bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract C123: Loss of tumor suppressor NDRG2 promotes cell proliferation and migration/invasion of oral squamous cell carcinoma cells
Authors: Yoon Hye-Jung , Ahn Chi-Hyun , Kim Ji-Hoon
Published: March 2020

More than 90% of oral cancers are diagnosed as oral squamous cell carcinoma (OSCC). Although there has been much understanding of the molecular pathogenesis of OSCC, the majority of patients still die from local recurrence and metastasis of tumors. Recently, N-myc downstream-regulated gene 2 (NDRG2) has been known as a tumor suppressor gene in various types of malignancies, but its function and molecular mechanism in OSCC are very little known. To verify the functional roles of NDRG2 in OSCC, we screened six OSCC cell lines first using western blotting. Among the 6 cell lines, HN22 cell line showing high expression of NDRG2 was selected and treated with anti-NDRG2 small interfering RNA (siRNA). The effect of NDRG2 on cell proliferation was evaluated at 24h, 48h, and 72h after treatment of 50nM of anti-NDRG2 siRNA. In addition, in vitro migration and invasion assays were performed after same treatment. Western blot analysis showed variable levels of NDRG2 in 6 OSCC cell lines. Normal human oral keratinocyte as a positive control expressed very high level of NDRG2. Among 6 OSCC cell lines, three cell lines had very low level or absence of NDRG2, but remaining three cell lines including HN22 cell had moderate to high level of NDRG2. After 24h treatment of anti-NDRG2 siRNA on the HN22 cells, NDRG2 expression was significantly downregulated. In cell proliferation assay after treatment of 50nM of anti-NDRG2 siRNA, there was a significant increase in cell growth of HN22 cells when compared with the control (p=.005). In wound healing assay, NDRG2-downregulated HN22 cell line showed much more closure of wound area when compared with the control (p=.007). From the invasion assay after treatment, there was a significant increase in invasion ability of HN22 cells (p<.001). These date indicate that loss of function of NDRG2 as a tumor suppressor can enhance the cell growth, migration, and invasion of OSCC cells. Our results suggest that loss of NDRG2 expression plays a role in tumor progression of OSCC by enhancing tumor cell growth, motility, and invasiveness. Therefore, recovering loss of function of NDRG2 may be a new therapeutic strategy for OSCC treatment.

Citation Format: Hye-Jung Yoon, Chi-Hyun Ahn, Ji-Hoon Kim. Loss of tumor suppressor NDRG2 promotes cell proliferation and migration/invasion of oral squamous cell carcinoma cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C123. doi:10.1158/1535-7163.TARG-19-C123
Abstract C132: Fusobacterium nucleatum in colorectal carcinoma and expression of multiple immune checkpoint molecules on T cells
Authors: Kim Han Sang , Kim Wonkyu , Kim Chang Gon , Yoo Jinseon , Paik Soonmyung , Shin Eui-Cheol , Kim Tae-Min , Kim Hoguen , Ahn Joong Bae
Published: March 2020

