bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2078: Mitochondrial DNA copy number correlates with trifluridine sensitivity of human tumor cell lines
Authors: Kobunai Takashi , Matsuoka Kazuaki , Nukatsuka Mamoru , Tsukihara Hiroshi , Takechi Teiji
Published: August 2019

Background: Trifluridine (FTD) is a key component of the novel oral antitumor drug trifluridine/tipiracil, which was approved for the treatment of patients with metastatic colorectal cancer refractory to standard chemotherapies. FTD, an antineoplastic thymidine analogue, is incorporated into the genomic DNA of tumor cells. However no detailed analysis of the relation between mitochondrial DNA (mtDNA) in human tumors and FTD’s cytotoxic effects has been reported yet. In this study, we analyzed mtDNA copy number of 23 human cancer cell lines and its relation with FTD sensitivity.

Method: Twenty-three human cancer cell lines, representing the nine main tumor types, were tested for sensitivity to FTD by WST-8 assay. The cell lines included lung (NCI-H226, NCI-H23), colon (COLO 205, HCT 116, HCT-15, SW620), pancreas (PANC-1), breast (Hs 578T, MCF7, MDA-MB-231, T-47D), melanoma (Malme-3M, SK-MEL-28, SK-MEL-5), prostate (DU 145, PC-3), renal (786-O, A-498), leukemia (CCRF-CEM, HL-60, MOLT-4), and ovary (OVCAR-3, SK-OV-3). Genomic DNA was purified using a silica membrane spin column-based centrifugation procedure. The copy number of mtDNA was quantitated by array-based digital PCR and real-time PCR. Furthermore, the mtDNA heterogeneity of each cell line was assessed by deep-sequencing as follows: a DNA panel targeting 222 amplicons covering the entire mitochondrial genome was used for NGS library preparation; NGS libraries were subjected to cluster generation in a flow cell and paired-end sequencing for 300 cycles; and finally, the resulting fastq files were uploaded to mtDNA-server for mapping and heteroplasmy evaluation.

Results: The IC50s of FTD ranged from 0.6 uM to 52 uM (Median: 5.6 uM). While cell lines were arbitrarily classified as FTD-high- and FTD-low sensitive cell lines according to their respective IC50 values at median, mtDNA copy numbers of FTD-high sensitive cell lines were significantly lower than those of FTD-low sensitive cell lines (p = 0.01). Median heteroplasmy level and counts were 0.057 and 137, respectively. Interestingly, significant negative correlation was found between mtDNA copy number and heteroplasmy level. (Spearman’s Rho = -0.46, p= 0.02). While 6 out of 23 cell lines were microsatellite instable, MSI status also significantly correlated with mtDNA copy number, FTD-sensitivity, and heteroplasmy level (p< 0.05).

Conclusions: Integrated analysis of mtDNA copy number and mutational profiling may be useful to predict FTD sensitivity of cancer cells.

Citation Format: Takashi Kobunai, Kazuaki Matsuoka, Mamoru Nukatsuka, Hiroshi Tsukihara, Teiji Takechi. Mitochondrial DNA copy number correlates with trifluridine sensitivity of human tumor cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2078.
Abstract 2087: Mechanistic studies reveal novel molecular insights on anticancer properties of F10 compound in human colon cancer cells
Authors: Mani Chinnadurai , Gmeiner William , Palle Komaraiah
Published: August 2019

Fluoropyrimidine (FP) drugs, mainly 5-Fluorouracil (5-FU), constitute the backbone of combination chemotherapy regimens (e.g. FOLFOX and FOLFIRI) for treating colorectal cancer (CRC) and provide a survival benefit for patients with stage II, III, and IV colorectal cancer. However, 5 year survival rate for CRC patients with metastatic disease is less than 10%. This dreadful situation suggests urgent need for novel and more potent FPs to improve the outcomes of these patients. We have developed novel fluoropyrimidine drug polymers (e.g. F10) that show promising antitumor activity in multiple pre-clinical models even while displaying reduced systemic toxicities. F10 is taken up by malignant cells via active transport compared to 5-Fluorouracil, which is taken up by diffusion in malignant and non-malignant cells. Additionally, F10 compound is primarily incorporated into DNA, and produced thymidylate synthase and topoisomerase toxic metabolites at greater levels, whereas 5-Fluorouracil is primarily incorporated into RNA, and only has about 5-10% production of active form of thymidylate synthase toxic metabolites. MMR is involved in repairing the DNA mismatches and FP drugs induced DNA damage. Interestingly, our studies show that F10 acts by different mechanisms compared to 5-FU. Particularly, F10 treated CRC cells exhibit replication stress as evidenced by paused and slow progression of replication forks and R-loops formation at the active transcriptional regions, suggesting replication and transcription dependent generation of DNA lesions in these cells. F10 treatment induced several fold increased DNA double strand breaks (DSB) as measured by COMET assays and about 1000 folds more cytotoxicity to CRC cells compared to 5-FU. Similarly, F10 treatment activated distinctive DNA damage responses in MMR proficient CRC cells compared MMR deficient cells. Additionally, our studies suggest F10 as a promising therapeutic agent for CRC therapy either alone or in combination with CHK1 and PARP inhibitors.

