bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2405: Metabolic plasticity of ovarian metastases in response to adherence and physiologically relevant culture conditions
Authors: Compton Stephanie Edwards , Grieco Joseph , Schmelz Eva M.
Published: July 2021

Ovarian cancer is typically diagnosed at late stages with a poor prognosis due to high recurrence. Exfoliation from the primary tumor results in aggregation of ovarian cancer cells into spheroids in the peritoneal cavity, which then adhere to secondary sites to form metastatic outgrowth. Spheroids in the peritoneal cavity are exposed to hypoxic, low glucose conditions. Sphingosine-1-phosphate (S1P), increased in the ascites fluid of ovarian cancer patients, has been shown to increase tumor cell survival and tumorigenesis through changes in mitochondrial function. However, little is known about these effects in ovarian cancer spheroids, particularly upon secondary adherence and outgrowth in physiological conditions. Changes in spheroid metabolism in response to treatment with S1P, hypoxia, low glucose, and obese peritoneal serous fluid (PSF) in both free and adherent states was investigated. Using a late-stage and tumor-initiating murine ovarian cancer model, spheroids were subjected to treatment with S1P or obese PSF as free or adherent spheroids. Using a Seahorse XFe96, we investigated changes in metabolism over time trials measured after 24 hour treatment for free spheroids and after different time points of adherence (4, 8, 12, and 24 hours). Data show changes in basal respiration, maximal oxygen consumption rate, and spare respiratory capacity of spheroids over adherence time when treated with S1P and PSF. This suggests that the cells respond to energetic demand with metabolic flexibility as the spheroids adhere to new surfaces. The identification of the regulation of these events may help elucidate the flexibility that takes place during metastasis in culture that more closely mimics physiological conditions, providing a foundation for future interventions for preventative therapies for ovarian cancer with dietary sphingolipids and possible treatment targets.

Citation Format: Stephanie Edwards Compton, Joseph Grieco, Eva M. Schmelz. Metabolic plasticity of ovarian metastases in response to adherence and physiologically relevant culture conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2405.
Abstract 2408: Targeted bioinformatics analysis identifies high ABAT transcript expression as a multi-cancer marker of favorable patient prognosis
Authors: Knott Erika L. , Leidenheimer Nancy J.
Published: July 2021

Unsupervised analysis of γ-aminobutyric acid (GABA) system genes has identified some genes as correlated with indicators of severity in several cancers. Central to GABA system function is the GABA shunt, a series of reactions bypassing two steps of the tricarboxylic acid (TCA) cycle, which encompasses the synthesis and metabolism of the non-proteinaceous amino acid GABA. Here, we have undertaken a targeted bioinformatics study utilizing publicly available databases - including cBioPortal, UALCAN, the Human Protein Atlas, and the Human Cancer Metastasis Database - to evaluate the expression of transcripts encoding GABA shunt enzymes, and their correlation with clinical outcomes, across nearly three dozen cancers from The Cancer Genome Atlas (TCGA) Pan-Cancer Atlas initiative dataset. Of the three genes encoding enzymes of the GABA shunt, we have focused on ABAT, which encodes the GABA-catabolizing enzyme γ-aminobutyrate aminotransferase (ABAT; aka GABA-transaminase or GABA-T). ABAT is widely expressed across all 32 TCGA studies, ranging from moderate to high expression in skin cutaneous melanoma and low-grade glioma (average ± SEM = 6.5 ± 0.08; 13.3 ± 0.03 log2 RSEM), respectively. For the majority of these tissues, the expression of ABAT in tumor is lower than its expression in adjacent normal tissue. Importantly, ABAT expression is prognostic in 11 cancers: upregulation is favorably prognostic for ACC, BLCA, BRCA, and PAAD, while downregulation is unfavorably prognostic for KIRC, KIRP, LGG, LIHC, LUAD, MESO, and SKCM (Kaplan-Meier survival estimates, z-score = 0.5, p < 0.05). A subset of these cancers, as well as 4 other TCGA cancers, show a decrease in ABAT transcript expression with increasing tumor stage (p < 0.05). Furthermore, 7 cancers express lower levels of ABAT transcripts in the primary tumors of patients with metastases vs the primary tumors of patients without metastases. Additionally, metastases of 4 cancers express elevated levels of ABAT transcripts vs their respective primary tumors. These results imply a dynamic regulation of ABAT expression throughout cancer progression. As very few studies have directly addressed the role of ABAT in cancer progression, the identification of ABAT transcript expression as a broadly applicable multi-cancer prognostic marker may ultimately benefit a wide variety of patients. We would like to thank the Feist-Weiller Cancer Center and LSU Health, Shreveport for supporting this work.

Citation Format: Erika L. Knott, Nancy J. Leidenheimer. Targeted bioinformatics analysis identifies high ABAT transcript expression as a multi-cancer marker of favorable patient prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2408.
Abstract 2410: The mutant p53 C-terminal domain assists in DNA interactions and cell cycle promotion
Authors: Lundine Devon , Syed Zafar , Ellison Viola , Annor George , Xiao Gu , Bargonetti Jill
Published: July 2021

Introduction: Triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer and has the worst prognosis of any breast cancer sub-type. More than 80% of TNBC patients harbor mutated TP53. Some mutant p53 proteins (mtp53) exhibit gain-of function (GOF) oncogenic characteristics, contributing to the decreased prognosis for TNBC patients, but also providing a potential therapeutic target for this aggressive disease. Our group previously found mtp53 R273H interacts with replicating DNA, and increases chromatin-bound replication proteins MCM2-7, PCNA, and PARP1. Mutant p53 retains an intact C-terminal domain (CTD), the domain which facilitates non-specific DNA binding. We hypothesize that the CTD of mtp53 facilitates increased DNA replication processivity.

