bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1671: Deregulation of mTOR signaling contributes to chemoradiation resistance in lung squamous cell carcinoma
Authors: Shen Changxian , Shyu Duan-Liang , Xu Min , Yang Linlin , Duan Wenrui , Williams Terence M.
Published: September 2020

Non-small cell lung carcinoma (NSCLC) represents ~ 85% of lung cancer, and ~1/3 of these patients present with locally-advanced, unresectable/medically-inoperable disease. Patients who present with locally-advanced NSCLC are commonly treated with definitive chemoradiation. Even though an initial response to concurrent chemoradiation is commonly observed, disease relapse in the radiation fields occurs in 1/3 of cases, likely driven by therapy-resistant tumor cells. However, the underlying molecular mechanisms of chemoradiation resistance of NSCLC remains largely unknown. We herein report that upregulation of mTOR signaling plays an important role in the chemoradiation resistance of NSCLC. Patient-derived xenograft (PDX) models of lung squamous cell carcinoma (LUSC) were established in the flanks of NOD-SCID gamma (NSG) mice. Chemoradiation resistant tumor models were developed by treating the tumors with a regimen of 2-4 weeks of chemoradiation (20, 30 or 40 Gy at 2 Gy per day, concurrent with cisplatin). Recurrent tumor models were collected and subjected to functional proteomics using reverse phase protein analysis (RPPA). Several LUSC tumors recurred in the 20 Gy+cisplatin, and 40 Gy+cisplatin groups. RPPA analysis of the recurrent and parental (untreated) PDX tumors showed that there were enhanced mTOR signaling in the chemoradiation resistant 20 Gy+cisplatin and 40 Gy+cisplatin tumors compared to that in the control tumors. Of note, phospho-AKT-S473, phospho-AKT-S308, phospho-S6-S235/6 and phospho-GSK3β-S9/21 were the highest upregulated signaling and were validated by immunoblotting. Consistently, pharmacological inhibition of mTOR kinase by Torin2 significantly sensitized LUSC cell lines to ionizing radiation. Our results demonstrate that utilization of PDX models is a useful method to elucidate the molecular mechanisms of chemoradiation resistance of NSCLC and deregulation of mTOR signaling contributes to the chemoradiation resistance of NSCLC. Our findings suggest that clonal selection of subpopulations with high mTOR signaling in heterogeneous NSCLC tumors might contribute to the relapse of NSCLC after chemoradiation and that mTOR kinase inhibitors are promising agents for the upfront treatment of NSCLC patients in combination with chemoradiation.

Citation Format: Changxian Shen, Duan-Liang Shyu, Min Xu, Linlin Yang, Wenrui Duan, Terence M. Williams. Deregulation of mTOR signaling contributes to chemoradiation resistance in lung squamous cell 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 1671.
Abstract 1674: Correlation of the mutational pattern and gene expression data of patient-derived Non-Hodgkin lymphoma xenografts (PDX) with the response to targeted therapies
Authors: Brzezicha Bernadette , Conrad Theresia , Becker Michael , Bittner Aitomi , Janz Martin , Schmitt Clemens A. , Keilholz Ulrich , Hoffmann Jens
Published: September 2020

Non-Hodgkin lymphomas (NHL) are lymphoid malignant neoplasms that represent a very heterogeneous group and still pose an important clinical challenge. In particular, the frequent development of resistance to the treatment with standard-of-care (SoC) drugs is associated with a high incidence of disease recurrence. Recent progress in molecular high-throughput profiling has helped to identify genetic drivers for many subtypes of B-cell lymphomas as well as T-cell lymphomas. Target validation and drug development depend on corresponding preclinical models representing the different subtypes. Therefore, we established and characterized a panel of patient-derived NHL xenografts. All lymphoma PDX were derived from peripheral blood, lymph node extirpations or core needle biopsies, respectively, and were routinely implanted subcutaneously into immunodeficient mice. For further characterization, established lymphoma PDX models were treated with SoC (e.g., cyclophosphamide, doxorubicine, vincristine, etoposide) and investigational drugs. To gain a deeper insight into the molecular biology of the lymphoma models, RNA sequencing was performed. More than 20 PDX models from different NHL entities have been successfully established and characterized. We observed heterogeneous individual responses to the SoC treatments as well as to target treatments such as rituximab or ibrutinib. Explorative analysis of RNA sequencing data confirmed the representation of the clinical lymphoma subgroups (e.g. DLBCL, Burkitt, AITL) in our panel of lymphoma models. Based on the RNA sequencing data, the individual Human Leukocyte Antigen (HLA) type of each lymphoma PDX was determined with the aim to enable personalized, HLA type-specific studies. Our lymphoma PDX portfolio provides an exceptional platform for the identification and validation of new targets and allows the preclinical screening of new combinations in translational research projects.