Citation Format: Chinnadurai Mani, William Gmeiner, Komaraiah Palle. Mechanistic studies reveal novel molecular insights on anticancer properties of F10 compound in human colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2087.
Abstract 2092: Metabolic approach to EGFR-targeted therapy in non-small cell lung cancer
Authors: Kam Yoonseok , Swain Pamela , Romero Natalia , Dranka Brian P.
Published: August 2019

Mutation of the epidermal growth factor receptor (EGFR) is a major genetic driver of non-small cell lung cancer (NSCLC). While first-line tyrosine-kinase inhibitors (TKIs) have improved patient survival, many patients eventually develop resistance to these drugs. Combination therapies are now being researched to overcome this resistance. Pathways that may synergize with EGFR signaling are of interest since a liability created with one compound may then be exploited by a second. It is well known that glycolysis is controlled by EGFR signaling in cancer cells. However, the interplay of cell metabolism and EGFR inhibitors is not fully understood. Using PC9 cells which have known constitutive EGFR activation, we examined the relative poise of ATP production from mitochondria and glycolysis. In response to any of four different TKIs: afatinib, CO-1686, dacomitinib and erlotinib, a rapid decrease in glycolytic activity was induced. However, the total ATP production rates were not changed significantly as a result of increased mitochondrial ATP production. We next examined the mitochondrial fuel sources which support this increase by selectively blocking individual fuel pathways. In the presence of either a mitochondrial pyruvate carrier or glutaminase inhibitor, total ATP production rate decreased, and cell viability followed. Taken together, these data suggest that a combinational application of EGFR-targeted therapy and mitochondrial-targeted therapy may provide metabolic stress to cancer cells. Further study is required to understand the interplay of the tumor microenvironment and metabolic niches that may be found therein.

Citation Format: Yoonseok Kam, Pamela Swain, Natalia Romero, Brian P. Dranka. Metabolic approach to EGFR-targeted therapy in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2092.
Abstract 2108: Chromatin state as a mechanism of anthracycline response in breast cancer
Authors: Seoane Jose A. , Kirkland Jacob G. , Caswell-Jin Jennifer L. , Crabtree Gerald R. , Curtis Christina
Published: August 2019

The aim of this study is to determine the effect of chromatin regulators on anthracycline response in breast cancer. Anthracyclines are a highly effective component of curative breast cancer chemotherapy, but can be associated with substantial side effects. Because anthracyclines work in part via inhibition of topoisomerase II (TOP2) on accessible DNA, we hypothesized that expression levels of chromatin regulatory proteins that mediate DNA accessibility might predict anthracycline response. We used RNA-sequencing data from the TCGA breast cancer cohort to infer a breast cancer chromatin regulatory network using the ARACNE algorithm. A panel of 45 breast cancer cell lines with accompanying gene expression and growth inhibitory (GI50) data was used to build a genome-wide signature of anthracycline response. Based on the GI50, cell lines were classified as sensitive or resistant. The VIPER algorithm was then used to identify chromatin regulatory genes (CRGs) from the ARACNE network whose targets were significantly enriched in the anthracycline resistance signature. We further compiled a metacohort of 1006 clinically annotated early-stage breast cancer patients with gene expression data, allowing us to identify CRGs whose expression levels were associated with outcome after adjuvant treatment with anthracyclines. 24 CRGs associated (P<0.1) with anthracycline response in vitro and 54 CRGs associated (P<0.05) with anthracycline response in the patient cohort, 9 of which overlapped with the in vitro CRGs. In the patient cohort, CRG expression associated strongly with anthracycline response in all major subgroups of breast cancer (ER+/HER2-, HER2+, and triple-negative). In general, CRGs that promoted open chromatin (BAF and COMPASS) were, at high levels of expression, associated with anthracycline sensitivity, while CRGs associated with closed chromatin (PRC2) were, at high levels of expression, associated with anthracycline resistance. We functionally validated KDM4B as a predictor of anthracycline, but not taxane, sensitivity in a breast cancer cell line model through shRNA knockdown. We present a set of CRGs that control the chromatin accessibility in breast cancer both in vitro and in vivo; we infer that those tumors with more open chromatin as determined by the expression of these CRGs are intrinsically more sensitive to anthracyclines, while those with more closed chormatin are more resistant. Future work into the predictive power of these CRGs may allow us to target anthracyclines to only those patients who will benefit.

Citation Format: Jose A. Seoane, Jacob G. Kirkland, Jennifer L. Caswell-Jin, Gerald R. Crabtree, Christina Curtis. Chromatin state as a mechanism of anthracycline response in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2108.
Abstract 2111: Increased heme influx dictates tumorigenic functions of non-small cell lung cancer cells
Authors: Sohoni Sagar Shashikant , Ghosh Poorva , Zhang Li
Published: August 2019

Heme, iron protoporphyrin IX, is a central metabolic and signaling molecule that regulates diverse molecular and cellular processes relating to oxygen utilization and metabolism. Heme serves as a cofactor or a prosthetic group for complexes II, III, and IV in electron transport chain. Hence, it has a direct role in oxidative phosphorylation and energy generation in cells. Most human cells can synthesize and uptake heme from circulation. A number of epidemiological studies have shown that high heme intake is associated with an increased risk of cancer, including lung cancer. Our lab has previously shown that an array of non-small cell lung cancer cells (NSCLCs) exhibit high heme synthesis and uptake. This increased heme flux correlates with an enhanced oxygen consumption and ATP production. NSCLCs also exhibited significantly elevated levels of hemoproteins and mitochondrial biogenesis proteins compared to normal lung cell lines. Expression levels of these proteins correlate with increased oxygen consumption and the total heme content of NSCLCs suggesting a possible role of heme in energy metabolism and mitochondrial biogenesis.

