bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2169: New telomeres and chromosomal arm fusions in cancer genomes revealed by long-read genome sequencing
Authors: Tan Kar-Tong , Slevin Michael K. , Garrity-Janger Max , Li Heng , Meyerson Matthew
Published: June 2022

Chromosomal rearrangements are widespread features of cancer genomes, and often lead to the generation of both larger and smaller chromosomes that are readily observed by cytogenetics analysis. The generation of these chromosomal alterations likely involves alterations in the structure and location of telomeres at chromosome ends, though these events have been difficult to assess due to the long and highly repetitive nature of telomeres. Here, we developed an analytic method (TeloFuse) to identify new telomeres and chromosomal arm fusions which are characterized by telomeric repeats at intra-chromosomal locations. We applied TeloFuse to identify telomere-containing fusions as well as novel sites of telomere addition using short-read genome sequencing data from 326 cancer cell lines followed by long-read genome sequencing and cytogenetic analysis of selected cell lines. We observed new telomeres at sites of terminal deletion in long read data (telomere length = 2-9 kb), and in 76/326 (23%) of cell line genomes with short read data. Telomere sequence-containing fusions at intra-chromosomal sites were detected in 75/326 (23%) of cancer cell lines and confirmed as chromosomal arm fusion events by long-read genome sequencing. Long-read genome sequencing further suggests that new telomeres have similar telomere length as telomeres found on normal chromosomal arms (median = 5kb). Conversely, telomeric repeats at sites of chromosomal arm fusions were found to be critically short (~200-500bp long). Extending our analysis to 97 lung adenocarcinoma patient samples from The Cancer Genome Atlas, we further found new telomeres in 17/97 (18%) and chromosomal arm fusions in 28/97 (29%) patients with short-read data. Notably, new telomeres and chromosomal arm fusion events were also found to disrupt protein-coding genes, suggesting that these events represent a poorly appreciated mode for gene inactivation in cancer. Our results suggest that the formation of new telomeres and chromosomal arm fusions are prevalent events during cancer genome evolution and are likely critical for the stabilization of new chromosomes in human cancers. More broadly, our findings also highlight the utility of long-read genome sequencing in delineating chromosomal-scale alterations that have been widely observed by cytogenetics analysis.

Citation Format: Kar-Tong Tan, Michael K. Slevin, Max Garrity-Janger, Heng Li, Matthew Meyerson. New telomeres and chromosomal arm fusions in cancer genomes revealed by long-read genome sequencing [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 2169.
Abstract 217: A novel class of dimeric quinacrine compounds induce DNA damage and increase the anticancer activity of radiation
Authors: Karagounis Ilias V. , Karar Jayashree , Thomas Christoforos , Koumenis Constantinos , Winkler Jeffrey , Amaravadi Ravi , Maity Amit
Published: June 2022

Radiation therapy is commonly used to treat cancers in the definitive setting, but the local control often remains suboptimal, thereby motivating us to search for novel radiosensitizers. Recently a series of novel quinacrines based upon a previously reported dimeric chloroquine has been synthesized. We studied one of these agents, DQ660. By itself, the drug upregulates both p53 protein and p53 transcriptional targets and also increases apoptosis in p53-wildtype H460 lung cancer cells. Furthermore, we found that DQ660 is a potent radiosensitizer in vitro. This radiosensitization is not contingent upon wildtype p53 as it occurs in cells null for p53 and containing mutant p53. The combination of radiation and DQ660 proded significant growth retardation of human xenografts grown in vivo compared to radiation or DQ660 alone. We saw higher levels of γ-H2AX foci with combined treatment compared to either alone, suggesting that the drug increases DNA damage induction. Using agarose gel electrophoresis and kinetoplast DNA (kDNA) as a substrate, we found that DQ660 can inhibit topoisomerase II activity. DQ660 did not potentiate the phosphorylation of ATM at S1981 seen after radiation. However, the drug did lead to an increase in Ser428 phosphorylation of ATR. This phosphorylation was not seen in response to radiation alone, suggesting that DQ660 and radiation activate different signaling pathways in response to DNA damage. We saw similar results with another dimeric quinacrine, DQ550, suggesting that this class of agents may be radiosensitizers. Our findings have uncovered a novel class of radiosensitizers that work at nanomolar concentrations and will spur investigations for its use in combination therapy for tumors treated with radiation

Citation Format: Ilias V. Karagounis, Jayashree Karar, Christoforos Thomas, Constantinos Koumenis, Jeffrey Winkler, Ravi Amaravadi, Amit Maity. A novel class of dimeric quinacrine compounds induce DNA damage and increase the anticancer activity of radiation [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 217.
Abstract 2220: Immunohistochemical detection of prostate cancer heterogeneity by using ETS and PTEN monoclonal antibodies
Authors: Schafer Cara , Young Denise , Song Yingjie , Jiang Jiji , Dobi Albert , Petrovics Gyorgy , Drake Bettina F. , Chesnut Gregory T. , Sesterhenn Isabell A. , Tan Shyh-Han
Published: June 2022

