bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract AP26: CANCER STEM CELLS: DISTINCT SEEDS FOR RECURRENT OVARIAN CANCER
Authors: Ahmed Nuzhat , Escalona Ruth , Kadife Elif , Leung Dilys , Kannourakis George
Published: June 2021

PURPOSE: The treatment of ovarian cancer (OC) with chemotherapy leaves resistant cancer cells which in a short time re-grow as recurrent cancer. A diverse array of resistance mechanisms for chemotherapy has been described but none have proven as viable targets in a clinical setting. Cancer stem cells (CSCs) are increasingly accepted as the putative mediators of chemoresistance and relapse of cancer. This study aimed to understand the molecular mechanisms involved with chemoresistance and recurrence by investigating the proteomic profile of ascites-derived tumor cells obtained from OC patients prior to (chemonaive, CN) and after chemotherapy treatments (recurrent, CR).

METHODS: Ascites collected from CN and CR OC patients diagnosed with advanced-stage serous OC were cultured using a novel in vitro method to obtain a distinct population of epithelial tumor cells. Flow cytometry and immunofluorescence were used to characterize the tumor population. High-resolution label-free quantitative proteomic profiling was used to define significantly differentially expressed proteins between CN and CR tumor cells. KEGG and DAVID software's were used to determine pathways associated with CR cells. The mechanisms of survival of cisplatin and paclitaxel-treated OC cancer cells were determined in vitro by mRNA analysis and in vivo mouse xenograft models by immunohistochemistry.

RESULTS: Proteomic profiling of CN and CR tumor cells showed significant differences in proteins encoding for CSC, immune surveillance, DNA repair mechanisms, cytoskeleton rearrangement, cell-cell adhesion, cell cycle pathways, cellular transport, and proteins involved with glycine/proline/arginine synthesis in tumor cells isolated from CR relative to CN patients. Pathway analyses revealed enrichment of chemoresistance markers, energy metabolism, DNA repair mechanisms and immune surveillance pathways in recurrent CR tumor cells. Validation of these pathways in OC cell lines and nude mice treated with chemotherapy demonstrated increased expression of CSC and chemoresistant markers, enhancement in glycolytic pathway and oxidative phosphorylation, suppression of immune surveillance in chemotherapy treated cell lines and recurrent mice xenografts compared to untreated control.

CONCLUSION: These findings unravel some of the molecular mechanisms by which chemoresistance and relapse occur in OC patients' post-chemotherapy treatment; and may be important in designing novel therapeutic options for advanced-stage OC patients.

Citation Format: Nuzhat Ahmed, Ruth Escalona, Elif Kadife, Dilys Leung and George Kannourakis. CANCER STEM CELLS: DISTINCT SEEDS FOR RECURRENT OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr AP26.
Abstract AP31: ATR INHIBITION TARGETS TREATMENT RESISTANT OVARIAN CANCER
Authors: Kim Hyoung , George Erin , Xu Haineng , Medvedev Sergey , Jagannathan Veena , Morgan Mark , Mills Gordon , Brown Eric , Simpkins Fiona
Published: June 2021

Introduction: Although new treatments for ovarian cancer have been developed with the introduction of Poly (ADP-ribose) polymerase inhibitors (PARPi) to standard of care platinum chemotherapy, the majority of tumors acquire drug resistance. Strategies to circumvent PARPi and platinum resistance are needed and our study addresses this gap. We hypothesize that PARPi resistant tumors are dependent on ATR/CHK1 for DNA repair, and by targeting ATR in combination with PARPi would be more effective in eradicating tumor growth.

Experimental Procedures: Drug resistant cells were developed by long term treatment of PEO1 (BRCA2 mutant) and JHOS4 (BRCA1 mutant) ovarian cancer cells with PARPi (AZD2281) or Carboplatin. Cell viability, colony formation, cell cycle and apoptosis were evaluated. Homologous recombination was evaluated by Rad51 foci immuno staining. Replication fork asymmetry was counted by DNA combing assay. PARPi resistant orthotopic PDX models (germline BRCA1/ 2 mutant) were developed by long term treatment with PARPi. PDX developed from a platinum-resistant HGSOC patient with CCNE1 amplification was also tested to evaluate PARPi +/- ATRi. Whole genome sequencing, Reverse-Phase Protein Array Analysis (RPPA) and IHC were performed on cells and xenografts to evaluate for biomarkers of response.

Results: ATR/CHK1 pathway was constitutively activated in treatment resistant cell lines. Monotherapy with ATRi alone in vitro modestly induced cell death and DNA damage in PARPi and platinum resistant cell lines. Treatment with ATRi in combination with PARPi is synergistic in reducing survival of these drug resistant cells. Combination treatment was more effective in targeting cell cycle mediators, and promoting apoptosis. HR was restored in PARPi and platinum treatment resistant PEO1 cells. ATRi treatment suppressed restored HR and increased stalled replication fork. PARPi and ATRi combination treatment was synergistic in causing tumor regression in PARPi/Carboplatin resistant PDX models.

Conclusions: Our work suggests treatment resistant cells are more dependent on ATR/CHK1 pathway and ATR is a promising target for augmenting PARPi and platinum response in treatment resistant HGSOCs.

