copy number, provide a compelling case for the routine use of cfDNA-HPV ddPCR in management of OPSCC and for clinical trials to assess its impact on treatment outcomes.

Citation Format: Martyna Joanna Adamowicz, Sophie J. Warlow, John P. Thomson, Lara M. Carey, Helen Thain, Robert Wescott, Kate Cuschieri, Lucy Q. Li, Brendan Conn, Ashley Hay, Iain J. Nixon, Timothy J. Aitman. Longitudinal measurement of HPV copy number in cell free DNA predicts progression free survival in HPV positive oropharyngeal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3408.
Abstract 3430: CD47 x ICAM-1 bispecific antibody represents a novel approach for treating ICAM-1 overexpressing tumors
Authors: Wang Xinhua , Wong Oi Kwan , Post Leonard , Chen Xiaocheng
Published: June 2022

CD47/SIRPa axis is an important checkpoint of innate immune system, CD47 interacts with its ligand signal regulatory protein-alpha (SIRPa) on myeloid cells, conveys a “don’t eat me” signal and blocks macrophage mediated phagocytosis. Tumor cells, which express high level of CD47, exploit this mechanism to evade from immune surveillance. CD47 is considered a prominent target for cancer treatment. However, the wide expression of CD47 on normal cells could cause antigen sink and lead to safety issues, such as anemia and thrombocytopenia. ICAM-1 is constitutively present at low levels on endothelial cells, but its overexpression has been observed on various types of tumors, such as primary myeloma, lymphoma and kidney cancer. Here we describe a novel CD47 x ICAM-1 bispecific antibody, which specifically targets CD47+/ICAM-1+ tumors in preclinical studies.A novel anti-CD47 antibody and an anti-ICAM-1 antibody were used for the generation of a CD47 x ICAM-1 bispecific antibody. In vitro assays including FACS binding, FACS-based SIRPa blocking, ADCP, RBC binding and hemagglutination were performed to characterize the bispecific CD47 x ICAM-1 antibody. In vivo efficacies of CD47 x ICAM-1 bispecific antibody were evaluated in xenograft tumor models with high ICAM-1 expression.The CD47 x ICAM-1 bispecific antibody selectively exhibited potent SIRPa blocking and antibody-dependent cellular phagocytosis (ADCP) activity on CD47+/ICAM-1+ tumor cells, but not on cells lacking ICAM-1 expression. Minimal RBC binding and RBC phagocytosis were observed upon treatment with CD47 x ICAM-1 bispecific antibody. The bispecific antibody did not cause any appreciable hemagglutination with up to 1µM antibody treatment. Most importantly, the CD47 x ICAM-1 bispecific antibody demonstrated potent anti-tumor activities in in vivo CDX and PDX models that overexpress both CD47 and ICAM-1. Our findings suggest that the novel CD47 x ICAM-1 bispecific antibody selectively binds to CD47 and blocks CD47/SIRPa binding on ICAM1 overexpressing tumor cells. The bispecific antibody has minimal RBC binding compared to the bivalent CD47 monoclonal antibodies and benchmark anti-CD47 antibodies. The bispecific antibody shows potent in vivo efficacy and specificity toward ICAM-1+/CD47+ positive tumor cells and represents a novel approach for treating ICAM-1+/CD47+ tumors.

Citation Format: Xinhua Wang, Oi Kwan Wong, Leonard Post, Xiaocheng Chen. CD47 x ICAM-1 bispecific antibody represents a novel approach for treating ICAM-1 overexpressing tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3430.
Abstract 3452: Morphological phenotyping of organotropic brain- and bone-seeking triple negative metastatic breast tumor cells
Authors: DeCastro Ariana Joy L. , Pranda Marina , Gray Kelsey , Merlo-Coyne John , Girma Nathaniel , Hurwitz Madelyn , Zhang Yuji , Stroka Kimberly
Published: June 2022

Triple negative breast cancer (TNBC) follows a non-random pattern of metastasis to the bone and brain tissue. Prior work has found that brain-seeking breast tumor cells display altered proteomic profiles, leading to alterations in pathways related to cell signaling, cell cycle, metabolism, and extracellular matrix remodeling. Given the unique microenvironmental characteristics of brain and bone tissue, we hypothesized that brain- or bone-seeking TNBC cells may have altered morphologic or migratory phenotypes from each other, or from the parental TNBC cells, as a function of the biochemical or mechanical microenvironment. In this study, we utilized TNBC cells (MDA-MB-231) that were conditioned to metastasize solely to brain (MDA-BR) or bone (MDA-BO) tissue. We quantified characteristics such as cell morphology, migration, and stiffness in response to cues that partially mimic their final metastatic niche. We have shown that MDA-BO cells have a distinct protrusive morphology not found in MDA-P or MDA-BR. Further, MDA-BO cells migrate over a larger area when on a collagen I (abundant in bone tissue) substrate when compared to fibronectin (abundant in brain tissue). However, migration in highly confined environments was similar across the cell types. Modest differences were found in the stiffness of MDA-BR and MDA-BO cells plated on collagen I vs. fibronectin-coated surfaces. Lastly, MDA-BO cells were found to have larger focal adhesion area and density in comparison with the other two cell types. These results initiate a quantitative profile of mechanobiological phenotypes in TNBC, with future impacts in helping to predict metastatic propensities to organ-specific sites in a clinical setting.

