bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4852: CBLC functions as a novel therapeutic target to enhance the efficacy of paclitaxel on EGFR wild-type lung adenocarcinoma by downregulating AURKA
Authors: Hong Shiao-Ya , Kao Yu-Rung , Lin Yi-Ping , Lee Meng-Hsuan , Wu Jyun-Yi , Wu Cheng-Wen
Published: September 2020

Patients with lung adenocarcinoma harboring driver genes can be treated with target therapies, such as tyrosine kinase inhibitors for epithelial growth factor receptor (EGFR) mutations, anaplastic lymphoma kinase (ALK) inhibitors for ALK gene rearrangements, and so on. However, a large portion of lung adenocarcinoma patients, especially for those with wild-type EGFR, still do not have valid target therapies in clinics. The CBL family proteins (CBL, CBLB, and CBLC) are E3 ubiquitin ligases of protein tyrosine kinases (PTKs), including EGFR, hepatocyte growth factor receptor (MET), and SRC proto-oncogene (SRC). It has been reported that the K63-linkaged ubiquitination mediated by CBL can negatively regulate activated EGFR (aEGFR) signaling through lysosomal degradation. Previously, we discovered that CBLC, unlike CBL, possesses an oncogenic function and positively regulates aEGFR stability through polyubiquitination via K6 and K11 linkages. The CBLC-mediated conjugation of polyubiquitin promotes aEGFR preferentially recycled back to the plasma membrane, or trafficked to the cell nucleus, leading to the sustained activation of EGFR signaling in lung adenocarcinoma. Aside from aEGFR dysregulation, we recently found that CBLC assumes a novel role in positively regulating the expression of Aurora kinase A (AURKA), which contributes to tumor development by promoting cell proliferation, metastasis, epithelial-mesenchymal transition, and drug resistance. In lung adenocarcinoma cells with wild-type EGFR, CBLC knockdown significantly reduced cell viability, delayed cell cycle progression and increased apoptotic death. Our study also showed that CBLC knockdown in EGFR wild type cells was associated with increased sensitivity to paclitaxel, probably through downregulating AURKA. Taken together, these findings indicated that CBLC may play a role in promoting cell cycle progression and may be a novel therapeutic target to enhance the efficacy of paclitaxel on EGFR wild-type lung adenocarcinoma.

Citation Format: Shiao-Ya Hong, Yu-Rung Kao, Yi-Ping Lin, Meng-Hsuan Lee, Jyun-Yi Wu, Cheng-Wen Wu. CBLC functions as a novel therapeutic target to enhance the efficacy of paclitaxel on EGFR wild-type lung adenocarcinoma by downregulating AURKA [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 4852.
Abstract 488: Establishing consistent methods for the generation of donor-derived iPSCs from T cell subsets
Authors: Landon Mark , MacArthur Chad C. , Hernandez Melissa E. , Lakshmipathy Uma , Guzman Jerry
Published: September 2020

T cell-based immunotherapies offer a potent and specific solution to grievous diseases such as cancer and viral infections. Expansion and re-infusion of antigen specific T cells has seen success in the treatment of metastatic melanoma and prevention of viral infections such as CMV and EBV. Additionally, engineered T cell therapies, especially chimeric antigen receptor (CAR) T cells, have been met with great clinical success and spurred innovation within the field of cellular immunotherapies. While promising, these therapies rely on sourcing T cells from donor derived blood. This process is invasive to the patient, especially when multiple or prolonged blood draws are required to obtain sufficient T cell numbers. Additionally, T cells sourced from diseased patients are often exhausted and display significantly perturbed growth and dysfunctional responses to antigen stimulation. An appealing alternative is somatic reprogramming of T cells to generate induced pluripotent stem cells (iPSCs) and differentiated into rejuvenated T cells that retain the original TCR, particularly with tumor infiltrating lymphocytes or viral specific T cells. We had previously reported a Sendai-virus based CytoTune 2.1 Reprogramming specifically designed for translational and clinical research. In this study we examine the effect of various cellular phenotypes and optimize methods for consistent reprogramming of multiple donor T cell reprogramming. Additionally, pan CD3+ cells isolated using different methods and further enriched for CD4+ and CD8+ were successfully reprogrammed. The resulting iPSCs were extensively characterized to ensure that pluripotency was achieved and that TCR sequences were preserved. This approach of reprogramming T cells and specific cell subtypes to derive iPSC with a known TCR sequence enables their use for downstream application as a renewable source of T cells with known antigen specificity.

Citation Format: Mark Landon, Chad C. MacArthur, Melissa E. Hernandez, Uma Lakshmipathy, Jerry Guzman. Establishing consistent methods for the generation of donor-derived iPSCs from T cell subsets [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 488.
Abstract 4896: Transcriptomic alterations by HPV-human fusion transcripts in HPV+ HNSCC cell lines
Authors: Pinatti Lisa M. , Sinha Hana , Brenner Chad , Walline Heather M. , Carey Thomas E.
Published: September 2020

