bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2998: Linear confined space migration induces treatment resistance and gene expression changes in glioblastoma multiforme
Authors: Babu Anish , McKernan Patrick , Cai Xue , Battiste James
Published: September 2020

Background: Glioblastoma Multiforme (GBM), an aggressive brain cancer, is known for its treatment resistant recurrence and lethality. The diffuse migration and invasion of GBM cells commonly occurs along brain white matter tracts in linear confined spaces between axons and has significant clinical implications as these cells survive initial therapies forming the main source of recurrent tumors resistant to therapies. In this context, this project aims to analyze the treatment resistance mechanism associated with linear confined space migrating cells (LCSM) to devise a better treatment strategy for GBM.

Methods: We used a combination of microfluidic technology (5 µM wide channels in PDMS block) emulating linear white matter tracts in brain and standard molecular analysis (confocal microscopy, high content imaging, western blot, and flow cytometry) to investigate LCSM cells compared to Standard Monolayer Culture cells (SMCs) for the mechanism of drug resistance. Both established and patient derived GBM cell lines were used in the study.

Results: LCSM cells were more resistant to chemo-drugs (Temozolomide and Doxorubicin) than SMCs as observed in cell viability assays. Enhanced drug efflux was observed in LCSM cells compared to SMC cells indicating the activation of drug efflux pumps. Protein expression analysis indicated that ABC drug efflux transporters (ABCB1, ABCC1, ABCG2) were activated or enhanced at varying levels in different LCSM cells compared to SMCs. Pharmacological inhibition of the prominently expressed ABCG2 protein using KO143 resulted in enhanced cell death by chemo drugs, suggesting that chemo resistance in LCSM, at least in part, is conferred by ABCG2 or other efflux transporters. In addition, CD133 (a marker of treatment resistant stem cell population in GBM) and AQP4 (Aquaporin 4; pro-migratory marker in GBM) were also enhanced in LCSM compared to SMC suggesting their possible roles in treatment resistance.

Conclusion: Preliminary studies reveal that the enhanced expression ABC transporters, prominently ABCG2, might be responsible for enhanced drug efflux and low therapeutic sensitivity in GBM- LCSM cells. The over expression of AQP4 and CD133, and differential expression of other genes suggest that more complex mechanisms might be involved, which are currently under investigation.

Citation Format: Anish Babu, Patrick McKernan, Xue Cai, James Battiste. Linear confined space migration induces treatment resistance and gene expression changes in glioblastoma multiforme [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 2998.
Abstract 3000: Evaluating the efficacy of a novel class of covalent microtubule stabilizers in taxane resistant ovarian cancer
Authors: Yee Samantha S.M. , Du Lin , Risinger April L.
Published: September 2020

Ovarian cancer has few targeted therapies and high rates of metastasis. Taxane microtubule stabilizers, including paclitaxel, are a mainstay in the treatment of adult solid tumors as single agents and in combination with other cytotoxic chemotherapies, targeted therapies, and immunotherapies. The standard of care for ovarian cancer patients comprises of combination chemotherapy consisting of a taxane with a platinum agent administered intravenously or intraperitoneally. Although this approach is highly effective initially, up to 70% of ovarian cancer patients will acquire taxane resistance. Therefore, it is essential to identify drugs that retain efficacy in clinically relevant models of taxane resistance, including those with elevated P-glycoprotein (Pgp) expression. The development of a microtubule stabilizer that retains efficacy in taxane resistant ovarian cancer would be optimal since microtubule disruption is proven to be an effective target in this disease. The taccalonolides are a novel class of covalent and irreversible microtubule stabilizers that circumvent clinically relevant forms of taxane resistance in vitro and in vivo breast cancer models. However, the taccalonolides have a narrow therapeutic window and short serum half-life that would benefit from a more targeted delivery. We hypothesized that localized intraperitoneal delivery of the taccalonolides in ovarian cancer models may be an optimal strategy for the treatment of intraperitoneally disseminated disease. We have found that the taccalonolides are less susceptible than paclitaxel to Pgp-mediated drug resistance in a human ovarian cancer cell line model. Additional experiments have demonstrated that the taccalonolides retain long-term antiproliferative and cytotoxic efficacy following acute treatment of human and murine ovarian cancer cells, demonstrating superior cellular persistence as compared to other classes of microtubule stabilizing drugs. We are currently evaluating the efficacy of the taccalonolides in ovarian cancer models using localized, intraperitoneal administration in a syngeneic and orthotopic murine model, which may be optimal for delivery of this irreversible microtubule stabilizer.