In order to assess the involvement of heme in tumorigenicity of the NSCLC cell lines, we overexpressed heme synthesis enzyme, ALAS1 or heme transporters, HCP1 or HRG1 using lentivirus system. NSCLC cell lines A549 and H1299 were infected with a lentivirus carrying ALAS1 or HCP1 or HRG1 plasmid. Stable clones were obtained with antibiotic selection and checked for overexpression with western-blots. Cells overexpressing ALAS1, HCP1 and HRG1 showed significantly higher rates of heme influx as well as intensified oxygen consumption and ATP formation compared to eGFP controls. Tumorigenic properties such as invasion, migration and colony formation were also found to be significantly increased in these cell lines compared to their eGFP counterparts. Subcutaneous xenografts generated with overexpression cell lines also showed significantly high tumor growth rates and mass compared to eGFP controls. Our data from both in vitro and in vivo experiments show that heme directly improves the tumorigenic properties of NSCLC cells by substantially improving the respiration rates and energy metabolism.

Citation Format: Sagar Shashikant Sohoni, Poorva Ghosh, Li Zhang. Increased heme influx dictates tumorigenic functions of non-small cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2111.
Abstract 2115: FOS contributes to 5-FU resistance and reversal effects of natural flavonoid GL-V9 in colon cancer
Authors: Zhao Li , Zhaohe Li , Youxiang Ding
Published: August 2019

5-FU is widely used in the treatment of colon cancers. Although 5-FU can improve response rates and survival, response rates for 5-FU-based chemotherapy as a first-line treatment for advanced colorectal cancer are only 10-15%. Meanwhile, cancer drug resistance often occurs, which is an important reason for poor treatment effect of 5-FU. Therefore, it is significant to uncover the mechanisms underlying 5-FU resistance and develop effective but nontoxic agent candidates for reversing cancer drug resistance. Here, we found that proto-oncogene FOS was activated in 5-FU-resistant colon cancer cells compared to parental cells. Additionally, FOS overexpression using lentivirus plasmid obviously triggered 5-FU resistance in colon cancer cells. Moreover, by analyzing two GEO datasets, we also found that FOS expression was associated with poor overall survival of colon cancer. Taken together, FOS confers 5-FU resistance to colon cancer and it is potential to be exploited as a therapeutical target. Lastly, we studied the reversal effects of natural flavonoid GL-V9 on the FOS activation and 5-FU resistance in order to develop candidates for FOS inhibitor which would be helpful for 5-FU-resistant colon cancer. Consequently, nontoxic doses of GL-V9 could significantly suppress activation of FOS and sensitize colon cancer cells to 5-FU. Therefore, the flavonoid is potential to be exploited as a synergetic agent for 5-FU-resistant colon cancer.

Note: This abstract was not presented at the meeting.

Citation Format: Li Zhao, Li Zhaohe, Ding Youxiang. FOS contributes to 5-FU resistance and reversal effects of natural flavonoid GL-V9 in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2115.
Abstract 2125: Genetic analysis for classification and treatment of poorly differentiated sinonasal cancer
Authors: Hermsen Mario , Riobello Cristina , Cabal Virginia N. , García-Marín Rocío , Potes-Ares Sira , Suárez-Fernández Laura , Vivanco Blanca , Franchi Alessandro , Büttner Reinhard , López Fernando , Llorente José Luis
Published: August 2019

Introduction: The sinonasal cavities harbour a wide variety of rare tumour types, including squamous-cell carcinoma (SNSCC), intestinal-type adenocarcinoma (ITAC), olfactory neuroblastoma (ONB), neuroendocrine carcinoma (SNEC), undifferentiated carcinoma (SNUC) and malignant mucosal melanoma (MMM). Histopathological classification is related to clinical outcome, but correct diagnosis is difficult in cases with poor differentiation. Five-year overall survival ranges from 20% to 60% while therapeutic options are limited. This study aims to identify genetic alterations that may aid diagnosis and indicate targets for modern specific therapies.

Experimental procedures: DNA was extracted from 80 frozen or paraffin-embedded tumor tissues collected from three different hospital centers. Mutations were screened by next generation sequencing of a panel of 120 cancer-related genes using the SureSelect QXT Target Enrichment Kit for Ilumina Multiplexed Sequencing, and recurrent hits were confirmed by Sanger sequencing and by analyzing their effect on protein expression.

Results: Frequently mutated genes were TP53, KRAS, PI3K, NTRK1, ATM and BRCA1 and BRCA2. Several mutations were found exclusive to only one sinonasal tumor-type: APC and B-catenin in ITAC, EGFR exon 20 in SNSCC, IDH2 in SNUC and NF1 in MMM. With exception of ONB, a majority of cases showed a high mutational burden.

Conclusions: A number of tumor-specific mutations were identified that may be used for differential diagnosis. In addition, many gene mutations may serve as targets for specific antibody or small molecule inhibitors. Finally, the finding that sinonasal tumors carry a high mutational burden suggests that therapy with immune checkpoint inhibitors may be effective in clinical management.