The multifocality and highly variable molecular pathology of prostate cancer are underlying features of this clinically heterogeneous disease. Most patients harbor multiple molecularly distinct cancer foci at diagnosis that likely arose as independent clones. There is a critical need for molecular biomarkers that can distinguish the multifocality and inter-focal heterogeneity of tumors and help stratify patients for treatment. PTEN loss and ETS gene rearrangements are among the most prevalent genomic alterations in prostate carcinogenesis. The detection of both ERG overexpression and PTEN protein loss by immunohistochemistry (IHC) have proven to be reliable substitutes for detecting genomic alterations by fluorescence in situ hybridization (FISH) assay. We developed monoclonal antibodies against ETV1 and ETV4 and evaluated their performance in prostate cancer specimens. The expression of ETV1, ETV4, were examined in relation to ERG and loss of PTEN expression in TMAs constructed from primary prostate cancer specimens of two independent patient cohorts. The first TMA was constructed from multiple 1 mm cores representing distinct tumor focus from multifocal tumors from 50 African American (AA) and 50 Caucasian American (CA) men. The second was constructed from a single 2 mm core of individual tumors from an independent cohort of 152 AA and 304 CA men. We present results on the expression of each protein biomarker in the context of patient race and their association with clinico-pathologic features, together with concurrence or mutual exclusiveness for each event. These results support the application of ETS monoclonal antibodies in IHC assays to detect prostate cancer tumor heterogeneity and to identify subsets of prostate cancer.

Citation Format: Cara Schafer, Denise Young, Yingjie Song, Jiji Jiang, Albert Dobi, Gyorgy Petrovics, Bettina F. Drake, Gregory T. Chesnut, Isabell A. Sesterhenn, Shyh-Han Tan. Immunohistochemical detection of prostate cancer heterogeneity by using ETS and PTEN monoclonal antibodies [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 2220.
Abstract 2249: A metabolomic analysis of adiposity measures and pre- and postmenopausal breast cancer risk in the Nurses’ Health Studies
Authors: Brantley Kristen D. , Zeleznik Oana A. , Dickerman Barbra A. , Balasubramanian Raji , Clish Clary B. , Avila-Pacheco Julian , Rosner Bernard , Tamimi Rulla M. , Eliassen A. Heather
Published: June 2022

Adiposity is consistently positively associated with postmenopausal breast cancer and inversely associated with premenopausal breast cancer risk. In this nested case-control study of 1651 breast cancer cases and 1651 matched controls from the Nurses’ Health Study (NHS) and the Nurses’ Health Study II (NHSII), we selected lipids and polar metabolites correlated with BMI, waist circumference, weight change since age 18, or derived fat mass, developed a metabolomic score for each measure using LASSO regression, and used logistic regression to investigate the association between this score and breast cancer risk adjusted for risk factors. Analyses were stratified by menopausal status at blood draw and diagnosis. We further explored risk of ER+ and ER- breast cancer.Metabolite correlations were similar across adiposity measures. Metabolite scores developed among only premenopausal or postmenopausal women were highly correlated with scores developed in all women (r=0.94-0.96). Higher metabolomic adiposity scores were generally inversely related to breast cancer risk among premenopausal women. Among postmenopausal women, significant positive trends with risk were observed for higher metabolomic adiposity scores (e.g., metabolomic waist circumference score OR Q4 v. Q1=1.55, 95% CI=1.08-2.22, p-trend=0.01). Metabolites associated with adiposity and breast cancer risk, glycerophosphocholines and branched-chain amino acids, are also associated with metabolic dysregulation. The same metabolites represented adiposity in pre- and postmenopausal women, though breast cancer risk associations still differed by menopausal status. Thus, metabolic dysregulation may have a differential association with pre- vs. postmenopausal breast cancer. Investigation of these metabolites and their role in adiposity-related breast cancer risk should be further explored.

Citation Format: Kristen D. Brantley, Oana A. Zeleznik, Barbra A. Dickerman, Raji Balasubramanian, Clary B. Clish, Julian Avila-Pacheco, Bernard Rosner, Rulla M. Tamimi, A. Heather Eliassen. A metabolomic analysis of adiposity measures and pre- and postmenopausal breast cancer risk in the Nurses’ Health Studies [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 2249.
Abstract 2255: Alu retroelement copy number alterations and non-Hodgkin lymphoma
Authors: Hu Wei
Published: June 2022

Background: Alu retrotransposons are repetitive DNA elements that make up nearly 10% of the human genome and reflect genomic instability. Genomic instability plays an important role in the pathogenesis of non-Hodgkin lymphoma (NHL). However, the relationship between Alu retrotransposons and future risk of NHL is unclear.

Methods: We conducted a nested case-control study of NHL risk and Alu retroelement copy number within the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) study, a prospective cohort with prediagnostic blood samples from 29,133 male Finnish smokers. We measured Alu copy number in 107 incident NHL cases and 1:1 matched controls (± 5 years age). DNA samples extracted from blood were assayed in triplicate using a monochrome multiplex quantitative polymerase chain reaction. We used the albumin gene for the single copy gene to control for genome content in samples. The odds ratios (OR) and 95% confidence intervals (CI) for all NHL were estimated using conditional logistic regression models, adjusting for age at randomization, number of cigarettes smoked per day and smoking duration. Polytomous logistic regression models were fit to compute ORs with Alu copy number for NHL subtypes. To determine if the association might be driven in part by elevated Alu copy number among undiagnosed cases at the time of blood sample collection, we excluded cases diagnosed within the first two years of follow-up.

