bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2582: Investigating the origin and function of circulating miRNA in lung cancer
Authors: Colangelo Tommaso , Mazzarelli Francesco , Cuttano Roberto , Dama Elisa , Melocchi Valentina , Graziano Paolo , Fiordelisi Fabiola , Bianchi Fabrizio
Published: August 2019

MicroRNAs (miRNAs) are small, highly conserved noncoding RNA molecules involved in the regulation of gene expression. MicroRNAs are resistant to harsh conditions and stably exist in body fluids (e.g. saliva, urine, breast milk, blood). Circulating cell-free miRNAs (cf-miRNA) were shown to be effective biomarkers for the early diagnosis of cancer. Furthermore, cf-miRNA were also found in exosomes that are nano-sized extracellular vesicles which exchange molecular information among cells. However, the understanding of origin of cf-miRNAs and of biological function still remains elusive. We recently identified a signature of cf-miRNAs diagnostic for lung cancer which we now hypothesize being released by lung cancer cells (EP-cf-miRNA) and by tumor microenvironment (TME-cf-miRNA). In particular, two cf-miRNAs, miR-29a and 223-3p, were selected as prototypes of EP- and TME-cf-miRNAs for further investigations.We found that the expression of miR-223-3p was higher in TME than in cancer cells of a cohort of 19 lung adenocarcinoma. In contrast, miR-29a-3p was frequently overexpressed (≥2 fold) in cancer cells. Interestingly, the serum concentration of cf-miR-29a was lower than miR-223-3p and this also in exosomes.We next investigated the expression profile of miR-29a/223-3p in a panel of lung normal and adenocarcinoma cell lines (N=14). miR-29a/223-3p were both expressed intracellularly and in exosomes. Of note, the miR-29a expression level resembles that observed in lung cancer cells from FFPE samples. Conversely, miR-223-3p expression was lower in the adenocarcinoma cell lines which further confirm a more TME origin. We are now expanding our analysis by screening the whole set of cf-miRNAs by high-throughput qPCR profile (OpenArray technology) and by applying laser capture microdissection to investigate single cancer epithelial cells and TME. Our work provides proof of principle demonstration of an effective approach to investigate cf-miRNA origin to unravel the role of cf-miRNA in lung adenocarcinoma.

Citation Format: Tommaso Colangelo, Francesco Mazzarelli, Roberto Cuttano, Elisa Dama, Valentina Melocchi, Paolo Graziano, Fabiola Fiordelisi, Fabrizio Bianchi. Investigating the origin and function of circulating miRNA in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2582.
Abstract 2612: Roles of USF1 in breast tumorigenesis and disease progression
Authors: Lewis Jessica , Mahmud Iqbal , Tian Guimei , Liao Daiqing
Published: August 2019

Breast cancer accounts for nearly one-quarter of all cancer diagnoses and is the principal cause of cancer-related mortality in women worldwide. Triple negative breast cancer (TNBC) is a clinically aggressive subtype of breast cancer commonly resistant to therapeutics that have been successful in increasing survival in patients with ER+, PR+ and HER2+ breast cancer subtypes. As such, identifying factors that contribute to poor patient outcomes and mediate the growth and survival of TNBC cells remain important areas of investigation. USF1 (upstream stimulatory factor 1), a gene linked to drive lipogenesis and cellular proliferation, is overexpressed in human malignancies, yet its contribution to cancer remains unclear. In analyzing large number of genomic datasets including The Cancer Genome Atlas (TCGA), we found that USF1 expression is significantly higher in TNBC patient samples. Also, USF1 gene expression positively correlates with key lipogenic enzymes. Significantly, we found that high expression of USF1 in breast cancer correlates with decreased patient survival. We therefore hypothesize that USF1 promotes breast tumorigenesis and progression by activating lipogenic gene expression. We conducted pilot in vitro studies to determine the influence of USF1 expression and cell proliferation. It was demonstrated that knockdown of USF1 decreased cellular proliferation in 2D cell culture of the TNBC cell line MDA-MB-231. We also assessed the effects on USF1 expression levels on in vivo tumor growth. We found that USF1 overexpression and knockdown enhanced and reduced tumor growth in vivo, respectively. Further studies are underway to determine the mechanisms by which USF1 promotes TNBC tumorigenesis and metastatic progression. Our studies will shed lights on roles of USF1 in breast cancer tumor biology. (Supported by McKnight Foundation, Bankhead-Coley Cancer Research Program and James and Esther King Biomedical Research Program, Florida Department of Health.)

Citation Format: Jessica Lewis, Iqbal Mahmud, Guimei Tian, Daiqing Liao. Roles of USF1 in breast tumorigenesis and disease progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2612.
Abstract 262: Anti-tumorigenic effect of ONC201 is enhanced by combination treatment with TRAIL or a DR5 agonist in endometrial cancer in vitro
Authors: Ray Jocelyn , Ralff Marie , Dicker David , El-Deiry Wafik
Published: August 2019

