bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1751: BPI-23314: A novel orally bioavailable small molecular BET inhibitor with target protein degradation activity
Authors: Guo Jing , Xu Yan , Yan Dan , Yu Bo , Yu Weiwei , Chen Haibo , Xu Xiaofeng , Liu Xiangyong , Lan Hong , Ding Lieming , Wang Jiabing
Published: September 2020

The Bromo- and Extra-Terminal (BET) proteins, BRD2, BRD3, and especially BRD4, play important roles in transcriptional regulation of many oncogenes like MYC or BCL2, thus makes it an attractive drug target in multiple pathological settings, particularly in cancer. Earlier BET inhibitors, while effectively bind and inhibit the activity of BRD4, adversely induce compensatory accumulation of the target protein, that may account for their limited preclinical and clinical efficacy. Herein, we report BPI-23314, a potent, selective, and orally available small molecular BET bromodomain inhibitor, which possesses potent target inhibition activity as well as target protein degradation property. In vitro, BPI-23314 induced dose-dependent growth inhibition in a wide range of cancer cell lines, including those from AML, MM, DLBCL, NSCLC, TNBC, and CRPC, etc. Quantitative RT-PCR revealed that BPI-23314 markedly affected the transcription of genes encoding MYC, BCL2, and HEXIM1, thus induced G1 cell cycle arrest and apoptosis in MV-4-11 AML cell line. In vivo, oral administration of BPI-23314 led to tumor growth suppression or regression in multiple xenograft models of AML and MM. Transcriptional modulation of MYC, BCL2 and HEXIM1 were confirmed in BPI-23314-treated xenografts and in normal monkey, supporting their potential as pharmacodynamic biomarkers in clinical trials. Unlike previously described BET inhibitors that induced BRD4 accumulation, BPI-23314 serendipitously decreased BRD4 protein in MV-4-11 cells and in tumors from MV-4-11 xenografts. The feature of target-protein degradation differentiates BPI-23314 from the other BET inhibitors, renders it good in vivo efficacy, and suggests a lower risk of drug resistance in future clinical development. In conclusion, our study establishes BPI-23314 as a novel small molecular BET inhibitor with target BRD4 protein degradation property. BPI-23314 has obtained IND approval from Chinese regulatory authority and is planned to enter Phase 1 clinical development in hematologic malignancies in early 2020.

Citation Format: Jing Guo, Yan Xu, Dan Yan, Bo Yu, Weiwei Yu, Haibo Chen, Xiaofeng Xu, Xiangyong Liu, Hong Lan, Lieming Ding, Jiabing Wang. BPI-23314: A novel orally bioavailable small molecular BET inhibitor with target protein degradation activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1751.
Abstract 1755: Prodigiosin inhibits the proliferation and migration of mcf-7 breast cancer cells through the demethylation of mir-200c gene
Authors: Shi Fengqin , Lu Lingeng , LV Peng , Li Ya , Li Hou , Chen Xinyi , Zhu Yong
Published: September 2020

Background: DNA methylation in microRNA (miRNA) host genes is associated with the down-regulation of gene expression in tumorigenesis and progression. Accumulating evidence shows that low levels of miR-200c were correlated with a worse prognosis in breast cancer. MiRNA promoter methylation has been reported as a major cause of miR-200c downregulation. Prodigiosin (PG), red bacterial pigment with a pyrrolylpyrromethene skeleton, has significant antibacterial, antifungal, antimalarial and antitumor activity via the modulation of multiple cell signaling pathways against a variety of human cancer cells. However, there is little information regarding the epigenetic-based effects of Prodigiosin in breast cancer. In this study, we hypothesize that Prodigiosin has anti-proliferative and anti-migration effects on breast cancer cells through the demethylation of miR-200c.

Material and Methods: MCF-10A breast epithelial cells and MCF-7 breast cancer cells were used to investigate the effects of Prodigiosin on the expression and methylation of miR-200c using real time-PCR (RT-qPCR) and methylation-specific PCR (qMSP), respectively. MTS proliferation assay, cell scratch assay and three-dimensional (3D) formation assay were performed to assess the effect of Prodigiosin on the proliferation and migration of MCF-7 breast cancer cells.

Result: MCF-7 breast cancer cells showed significant hypermethylation of miR-200c, and significantly down-regulated miR-200c expression compared to MCF-10A breast epithelial cells. Prodigiosin treatment led to significant inhibition of proliferation and migration, and significantly up-regulated miR-200c expression, whereas the methylation level of miR-200c significantly decreased in MCF-7 breast cancer cells.

Conclusion: Prodigiosin, a secondary metabolite of bacteria, inhibits the proliferation and migration of breast cancer cells, and restores the demethylation status and expression of miR-200c. These findings elucidate that the demethylation of miR-200c may be a molecular mechanism underlying the anti-tumor of Prodigiosin in breast cancer.

Citation Format: Fengqin Shi, Lingeng Lu, Peng LV, Ya Li, Hou Li, Xinyi Chen, Yong Zhu. Prodigiosin inhibits the proliferation and migration of mcf-7 breast cancer cells through the demethylation of mir-200c gene [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1755.
Abstract 1757: Developing sensitive assays to facilitate hit identification of RNA modifying enzymes
Authors: Tomlinson Chris , Lawrence Rachel , Best Stuart , Boden Grace , Alanine Thomas , Wasp James , Smith Bethany , Wilkie Ross , Thomson Stuart , Unitt John , Pollack Scott , Jordan Allan M.
Published: September 2020

N-6-methyladenosine (m6A) is the most abundant RNA chemical modification found in the eukaryotic transcriptome. It is a dynamic and reversible modification which plays a role in RNA function across a wide range of biological processes. Dysregulation of the m6A modification and the readers, writers and erasers involved in these processes have been implicated in the progression of multiple types of cancer and metabolic disease. RNA modifying enzymes are therefore emerging as exciting therapeutic targets.

