bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5116: Benchmarking the HLA typing performance of three HLA assay and two software
Authors: Meng Rui , Zhu Tao , Gong Yu , Chen Yanan , Dong Hua , Yizhou Ye , Li Fugen , Liu Ping , Yao Minya
Published: August 2019

Background:The adaptive immune response intrinsically depends on hyper variable human leukocyte antigen (HLA) genes. Correct HLA typing is crucial for successful donor-patient matching in organ transplantation and recently is also an emerging biomarker for immune checkpoint inhibitor. With the great progress on next generation sequencing (NGS) technology, sequencing accuracy and throughput have increased while the cost for data have decreased faster than Moore's Law, thus HLA tying by NGS has been widely used from laboratory to clinic. Method:We tested three NGS assay for HLA typing and two recently published HLA typing software from 24 samples. Three assays involve two target capture methods (WES and HLA target sequencing) and one amplification sequencing method. Two software involve Xhla and HLAHD, respectively. First, we compared 3 assay performance based on PE150 read length and xHLA software by both 4 alleles and 8 alleles. Second, we compared the performance of two widespread HLA typing software xHLA and HLA-HD according to different assay type, read depth, read length and speed. Results:First of all, for PE150 datasets on three assay, the accuracy rate of HLAHD is all 100%, while xhla in WES is 99%, amplicom 98.6%, target HLA sequencing 100%. On reads length evaluation, the accuracy of HLAHD decreases gradually from PE150 to PE100 and PE75, and the capture method is more sensitive to reads length than the amplification assay, while Xhla is very robust on read length. On coverage evaluation, when the amplicom sequencing data was down-sampled 500X to 10X, the accuracy of HLAHHD and XHLA is both 100% in 50X and above and decrease gradually when less than 50X. In terms of time, with the increase in sequencing depth, running time for both software increase. HLAHD prediction from fastq file is longer than that from bam file, especially when the panel data is relatively large, the time will increase exponentially. Conclusions:Our benchmarking dataset show that current HLA assay and computational methods for HLA typing generally provide satisfactory results, while different assay type matched with different software, read depth and read length do affect the performance.

Citation Format: Rui Meng, Tao Zhu, Yu Gong, Yanan Chen, Hua Dong, Ye Yizhou, Fugen Li, Ping Liu, Minya Yao. Benchmarking the HLA typing performance of three HLA assay and two software [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 5116.
Abstract 512: Adrenergic modulation of inflammatory cytokines in ovarian cancer
Authors: Colon-Echevarria Claudia B. , Ortiz Tatiana , Maldonado-Laboy Lizette , Hidalgo-Vargas Melanie J. , Aquino Alexandra N. , Armaiz-Pena Guillermo N.
Published: August 2019

Work from our group and others suggest that chronic stress accelerates growth of existing tumors by activating the sympathetic nervous system. Specifically, our data suggest that sustained adrenergic signaling can induce tumor growth, secretion of pro-inflammatory cytokines and macrophage infiltration. Moreover, increased macrophage infiltration was associated with decreased survival in ovarian cancer patients. Hence, we investigated role of adrenergic-stimulated macrophages in ovarian cancer biology. To assess the effect of adrenergic stimulation in pro-tumoral functional outputs, we used cytokine arrays and invasion assays. A pre-clinical orthotopic model of ovarian cancer was used to assess the in vivo effect of daily restraint stress on tumor growth and adrenergic-induced macrophages. Cytokine array analyses showed that adrenergic stimulation modulated pro-inflammatory cytokine secretion in a SKOV3ip1 (ovarian cancer cells) /U937 (macrophages) co-culture system. Among these, 23 significantly upregulated cytokines were identified in both, epinephrine and norepinephrine, treated co-culture systems. PDGF-AA, ENA-78, Angiogenin, VEGF, GM-CSF, IL-5, Lipocalin-2, MIF, and TfR were among upregulated cytokines shared between treatment groups. In addition, daily restraint stress resulted in increased ovarian cancer growth and infiltration of CD68+ macrophages in two orthotopic models of ovarian cancer (SKOV3ip1 and HeyA8), while zoledronic acid (a bisphosphonate that has been shown to impair macrophage activity) abrogated this effect. Immunohistochemistry analyses showed restraint stress increased expression of PDGF-AA and ENA-78 inflammatory cytokines. Here, we show that adrenergic regulation of macrophages might play a key role in the progression of ovarian cancer.

Citation Format: Claudia B. Colon-Echevarria, Tatiana Ortiz, Lizette Maldonado-Laboy, Melanie J. Hidalgo-Vargas, Alexandra N. Aquino, Guillermo N. Armaiz-Pena. Adrenergic modulation of inflammatory cytokines in ovarian 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 512.
Abstract 5122: Comprehensive detection of germline and somatic structural mutation in cancer genomes by Bionano Genomics optical mapping
Authors: Pang Andy
Published: August 2019

In cancer genetics, the ability to identify constitutive and low-allelic fraction structural variants (SVs) is crucial. Conventional karyotype and cytogenetics approaches are manually intensive. Microarrays and short-read sequencing cannot detect calls in segmental duplications and repeats, often miss balanced variants, and have trouble finding low-frequency mutations.