Citation Format: Bernadette Brzezicha, Theresia Conrad, Michael Becker, Aitomi Bittner, Martin Janz, Clemens A. Schmitt, Ulrich Keilholz, Jens Hoffmann. Correlation of the mutational pattern and gene expression data of patient-derived Non-Hodgkin lymphoma xenografts (PDX) with the response to targeted therapies [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 1674.
Abstract 1679: Characterization and establishment of triple negative breast cancer patient derived xenograft derived from a Nigerian woman
Authors: Okoli Uzoamaka Adaobi , Okafor Michael Tochukwu , Agu Kenneth A. , Ohayi Samuel R. , Nna Emmanuel O. , Chijioke Chioli P. , Udeniya Iroka J.
Published: September 2020

Extensive literature has demonstrated the use of fresh human breast tumour tissue surgically implanted in immunodeficient mice to establish patient-derived xenograft (PDX) as a therapeutic assessment tool. Unfortunately, there is a paucity of genomic research using PDX as a preclinical tool for breast cancer metastasis in Nigerian women. Previous studies of rodent models have concentrated on cell line xenograft of other tumours usually of Caucasian or African American counterpart. To overcome this limitation, Triple-negative breast cancer (TNBC) was derived from a consenting 32-year-old Nigerian woman who had neoadjuvant therapy and had undergone a mastectomy. Triple-negative breast cancer lacks expression of hormone receptors including human epidermal receptor (HER2-), estrogen receptor (ER-) and progesterone receptor (PR-) genes. TNBC xenograft was established by orthotopic surgical implantation of breast cancer tissue fragments of 2mm3 in size with cultrex in the cleared breast pads of immunosuppressed mice and a further two passages. The size of the implanted tumour was checked twice weekly. Haematology & eosin histology and immunohistochemistry analysis were used to structurally and functionally characterize the tumour xenograft and primary tumour respectively. Real-time quantitative polymerase chain reaction (RQ-PCR) was used to evaluate the genomic correlation between primary tumour and tumour xenograft. Target genes compared between the tumour xenograft and primary tumours included CXCL16; CLD10; CDC42EP3; CXCL16; EPSTI1; PALMD and ZAN. Reference genes RLP13A and GAPDH were selected as assays for endogenous controls. All tumour tissues were mechanically homogenized and lysed in guanidine isothiocyanate buffer from which RNA was extracted and converted to cDNA by random hexamer priming. RQ-PCR was performed using TaqMan chemistries. This was validated by DNA sequencing of the xenografts tumour and primary tumour using Nanopore DNA sequencing, and Ligation Sequencing ID2 chemistry. The DNA sequences were aligned with Meg Align Pro The xenografts appear to maintain characteristic human histopathology, tumour-marker production. None of the tumours showed hormone sensitivity. There was a local invasion of breast cancer in the bone. The pairwise alignment yielded significant percentage relativity.

Citation Format: Uzoamaka Adaobi Okoli, Michael Tochukwu Okafor, Kenneth A. Agu, Samuel R. Ohayi, Emmanuel O. Nna, Chioli P. Chijioke, Iroka J. Udeniya. Characterization and establishment of triple negative breast cancer patient derived xenograft derived from a Nigerian woman [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 1679.
Abstract 1714A: TGF-beta signaling on myeloid cells regulates ECM deposition in mammary carcinoma via adenosine dependent mechanisms
Authors: Vasiukov Georgii , Novitskaya Tatiana , Schwager Samantha , Moses Harold , Blackwell Timothy , Feoktistov Igor , Novitskiy Sergey
Published: September 2020

TGF-beta plays a crucial role in tumor microenvironment by regulating cell-cell and cell-stroma interactions. We previously demonstrated that TGF-beta signaling on myeloid cells regulates expression of CD73, a key enzyme for production of adenosine, a pro-tumorigenic metabolite implicated in regulation of tumor cell behaviors, immune response and angiogenesis. Using MMTV-PyMT mouse mammary tumor model, we discovered that deletion of TGF-beta signaling on myeloid cells (PyMT/TGFbRII-LysM) affects ECM formation in tumor tissue, specifically increases collagen and decreases fibronectin deposition, and these changes associate with mitigated tumor growth and reduced metastases. Using multiplexed five-color immunofluorescent staining and spatial analysis on a single-cell level, we discovered that reduced TGF-beta signaling on fibroblasts associates with their proximity to CD73 positive myeloid cells in tumor tissue. Consistent with these findings, in vitro gel contraction assay and Western blotting for Collagen I and pSMAD proteins confirmed that adenosine significantly downregulates TGF-beta signaling on fibroblasts. Using in vitro pharmacological approach, we found that this effect is regulated by A2a and A2b adenosine receptors. TCGA data base analysis revealed that patients with triple negative breast cancer and basal type have similar signature of adenosine and ECM profiles: high expression of A2b adenosine receptors correlates with decreased expression of Col1 and is associates with poor outcome. Taken together, our studies reveal a new role for TGF-beta signaling on myeloid cells in tumorigenesis. Discovered crosstalk between TGF-beta/CD73 on myeloid cells and TGF-beta signaling on fibroblasts can contribute to ECM remodeling and pro-tumorigenic actions of CAFs.

Citation Format: Georgii Vasiukov, Tatiana Novitskaya, Samantha Schwager, Harold Moses, Timothy Blackwell, Igor Feoktistov, Sergey Novitskiy. TGF-beta signaling on myeloid cells regulates ECM deposition in mammary carcinoma via adenosine dependent mechanisms [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 1714A.
Abstract 1719: Drug-loaded porphyrin nanoparticles as a platform for targeted and photodynamic combination therapy
Authors: Cheung Justin M. , Baker Hanan , Li Daniel , Stor Daniel , Heller Daniel A.
Published: September 2020