Human papillomavirus (HPV) genomic integration is frequently seen in oropharyngeal squamous cell carcinoma (OPSCC). Integration into cellular genes can cause the generation of HPV-host fusion transcripts, which have been associated with altered gene expression of critical cellular pathways and worse survival in OPSCC patients. The mechanism of these transcriptomic alterations by fusion transcripts and how they contribute to poor outcome are unknown. We began our analysis by investigating whether our cell lines models of HPV+ OPSCC recapitulate these gene expression alterations using a qRT-PCR profiling system. We compared the expression of 128 genes in relevant pathways in four fusion transcript positive cell lines versus two fusion transcript negative cell lines and found a number of genes that were differentially expressed between the two groups. This suggests that these cell lines are appropriate models to study the mechanism of fusion transcript-dependent transcriptional regulation. To confirm our results in another system, we created fusion transcript positive/negative cell lines by cloning two pairs of either an HPV-human fusion transcript or an HPV only transcript into normal keratinocytes. These cloned cells were then run through the same qRT-PCR profiling system, and we saw differentially expressed genes between the two groups. There was an overlapping set of gene pathways between the HPV+ OPSCC cell lines and cloned keratinocytes, suggesting HPV-human fusion transcripts are sufficient to alter cellular gene expression. Subsequent in-depth molecular characterization and in vitro studies will help clarify the mechanism of these changes and their effects on cancer formation and metastasis. These studies will advance our understanding of how fusion transcripts lead to worse patient survival and help develop new therapies for these patients.

Citation Format: Lisa M. Pinatti, Hana Sinha, Chad Brenner, Heather M. Walline, Thomas E. Carey. Transcriptomic alterations by HPV-human fusion transcripts in HPV+ HNSCC cell lines [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 4896.
Abstract 4897: Genome-wide structural variation analysis and characterization of Hepatitis B Virus integration sites in hepatocellular carcinomas using Bionano Genomics whole genome imaging
Authors: Hong Karl , Péneau Camille , Bayard Quentin , Imbeaud Sandrine , Pang Andy , Hastie Alex , Letouzé Eric , Zucman-Rossi Jessica
Published: September 2020

Large genomic rearrangements are known contributors to cancer formation. Hepatitis B virus (HBV) integration is a frequent driver of hepatocellular carcinoma, frequently associated with genomic rearrangements. Next generation sequencing (NGS) is commonly used to study these integration sites and rearrangements; however, due to short read lengths, NGS is often unable to characterize large and complicated genomic rearrangements. To solve this problem, whole genome imaging was used to perform genome-wide detection of large structural variants (SVs) in DNA extracted from liver tumors, including characterization of HBV viral integration sites and nearby rearrangements. Ultra-high molecular weight DNA was extracted from liver tissue of four patients diagnosed with hepatocellular carcinoma. The DNA was fluorescently labeled at the motif CTTAAG, linearized, and imaged in nanochannel arrays using a Bionano Genomics Saphyr platform. Images of molecules were converted into digital single-molecule reads. 400X coverage of DNA molecules at least 150 kbp in length was collected for each sample. For three of the patients, somatic structural variants (SV) were identified by comparing SV calls and single molecule DNA reads of liver tumor resections against those of matching physiologically normal liver tissue. For the fourth patient, putative somatic SVs were identified by comparing SV calls against a database of 59,490 known variants from 57 physiologically normal individuals. Among the matched-pair samples, 511 large tumor-unique structural variants were identified. For the fourth sample, 619 putative somatic events were identified. HBV viral integration sites, which had previously been identified via NGS, and nearby SVs were characterized. Complex SVs were resolved, including translocation events with several loci. These findings demonstrate the utility of whole genome imaging for studying the relationship between viral integration and cancer.

Citation Format: Karl Hong, Camille Péneau, Quentin Bayard, Sandrine Imbeaud, Andy Pang, Alex Hastie, Eric Letouzé, Jessica Zucman-Rossi. Genome-wide structural variation analysis and characterization of Hepatitis B Virus integration sites in hepatocellular carcinomas using Bionano Genomics whole genome imaging [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 4897.
Abstract 492: A Novel QUATRAMER sustained injectable suspension for the intracellular delivery of Salinomycin, a stem cell inhibitor (HSB-1216), for the treatment of triple negative breast cancer
Authors: Kharbanda Surender M. , Mohammad Anees , Gupta Bhawana , Appajosyula Sireesh , Laumas Sandeep , Jain Jugnu , Kufe Donald , Singh Harpal
Published: September 2020

Triple-negative breast cancer (TNBC) accounts for approximately 15-20 % of all breast cancers, is an aggressive disease with a poor prognosis, and lacks targeted therapy. Recent studies suggest that cancer stem cells (CSCs) play an important role in TNBC progression and tumorigenesis and that targeting CSCs may be a promising, novel strategy for the treatment of this disease. Both CD44+/CD24- and CD44+/CD24 low CSCs are enriched in TNBCs and may contribute to chemotherapy resistance, emphasizing the need to target this population. In this regard, recent studies have shown that salinomycin (SAL) preferentially targets CSCs; however, the preclinical toxicity associated with this agent has limited its clinical development.In the current studies, we have generated novel biodegradable QUATRAMER polymeric nanoparticles comprised of a polyethylene glycol (PEG)-polypropylene glycol (PPG)-PEG modified polylactic acid (PLA)-tetra-block copolymer (SAL-NPs; HSB-1216) for the intracellular delivery of SAL. Treatment of human TNBC MDA-MB-231 cells with HSB-1216 was associated with significant cell death with an IC50 of around 2 uM. Similar results were obtained when other TNBC cell line SUM149 was treated with HSB-1216 (IC50 ~ 0.5 uM). Based on the characteristic formation of TNBC mammospheres in in vitro 3D culture conditions, the mammosphere formation assay has become an essential tool for quantifying CSC activity. Cultured MDA-MB-231 mammospheres were therefore exposed to different concentrations and durations of HSB-1216. Our results demonstrate dose-dependent inhibition of mammosphere formation in response to HSB-1216. Additionally, CD44+/CD24- enriched CSCs were isolated from a TNBC patient tumor biopsy, grown as 3D culture/mammospheres and then treated with HSB-1216. Interestingly, in contrast to Paclitaxel, treatment with HSB-1216 was associated with nearly complete inhibition of mammosphere formation. Taken together, these results demonstrate an effective method for sustained delivery of a potent stem cell inhibitor, SAL, and its potential for the treatment of TNBC.