We describe the use of Bionano Genomics’s Saphyr platform to comprehensively identify SVs for studying cancer genomes. DNA >100 kbp is extracted, labelled at specific motifs, and linearized through NanoChannel arrays for visualization. Molecule images are digitized and de novo assembled, creating chromosomal arm scale genome maps. Somatic mutations can be identified by running the variant annotation pipeline that compares the cancer sample’s assembly SVs against >600,000 SVs in Bionano’s control sample SV database, and against a matched control sample’s SVs, if avaliable. Also, two new Bionano pipelines leverage these long molecules to identify additional somatic SVs: the copy number variation (CNV) and the molecule mapping pipelines. By examining the coverage-depth of molecules alignment to the public reference, the pipeline can identify megabases long CNVs. Similarly, clusters of split-molecule alignments can reliably find translocations and other rearrangements.

We applied this suite of discovery tools to identify SVs in a well-studied melanoma cell line COLO829. We collected data from the tumor and the matched blood cell line, constructed contiguous assemblies (N50 >50 Mbp), and called >6,000 SVs in each genome. Then, we classified 51 as somatic by comparing the tumor and the blood control. The two new pipelines further increased sensitivity to rearrangements, for example they captured a BRAF duplication, and other chromosome-arm CNVs. We apply these thorough approaches to multiple well-studied cancer lines to identify novel SVs missed by previous studies. In conclusion, with one comprehensive platform, Saphyr can discover a broad range of traditionally refractory but relevant SVs, and further improves our understanding of cancer.

Citation Format: Andy Pang. Comprehensive detection of germline and somatic structural mutation in cancer genomes by Bionano Genomics optical mapping [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 5122.
Abstract 5127: Association of inactivating calcium sensing receptor exon 7 SNPs with hypercalcemia-related disease phenotypes
Authors: Actkins Ky'Era , Beasley Heather , Faucon Annika , Davis Lea , Sakwe Amos
Published: August 2019

The calcium sensing receptor (CaSR) is a ubiquitously expressed G-protein coupled receptor known to be a major calcium sensor in most tissues and organ systems. It is known to regulate systemic calcium homeostasis by sensing slight increases in systemic calcium and by influencing the secretion of parathyroid hormone by parathyroid chief cells. Unfortunately, the CaSR is known to be invariably mutated and inactivating mutations in particular, are associated with the development of cancer-induced hypercalcemia (CIH). Among the several inactivating CASR polymorphisms described thus far, the rs1801725 (A986S SNP) is common among Caucasian subjects while the rs1801726 (Q1011E SNP) is common among African American subjects. However, whether these mutations contribute to other hypercalcemia-related disease phenotypes remains unclear. In this study, we carried out a phenome-wide association study (PheWAS) using the Synthetic Derivative (SD), a database at VUMC containing de-identified electronic health records (EHRs) linked to patient DNA samples in the BioVU biorepository. Using 39,893 genotyped individuals in the Multi-Ethnic Genotyping Array (MEGA), we observed that SNP rs1801725 was significantly associated with disorders of bone and cartilage (p = 5.93e-6, OR = 1.28) as well as with secondary malignancy of bone (p = 1.46e-5, OR = 1.21); whereas SNP rs1801726 was significantly associated with osteoporosis related phenotypes (p = 5.93e-6, OR = 1.28). Given that breast cancer and other solid tumors frequently metastasize to skeletal tissues, these data suggests that the inactivating rs1801725 and rs1801726 CaSR variants differentially influence hypercalcemia-related comorbidities and cancer metastasis to bone. We will further verify these effects using the associated clinical data and perform genetic risk analyses for the expression of these SNPs and the associated disease phenotypes.

Citation Format: Ky'Era Actkins, Heather Beasley, Annika Faucon, Lea Davis, Amos Sakwe. Association of inactivating calcium sensing receptor exon 7 SNPs with hypercalcemia-related disease phenotypes [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 5127.
Abstract 5144: The potential biomarkers and rational combination of immunotherapy in microsatellite stable colorectal cancer
Authors: Lu Xuman , Ji Zhe , Zhao Jing , Zhang Yuzi , Huang Mengli , Cai Shangli , Huang Jun
Published: August 2019

Purpose Immune checkpoint blockades (ICBs) have been reported to be used in early stage colorectal cancer (CRC) as neoadjuvant therapy. However, it seems that microsatellite stable (MSS) CRC patients obtained no significant clinical benefit in studies with small sample size. We aimed to explore the potential biomarkers and rational immunotherapy to optimize response to ICBs in MSS CRC.

Procedures Whole exome sequencing and RNAseq of 612 CRC patients from The Cancer Genome Atlas were analyzed. T cell dysfunction/exclusion signatures and Tumor infiltrating lymphocytes were calculated from TIDE model and TIMER algorithm, respectively. Mann Whitney U test and Kruskal test were used to compare the difference among two or more than two groups. P value was adjusted by BH method for multiple comparisons.

