bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract LB-195: Identifying cooperative effects of tumor suppressor gene mutations in lung adenocarcinoma using paired CRISPR-Cas9
Authors: Perez-Oquendo Mabel G. , Feldser David M. , Walter David
Published: August 2018

Cancer is a heterogeneous disease that arises from the accumulation of genetic mutations that lead to a loss of tumor suppressor genes and activation of oncogenes. Mutational inactivation of genes that regulate epigenetic mechanisms, such as post translational modifications to DNA and histones, also contribute to cancer progression but the mechanisms are poorly understood. Trimethylation on histone 3 lysine 36 (H3K36me3) is encoded by the histone lysine methyltransferase (SETD2). SETD2 is recurrently inactivated by missense and frameshift mutations in multiple tumor types. Previously, we identified that Setd2 loss has potent tumor promoting activities in a mouse model of oncogenic Kras-induced lung adenocarcinoma. However, whether Setd2 loss has cooperating effects with other tumor suppressor gene mutations remains unstudied. To simultaneously inactivate distinct tumor suppressor genes, we use a lentiviral-based CRISPR/Cas gene editing approach to express a combination of specific gRNA-pairs along with Cas9 and Cre recombinase. Inhalation of these lentiviral particles leads activation of an oncogenic KrasLSL-G12D allele and simultaneously inactivation two gRNA-targeted genes. We hypothesize that SETD2 will collaborate with other tumor suppressor gene mutations in lung adenocarcinoma. To clone lentivirus vectors that have two different gRNA we use oligonucleotides encoding gRNAs targeting Setd2 and either Lkb1, Apc, or Rb1 to PCR amplify the 3' region of the murine U6 (mU6) promoter and the 5' region of the gRNA scaffold. Overlapping sequences in the 148-bp PCR amplicon, facilitate recombination with a 415-bp donor fragment derived from BbsI-digested pDonor plasmid using the Gibson cloning method. The newly circularized fragment is then re-linearized via BbsI digestion and cloned into LentiCRISPRv2Cre vector through the Golden Gate method using BsmBI type IIs restriction endonuclease and T4 DNA ligase. Our data demonstrate the development and presence of dual gRNA nucleotides through NotI/EcoRI restriction enzymes digestion and DNA Sequencing. Moreover, to study the cooperative effects of these mutations in mice, we created viruses and corroborated through Flow cytometry and Fluorescence microscopy. In conclusion, we generate lentiviral particles expressing pairs of gRNAs targeting distinct tumor suppressor genes, to identify cooperative effects of their mutation in lung adenocarcinoma models.

Citation Format: Mabel G. Perez-Oquendo, David M. Feldser, David Walter. Identifying cooperative effects of tumor suppressor gene mutations in lung adenocarcinoma using paired CRISPR-Cas9 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-195.
Abstract LB-198: Novel somatic mutations of PRKAR1A in sporadic cardiac myxomacardiac myxoma
Authors: He Jian
Published: August 2018

Background: Cardiac myxomas are benign tumors that commonly arise within the left atria. Familial cardiac myxomas are a part of Carney Complex (CNC), an autosomal dominant multiple neoplasia syndrome caused by germline mutations in PRKAR1A. Seven percent of cardiac myxomas are associated with CNC. To date, the genetic basis of isolated cardiac myxomas (ICM), however, has not been fully elucidated

Methods: We investigated the genetic profile of ICM using whole exome sequencing (WES). Suspected mutations were confirmed using targeted sanger sequencing. To further examine the presence of PRKAR1A mutations in ICM, we performed targeted sequencing in an additional 61 ICM specimens.

Results: 87.5% (7/8) of ICM harbored mutations in PRKAR1A. Three of the 8 ICM harbored biallelic somatic mutations of PRKAR1A, including c.607_610del:p. Leu203fs (pathogenic) + c.C896G:p.Ser299X (pathogenic), c.952delT:p.Leu318fs (pathogenic) + c.769-2 A>G (pathogenic) and c.178-1 G>C (pathogenic) + c. 550+1 G>C (pathogenic). Four of 8 tumors harbored monoallelic PRKAR1A mutations, including c.523_524insG:p.Tyr175_Val176delinsX (pathogenic), c.C920A:p.Ser307X (pathogenic), c.30delG:p.Glu10fs (pathogenic) and c.C289T:p.Arg97X (pathogenic). No identical variants were observed across the 8 ICM samples. Interestingly, none of these variants have been previously described in familial cardiac myxomas. In order to confirm our findings, directed sequencing of 61 ICM specimens was subsequently performed. Sixty-four percent (39/61) of ICMs tumors contained inactivating PRKAR1A mutations.

Conclusion: Our findings suggest that loss-of-function mutations of PRKAR1A may play a vital role in the formation of isolated cardiac myxomas.

