bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2288: A novel enzymatic fragmentation library preparation chemistry that greatly reduces sequencing artifacts
Authors: Jaafar Zane , Haimes Josh , Harrison Thomas , Peterkin Lindsay , Ranik Martin , Scott Kristin , Kudlow Brian A.
Published: June 2022

After a decade of NGS technology and workflow innovation, personalized medicine is starting to become a reality. DNA fragmentation is still a critical bottleneck during library preparation. Sonication methods have been the gold standard for consistent fragmentation and uniform GC coverage, but are associated with a high upfront investment, expensive consumables, and are prone to oxidative DNA damage. On the other hand, enzymatic fragmentation methods hold the potential to be scalable, automation-friendly workflows that minimize DNA damage. Yet, typical enzymatic fragmentation methods have been shown to introduce systematic hairpin artifacts, exhibit suboptimal uniformity, and have a narrow range of input mass compatibility. We have developed a novel enzymatic fragmentation-based library preparation technology which, together with our high-fidelity library amplification module, effectively overcomes many of the key limitations of related chemistries. Libraries generated using our workflow reduced chimeric reads and terminal hairpin artifacts 10-fold compared to other enzymatic methods, and reached comparable levels of mechanically sheared DNA controls. Furthermore, we observed consistently even coverage uniformity and low sequence-specific bias for human whole genome sequencing. Fragmentation was tested across a broad sample input range from 100 pg to 500 ng. Library insert sizes were highly tunable from 150 bp to 550 bp and consistent across the input titration. High quality libraries with minimal adapter dimer were prepared from as little as 1 picogram of gDNA by adjusting the post amplification clean up conditions. Taken together, this enzymatic fragmentation and library preparation workflow avoids library preparation artifacts that convolute variant calling, is highly scalable, and suitable for ultra-low input samples.

Citation Format: Zane Jaafar, Josh Haimes, Thomas Harrison, Lindsay Peterkin, Martin Ranik, Kristin Scott, Brian A. Kudlow. A novel enzymatic fragmentation library preparation chemistry that greatly reduces sequencing artifacts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2288.
Abstract 2298: OTUD6B is a dependency in multiple myeloma that drives S-phase entry via MYC activation
Authors: Spallek Ria , Paulmann Carmen , Karpiuk Oleksandra , Zecha Jana , Klaeger Susan , Schaeffer Isabell , Öllinger Rupert , Engleitner Thomas , Krönke Jan , Wirth Matthias , Keller Ullrich , Rad Roland , Kuster Bernahrd , Bassermann Florian
Published: June 2022

Multiple myeloma (MM) is the second most common hematological malignancy and remains incurable, thus demanding for new therapeutic targets. While the pathophysiology of MM is poorly understood, the substantial responsiveness of MM patients to proteasomal inhibitors (PIs) like bortezomib or carfilzomib hints towards a central role of the ubiquitin proteasome system (UPS). Deubiquitylases (DUBs) are therapeutically targetable components of the UPS, whose inhibition can destabilize oncoproteins. However, the identities of oncoprotein-regulating DUBs remain largely elusive. To identify new vulnerabilities in MM, a CRISPR/Cas9 screen targeting all human DUBs was performed. For validated candidates, phenotypical analysis regarding proliferation and cell cycle progression was performed, as well affinity and non-affinity mass spectrometry-based screens to identify substrates. We thereby identified OTUD6B as a novel oncogene that drives G1/S-transition. LIN28B, a suppressor of microRNA biogenesis, was delineated as both a cell cycle-specific deubiquitylation substrate and activator of OTUD6B. RNA-Seq and qPCR analyses of OTUD6B and LIN28B depleted MM cells revealed that the stabilization of LIN28B drives MYC expression and activity at the G1/S transition, which in turn allows for rapid S-phase entry. Thus, silencing of OTUD6B as well as LIN28B inhibited MM outgrowth in xenograft experiments. Analyses of large MM patient cohorts revealed a progressive increase of OTUD6B expression along the transition from normal plasma cells to MGUS to MM and that high expression of OTUD6B was associated with a significantly adverse overall survival. Furthermore, OTUD6B expression was found to strongly correlate with MYC expression and significantly reduced progression-free survival in patients treated with the PI bortezomib. Knockout of OTUD6B in MM cells significantly enhance the anti-myeloma activity of the drug when using sub-lethal doses. Together, these results validate OTUD6B as a new therapeutically targetable oncogene, dependency, and prognostic factor in MM, that eventually serves as a master regulator of MYC activity to drive cell cycle progression.

Citation Format: Ria Spallek, Carmen Paulmann, Oleksandra Karpiuk, Jana Zecha, Susan Klaeger, Isabell Schaeffer, Rupert Öllinger, Thomas Engleitner, Jan Krönke, Matthias Wirth, Ullrich Keller, Roland Rad, Bernahrd Kuster, Florian Bassermann. OTUD6B is a dependency in multiple myeloma that drives S-phase entry via MYC activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2298.
Abstract 2302: The pan-cancer regulator of the mutant TERT promoter and a new model of the GABP-TERT axis
Authors: Stevers Nicholas O. , Barger Carter , Lenzo Olivia , Hong Chibo , Soczek Katarzyna , Wu Samuel H. , McKinney Andrew M. , Suwala Abigail , Doudna Jennifer A. , Costello Joseph F.
Published: June 2022