Results: Higher Alu copy number (using the median as the cut-point) was associated with an increased risk of NHL, both overall [OR, 95% CI=3.80 (1.76-8.17)] as well as for the diffuse large B-cell lymphoma [OR, 95% CI=4.83 (1.50-15.55)] and chronic lymphocytic leukemia/small lymphocytic lymphoma (SLL) [OR, 95% CI=2.66 (1.10-6.40)] subtypes. These associations were similar after excluding individuals diagnosed with NHL within two years after blood draw.

Conclusions: These findings, which require replication, suggest that higher Alu copy number in prediagnostic leukocytes may play a role in the pathogenesis of NHL.

Citation Format: Wei Hu. Alu retroelement copy number alterations and non-Hodgkin lymphoma [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 2255.
Abstract 226: Recurrent trisomies, variable selection and precancerous evolution in the normal gastric epithelium
Authors: Coorens Tim H. , Collord Grace , Jung Hyungchul , Wang Yichen , Zumalave Sonia , Leongamornlert Daniel , Moore Luiza , Mahbubani Krishnaa , Saeb-Parsy Kourosh , Leung Suet Yi , Stratton Michael R.
Published: June 2022

Gastric cancer is the third leading cause of cancer-related mortality globally and is often associated with infectious agents such as the bacterium Helicobacter pylori. However, the background mutational landscape in normal gastric epithelium and the first genomic steps towards the formation of gastric cancer remain poorly understood. Here, we use whole-genome sequencing of microdissected gastric glands (n=271) from 30 patients, 18 of whom had gastric cancer. We show that gastric glands are clonal structures and accrue approximately 27 single base substitutions per year. While the mutational signatures in most normal glands reflect age-related mutagenesis, gastric glands sampled close to a tumor showed exposure to a mutagenic process enriched in tumors (COSMIC reference mutational signature SBS17). Phylogenetic analysis shows that acquisition of SBS17 substitutions is closely linked to overt malignant transformation. We also observe widespread trisomies of specific chromosomes, which are recurrently and independently acquired in many gastric glands of the same patient. Mutations in genes encoding epigenetic modifiers and chromatin remodelers showed evidence of positive selection and were highly enriched in some patients. This was confirmed by targeted sequencing of cancer genes in a further 1008 gastric glands. Strikingly, glands that exhibit driver mutations, a recurrent trisomy or elevated mutation loads only overlap minimally, suggesting a highly variable and patient-specific mutation and selection landscape in the normal gastric epithelium. Taken together, these results give novel insights into the preclinical evolution of gastric malignancies.

Citation Format: Tim H. Coorens, Grace Collord, Hyungchul Jung, Yichen Wang, Sonia Zumalave, Daniel Leongamornlert, Luiza Moore, Krishnaa Mahbubani, Kourosh Saeb-Parsy, Suet Yi Leung, Michael R. Stratton. Recurrent trisomies, variable selection and precancerous evolution in the normal gastric epithelium [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 226.
Abstract 227: Kethoxal-assisted single-stranded DNA sequencing (KAS-seq) reveals dynamic and temporal changes in transcription in response to UVB exposure
Authors: Wilkinson Emma A. , Wu Tong , Park Gayoung , Cui Yan-Hong , He Chuan , He Yu-Ying
Published: June 2022

Skin cancer is the most common form of cancer, with over 5 million cases reported in 2021. UVB exposure derived from sunlight is the major carcinogenic driver of skin cancer and poses a major public health risk. In response to UVB exposure, keratinocytes must activate DNA repair pathways to avoid mutagenesis. In addition to DNA repair pathways, cells initiate well-coordinated DNA damage response pathways to facilitate the repair process, including arresting global transcription. Here we report the use of kethoxal-assisted single-strand DNA-Sequencing (KAS-Seq) technology to profile the transcriptional dynamics that underlie the DNA damage response upon UVB exposure. Mechanistically, KAS-Seq involves the addition of an azide-tagged kethoxal group (N3-kethoxal) to DNA samples, which then reacts with guanines on single-stranded DNA (ssDNA). A “KAS signal” is therefore only obtained when DNA is unwound, allowing the N3-kethoxal reaction with accessible guanines to occur. Accordingly, in response to DNA damage, DNA can form ssDNA bubbles in order to facilitate DNA damage recognition and repair processes. KAS-Seq is therefore a suitable method to profile the transcriptional dynamics that occur in response to UVB-induced DNA damage. Here we show that UVB exposure in normal human keratinocytes (NHEK) cells induces a temporal and dynamic reprogramming in transcription using KAS-Seq. In terms of global transcription, our data show that the greatest loss of KAS signal occurs rapidly between 30mins and 3hrs post-UVB exposure, signifying transcriptional arrest. Interestingly, KAS signal is regained between 6-24hrs post-UVB, which is representative of a transition towards transcriptional recovery and induction of DNA damage processes. Furthermore, a more discrete and targeted analysis shows that the gain and loss of KAS signals primarily occur within the gene body, followed by promoter and intergenic regions. Overall, our data shows that transcriptional control in response to UVB-induced DNA damage is both dynamic and temporal. Further analysis is required to identify which transcriptional phenotype (transcriptional arrest or transcriptional recovery) is the most deleterious to the cell and DNA damage repair processes when disrupted. Additionally, we aim to further elucidate the molecular mechanisms that underlie these global changes in transcription, which will provide expanded insights into the wide-ranging oncogenic mechanisms that underlie UVB exposure.