Citation Format: Jian He. Novel somatic mutations of PRKAR1A in sporadic cardiac myxomacardiac myxoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-198.
Abstract LB-223: Examination of intra-assay variability in a commercial ctDNA NGS assay with triplicate clinical plasma and pooled normal samples
Authors: Stetson Daniel , Dougherty Brian , Ahmed Ambar , Nuttall Barrett , Lubinski Tristan J. , Afshar Fakhera , Raymond Amelia , Barrett Carl
Published: August 2018

The increased reliance on liquid biopsy next generation sequencing assays for oncology clinical decisions has highlighted the importance of understanding the factors that impact the analytical validation and clinical utility of these tests. Although recent studies have been published examining the concordance between tumor and plasma sequencing data from the same patient, there has been a lack of insight into the intra-assay variability within these tests. We have previously performed a replicate study across multiple ctDNA sequencing vendors and found a high degree of variability among assays particularly at low allele fractions (AF<1%). To further extend our analysis, we examined the variability of ctDNA sequencing results from replicate patient plasma samples with the same vendor assay. The cohort consisted of 25 total samples: seven cancer patient plasma samples, representing triplicate 2 ml baseline plasma samples, plus four healthy normal samples, representing two different pools of six subjects in duplicate. The plasma names were blinded and shipped to Guardant Health for Guardant360 panel testing and both the raw data and reported variant calls were returned to AstraZeneca for comparative analysis. A total of 17 unique variants were called in this study, of which four variants were concordant for all three replicate plasmas and 13 variants were reported in only one or two replicates. Examining the raw data for all samples, the majority of the discordant variants were able to be identified at low levels. Plasmas had low amounts of ctDNA, and two patients had low unique molecule counts, with allele fractions ranging from 0.10-1.13%. Interestingly, the reported yield of the extracted DNA did not correlate with the ability to detect low frequency somatic variants. Data will be presented showing the unblinded comparisons between the replicate samples for both the cancer patient plasma data and the pooled normal plasmas. This study highlights the importance of reproducibility studies for ctDNA samples with variants at low allele fractions to discern between false negative and false positive finding.

Citation Format: Daniel Stetson, Brian Dougherty, Ambar Ahmed, Barrett Nuttall, Tristan J. Lubinski, Fakhera Afshar, Amelia Raymond, Carl Barrett. Examination of intra-assay variability in a commercial ctDNA NGS assay with triplicate clinical plasma and pooled normal samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-223.
Abstract LB-244: Overcoming resistance to DNA targeted agents by epigenetic activation of Schlafen 11 (SLFN11) expression with class I histone deacetylase inhibitors
Authors: Ghafoor Azam , Tang Sai-Wen , Thomas Anish , Murai Junko , Trepel Jane B. , Bates Susan E. , Rajapakse Vinodh N. , Pommier Yves
Published: August 2018

Purpose: Schlafen 11 (SLFN11), a putative DNA/RNA helicase is a dominant genomic determinant of response to DNA damaging agents and is frequently not expressed in cancer cells. Whether histone deacetylase (HDAC) inhibitors can be used to release SLFN11 and sensitize SLFN11-inactivated cancers to DNA-targeted agents is tested here. Experimental Design: SLFN11 expression was examined in The Cancer Genome Atlas (TCGA), in cancer cell line databases and in patients treated with romidepsin. Isogenic cells overexpressing or genetically inactivated for SLFN11 were used to investigate the effect of HDAC inhibitors on SLFN11 expression and sensitivity to DNA damaging agents. Results: SLFN11 expression is suppressed in a broad fraction of common cancers and cancer cell lines. In cancer cells not expressing SLFN11, transfection of SLFN11 sensitized the cells to camptothecin, topotecan, hydroxyurea and cisplatin but not to paclitaxel. SLFN11 mRNA and protein levels were strongly induced by class I (romidepsin, entinostat), but not class II (roclinostat) HDAC inhibitors in a broad panel of cancer cells. SLFN11 expression was also enhanced in peripheral blood mononuclear cells of patients with circulating cutaneous T-cell lymphoma treated with romidepsin. Consistent with the epigenetic regulation of SLFN11, camptothecin and class I HDAC inhibitors were synergistic in many of the cell lines tested. Conclusion: This study reports the prevalent epigenetic regulation of SLFN11 and the dominant stimulatory effect of HDAC inhibitors on SLFN11 expression. Our results provide a rationale for combining class I HDAC inhibitors and DNA damaging agents to overcome epigenetic inactivation of SLFN11-mediated resistance to DNA-targeted agents.

Citation Format: Azam Ghafoor, Sai-Wen Tang, Anish Thomas, Junko Murai, Jane B. Trepel, Susan E. Bates, Vinodh N. Rajapakse, Yves Pommier. Overcoming resistance to DNA targeted agents by epigenetic activation of Schlafen 11 (SLFN11) expression with class I histone deacetylase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-244.
Abstract LB-264: Alcohol-aversion drug disulfiram targets cancer via p97 segregase adaptor NPL4
Authors: Mistrik Martin , Skrott Zdenek , Hajduch Marian , Friis Søren , Dzubak Petr , Gursky Jan , Majera Dusana , Ozdian Tomas , Vaclavkova Jana , Michalova Martina , Pouckova Pavla , Cvek Boris , Andersen Klaus Kaae , Bartek Jiři
Published: August 2018

Cancer incidence is rising and this global challenge is further exacerbated by resistance of advanced tumors to standard-of-care and emerging precision medicines. A promising approach to such unmet need for innovative and cost-effective cancer treatments is drug repurposing, exploitation of drugs already approved for other indications that show anticancer activity. Here we highlight the potential for repurposing disulfiram (Antabuse), an old alcohol-aversion drug that kills a broad range of cancer types in preclinical studies. Our nationwide population study reveals that patients who continued using disulfiram have a lower risk of death from cancer compared to those who stopped using the drug at their diagnosis. More importantly, we identify ditiocarb-copper complex as the metabolite of disulfiram responsible for anticancer effects, present methods to detect its preferential accumulation in tumors and candidate biomarkers of impact in cells and tissues. Finally, our functional and biophysical analyses reveal the long-thought molecular target: NPL4 adapter of the p97/VCP segregase essential for protein recycling involved in multiple stress-response cellular pathways.

