bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1621: Radotinib, a medicine for chronic myeloid leukemia, induces cell death of mantle cell lymphoma cells
Authors: Heo Sook-Kyoung , Noh Eui-Kyu , Jeong Yoo Kyung , Kim Jeong Yi , Choi Yunsuk , Cheon Jaekyung , Koh SuJin , Baek Jin Ho , Min Young Joo , Jo Jae-Cheol
Published: August 2018

Background: Radotinib is a medicine for the treatment of some types of cancer. It is approved in South Korea for use as a second-line treatment of chronic myeloid leukemia (CML). Its mechanism of action involves inhibition of the tyrosine kinase Bcr-Abl and of platelet-derived growth factor receptor (PDGFR). It has been little known the effects of radotinib on mantle cell lymphoma cells (MCL). Methods: First, we examined cytotoxicity of radotinib on MCL cell line, MAVER-1 and REC-1. Also, cytotoxicity of radotinib on both cell lines which have a different genetic back ground. Annexin V positive cell, caspas-3 and -9 activities, cell cycle distribution and mitochondrial membrane potential (MMP) were observed by analyzed with flow cytometric analysis. And diverse signaling pathways were investigated by Western blotting in MCL cells.

Results: Interestingly, radotinib caused cell death of MCL cells. And radotinib induces G1-phase arrest in MAVER-1 and REC-1 cells. And it also inhibited the expression of CDK2, CDK4, and cyclin D1, D3 and E. Therefore, radotinib induces G1-phase arrest via suppression of CDK2, CDK4, and cyclin D1, D3 and E. Generally, the intrinsic apoptotic pathway involves mitochondrial activation and caspase and phosphatidylserine externalization. Radotinib induced Annexin V positive cells, and caspase pathway activation including caspase-3, -7 and -9. And its treatment remarkably decreased MMP in MCL cells at 72 h. As well as we observed that cytochrome c accumulated dose dependently in the cytosol of radotinib-treated MAVER-1 and REC-1 cells. Moreover, radotinib decreased the expression of Bcl-xL and Bcl-2, and increased the expression of Bax in MCL cells. These results indicate that radotinib induces cell death of MCL cells by induction of G1-phase arrest and mitochondrial-dependent apoptosis. Furthermore, the expression of p-AKT, AKT and ERK was significantly reduced by radotinib in MCL cells. Conclusion: Radotinib may play an important role as a candidate or chemosensitizer for treatment agent in MCL. These data indicate that radotinib has a potential for anti-cancer therapy in MCL.

Citation Format: Sook-Kyoung Heo, Eui-Kyu Noh, Yoo Kyung Jeong, Jeong Yi Kim, Yunsuk Choi, Jaekyung Cheon, SuJin Koh, Jin Ho Baek, Young Joo Min, Jae-Cheol Jo. Radotinib, a medicine for chronic myeloid leukemia, induces cell death of mantle cell lymphoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1621.
Abstract 1626: Sensitive detection of MET exon 14 skipping by RT-qPCR and next generation sequencing
Authors: Patel Mukund , Polur Honey , Hurban Patrick
Published: August 2018

The MET receptor tyrosine kinase (MET) gene is a proto-oncogene whose abnormal activation can trigger tumor growth, angiogenesis, and metastasis. Numerous mutations have been identified that lead to over-activation of the gene, including well characterized amplification and somatic mutations. Mutations that lead to the skipping of MET exon 14 are likely to be highly predictive of response to MET inhibition. Exon 14 contains a motif that is required for the efficient recruitment of the ubiquitin ligase, CBL, which targets MET for degradation. Loss of exon 14 leads to prolonged stability of the MET protein which causes increased signaling upon stimulation by hepatocyte growth factor. Over 200 distinct mutations have been identified that cause MET exon 14 skipping ranging from single nucleotide mutations to large insertion/deletions. Due to this complexity, detecting MET exon 14 skipping mutations using DNA-based NGS hybridization capture methods is challenging. RNA-based testing can circumvent the complexity of DNA-based changes. We developed a RT-qPCR assay that detects MET exon 14 skipping independent of the causal mutation. Analytical validation using RNA admixtures demonstrated 1% sensitivity of detecting MET exon 14 mutant transcripts. 100 FFPE lung adenocarcinomas were screened with the RT-qPCR assay. Of 100, two samples (2%) were identified as positive for MET exon 14 skipping. The two positive samples along with three negative samples were orthogonally confirmed using the Illumina TruSight Tumor 170 hybridization capture-based targeted panel assay that simultaneously analyzes DNA and RNA. Overall, the number of samples identified positive for MET exon 14 by RT-qPCR test is in line with the frequency observed from other studies in lung adenocarcinomas of about ~3%. The assay can detect as little as 1% transcripts with MET exon 14 skipping suggesting that we have developed an accurate and sensitive RNA-based assay for use in clinical trials.