Methods: To test this hypothesis, we have transiently transfected human cells to compare wild-type p53, and R273H mtp53 full-length and CTD-truncated variants. We used CRISPR/Cas9 to generate endogenous CTD truncations of mtp53 R273H in the breast cancer cell line MDA-MB-468. Specifically, clones were used which have mtp53 CTD truncations (R273HΔ381-388 and R273HΔ347-393) and one clone with a frameshift resulting in no detectable mtp53 protein (R273Hfs387). Various western blotting techniques were used to assess protein level and sub-cellular localization, including the iPOND (isolation of proteins on nascent DNA) assay. Several microscopy techniques were used to visualize protein-protein interactions in cells including immunofluorescence, proximity ligation, and DNA fiber assays.

Results: Our data show mtp53 R273H and wild-type p53 proteins with CTD truncations tether less to chromatin than their full-length counterparts. Also, our data shows that the truncated R273HΔ381-388 mtp53 is in close proximity to PARylated proteins (and potentially even PARylated itself), but the R273HΔ347-393 truncation is not in close proximity to PARylated proteins. Additionally, R273HΔ381-388 and R273HΔ347-393 demonstrate delayed cell cycle progression and reduced proliferation compared to MDA-MB-468 cells. We observe DNA replication processivity is decreased in 468 R273HΔ381-388 and R273Hfs387 cells. Processivity, but not proliferation rate, is rescued in 468 R273HΔ347-393 cells.

Conclusions: The combination of reduced cell proliferation in all CTD-truncated cells, and reduced DNA replication processivity in R273HΔ381-388 and R273Hfs387 cells, but not in R273HΔ347-393 indicates a nuanced role for the CTD in DNA replication and S-phase progression.

Citation Format: Devon Lundine, Zafar Syed, Viola Ellison, George Annor, Gu Xiao, Jill Bargonetti. The mutant p53 C-terminal domain assists in DNA interactions and cell cycle promotion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2410.
Abstract 2420: Characterizing the oncogenic activity of ERBB4 mutations and their sensitivity to neratinib
Authors: Suominen Peppi , Chakroborty Deepankar , Kurppa Kari J. , Diala Irmina , Eli Lisa D. , Lalani Alshad S. , Elenius Klaus
Published: July 2021

Hundreds of somatic ERBB4 mutations have been described in cancer tissues with very limited information about their functional significance. However, analyses of individual mutants have indicated that activating ERBB4 mutations, such as ERBB4 K935I, do exist. Understanding the functional consequences of ERBB4 mutations is needed in order to assess the relevance of targeting ERBB4 in human cancers with matched therapies such as neratinib. Neratinib is an irreversible pan-HER tyrosine kinase inhibitor that binds to and potently inhibits ERBB4 kinases activity in-vitro, and is currently approved clinically for the treatment of early stage and metastatic HER2+ breast cancers.

We set up to address the transforming potential of individual ERBB4 variants by selecting 18 mutations (Table 1) from cBioPortal data (www.cbioportal.org) using the following criteria: 1) the mutations were recurrent, 2) they were analogous to activating mutations described for other oncogenic ERBB family members and/or 3) their position at receptor dimerization interfaces suggested functional relevance.

Retroviral pBABE-puro-gateway vector was used to achieve functional, but still near-physiological expression levels of ERBB4 in different cell backgrounds. A screen for optimal cellular background to assess the transforming potential of ERBB4 variants indicated that 1) IL-3 independent growth of mouse lymphoid Ba/F3 cells, 2) focus formation analysis of mouse NIH-3T3 fibroblasts, and 3) ligand-induced proliferation of human mammary epithelial MCF-10A provided read-outs with robust differences when the effects of the positive control mutant ERBB4 K935I was compared to wild-type ERBB4. These models are now being used to address the transforming potential of the 18 mutations (Table 1).

Table 1.Somatic ERBB4 mutations chosen for functional analyses.R106CE452KR711CL798RG870RK1223TS303FR524CG741EV840IG907ES1289AR393WR544WS774GR847HR992CR1304W

Citation Format: Peppi Suominen, Deepankar Chakroborty, Kari J. Kurppa, Irmina Diala, Lisa D. Eli, Alshad S. Lalani, Klaus Elenius. Characterizing the oncogenic activity of ERBB4 mutations and their sensitivity to neratinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2420.
Abstract 243: Identification of novel epitopes of NY-ESO-1 for solid malignancies by Kiromic proprietary search engine Diamond
Authors: Piccotti Lucia , Mirandola Leonardo , Chiriva-Internati Maurizio
Published: July 2021