Results There were 109 stage I, 228 stage II, 184 stage III and 91 stage IV in 612 CRC patients. The MSI-H prevalence, tumor mutation burden, PD-L1 mRNA and CD8 T cell infiltration decreased with the increase in pathologic stage (all P < 0.01). In MSS CRC, mRNA expression of immune checkpoint related genes including PD-L1, CTLA4 and LAG3 were significantly higher in stage I-III than stage IV (adjusted P < 0.01, < 0.01 and 0.04, respectively). For immune activation genes (CD8A, CXCL9, CXCL10, GBP1, GZMB, GZMA, IFNG, FAS, STAT1, TAP1), their mRNA expression levels increased with the decrease of stage (All adjusted P < 0.01). Besides, stage I-III MSS CRC had lower T cell exclusion signature score than stage IV (adjusted P = 0.02). No similar results were observed in MSI-H. Compared with MSI-H, MSS had higher M2 macrophage signature score (P < 0.001), but no association were observed between M2 macrophage signature and stage.

Conclusions Early stage CRC with MSS had higher levels of positive predictive features for ICBs treatment, which suggested stronger immune responsiveness. Combination of M2 macrophage inhibitor and ICBs has the potential to turn “cold tumor” into “hot tumor” and acts as a promising therapeutic regimen, especially for early stage CRC. More clinical trials are needed to confirm this hypothesis.

Note: This abstract was not presented at the meeting.

Citation Format: Xuman Lu, Zhe Ji, Jing Zhao, Yuzi Zhang, Mengli Huang, Shangli Cai, Jun Huang. The potential biomarkers and rational combination of immunotherapy in microsatellite stable colorectal 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 5144.
Abstract 5152: EMT cells increase the metastatic potential of neighboring carcinoma cells via non-canonical activation of GLI signaling through secretion of VEGF-C
Authors: Kong Deguang , Neelakantan Deepika , Zhou Hengbo , Lewis Michael T. , Ford Heide L.
Published: August 2019

Breast cancer is a devastating disease that claims around 50 million lives per year worldwide, and almost all of these deaths result from metastatic disease rather than from primary tumor burden. Recent studies clearly demonstrate that there is significant intratumor heterogeneity in breast cancer, and that this heterogeneity contributes to malignant progression. Our laboratories previously demonstrated that breast cancer cells that had undergone an oncogenic epithelial-to-mesenchymal transition (EMT) could increase metastasis of neighboring cells via activation of non-canonical GLI transcription factor activity in a paracrine manner that is dependent on Six1 expression in the EMT cells. However, the mechanism by which these Six1-expressing EMT cells activate GLI signaling thereby imparting aggressive properties on neighboring cells remained unknown. Herein we describe the novel discovery that VEGF-C, which is transcriptionally upregulated by Six1, mediates paracrine non-canonical activation of GLI and resultant enhancement of metastatic properties in cells that do not express Six1. Our data demonstrate that VEGF-C is upregulated in HMLER-Snail1 cells, MCF7-Six1 cells and in Met-1 cells endogenously expressing Six1, in a manner that depends on Six1 expression, and that VEGF-C is secreted into the conditioned media (CM) of these cells. Inhibition of VEGF-C in the HMLER-Snail1, MCF7-Six1, and the Met-1 models abrogates paracrine GLI activation and attenuates non-cell autonomous induction of proliferation, migration, and invasion in vitro. In vivo, we show that cells expressing Six1 can enhance the growth and metastasis of those not expressing Six1, and studies will be discussed that examine whether VEGF-C inhibition in Six1 expressing cells disrupts the crosstalk and inhibits non-cell autonomous induction of metastasis by Six1. Finally, by interrogating the TCGA dataset we find that VEGF-C and GLI1 expression significantly positively correlate in human breast cancer encompassing all molecular subtypes. Taken together, these data suggest that VEGF-C secretion may be a novel and conserved paracrine means by which EMT cells activate GLI in neighboring tumor cells that do not express these EMT-inducing transcription factors (TF), ultimately enhancing overall metastasis of heterogenous breast tumors.

Citation Format: Deguang Kong, Deepika Neelakantan, Hengbo Zhou, Michael T. Lewis, Heide L. Ford. EMT cells increase the metastatic potential of neighboring carcinoma cells via non-canonical activation of GLI signaling through secretion of VEGF-C [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 5152.
Abstract 5153: Measuring T-cell avidity and enrichment using an acoustic force based technology
Authors: Raja Ali , Scheper Wouter , Merino Elena , Braster Rens , Sitter Gerrit , Oswald Felix , Schumacher Ton , Candelli Andrea , Anderson Leif
Published: August 2019

The key driver for effective immunotherapies is the overall strength binding of the immune cell and the target cell (e.g. tumor cells). The overall strength is known as ‘avidity’, a parameter reflecting interaction efficiency. One of the main obstacles has been the generation of immunotherapies promoting effective and long-lasting immune responses, due to the lack of tools measuring this parameter. Here, we have made used of acoustic forces as a novel method to isolate immune cells based on their avidity to specific targets, such as (tumor) cells. The force that cell-target need to experience to be separated is called ‘rupture force’. In this study, we were able to identify the rupture forces of tumor specific and non-specific T cells and separate these different populations applying controllable acoustic forces. T cells engineered with a melanoma antigen recognizing T-cell receptor needed 6 times more relative force than the control, non-specific T cells, to be separate from their melanoma cells. These findings indicate that the specific melanoma T cells bind with higher efficiency and higher avidity than the non-specific ones, and that they can be separated with this method. Furthermore, 1.4 to 3.6-fold enrichment of high avidity T cells from a mixed specific: non-specific (1:10) T cell population was obtained after their isolation using acoustic forces and confirmed using FACS measurements. Therefore, we demonstrate here that acoustic forces can be use as a tool to measure avidity.