Citation Format: Martin Mistrik, Zdenek Skrott, Marian Hajduch, Søren Friis, Petr Dzubak, Jan Gursky, Dusana Majera, Tomas Ozdian, Jana Vaclavkova, Martina Michalova, Pavla Pouckova, Boris Cvek, Klaus Kaae Andersen, Jiři Bartek. Alcohol-aversion drug disulfiram targets cancer via p97 segregase adaptor NPL4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-264.
Abstract LB-311: Inhibition of ubiquitin-specific protease 34 (USP34) regenerates mammary glands via induction of epithelial-mesenchymal transition (EMT) and stemness
Authors: Oh Eunhye , Sung Daeil , Kim Yoon-Jae , Cho Tae-Min , Jang Seojin , Kim Ji Young , Seo Jae Hong
Published: August 2018

Mammary stem cells (MaSCs) possess specific biological properties including self-renewal capacity and the ability to differentiate into two primary cell lineages; luminal cells and myoepithelial cells. Epithelial-mesenchymal transition (EMT) is a key process during mammary gland development. Recent study demonstrates that ubiquitin-specific protease 34 (USP34), as a member of the USP family plays a critical role in Wnt/b-catenin signaling pathway. Our objective was to investigate possible roles of USP34 on induction of EMT and properties of mammary epithelial stem cells as well as mammary gland regeneration. We observed that USP34 level was relatively lower in mesenchymal-like phenotype MDA-MB-231 and 4T1 cells, compared to epithelial-like phenotype cells. Inhibition of USP34 induced EMT in NMuMG cells, which was accompanied with upregulation of EMT markers, such as N-cadherin, phospho-smad3, snail and active-β-catenin as well as downregulation of E-cadherin. NMuMG cells exhibited a typical epithelial phenotype with an absence of invasive behavior, while USP34 knockdown NMuMG cells acquired an invasive property. This observation was associated with a significant increased MMP2 level. Furthermore, inhibition of USP34 promotes stemness as evidenced by increased mammosphere-forming ability, concomitant with upregulation of Nanog, Oct4 and Sox2 mRNA levels in NMuMG cells. To examine the importance observations of our in vitro studies physiologically, the CTL-KD or USP34-KD cells were transplanted into the cleared mammary fat pads (CFP), and then mammary gland regeneration was observed for 10 months. Our in vivo data showed that USP34-KD cells regenerated elongated ductal branching and TEB of mammary gland in CFP. Our findings suggest that USP34 may be potentially involved in development of mammary glands.

Citation Format: Eunhye Oh, Daeil Sung, Yoon-Jae Kim, Tae-Min Cho, Seojin Jang, Ji Young Kim, Jae Hong Seo. Inhibition of ubiquitin-specific protease 34 (USP34) regenerates mammary glands via induction of epithelial-mesenchymal transition (EMT) and stemness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-311.
Abstract LB-313: CXCR2 inhibitor Reparixin enhances local tumor control and reduces distant metastases in mouse tumor radiotherapy
Authors: Chang Chia-lun , Liang Yi-Hsin , Wu Szu-yuan
Published: August 2018

Inadequately irradiated primary tumor of primary Lewis lung carcinoma (LLC-LM) in vivo is a mechanism of radiation-enhanced LLC-LM cells in pulmonary metastasis. We observed a CXCL1/GRO alpha, is a member of the CXC family of chemokines is elevated after radiation therapy. CXCL1 protein is function with to bind the G-protein coupled receptor chemokine (C-X-C motif) receptor 2 (CXCR2) to recruit neutrophils. Reparixin is an inhibitor of CXCR2, and demonstrates activity against malignant behavior of human cancer cells xenografts. The effect of Reparixin on the radiotherapy of LLC-LM primary tumor and the effects on distant metastases have not been studied. The right thighs of C57BL/6 mice (n = 40) were injected s.c. with 1 x 10(6) LLC-LM cells. Animals were randomized to one of four groups: no irradiation, 10 Gy in one fraction, Reparixin (0.1mg/kg/day, for 5days) plus 10 Gy in one fraction, or Reparixin( 0.1mg/kg/day, for 5days) only . Tumors were clinically irradiated in each treatment group. All of the mice were killed within 21 days after the completion of radiation therapy. Examination of their lungs revealed >13 (range, 13-22) surface metastases in the 10 Gy treated mice with 3 (range, 2-7) in the no irradiation mice, in Reparixin plus 10 Gy treated mice with 6(range, 4-13), in Reparixin treated mice with 2 (range, 2-6). The lung weights had increased from 0.22 g (range, 0.21-0.24 g) in the no irradiation mice to 0.43 g (range 0.37-0.61) in the 10 Gy treated mice, in Reparixin plus 10 Gy treated mice with 0.31(range, 0.26-0.41), in Reparixin treated mice with 0.23 (range, 0.2-0.25 g). In the surface metastases and lung weight, Reparixin plus irradiation treatment were statistically significant with P < 0.001 when compare with irradiation alone. Other hand, the tumor growth curve also demonstrated that the Reparixin enhanced the tumor control of irradiation of LLC-LM tumor. In this model, the use of inadequately radiation to eradicate a primary LLC-LM tumor results in the growth of previously dormant lung metastases and suggests that combining CXCR2 inhibitor with radiation therapy may enhance local tumor control and reduce distant metastases.