Adoptive cell therapy has been proven a powerful approach for the cure of cancer and other diseases. In particular, the selection of appropriate immunogenic targets has been key to positive outcomes in clinical settings. The availability of RNA-Seq analysis, the accessibility to large data repositories such as TCGA and GTEx, and the creation of new bioinformatic tools have accelerated the process of neoantigen discovery. However, most of the current algorithms are encumbered by the intrinsic complexity of predicting antigen immunogenicity. Diamond™ is a novel artificial intelligence and cognitive machine and deep learning platform to predict peptide processing, HLA binding, and T cell activation. To validate the predictive value of DIAMOND algorithms, the meta-analyses of expression data of cancer-testis antigen New York Esophageal Squamous Cell Carcinoma 1 (NY-ESO-1) and predictions for the immunogenic peptides were compared to experimental data in the literature. In agreement with published clinical observations, DIAMOND metanalysis showed NY-ESO-1 genic overexpression in skin cutaneous melanoma, lung adenocarcinoma, and sarcoma. Moreover, DIAMOND predicted an MHC binding affinity of 0.289 with Supertype A2 for a new NY-ESO-1 peptide, which has been successfully targeted in clinical trials for patients with HLA-A*02:01, as well as it mirrored published data in its prediction of peptide affinity binding in NY-ESO-1–specific MHC II–restricted T cell receptors. Taken together these data support DIAMOND as a reliable platform for the discovery of new immunogenic targets for cancer therapy.

Citation Format: Lucia Piccotti, Leonardo Mirandola, Maurizio Chiriva-Internati. Identification of novel epitopes of NY-ESO-1 for solid malignancies by Kiromic proprietary search engine Diamond [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 243.
Abstract 2433: Core regulatory circuits of clear cell ovarian and kidney cancers
Authors: Scott Marla Elizabeth , Nameki Robbin , Abbasi Forough , Fonseca Marcos Abraao de Souza , (Ivetth) Corona de la Fuente Rosario , Lawrenson Kate
Published: July 2021

Clear cell ovarian and kidney cancers are rare but aggressive cancers characterized by resistance to traditional chemotherapies. The transcriptional drivers of these tumors are poorly understood, but the histologic similarities suggest that a subset of common factors may drive differentiation of the clear cell phenotype, agnostic of anatomic origin. Master transcription factors (MTFs) are the essential transcriptional regulators that create interconnected auto-regulatory circuits critical for tumor cell survival. Candidate MTFs can be identified due to their characteristic high level, tissue-specific expression, functional essentiality in tumor models, and participation in interconnected core regulatory transcriptional circuits. We collated 5 high grade serous ovarian cancer, 5 clear cell ovarian cancer, 5 papillary renal cell cancer and 11 clear cell renal cell cancer primary tissue and 27 cell line data sets for clear cell ovarian and kidney cancers, and non-clear cell tumors of the same organs. We integrated gene expression, super-enhancer landscapes and global maps of open chromatin to identify candidate MTFs specific to clear cell tumors. This identified known Hepatocyte nuclear factor-1-beta (HNF1B), commonly over-expressed in >90% of clear cell cancers and other novel candidates, including RARA, which encodes retinoic acid receptor alpha. Clear cell kidney and ovarian cancer cell lines are often co-dependent on sustained expression of HNF1B and RARA for survival, suggesting perturbed retinoic acid signaling represents a novel therapeutic target for clear cell tumors. Transcriptional inhibitors provide a novel therapeutic target for many cancers that may otherwise be chemoresistant. Clear cell lines are particularly sensitive to general transcriptional inhibitors in in-vitro models; functional analyses are ongoing to determine the role of RARA, HNF1B and other factors in explaining these responses.

Citation Format: Marla Elizabeth Scott, Robbin Nameki, Forough Abbasi, Marcos Abraao de Souza Fonseca, Rosario (Ivetth) Corona de la Fuente, Kate Lawrenson. Core regulatory circuits of clear cell ovarian and kidney cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2433.
Abstract 2441: Polysome profiling suggests VHL-dependent translational control in clear cell renal cell carcinoma
Authors: Vassalakis Julia A. , Zequi Stenio C. , Bezerra Stephania M. , da Costa Walter H. , Larsson Ola , Topisirovic Ivan , Hajj Glaucia N.
Published: July 2021

ccRCC is the most common type of renal carcinoma with 80% of incidence among all types of kidney neoplasms. Most cases are localized in the kidney and potentially curable after nephrectomy however about 30% of patients will relapse with distant metastasis. Metastatic patients comprehend one third of all cases and, despite the advances in therapies, they still have low response rates. The identification of molecular mechanisms associated with ccRCC is essential to understand disease progression and treatment resistance. Genes frequently mutated in ccRCC affect the activation of signaling pathways including the mTOR pathway which can cause an unbalance in translational control. Another frequent mutation is in the tumor suppressor gene VHL which regulates response under hypoxia. Hypoxia affects gene expression by both translational and transcriptional controls that contributes to tumor formation and disease progression. Here we aim to understand how translational control can contribute to ccRCC development. We evaluated the activity of mTOR pathway and translational control in cell lines and PDX models with VHL mutation through polysome profiling. We observed lower global translational rates in both VHL mutated models suggesting an important role in translational control. Differentially translated genes identified from polysome associated RNA show a specific translational signature in response to VHL deletion. For human tumors, a cohort of 118 cases was selected between metastatic and non-metastatic patients available at A.C. Camargo Cancer Center Tumor Tissue Biobank. Polysome profiling was performed for all cases and show that increased translational rates are associated with reduced overall and progression-free survival.