To facilitate and accelerate drug discovery efforts in this area, we have developed several sensitive and robust assays suitable for identifying and assessing chemical matter against the RNA modifying enzymes, fat mass and obesity associated protein (FTO) and methyltransferase-like 3/14 (METTL3/14). Specifically, a robust Microscale Thermophoresis (MST) assay was developed, validated, and used to screen 100 compounds from the LOPAC for novel human FTO small molecule binders. Five compounds, which displayed reproducible, concentration-dependent binding in the MST screen, were tested for inhibition of human FTO RNA demethylation activity and revealed a single, novel FTO inhibitor. To further profile emerging FTO hit matter, we are also developing sensitive and robust mass spectrometry assays to demonstrate target inhibition and proof of mechanism. Our progress in this area will be reported. Additionally, we have developed a highly sensitive and robust LC-MS/MS assay to monitor the METTL3/14 catalysed conversion of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH). This assay is suitable for identifying either substrate competitive or allosteric METTL3/14 inhibitors.

In summary, our proof of concept studies describe the development of several robust and sensitive assays suitable for hit identification and further characterisation of inhibitors of key RNA modifying enzymes and offer a platform for the discovery and development of novel therapeutics in this emerging area of research.

Citation Format: Chris Tomlinson, Rachel Lawrence, Stuart Best, Grace Boden, Thomas Alanine, James Wasp, Bethany Smith, Ross Wilkie, Stuart Thomson, John Unitt, Scott Pollack, Allan M. Jordan. Developing sensitive assays to facilitate hit identification of RNA modifying enzymes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1757.
Abstract 1759: KDM5 inhibitor regulates growth of early stage and advanced human prostate cancer cells
Authors: Smith Tunde , Chen Zhenbang , Stewart LaMonica V.
Published: September 2020

Prostate cancer (PCa) remains a leading cause of cancer deaths because many patients ultimately develop an incurable castration-resistant form of metastatic PCa. While chromatin alterations are linked to PCa, the role of histone demethylases in the PCa progression has not been fully elucidated. Members of the Jumonji AT-rich interactive domain 1 (JARID1) or lysine demethylase 5 (KDM5) family of lysine demethylases are known for their ability to demethylate H3K4me3. However, the genes encoding these proteins are amplified in metastatic PCa. Literature suggests members of the KDM5 family enhance expression and function of androgen receptor (AR), a protein that drives growth of human PCa cells. The KDM5 family inhibitor 2-4(4-methylphenyl)-1,2-benzisothiazol-3(2H)-one (PBIT) has been suggested as a potential lead compound for cancer therapy since it suppresses proliferation of human breast cancer cells. However, to date no studies have tested the effects of PBIT in PCa. The goal of this study was to characterize the effects of PBIT alone and in combination with a PPARγ agonist, 15-Deoxy-Δ-12,14-prostaglandin J2 (15-dPGJ2) on PCa cells. Presto blue analyses were conducted in AR positive, castration-sensitive LNCaP cells and the castration-resistant PC3 cells expressing little to no AR. Our studies revealed that PBIT treatment decreased the proliferation of both LNCaP and PC3 cells in a dose-dependent manner. Significant decreases in cell proliferation were detected when PBIT concentrations were greater than or equal to 5 μM. In addition, 15-dPGJ2 was able to decrease the proliferation of PCa cells. Importantly, the combination of PBIT and 15-dPGJ2 significantly suppressed the growth of PC3 and LNCaP cells. The anti-proliferative effect of the combined inhibition was at least additive, as the combination index was less than 1. Furthermore, PBIT at concentrations ≥ 5 μM also reduced migration of PC3 cells. Together, our data suggest that PBIT and other KDM5 inhibitors can reduce the proliferation and migration of multiple types of PCa cells. Our findings also provide evidence that the combination of KDM5 family inhibitors and PPARγ agonists might serve as an effective therapy for advanced human PCa.

Citation Format: Tunde Smith, Zhenbang Chen, LaMonica V. Stewart. KDM5 inhibitor regulates growth of early stage and advanced human prostate cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1759.
Abstract 1761: Examination of therapeutic potential of epigenetic modulation by dual HDAC-LSD1 inhibition in murine models of triple negative breast cancer (TNBC)
Authors: Messerli Shanta M. , Hoffman Mariah M. , Zylla Jessica S. , Gnimpieba Etienne , Miskimins Keith W.
Published: September 2020