Citation Format: Ali Raja, Wouter Scheper, Elena Merino, Rens Braster, Gerrit Sitter, Felix Oswald, Ton Schumacher, Andrea Candelli, Leif Anderson. Measuring T-cell avidity and enrichment using an acoustic force based technology [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 5153.
Abstract 516: Metformin inhibits monocyte differentiation to tumor associated macrophage (TAM) and may restore anti-tumor immunity
Authors: Saito Akira , Ohzawa Hideyuki , Tojo Mineyuki , Kumagai Yuko , Kanamaru Rihito , Tsukui Hidenori , Shiba Satomi , Ito Homare , Sata Naohiro , Kitayama Joji
Published: August 2019

Background: Tumor-associated macrophages (TAMs) play pivotal roles to regulate tumor cell behavior in tumor microenvironment and a high density of TAMs is associated with poor prognosis in various cancers. Recent studies have demonstrated that metformin, the first-line medication for the treatment of type 2 diabetes, have suppressive effects on tumor growth. Here, in this study, we investigated the effect of metformin on monocyte differentiation to TAMs.

Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers and CD14+ peripheral blood monocytes(PBMo) were purified by positive selection using the magnetic cell separation system. The PBMo were co-cultured with Panc-1, a human pancreas cancer cell, with metformin (10μM~10mM) using double chamber inserts of 0.4-μm sized pores at 37°C in 5% CO2 conditions for 5 days. Separately, PBMo were cultured with M-CSF for 5 days and then with IL-10 (20ng/ml)+TGF-β(20ng/ml) for addition 2 days with or without metformin. The differentiation to TAM were evaluated by the expression of M1/M2 markers as well as PD-L1/L2 by flowcytometry. The macrophages were co-cultured with autologous T cells stimulated with anti-CD3 and anti-CD3 and anti-CD28 microbeads and the effect on T cell proliferation was evaluated with CFSE dilution assay.

Results: PBMo co-cultured with Panc-1 showed significantly enhanced expression of CD163, CD206, as well as markedly increased side scatter, suggesting the differentiation to M2-type macrophages. The macrophage also highly expressed PD-L1 and strongly inhibited the proliferation of autologous T cells. However, the expression of these antigens and inhibition of T cell growth were significantly suppressed by the presence of metformin with dose dependent manner. Metformin was effective at 100μM and almost nullified the effects of Panc-1 at 1mM. Metformin did not affect the antigen expression pattern during the treatment by M-CSF, but did downregulate the expression of CD206 and PD-L1 if added together with IL-10 +TGF-β.

Conclusion: During the co-culture with tumor cells, metformin suppresses the monocyte differentiation to TAM at relatively late stage. Metformin can restore T cell mediated-tumor immunity in tumor microenvironment, at least partly, through the effects on TAM, that may result in tumor inhibition.

Citation Format: Akira Saito, Hideyuki Ohzawa, Mineyuki Tojo, Yuko Kumagai, Rihito Kanamaru, Hidenori Tsukui, Satomi Shiba, Homare Ito, Naohiro Sata, Joji Kitayama. Metformin inhibits monocyte differentiation to tumor associated macrophage (TAM) and may restore anti-tumor immunity [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 516.
Abstract 5167: Cell autonomous and non-autonomous actions of CCL22 in oral carcinogenesis
Authors: Wu Li-Wha
Published: August 2019

Oral cancer, a subtype of head and neck cancer, has been the 4th leading cause of male cancer death in Taiwan for the past decade. Smoking, alcohol consumption, and betel quid chewing are major risk factors for oral cancer in Taiwan. The crosstalk between immunity and cancer cells can both inhibit and enhance tumor growth and is now classified as a cancer hallmark. The accumulation of regulatory T cells (Tregs) in several tumor types potentially plays a critical role in tumor evasion of immune surveillance. Oral cancer is also characterized by an increase in the number of infiltrating Tregs; however, the significance of the increase on the patient prognosis remains controversial. We found that the expression of CCL22, a chemokine for Treg recruitment, was not only positively with poor prognosis in oral cancer patients but also significantly correlated with that of FOXP3, a master regulator for Treg development and functions, in the same patients. The correlation is also validated in TCGA database analysis of head and neck cancer patients. The depletion of CCL22 expression reduced oral cancer cell proliferation, migration and invasion, suggesting an oncogenic role of CCL22 in oral cancer. Since CCR4 is the receptor for CCL22, we studied the in vitro and in vivo impact of deregulated CCL22-CCR4 signaling axis in oral carcinogenesis. We first confirmed the surface expression of CCR4 in oral cancer cells by flow cytometry. Ectopic CCL22 overexpression increased oral cancer cell migration and invasion without the effect on proliferation. Through injection of CCL22-altered oral cancer cells in both syngeneic and athymic backgrounds, the cell autonomous and non-autonomous actions of CCL22 and the mechanism responsible for the CCL22 deregulation in oral cancer will be discussed. This study will not only further the role of CCL22-CCR4 signaling axis in modulating the crosstalk between oral cancer and its microenvironment but also provide a novel therapeutic strategy for treating oral cancer.

