bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4963: Notch1 non-canonical pathway drives breast cancer stem cell function
Authors: Sui Lufei , Wang Suming , Sim Danielle , Watnick Randolph S.
Published: September 2020

Current treatment of triple negative breast cancer is hindered by a high incidence of chemoresistance (30-50%). The prevailing theory is that resistance and subsequent recurrence is driven by cancer stem cells. Unfortunately, the functional characterization of cancer stem cells at the molecular level is still incomplete. We show here, that within the canonical breast cancer stem cell population (CD24low/CD44+/ESA+) of triple negative breast cancer cells, a subset of cells characterized by high Notch1 expression possesses the tumor-initiating and chemoresistance properties associated with cancer stem cells. Moreover, the tumor initiating property of these Notch1-high expressing breast cancer stem cells is mediated by the repression of SIRT1. We also demonstrate that Notch1 represses SIRT1 through a non-canonical, cleavage independent pathway via PI3K/Akt/mTORC2. Strikingly, the repression of SIRT1 obviates the requirement for high Notch1 expression. These findings provide the first functional mechanistic requirements for breast cancer stem cell tumor initiation and chemoresistance. Our results also identify a novel non-canonical Notch1 signaling pathway regulating breast cancer stem cell function. We postulate that this pathway can be exploited as a potential therapeutic strategy to eradicate cancer stem cells and reduce the incidence of chemoresistance and recurrence.

Citation Format: Lufei Sui, Suming Wang, Danielle Sim, Randolph S. Watnick. Notch1 non-canonical pathway drives breast cancer stem cell function [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 4963.
Abstract 4966: MyD88 in myofibroblasts enhances colitis-associated tumorigenesis via promoting macrophage M2 polarization in mice
Authors: Zhang Jinhua , Tian Tian , Yuan Qi
Published: September 2020

Background & Aims: The signal adapter MyD88, an essential component of TLR signaling, plays an important role in gut-microbiome interactions. However, its contribution to colitis-associated cancer (CAC) is still controversial. We investigated whether MyD88 signaling in myofibroblasts contributes to an environment that promotes CAC development.

Methods: C57BL/6J mice (control) and mice with myofibroblasts-specific deletion of MyD88 (Fib-MyD88 KO) were performed azoxymethane (AOM)/dextran sodium sulfate (DSS) model or DSS model. Colon tissue was collected and analyzed for inflammation, cell proliferation and macrophage polarization. Primary normal fibroblasts (NFs) were isolated and measured for mechanisms that induced macrophage polarization.

Results: Myofibroblasts MyD88-deficient mice were resistant to AOM/DSS-induced tumorigenesis, as evidence by the decrease in the number and sizes of tumors. MyD88 deficiency in myofibroblasts did not affect the inflammatory response but attenuated intestinal epithelial cell (IEC) proliferation after acute DSS-induced colitis. Furthermore, MyD88 signaling in myofibroblasts increased the secretion of osteopontin (OPN), which promoted macrophage M2 polarization through binding to αvβ3 and CD44, leading to activation of the STAT3 and PPARγ pathways.

Conclusions: MyD88 signaling in myofibroblasts crucially contributes to colorectal cancer development and provides a promising therapeutic target for the prevention of colitis-associated carcinogenesis.

Citation Format: Jinhua Zhang, Tian Tian, Qi Yuan. MyD88 in myofibroblasts enhances colitis-associated tumorigenesis via promoting macrophage M2 polarization in mice [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 4966.
Abstract 4970: Prognostic role of CD112R, PD-1 and Ki67 expression in CD8+cytotoxic T cells in colorectal cancer
Authors: Simon Ronald , Blessin Niclas Christian , Mandelkow Tim , Bady Elena , Hube-Magg Claudia , Sauter Guido , Lennartz Maximilian , Fraune Christoph , Weidemann Sören A. , Möller Katharina , Büscheck Franziska
Published: September 2020

Expression of inhibitory immune checkpoint receptors such as CD112R (Poliovirus Receptor-Related Immunoglobulin Domain-Containing Protein, PVRIG) and PD-1 on CD8+ lymphocytes are considered to indicate an “exhausted” stage of T cell activation while Ki67 protein expression in these cells is viewed as sign of activity or expansion. To assess the prognostic impact of the clinical impact of CD112R, PD-1, and Ki67 expression of CD8+ lymphocytes, expression of these proteins was analyzed by fluorescent multiplex immunohistochemistry on a tissue microarray containing 384 colorectal cancers with clinical follow-up data. An elevated fraction of CD8+Ki67+ cells was linked to the absence of nodal metastasis and longer survival in these tumors (p≤0.0039 each). CD112R and PD-1 expression was detected at variable intensity levels and were separately quantitated in both ki67+ (expanding) and Ki67 negative (non-expanding) CD8+ cytotoxic lymphocytes. Elevated PD-1 expression in CD8+ T cells was associated with an absence of nodal metastasis (p=0.004) and longer overall survival in all CD8+ cells (p=0.005). This association was more evident in the subset of expanding (p<0.0001) than in non-expanding (p=0.034) cytotoxic T cells. Elevated CD112R expression level in CD8+ T cells were linked to a low tumor grade (p=0.048), absence of nodal metastasis (p=0.023) and a prolonged overall survival (p=0.041) but these associations were not retained in subgroups of either expanding or non-expanding CD8+ cells. Patients with an elevated fraction of CD8+ki67+CD112R+PD-1+cells exhibited a particularly good prognosis. In conclusion, our data reveal that “exhausted” CD112R+/ PD-1+CD8+ cytotoxic T lymphocytes are able to expand (ki67+). This observation in combination with the good prognosis of cancers with high number of expanding exhausted cells argues for retained cytotoxic activity of CD112R+/ PD-1+ CD8+ T lymphocytes.