Citation Format: Mukund Patel, Honey Polur, Patrick Hurban. Sensitive detection of MET exon 14 skipping by RT-qPCR and next generation sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1626.
Abstract 1627: Custom VariantFlex technology allows rapid design and testing of reference materials for tumor genotyping assays
Authors: Huang Catherine , Santhanam Ram , Brudzewsky Dan , Brown Trevor , Garlick Russell , Anekella Bharathi
Published: August 2018

Introduction: Development and validation of tumor genotyping assays requires highly characterized reference materials for effective assessment of accuracy, precision, reproducibility and limits of detection. Highly multiplexed reference materials can facilitate and expedite these studies; however, there is not a single reference material that is appropriate for all assays. Traditional cancer cell line methods or genetic engineering methods can take 6 - 9 months, so more flexible solutions for reference materials are needed. Here we describe the development of VariantFlex technology, which enables a laboratory to configure a reference material to contain the variants of interest at the allele frequency of relevance to their studies in rapid time. Methods: A library of biosynthetic constructs was generated that contains ~140 somatic cancer DNA variants (SNVs, small and large indels and tandem duplications), ~27 RNA fusions, and 6 very large gene region constructs for copy number gains. TaqMan® digital PCR assays were developed to precisely quantify the variant allele fraction, fusion RNA concentration or copy number gain. As an example, biosynthetic constructs bearing multiple SNVs in TP53, PIK3CA and EGFR, large deletions in EGFR, and the structural variants NCOA4-RET and TPR-ALK, were selected from the VariantFlex library and spiked into DNA extracted from the highly characterized GM24385 cell line. The mixture was processed to obtain ctDNA-like profile, encapsulated to provide stability, and then diluted in artificial plasma to generate a customized reference material for liquid biopsy. Results: The ctDNA reference material was quantified by digital PCR and performance was verified on hybrid capture and amplicon based NGS assays. Variant allele frequency as low as 0.125% VAF was accurately targeted (range for 0.125% target was +/- 0.025% by digital PCR). The VariantFlex technology has been validated to work with amplified NGS workflows, hybrid-capture based NGS assays as well as non-NGS assays such as qPCR and array based methods. Conclusions: This study shows how a large library of cancer-related variants can facilitate rapid production of customized reference materials. The flexibility to include exactly the variants of interest, at the critical allele frequencies, and in the most patient-like format will facilitate assay development and validation.

Citation Format: Catherine Huang, Ram Santhanam, Dan Brudzewsky, Trevor Brown, Russell Garlick, Bharathi Anekella. Custom VariantFlex technology allows rapid design and testing of reference materials for tumor genotyping assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1627.
Abstract 164: Ston2 knockdown promotes stem-like properties via dnmt1-mediated muc1 upregulation in ovarian cancer
Authors: Xu Shanshan
Published: August 2018

Cancer stem cells possess the ability of self-renewal, unlimited proliferation,and non-sensitivity to physical and chemical factors, which is regarded to be a cause of ovarian cancer recurrence, but the mechanism is currently unclear. We found that the expression of STON2 was significantly lower in ovarian cancer-stem like cells by LC-MS/MS-based label free quantitative proteomics approach and western blot validation. STON2 knockdown upregulated the expression of the CSC-related markers in protein level, increased the spheroids number and CD44+CD24−proportion. Besides, in vivo xenograft experiments, the volume of tumors from the shSton2 group were much bigger compared with the shNC group. Moreover, STON2 overexpression decreased the CSC-related markers and inhibited the spheroids number compared with the control group. Transcriptome sequencing data showed that MUC1 is negative correlation with STON2, which were verificated by RT-PCR and western blot analysis. Subsequently, we found that MUC1 mediated the role of STON2 in regulation of stem-like properties in ovarian cancer. MUC1 knockdown resulted in a significant decrease in the sphere-forming ability, CSC-related markers and CD44+CD24−proportion. In addition, MUC1 overexpression caused an increment of CSC-related markers expression and CD44+CD24−proportion. Furthermore, we found that the reducing of sphere-forming ability caused by STON2 overexpression was rescued by MUC1 overexpression. As MUC1 can be regulated through epigenetic mechanisms, we found that DNA methylation inhibitor Azacitidine was able to rescue the upregulation of MUC1 by STON2. The DNA methylation levels of MUC1 in one CpG-rich region were then evaluated with pyrosequencing, and the data displayed that the siSton2 group is lower than the control group. Furthermore, we found that the DNMT1 as a member of the DNA methyltransferase family decreased following with STON2 knockdown in protein level, rather than in mRNA level. We thus knock down DNMT1 using specific siRNA, and found that MUC1 overexpression both in mRNA level and protein level. In this study, we focused primarily on STON2 repression leading to DNMT1 downregulation and MUC1 activation is a main mechanism underlying the maintenance of stem-like properties in ovarian cancer.

Citation Format: Shanshan Xu. Ston2 knockdown promotes stem-like properties via dnmt1-mediated muc1 upregulation in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 164.
Abstract 1645: Innovative discovery of therapeutic plateletpheresis in patients with thrombocytosis using the Fresenius COM.TEC
Authors: Jin Yanxia , Yuan Guolin , Zhou Fuling
Published: August 2018

Plateletpheresis is an important therapeutic method for treatment of patients with myeloproliferative neoplasms (MPN) presenting with thrombocytosis. This study analysed the collection efficiency of plateletpheresis (CEPP) for 94 patients with thrombocytosis who underwent plateletpheresis by Fresenius COM.TEC machine, especially analysed the CEPP for enrolled patients with PLT count at 500-1000 × 109/l. The results indicated that this device could significantly decreased PLT count, and with mean CEPP of 24.23±17.42% that ranges from 3.18% to 78.46%. Further analysis shows that the PLT counts were 500-800 × 109/l for patients, which CEPP was above 42.1% with a lower pooled WBC and RBC. Paired t-test suggested that the red blood cell (RBC) counts, PLT counts, white blood cell (WBC) counts, monocyte counts and haematocrit (HCT) significantly decreased after plateletpheresis. Bivariate correlation and multiple logistic regression analysis showed that PLT count groups, hemoglobin (HGB) before apheresis were greatly correlated with CEPP. We suggest that the device is effective at reducing platelet counts with acceptable efficiency and it is available for plateletpheresis when the PLT counts about 500-800 × 109/l for patients with higher HGB pre-apheresis in female patients.