Citation Format: Surender M. Kharbanda, Anees Mohammad, Bhawana Gupta, Sireesh Appajosyula, Sandeep Laumas, Jugnu Jain, Donald Kufe, Harpal Singh. A Novel QUATRAMER sustained injectable suspension for the intracellular delivery of Salinomycin, a stem cell inhibitor (HSB-1216), for the treatment of 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 492.
Abstract 4962: Tumor heterogeneity driven by cancer stem cell expressing POU5F1
Authors: Miyoshi Norikatsu , Fujino Shiki , Sasaki Masaru , Ogino Takayuki , Takahashi Hidekazu , Uemura Mamoru , Matsuda Chu , Mizushima Tsunekazu , Eguchi Hidetoshi , Doki Yuichiro
Published: September 2020

Background: The gene POU5F1, encoding the POU domain, class 5, is a transcription factor. and the expression is found in embryonic stem cells. In these pluripotent stem cells, POU5F1 gene controls self-renewal and differentiation, regulating the pluripotency and proliferation. Regarding the tumor heterogeneity and microenvironment, primary culture of cancer cells derived from the patient's tumor can provide important information of the “individual tumor,” representing the tumor heterogeneity. We focused on the gene expression and the relationship between colorectal cancer stem cells and the tumor heterogeneity, in the primary culture model.

Methods: Using POU5F1 promoter-containing sequence, POU5F1-EGFP-positive cells were established by the construct with lentiviral vector, and we examined their sub-populations. The capacity to form distant metastasis in vivo was examined using the primary culture models.

Results: We obtained clinical samples from surgically resected gastrointestinal cancers. They were mechanically and enzymatically digested and collected using customized filters. Obtained cells were cultured on a matrix-coated plate with chemically defined stem-cell culture medium. All these primary cultured cells grew and about 90% of them were successfully passaged. The morphology and gene expressions were similar to each parental clinical tumor. Using these primary tumors, POU5F1 expressing cells were examined. The POU5F1-EGFP-positive cells highly expressed stem cell-associated markers and they had self-renewal and differentiation abilities. POU5F1-high cells actively formed liver metastasis.

Conclusion: POU5F1-positive cells have the self-renewal and differentiation abilities as cancer stem cells. The positive cells produce tumor heterogeneity. POU5F1 contributes to the ability forming liver metastasis in CRC. Keywords: colorectal cancer, tumor heterogeneity, metastasis, POU5F1.

Citation Format: Norikatsu Miyoshi, Shiki Fujino, Masaru Sasaki, Takayuki Ogino, Hidekazu Takahashi, Mamoru Uemura, Chu Matsuda, Tsunekazu Mizushima, Hidetoshi Eguchi, Yuichiro Doki. Tumor heterogeneity driven by cancer stem cell expressing POU5F1 [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 4962.
Abstract 4986: Utilizing image analysis to characterize an 8-plex immunofluorescence immune checkpoint biomarker panel within the tumor tissue microenvironment
Authors: Bigley Alison L. , Sherry Lorcan A. , Stillie Nicole , Wood Douglas , Downing Sean
Published: September 2020

Multiplex staining and image analysis are being increasingly deployed for immuno-oncology (I/O) evaluations to investigate and understand the complexity of immune cell / tumor cell interactions in the tumor microenvironment (TME) and in discovering novel predictive biomarkers for targeted immunotherapy. Immune checkpoint inhibitor treatment, guided by immunohistochemically labelled tissue expression of PD-L1 and PD-1, have already been shown to improve outcomes, in the metastatic setting, for non-small cell lung carcinoma (NSCLC) and colorectal cancer (CRC) patients. The purpose of the current study was to phenotype and compare checkpoint and immune cell interactions within NSCLC and CRC tumor types. The study included samples from NSCLC, CRC, and normal tonsil tissues. Sections were stained using InSituPlex® multiplex immunofluorescence (mIF), for studying PD-L1/PD-1 checkpoint expression including immune cell markers for CD3, CD4, CD8, CD68, FoxP3, and pan-CK as an epithelial tumor indicator. Slides were scanned at 20X magnification on the ZEISS Axio Scan.Z1 platform, with individual .tiff format files being generated per marker. Image analysis was performed using the Visiopharm® Oncotopix® platform. Individual marker image layers were aligned, tumor/stroma regions of interest (ROI) classified, and cell formations detected. Quantification of markers was determined across the image layers to evaluate positive cell staining within both cancer indications. Quantitative image analysis data generated included total positive and negative cell counts per marker, per mm2 ROI. Co-localization analysis provided relevant immune cell phenotypes (T-cytotoxic, T-helper, T-regs, and macrophages) and immune cell functional status. Cell spatial analysis relationships were generated, including CD8+/PD-1− and CD8+/PD-1+ to CK+/PD-L1+ tumor cells. This study highlighted the benefits of utilizing an 8-plex staining protocol and detailed image analysis approach for the comprehensive assessment of the immune cell types and interactions within the TME. The ability to carry out multiparameter cellular analysis facilitates the delivery of key phenotypic data, thereby leading to greater understanding of tumor biology.