Citation Format: Ronald Simon, Niclas Christian Blessin, Tim Mandelkow, Elena Bady, Claudia Hube-Magg, Guido Sauter, Maximilian Lennartz, Christoph Fraune, Sören A. Weidemann, Katharina Möller, Franziska Büscheck. Prognostic role of CD112R, PD-1 and Ki67 expression in CD8+cytotoxic T cells in colorectal 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 4970.
Abstract 4976: Combining measures of immune infiltration uncovers predictors of survival in high-grade serous ovarian cancer
Authors: Montfort Anne , Owen Steph J. , Piskorz Anna M. , Supernat Anna , Moore Luiza , Al-Khalidi Sarwah , Boehm Steffen , Pharoah Paul , McDermott Jacqueline , Balkwill Frances R. , Brenton James D.
Published: September 2020

Background: In colorectal and breast cancer, the density and localization of immune infiltrates provides strong prognostic information. We asked whether similar automated quantitation and combined analysis of immune infiltrates could refine prognostic information in high-grade serous ovarian carcinoma (HGSOC) and tested associations between patterns of immune response and genomic driver alterations.

Methods and Findings: Epithelium and stroma were semi-automatically segmented and the infiltration of CD45RO+, CD8+ and CD68+ cells was automatically quantified from images of 332 HGSOC patient tissue microarray cores. Epithelial CD8 (p=0.027, HR=0.83), stromal CD68 (p=3 × 10-4, HR=0.44) and stromal CD45RO (p=7 × 10-4, HR=0.76) were positively associated with survival and remained so when averaged across the tumor and stromal compartments.Using principal component analysis (PCA), we identified optimized multiparameter survival models that combined information from all immune markers (p=0.016, HR=0.88). There was no significant association between PTEN expression, type of TP53 mutation or presence of BRCA1/BRCA2 mutations and immune infiltrate densities or principal components.

Conclusions: Combining measures of immune infiltration provided improved survival modeling and evidence for the multiple effects of different immune factors on survival. The presence of stromal CD68+ and CD45RO+ populations was strongly associated with survival, underscoring the benefits that evaluating stromal immune populations may bring for prognostic immunoscores in HGSOC.

Citation Format: Anne Montfort, Steph J. Owen, Anna M. Piskorz, Anna Supernat, Luiza Moore, Sarwah Al-Khalidi, Steffen Boehm, Paul Pharoah, Jacqueline McDermott, Frances R. Balkwill, James D. Brenton. Combining measures of immune infiltration uncovers predictors of survival in high-grade serous ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4976.
Abstract 5005: Impact of H3.3K27M mutation on diffuse intrinsic pontine glioma's resistance to treatment
Authors: Bailleul Quentin , Arcicasa Mélanie , Hochart Audrey , Rakotomalala Andria , Castets Marie , Pasquier Eddy , Angrand Pierre-Olivier , Adriaenssens Eric , Bourhis Xuefen Le , Leblond Pierre , Meignan Samuel
Published: September 2020

Diffuse Intrinsic Pontine Glioma is one of the worst pediatric brain tumors regarding prognosis due notably to intrinsic cell resistance to radio and chemotherapy. One of the main characteristics of DIPG cells is the presence of a mono-allelic mutation on the lysine 27 of histone H3 (H3K27M). This mutation inhibits the trimethylation of this lysine that leads to strong modifications of gene expression. Until now, even though this mutation seems to be a driver event in tumorigenesis, its role in cell resistance to treatment has not been deciphered, due to a lack of relevant cellular models. This way, in order to evaluate the role of the mutation on resistance to treatment, we first induced the mutation in three H3K27-unmutated pediatric glioma cell lines. In parallel, using the CRISPR/Cas9 technology, we are establishing DIPG cellular models in which the mutation will be reversed. By gene trapping approach, we aim to restore an H3F3Awt/wt genotype. After validation, these models would result in original tools to study the impact of H3K27M mutation in DIPG cells resistance to treatment. For the model of induction, the transfected cell lines exhibit the mutation accompanied by a loss of H3K27me3 mark and H3.3 overexpression. For now, we showed an increased cell growth due to the mutation in two cell lines, under normoxia as well as under hypoxia. On contrary there was no impact on resistance to chemotherapy or ionizing radiation. In the third cell line, we didn't observe any impact on cell growth, but an increase of cell radioresistance. Concerning the mutation reversion, our preliminary results show homologous recombination at the right locus in the genome, and some clones present a loss of the mutation confirmed by sequencing. After the removal of resistance cassette by action of a recombinase protein, we will be able to evaluate the biological effects of mutation reversion. To sum up, these different models would allow us to decipher cellular and molecular mechanism induced by the H3.3K27M mutation in DIPG cells including resistance to treatment, and thus, to possibly identify putative therapeutic targets.