Background: Fusobacterium nucleatum (Fn) appears to play a role in colorectal cancer carcinogenesis through suppression of antitumor immune response. This study aims to characterize immune-related signatures, including the expression of multiple immune checkpoint molecules in tumor-infiltrating T cells. Methods: We measured Fn DNA by quantitative PCR in a total of 155 colorectal cancer tissues. Gene expression profile was studied using microarray technology to determine the consensus molecular subtypes (CMS) and immune-related signatures using CIBERSORT and Gene Set Enrichment Analysis (GSEA), respectively. Expression of immune checkpoint molecules (PD-1, TIM-3, and TIGIT) on CD8+ cytotoxic T cells and the ratio of FOXP3-CD4-/FOXP3+CD4 were examined using multicolor flow cytometric analysis. Results: All patients had stage III disease underwent curative surgery. Fn infection was observed in 43% of patients and was associated with recurrence (Fisher’ exact test, P=0.002) and decreased overall survival (log-rank test, P<0.001). The distribution of CMS subtypes was CMS1/2/3/4/mixed (14%/24%/15%/27%/20%), which is comparable to previous data. Fn infection was associated with the platinum-resistant signature, hypoxia pathway, and stem cell pathways in the GSEA. Interestingly, Patients with Fn infection showed a trend toward enrichment in CMS4, mesenchymal subtype with the inflamed immune phenotype (9% vs. 17% with Fn-infection, P=0.24). The number of cytotoxic CD8+ T cells showed a trend toward decreased expression in patients with Fn infection. Furthermore, higher expression of inhibitory immune checkpoints (PD-1, P<0.01; TIM-3, P<0.05; TIGIT, P<0.05) was observed on CD8+ cytotoxic T cells of patients with Fn infection, compared to that of Fn negative cases. Besides, the regulatory FOXP3+CD4+ cells were increased in patients with Fn infection, supporting the suppressive role of Fn infection in the tumor immune microenvironment (P<0.05). Conclusions: The adverse prognostic impact of Fn infection depends on the suppression of T cell-mediated immune response by increased expression of inhibitory immune checkpoints on cytotoxic T cell and by increased regulatory T cells.

Citation Format: Han Sang Kim, Wonkyu Kim, Chang Gon Kim, Jinseon Yoo, Soonmyung Paik, Eui-Cheol Shin, Tae-Min Kim, Hoguen Kim, Joong Bae Ahn. Fusobacterium nucleatum in colorectal carcinoma and expression of multiple immune checkpoint molecules on T cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C132. doi:10.1158/1535-7163.TARG-19-C132
Abstract C133: Effective targeting of KRAS-driven tumors by the first-in-class, orally bioavailable SOS1::KRAS inhibitor BI-3406
Authors: Hofmann Marco H , Gmachl Michael , Ramharter Jürgen , Savarese Fabio , Sanderson Michael P , Gerlach Daniel , Trapani Francesca , Botesteanu Dana-Adriana , Kessler Dirk , Ettmayer Peter , Arnhof Heribert , Gerstberger Thomas , Schnitzer Renate , Kofink Christiane , Stadtmueller Heinz , Wunberg Tobias , O’Connell Jonathan C. , Mendes Rachel L , Richard David , Saldanha Adrian S , Moll Juergen , Pearson Mark , McConnell Darryl B , Kraut Norbert
Published: March 2020

KRAS is the most frequently mutated oncogene with high prevalence in pancreatic, colorectal, and non-small cell lung cancers. KRAS signaling is tightly regulated and release of negative feedback pathways have limited the clinical efficacy of inhibitors of the downstream MAPK signaling in the KRAS mutant context. Here we report the discovery of BI-3406, a highly potent and selective, first-in-class, orally bioavailable SOS1::KRAS inhibitor that binds to the catalytic domain of the guanine exchange factor SOS1 thereby preventing the interaction with KRAS-GDP. In KRAS-dependent cancers, BI 3406 potently reduces the formation of GTP-loaded KRAS, and inhibits MAPK pathway signaling. Down-modulation of this signaling cascade by BI-3406 in KRAS G12 or G13 mutant cells results in a cytostatic effect. In monotherapy, the observed pathway modulation translates into marked anti-tumor efficacy in KRAS mutant xenografts. BI-3406 blocks the negative feedback induced by MAPK inhibition and consequently sensitizes KRAS-dependent cancers to MEK inhibitor treatment resulting in complete blockade of the pathway, which translates into tumor regression in vivo. Collectively, our data suggest that SOS1 inhibition is a promising therapeutic strategy to treat KRAS dependent cancers in combination with MEK inhibition, with the aim to deliver meaningful survival benefits for cancer patients.