Citation Format: Hyoung Kim, Erin George, Haineng Xu, Sergey Medvedev, Veena Jagannathan, Mark Morgan, Gordon Mills, Eric Brown, Fiona Simpkins. ATR INHIBITION TARGETS TREATMENT RESISTANT OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr AP31.
Abstract DP-015: BIOTINYLATED ESTROGENS A NOVEL TOOL FOR EARLY DETECTION OF ADDUCT IN OVARIAN CANCER
Authors: Tripathi Kaushlendra , Mani Chinnadurai , Clark David W. , Palle Komaraiah
Published: June 2021

Elevated estrogens exert their carcinogenic effects by at least three different mechanisms; they promote cell proliferation by transcriptional regulation of estrogen responsive genes, cause generation of reactive oxygen species and other free radicals, and directly react with DNA and form potentially mutagenic adducts. However, unbalanced estrogen metabolism has been linked to development of several malignant diseases including breast, endometrial and ovarian cancer and has been identified as a risk factor for these cancers. The metabolic breakdown of estogens is regulated by a series of reactions mediated by hepatic and peripheral enzymes that balances the induction and reduction of cellular stress. An imbalance in this enzymes and processes can lead to highly reactive catechol estrogen metabolites that can react with DNA, form carcinogenic DAN-adducts and lesions. Although several studies established a connection between estrogen-induced DNA damage and carcinogenesis, the underlying molecular mechanisms have been difficult to study because of the technical challenges in detecting and analyzing the variety of different DNA lesions that are formed by estrogen compounds. Moreover, detection and analysis of these adducts are important to directly monitor estrogen metabolites induced cellular responses in the cells. Towards this, we developed a novel method using biotinylated-estrogens that allows immunodetection of estrogen-induced DNA adducts by Slot-blot and single-cell molecular combing and proximity ligation assays. Using these modified estrogens we first time quantitatively detected these adducts on DNA by immune Slot-blot techniques and on DNA fibers. Furthermore, similar to other environmental carcinogens estrogens activates replication associated DNA damage responses and induces chromosomal instability. Hence, first time our studies demonstrate that biotin-labeled estrogens could be a powerful tool to detect estrogen adducts and to probe associated DNA damage responses and cellular responses.

Citation Format: Kaushlendra Tripathi, Chinnadurai Mani, David W. Clark, Komaraiah Palle. BIOTINYLATED ESTROGENS A NOVEL TOOL FOR EARLY DETECTION OF ADDUCT IN OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr DP-015.
Abstract GMM-026: AMPLIFICATION OF USP13 DRIVES OVARIAN CANCER METABOLISM
Authors: Han Cecil
Published: June 2021

Dysregulated energetic metabolism has been recently identified as a hallmark of cancer. Although mutations in metabolic enzymes hardwire metabolism to tumourigenesis, they are relatively infrequent in ovarian cancer. More often, cancer metabolism is re-engineered by altered abundance and activity of the metabolic enzymes. Here we identify ubiquitin-specific peptidase 13 (USP13) as a master regulator that drives ovarian cancer metabolism. USP13 specifically deubiquitinates and thus upregulates ATP citrate lyase and oxoglutarate dehydrogenase, two key enzymes that determine mitochondrial respiration, glutaminolysis and fatty acid synthesis. The USP13 gene is co-amplified with PIK3CA in 29.3% of high-grade serous ovarian cancers and its overexpression is significantly associated with poor clinical outcome. Inhibiting USP13 remarkably suppresses ovarian tumor progression and sensitizes tumor cells to the treatment of PI3K/AKT inhibitor. Our results reveal an important metabolism-centric role of USP13, which may lead to potential therapeutics targeting USP13 in ovarian cancers.

Citation Format: Cecil Han. AMPLIFICATION OF USP13 DRIVES OVARIAN CANCER METABOLISM [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-026.
Abstract GMM-045: PAX8 PLAYS AN ESSENTIAL ROLE IN HIGH GRADE SEROUS OVARIAN CANCER VIA ACTIVATION OF MUTANT P53 AND MISLOCALIZED P21
Authors: Ghannam Dima , Jacob Eyal , Kakun Reli , Wasserman Tanya , Korsensky Lina , Sternfeld Ofir , Kagan Julia , Bublik Debora Rosa , Aviel-Ronen Sarit , Levanon Keren , Sabo Edmond , Larisch Sarit , Oren Moshe , Hershkovitz Dov , Perets Ruth
Published: June 2021

High grade serous carcinoma (HGSC) is the most common subtype of ovarian cancer, and a very deadly disease, mostly due to lack of early detection methods. Wide spread disease at diagnosis has delayed the understanding that it often originates from the fallopian tube. This novel understanding has led to increased interest in fallopian tube developmental lineage markers, such as PAX8. In the adult fallopian tube, PAX8 is expressed in the fallopian tube secretory epithelial cell (FTSEC) and its expression is maintained through the process of FTSEC transformation Serous Tubal Intraepithelial Carcinoma (STIC) and to HGSC. We show that PAX8 has an essential pro-proliferative and anti-apoptotic role in HGSC. This role is mediated through direct transcriptional activation of mutated TP53, which often carries missense mutations that potentially lead to gain of function (GOF) oncogenic activities. Surprisingly, mutant p53 binds the p21 promoter and transcriptionally activates the expression of p21, which localizes to the cytoplasm of cells where it plays a non-canonical, pro-proliferative role. While in normal fallopian tube p21 is nuclear as expected, and STIC shows a mix of nuclear and cytoplasmic staining, an analysis of p21 staining in EOC shows that cytoplasmic p21 expression correlates with bad prognostic factors. Together, our findings illustrate that in HGSC TP53 mutations and abnormal p21 localization subvert a normal developmental pathway into a driver of tumor progression.