Citation Format: Mario Hermsen, Cristina Riobello, Virginia N. Cabal, Rocío García-Marín, Sira Potes-Ares, Laura Suárez-Fernández, Blanca Vivanco, Alessandro Franchi, Reinhard Büttner, Fernando López, José Luis Llorente. Genetic analysis for classification and treatment of poorly differentiated sinonasal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2125.
Abstract 2129: CPTAC: Biospecimen accrual for proteogenomics
Authors: Thiagarajan Mathangi
Published: August 2019

The Clinical Proteomics Tumor Analysis Consortium (CPTAC) is a National Cancer Institute initiative that seeks to uncover the molecular basis of cancer using a proteogenomic approach to study prospective cancer specimens from treatment naïve patients. Leidos Biomed provides an infrastructure for supporting the collection of high quality biospecimens specific to proteogenomics and the corresponding clinical data, in addition to project and subcontract management for the program. The proportion of biospecimens so far from racial and ethnic minority patients are underrepresented, which preclude equitable research across all patient groups for cancer treatment. CPTAC program seeks diversity in the accrual cohort and an effort is ongoing to encourage the participation of tissue source sites serving racial and ethnic minorities that can contribute high quality biospecimens towards the collection. CPTAC applies the understanding of the molecular basis of cancer to identify biomarker candidates. CPTAC Phase II completed in 2016, collecting over 350 cases from breast, colon and ovarian patients. CPTAC Phase III that is in progress began to collect and analyze 200 cases from each of ten additional cancers. Analysis is close to completion for uterine corpus endometrial carcinoma, kidney clear cell renal cell carcinoma and lung adenocarcinoma while accruals are ongoing for 7 other cancer types. The study entails collection and pathology evaluation of biospecimens, high-quality clinical data and images from clinical sites from around the world. A biorepository evaluates and processes the biospecimens, sending nucleic acids to a sequencing center and tissues to proteomics groups. Data are combined and analyzed by analysis and translational centers. Genomic data are made available to the research community through the NCI Genomic Data Commons(GDC). Proteomic data are made available through CPTAC’s Data Coordinating Center(DCC). Imaging data are made available through The Cancer Imaging Archive (TCIA). The program is expanding and initiating biospecimen collection for proteogenomic analysis of ten additional rare tumor types.

Citation Format: Mathangi Thiagarajan. CPTAC: Biospecimen accrual for proteogenomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2129.
Abstract 2134: Niraparib combined with abiraterone acetate inhibits the growth of BRCA2wt prostate tumors
Authors: Damle Rajendra N. , Hawkins Rebecca , Hosbach Jennifer , Ndikuyeze Georges Habineza , Driscoll Jenny , Fulton Natalie L. , Derosa David , Hughes Anna , Chu Gerald , Calara-Nielsen Karl , Smirnov Denis , Shen Dong , Snyder Linda A.
Published: August 2019

Patients with metastatic prostate cancer may initially respond well to first- and second-generation androgen receptor (AR)-targeted therapies, but many will experience disease progression, indicating a need for alternative therapies. A subset of these patients bears prostate tumors that harbor deleterious mutations in DNA damage repair (DDR) pathway genes, such as BRCA2. These DDR mutations render the tumors sensitive to drugs that inhibit poly (ADP-ribose) [PAR] polymerase-1 (PARP1), inducing synthetic lethality. Studies indicate that there are interactions between AR signaling pathways and DDR pathways in prostate cancer cells, supporting the hypothesis that dual AR/PARP inhibition could inhibit prostate tumor growth more potently than single agents, regardless of the DDR status of the tumor. This hypothesis was tested using well-known human and mouse prostate tumor models that are sensitive to androgen deprivation. Genotyping of the models confirmed that none of them carried homozygous pathogenic mutations in DDR genes, including BRCA1, BRCA2, and ATM. When the cell lines were incubated in vitro with niraparib, a potent and highly selective PARP1/2 inhibitor, PAR formation was significantly inhibited, and there was induction of γH2AX in treated cells, indicating the drug induced DNA double-stranded breaks. In addition, the cytotoxicity IC50 values for niraparib were in the range of 1-10 µM, as expected for cells that are DDRwt. In vivo efficacy studies demonstrated that the VCaP and Myc-CaP tumor models were insensitive or modestly sensitive to single agent treatment with niraparib or abiraterone acetate (AA). In contrast, the combination treatment of niraparib plus AA significantly reduced tumor growth and increased survival (p<0.05) as compared with control or single agent arms. Studies are ongoing to understand the mechanisms of tumor growth control mediated by the combination of these two agents. The results support the hypothesis that prostate tumors may respond to a combination treatment with niraparib, a PARP inhibitor, and AA, an androgen synthesis inhibitor, even in the absence of DDR anomalies. A Phase 3 study is planned to evaluate this hypothesis and the safety and efficacy of this combination in humans.

Citation Format: Rajendra N. Damle, Rebecca Hawkins, Jennifer Hosbach, Georges Habineza Ndikuyeze, Jenny Driscoll, Natalie L. Fulton, David Derosa, Anna Hughes, Gerald Chu, Karl Calara-Nielsen, Denis Smirnov, Dong Shen, Linda A. Snyder. Niraparib combined with abiraterone acetate inhibits the growth of BRCA2wt prostate tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2134.
Abstract 2136: Doublecortin-like Kinase 1 (DCLK1) correlates with the cell proliferation in malignant pleural mesothelioma
Authors: Miura Akihiro , Yamamoto Hiromasa , Tao Hiroyuki , Suzawa Ken , Miyauchi Syunsaku , Araki Kota , Takahashi Yuta , Kurihara Eisuke , Ogoshi Yusuke , Shien Kazuhiko , Soh Junichi , Okabe Kazunori , Tomida Shuta , Sakaguchi Masakiyo , Toyooka Shinichi
Published: August 2019