Many traditional nanoparticle delivery mechanisms require the use of an inert vehicle to deliver single therapeutic payloads. Here, we describe a novel, dual-therapeutic, self-assembled nanoparticle class wherein both the payload and encapsulating vehicle may potentiate anti-neoplastic effects. The nanoparticles consist of photoactive porphyrin dyes encapsulating small molecule drugs. We screened 9 porphyrins with 27 small molecules for the formation of porphyrin-drug nanoparticles; a total of 243 nanoparticle candidates, of which 25 formed nanoparticles as dictated by pre-determined cutoffs for size, polydispersity, and porphyrin/drug concentrations. Logistic regression based on formation data and molecular descriptors suggested that the presence of electronegative groups positively influenced nanoparticle formation. Chemical inhibition of various uptake pathways in endothelial cells indicated that the uptake of these porphyrin-drug nanoparticles is largely mediated by clathrin and macropinocytosis. Stability testing demonstrated that porphyrin-drug nanoparticles remained stable at 37°C for at least 6 hrs. This work outlines the synthesis and characterization of a new class of porphyrin nanoparticles with the capacity to deliver targeted therapy. Furthermore, the simplicity of the self-assembly process highlights the potential for this novel nanoparticle paradigm to be easily tested in disease models.

Citation Format: Justin M. Cheung, Hanan Baker, Daniel Li, Daniel Stor, Daniel A. Heller. Drug-loaded porphyrin nanoparticles as a platform for targeted and photodynamic combination therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1719.
Abstract 1722: Nanoparticle pro-drug to overcome the stromal barrier in pancreatic ductal adenocarcinoma
Authors: Alzhrani Rami , Sau Samaresh , Aboukameel Amro , Alsaab Hashem , Azmi Asfar , Iyer Arun
Published: September 2020

Pancreatic ductal adenocarcinoma (PDAC) is the third-highest cause of cancer-related death in the United States. The heterogenicity of the PDAC is a major challenge in the success of anti-PDAC therapy. PDAC is comprised of >90 % dense desmoplastic tumor stroma that impedes the delivery of drugs into the core of the tumor, resulting development of drug resistance. Thus, we developed a nanoparticle pro-drug to overcome the stromal barrier of PDAC. We utilized nanoengineering to target dual CD44 and c-Met biomarkers that have a significant role in developing drug resistance on PDAC. Western blot analysis in various PDAC cell lines, such as PANC-1, AsPc-1, and BxPc3 revealed a high abundance of CD44 and cMet, strongly supporting our hypothesis. Therefore, targeting the CD44 and c-Met biomarker and selectively delivering anti-PDAC drug is a smart strategy to improve the anti-PDAC outcome. We developed dual CD44/c-Met targeted nanoparticle, called D44M that can efficiently penetrate the core of tumor as well as home into CD44 and cMet biomarker. We evaluated tumor selectivity of the near infrared (NIR) fluorescent dye conjugated D44M, D44M-NIR in the orthotopic PDAC model. The biodistribution of D44M-NIR in orthotopic animal model proved superior tumor penetration of D44M-NIR with 7-fold higher than the control NIR dye treatment. Moreover, D44M-NIR treated showed minimum liver accumulation, suggesting an outstanding approach of overcoming nanoparticle mediated liver toxicity. Finally, the D44M was chemically conjugated with gemcitabine (GEM), namely D44M-GEM pro-drug that showed excellent PDAC killing. The PDAC cell killing study of D44M-GEM in combination with Everolimus revealed high synergistic combination effect with CI value of 0.2-0.5. The anti-PDAC therapy in ASPC-1 xenograft model demonstrated significant tumor growth inhibition for treatment of D44M-GEM and D44M-GEM+Everolimus as compared to control. Supported with data, our work provides a perspective towards the implementation of nanotherapeutic to overcome the bottleneck barrier of therapeutic delivery and efficacy of PDAC management.

Citation Format: Rami Alzhrani, Samaresh Sau, Amro Aboukameel, Hashem Alsaab, Asfar Azmi, Arun Iyer. Nanoparticle pro-drug to overcome the stromal barrier in pancreatic ductal adenocarcinoma [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 1722.
Abstract 174: Genomic cytometry characterization of preclinical models for development of immune-oncology therapeutics
Authors: Lee Joon Sang , McGrath Shannon , Wang Emma , Rogers-Grazado Maximilian , Zhang Yu-an , Malkova Natalia , Pollard Jack , Protopopov Alexei
Published: September 2020

Single cell characterization of protein epitopes is usually associated with fluorescent Flow Cytometry, which can be both costly and time consuming to optimize for multiple epitopes. To overcome the limitations of this method, we introduce Genomic Cytometry that utilizes cellular indexing of transcriptomes and protein epitopes by next-generation sequencing (CITE-seq) technology instead of flow. Genomic Cytometry is based on staining cells with antibodies conjugated to unique 15-bp DNA barcodes instead of fluorophores. After sequencing and data analysis the barcode read number can be linked to protein abundance. We utilized Genomic Cytometry to simultaneously analyze over 50 different protein biomarkers in a single scalable workflow. The application is ideal for characterization of heterogeneous cell populations such as tumors including their immune content. In addition to profiling a number cell surface protein markers that exceeds capabilities of traditional flow cytometry the technology allows for assessment of single cell gene expression data to link protein and genomic data sets.