ONC201 is a well-tolerated, orally active small molecule in the novel imipridone class that has anti-tumorigenic properties in a number of solid tumors, but not in non-neoplastic cells. ONC201 has demonstrated promising activity as a single agent in patients with advanced endometrial cancer in the first-in-human clinical trial with Phase II trials in endometrial cancer patients now underway. Although the mechanism of action in endometrial cell pathology has not been well studied, ONC201’s anti-tumor effect has been shown in other model systems to be p53-independent and mediated through activation of the integrated stress response (ISR) leading to DR5 activation and through induction of cell death or growth arrest. ONC201 inhibits ERK and AKT leading to upregulation of TRAIL in many systems. We hypothesized that ONC201 upregulation of DR5 could sensitize tumor cells to TRAIL and that TRAIL could convert growth arrest to cell death in ONC201-treated cells. Three endometrial cancer cell lines AN3CA, HEC1A and KLE were treated with ONC201 alone or in combination with TRAIL, or a DR5 agonist. Effects on cell viability were assessed by the Cell Titer-Glo cell viability assay, colony formation assays and cell cycle analysis by propidium iodide staining. QPCR and Western blot analysis were used to evaluate mRNA and protein expression, respectively. Assessment of TRAIL and DR5 cell surface expression was evaluated by surface staining using flow cytometry and FlowJo data analysis. ONC201 decreased the cell viability of all three endometrial cancer cell lines at clinically achievable low micro-molar concentrations. ONC201 activated the ISR and an anti-proliferative effect involving a G1 phase arrest and little cell death as indicated by increased Sub-G1 analysis and PARP cleavage. DR5 mRNA and protein expression at the cell surface were induced by ONC201. All three endometrial cancer cell lines were resistant to TRAIL alone, however, pre-treatment with ONC201 sensitized the AN3CA and KLE to TRAIL and a DR5 agonist, leading to potent cell death induction. The combination of ONC201 plus TRAIL did not cause appreciable cell death in normal human fibroblast. ONC201 decreases cell viability in endometrial cancer cells lines primarily through growth arrest while the combination of ONC201 and TRAIL or a DR5 agonist promotes cell death in AN3CA and KLE cells but not in HEC1A endometrial cancer cells. Our results suggest a novel cancer therapeutic strategy that can be exploited in the clinic.

Citation Format: Jocelyn Ray, Marie Ralff, David Dicker, Wafik El-Deiry. Anti-tumorigenic effect of ONC201 is enhanced by combination treatment with TRAIL or a DR5 agonist in endometrial cancer in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 262.
Abstract 2620: Global epigenetic regulation of gene expression and tumor suppression in T-cell leukemia by Ikaros
Authors: Ding Yali , Zhang Bo , Payne Jonathon L. , Payne Kimberly J. , Song Chunhua , Gowda Chandrika , Iyer Soumya , Dhanyamraju Pavan K. , Desai Dhimant , Huang Suming , Yue Feng , Dovat Sinisa
Published: August 2019

Ikaros encodes a DNA-binding zinc finger protein that functions as a tumor suppressor. Loss of Ikaros function results in the development of high-risk B-cell and T-cell acute lymphoblastic leukemia. Previous studies suggested that Ikaros controls expression of its target genes via chromatin remodeling, and by forming complexes with the histone deacetylase, HDAC1. However, the mechanisms through which Ikaros regulates the global epigenetic signature in leukemia is unknown. We studied the role of Ikaros in regulating tumor suppression in Ikaros-defficient T-cell acute lymphoblastic leukemia (T-ALL) cells. The re-introduction of Ikaros at physiological levels via retroviral transduction into T-ALL cells, results in a cessation of cellular proliferation and cell cycle arrest within 4 days. To determine the dynamics of Ikaros-mediated epigenetic regulation of gene expression in T-ALL, we performed ChIP-seq of Ikaros, HDAC1, and histone modifications, along with microarray and ATAC-seq in wild-type T-ALL (that lacks Ikaros), and during each of 3 days following the re-introduction of Ikaros. Our results demonstrated that Ikaros directly regulates the global epigenomic landscape including genome wide de novo formation of enhancers in T-ALL. Further analysis showed that Ikaros induces the re-distribution of HDAC1 via direct recruitment of HDAC1 to promoter and enhancer regions of different target genes resulting in their transcriptional repression. Expression analysis identified a large number of novel signaling pathways that are directly regulated by Ikaros and HDAC1, and are responsible for the cessation of proliferation and the cell cycle arrest of T-ALL cells. Our results identified novel functions of Ikaros in the epigenetic regulation of gene expression and tumor suppression in T-ALL.

Citation Format: Yali Ding, Bo Zhang, Jonathon L. Payne, Kimberly J. Payne, Chunhua Song, Chandrika Gowda, Soumya Iyer, Pavan K. Dhanyamraju, Dhimant Desai, Suming Huang, Feng Yue, Sinisa Dovat. Global epigenetic regulation of gene expression and tumor suppression in T-cell leukemia by Ikaros [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2620.
Abstract 2621: OLIG2 regulates stem cell maintenance and cell cycle in glioma stem cells
Authors: Saito Norihiko , Hirai Nozomi , Aoki Kazuya , Fujita Satoshi , Nakayama Haruo , Hayashi Morito , Sakurai Takatoshi , Iwabuchi Satoshi
Published: August 2019

The Cancer Genome Atlas project described a robust gene expression-based molecular classification of glioblastoma with the functional and biological significance of the subclasses yet to be determined. Here, we show that a comprehensive analysis of a panel of glioma initiating cell (GIC) lines can identify a group of stem cells with high OLIGg2 expression as in Proneural-like GBM subtype. In vitro differentiation studies showed that proneural GIC lines possess the potential to differentiate into astrocytic, neuronal, and oligodendrocytic lineages, whereas mesenchymal GICs exhibited limited potential for neural lineage differentiation following retinoic acid induction. A considerable decline of OLIG2 in proneural GIC lines was observed following retinoic acid treatment. We also showed that OLIG2 is a functional marker associated with cell proliferation in Olig2-high GIC lines. In addition, OLIG2 inhibiton disrupted cell-cycle control mechanism by decreasing CDK2 and CDK4 and elevating apoptosis-related molecules. Mechanistic investigations revealed molecular interactions between CDK2/CDK4 and OLIG2. Inhibition of CDK2/CDK4 activity disrupted OLIG2-CDK2/CDK4 interactions and attenuated OLIG2 protein stability. Further investigation on these mechanisms may lead to novel targeted therapy on GBMs with high OLIG2 expression.