This study examines the effects of the epigenetic modulator 4SC-202, a dual histone deacetylase (HDAC) /lysine-specific histone demethylase 1A (LSD1) small molecule inhibitor, on tumor growth and metastasis in a mouse model of breast cancer. 4SC-202 differs from clinically available HDAC inhibitors such as Vorinostat (SAHA) by specifically targeting class I HDACs including HDAC1, HDAC2, and HDAC3 and the histone demethylase LSD1. The hypothesis to be examined is that epigenetic modulation via dual inhibition of HDAC1 and LSD1 improves disease control in breast cancer. In vitro data indicates that 4SC-202 is significantly cytotoxic and cytostatic to the highly metastatic murine Triple Negative Breast Cancer (TNBC) murine model 4T1 and the human TNBC cell line MDA-MB-231 and reduces cancer cell migration. In vivo studies conducted in the syngeneic 4T1 model which closely mimics human TNBC in terms of sites of metastasis, show increased necrosis in 4T1 tumors, reduced tumor burden in vivo as compared to Vorinostat treatment, and significantly reduced metastasis. Furthermore, multiple studies in different cell types indicate that the mechanism of action of 4SC-202 may involve effects on cancer stem cells, which are able to self-renew and form metastatic lesions. It has been shown that a marker of cancer stem cells (CSC's) in breast cancer is high aldehyde dehydrogenase (ALDH) activity and that ALDH high cells mediate metastasis. In the 4T1 syngeneic mouse model of breast cancer, inhibition of ALDH activity suppresses stem-like cell properties. Preliminary data suggests 4SC-202 substantially reduces the ALDH high 4T1 cancer stem population. Transcriptome bulk RNA -sequencing analysis from 4T1 tumors reveal changes in metastatic related pathways in 4SC-202 treated tumors, including significant changes to the expression levels of genes implicated in focal adhesion and ECM-receptor interaction pathways. Furthermore, 4SC-202 appears to alter expression of cytokines which serve as chemokines, potentially leading to increased expression of tumor infiltrating lymphocytes. In summary, treatment of a highly metastatic murine model of TNBC with 4SC-202 significantly reduces metastasis, and warrants further preclinical studies in conjunction with standard of care chemotherapy treatment.

Citation Format: Shanta M. Messerli, Mariah M. Hoffman, Jessica S. Zylla, Etienne Gnimpieba, Keith W. Miskimins. Examination of therapeutic potential of epigenetic modulation by dual HDAC-LSD1 inhibition in murine models of triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1761.
Abstract 1773: Targeting HER2 and Src in HER2-driven ovarian and esophagogastric cancers
Authors: Conlon Neil T. , Kroh Keshia , Crown John , Collins Denis M.
Published: September 2020

Background: Neratinib, an oral, irreversible pan-HER tyrosine kinase inhibitor, has exhibited clinical activity against HER2-mutant and HER2 over-expressing cancers. Our previous pre-clinical studies have shown that combining the multi-kinase inhibitor dasatinib with neratinib is highly synergistic in HER2-positive breast cancer. Therefore, the aim of this study was to examine the potential of combining neratinib and dasatinib in HER2 over-expressing/mutant esophagogastric (EG) and ovarian cancer.

Methods: The prevalence of HER2 mutation and amplification levels in ovarian and EG cancers was determined using the AACR Project GENIE database. The anti-proliferative effects of neratinib and dasatinib were assessed by acid phosphatase assay in a panel of HER2-altered ovarian (Caov3, OVCAR8, IGROV-1) and EG (NCI-N87, OE19) cancers. Synergy was assessed using Combenefit software with the Loewe additivity model, with additivity and synergy > 0 and antagonism < 0. Western blotting was used to examine the basal expression of HER2 and downstream signalling activity and to investigate the signalling effects of drug treatment in HER2-mutant OVCAR8 cells.

Results: HER2 was the most commonly amplified gene in EG cancers (14.1%) and was also amplified in 2.4% of ovarian cancers. HER2 mutations occurred in 6.1% of EG and 1.5% ovarian cancers. All five cell lines tested displayed nanomolar sensitivity to single agent neratinib or dasatinib (Table). The combination of neratinib and dasatinib was synergistic in all cell lines. The greatest synergy was observed with low concentrations of neratinib in EG cell lines. Synergistic interactions occurred across a range of concentrations with both drugs in ovarian cancer cell lines. Antagonism was not observed at any concentration tested. Neratinib plus dasatinib inhibited Src and MAPK activity in OVCAR8 cells.

Neratinib and dasatinib in ovarian and EG cancersCell linesCancertypeNeratinib EC50 value (nM)Dasatinib EC50 value (nM)Combination effectNCI-N87Esophagogastric0.188979SynergismOE19Esophagogastric0.432284SynergismOVCAR8Ovarian16281SynergismIGROV-1Ovarian1706SynergismCaov3Ovarian13730Synergism

Conclusions: These data suggest that the addition of dasatinib may enhance the anti-cancer effect of neratinib in HER2-driven ovarian and EG cancers and warrants further investigation.

Citation Format: Neil T. Conlon, Keshia Kroh, John Crown, Denis M. Collins. Targeting HER2 and Src in HER2-driven ovarian and esophagogastric cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1773.
Abstract 1793: Interaction of MYC and SUMOylation machinery in an aggressive pancreatic cancer subtype
Authors: Hassan Zonera , Biederstädt Alex , Schneeweis Christian , Schick Markus , Jonas Nilsson , Wirth Mathias , Coluccio Andrea , Orben Felix , Steiger Katja , Slawska Jolanta , Langston Steve , Saur Dieter , Rad Roland , Reichert Maximilian , Schneider Guenter , Keller Ulrich
Published: September 2020

Despite recent improvements in treatment regimen such as combination chemotherapies, pancreatic ductal adenocarcinoma still carries a dismal prognosis with an overall survival rate of 9%. The oncogenic transcription factor MYC is amplified in about 12% of all PDAC, which is connected to a worse survival. It is clear that hyperactivation of MYC generates dependencies that can be exploited therapeutically. Therefore, the aim of the study was to find and to target MYC-associated dependencies.

Human PDAC gene expression data sets were analyzed. Moreover, analysis of the SUMO pathway in large cohort of PDAC was performed by IHC. Furthermore, a novel, pharmacological SUMO inhibitor was used and characterized by using human and murine 2D-, organoid-, and in vivo-models of PDAC. In addition, this connection was also verified by the genetically modified human and murine models.