Citation Format: Quentin Bailleul, Mélanie Arcicasa, Audrey Hochart, Andria Rakotomalala, Marie Castets, Eddy Pasquier, Pierre-Olivier Angrand, Eric Adriaenssens, Xuefen Le Bourhis, Pierre Leblond, Samuel Meignan. Impact of H3.3K27M mutation on diffuse intrinsic pontine glioma's resistance to treatment [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 5005.
Abstract 5042: Targeting the ERK5 and ERK1/2 pathways simultaneously induces mesenchymal to epithelial transition in TNBC
Authors: Bhatt Akshita , Wright Thomas , Barnes Van , Chakrabarty Suravi , Flaherty Patrick , Burow Matthew , Cavanaugh Jane
Published: September 2020

Extracellular signal-regulated kinase (ERK) 5, a member of mitogen activated protein kinase (MAPK) family, is an emerging target in cancer therapeutics. Activation of ERK5 via overexpression induces EMT and hormone-independent growth of breast cancer. EMT leads to the loss of cell polarity, downregulation of E-cadherin, and upregulation of mesenchymal markers snail, zinc-finger E-box binding homeobox (ZEB1), and vimentin. EMT is also associated with drug resistance. Although ERK1/2 and ERK5 activation is known to mediate EMT, the effect of ERK1/2 and ERK5 inhibition on mesenchymal to epithelial transition (MET), the reverse of EMT, is poorly understood in cancer.Triple negative breast cancer (TNBC) cells have a mesenchymal phenotype and show poor sensitivity to chemotherapy agents. The loss of estrogen, progesterone hormone receptors and human epidermal growth factor receptors (HER2) contributes to the aggressive state of the disease and lack of targeted therapy. Activation of the intracellular signaling pathways such as the MAPK pathway mediates tumorigenesis in TNBCs. MEK1/2 inhibitors have been successful clinical drug candidates; however, there is emerging evidence that the activation of the MEK5-ERK5 pathway mediates resistance to the MEK1/2 inhibitors in several BRAF and KRAS-mutant cancers. The effect of MEK1/2 inhibition on MEK5-mediated EMT, cell survival, and migration in TNBC is less well understood. Therefore, we hypothesize that dual inhibition of the ERK1/2 and ERK5 pathways is a relevant strategy to target TNBCs.In the present study, the effect of dual ERK1/2 and ERK5 inhibition on MET, cell viability, migration, and anchorage-independent growth was evaluated in TNBC. ERK1/2 and ERK5 activities were modulated via pharmacological inhibitors and molecular tools. Cell morphology and protein expression of EMT markers E-cadherin, ZEB1, snail, and vimentin were evaluated. XMD8-92, an ERK5 inhibitor was found to synergize with doxorubicin in lung and cervical cancer cells. Therefore, the effect of dual ERK1/2 and ERK5 on doxorubicin sensitivity was evaluated.

Citation Format: Akshita Bhatt, Thomas Wright, Van Barnes, Suravi Chakrabarty, Patrick Flaherty, Matthew Burow, Jane Cavanaugh. Targeting the ERK5 and ERK1/2 pathways simultaneously induces mesenchymal to epithelial transition in TNBC [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 5042.
Abstract 5044: Integrin-linked kinase gene knockdown results in decreased growth of ovarian cancer xenograft models
Authors: Redfern Tiffany , Wilson Benjamin , Ulm Michael , ElNaggar Adam , Ponnusamy Suriyan , Wang Yinan , Asemota Sarah , Narayanan Ramesh
Published: September 2020

Integrin-linked kinase (ILK) is overexpressed in ovarian cancer cells. In the present study, we sought to assess the effect of ILK silencing on ovarian cancer cell proliferation using shRNA. Our lab previously demonstrated that phosphorylation of ILK acts as a proliferative signal in a variety of ovarian cancer cell lines. Cytotoxicity of ILK knockdown using shRNA was analyzed in vitro using SKOV3 cell lines. Transfected cells were incubated in Incucyte®, which allowed for simultaneous assessment of viable cells at the end of the incubation period using CellTiter glo assay. Knockdown of ILK using shRNA significantly inhibited the proliferation of SKOV3 cells. SKOV3 cells were then infected with lentivirus expressing ILK shRNA to obtain stable knockdown of ILK, which was confirmed with Western blot. Both parental and ILK shRNA infected cells were injected subcutaneously in NOD SCID Gamma immunocompromised mice. Tumor growth was assessed by sequential tumor measurements and final weights. Xenograft data indicated significantly decreased tumor volume over multiple time points in the transfected cells (p <0.0001) (Figure 1). Average final volume for the ILK-shRNA group (n = 15) and control group (n = 13) was 660mm3 (SD = 366.7, range 271 - 1667 mm3) and 1165 mm3 (SD = 668.7, range 506 - 2664 mm3), respectively. Similarly, average tumor weight was less for the ILK-shRNA group (404.4mg [range 226.1 - 801mg]) relative to control (584.3mg [range 293.2 - 1033.4mg]). Our findings demonstrate that knockdown of ILK in ovarian cancer cell lines results in decreased cell proliferation, as well as decreased tumor growth in xenograft models. This supports the role for further investigation into ILK as a potential therapeutic target in ovarian cancer.