Citation Format: Norihiko Saito, Nozomi Hirai, Kazuya Aoki, Satoshi Fujita, Haruo Nakayama, Morito Hayashi, Takatoshi Sakurai, Satoshi Iwabuchi. OLIG2 regulates stem cell maintenance and cell cycle in glioma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2621.
Abstract 2623: Modulating native GABAA receptors in medulloblastoma with positive allosteric benzodiazepine-derivatives induces cell death
Authors: Kallay Laura , Keskin Havva , Ross Alexandra , Rupji Manali , Moody Olivia A. , Wang Xin , Li Guanguan , Ahmed Taukir , Rashid Farjana , Stephen Michael Rajesh , Cottrill Kirsten A. , Nuckois Austin , Xu Maxwell , Martinson Deborah E. , Tranghese Frank , Pei Yanxin , Cook James M. , Kowalski Jeanne , Taylor Michael D. , Jenkins Andrew , Krummel Daniel Pomeranz , Sengupta Soma
Published: August 2019

Pediatric brain cancer medulloblastoma standard-of-care results in numerous comorbidities. Medulloblastoma is comprised of four molecular subgroups. Group 3 subgroup patients have the highest relapse rates and 20% survival after standard-of-care. Group 3 tumors have high expression of GABRA5, which codes for the α5 subunit of the γ-aminobutyric acid type A receptor (GABAAR). We are advancing a therapeutic approach for group 3 based on GABAAR modulation using benzodiazepine-derivatives. We performed analysis of GABR and MYC expression in medulloblastoma tumors and used molecular, cell biological, and whole-cell electrophysiology approaches to establish presence of a functional ‘druggable’ GABAAR in group 3 cells. Analysis of expression of 763 medulloblastoma tumors reveals that group 3 tumors share high subgroup-specific and correlative expression of GABR genes, which code for GABAAR subunits α5, β3 and γ2 and 3. There are ~1000 functional α5-GABAARs per group 3 patient-derived cell that mediate a basal chloride-anion efflux of 2x109 ions/sec. Benzodiazepines, designed to prefer α5-GABAAR, impair group 3 cell viability by enhancing chloride-anion efflux with subtle changes in their structure having significant impact on potency. A potent, non-toxic benzodiazepine (‘KRM-II-08’) binds to the α5-GABAAR (0.8 μM EC50) enhancing a chloride-anion efflux that induces mitochondrial membrane depolarization and in response, TP53 upregulation and p53, constitutively phosphorylated at S392, cytoplasmic localization. This correlates with pro-apoptotic Bcl-2-associated death promoter (BAD) protein localization. GABR expression can serve as a diagnostic biomarker for group 3 tumors, while α5-GABAAR is a therapeutic target for benzodiazepine binding, enhancing an ion imbalance that induces apoptosis.

Citation Format: Laura Kallay, Havva Keskin, Alexandra Ross, Manali Rupji, Olivia A. Moody, Xin Wang, Guanguan Li, Taukir Ahmed, Farjana Rashid, Michael Rajesh Stephen, Kirsten A. Cottrill, Austin Nuckois, Maxwell Xu, Deborah E. Martinson, Frank Tranghese, Yanxin Pei, James M. Cook, Jeanne Kowalski, Michael D. Taylor, Andrew Jenkins, Daniel Pomeranz Krummel, Soma Sengupta. Modulating native GABAA receptors in medulloblastoma with positive allosteric benzodiazepine-derivatives induces cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2623.
Abstract 2627: Inhibition of TGFβ in colorectal cancer cells is associated with a compensatory activation of AXL and p38 MAPK signaling pathways
Authors: Ciardiello Davide , Vitiello Pietro Paolo , Matrone Nunzia , Belli Valentina , Cardone Claudia , Poliero Luca , Borrelli Carola , Arrichiello Gianluca , Martini Giulia , Ciaramella Vincenza , Barra Giusi , Morgillo Floriana , Troiani Teresa , Melisi Davide , Ciardiello Fortunato , Martinelli Erika
Published: August 2019

According to the consensus molecular subtypes (CMS), almost 23% of human colorectal cancer (CRC) are classified in the CMS4 group, characterized by a mesenchymal signature and the activation of transforming growth factor β (TGF-β) signaling. AXL is a tyrosine kinase receptor involved in epithelial to mesenchymal transition, angiogenesis and immune modulation in CRC. We have previously demonstrated that AXL gene is amplified in approximately 5% of human CRC, and that AXL inhibition causes a significant blockade of CRC cell proliferation and migration. Here we have evaluated the role of TGF-β signaling and the potential interaction between TGF-β and AXL in human CRC cell lines. We assessed the expression and activation of TGF-β and AXL in a panel of six human CRC cell lines by western blot (WB) and real time PCR. We tested the sensitivity of HCT116 and LOVO cells to treatment with galunisertib ( LY21209761), a selective TGF-β R1 inhibitor, by MTT, cell invasion, wound healing and soft-agar colony forming assays. Further, intracellular CRC cell signaling was evaluated following galunisertib treatment by WB. TGF-β receptors 1 and 2 were expressed in all human CRC cell lines (HCT116, SW480, LOVO, LIM 1215, SW48 and CaCo2), whereas AXL expression was restricted to HCT116, SW480, LOVO cells. Treatment with galunisertib had little or no effect on cancer cell growth, whereas it partially reduced TGF-β induced cell migration, invasion and colony formation in HCT116 and LOVO cells (that co-expressed both TGF-β receptors and AXL). Interestingly, TGF-β inhibition resulted in a concomitant significant activation of AXL and p38 MAPK in both cell lines, that could represent a cancer cell adaptive mechanism of resistance to galunisertib treatment. These results suggest a potential functional cross talk between TGF-β, AXL and p38 MAPK signals in human CRC cells. In this respect, further experiments are ongoing to better understand this interaction with the aim of identifying potential novel anti-AXL and anti-TGF-β therapeutic strategies.