Large-scale gene expression datasets of human PDACs analysis revealed a connection of MYC to the SUMOylation machinery in PDAC. Moreover, results were also corroborated by the hyperactivation of SUMO pathway in a large patient cohort which characterized a PDAC subtype with a dismal prognosis. We furthermore corroborated these results by using the novel SUMO inhibitor ML093 by in vitro studies that hyperactivation of MYC is connected to an increased sensitivity to pharmacological SUMO inhibition. In conclusion, SUMO inhibitor-based therapies should be further developed for an aggressive PDAC subtype.

Citation Format: Zonera Hassan, Alex Biederstädt, Christian Schneeweis, Markus Schick, Nilsson Jonas, Mathias Wirth, Andrea Coluccio, Felix Orben, Katja Steiger, Jolanta Slawska, Steve Langston, Dieter Saur, Roland Rad, Maximilian Reichert, Guenter Schneider, Ulrich Keller. Interaction of MYC and SUMOylation machinery in an aggressive pancreatic cancer subtype [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1793.
Abstract 181: Pan-cancer clinical genomic profiling in circulating tumor DNA from 7345 Chinese advanced cancer patients
Authors: Li Wenhua , Chen Hui , Zhou Yifan , Sun Danpeng , Zhang Bei , Bai Yuezong , Ding Qingqing
Published: September 2020

Purpose: Circulating tumor DNA (ctDNA) sequencing was implemented widely in advanced cancer patients for guiding matched therapy, which could avoid invasive specimen collection and tumor heterogeneous. However, clinical ctDNA genomic profiling of Chinese advanced pan-cancer patients is still unknown.

Methods: 7624 blood samples were obtained from 7345 Chinese patients with advanced solid tumor, including 3506 lung cancer, 860 liver cancer, 674 colorectum cancer, 343 stomach cancer, 329 biliary tract cancer, 324 breast cancer, and 1588 other 17 tumor types. Genomic alterations (GAs), including SNV/Indels, CNVs, and rearrangements were identified via ctDNA sequencing with a 150 cancer-related gene panel. Potentially actionable GAs was annotated according to OncoKB database.

Results: There are 85.2% (6497/7624) samples harbors maximum somatic allele frequency (MSAF) >0.3% and average 5.5 GAs per samples. 21253 known or likely deleterious alterations were identified in 137 genes, and average 3.3 known or likely deleterious GAs per samples. Additionally, 38.4% (2930/7624) samples had at least one potentially actionable GA (PAGA). The most frequently and potentially actionable altered genes were EGFR (13.2%), KRAS (8.6%), PIK3CA (5.1%), ERBB2 (4.0%), FGFR1 (3.6%) and BRAF (3.3%). Importantly, PAGAs in RTK-RAS-MAPK pathway was the most common with 33.7% samples (19.2% SNV/Indels only, 9.8% CNVs only, 1.4% rearrangements only, 3.4% either two or more types of alterations), followed by 5.9% PI3K/AKT/mTOR pathway, 2.1% TP53/cell cycle pathway, 0.8% Epigenetic pathway, 0.1% Hedgehog pathway, and 0.1% DDR pathway. The samples of at least one PAGA in aforementioned pathways were 53.1% in breast cancer, 49.6% in colorectum cancer, 44.0% in bladder/urinary tract cancer, 43.5% in lung cancer, 39.4% in stomach cancer, respectively. In all, 287 cases with PAGAs in RTK-RAS-MAPK pathway (ROS1, ALK, MET) could benefit from crizotinib; 49 cases with PAGAs in RTK-RAS-MAPK pathway (BRAF) could benefit from vemurafenib; 447 cases with PAGAs in PI3K/AKT/mTOR pathway (PIK3CA, PTEN, AKT1, mTOR) could benefit from everolimus; and 6 cases with PAGAs in DDR pathway (ATM) could benefit from olaparib.

Conclusions: The study revealed a comprehensive ctDNA genomic profiling of Chinese patients with advanced solid tumors. And further larger cohorts validation for ctDNA sequencing as a diagnostic tool in target-therapy is needed.

Citation Format: Wenhua Li, Hui Chen, Yifan Zhou, Danpeng Sun, Bei Zhang, Yuezong Bai, Qingqing Ding. Pan-cancer clinical genomic profiling in circulating tumor DNA from 7345 Chinese advanced cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 181.
Abstract 1816: β-catenin antagonist peptide attenuates oncogenic gene transactivation and promotes antitumor activity in breast cancer models
Authors: Rotolo Jim A. , Ghamsari Lila , Gallagher Erin , Ramirez Rick , Koester Mark , Leong Siok , Merutka Gene , Kappel Barry J.
Published: September 2020

