bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2751: Development of a specific Wee-1 inhibitor
Authors: Watson Mandy , Pesnot Tom , Scott Andrew , Huxley Anthony , Nelson Gary , Shelbourne Montserrat , Morton Jen , Bingham Tilly
Published: August 2019

Somatic mutations in the TP53 gene are one of the most frequent alterations in human cancers. The resulting TP53-deficient cancer cells rely on WEE1 to trigger a G2/M arrest, which allows for DNA repair and survival. Inhibition of WEE1 has therefore emerged as an attractive therapeutic strategy to selectively sensitise TP53-deficient tumours to DNA damaging agents. The WEE1 kinase inhibitor adavosertib is the only agent undergoing evaluation in a range of clinical trials to validate this hypothesis. It has also been the tool compound of choice to interrogate WEE1 biology for over a decade.

However, recent reports show adavosertib has antiproliferative single agent activity, which is counter intuitive considering its postulated mode of action. Other studies suggest adavosertib exerts poor kinase selectivity, and inhibits PLK1 with similar potency as WEE1. Since PLK1 is a well-established mitotic regulator with biological functions throughout the cell cycle, its inhibition may contribute to the clinical efficacy and toxicity observed for adavosertib. Other reported WEE1 inhibitors also suffer from low selectivity and single agent toxicity, making then unsuitable tool compounds. There is a clear need to identify a selective Wee1 tool compound.

Using a structure-based drug design approach, we exploited subtle active site differences to identify novel, potent WEE1 inhibitors that display high selectivity over PLK1. These compounds allowed us to demonstrate the distinct effects of WEE1 and PLK1 inhibition on cellular toxicity, and confirm that adavosertib’s single agent efficacy is unlikely to be mediated solely through the inhibition of WEE1. Our ongoing studies aim to provide a WEE1 selective tool compound that more rigorously probes WEE1 biology, and eventually leads to the development of less toxic therapeutic agents.

Citation Format: Mandy Watson, Tom Pesnot, Andrew Scott, Anthony Huxley, Gary Nelson, Montserrat Shelbourne, Jen Morton, Tilly Bingham. Development of a specific Wee-1 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2751.
Abstract 2752: Recognition of the hybrid-1 human telomeric G-quadruplex by a platinum(II)-based compound
Authors: Liu Wenting , Lin Clement , Mao Zong-wan , Yang Danzhou
Published: August 2019

The human telomeric DNA, which consists of tandem repeat sequences of d(TTAGGG)n, plays an important role in cancers and cell aging. Previous studies in human cancer cells have demonstrated DNA G-quadruplex (G4) structure formation in telomeres. G-quadruplexes are four-stranded structures formed in guanine-rich sequences that are held together by guanine-guanine Hoogsteen hydrogen bonding, and G-quadruplex stabilization by small molecules induces tumor cell senescence and apoptosis by repressing telomerase activity and the DNA damage response pathway. Additionally, telomeric-overhang DNA can form biologically relevant higher-order DNA structures containing consecutive G-quadruplexes which provides additional binding sites for small molecules. Thus, telomeric DNA G-quadruplexes have attracted increasing interest as potential drug targets for cancer therapy. Here, we found that a novel platinum (II)-based tripod (Pt-tripod) specifically recognizes the hybrid-1 human telomeric G4, which is formed in the physiologically relevant K+ solution. Pt-tripod strongly represses telomerase activity and exhibits DNA-targeted photodynamic therapy anticancer activity. Using NMR, we show that Pt-tripod binds the hybrid-1 human telomeric G4 over other G-quadruplex structures and dsDNA. We determined solution structures of the 1:1 and the dimeric 4:2 Pt-tripod-hybrid-1 telomeric G4 complexes by NMR. Our 1:1 complex structure shows preferential binding of the Pt-tripod to the 5′-end of hybrid-1 telomeric G4. At higher ligand ratio, Pt-tripod binds to the 3′-end of the hybid-1 G4 and induces an unprecedented dimeric 4:2 structure interlocked by a non-canonical A:A pair at the 3′-end. The specific binding of the Pt-tripod with the hybrid-1 G4 is achieved by unique interaction modes including the π-π stacking, hydrogen bonding, and electrostatic interactions with the loop and flanking segments. The non-planar tertiary amine conformation and three properly sized cationic platinum arms are critical for the specific and strong binding. Our structures provide significant insights into understanding the dynamic binding of small molecules with G-quadruplexes, and a structural basis for future rational anticancer drug design of non-planar small molecules targeting the human telomeric G4. Our studies also provide a potential handle to study the specific protein interactions and biological functions of hybrid-1 human telomeric G4.