Days From Tumor Implantation1214161921232628313438VectorAverage Tumor Volume (mm^3)142.4151.6203274.7379.3482.1671.4741.2845.11083.81164.6Standard Error26.983.430.835.759.682112.3110.7133.9172.2185.5ILK-shRNAAverage Tumor Volume (mm^3)45.9107.282.4113150.9177263.4345.2397.3568.8659.9Standard Error6.39.99.915.225.822.841.558.455.19994.7

Citation Format: Tiffany Redfern, Benjamin Wilson, Michael Ulm, Adam ElNaggar, Suriyan Ponnusamy, Yinan Wang, Sarah Asemota, Ramesh Narayanan. Integrin-linked kinase gene knockdown results in decreased growth of ovarian cancer xenograft models [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 5044.
Abstract 5051: RenMab Mouse: A leading platform for fully human antibody generation
Authors: Ng Gary , Zhao Huizhen , Zhang Yabo , Liu Lili , Lu Hui , Huang Shuwen , Shen Yuelei
Published: September 2020

With the development of immune-oncology, therapeutic antibodies have been proven to be extraordinarily effective for cancer treatment. Conventional human antibody discovery process can be divided into steps including target selection, target validation, screening preparation, hits generation, leads selection, lead optimization, and clinical candidate selection. To accelerate antibody development process, Biocytogen has developed RenMab Mouse, a fully human antibody mouse whose entire variable regions were replaced by human Ig heavy chain and κ light chain through Biocytogen's unique Mb-scale chromosome engineering technology. RenMab Mouse provides a remarkably efficient therapeutic antibody discovery platform for fully human antibody generation, characterization, and rapid in vivo efficacy screening. We have characterized RenMab Mouse with the following features.

1. The entire mouse variable regions were replaced with complete human genome DNA in situ for complete human & mouse regulatory elements. The gene regulation of RenMab mouse is highly consistent with that of human.

2. Mouse constant region remains to ensure normal immune cell population, development and maturation. Immune system of RenMab Mouse has been proven to be almost identical to that of wild type mice. RenMab Mouse showed normal antibody immune responses to antigens.

3. RenMab Mouse generates a highly diverse repertoire of fully human antibody variable regions through V(D)J recombination. This capability can lead to improved hit rates in antibody drug discovery.

In conclusion, RenMab Mouse, a fully human antibody mouse developed by Biocytogen's chromosome engineering technology, has been proven to exhibit normal immune cell profile and rapid immune response. Combined with existing inventory of single, double or triple target humanized mouse models and a world-leading proprietary gene editing platform, Biocytogen is dedicated to providing a one-stop solution for antibody discovery and development from target validation to IND application.

Citation Format: Gary Ng, Huizhen Zhao, Yabo Zhang, Lili Liu, Hui Lu, Shuwen Huang, Yuelei Shen. RenMab Mouse: A leading platform for fully human antibody generation [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 5051.
Abstract 5089: The origin and contribution of the tumor stroma in colorectal cancer
Authors: Kobayashi Hiroki , Gieniec Krystyna A. , Lannagan Tamsin RM , Wang Tongtong , Asfaha Samuel , Hayakawa Yoku , Leedham Simon J. , Arpaia Nicholas , Mukherjee Siddhartha , Wang Timothy C. , Enomoto Atsushi , Takahashi Masahide , Woods Susan L. , Worthley Daniel L.
Published: September 2020

Cancer-associated fibroblasts (CAFs) are a major constituent of the tumor microenvironment and play a critical part in cancer progression. However, the precise origin of the tumor stroma remains unknown, making it challenging to effectively target the cancer mesenchyme. Here, employing 4 different genetic fate mapping mouse models and a bone marrow transplantation model in combination with BrdU labeling, we uncovered a key contributor to the tumor stroma in colorectal cancer (CRC). We found that approximately half of a-smooth muscle actin (aSMA)+ CAFs emerge through proliferation in an AOM/DSS mouse model of CRC. Lineage tracing experiments revealed that intestinal leptin receptor (Lepr)-lineage stromal cells expanded and contributed to 75% of the aSMA+ proliferating CAFs. Notably, no aSMA+ CAFs in the tumor were derived from Krt19-lineage epithelial cells or bone marrow-transplanted cells, indicating no involvement of epithelial-mesenchymal transition and bone marrow recruitment to the tumor in this model. Moreover, RNA-sequencing of FACS-purified CRC mesenchymal cells identified MCAM (also known as CD146) as a CRC stroma-specific marker, which is expressed by Lepr-lineage cells. Analysis of human CRC samples showed that high MCAM expression was associated with a mesenchymal subtype of CRC and was independently prognostic of poor overall survival. Our data identify Lepr-lineage cells as a major source of the tumor stroma in CRC and suggest that targeting MCAM+ cells may serve as a novel therapeutic approach to restrain CRC progression.