Citation Format: Dima Ghannam, Eyal Jacob, Reli Kakun, Tanya Wasserman, Lina Korsensky, Ofir Sternfeld, Julia Kagan, Debora Rosa Bublik, Sarit Aviel-Ronen, Keren Levanon, Edmond Sabo, Sarit Larisch, Moshe Oren, Dov Hershkovitz, Ruth Perets. PAX8 PLAYS AN ESSENTIAL ROLE IN HIGH GRADE SEROUS OVARIAN CANCER VIA ACTIVATION OF MUTANT P53 AND MISLOCALIZED P21 [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-045.
Abstract GMM-055: BMI1 AND RING1A ARE INVOLVED IN H2A UBIQUITINATION AT SITES OF PLATINUM-INDUCED DAMAGE
Authors: Sriramkumar Shruthi , O'Hagan Heather M.
Published: June 2021

Ovarian cancer is the 5th leading cause of death among women with the five year survival rate for ovarian cancer patients being only 46%. The standard of care for treatment of ovarian cancer patients is debulking surgery followed by platinum-taxane based chemotherapy. However, the major obstacle in the treatment of these patients with platinum based agents is development of chemoresistance. The platinum agents, cisplatin and carboplatin damage DNA by forming adducts with adjacent guanines, a high density of which are found in the promoter CpG islands of genes. Aberrant promoter DNA hypermethylation of genes and their subsequent transcriptional repression has been associated with development of cisplatin resistance. However, the mechanism of initiation of this aberrant DNA methylation and subsequent transcriptional repression is not known. Our overall hypothesis is that platinum induced DNA damage or repair of the damage leads to recruitment of repressive proteins to sites of damage. These repressive proteins transiently repress transcription in vicinity of damage to promote repair. However, similar to our findings with enzyme induced double strand breaks, repressive proteins may be retained at some key loci causing persistent transcriptional repression and gene silencing contributing to the development of platinum resistance. Our preliminary data demonstrates that BMI1, a member of polycomb repressive complex 1, localizes to sites of damage after cisplatin treatment. We also observe mono-ubiquitination of H2AX after cisplatin treatment and we hypothesize that this ubiquitination is occurring on K119. Mono-ubiquitination of H2A/H2AX at K119 has been associated with transcriptional repression and gene silencing during development and differentiation and also during repair of ionizing radiation and enzyme induced double strand breaks. Our data suggests that knockdown of RING1A reduces the cisplatin induced H2AX ubiquitination. We are currently determining which repair pathways and/or proteins result in recruitment of BMI1, RING1A to sites of damage. Importantly, after acute cisplatin treatment we also observe a reduction in expression of candidate genes which are known to be methylated in cisplatin resistant cells. We plan to determine if RING1A mediated ubiquitination is responsible for reduction in expression of target genes after acute cisplatin treatment and their methylation in cisplatin resistant cells. Understanding the mechanism of platinum induced gene silencing will enable us to design therapies to avert the development of drug resistant ovarian cancers.

Citation Format: Shruthi Sriramkumar, Heather M. O'Hagan. BMI1 AND RING1A ARE INVOLVED IN H2A UBIQUITINATION AT SITES OF PLATINUM-INDUCED DAMAGE [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-055.
Abstract GMM-061: MOLECULAR CHARACTERIZATION OF MOUSE MODELS OF HIGH-GRADE SEROUS CARCINOMA ARISING IN THE OVIDUCT
Authors: Zhai Yali , Wang Yisheng , Schulman Stephanie M. , Hu Kevin , Liu Albert , Tomlins Scott A. , Fearon Eric R. , Cho Kathleen R.
Published: June 2021

We recently developed genetically engineered mouse models (GEMMs) of oviductal high-grade serous carcinoma (HGSC) based on conditional inactivation of several different combinations of tumor suppressor genes (Brca1, Trp53, Rb1, and Nf1) that are recurrently mutated in human HGSCs. The histopathological features of the mouse tumors closely mimic their human tumor counterparts. In order to characterize how well the mouse tumors recapitulate the molecular characteristics of human HGSCs, targeted exome sequencing was used to analyze the 32 most commonly mutated genes in human HGSC, in 60 mouse tumors arising in the context of Brca1, Trp53, Rb1, and/or Nf1 inactivation. We employed the sequence data to assess DNA copy number alterations (CNAs) and single nucleotide variants (SNVs) in the mouse tumors.

Compared to 14 normal tissues and 8 oviductal tumors arising in the context of Apc, Pten, ± Arid1a inactivation, the mouse HGSCs showed a high level of genomic instability, with many widely distributed CNAs – very similar to the widespread CNAs observed in human HGSCs. Targeted exome sequencing also showed that a subset of the mouse tumors acquired alterations observed in human HGSCs, including amplification of cMyc, and deletion of Pten. Variant analysis identified nonsynonymous SNVs in Csmd3, Crebbp, Pten, Mettl17, and Zymynd8 and a frameshift deletion of Pten. Sanger sequencing confirmed the presence of these somatic mutations in the mouse tumors and their absence in matched normal tissues. Loss of PTEN expression was observed in those tumors that acquired somatic Pten alterations.