Citation Format: Yanxia Jin, Guolin Yuan, Fuling Zhou. Innovative discovery of therapeutic plateletpheresis in patients with thrombocytosis using the Fresenius COM.TEC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1645.
Abstract 1653: Design and synthesis of basic amino selective estrogen receptor degraders (BA-SERDs) for treatment-resistant breast cancer
Authors: Lu Yunlong , Thatcher Gregory R.
Published: August 2018

About 1 in 8 United States women (about 12%) will develop invasive breast cancer during their lifespan. Approximately 40,000 women are expected to die from breast cancer each year. The largest single breast cancer subtype is defined as estrogen receptor alpha (ERα; ESR1) positive breast cancer (75%), wherein ERα played a critical role in cancer cell survival, proliferation and metastasis. The dysregulation of ER signaling formed the most important molecular event in the ER positive (ER+) breast cancer. Endocrine therapy (ET) had been successfully employed on such breast cancer patients and most of them had substantial reduced risks of breast cancer recurrence and mortality in the initial 5-10 years. However, the development of resistance to ET has the greatest impact on clinical use. The emergence of resistance is inevitable over time and will eventually become metastatic disease after long term of ET. Recent studies confirmed treatment failure of SERMs and AIs in ER+ metastatic settings, whereas ERα remains engaged in the advanced disease and contributes to disease pathogenesis. Fortunately, the treatment resistant breast cancer patients remain responsive to fulvestrant (ICI182,780) which has been described as a “pure antagonist” and caused ERα degradation as a SERD. Nonetheless, the pharmaceutical liabilities of fulvestrant including poor solubility and pharmacokinetics (PK) resulted in poor compliance. The clinical development of orally bioavailable SERDs i.e. GDC-0810, AZD9496, RAD1901 is needed to overcome these shortcomings. Herein, our lab identified a novel class of BA-SERDs to circumvent drug resistance.

Citation Format: Yunlong Lu, Gregory R. Thatcher. Design and synthesis of basic amino selective estrogen receptor degraders (BA-SERDs) for treatment-resistant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1653.
Abstract 1661: Discovery of potent Abelson kinase inhibitors from the scaffold of pyrano[2,3-d]pyrimidin-7-one
Authors: Wu Zhixing , Fu Boqiao , Qiao Changjiang , Nagaraju Ashila , Lu Dai
Published: August 2018

Tyrosine kinases regulate cell proliferation, survival, invasion and angiogenesis during tumor initiation and progression. The Abelson (ABL) family of protein kinases comprises ABL1 and ABL2, which link diverse extracellular stimuli to signaling pathways that control cell growth, survival, invasion, adhesion and migration. Inhibition of ABLs has been implied in many type of hematopoietic malignancies and solid tumors. The compound PD173955 is a potent ABL kinase inhibitor. It was developed from the scaffold pyrido[2,3-d]pyrimidin-7-one by Parke-Davis (Pfizer). To discover novel ABL inhibitors, we investigated the scaffold pyrano[2,3-d]pyrimidin-7-one, which is structurally analogous to pyrido[2,3-d]pyrimidin-7-one. This novel scaffold has not been extensively studied in synthetic chemistry and pharmacology. The compound LDK1504 generated from this scaffold is a close analog of PD173955. It was synthesized as a prototypical compound from the pyrano[2,3-d]pyrimidin-7-one scaffold. In the enzymatic assays, LDK1504 was demonstrated with an inhibitory activity comparable to PD173955 for ABL1 kinase. It can inhibit the ABL1 kinase with an IC50 of 18.7 nM. Further optimization leads to the identification of LDK1512 (Kd = 0.38 nM; IC50 = 1.27 nM) against ABL1. The preparation of this class of compounds was achieved through a facile synthesis of 6-bromo-2-(methylthio)-7H-pyrano[2,3-d]pyrimidin-7-one followed by Suzuki coupling reaction and derivatization of the C-2 position of the pyrimidine ring. In this presentation, our synthesis, preliminary SAR of this class of compounds, and kinase selectivity profiling will be discussed.