Corresponding pre-analytical and 10X Genomics-based analytical workflows were established for human PBMC samples, as well as for solid tissue including tumor specimens derived from mouse syngeneic tumor models. Data analysis pipelines were developed and validated that includes custom scripts. We ran a number of cross-platform validation studies (mouse and human cells) and found that the Genomic Cytometry is highly correlative to fluorescent flow. Multiplex methods were implemented to allow pooling of samples to reduce biases related to sample manipulation. The method enables large-scale single-cell sequencing experiments. In proof of concept experiments, a number of preclinical models, such as MC38 and H22, were deeply analyzed using CITE-seq approach. Genomic Cytometry enables rapid and cost effective characterization of both immune-oncology (I-O) models and modes of action of new I-O molecules in order to develop more differentiated therapeutics.

Citation Format: Joon Sang Lee, Shannon McGrath, Emma Wang, Maximilian Rogers-Grazado, Yu-an Zhang, Natalia Malkova, Jack Pollard, Alexei Protopopov. Genomic cytometry characterization of preclinical models for development of immune-oncology therapeutics [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 174.
Abstract 1762: Dual HDAC-BET proteins inhibition by TW9 hold promise for breast cancer
Authors: Xavier Pedro Luiz P. , Lucas Romain , Weiser Tim , Knapp Stefan , Fukumasu Heidge , Müller Susanne
Published: September 2020

Epigenetic proteins play a key role in gene expression regulation and cellular states and, consequently, abnormalities in these mechanisms result in the development and progression of several types of cancer. Thus, inhibitors against epigenetic targets have recently entered clinical trials as potential and innovative therapies against this disease. Both histone deacetylases (HDACs) and bromodomain and extraterminal proteins (BET) inhibitors have been shown to be efficient against several different cancer types, including breast cancer. Although synergistic effects of HDAC and BET inhibitors have been observed in different cancer types, few studies have been performed in breast cancer. However, combination studies often result in increased toxicity, thus new strategies are necessary to overcome these limitations. Therefore, we designed and synthesized a dual HDAC-BET inhibitor, TW9. In this study, we determined the effects of TW9 on two HR-positive cell lines MCF7 and T-47D and two triple-negative breast cancer cell lines, MDA-MB-231 and HCC1806 in 2D and 3D in vitro models. To determine the cytotoxic effects, cells were treated with (+)-JQ1 (BET inhibitor), CI994 (HDAC inhibitor) and TW9 (dual HDAC-BET inhibitor). After 72h, cell viability was tested using XTT assay. Primary and secondary tumorspheres were grown in ultra-low attachment multiwell plates and were treated with DMSO (control) or different concentrations of TW9. The triple-negative cell lines were more resistant to all inhibitors and TW9 was more potent compared to (+)-JQ1 or CI994 in all the breast cancer cells (p<0.05). Tumorspheres of MCF7 cells were treated with TW9 ranging from 100 nM to 500 nM. Significant decrease of primary and secondary tumorspheres was observed at all doses (p<0.0001). In addition, TW9 was able to inhibit the formation of secondary tumorspheres, even after removal of the treatment during dissociation of primary tumorspheres (p<0.0001), pointing to an epigenetic effect. In summary, our findings showed that dual HDAC-BET inhibition by TW9 shows positive effects regarding tumorigenicity and self-renewal of cancer cells and thus may provide a promising treatment against breast cancer, in particular for the more resistant triple-negative cells.

Citation Format: Pedro Luiz P. Xavier, Romain Lucas, Tim Weiser, Stefan Knapp, Heidge Fukumasu, Susanne Müller. Dual HDAC-BET proteins inhibition by TW9 hold promise for 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 1762.
Abstract 1768: Identification of a potent, orally-available SMARCA2/4 inhibitor with in vitro and in vivo activity in preclinical models of SMARCA4-mutant NSCLC
Authors: Drew Allison E. , Jacques Suzanne L. , Eichinger Lindsey W. , Pantano Chloe , Motwani Vinny , Tang Cuyue , Tang Cuyue , Farrow Neil , Brach Dorothy , Howe Selene , Raimondi Alejandra , Dransfield Daniel T. , Duncan Kenneth W. , Stickland Kim , Huang Liyue , Lampe John
Published: September 2020

SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin subfamily A member 2 (SMARCA2) is an ATP-dependent DNA helicase and a catalytic component of the SWI/SNF complex. SWI/SNF-mediated nucleosome remodeling is a critical regulator of chromatin accessibility resulting in transcriptional regulation of gene sets that determine and maintain cell state. The role of SMARCA2 as the catalytic driver of SWI/SNF activity is mutually exclusive with that of its close paralog, SMARCA4. Loss of functional SMARCA4, as is reported in a subset of NSCLC and other cancer types generally confers a dependency on SMARCA2. Cancers lacking functional SMARCA4 have been shown to be sensitive to loss of SMARCA2 function either through genetic ablation or the use of a small molecule SMARCA2/4 inhibitor. We describe the identification and characterization of a small molecule SMARCA2/4 inhibitor Compound 1 and its selective activity in the SMARCA4-mutant setting both in in vitro and in vivo model systems. The compound demonstrates inhibition of SMARCA2 and SMARCA4 enzymatic activity in biochemical assays and broad selectivity against other helicases. Selective sensitivity in anti-proliferative assays of 15-fold was observed in NSCLC cell lines with SMARCA4 protein loss compared to those harboring WT SMARCA4 protein. Oral dosing demonstrates dose-dependent in vivo SMARCA2 inhibition and anti-tumor activity in SMARCA4-mutant NSCLC xenograft models. This compound is suitable for further exploration of the role of SMARCA2/4 and SWI/SNF in vitro and in vivo in cancer and other indications.