Citation Format: Chia-lun Chang, Yi-Hsin Liang, Szu-yuan Wu. CXCR2 inhibitor Reparixin enhances local tumor control and reduces distant metastases in mouse tumor radiotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-313.
Abstract LB-316: Dynamic live cell imaging of tumor migration and invasion
Authors: Booth Ross , Chen Cindy , Yeh Victor , Verma Deepika , Park Jun
Published: August 2018

Migration and invasion activities of metastasizing cancer cells are of immense significance to the field of cancer research, often leading to fatal spread of the disease. While much understanding has been gained regarding the underlying molecular and cellular mechanisms governing these processes, the direct observations of associate morphological and biochemical features are limited by the current methodologies which largely rely on endpoint micro-porous membrane-based assays or various live cell imaging assays performed on 2D/3D substrates- neither of which enable dynamic control over the tumor microenvironment. Microfluidic technologies enable high-resolution time-lapse imaging while providing exceptional experimental control to maintain a physiologically relevant microenvironment. Here we demonstrate the value of the CellASIC ONIX2 “dynamic live cell imaging system”, where microenvironmental parameters such as flowrates, the perfusion of nutrients and reagents, and temperature and gas compositions can be precisely controlled on demand by software during the entire duration of a given experiment. We now show the value of this system for imaging of metastatic processes with a novel microfluidic design comprising two parallel chambers separated by a micro-porous wall. One chamber contains MDA-MB-231 breast cancer cells embedded in 3D hydrogel culture or simply grown in 2D fashion, and the other chamber contains HUVEC endothelial cells in a monolayer. The device enables exceptionally clear real-time imaging of the migration of MDA-MB-231 cells, as well as invasion of these cells through and out of the gel into the “vascular” channel, enabling the monitoring of the adhesion and interaction of these cells with the HUVEC cells once the tumor cells had left the gel. Unlike the state-of-the-art endpoint or static culture imaging assays, the microfluidics system described herein enables the investigation of wider microenvironmental parameters and their interplay with cellular processes in unprecedented precision and permutations, and is also suitable for the studies of wider biological applications outside of cancer biology.

Citation Format: Ross Booth, Cindy Chen, Victor Yeh, Deepika Verma, Jun Park. Dynamic live cell imaging of tumor migration and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-316.
Abstract LB-325: New synergistic mTOR-based combinations for the treatment of Ewing sarcoma
Authors: Menegaz Brian A. , Cuglievan Branko , Admane Prasad S. , Yang Feng , Dimiter Dimitrov , Landesman Yosef , Cherradi Salah-Eddine Lamhamedi , Ludwig Joseph A.
Published: August 2018

Ewing sarcoma (ES), an aggressive bone cancer in children and young adults, is dependent upon the insulin-like growth factor receptor 1 (IGF-1R) and mammalian target of rapamycin (mTOR) cell-signaling axis for malignancy, survival, and metastasis. Individual targeting of mTOR has negligible anti-tumor activity in ES, and single-agent IGF-1R targeting leads to only transient effects that benefit only 10-14% of patients. Co-targeting strategies using IGF-1R Abs combined with mTOR inhibitors were shown in 2012 to increase response rates to 29% but the high toxicity profile limited subsequent clinical development. While an ongoing COG protocol combines IGF-1R Abs with standard-of-care cytotoxic chemotherapy in newly diagnosed ES patients harboring metastatic disease, this combination is unlikely to benefit those who have recurred or developed chemoresistant disease. Herein, we present several highly synergistic drug combinations using novel biologically-targeted therapies aimed at the IGF-1R/mTOR pathway. A dual-specific anti-IGF-1/IGF-2 human monoclonal antibody (m708.5), which binds human IGF-1 and IGF-2 with high pico-molar affinity, potently inhibited phosphorylation of the IGF-1R and the insulin receptor (IR) in an EW5 xenograft animal model of ES. In concert with mTOR inhibitors (e.g., everolimus or ridaforolimus), m708.5 demonstrated striking tumor regression and prolonged mouse survival. Additionally, we expand upon the synergistic role of CRM1 (XPO1) in combating ES tumor growth. A small molecule inhibitor of this protein (KPT-330) was previously shown to attenuate IGF-1-induced activation of IGF-1R when combined with a small-molecule inhibitor of IGF-1R and IR (Linsitinib, OSI906). Our results indicate superior preclinical anti-tumor activity when it is combined with everolimus in our xenograft ES models. Though these combinations have not yet been assessed in the clinical setting, early-phase clinical trials have begun testing KPT-330 in select sarcoma subtypes, and a host of mTOR inhibitors are FDA-approved and widely available. Our data supports several novel drug combinations that could rapidly be explored in the clinic to improve survival of ES patients.