Citation Format: Emma A. Wilkinson, Tong Wu, Gayoung Park, Yan-Hong Cui, Chuan He, Yu-Ying He. Kethoxal-assisted single-stranded DNA sequencing (KAS-seq) reveals dynamic and temporal changes in transcription in response to UVB exposure [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 227.
Abstract 2273: Unsupervised clustering of multi-omics molecular layers reveals consensus molecular subtypes showing potential therapeutic opportunities for pancreatic cancer
Authors: Lee Sung Hwan , Park Jiyeon
Published: June 2022

Introduction: Pancreatic cancer is a lethal disease showing dismal prognosis and therapeutic resistance. Previous molecular subtypes from genome or transcriptome did not show clinical relevance regarding precision strategy for optimal therapeutic options followed by precise patient classification. This study aims to uncover consensus molecular subtypes from cancer-specific multi-omics data showing clinically relevant therapeutic opportunities.

Methods: We performed comprehensive analyses using the dataset from the cancer dependency map (DepMap) project, including cancer-specific molecular characterization with multi-omics data, genome-wide loss-of-function screening using the CRISPR-Cas9 system, and cancer drug sensitivity. The subtype-specific molecular signatures were validated in independent translational cohorts (TCGA-PAAD; n=150, ICGC-PACA-AU; n=461, ICGC-PACA-CA; n=317).

Results: Integrative profiling of multi-omics molecular layers (Mutational signature, Copy number alteration, Transcriptome, MicroRNA, Chromatic profile, Proteome, and Metabolome) from pancreatic cancer cell lines (n=59) from the Cancer Cell Line Encyclopedia (CCLE) revealed a total of three cancer-specific molecular subtypes showing distinct tumor biology through all omics layer as well as clinical relevance with unique molecular dependency. Major molecular features of each subtype were reproducible in the validation cohorts. Subtype-specific molecular biomarkers, including mutational signature and metabolites, were identified. Finally, the target drug with subtype-specific genetic dependency was analyzed to provide precision strategy according to distinct subtypes’ molecular characterization.

Conclusions: Integrative profiling from multi-omics molecular layers revealed precision strategies based on cancer-specific molecular subtypes of pancreatic cancer in terms of tumor classification and discriminative therapeutic opportunities. Prospective translational studies companion with clinical trials based on cancer-specific molecular subtypes is mandatory to establish the precision strategy for managing pancreatic cancer.

Citation Format: Sung Hwan Lee, Jiyeon Park. Unsupervised clustering of multi-omics molecular layers reveals consensus molecular subtypes showing potential therapeutic opportunities for pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2273.
Abstract 2285: Co-expressed gene networks in CRC CMS1 subtype
Authors: Nunez Sha'Kayla K. , Young Corey D. , Griffen Ti'ara L. , Kopetz Scott , Lillard James W.
Published: June 2022

Colorectal cancer (CRC) is a heterogenous disease driven by dysregulated Wnt, Ras-Raf-MAPK, TGF-β, and PI3K-Akt signaling, which leads to an upregulation of proliferative and survival signals from the tumor microenvironment. Molecular and genomic alterations, immune signatures and gene mutations contribute to the progression of CRC. The consensus molecular subtype (CMS), an RNA-based classification system with four classes, is used to better understand CRC heterogeneity. CMS1 is the immunogenic subtype, enriched with MSI; CMS2-CMS3 has epithelial characteristics; and CMS4 is characterized by mesenchymal transitions associated gene expression. CMS1 tumors display immune infiltration, hypermethylation and microsatellite instability, and are known to have a poorer prognosis. We investigated gene signatures and associated coding mRNAs, which are co-expressed by CMS1 CRC. We acquired CRC primary tumor samples from The Cancer Genome Atlas (TCGA). CXCL13, CXCR5, IL10, PIK3R5, PIK3AP1, CCL19 and other co-expressed genes were identified to be positively correlated with CMS1. The canonical pathways and their upstream regulators of this co-expressed network were analyzed using Gene Ontology (GO). The co-expressed network associated with CMS1 was significantly and positively correlated with PI3K, WNT, ERK1 and ERK2 activation. This network was also positively correlated to right-sided tumors, microsatellite instability, chemokine-mediated signaling pathways, and immune responses. Our findings have confirmed and identified new gene signatures significantly correlated with CMS1, which in no doubt activate key pathways associated with CRC progression.