Citation Format: Samantha S.M. Yee, Lin Du, April L. Risinger. Evaluating the efficacy of a novel class of covalent microtubule stabilizers in taxane resistant ovarian 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 3000.
Abstract 3008: A new mechanism of drug resistance in cancer: Extracellular ATP-induced resistance through ATP internalization and upregulation of ATP-binding cassette multidrug transporters
Authors: Zhang Haiyun , Wang Xuan , Chen Xiaozhuo
Published: September 2020

Cancer is the second leading cause of death in the US. Multidrug resistance (MDR) and the resulting ineffectiveness of the drug treatment are responsible for most relapses of cancer and cancer-related death. One major mechanism of MDR is the efflux of multiple drugs mediated by ATP-binding Cassette (ABC) multidrug transporters. ABC transporters pump drugs with different structures and mechanisms of action out of cancer cells using ATP-provided energy. And ABC activities is also intimately linked to epithelial mesenchymal transition and metastasis. The opportunistic uptake of extracellular molecules has been named as a key emerging hallmark of cancer metabolism. Intratumoral extracellular ATP levels of various cancer types are 103 to 104 times higher than those in their corresponding normal tissues. Our previous studies have shown that extracellular ATP is internalized by cancer cell via macropinocytosis, and promotes cancer drug resistance by directly fueling ABC transporters and increase its drug efflux activities. However, whether extracellular ATP can induce change in the expression of ABC transporters and the underlying mechanisms remain unclear. Our results from quantitative reverse transcription PCR and flow cytometry show that extracellular ATP significantly alters mRNA levels and cell surface expression of ABC transporters. These results suggest that extracellular ATP has profound regulatory effects on ABC transporters and thus promotes drug resistance, linking the ATP-rich tumor microenvironment with cancer drug resistance. On-going studies include identification of mechanisms for the extracellular ATP-induced drug resistance via ABC transporters. The completion of the study will expand our understanding of the roles of extracellular ATP in cancer, and reveal new therapeutic targets for reducing drug resistance and enhancing anticancer efficacy of chemotherapeutics and targeted drugs.

Citation Format: Haiyun Zhang, Xuan Wang, Xiaozhuo Chen. A new mechanism of drug resistance in cancer: Extracellular ATP-induced resistance through ATP internalization and upregulation of ATP-binding cassette multidrug transporters [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 3008.
Abstract 3009: Targeting MDM2-amplified tumors with AMG-232 to overcome drug resistance, inflammatory pro-tumorigenic cytokine production and hyperprogression with immune checkpoint therapy
Authors: Sahin Ilyas , Zhang Shengliang , Zhou Lanlan , Dizon Don , Safran Howard , El-Deiry Wafik S.
Published: September 2020

Mouse double minute 2 homolog (MDM2) is E3 ubiquitin-protein ligase which facilitates proteasomal degradation of p53. MDM2 amplification has been reported in different tumor types and recently been implicated as a potential marker for accelerated tumor growth or hyperprogression in various types of cancer after receiving immune checkpoint inhibitors. Thus, targeting MDM2 might be a promising treatment strategy in the era of cancer immunotherapy especially for tumor with MDM2 amplification or overexpression that may be predisposed to hyperprogression after exposure to immune checkpoint therapy. To investigate the potential of targeting MDM2 strategy, we explored the role of inhibition of MDM2 by AMG-232, an investigational oral selective MDM2 inhibitor. MDM2-amplified and MDM2-non-amplified ovarian clear cell carcinoma cell lines carrying p53 wild-type were treated with AMG-232 and showed a dose-dependent reduction in cancer cell viability. MDM2 amplification showed more resistance to the treatment with AMG-232. Consistent with previous data, treatment with AMG-232 led to activation of the p53 signaling pathway in cancer cells in a dose-dependent manner. Importantly, the effect of MDM2 inhibition on cytokines expression was tested and significant reduction in Interleukin-6 (IL-6), a proinflammatory cytokine which promotes tumorigenesis by multiple signaling pathways, was observed in a dose-dependent manner. Ongoing experiments are examining the role of targeting MDM2 using AMG-232 in combination with immunotherapy, including the impact on killing of MDM2-amplified ovarian cancer cells by T-cells in the presence of immune checkpoint therapy. Overall, our data suggest a potential therapeutic role of targeting MDM2 especially in tumors with overexpression or amplification of MDM2 to overcome drug resistance including hyperprogression in the context of immune checkpoint therapy.

Citation Format: Ilyas Sahin, Shengliang Zhang, Lanlan Zhou, Don Dizon, Howard Safran, Wafik S. El-Deiry. Targeting MDM2-amplified tumors with AMG-232 to overcome drug resistance, inflammatory pro-tumorigenic cytokine production and hyperprogression with immune checkpoint therapy [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 3009.
Abstract 3012: ONC201 induces apoptosis in gastric adenocarcinoma and exhibits synergy with rhTRAIL
Authors: Parker Cassanda Susan , Zhou Lanlan , El-Deiry Wafik S.
Published: September 2020

Gastric adenocarcinoma is highly lethal with a 5-year survival of only 31.5%, largely due to late presentation of disease and ineffective chemotherapy options. ONC201 is a first-in-class small molecule anti-cancer therapy that induces cell death in many different types of malignant tumors via a multitude of mechanisms including up-regulation of TNF-related apoptosis-inducing ligand (TRAIL) by Foxo3a and death receptor 5 (DR5) via activation of the integrated stress response. We investigated the efficacy of ONC201 in inducing cell death in gastric adenocarcinoma cells as monotherapy and in combination with recombinant human TRAIL (rhTRAIL).

AGS (caspase 8 and PIK3CA mutant, HER2 amplified) and SNU-16 (p53 mutant) gastric adenocarcinoma cell lines were treated with ONC201 and rhTRAIL and viability assays were performed to determine cell line sensitivities. Both cell lines were then treated with combination therapy based on sensitivities and viability assays were performed in order to determine combination indices. Cell death was verified with sub-G1 analysis via flow cytometry and protein expression was established via western blotting.