SELC & JAL: Co-senior Authors

Citation Format: Brian A. Menegaz, Branko Cuglievan, Prasad S. Admane, Feng Yang, Dimitrov Dimiter, Yosef Landesman, Salah-Eddine Lamhamedi Cherradi, Joseph A. Ludwig. New synergistic mTOR-based combinations for the treatment of Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-325.
Abstract LB-329: Multiple-resolution characterization of tumor heterogeneity as associated with disease progression in a mouse model of colorectal cancer
Authors: Farhoud Mohammed , Leystra Alyssa , Albrecht Dawn M. , Brevard Mathew , Dimant Hemi , Holt Robert , Halberg Richard B.
Published: August 2018

The ability to engineer models to express endogenous fluorophores has become an invaluable tool for studying tumor biology. The utility of these models is limited by the sensitivity and resolution of currently available imaging platforms. In vivo techniques enable longitudinal studies in which changes in fluorescent markers, tumors, and whole organs can be monitored over time. In vivo fluorescence suffers from poor sensitivity and resolution owing to the high absorption and scatter of light in biological tissue. While ex vivo fluorescence techniques allow for high resolution evaluation and characterization, these techniques typically only focus on a small samples of tissue instead of whole animals. Using a Cryo-Fluorescence Tomography (CFT) imaging approach, an imaging modality based on serial slicing and off-the-block fluorescence imaging, we can bridge the gap between in vivo and ex vivo resolution of the entire animal. As proof of concept, a novel mouse model expressing fluorophores was evaluated and characterized. Individual cells were labeled with either EGFP or tdTomato prior to tumor initiation. Intestinal tumors were induced through loss of Apc, the activation of PI3K, or both. Tumor heterogeneity and grade were assessed by traditional histopathology, individual-tissue CFT, and whole-animal CFT. Using CFT, serial sectioning and 3D-reconstruction of whole tissues were achieved at 10-50 µm resolution.The multi-resolution imaging approach of this model enabled visualization of molecular and histopathological information from a common in vivo resolution (1mm) to whole-tissue resolution. The CFT platform enabled a 3D data set at a resolution that was not previously available in traditional histopathology. CFT as a molecular tissue imaging technique has proven its utility as a complimentary assay in preclinical oncology studies allowing several characteristics of tumors including heterogeneity, growth, and malignant spread to be readily assessed.

Citation Format: Mohammed Farhoud, Alyssa Leystra, Dawn M. Albrecht, Mathew Brevard, Hemi Dimant, Robert Holt, Richard B. Halberg. Multiple-resolution characterization of tumor heterogeneity as associated with disease progression in a mouse model of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-329.
Abstract LB-330: Investigating how extracellular signals contribute to single cancer cell heterogeneity using near-infrared quantum dots
Authors: Le Phuong , Baculis Brian C. , Chung Hee Jung , Kilian Kristpher , Smith Andrew M.
Published: August 2018

Cancer cells heterogeneity presents a major roadblock in clinical diagnostics and therapy. Investigations of tumor cell heterogeneity using single-cell analytical techniques have revealed not only the presence of multiple clonal subpopulations but also phenotypic variability among genetically identical cells. This heterogeneity plays a critical role in therapeutics, as population extrema, rather than cells near the ensemble mean, can dominate pathogenesis as well as drug resistance. To characterize single cell heterogeneity, multiple techniques quantifying single-cell gene expression have been developed. However, there's a lack of experimental techniques to measure how cellular decision-making processes underlying population variability derive from extracellular biochemical input signals, such as peptide growth factors, which cannot be measured at the single-cell level. Here, to digitally count growth factors in single cells, we develop a novel method combining fluorescent quantum dots and calibrated three-dimensional deconvolution microscopy (QDC-3DM). Using quantum dots with near-infrared emission to overcome intrinsic cellular autofluorescence, we were able to detect and accurately count individual quantum dots conjugated to epidermal growth factor (EGF) using their fluorescent intensities. Analyzing triple-negative breast cancer cells (MDA-MB-231) with QDC-3DM, we observed that single-cell heterogeneity in growth factor stimulation led to heterogeneity in receptor activation. When treating cells with increasing concentration of phamarcological inhibitor blocking receptor activation, we observed a proportional increase in receptor activation heterogeneity. Together, our results indicate that external stimulation contributes to signaling variation and drug response variation at the single-cell level. We anticipate that QDC-3DM can be applied to any peptidic ligands to study how extracellular signaling stimulation contributes to phenotypic variability to provide new insight into cancer cell heterogeneity that plays a critical role in therapeutic resistance.

Citation Format: Phuong Le, Brian C. Baculis, Hee Jung Chung, Kristpher Kilian, Andrew M. Smith. Investigating how extracellular signals contribute to single cancer cell heterogeneity using near-infrared quantum dots [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-330.
Abstract LB-332: Evidence for cutaneous squamous tumors derived from carcinogen-exposed bone marrow
Authors: Lad Sonali , Johnson Kelly , Boland Kelsey , Readio Nyssa , Schuster Todd , Li Yong , Gordon Derek , Morris Rebecca J.
Published: August 2018