Citation Format: Hiroki Kobayashi, Krystyna A. Gieniec, Tamsin RM Lannagan, Tongtong Wang, Samuel Asfaha, Yoku Hayakawa, Simon J. Leedham, Nicholas Arpaia, Siddhartha Mukherjee, Timothy C. Wang, Atsushi Enomoto, Masahide Takahashi, Susan L. Woods, Daniel L. Worthley. The origin and contribution of the tumor stroma in colorectal 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 5089.
Abstract 5142: Proteome-wide analysis identifies three subgroups of diffuse-type gastric cancer
Authors: Huang Wenwen , Xia Xia , Zhan Dongdong , Shen Lin , Qin Jun
Published: September 2020

Background: Gastric cancer (GC) is highly heterogeneous and the leading cause of cancer death. Diffuse-type gastric cancer (DGC) is one of poor-prognosis GC subtypes with few treatment options. Genomic subtyping classifies overall gastric cancers into different subgroups that associate with prognosis and therapeutic targets, but it has not translated into clinical benefit. Proteomic subtyping with DGC may offer the potential for personalized risk stratification and treatment recommendation.

Methods: We developed a workflow using fast mass spectrometry and ConsensusClusterPlus (CCP) consensus clustering for proteomic subtyping and applied it to 99 DGC formalin-fixed, paraffin-embedded (FFPE) samples.

Results: In this study, 99 FFPE tumor samples were analyzed and classified into three proteomic subtypes (DGCP1-3) based on proteome-wide data, which were significantly associated with prognosis and response to chemotherapy. DGCP1 is with the best survival and DGCP3 is with the worst survival. DGCP2 derives benefit from chemotherapy. The rest of the subtypes do not receive benefit from chemotherapy. Specific signaling pathways are enriched in the subtypes: DGCP1/innate immune system; DGCP2/metabolism of RNA plus cell cycle; DGCP3/Extracellular Matrix (ECM). The classification with subtype-specific proteins provides a roadmap for stratifying patients into subgroups in trials of chemotherapy, targeted, and immune therapies.

Conclusions: Proteomic subtyping of DGC identified three subgroups that have significant associations with distinct clinical characteristics. The DGCP subtyping can be further verified in more clinical cohorts and used to predict prognosis and recommend clinical actions in DGC treatment.

Citation Format: Wenwen Huang, Xia Xia, Dongdong Zhan, Lin Shen, Jun Qin. Proteome-wide analysis identifies three subgroups of diffuse-type gastric 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 5142.
Abstract 5173: Identification of new targets in SMAD4-deficient cancers by whole genome CRISPR screening
Authors: Marcireau Christophe , Erasimus Hélène , Kolnik Vanessa , Lacroix Frederic , D'agostino Stéphane , Lemaitre Olivier , Rohaut Alexandre , Sanchez Isabelle , Sidhu Sukhvinder , Thill Gilbert , Didier Michel , Debussche Laurent
Published: September 2020

The TGF-β signaling mediator SMAD4 is frequently mutated or deleted in colorectal cancer (CRC) and pancreatic cancers (PC) and is associated with poorer prognosis. This makes SMAD4 loss interesting to harness for synthetic lethality-based therapy. In this study, we first restored SMAD4 in SMAD4-deficient cell lines (HT29 and SW620) and showed that SMAD4 restoration reestablished the TGF-β-induced growth inhibition in vitro and in vivo, thereby confirming the tumor suppressive role of SMAD4 in these cell lines. In order to identify genetic vulnerabilities of SMAD4-deficient cells, we conducted pooled genome-wide loss-of-function CRISPR/Cas9 screens across four non-isogenic cell lines harboring altered (HT29 and SW620) or wild-type (DLD1 and MIAPACA2) SMAD4. We identified candidate genes, including the Krüppel-like factor 5 (KLF5), which has already been suggested as a potentially interesting target for drug therapy in SMAD4-deficient cancers. Novel candidates that might be interesting synthetic lethal targets in SMAD4-negative cancers will be presented.

Citation Format: Christophe Marcireau, Hélène Erasimus, Vanessa Kolnik, Frederic Lacroix, Stéphane D'agostino, Olivier Lemaitre, Alexandre Rohaut, Isabelle Sanchez, Sukhvinder Sidhu, Gilbert Thill, Michel Didier, Laurent Debussche. Identification of new targets in SMAD4-deficient cancers by whole genome CRISPR screening [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 5173.
Abstract 5180: Nuclear receptor tyrosine kinase c-MET restrains efficacy of PARP inhibitor in pancreatic cancer cells
Authors: Gao Yuan , Chen Mei-Kuang , Yu Dihua , Hung Mien-Chie
Published: September 2020

Pancreatic tumors commonly have intra-tumor hypoxia and high reactive oxygen species (ROS) production and have become the fourth leading cause of death in the United States. Although ROS leads to oxidation of macromolecules such as DNA, protein, and lipids, pancreatic cancer cells are capable of surviving from the ROS-induced stress at relatively high tolerable levels. Therefore, identifying potential molecular mechanisms for pancreatic cancer cells to overcome ROS-induced cellular stress is important for develop therapeutic strategies in treating pancreatic cancer patients. While poly (ADP-ribose) polymerase 1 (PARP1) is a key protein for repairing ROS-induced DNA damages, we intended to include PARP inhibitors in targeted therapy strategy. Recent clinical trial showed that PARP inhibitor benefits pancreatic patients with BRCA mutation. However, our goal is to expand PARP inhibitor targeted therapy to pancreatic cancer patients without BRCA mutation with PARP inhibitor using strategies of combining kinase inhibitors as we demonstrated in breast cancer in our previous publications. Here, we used hydrogen peroxide (H2O2) as ROS stimuli and demonstrated that H2O2 induces nuclear transport of the receptor tyrosine kinase c-MET in AsPC-1 and BxPC-3 pancreatic cancer cells. We further showed that nuclear-located c-MET interacts with PARP1, and the interaction can be partially interrupted by crizotinib, a kinase inhibitor targeting c-MET. Moreover, combination of crizotinib and PARP inhibitor, olaparib, has synergistic anti-tumor effect in eliminating pancreatic cancer cells. Our data suggest that the intra-tumor hypoxia micro-environment of pancreatic cancer may promote nuclear localization of c-MET, which results in resistance to PARP inhibitor therapy. We further proposed a new therapeutic strategy of combining c-MET inhibitors and PARP inhibitors for advanced pancreatic cancer patients.