Citation Format: Julia A. Vassalakis, Stenio C. Zequi, Stephania M. Bezerra, Walter H. da Costa, Ola Larsson, Ivan Topisirovic, Glaucia N. Hajj. Polysome profiling suggests VHL-dependent translational control in clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2441.
Abstract 245: A comprehensive characterization of hyper-morph, hypo-morph, and neo-morph mutations in cancer
Authors: Tagore Somnath , Califano Andrea
Published: July 2021

Although annotation of genomic mutations is a highly relevant and complex segment of the analysis of sequence-based genomic analyses, currently more than ten million variants lack functional annotation. While computational predictions of variant function are usually integrated into gene-based analyses of rare-variants, there is limited information for assessing variant function in the context of a particular disease. The goal of this study is to assess the mechanistic characterization of context-dependent hyper-, hypo-, and neomorph mutations in cancers. We developed a computational approach to characterize these mutations and assess the functional effects of mutations occurring in the same or different protein domains, and differentiate mutations in terms of their hyper-morph (gain-of-function), hypo-morph (loss-of-function), neutral, or neo-morph (establishing a novel function) potential. To elucidate the functional consequence of such mutations, we based our computational pipeline on inferring the activity of regulatory proteins such as transcription factors (TFs) and co-factors (co-TFs) using the VIPER algorithm. Our analytical pipeline integrates structural and functional information encompassing six topics : 1) structural domains affected by the mutation, 2) the overlap between mutation-specific TF/co-TFs, 3) differential activity signatures and signatures induced by established hyper-morph, hypo-morph and neutral or neo-morph mutations, 4) in vitro data generated by reporter assays, 5) the VIPER-inferred activity of each protein relative to a validated control, and 6) the fraction of proteins in a sample that are not affected by established hyper-morphs and hypo-morphs (candidate neo-morphs).The current repertoire considers 25 TCGA cohorts for 3830 proteins (oncoproteins/tumor suppressors). As an example, we validated our method on the TCGA Breast Cancer dataset (TCGA-BRCA) by predicting with very high confidence several neo-morphic phenotypes, including the previously-described PIK3CAE545, PIK3CAE542, PIK3CAH1047, PIK3CAQ546K and PIK3CAG1049R. Interestingly, PIK3CAE545K, classified previously as a gain-of-function mutation (in one TCGA-BRCA sample) or a loss-of-function mutation (in two other TCGA-BRCA samples), is predicted to be a neo-morph based on our approach. Further validation of these mutations using PIK3CA reporter assays led to the identification of several significant hypo-morphic signals in TP53 mutant samples. We defined this phenomenon as mutational mimicry (i.e. mutations in proteins mimicking those in established oncogenes) and we propose it as a tool for predicting tumor sensitivity/resistance to drugs.

Citation Format: Somnath Tagore, Andrea Califano. A comprehensive characterization of hyper-morph, hypo-morph, and neo-morph mutations in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 245.
Abstract 2450: A systemic approach to decipher the interactome of RET receptor isoforms
Authors: Kheitan Samira , Pedersen Annika E. , Hyndman Brandy D. , Drecun Luka , Saraon Punit , Stagliar Igor , Mulligan Lois M.
Published: July 2021

The REarranged during Transfection (RET) receptor tyrosine kinase is pivotal for normal tissue development, but is also an oncogene driver involved in several human cancers. Alternative splicing at the 3' end of the RET gene leads to expression of two conserved protein isoforms, RET9 and RET51, that differ in their subcellular localization and protein trafficking, as well as their functional roles in tumorigenesis and metastatic processes. Importantly, RET9 and RET51 have unique C-terminal phospho-tyrosine binding sites, suggesting that they may also differ in their interactomes. We used a combination of cell-based screening and in silico approaches to identify novel potential interaction partners of RET isoforms. We performed a Mammalian Membrane Two-Hybrid (MaMTH) screen using a library of SH2 domain-containing adaptor and signaling proteins, to identify interactions with each RET isoform. We complemented these studies by using sequence homology detection models (HMM, PSSM), based on SH2 domain sequences known to interact with RET, to rank the SH2 library members and predict novel interactions. Independently, we compared published consensus binding sequences for each SH2 domain library member with predicted RET phosphotyrosine motifs to identify potential interactors. Predicted interactions were validated in co-immunoprecipitation assays. We confirmed previously known interactions of RET with SH2 domain proteins including SHC1, GRB2 and GRB10, and identified additional novel RET-binding proteins, a subset of which showed differential interactions that were mediated through RET isoform-specific docking sites. Our results suggest that combinations of distinct interaction partners may contribute to RET isoform-specific functions. Together, our research has developed a systematic approach to map and characterize RET isoform interactions. Our data suggest that no single method identified all confirmed RET interactions, and that a combination of multiple approaches improves characterization of growth factor receptor interactomes.