Citation Format: Zhixing Wu, Boqiao Fu, Changjiang Qiao, Ashila Nagaraju, Dai Lu. Discovery of potent Abelson kinase inhibitors from the scaffold of pyrano[2,3-d]pyrimidin-7-one [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1661.
Abstract 1666: Novel glucose transporter inhibitors decrease glioblastoma growth and glucose uptake
Authors: Libby Catherine J. , Zhang Sixue , Benavides Gloria A. , Li Yanjie , Redmann Matthew , Tran Anh N. , Otamias Arphaxad , Darley-Usmar Victor , Napierala Marek , Zhang Jianhua , Zhang Wei , Hjelmeland Anita
Published: August 2018

Glioblastoma multiform (GBM) is the most common primary malignant adult brain tumor and one of the deadliest cancers. Patients with GBM typically undergo surgical resection, radiation and chemotherapy with temozolomide. Complete tumor resection is virtually impossible due to tumor location and the highly infiltrative nature of GBM, which leads to disease recurrence near the original tumor site. As such, these patients have a dramatically decreased life expectancy. Therapeutic development to prolong survival has been hampered by a high degree of inter- and intra-tumoral heterogeneity. Contributing to tumor heterogeneity is a subset of highly tumorigenic cells, termed brain tumor initiating cells (BTICs), that are able to self-renew and can be highly invasive and therapy resistant. BTICs are often enriched in perinecrotic regions, a GBM hallmark, where they can survive under nutrient restriction via increased glucose transporter 3 expression (GLUT3). GLUT3 has a 5-fold greater capacity for glucose transport than the other major glucose transporter isoform in the brain, GLUT1, and is typically restricted to neurons, testis, preimplantation embryos, and stem cells. GLUT3 expression is elevated in many solid tumor types, including GBM, and correlates with poor patient prognosis. Previously, we have shown that knockdown of GLUT3 in BTICs significantly inhibits their growth both in vitro and in vivo, indicating GLUT3 is a possible target for therapeutic intervention. Using structure based virtual screening, we have identified novel GLUT inhibitors that preferentially decrease the growth and glucose uptake of BTICs with minimal toxicity to non-malignant cells in vitro. Preliminary in vivo assessment of these compounds has not indicated toxicity. Our goal is to identify a potential new therapeutic option targeting metabolic reprogramming for the treatment of glioblastoma, as well as other tumor types.

Citation Format: Catherine J. Libby, Sixue Zhang, Gloria A. Benavides, Yanjie Li, Matthew Redmann, Anh N. Tran, Arphaxad Otamias, Victor Darley-Usmar, Marek Napierala, Jianhua Zhang, Wei Zhang, Anita Hjelmeland. Novel glucose transporter inhibitors decrease glioblastoma growth and glucose uptake [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1666.
Abstract 1670: Discovery of novel water-soluble derivatives of mebendazole as selective CLK1/4 kinase inhibitors and their anticancer cancer activity
Authors: Sun Lijun , Cheong Jae Eun , Zaffagni Michela , Zhou Kun , Zetter Bruce
Published: August 2018

Metastatic cancers remain clinically challenging and account for more than 90% of all cancer deaths. Drugs used to treat advanced metastatic cancers often generate drug resistance and relapse. Therefore, there is a critical need for novel therapeutic approaches for patients with advanced stage cancers that do not respond to any currently available anticancer therapies. Mebendazole and structurally related benzimidazole analogues, which are FDA approved compounds used to treat helminthic infections in the gastrointestinal track, are effective in inhibiting in vitro cancer cell proliferation. Unfortunately, their therapeutic applications in metastatic cancer are limited by their extremely low solubility and poor bioavailability. Further, the mechanism of the anticancer activities of this class of compounds is poorly defined. Here, we report the design and synthesis of water-soluble benzimidazoles as novel anticancer agents. Among them, the novel oxetanyl substituted compound, OBD9 (Methyl (5-(4-(methyl(oxetan-3-yl)amino)benzoyl)-1H-benzo[d]imidazol-2-yl)carbamate), demonstrated potent cytotoxicity towards a variety of highly aggressive cancer lines including prostate, lung, and ovarian cancers (IC50: 0.9-3.8 μM). In the NCI60 cancer cell panel screen, OBD9 broadly inhibited the proliferation of leukemia, melanoma, and breast and colon cancers. The aqueous solubility of OBD9 achieved 361 μM vs <1μM for mebendazole. In a mouse xenograft model of the highly metastatic human prostate cancer PC3MLN4, OBD9 (30 mg/kg/day, three times/week for two weeks) significantly inhibited the growth of established tumors (treatment-to-control ratio: 0.36) without noticeable toxicity. We performed broad kinase screening (KINOMEscan, DiscoverX) to explore the mechanism of action and discovered OBD9 as a potent and highly selective inhibitor of Cdc-like kinase 1 and 4 (CLK1 and 4). The Selectivity Score of OBD9 at 10 μM concentration in the 403 non-mutant kinases is 0.002; and its IC50 is 1.5 and 1.2 μM for CLK1 and CLK4, respectively. CLKs are nuclear serine/threonine (S/T) kinases that regulate gene splicing and are frequently over activated in cancers. Our results suggest that OBD9 impedes cancer growth at least in part by inhibiting CLK1/4.