Citation Format: Ariana Joy L. DeCastro, Marina Pranda, Kelsey Gray, John Merlo-Coyne, Nathaniel Girma, Madelyn Hurwitz, Yuji Zhang, Kimberly Stroka. Morphological phenotyping of organotropic brain- and bone-seeking triple negative metastatic breast tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3452.
Abstract 3496: Transcriptomic classification of lung adenocarcinoma identifies a novel subgroup with a poor survival prognosis
Authors: Satter Khaled Bin , Tran Paul MH , Tran Lynn KH , Nechtman John , Patel Nikhil , Santos Bruno , Hopkins Diane , Karim Nagla Abdel , She Jin-Xiong , Purohit Sharad B.
Published: June 2022

Lung adenocarcinoma is one-half of all lung cancer and the third deadliest cancer in the world. The traditional classification of lung adenocarcinoma involves invasiveness, growth pattern, presence of mucin, and other histological features. However, this classification provides limited prognostic predictability. Molecular profiles based The Cancer genome Atlas lung adenocarcinoma cohort classified the tumors into the terminal respiratory unit (TRU), proximal proliferative (PP), and proximal infiltrative (PI) based on unsupervised classification, which did not address clinical applicability to the classification. Here, we developed an unsupervised classification with Density-based UMAP (DBU), which identified four transcriptomic profiles for lung adenocarcinoma (Transcriptomic profile, TP1-TP4). We identified a gene signature with 299 genes to develop a supervised classifier, called Ensemble Transcriptomic classifier (ETC), to identify these four subgroups within the total cohort. All TP1 is PI. TP2 is 49.0% TFU and 45.0% PP. TP3 is 76.2% PI and 23.8% PP. TP4 is 54.5% TFU and 45.5% PI. TP1 is characterized by neuroendocrine-like features and clusters with large cell neuroendocrine tumors in cluster analysis. TP3 is squamous-like enriched with cell cycle and DNA repair high and worse prognosis and groups with squamous cell carcinoma in cluster analysis. TP2 has high immune cell infiltration, and TP4 showed low mutational burden and low immune cell infiltration. We validated these subgroups identified in the TCGA cohort in a microarray meta-dataset with DBU, ETC. This classification showed four subgroups in lung adenocarcinoma, which shows potential clinical actionable findings.

Citation Format: Khaled Bin Satter, Paul MH Tran, Lynn KH Tran, John Nechtman, Nikhil Patel, Bruno Santos, Diane Hopkins, Nagla Abdel Karim, Jin-Xiong She, Sharad B. Purohit. Transcriptomic classification of lung adenocarcinoma identifies a novel subgroup with a poor survival prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3496.
Abstract 363: Intracellular redox milieu regulates Mcl-1 and decreases overall mitochondrial priming in hematopoietic cancers
Authors: Lai Jolin Xiao , Chong Stephen Jun , Davids Matthew , Pervaiz Shazib
Published: June 2022

Background: The dichotomy of redox signaling in cancer cell fate decisions has emerged as a topic of importance over the years. In particular, our group has highlighted the role of superoxide (O2 .-)-mediated signaling in the acquisition of drug resistance and cancer survival. Mechanistically, this involves post-translational modification of proteins involved in oncogenic signaling, such as c-Myc and NF-κB, as well as apoptosis resistance like Bcl-2 at the serine 70 phosphorylation site. Here we present evidence that O2 .--mediated signaling stabilizes anti-apoptotic protein Mcl-1, a commonly amplified gene in human cancers and is implicated in resistance to venetoclax, the Bcl-2 specific BH3 mimetic.

Methods: Intracellular O2 .- levels was increased either pharmacologically or by siRNA mediated knockdown of SOD1 in MOLM14, RPMI8226, OCI-AML3 hematopoietic cell lines. Cell viability and apoptosis were assessed by MTT and Annexin V/DAPI staining. Protein expression was determined by Western blot analysis. BH3 profiling of patient-derived CLL cells was used to assess mitochondrial priming.

Results: Elevated O2 .- levels increased Mcl-1 stability through Thr163 phosphorylation of Mcl-1, which in turn reduced ubiquitination-dependent degradation. Furthemore, venetoclax resistant cells possessed higher O2 .- levels and increase in intracellular O2 .- levels promoted resistance to Mcl-1 inhibitor, S63845 through increased binding between Mcl-1 and pro-apoptotic proteins NOXA and Bak. Contrarily, scavenging of O2 .- re-sensitized venetoclax-resistant leukemia cells to venetoclax. Importantly, BH3 profiling revealed that increase in intracellular O2 .- levels decreased overall mitochondrial priming in primary CLL cells by promoting sequestration of pro-apoptotic proteins that specifically target Mcl-1.

Conclusion: Oncogenic activity of O2 .- is mediated by increased Mcl-1 stability through Thr163 phosphorylation, which resulted in a reduction in overall mitochondrial priming and increased binding of Mcl-1 to NOXA and Bak. Scavenging O2 .- reduces Mcl-1 levels and sensitizes resistant cells to venetoclax-induced apoptosis thereby demonstrating that manipulation of the redox microenvironment could be a potential vulnerability to be exploited in venetoclax resistant cancers

Citation Format: Jolin Xiao Lai, Stephen Jun Chong, Matthew Davids, Shazib Pervaiz. Intracellular redox milieu regulates Mcl-1 and decreases overall mitochondrial priming in hematopoietic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 363.