Citation Format: Samira Kheitan, Annika E. Pedersen, Brandy D. Hyndman, Luka Drecun, Punit Saraon, Igor Stagliar, Lois M. Mulligan. A systemic approach to decipher the interactome of RET receptor isoforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2450.
Abstract 2452: Metformin administration in hypoglycemia compromises cancer cell viability in glioblastoma initiating cells
Authors: Cianci Francesca , Cazzoli Riccardo , Verduci Ivan , Minucci Saverio , Mazzanti Michele
Published: July 2021

Cancer cells show higher energy and biomolecules requirements than healthy cells, making the targeting of cancer metabolic dysfunctions an appealing therapeutic approach. Unfortunately, tackling the tumor metabolism is a daunting task, due to the ability of cancer cells to re-organize their metabolism switching between glycolysis and oxidative phosphorylation. Cancer metabolic plasticity and re-programming is partially supported by altered ionic channels expression and activity, our laboratory has described Chloride Intracellular Channel 1 (CLIC1) as a marker of malignancy in tumor cells and a possible therapeutic target. CLIC1 is a metamorphic protein able to switch between cytoplasmic and transmembrane forms (tmCLIC1), the latter characterized by chloride conductance. TmCLIC1 was found to be overexpressed in several solid tumors where supports cancer proliferation and growth, while it is mainly absent in healthy cells. Recently, it has been demonstrated that tmCLIC1 activity is affected by biguanide compounds, in particular metformin. Metformin was recently repurposed as an anticancer drug with unclear mechanisms. We demonstrated that in hypoglycemia, metformin displays increased toxic effect on cancer cells, inducing apoptosis. Metformin inhibits Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A), and hypoglycemia upregulates PP2A subunit B56δ targeting the Glycogen Synthase Kinase 3 Beta (GSK3b). Metformin treatment in combination with hypoglycemia leads to de-phosphorylation of GSK3b protein resulting in degradation of induced myeloid leukemia cell differentiation protein (MCL-1), a pro-survival protein, and cell death (Elgendy et al., Cancer Cell 2019). In primary glioblastoma cell lines enriched in tumor-initiating-cells (GSCs), metformin has proven to be effective in reducing cell growth without affecting viability (Gritti et al., Oncotarget 2014). Here, we are going to show studies trying to establish whether CLIC1 is a component of the established PP2A-mediated signaling network. Our work aims to clarify metformin molecular mechanism of action and to assess a new potential therapeutic approach for solid tumors in which tmCLIC1 is found to be overexpressed.

Citation Format: Francesca Cianci, Riccardo Cazzoli, Ivan Verduci, Saverio Minucci, Michele Mazzanti. Metformin administration in hypoglycemia compromises cancer cell viability in glioblastoma initiating cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2452.
Abstract 2454: Vitamin D-mediated anti-cancer activity involves iron homeostatic balance disruption and oxidative stress induction in breast cancer
Authors: Bajbouj Khuloud , Shafarin Jasmin , Sahnoon Lina , Al-Ali Abeer , Muhammad Jibran Sualeh , Guraya Salman Y. , Hamad Mawieh
Published: July 2021

Background: Vitamin D deficiency has long been associated with increased risk of breast cancer (BC). Moreover, increased cellular iron content is emerging as a biomarker of BC metastasis. Although the connection between vitamin D and cellular iron content in BC remains ambiguous, recent studies have shown that vitamin D exerts some of its anti-cancer effects by modulating the expression of key iron regulatory genes (IRGs). Herein, we addressed the question of whether vitamin D signaling plays a role in cellular iron homeostasis and whether this has a effects on breast cancer cell survival and growth.

Experimental Methods: Bioinformatics analysis from publicly available transcriptomic data set has been utilized to profile the expression status of key IRGs in vitamin D-treated breast cancer cells. Additionally, hormonal-responsive (MCF-7) and triple negative (MDA-MB-231) BC cells were treated with 1α,25-dihydroxyvitamin D3 (calcitriol; the vitamin D active form) and evaluated for labile iron content, expression of IR proteins, oxidative stress, and cell survival.

Results and Discussions: Bioinformatics analysis revealed that several IRGs including hepcidin, ferroportin, ferritin and transferrin receptor as well as genes related to metabolic cellular stress are differentially expressed in BC cells. Vitamin D treatment resulted in cellular iron depletion and differentially affected the expression of key IR proteins. Moreover, vitamin D induced oxidative stress and altered the redox balance in treated cells by reducing catalase protein levels and increasing the expression of several stress-related markers including γ-H2AX, hypoxia-inducible factor 1 alpha and hemoxygenase 1. Consecutively, BC cell survival was significantly decreased in vitamin D-treated cells.

Conclusion: Collectively, data presented here demonstrate that vitamin D disrupts iron homeostasis, augments oxidative damage and reduces BC cell survival and growth.

Citation Format: Khuloud Bajbouj, Jasmin Shafarin, Lina Sahnoon, Abeer Al-Ali, Jibran Sualeh Muhammad, Salman Y. Guraya, Mawieh Hamad. Vitamin D-mediated anti-cancer activity involves iron homeostatic balance disruption and oxidative stress induction in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2454.
Abstract 2457: NANOG bindsPRC2 to reprogram metabolismandpromotelabel-retaining populationin experimental mouse and clinical carcinoma
Authors: Yeh Da-Wei , Hernandez Juan Carlos , Machida Keigo
Published: July 2021

Background: Alcohol and HCV synergistically promote hepatocarcinogenesis. Our previous studies indicated that this synergistic effect of HCV and alcohol was mediated by TLR4, which increases the expression of NANOG, a transcription factor important for the stemness of TICs. Our recent studies further indicated that NANOG could repress mitochondrial oxidative phosphorylation (OXPHOS) genes to support the self-renewal and drug resistance of tumor-initiating stem cell-like cells (TICs), leading to relapse and metastasis (Chen et al., Cell Metabolism). In addition, approximately 39% of HCV-alcohol-associated HCCs have mutations in ARID1A, a component of the chromatin remodeling complex SWI/SNF.