Ninety percent of human tumors reactivate telomerase reverse transcriptase (TERT) to achieve cellular immortality. The route to immortality found in over 50 cancer types is mutation of the TERT promoter (TERTp) mutations. Studies in glioblastoma have shown the two most common TERTp mutations, G228A and G250A, reactivate TERT expression through de novo E26 transformation specific (ETS) site generation that, in tandem with a native TERTp ETS site, recruits the tetrameric GA-binding protein (GABP) complex formed by two GABP alpha (GABPA) subunits and two tetramer specific GABPB1 (B1) subunits, GABPB1L (B1L). While ten additional TERTp mutations have been described across cancer types, functional studies have been limited to select mutations and cancer types. We found that each de novo ETS motif generating TERTp mutation increases TERTp transcriptional activity in a GABP-dependent manner. Furthermore, our pan-cancer analysis demonstrated selective enrichment of GABPA at the mutant TERTp across 14 cancer types and further determined that this recruitment is necessary for TERT expression. Prior investigations found that CRISPR-cas9 mediated mutagenesis of the B1 ninth exon, specific to B1L, reduces TERT transcriptional activity in a TERTp-mutation dependent manner. We explored B1L targeting in the reversal of tumor cell immortality in several cancers. We found that the B1 dimer restricted isoform, GABPB1S (B1S), is consistently and significantly increased following B1L reduction, which we determined to be the consequence of a disengagement of a GABP tetramer mediated negative feedback loop acting on the B1 promoter. Interestingly, this upregulated B1S expression enables GABP dimer binding and activation of the mutant TERTp thereby compensating for B1L loss. Indeed, knockout of B1L and B1S together resulted in a near complete elimination of GABPA TERTp recruitment and a striking reduction in TERT expression, leading to telomere shortening and ultimately resulting in tumor cell death and senescence. This data identifies GABP as the master regulator of the mutant TERTp across cancer type and de novo ETS mutation type and suggests a new model of the GABP-TERT axis involving both the GABP tetramer, and the GABP dimer.

Citation Format: Nicholas O. Stevers, Carter Barger, Olivia Lenzo, Chibo Hong, Katarzyna Soczek, Samuel H. Wu, Andrew M. McKinney, Abigail Suwala, Jennifer A. Doudna, Joseph F. Costello. The pan-cancer regulator of the mutant TERT promoter and a new model of the GABP-TERT axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2302.
Abstract 2313: Fenbendazole induces cell cycle arrest in colorectal cancer cells and patient-derived colon cancer organoids
Authors: Kang Kyung-Sun , Kim Da-Hyun
Published: June 2022

Colorectal cancer is one of leading cause of cancer-related deaths. Therefore, there have been various attempts to cure the cancer by developing new efficient anti-cancer therapy in addition to surgical resection and chemotherapy. In this study, we investigated the effects of fenbendazole, an anti-helminthic drug, both colon cancer cells and patient-derived colon tumor organoids. Notably, we employed 3D tumor organoid models because 2D-cultured cell lines were not able to recapitulate the physiology of solid tumors. We first observed that treatment of fenbendazole to colon cancer cells induced apoptosis within 24 hours, which was extended for a long-term. We revealed that fenbendazole markedly suppressed proliferation rate via cell cycle arrest. Cell cycle progression is elaborately regulated by multiple genes, such as cyclins and cyclin-dependent kinases (CDKs). From a screening of cell cycle-related factors, we found that the protein levels of CDK1 phosphorylated at Tyr15 and cyclin B1 which was known to regulate M phase transition, were drastically downregulated when the tumor cells were exposed to fenbendazole. Next, colorectal tumor-bearing mouse model was established using AOM/DSS. Oral administration of fenbendazole into the mouse not only reduced the number of tumor cells but also lowered tumor grades. Overall, our study suggested a possibility that fenbendazole could be applied for anti-cancer therapy by targeting cell cycle arrest.

Citation Format: Kyung-Sun Kang, Da-Hyun Kim. Fenbendazole induces cell cycle arrest in colorectal cancer cells and patient-derived colon cancer organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2313.
Abstract 2324: Small-scale microwave-assisted acid hydrolysis method for glycogen determination and turnover in tumors using Stable Isotope Resolved Metabolomics
Authors: Lane Andrew N. , Scott Timothy L. , Zhu Juan , Cassel Teresa A. , Vicente-Munoz Sara , Lin Penghui , Higashi Richard M. , Fan Teresa W-M
Published: June 2022

Introduction: Glycogen is a readily deployed intracellular energy storage macromolecule composed of branched chains of glucose. Although glycogen primarily occurs in the liver and muscle, it can be found in most tissues throughout the body, and its metabolism has been shown to be important in cancers and immune cells. Robust analysis of glycogen turnover requires stable isotope tracing plus a reliable means of quantifying total and labeled glycogen derived from precursors such as 13C6-glucose. Current methods for analyzing glycogen are time- and sample-consuming, at best semi-quantitative, and unable to measure stable isotope enrichment.

Methods: We have developed a microscale method for quantifying both intact and acid-hydrolyzed glycogen by ultra-high-resolution Fourier transform mass spectrometric (UHR-FTMS) and/or NMR analysis in stable isotope resolved metabolomics (SIRM) studies. Polar metabolites, including intact glycogen and their 13C positional isotopomer distributions were first measured in crude biological extracts by high resolution NMR, followed by rapid and efficient acid hydrolysis to glucose in 1 N HCl for 10 minutes at 110 °C under a N2 atmosphere in a microwave-assisted synthesis reactor. The resulting glucose and its 13C isotopologues were then analyzed by UHR-FTMS and/or NMR.