Citation Format: Davide Ciardiello, Pietro Paolo Vitiello, Nunzia Matrone, Valentina Belli, Claudia Cardone, Luca Poliero, Carola Borrelli, Gianluca Arrichiello, Giulia Martini, Vincenza Ciaramella, Giusi Barra, Floriana Morgillo, Teresa Troiani, Davide Melisi, Fortunato Ciardiello, Erika Martinelli. Inhibition of TGFβ in colorectal cancer cells is associated with a compensatory activation of AXL and p38 MAPK signaling pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2627.
Abstract 2629: Constitutive AhR signaling enhances androgen receptor signaling and growth in prostate cancer cells
Authors: Ghotbaddini Maryam , McLaughlin Sakura , Turner Kailen , Pirtle Devyn , Powell Joann B.
Published: August 2019

The aryl hydrocarbon receptor (AhR) has been reported to interact with multiple signaling pathways during prostate cancer progression to castration resistant prostate cancer (CRPC) including the androgen receptor. Previously published data from our lab shows that AhR protein is overexpressed and constitutively active in castration resistant (DU145, PC3 and PC3M) prostate cancer cells. Pharmacological inhibition of AhR in these advanced prostate cancer cell lines reduced the growth rate under androgen depleted conditions. Furthermore, shRNA mediated depletion of AhR expression in castration resistant C4-2 cells, which were derived from hormone sensitive LNCaP cells, resulted in decreased expression of androgen responsive genes as well as a decreased growth rate which was comparable to LNCaP cell growth. In the present study, we evaluate cloneA (moderate increase in AhR) and CloneB (high increase in AhR) for alterations in AhR and AR signaling. AhR was overexpressed in LNCaP using PLNCX2 retrovirus vector containing AhR cDNA to determine if ectopic overexpression alone induces a castrate resistant phenotype. The highly overexpressed AhR clone (CloneB) illustrated further increase in transcriptional and promotor activity for AhR and AR compared to the moderately overexpressed AhR clone (Clone A) and control. Western blot analysis showed more AhR, AR, cSrc, and pSrc protein expression in clones compared to empty vector control. AhR overexpression was found to induce several biological properties such as migration, invasion, proliferation, and promotion of G1 to S phase during the cell cycle. Bicalutamide treatment had no effect on AR transcriptional activity in either clone, proving resistance to anti-androgen therapy. Our results confirm that overexpression of AhR induces constitutive AhR activity and stimulates androgen receptor signaling. This suggests a role for AhR in the development of CRPC.

Citation Format: Maryam Ghotbaddini, Sakura McLaughlin, Kailen Turner, Devyn Pirtle, Joann B. Powell. Constitutive AhR signaling enhances androgen receptor signaling and growth in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2629.
Abstract 2634: The role of Eph A receptor 3 tyrosine kinase signaling in prostate cancer progression
Authors: Al-Mathkour Marwah M. , Cinar Bekir
Published: August 2019

Dysregulation of the receptor tyrosine kinases (RTKs) by means of mutation, amplification or overexpression plays a crucial role in cell growth, cell survival, cell motility during cancer progression and metastasis. EPHA3 (erythropoietin-producing hepatocellular carcinoma cell surface type A receptor 3) is a member of the RTKs. Evidence indicates that the upregulation of the EPHA3 activity is implicated in the pathobiology of various cancers, including prostate cancer, and thus, it is a prime therapeutic target in cancer. However, the role of EPHA3 signaling in prostate cancer progression remains obscure. Currently, the development of castration-resistant prostate cancer (CRPC) poses a clinical challenge because it is lethal. The molecular mechanisms that contribute to lethal prostate cancer are largely unknown. The objective of this study is to investigate whether EPHA3 signaling plays a critical role in prostate cancer progression and therapeutic relapse. Our analysis of the prostate cancer public datasets revealed that the EPHA3 gene was amplified up to 19% of metastatic CRPC cases with the neuroendocrine phenotype. Our immunological assay confirmed the positive staining of EPHA3 protein in human prostate cancer specimens. Our semi-quantitative and quantitative PCR assays demonstrated that the levels of EPHA3 varies among established prostate cancer cell lines. Nevertheless, we consistently found that the levels of EPHA3 mRNA in CRPC cell line, C4-2, were 3-fold higher than its castration-sensitive parental LNCaP cells. Furthermore, we demonstrated that increase in expression of EPHA3 mRNA in C4-2 compared with LNCaP cells coincided with the upregulation of the EPHA3 protein, as independently confirmed by western blotting and immunofluorescence imaging. These findings indicate that EPHA3 may confer an aggressive prostate cancer cell phenotype. Because androgen receptor (AR) signaling is a potent mediator of CRPC cell growth and survival, the targeting of EPHA3 signaling alone or together with AR may improve the efficacy of current therapies for patients with advanced prostate cancer.