These data show that HGSCs arising in our GEMMs have very similar molecular characteristics to their human tumor counterparts. The somatic alterations are likely acquired during the relatively lengthy period (several months) between tumor initiation and progression to overt malignancy. These features render the models particularly well suited for studying the early phases of HGSC development and for translational applications aimed at identifying effective strategies for HGSC prevention and early detection.

Citation Format: Yali Zhai, Yisheng Wang, Stephanie M. Schulman, Kevin Hu, Albert Liu, Scott A. Tomlins, Eric R. Fearon, and Kathleen R. Cho. MOLECULAR CHARACTERIZATION OF MOUSE MODELS OF HIGH-GRADE SEROUS CARCINOMA ARISING IN THE OVIDUCT [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-061.
Abstract NT-091: TGF-β1-INDUCED CHEMORESISTANCE IS REVERSED BY BG-4 PEPTIDE
Authors: Dia Vermont P
Published: June 2021

Transforming growth factor-β (TGF-β) superfamily plays a significant role in ovarian physiology but dysregulated TGF-β signaling is associated with promotion of ovarian carcinogenesis. TGF-β signaling in ovarian cancer favors cancer invasion and metastasis and controls ovarian cancer cell proliferation. The objective of this study is to determine the ability of a food-derived peptide, called BG-4, to suppress TGF-β1 signaling in ovarian cancer cells. BG-4 is a novel bioactive peptide isolated from the seeds of Momordica charantia with demonstrated anticancer and strong trypsin inhibitory properties. Different ovarian cancer cells (A2780-1A9, BR and PEO4) were exposed to 10 ng/mL TGF-β1 with or without non-cytotoxic dose of BG-4 (100 µg/mL) and response to chemotherapeutic reagents paclitaxel and cisplatin was evaluated. In addition, markers of epithelial-to-mesenchymal transition (EMT) and apoptosis were evaluated using western blot and immunofluorescence microscopy. TGF-β1 treatment resulted in cisplatin resistance in three cell lines as evidenced by increase in ED50 values from 42.9 µM to 88.5 µM, 53.7 µM to 108.3 µM and 44.8 µM to 67.3 µM for BR, PEO4 and A2780-1A9 cells, respectively. In the presence of BG-4, ED50 values of 35.6 µM, 56.4 µM and 46.6 µM were obtained for BR, PEO4 and A2780-1A9 cells, respectively. These values are similar to non-TGF-β1-treated cells indicating the ability of BG-4 to reverse TGF-β1-induced chemoresistance in ovarian cancer cell lines. Mechanistic studies showed that TGF-β1 signaling induces EMT in ovarian cancer cells as shown by differential expression of epithelial marker E-cadherin and mesenchymal markers N-cadherin, vimentin and transcription factors Snail, Slug and Twist1. In addition, the expression of apoptosis markers were affected by TGF-β1 signaling. On the other hand, treatment of TGF-β1 in the presence of 100 µg/mL BG-4 led to reversal of the EMT process. These results showed that the ability of BG-4 to reverse TGF-β1-induced chemoresistance in ovarian cancer cell lines may be associated with inhibition of the EMT process.

Citation Format: Vermont P Dia. TGF-β1-INDUCED CHEMORESISTANCE IS REVERSED BY BG-4 PEPTIDE [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-091.
Abstract NT-095: ERADICATION OF CHEMORESISTANT METASTATIC OVARIAN CANCER WITH IMMUNOTARGETED EPIGENOMIC NANOSURGERY (IEN)
Authors: Giannios John N.
Published: June 2021

INTRODUCTION: Resistance to conventional chemotherapy such as cisplatin, and radiotherapy in ovarian cancer may be caused by deregulated oncogenic miRNA-221/222 inhibiting induction of apoptosis.

METHODS: Chemoradioresistant cells overexpressing EphA2 were obtained for developing an ovarian metastatic cancer model, which was treated with anti-EphA2 MAbs immunotargeted pegylated nanosomes with encapsulated paired guide-RNAs targeting 3' and 5' of the precursor microRNA 221/222.

RESULTS: Post-treatment, the paired guide-RNAs caused deletions in the targeted pre-microRNA 221/222 genomic-sequences removing the stem-loop hairpins after blockade of RNase-III Drosha and DGCR8 (microprocessor-complex), subsequent transport by exportin-5/RanGTP and cleavage by Dicer inhibiting miRNA221/222 biogenesis, and miRNP circumventing their incorporation in RISC, and subsequent mRNA cleavage of coding genes regulating according to NGS the PTEN, GJA1, SEMA3B, TIMP3, PTPRM, CDKN1C, MGMT, CDKN1B, PUMA, connexin-43 (CX-43), c-Jun, MMP, NF-kB, PI3K, p27/Kip1, PTPμ, NIAP, ICAM-1, STAT1, STAT2, STAT3/Akt, CDK4, CDKN1C/p57, p53, and p57 inhibiting metastatic ovarian cancer cell proliferation, chemo/radioresistance, migration, angiogenesis, invasion, metastasis and maintenance of MET expressing ovarian metastatic cancer stem cells, while inducing cytotoxic immune responses (CTLs), and D2 irreversible stage of apoptosis or programmed cell death type-I leading to a bystander killing effect (BKE) due to phagocytosis of adjacent tumor cells according to transmission electron micrographs.