Citation Format: Allison E. Drew, Suzanne L. Jacques, Lindsey W. Eichinger, Chloe Pantano, Vinny Motwani, Cuyue Tang, Cuyue Tang, Neil Farrow, Dorothy Brach, Selene Howe, Alejandra Raimondi, Daniel T. Dransfield, Kenneth W. Duncan, Kim Stickland, Liyue Huang, John Lampe. Identification of a potent, orally-available SMARCA2/4 inhibitor with in vitro and in vivo activity in preclinical models of SMARCA4-mutant NSCLC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1768.
Abstract 1776: Exploring anetumab ravtansine in a preclinical model of thymic carcinoma
Authors: Chen Vincent , Sommer Anette , Schatz Christoph , Zitzmann-Kolbe Sabine , Kim In-Kyu , Giaccone Giuseppe
Published: September 2020

Thymic epithelial tumors (TETs) are a group of rare tumors (0.13 per 100,000 persons), comprised of thymomas and thymic carcinoma, arising from the epithelial cells of the thymus. The biology of these tumors is not well understood, and there are no approved targeted therapies against TETs. One promising drug target is mesothelin (MSLN), which is a surface glycoprotein that is cleaved to produce two proteins: mature MSLN (mMSLN) and megakaryocyte potentiating factor (MPF). In normal physiology, expression of MSLN is limited to mesothelial cells lining the pleura, pericardium, and peritoneum. Its expression is also present in various cancers, including thymic carcinoma, where it is expressed in 79% of cases. In this study, we investigate the ability of the MSLN targeting, fully human monoclonal antibody-drug conjugate (ADC) anetumab ravtansine (ARav) to inhibit growth of thymic carcinoma in in-vitro and in-vivo models.

We utilized immunoblotting and RT-qPCR to characterize MSLN expression in human thymoma and thymic carcinoma derived cell lines. Binding of anetumab, the antibody moiety in ARav, to thymoma and thymic carcinoma cell lines was assessed by flow cytometry. In-vitro, ARav's effect on cell proliferation was tested against MP57 cells (thymic carcinoma), stably transfected with MSLN, utilizing CellTiter-Glo. ARav's activity in-vivo as a monotherapy, and as a combination therapy with cisplatin, was tested against MSLN expressing T1889 xenograft tumors in athymic nude mice and compared to vehicle, isotype control ADC, and standard-of-care (SoC) cisplatin.

Using the anetumab antibody, thymoma (T1682) and thymic carcinoma (T1889) cell lines were shown to express MSLN on the cell surface. In-vitro, ARav was effective at inhibiting growth of MP57 cells stably transfected with mMSLN. In-vivo, 15 mg/kg ARav inhibited T1889 xenograft tumor growth more efficaciously than vehicle, the isotype control ADC (all given every 2 weeks), and 4 mg/kg cisplatin (given every week). Combining ARav at a dose of 7.5 mg/kg with 4 mg/kg cisplatin yielded an additive effect on inhibiting tumor growth.

MSLN is expressed in thymoma and thymic carcinoma derived cell lines. Anetumab ravtansine inhibits growth of MSLN positive thymic carcinoma in our preclinical model; when combined with SoC cisplatin, ARav confers enhanced tumor growth inhibition.

Citation Format: Vincent Chen, Anette Sommer, Christoph Schatz, Sabine Zitzmann-Kolbe, In-Kyu Kim, Giuseppe Giaccone. Exploring anetumab ravtansine in a preclinical model of thymic 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 1776.
Abstract 1788: A high-throughput chemical genetic screen reveals SALL4-induced metabolic vulnerabilities in cancer
Authors: Tan Justin L. , Li Feng , Yeo Joanna Z. , Yong Kol Jia , Bassal Mahmoud A. , Ng Guo Hao , Lee May Yin , Leong Chung Yan , Tan Hong Kee , Wu Chan-Shuo , Liu Bee Hui , Chan Hon Man , Tan Zi Hui , Chan Yun Shen , Wang Siyu , Lim Zhi Han , Toh Tan Boon , Hooi Lissa , Low Kia Ngee , Ma Siming , Kong Nikki R. , Stein Alicia J. , Wu Yue , Thangavelu Matan T. , Suzuki Atsushi , Periyasamy Giridharan , Asara John M. , Dan Yock Young , Bonney Glenn K. , Chow Edward K. , Lu Guo-Dong , Ng Huck Hui , Kanagasundaram Yoganathan , Ng Siew Bee , Tam Wai Leong , Chai Li , Tenen Daniel G.
Published: September 2020

Transcription factors are important drivers of cancer but the development of therapeutics against these factors has had limited success. We developed a stringent high-throughput chemical genetic screening platform to identify compounds that target oncogenic transcription factor SALL4 dependency in liver cancer. The platform comprises SALL4 low- and high-expressing endogenous and engineered isogenic liver cancer cell lines. Unexpectedly, from screening 21,575 natural product extracts, the top hits were four oxidative phosphorylation inhibitors that selectively reduced SALL4-dependent cell viability. The ATP synthase inhibitor oligomycin suppressed SALL4-expressing cancer in lung and liver cancer cell culture models, and in patient-derived xenograft models of liver cancer. Oligomycin also synergized with sorafenib, the standard-of-care targeted therapy in liver cancer, to effectively suppress SALL4-driven tumorigenesis in vivo. When aberrantly expressed in cancer, SALL4 binds ~50% of mitochondrial genes, including many oxidative phosphorylation genes, to predominantly upregulate their expression. SALL4 upregulation also alters the levels of oxidative phosphorylation-related metabolites and functionally increases oxidative phosphorylation activity. Application of our endogenous/isogenic transcription factor-screening platform revealed a therapeutically actionable oxidative phosphorylation vulnerability in SALL4-expressing cancers.