Hypothesis: The efficacy of the combined FAOi and PRC2 inhibition (PRC2i) therapy relies on activation of cell death signaling.

Aims: We (1) tested if NANOG together with the PRC2 complex inhibits OXPHOS and generates TICs in the presence of ARID1A mutations; (2) tested if specific genetic alterations and NANOG cooperate to generate different classes of TICs for the development of HCC; (3) tested if ARID1A mutations with NANOG induction generate TICs.

Methods: We transduced LPCs with a lentiviral vector that expresses mCherry and used the CRISPR/Cas9 system to target Arid1a or Ctnnb1. EZH2, SUZ12 and EED were silenced in patient-derived TICs (with or without ARID1A mutations). Surviving cells are subjected to the spheroid formation assay on ultra-low attachment plates for analysis of their self-renewal ability.

Results: Mutant Arid1a HCCs with Nanog overexpression caused HCC metastasis to the lung. The humanized FRG mice subjected to alcohol feeding and/or HCV infection developed HCCs, especially in the CTNNB1 or ARID1A mutant hepatoblast groups, six months post-HCV infection and/or alcohol feeding. HCV-infected humanized livers in these mice contained HCV RNA, but not in the UV-irradiated HCV infected group.

Conclusion: ARID1A gene loss promotes sorafenib-resistance phenotype and self-renewal through PRC2 complex genes. We demonstrated that sorafenib induced cytochrome c release from mitochondria to cause apoptosis of TICs, that restoration of OXPHOS was overexpressed in TICs. The studies established a novel therapy to overcome the chemoresistance of TICs for HCC treatment.

Citation Format: Da-Wei Yeh, Juan Carlos Hernandez, Keigo Machida. NANOG bindsPRC2 to reprogram metabolismandpromotelabel-retaining populationin experimental mouse and clinical carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2457.
Abstract 2466: Stochastic loss of GLUL expression correlates with STK11-loss-dependent glutamine addiction and may impact anti-PD1 therapy resistance in NSCLC
Authors: Lenahan Sean M. , Sarausky Hailey M. , Seward David Joseph
Published: July 2021

Our work aims to delineate the molecular interactions linking somatic disruption of Serine-Threonine Kinase 11 (STK11) with anti-PD1 therapy resistance in KRAS-driven lung adenocarcinoma (LUAD). Data presented here suggests glutamine synthetase (GLUL) may modulate this phenotype. Immune checkpoint inhibitors (ICIs), including anti-PD1 monoclonal antibody therapy, represent a promising therapeutic option for non-small cell lung cancer (NSCLC) patients. Unfortunately, efficient use of these drugs is hampered by an inability to accurately predict which patients will respond. Clinical observations have correlated anti-PD1 therapy resistance in KRAS-driven LUAD with somatic STK11 disruption, but the mechanism(s) responsible remains unknown. To investigate this connection we knocked out STK11 in three different human KRAS-driven LUAD cell lines and performed RNA-seq analysis to identify STK11-loss-dependent differentially expressed genes. Intriguingly, GLUL expression was reproducibly lost in a subset of KRAS-driven/STK11 null clones across two different human lung adenocarcinoma cell lines. GLUL is an enzyme that converts glutamate to glutamine. Glutamine is a critical metabolite supplying carbon and nitrogen necessary for amino acid, nucleotide, and lipid synthesis. Additionally, glutamine plays an important role in redox homeostasis, chromatin organization, and intra-tumoral signaling. Glutamine dependency is a common phenotype across many solid tumor types, and has previously been linked with STK11-deficiency in the context of polycystic kidney disease. Our current work suggests loss of GLUL expression correlates with an altered tumor-intrinsic response to nutrient stress in KRAS-driven LUAD culminating in immune evasion and anti-PD1 therapy resistance. Ongoing experiments aimed to define the relationship between STK11 loss and GLUL expression suggest this phenomenon is stochastic in nature and may provide tumor cells with a selective advantage. In conclusion, understanding the biology underlying the molecular connection between STK11 and GLUL may reveal reliable biomarkers for anti-PD1 therapy response and suggest a method for restoring sensitivity to current therapies.

Citation Format: Sean M. Lenahan, Hailey M. Sarausky, David Joseph Seward. Stochastic loss of GLUL expression correlates with STK11-loss-dependent glutamine addiction and may impact anti-PD1 therapy resistance in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2466.
Abstract 2467: Adiponectin receptor agonism induces a metabolic switch in pancreatic cancer cells from oxidative phosphorylation to glycolysis
Authors: Manley Sharon J. , Jack Jarrid , Ambrose Joseph , Olou Appolinaire A. , VanSaun Michael N.
Published: July 2021

