bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4955: Heterogeneous nuclear ribonucleoprotein C as a novel therapeutic target for acute myeloid leukemia
Authors: Vijay Vindhya , Meacham Amy , Terrell Jesse , Katzell Lauren , Drusbosky Leylah , Cogle Christopher
Published: August 2018

Acute myeloid leukemia (AML) is a highly refractory cancer despite conventional treatment. Previously, we found that blood vessels are sanctuary sites for chemo-refractory AML cells. To target AML cells sequestered in the vascular niche, we developed an in vitro co-culture assay consisting of AML cells on bone marrow- derived endothelial cells (BMECs), mimicking the chemo-protective effect of the vascular niche in the bone marrow microenvironment. Using this assay, we performed high-throughput chemical-phenotypic screening of 31 million compounds and identified a novel polyamine sulfonamide (2470-51) that selectively killed AML cells, while sparing the underlying BMECs. 2470-51 was also cytotoxic to CD34+CD38-CD123+ AML stem cells; however, the compound was non-toxic to hematopoietic stem/progenitor cells and T lymphocytes from healthy volunteers. In vivo AML patient-derived xenograft modeling further validated the efficacy of 2470-51 as a selective anti-leukemic agent compared to cytarabine (conventional control) and vehicle control. Protein target identification experiments using unbiased label-free shotgun proteomic analysis in combination with targeted selected ion monitoring revealed heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNPC) as the covalent target of 2470-51. These findings were further validated by shRNA mediated knock-down of HNRNPC (HNRNPC-kd) in AML cell lines THP-1 and K562. HNRNPC-kd significantly reduced AML cell proliferation as well as AML cell viability by inducing apoptosis, compared to scramble controls. HNRNPC-kd also significantly reduced the clonogenic potential of both AML cell lines. In contrast, normal mesenchymal/fibroblastic (HS5) and endothelial cells (BMECs) were unaffected by HNRNPC-kd, indicating the selective dependence of AML cells on hnRNPC. RNA-Seq analyses revealed alternative splice events associated with HNRNPC-kd. Functional enrichment analyses followed by pathway analyses indicated the involvement of hnRNPC in key oncogenic and cell survival pathways. Depleting hnRNPC significantly altered the post-transcriptional landscape, leading to induction of apoptosis. Clinical studies using TCGA AML datasets showed significant survival advantage associated with lower HNRNPC expression. Collectively, our data indicate that hnRNPC is a critical factor in AML and that inhibiting this splicing repressor may be a new therapeutic strategy.

Citation Format: Vindhya Vijay, Amy Meacham, Jesse Terrell, Lauren Katzell, Leylah Drusbosky, Christopher Cogle. Heterogeneous nuclear ribonucleoprotein C as a novel therapeutic target for acute myeloid leukemia [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 4955.
Abstract 4967: HDAC11 function as a transcriptional regulator in immature myeloid cells to myeloid-derived suppressor cells transition
Authors: Chen Jie , Cheng Fengdong , Sahakian Eva , Powers John , Wang Zi , Villagra Alejandro , Pinilla-Ibarz Javier , Sotomayor Eduardo M.
Published: August 2018

In normal myelopoiesis, immature myeloid cells (IMCs) differentiate into macrophages, neutrophils or dendritic cells, a process that is tightly controlled by transcription factors and epigenetic regulators. However, under tumor burden, IMCs differentiate into myeloid derived suppressor cells (MDSCs) and with subsequent up-regulation of immune suppressive factors and a pro-tumor effect. In prior studies, we found that MDSCs from HDAC11 KO mice displayed an increased T-cell suppressive activity that was associated with a more aggressive tumor growth as compared to MDSCs from wild type control mice. Unlike MDSC's in which absence of HDAC11 is associated with a suppressive phenotype, T-cell lacking HDAC11 are hyper-reactive and endowed with strong antitumor activity. To assess which phenotype will be the dominant one in vivo, we performed adoptive immune cell transfer experiments of MDSC and/or T-cells from HDAC11 KO mice into C57BL/6 tumor-bearing mice. The transfer of HDAC11KO MDSCs was able to eliminate, at least partially, the anti-tumor effect elicited by the adoptive transfer of HDAC11KO T cells.

Mechanistically we have found that MDSCs lacking HDAC11 displayed up-regulation of expression and enzymatic activity of arginase 1 and Nos2, two enzymes that are crucial in regulating MDSCs suppressive function. The aberrant enzymatic activities of Arg1 and Nos2 in HDAC11KO MDSCs correlate with over-expression of the lineage-specific transcription factor C/EBPβ, which has been shown to be essential for the differentiation of functional MDSCs. Furthermore, ChIP analysis confirmed that HDAC11 is recruited to the C/EBPβ gene promoter where exerts a negative regulatory effect upon gene transcription.

Taken together, we have uncovered a previously unknown role for HDAC11 as a transcriptional regulator of MDSCs function. A better understanding of this novel role of HDAC11 in myeloid biology will lead to targeted epigenetic therapies to manipulate the suppressive effect of these immunoregulatory cells.