Citation Format: Alison L. Bigley, Lorcan A. Sherry, Nicole Stillie, Douglas Wood, Sean Downing. Utilizing image analysis to characterize an 8-plex immunofluorescence immune checkpoint biomarker panel within the tumor tissue microenvironment [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 4986.
Abstract 5000: YB-1 based oncolytic virotherapy in combination with small molecular inhibitors for treatment of refractory osteosarcoma
Authors: Middendorf Charlotte Friederike , Schober Sebastian Johannes , Kordass Corazón , Nawroth Roman , Burdach Stefan E. , Holm Per Sonne , Thiel Uwe
Published: September 2020

This in vitro study evaluates YB-1 based oncolytic virotherapy in combination with molecular target therapy as a novel therapeutic strategy for refractory osteosarcoma.

In vitro combination approaches include CDK4/6-, MDM2- and PARP-inhibitors. The treatment strategy was explored in four established osteosarcoma cell lines (143B, MG-63, SJSA, U2OS). Intracellular YB-1 expression was evaluated by immunofluorescence for oncolytic therapy with YB-1 selective adenovirus XVir-N-31. Efficacy of mono- and combination therapy is assessed using cell survival assays. The Chou Talalay method is used to determine synergistic effects between combinational components. Characterization of molecular pathways associated with oncolytic cytotoxicity is done using cell cycle analysis, western blotting, hexon titer test, sqPCR and ELISA.

We observe a potent preclinical anti-tumor effect using XVir-N-31 monotherapy on all tested osteosarcoma cell lines. However, most cell lines were resistant towards inhibitor monotherapy in clinically achievable concentrations. In contrast, all dual combinations resulted in significantly enhanced cytotoxicity. These results require reduced numbers of infectious viral particles and are observed at clinically implementable inhibitor concentrations. Increased oncolytic cytotoxicity is associated with changes of tumor cell cycle regulation due to inhibitor treatment and modulation of infectious viral particle formation.

In summary, we determine XVir-N-31 as a potent oncolytic agent for the treatment of refractory osteosarcoma. We provide a strong preclinical rationale for the combination of oncolytic adenovirus with molecular target therapy. A phase I clinical trial with XVir-N-31 in combination with molecular target therapy for refractory childhood sarcoma is planned.

Citation Format: Charlotte Friederike Middendorf, Sebastian Johannes Schober, Corazón Kordass, Roman Nawroth, Stefan E. Burdach, Per Sonne Holm, Uwe Thiel. YB-1 based oncolytic virotherapy in combination with small molecular inhibitors for treatment of refractory osteosarcoma [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 5000.
Abstract 5029: Targeting fatty acid oxidation to promote anoikis and inhibit ovarian cancer progression
Authors: Bitler Benjamin Guy , Sawyer Brandon , Qamar Lubna , Richer Jennifer K. , Behbakht Kian , Schlaepfer Isabel R.
Published: September 2020

Purpose: Epithelial-derived high-grade serous ovarian cancer (HGSOC) is the deadliest gynecologic malignancy and in the US accounts for over 14,000 deaths per year. Roughly 80% of patients are diagnosed with late-stage disease, which is defined by wide-spread cancer dissemination throughout the pelvic and peritoneal cavities. HGSOC dissemination is dependent on tumor cells acquiring the ability to resist anoikis (apoptosis triggered by the loss of attachment). Epithelial cell detachment from the underlying basement membrane or extracellular matrix leads to cellular stress, including nutrient-deprivation. Reports describe metabolic reprogramming that promotes fatty acid oxidation (FAO) potentially supports anoikis resistance.

Experimental Design: Examination of a panel of HGSOC cell lines cultured in adherent and suspension conditions. Evaluated anoikis by caspase 3/7 activity, cleaved caspase 3 immunofluorescence, and flow cytometry. Using the Seahorse Bioanalyzer examined mitochondria activity in suspension. Tested the role of exogenous free fatty acids in anoikis resistance and metabolism. In a patient-derived xenograft model, administered an FAO inhibitor and/or platinum-based chemotherapy.

Results: HGSOC cultured in suspension upregulated the FAO rate-limiting enzyme, Carnitine Palmitoyltransferase 1A (CPT1A). CPT1A is overexpressed in HGSOC and correlates with poor overall survival. The knockdown of CPT1A promoted anoikis. HGSOC cells in suspension culture have decreased oxygen consumption that is rescued by exogenous fatty acids. In a patient-derived xenograft of HGSOC, a CPT1A inhibitor, etomoxir, significantly inhibited tumor growth and reduced tumor distribution.

Conclusions: Targeting FAO in HGSOC to promote anoikis and attenuate dissemination is a potential approach to promote a more durable anti-tumor response and improve patient outcomes.