Citation Format: Marwah M. Al-Mathkour, Bekir Cinar. The role of Eph A receptor 3 tyrosine kinase signaling in prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2634.
Abstract 2637: Novel targeted therapy-resistant FGFR3 splice variant increases oncogenic phenotype in African American prostate cancer
Authors: Moy Jacqueline , Wang Bi-Dar , Lee Norman H.
Published: August 2019

Introduction: In this study, we investigate the role of alternative splicing in the receptor tyrosine kinase family on prostate cancer health disparities in African American men.

Background: Prostate cancer (PCa) is the most diagnosed cancer in men and the second leading cause of male-cancer related deaths in the U.S. Dramatic ethnic disparities have been observed in PCa patients, as African American (AA) men are 60% more likely to be diagnosed with PCa and have a 2.4 fold higher mortality rate compared to European American (EA) men. Increasing evidence suggests that, after accounting for epidemiological factors, a remaining component of this disparity is due to intrinsic genetic and biological factors. Interestingly, exon array data from our lab suggest that differential alternative splicing (dAS) may be occurring in AA PCa. We hypothesize that dAS involving exon 14 of FGFR3 is generating a shorter, more oncogenic variant in AA PCa, which is absent or weakly expressed in EA PCa. Differential splicing of FGFR3 in AA patients may be mechanisms contributing to AA PCa health disparities.

Results: Cloning confirmed the presence of the FGFR3-L variant (containing exon 14) and the FGFR3-S variant (without exon 14) from EA and AA PCa cell lines, respectively. RNA-seq data analysis suggests decreased survival of PCa patients with high FGFR3-S/-L expression ratios. EA cell lines overexpressing FGFR3-S show increased small molecule inhibitor (SMI) TKI258-mediated resistance to proliferation, invasion, and apoptosis in vitro. Additionally, mice injected with cells overexpressing FGFR3-S have increased tumor growth, metastasis, and resistance to TKI258 treatment.

Conclusions: We have identified an oncogene of interest, FGFR3, which undergoes exon skipping specific to AA PCa. We have successfully cloned this novel FGFR3 variant in cell lines and created stable cell lines overexpressing both short and long variants. In vitro and in vivo studies suggest FGFR3-S is more resistant to SMIs. Thus, dAS in FGFR3 may be one mechanism contributing to the increased aggressiveness of PCa in AA patients.

Citation Format: Jacqueline Moy, Bi-Dar Wang, Norman H. Lee. Novel targeted therapy-resistant FGFR3 splice variant increases oncogenic phenotype in African American prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2637.
Abstract 2655: Oxygen tension regulates lysosomal activation and receptor tyrosine kinase degradation
Authors: Hong Jaewoo , Lin P. Charles , Wuest Todd , Min Yongfen
Published: August 2019

Oxygen sensing is crucial for adaptation to variable habitats and physiological conditions. Low oxygen tension, or hypoxia, is a common feature of solid tumors and hypoxic tumors are often more aggressive and resistant to therapy. Here we show that, in mammalian tissue culture cells, hypoxia suppressed lysosomal acidification/activation and receptor tyrosine kinase (RTK) degradation. Hypoxia down-regulated mTORc1, reducing its ability to activate transcription factor EB (TFEB), a master regulator of v-ATPase, the lysosomal proton pump. Hypoxia prevented epidermal growth factor receptor (EGFR) degradation in tumor tissues, whereas activation of lysosomes enhanced tumor cell response to anti-EGFR treatment. Our results linkoxygen tension and lysosomal activity, provide a molecular explanation of the malignant phenotype associated with hypoxic tumors, and suggest activation of lysosomes may provide therapeutic benefit in RTK-targeted cancer therapy.

Citation Format: Jaewoo Hong, P. Charles Lin, Todd Wuest, Yongfen Min. Oxygen tension regulates lysosomal activation and receptor tyrosine kinase degradation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2655.
Abstract 2677: MSIClass–Identification and classification of microsatellite unstable using cell-free DNA from ultra-low-pass sequencing
Authors: Maruvka Yosef E. , Frazer Ruslana , Grimsby Jonna , Cibulskis Carrie , Adalsteinsson Viktor , Corcoran Ryan , Getz Gad
Published: August 2019

The acquisition of mismatch repair deficiency is common in many tumor types. A unique subtype of mismatch repair deficient tumors, called tumors with microsatellite instability (MSI), has been recently approved for treatment with PD-1\PD-L1 blockade immunotherapy. Therefore, developing a quick, inexpensive, and easy-to-implement technique for classifying tumors as MSI is of utmost importance. Here, we describe such a method using next-generation sequencing data, and show that it can detect MSI from cell-free DNA (cfDNA), even when there is low fraction of tumor DNA in the cfDNA.

We first analyzed the unique mutational features of MSI vs. microsatellite stable (MSS) tumors. Using our SignatureAnalyzer tool, we analyzed the signatures of the different insertions and deletions within microsatellite loci (MS-indels), and found that MSI tumors harbor elevated MS-indels of certain types, which we further validated using whole genome sequencing data from The Cancer Genome Atlas.

We next used this information to develop a method (MSIClass) that analyzes the WGS data of a given sample by capturing the features unique to MSI tumors. This method assigns each sample a score based on aggregating information from all MS loci simultaneously; since MSIClass is a sample-based tool, it can call the MSI status of tumor samples that have no patient-matched normal sample. We further demonstrate that MSIClass requires minimal genomic information; indeed, sequencing coverage as low as 0.05x (current sequencing cost of ~$2/sample) is sufficient for accurate classification by MSIClass.