Cytokeratins are frequently found in the blood and bone marrow of patients with epithelial cancers and are attributed to metastasis. We wondered whether we could find keratin expression in blood and bone marrow in untreated adult mice. To address this problem, we have used classical immunoreactivity, Krt1-14;mTmG transgenic mice together with fluorescence activated cell sorting, and quantitative reverse transcriptase polymerase chain reaction. We have made several novel findings. First, we discovered, rare but distinctive, keratin-14 and keratin-15 immunoreactive cells the size of small lymphocytes in blood and bone marrow of untreated mice. Second, using Krt1-14;mTmG transgenic mice, we found low (8.6 GFP positive cells per 10^6 cells analyzed), but significant numbers (p<0.0005) of GFP positive cells in bone marrow of normal adult mice when compared with negative controls. Third, qRT-PCR demonstrated very low but reproducibly detectable expression of keratin-14 mRNA in blood and bone marrow when compared with epidermal keratinocytes: with blood expressing one thousand times, and bone marrow, one hundred thousand times, less than epidermal keratinocytes. Moreover, FACS analysis of fresh bone marrow disclosed a subpopulation of keratin-14 immunoreactive cells that was negative for hematopoietic lineage markers. Furthermore, and even more curious, recipients of bone marrow transplants from DMBA-treated UBC/GFP mice (“toxic bone marrow”) developed GFP-immunoreactive skin tumors upon TPA tumor promotion: four of fifteen mice developed GFP-immunoreactive skin tumors. Of these tumors, four were squamous papillomas, and one was a squamous cell carcinoma. None of the ten control mice receiving normal whole bone marrow developed any skin tumors upon tumor promotion. We conclude from these observations that keratin-14 protein and mRNA are expressed at low, but detectable levels in the blood and bone marrow of mice, and that a subset of “toxic bone marrow” derived cells can be recruited to form epithelial benign and malignant squamous tumors. Building upon these observations should open new avenues of understanding for cutaneous biology, non-melanoma skin cancer, and other epithelia and their cancers.

Citation Format: Sonali Lad, Kelly Johnson, Kelsey Boland, Nyssa Readio, Todd Schuster, Yong Li, Derek Gordon, Rebecca J. Morris. Evidence for cutaneous squamous tumors derived from carcinogen-exposed bone marrow [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-332.
Abstract LB-338: SHP2 inhibition enhances sensitivity to MEK inhibitors in multiple resistant cancer models
Authors: Fedele Carmine , Tang Kwan Ho , Ran Hao , Wei Wei , Diskin Brian , Miller George , Neel Benjamin G.
Published: August 2018

Targeting tumors with kinase inhibitors induces complex adaptive programs that enable the persistence of a fraction of the original cell population, facilitating the eventual outgrowth of inhibitor-resistant tumor clones. Most tumors developing resistance to MEK inhibitors upregulate and/or activation of at least one of several different receptor tyrosine kinases (RTKs), which drive resistance. Notably, different RTKs or RTK combinations are upregulated in different cancer cells, even those from the same histotype. SHP2 (encoded by PTPN11) is required for RAS/ERK pathway activation downstream of most RTKs, suggesting that it might provide a common targetable resistance node in multiple resistant cancer cell lines. We show that the addition of a newly identified SHP2 inhibitor, SHP099, to MEK therapy enhances cell killing and impedes the emergence of drug-resistant cell populations in diverse cancer models in vitro and in vivo. These effects occurred in a wide range of malignancies, including KRAS mutant pancreas, lung cancer, triple negative breast cancer, and serous ovarian cancer cell lines, as well as in PDX and GEMM models. SHP099 effects were overcome by expressing an SHP2 mutant that loses drug binding but retain catalytic activity and regulation, demonstrating that on target effects of the inhibitor. Mechanistic studies across multiple cellular cancer models suggest that SHP2 inhibition by SHP099 prevents the compensatory RAS and ERK1/2 reactivation in response to MEK therapy and impedes the engagement of the ERK1/2 transcriptional programs (i.e., MYC, ETV1, FOSL1, etc.) and signaling outputs that characterize the drug-tolerant state. Combining full doses of MEK inhibitors and SHP099 causes significant toxicity in mice, but non-toxic, efficacious dosing regimens can be achieved. Our findings suggest that SHP099, in combination with MEK inhibition is a promising therapeutic strategy for enhancing the effectiveness of targeted therapies.

Citation Format: Carmine Fedele, Kwan Ho Tang, Hao Ran, Wei Wei, Brian Diskin, George Miller, Benjamin G. Neel. SHP2 inhibition enhances sensitivity to MEK inhibitors in multiple resistant cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-338.
Abstract LB-350: IgG4 expressing B cells associate with poor survival in triple negative breast cancer
Authors: Flynn Nicole , Amadi Ugochukwu , Somasundaram Rajasekharan , Sims-Mourtada Jennifer
Published: August 2018