SNU-16 cells were sensitive to ONC201, while AGS cells were shown to be resistant to monotherapy. Both cell lines were sensitive to rhTRAIL and exhibited synergy when treated in combination with ONC201, resulting in combination indices of less than 0.5 at doses that did not induce cell death in fibroblasts. The proportion of cells in sub-G1 phase of the cell cycle increased in a dose-dependent manner with combination treatment. PARP cleavage increased in a dose-dependent manner after combination therapy, indicating apoptosis and ATF4 and CHOP expression also increased with increasing doses of combination therapy, indicating activation of the integrated stress response.

These results suggest that ONC201 may be an effective therapeutic agent for gastric adenocarcinoma in combination with rhTRAIL by inducing apoptosis via the integrated stress response. We are currently expanding the in vitro model to include additional cell lines SNU-1(Ras mutant, microsatellite instability) and SNU-5 (p53

and CDH1 mutant). We are further exploring the role of ONC201 in priming cells via up-regulation of DR5 receptors such that they have increased sensitivity to rhTRAIL when pretreated with ONC201 as a potential source for the established synergy in gastric adenocarcinoma cells. We will also apply this study regimen to an organoid and in vivo model.

Citation Format: Cassanda Susan Parker, Lanlan Zhou, Wafik S. El-Deiry. ONC201 induces apoptosis in gastric adenocarcinoma and exhibits synergy with rhTRAIL [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 3012.
Abstract 304: Androgen deprivation induces epithelial to mesenchymal transition and neuroendocrine differentiation of prostate cancer via LIF-mediated phosphorylation of Smad2
Authors: Liu Yen-Nien , Lin Shian-Ren , Mokgautsi Ntlotlang , Yeh Hsiu-Lien , Huang Jiaoti
Published: September 2020

The current therapies for prostate cancer, which can either be androgen-sensitive or androgen-insensitive ones, are based on AR-targeting approaches; however, there is no available treatment for neuroendocrine prostate cancer (NEPC). We previously identified the leukemia inhibitory factor (LIF), which is stimulated by androgen deprivation therapy (ADT). Here, we further elucidated the physiological relevance of LIF, whereby the high LIF output is associated with the activation of epithelial-to-mesenchymal transition (EMT), and neuroendocrine differentiation of prostate cancer cells through stimulation of transforming growth factor (TGF)-β signaling. We have elucidated the effect of LIF on inducing the EMT and NEPC progression through the stimulated TGF-β signaling via phosphorylation of Smad2. We also demonstrated that the inverse relationship between inhibition of AR signaling and induction of LIF-Smad2 signaling may predispose prostate cancer to metastasis. Importantly, an established activation in prostate cancer cells was shown between recombinant LIF protein and phosphorylation of Smad2, supporting the connection between LIF and p-Smad2. This was supported by the results obtained in high-LIF samples with elevated expression of p-Smad2 in tissues from high-grade prostate cancer. This study highlights the role of the important crosstalk between the LIF and TGF-β signaling pathways may exist and provide a new direction for investigating NEPC development.

Citation Format: Yen-Nien Liu, Shian-Ren Lin, Ntlotlang Mokgautsi, Hsiu-Lien Yeh, Jiaoti Huang. Androgen deprivation induces epithelial to mesenchymal transition and neuroendocrine differentiation of prostate cancer via LIF-mediated phosphorylation of Smad2 [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 304.
Abstract 3051: A potent pyrimidinone Axl inhibitor DW17401
Authors: Zhang Hefeng , Peng Xia , Ji Yinchun , Dai Yang , Shen Yanyan , Ai Jing , Duan Wenhu
Published: September 2020

Aberrant signaling of Axl frequently occurs in many cancer types. Axl activation has been identified as a key trigger in epithelial to mesenchymal transition (EMT), which accounts for acquired drug resistance to many tyrosine kinase inhibitor therapies. It also has been revealed that Axl signaling is associated with immunosuppression. Therefore, Axl inhibitors may present a promising strategy for cancer therapeutics. We have designed and synthesized a series of pyrimidinone Axl inhibitors, and DW17401 was identified as a highly potent, orally available inhibitor against Axl, with IC50 values of 0.9 nM. DW17401 exhibited significant inhibitory effect against Axl driven cell proliferation, with an IC50 value of less than 0.02 nM for BaF3/TEL-AXL cell line. Oral dosing of DW17401 25 mg/kg (QD) resulted in complete tumor regressions in subcutaneous transplanted model of BaF3/TEL-AXL in nude mice. In addition, DW17401 could also inhibited c-Met kinase and c-Met-mediated tumor cell proliferation and growth with high potency, indicated it therapeutic potential in MET aberrant cancer. Of note, DW17401 presented favorable pharmacokinetic parameters on SD rat with a half-life time of 13.8 h and a plasma exposure value of 32647 ng.h/mL (p.o. at 3 mg/kg). All these data indicated that DW17401 was an effective promising Axl inhibitor for cancer treatment.