Citation Format: Jie Chen, Fengdong Cheng, Eva Sahakian, John Powers, Zi Wang, Alejandro Villagra, Javier Pinilla-Ibarz, Eduardo M. Sotomayor. HDAC11 function as a transcriptional regulator in immature myeloid cells to myeloid-derived suppressor cells transition [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 4967.
Abstract 4986: Association of sirtuins and diet in cancer development: Studying the roles of SIRT2/3
Authors: Ahmed Mohamed A. , O'Callaghan Carol , Vassilopulos Athanasios
Published: August 2018

Lifestyle factors, especially diet, affects health and lifespan. Dietary restriction (DR) has

been shown as a beneficial regimen to increase longevity and protect from cancer. On the other

hand, high fat intake has been found to be associated with greater risk of several cancer types.

In this regard, several studies suggest that the beneficial effect of calorie restriction (CR) is

mediated by the increased activity of sirtuins, a family of NAD-dependent deacetylases that plays

a role in aging and age-related diseases. However, the specific role of individual family members

in the inhibitory role of CR in tumorigenesis has not been elucidated. With the exception of SIRT1,

there is no evidence for the contribution of other sirtuins in the diet-induced effects on

tumorigenesis. In this study, we investigated, for the first time, the role of SIRT2 and SIRT3, which

are found predominantly in the cytoplasm and mitochondria respectively, in this context. To do

this, Sirt2-/- and Sirt3-/- mice were crossed with p53-/- mice to generate: p53+/-, (Sirt2-/-; p53+/-),

(Sirt3-/-; p53+/-), p53-/-, (Sirt2-/-; p53-/-), and (Sirt3-/-; p53-/-) mice. Subsequently, mice with the

different genotypes were subjected to 3 dietary regimes; ad libitum control diet, 30% calorie

restriction diet (CR), and high-fat diet (HFD). As expected, CR reduced tumor incidence and

increased survival of mice wild type for both SIRT2 and SIRT3. In contrast, HFD significantly

increased body weight, tumor incidence and decreased overall survival. Interestingly, the

protective effect of CR was enhanced in (Sirt3-/-; p53-/-) compared to (Sirt3+/+; p53-/-) mice, as

evidenced by delayed tumor incidence (median age 257 days vs 189 days) and increased survival.

On the other hand, Sirt2 deletion in both p53+/- and p53-/- backgrounds abolished the protective

effect of CR. In addition, loss of Sirt2 decreased tumor incidence age (111 days vs 184 days) and

accelerated the death of p53 deficient mice on HFD. Our results highlight a new role of SIRT2 in

mediating the diet-induced effect on tumorigenesis.

Citation Format: Mohamed A. Ahmed, Carol O'Callaghan, Athanasios Vassilopulos. Association of sirtuins and diet in cancer development: Studying the roles of SIRT2/3 [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 4986.
Abstract 5031: Organotypic brain explant culture as a drug evaluation system for malignant brain tumors
Authors: Minami Noriaki
Published: August 2018

Background: Inhibition of major metabolic pathways is being assessed for several intractable tumors, including glioblastoma. Following screening in 2D-systems, evaluating metabolic inhibitors in assays that include both tumor and normal brain tissue is indispensable for selecting compounds that can affect gliomas within their microenvironment. Here we asked whether organotypic brain slices from adult mice can be used to evaluate the effects of metabolic inhibitors on malignant glioma cells. Methods: Retroviral expression of H-RasV12 and dsRed in Ink4a/Arf-/- neural stem / progenitor cells was adopted to establish glioma initiating cells (GICs). GICs with different extracellular acidification potential were derived by single cell cloning and used after one in vivo passage. GICs or their clones were implanted into the forebrain of 6-weeks-old wild-type mice. Tumor-bearing brain explants were established as 200 µm coronal brain slices, treated with cisplatin, dichloroacetate or phenformin. The effects of the drugs were assessed by fluorescence microscopy of the live explants, as well as immunohistochemical staining at the end of the treatment. Results: The feasibility of assessing drug effects in brain tumor explants from adult mice was confirmed with the use of the DNA-damaging agent cisplatin. Treatment of brain slices carrying tumors formed by GICs with 50 μM cisplatin for 4 days effectively decreased the tumor burden, as shown by a significant reduction of the tumor area on fluorescence imaging and a marked increase in tumor cells positive for cleaved caspase 3 on immunohistochemical analysis. The action of metabolic inhibitors was assessed in explants with tumors formed by GIC-derived populations that predominantly use either glycolysis or mitochondrial respiration. Treatment of the brain slices with dichloroacetate, an inhibitor of pyruvate dehydrogenase kinase, resulted in a marked increase in cleaved caspase 3-positive cells in glycolytic tumors. In contrast, phenformin, which inhibits respiratory complex I, had a pronounced pro-apoptotic effect in tumors formed by cells with a high mitochondrial activity. Conclusion: Inhibitors of both glycolysis and oxidative phosphorylation effectively and selectively induced apoptosis of the targeted tumor cells in cultured brain slices. Our results suggest that brain explants from adult mice can be used to assess the effects of anti-metabolic drugs on the survival of glioma initiating cells within a syngeneic microenvironment. Explants could thus be useful in the evaluation of new metabolic inhibitors.