Citation Format: Yuan Gao, Mei-Kuang Chen, Dihua Yu, Mien-Chie Hung. Nuclear receptor tyrosine kinase c-MET restrains efficacy of PARP inhibitor in pancreatic 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 5180.
Abstract 5183: The preclinical characterization of TST001, a novel humanized anti-claudin18.2 mAb with enhanced binding affinity and anti-tumor activity
Authors: Teng Fei , Gu Yi , Chai Hui , Guo Huanhuan , Li Hongjun , Wu Xiwen , Yao Xinlai , Xu Fei , Shi Lei , Yan Zhenzhi , Zi Xiaoli , Dai Zheng , Liao Timethy , Zheng Lisa , Fan Francis , Li Zhen , Yang Jerry , Qian Xueming
Published: September 2020

Claudin-18 isoform 2 (CLDN18.2) is a member of the human claudin family of tetraspan membrane proteins that are crucial structural and functional components of tight junctions. Unlike other family members, CLDN18.2 expression is strictly limited to differentiated epithelial cells of gastric mucosa. Interestingly CLDN18.2 was ectopically expressed at a significant level in multiple tumor types including gastric, esophageal, pancreatic and lung cancers, which makes it as an attractive anti-cancer target. TST001 is a novel humanized IgG1 monoclonal antibody, which specifically binds to cells expressing human CLDN18.2 with high affinity but not to the closely related Claudin 18.1. TST001 can compete with IMAB362, the clinical stage anti-CLDN18.2 mAb, for its binding to CLDN18.2 yet engages distinct epitope for binding. By reducing fucosylation during cell culture process, TST001 has further enhanced binding affinity to FcγRIIIa, which translates into more potent ADCC activity. Indeed, TST001 showed sub-nanomolar ADCC activity against gastric cancer cells expressing medium to low CLDN18.2 in the presence of human PBMC and NK cells, which is significantly more potent than IMAB362. TST001 also showed more potent CDC and ADCP activities against CLDN18.2 expressing cells than IMAB362. In both Sprague Dawley Rat and Cynomolgus Monkey, the systemic exposure of TST001 increased proportionally in a dose-dependent manner. In gastric cancer cell line and patient derived xenograft tumor models, TST001 showed more potent anti-tumor activity as compared with IMAB362. Furthermore, the combination of TST001 with chemo agents resulted in synergistic anti-tumor effect in these tumor models. In addition, we have also generated and characterized an antibody that is suitable for IHC based detection and is selective to CLDN18.2 over CLDN18.1. Altogether, these preclinical findings warrant further clinical evaluation of TST001 in patients with CLDN18.2 positive tumors.

Citation Format: Fei Teng, Yi Gu, Hui Chai, Huanhuan Guo, Hongjun Li, Xiwen Wu, Xinlai Yao, Fei Xu, Lei Shi, Zhenzhi Yan, Xiaoli Zi, Zheng Dai, Timethy Liao, Lisa Zheng, Francis Fan, Zhen Li, Jerry Yang, Xueming Qian. The preclinical characterization of TST001, a novel humanized anti-claudin18.2 mAb with enhanced binding affinity and anti-tumor activity [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 5183.
Abstract 5186: Human stem cell humanized mouse model for in vivo evaluation of bispecific antibody efficacy and drug-induced cytokine release
Authors: Yao Li-Chin , Cai Danying , Aliperta Roberta , Colthart Allison , Meier Werner , Cheng Mingshan , Keck James G.
Published: September 2020

Bispecific antibodies have shown several promising advantages over monoclonal antibodies and are emerging as one of the growing cancer immunotherapies. Preclinical in vivo models to assess efficacy and cytokine release associated with immune cell engagement are needed. We have developed a human stem cell (HSC) humanized mouse model for testing efficacy and for quantifying cytokine release in response to bispecific antibody treatment. Here we evaluated an EGFR bispecific T cell engager (BiTE), using EGFR-positive MDA-MB-231 orthotopic breast cancer model in CD34+ HSC humanized NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG™) mice. BiTE treatment for 2 weeks resulted in complete tumor regression when dosing was initiated at an average tumor volume of around 100 mm3. In addition, BiTE treatment for 4 weeks effectively inhibited the tumor growth when dosing mice bearing larger tumors of around 400mm3 in volume. Sixteen hours after the first two doses as compared to PBS control, human cytokines IFN-γ, IL-6, and IL-10 were elevated. Cytokine levels then returned to the baselines following continuous dosing. We observed a similar time course and levels of cytokine release between these two BiTE-treated groups with different tumor sizes. T cell activation in the peripheral blood after the last dose was assessed by flow cytometry analysis. In the BiTE-treated group, T cell percentage decreased, as compared to the PBS control. Both CD4+ and CD8+ T cell subsets showed an increased frequency of CD69+ cells after BiTE treatment. Evaluation of drug efficacy and cytokine release in response to BiTE treatment using PBMC-humanized NSG™ mice with and without MDA-MB-231 tumors are underway and results will be discussed.