Citation Format: Hefeng Zhang, Xia Peng, Yinchun Ji, Yang Dai, Yanyan Shen, Jing Ai, Wenhu Duan. A potent pyrimidinone Axl inhibitor DW17401 [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 3051.
Abstract 3071: VERU-111, a novel orally bioavailable tubulin inhibitor, overcomes taxane resistance in metastatic triple-negative breast cancer
Authors: Deng Shanshan , Krutilina Raisa I. , Parke Deanna N. , Chen Hao , Miller Duane D. , Seagroves Tiffany N. , Li Wei
Published: September 2020

Triple-negative breast cancer (TNBC) has aggressive clinical features strongly associated with poorer overall prognosis and higher mortality rates relative to other molecular subtypes. FDA-approved drugs, such as paclitaxel, are effective in treating TNBC. Yet, treatment failure is commonly observed due to the development of acquired chemoresistance, which remains a clinical challenge for TNBC therapy. We have shown that orally administered VERU-111 inhibits MDA-MB-231 xenograft growth with similar efficacy to paclitaxel, but without acute toxicity, and represses metastases originating from the mammary fat pad. To better understand the effect of VERU-111 on taxane-resistant TNBC, we generated a panel of paclitaxel-resistant TNBC sublines (MDA-MB-231/TxR, MDA-MB-468/TxR, SUM159/TxR and Hs578T/TxR) by gradually exposing paclitaxel to parental lines. MTS assays were used to confirm the chemoresistance of each subline to paclitaxel. VERU-111 maintained its potency against all four taxane-resistant sublines. We further investigated the underlying mechanisms of VERU-111 action and found that it inhibited taxane-resistant TNBC cell proliferation by inducing mitotic arrest and apoptosis via disruption of microtubule stability. In vivo, relative to paclitaxel, VERU-111 significantly inhibited tumor growth in an orthotopic MDA-MB-231/TxR model and reduced lung metastases originating from the mammary fat pad. VERU-111 induced tumor necrosis, anti-angiogenesis and apoptosis in both taxane-sensitive and taxane-resistant TNBC models in vivo. In addition to the MDA-MB-231/TxR model, we also tested the efficacy of VERU-111 in a multidrug-resistant PDX model, HCI-10. VERU-111 significantly inhibited HCI-10 primary tumor growth and suppressed the growth of lymph node metastases present prior to therapy and suppressed lung, liver and kidney metastases at the endpoint. In contrast, paclitaxel had no efficacy to repress tumor growth or metastasis in this model, and in fact, stimulated liver metastasis. Taken together, our data demonstrate that VERU-111 is a new generation of oral tubulin inhibitor that potently inhibits the growth of taxane-sensitive and taxane-resistant TNBC. Therefore, VERU-111 therapy is a promising strategy for the treatment of metastatic TNBC patients, including those patients who progress on taxanes.

Citation Format: Shanshan Deng, Raisa I. Krutilina, Deanna N. Parke, Hao Chen, Duane D. Miller, Tiffany N. Seagroves, Wei Li. VERU-111, a novel orally bioavailable tubulin inhibitor, overcomes taxane resistance in metastatic triple-negative breast 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 3071.
Abstract 3126: Serum miR-4687-3p as the diagnostic biomarker for NSCLC
Authors: Liu Man , Ouyang Jun , Si Qiufang , Wang Yulin , Yang Ting , Zhang Xue , Jiang Di , Zhang Jianying , Dai Liping
Published: September 2020

Objective: The lack of useful biomarker contributes to the increased morbidity and mortality of non-small cell lung cancer (NSCLC) partly. However, biomarkers in blood have become increasingly appreciated in diagnosis of NSCLC. This study, we aimed to identify serum miRNAs for diagnosing NSCLC.

Methods: Microarray were used to detect 2549 miRNAs in serum from NSCLC patients (n=10) and healthy individuals (n=10) and gain differentially expressed miRNAs. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) assay were applied to validate the expression of selected miRNAs in validation set consisting of sera from 60 participants. Area under the receiver operating characteristic curve (AUC) was used to evaluate diagnostic accuracy of the chosen miRNAs. Tissue expression of the selected miRNA was analyzed based on TCGA database. Subsequently, the target gene of the miRNAs were predicted by TargetScan and Gene ontology (GO) and KEGG pathway enrichment analysis tested by R (clusterProfiler package). The effect of miR-4687-3p on proliferation, invasion, and metastasis of lung cancer cells were investigated through CCK-8 and Transwell assay.

Results: We identified serum miR-4687-3p that provided a high diagnostic accuracy of NSCLC (AUC=0.69, 95%CI: 0.543-0.815) in validation data set. According to TCGA database, we also found that the tissue miR-4687-3p level were significantly higher in NSCLC tissues than normal tissues (p<0.05). GO and KEGG pathway enrichment analysis showed that postsynaptic specialization and TGF-β signaling pathway were significantly enriched. Up-regulated miR-4687-3p by miR-4687-3p mimic could promote the proliferation, invasion, and metastasis of the NSCLC cells, compared with mimic negative control. Meanwhile, applying miR-4687-3p inhibitor could suppressed the proliferation, invasion, and metastasis of the NSCLC cells.

Conclusion: Our study on the first reported that serum miR-4687-3p levels may have clinical potentials as a novel non-invasive diagnostic biomarker for NSCLC patients.