Citation Format: Wenting Liu, Clement Lin, Zong-wan Mao, Danzhou Yang. Recognition of the hybrid-1 human telomeric G-quadruplex by a platinum(II)-based compound [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2752.
Abstract 2753: The molecular basis of blocking the TIM-3 checkpoint with the LY3321367 mAb in cancer immunotherapy
Authors: Haidar Jaafar N. , Antonysamy Stephen , Mathew Sneha , Wu Lan , Zhang Yi , Kearins Margaret C. , Shen Leyi , Sauder J. Michael , Schaer David , Driscoll Kyla E. , Kalos Michael
Published: August 2019

The anti-T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) antibody LY3321367 is a promising immune-checkpoint inhibitor that is being clinically tested for the treatment of multiple solid tumors (NCT03099109). LY3321367 was phenotypically selected from a large set of phage library Fabs that are TIM-3 binders. Tim3 has multiple protein and non-protein binding partners; hence, we attempt in this study to deconvolute the molecular mechanism of action of LY3321367. Surprisingly, LY3321367 in ELISA assays partially blocks the TIM-3/GAL-9 complex but does not block the TIM-3/CEACAM-1 complex. We also utilized X-ray crystallography to solve the structure the LY3321367-Fab complex with the IgV domain of TIM-3 at 2.0Å resolution. After solving the unbound structure of the LY3321367-Fab, we demonstrated that its CDRs do not undergo any conformational changes upon binding human TIM-3. The structural alignment of the human TIM-3/LY3321367-Fab complex with the available murine TIM-3 structure unexpectedly suggests that the 6.0Å epitope of LY3321367 overlaps the phosphatidylserine (PS) binding site on TIM-3. Consequently, we developed a cell-based assay to demonstrate that LY3321367 completely blocks the binding of soluble human TIM-3 to PS displayed on the surface of Campothecin-treated DO11.10 cells.

Our structural results suggest an important role for PS in TIM-3 biology which is blocked by LY3321367. When combined with the partial blocking of GAL-9, the structural data of LY3321367 suggests that the binding sites of PS and GAL-9 may be proximal to each other on TIM-3.

Citation Format: Jaafar N. Haidar, Stephen Antonysamy, Sneha Mathew, Lan Wu, Yi Zhang, Margaret C. Kearins, Leyi Shen, J. Michael Sauder, David Schaer, Kyla E. Driscoll, Michael Kalos. The molecular basis of blocking the TIM-3 checkpoint with the LY3321367 mAb in cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2753.
Abstract 2754: Affinity-based probe reveal Bim negatively regulates the chaperone functions of Hsp70, a non-Bcl-2 BH3 receptor
Authors: Wang Ziqian , Zhang Zhichao , Song Ting , Guo Zongwei
Published: August 2019

The identification of novel non-Bcl-2 BH3 receptors is still challenge owing to frequently weak, transient and reversible binding character of the PPIs mediated by them. Herein, we designed and synthesized an affinity-based probe (AfBP), S1b-probe, by introducing a photo-crosslinker to an artificial BH3 mimetic. Hsp70 proteins were identified as novel BH3 receptors in situ. The PPIs between Bcl-2 proteins and Hsp70 proteins were further validated by fluorescent polarization (FP), isothermal titration calorimetry (ITC) and Co-immunoprecipitation (Co-IP) experiments. Structural and functional analysis by photo-crosslinking/LC-MS/MS sequencing, 1H-15N transverse relaxation optimized spectroscopy (TROSY-HSQC), trypsin proteolysis, single-turnover ATPase rates and denatured rhodanese aggregation measurement demonstrated that BimBH3 bind in the NBD domain of Hsp70 proteins to act as a co-chaperone that negatively regulates the ATPase activity and chaperone functions. S1g, derived from BH3 mimetics, was screened as a specific Hsp70 inhibitor in native physiological context by using S1b-probe.

Citation Format: Ziqian Wang, Zhichao Zhang, Ting Song, Zongwei Guo. Affinity-based probe reveal Bim negatively regulates the chaperone functions of Hsp70, a non-Bcl-2 BH3 receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2754.
Abstract 2758: Domain swapping and SMYD1 interactions with the PWWP domain of human hepatoma-derived growth factor
Authors: Chen Chun-Jung , Hsu Li-Jin
Published: August 2019

The human hepatoma-derived growth factor (HDGF), containing the chromatin-associated N-terminal PWWP domain capable of binding the SMYD1 promoter, participates in various cellular processes and is involved in human cancers, including hepatocellular carcinoma, pancreatic cancer, oral cancer, breast cancer and non-small cell lung cancer. However, the functional mechanisms of human HDGF had been puzzling because of a lack of essential knowledge of its intact structure in complex with DNA. We present the first crystal structures of the human HDGF PWWP domain (residues 1 - 100) in a complex with SMYD1 of 10 bp at 2.84 Å resolution and its apo form at 3.3 Å, respectively. The structure of the apo PWWP domain comprises mainly four β-strands and two α-helices. The PWWP domain undergoes domain swapping to dramatically transform its secondary structures, altering the overall conformation from monomeric globular folding into an extended dimeric structure upon DNA binding. The flexible loop2, as a hinge loop with the partially built structure in the apo PWWP domain, notably refolds into a visible and stable α-helix in the DNA complex. The swapped PWWP domain interacts with the minor grooves of the DNA through residues Lys19, Gly22, Arg79 and Lys80 in varied ways on loops 1 and 4 of the two chains, and the structure becomes more rigid than the apo form. These novel structural findings, together with physiological and activity assays of HDGF and the PWWP domain, provide new insights into the DNA-binding mechanism of HDGF during nucleosomal functions.

Note: This abstract was not presented at the meeting.