Aberrant activation of the canonical Wnt signaling pathway occurs in a variety of cancers, resulting in overactivation of β-catenin-mediated transcriptional activity and increased expression of genes that promote tumor survival, proliferation and inhibit differentiation. Disruption of the β-catenin transcription complex, by inhibiting protein-protein interactions required for its formation, represents a powerful approach to inhibit this previously ‘undruggable' target. The goal of these experiments is to characterize the anti-tumor activity of β-catenin antagonist peptides, BCA1 and BCA2, designed to disrupt the β-catenin transcription complex, in breast cancer models. In in vitro experiments, MCF7 (HER2neg/HRpos) breast adenocarcinoma cells were exposed to β-catenin antagonist peptides, and the impact on β-catenin-mediated gene transactivation was determined by quantitative polymerase chain reaction (qPCR). Results indicate that β-catenin antagonist peptide attenuated oncogenic gene transactivation in MCF7 cells, significantly decreasing the expression of direct β-catenin target genes including Myc, Cyclin D1 and Cyclin-dependent kinase 4 (p<0.05). The impact of β-catenin antagonist peptides on cancer cell viability was quantified by annexin V/PI flow cytometry analysis, indicating a dose-dependent decrease in overall cell viability 48 hours post peptide exposure. Additionally, BCA1 was administered to primary breast cancer tumoroids (HER2pos/HRneg or HER2neg/ HRneg), and the impact on tumoroid viability was quantified by histology and immunofluorescence microscopy. β-catenin antagonist peptide BCA1 resulted in dose-dependent decrease in surviving HER2pos/HRneg and HER2neg/ HRneg breast cancer tumoroids, with EC50 values of 5.2 and 6.9 µM, respectively. Finally, the impact of BCA2, an analogue of BCA1 with an additional N-terminal modification, on subcutaneous MCF7 xenograft tumors was determined in vivo. Administration of 5 mg/kg BCA2 via subcutaneous injection at a dosing frequency of 3x/week for 3 weeks resulted in 18-day tumor regression from the start of treatment and a 63.1% tumor growth inhibition compared to vehicle-treated controls (p<0.05). Taken together, these data demonstrate the significant anti-breast cancer activity of β-catenin antagonist peptides in vitro and in vivo.

Citation Format: Jim A. Rotolo, Lila Ghamsari, Erin Gallagher, Rick Ramirez, Mark Koester, Siok Leong, Gene Merutka, Barry J. Kappel. β-catenin antagonist peptide attenuates oncogenic gene transactivation and promotes antitumor activity in breast cancer models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1816.
Abstract 1824: Coordinate translational control of the kras signaling pathway
Authors: Singh Kamini , Lin Jianan , Lecomte Nicolas , Mohan Prathibha , Leach Steve D. , Ouyang Zhengqing , Wendel Hans-Guido
Published: September 2020

G-quadruplex (GQ) elements are conserved translational control elements that induce a requirement for RNA helicase activity for efficient mRNA translation. Computational structure analyses identify multiple GQ elements in the 5'UTRs of mRNAs encoding KRAS and key downstream signalling molecules. KRAS is a key oncogene in pancreatic ductal adenocarcinoma (PDAC) and many other cancers and except for the specific G12C KRAS mutation we do not have pharmacological inhibitors. We find that KRAS and downstream signalling molecules including PI3K, RALA, RAC2, MYC, MET, and YAP1 strictly depend on the RNA helicase eIF4A for their translation. This implies a potential utility for eIF4A/DDX2 inhibitors. CR31B is a silvestrol analogue that efficiently and selectively blocks eIF4A. CR31B kills PDAC cells at nanomolar concentrations and has significant single agent in vivo efficacy across xenograft, primary PDX, and murine models of PDAC. Together, our findings reveal coordinate, GQ- and eIF4A-mediated control of the translation of key RAS signalling molecules as a vulnerability in KRAS-driven pancreatic cancer.

Citation Format: Kamini Singh, Jianan Lin, Nicolas Lecomte, Prathibha Mohan, Steve D. Leach, Zhengqing Ouyang, Hans-Guido Wendel. Coordinate translational control of the kras signaling pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1824.
Abstract 1877: Hypoxia induces EGFR inhibitor resistance in lung cancer cells by upregulation of fibroblast growth factor receptor 1 (FGFR1) via MAPK pathway
Authors: Lu Yuhong , Liu Yanfeng , Oeck Sebastian , Zhang Gary J. , Glazer Peter M.
Published: September 2020

The development of small molecule tyrosine kinase inhibitors (TKIs) specific for epidermal growth factor receptors (EGFRs) with activating mutations has led to a new paradigm in the treatment of non-small cell lung cancer (NSCLC) patients. However, most patients eventually develop resistance. Hypoxia is a key micro-environmental stress in solid tumors that is associated with poor prognosis due in part to acquired resistance to conventional therapy. In our previous study, we showed that long-term, moderate hypoxia promotes resistance to the EGFR TKI, gefitinib, in the NSCLC cell line, HCC827, which harbors an activating EGFR mutation. In this study, we found that hypoxia also induces third generation EGFR TKI, osimertinib (AZD9291), resistance in the NSCLC cell line, H1975, which had developed resistance to first and second generation of EGFR TKIs by harboring second EGFR mutation, T790M. Following growth in hypoxia, hypoxia-induced gefitinib resistant HCC827 clones and hypoxic H1975 cells show increased N-cadherin expression and decreased E-cadherin expression, characteristics of an epithelial-mesenchymal transition (EMT), coordinated by an increased expression of ZEB1, an EMT activator. Mechanistically, we show that hypoxia induces increased fibroblast growth factor receptor 1 (FGFR1) expression in HCC827 and H1975 cells, and that knockdown of the FGFR1 by shRNA can attenuate hypoxia induced EGFR TKI resistance in both HCC827 and H1975 cells. We also found the upregulated expression of FGFR1 by hypoxia is mediated through the MAPK pathway to lead to EGFR TKI resistance. In keeping with this, inhibition of FGFR1 function by the selective small molecular inhibitor, BGJ398, attenuates hypoxia-induced EGFR TKI resistance and promotes upregulation of BIM levels. Similarly, inhibition of MEK activity by the inhibitor, trametinib, shows similar effects. In vivo in tumor xenografts in mice, BGJ398 treatment or trametinib treatment suppresses tumor growth and enhances AZD9291 response. These results suggest that hypoxia is a driving force for acquired resistance to EGFR TKIs through increased FGFR1 expression and coordination of EMT in NSCLC. The combination of EGFR TKIs and FGFR1 or MEK inhibitors may offer an attractive therapeutic strategy for NSCLCs.