Key words: NSCLC, miR-4687-3p, microarray, bioinformatics, biomarker

Citation Format: Man Liu, Jun Ouyang, Qiufang Si, Yulin Wang, Ting Yang, Xue Zhang, Di Jiang, Jianying Zhang, Liping Dai. Serum miR-4687-3p as the diagnostic biomarker for NSCLC [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 3126.
Abstract 3127: To investigate the role of BRAF V600E co-occurring mutations in Langerhans cell histiocytosis (LCH)
Authors: Karmakar Subhradip , Dhar R , Purkayastha K , Seth R , Karmakar S
Published: September 2020

a. Introduction :

Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia caused by alterations of several genes in the MAPKinase pathway. The cell of origin is a white blood cell known as a dendritic cell priviously called as histiocyte characterized by a unique cytoplasmic organelle, the Birbeck granule, and the expression of CD1a and class II major histocompatibility complex molecules . The mutations cause these dendritic cells to attract other white blood cells and cause a “lesion” in any organ of the body. LCH has multi-orgnan involvement affecting skin bones, lymph nodes, brain etc. Systemic symptoms may include fever, bone pain, weight loss, jaundice, diabetes insipidus.

b. Description:

LCH is a rare sporadic, non-hereditary and non-malignant disease with unknown etiology characterized with a clonal proliferation of pathologic cells.Recent discovery of recurrent somatic mutations, BRAFV600E in MAPK pathway genes at critical stages of myeloid hematopoietic differentiation in LCH patients supports redefinition of the disease as a myeloproliferative disorder and provides opportunities to develop novel approaches to diagnosis and therapy. In the present proposal, we intend to analyze the BRAFV600E status along with other co-occurring mutations in a cohort of pediatric LCH patients by DNA sequencing in bone marrow samples or Peripheral Blood. Additionally we approach the above unanswered questions by applying an improved machine learning approach for classification and defining an algorithm for disease into different clusters for immediate clinical implication and to address its clinical heterogeneity.

c. Results :

We screened about 5 LCH patients and performed tageted exome seq to asertain the mutational profile of these patients. Sequencing detected BRAF V600E mutation in 3 of 5 samples for a mutation frequency of 60%. IHC based immunofloroscence shows that the intensity of phospho-MEK and phospho-ERK staining shows no significant alterations samples with mutation in BRAF. We are currently investigating mutations in other members of MAK pathways

d. Conclusion :

BRAF-V600E mutations were detected in few of our LCH patients. Neverless seeing the disease heterogeneity, we expect additionall mutations. More studies are needed to investigate mutations in the MAPK pathways that could efffect the course of disease management.We further plan to integrate the clinical and genomic findings to design a predictive model for the disease outcome.

Citation Format: Subhradip Karmakar, R Dhar, K Purkayastha, R Seth, S Karmakar. To investigate the role of BRAF V600E co-occurring mutations in Langerhans cell histiocytosis (LCH) [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 3127.
Abstract 315: Multifaceted functional assessment of checkpoint inhibitor efficacy using 3D tumor spheroids
Authors: Lassahn Katy A. , Appleton Kathryn M. , Elrod Ashley , Carlson Aaron L. , DesRochers Teresa M.
Published: September 2020

Three-dimensional (3D) culture of immortalized cell lines and patient-derived primary cells has been shown to be a more representative in vitro model of tumor biology compared to standard 2D techniques. For immune-oncology, animal models continue to lack the ability to fully recapitulate the human immune system. The use of 3D models to study immunotherapies provides the opportunity to mimic the complex interactions between immune cells and the tumor microenvironment in a fully human system. However, standard well-based assays that measure cell viability prevent the obtainment of useful knowledge on tumor-immune cell interactions and immunotherapy effect on immune cell numbers and viability. To address these limitations, we have developed an in vitro based assay for the visualization and quantitation of T-cell-mediated cell death using fluorescently labeled live tumor cells in a 3D spheroid platform. Enhanced T-cell mediated tumor cell killing using immune checkpoint inhibitors can be detected allowing for real-time evaluation of tumor-specific apoptosis in the presence of cytotoxic T-cells. Moreover, the cytotoxic effects can be inhibited by co-treatment with a blocking anti-CD8 antibody, demonstrating a specific requirement for CD8+ T-cells. In order to detect changes to tumor biology induced by T-cells as well as immune-oncology drug treatments, we monitored dynamic fluctuation of PD-L1 and MHC-I expression using immunofluorescence in the spheroids. Finally, we detected changes in T-cell populations, such as T-cell activation, and changes in CD8+:Treg ratios, induced by tumor spheroids with or without drug treatment using flow cytometry. In summary, we can observe T-cell mediated tumor cell killing and relate drug efficacy back to identified changes in tumor cell and T-cell phenotypes. This multifaceted approach is ideal for the functional evaluation of preclinical I/O agents and for identifying drug combinations as it represents a holistic perspective of drug response.

Citation Format: Katy A. Lassahn, Kathryn M. Appleton, Ashley Elrod, Aaron L. Carlson, Teresa M. DesRochers. Multifaceted functional assessment of checkpoint inhibitor efficacy using 3D tumor spheroids [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 315.
Abstract 3163: Cell-based reporter bioassays for development of Fc-functional and Fc-silent SIRPa/CD47 checkpoint inhibitors
Authors: Mitchell Jonathan , Grailer Jamison , Hartnett Jim , Fan Frank , Cong Mei , Cheng Zhijie Jey
Published: September 2020