Citation Format: Chun-Jung Chen, Li-Jin Hsu. Domain swapping and SMYD1 interactions with the PWWP domain of human hepatoma-derived growth factor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2758.
Abstract 2773: Prognostic value of KRASP53PIK3CA in non-small cell lung cancer patients
Authors: Han Yiping
Published: August 2019

We explored the relationship of KRAS, PIK3CA, and TP53 mutations with the clinical features and survival prognosis in 50 non-small cell lung cancer (NSCLC) patients. The most common concurrent single gene mutation was TP53, followed by KRAS and PIK3CA. Coexisting mutations were found in 17 patients. KRAS, PIK3CA, and TP53 mutations were related to Ca19-9 expression, invasive growth, vacuolar signs, and margin lobulation in chest CT imaging. The incidence of distant metastasis (bone and adrenal) with KRAS and TP53 mutations was greater than that of local metastasis (pleura). Patients the wild-type gene had longer progression-free survival than those with KRAS, TP53, KRAS/TP53, or PIK3CA/TP53 mutations. Patients with KRAS/TP53 or PIK3CA/TP53 mutations had shorter progression free survival than those with a single KRAS or TP53 mutation. KRAS, PIK3CA, and TP53 mutations were associated with distant metastases and poor prognosis. NSCLC patients should receive routine KRAS, PIK3CA, and TP53 gene sequencing to determine mutations for the analysis of clinical characteristics and prognosis.

Note: This abstract was not presented at the meeting.

Citation Format: Yiping Han. Prognostic value of KRASP53PIK3CA in non-small cell lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2773.
Abstract 2774: Pain signals oral cavity cancer metastasis
Authors: Bhattacharya Aditi , Janal Malvin N. , Kim Hyesung , Wang Susanna , Wu Angie K. , Hagiwara Mari , Kerr Alexander R. , Schmidt Brian L. , Albertson Donna G.
Published: August 2019

Metastasis to the cervical (neck) lymph nodes is the primary determinant of survival for patients diagnosed with oral cavity cancer. Imaging (MRI, PET, CT) and clinical examination lack sensitivity to accurately detect cervical metastasis. Therefore most clinically node negative patients receive surgery to remove the cervical lymph nodes (elective neck dissection) at the time of surgical tumor removal. Half of these patients do not benefit from this additional surgery, highlighting the need for improved preoperative assessment in predicting metastatic risk. Oral cancer patients suffer severe chronic and mechanically-induced pain at the site of the cancer. We asked whether patient reported pain measured prior to surgery by a validated instrument, the University of California San Francisco Oral Cancer Pain Questionnaire (UCSFOCPQ), was an indicator of risk for lymph node metastasis. Seventy-two oral cancer patients were consented and enrolled, patients rated their pain using the UCSFOCPQ. Clinical and pathological characteristics of the patient cohorts were collected from pathology reports and medical records. Inclusion criteria included diagnosis of an oral squamous cell carcinoma, planned curative resection of the cancer and completion of the UCSFOCPQ within six weeks of surgery. Exclusion criteria included prior chemo- or radiation therapy for cancer, failure to complete or understand the UCSFOCPQ and less than one year post surgery follow-up. Sixty-six patients met the inclusion/exclusion criteria (35 node negative and 31 node positive). Patients with metastasis reported higher pain scores prior to surgery. Low pain score and depth of invasion each predicted low metastatic risk with high sensitivity and negative predictive values. Amongst patients who were clinically staged N0 by current standard of care imaging and physical exam prior to surgery, low pain scores correctly identified patients determined by pathology to be N0. Conclusions: Low pain scores assessed prior to surgery could identify patients at low risk for metastases who would not benefit from neck dissection. Pain score might be added to the standard of care preoperative assessments and decision making processes for determining whether to recommend an elective neck dissection.

Citation Format: Aditi Bhattacharya, Malvin N. Janal, Hyesung Kim, Susanna Wang, Angie K. Wu, Mari Hagiwara, Alexander R. Kerr, Brian L. Schmidt, Donna G. Albertson. Pain signals oral cavity cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2774.
Abstract 2775: Investigating myeloid-derived suppressor cells (MDSC) gene expression in predicting outcome in stage matched colorectal cancer patients
Authors: Heneidi Saleh G. , Bloomer Chance , Ahluwalia Pankaj , Mondal Ashis , Kolhe Ravindra
Published: August 2019

Colorectal cancer (CRC) has emerged as the third most commonly diagnosed cancer in males and second most common in females. Although surgery, chemotherapy, and emerging immunotherapies have reduced mortality rates, tools that can assist in tailoring individualized treatments are needed. MDSC are immune-suppressive cells that interfere with the functioning of T cells and immune-inflammatory pathways. An extensive literature search identified 8 MDSC-centric genes which were tested for their prognostic significance in CRC. These genes; CEBPB, IL10, NOS2, RORC, S100A8, SOCS1, SOCS3, and TGFB1, along with their expression in tumor microenvironments could give new insights when correlated with clinical-pathologic features of colon cancer patients. The aim of the study is to quantify MDSC gene signatures in colorectal cancer patients. Under an IRB approved protocol, a total of 750 colon cancer patients at the Medical College of Georgia with 5 years of follow-up were initially selected. A total of 88 patients fit in our inclusion criteria, on the basis of survival duration after diagnosis. The FFPE blocks were acquired and patients were stratified on the basis of overall survival in two groups, with higher (>5 years) and lower survival (<1 year), as well as AJCC staging (I to IV), grade, gender and age. Total RNA was isolated and quantified through Nanodrop method. The statistical analysis of data was performed using student t-test and Pearson correlation. SOCS1 (p = 0.00*) was expressed in higher levels in patients with high survival (>5 years). Patients with low expression of TGFB1 survived longer compared to high expression group (p = 0.02*). On race comparison, CEBPA (p = 0.01*) showed significant expression in Caucasian population while NOS2 (p = 0.01*) expressed at higher levels in African Americans. These findings points to the clinical significance of MDSC based genes and tested the utility of a MDSC-related gene expression based prognostic biomarker panel for CRC patients.