Citation Format: Benjamin Guy Bitler, Brandon Sawyer, Lubna Qamar, Jennifer K. Richer, Kian Behbakht, Isabel R. Schlaepfer. Targeting fatty acid oxidation to promote anoikis and inhibit ovarian cancer progression [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 5029.
Abstract 5030: Vitamin D exhibits therapeutic anti-tumor and anti-angiogenic potential by reducing VEGF levels and altering TIMP/MMP system in breast cancer
Authors: Bajbouj Khuloud , Shafarin Jasmin , Sawan Ahmad , Shehada Ahmed , Elkhalifa Walaa Eldin
Published: September 2020

Background: Clinical and experimental observations suggest that Vitamin D deficiency associates with increased risk of breast cancer. Furthermore, studies concluded that Vitamin D precipitates in significant anti-neoplastic effects against breast, and inducing apoptosis and affecting angiogenesis in other types of cancer. However, the exact mechanism underlying this anti-angiogenic effect of Vitamin D is still elusive. This study aims to investigate the anti-cancer role of Vitamin D anti-cancer and its association with modulation of major factors involved in angiogenesis and metastasis in breast cancer cell lines.

Experimental methods: The hormonally active form of vitamin D, 1α,25-dihydroxyvitamin D3 (calcitriol), was used to evaluate anti-proliferative effect, inhibition of cell cycle progression and apoptosis induction in two breast cancer cell lines, MCF-7 and MDA-MB-231. Furthermore, impact of calcitriol on expression and activity of tissue inhibitors of metalloproteinases (TIMP), major groups of the matrix metalloproteinases (MMPs), vascular endothelial growth factor (VEGF), and tumor growth factor (TGF-β1) among others involved in tumor progression and angiogenesis were also examined.

Results and Discussions: Calcitriol treatment (10 µM) significantly inhibited cell proliferation in a timely dependent manner. Both cell lines showed G1 cell cycle arrest, decreased levels of the cell death inhibitor, survivin, and apoptosis induction in response to calcitriol treatment. Proteome Profiler array analysis revealed that calcitriol resulted in reducing the levels of major pro-angiogenesis regulators, especially VEGF, TGF-β1, Urokinase-type plasminogen activator (uPA), and Amphiregulin. Analysis of mRNA and proteins expression upon calcitriol exposure showed an increase of TIMP1, TIMP2 and a decrease in MMP 2, 3 and 9 levels. Finally, quantitative analysis using ELISA revealed that calcitriol treatment reduced MMP2 and MMP9 activities.

Conclusion: Our study demonstrates potential therapeutic property of calcitriol through inhibiting of pro-angiogenetic markers in breast cancer.

Citation Format: Khuloud Bajbouj, Jasmin Shafarin, Ahmad Sawan, Ahmed Shehada, Walaa Eldin Elkhalifa. Vitamin D exhibits therapeutic anti-tumor and anti-angiogenic potential by reducing VEGF levels and altering TIMP/MMP system in 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 5030.
Abstract 5050: Novel TNFR2 humanized mouse model for human TNFR2 antibody evaluation
Authors: Guo Yanan , Li Yanan , Zhou Xiaofei , Su Youhong , Lin Qingcong
Published: September 2020

In recent years, immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment. Tumor necrosis factor receptor 2 (TNFR2), also known as tumor necrosis factor receptor superfamily member 1B (TNFRSF1B), is a transmembrane receptor that has been linked into immune modulation and tissue regeneration. TNFR2 is mainly expressed on the surface of a subset of potent regulatory T cells (Tregs) and promote the proliferation of Tregs through nuclear factor kappa B (NF-κB). Anti-TNFR2 antibodies have been developed to inhibit NF-κB driven growth and have revived excitement for the use of anti-TNFR2 antibodies in the clinic.

To investigate the role of TNFR2, Biocytogen generated TNFR2 humanized mouse for both in vitro function validation of signaling pathway and in vivo efficacy evaluation of TNFR2 antibodies. In this model, the exon 2~6 of mouse Tnfr2 gene which encode the extracellular was replaced by human TNFR2 counterparts. Human TNFR2 is detectable on the Tregs in spleen and the TNFR2 antibodies bind well to the splenocytes of the TNFR2 humanized mice. Basal leukocyte subpopulations of TNFR2 humanized mice are comparable to that of wild-type mice, including T/B cells, NK cells, DC, granulocytes and monocytes/macrophages. Anti-human TNFR2 antibodies can repress the growth of colon cancer cells engrafted on TNFR2 humanized mice. The TNFα/TNFR2 signaling pathway is under validation on TNFR2 humanized mice and TNFα/TNFR2 double humanized mice due to the uncertain cross-reactivity between mouse and human TNFα/TNFR2.

Taken together, TNFR2 humanized mice is a useful tool for in vivo efficacy evaluation of human TNFR2 antibodies that can be advanced to human clinical trials.

Citation Format: Yanan Guo, Yanan Li, Xiaofei Zhou, Youhong Su, Qingcong Lin. Novel TNFR2 humanized mouse model for human TNFR2 antibody evaluation [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 5050.
Abstract 5052: CD44 inhibits the transition of ductal carcinoma in situ to invasive breast carcinoma
Authors: Liu Fang , Zhou Jun , Kundu-Roy Tanima , Wahler Joseph , So Jae Young , Lin Yong , Lou You-Rong , Barnard Nicola J. , Matsuura Isao , Suh Nanjoo
Published: September 2020

Ductal carcinoma in situ (DCIS) transition to invasive tumor is a big problem for breast cancer. The human MCF10DCIS.com cell line (abbreviated as MCF10DCIS) is derived from the human MCF10A breast epithelial cell line. The MCF10DCIS xenografts are similar to human high grade comedo DCIS. In the xenograft assay, MCF10DCIS cells form DCIS-like lesion, remain as DCIS-like lesion in the first several weeks, and then progress into invasive carcinoma. Thus, it is an excellent model to study human breast cancer transition from DCIS to invasive carcinoma. We investigated the role of CD44 in breast cancer transition from DCIS to invasive carcinoma by knockdown the expression of CD44 in the MCF10DCIS xenograft model. Our results show that knockdown of CD44 expression in the MCF10DCIS cells promotes DCIS transition to invasive carcinoma. Our finding is supported by a previous study that showed CD44 expression is higher in DCIS than in invasive breast carcinomas for human patients. Furthermore, we will present our ongoing studies for the underlying mechanisms. Our findings have important implications for breast cancer.