Citation Format: Li-Wha Wu. Cell autonomous and non-autonomous actions of CCL22 in oral carcinogenesis [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 5167.
Abstract 5186: Discovery of a novel histone deacetylase 6 inhibitor that kills drug-resistant breast cancer
Authors: Dowling Catríona , Dillion Eugene , Hemann Michael , Cagney Gerard , Letai Anthony , Chonghaile Triona Ni
Published: August 2019

Cytotoxic chemotherapy is the standard of care for patients with triple negative breast cancer (TNBC). Most patients with advanced TNBC progress after chemotherapy and die from metastatic disease. Currently, no established targeted therapeutics or biomarkers of outcome/response have been clinically approved in the context of TNBC. The aggressive nature of TNBC is reflected by an increased likelihood of distant recurrence and death within five years following primary intervention and a shorter survival once diagnosed with metastatic disease. To identify novel therapeutics for the treatment of chemoresistant TNBC, we performed a high-throughput screen to identify small molecules that are cancer selective but can kill drug-resistant TNBC cells. We screened a total of 30,000 compounds in duplicate across the TNBC cell line MDA-MB-321 in which the pro-death proteins BAX/BAK were knocked down, and the non-transformed "normal" MCF10A cell line. There was a hit rate of 0.3% in the screen and 85 compounds were retested in the validation cherry pick screen. From this screen 18 compounds were further validated with low-throughput assessment for mitochondrial independent killing and selectivity for cancer cells. To identify the mechanism of action of the lead compound we used a genetic approach to generate an RNAi signature for the compound, which indicated the lead compound was most likely that of histone deacetylase inhibitor (HDAC). Using an in vitro HDAC inhibitor screen, we identified that the compound specifically inhibited HDAC6. Using a series of cell based assays, we determined that treatment of cells with the compound BAS-2 resulted in the same phenotypical response (inhibition of migration, invasion and 3D spheroid formation) as HDAC6 knockout. To identify substrates of HDAC6 in cells we analyzed the proteome for increased acetylation by mass spectrometry following HDAC6 knockout or inhibition with the specific inhibitor, BAS-2. Remarkably, we identified a series of new substrates for HDAC6 that are common in both the HDAC6 knockdown and BAS-2 treated cells. In conclusion, we have identified a novel HDAC6 specific inhibitor that selectively kills cancer cells independent of mitochondrial apoptosis, alters acetylation of over a 50 common proteins and inhibits spheroid formation of TNBC.

Citation Format: Catríona Dowling, Eugene Dillion, Michael Hemann, Gerard Cagney, Anthony Letai, Triona Ni Chonghaile. Discovery of a novel histone deacetylase 6 inhibitor that kills drug-resistant breast 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 5186.
Abstract 5214: Targeting the estrogen receptor pathway in luminal breast cancer through inhibition of p300/CBP
Authors: Waddell Aaron , Mahmud Iqbal , Tian Guimei , Liao Daiqing
Published: August 2019

Luminal breast cancer represents approximately two thirds of all breast cancer cases and is characterized by the expression of hormone receptors, such as the estrogen receptor alpha (ER). ERα is a member of the steroid nuclear receptor family and is involved in a hormonal signaling pathway that drives tumor growth through upregulation of genes involved in cell cycle progression, such as MYC and CCND1. Antagonists of ER function are the standard of care treatment for ER+ luminal breast cancer. However, resistance to ER antagonists is common in advanced breast cancer cases. Therefore, there is an urgent need to investigate new therapeutics that can be utilized to treat tumors that are resistant to traditional therapies. p300/CBP are two paralogous acetyltransferases that catalyze histone 3, lysine 18 and 27 acetylation (H3K18ac and H3K27ac) at promoters and enhancers to promote gene expression. ER mediated transcription is critically reliant upon the recruitment of co-activators, including p300/CBP. However, the mechanistic role of p300/CBP catalytic activity in globally regulating ER mediated transcription remains poorly understood. Inhibition of p300/CBP, as a critical co-activator of ER, potentially represents an applicable strategy for inhibiting ER function in luminal breast tumors. Importantly, the catalytic histone acetyltransferase (HAT) domain of p300/CBP is under intense investigation as a target of small molecule therapeutics in cancer. We analyzed publicly available data and report that p300/CBP are upregulated at the mRNA level in breast cancer, including the luminal subtypes, and that their expression negatively correlates with patient survival. We also found that pharmacologic inhibition of p300/CBP HAT activity in ER+ cell lines potently suppresses ER mediated transcription, downregulates ER, c-Myc and Cyclin D1 protein levels and prevents estrogen induced growth in vitro. Studies are underway to understand how p300/CBP regulates ER signaling using genetic, pharmacologic, and biochemical methods to assess the therapeutic effects of pharmacologic p300/CBP inhibition on ER+ breast cancer. (Supported by Bankhead-Coley Research Program, and James and Esther King Biomedical Research Program, Florida Department of Health)

Citation Format: Aaron Waddell, Iqbal Mahmud, Guimei Tian, Daiqing Liao. Targeting the estrogen receptor pathway in luminal breast cancer through inhibition of p300/CBP [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 5214.
Abstract 5266: Kinetic measurements of intracellular ATP levels in co-culture models using live-cell analysis
Authors: Schramm Cicely L. , Bowe Michael L. , Skerlos Laura A. , Filonov Grigory S. , Chen Yong X. , Appledorn Daniel M.
Published: August 2019