Citation Format: Saleh G. Heneidi, Chance Bloomer, Pankaj Ahluwalia, Ashis Mondal, Ravindra Kolhe. Investigating myeloid-derived suppressor cells (MDSC) gene expression in predicting outcome in stage matched colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2775.
Abstract 2783: OMICS analysis of breast cancer PDX tumors to determine CTC-cluster-specific signature in predicting breast cancer metastasis
Authors: Thangavel Hariprasad , Angelis Carmine De , Vasaikar Suhas , Bhat Raksha , Jolly Mohit Kumar , Dobrolecki Lacey Elizabeth , George Jason T. , Giuliano Mario , Lewis Michael , Levine Herbert , Zhang Bing , Schiff Rachel , Trivedi Meghana V.
Published: August 2019

Circulating tumor cell clusters (CTCcl) have been shown to have a significantly higher metastatic potential compared to single CTCs and to predict long-term outcomes in breast cancer patients. A characterization of primary tumors that give rise to CTCcl hold significant promises for better diagnosis and may uncover targets for prevention and treatment of metastatic breast cancer. In our study, we utilized the available transcriptomic and proteomic data (RNA-Seq and RPPA) of 10 triple-negative breast cancer patient-derived xenograft (TNBC-PDX) transplantable models. The sample set consisted of 6 CTCcl-negative (-) and 4 CTCcl-positive (+) models. Genome-wide transcriptomic analysis revealed 549 differentially expressed genes in CTCcl+ TNBC-PDX models vs. CTCcl- models (316 up-regulated and 233 down-regulated, p<0.05). Gene set enrichment analysis (GSEA) terms significantly enriched (FDR<0.05) in CTCcl+ TNBC-PDXs included Cytoplasmic Ribosomal Proteins (NES=2.53), Angiogenesis (NES=1.95), Type II Interferon signaling (NES=-2.52), Antigen processing and presentation (NES=-2.38), TNF signaling pathway (NES=-2.04), Jak-STAT signaling pathway (NES=-1.91), and apoptosis (NES=-1.69). The proteomic analysis revealed elevated levels of Bcl2 expression in PDX tumors associated with CTCcl positivity, which substantiated the inhibited apoptosis pathway as seen in GSEA analysis. The increase in Bcl2 was also validated independently by IHC staining in primary PDX tumors that were CTCcl+. The distribution of EMT score based on the inferential EMT metric using canonical epithelial and mesenchymal markers was not significantly different between CTCcl+ and CTCcl- TNBC-PDX tumors suggesting no differential regulation of EMT in tumors that give rise to clusters versus only single cells. In summary, our results suggest that primary tumors with active anti-apoptotic and/or survival pathways may promote CTC clusters formation and increase the risk of distant metastasis.

Citation Format: Hariprasad Thangavel, Carmine De Angelis, Suhas Vasaikar, Raksha Bhat, Mohit Kumar Jolly, Lacey Elizabeth Dobrolecki, Jason T. George, Mario Giuliano, Michael Lewis, Herbert Levine, Bing Zhang, Rachel Schiff, Meghana V. Trivedi. OMICS analysis of breast cancer PDX tumors to determine CTC-cluster-specific signature in predicting breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2783.
Abstract 2792: PD-1 modulates CD8+ T cell function in prostate tumor microenvironment
Authors: Stokes James , Berry Eric , Singh Rajesh , Manne Upender , Mishra Manoj K.
Published: August 2019

In general, the Regulatory T (Treg) suppresses the immune response. However, their role and immunosuppressive mechanism(s) in context to programmed death 1 (PD-1) are not completely understood. This study aims to determine the role of PD-1 on Treg cells and their impact on CD8+ T cell function in prostatic tumor microenvironment using transgenic adenocarcinoma of the mouse prostate (TRAMP) cells (TRAMP C1, C2, and C3) as a model system. To execute the above aim, tumor induction studies were performed. Briefly, the C57BL/6 mice were administered with serial log concentrations of TRAMP (C1-3) cells. Interestingly, the TRAMP-C3 cells do not form a tumor, however, TRAMP-C1 and TRAMP-C2 cells do form a tumor. After tumor development, mice were sacrificed by cervical dislocation; tumor, lung, spleen, and draining lymph nodes were harvested when the tumor size reached approximately 20mm. Single cell suspensions were prepared from different organs, cells were then stained with specific antibodies, and flow analyzed for the expression of different immune markers. Our preliminary findings demonstrated that PD-1 expression on Foxp3+ Treg cells displayed greater suppressive capacity against CD8+ T cell function in tumor, lung, spleen, and draining lymph node when compared to the control. More importantly, Foxp3+ Treg(high) PD-1(high) interaction with PDL-1 induced immunosuppression by blocking CD8+ T cells function in the prostatic tumor microenvironment. Our data suggest that the expression of Foxp3 and PD-1 may enhance tumor progression; thus, targeting the PD-1 on Treg cells may be a possible therapy to treat prostate cancer.