Background: Malignant pleural mesothelioma (MPM) is a refractory disease with poor prognosis despite multimodality therapy. Because standard therapy is unsatisfactory, it is expected to establish a novel strategy such as molecular-targeting therapy for MPM. Doublecortin-like kinase 1 (DCLK1), a microtubule-associated protein, is one of the tumor-specific stem cell marker and is revealed that the cell survival is reduced by inhibiting this protein in pancreatic and colorectal cancers. DCLK1 is known to have several isoforms named as isoform #1-#4, although the significance of them in MPM has not been clarified. We hypothesized that DCLK1 was a potential therapeutic target for MPM.<Materials and

Methods: Immunohistochemical staining was performed using surgically resected formalin-fixed paraffin-embedded tissue of MPM to evaluate the expression of DCLK1. Additionally, MPM cell lines were analyzed to characterize the protein and mRNA expression of DCLK1 by western blotting and RT-PCR. The expression of DCLK1 isoforms were selectively knocked down by small interfering RNA (siRNA) and cell viability was analyzed by colony formation and cell count assays.

Results: Immunohistochemical staining demonstrated that the expression of DCLK1 in MPM specimens was positive in 24 out of 27 samples (89%). Western blotting and RT-PCR revealed that DCLK1 was expressed in seven out of nine MPM cell lines. The results of RT-PCR revealed that isoform #2 and isoform #4 of DCLK1 were expressed in H2052, isoform #4 was expressed in H28, and isoform #2 was expressed in YUMC44 cell lines. Expressions of DCLK1 isoforms were selectively suppressed by isoform-specific siRNAs. Colony formation and cell count assays revealed that siRNA-mediated knockdown of both isoforms #2 and #4 suppressed cell growth compared to knockdown by negative control siRNA and knockdown of only isoform #2 or #4.

Conclusion: Our results demonstrate that simultaneous knockdown of both isoforms #2 and #4of DCLK1 results in the inhibition of cell growth and suggest its potential as a therapeutic target in MPM.

Citation Format: Akihiro Miura, Hiromasa Yamamoto, Hiroyuki Tao, Ken Suzawa, Syunsaku Miyauchi, Kota Araki, Yuta Takahashi, Eisuke Kurihara, Yusuke Ogoshi, Kazuhiko Shien, Junichi Soh, Kazunori Okabe, Shuta Tomida, Masakiyo Sakaguchi, Shinichi Toyooka. Doublecortin-like Kinase 1 (DCLK1) correlates with the cell proliferation in malignant pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2136.
Abstract 214: Preclinical evaluation of a novel MET antibody-drug conjugate in uveal melanoma models
Authors: Surriga Oliver , DaSilva John , Schwartz Gary K.
Published: August 2019

Uveal melanoma is the most common primary intraocular malignant tumor in adults. These tumors are characterized by mutations in G-proteins (GNAQ and GNA11) and high expression of MET, a receptor tyrosine kinase. While inhibition of MET signaling delays the development of uveal melanoma metastases in preclinical models (possibly by inhibiting cell migration), primary tumor growth is not inhibited, indicating a lack of dependence on the MET pathway. Thus, treatment of established metastatic uveal melanoma with MET signal blockers is unlikely to be effective. However, the high expression of MET protein in metastatic uveal melanoma presents an opportunity to directly kill tumor cells with a MET-targeted antibody drug conjugate (ADC). METxMET-ADC is a novel bispecific antibody that binds to two distinct epitopes on MET and is conjugated to a maytansinoid payload via a cleavable linker. The METxMET-ADC potently induces receptor internalization and mitotic arrest of cancer cells. Our in vitro studies show that the METxMET-ADC significantly and specifically decreases the viability of MET expressing uveal melanoma cell lines in a dose-dependent manner with an IC50 of less than 1 nM. A longer 7-day cell viability assay shows significantly enhanced inhibition of proliferation of several MET expressing cell lines (Mel270, Mel202, OMM1.3 and MP65) but not of OCM3 cells which express low levels of MET. Apoptosis is also induced up to 40% in the MET-expressing cell lines when treated with 10 nM of the METxMET-ADC for 48 hours. Cell cycle analysis shows a significant increase of OMM1.3 and Mel202 cells in mitosis after 6-24 hours of treatment and appearance of a sub-G1 population after 24-48 hours. Western blots confirm a decrease of MET protein levels, presumably due to receptor internalization and trafficking to lysosomes. Finally, treatment with the METxMET ADC is associated with induction of PARP cleavage and phosphorylation of histone H3 after 24 hours in OMM1.3 and Mel202 cells, but not in OCM3 cells. In summary, conjugation of a METxMET bispecific antibody with a cytotoxic payload allows for potent and selective killing of MET-expressing uveal melanoma cells. The METxMET-ADC demonstrates promising therapeutic potential for the treatment of uveal melanoma.