CONCLUSION: Immunotargeted epigenomic nanosurgery may eradicate metastatic ovarian cancer after inhibition of oncogenic miRNA 221/222 expression, and downstream signaling pathways involving interactions with coding RNA genes leading to apoptosis or PCD type I and BKE after the circumvention of chemo/radioresistant mechanisms.

Citation Format: John N. Giannios. ERADICATION OF CHEMORESISTANT METASTATIC OVARIAN CANCER WITH IMMUNOTARGETED EPIGENOMIC NANOSURGERY (IEN) [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-095.
Abstract TMIM-074: IGF2 IN HUMAN FOLLICULAR FLUID PROMOTES LYMPHOMAGENESIS IN MAMMARY FAT PAD OF TRP53-NULL MICE, DEPENDING ON ESTROGEN IN MICROENVIRONMENT
Authors: Huang Hsuan-Shun , Chu Sung-Chao , Chu Tang-Yuan
Published: June 2021

Ovulation overdrive is the most important risk factor of ovarian cancer. In previous study we discovered that preovulatory follicular fluid (FF) collected from IVF women, upon weekly injections into the mammary fat pad of Trp53-null mice for 7 weeks, could induce early onset of B cell lymphomas. In subsequent studies (Abstract by Chu TY et al.), we disclosed the IGF-axis signaling conferred by FFs is responsible for the stemness, clonal expansion and transformation of fallopian tube fimbrial epithelial cells where IGF2 in FF bound IGF-1R on secretory cells to activate the downstream PI3K/Akt/mTOR and PI3K/Akt/NANOG signaling pathways for the transformation.

In this study, we identified the same IGF2 is also responsible for the FF-induced lymphomagenesis in Trp53-null mice. When IGF2 was depleted from FF, the tumorigenesis rate reduced from 10/18 (56%) to 1/6 (17%). On the other hand, injections with pure IGF2 (100 mg/ml) grew tumors in 33% (2/6) of mice. The same injection to Trp53 wild type did not grow tumor.

We also found a microenvironment of adipose tissue seemed to be important for the FF-induced lymphomagenesis. The same injections of FF into the subcutis and other sites did not grow tumor.

Additionally, tumor development depends not only on the injection site but also on the female sex hormone and ER function. No tumor growth was observed in castrated female mice and in male mice. Adding fulvastrant to the injections resulted in complete shrinkage of tumor. Meanwhile, extended injection of FF from 7 weeks to 13 weeks promoted tumor progression but did not increase tumor incidence.

The findings suggest a transformation activity of human preovulatory FF largely conferred by IGF2. In the context of germline Trp53 loss, this activity likely promotes expansion of the lymphocytic progenitors that have already been transformed. Meanwhile, we found ER is expressed in the tumor adjacent adipocytes but not in the lymphoma cells. Also, ER antagonist, although largely confined tumor growth, did not decrease the tumor incidence. In this tumorigenesis model conferred by Trp53 loss and FF-IGF2, estrogen seems to play an essential role in tumor progression but not in tumor initiation, and this promotion effect is through the adipocyte microenvironment.

Citation Format: Hsuan-Shun Huang, Sung-Chao Chu, Tang-Yuan Chu. IGF2 IN HUMAN FOLLICULAR FLUID PROMOTES LYMPHOMAGENESIS IN MAMMARY FAT PAD OF TRP53-NULL MICE, DEPENDING ON ESTROGEN IN MICROENVIRONMENT [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr TMIM-074.
Abstract TMIM-081: THE HYPOXIC TUMOR-MESOTHELIAL NICHE PROMOTES OVARIAN CANCER METASTASIS THROUGH COLLAGEN REMODELING
Authors: Natarajan Suchitra , Foreman Katie , Soriano Michaela , Shehade Hussein , Fregoso Daniel , Eggold Joshua , Rosen Ninna S. , Heilshorn Sarah , Krieg Adam J. , Krishnan Venkatesh , Dorigo Oliver , Sinha Subarna , Fuh Katherine C. , Rankin Erinn B.
Published: June 2021

High grade serous ovarian cancer (HGSOC) is a leading cause of cancer related deaths among women. The primary cause of morbidity and mortality in HGSOC patients is metastasis to organs within the peritoneal cavity. Mesothelial cells line the surface of peritoneal organs and are a key stromal cell in HGSOC metastatic niche. However, the mechanisms by which mesothelial cells promote ovarian cancer peritoneal metastasis remain largely unknown. Here we demonstrate that tumor associated mesothelial cells promote tumor invasion by increasing collagen deposition and remodeling. Mechanistically, we demonstrate the tumor-mesothelial niche is hypoxic where both tumor cells and mesothelial cells express the hypoxia inducible factors HIF-1 and HIF-2 and their downstream target gene lysyl oxidase (LOX). LOX is an enzyme that crosslinks collagen fibrils to promote collagen remodeling. In tumor-mesothelial co-culture experiments, we demonstrate that hypoxia enhances extracellular fibrillar collagen deposition by mesothelial cells. Conditioned media from hypoxic tumor-mesothelial co-cultures promotes collagen remodeling and HGSOC tumor cell invasion. Genetic inactivation of either HIF-1 and HIF-2 or LOX reduces the hypoxic induction of collagen remodeling and tumor cell invasion. Importantly, pharmacologic inhibition of HIF-1 and HIF-2 with digoxin reduces the colonization of disseminated HGSOC cells to the omentum, the preferential migration site of peritoneal cancer metastasis. Moreover, pharmacologic inhibition of LOX with BAPN (beta-aminopropionitrile) is sufficient to inhibit metastatic tumor burden and collagen remodeling at metastatic sites in preclinical models of HGSOC metastasis. These data reveal a novel role for mesothelial cells in the production of type I collagen and collagen remodeling in the HGSOC metastatic microenvironment. Furthermore, these studies demonstrate a role for the HIF/LOX signaling axis in the HGSOC tumor-mesothelial niche that can be therapeutically targeted to inhibit collagen remodeling and ovarian cancer metastatic progression.