Triple negative breast cancer (TNBC) is an aggressive form of breast cancer with high levels of lymphocyte infiltration. Although high levels of infiltrating B cells are present in TNBC, their role in progression of this disease is unclear. IgG4 is one of four subclasses of IgG and is the least abundant in healthy human serum. IgG4 has been found at higher percentages in both tissue and serum in cases of chronic inflammation, which promotes class switching to IgG4. Expression and secretion of IgG4 by B cells may be non-beneficial and potentially harmful in the cancer setting. Unlike the other subclasses of IgG, IgG4 does not activate complement dependent cytotoxicity and has a lower affinity for FcγR, thus resulting in a poor ability to engage immune effector cells and activate antibody-depended cell-mediated cytotoxicity. Additionally, IgG4 can compete with other IgG subclasses by binding to antigens and can sequestering IgG through Fc-Fc interactions, thus impairing potential anti-tumor antibody-mediated responses. As IgG4 expressing B cells have been shown to be associated with poor outcomes in other aggressive cancers such melanoma, glioblastoma and pancreatic cancer, we sought to determine the expression of IgG4 in TNBC. We performed immunohistological staining of IgG4 in paraffin-embedded tissue microarrays from 75 treatment-naive TNBC specimens obtained from women who underwent surgical resection of their tumors at the Helen F Graham Cancer Center and Research Institute from 2006-2007. We found that 69 % of TNBC cases contain IgG4+ cell infiltration. Moreover, the presence of IgG4+ cells is significantly correlated with poorer progression free (p<0.0003) and overall survival (p<0.0001) as determined by a Log-rank (Mantel-Cox) test. These findings indicate the presence of an immunosuppressive B cell population. Further study is needed to characterize these cells and to determine the significance of IgG4+ tumor infiltrating B cells, and their potential immunosuppressive role in the progression of TNBC.

Citation Format: Nicole Flynn, Ugochukwu Amadi, Rajasekharan Somasundaram, Jennifer Sims-Mourtada. IgG4 expressing B cells associate with poor survival in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-350.
Abstract LB-353: Spatiotemporal quantification of tumor-associated tertiary lymphoid structures in murine lung adenocarcinoma
Authors: Nader Mursal , Joshi Nikhil S.
Published: August 2018

In many human cancer types (i.e., lung, breast, melanoma), infiltrating immune cells are found in complex, lymph node (LN)-like structures known as the tumor-associated tertiary lymphoid structures (TA-TLSs). TA-TLSs are associated with longer patient survival and perform critical functions of LNs such as facilitating recruitment of T cells into the tumor microenvironment, mediating interactions between T cells and tumor-antigen presenting dendritic cells (DCs), and enabling local activation of T cells. Furthermore, using a genetically engineered mouse model (GEMM) of lung adenocarcinoma, we have previously shown that TA-TLSs form as tumors develop and that therapeutically depleting regulatory T cells (Tregs) from TA-TLSs elicits the anti-tumor immune response and results in tumor destruction. Hence, a powerful anti-tumor immune response can be both stimulated and suppressed in TA-TLSs, suggesting that therapeutically targeting the formation and maintenance of TA-TLSs would result in strong anti-tumor outcomes. Understanding how TLS formation is altered by experimental manipulation requires quantitative spatial and temporal analysis of the location, number, and size of TLSs in the tumor-bearing lung. Visualizing the presence of TA-TLSs throughout a tumor-bearing lung lobe presents several technical challenges, thus we have developed a high throughput immunofluorescence (IF) analysis pipeline for visualization and quantification of TA-TLSs in our model. This streamlined process requires 1-2 weeks per lung lobe. We are analyzing tertiary lymphoid structures in influenza-infected mice as a comparison to tumor-associated TLSs. Here, we hypothesize that there are key differences in the location and composition of influenza associated TLSs and TA-TLSs, which will provide substantial mechanistic insight into the biology and formation of TLSs. Using this spatiotemporal quantification technique, detailed interrogation of TLSs in lung adenocarcinoma will provide cellular and molecular targets for therapeutic modulation of TA-TLSs. Furthermore, this method can be expanded and adapted for the study of TA-TLSs in other disease models.

Citation Format: Mursal Nader, Nikhil S. Joshi. Spatiotemporal quantification of tumor-associated tertiary lymphoid structures in murine lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-353.
Abstract LB-355: Loss of keratinocyte desmoglein 1 occurs during melanoma development and alters crosstalk between keratinocytes and melanocytes
Authors: Arnette Christopher , Johnson Jodi L. , Burks Hope E. , Koetsier Jennifer L. , Green Kathleen J.
Published: August 2018

Ultraviolet (UV)-induced mutations in melanocytes are a well-established cause of melanoma. However, histologically normal “fields” of altered keratinocytes present in sun-exposed epidermis could also drive initiation and progression of melanoma. We previously demonstrated that UV radiation reduced expression of the keratinocyte desmosomal cadherin, desmoglein 1 (Dsg1), and that loss-of-function mutations in Dsg1 increased keratinocyte cytokine production in Severe dermatitis, multiple Allergies, and Metabolic wasting (SAM) syndrome. Based on these observations we hypothesized that loss of Dsg1 alters the microenvironment through increased cytokine signaling. Here, we show that Dsg1 expression is progressively reduced in regions surrounding human dysplastic nevi, melanoma in situ, and stage I/II melanomas. In contrast, keratinocyte E-cadherin was not significantly altered in early tumors. Dsg1 knockdown in keratinocytes in vitro increased phospho-EGFR, phospho-Stat3, and nuclear NFκB, while increasing keratinocyte mRNAs encoding IL-1α, IL-1β, IL-6 and IL-8 pro-inflammatory cytokines. Re-expressing silencing-resistant Dsg1 returned cytokine levels to control levels. To determine whether Dsg1 loss in keratinocytes elicits paracrine-mediated changes in melanocyte behavior, we treated normal primary melanocytes with conditioned media from Dsg1-silenced keratinocytes. Short-term (overnight) exposure to Dsg1-deficient conditioned media resulted in significantly increased melanocyte dendrite length, similar to morphology changes induced by UV exposure; however, long-term (7 day) exposure resulted in significantly shorter dendrites. Melanocytes exposed to Dsg1-deficient media increased melanin secretion and decreased melanin retention, a phenomenon associated with the process of transformation. Melanocytes incorporated into Dsg1-deficient 3D human skin equivalents exhibited aberrant behavior, including increased numbers and mis-localization to suprabasal layers. Based on these observations, we propose that under homeostatic conditions Dsg1 functions to maintain baseline levels of cytokine signaling, but that Dsg1 loss due to environmental stress disrupts keratinocyte:melanocyte communication through altered paracrine signaling to promote melanoma initiation.