Understanding the impact of risk factors on the progression of pancreatic cancer is critical for the development of novel therapeutics. Multiple studies have clearly shown that obesity positively correlates with increased progression of pancreatic adenocarcinoma (PDAC). We have demonstrated that individual adipose derived cytokines (aka adipokines) can elicit either pro-tumorigenic or anti-tumorigenic effects on PDAC progression. We previously demonstrated that adiponectin receptor agonism effectively suppresses pancreatic cancer growth in vitro as well as in vivo, through suppression of MAPK as well as STAT3 pathways. Yet, adiponectin is well known to stimulate the AMPK pathway and therefore we hypothesized that adiponectin agonism could also be affecting metabolic function in PDAC. In our current studies, we defined multiple metabolic alterations in pancreatic cancer cells in response to adiponectin receptor agonism using a seahorse-based assay. Human and murine PDAC cell lines were seeded into XFe96 microplates and subjected to a mitochondrial stress test as well as the glycolysis stress test. Our results demonstrate that adiponectin receptor activation impairs pancreatic cancer mitochondrial function leading to a metabolic switch from mitochondrial respiration to glycolysis. We further demonstrate that this switch is associated with AMPK phosphorylation-mediated inhibition of Acetyl Coenzyme A Carboxylase (ACC), the lipogenic enzyme whose inhibition induces fatty acid breakdown. Thus, our results support a role for adipose-secreted factors to influence the metabolic phenotype of pancreatic cancer. Since metabolic rewiring is often associated with development of chemotherapeutic resistance in cancer, we are currently testing whether dual therapeutic targeting of the mitochondrial respiration, via adiponectin receptor agonism, and glycolysis would provide an efficacious therapeutic.

Citation Format: Sharon J. Manley, Jarrid Jack, Joseph Ambrose, Appolinaire A. Olou, Michael N. VanSaun. Adiponectin receptor agonism induces a metabolic switch in pancreatic cancer cells from oxidative phosphorylation to glycolysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2467.
Abstract 2468: The role of autophagy in lymphangiosarcoma development
Authors: Yang Fuchun , Haas Michael , Guan Jun-Lin
Published: July 2021

Lymphangiosarcoma is a rare malignant vascular tumor which is formed from aberrant proliferation of endothelial cells (ECs). We previously established an endogenous mouse model of lymphangiosarcoma by conditional knockout of Tsc1 gene in ECs and revealed that hyper-activated mTORC1 is required for the vascular tumor development and progression through increased VEGF autocrine signaling. Using this model, we investigated the role of an essential autophagy gene FIP200 (FAK family interacting protein of 200 kDa) and its canonical autophagy function in lymphangiosarcoma development and progression by generating a FIP200 knockout (designated as Tsc1/Fip200 2cKO mice) or introducing a knockin allele that specifically blocks its canonical autophagy function (designated as Tsc1/Fip200 cKI mice) in combination with Tsc1 cKO in this study. We found that while both Tsc1/Fip200 cKO and Tsc1/Fip200 cKI mice exhibited lymph edema phenotype as Tsc1 cKO mice, they did not develop lymphangiosarcoma, suggesting that autophagy is required for lymphangiosarcom formation. Consistent with the above results, we also found that CRISPR-Cas9 mediated Fip200 KO in an immortalized vascular tumor cell line Tsc1ΔEC from Tsc1 cKO mice blocked their ability to grow tumors in xenograft transplants. Mechanistically, we found that autophagy was required for maintaining mTORC1 hyper-activation under nutrient stress condition. Moreover, the impaired lipophagy by Fip200 knockout affected lipid catabolism under glucose free condition and thus reduced the substrate for fatty acid oxidation (FAO). Our data showed that Fip200, Atg5 or Atg7 ablation in Tsc1ΔEC inhibited mitochondria respiration and decreased ATP contents as measured by Seahorse assays. Either lipase inhibitor Orlistat or carnitine palmitoyltransferase-1 (Cpt1) inhibitor Etomoxir treatment decreased Tsc1ΔEC mitochondria respiration and ATP production. Collectively, our data suggested that autophagy is required for lymphangiosarcoma initiation and progression by modulating FAO to supply cellular energy under stress for sustaining mTORC1 hyper-activation for tumor growth.

Citation Format: Fuchun Yang, Michael Haas, Jun-Lin Guan. The role of autophagy in lymphangiosarcoma development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2468.
Abstract 2472: Effective ER antagonists resist conformational restrictions imposed by somatic mutation but do not correlate with effects on receptor stability in live cells
Authors: Hosfield David , Li Nan-Sheng , Han Ross , Laine Muriel , Weber Sandra , Sauvage Madaline , Madar Sylvie , Greene Geoffrey , Fanning Sean
Published: July 2021

Approximately 50% of luminal breast cancer patients become resistant to first-line endocrine therapies after an average of 5 years. Acquired activating mutations to the ESR1 ligand binding domain enable hormone therapy resistance in approximately 25-40% of these patients. Competitive estrogen antagonists function by maintaining the receptor in a transcriptionally inactive state by forcing estrogen receptor alpha ligand binding domain (ERα LBD) helix 12 into the AF-2 cleft to prevent the docking of LXXLL-containing transcriptional coactivators. Multiple next-generation ERα antagonists that function as molecular degraders of the receptor have been described and shown activity in breast cancers harboring the activating ESR1 mutations. To understand whether ERα antagonism correlates with receptor stability, we examined a comprehensive panel of structurally diverse antagonists in live breast cancer cell assays using WT, Y537S, and D538G-mutant receptors. We found that potent antagonists are able to both stabilize and degrade the receptor and further demonstrate that the D538G mutation reduces turnover with ICI due to specific defects in receptor ubiquitination and SUMOylation. High resolution x-ray crystal structures of WT and Y537S ERα LBD in complex with stabilizing and degrading antiestrogens suggest that modulators that enforce the classical antagonist-like helix 12 conformation in the mutant receptor achieve the greatest activity in transcriptional assays, regardless of their effects on receptor stability