Citation Format: Suchitra Natarajan, Katie Foreman, Michaela Soriano, Hussein Shehade, Daniel Fregoso, Joshua Eggold, Ninna S. Rosen, Sarah Heilshorn, Adam J. Krieg, Venkatesh Krishnan, Oliver Dorigo, Subarna Sinha, Katherine C. Fuh, Erinn B. Rankin. THE HYPOXIC TUMOR-MESOTHELIAL NICHE PROMOTES OVARIAN CANCER METASTASIS THROUGH COLLAGEN REMODELING [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr TMIM-081.
Longitudinal Single-Cell Dynamics of Chromatin Accessibility and Mitochondrial Mutations in Chronic Lymphocytic Leukemia Mirror Disease History
Authors: Penter Livius , Gohil Satyen H. , Lareau Caleb , Ludwig Leif S. , Parry Erin M. , Huang Teddy , Li Shuqiang , Zhang Wandi , Livitz Dimitri , Leshchiner Ignaty , Parida Laxmi , Getz Gad , Rassenti Laura Z. , Kipps Thomas J. , Brown Jennifer R. , Davids Matthew S. , Neuberg Donna S. , Livak Kenneth J. , Sankaran Vijay G. , Wu Catherine J.
Published: June 2021

While cancers evolve during disease progression and in response to therapy, temporal dynamics remain difficult to study in humans due to the lack of consistent barcodes marking individual clones in vivo. We employ mitochondrial single-cell assay for transposase-accessible chromatin with sequencing to profile 163,279 cells from 9 patients with chronic lymphocytic leukemia (CLL) collected across disease course and utilize mitochondrial DNA (mtDNA) mutations as natural genetic markers of cancer clones. We observe stable propagation of mtDNA mutations over years in the absence of strong selective pressure, indicating clonal persistence, but dramatic changes following tight bottlenecks, including disease transformation and relapse posttherapy, paralleled by acquisition of copy-number variants and changes in chromatin accessibility and gene expression. Furthermore, we link CLL subclones to distinct chromatin states, providing insight into nongenetic sources of relapse. mtDNA mutations thus mirror disease history and provide naturally occurring genetic barcodes to enable patient-specific study of cancer subclonal dynamics.

Significance:

Single-cell multi-omic profiling of CLL reveals the utility of somatic mtDNA mutations as in vivo barcodes, which mark subclones that can evolve over time along with changes in accessible chromatin and gene expression profiles to capture dynamics of disease evolution.

See related commentary by Hilton and Scott, p. 2965.

This article is highlighted in the In This Issue feature, p. 2945
Abstract A17: Predictions of comparative clinical outcomes for checkpoint inhibitor combo therapies and mechanistic targets in first-line NSCLC
Authors: Vanderman Kadie , Stine Andrew , Chang Steven
Published: June 2021

The success of checkpoint inhibitors for immunotherapy has ushered in a new era of cancer treatment. While response rates of approximately 20% are observed with checkpoint monotherapy in NSCLC, it remains one of the most deadly forms of cancer. Combinatory therapy approaches have been rapidly implemented, with the selection of agents largely based on theoretical considerations (Kargl, 2017 #19227). The high number of new clinical trials of anti-PD1 with a combination therapy raises the concern that adequate patient enrollment may become a barrier to trial completion and approval. Hence, drug candidate selection and identification of patients most likely to benefit from treatment are emerging needs of the field. Further, irregularity in patient enrollment criteria and trial designs prevents the direct comparison of regimens, impeding clinical decision making. To address rationale immunotherapy decisions for first- line NSCLC patients, we developed a mechanistic, systems biology model to compare the efficacy of 4 candidate therapies in combinations with an anti-PD1 therapy. Our model captured mechanisms underlying the pro- and anti-inflammatory influences of the innate immune system on Tregs and T cell effector function. “Simulated patients” were then created based on patient level data of 30-60 baseline measurements from preclinical and clinical publications in squamous and nonsquamous NSCLC patients. The model permeated all vectors and combinations of data. Each unique set of permutations that fit within clinical monotherapy data from platinum doublet chemotherapy SOC, anti-PD1, anti-CTLA4, and EGFR inhibitor trials in NSCLC represented a simulated patient. Over 200 patients were simulated to create a virtual trial population. As a performance validation, our model blindly predicted the NSCLC results from published combination trials of anti-PD1 + CTLA4 and anti-PD1 + EGFR. The model then was used to predict results for an anti-PD1 + Lag3 and anti-PD1 + CCL2, for which no trial results are available. We conducted a direct comparison between the co-therapy regimens, controlling for trial design and using the same simulated patient population for each trial arm. The large and diverse simulated patient population allowed us to evaluate trends in the characteristics of the tumor microenvironment of differential responders/nonresponders on one regimen vs. another, such as the immune cell composition, biomarker levels, and cytokine levels.