CD47, a membrane glycoprotein commonly overexpressed in human cancers, interacts with its cognate receptor SIRPα on myeloid cells to deliver a “don't eat me” signal that inhibits phagocytosis. This SIRPα/CD47 interaction has emerged as a critical and potentially druggable immune checkpoint. Blockade of SIRPα/CD47 interaction promotes phagocytosis of tumor cells in vitro, and SIRPα/CD47 inhibitors exhibit anti-tumor activity, including synergistic activity with other tumor-targeted mAbs (e.g. rituximab), in vivo. These encouraging pre-clinical data have sparked widespread development of SIRPα/CD47 inhibitors and driven a need for robust functional assays to measure the activity of these drug candidates. Biological activity of SIRPα/CD47 inhibitors is typically assessed via in vitro phagocytosis assays based on imaging or flow cytometry. These assays often use primary monocyte-derived macrophages and are inherently prone to donor variability, are low-throughput, and difficult to implement in drug development settings. Additionally, many SIRPα/CD47 inhibitors under development are designed to minimize Fc function to enhance safety and are thus incapable of driving phagocytosis directly. To overcome these limitations, we have developed a pair of reporter-based bioassays for measuring the biological activity of Fc-functional and Fc-silent SIRPα/CD47 inhibitors. These bioassays utilize a SIRPα-positive monocyte effector cell-line that expresses multiple Fc gamma receptors (FcγRs) and an FcγR-responsive NanoLuc luciferase reporter that is inhibited by SIRPα/CD47 interaction upon co-culture of SIRPα effector cells with CD47-positive target cells. Addition of CD47 inhibitors with Fc functional activity simultaneously disrupts the SIRPα/CD47 interaction and engages FcγRs on the SIRPα effector cells, resulting in NanoLuc reporter activation. To enable testing of Fc-silent inhibitors, we have engineered a second CD47 target cell-line which provides a constitutive activating stimulus to SIRPα effector cells that is restricted by SIRPα/CD47 interaction. Addition of Fc-silent SIRPα/CD47 blockers releases inhibition by SIRPα/CD47, resulting in NanoLuc reporter activation. These newly developed reporter bioassays provide a robust, high-throughput platform to facilitate discovery and development of diverse SIRPα/CD47 checkpoint inhibitors.

Citation Format: Jonathan Mitchell, Jamison Grailer, Jim Hartnett, Frank Fan, Mei Cong, Zhijie Jey Cheng. Cell-based reporter bioassays for development of Fc-functional and Fc-silent SIRPa/CD47 checkpoint inhibitors [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 3163.
Abstract 3173: Predictive biomarker evaluation for the anti-cancer imipridone ONC201
Authors: Morrow Sara , Prabhu Varun Vijay , Allen Joshua E. , Oster Wolfgang
Published: September 2020

Small molecule imipridone ONC201 is a selective, mixed competitive and non-competitive antagonist of dopamine receptor D2 (DRD2), a GPCR that is overexpressed in a broad range of malignancies (Madhukar et al, Nature Communications, 2019). ONC201 was recently found to also act as an allosteric agonist of the mitochondrial protease ClpP that degrades members of the electron transport chain and other mitochondrial substrates (Ishizawa et al, Cancer Cell 2019). ONC201 is currently being tested in several Phase II clinical trials with evidence of clinical activity in DRD2-dysregulated tumor types, including high-grade glioma, endometrial cancer, mantle cell lymphoma and adrenal tumors. Previously, we reported that DRD2 and DRD5 expression correlates with ONC201 efficacy in the NCI60 and GDSC panels (>1000 cancer cell lines) (Prabhu et al, Clinical Cancer Research, 2019). In this study, we explored additional predictive biomarkers of response for ONC201, including ClpP and known downstream effects of ONC201 across the NCI60 and GDSC cancer cell line panels. Elevated ClpP expression emerged as a top predictive biomarker for ONC201 anti-cancer efficacy (low IC50) in the NCI60 and GDSC (p = 0.0066 and p = 0.00071, respectively) panels. Upregulation of HIF1 expression is known to drive cancer cells toward glycolysis; conversely, its loss shunts cellular energetics toward oxidative phosphorylation as the primary mechanism of ATP generation (Clark et al, Cell, 2019) that is inhibited by ONC201 via ClpP activation. Accordingly, decreased HIF1a expression correlated with increased anti-cancer efficacy in the NCI60 (p = 0.058) and GDSC (p = 0.0021) panels. ONC201 is known to cause downstream activation of the integrated stress response pathway, triggering induction of ATF4 (Ishizawa et al, Science signaling, 2016). High baseline ATF4 expression correlated with lower ONC201 IC50s in the GDSC panel (p = 0.027). Proteasomal degradation of c-myc by ONC201 has been reported in association with DRD2 antagonism and inhibition of oxidative phosphorylation in GBM (Ishida et al Clinical Cancer Research, 2018). High c-myc expression correlated with lower ONC201 IC50s in the GDSC panel (p = 2.5e-6). Ongoing studies are investigating biomarker combinations and identifying tumor types that are enriched for these predictive biomarkers. In conclusion, a series of target and downstream signaling biomarkers can be used to predict innate ONC201 tumor cell sensitivity that may provide guidance for patient and tumor type selection in future ONC201 clinical trials.