Results: We optimized the microwave digestion time, temperature, and oxygen purging in terms of recovery versus degradation and found 10 minutes at 110-115 °C to give > 90% recovery. The method was applied to track the fate of 13C6-glucose in primary human lung BEAS-2B cells, human macrophages, murine liver and patient-derived tumor xenograft (PDTX) in vivo, and the fate of 2H7-glucose in ex vivo lung organotypic tissue cultures of a lung cancer patient. We showed the incorporation of 13C6-glucose into glycogen and its metabolic intermediates, UDP-Glucose and glucose-1-phosphate, both in terms of the 13C levels and fractional enrichment, thereby demonstrating the utility of the method in tracing glycogen turnover in cells and tissues.

Conclusions: The method offers a quantitative, sensitive, and convenient means to analyze glycogen turnover in mg amounts of complex biological materials.

Keywords: glycogen turnover; 13C6-glucose, stable isotope resolved metabolomics (SIRM); microwave-assisted hydrolysis.

Citation Format: Andrew N. Lane, Timothy L. Scott, Juan Zhu, Teresa A. Cassel, Sara Vicente-Munoz, Penghui Lin, Richard M. Higashi, Teresa W-M Fan. Small-scale microwave-assisted acid hydrolysis method for glycogen determination and turnover in tumors using Stable Isotope Resolved Metabolomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2324.
Abstract 2328: Metabolomics pathways and biomarkers in predicting breast cancer prognosis
Authors: Hu Jennifer J. , Takita Cristiane , Reis Isildinha M. , Yang George , Zhao Wei , Lee Eunkyung
Published: June 2022

Breast cancer is the most frequently diagnosed cancer in women and the second leading cause of cancer death in Americans. With more than 3 million breast cancer survivors in the US, a number that is projected to increase, it is important to identify targets for precision intervention to improve breast cancer prognosis. With the rapid advancement of technology for metabolomics, the results from several recent studies have shown that metabolomics may have applications in breast cancer diagnosis and subtype analysis, characterization of heterogeneity of breast cancer, and prognosis. In the current study, we performed a global urinary metabolomic analysis of 120 breast cancer patients: 60 progression-free (PF) cases as the reference group and 60 with progressive disease (PD: recurrence, second primary, metastasis, or death). The urine samples were collected immediately after radiotherapy. Using UPLC-MS/MS and GC-MS, Metabolon Inc. identified a robust set of 1,742 biochemicals (1,258 known and 484 unknown structure). The most notable differences between PF and PD patients involved multiple pathways and metabolites include: carbohydrate metabolism (e.g., glucose, sedoheptulose, and N6-carboxymethyllysine), branch-chain amino acid metabolism (e.g., alpha-hydroxyisocaproate and beta-hydroxyisovalerylglycine), phosphatidylcholine metabolism (e.g., 1-palmitoyl-2-oleoyl-GPC (16:0/18:1) and 1-palmitoyl-2-linoleoyl-GPC (16:0/18:2)), arginine metabolism (e.g., dimethylarginine, N-acetylcitrulline, and homocitrulline), oxidative stress-related metabolites (e.g., cysteine-glutathione disulfide, gamma-glutamylisoleucine, and gamma-glutamylthreonine), androgenic steroids (Dehydroepiandrosterone sulfate (DHEA-S) and 16a-hydroxy DHEA 3-sulfate), and nucleotide metabolism. Some of these identified metabolomic differences may serve as potential predictive biomarkers of breast cancer prognosis. In summary, with increasing interest in targeting tumor metabolism in precision medicine and our pilot data suggesting multiple metabolic pathways in predicting breast cancer prognosis, future research is warranted to validate our findings and identify metabolomic targets for precision interventions.

Citation Format: Jennifer J. Hu, Cristiane Takita, Isildinha M. Reis, George Yang, Wei Zhao, Eunkyung Lee. Metabolomics pathways and biomarkers in predicting breast cancer prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2328.
Abstract 235: Differential polyamine expression in human pancreatic cancer supports the use of polyamine blockade therapy to modulate the in vivo pancreatic tumor microenvironment
Authors: Nakkina Sai Preethi , Gitto Sarah B. , Pandey Veethika , Parikh Jignesh , Geerts Dirk , Maurer Carlo , Olive Kenneth P. , Phanstiel Otto , Altomare Deborah A.
Published: June 2022

Pancreatic cancer has a poor five-year survival rate of less than 8% and is projected to be the second leading cause of cancer related deaths in the US by the year 2030. Polyamine metabolism is an underexplored therapeutic target in pancreatic ductal adenocarcinoma (PDAC). The current investigation profiled RNA expression of genes associated with polyamine metabolism, transport and homeostasis in clinical pancreatic cancer samples to reveal evidence for dysregulation of polyamine-based pathways in PDAC. Increased expression of select pro-polyamine genes was associated with poorer patient prognosis in data obtained from the TCGA dataset. Preclinical experimental data supported the use of polyamine blockade therapy (PBT) in pancreatic tumor bearing mice, and revealed that PBT increased survival and decreased tumor weights in comparison to control treatment. PBT therapeutic effects were associated with increased expression of CD86 T cell co-stimulatory marker present on antigen presenting cells in the tumor microenvironment. Collectively, polyamine dysregulation in human pancreatic cancer is associated with poorer patient prognosis, whereas PBT treatment resulted in improved in vivo therapeutic outcomes in part through immune modulation in the tumor microenvironment.