Citation Format: Noriaki Minami. Organotypic brain explant culture as a drug evaluation system for malignant brain tumors [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 5031.
Abstract 5044: Molecular analysis of HBV in histologically confirmed hepatocellular carcinoma in a tertiary hospital in Ghana
Authors: Duduyemi Babatunde M. , Asante Du-bois , Titiloye Nicholas , Afihene Mary
Published: August 2018

Background: Hepatocellular carcinoma (HCC) is a leading cause of death in Africa. Viral hepatitis B is a leading cause of hepatocellular cancer in Ghana and most African countries except Egypt where hepatitis C virus is more prevalent. This study aims at reviewing the histopathological patterns of HCC and its association with hepatitis B virus in our environment.

Method: Demographics and histological diagnosis were retrieved from the surgical daybook and archival FFPE tissue samples with histopathologically confirmed HCC were used for this study. Sections (10µm) were taken from the tissues and digested to obtain DNA lysates. The DNA lysates were used in polymerase chain reaction (PCR) to determine the prevalence of HBV in the biopsies.

Result: Of the 24 confirmed cases of HCC seen in the 5 year period, there were 17 males and 7 females with M:F ratio of 2.4:1. The mean age of our patients was 39.92 ± 1.98 years with age range 13-85 years. 50% of the cases were moderately differentiated while 25% each were well and poorly differentiated. Out of the 24 archival HCC biopsies screened, HBV DNA PCR amplification was achieved in 11 after the restriction fragment length polymorphism PCR reaction. Out of the 24 archival HCC biopsies screened, HBV DNA PCR amplification was achieved in 11 (45.83%) after the restriction fragment length polymorphism PCR reaction. Eight of the 11 cases were found in the male and 3 in females. Of the 11 (45.83%) samples that were positive for HBV DNA, 3 were above 40 years and 8 were 40 years and below.

Conclusion: The overall prevalence of HBV DNA in our study was 45.83% and a greater proportion seen in less than 40 years. This suggests that most of our patients are infected with HBV early in life in our environment.

Citation Format: Babatunde M. Duduyemi, Du-bois Asante, Nicholas Titiloye, Mary Afihene. Molecular analysis of HBV in histologically confirmed hepatocellular carcinoma in a tertiary hospital in Ghana [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 5044.
Abstract 5060: Exosomes derived from HIV-infected T cells promote cancer cell growth and progression via the HIV TAR RNA
Authors: chen Lechuang , Feng Zhimin , Yue Hong , Sieg Scott , Wang Bingcheng , Huang Alex Y. , Jin Ge
Published: August 2018

Background: Residual and persistent HIV replication plays key roles in the development and progression of non-AIDS-defining cancers (NADCs), including head and neck as well as lung cancers. However, the underlying mechanisms remain elusive. HIV-infected T cells produce immunologically active exosomes to influence intercellular communication and regulate immune response at both local and distant sites, thus potentially contribute to enhanced risk for tumorigenesis. We investigated the role of exosomes derived from HIV-infected T cells in growth and progression of head and neck cancer (HNC) and lung cancer cells. Methods: Exosomes were isolated from conditioned media of HIV-1-infected J1.1 cells and non-infected control Jurkat cells using the ultracentrifugation protocol. Exosomes were validated by western blotting for tetraspanins (CD9, CD63 and CD81) and transmission electron microscope. The acetyl-CoA acetylcholinesterase (AChE) enzyme activity was used to quantify exosomes. HNC cells UM-SCC-104, TR146, HSC3, SCC9 and lung cancer cells NCI-H1299, NCI-H1437 were treated with exosomes for cell growth, migration and gene expression in vitro and in vivo. Exosome HIV RNA was determined by quantitative RT-PCR. Results: Exosomes isolated from HIV-1-infected T cells, but not those from normal control T cells, significantly stimulated HNC and lung cancer cell proliferation, migration and invasion in vitro. Exosomes purified from plasma of HIV-positive individuals also stimulated proliferation of cancer cells. HNC and lung cancer cells co-inoculation with exosomes from HIV-1-infected T cells into nude mice promoted tumor growth compared to those from non-HIV control T cells. Mechanistically, we found that the HIV trans-activation response (TAR) element RNA, which exists in excess of other HIV RNAs in exosomes from HIV-1-infected T cells, enhanced cancer cell proliferation and stimulated EGF- and TLR3-inducible gene expression. The TAR RNA mutant with nucleotide replacements in the loop/bulge region failed to induce gene expression. Moreover, co-transfection of the TAR RNA aptamer blocked induction of gene expression induced by HIV-positive T-cell exosomes. Conclusion: HIV TAR RNA-containing exosomes derived from HIV-1-infected T cells promote growth and progression of NADCs through interaction with EGFR and TLR3.

Citation Format: Lechuang chen, Zhimin Feng, Hong Yue, Scott Sieg, Bingcheng Wang, Alex Y. Huang, Ge Jin. Exosomes derived from HIV-infected T cells promote cancer cell growth and progression via the HIV TAR RNA [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 5060.
Abstract 5072: Immunohistochemical expression of CC-chemokine receptor-like 2/CCRL2 in a prostate cancer tissue microarray
Authors: Reyes Niradiz , Benedetti Ines , Geliebter Jan
Published: August 2018

Background: CCRL2 is a presumed member of the atypical chemokine receptor family, whose members are increasingly recognized as key components of the regulatory network of inflammation and immunity in cancer and may have a major effect on anti-inflammatory and immunotherapeutic strategies. Recent studies have reported the expression of CCRL2 in different human cancer cell lines and tissues. However, its function and expression in prostate cancer has not been previously addressed. The aim of this work was to determine the protein expression profile of the atypical chemokine receptor CCRL2 in human prostate cancer tissue using a tumor microarray approach.