Using in silico and in vitro DNA mixing we show that MSIClass can accurately identify MSI cases in very low purity cases, at 0.1% tumor DNA (using 0.5x sequencing coverage). We show that MSIClass, applied on cfDNA, could accurately classify 29 colon cases – 22 MSS and 7 MSI cases, 3 of which are early stage disease and with low tumor content.

These results demonstrate that MSIClass is an inexpensive MSI test that can be applied in various cases: bulk sequencing where a matched normal is not available, cases of very low purity tumors, and even using cfDNA from blood in cases a biopsy is not available or possible. Finally, this methodology can be the basis for monitoring minimal residual disease, and potential be used for early detection of MSI tumors.

Citation Format: Yosef E. Maruvka, Ruslana Frazer, Jonna Grimsby, Carrie Cibulskis, Viktor Adalsteinsson, Ryan Corcoran, Gad Getz. MSIClass–Identification and classification of microsatellite unstable using cell-free DNA from ultra-low-pass sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2677.
Abstract 270: Temozolomide resistant MGMT negative/MMR proficient cancer cells rely on ATR signaling and homologous recombination for DNA repair and survival
Authors: Touny Lara El , Harris Erik , Hose Curtis , Connelly John , Vengsarkar Diana , Monks Anne , Parchment Ralph E. , Doroshow James H. , Teicher Beverly A. , Rapisarda Annamaria
Published: August 2019

MGMT loss is used to predict response to temozolomide (TMZ), however, some patients do not benefit, suggesting that additional criteria for selection remain to be identified. Using patient derived and well-established cancer lines cancer lines we observed a 100% association between mismatched repair (MMR) defects and TMZ resistance; however, only 16% of MGMT-/MMR+ lines were sensitive to TMZ at clinically relevant concentrations. Activation of DNA damage and repair signaling was noted in both sensitive and resistant bladder cancer cell lines in response to TMZ but protracted DNA damage, accompanied by persistent G2/M arrest, was observed only in the sensitive models. ATR inhibition sensitized MGMT-/MMR proficient resistant models to TMZ resulting in measurable DNA damage, prolonged G2/M arrest and persistent growth inhibition: an effect that was mediated by Chk1. Homologous recombination and repair has been shown to play an important role in repairing TMZ-induced DNA damage. We demonstrate that ATR inhibition abrogated homologous recombination (HR) activity in a MGMT-/MMR proficient TMZ resistant bladder model and that a TMZ sensitive bladder model had minimal HR (assessed by pDR-GFP/SceI assay). Moreover, HR -deficiency (mediated by BRCA1 defects or RAD51 inhibition) conferred sensitivity to TMZ in combination with an ATR inhibitor. Our in vitro data suggest that a MGMT-/MMR+/HR-Deficiency profile identifies models that respond to TMZ/ATR inhibitor combination. Indeed, one Patient-derived Xenograft (PDX) model (available in the Patient Derived Models Repository), selected by this criterion (MGMT-/MMR+ and LOH>40%) showed delayed tumor growth and increased survival in the TMZ-VX970 combination arm, compared to single agents. Overall, our data suggest that MGMT-/MMR+ cells rely on ATR-dependent signaling for repair of TMZ-induced DSBs and that HR defects should be evaluated as criteria for selecting patients that will benefit from an ATR inhibitor/TMZ combination regimen. Funded by NCI Contract No. HHSN261200800001E. This research was supported, in part, by the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis of the National Cancer Institute.

Citation Format: Lara El Touny, Erik Harris, Curtis Hose, John Connelly, Diana Vengsarkar, Anne Monks, Ralph E. Parchment, James H. Doroshow, Beverly A. Teicher, Annamaria Rapisarda. Temozolomide resistant MGMT negative/MMR proficient cancer cells rely on ATR signaling and homologous recombination for DNA repair and survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 270.
Abstract 2712: Effect of combined PD-1 and IL-6 blockade on K-ras mutant lung cancer
Authors: Ramos-Castaneda Marco A. , Daga Neha , Peng Stephen , Deng Shanshan , Velazco Walter , Caetano Mauricio Da Silva , Moghaddam Seyed
Published: August 2019

Lung cancer is the leading cause of cancer death and the second most common cancer diagnosed worldwide in both males and females. K-ras gene mutations are among the most common mutations encountered in non-small cell lung cancer (NSCLC), encompassing up to 30% of them, and are unfortunately associated with chemoresistance and poor prognosis. Recently, immunotherapy treatment of several cancers including NSCLC, particularly by targeting the co-inhibitory molecules program cell death protein 1 (PD-1) and program death-ligand 1 (PD-L1), have shown promising results given either in combination with standard chemotherapy or as neoadjuvant therapy. We have previously shown an important role for IL-6 in K-ras mutant lung tumorigenesis by reprogramming myeloid contexture in the lung tumor microenvironment (TME) and promoting tumor cell proliferation and angiogenesis through the activation of STAT3 pathway. Accordingly, we hypothesized that targeting both immunosuppressive TME and IL-6 driven pro-tumor immune response, through the use of immune checkpoint blockade (ICB), and inhibition of IL-6 would give us a synergistic/additive response and increases the tumor inhibitory effect of ICB. Briefly, starting at the age of 6 weeks, 4 cohorts of K-ras mutant mice (LSL-K-rasG12D/ CCSPCre) (CC-LR) were injected intraperitoneally with vehicle alone (PBS1x), anti-PD-1 (CD279) antibody alone (Clone: 29F.1A12- Bioxcell - BE0273-CUST) (200µg), anti-IL-6 antibody alone (Clone: MP5-20F3 - Bioxcell - BE0046-CUST) (20 mg/kg), or a combination of both. After 8 weeks of treatment, tumor burden and inflammation were studied and quantified. We found a significant (2-fold) decrease in tumor burden in the anti-PD1 alone treated group which was further reduced in the combined anti-PD-1 and anti-IL-6 treated group compared to age and sex matched vehicle treated group. There was also a significant difference in the lung macrophage infiltration in combined anti-PD1/anti-IL-6 treated group similar to what we previously observed in anti-IL-6 alone treated group. Our results suggest that immunomodulatory approaches by targeting cytokine networks like IL-6 blockade could be used to increase the efficacy of immunotherapy, i.e. PD-1 blockade, as a preventive and/or therapeutic strategy for K-ras mutant lung cancer.