Citation Format: Justin L. Tan, Feng Li, Joanna Z. Yeo, Kol Jia Yong, Mahmoud A. Bassal, Guo Hao Ng, May Yin Lee, Chung Yan Leong, Hong Kee Tan, Chan-Shuo Wu, Bee Hui Liu, Hon Man Chan, Zi Hui Tan, Yun Shen Chan, Siyu Wang, Zhi Han Lim, Tan Boon Toh, Lissa Hooi, Kia Ngee Low, Siming Ma, Nikki R. Kong, Alicia J. Stein, Yue Wu, Matan T. Thangavelu, Atsushi Suzuki, Giridharan Periyasamy, John M. Asara, Yock Young Dan, Glenn K. Bonney, Edward K. Chow, Guo-Dong Lu, Huck Hui Ng, Yoganathan Kanagasundaram, Siew Bee Ng, Wai Leong Tam, Li Chai, Daniel G. Tenen. A high-throughput chemical genetic screen reveals SALL4-induced metabolic vulnerabilities in 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 1788.
Abstract 1796: Requirement for LIM kinases in acute myeloid leukemia
Authors: Jensen Patrizia , Carlet Michela , Kress Jana , Weber Andrea , Brunner Ines , Slabicki Mikolaj , Grill Gregor , Weisemann Simon , Cheng Ya-Yun , Jeremias Irmela , Scholl Claudia , Fröhling Stefan
Published: September 2020

Acute myeloid leukemia (AML) is a highly aggressive disease which, in spite of extensive research in the last decades, is still treated with conventional chemotherapeutic regimens in most cases. Recent approvals of FLT3 and IDH1/2 inhibitors have highlighted the promise of targeted approaches for personalized AML therapy. However, the majority of AML driver mutations currently remain “undruggable”, making the search for additional molecular targets imperative. LIM kinases 1 and 2 (LIMK1/LIMK2), which are primarily known for regulating actin dynamics via phosphorylation of the actin-depolymerizing factor cofilin, have previously been implicated in progression and metastasis formation of several epithelial tumors. However, their function in leukemia, and AML in particular, has not been addressed in detail. Using high-throughput RNA interference (RNAi) screens in AML cell lines, we identified LIMK1 as a potential novel molecular target for AML treatment. High LIMK1 expression was significantly correlated with shorter survival of AML patients, and was associated with NPM1 and DNMT3A mutations, MLL rearrangements, and elevated HOX gene expression. RNAi- and CRISPR/Cas9-mediated suppression, as well as pharmacologic inhibition of LIMK1 and its close homolog LIMK2, reduced colony formation and decreased proliferation due to slowed cell cycle progression of MLL-rearranged AML cell lines and patient-derived xenograft samples. This was accompanied by morphologic changes indicative of myeloid differentiation. Transcriptome analysis showed upregulation of several tumor suppressors as well as downregulation of HOXA9 targets and mitosis-associated genes in response to LIMK1 suppression, providing a potential mechanistic explanation for the anti-leukemic phenotype. Finally, we observed a mutual regulation between LIMK1 and CDK6, a kinase known to be involved in the differentiation block of MLL-rearranged AML, and addition of the CDK6 inhibitor palbociclib further enhanced the anti-proliferative effect of LIMK1 depletion. Our data therefore suggest that LIM kinases are promising targets for AML therapy.

Citation Format: Patrizia Jensen, Michela Carlet, Jana Kress, Andrea Weber, Ines Brunner, Mikolaj Slabicki, Gregor Grill, Simon Weisemann, Ya-Yun Cheng, Irmela Jeremias, Claudia Scholl, Stefan Fröhling. Requirement for LIM kinases in acute myeloid leukemia [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 1796.
Abstract 18: The CXCR4/CXCL12 axis is a target of a polyphenol extract from olive oil processing with potential cancer prevention and interception activities
Authors: Gallazzi Matteo , Festa Marco , Sansone Clementina , Corradino Paola , Noonan Douglas , Albini Adriana
Published: September 2020

The olive mill wastewater polyphenol extracts exhibit chemopreventive properties in lung cancer cell lines, acting on the CXCR4/CXCL12 axis.

Lung cancer still is a leading cause of cancer death worldwide. Although avoidance of tobacco use and smoking cessation will have the greatest impact on lung cancer development, (chemo)prevention could prove to be very effective, particularly in high risk individuals. Diverse diet-derived compounds have been reported to exert antioxidant, anti-proliferative and anti-angiogenic effects. Olive oil production involves the generation of an aqueous phase, defined as olive mill wastewater (OMWW), a polluting effluent rich in soluble polyphenols. We established a polyphenol rich, purified extract, from OMWW, that we termed A009. The effect of A009 on cell proliferation was determined by the crystal violet assay, while induction of apoptosis was measured by flow cytometry. The ability of the A009 to regulate the cell migration/invasion axis CXCR4/CXL12 and the production of pro angiogenic factors (VEGF, CXCL8, CCL2), was investigated by flow cytometry. The sprouting assay on Matrigel was used to functional verify the ability of A009 to limit invasive properties. Hydroxytyrosol, the most abundant polyphenols present in our A009, was used as reference molecules in all the assays performed.