Note: This abstract was not presented at the meeting.

Citation Format: James Stokes, Eric Berry, Rajesh Singh, Upender Manne, Manoj K. Mishra. PD-1 modulates CD8+ T cell function in prostate tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2792.
Abstract 28: Dihydrolipoyl dehydrogenase is a key molecule inducing ferroptotic cell death
Authors: Lee Jaewang , Shin Daiha , Roh Jong-Lyel
Published: August 2019

Ferroptosis is iron-dependent, non-apoptotic cell death from lipid peroxidation. Cystine-glutamate transporter (xCT) and glutathione peroxidase 4 (GPX4) are recognized as the key regulators of ferroptosis. Glutathione depletion via cystine deficiency or inhibition of xCT or GPX4 accumulates cellular lipid peroxidation and ferrous iron, resulting in ferroptosis. Glutamine is an amino acid essential for growth of cancer cells as well as induction of ferroptotic cell death via the production of alpha-ketoglutarate (αKG) from glutaminolysis. However, it is still unclear whether αKG or its relevant metabolic process is engaged in ferroptotic cell death. Therefore, we examined the role of αKG dehydrogenase (αKGDH) in induction of ferroptosis in head and neck cancer (HNC) cells. Cystine-deficient conditioned medium or xCT inhibition with sulfasalazine induced typical ferroptotic cell death, which was prevented by the inhibition of glutaminolysis or αKG production. Next, we focused the dihydrolipoyl dehydrogenase (DLD) and dihydrolipoyl succinyltransferase (DLST), consisting of the E3 and E2 components of αKGDH respectively. Ferroptosis was significantly prevented by genetic inhibition of the DLD but not the DLST, which was rescued by re-insertion of the DLD gene. Cellular reactive oxygen, lipid peroxidation, and ferrous iron accumulation were induced by the increased function of αKGDH, that is mainly the DLD, when αKG was converted to succinate in the tricarboxylic acid cycle. Sulfasalazine did not significantly suppress the in vivo growth of the shDLD-transfected HNC cells but vector control transplanted in nude mice. Taken together, our data support that the DLD is a key molecule inducing ferroptosis in cancer cells.

Note: This abstract was not presented at the meeting.

Citation Format: Jaewang Lee, Daiha Shin, Jong-Lyel Roh. Dihydrolipoyl dehydrogenase is a key molecule inducing ferroptotic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 28.
Abstract 2803: CSF-1 receptor-mediated macrophage depletion can induce immunomodulatory resistance mechanisms in murine tumor models
Authors: O'Brien Sarah A. , Skrzypczynska Katarzyna , Orf Jessica , Belmontes Brian , Tan Hong , Lu Daniel , Driver Ian , Egen Jackson
Published: August 2019

Tumor associated macrophages (TAMs) are present within the stroma of most solid tumors where they can act to inhibit immune responses and promote disease progression through expression of immunosuppressive factors and modulation of tumor stroma. Tumor immunotherapies aimed at depletion of TAMs, for instance through the use of blocking antibodies against the CSF1 receptor (CSF1R), have been widely explored, with multiple studies demonstrating that blocking TAM function can inhibit tumor growth/metastasis and stimulate anti-tumor immunity. In order to further dissect the functions of TAM populations and define conditions under which antiCSF1R immunotherapy may have benefit, we evaluated the effect of antiCSF1R treatment on multiple subcutaneous murine tumor models with varying dosing parameters. Despite robust macrophage depletion, some tumor models, such as Renca renal cell adenocarcinoma, showed only a modest change in tumor volume and minimal effector T cell recruitment or activation in response to anti-CSF1R therapy. Comprehensive flow cytometry-based immunophenotyping and single-cell RNA sequencing on immune cells isolated from antiCSF1R and isotype control-treated Renca tumors revealed shifts in gene expression patterns related to vascular remodeling, hypoxic responses, and enhanced regulatory T cell activity. These studies indicate macrophage depletion may, under some conditions, result in compensatory anti-tumor immune responses that could limit the clinical benefit of this therapeutic approach.

Citation Format: Sarah A. O'Brien, Katarzyna Skrzypczynska, Jessica Orf, Brian Belmontes, Hong Tan, Daniel Lu, Ian Driver, Jackson Egen. CSF-1 receptor-mediated macrophage depletion can induce immunomodulatory resistance mechanisms in murine tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2803.
Abstract 2808: Investigating the role of macrophage-mediated suppression in the efficacy of NK cell immunotherapy for metastatic outgrowth of breast cancer cells
Authors: Brownlie Demi , Doughty-Shenton Dahlia , Pollard Jeffrey W. , Kitamura Takanori
Published: August 2019

Breast cancer is the leading cause of cancer-related death in females worldwide. Although 5-year survival of breast cancer patients in early stages is 89-100%, that of patients with metastatic tumours is reduced to just 21%, suggesting the requirement of more effective therapies for metastatic breast cancer (MBC). As MBC is refractory to current therapies, NK cell infusion has been suggested as an alternative therapy. However, its efficacy is modest in MBC possibly due to the suppressive nature of the tumour microenvironment, especially through the tumour-associated macrophages (TAMs) within it. Here we investigated the effects of TAMs in metastatic tumours on NK cell cytotoxicity, as well as the mechanism behind TAM-mediated NK cell suppression.