Citation Format: Li-Chin Yao, Danying Cai, Roberta Aliperta, Allison Colthart, Werner Meier, Mingshan Cheng, James G. Keck. Human stem cell humanized mouse model for in vivo evaluation of bispecific antibody efficacy and drug-induced cytokine release [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 5186.
Abstract 5251: Reproducible, MoA-reflecting reporter-based bioassays to enable drug development of biosimilars and biobetters
Authors: Edenson Steven , Moravec Richard , Li Dun , Wilkinson Jennifer , Fan Frank , Cong Mei
Published: September 2020

Cytokines and growth factors are small immunomodulatory proteins secreted by a wide variety of cells including fibroblasts, endothelial and stromal cells, whose role is to regulate surrounding cells in an autocrine, paracrine or endocrine fashion. Immunocytokines represent a promising class of activators of the immune system, with the potential to be used alone or in combination with other therapeutic modalities. Many are currently FDA approved therapy agents (e.g. IFN, IL-2 and Epo), while others are targets for approved biological blocking therapies to treat a variety of diseases. Examples of cytokine blocking agents include basiliximab (IL-2R), tocilizumab and sarilumabad (IL-6R), siltuximab (IL-6), ustekinumab and its biosimilars (IL-12/IL-23 p40), secukinumab (IL-17A), bevasizumab (VEGF), and denosumab (RANKL). Many more are in development and trials as biosimilars and biobetters. IL-2 and IL-15 are still clinically important cytokines as researchers look to improve potency, patient tolerance and response by developing new molecules with sustained and targeted activities. We have developed luciferase reporter bioassays which individually can be used for the quantitation of a variety of cytokines and growth factors including IL-2, IL-6, IL-10, IL-12, IL-15, IL-17, IL-23, BCMA, TGF-b, VEGF and RANKL using respective mechanism of action pathways. The bioassay format is based on thaw-and use cells, eliminating the need to establish and pre-culture cytokine responsive cell lines which provides the benefits of convenience, reproducibility, and transferability. We demonstrate these assays measure cytokine response and inhibition with blocking drugs or potency changes from stressed samples. In summary, these reporter-based bioassays provides valuable tools for the development, stability testing, and potency determination in the manufacture of cytokine biosimilars and biobetters.

Citation Format: Steven Edenson, Richard Moravec, Dun Li, Jennifer Wilkinson, Frank Fan, Mei Cong. Reproducible, MoA-reflecting reporter-based bioassays to enable drug development of biosimilars and biobetters [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 5251.
Abstract 5254: Combined in vitro TTFields and paclitaxel reduce proliferation and clonogenicity in non-small cell lung cancer (NSCLC) cells from a patient previously treated with standard-of-care
Authors: Michelhaugh Sharon K. , Mittal Sandeep
Published: September 2020

TTFields therapy in conjunction with chemotherapy has been FDA approved as a first line treatment regimen for patients with unresectable malignant pleural mesothelioma. Clinical trials are underway to test the efficacy of TTFields in patients with progressive NSCLC or NSCLC brain metastases following standard-of-care or radiosurgery, respectively. Our study utilized tumor cells extracted from a NSCLC metastatic brain tumor from a patient previously treated with standard-of-care chemoradiation prior to progression to brain metastasis. These patient-derived NSCLC cells were subjected to TTFields application with and without paclitaxel to determine if the combination of TTFields with paclitaxel would be more effective than either treatment alone. Written informed consent was obtained from the patient, who was 60 years-old and female. She received concurrent carboplatin/paclitaxel and radiotherapy to the upper lobe of her left lung prior to discovery and resection of a solitary brain lesion. Cells isolated from the metastatic brain tumor were cultured for 3 passages prior to plating on coverslips (4 × 104 each) in DMEM/F12 media with 10% fetal bovine serum. TTFields were applied at ~1.6 V/cm at 150 kHz. Paclitaxel was added to the media to a final concentration of 5 nM. Cells were treated for 14 days. At the end of the treatment period, some coverslips of cells were lysed and assayed for lactate dehydrogenase (LDH) to represent cell number (n=5) and another set of coverslips of cells were harvested and replated in triplicate for the clonogenic assay (n=3). Groups were compared with one-way ANOVA. Data shown in table are mean ± SD. For the LDH assay, ANOVA p<0.0001. For the clonogenic assay, ANOVA p=0.0003. Overall, the combination of TTFields with paclitaxel produced additive reductions in cell proliferation (75%) and reduced the clonogenic potential (95%) compared to untreated controls. The additive effects of TTFields and paclitaxel suggest that they target different cell populations within a heterogeneous tumor, and that lung cancer patients previously treated with standard-of-care may benefit from TTFields therapy.