Methods: A tissue microarray was constructed using 231 cores of matched prostate cancer and benign prostatic tissue from 47 patients with a previous diagnosis of localized prostate cancer, which underwent surgical resection as their primary treatment. CCRL2 expression was evaluated by immunohistochemistry using a monoclonal, anti-human-CCRL2 antibody. An H score was calculated in each spot as the sum of expression intensity (0-3+) by extent (0%-100%). Differences in CCRL2 protein expression were assessed by comparing H-Scores between benign and cancerous tissues using Mann-Whitney test for non-Gaussian distribution.

Results: Immunohistochemical evaluation of CCRL2 at the protein level in a prostate tissue microarray constructed with clinical specimens confirmed its overexpression in malignant prostatic tissue. CCRL2 staining was present predominantly in the cytoplasm of prostate cancer tumor cells. Compared to benign epithelial cells, CCRL2 expression was significantly higher in epithelial tumor cells in cores with prostate cancer compared to cores with benign tissue (p=0.0001).

Conclusions: Immunohistochemistry of prostate cancer tissue specimens arrayed in a tissue microarray revealed that CCRL2 expression was significantly stronger in epithelial cells in cancerous acini compared to epithelial cells in matched adjacent benign acini from the same patient. To this point, the physiological effects of CCRL2 expression in epithelial cancer cells are unclear.

Citation Format: Niradiz Reyes, Ines Benedetti, Jan Geliebter. Immunohistochemical expression of CC-chemokine receptor-like 2/CCRL2 in a prostate cancer tissue microarray [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 5072.
Abstract 5080: ADAM10 promotes breast cancer via CXCL16 constitutive cleavage and CXCR6 signaling
Authors: Gales Dominique N. , Mir Hina , Kapur Neeraj , Lillard James W. , Singh Shailesh
Published: August 2018

Despite the survival of breast cancer (BrCa) patients, there is still demand to better understand metastasis as the obvious mark for most aggressive breast cancers. Recent evidence highlighted the possible involvement of chemokines and their cognate receptors in BrCa progression and metastasis. Expression and functional role of CXCR6 and CXCL16 have been investigated in different types of cancer. ADAM10 is also known to regulate the metastatic process by the proteolytic shedding of CXCL16 creating a soluble form of CXCL16. High levels of soluble CXCL16 could support tumor progression by promoting migration and invasion of CXCR6 expressing BrCa cells. Here, we demonstrate the significance of CXCR6-CXCL16 axis in BrCa progression. In addition to increased CXCR6 expression, our tissue microarray analysis (TMA) showed increased expression of CXCL16 and ADAM10 in BrCa tissues. BrCa cell lines (MCF-7 and MBA-231) also showed higher expression of ADAM10 as compared to the normal epithelial cells (MCF-10A). Further, these BrCa cells showed increased migration and invasion towards CXCL16 gradient that is due to high expression of CXCR6 and ADAM10. Hence, our findings suggest the clinical and biological significance of ADAM10 and soluble CXCL16 in BrCa progression. We also highlight the potential of ADAM10 and soluble CXCL16 as prognostic markers and new therapeutic target for BrCa.

Citation Format: Dominique N. Gales, Hina Mir, Neeraj Kapur, James W. Lillard, Shailesh Singh. ADAM10 promotes breast cancer via CXCL16 constitutive cleavage and CXCR6 signaling [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 5080.
Abstract 5095: Concurrent Dnmt3a haploinsufficiency partially abrogates competitive disadvantage in Smc3 haploinsufficient myeloid cells
Authors: Wang Tianjiao , Welch John S.
Published: August 2018

SMC3 encodes a subunit of cohesin associated with recurrent heterozygous mutations in acute myeloid leukemia (AML) and other myeloid malignancies. About one-third of these mutations are predicted loss-of-function (nonsense and splice-site mutations) and the rest are missense with unclear function. To understand whether these missense mutations might have dominant-negative effects or phenocopy loss-of-function effects, we compared the consequences of Smc3 deficient and haploinsufficient mouse models. We found that both embryonic and somatic deletion of homozygous Smc3 alleles led to complete hematopoietic failure. Furthermore, in competitive transplantation, following engraftment and subsequent tamoxifen treatment, the Smc3fl/fl/ERT2-Cre+/- BM cells were rapidly outcompeted, with earliest cell loss in the Gr1+ myeloid compartment. Hence, Smc3 is indispensable for hematopoiesis and AML-associated SMC3 missense mutations are unlikely to have dominant-negative effects. We examined the consequences of somatic Smc3 haploinsufficiency using Smc3fl/+/ERT2-Cre+/-. We found that Smc3 haploinsufficiency did not lead to increased number of initial methylcellulose colonies, nor did these cells replate beyond two weeks. In competitive transplantations, we again observed a significant competitive disadvantage in the Smc3fl/+/ERT2-Cre+/- BM cells, most pronounced in the Gr1+ myeloid cells. Competitive disadvantage is counter-intuitive for a leukemia-associated mutation. SMC3 mutations frequently co-occur with DNMT3A mutations. We, therefore, asked whether Smc3 haploinsufficiency might lead to a competitive advantage if it occurred in the background of Dnmt3a haploinsufficiency. With the addition of Dnmt3a haploinsufficiency, the severe myeloid competitive disadvantage was partially ameliorated, but the significant competitive disadvantage in other lineages remained intact. In summary, our data demonstrate that homozygous Smc3 deletion was incompatible with embryonic or adult hematopoiesis, with the greatest defects noted within myeloid compartment, indicating that AML-associated SMC3 mutations are unlikely to have dominant-negative effects. Smc3 haploinsufficiency resulted in a myeloid competitive disadvantage, which was partially abrogated in the presence of concurrent Dnmt3a haploinsufficiency, suggesting lineage-specific interactions between the mutations.