To evaluate NK cell cytotoxicity, we established a method whereby mouse breast cancer cells were cultured with splenic NK cells, and the resultant tumour cell apoptosis was determined by quantitative fluorescence microscopy. To investigate the effects of TAMs on this NK cytotoxicity, we purified them from lungs with metastatic tumours and added them to this co-culture. We also investigated the suppressive effects of bone marrow-derived macrophages cultured with M-CSF (M-BMMs). To further investigate the mechanism, we depleted TAMs in mice with metastatic tumours and detected inhibitory receptor expression on NK cells as well as NK cell inhibitory ligands on TAMs in the metastatic lung and on M-BMMs by flow cytometry.

TAMs from metastatic tumours significantly reduced NK cell cytotoxicity toward tumour cells. M-BMMs (resembling TAMs) also reduced NK cell cytotoxicity. We also found that TAMs and M-BMMs expressed high levels of NK cell inhibitory ligands. There was no difference in the expression of inhibitory receptors on NK cells between tumour bearing lungs with or without TAMs.

To conclude, we have shown that TAMs in metastatic tumours, as well as their mimetic M-BMMs, can suppress NK cell cytotoxicity towards breast cancer cells in vitro. Our data suggest that TAMs express higher levels of inhibitory ligands and thereby transmit suppressive signals possibly through direct contact with NK cells. Further investigation of mechanisms behind TAM-mediated NK suppression might lead to the improvement of NK cell-based immunotherapy for MBC.

Citation Format: Demi Brownlie, Dahlia Doughty-Shenton, Jeffrey W. Pollard, Takanori Kitamura. Investigating the role of macrophage-mediated suppression in the efficacy of NK cell immunotherapy for metastatic outgrowth of breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2808.
Abstract 2812: Triple-negative breast cancer cell-intrinsic glucocorticoid receptor activity and modulation of the immune microenvironment
Authors: Dolcen Deniz N. , Szymanski Diana West , Panchamukhi Ananth , Wang Xiaomeng , Sweis Randy F. , Nanda Rita , Conzen Suzanne
Published: August 2019

Background: Variation in the infiltration of TNBC immune microenvironment is not well understood. Our lab previously demonstrated that high glucocorticoid receptor (GR; nuclear hormone receptor) activity in TNBC models is associated with tumor cell-intrinsic anti-inflammatory gene expression pathways. In addition, high GR expression in early-stage TNBC is associated with a significantly lower rate of recurrence-free survival suggesting a role for GR-associated mechanisms in TNBC progression. GR activation in lymphocytes is well known to result in their apoptosis; however, a current gap in knowledge regarding GR biology is whether tumor-cell intrinsic GR activity modulates immune cells in the microenvironment. We hypothesized that TNBC cell-intrinsic GR activity may inhibit antitumor T cell activity in the tumor microenvironment.

Methods and Results: We divided TCGA primary TNBC RNAseq expression data based on GR expression and determined enrichment for immune cells using two transcriptome parsing algorithms. We found that TNBCs with high GR expression are significantly enriched for the T cell-inflamed gene expression signature, suggesting the presence of T cells in the microenvironment. The xCell algorithm found that TNBCs with high GR expression are significantly enriched for the CD4+ naïve T cell transcriptome, which is absent in TNBCs with low GR-expression. To begin to determine whether GR activation in TNBC cells induces the secretion of cytokines and/or growth factors that result in the chemotaxis of CD4+ naïve T cells, we collected conditioned cell culture media from control versus GR-active TNBC cells and performed multiplex cytokine ELISA. We found that G-CSF secretion by TNBC cells was significantly increased following GR activation; G-CSF has been shown to promote immunosupressive Treg cell differentiation.

Conclusions: Prior data suggest that breast tumor-infiltrating immunosuppressive Tregs arise from chemotaxis of circulating naive CD4+ T cells that differentiate into Tregs in situ. Thus targeting GR in TNBC may be an important therapeutic approach to potentiate antitumor T cell responses and suppress the Treg population in the tumor microenvironment. Ongoing work is aimed at further delineating a mechanism by which GR can modulate the immune tumor microenvironment.

Citation Format: Deniz N. Dolcen, Diana West Szymanski, Ananth Panchamukhi, Xiaomeng Wang, Randy F. Sweis, Rita Nanda, Suzanne Conzen. Triple-negative breast cancer cell-intrinsic glucocorticoid receptor activity and modulation of the immune microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2812.
Abstract 2823: Sample storage conditions alter microbiome profiles - its potential relevance for cancer patients undergoing fecal microbial transplants
Authors: Jenkins Samir V. , Vang Kieng B. , Gies Allen , Griffin Robert J. , Jun Se-Ran , Nookaew Intawat , Dings Ruud Petrus
Published: August 2019

Background and Objective: Fecal microbiota transplantation helps restore beneficial bacteria in cancer patients after intense antibiotics administration. Here we investigated the influence of different stabilization and storage strategies on the quality and composition of the fecal microbial community.