Proliferation and clonogenic assay results from combined TTFields and paclitaxelAssayControlPaclitaxel (5 nM)TTFieldsTTFields ± Paclitaxel (5 nM)Proliferation (LDH assay)1.83±0.090.81±0.041.34±0.150.46±0.21Clonogenicity (cell counts)26641±46258697±161717399±59981598±598

Citation Format: Sharon K. Michelhaugh, Sandeep Mittal. Combined in vitro TTFields and paclitaxel reduce proliferation and clonogenicity in non-small cell lung cancer (NSCLC) cells from a patient previously treated with standard-of-care [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 5254.
Abstract 5255: The SAGE Direct TestTM: A reliable way of functional profiling to predict the most effective treatment for cancer patients
Authors: Mishra Dhruva , Moy Jennifer , Apfel Chris
Published: September 2020

Cancer is a genetic disease with increasing heterogeneity during disease progression. However, despite the widespread use of genomic testing, a recent publication estimated that only 5% of cancer patients in the US benefit from genome-driven targeted therapies, largely because “actionable mutations” are rare.[1] Thus, SageMedic has developed the SAGE Direct Test™, leading the next generation of functional precision medicine to accurately predict the most effective treatment for virtually all cancer patients without limitations of genomic sequencing or having to identify a specific pathway. Our SAGE Direct Test™ consists of four critical steps, i.e., tissue processing, creation of 3D microtumors, live/dead cell imaging, and metabolic profiling. Using our proprietary technology, we can create hundreds of 3D microtumors from a core needle biopsy sample that recapitulate the in vivo tumor heterogeneity and microenvironment of the tumor biopsy. After these microtumors have been treated with a panel of drugs, we quantify the number of live and dead cells in those microtumors using our proprietary technology and scalable automatic stage confocal imaging (Molecular Devices). The advantage of our SAGE Direct Platform™ is the ability to multiplex our image-based results with the metabolic profile of our 3D microtumors, which cross validates our image-based readout. Our SAGE Direct Test™ has been optimized with ATCC cell lines and further validated with patient-derived tumor cells from lung, ovarian, and colon cancer tumor samples. Validation of high-quality confocal imaging samples followed by quantitative metabolic analysis suggests accurate quantification of viable cells, as reflected in a positive correlation between imaging quantitation and metabolic analysis (correlation coefficient r=0.84, p<0.01). In summary, SageMedic has developed the SAGE Direct Test™, a simple, highly sensitive, robust, and straightforward functional profiling assay with significant clinical relevance, as it can model the natural physiological conditions of tumors.

[1] Marquart et al. Estimation of the Percentage of US Patients With Cancer Who Benefit From Genome-Driven Oncology. JAMA Oncology 2018 Aug 1;4(8):1093-1098

Citation Format: Dhruva Mishra, Jennifer Moy, Chris Apfel. The SAGE Direct TestTM: A reliable way of functional profiling to predict the most effective treatment for cancer patients [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 5255.
Abstract 5256: Toward biomarkers discovery: Profiling triple-negative breast cancer cells by cell-SELEX
Authors: Camorani Simona , Leonetti Francesco , Collina Francesca , Cantile Monica , Botti Gerardo , Zannetti Antonella , Fedele Monica , Cerchia Laura
Published: September 2020

Triple-negative breast cancer (TNBC) is a high heterogeneous group of tumors with a distinctly aggressive nature and high rates of relapse. So far, the lack of any known targetable proteins has not allowed a specific anti-tumor treatment, thus the identification of novel agents for specific TNBC recognition and treatment is desperately needed. Nucleic acids aptamers are emerging as the most promising candidates for targeting tumor cells. They are also termed "chemical antibodies" because their form unique tertiary structures, which allow specific binding to their target molecules. Recently, by cell-SELEX integrated to advanced Next Generation Sequencing and bioinformatics, we isolated a panel of six nuclease-resistant RNA aptamers able to bind at high affinity to TNBC cells, discriminating them from both normal cells and non-TNBC breast cancer cells, and interfere with their growth as mammospheres. Also, they diversify TNBC histological specimens in a tissue microarray without recognizing samples from both ER+, PR+, HER2+ breast cancers and normal adjacent tissues. Here, short versions of the aptamers have been generated, by truncating their length from 84 nt up to nearly 30 nt, which preserve efficacious targeting and anti-tumor properties as the parental moieties. In order to identify the targets of the above aptamers, which might be important surface cancer biomarkers crucial for the malignant phenotype, different biochemical approaches are harnessed that include aptamer-mediated affinity purification coupled to Mass Spectrometry and/or antibody array for human soluble receptors. Binding analyses on recombinant proteins, cells or tissues are used to verify the putative targets. Results obtained so far indicate that the target of one of the six aptamers is a secreted protein tightly and specifically associated to TNBC cell surface and with a poor prognosis. This protein identification and the functional role of its targeting by the aptamer will be shown here in detail.

The proposed cell-targeting aptamer strategy could provide an important new avenue for selective TNBC therapy.

Citation Format: Simona Camorani, Francesco Leonetti, Francesca Collina, Monica Cantile, Gerardo Botti, Antonella Zannetti, Monica Fedele, Laura Cerchia. Toward biomarkers discovery: Profiling triple-negative breast cancer cells by cell-SELEX [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 5256.