Citation Format: Kadie Vanderman, Andrew Stine, Steven Chang. Predictions of comparative clinical outcomes for checkpoint inhibitor combo therapies and mechanistic targets in first-line NSCLC [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A17.
Abstract A54: Epigenetic activated IL1B promotes tumor progression of tongue cancer
Authors: Zheng Dali , Chen Weitao , Lu Youguang
Published: June 2021

Alterations in host immunity, inflammation, angiogenesis, and metabolism have been noted to be prominent clinical features in patients with head and neck squamous cell carcinoma (HNSCC). IL-1β, a prototypical inflammatory cytokine, is induced and activated following infection and tissue damage. High IL-1 levels in the tumor microenvironment are associated with a more aggressive tumor phenotype and generally poor prognoses. It was reported that single-nucleotide polymorphisms (SNPs) identified in IL1B gene influence the risk of many kinds of cancer, but the role of IL1B in the development and progress of HNSCC and the related molecular mechanism were not well understood. In the present study, we found IL1B was upregulated in tongue cancer tissues according to our RNA-Seq data and was validated in other 60 paired clinical samples. Using data from The Cancer Genome Atlas (TCGA) head and neck squamous cell carcinoma (HNSC), we found that the expression of IL1B in HNSCC tumor samples (n=501) is significantly higher than the adjacent noncancer tissues (n=44) and hypomethylation of CpG sites was found around the promoter region of IL1B in the cancer samples. Regression analysis identified a moderately negative correlation between IL1B expression and the DNA methylation of these CpG sites, even though there is not CpG island around its promoter. Knockdown of IL1B in Cal-27 and TCA-8113 tongue cancer cells inhibited cell proliferation, migration and invasion ability, and overexpression of IL1B or recombinant IL1B protein can promote proliferation, migration and invasion of tongue cancer cells. The results indicated that aberrant IL1B expression might be modulated by its DNA hypomethylation and IL1B produced by cancer cells can promote tumor progression and metastasis, and the inhibition of IL1B pathway may be a novel therapeutic approach in the treatment of HNSCC.

Citation Format: Dali Zheng, Weitao Chen, Youguang Lu. Epigenetic activated IL1B promotes tumor progression of tongue cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A54.
Abstract A56: Alteration of tumor metabolism by antitumor CD4+ T cells leads to tumor rejection
Authors: Zhou Gang , Habetesion Tsadik , Ding Zhi-Chun
Published: June 2021

Mounting evidence has revealed a dynamic metabolic crosstalk between cancer cells and T cells. It is evident that the metabolic constraints imposed by cancer cells compromise T-cell metabolic fitness and render T cells dysfunctional even in the face of antigenic stimulation. However, whether and how T cells affect tumor cell metabolism remains unclear. In the present study, we report that adoptive transfer of tumor-specific CD4+ T cells following CTX preconditioning gave rise to polyfunctional CD4+ effector cells capable of concomitantly producing multiple inflammatory cytokines, including tumor necrosis factor alpha (TNF-α) and interferon gamma (IFNγ). These CD4+ effector cells induced profound metabolic changes in tumors before tumor regression was evident. Disruptions in multiple metabolic pathways converged to cause defective synthesis of the major cellular antioxidant glutathione (GSH), resulting in severe GSH deficiency, heightened reactive oxygen species (ROS) accumulation, and oxidative DNA damage in tumor cells. We demonstrated that tumor cell-intrinsic TNF-α signaling was required to synergize with chemotherapy to intensify ROS production in tumor via a mechanism involving nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidases. Our data provide new insight into the impact of an effective immunotherapy on tumor metabolism and identify tilting tumor redox toward excessive oxidative stress as an important mechanism underlying the efficacy of adoptive immunotherapy.

Citation Format: Gang Zhou, Tsadik Habetesion, Zhi-Chun Ding. Alteration of tumor metabolism by antitumor CD4+ T cells leads to tumor rejection [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A56.
Abstract A71: Resistance mechanisms limiting the immunostimulatory and antitumor activity of anti-CSF-1 receptor-mediated macrophage depletion
Authors: Skrzypczynska Katarzyna M. , O'Brien Sarah A. , Belmontes Brian , Tan Hong , Orf Jessica , Lu Daniel , Driver Ian , Egen Jackson G.
Published: June 2021

Tumor-associated macrophages (TAMs) are present within the stroma of most solid tumors where they contribute to disease progression through production of tumor and angiogenic growth factors, extracellular matrix remodeling, and suppression of immune responses. As such, depletion of tumor-associated macrophages, for instance through the use of blocking antibodies against the CSF-1 receptor (CSF1R), has been widely explored as a therapeutic approach to both inhibit tumor growth/metastasis and stimulate antitumor immune responses. In the context of immunotherapy, multiple preclinical murine studies have demonstrated that anti-CSF1R meditated macrophage depletion can promote antitumor T-cell responses, synergizing with immune checkpoint inhibitor therapy, such as PD1 blockade, and leading to tumor regression. However, some tumor models appear resistant to these beneficial effects of CSF1R blockade. In order to define potential mechanisms of resistance to CSF1R inhibitors, we evaluated the effects of anti-CSF1R treatment in mice bearing subcutaneous Renca renal cell adenocarcinoma tumors. Despite robust macrophage depletion, this model shows only a modest decrease in tumor volume and minimal effector T-cell recruitment or activation in response to anti-CSF1R therapy. Comprehensive flow cytometry-based immunophenotyping and single-cell RNA sequencing on immune cells isolated from anti-CSF1R- and isotype control-treated Renca tumors revealed shifts in gene expression patterns related to vascular remodeling, hypoxic responses, and enhanced regulatory T-cell activity. These studies indicate macrophage depletion may, under some conditions, have negative effects on antitumor immune responses that could limit the clinical benefit of this therapeutic approach.