Citation Format: Lijun Sun, Jae Eun Cheong, Michela Zaffagni, Kun Zhou, Bruce Zetter. Discovery of novel water-soluble derivatives of mebendazole as selective CLK1/4 kinase inhibitors and their anticancer cancer activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1670.
Abstract 169: Identification of RNA aptamers selectively recognizing and affecting glioblastoma stem cells
Authors: Affinito Alessandra , Quintavalle Cristina , Albero Maurizio , Vilardo Claudia , Palma Francesco , Roscigno Giuseppina , Vitiani Lucia Ricci , Pallini Roberto , Esposito Carla Lucia , Franciscis Vittorio de , Condorelli Gerolama
Published: August 2018

Glioblastoma (GBM) is the most frequent and aggressive primary brain tumour in adults. Despite advances in surgical and medical neuro oncology, prognosis for GBM patients remains dismal, with a median survival of about 15 months. It has been demonstrated that the modest benefit of conventional therapies depends on a small population of cancer stem cells within the tumor, named Glioma Stem Cells (GSCs) that cause tumor relapse and chemoresistance and therefore could play a key role in GBM recurrence. Thus, the identification of new specific ligands for GSCs could be a fundamental challenge for the development of effective glioma therapies. Here, we developed an in vitro evolution based approach, named differential whole cellSELEX; it is used to generate nucleic acid ligands, named aptamers, with high affinity and specificity for GSCs. Aptamers, were obtained through the iterative evolution of a random pool of sequences using human primary GSCs as target. Among different potential candidates we focused on one sequence, named 40L. The 40L aptamer and its truncated form, 40S, were selective for human GSCs distinguishing them from tumor differentiated cells, obtained from the stem cells induced to differentiate. 40L revealed to be functionally active on target cells and able to inhibit stemness, cell growth and migration. 40s preserves binding ability of 40L sequence and it has further proven to strongly reduce tumor proliferation in in vivo experiment. Moreover, both 40L and 40s were able to rapidly internalize upon target binding and therefore may serve as selective vehicle for therapeutics.In conclusion, our results indicate that 40L and its short form 40s can selectively target GSCs both in vitro and in vivo. Given the crucial role of these cells in GBM recurrence and therapy resistance, 40L and 40s represent innovative drug candidates with a great potential in the GBM treatment.

Citation Format: Alessandra Affinito, Cristina Quintavalle, Maurizio Albero, Claudia Vilardo, Francesco Palma, Giuseppina Roscigno, Lucia Ricci Vitiani, Roberto Pallini, Carla Lucia Esposito, Vittorio de Franciscis, Gerolama Condorelli. Identification of RNA aptamers selectively recognizing and affecting glioblastoma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 169.
Abstract 1692: The impact of neutrophil/lymphocyte ratio as the predictive marker to anti-PD-1 antibody treatment in NSCLC patients
Authors: Tanimura Keiko , Yamada Tadaaki , Tamiya Nobuyo , Kaneko Yoshiko , Takeda Takayuki , Uchino Junji , Kubota Yutaka , Takayama Koichi
Published: August 2018

Background: Anti-Programmed death 1 (PD-1) antibody which enhances anti-tumor activity of cytotoxic-T-lymphocytes by blockade of PD-1/PD-L1 pathway has demonstrated improvement of survival in the patients with advanced non-small-cell lung cancer (NSCLC). PD-1 and its ligand PD-L1 is also known as key players in the formation of tumor microenvironment, closely related with inflammatory cytokines. Here, we retrospectively analyzed the peripheral blood cells as a surrogate marker for predicting outcome of immune checkpoint inhibitors (ICI). Method: Total of 78 cases treated with anti-PD-1 antibodies, nivolumab or pembrolizumab from February 2016 to September 2017 were registered retrospectively. We investigated the differential leukocyte counts, C-reactive protein (CRP) and other peripheral blood parameter on the treatment progress with ICI, and evaluated correlation between response to ICI and peripheral blood cells. Result: Out of 78 cases, 56 cases (77%) were treated with nivolumab, 18 cases (23%) were treated with pembrolizumab, and 4 cases (5%) have treatment history of both agents. Median follow up period was 5.7 months (range: 0.4-18.7 months). Median progression free survival (PFS) and median overall survival (OS) were 2.4 and 8.7 months, respectively. Overall response rate was 21.8%. Of some parameters in peripheral blood cells, higher absolute neutrophil count (ANC), CRP at baseline were associated with shorter OS. Furthermore, we determined neutrophil / lymphocyte ratio (NLR), platelet / lymphocyte ratio (PLR), lymphocyte / monocyte ratio (LMR) and prognostic nutritional index (PNI), which were known as predictive biomarkers in various tumor type. In patients with longer PFS for ICI treatment, ANC, NLR and CRP remained low level until disease progression, whereas these markers increased during ICI treatment in the patients with shorter PFS for ICI. Conclusion: Our retrospective analysis showed that ANC, NLR and CRP may predict response and acquisition of resistance to anti-PD-1 therapy in NSCLC patients. These results suggest that peripheral blood findings might be promising as a predictive biomarker of ICI treatment.

Citation Format: Keiko Tanimura, Tadaaki Yamada, Nobuyo Tamiya, Yoshiko Kaneko, Takayuki Takeda, Junji Uchino, Yutaka Kubota, Koichi Takayama. The impact of neutrophil/lymphocyte ratio as the predictive marker to anti-PD-1 antibody treatment in NSCLC patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1692.
Abstract 1700: Metabolic adaptations confer immunotherapy resistance in melanoma
Authors: Jaiswal Ashvin R. , Pudakalakatti Shivanand , Prasanta Dutta , Liu Arthur , Bartkowiak Todd , Ager Casey , Davies Michael A. , Davis Richard E. , Wargo Jennifer , Bhattacharya Pratip K. , Hong David S. , Curran Michael A.
Published: August 2018

Background: Despite the success of T-cell checkpoint blockade antibodies in treating an array of cancers, the majority of patients still fail to respond to these therapies, or respond transiently and then relapse. The molecular mechanisms that drive lack of response to checkpoint blockade, whether pre-existing or evolved on therapy, remain unclear.