Citation Format: Fang Liu, Jun Zhou, Tanima Kundu-Roy, Joseph Wahler, Jae Young So, Yong Lin, You-Rong Lou, Nicola J. Barnard, Isao Matsuura, Nanjoo Suh. CD44 inhibits the transition of ductal carcinoma in situ to invasive breast carcinoma [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 5052.
Abstract 5097: Pancreatic cancer cells derived exosomes stimulate pericyte phenotype switching in the tumor microenvironment
Authors: Delgado Alexander , Jacobson Reed , Wrede Dylan , Ha Sangdeuk , Kim Jiha
Published: September 2020

In pancreatic cancer (PC), most conventional and targeted therapies fail to provide substantial response partly due to the limited delivery efficacy of cytotoxic drugs. Poor vascular structure and stroma-rich tumor microenvironment (TME) likely contribute to such failure. We have previously shown that in breast cancer, differential perivascular investment significantly impacts vascular functionality and drug delivery efficacy. Considering unique pancreatic cancer TME, it is a promising approach to "re-invest" perivascular cells in which blood vessels are in their optimal functional status.

As a first attempt to understand the perivascular phenotype of pancreatic tumors, we utilized a multiplex imaging analysis technique to map out differential pericyte markers expression on several mouse model systems for PC as well as tumor microarray (TMA) of patients with PC. Among pericyte markers examined, alpha-smooth muscle actin (α-SMA) expression was significantly increased in all cases of PC compared to normal pancreas tissues, which skewed the ratio between Desmin (mature pericyte marker) and α-SMA.

To determine if eliminating pathological pericytes can normalize vascular phenotype of pancreatic tumor, we utilized PDGFRβ-TK transgenic mice in which proliferating pericytes that express PDGFRβ are depleted. However, eliminating these pericytes resulted in increased metastasis accompanied by increased vascular leakiness. This result indicates that elimination tumor-associated pericyte might result in an adverse effect instead of vascular normalization. Therefore, we hypothesize that normalizing vascular phenotype by re-investment of perivascular cells will likely contribute to improving TME for a better outcome.

To understand the molecular mechanism governing pericyte phenotype switching, we established in vitro vascular cell culture system. The results indicate that PC cells derived exosomes can modify pericyte phenotype that resembles tumor-associated pericyte. Such phenotype conversion was even further enhanced in a hypoxic environment. Currently, we are investigating global gene expression changes of pericyte upon exposure to PC cell exosomes using a single-cell RNA sequencing method to have a comprehensive understanding of pericyte phenotype switching. Currently, we are performing in vivo perivascular re-investment using our candidate molecules to prove our hypothesis that perivascular re-investment will improve vascular performance and consequently enhance drug delivery efficacy.

Citation Format: Alexander Delgado, Reed Jacobson, Dylan Wrede, Sangdeuk Ha, Jiha Kim. Pancreatic cancer cells derived exosomes stimulate pericyte phenotype switching in the tumor microenvironment [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 5097.
Abstract 5166: In silico identification of specific inhibitor(s) of tumor associated Vacuolar ATPase ‘a2' isoform: A critical target for anti-cancer drug development
Authors: Kulshrestha Arpita , Katara Gajendra K. , Kumar Awanish , Beaman Kenneth D.
Published: September 2020

The discovery of effective anti-cancer therapies has immense consequences on global health. Vacuolar (H+)-ATPases (V-ATPases) are multi-subunit nano-motors that acidify the lumen of intracellular organelles in normal physiology. In cancer cells, the plasma membrane V-ATPases acidify the extracellular space. This is critical in tumor invasion, metastasis, and chemo-resistance. The `a2` isoform of V-ATPase (a2V) is the major pH sensing unit of this complex and is distinctly overexpressed on the surface of cancer cells making it an excellent drug target. The chemical inhibitors such as bafilomycin/concanamycin target V-ATPase subunits also critical in normal cell function. Currently, there are no selective a2V inhibitors commercially available as anti-cancer agents. Here, we employed in silico molecular modeling to analyze specific a2V binding sites in order to design targeted small molecules a2V inhibitors. We have identified several natural compounds as a2V ligands which inhibit specifically a2V in cancer. Employing human ovarian and breast epithelial cells (normal or tumor), in vitro studies measured each a2V ligand(s) effects on cancer cell proliferation. We selected five a2V ligands showing the most potent anti-proliferative activity in cancer cells and were least toxic to normal cells. We further measured each ligand(s) effects on specific ATPase activity in vitro by determining the decrease in inorganic phosphate levels. Next, the two most potent a2v ligands (KDB001, KDB002) which belonged to alkaloid and mono-terpenoid phenol groups, were selected to measure the changes in cytosolic and extracellular pH- SNARF assay and pH of cell supernatants. KDB001 induced cytosolic pH acidification in a dose-dependent manner indicating inhibition of V-ATPase activity. Based on the above experiments, the best ligand with most prominent effect on a2V activity (KDB-001) was further validated in vivo for anti-ovarian cancer efficacy in an athymic nude mouse model. KDB001 administration resulted in a significant delay in in vivo ovarian tumor growth. Studies are underway to design and synthesize more efficient analogs for KDB001 molecular modelling, toxicity assessment and chemical synthesis approach. In conclusion, the study has discovered new inhibitors of V-ATPase-a2V, positioning these promising compounds to enter the anti-cancer drug development pipeline.