Methods: Same-day isolated murine DNA was compared to samples stored for one month in air at ambient temperature, with or without preservative buffers (i.e. EDTA and lysis buffer), different temperatures (i.e. 4°C, -20°C, and -80°C), and hypoxic conditions.

Results: Only storage in lysis buffer significantly reduced DNA content, yet without integrity loss. Storage in EDTA affected alpha diversity the most, which was also reflected in cluster separation. Distinct changes were also seen in the phyla and bacterial species abundance per storage strategy. Metabolic function analysis showed 22 pathways not significantly affected by storage conditions, whereas the tyrosine metabolism pathway was significantly changed in all strategies except by EDTA.

Conclusions: Overall, each long-term storage strategy introduced a unique post-collection bias, which is important to take into account when interpreting data. Funded by COBRE P20GM103625.

Citation Format: Samir V. Jenkins, Kieng B. Vang, Allen Gies, Robert J. Griffin, Se-Ran Jun, Intawat Nookaew, Ruud Petrus Dings. Sample storage conditions alter microbiome profiles - its potential relevance for cancer patients undergoing fecal microbial transplants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2823.
Abstract 283: Combination treatment of PARP and SRC inhibitors in BRCA2 mutated prostate cancer
Authors: Khan Nabeela , Chakraborty Goutam , Nandakumar Subhiksha , Mazzu Ying Z. , Atiq Mohammad , Yoshikawa Yuki , Lee Gwo-Shu Mary , Kantoff Philip
Published: August 2019

Background: Recently, poly (adenosine diphosphate [ADP]-ribose) polymerase inhibitors (PARPi) have shown promise in metastatic castration resistant prostate cancers (mCRPC) patients harboring DNA repair defects due to mutations of BRCA1/2 and ATM. Despite responses, resistance is common and treatment modalities for PARPi-resistant patients are limited. We hypothesized that combining PARPi with other agents in BRCA2 altered tumors would create synergy and greater response rate and durability.

Methods: We established a transcriptomic profile associated with genomic deletion of BRCA2 (homozygous and heterozygous) using publicly available datasets from mCRPC patient tumors. To identify the significantly unregulated oncogenic signaling pathways associated with BRCA2 loss, we used gene-set enrichment analysis (GSEA). We also analyzed this transcriptomic profile by Toppgene suite to identify potential drug targets for BRCA2 deleted tumors. We used experimental human prostate cancer cell lines to validate our current observations from human clinical datasets and pathway analysis.

Results: Our GSEA analysis showed significant enrichment of the SRC signaling pathway in BRCA2-altered tumors, and based on the Toppgene suite, we also identified dasatinib (pharmacological inhibitor of SRC) as a potential agent for BRCA2-deleted tumors. Similarly, we observed significant up regulation of SRC phosphorylation in human prostate cancer cells (LNCaP-abl and PC3M) that harbor genomic deletion of BRCA2. We performed cell growth assays in these BRCA2 deleted cells treated with PARPi (olaparib, talazoparib), and dasatinib alone or in combination and calculated drug synergy based on the Chou-Talay Method. PC3M and LNCaP-abl prostate cancer cell lines showed different sensitivities to olaparib and talazoparib. We used olaparib for PC3M and talazoparib for LNCaP-abl cells. Co-administration of the PARPi and SRCi showed significant synergy in both cell lines compared to either inhibitors alone. We will develop the rationale for combining PARPi and SRCi in CRPC patients who harbor defects of BRCA2 after completion of our study in xenograft models.

Conclusion: Our study reveals the synergistic effect of PARPi and SRCi in prostate cancer cell lines. We believe this shows great potential for future clinical trials in patients with mCRPC harboring BRCA2 deletions.

Citation Format: Nabeela Khan, Goutam Chakraborty, Subhiksha Nandakumar, Ying Z. Mazzu, Mohammad Atiq, Yuki Yoshikawa, Gwo-Shu Mary Lee, Philip Kantoff. Combination treatment of PARP and SRC inhibitors in BRCA2 mutated prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 283.
Abstract 2833: Alterations in intestinal microbiota of colorectal cancer patients receiving radical surgery combined with adjuvant CapeOx therapy
Authors: Kong Cheng , Gao Renyuan , Qin Huanlong
Published: August 2019

Background: An intricate relationship exists and interactions occur between gut microbiota and colorectal cancer (CRC). Radical surgery combined with adjuvant chemotherapy (AC) serves as the mainstream therapeutic scheme for most CRC patients. However, the impact of surgery or chemotherapy on gut microbiota remains elusive.

Methods: Forty-three CRC patients who received radical surgery and AC were enrolled in the present study. Fecal samples of these patients were collected preoperatively, postoperatively, and after the first to fifth cycles of postoperative chemotherapy. The microbial community of each sample was analyzed using high throughput 16S rRNA amplicon sequencing.