Citation Format: Sai Preethi Nakkina, Sarah B. Gitto, Veethika Pandey, Jignesh Parikh, Dirk Geerts, Carlo Maurer, Kenneth P. Olive, Otto Phanstiel, Deborah A. Altomare. Differential polyamine expression in human pancreatic cancer supports the use of polyamine blockade therapy to modulate the in vivo pancreatic tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 235.
Abstract 2379: Gender-specific metabolome in bladder cancer: Role of EPHX2 in bladder cancer
Authors: Hassan Mohammed Khurshidul , Borkar Roshan , Reddy Karthik Reddy Kami , Piyarathna Danthasinghe Waduge Badrajee , Amara Chandra Shekar , Bellman Allison , Ambati ChandraShekar R. , Putluri Vasanta , Kamal Abu Hena Mostafa , Bollag Roni J. , Terris Martha K. , Ballester Leomar Y. , Lotan Yair , Coarfa Cristian , Sreekumar Arun , Putluri Nagireddy
Published: June 2022

Bladder cancer (BLCA) is the fourth most common cancer diagnosed in American men. In the U.S., men are four times more likely to be diagnosed with bladder cancer than women. Our research group has previously identified alterations in xenobiotic metabolism in BLCA. In this study, using a metabolomics approach, we identified alterations in the arachidonic acid pathway in bladder tumors from male versus female patients. Included within this altered arachidonic acid pathway was downregulation of EPHX2, an enzyme that converts epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids (DiHETs). Consistent with this, levels of EETs were significantly elevated in bladder tumors derived from male versus female patients. Cox proportional hazard regression analysis further revealed that reduced expression of EPHX2 was significantly associated with poor clinical outcome across multiple publicly available datasets only in male BLCA patients but not in females. Mechanistic studies revealed that EPHX2 over-expression in male-derived BLCA cell line reduced cell growth in vitro and tumor growth in vivo. Overall, these studies nominate EPHX2 as a potential tumor suppressor in male patients with BLCA.

Citation Format: Mohammed Khurshidul Hassan, Roshan Borkar, Karthik Reddy Kami Reddy, Danthasinghe Waduge Badrajee Piyarathna, Chandra Shekar Amara, Allison Bellman, ChandraShekar R. Ambati, Vasanta Putluri, Abu Hena Mostafa Kamal, Roni J. Bollag, Martha K. Terris, Leomar Y. Ballester, Yair Lotan, Cristian Coarfa, Arun Sreekumar, Nagireddy Putluri. Gender-specific metabolome in bladder cancer: Role of EPHX2 in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2379.
Abstract 2391: Alterations in metformin mediated metabolism in chemoresistant ovarian cancer cells
Authors: Jenkins Brenita Colette , Roy Debarshi , Maner-Smith Kristal , Shridhar Viji
Published: June 2022

Ovarian cancer (OVCA) is the fifth leading cause of death in post-menopausal women in comparison to all other gynecologic malignancies. Late detection of OVCA is common as screening is done mostly in women over 63. This delay in screening coupled with poor prognosis presents the need for novel treatment measures. Repurposing of Metformin (Met), a biguanide used to increase insulin responses in the body, has been identified as a promising approach for therapeutic treatment in OVCA patients. The purpose of this study is to isolate metabolic pathways used by Met to alter OVCA onset. Preliminary results show that Met inhibits cell proliferation and clonogenic growth in both HeyA8 and HeyA8MDR which are drug-sensitive and drug-resistant cell lines, respectively. In combination with carboplatin, Met treatment enhanced chemosensitivity in both cell lines. In addition, lipidomic analysis was done to monitor the impact of lipid metabolites on OVCA cells. Our analysis showed differences in neutral lipid composition in drug-sensitive and resistant cells which indicates the role of various lipid species play in mediating drug-resistance. Furthermore, Met treatment triggered anti-cancerous pathways through syntheses of lipid metabolites. This data indicates that cell proliferation, along with clonogenic growth are inhibited dose dependently by Met treatment in drug-resistant and drug-sensitive cell lines. Chemosensitivity is enhanced by Met treatment in combination with carboplatin in HeyA8 and HeyA8MDR cell lines. Together, the data suggests that autophagy and lipid biogenesis can be altered through metabolic pathways by Met treatment. Through the use of repurposed Metformin, this approach shows promising results of a treatment regimen and potential prevention of OVCA.