Citation Format: Arpita Kulshrestha, Gajendra K. Katara, Awanish Kumar, Kenneth D. Beaman. In silico identification of specific inhibitor(s) of tumor associated Vacuolar ATPase ‘a2' isoform: A critical target for anti-cancer drug development [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 5166.
Abstract 5300: A survey of EphA2 expression by immunohistochemistry (IHC) in tumor tissue microarrays (TMAs) to support BT5528 indication selection
Authors: Campbell Carly T. , Smale Robert , Lee James , Ragheb Josette William , Rigby Michael , Gelb Tara , Mahnke Lisa , Bennett Gavin , Blakemore Stephen J.
Published: September 2020

Introduction: BT5528 is a Bicycle® Toxin Conjugate (BTC) which contains an EphA2 binding peptide (Bicycle) conjugated through a molecular spacer and cleavable linker to the antimitotic toxin MMAE. EphA2 is an attractive tumor target; overexpression has been reported in multiple tumor types. Preclinical activity of BT5528 has been described previously, demonstrating increased efficacy is associated with high EphA2 tumor cell expression levels in xenograft models. Enrollment to a Phase I dose escalation study with BT5528 (BT5528-100) has started in patients with advanced solid tumors associated with EphA2 expression. Here we describe the analysis of TMAs stained using an EphA2 IHC assay developed for retrospective analysis of archive tumor from the BT5528-100 clinical trial.

Methods: A clinical grade EphA2 IHC assay was developed on the Dako platform using R&D Systems EphA2 primary antibody (AF3035) at 10 µg/mL and the Dako FLEX detection kit. TMAs of indications reported to have high EphA2 including pancreatic, bladder, head & neck, stomach, NSCLC, TNBC, and ovarian cancer were stained and scored for EphA2 expression. H-scores (the product of stain intensity on a scale of 0-3 and % positive tumor cells) were generated by a pathologist independently for tumor cell membrane (TM) and tumor cytoplasm (TC). H-scores ≥20 were considered positive for EphA2.

Results: The pattern of EphA2 expression was different across indications tested with pancreas having the greatest frequency of EphA2 expression. In contrast to the other indications assessed, pancreas and bladder had a greater percentage of cores positive for TM over TC. These differences may be relevant for BT5528 indication selection given that the mechanism of BTCs may be enhanced in cases which are TM positive.

Conclusion: Use of an IHC assay assessing expression of EphA2 across multiple TMAs and scoring TM and TC individually may help guide indication selection for the BT5528 clinical program.

EphA2 expression by IHC in multiple indicationsIndicationTotal cores (N)TM Positive N (%)TC Positive N(%)TM or TC Positive N (%)Pancreatic9248 (52)14 (15)48 (52)Bladder14242 (30)37 (26)51 (36)Head & Neck6914 (20)17 (25)22 (32)Stomach5310 (19)15 (28)15 (28)NSCLC22145 (20)52 (24)60 (27)Ovarian838 (10)13 (16)17 (20)TNBC1243 (2)25 (20)25 (20)

Citation Format: Carly T. Campbell, Robert Smale, James Lee, Josette William Ragheb, Michael Rigby, Tara Gelb, Lisa Mahnke, Gavin Bennett, Stephen J. Blakemore. A survey of EphA2 expression by immunohistochemistry (IHC) in tumor tissue microarrays (TMAs) to support BT5528 indication selection [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 5300.
Abstract 5363: A direct amplicon-based targeted sequencing assay for mutation analysis of single circulating tumor cells and correlation with circulating tumor DNA
Authors: Ericson Nolan G. , Kapoor Vidushi , Liu Guoying , Clein Alisa C. , Sabath Daniel E. , Kaldjian Eric P. , George Tad
Published: September 2020