Citation Format: Tianjiao Wang, John S. Welch. Concurrent Dnmt3a haploinsufficiency partially abrogates competitive disadvantage in Smc3 haploinsufficient myeloid cells [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 5095.
Abstract 5097: Knock-in of a human tumor suppressor (25-kilobase pairs) by traditional and CRISPR/Cas9-stimulated homologous recombination
Authors: Leidy-Davis Tiffany , Cheng Kai , Goodwin Leslie , Morgan Judith , Juan Wen Chun , Roca Xavier , Ong Sin-Tiong , Bergstrom David E.
Published: August 2018

Here we describe an expansion of the DNA size limitations associated with CRISPR knock-in technology, more specifically, the physical extent to which mouse genomic DNA can be replaced with donor (in this case, human) DNA at an orthologous locus. Driving our efforts was the desire to create a whole animal model inserting a 25-kbp segment of a human tumor-suppressor gene in place of 17 kbp of its corresponding mouse orthologue. Included within the human fragment is a conditionally removable (by Cre-loxP) segment of the gene's second intron, a cryptic exon immediately 3' of this, and a native exon some 20 kbp downstream. Using two methods, we first carried out the replacement by employing a combination of bacterial artificial chromosome recombineering, classic ES cell targeting, dual selection, and recombinase-driven cassette removal (traditional approach). Using a unique second method, we employed the same vector (devoid of its selectable marker cassettes), microinjecting it along with CRISPR RNA guides and Cas9 into mouse zygotes (CRISPR approach). In both instances, we were able to achieve humanization of the tumor suppressor to the extent designed, remove all selection cassettes, and demonstrate the functionality of the conditionally removable, loxP-flanked, intronic segment.

Citation Format: Tiffany Leidy-Davis, Kai Cheng, Leslie Goodwin, Judith Morgan, Wen Chun Juan, Xavier Roca, Sin-Tiong Ong, David E. Bergstrom. Knock-in of a human tumor suppressor (25-kilobase pairs) by traditional and CRISPR/Cas9-stimulated homologous recombination [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 5097.
Abstract 5099: Evaluating Ago2 as an oncogene in Kras-dependent lung carcinoma
Authors: Goodrum Andrew E. , Wang Lisha , Xu Alice , Juckette Kristin M. , Chinnaiyan Arul M. , Tien Jean C.
Published: August 2018

Background: Lung cancer is the deadliest malignancy in the United States. Non-small cell lung cancer (NSCLC) accounts for 85% of cases, and is frequently driven by activating mutations in the gene encoding the KRAS GTPase (e.g. KRASG12D). Our previous work demonstrated that Argonaute 2 (AGO2)—a component of the RNA induced silencing complex (RISC)—physically interacts with KRAS and stabilizes it at the protein level. In NSCLC cell lines, AGO2 knockdown reduces KRAS protein level and attenuates cell proliferation. We hypothesized that AGO2 acts as an oncogene in KRASG12D-dependent NSCLC in vivo.

Methods: To test the hypothesis, we generated a NSCLC model by intercrossing mice harboring a stop-floxed KRASG12D allele with animals containing a tamoxifen-responsive Cre recombinase gene driven by the (clara cell-specific) CCSP promoter. These animals, which frequently die within 10 weeks of tamoxifen treatment, display pulmonary lesions including hyperplasia, adenoma and frank adenocarcinoma. We evaluated whether concomitant ablation of one or both copies of (floxed) AGO2 influenced the NSCLC phenotype. After tamoxifen administration, we monitored mice daily and harvested tissues when mice were clinically moribund or reached 16 weeks of post-treatment. We harvested lungs for gross/ pathological examination and gene/ protein expression analysis.

Results: While pathologically discernable adenocarcinoma was detectable in AGO2+/+ and AGO2f/+ animals, AGO2f/f mice were free of these lesions. Despite this, deletion of AGO2 had no impact on frequency or extent of hyperplastic or adenomatous lesions. Immunohistochemistry demonstrated absence of AGO2 in tumor tissue of AGO2f/f mice.

Conclusion: AGO2 promotes progression but not initiation of cancerous lesions in a mouse model of KRASG12D-dependent NSCLC.