Citation Format: Brenita Colette Jenkins, Debarshi Roy, Kristal Maner-Smith, Viji Shridhar. Alterations in metformin mediated metabolism in chemoresistant ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2391.
Abstract 2401: Investigating the role of type II IL4 receptor in breast cancer brain metastasis
Authors: Bindeman Wendy , Williams Demond , Hargrove-Wiley Ebony , Fingleton Barbara
Published: June 2022

Breast-to-brain metastasis is a devastating disease complication for which there are currently few treatment options. Among breast cancer subtypes, patients with triple-negative breast cancer (TNBC) and HER2+ breast cancer experience elevated rates of brain metastasis. The cytokines IL4 and IL13 are overexpressed by many human solid tumors. Both are associated with invasive and metastatic phenotypes; IL13 is additionally consequential in primary brain tumors. These cytokines signal through type I (IL4 only) and type II (IL4/IL13) IL4 receptors (IL4R). Importantly, expression of type II IL4R correlates with poor patient prognosis in basal-like (i.e., triple-negative) breast cancer, and HER2+ tumors display elevated type II IL4R expression. Our laboratory has shown that genetic loss of IL4R signaling in tumor cells attenuates lung and liver metastasis in mice. Additionally, we recently identified a role for IL4R signaling in modulating glycosylation in tumor cells. We hypothesize that IL4R signaling influences TNBC brain metastasis at least in part via modulating glycosylation. In a pilot mouse study, pharmacological blockade of IL4R after intracardiac injection of syngeneic TNBC cells resulted in attenuation of brain metastatic growth and circulating tumor cell burden. We have also observed significant changes in the expression of a sialyltransferase (ST8SIA1) that has been implicated in primary brain tumors, as well as in the abundance of several sialic acid-containing protein species in breast cancer cells following treatment with IL4 or IL13. Further, IL4 stimulation increases the overall glycosylation of several proteins of interest, including a glucose transporter and a glutamine transporter, without altering gene expression. Overall, our data suggest that type II IL4R signaling promotes metastasis, potentially through a mechanism involving protein glycosylation. Blockade of this receptor may be a useful therapeutic approach for patients with breast cancer brain metastases.

Citation Format: Wendy Bindeman, Demond Williams, Ebony Hargrove-Wiley, Barbara Fingleton. Investigating the role of type II IL4 receptor in breast cancer brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2401.
Abstract 2407: Transmembrane chloride intracellular channel 1 (tmCLIC1) protein is a key regulator of colorectal cancers' metastasis
Authors: Cianci Francesca , Verduci Ivan , Carlini Valentina , Tacconi Carlotta , Ranucci Matteo , Fantin Alessandro , Florio Tullio , Mazzanti Michele
Published: June 2022

Colorectal cancer (CRC) is the third most common tumor worldwide and the fourth cause of cancer-related death. The presence of distal metastasis at the time of the first diagnosis represents a worst prognosis factor for patients’ long-term survival. Therefore, finding new targets to arrest the metastatic process is a main challenge for successful CRC therapy. Our laboratory focused its attention on the Chloride Intracellular Channel 1 (CLIC1), a metamorphic protein able to switch from a cytoplasmic to a transmembrane form associated to ion channel activity. Previous experiments demonstrate that tmCLIC1 functional activity is negligible in normal colon cells, while increases proportionally to CRC cells’ aggressiveness. Moreover, we also reveal that tmCLIC1 supports CRC cells abnormal proliferation rate. Here, we show that tmCLIC1 pharmacological inhibition or its stable downregulation led to a significative reduction of CRC cells migrative and invasive potential, in vitro. To further confirm our hypothesis, we analysed downregulation of metalloproteinase-7 (MMP-7) protein and p38 mitogen-activated protein kinase (MAPK) signalling, both involved in migration and metastatic processes. Moreover, we demonstrate in murine model that CLIC1 knock-down impairs tumor growth and limits cancer cells dissemination. We tried to investigate the alteration of metastatic pathways after CLIC1 silencing. We obtained that many different genes are downregulated when CLIC1 protein is absent; in particular, genes regulated by Reactive Species of Oxygen (ROS) levels. In this scenario, we tried to define the mechanism of action in which CLIC1 is involved. Altogether, all the results obtained in the present work could be considered a starting point to design a new therapeutic strategy to counteract against metastatic colorectal cancer.

Citation Format: Francesca Cianci, Ivan Verduci, Valentina Carlini, Carlotta Tacconi, Matteo Ranucci, Alessandro Fantin, Tullio Florio, Michele Mazzanti. Transmembrane chloride intracellular channel 1 (tmCLIC1) protein is a key regulator of colorectal cancers' metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2407.
Abstract 2409: EGFR-phosphorylated GDH1 harmonizes with RSK2 to drive CREB activation and tumor metastasis in EGFR mutated cancers
Authors: Kang JiHoon , Hwang Jung Seok , Boese Austin , Malin Courteney , Kang Sumin
Published: June 2022

Cancer metastasis process involves dysregulated oncogenic kinase signaling, but the precise mechanism by which this orchestrates metabolic networks and signal cascades for metastasis is largely unclear. Here we report that pharmacological inhibition of a glutaminolytic enzyme glutamate dehydrogenase 1 (GDH1) and ribosomal S6 kinase 2 (RSK2) synergistically attenuates cell invasion, anoikis resistance, and immune escape more evidently in tumors harboring EGFR activating or EGFR inhibitor-resistant mutations. Mechanistically, GDH1 is activated by EGFR through phosphorylation at tyrosine 135, and together with RSK2 enhances transcriptional activity of CREB via CaMKIV signaling fostering metastasis. Moreover, GDH1, RSK2, and CREB phosphorylation positively correlate with EGFR mutation status and metastatic cancer progression in primary patient tumors. Our finding reveals a crosstalk between kinase, metabolic, and transcription machinery in metastasis and offers an alternative combinatorial therapeutic strategy to target EGFR therapy-resistant metastatic cancers.