Citation Format: Oliver Surriga, John DaSilva, Gary K. Schwartz. Preclinical evaluation of a novel MET antibody-drug conjugate in uveal melanoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 214.
Abstract 2146: High expression of Olfactomedin-4 correlates with chemoresistance and poor prognosis for pancreatic cancer
Authors: Ohkuma Ryotaro , Yada Erica , Kubota Yutaro , Hamada Kazuyuki , Ishida Hiroo , Hirasawa Yuya , Ariizumi Hirotsugu , Satoh Etsuko , Tsurutani Junji , Yoshimura Kiyoshi , Sasada Tetsuro , Aoki Takeshi , Murakami Masahiko , Norose Tomoko , Ohike Nobuyuki , Takimoto Masafumi , Tsunoda Takuya , Wada Satoshi
Published: August 2019

Background: Pancreatic cancer has an extremely poor prognosis and novel therapeutic strategies are desperately needed. It is considered that suitable molecules as therapeutic targets are resistant to chemotherapy and correlate with poor prognosis. In this experiment, we aimed to identify the optimal therapeutic targets in pancreatic cancer.

Materials and Methods: We established 10 lines of patient derived xenografts (PDXs) for pancreatic cancer. Next generation sequencer (NGS) analysis was performed for PDXs which were administered chemotherapy and untreated PDXs as a control. Furthermore, we performed immunohistochemistry (IHC) analysis for the protein expression of identified molecules from PDXs and post-operative pathologic specimens from 80 patients with pancreatic cancer. When chemotherapy was added on tumor cell lines forcibly expressing the molecule, antitumor effect was compared with the control.

Results: As a result of NGS analysis, we identified Olfactomedin-4 (OLFM4) molecule which was commonly shown high expression in chemotherapy administered PDXs. In IHC analysis, OLFM4 molecule expression was shown high expression with chemotherapy administered PDXs compared to untreated PDXs. For the analysis to use tumor cell lines forcedly expressing OLFM4 in vitro, the reduction rate of the tumor cell numbers was significantly lower than control vector, even if the concentration of the chemotherapy agent was increased. In a study using tissue specimens of 80 pancreatic cancer patients, Kaplan-Meier analysis showed that low expression of OLFM4 had a better survival rate than patients with high expression (p=0.0926, log-rank test). In multivariate analysis, it was shown that the high expression of OLFM4 was an independent poor prognostic factor with a statistically significant difference (p=0.044, Cox proportional hazards).

Conclusions: It was demonstrated that high expression of OLFM4 was involved in chemoresistance and was an independent prognostic factor for pancreatic cancer. This molecule might be a candidate of therapeutic target in pancreatic cancer.

Citation Format: Ryotaro Ohkuma, Erica Yada, Yutaro Kubota, Kazuyuki Hamada, Hiroo Ishida, Yuya Hirasawa, Hirotsugu Ariizumi, Etsuko Satoh, Junji Tsurutani, Kiyoshi Yoshimura, Tetsuro Sasada, Takeshi Aoki, Masahiko Murakami, Tomoko Norose, Nobuyuki Ohike, Masafumi Takimoto, Takuya Tsunoda, Satoshi Wada. High expression of Olfactomedin-4 correlates with chemoresistance and poor prognosis for pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2146.
Abstract 215: Generation of site-specific DARPin® drug conjugates using EGFR as a model system
Authors: Laviolette Laura A. , Guidi Cynthia J. , Reichen Christian , Qiu Qifeng , Harris Luke , Schildknecht Patricia , Fischer Stefanie , Arany Zita , Hospodarsch Tanja , Skaletskaya Anna , Fuller Megan , Abbott Stephen , McCarthy Rebecca , Lee Jenny , Francisco Katherine , Sinkevicius Kerstin , Adams Sharlene , Espelin Christopher , Reid Emily , Li Wei , Marashio Carla , Donahue Kerry , Hicks Stuart , Snell Dan
Published: August 2019

DARPin® molecules are small engineered proteins, derived from natural ankyrin repeat proteins that are selected to bind to specific targets with high affinity. Individual DARPin® molecules can be linked together genetically in order to create multi-specific drug molecules. This versatility of DARPin® molecules makes them an attractive alternative to antibodies for the development of drug conjugates. We have developed DARPin® drug conjugates (DDCs) using a model EGFR multi-specific DARPin® molecule, consisting of four DARPin® domains linked together. DARPin® constructs were conjugated to the indolinobenzodiazepine mono-imine DGN549, a potent DNA alkylating payload. Biophysical characterization showed the DDCs to be well behaved in stability and solubility assays. The in vitro binding and cytotoxicity of these DDCs were evaluated. The DARPin® conjugates displayed in vitro potency in direct cytotoxicity assays across a panel of cell lines expressing EGFR. The impact of conjugation on PK parameters and in vivo efficacy is currently under evaluation. DARPin® drug conjugates combine the potency observed with antibody drug conjugates and the modular design of DARPin® molecules to create designer therapeutics.

Citation Format: Laura A. Laviolette, Cynthia J. Guidi, Christian Reichen, Qifeng Qiu, Luke Harris, Patricia Schildknecht, Stefanie Fischer, Zita Arany, Tanja Hospodarsch, Anna Skaletskaya, Megan Fuller, Stephen Abbott, Rebecca McCarthy, Jenny Lee, Katherine Francisco, Kerstin Sinkevicius, Sharlene Adams, Christopher Espelin, Emily Reid, Wei Li, Carla Marashio, Kerry Donahue, Stuart Hicks, Dan Snell. Generation of site-specific DARPin® drug conjugates using EGFR as a model system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 215.
Abstract 2151: Monitoring AMP Concentration,the universal product of diverse enzymatic reactions
Authors: Goueli Said A. , Hsiao Kevin , Mondal Subhanjan
Published: August 2019