We found that the A009 limit A549 and H1560 cell proliferation, in a dose and time dependent manner. These effects were associated with induction of apoptosis at 24 and 48 hours of treatment, that was higher in H1650, as compared to A549 cells. The A009 extracts were effective in inhibiting the production of CXCR4 and CXCL12 in both the A549 and H1650 cells, accompanied with reduction in H1650 cell capability to generate invasive sprouts. in a Matrigel layer. Finally, A009 interfere with the production of VEGF, CXCL8 and CCL2 in both the A549 and H1650 cells.

Our preliminary results suggest that A009 exhibit promising preventing and interceptive properties on lung cancer cell lines in vitro.

Citation Format: Matteo Gallazzi, Marco Festa, Clementina Sansone, Paola Corradino, Douglas Noonan, Adriana Albini. The CXCR4/CXCL12 axis is a target of a polyphenol extract from olive oil processing with potential cancer prevention and interception activities [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 18.
Abstract 1805: The therapeutic potential of 5-Azacitidine plus various BH3 mimetics in melanoma
Authors: Dart Chiara , Mukherjee Nabanita , Amato Carol , Van Gulick Robert J. , MacBeth Morgan , Couts Kasey , Turner Jacqueline , Bagby Stacey , Fujita Mayumi , Robinson William , Shellman Yiqun
Published: September 2020

Treatment options for melanoma have progressed in recent years, with the rise of targeted therapy and immunotherapy. Options are still limited for patients that are BRAF wildtype, treatment refractory, and patients with non-sun exposed melanoma subtypes that typically do not respond to standard therapies. Therefore, it is necessary to explore other biological processes that may provide new therapeutic approaches. One of most appealing is the BCL-2 family of apoptotic/anti-apoptotic system that is effective in leukemias, with BH3 mimetics venetoclax alone or in combination with the hypomethylating agent 5-azacitidine. We examined similar combinations on the group of eleven patient-derived and commercially available melanoma cell lines. The BH3 mimetics we tested include MCL1 inhibitors (S63845, AMG 176, AZD5991), an inhibitor of BCL2, BCLXL, and BCLW (navitoclax/ABT-263), a BCL2 inhibitor (venotoclax/ABT-199), and a BCLXL inhibitor (A1331852). Our data showed that treatments with MCL1 inhibitors plus 5-azacitidine are effective in reducing melanoma cell viability. S63845 was the most potent compound when combined with 5-azacitidine, with nine out of eleven (~80%) melanoma cell lines achieving less than 50% viability at the highest dose. There was an overall weaker response to treatment with 5-azacitidine in combination with ABT-263 or A1331852, with only 54% and 45% of cell lines responding at the highest dose, respectively. Response to ABT-199 with 5-Azacitidine was highly limited, 18% of cell lines responded at the highest concentration. Our data demonstrate that treatment with S63845 in combination with 5-azacitidine is highly effective in cell lines from a variety of melanoma subtypes, including the rare uveal and mucosal subtypes. In vivo and mechanistic experiments are planned. The novel combination of the MCL1 inhibitor S63845 and 5-azacitidine may be a promising alternative therapeutic target in melanoma.

Citation Format: Chiara Dart, Nabanita Mukherjee, Carol Amato, Robert J. Van Gulick, Morgan MacBeth, Kasey Couts, Jacqueline Turner, Stacey Bagby, Mayumi Fujita, William Robinson, Yiqun Shellman. The therapeutic potential of 5-Azacitidine plus various BH3 mimetics in melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1805.
Abstract 1832: A real-time extracellular ATP assay to identify modulators of the innate immune response
Authors: Niles Andrew L. , Kupcho Kevin R. , Lazar Dan F. , Cali James J.
Published: September 2020

Damage-associated molecular patterns (DAMPs) liberated or exposed by dying cells after chemotherapeutic treatment augment innate immune responses and improve clinical outcomes as a result of the immunogenic cell death (ICD) phenotype. Extracellular ATP (eATP) is a potent DAMP that acts as a chemotactic agent for dendritic cells and as an immunomodulator for fine-tuning the inflammatory response. Current in vitro methods for identifying eATP inducers are procedurally difficult and laborious and are hampered by the instability of eATP in cell culture environments. To overcome these challenges, we have developed an easy-to-use, homogeneous, bioluminescent chemistry that can be introduced with test articles to measure dose-dependent ATP release kinetics and magnitudes of response in real time. We tested the assay using U20S and U937 cells dosed with serial dilutions of doxorubicin, idarubicin, mitoxantrone, and romidepsin. Luminescent data were continuously collected throughout the 24h exposure in 5- or 10- minute increments using a conventional multimode plate reader with CO2-independent medium, or with readers equipped with atmospheric control. When coupled with parallel cell health assays designed to measure either viability or loss of membrane integrity, the data can reveal agents that potentiate ATP release during early or late apoptosis. Our real-time data suggest the potency of eATP release is dependent upon cell model and agent and can be reproducibly and robustly measured in 96 and 384 well environments with kinetic resolution not possible by other methods. This new eATP measurement workflow may help define the capacity of new chemical entities to induce apoptosis and immunogenic cell death.