Citation Format: Sha'Kayla K. Nunez, Corey D. Young, Ti'ara L. Griffen, Scott Kopetz, James W. Lillard. Co-expressed gene networks in CRC CMS1 subtype [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 2285.
Abstract 2301: Targeting the MUVB complex or downstream G2/M proteins potentiates CDK4/6 inhibitor efficacy in breast cancer
Authors: Cuellar-Vite Leslie , Weber-Bonk Kristen , Keri Ruth A.
Published: June 2022

Uncontrolled progression through the cell cycle sustains the growth and progression of cancer. The cell cycle is comprised of 4 phases that are tightly regulated by two key transcriptional modulators, RB-E2F and MUVB complexes that regulate gene expression during the G1/S and G2/M cell cycle transitions, respectively. Target transcriptomes of both complexes overlap, suggesting that maximal inhibition of cell cycle progression, requires targeting both. Inhibitors that regulate the RB-E2F signaling axis have been FDA approved for treatment of a subset of breast cancers. These drugs specifically inhibit two kinases involved in the cell cycle known as CDK4 and CDK6. Palbociclib and abemaciclib, CDK4/CDK6 inhibitors (CDK4/6i), decrease cell proliferation by inducing a G1 cell cycle arrest. In contrast to CDK4/6, we previously reported that the activity of MUVB complex can be repressed using inhibitors of BET proteins, including the tool compound, JQ1. We hypothesized that dual targeting of MUVB and CDK4/CDK6 will synergize in breast cancer models to further decrease growth. To test this hypothesis, we assessed the impact of growth by adding JQ-1 to CDK4/6i in multiple intrinsically resistant cell lines. We found that addition of JQ-1 reduced IC50 of CDK4/6i by 60-75% and that the combination displayed synergistic growth suppression. Mechanistically, analysis of mRNA and protein expression revealed that the combination had greater ability to suppress expression of proteins that are MUVB target genes, G2/M, compared to single agents. We found through genetic and pharmacological approaches that inhibition of G2/M proteins Aurora Kinase A and NEK2 mediated CDK4/6i inhibitor response. Preclinical xenograft models confirmed that combined inhibition of CDK4/6 and MUVB, NEK2, or Aurora Kinase A can synergistically suppress tumor growth. Together, these results indicate that dual targeting of distinct phases of the cell cycle should be a more efficacious approach for suppressing breast cancer growth and provide a potential approach for blocking the acquisition of resistance to CDK4/6 inhibitors, a major clinical challenge for the treatment of luminal breast cancer.

Citation Format: Leslie Cuellar-Vite, Kristen Weber-Bonk, Ruth A. Keri. Targeting the MUVB complex or downstream G2/M proteins potentiates CDK4/6 inhibitor efficacy in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2301.
Abstract 2318: Real-time quantitative PCR based analysis of transcriptional effects of CDK8/Cyclin C inhibitors
Authors: Jordt Laura M. , Totzke Frank , Lauterwasser Joachim , Ehlert Jan E. , Hekking Koen , Aerts Bas , Obodozie Cynthia , Weber Holger , Müller Gerhard , Kubbutat Michael H.
Published: June 2022

CDK8 has been identified as being frequently upregulated in various types of human cancer such as colorectal cancer, certain sub-types of breast cancer and acute myeloid leukemia. In different tumor cell lines RNA interference mediated knockdown of CDK8 results in significant inhibition of tumor cell growth in vitro and in vivo. CDK8, therefore, represents an interesting target for the development of novel anti-cancer drugs.

Various compounds targeting CDK8/Cyclin C with high potency in low nanomolar range have been reported. These compounds differ with respect to their binding modes, but all of them show high potency in biochemical assays, inhibition of tumor cell growth, and for many in vivo activities in different tumor cell xenograft models have been demonstrated.

CDK8 binds to Cyclin C resulting in the active protein kinases complex. CDK8/Cyclin C interacts with MED12 and MED13, two proteins that regulate CDK8/Cyclin C activity and its interaction with the mediator complex. Mediator consists of 26 subunits and the interaction of the CDK8/Cyclin C/MED12/Med13 complex with mediator enables CDK8/Cyclin C to regulate gene transcription via C-terminal phosphorylation of RNA-polymerase II. This mechanism mediates an indirect regulation of transcription factors such as ß-catenin. In addition of its function via phosphorylation of RNA-polymerase II, CDK8/Cyclin C can also directly regulate the activity of transcription factors by phosphorylation. Best known examples are STAT1 and STAT5 which are phosphorylated by CDK8/Cyclin C on serine 727 and Serine 726, respectively. Recent data indicates that mutations of MED12 can result in a reduced sensitivity of CDK8/Cyclin C towards certain published CDK8/Cyclin C inhibitor in a cellular context. In this study we performed real-time quantitative PCR (qPCR) analyses to characterize the effect of different type I and type II CDK8/Cyclin C inhibitors on the cellular expression of selected genes controlled by two CDK8/Cyclin C dependent pathways, namely the WNT/ß-catenin- and the STAT-pathway. Furthermore, results of qPCR analyses of tumors derived from human xenograft in vivo models treated with type I and type II CDK8/Cyclin C inhibitors will be presented.

Citation Format: Laura M. Jordt, Frank Totzke, Joachim Lauterwasser, Jan E. Ehlert, Koen Hekking, Bas Aerts, Cynthia Obodozie, Holger Weber, Gerhard Müller, Michael H. Kubbutat. Real-time quantitative PCR based analysis of transcriptional effects of CDK8/Cyclin C inhibitors [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 2318.
Abstract 2326: Preclinical characterization of MTB-9655, a first-in-class, clinical stage ACSS2 inhibitor
Authors: Goutopoulos Andreas , Amanati Iris , Fattaey Ali , Hay-Koren Avital , Nakache Philippe , Erez Omri , Kovalerchik Dimitri , Paz Dikla , Botti Simone , Oppenheimer Hannah , Fainer Irit
Published: June 2022