Citation Format: Marco A. Ramos-Castaneda, Neha Daga, Stephen Peng, Shanshan Deng, Walter Velazco, Mauricio Da Silva Caetano, Seyed Moghaddam. Effect of combined PD-1 and IL-6 blockade on K-ras mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2712.
Abstract 2713: Discovery and characterization of AZD8701, a high affinity antisense oligonucleotide targeting FOXP3 to relieve immunosuppression in cancer
Authors: Sinclair Charles , Revenko Alexey S. , Johnson R B. , Peter Alison , Taylor Molly A. , Hettrick Lisa A. , Klein Stephanie , Solanki Anisha , Chapman Melissa , Yates James , Angell Helen K. , Watt Andrew , Gattis Danielle , Monia Brett P. , Barry Simon T. , Lyne Paul , Edbrooke Mark , Goldberg Frederick , Macleod A R.
Published: August 2019

Regulatory T cells (Treg) critically maintain immuno-suppression in the tumor microenvironment, representing an attractive immuno-oncology target. The Treg lineage is defined by expression of the FOXP3 transcription factor, which controls immune-suppressive functions. We have developed the clinical candidate AZD8701, a next-generation antisense oligonucleotide inhibitor of FOXP3 (utilizing the Ionis Gen 2.5 cEt-modified ASO platform). AZD8701 treatment knocked down FOXP3 in primary human Tregs via free uptake (IC50 65nM), which was also associated with modulation of known FOXP3 target genes including 25-50% reduction in CTLA4, ICOS, CCR8 and GITR. Tregs treated with AZD8701 further exhibited reduced suppressive functions in in vitro suppression assays, which confirmed the functional effects of FOXP3 modulation. Finally, AZD8701 promoted dose-dependent knockdown of FOXP3 in humanized mice, including >50% FOXP3 knockdown at doses that can be feasibly achieved with the Gen 2.5 ASO platform in the clinic. To support the importance of FOXP3 in immuno-oncological settings, we characterized murine surrogate FOXP3 ASOs in the context of syngeneic tumour bearing mice. Murine FOXP3 ASOs similarly promoted >50% FOXP3 knockdown in mice and were well tolerated with no overt toxicological findings at high doses, over a maximum of 12 weeks of treatment. Murine FOXP3 ASOs significantly attenuated tumour growth in A20 and ID8-VEGF syngeneic models, which was associated with some complete tumour regressions. Moreover, we found that mouse surrogate FOXP3 ASOs promoted additive/enhanced therapeutic effects when combined with immune checkpoint blockade. Collectively, FOXP3 ASOs represent a first-in-class strategy to target Tregs in cancer in a highly selective manner. The clinical application of AZD8701 may provide therapeutic benefit to patients either as a monotherapy or in combination with immune checkpoint blocking agents.

Citation Format: Charles Sinclair, Alexey S. Revenko, R B. Johnson, Alison Peter, Molly A. Taylor, Lisa A. Hettrick, Stephanie Klein, Anisha Solanki, Melissa Chapman, James Yates, Helen K. Angell, Andrew Watt, Danielle Gattis, Brett P. Monia, Simon T. Barry, Paul Lyne, Mark Edbrooke, Frederick Goldberg, A R. Macleod. Discovery and characterization of AZD8701, a high affinity antisense oligonucleotide targeting FOXP3 to relieve immunosuppression in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2713.
Abstract 2719: Metabolic product of inositol-trisphosphate 3-kinase B confers chemotherapy resistance by managing redox homeostasis
Authors: Pan Chaoyun , Chun Jaemoo , Boese Austin , Kang Jihoon , Li Jie , Jin Lingtao , Kang Sumin
Published: August 2019

How altered metabolism contributes to chemotherapy resistance in cancer cells remains unclear. Through a metabolism-related kinome RNAi screen, we identified inositol-trisphosphate 3-kinase B (ITPKB) as a critical enzyme that contributes to cisplatin-resistant tumor growth. We demonstrate that inositol (1,3,4,5) tetrakisphosphate (IP4), the product of ITPKB, plays a critical role in redox homeostasis upon cisplatin exposure by reducing cisplatin-induced ROS through inhibition of a ROS-generating enzyme NADPH oxidase 4 (NOX4), which promotes cisplatin- resistant tumor growth. Mechanistically, IP4 competes with the NOX4 cofactor NADPH for binding and consequently inhibits NOX4. Targeting ITPKB with shRNA or its small molecule inhibitor resulted in attenuation of NOX4 activity, imbalanced redox status, and sensitized cancer cells to cisplatin treatment in patient-derived xenografts. Our findings provide insight into the crosstalk between kinase-mediated metabolic regulation and platinum-based chemotherapy resistance in human cancers. Our study also suggests a distinctive signaling function of IP4 that regulates NOX4. Furthermore, pharmaceutical inhibition of ITPKB displayed synergistic attenuation of tumor growth with cisplatin suggesting ITPKB as a promising synthetic lethal target for cancer therapeutic intervention to overcome cisplatin resistance.