Citation Format: Sara Morrow, Varun Vijay Prabhu, Joshua E. Allen, Wolfgang Oster. Predictive biomarker evaluation for the anti-cancer imipridone ONC201 [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 3173.
Abstract 3177: Panel-derived tumor mutational burden (TMB) is associated with the response to the immune checkpoint inhibitors (ICIs) in urothelial cancers
Authors: Hsieh Yi-Lin , Yu Pei-Ning , Jan Yi-Hua , Lai Meng-Shao , Wang Woei-Fuh , Zhuo De-Wei , Chen Shu-Jen , Cheng Jen-Hao , Tan Kien Thiam , Su Yu-Li
Published: September 2020

Background: Tumor mutational burden (TMB) has been emerging as a relatively new biomarker that is independent of PDL1 for the prediction of response to the immune checkpoint inhibitor (ICI) treatment. A recent study has shown that whole exome sequencing (WES)-derived TMB correlates well with panel-derived TMB that is estimated using targeted sequencing. Here, we evaluate the correlation between panel-derived TMB with response to ICI treatment in urothelial cancers.

Methods: FFPE tumor tissues from 30 patients with urothelial cancers who had previously received ICI as monotherapies or combination with chemotherapies at Chang Gung Memorial Hospital were retrospectively underwent targeted next-generation sequencing (ACTOncoTM) for the identification of nonsynonymous variants across 440 cancer- associated genes. Tumor mutational burden (TMB) was calculated by using the sequenced regions of ACTOncoTM to estimate the number of somatic nonsynonymous mutations per megabase of all protein-coding genes. The TMB calculation predicted somatic variants and applied a machine learning model with a cancer hotspot correction. RECIST criteria were used to categorize tumor response to the treatment.

Results: Patients were defined as responders (CR/PR) and non-responders (SD/PD) according to the RECIST evaluation. In the cohort receiving ICI monotherapy, the responders (CR/PR, n=8) had significantly higher TMB than non-responders (SD/PD, n=15) (Median 16.2 muts/Mb vs. 6.5 muts/Mb, p=0.0035). One patient with moderate TMB (11.0 muts/Mb) who exhibited disease progression following ICI therapy harbored biallelic JAK2 inactivation. Three high-TMB tumors exhibited mutational signature of aristolochic acid exposure.

Conclusions: Although the cohort size is small, this study showed that panel-derived TMB can serve as an independent predictive biomarker to identify urothelial cancer patients for immune checkpoint inhibitor therapy.

Citation Format: Yi-Lin Hsieh, Pei-Ning Yu, Yi-Hua Jan, Meng-Shao Lai, Woei-Fuh Wang, De-Wei Zhuo, Shu-Jen Chen, Jen-Hao Cheng, Kien Thiam Tan, Yu-Li Su. Panel-derived tumor mutational burden (TMB) is associated with the response to the immune checkpoint inhibitors (ICIs) in urothelial 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 3177.
Abstract 319: Impact of chemotherapy on lung cancer cells and their microenvironment, evaluated in tridimensional multicellular tumor spheroids
Authors: Hulo Pauline , Deshayes Sophie , Dehame Virginie , Grégoire Marc , Bennouna Jaafar , Blanquart Christophe
Published: September 2020

Introduction: Pembrolizumab, an anti-PD1 antibody, is approved in non pretreated advanced Non Small Cell Lung Cancer (NSCLC) with high expression of PD-L1, more than 50 %, on tumor cells. Conversely, the combination of platinum-based chemotherapy + pembrolizumab in this indication is not restricted to the level of PD-L1 expression. This suggests that chemotherapy could sensitize tumor tissue to Immune Checkpoint Inhibitors (ICIs). Based on those clinical datas, we propose to evaluate the impact of chemotherapy on tumor cells and on their microenvironment by using tridimensional multicellular tumor spheroids (MCTS).

Material and methods: MCTS integrated lung adenocarcinoma cells expressing GFP, fibroblasts with ruby fluorescent protein and monocytes/macrophages from healthy donors. MCTS were treated with repeated and combined chemotherapy drugs (carboplatin and gemcitabine, or carboplatin and paclitaxel), reproducing therapeutic schedules. Different biological factors were analysed : impact of chemotherapy on MCTS morphology, cell viability, invasion abilities, modifications in genes and protein expression profiles using differential gene expression sequencing (DGE-Seq) and flow cytometry, respectively.

Results: Chemotherapy decreased MCTS size and cell viability. Whereas carboplatin/gemcitabine mainly affected lung cancer cells inside MCTS, carboplatin/paclitaxel seemed to be cytotoxic mainly on peripheral malignant cells. Fibroblasts were resistant to chemotherapy, unlike macrophages which were strongly affected. A large RNA analysis was performed using DGE-Seq and showed that expression of several genes, and notably of CD274 (PD-L1) and CD273 (PD-L2), were induced after treatment. We confirmed PD-L1 up-regulation byflow cytometry.

Conclusion: Platinium-based chemotherapy up-regulate immune checkpoints expression in lung cancer cells. Our results are in line with the latest studies performed in mice. They underline that our MCTS model is closed to in vivo conditions. It could be developedas a drug screening tool.