Many enzymatic reactions use Adenosine triphosphate (ATP) as a substrate and produce adenosine monophosphate (AMP) as the common product of these reactions. AMP also functions as a modulator of many enzymes and a key cellular metabolite regulating energy homeostasis and signal transduction. For example, by activating AMP-Dependent protein kinase (AMPK), it maintains the energy charge ratio at optimal level during metabolic activities; and its concentration is altered during disregulated disease conditions. Thus, monitoring the activities of these enzymes is a primary goal for developing modulators for these enzymes. Here, we demonstrate the versatility of an enzyme coupled assay that quantifies the amount of AMP produced by any enzymatic reactions regardless of its substrates in a homogenous and high through-put formatted form. We successfully implemented it to enzyme reactions that use ATP as a substrate (aminoacyl tRNA synthetase and DNA ligase) by first depleting the remaining residual ATP substrate and converting AMP produced into ATP, the later can be quantified using luciferase/luciferin and generating light. We also tested this assay to measure direct activities of AMP generating enzymes that do not require ATP as substrate, including phosphodiesterases (cAMP) and E.coli DNA ligases (NAD+). In a further elaboration of the AMP-Glo platform, we coupled it to E.coli DNA ligase, enabling measurement of NAD+ and enzymes that use NAD+ like mono- and poly-ADP-ribosyltransferases. Sulfotransferases use 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as the universal sulfo-group donor and phosphoadenosine-5'-phosphate (PAP) is the universal product. PAP can be quantified by converting PAP to AMP by a Golgi-resident PAP-specific phosphatase, IMPAD1. By coupling IMPAD1 to the AMP-Glo system we can measure the activities of sulfotransferases. In summary, we have developed a very strong platform to monitor AMP concentrations that is applicable to diverse enzymatic reactions.

Citation Format: Said A. Goueli, Kevin Hsiao, Subhanjan Mondal. Monitoring AMP Concentration,the universal product of diverse enzymatic reactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2151.
Abstract 2159: Label-free impedance analysis as a versatile tool for dynamic screening of cancer molecular therapeutics
Authors: Cerignoli Fabio , Xi Biao , Lamarche Brandon , Zhao Leyna , Abassi Yama
Published: August 2019

In vitro monitoring cancer cells response to treatment often involves laborious sample processing and collects single data points that are against the dynamic nature of cancer cells. Here we present the adaptation of an impedance-based methodology to dynamically monitor cancer cell behavior and therapeutic response. The technology detects cell death, proliferation or migration by measuring changes in conductance of microelectrodes embedded in 96 and 384-wells cell culture plates. It avoids sample labeling and processing and allows continuous monitoring of cell response. Our data shows validation with cancer cell lines and primary cells monitored for cell invasion/migration through transwells or after a scratch has been performed with a dedicated device, anticancer drug response and receptor signaling activation, with similar results over end point assays. Easy experiment set up and minimal sample processing make the technology ideal for applications in large screening campaigns, while label-free technology allows less artifacts and further analysis on the same samples through orthogonal assays.

Citation Format: Fabio Cerignoli, Biao Xi, Brandon Lamarche, Leyna Zhao, Yama Abassi. Label-free impedance analysis as a versatile tool for dynamic screening of cancer molecular therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2159.
Abstract 216: pro-Pyrrolobenzodiazepine (pro-PBD) bioconjugates, the legacy: Design and synthesis of pro-PBD conjugates containing self-immolative substituted disulfide linkers
Authors: Vlahov Iontcho , Qi Longwu , Santhapuram Hari K. , Parham Garth , Wang Kevin , Vaughn Jeremy F. , Hahn Spencer J. , Vetzel Marilynn , Nelson Melissa , Reddy Joseph A. , Leamon Christopher P.
Published: August 2019

Naturally occurring pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a group of highly potent antitumor antibiotics, isolated from various streptomycin species. PBDs covalently bond and cross-link with discrete sequences of base pairs in the minor groove of DNA. A wide range of synthetic PBDs and PBD dimers have been shown to be highly potent cytotoxic agents, and have been used as payloads in antibody drug conjugates (ADCs). However, all PBDs as stand-alone agents or as delivered via small molecule drug conjugates (SMDCs), cause undesired off-target toxicity. Recently, we designed folate conjugated pro-PBDs, masking the imine moiety with its synthetic precursors: namely, 1,3-oxazolidine carbamate and an aromatic amine. The 1,3-oxazolidine carbamate provided a conjugation site for attachment of pro-PBD to a targeting ligand. After delivery to the targeted cell and internalization, reductive cleavage of the linker system results in the generation of a 1,3-oxazolidine. The oxazolidine reacts with water to form an aldehyde which subsequently reacts intramolecularly to ultimately form the PBD diazepine ring. Herein, we report the design and synthesis of folate conjugates of pro-PBDs and self-immolative sterically hindered disulfide linker systems, which pave the way toward novel, highly-specific therapeutics which have the potential to minimize off-target toxicities.

One of the so designed conjugates, EC2629, satisfied all of our criteria for selection for pre-clinical development:

• EC2629 shows high affinity towards FR mediated binding.

• Cross-links DNA only following self-immolative release of free drug.

• Picomolar potency against FR-expressing cells.

• Curative activity with no weight loss in animal models.

• Active against drug resistant tumor xenograft models in-vivo.

• EC2629 is active against patient derived xenograft models in-vivo.

• Based on high cytotoxicity and minimal off target toxicity (in-vitro & in-vivo), EC2629 is selected for pre-clinical development.