Citation Format: Katarzyna M. Skrzypczynska, Sarah A. O'Brien, Brian Belmontes, Hong Tan, Jessica Orf, Daniel Lu, Ian Driver, Jackson G. Egen. Resistance mechanisms limiting the immunostimulatory and antitumor activity of anti-CSF-1 receptor-mediated macrophage depletion [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A71.
Abstract A94: Identification of potent, insurmountable A2a antagonists for modulation of the tumor microenvironment
Authors: Pettipher Roy , White Jonathan , Patel Viral , Moulton Ben , Pores Soraya , Assuncao Marta , Gherbi Karolina , Sengmany K. , Rosethorne Elisabeth , Finan Peter , Charlton Steven , McCarthy Clive
Published: June 2021

Adenosine is elevated in the tumor microenvironment and plays a critical role in suppressing T-cell function through high-affinity interaction with the A2a receptor. Genetic deficiency of A2a in mice is associated with enhanced cytotoxic responses and reduced tumor burden in syngeneic models. These effects are mimicked by small-molecule A2a antagonists and some of these compounds are currently being evaluated in clinical trials for the treatment of solid tumors, particularly in combination with checkpoint inhibitors. However, high levels of adenosine in the tumor microenvironment may pose a challenge for the development of A2a antagonists unless they possess the appropriate pharmaceutical profile. A series of potent antagonists were identified that displayed sub-nanomolar binding activity and selectivity over other adenosine receptors. Within this series, compounds were identified that maintained activity across a broad range of adenosine concentrations. These compounds behaved as insurmountable antagonists in a functional assay utilizing CHO cells expressing human recombinant A2a and displayed slow dissociation kinetics in a FRET-based receptor-binding assay. This series is exemplified by ARX1598, which had a Ki of 0.08nM in the A2a receptor-binding assay and an estimated KB of 0.06 nM in the recombinant cell-based assay. Schild analysis revealed that this compound behaved as an insurmountable antagonist and had a markedly slower off-rate than comparator competitive antagonists. The adenosine receptor agonist NECA suppressed cytokine production by peripheral blood mononuclear cells activated by anti-CD3/anti-CD28. ARX1598 prevented NECA-mediated suppression of cytokine production by human peripheral blood mononuclear cells (EC50= 6 nM) and prevented elevation of pCREB induced by high concentrations of NECA (3μM) in human whole blood (IC50=22nM). The potency of ARX1598 in the human whole blood assay was 50-100 times higher than comparator compounds currently being evaluated in cancer trials. These data illustrate that it is possible to identify potent compounds that have the potential to modulate the adenosine pathway in the tumor microenvironment.

Citation Format: Roy Pettipher, Jonathan White, Viral Patel, Ben Moulton, Soraya Pores, Marta Assuncao, Karolina Gherbi, K. Sengmany, Elisabeth Rosethorne, Peter Finan, Steven Charlton, Clive McCarthy. Identification of potent, insurmountable A2a antagonists for modulation of the tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A94.
Abstract A97: The inhibitory receptor NKG2A acts as a checkpoint on CD8 T cells in the context of cancer vaccines
Authors: van Hall Thorbald , van Montfoort Nadine , Borst Linda , Korrer Michael J. , Kim Young , André Pascale , Wagtmann Nicolai , Piersma Sytse , van der Burg Sjoerd H.
Published: June 2021

We previously demonstrated that high HLA-E expression in ovarian carcinoma and NSCLC may neutralize the survival benefit of T-cell infiltration, suggesting NKG2A could represent an immune checkpoint. Here, we demonstrate higher frequencies of NKG2A+CD8+ T cells in TIL samples obtained from human HNSCC biopsies in patients with measurable immune reactivity to HPV16-viral antigens. CyTOF analysis suggested that this T-cell subset harbored an CD103+ effector phenotype. In mouse tumor models, therapeutic cancer vaccines increased the frequency of NKG2A+-positive CD8+ TIL up to 80%. Expression of Qa-1, the mouse homologue of HLA-E, on tumor cells also increased after cancer vaccinations. In vitro studies revealed that upregulation of Qa-1 was mediated by T cell-derived IFNγ. Blockade of this inhibiting NKG2A-Qa-1 axis by antibody improved the T-cell mediated rejection of tumors and, in addition, genetic knockdown of Qa-1 in three different tumor models recapitulated this finding. Blocking of NKG2A as a stand-alone treatment was not effective, indicating that NKG2A constitutes an adaptive resistance mechanism in response to CD8 T-cell activation. In conclusion, NKG2A is enriched on CD8+ TIL and functions as an immune checkpoint that restrains therapeutic efficacy of cancer vaccines.

Citation Format: Thorbald van Hall, Nadine van Montfoort, Linda Borst, Michael J. Korrer, Young Kim, Pascale André, Nicolai Wagtmann, Sytse Piersma, Sjoerd H. van der Burg. The inhibitory receptor NKG2A acts as a checkpoint on CD8 T cells in the context of cancer vaccines [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A97.