Citation Format: JiHoon Kang, Jung Seok Hwang, Austin Boese, Courteney Malin, Sumin Kang. EGFR-phosphorylated GDH1 harmonizes with RSK2 to drive CREB activation and tumor metastasis in EGFR mutated cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2409.
Abstract 2425: Mechano-response via ATP alters calcium signaling in breast epithelial cells with oncogenic KRas mutation
Authors: Mull Makenzy , Pratt Stephen , Lee Rachel , Gad Abanoub , Chang Katarina , Annis David , Thompson Keyata , Vitolo Michele , Boyman Liron , Martin Stuart
Published: June 2022

The majority of breast cancer patient deaths occur when tumor cells migrate away from the primary tumors and disseminate metastatically. Cancer cells at the invasive front mimic the behaviors of non-tumorigenic epithelial cells at wound edges but show less coordinated migration comparatively. While most wound healing studies use a timeframe of 24-48 hours, our previous studies have shown that ATP released from wounded cells initiates a calcium (Ca2+) signal within seconds that spreads to neighboring cells through activation of the P2Y2 surface receptor. RNAseq data of MCF10A mammary epithelial cells with stepwise mutations of PTEN knockout and KRas activation alone or in combination, demonstrate that P2Y2 is downregulated specifically by active KRas. Fluorescence microscopy was used to measure Ca2+ signaling and mitochondrial membrane potential in parental and mutant MCF10A cells. Change in total fluorescence was measured and compared to baseline using Fluo-4, a Ca2+ fluorescent indicator, and TMRM (tetramethylrhodamine, methyl ester), a fluorescent mitochondrial membrane potential dye on Nikon analysis software. We show that KRas activation disrupts ATP stimulation and Ca2+ signaling. This change was further quantified using a FlexStation III plate reader with Fluo-4 to read specific fluorescent changes in Ca2+ after ATP addition. ATP-stimulated Ca2+ was also disrupted in other breast tumor cell lines harboring Ras activating mutations, MDA-MB-231 and MDA-MB-436, both showing a similar decrease in P2Y2 expression. These data show that ATP stimulation can be used to further understand the differences in Ca2+ signaling and mechanical-response between normal breast epithelial cells and cancer cells. Clarifying these molecular mechanisms could reveal targets for new cancer treatments, especially since 90% of human tumors are epithelial carcinomas.

Citation Format: Makenzy Mull, Stephen Pratt, Rachel Lee, Abanoub Gad, Katarina Chang, David Annis, Keyata Thompson, Michele Vitolo, Liron Boyman, Stuart Martin. Mechano-response via ATP alters calcium signaling in breast epithelial cells with oncogenic KRas mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2425.
Abstract 2528: PDLIM2 expression in triple negative breast cancer stromal macrophages
Authors: Cox Orla T. , Ward Stephanie , Tresse Emilie , O'Connor Rosemary
Published: June 2022

PDLIM2 is a PDZ-LIM domain protein expressed in hematopoietic and epithelial cells that regulates the stability of transcription factors including NFΚB, STATs and beta catenin. PDLIM2 function is associated with tumour suppression and also with the acquisition of a metastatic or epithelial mesenchymal transition (EMT) phenotype. We recently showed that PDLIM2 is more highly expressed in Triple Negative Breast Cancer (TNBC) tumours than in other breast cancer types or normal breast tissue, and that this is associated with active beta catenin in TNBC (1). Interestingly, in PDLIM2-positive TNBC tumours, PDLIM2 was also present in the tumour-associated stroma. However, whether stromal PDLIM2 expression contributes to TNBC cancer progression is unknown.

To investigate this further we assessed the phenotype of PDLIM2-expressing stromal cells and determined that they most strongly represented a M2-like macrophage population. We then investigated the requirement for PDLIM2 in polarization towards either the M1 and M2-like macrophage phenotype using cultured bone marrow-derived macrophages from PDLIM2 knockout mice compared with wild type. This demonstrated that PDLIM2 is required for full adoption of IL-4-induced M2 macrophage polarization and function, including cell adhesion and migration. M1 polarization was less dependent on PDLIM2, although potential for activation of STATs and NFKB was similar in both macrophage populations.We conclude that high expression of PDLIM2 in TNBC influences the tumour microenvironment through the presence of an M2 macrophage population.

Reference: (1) Cox, O. T., Edmunds, S. J., Simon-Keller, K., Li, B., Moran, B., Buckley, N. E., Bustamante-Garrido, M., Healy, N., O'flanagan, C. H., Gallagher, W. M., Kennedy, R. D., Bernards, R., Caldas, C., Chin, S. F., Marx, A. & O'Connor, R. 2019. Pdlim2 Is A Marker Of Adhesion And Beta-Catenin Activity In Triple-Negative Breast Cancer. Cancer Res, 79, 2619-2633.