ACSS2 is a nucleo-cytoplasmic enzyme that converts acetate into Acetyl-CoA. In many tumor types ACSS2 is dramatically upregulated in response to the hypoxic and lipid-depleted conditions often encountered in the tumor microenvironment. In these conditions, ACSS2 allows tumor cells to switch to acetate as key carbon source to meet their anabolic and energetic needs and thereby they become dependent on both acetate and ACSS2. MTB-9655 is a novel, potent and selective ACSS2 inhibitor discovered at Metabomed. It is currently in Phase I clinical investigation on patients with tumors with high levels of ACSS2. The antitumor effect of MTB-9655 was characterized in a number of PDX models across various tumor types. PDX tumors that exhibit over 50 TPM copies of ACSS2 mRNA showed significant sensitivity to ACSS2 inhibition in vivo. TCGA analyses suggests that most GU tumors, such as hepatocellular, gastric, colorectal show high frequency of ACSS2 upregulation and therefore present an opportunity for treatment with MTB-9655. In addition, PDX studies revealed a significant combination effect with cisplatin and gemcitabine. This opens opportunities for combination treatments of several GU cancers expressing high levels of ACSS2, such as ovarian and breast cancers.As a potential PD biomarker assay, 11C acetate PET imaging was explored preclinically in tumor-bearing mice as a way to assess inhibition of acetate flux in the presence of ACSS2 inhibitor. MTB-9655 resulted in robust inhibition of 11C acetate uptake in tumors in a dose-dependent manner opening the possibility of using 11C acetate PET as a clinical PD biomarker.

Citation Format: Andreas Goutopoulos, Iris Amanati, Ali Fattaey, Avital Hay-Koren, Philippe Nakache, Omri Erez, Dimitri Kovalerchik, Dikla Paz, Simone Botti, Hannah Oppenheimer, Irit Fainer. Preclinical characterization of MTB-9655, a first-in-class, clinical stage ACSS2 inhibitor [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 2326.
Abstract 2342: Quinolinate promotes alternatively activated macrophage-induced immune tolerance in glioblastoma through the NMDA/PPARy signaling axis
Authors: Kesarwani Pravin , Kant Shiva , Prabhu Antony , Buelow Katie L. , Miller C. Ryan , Chinnaiyan Prakash
Published: June 2022

There has been considerable scientific effort dedicated to understanding the biologic consequence and therapeutic implications of aberrant tryptophan metabolism in brain tumors and neurodegenerative diseases. An overwhelming majority of this work has focused on the upstream metabolism of tryptophan, however, this has not resulted in clinical application. Using global metabolomic profiling of patient-derived brain tumors, we identify the downstream metabolism of tryptophan and accumulation of quinolinate (QA) as a metabolic node in glioblastoma and went on to demonstrate its critical role in promoting immune tolerance. QA acts as a “metabolic checkpoint” in glioblastoma by inducing NMDA receptor activation and Foxo1/PPARγ signaling, resulting in amplification of immune suppressive macrophages. Using a genetically-engineered mouse model designed to inhibit production of QA, we identify kynureninase as a promising therapeutic target to revert the potent immune suppressive microenvironment in glioblastoma. These findings offer the scientific community an opportunity to revisit the biologic consequence of this pathway as it relates to oncogenesis and neurodegenerative disease and a framework for developing new immune modulatory agents to further clinical gains in these otherwise incurable diseases.

Citation Format: Pravin Kesarwani, Shiva Kant, Antony Prabhu, Katie L. Buelow, C. Ryan Miller, Prakash Chinnaiyan. Quinolinate promotes alternatively activated macrophage-induced immune tolerance in glioblastoma through the NMDA/PPARy signaling 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 2342.
Abstract 2344: BRAF inhibitor resistant thyroid cancer cells exhibit altered cellular energetics
Authors: Bolf Eric , Schweppe Rebecca
Published: June 2022

Thyroid cancer is the most commonly diagnosed endocrine malignancy. Although prognosis for patients is positive overall, dedifferentiated thyroid tumors and invasive thyroid tumors are difficult to treat. There is one FDA approved targeted therapy in use for in BRAF mutant, highly dedifferentiated anaplastic thyroid cancer, a combination of the BRAF inhibitor dabrafenib and the ERK inhibitor trametinib. While this treatment improves outcomes, resistance is of great concern. In other cancers driven by BRAF mutations, particularly melanoma, a known driver of BRAF inhibitor resistance is enhanced oxidative phosphorylation. We therefore investigated the energetics of thyroid cancer cells to determine whether metabolic changes can drive resistance in thyroid cancer. We have previously determined sensitivity to dabrafenib by area under the curve to classify cell lines as sensitive or resistant (Hicks HM et al, 2021). We used CUTC5 cells as our primary model of intrinsically sensitive cells and CUTC60 cells to model intrinsic resistance. We observed both cell lines had similar basal oxygen consumption rates (OCR), however dabrafenib treatment reduced this in the sensitive CUTC5 cells and did not in the resistant CUTC60 cells. Additionally, maximum OCR was reduced in the CUTC5 cells with as little as 10 nM dabrafenib. In a preliminary experiment, CUTC60 cells were co-treated with dabrafenib and rotenone, an inhibitor of the electron transport chain, and synergistic inhibition of proliferation was observed. We further investigated energetics in the dabrafenib sensitive cell line KTC1 and KTC1-VA7, which are derived from KTC1 and BRAF-inhibitor resistant (Danysh BP et al, 2016). Unlike the other cells evaluated these cells did not have spare respiratory capacity. Additionally, dabrafenib treatment did not alter basal OCR except at the highest doses. Although, we did observe a reduction in glycolysis in the parental KTC1 upon dabrafenib treatment, which was not observed in the KTC1-VA7 cells. Sensitive CUTC5 cells also exhibited a greater decrease in glycolysis than resistant CUTC60 cells. From this, we conclude that glycolysis may contribute to dabrafenib resistance and merits further investigation. With regards to oxidative metabolism, we only observed a reduction in OCR in a dabrafenib sensitive cell line and there was synergistic inhibition in the resistant CUTC60 cells treated with both dabrafenib and rotenone, suggesting that oxidative metabolism aids the cells in tolerating BRAF inhibition. Further screening of dabrafenib sensitive and resistant cells is necessary to determine if this is a function of dabrafenib sensitivity.