Materials and Methods: To address this critical gap in clinical knowledge, we established a mouse model of melanoma designed to elucidate the molecular mechanisms underlying immunotherapy resistance. Through multiple in vivo passages, we selected a B16 melanoma tumor line that evolved complete resistance to combination blockade of CTLA-4, PD-1, and PD-L1, which cures ~75% of mice of the parental tumor. Using gene expression analysis, proteomics, and immunogenomics, we determined the adaptations engaged by this melanoma to become completely immunotherapy resistant. NMR spectroscopy, Seahorse XF Analysis, flow cytometry, confocal microscopy and Western blot analysis provided further insight into the mechanisms driving checkpoint blockade resistance.

Results: Acquisition of immunotherapy resistance by these melanomas was driven by acquisition of a hypermetabolic state through coordinate upregulation of both glycolysis and oxidative phosphorylation (OxPhos). Depletion of essential nutrients from the tumor microenvironment by these resistant tumors functionally compromises infiltrating T cells to an extent that cannot be rescued by even triple checkpoint blockade. The glycolysis and OxPhos-associated genes most highly induced by these resistant melanomas were capable of conferring enhanced resistance to checkpoint blockade when reintroduced monogenically into the parental tumor. We have validated upregulation of these pathways in a unique cohort of melanoma patients who failed dual checkpoint blockade. Additionally, we employed a novel MRI imaging technique to visualize metabolic changes acquired by resistant tumors in live mice. Clinical application of this technique could provide a much-needed noninvasive tool to predict immunotherapeutic sensitivity of patient cancers.

Conclusion: Acquisition of a hypermetabolic state by melanoma tumor cells cripples T cells in the microenvironment and confers complete resistance to checkpoint blockade.

Citation Format: Ashvin R. Jaiswal, Shivanand Pudakalakatti, Dutta Prasanta, Arthur Liu, Todd Bartkowiak, Casey Ager, Michael A. Davies, Richard E. Davis, Jennifer Wargo, Pratip K. Bhattacharya, David S. Hong, Michael A. Curran. Metabolic adaptations confer immunotherapy resistance in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1700.
Abstract 1705: Systemic analysis of innate responses to anti-PD1/PD-L1 treatment in a melanoma model
Authors: Gassen Rodrigo , Welm Alana , Bonorino Cristina
Published: August 2018

Response to anti-PD1 therapy for tumors is often thought to target T cells, directly and exclusively. However, while some patients, even with metastasis, respond completely, most patients do not respond to treatment as predicted. The mechanisms of response ad resistance to anti-PD-1 therapy need to be elucidated, in order to optimize treatment and prevent unnecessary adverse effects. Most innate cells, especially in tumor bearing individuals, express PD-1, not only in the tumor microenvironment, but systemically. Among those are macrophages and dendritic cells. The implications of PD-1 expression by these cells in their immune function are still unknown, and so are the signaling pathways activated by agonists or antagonists of this receptor. Although recent data suggest that PD-1+ tumor associated macrophages (TAMs) promote advanced tumor growth, little is known of the kinetics of PD-1 expression by innate cells during tumor progression. Using an immunocompetent murine melanoma model, we characterized PD-1 and PD-L1 expression in different innate cells, in lymphoid organs, blood, and tumor microenvironment. In NSG mice, using PDX grafts of triple negative breast cancers, we determined that such expression is independent of lymphocytes and NK cells. Treatment with anti-PD-1 mobilized an early (7 days) wave of DC migration that can be seen in the blood, preceding a CD4+ T cell mobilization that peaked ad day 14 after treatment. These early responses correlated with CD8+ T cell infiltration to the tumor microenvironment as well as tumor regression. PD-1 expression and migration in the blood was different among the DC subpopulations, and anti-PD-1 treatment enhanced survival as well as activation of dendritic cells. These findings are relevant to understanding the different outcomes of immunotherapy with anti-PD-1 and anti-PD-L1.

Citation Format: Rodrigo Gassen, Alana Welm, Cristina Bonorino. Systemic analysis of innate responses to anti-PD1/PD-L1 treatment in a melanoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1705.
Abstract 1706: Inhibition of fibulin-3 reverses macrophage polarization in glioblastoma and increases anti-tumor inflammatory responses
Authors: Longo Sharon L. , Behera Prajna , Viapiano Mariano S. , Nandhu Mohan Sobhana
Published: August 2018