Citation Format: David Hosfield, Nan-Sheng Li, Ross Han, Muriel Laine, Sandra Weber, Madaline Sauvage, Sylvie Madar, Geoffrey Greene, Sean Fanning. Effective ER antagonists resist conformational restrictions imposed by somatic mutation but do not correlate with effects on receptor stability in live cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2472.
Abstract 2487: disrupting binding of p53 mutants to PEPD unleashes their tumor suppressor activities
Authors: Yang Lu
Published: July 2021

Background & Goals: The p53 tumor suppressor is one of the most important determinants of cell fate and is critical for tumor suppression. It functions by regulating the expression of a plethora of genes via sequence-specific DNA binding and also by transcription-independent, mitochondria-mediated mechanisms. Not surprisingly, the TP53 gene, which encodes p53, is frequently mutated in human cancer. For example, TP53 is mutated in about 75% of HER2-positive breast cancer and about 85% of triple negative breast cancer. The majority of TP53 mutations are missense mutations, changing one amino acid per mutant, most of which occur in its DNA-binding domain. p53 mutants show loss of tumor suppressor functions or gain of oncogenic functions. We recently found that peptidase D (PEPD), also known as prolidase, binds via its C-terminal sequence to the proline-rich domain (PRD) of wild type p53 and that disrupting the binding activates p53. The enzymatic activity of PEPD is not required for this function. Our objective in the present study is to determine whether PEPD also regulates p53 mutants. We focused on some of the most common oncogenic p53 mutants, including R175H, R248Q, R273H and R280K.

Results: Our study shows that 1) each p53 mutant binds directly to PEPD and binds to PEPD in cells in a significant quantity, 2) disrupting their binding in cells by PEPD knockdown reactivates each p53 mutant, unleashing both transcription-dependent and -independent tumor suppressing activities, 3) after leaving PEPD, p53 mutants undergo acetylation, mono-ubiquitination and phosphorylation, which drive their refolding and reactivation, 4) Lysine 373 acetylation is essential for their reactivation, and 5) p53 mutants and p53WT show almost no difference in PEPD-binding and response to PEPD KD.

Conclusions & Therapeutic Relevance: In conclusion, we have identified a cellular mechanism by which the tumor suppressor functions of p53 mutants are strongly reactivated, which is neither cell-specific nor mutant-specific. Mainly, p53 mutants bind to PEPD in cancer cells, and disrupting the complex causes posttranslational modifications of mutants freed from PEPD, which drive their refolding and unleash their tumor suppressor activities. This discovery advances our understanding of the function and regulation of p53 mutants and breaks new ground for developing novel cancer treatment strategies.

Citation Format: Lu Yang. disrupting binding of p53 mutants to PEPD unleashes their tumor suppressor activities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2487.
Abstract 2497: Inositol Polyphosphate 4-Phosphatase Type-II B protects mice from benign prostatic hyperplasia
Authors: Ceyhan Yasemin , Zhang Manqi , Agoulnik Irina U.
Published: July 2021

BPH is the most common tumor-like condition that afflicts more than 50% of men between the ages of 51-60 and over 90% in men older than 80. While connection between obesity and BPH is well established, the driving signaling pathways are not clear. BPH is a nonmalignant enlargement of prostate epithelium that requires androgen receptor (AR) signaling. INPP4B and EZH2 are highly expressed in prostate epithelium where they regulate AR signaling and play an important role in prostate cancer etiology. EZH2 is an epigenetic silencer that methylates histone H3 on K9 and K27 residues and INPP4B is a dual specificity phosphatase that dephosphorylates PI(3,4)P2 and PI(4,5)P2, membrane lipids that activate Akt and PKC signaling pathways respectively. We discovered that Inpp4b-/- male mice develop BPH at 3 months old of age and subsequently prostate intraepithelial neoplasia (PIN) when fed high fat diet. While expression of AR in the prostates of these mice is similar, AR activity is reduced. The loss of INPP4B caused reduction in EZH2 protein levels in prostate cancer cell lines and in mouse prostate. Inhibition of Akt and PKC signaling reduced EZH2 levels in ventral and dorsolateral prostate lobes of Inpp4b-/- males, suggesting that INPP4B upregulates EZH2 levels by suppressing Akt and PKC signaling pathways. Consistent with decline in EZH2 levels, Inpp4b-/- males have reduced levels of H3K27me3 and H3K9me2 in the ventral prostate, and high fat diet exacerbated this phenotype. Our data suggests that INPP4B modulates epigenetic profile of prostate epithelium by stabilizing EZH2 levels. Both loss of INPP4B and high fat diet treatment lead to decline in EZH2 levels and activity and contribute to development of BPH in mouse model.

Citation Format: Yasemin Ceyhan, Manqi Zhang, Irina U. Agoulnik. Inositol Polyphosphate 4-Phosphatase Type-II B protects mice from benign prostatic hyperplasia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2497.