Citation Format: Eric Bolf, Rebecca Schweppe. BRAF inhibitor resistant thyroid cancer cells exhibit altered cellular energetics [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 2344.
Abstract 2345: Specificity of catechol-O-methyltransferase (COMT) for hydroxyestrogens favors 2-OHEs over 4-OHEs
Authors: Krzizike Daniel D. , Clapper Margie L. , Andrews Andrew J.
Published: June 2022

The contribution of estrogen receptor-mediated signaling to cancer progression and metastasis has been well documented. However, much less attention has been given to the role of estrogen metabolism in carcinogenesis. Parent estrogens are metabolized to 2- and 4-hydroxyestrogens (2-OHEs and 4-OHEs) that can retain their estrogenic activity, form reactive quinones that cause DNA damage, or become inactivated primarily by the rate-limiting conjugation enzyme catechol-O-methyltransferase (COMT). The ability of 4-OHEs to induce genomic instability and the malignant transformation of human breast epithelial cells has been reported. Glucuronidation or sulfation of methylated hydroxyestrogens, produced by COMT, increases their solubility and facilitates their excretion via urine or feces. The extent to which catechol estrogens are methylated dictates, in part, their ability to initiate carcinogenesis verses protect against tumor formation. COMT is a ubiquitous polymorphic enzyme that is expressed in humans in both peripheral tissues (soluble, s-COMT) and the central nervous system (membrane-bound). The goal of the present study was to compare the rate at which human wild type (WT) s-COMT and its major polymorphic variant (V108M s-COMT) transfer the methyl group from S-adenosylmethionine to 2-OHE and 4-OHE. This was accomplished by expressing COMT in E. coli, followed by protein purification and steady state Michaelis-Menten kinetics. Production of methylated hydroxyestrogens (2-MeOEs and 4-MeOEs) was quantified using LC-MS/MS methodology. The resulting data indicate that WT s-COMT methylates catechol estrogens more efficiently than V108M s-COMT. In addition, the specificity constant (kcat/KM) of WT s-COMT for methylation of 4-OHE2 was 12-fold greater than that of V108M s-COMT. The kcat/KM of V108M s-COMT was 4-fold greater for 2-OHE2 vs. 4-OHE2. Based on the enhanced catalytic efficiency of WT s-COMT in converting 2-OHEs to 2-MeOEs, 4-OHEs may have greater potential to cause DNA damage. Additional studies are needed to determine if the V108M s-COMT variant contributes to increased risk for cancer. These data provide novel insight into the potential mechanism by which 4-OHEs promote hormone-induced carcinogenesis.

This work was supported by an In Vino Vita Award from Fox Chase Cancer Center.

Citation Format: Daniel D. Krzizike, Margie L. Clapper, Andrew J. Andrews. Specificity of catechol-O-methyltransferase (COMT) for hydroxyestrogens favors 2-OHEs over 4-OHEs [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 2345.
Abstract 2358: Impact pathway analysis of FOXC2-correlated genes in melanoma: A meta-analysis approach to understand the tumor-promoting functions of the FOXC2 transcription factor
Authors: Wall James B. , Hargadon Kristian M.
Published: June 2022

Melanoma is a cancer that arises from pigment-producing melanocytes in the skin. Because of the increasing incidence of melanoma and its resistance to many therapies, insights into melanoma progression are needed. FOXC2, a member of the forkhead box family of transcription factors, has been shown to have oncogenic activity in many epithelial cancer types, and we have recently reported a role for FOXC2 in melanoma progression as well. To gain insight into the role of FOXC2 in melanoma progression, we analyzed publicly available RNA-sequencing data from tumor biopsies of melanoma patients, including a cohort of 479 patients catalogued in The Cancer Genome Atlas as well as additional cohorts from other independent studies. We performed Spearman correlation analyses to identify genes positively and negatively co-expressed with FOXC2, and we performed Impact Pathway Analysis and Gene Ontology Analysis to determine which biological pathways and processes are most impacted by FOXC2-associated genes in melanoma. Our data highlight critical pathways impacted by the expression of FOXC2, and they identify specific genes influenced by this transcription factor that provide mechanistic insight into the oncogenic activity of FOXC2 in melanoma and potentially other cancers.