Glioblastomas (GBMs) are the most common primary tumors of the Central Nervous System and one of the most aggressive and difficult to treat forms of cancer. Our laboratory was the first to identify the extracellular protein fibulin-3 (EFEMP1) in GBMs and demonstrate the pro-tumoral functions of this protein in solid tumors. We have recently developed a function-blocking antibody ("mAb428.2") against fibulin-3, which inhibits the enhancing effect of this protein on ADAM17 activity and NFkB signaling (Nandhu et al., Clin Cancer Res, 2017). Using mAb428.2 we observed a significant anti-tumor effect and prolongation of survival in nude mice carrying patient-derived GBM xenografts implanted subQ or intracranially (antibody treatment: 8x 30 mg/kg, q24h, IV). Interestingly, reduction of tumor growth during mAb428.2 treatment was accompanied with significant tumor necrosis and increased infiltration of Iba1-positive tumor-associated macrophages (TAMs) both in subQ and orthotopic tumors. Remarkably, TAMs in mAb428.2-treated GBMs showed very low co-expression of Arginase-I, a bona-fide marker of M2 ("tumor promoting") TAM polarization. Further analysis of mouse-specific gene expression in the tumor mass and in recovered macrophages (CD11b-positive cells) showed that TAMs in mAb428.2-treated GBMs had significantly increased expression of inflammatory cytokines (IFN-gamma, IL-1b, IL-10) and downregulation of M2 markers (CD163, CD206, ARG1), suggesting that our anti-fibulin-3 treatment prevented the expected M2 polarization of TAMs. Accordingly, mAb428.2-treated tumors showed drastically decreased expression of CSF-1a, an M2-inducing cytokine that may be regulated by fibulin-3. Co-culture experiments of PMA-activated U937 macrophages with GBM stem-like cells did not affect tumor cell viability when a control IgG1 was added to the cultures. However, addition of mAb428.2 caused significant decrease of GBM cell viability, suggesting that anti-fibulin-3 treatment enables TAMs to attack tumor cells. Taken together, our results strongly suggest that anti-fibulin-3 and possibly other anti-ECM approaches may induce a marked inflammatory reaction driven by innate immune cells, potentiating anti-tumor therapy.

Citation Format: Sharon L. Longo, Prajna Behera, Mariano S. Viapiano, Mohan Sobhana Nandhu. Inhibition of fibulin-3 reverses macrophage polarization in glioblastoma and increases anti-tumor inflammatory responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1706.
Abstract 1713: Immunomodulatory effect of statins on Tregs within human colorectal cancer is determined by the stage of disease
Authors: Al-Husein Belal
Published: August 2018

Colorectal cancer (CRC) is a public health problem worldwide and in Jordan. Statins are best seller cholesterol lowering agents. Beyond their cholesterol-lowering effects, statins use has been reported to reduce the risk of several malignances including CRC. This study aimed to assess the effect of statins on CRC risk by studying cellular infiltration of Tregs into CRC tissues and their effect on Transforming growth factor beta 1 (TGF-β1) level and angiogenesis. A total 47 specimens (25 statins users, and 22 statins nonusers) of CRC were used. Immunohistochemical analysis was performed to study Tregs infiltration by studying their specific marker fork head transcription factor (FOXP3), and angiogenesis by studying CD31 positive blood vessels. TGF-β level was measured using commercial ELISA kits. Results revealed that in case of statins use was associated with more Tregs infiltration, more angiogenesis but no difference in TGF-β1 content in tumor tissue. When further categorized analysis according to stage of disease, angiogenesis effect was more pronounced in early disease and wasn't significant in advanced disease.In addition, more Tregs infiltration was significantly noticed in advanced disease but not in early disease. Same stage-dependence wasn't notices with TGF-β1. Since statins are associated with reduced risk of death in colorectal cancer patients, we could suggest some immunologic mechanism for that. Tregs infiltration into tumor and reduction of inflammation at tumor site might reduce cancer aggressiveness in advanced disease. In advanced disease, however, reduction of angiogenesis would be the cause of tumor aggressiveness. These results suggest that statins might be used in prophylaxis or treatment of CRC. Further studies are needed to evaluate the effect of various types and doses of statins.

Citation Format: Belal Al-Husein. Immunomodulatory effect of statins on Tregs within human colorectal cancer is determined by the stage of disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1713.
Abstract 172: Collagen increases tumorigenic characteristics of papillary thyroid cancer cells harboring BRAFV600Emutations
Authors: Sharabura Anna , Jenkins Jonathan , MacDonald Laura
Published: August 2018

Thyroid cancer is the most common endocrine cancer, and incidence is increasing worldwide. Thyroid cancer can be classified as either well differentiated or poorly differentiated. Of well-differentiated thyroid cancers, papillary thyroid cancer is most common, and is associated with activating BRAF mutations. While our understanding of the genetic basis for thyroid cancer is fairly extensive, less is known about how the tumor microenvironment alters characteristics of tumor cells. Recently, Jolly and others reported that papillary thyroid tumors derived from cells harboring BRAFV600E mutations and PTEN deletions are enriched with fibrillar collagen. Notably, overexpression of collagen was also associated with decreased survival and worse overall prognosis in patient samples. In this study, we investigated whether growth on collagen enhanced tumorigenic characteristics of papillary thyroid cancer cell lines with BRAFV600E mutations. Three distinct cell lines derived from mouse papillary thyroid cancer tumors were grown in the presence and absence of collagen and assessed for changes in morphology, proliferation, and drug resistance. Interestingly, our results suggest that growth on collagen contributes to a more mesenchymal morphology, increased proliferation, and decreased sensitivity to chemotherapy drugs. These and other results implicate an important role for collagen in the progression of thyroid cancer.