Citation Format: Yuhong Lu, Yanfeng Liu, Sebastian Oeck, Gary J. Zhang, Peter M. Glazer. Hypoxia induces EGFR inhibitor resistance in lung cancer cells by upregulation of fibroblast growth factor receptor 1 (FGFR1) via MAPK pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1877.
Abstract 1884: The activation of YES1 leads to the acquired resistance to neratinib in HER2-amplified breast and lung cancers
Authors: Yamamoto Hiromasa , Takeda Tatsuaki , Suzawa Ken , Tomida Shuta , Miyauchi Shunsaku , Araki Kota , Nakata Kentaro , Miura Akihiro , Shien Tadahiko , Kitamura Yoshihisa , Sendo Toshiaki , Toyooka Shinichi
Published: September 2020

Background: Molecular-targeted therapies directed against human epidermal growth factor receptor 2 (HER2) are evolving for various cancers. Neratinib is an irreversible pan-HER tyrosine-kinase inhibitor, and was approved by the FDA as an effective drug for HER2-positive breast cancer. However, acquired resistance of various cancers to molecular-targeted drugs is an issue of clinical concern, and emergence of resistance to neratinib is also considered inevitable.

Methods: We established various types of neratinib-resistant cell lines from HER2-amplified breast and lung cancer cell lines using various drug exposure conditions. Then we analyzed the mechanisms of emergence of the resistance in these cell lines and explored effective strategies to overcome the resistance.

Results: YES1, which is a member of the SRC family, was amplified in two neratinib-resistant breast cancer cell lines and one lung cancer cell line. Knockdown of YES1 by siRNA and pharmacological inhibition of YES1 by dasatinib restored the sensitivity of the YES1-amplified cell lines to neratinib in vitro. Combined treatment with dasatinib and neratinib inhibited tumor growth in vivo. Moreover, this combination also induced downregulation of signaling molecules such as HER2, AKT and MAPK.

Conclusion: YES1 plays an important role in the emergence of resistance to HER2-targeted drugs, and that dasatinib enables such acquired resistance to neratinib to be overcome.

Citation Format: Hiromasa Yamamoto, Tatsuaki Takeda, Ken Suzawa, Shuta Tomida, Shunsaku Miyauchi, Kota Araki, Kentaro Nakata, Akihiro Miura, Tadahiko Shien, Yoshihisa Kitamura, Toshiaki Sendo, Shinichi Toyooka. The activation of YES1 leads to the acquired resistance to neratinib in HER2-amplified breast and lung cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1884.
Abstract 1894: Role of IGF1R in mediating resistance to MAPK pathway inhibitors in BRAF-mutant melanoma
Authors: Patel Hima , Mishra Rosalin , Broit Natasa , Yacoub Nour , Yuan Long , Alanazi Samar , Hayward Nicholas , Johansson Peter , Garrett Joan T.
Published: September 2020

Melanoma is the most aggressive form of skin cancer originating in the pigment producing melanocytes of the skin. The five year survival rate for patients diagnosed with metastatic melanoma is only 23%. Around 60% of the melanomas encompass the active BRAFV600E oncoprotein, which is critical for survival and proliferation of the melanoma cells as a consequence of hyperactivation of theMAPK pathway. This opened up avenues for development of BRAF inhibitors and subsequently the development of MEK inhibitors. Co-targeting of BRAF and MEK with Dabrafenib and Trametinib respectively, results in a response rate of about 70% in patients harboring the BRAF mutation. The use of these inhibitors is limited as patients relapse on treatment typically within 6 months due to the development of acquired resistance. Recent studies have identified various resistance mechanisms including involvement of various receptor tyrosine kinases. In order to decipher novel mechanisms of resistance, we generated double resistant cell lines to BRAF and MEK inhibitor in WM115, WM983 and A375 human melanoma cell lines harboring BRAFV600 mutations using Dabrafenib and Trametinib over a course of 9-11 months. The drug resistant cell lines exhibited increased viability and maintenance of downstream p-ERK protein as compared to their drug sensitive counterparts. Receptor tyrosine kinase array revealed an increase in phosphorylated HER3, AXL, EGFR and IGF1R in the drug resistant cells versus drug sensitive cells. Quantitative PCR data revealed an upregulation of these molecules along with an increase in the expression of some of their ligands at the mRNA level. RNA-seq analysis identified IGF1R and IGF2BP2 to be upregulatedin the resistant cell lines compared to parental cell lines.IGF2BP2 is a tumor promoter and drives cancer proliferation in a variety of malignancies and is correlated with poor survival.Our next experiments are focused on testing the efficacy of IGF1R inhibitors in the drug resistant cell lines and their downstream effector signaling molecules in vitro and in vivo. Further, we aim to decipher the mechanism by which IGF2BP2 regulates the IGF1R signaling pathway in MAPK inhibitors resistant melanoma setting. Delineating the role of IGF1R in mediating resistance to BRAF and MEK inhibitors will help expand possible treatment options to aid in long-term success for BRAF-mutant melanoma patients.