Differential metabolic requirements of tumor cells have gained recognition as attractive therapeutic targets. For example, clinical trials are currently underway to test the efficacy of the glutaminase-1 (GLS1) inhibitor CB-839 in a variety of indications, including triple-negative breast cancer (TNBC). Standard approaches to monitoring drug induced metabolic perturbations are limited to endpoint assays that lack cell-specific data in complex co-culture models and provide limited kinetic information. Here we describe a live-cell approach to image and analyze intracellular ATP levels of tumor cells in mono- or co-culture over time using the IncuCyte® CytoATP Sensor. A panel of breast cancer cell lines stably expressing a genetically encoded, fluorescent ATP sensor or a control (non-ATP binding) sensor were generated and cultured alone or with a monolayer of stromal cells. The effect of CB-839 treatment on tumor cell ATP levels was monitored over days and analyzed using the IncuCyte® S3 for Metabolism, which is equipped with a specialized optical module and image acquisition mode. In concordance with previous results, TNBC cell lines were more sensitive to GLS1 inhibition than their receptor-positive counterparts. Across all TNBC lines tested, CB-839 treatment resulted in a reduction in intracellular ATP that was sustained for the duration of the 3-day time course. In contrast, most receptor-positive cell lines displayed little to no effect of GLS1 inhibition. In some cases, a transient reduction followed by recovery within 48 hours was observed, highlighting the value of kinetic analysis. Co-culture with CCD-1086SK fibroblasts attenuated the effect of CB-839 in TNBC cell lines. This was not due to drug buffering, as the reduction in ATP induced by CB-839 treatment of MCF7 cells was unaffected by co-culture with stromal cells. Visualization tools included in the IncuCyte® ATP Analysis Software Module provide a quick, qualitative assessment of data across the assay plate and a view into the heterogeneity of responses to treatment. Measurements of proliferation, cell death, and mitochondrial membrane potential provided additional data supporting the observations generated via ATP readout. Overall, these data highlight the value of live-cell, kinetic analysis of metabolic and cell health measurements.

Citation Format: Cicely L. Schramm, Michael L. Bowe, Laura A. Skerlos, Grigory S. Filonov, Yong X. Chen, Daniel M. Appledorn. Kinetic measurements of intracellular ATP levels in co-culture models using live-cell analysis [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 5266.
Abstract 527: High-dimensional profiling of the systemic immune response informs on optimal sequencing of radiotherapy (RT) and immune checkpoint blockade (ICB)
Authors: Chua Kevin L. , Fehlings Michael , Chu Pek Lim , Lin Xiao-Tian , Yeo Eugenia , Low Kar Perng , Poon Dennis , Ong Enya , Woo Wai Yee , Wee Joseph , Nardin Alessandra , Iyer Gopalakrishna , Tan Daniel S. , Soo Kee Chee , Newell Evan , Chua Melvin
Published: August 2019

Background: Combinatorial RT-ICB potentiates anti-tumour reactivity by modulating the immune response. We therefore performed in-depth phenotypic profiling of the systemic T cell compartment following treatment with RT-ICB.

Methods: We recruited 20 patients with biopsy-proven metastatic renal cell and non-small cell lung carcinoma, who were treated with a sandwich regime of ICB-RT-ICB under a prospective observational study protocol, and compared against a RT alone-treated cohort (N=10). All patients received ablative RT (8-50Gy/1-5fr) for oligoprogression and/ or local palliation. Blood samples were longitudinally collected at pre-RT, 14 d post-RT and cycle 2 ICB post-RT. Deep T cell profiling was performed by mass cytometry using a customised 41 parameter panel, together with high dimensional analysis tools.

Results: Median follow-up of the overall cohort was 18mo; median duration of ICB received in the ICB-RT-ICB arm was 15mo. We observed significant diversity of the systemic T cell repertoire between patients at baseline, and this corresponded to significant interpatient heterogeneity in T cell responses specific to the central/ effector memory, EMRA and Treg subsets post-RT. Dramatic local response (complete response at 1 mo post-RT) was significantly higher in the ICB-RT-ICB cohort compared to the RT alone cohort (12/20 vs 1/10, P<0.01). This clinical phenomenon corresponded to an increased %Ki67high CD8 and CD4 T cells post-RT exclusively in the combinatorial treated cohort, which was further enhanced upon resumption of ICB (mean = 10% vs 3% [CD8]; 13% vs 2% [CD4]; P<0.01). Deeper immunophenotyping of the Ki67high subsets revealed associated high expression of GranzymeB and Eomes.

Conclusions: Here, we observed changes in the T cell phenotypes that varied remarkably across all patients following RT. We further highlight a RT-dependent T cell proliferation amongst all RT-ICB-treated patients that was further enhanced by ICB in prior responders. This immune phenomenon may account for the dramatic responses to combinatorial treatment, and informs on optimal sequencing strategies for combining RT and ICB.