Citation Format: Chaoyun Pan, Jaemoo Chun, Austin Boese, Jihoon Kang, Jie Li, Lingtao Jin, Sumin Kang. Metabolic product of inositol-trisphosphate 3-kinase B confers chemotherapy resistance by managing redox homeostasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2719.
Abstract 2739: EZH2 promotes breast cancer bone metastasis
Authors: Qu Jingkun , Zhang Lin , Zhou Zhifen , Yu Dihua
Published: August 2019

Metastasis causes about 90% of cancer deaths and approximately 55% of breast cancer patients develop bone metastases. Although significant advances in the cure of breast cancer, effective therapies targeted bone metastases are still not available. To develop novel therapies for patients with bone metastasis, better understanding of underlying mechanisms of bone metastasis is critical. EZH2 is a classic histone methyl transferase that generally suppresses downstream target genes through tri-methylation of H3K27. EZH2 is considered as a breast cancer oncogene and is frequently over expressed in breast cancer, but the effect of EZH2 on bone metastasis is unknown. To explore the function of EZH2 in bone metastasis, we intracardially injected into nude mice control MDA.MB.231 breast cancer cells expressing readily detectable EZH2 or a MDA.MB.231 subline having EZH2 knocked out (231.EZH2.ko). We found that the mice injected with 231.EZH2.ko had prolonged bone metastasis-free survival compared to the control group of mice. To gain initial insight on the underlying mechanism of EZH2-mediated bone metastasis, we co-cultured MDA.MB.231 cells or 231.EZH2.ko with pre-osteoclasts (RAW264.7) and osteoblasts (MC3T3) under TGF-β treatment (triple co-culture) to mimic bone metastasis tumor microenvironment. We examined osteoclast maturation and breast cancer cell proliferation because osteolysis induced by mature osteoclasts is critical for breast cancer cell growth in the bone metastasis. In triple co-culture of MDA.MB.231 cells, more RAW264.7 pre-osteoclasts were differentiated to mature osteoclasts, as measured by TRAP+ staining, a marker of mature osteoclasts, and MDA.MB.231 cells grew faster than the triple co-culture of 231.EZH2.ko cells. Furthermore, we found that the mRNA level of PTHrP, a master regulator of bone metastasis and a TGF-β downstream cytokine, was expressed at significantly higher level in MDA.MB.231 cells than that in 231.EZH2.ko cells under TGF-β stimulation. PTHrP was known to promote the “vicious cycle” of bone destruction in bone metastasis. The data suggests EZH2 promotes breast cancer bone metastasis through PTHrP. Currently, we are further exploring how EZH2 regulates PTHrP in breast cancer cells to promote bone metastasis. Our studies may reveal novel biomarkers and/or therapeutic targets for bone metastasis.

Citation Format: Jingkun Qu, Lin Zhang, Zhifen Zhou, Dihua Yu. EZH2 promotes breast cancer bone metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2739.
Abstract 2744: Blocking CXCR4 alleviates desmoplasia, increases T-lymphocyte infiltration, and improves immunotherapy in metastatic breast cancer
Authors: Chen Ivy X. , Chauhan Vikash P. , Posada Jessica , Ng Mei R. , Wu Michelle , Adstamongkonkul Pichet , Huang Peigen , Lindeman Neal , Langer Robert , Jain Rakesh K.
Published: August 2019

Metastatic breast cancers (mBCs) are largely resistant to immune checkpoint blockade, but the mechanisms remain unclear. Primary breast cancers are characterized by a dense fibrotic stroma, which is considered immunosuppressive in multiple malignancies, but the stromal composition of breast cancer metastases and its role in immunosuppression are largely unknown. Here we show that liver and lung metastases of human breast cancers tend to be highly fibrotic, and unlike primary breast tumors, they exclude cytotoxic T-lymphocytes (CTLs). Unbiased analysis of the TCGA database of human breast tumors revealed a set of genes that are associated with stromal T-lymphocyte exclusion. Among these we focused on CXCL12 as a relevant target based on its known roles in immunosuppression in other cancer types. We found that the CXCL12 receptor CXCR4 is highly expressed in both human primary tumors and metastases. To gain insight into the role of CXCL12/CXCR4 axis, we inhibited CXCR4 and found that CXCR4 blockade decreases fibrosis, alleviates solid stress, decompresses blood vessels, increases CTL infiltration, and decreases immunosuppression in murine mBC models. We confirmed that these immunosuppressive effects are dependent on cancer-associated fibroblast (CAF) signaling through genetic deletion of Cxcr4 in aSMA+ cells. Accordingly, CXCR4 inhibition more than doubles the response to immune checkpoint blockers in mice bearing mBCs. These findings demonstrate that CXCL12/CXCR4-mediated desmoplasia in mBC promotes immunosuppression and is a potential target for overcoming therapeutic resistance to immune checkpoint blockade in mBC patients.

Citation Format: Ivy X. Chen, Vikash P. Chauhan, Jessica Posada, Mei R. Ng, Michelle Wu, Pichet Adstamongkonkul, Peigen Huang, Neal Lindeman, Robert Langer, Rakesh K. Jain. Blocking CXCR4 alleviates desmoplasia, increases T-lymphocyte infiltration, and improves immunotherapy in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2744.