There is increasing interest in sequence analysis of circulating tumor cells (CTCs) to identify actionable mutations that may complement circulating tumor DNA (ctDNA) findings. Because the amount of DNA present in a single cell is miniscule (~6 pg), whole genome amplification (WGA) is typically performed prior to next generation sequencing (NGS) library preparation. Existing WGA methods have inherent amplification biases leading to non-uniform genome coverage that can cause dropout of desired targets, as well as replication errors that can lead to false positive results. Targeted sequencing is an alternative approach that does not require WGA. We modified the CleanPlex® OncoZoom® Cancer Hotspot Panel protocol to perform targeted sequencing of single CTCs isolated by the RareCyte platform using A549 cell line spike-in and patient samples. We also used the CleanPlex panel to evaluate ctDNA from plasma isolated by blood collection tubes from two manufacturers (RareCyte and Streck). In both cases we compared OncoZoom results with and without prior WGA. Increased uniformity of coverage with decreased target dropout was observed when CleanPlex NGS libraries were prepared directly from cell lysates without WGA. Median read depth increased 48-fold when compared to the WGA method. On average, 23 out of 29 (79%) variants present in bulk A549 genomic DNA were observed without WGA in single model CTCs, while 15 of 29 (52%) were observed after WGA. Additionally, the false positive error frequency of non-WGA samples was 8-fold lower than in the WGA samples. CTCs and cell-free DNA from two metastatic breast cancer patients were also sequenced using OncoZoom. In one patient, PIK3CA E542K, a well-documented oncogenic mutation, was observed in 3 of 5 CTCs. In the other, ERBB2 L755S, a known HER2-reactivating mutation associated with chemoresistance, was found in 3 of 5 CTCs. Both patients' variants were present at similar allelic frequencies in plasma isolated from RareCyte and Streck cell-free DNA BCT tubes. Single-cell CleanPlex amplicon-based sequencing without prior WGA resulted in libraries with more complete and consistent coverage and lower error frequencies, enabling efficient and accurate assessment of somatic mutations in CTCs. This approach shows promise for cell-based liquid biopsy diagnostic applications.

Citation Format: Nolan G. Ericson, Vidushi Kapoor, Guoying Liu, Alisa C. Clein, Daniel E. Sabath, Eric P. Kaldjian, Tad George. A direct amplicon-based targeted sequencing assay for mutation analysis of single circulating tumor cells and correlation with circulating tumor DNA [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 5363.
Abstract 5402: Inhibition of RET via BLU-667 in an in vitro model of neuroblastoma
Authors: Gutierrez Jessica , Flynn Sean , Huo Yuchen , Lesperance Jacqueline , Zage Peter
Published: September 2020

Neuroblastoma is the most common extracranial solid tumor of childhood. Despite aggressive treatment, outcomes in patients with high-risk disease remains poor and new therapeutics are needed to address their needs. Rearranged during Transfection (RET) is a tyrosine kinase that is a known oncogenic driver of multiple human tumors. RET is expressed on neural crest derived cells, including neuroblastoma cells, and has been shown to play a role in neuroblastoma proliferation and survival as well. Recent studies have shown RET inhibition to be a promising new therapeutic target in neuroblastoma. BLU-667 is a selective RET inhibitor with a higher potency and specificity for RET than the current inhibitors that have been previously tested. We hypothesize that this increased potency and specificity will result in greater efficacy against neuroblastoma in preclinical models with fewer off target effects. Thus, to investigate this we evaluated BLU-667 at different concentrations against a panel of established human neuroblastoma cell lines to assess its effects on neuroblastoma cell proliferation, differentiation, and viability. We found that inhibition of RET resulted in a significant decrease in cell proliferation and viability in vitro. Further, given the increased specificity of BLU-667 to RET, we hope to use western blot protein analysis to establish the specific downstream pathways through which RET inhibition leads to decreased neuroblastoma cell survival, potentially revealing further therapeutic targets.

Citation Format: Jessica Gutierrez, Sean Flynn, Yuchen Huo, Jacqueline Lesperance, Peter Zage. Inhibition of RET via BLU-667 in an in vitro model of neuroblastoma [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 5402.
Abstract 5430: Deep learning-based prognosis prediction in T3N1 nasopharyngeal carcinoma patients treated with induction chemotherapy followed by concurrent chemoradiotherapy
Authors: Zhong Lianzhen , Dong Di , Tang Linglong , Han Shuyan , Tian Jie
Published: September 2020

Purpose: To estimate the value of deep learning on magnetic resonance (MR)-base radiomics for T3N1 nasopharyngeal carcinoma (NPC) patients receiving induction chemotherapy (ICT) followed by concurrent chemoradiotherapy (CCRT).

Methods: A total of 672 NPC patients with T3N1 disease (training cohort: n = 470; test cohort: n = 202) were enrolled and underwent MRI scan prior to receiving ICT+CCRT. Primary endpoint was disease-free survival (DFS). From each of three sequences of MR images of the primary tumor at baseline, a survival model based on SE-ResNeXt was developed to predict DFS end-to-end. Incorporating three deep learning-based radiomic signatures, a radiomic model was built using multivariable Cox proportional hazard model. Harrell's concordance indices (C-index) were applied to evaluate the predictive power of radiomics nomogram.

Results: Three deep learning-based radiomic signatures were significant associated with DFS in the training (C-index: 0.609-0.616) and test (C-index: 0.587-604) cohorts. The radiomic model had the best result for predicting DFS (train cohort: C-index, 0.647, 95% CI, 0.587-0.708, p < 0.001; test cohort: C-index, 0.647, 95% CI, 0.552-0.741, p = 0.002). In addition, the radiomic model also had a significant predictive value for secondary endpoints in the entire dataset (overall survival: C-index, 0.622, p < 0.001; distant metastasis-free survival: C-index, 0.578, p = 0.044; locoregional relapse-free survival: C-index, 0.648, p < 0.001).

Conclusion: Deep learning MR-based radiomic signatures may serve as a powerful tool for prognosis prediction in T3N1 nasopharyngeal carcinoma patients receiving ICT + CCRT treatment.

Citation Format: Lianzhen Zhong, Di Dong, Linglong Tang, Shuyan Han, Jie Tian. Deep learning-based prognosis prediction in T3N1 nasopharyngeal carcinoma patients treated with induction chemotherapy followed by concurrent chemoradiotherapy [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 5430.