Citation Format: Kevin L. Chua, Michael Fehlings, Pek Lim Chu, Xiao-Tian Lin, Eugenia Yeo, Kar Perng Low, Dennis Poon, Enya Ong, Wai Yee Woo, Joseph Wee, Alessandra Nardin, Gopalakrishna Iyer, Daniel S. Tan, Kee Chee Soo, Evan Newell, Melvin Chua. High-dimensional profiling of the systemic immune response informs on optimal sequencing of radiotherapy (RT) and immune checkpoint blockade (ICB) [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 527.
Abstract 5283: Prognostic value of resectable hepatocellular carcinoma: Clinico-pathologic findings
Authors: Kim Jin Young , Heo Mi Hwa , Shin So Jin
Published: August 2019

We investigated the prognostic factors of resectable hepatocellular carcinoma (HCC). Preoperative peripheral blood factors, fluorine-18 fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) and pathologic parameters were investigated for progression free survival (PFS). From February 2008 to December 2012, a retrospective analysis was conducted on 65 patients who had received liver resection. Peripheral blood parameters were collected within 7 days based on operation date. The 18F-FDG uptake was calculated based on the maximum standardized uptake value of the tumor (SUVmax), the liver mean SUV and the tumor-to-normal SUV ratio (TNR). Immuno-histochemical (IHC) stain results were reviewed by a pathologist through specimen. A total of 65 patients were included (46 males, 19 females; average age, 57.5 ± 8.9 years). The median PFS of HCC was 52.9 months. SUVmax was correlated with PFS and the optimal cut off value of SUVmax was 3.25 (p = 0.005). Median PFS of low SUVmax was 76.4 months and high SUVmax was 8.5 months. The optimal cut-off value of TNR was 1.77 for PFS. The median PFS of HCC in patients with low TNR (< 1.77) and high TNR (≥ 1.77) was 82.3 and 7.0 months, respectively (p = 0.002). AST to platelet ratio index (APRI) could be a predictable marker for recurrence of HCC (p = 0.034). The median PFS of high PNI and low PNI were 64.9 and 10.9 months, respectively (p = 0.004). The IHC expression of glucose uptake transporter 2 was correlated with SUVmax. Preoperative SUVmax, TNR, APRI and PNI could predict recurrence after curative resection of HCC.

Citation Format: Jin Young Kim, Mi Hwa Heo, So Jin Shin. Prognostic value of resectable hepatocellular carcinoma: Clinico-pathologic findings [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 5283.
Abstract 5285: The role of HIV-1 TAT interactive protein 2 in modulating tumor response to hypoxia
Authors: Zhou Qingyu Stephanie , Guo Xiaofang , Li Minghua , Pan Hua
Published: August 2019

While there is ample evidence that HIV-1 TAT interactive protein 2 (HTATIP2) is a tumor suppressor exerting its pro-apoptotic and anti-metastatic activities by regulating the expression of a subset of pro-apoptotic and anti-apoptotic genes and metastasis suppressor genes, it remains unclear if HTATIP2 has a role in tumor response to hypoxia. The objective of this study was to define the role of HTATIP2 in mediating certain molecular and cellular changes in response to hypoxia in tumors. We employed a series of isogenic A549 human lung adenocarcinoma sublines that were genetically deficient for HTATIP2 and/or HIF-1α to examine the potential modulatory effect of HTATIP2 on oncogenic signaling pathways mediated by HIF-1α and HIF-2α, and established the HTATIP2-deficient A549 xenograft model to explore the impact of deletion of HTATIP2 on tumor response to hypoxia. Results from the human HIF-regulated cDNA plate array study revealed that under hypoxic condition (0.5% O2), several genes promoting tumor metastasis and resistance to apoptosis and anticancer treatment, including HIF1α, CYSD, MMP1, MXI1, NDRG2, PAI1 and NDRG1, were upregulated to a greater extent in HTATIP2 knockdown A549 cells than in the control A549 cells. In contrast, genes involved in angiogenesis, glucose metabolism and protection of apoptosis under hypoxia, such as PDGF, PDK1, CA9, and SAG, were upregulated to a greater extent in control A549 cells than in HTATIP2-knockdown A549 cells. Results from the in vivo study demonstrated that the mean growth rate of HTATIP2-deficient A549 xenograft tumors was significantly increased as compared with that of parental A549 xenograft tumors irrespective of the presence or absence of sorafenib treatment. Moreover, the data of photoacoustic imaging (PAI) of tumor hypoxia revealed that the parental A549 tumors were better oxygenated than the counterpart HTATIP2-deficient A549 tumors. After the 10-day sorafenib treatment, the oxygenated regions in sorafenib-treated parental A549 tumors were significantly reduced as compared with the vehicle-treated parental A549 tumors, whereas no significant change in the oxygenated regions was found between vehicle- and sorafenib-treated HTATIP2-deficient A549 tumors. The overall finding of our study suggests that downregulation of HTATIP2 affords growth advantage to tumor cells by fine-tuning tumor adaptation to hypoxia, leading to tumor progression and aggression.