Results: Compared with preoperative samples, fecal samples collected postoperatively exhibited a significant decrease of obligate anaerobes (Bacteroides, Parabacteroides, Faecalibacterium, Prevotella_9 and Bifidobacterium), tumor-related bacteria (Enterococcus and Fusobacterium), and butyric acid-producing bacteria (Bacillus, Bilophila, Barnesiella and Enterococcus). However, a significant increase of some conditional pathogens was observed (Escherichia-Shigella, Enterobacteriaceae_unclassified, Veillonella, Morganella, Streptococcus, and Proteus). In addition, the AC regimen (CapeOx) was found to alter intestinal microbiota dramatically. In particular, several changes were observed after chemotherapy including an increase of pathogenic bacteria (Collinsella, Anaerostipes, Bilophila, Comamonas, Weissella, and Eggerthella), the “rebound effect” of chemotherapy-adapted bacteria (Dorea, Ruminococcaceae_UCG-010, Streptococcus, Prevotella_9, Mogibacterium and Roseburia), the shift of lactate-utilizing microbiota from Veillonella to Butyricimonas and Butyricicoccus, as well as the decrease of probiotics (such as Lactobacillus).

Conclusions: Both radical surgery and CapeOx chemotherapy exert a non-negligible effect on the gut microbiota of CRC patients. Microbiota-based intervention may be beneficial for these patients during postoperative clinical management.

Citation Format: Cheng Kong, Renyuan Gao, Huanlong Qin. Alterations in intestinal microbiota of colorectal cancer patients receiving radical surgery combined with adjuvant CapeOx therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2833.
Abstract 284: Combination therapies to prevent resistance to androgen deprivation therapies in prostate cancer
Authors: Li Zhenghong , Sun Carrie Qi , Arnold Rebecca , Petros John A. , Moreno Carlos S.
Published: August 2019

Androgen receptor (AR) signaling is a distinctive feature of prostate cancer (PCa) and represents a major therapeutic target for treating metastatic prostate cancer (mPCa). Thus, androgen deprivation therapy (ADT) is a first-line treatment for mPCa. Although initially highly effective as a treatment for mPC, ADT is characterized by the frequent emergence of resistance, a disease state termed castration-resistant prostate cancer (CRPC) and is generally incurable after progression to metastatic disease. Therefore, understanding the mechanisms underlying CRPC and subsequent progression to metastatic disease is critical. In our previous study, which was mainly focused on how transcriptional networks change in response to ADT and lead to metastasis, we analyzed matched pre-ADT and post-ADT tissue samples via RNAseq analysis of 40 formalin-fixed paraffin-embedded (FFPE) patient-matched pre-ADT biopsy (Bx) and post-ADT radical prostatectomy (RP) prostate cancer samples. We observed strong upregulation of components of the MAPK pathway including FOS, FOSB, and JUN, as well as downstream targets of MAPK signaling. These data suggest that ADT may induce a compensatory increase in MAPK signaling in response to the decrease in androgen signaling. Thus, we hypothesize that combination therapies targeting AR and the MAPK pathway may synergistically kill prostate cancer cells and prevent recurrence and progression to CRPC. In the current study, we have tested the effects of the MEK inhibitors PD0325901 and GSK1120212, ERK1/2 inhibitor GDC-0994, and the JNK inhibitor AS602801 alone and in combination with enzalutamide in androgen-sensitive LNCaP and MDA-PCa-2b cells. Cell viability assays indicated that enzalutamide combined with MEK and JNK inhibitors synergistically killed LNCaP and MDA-PCa-2b cells, and decreased migration and invasion of LNCaP cell more than any of the drugs alone. We therefore propose that combination therapy targeting AR and MEK and/or JNK signal pathways may be an effective treatment for recurrent prostate cancer. We are currently investigating the most promising combinations of enzalutamide with JNK inhibitors for anti-tumorigenic effects in vivo using a mouse xenograft model.

Citation Format: Zhenghong Li, Carrie Qi Sun, Rebecca Arnold, John A. Petros, Carlos S. Moreno. Combination therapies to prevent resistance to androgen deprivation therapies in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 284.
Abstract 2846: Chemerin, as a serum biomarker, promotes tumorigenesis and metastasis in oral squamous cell carcinoma
Authors: Wang Anxun
Published: August 2019

Chemerin encoded by retinoic acid receptor responder 2 (RARRES2) has been found to be related with malignant tumors, but its causal role in the development of oral squamous cell carcinoma (OSCC) is largely unexplored. In the present study, clinical data showed that higher serum levels of chemerin were observed in OSCC patients compared to the healthy and significantly decreased after tumor resection, and were positively associated with advanced tumor stage and lymph node metastasis. The expression level of chemerin was positive correlated with the migration and invasion of OSCC cell lines. Recombinant chemerin dose-dependently enhanced the migration, invasion and proliferation of OSCC cells in vitro, and shRNA targeting RARRES2 inhibited chemerin expression, OSCC cells metastasis and proliferation in vitro and vivo. Additionally, Chemerin activated the Manganese superoxide dismutase (SOD2) activity and increased intracellular H2O2, led to a significant decrease of E-cadherin expression and a dramatically increasing expression of p-ERK1/2, Slug, Vimentin and N-cadherin. This study suggested that chemerin was a novel biomarker for OSCC patients. Chemerin promoted the tumorigenesis and metastasis of OSCC and would be a new therapeutic target for OSCC.

Keywords: Chemerin; Oral squamous cell carcinoma; Tumorgenesis; Metastasis; SOD2

Citation Format: Anxun Wang. Chemerin, as a serum biomarker, promotes tumorigenesis and metastasis in oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2846.