Citation Format: James B. Wall, Kristian M. Hargadon. Impact pathway analysis of FOXC2-correlated genes in melanoma: A meta-analysis approach to understand the tumor-promoting functions of the FOXC2 transcription factor [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 2358.
Abstract 2361: MYC-driven breast cancer tumorigenesis is dependent on normal mitochondrial function
Authors: Moyer Sydney M. , Ilic Nina , Gang Sydney , Arnoff Taylor E. , Hahn William C.
Published: June 2022

While the transcription factor MYC is amplified in all breast cancer subtypes, nearly 60% of patients with triple-negative tumors have elevated MYC copy number and expression. Patients with triple-negative breast cancer (TNBC) typically have increased metastasis, decreased response to therapies, and poor outcomes, highlighting MYC’s causal association with disease aggressiveness and low survival rates. Unfortunately, MYC is not considered directly pharmacologically tractable. Therefore, we sought to identify collateral “genetic dependencies,” downstream of oncogenic MYC. Using comparative genome-scale CRISPR/Cas9 screening in isogenic human mammary epithelial cells (HMECs), we have identified two mitochondrial membrane transporter genes, TIMM17A and MTCH2, specifically required for MYC-dependent proliferation and survival.

Since MYC is suspected to drive metabolic reprogramming in cancers, we assessed how MYC affected mitochondrial protein content by quantitative mass spectrometry. This revealed an increase in N-acetylaspartate (NAA) in HMECs with MYC amplification compared to other genetic backgrounds. Interestingly, increased NAA levels are dependent on TIMM17A and MTCH2 presence. Loss of either of these genes results in cell death coupled with decreased NAA. NAA supplementation in the media of MYC-HMECs following loss of TIMM17A or MTCH2 can rescue the cell death. Importantly, NAA supplementation in cells with guides targeting GFP (negative control) or general essential gene RPL11 did not show increased cell growth/viability - showing that the NAA supplementation is specifically compensating for loss of mitochondrial transport function in MYC-HMECs.

Based on these data, it appears that MYC-amplified TNBCs are uniquely dependent on TIMM17A and MTCH2 mitochondrial transporter function because they drive metabolic reprogramming resulting in addiction to N-acetylaspartate production. To conclude these studies, we are evaluating the dependence on mitochondrial transport and NAA synthesis in MYC-amplified TNBCs by assessing the function of TIMM17A and MTCH2 as MYC-specific genetic dependencies in patient derived xenografts of TNBC and determining if TNBCs are addicted to increased NAA synthesis by overexpressing aspartoacylase to breakdown NAA. Successful completion of this work will provide novel drug targets required for survival of aggressive MYC-amplified breast cancers.

Citation Format: Sydney M. Moyer, Nina Ilic, Sydney Gang, Taylor E. Arnoff, William C. Hahn. MYC-driven breast cancer tumorigenesis is dependent on normal mitochondrial function [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 2361.
Abstract 2365: Mechanisms of mutant p53 targeting to the genome
Authors: Metcalf Stephanie , Morris Stevie , Hollenhorst Peter
Published: June 2022

Mutations in genes often result in tumor formation, especially when these mutations occur in tumor suppressors. TP53 is a tumor suppressor that is frequently mutated in cancer, with mutations in this gene occurring in nearly 50% of all cancer cases. Once mutated, p53 loses its tumor suppressive function while simultaneously gaining oncogenic function. One of the functions that mutant p53 loses is the ability to directly bind to chromatin, however, it has been reported that mutant p53 can still affect the transcriptome of cancer cells via interactions with other transcription factors. One of these interacting partners is ETS2. ETS2 belongs to the ETS transcription factor family, which has 28 family members. This family is characterized by their affinity for an ETS binding site (EBS). EBS’s are present in 50% of all mutant p53 occupied promoters. Other ETS family members have also been linked to mutant p53 but these interactions have either been deemed as weak (ETS1) or have yet to be identified as direct (ERG). To determine which ETS proteins interact with mutant p53 I conducted affinity pull-down assays using purified ETS proteins and purified mutants of p53. My data shows that several ETS proteins interact with mutant p53 better than ETS2. I then sought to determine which residues are important for this interaction through truncation studies in which I used purified truncations of ETS proteins and purified mutants of p53. I found that ERG, one protein that strongly interacted with mutant p53, had two interaction interfaces. This may explain why the interaction is strong. My next step was to determine which ETS proteins are responsible for the targeting of mutant p53 to the genome. To address the requirement for ETS to recruit mutant p53 to chromatin I performed chromatin immunoprecipitation sequencing studies in the presence or absence of different ETS factors to determine differences in mutant p53 binding. For these studies I knocked down ETS2 or ERG prior to performing p53 ChIP-Seq to determine differences in p53 binding to chromatin under these conditions. My analyses of these data indicate that each of the conditions resulted in different p53 binding patterns in the ChIP-Seq and that there is a requirement for ETS in mutant p53 binding.My studies have demonstrated that ETS proteins interact with mutant p53 and that this interaction seems to be required for mutant p53 binding to the genome. My future work will test phenotypes related to ETS/mutant p53 interactions. Ultimately, if ETS/mutant p53 interactions are deemed important for oncogenic function, these will be attractive targets for future drug development.

Citation Format: Stephanie Metcalf, Stevie Morris, Peter Hollenhorst. Mechanisms of mutant p53 targeting to the genome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2365.