Citation Format: Qingyu Stephanie Zhou, Xiaofang Guo, Minghua Li, Hua Pan. The role of HIV-1 TAT interactive protein 2 in modulating tumor response to hypoxia [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 5285.
Abstract 5288: Evaluating the potential to repurpose statins for ovarian cancer therapy
Authors: Kroeger Paul T. , Drapkin Ronny
Published: August 2019

The recent exciting results using primary PARP inhibitor maintenance after upfront treatment for advanced ovarian cancer in BRCA1/2 mutation carriers highlight the importance of identifying druggable vulnerabilities in this disease. The fact that not all ovarian cancer patients will benefit from PARP inhibitors or experience durable responses, places great emphasis on the need to identify additional therapies. Re-purposing existing FDA-approved drugs offers an opportunity to accelerate progress in this area. Statins are drugs primarily prescribed for hypercholesterolemia and have been intensely researched for decades. Some of this work has demonstrated, through epidemiologic approaches and limited experimental studies, that statins have anticancer properties. We hypothesized that statins can be repositioned as part of a therapeutic regimen for HGSOC. Here, we demonstrate that certain ovarian cancer cell lines are exquisitely sensitive to treatment with statins. Our ongoing studies reveal that HGSOC cells that are sensitive to stain treatment manifest an amplification of chromosome 1q21. Within this genomic region are the S100 proteins, previously identified as potential biomarkers in several cancer types, as well as the anti-apoptotic protein MCL1. We performed a Reverse Phase Protein Array (RPPA) analysis on higher dose/short exposure, and low dose/longer exposure statin-treated cells. In both conditions, MCL1 levels were altered in statin-sensitive cell lines, while there was no such change in non-sensitive lines. After identifying MCL1 as a potential marker of response, Western blot analysis demonstrated a dose-dependent decrease in protein level of MCL1 with increasing statin concentration. Additionally, a short pro-apoptotic splice variant of MCL1 was produced upon statin treatment, but only in the sensitive lines. Mechanistically, we find that phospho-YAP (inactive) is upregulated after statin treatment. This data corroborated previous research that suggested that multiple different statins decrease nuclear YAP. Given its role as an oncogene, the apparent ability of statins to deactivate YAP and downregulate MCL1 steers statins in a promising direction for their repositioning for future therapeutic use.

Citation Format: Paul T. Kroeger, Ronny Drapkin. Evaluating the potential to repurpose statins for ovarian cancer 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 5288.
Abstract 5299: Role of short chain free fatty acid receptors in colorectal cancer
Authors: AlGhamdi Amal , AlMahri Saeed , AlAkiel Maaged , Mohammad Sameer , Aziz Mohammad
Published: August 2019

One of the well established risk factors for colorectal cancer is dietary profile. A well recognized negative association of dietary fiber with colorectal cancer warrants further understanding of the underlying mechanism. Bacterial fermentation of dietary fibre results in formation of short chain free fatty acids in the gut. There are two receptors for these fatty acids one of which has been implicated in colorectal cancer. Free fatty acid receptor 2 has been associated with development of colorectal adenoma in a mouse model. We analyzed the differential expression of FFAR2 gene in colorectal cancer patients. Further, we generated genetically engineered colorectal cancer cell line with reduced level of expression of these receptors. These engineered cells were found to have enhanced rate of cell proliferation. In order to understand the mechanism of increased proliferation, we explored the involevement of glucose uptake and cAMP pathways. While there was an increased level of glucose uptake in engineered cells, there was no evidence of involvement of Protein kinase A mediated cAMP pathway. Further studies are needed to fully establish the connection between dietary fibre, gut microbiota and colorectal cancer.

Citation Format: Amal AlGhamdi, Saeed AlMahri, Maaged AlAkiel, Sameer Mohammad, Mohammad Aziz. Role of short chain free fatty acid receptors in colorectal 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 5299.
Abstract 5310: Novel, single-tube, multiplex rhPCR technology for detection of low-frequency variants in BRCA1 and BRCA2
Authors: Sponer Nicole , Wang Yu , Wang Junzhou , Chen Shengyao , Lai Kevin , Rose Scott , Lau Patrick
Published: August 2019

Targeted next-generation sequencing (NGS) technologies serve as critical tools for cancer diagnostics. For example, individuals with pathological BRCA1 and BRCA2 (BRCA1/2) mutations have a dramatically increased risk for certain cancers, including breast and ovarian cancer. Those mutations are either germline or somatically acquired, and play an important role in cancer risk assessment, prevention of malignancy, and management of disease. A variety of methodologies are currently available to screen for BRCA1/2 mutations. Amplicon-based sequencing technologies are prevalent due to their easy workflow but have specific challenges due to inherent limitations of PCR, such as difficulties with amplification of overlapping amplicons for contiguous coverage in a single-tube or with detection of somatic low-frequency variants due to high polymerase error rates. We developed a robust, highly multiplex rhPCR workflow to screen for BRCA1/2 mutations across their entire coding regions in a single-tube. This method allows for detection of low-frequency variants through use of unique molecular identifiers (UMIs), which correct for PCR and sequencing errors and dramatically increase specificity. This method supports a wide DNA input range (1 to 50 ng) from cell-free, formalin-fixed paraffin-embedded (FFPE), or small biopsy samples. Our rhAmpSeq™ chemistry uses a two-enzyme system coupled with uniquely designed RNA-DNA hybrid primers, which enable the amplification of overlapping, multiplexed amplicons. Thus, we eliminate the need to split precious DNA samples into multiple tubes and prevent UMI hopping. Here, we demonstrate the performance of our novel, highly specific amplicon-based technology for detection of low-frequency variants of BRCA1/2 and compare our technology to current industry standards.

Citation Format: Nicole Sponer, Yu Wang, Junzhou Wang, Shengyao Chen, Kevin Lai, Scott Rose, Patrick Lau. Novel, single-tube, multiplex rhPCR technology for detection of low-frequency variants in BRCA1 and BRCA2 [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 5310.