• Biological evaluation of EC2629 was presented at World ADC, Berlin, Germany (March, 2018).

Citation Format: Iontcho Vlahov, Longwu Qi, Hari K. Santhapuram, Garth Parham, Kevin Wang, Jeremy F. Vaughn, Spencer J. Hahn, Marilynn Vetzel, Melissa Nelson, Joseph A. Reddy, Christopher P. Leamon. pro-Pyrrolobenzodiazepine (pro-PBD) bioconjugates, the legacy: Design and synthesis of pro-PBD conjugates containing self-immolative substituted disulfide linkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 216.
Abstract 2160: Microfragmented human fat tissue is a natural scaffold for drug delivery: potential application in cancer chemotherapy
Authors: Alessandri Giulio , Coccè Valentina , Pastorino Fabio , Paroni Rita , Deicas Michele , Restelli Francesco , Tremolada Carlo , Berenzi Angiola , Parati Eugenio , Brini Anna Teresa , Bondiolotti Giampietro , Pessina Augusto , Ponzoni Mirco
Published: August 2019

Localization of chemotherapy at the tumor site can improve therapeutic efficacy and reduce systemic toxicity. In previous studies we have shown that mesenchymal stromal cells (MSCs) isolated from bone marrow or fat tissue, can be loaded with the anti-cancer drug Paclitaxel (PTX) and kill cancer cells when localized nearby. We here investigated the capacity of human micro-fragmented adipose tissue (MFAT), used as a natural container of MSCs, to delivery PTX with the idea to improve local drug concentration and to prolong the therapeutic activity. Surprisingly, we found that both fresh but also devitalized MFAT (DMFAT) (by freezing/thawing procedure) were very effective biomaterials able to deliver and release significant amount of PTX, killing several human cancer cell lines in vitro with an impressive long lasting activity. In an orthotopic mice model of Neuroblastoma (NB) transplant, we found that DMFAT loaded with PTX prevents or delay NB relapse when placed in the surgical area of tumor resection, without any collateral toxicity. We concluded that MFAT, but also DMFAT, may work as natural biomaterials that can be used to localize and release anti-cancer molecules at the tumor site. In addition, the procedure to easily prepare DMFAT did not alter its natural scaffold structure. This suggest that such biomaterial may have a prominent role in the near future in many oncological applications to delivery anti-cancer drugs, helping to fight cancer in human.

Citation Format: Giulio Alessandri, Valentina Coccè, Fabio Pastorino, Rita Paroni, Michele Deicas, Francesco Restelli, Carlo Tremolada, Angiola Berenzi, Eugenio Parati, Anna Teresa Brini, Giampietro Bondiolotti, Augusto Pessina, Mirco Ponzoni. Microfragmented human fat tissue is a natural scaffold for drug delivery: potential application in cancer chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2160.
Abstract 2166: A novel etoposide-bound magnetic nanoparticle for remote targeting of cancer cells
Authors: Willis Alexander , Karumudi Bhargava , Liu Bing , Sough Ashley , Hwang Sangyeul , Liu Ying , Engelhard Herbert
Published: August 2019

Background: Magnetic nanoparticles (MNPs) have potential for enhancing drug delivery in selected cancer patients. To date, successful clinical use of MNPs has been limited due to the: 1) limited capacity for targeting at human-sized distances, 2) route of delivery, and 3) choice of therapeutic component. Here, we present data related to the creation and in vitro use of a new two-part MNP system consisting of: 1) an AuFe base particle having streptavidin binding sites, and 2) biotinylated etoposide, which was bound to the base particle. Etoposide was chosen in part due to its previous clinical use within cerebrospinal fluid, which could serve as a delivery medium for the new particle.

Methods: Base MNPs were manufactured by IMRA America, Inc., and combined with biotinylated etoposide to create AuFe-etoposide particles (etop-MNPs), which have not been previously described. Etop-MNPs were characterized by electron microscopy, UV-vis spectroscopy, and DLS. Etop-MNPs could be moved at a distance (e.g. 10 cm) by means of a “surface walking” phenomenon, occurring in response to a rotating neodymium-boron-iron permanent magnet, kindly provided by Pulse Therapeutics (St. Louis, MO). Cell lines (including D283, U138, and normal endothelial cells) were maintained using standard tissue culture technique. Viability of the cells in response to the MNP base particles, etoposide alone, and etop-MNPs, was studied by light microscopy and MTT assay.

Results: Etoposide was successfully bound to the IMRA base particle through the streptavidin-biotin interaction. Typically, 100uM etoposide was used. Etop-MNPs could be moved remotely by the rotating magnet, which produced a tangential particle velocity of 0.15 +/- 0.026 cm/sec. With 2 hours of treatment, etop-MNPs caused morphologic changes and decreased cancer cell viability to 0.50 +/- 0.03 percent that of control values.

Conclusions: These findings indicate that our novel etop-MNPs have tumoricidal effect, and can be moved relatively rapidly at physiologic distances, using a rotating magnet. Etop-MNPs, may therefore prove to be useful for magnetic drug targeting in clinical settings, such as intrathecal use within the spinal subarachnoid space.

Citation Format: Alexander Willis, Bhargava Karumudi, Bing Liu, Ashley Sough, Sangyeul Hwang, Ying Liu, Herbert Engelhard. A novel etoposide-bound magnetic nanoparticle for remote targeting of cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2166.