Citation Format: Andrew E. Goodrum, Lisha Wang, Alice Xu, Kristin M. Juckette, Arul M. Chinnaiyan, Jean C. Tien. Evaluating Ago2 as an oncogene in Kras-dependent lung carcinoma [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 5099.
Abstract 510: MicroRNA-8073 as a tumor suppressor and a potential diagnostic and therapeutic target
Authors: Mizoguchi Atsuko , Takayama Aiko , Arai Taiga , Kida Yuho , Kawauchi Junpei , Sudo Hiroko
Published: August 2018

MicroRNAs (miRNAs) generated inside cells are also present in peripheral blood. MiR-8073 has been studied in various disease model, but its molecular function had been unknown. In this study, we demonstrated that miR-8073 regulates target genes that participate in cell survival and works as a tumor suppressor controlling only cancer cells.

First, we observed that miR-8073 expression was evidently decreased in multiple types of cancer cells compared to normal cells, but was increased in cancer exosome secreted into cultured supernatant. We then transfected synthetic miR-8073 mimic into various cancer cell lines and found dramatic reduction in cell proliferation, which was not detected in normal cells. Additionally, in tumor xenograft mice model, three-time subcutaneous injections of miR-8073 mimic at 2nM suppressed 57% growth of colorectal tumor weight.

Through mRNA expression analysis and in silico sequence analysis, we further identified five potential miR-8073 targets that plays critical roles in regulation of cell proliferation, cycle and apoptosis, and demonstrated that miR-8073 indeed bound with the 3'-UTR of these target mRNAs and reduced both mRNA and protein expression.

These results suggest that miR-8073 is a potential tumor suppressor, possibly discarded from cancer cells for their survival out to extracellular space, and circulates in bloodstream in the form of exosome. Our findings promise miR-8073 as a novel cancer diagnostic and therapeutic target.

Citation Format: Atsuko Mizoguchi, Aiko Takayama, Taiga Arai, Yuho Kida, Junpei Kawauchi, Hiroko Sudo. MicroRNA-8073 as a tumor suppressor and a potential diagnostic and therapeutic target [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 510.
Abstract 5106: CRISPR/Cas9-based development of RNAi rat models for drug discovery
Authors: Premsrirut Prem K. , Wang Chia-Lin , Yang Yu-Ting , Dow Lukas E. , Zuber Johannes , Lowe Scott W.
Published: August 2018

The cost of drug development has skyrocketed to an estimated $2.6B for every FDA approved drug primarily due to failures from lack of efficacy or safety. This suggests that our current preclinical validation process has been insufficient in predicting therapeutic potential and toxicity in humans. While genetically engineered animal models have been highlighted as the gold standard for dissecting cancer mechanisms and evaluating novel drug targets in vivo, the cost and long lead time to develop them previously prevented their routine use in the oncology drug discovery process. Now, with the advent of CRISPR/Cas9 technology, the speed and precision in which genetically engineered mouse models can be created is unprecedented. When combined with inducible RNA inteference technology, we we can not only generate cancer de novo in a few weeks time, but also mimic drug therapy via RNAi in the same mice, giving us advanced capabilities to perform preclinical studies in vivo. Despite the utility of mouse models, the rat has historically been the major model species in many biomedical fields, notably toxicology and carcinogenicity testing; and for many scientists, the rat still remains the preferred rodent due to their larger size for surgical manipulation, repeat blood sampling, and their cognitive and physiological characteristics that more closely resemble humans than their mouse counterparts. Here, we take advantage of our two-step engineering approach and exploit the efficiency of CRISPR-based targeting to develop RNAi rat models that enable inducible and reversible gene silencing to simulate therapeutic regimes. We demonstrate that our approach allows us to rapidly generate RNAi rat models and mimic the function of the targeted small molecule inhibitors, such as BET inhibitors targeting Brd4. We compare our results to our Brd4 RNAi mice and demonstrate organism variances that provide valuable insight to cross-species differences. These results demonstrate that our high-throughput system currently used to generate RNAi mice is also applicable to the rat system and, by extension, other mammalian models. Inducible RNAi rat models will undoubtedly be powerful tools that can be used to model human cancers, to mimic the action of putative drugs, and to assess the potential of therapeutic targeting strategies in vivo prior to the costly drug development, ultimately guiding the development of safer and more effective drugs.

Citation Format: Prem K. Premsrirut, Chia-Lin Wang, Yu-Ting Yang, Lukas E. Dow, Johannes Zuber, Scott W. Lowe. CRISPR/Cas9-based development of RNAi rat models for drug discovery [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 5106.
Abstract 5109: Somatic CRISPR/Cas9-mediated gene editing enables versatile brain tumor modeling
Authors: Zuckermann Marc , Ismer Britta , Hovestadt Volker , Knobbe-Thomsen Christiane B. , Zapatka Marc , Northcott Paul A. , Roussel Martine F. , Reifenberger Guido , Lichter Peter , Pfister Stefan M. , Kawauchi Daisuke , Gronych Jan , Jones David T.
Published: August 2018

Despite substantial advances in the molecular classification of pediatric brain tumors over the last decade, many tumors are still incurable, have a poor prognosis or therapy-related detrimental long term effects. To develop innovative and modern therapy approaches, sophisticated mouse models, faithfully reflecting the human disease are urgently needed.

However, the development of transgenic mouse lines is a time consuming process that is confronted with an increasing number of candidate genes resulting from next generation sequencing of tumor specimens. To cope with this discrepancy, more adaptable animal models are needed. Using somatic gene transfer to manipulate cells in situ holds great advantages in terms of speed and flexibility.