Citation Format: Hima Patel, Rosalin Mishra, Natasa Broit, Nour Yacoub, Long Yuan, Samar Alanazi, Nicholas Hayward, Peter Johansson, Joan T. Garrett. Role of IGF1R in mediating resistance to MAPK pathway inhibitors in BRAF-mutant melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1894.
Abstract 1906: Combination MEK and mTORC1/2 inhibition overcomes resistance to single agent therapy in pancreatic cancer
Authors: Brown Wells , Nemirovsky Oksana , Gillespie Jordan , McDonald Paul , Dedhar Shoukat
Published: September 2020

Activating mutations in the KRAS oncogene are found in greater than 90% of pancreatic ductal adenocarcinoma (PDAC), yet KRAS remains difficult to inhibit pharmacologically. Small molecule inhibitors of RAF/MEK/ERK (MAPK) or mTOR are first line treatments for several human malignancies, however resistance to these targeted therapies continues to be a major clinical problem. We show in vitro dose dependent inhibition of proliferation and pERK1/2 in human PDAC cell lines and a KRAS-driven genetically engineered mouse model (GEMM) of pancreatic cancer in response to Trametinib, a small molecule MEK-inhibitor (MEKi). Similarly, Trametinib elicited a dose dependent inhibition of the in vivo tumor growth of a subcutaneous human PDAC xenograft, however these tumors rapidly developed resistance and contained areas of increased activation of pAkt(Ser473). We determined that genetic depletion of KRAS using doxycycline-inducible shRNA or small molecule MEKi leads to increased pAkt(Ser473) and activation of mTORC2 by western blot, implicating activation of the Akt/mTORC1/2 pathway may be a mechanism of resistance to MEKi. Interestingly, treatment of PDAC cell lines with Torin1, a small molecule inhibitor of mTORC1/2, results in the increased activation of pERK1/2, suggesting crosstalk and compensation of these two pathways as mechanisms of resistance. The combination of Trametinib and Torin1 leads to the inhibition of both pERK1/2 and pAkt(Ser473) that is accompanied by a dose dependent, synergistic increase in cytotoxicity not observed with either single agent. In human PDAC xenografts and a syngeneic GEMM, combination treatment of Trametinib and Torin1 dramatically reduced tumor burden and increased survival, without serious toxicity. These results demonstrate that combined inhibition of MEK/ERK and mTORC1/2 is a potent and viable therapeutic strategy for PDAC.

Citation Format: Wells Brown, Oksana Nemirovsky, Jordan Gillespie, Paul McDonald, Shoukat Dedhar. Combination MEK and mTORC1/2 inhibition overcomes resistance to single agent therapy in pancreatic cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1906.
Abstract 198: Molecular genetic characterization of primary breast cancer via clinical next-generation sequencing
Authors: Park Jung Ho , Ko Seung Yeon , Kim Sang Hwa , Kim Ho Young , Ko Sung Hoon , Kim Lee Su
Published: September 2020

Targeting appropriate driver genes is crucial in cancer precision medicine. To find oncogenic drivers, targeted next-generation sequencing is becoming widely used in the clinical field. Using targeted next-generation sequencing, we aimed to detect targetable genetic alterations and characterize mutational profiles of breast cancer. We analyzed breast cancer biopsy specimens using customized 50-gene panel. In total, 196 patients of primary operable breast cancer patients were selected from single institution in South Korea. Among the 196 patients, 22 kinds of genes were significantly mutated or amplified. They comprised 161 nonsynonymous missense mutations, 54 frameshift mutations, 29 missense mutations and 5 small indels; 64 gene amplifications and 2 deletions. The most frequently mutated gene was PIK3CA (n=81/196; 41.3%), followed by TP53 (n=62; 31.6%), AKT1 (n=21; 10.7%), GATA3 (n=17; 8.7%) and PTEN (n=14; 7.1%). The most frequently amplified gene was ERBB2 (n=25/196; 12.8%), followed by FGFR1 (n=11; 5.6%), MYC (n=9; 4.6%), FGFR2 (n=3; 1.5%) and FGFR3 (n=3; 1.5%). Targeted next-generation sequencing can be readily performed in daily clinical practice. The most commonly found targetable genetic alterations were mutations of PI3K/AKT/mTOR pathway genes and FGFR family gene amplifications. Despite the limited number of available targeted agents, our study shows the feasible application of precision medicine in breast cancer.

Citation Format: Jung Ho Park, Seung Yeon Ko, Sang Hwa Kim, Ho Young Kim, Sung Hoon Ko, Lee Su Kim. Molecular genetic characterization of primary breast cancer via clinical next-generation sequencing [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 198.
Abstract 1986: Mutation analysis in cell-free DNA upon development of resistance to afatinib
Authors: Terada Nanao , Kimura Hideharu , Miyakawa Mako , Koba Hayato , Yoneda Taro , Takeda Takayuki , Shirasaki Hiroki , Shibata Kazuhiko , Nishi Koichi , Takayama Koichi , Kasahara Kazuo
Published: September 2020

Introduction: EGFR-mutant lung cancers are genomically heterogeneous in terms of compound EGFR mutations and mutations in other genes. The purpose of this study was to explore gene mutations in circulating cell-free DNA that are related to resistance to afatinib.

Patients and methods: Patients with EGFR-mutant non-small cell lung cancer who received afatinib monotherapy were enrolled in this trial. Plasma samples were longitudinally collected before afatinib treatment, during treatment, and at the time of development of afatinib resistance. Written informed consent was obtained from all patients. Cell-free DNA was extracted from plasma using the QIAamp Circulating Nucleic Acid Kit (QIAGEN). Mutation profiles were determined by next-generation sequencing and analyzed using a human lung cancer panel (QIAGEN).

Results: A total of 71 patients were enrolled in this study. Plasma samples both before treatment and at the time of resistance to afatinib were collected from 21 patients. Although currently under analysis, the mutation profiles determined by next-generation sequencing revealed the following changes. 1) The number of gene mutations per patient that newly appeared at resistance development ranged from 0 to 7. 2) The allele frequency of the T790M EGFR mutation, a mutation responsible for resistance to EGFR tyrosine kinase inhibitors, was increased after development of afatinib resistance in approximately half of the patients.