Citation Format: Anna Sharabura, Jonathan Jenkins, Laura MacDonald. Collagen increases tumorigenic characteristics of papillary thyroid cancer cells harboring BRAFV600Emutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 172.
Abstract 1730: A translational platform using primary human immune cells in vitro, syngeneic and humanized models in vivo to support and advance immuno-oncology drug discovery
Authors: O'Rourke Martin , Roman Shilina , Salmon Gary , Waddell Ian , Schueler Julia , Wood Edgar
Published: August 2018

Charles River Laboratories (CRL) has established a powerful translational immuno-oncology platform with the capability of progressing biologics or small-molecule modulators of immune response from in vitro to in vivo assays using human and mouse variants of current checkpoint inhibitors and small molecules. Utilizing a blood donor panel, our in vitro platform includes primary human immune cell assays that profile T-cell activation, T cell mediated-cancer cell kill, expansion of T-cell populations, mixed lymphocyte reactions (MLR), T-cell invasion, and antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The platform is currently being expanded to determine the effect of activated immune cell populations on tumor cell spheroid cultures. It has been validated with standard-of-care chemotherapeutics, including anti-CTLA4, anti-PD1 and a selection of small-molecule inhibitors of targets known to modulate immune responses including IDO inhibitors. Ex vivo analysis of activated mouse splenocyte response to checkpoint inhibitors, measured as cytokine release, and modulation of immune cell populations, as determined by flow cytometry, supports the translation of important compounds from the bench to preclinical models. Syngeneic mouse tumor models have frequently been used to profile immune responses in tumors; CRL has optimized and profiled existing checkpoint inhibitors to support immuno-oncology drug discovery using mouse and rat antibody variants of anti-CTLA4 and anti-PD1. To confirm the translational development of our platform in human tissue, CRL has developed and optimized humanized mouse models using subcutaneous implanted patient-derived xenografts (PDX) with human engraftment via CD34+ haematopoeitic stem cells in NOG mice, which were treated with anti-CTLA4 and anti-PD1. Infiltration of human immune cells and PDL-1 expression was detected by flow cytometry (FC) and immunohistochemistry (IHC) in hematopoietic organs and tumor tissue, supporting the initial in vitro response in primary immune cells. Here we present a screening platform, validated with standard-of-care chemotherapeutics, which will support translation of compounds from in vitro primary immune cell assays to modulation of mouse immune cell population in spleen and tumors, resulting in efficacy and tumor immune cell activation in humanized mouse models.

Citation Format: Martin O'Rourke, Shilina Roman, Gary Salmon, Ian Waddell, Julia Schueler, Edgar Wood. A translational platform using primary human immune cells in vitro, syngeneic and humanized models in vivo to support and advance immuno-oncology drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1730.
Abstract 1732: Gp96-Ig/costimulator combination platform improves T cell priming and enhances immunity, memory, and tumor elimination
Authors: Giffin Louise , Gonzalez Louis , Rose Jason , Hutchins Jeff
Published: August 2018

In this study, we report that addition of checkpoint inhibition and T cell costimulators into the Heat Biologics gp96-Ig-based vaccine platform elicits stronger antigen-specific CD8+ T cell activation and expansion, stimulates memory precursor cell activation, and enhances rejection of murine tumors. Despite the dramatic successes of checkpoint inhibitors in limited populations of cancer patients, 60-90% of patients are still failing to respond to these therapies. It is widely believed that approaches such as ours that target multiple facets of the immune system, including combinations with checkpoint inhibition and costimulator agonism, will be required to improve patient outcomes. Heat Biologics' first-generation ImPACT vaccine is a cell-based therapy that utilizes representative tumor cell lines expressing the antigen chaperone, gp96-Ig, to efficiently prime antigen-specific CD8+ T cells. Heat recently developed a next generation vaccine called ComPACT (COMbination Pan-Antigen Cytotoxic Therapy) that incorporates expression of a T cell costimulator (OX40L-Ig) and gp96-Ig within a single vaccine cell line. In multiple murine tumor and tumor-naive models, ComPACT elicits more potent activation of antigen-specific T cells as well as expansion of CD127+KLRG1- memory precursor cells, leading to a detectable memory response. Furthermore, ComPACT lacks the toxic systemic inflammatory cytokine production and proliferation of non-specific CD4+ T cells and Tregs observed with systemic administration of OX40 agonist antibodies. ComPACT also leads to high frequencies of IFNγ+, TNFα+, granzyme B+ and IL-2+ antigen-specific CD8+ T cells in mice at both priming and boosting, which increases rejection of established murine melanoma (B16.F10) and colon cancer (CT26) tumors and increased overall survival. Here, we have further assessed ImPACT and ComPACT-OX40L in combination with the anti-PD1 checkpoint inhibitor and with expression of an additional cell-secreted T cell costimulator, TL1A-Ig. In various murine models we show that these treatments synergize effectively to amplify antigen-specific T cells, program a functional memory response, and eliminate tumors. The combination of ComPACT-OX40L/TL1A with αPD1 or αPD-L1 may therefore translate into an efficacious approach to treat human cancers.

Citation Format: Louise Giffin, Louis Gonzalez, Jason Rose, Jeff Hutchins. Gp96-Ig/costimulator combination platform improves T cell priming and enhances immunity, memory, and tumor elimination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1732.