We therefore established a system in which we are using the CRISPR/Cas9 system for somatic disruption of candidate tumor suppressor genes (TSGs) in combination with transposon-mediated overexpression of candidate oncogenes to induce murine brain tumors. By deleting Ptch1 in murine cerebella, we induced Sonic Hedgehog (SHH) subgroup medulloblastoma with high penetrance and short latency. We characterized the induced mutations in depth and did not detect recurrent off-targets using unsupervised whole genome sequencing. We further demonstrated that this method is suited to induce other brain tumor entities by targeting multiple TSGs at the same time or by combining TSG targeting with overexpression of certain oncogenes. To date we established five novel mouse models using this system. Two of these resulted from simultaneous deletion of Nf1, Pten and Trp53 or from combining overexpression of a MET fusion with a loss of Trp53, respectively. Both of these combinations led to efficient glioblastoma induction in the murine forebrain. Our results demonstrate a fast and flexible system for validating novel candidate genes and for generating faithful animal models of human cancer.

Citation Format: Marc Zuckermann, Britta Ismer, Volker Hovestadt, Christiane B. Knobbe-Thomsen, Marc Zapatka, Paul A. Northcott, Martine F. Roussel, Guido Reifenberger, Peter Lichter, Stefan M. Pfister, Daisuke Kawauchi, Jan Gronych, David T. Jones. Somatic CRISPR/Cas9-mediated gene editing enables versatile brain tumor modeling [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 5109.
Abstract 513: Antitumor miR-451a regulatory network is associated with poor prognosis of renal cell carcinoma
Authors: Yamada Yasutaka , Sugawara Sho , Arai Takayuki , Okato Atsushi , Kojima Satoko , Naya Yukio , Ichikawa Tomohiko , Seki Naohiko
Published: August 2018

Renal cell carcinoma (RCC) is the most common neoplasm of the kidney, and approximately 70-80% of RCCs are classified as clear cell-type (ccRCC). Approximately 350,000 new cases are diagnosed worldwide, with over 140,000 deaths, every year. Although surgical resection remains the only known curative treatment for localized RCC, a significant percentage of these patients develop relapses or metastatic disease, which are associated with a poor prognosis. Recently developed targeted molecular therapies and immunomodulatory agents are currently being widely used for patients with metastatic or recurrent RCC. However, those advanced treatments are restricted to RCC patients in advanced stages of disease and the 5-year survival rate of the patients is only 5-10%.

MicroRNAs (miRNAs) belong to a class of noncoding RNA molecules that fine-tune the expression of protein coding/noncoding RNAs by repressing translation or cleaving RNA transcripts in a sequence-dependent manner. Aberrantly expressed miRNAs contribute to the initiation, development, and metastasis of several types of cancers, including RCC. Current advanced technologies can be used to identify dysregulated miRNAs in HNSCC clinical specimens. Identification of differentially expressed miRNAs is the first step to elucidating novel miRNA-mediated RNA networks in cancer cell.

Analyses of miRNA expression signature by RNA sequencing revealed that miR-451a is significantly downregulated in RCC tissues. Here, we aimed to identify novel RNA networks for miR-451a regulation in RCC cells. Ectopic expression of miR-451a significantly inhibited cancer cell aggressiveness in RCC cells. The Cancer Genome Atlas (TCGA) showed that low expression of miR-451a was significantly associated with poor survival of patients with RCC (p=0.00305). Genome-wide gene expression analyses were applied to identify miR-451a target genes. A total of 16 genes were identified as putative targets of miR-451a in

RCC cells. Among these targets, high expression of 8 genes (F2, CRELD2, MSC, LENG8, CLEC2D, SPC25, PMM2, BST2 and NSMAF) were significantly associated with poor prognosis of the patients with RCC by large numbers of cohort analyses.

Downregulated miR-451a acted as an antitumor miRNA in RCC cells. Antitumor miR-451a regulatory network was contributed to RCC aggressiveness. The identification of antitumor-mediated oncogenic networks may lead to a better understanding of RCC pathogenesis.

Citation Format: Yasutaka Yamada, Sho Sugawara, Takayuki Arai, Atsushi Okato, Satoko Kojima, Yukio Naya, Tomohiko Ichikawa, Naohiko Seki. Antitumor miR-451a regulatory network is associated with poor prognosis of renal cell carcinoma [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 513.
Abstract 5131: Obese conditions induce changes in stromal fibroblast phenotype
Authors: Harlow Brittany , Davalos Albert , Brenner Andrew , Jolly Christopher , Hursting Stephen , deGraffenried Linda
Published: August 2018

Background: Over 40% of American women present a BMI ≥30, which is particularly alarming since obesity confers a worse prognosis for both pre- and postmenopausal breast cancer patients. Although the molecular mechanisms behind this remain unclear, obese conditions have been shown to upregulate certain components of the cancer-associated fibroblast (CAF) secretome and senescence associated secretory phenotype (SASP), both directly correlated with tumorigenesis. However, studies have yet to determine whether obesity induces a cohesive SASP or CAF-like phenotype from any one cell type of the breast tumor microenvironment. Because fibroblasts represent the most common breast tumor cell type, it is of particular importance to investigate obesity-induced changes in this cellular compartment and their effects on cancer cell behavior. Methods: Direct effects of obesity were examined by exposing IMR-90 and HCA2 human fibroblasts to media supplemented with 2% sera from obese women and measuring changes in gene and protein expression, while indirect effects were evaluated by exposing the fibroblasts to conditioned media (CM) from obesity-stimulated MCF-7 and T47D cells and assessing changes in expression of proinflammatory cytokines. Breast cancer cells were also exposed to CM from obesity-stimulated fibroblasts and measured for changes in proliferation, survival, motility, and invasion. Results and Conclusions: Obese conditions induced proinflammatory changes in fibroblasts both directly and indirectly and stimulated changes in the paracrine signaling between fibroblasts and breast cancer cells. These data contribute to the growing evidence implicating obesity in the promotion of breast cancer progression. Additionally, these findings provide a new line of research to develop potential therapeutic targets to improve outcome.