Conclusions: The T790M allele frequency tended to increase after the development of resistance to afatinib. Some mutations were detected after resistance to afatinib. The role of this mutation needs to be investigated in a further study.

Citation Format: Nanao Terada, Hideharu Kimura, Mako Miyakawa, Hayato Koba, Taro Yoneda, Takayuki Takeda, Hiroki Shirasaki, Kazuhiko Shibata, Koichi Nishi, Koichi Takayama, Kazuo Kasahara. Mutation analysis in cell-free DNA upon development of resistance to afatinib [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1986.
Abstract 1988: Enabling personalized biomarker discovery in challenging oncology samples by coupling a novel library preparation chemistry with hybridization capture
Authors: Scott Karissa , Chakravarty Ushati Das , Huang Hsiao-Yun , Barnes Timothy , Lai Kevin , Sheu Jessica , Chen Tzu-Chun , Lopez Ramses , Lewis Lynette , Potts Anastasia , Henck Steven
Published: September 2020

Discovery and identification of new, targetable biomarkers is driven by comprehensive tumor profiling using next generation sequencing (NGS). However, converting tissue samples into NGS libraries is often challenging due to the low quantity and quality of DNA in such samples. Here we present sensitive and accurate detection of low-frequency variants by combining a novel library preparation optimized for low-input and degraded samples with IDT's xGen hybridization capture. The library preparation is enabled by an engineered mutant ligase and proprietary sequencing adapters that together prevent chimeras, suppress dimer-formation, enable double strand consensus calling, and maximize conversion. We demonstrate the performance of these reagents with archived tissue samples collected from five individuals with lung cancer. These matched samples consisted of DNA isolated from formalin-fixed paraffin-embedded (FFPE) tumor, DNA isolated from fresh-frozen normal tissue, and cell-free DNA (cfDNA) isolated from plasma. Sequencing libraries were prepared from 250 ng of tumor and healthy samples and 10 ng of cfDNA. Libraries were captured using a pan-cancer panel designed to simultaneously detect copy number variations, indels, rearrangements, and microsatellite instability across 532 oncogene targets. For each individual, tumor-associated mutations were identified using single-stranded consensus calling of variants found in tumor but not normal samples. As cfDNA has been correlated to disease progression, we wanted to determine if tumor-associated variants could be identified in the matched cfDNA samples. Custom capture panels were designed and delivered within 5 business days to target each individual's tumor-associated mutations. Relevant cfDNA samples were captured with these custom panels, sequenced, and variants were called after double stranded consensus calling. Mutations called from both FFPE tumor and cfDNA samples were verified by droplet digital PCR. By combining a high conversion library preparation with efficient hybridization capture and double stranded consensus calling, we have demonstrated an effective approach to extract information from difficult samples.

Citation Format: Karissa Scott, Ushati Das Chakravarty, Hsiao-Yun Huang, Timothy Barnes, Kevin Lai, Jessica Sheu, Tzu-Chun Chen, Ramses Lopez, Lynette Lewis, Anastasia Potts, Steven Henck. Enabling personalized biomarker discovery in challenging oncology samples by coupling a novel library preparation chemistry with hybridization capture [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1988.
Abstract 2075: GPCRs for pituitary adenylate cyclase activating polypeptide regulate transactivation of HER3 in non-small cell lung cancer cells
Authors: Moody Terry W. , Lee Lingaku , Jensen Robert T.
Published: September 2020

HER3, which lacks intrinsic kinase activity, plays an important role in cancer proliferation by formation of heterodimers with the EGFR or HER2 (Kiavue et al., Oncogene, 2019). Peptide GPCRs for pituitary adenylate cyclase activating polypeptide (PACAP) stimulate growth by transactivation of the EGFR and HER2 (Moody et al., JPET, 2012 and Peptides, 2019), but its effect on HER3 is unknown. The ability of PACAP to cause tyrosine phosphorylation of the EGFR, HER2 and HER3 was investigated. Using a panel of 17 non-small cell lung cancer (NSCLC) cell lines, mRNA for EGFR, HER2, HER3 was detected in 94%, 88%, and 100% of the cell lines, respectively. Addition of 100 nM PACAP but not vasoactive intestinal peptide to A549 cells increased EGFR, HER2 and HER3 tyrosine phosphorylation by 420, 240 and 190%, respectively. The increase is EGFR tyrosine phosphorylation was blocked by gefitinib or PACAP(6-38), a PAC1 antagonist. The increase in HER2 tyrosine phosphorylation caused by PACAP was inhibited by Herceptin or PACAP(6-38). The increase in HER3 tyrosine phosphorylation caused by PACAP was inhibited by the HER3 blocking antibody mAb3481 or PACAP(6-38). Immunoprecipitation experiments indicated the PACAP addition to A549 cells resulted in the formation of EGFR/HER2 and HER2/HER3 heterodimers, whereas addition of EGF resulted in EGFR/HER2 heterodimers and addition of the HER3 agonist neuregulin-1 resulted in HER2/HER3 heterodimers. Addition of N-acetyl-cysteine (antioxidant), Tiron (superoxide scavenger) or diphenylene iodonium (Nox/Duox inhibitor) impaired the ability of PACAP to cause EGFR, HER2 or HER3 transactivation. The results indicate that reactive oxygen species are essential for PACAP to cause EGFR, HER2 or HER3 transactivation in non small cell lung cancer cells.

Citation Format: Terry W. Moody, Lingaku Lee, Robert T. Jensen. GPCRs for pituitary adenylate cyclase activating polypeptide regulate transactivation of HER3 in non-small cell lung cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2075.