Citation Format: Orla T. Cox, Stephanie Ward, Emilie Tresse, Rosemary O'Connor. PDLIM2 expression in triple negative breast cancer stromal macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2528.
Abstract 2534: Neural control of glioma infiltration
Authors: Huang-Hobbs Emmet J. , Cheng Yi-Ting , Ko Yeungjun , Woo Junsung , Harmanci Akdes , Rao Ganesh , Deneen Benjamin
Published: June 2022

Adult glioblastoma multiforme (GBM) is the most common and deadly form of malignant brain cancer. A broad relationship between tumor progression and neuronal stimulation in the microenvironment is known in peripheral tumors, but only a few interactions between neuronal cues and GBM cells have been characterized. By combining in utero electroporation (IUE) with CRISPR/Cas9 and piggyBac transposase genetics, we have developed an endogenous, immunocompetent, in vivo model with which to study these interactions in a native tumor context. Combining this model with Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) we interrogated tumor-neuron interactions. We assessed molecular and physiological changes in tumors during neural stimulation through tumor development with single cell sequencing and spatial transcriptomics. We examined expression data in populations present under stimulation that did not appear or appeared later in control tumors. These data showed axon guidance genes, a family of genes associated with directed cell motility, were upregulated in these populations. To functionally test these findings, we performed a barcoded screen of axon guidance genes in the IUE model followed by individual confirmation with gain and loss of function of individual genes. We found Sema4F affects infiltration of tumors as well as overall survival. Currently we wish to study the abrogation of this infiltration with Sema4F knockout in the context of DREADD stimulation. Completion of this study will provide new insights into neuronal interactions driving tumor progression in GBM.

Citation Format: Emmet J. Huang-Hobbs, Yi-Ting Cheng, Yeungjun Ko, Junsung Woo, Akdes Harmanci, Ganesh Rao, Benjamin Deneen. Neural control of glioma infiltration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2534.
Abstract 2585: Targeted protein upregulation strategy potentiates STING agonist immunotherapy
Authors: Cho Wansang , Won Solchan , Choi Yoona , Park Jong Beom , Park Jun-Gyu , Yi Sihyeong , Kim Caroline E. , Narayana Chintam , Lee Dong-Sup , Park Seung Bum
Published: June 2022

As cancer immunotherapy has been emerged as a new pillar of cancer therapy, many strategies modulating host immunity were suggested for cancer treatment. Stimulator of interferon genes (STING) is a promising target for anticancer immunotherapy. However, dysregulated STING expression, or poor pharmacokinetic profiles of STING agonists pose major challenges. Recently, modulating target protein levels via the ubiquitin-proteasome system, such as proteolysis-targeting chimera (PROTAC), has broadened the scope of pharmacological inventions. Herein, we propose UPPRIS (upregulation of target proteins by protein-protein interaction strategy) to overcome these limitations. We discovered the small molecule SB24011 that inhibits STING-E3 ligase interaction, thereby induces the blockade of E3 ligase-mediated STING degradation. As a result, SB24011 enhanced the STING agonist-mediated immune responses by upregulating cellular STING protein level. Thus, co-administration of SB24011 markedly improved the immuno-oncological efficacy of STING agonist cGAMP and anti-PD-1 therapy for tumor regression and robust systemic antitumor response. Taken together, we successfully demonstrated that targeted STING protein upregulation is a promising strategy for cancer immunotherapy.

Citation Format: Wansang Cho, Solchan Won, Yoona Choi, Jong Beom Park, Jun-Gyu Park, Sihyeong Yi, Caroline E. Kim, Chintam Narayana, Dong-Sup Lee, Seung Bum Park. Targeted protein upregulation strategy potentiates STING agonist immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2585.
Abstract 2611: The NEDD8 pathway as a therapeutic target in HER2-amplified colorectal cancer
Authors: Invrea Federica , Carugo Alessandro , Arena Sabrina , Bardelli Alberto , Draetta Giulio F. , Medico Enzo
Published: June 2022

Colorectal cancer (CRC) is a heterogeneous disease with a wide spectrum of clinical outcomes, from indolent resectable disease to aggressive-metastatic cases. Primary and acquired resistance limits the efficacy of available treatments, and the identification of effective drug combinations is needed to further improve patients' outcomes. We previously found that the NEDD8-activating enzyme inhibitor pevonedistat induced tumor stabilization in preclinical models of poorly differentiated, clinically aggressive CRC resistant to available therapies. To identify drugs that can be effectively combined with pevonedistat, we performed a "drop-out" loss-of-function synthetic lethality screening with an shRNA library covering 200 drug-target genes in four different CRC cell lines. Multiple screening hits were found to be involved in the EGFR signaling pathway, suggesting that, rather than inhibition of a specific gene, interference with the EGFR pathway at any level could be effectively leveraged for combination therapies based on pevonedistat. Exploiting both BRAF-mutant and RAS/RAF wild-type CRC models, we validated the therapeutic relevance of our findings by showing that combined blockade of NEDD8 and EGFR pathways led to increased growth arrest and apoptosis both in vitro and in vivo. Pathway modulation analysis showed that compensatory feedback loops induced by single treatments were blunted by the combinations. Our results suggested possible therapeutic opportunities in specific CRC clinical settings. We further focused on HER2 amplified CRC cells and we observed cooperation between pevonedistat and HER2/EGFR blockade by the trastuzumab + lapatinib combination -the current standard treatment for HER2-amplified CRC. In addition, to confirm cooperation in reducing colony formation in vitro, we explored the effect of pevonedistat on long-term persisters, i.e., cell surviving to several weeks of HER2/EGFR blockade. Interestingly, we observed a marked decrease of cell colonies when pevonedistat was added to persisters survived to three weeks of trastuzumab + lapatinib treatment. These results unveil the possibility of testing in vivo the addition of pevonedistat subsequently to tumor stabilization or reduction by HER2/EGFR blockade, to promote further tumor regression.