Citation Format: Brittany Harlow, Albert Davalos, Andrew Brenner, Christopher Jolly, Stephen Hursting, Linda deGraffenried. Obese conditions induce changes in stromal fibroblast phenotype [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 5131.
Abstract 5132: Lactic acidosis-mediated transcriptomic and metabolomic rewiring in pancreatic cancer cells
Authors: Murthy Divya , Abrego Jaime , King Ryan James , Singh Pankaj Kumar
Published: August 2018

Tumor microenvironment plays a critical role in regulating tumor cell metabolism and progression. The tumor cells are constantly exposed to hypoxia, nutrient starvation, and extracellular acidification that contribute to tumor malignancy. These malignant cells exhibit Warburg effect by maintaining glycolytic phenotype even in the presence of oxygen and excrete lactate and protons via monocarboxylate transporters, and sodium-hydrogen antiporter 1 (NHE1) causing constant acidification of the tumor microenvironment. The tumor cells must thus adapt to acidosis to thrive in such hostile microenvironment. The purpose of the study is to identify the functional response of pancreatic cancer cells to the acidic tumor microenvironment. We performed integrated transcriptomic and metabolomic analyses to identify the existence of low pH-responsive transcriptional and metabolic regulators. Culturing the cells under low pH resulted in a reduction in cell growth and an increase in oxidative stress within the cells. RNA-seq-based transcriptomic analyses revealed that acidosis drives distinct gene expression patterns in PDAC cells with the expression of 499 and 496 genes up- and downregulated, respectively in PDAC cells. GSEA analyses of significantly modulated genes identified pathways including cell cycle, DNA repair, metabolic pathways, ABC transporters, glutathione metabolism, and genes involved in signaling cascades such as the inositide signaling pathway. As the transcriptomic analyses showed an enrichment of metabolic genes, intracellular metabolomic analyses revealed that chronic acidosis leads to a shift in cellular metabolism towards oxidative metabolism. Our studies demonstrate that while chronic acidosis in the tumor microenvironment leads to reduced proliferation and glycolysis in tumor cells, it also results in increased expression of genes involved in cellular migration, signaling pathways and switching to anaplerotic glutamine metabolism in response to an acidic tumor microenvironment. Overall, our studies demonstrate metabolic reprogramming in PDAC cells in response to low pH microenvironments creating a metabolic vulnerability that could be exploited for therapeutic targeting of pancreatic cancer.

Citation Format: Divya Murthy, Jaime Abrego, Ryan James King, Pankaj Kumar Singh. Lactic acidosis-mediated transcriptomic and metabolomic rewiring in pancreatic cancer cells [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 5132.
Abstract 5145: PAH-metabolizing microorganism in the oral cavity of smokers
Authors: Adami Guy R. , Tao Lin , Pavlova Sylvia , Green Stefan J. , Naqib Ankur , Salameh Benjamin , Tang Jessica L. , Schwartz Joel L.
Published: August 2018

Certain soil microorganisms have been identified as being not only resistant to polycyclic aromatic hydrocarbons (PAHs) but also capable of metabolizing these compounds. We hypothesized that a subset of oral bacteria may be enriched by the altered oral environment of the tobacco smoker with relatively high levels of PAHs. We identified microorganisms that survived long term in minimal media with PAH as the sole carbon source and showed they were much more likely to be isolated from smokers. To start to determine their contribution to oral health, we measured their level in vivo on two mucosal surfaces. On both gingival and lateral border of the tongue mucosa, a group of bacteria were seen to be at lower relative numbers in those exposed to cigarette smoke, while a minority increased. Three taxons were shown to be enriched on gingiva on the family, Streptococcaceae, or genus level, Staphylococcus and Granulicatella. On tongue, genera Mogibacterium and Dialister were enriched. Of bacteria genera that survived harsh PAH exposure in vitro, one was found at substantial levels in the oral mucosal microbiome. Staphylococcus was enriched approximately 6x in smokers, suggesting that the reason for its increase in smokers is due to its resistance to PAH. We will report on this and other oral microbes that can metabolize PAHs. This study highlights the possibility that the oral microbiome can metabolize PAHs like those in cigarette smoke, resulting in carcinogenic products that may contribute to the cancer process in vivo.

Citation Format: Guy R. Adami, Lin Tao, Sylvia Pavlova, Stefan J. Green, Ankur Naqib, Benjamin Salameh, Jessica L. Tang, Joel L. Schwartz. PAH-metabolizing microorganism in the oral cavity of smokers [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 5145.