Citation Format: Andrew L. Niles, Kevin R. Kupcho, Dan F. Lazar, James J. Cali. A real-time extracellular ATP assay to identify modulators of the innate immune response [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 1832.
Abstract 1840: NUC-1031 causes release of DAMPs and upregulates PD-L1 expression in lung cancer cells
Authors: Bré Jennifer , Read Oliver J. , Harrison David J.
Published: September 2020

Background: The nucleoside analog gemcitabine is used for the treatment of patients with lung cancer. Its cytotoxic effect is largely attributed to generation of the active metabolite dFdCTP, which causes DNA damage. NUC-1031, a ProTide transformation of gemcitabine, is specifically designed to overcome the key cancer resistance mechanisms associated with poor survival following gemcitabine treatment. NUC-1031 has shown high response rates in clinical studies, and efficacy in patients who were refractory to or had relapsed on gemcitabine. Certain cytotoxic agents are known to promote the release of damage-associated molecular patterns (DAMPs) from cancer cells, including cell surface exposure of calreticulin (CRT) and re-localization of nuclear high mobility group box protein 1 (HMGB1). DAMPs enhance cancer cell recognition by the immune system, promoting immunogenic cell death (ICD). Conversely, studies have shown that cancer cells can upregulate programmed death-ligand 1 (PD-L1) as an adaptive, protective response to cell stress resulting from drug exposure, potentially leading to evasion from immune cell-mediated cytotoxicity. This study investigates the effect of NUC-1031 on the release of DAMPs and modulation of PD-L1 in lung cancer cells.

Methods: Non-small cell lung cancer (NSCLC) cell lines were treated with NUC-1031 for 24h and cytotoxicity was assessed at 96h post-treatment to determine half-maximal inhibitory concentrations (IC50). Cells were treated with increasing concentrations of NUC-1031 and cell surface expression of CRT and PD-L1 were analyzed by flow cytometry and complemented with RT-qPCR. IFN-γ was used as a positive control for PD-L1 induction. Localization of HMGB1 was assessed by immunofluorescence microscopy.

Results: NUC-1031 induced the release of HMGB1 from the nucleus and increased cell surface expression of CRT in a dose-dependent manner. We confirmed that IFN-γ strongly and uniformly induced cell surface PD-L1 (270% increase). By comparison, NUC-1031-induced cell surface expression of PD-L1 was modest (67% increase). This effect was dose-dependent, with no further increase in PD-L1 expression above 1 μM.

Conclusions: NUC-1031 is a potent cytotoxic agent that directly causes DNA damage through generation of dFdCTP. In addition, these data demonstrate the NUC-1031 promotes adaptive changes that alter the recognition of cancer cells by the immune system. The release of DAMPs by NUC-1031 and increase in PD-L1 is predicted to engender an immune response, which might be further potentiated by immune checkpoint inhibition.

Citation Format: Jennifer Bré, Oliver J. Read, David J. Harrison. NUC-1031 causes release of DAMPs and upregulates PD-L1 expression in lung cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1840.
Abstract 1848: NUC-3373 induces ER stress and the release of damage associated molecular patterns in colorectal cancer cells
Authors: McKissock Fiona G. , Read Oliver J. , Harrison David J.
Published: September 2020

Background: NUC-3373 is a ProTide transformation of fluorodeoxyuridine monophosphate (FUDR-MP), the active anti-cancer metabolite of 5-fluorouracil (5-FU). NUC-3373 can overcome key cancer resistance mechanisms and the rapid degradation known to impact 5-FU's clinical utility. Clinical studies are currently investigating NUC-3373 as monotherapy (solid tumors) and in combination with agents commonly used to treat patients with colorectal cancer (CRC). Previous studies revealed ultrastructural adaptations in CRC cell lines exposed to NUC-3373 that were indicative of endoplasmic reticulum (ER) stress. NUC-3373 was also shown to induce the formation of long-lasting ternary thymidylate synthase (TS) complexes. The induction of ER stress may promote the release of damage-associated molecular patterns (DAMPs) which have the potential to evoke immunogenic cell death (ICD). This study examines the ability of NUC-3373 to induce both ER stress and the release of DAMPs in CRC.

Methods: Immunoglobulin-binding protein (BiP) was used as a marker of ER stress induction. BiP and TS (free and ternary complex) expression were measured by Western blot and quantified using Licor Odyssey. TS was knocked down using TYMS-targeting siRNA and lipofectamine-based transfection. DAMPs, active release of nuclear high mobility group box protein 1 (HMGB1) and cell surface exposure of calreticulin (CRT), were assessed by fluorescence microscopy and flow cytometry respectively.

Results: NUC-3373 resulted in the rapid formation of TS ternary complexes, which was associated with ER stress as assessed by BiP protein expression. siRNA knockdown studies confirmed that TS ternary complex formation is necessary for the induction of ER stress. A strong correlation between BiP and TS ternary complex formation (R2=0.97) was observed. Furthermore, NUC-3373 induced the release of DAMPs, with increased expression of CRT at the cell surface and concomitant loss of nuclear HMGB1.

Conclusions: NUC-3373, a potent inhibitor of TS activity, also induces ER stress, upregulates cell surface CRT, and decreases nuclear HMGB1 in CRC cells. The aforementioned DAMPs have been shown to cause ICD. In addition to being an effective cytotoxic agent, these findings suggest that NUC-3373 has the potential to evoke a host immune response and enhance the clinical utility of immunotherapy.

Citation Format: Fiona G. McKissock, Oliver J. Read, David J. Harrison. NUC-3373 induces ER stress and the release of damage associated molecular patterns in colorectal cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1848.