Citation Format: Pauline Hulo, Sophie Deshayes, Virginie Dehame, Marc Grégoire, Jaafar Bennouna, Christophe Blanquart. Impact of chemotherapy on lung cancer cells and their microenvironment, evaluated in tridimensional multicellular tumor spheroids [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 319.
Abstract 3193: FGFR4 mutationas a novel biomarker for immunotherapy in patients with non-small cell lung cancer
Authors: Jin Xiangfeng , Fan Xinglong , Tian Kaihua , Xu Hanlin , Ge Nan , Wang Yafei , Zhang Shiyue , Chen Hui , Yuan Shaohua , Wang Kai
Published: September 2020

Background: Cancer immunotherapy, especially immune checkpoint inhibitors (ICIs), has provided a remarkable antitumor effect in non-small cell lung cancer (NSCLC), but only a limited number of patients can derive durable benefit. Therefore, more accurate biomarkers are highly needed. The fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases is comprised of 4 members (FGFR1-4) that mediate the function of the FGFR ligand family. Although the genomic alterations (GAs) of FGFRs were frequently detected by targeted next-generation sequencing (NGS) and considered as potent oncogenes, their correlation with recognized immune biomarkers and predictive value for ICIs response in NSCLC remains unclear.

Methods: Formalin-fixed, paraffin-embedded (FFPE) tumor and matched blood samples of 3433 NSCLC patients from OrigiMed were collected for targeted NGS panel sequencing from December 2017 to January 2019. GAs including single nucleotide variations, short and long insertions/deletions, copy number variations, and gene rearrangements were assessed. Genomic data and ICIs treatment outcome of 2 cohorts of NSCLC patients were derived from The Cancer Genome Atlas (TCGA) and Memorial Sloan Kettering Cancer Center (MSKCC) databases.

Results: GAs of FGFR gene family were detected in 5% (172/3433) of patients with NSCLC in the OrigiMed database, including 84 FGFR1 GAs, 34 FGFR4 GAs, 28 FGFR2 GAs, and 26 FGFR3 GAs. No significant difference was observed in the frequency of FGFR4 GAs within the histologic subtypes. Compared with wild-type (WT), FGFR4 GAs were associated with a significantly higher tumor mutational burden (TMB) (6.55 vs. 4.6 muts/Mb, respectively, p<0.001). Survival analysis from 2 independent cohorts confirmed that patients with FGFR4 GAs had remarkable clinical benefit to ICIs compared with WT patients in both progression free survival (PFS) (13.17 months vs 3.17 months, respectively, p=0.025) and durable clinical benefit (DCB) (80% vs 29.3%, respectively, p=0.03). Furthermore, FGFR4 GAs were an independent risk factor of PFS (HR: 0.22, 95%CI: 0.055-0.92, p=0.037) and overall survival (OS) (HR: 0.23, 95%CI: 0.057-0.92, p=0.038).

Conclusion: Our results supported that the FGFR4-mutated NSCLC patients were associated with an increased TMB and favorable response to ICIs. This suggests that GAs of FGFR4 may have implications as a biomarker for guiding ICI treatment.

Citation Format: Xiangfeng Jin, Xinglong Fan, Kaihua Tian, Hanlin Xu, Nan Ge, Yafei Wang, Shiyue Zhang, Hui Chen, Shaohua Yuan, Kai Wang. FGFR4 mutationas a novel biomarker for immunotherapy in patients with non-small cell lung 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 3193.
Abstract 3223: The Cancer Genomics Cloud enables complex and multi-omic data science in the cloud
Authors: Ray Manisha , DiGiovanna Jack , Radenkovic Jelena , Tosic Marko , Mirkovic Nikola , Majic Boris , Andjus Vladan , Davis-Dusenbery Brandi
Published: September 2020

The Cancer Genomics Cloud (CGC), powered by Seven Bridges, is an NCI-funded platform to make the analysis of cancer data faster, easier, and more accessible. As datasets grow larger, more diverse, and more complex, they have become increasingly challenging for many cancer researchers. The CGC provides a centralized resource for analysis by co-localizing three components within the same cloud platform: 1) large cancer datasets like The Cancer Genome Atlas (TCGA); 2) tools and workflows for analyzing public and private data; and 3) the computational capabilities to do large-scale analyses. In addition to the simplicity of data access; the CGC also contains >400 of best-practice workflows, the flexibility to bring private tools and notebooks, and the ability to complete interactive analyses, all with the speed of cloud computing. The number of available datasets and tools are rapidly increasing, connecting researchers to datasets from a wide range of sources and data types, such as proteomics and imaging.

The platform has been continuously expanded to include new applications and features since its launch in 2016. Here we describe how new features have been implemented to broaden the data and workflows accessible to cancer researchers. First, we have integrated with several new nodes within the Cancer Research Data Commons to enable access and analysis on proteomics, canine, and other cancer datasets, which alongside with the genomics data on the CGC, enables true multi-omic analysis. Second, we have expanded our infrastructure to enable computation on both Google and Amazon cloud environments, allowing for analysis of data held in either environment. We have also added support for RStudio within the CGC, along with Python and Julia notebooks. Researchers can complete their entire workflow on the platform, from data discovery through visualizations, while streamlining collaboration and speeding the time from hypothesis to conclusion. Altogether, these added features enable a network of Findable, Accessible, Interoperable, and Reusable (FAIR) datasets towards making cancer data analysis faster, easier, and more accessible for all.

Citation Format: Manisha Ray, Jack DiGiovanna, Jelena Radenkovic, Marko Tosic, Nikola Mirkovic, Boris Majic, Vladan Andjus, Brandi Davis-Dusenbery. The Cancer Genomics Cloud enables complex and multi-omic data science in the cloud [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 3223.