Citation Format: Christopher Arnette, Jodi L. Johnson, Hope E. Burks, Jennifer L. Koetsier, Kathleen J. Green. Loss of keratinocyte desmoglein 1 occurs during melanoma development and alters crosstalk between keratinocytes and melanocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-355.
Abstract LB-356: Maintenance of breast cancer stem-like cells by cancer associated fibroblast-derived soluble factors
Authors: Murayama Takahiko , Nishimura Tatsunori , Tojo Arinobu , Gotoh Noriko
Published: August 2018

Accumulating evidence indicates the presence of cancer stem cells (CSCs) or tumor initiating cells in many types of tumors, which would be the leading cause of poor prognosis of tumor patients. They are defined as cell populations which have self-renewal ability and multi-differentiation capacity, and have been thought to greatly contribute to tumor initiation, recurrence and metastasis. CSCs are also known to be highly resistant to conventional therapies. Therefore, developing CSC targeting therapy is in urgent need to eradicate cancer cells efficiently. Stem cell properties of CSCs are thought to be maintained by the interaction with the tumor microenvironment, suggesting that it is important to identify factors which mediate such an interaction between cancer cells and the microenvironment. In this study, we focused on cancer-associated fibroblasts (CAFs) that forms the great part of breast tumor microenvironment and investigated how CAFs influence the functions of CSCs. By sphere forming assay, we found that some soluble factors secreted by CAFs induced growth of CSC-like populations. Then, to identify which factors are upregulated when fibroblasts get CAF-like properties, we compared transcriptomes of fibroblasts which were cultured with or without clinical sample-derived breast cancer cells by RNA-seq analysis. We found two genes in the same gene family were highly upregulated in co-cultured cells and they could induce sphere forming ability of CSCs. We are advancing our research based on a hypothesis that these two factors contribute to maintenance of stem cell functions of CSCs. Here, we will introduce results obtained by recent experiments.

Citation Format: Takahiko Murayama, Tatsunori Nishimura, Arinobu Tojo, Noriko Gotoh. Maintenance of breast cancer stem-like cells by cancer associated fibroblast-derived soluble factors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-356.
Abstract LB-373: Comprehensive analysis of cancer stemness
Authors: Malta Tathiane M. , Sokolov Artem , Gentles Andrew J. , Burzykowski Tomasz , Poisson Laila , Weinstein John , Kamińska Bożena , Huelsken Joerg , Omberg Larsson , Gevaert Olivier , Colaprico Antonio , Czerwińska Patrycja , Mazurek Sylwia , Mishra Lopa , Heyn Holger , Krasnitz Alex , Godwin Andrew K. , Lazar Alexander J. , Network The Cancer Genome Atlas Research , Stuart Joshua M. , Hoadley Katherine , Laird Peter W. , Noushmehr Houtan , Wiznerowicz Maciej
Published: August 2018

Cancer progression involves the gradual loss of a differentiated phenotype and acquisition of progenitor and stem cell-like features. Here, we provide new stemness indices for assessing the degree of oncogenic dedifferentiation. We took advantage of an innovative one-class logistic regression machine learning algorithm (OCLR) to extract transcriptomic and epigenetic feature sets derived from non-transformed pluripotent stem cells and their differentiated progenies. Using OCLR, we were able to sort TCGA tumor samples by stemness phenotype and identify previously undiscovered biological mechanisms associated with the dedifferentiated oncogenic state. Analyses of tumor microenvironment revealed the correlation of cancer stemness with immune checkpoint expression and infiltrating immune system cells not previously anticipated. We have shown the de-differentiated oncogenic phenotype increased in the metastatic tumor that further justify their more aggressive phenotype. Application of our stemness indices reveals features of intra-tumor heterogeneity in molecular profiles obtained from the single-cell analyses. Finally, the machine learning-based indices allowed for the identification of chemical compounds and novel targets for the cancer therapies aiming at tumor differentiation. Our findings provide new prognostic signatures that enable cancer biologists and oncologists to quantify the impact of tumor stemness on outcome across cancer types and may help to pave the way for progress in treatment strategies for cancer patients.

Citation Format: Tathiane M. Malta, Artem Sokolov, Andrew J. Gentles, Tomasz Burzykowski, Laila Poisson, John Weinstein, Bożena Kamińska, Joerg Huelsken, Larsson Omberg, Olivier Gevaert, Antonio Colaprico, Patrycja Czerwińska, Sylwia Mazurek, Lopa Mishra, Holger Heyn, Alex Krasnitz, Andrew K. Godwin, Alexander J. Lazar, The Cancer Genome Atlas Research Network, Joshua M. Stuart, Katherine Hoadley, Peter W. Laird, Houtan Noushmehr, Maciej Wiznerowicz. Comprehensive analysis of cancer stemness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-373.