Citation Format: Federica Invrea, Alessandro Carugo, Sabrina Arena, Alberto Bardelli, Giulio F. Draetta, Enzo Medico. The NEDD8 pathway as a therapeutic target in HER2-amplified colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2611.
Abstract 2612: Depleting EGFR and HER2 overcomes resistance to EGFR inhibitors in colorectal cancer
Authors: Yang Lu , Bhattacharya Arup , Li Yun , Sexton Sandra , Ling Xiang , Li Fengzhi , Zhang Yuesheng
Published: June 2022

Background & Goals: Epidermal growth factor receptor (EGFR), an oncogenic receptor tyrosine kinase, is a major therapeutic target in several types of cancers. Several EGFR inhibitors are used to treat EGFR-positive colorectal cancer (CRC). Unfortunately, primary and acquired resistance to the inhibitors is very common. Current approaches to combating the resistance include combining EGFR inhibitors with agent(s) that targets compensatory signaling molecules (HER2, HER3, IGF1R, MET, KRAS, BRAF and PIK3CA). However, responses to such combination treatments are modest, if any. Overcoming drug resistance remains a major unmet medical need in EGFR-positive CRC.

Results: Here, we show the main reason for CRC resistance to EGFR inhibitors and a therapeutic strategy that overcomes it. The sensitivity of CRC cells to EGFR inhibitors correlates with EGFR downregulation. PEPDG278D, a recombinant human protein that induces the degradation of both EGFR and HER2, strongly inhibits oncogenic signaling and growth of CRC in vitro and in vivo (subcutaneous tumors, orthotopic tumors, and patient-derived xenografts), regardless of activating mutations of KRAS, BRAF and PIK3CA. siRNA knockdown of EGFR or HER2 also inhibits CRC cells resistant to EGFR inhibitors. PEPDG278D indirectly suppresses HER3, IGF1R and MET by disrupting their heterodimerization with EGFR or HER2. While excessive tumor-generated EGFR ligands may block target engagement by PEPDG278D, inhibiting ligand shedding by aderbasib enables PEPDG278D to exert strong antitumor activity, and fluorouracil further enhances tumor inhibition. Collectively, our study shows that CRC resistance to EGFR inhibitors result primarily from their inability to eliminate their target and a PEPDG278D-centered combination treatment overcomes the resistance.

Conclusions & Therapeutic Relevance: The therapeutic activity of PEPDG278D highlights that EGFR and HER2 remain indispensable to the survival of CRC cells resistant to EGFR inhibitors. The strong therapeutic activity of PEPDG278D-centered combination treatment in preclinical models of CRC, which carry clinically relevant gene mutations that confer resistance to current EGFR inhibitors, provides the scientific premise for potential evaluation of this recombinant human protein in patients with drug-resistant CRC.

Citation Format: Lu Yang, Arup Bhattacharya, Yun Li, Sandra Sexton, Xiang Ling, Fengzhi Li, Yuesheng Zhang. Depleting EGFR and HER2 overcomes resistance to EGFR inhibitors in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2612.
Abstract 2625: Personalized chemotherapy treatments for microsatellite instable tumors
Authors: Hoffman Adam , Pal Sanjima , Sangwan Veena , Ferri Lorenzo
Published: June 2022

Introduction: Esophagogastric adenocarcinomas with microsatellite instability (MSI) high status often display chemoresistance, as the current therapies are ineffective. It is hypothesized that the poor response of MSI-high esophagogastric adenocarcinomas to therapies is due to the extensive genomic mutational heterogeneity, which can be overcome by identifying, stratifying, and targeting multiple genetic alterations concurrently. The goals of this project are to: 1) Identify a matched retrospective cohort of 20 MSI-high to 20 microsatellite stable organoids, derived from biobanked tumor tissue. 2) Analyze mismatch repair protein function/loss, to confirm that the primary tumor microsatellite status is recapitulated in 3D organoid models. 3) Test standard of care chemotherapies, and explore alternative approaches to prevent chemoresistance by evaluating targeted agents based on genomic sequencing.

Methods: Patient-derived organoids grow for ~14 days, after which they are enzymatically disrupted and plated in Matrigel. I will confirm that the organoid models accurately represent clinal response, by treating them with the same chemotherapy received by the patient. 10 different drug concentrations are added in triplicate using a drug dispenser, then after 72 hours, organoid viability is determined with a plate reader.

Results: Genomic characterization of 13/226 MSI-high cases has been conducted with a 234 gene gastric cancer specific panel to identify and target genomic alterations. Preliminary robotic drug screens guided by these mutation profiles have been conducted, yielding promising alternative treatments.

Conclusions: By targeting the particular mutations found in MSI-high patients, it could help explain the chemoresistance mechanism, so promising personalized treatment approaches for esophagogastric adenocarcinomas can be clinically implemented.

Sources of Funding: This project is funded by the Inez and Willena Beaton Award in Oncology, valued at $7,500 and provided by the RI-MUHC. The operating grants that support the research are $120,000 over 2 years from the Cancer Research Society, and $1,200,000 USD over 3 years given by the Department of Defense.

Citation Format: Adam Hoffman, Sanjima Pal, Veena Sangwan, Lorenzo Ferri. Personalized chemotherapy treatments for microsatellite instable tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2625.