Citation Format: Marco H Hofmann, Michael Gmachl, Jürgen Ramharter, Fabio Savarese, Michael P Sanderson, Daniel Gerlach, Francesca Trapani, Dana-Adriana Botesteanu, Dirk Kessler, Peter Ettmayer, Heribert Arnhof, Thomas Gerstberger, Renate Schnitzer, Christiane Kofink, Heinz Stadtmueller, Tobias Wunberg, Jonathan C. O’Connell, Rachel L Mendes, David Richard, Adrian S Saldanha, Juergen Moll, Mark Pearson, Darryl B McConnell, Norbert Kraut. Effective targeting of KRAS-driven tumors by the first-in-class, orally bioavailable SOS1::KRAS inhibitor BI-3406 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C133. doi:10.1158/1535-7163.TARG-19-C133
Abstract CN01-02: Maximizing the potential of bispecific antibodies for cancer drug development
Authors: Parren Paul W H I
Published: March 2020

Landmark advances in the engineering and development of bispecific antibodies (bsAbs) are enabling unprecedented versatility in therapeutic antibody concepts. Currently more than 20 different commercialized technology platforms are available for bsAb creation and development, two bsAbs are marketed, of which one in cancer, and over 85 are in clinical development. A defining bsAb feature is their potential for novel functionalities — that is, activities that do not exist in mixtures of the parental or reference antibodies. These so-called obligate bsAb have high potential for the development of novel and differentiating cancer drugs. Bispecific T cell engagers (bs-TCE) are a prominent class of obligate bsAb for targeted cancer immunotherapy. Classical bs-TCE designs consist of a tumor antigen-binding domain combined with a binding domain against the CD3 T-cell receptor-signaling complex. Irrespective of the bispecific antibody format used, CD3-based bs-TCEs have a number of disadvantages, which in part are explained by the fact that they activate all T-cells irrespective of lineage, which associates with serious adverse events as a result of exaggerated T cell activation and cytokine release in some patients, and limited efficacy due to T suppressor cell activation in others. The development of bs-TCEs with increased tumor selectivity to widen the therapeutic window has high potential. The engagement of γδ T cells, and Vγ9Vδ2-T cells in particular, is in this context of particular interest. This γδ T cell subset has been shown to display powerful innate anti-tumor immune effector activity with an ability to infiltrate human tumors in which its abundance has been shown to positively correlate with patient survival. The potential of bsTCEs designed to engage Vγ9Vδ2-T cells as an approach for the development of novel cancer immunotherapies will be discussed.

Citation Format: Paul W H I Parren. Maximizing the potential of bispecific antibodies for cancer drug development [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr CN01-02. doi:10.1158/1535-7163.TARG-19-CN01-02
Abstract CN07-02: Immunotoxins targeting GPC3 for liver cancer
Authors: Ho Mitchell , Fleming Bryan D , Greten Tim F , Pastan Ira
Published: March 2020

Glypican-3 (GPC3) is a cell surface glypican that serves as a co-receptor for Wnt. Our laboratory has generated two high affinity antibodies targeting GPC3, HN3 and YP7. The HN3 human nanobody recognizes the N-lobe of GPC3, whereas the YP7 antibody recognizes the C-lobe of GPC3. Mutating residue F41 on the N-lobe of GPC3 inhibits binding of Wnt and the HN3 nanobody and inhibits activation of β-catenin. Both antibodies are fused to a fragment of Pseudomonas exotoxin A (PE38) to create recombinant immunotoxins. Interestingly, the HN3-derived immunotoxin (HN3-PE38) has superior antitumor activity as compared with the YP7-derived immunotoxin (YP7-PE38). Intravenous administration of HN3-PE38 causes regression of hepatocellular carcinoma (HCC) tumor xenografts in mice. Our study establishes GPC3 as a promising target for immunotoxin-based liver cancer therapy and demonstrates immunotoxin-induced tumor regression via dual mechanisms: inactivation of Wnt signaling via the HN3 nanobody and inhibition of protein synthesis via the PE bacteria toxin. However, immunogenicity and a short serum half-life may limit the ability of immunotoxins to transition to the clinic. To address these issues, we have recently engineered HN3-based immunotoxins to contain various deimmunized PE toxins. These new immunotoxins include HN3-T20, which is modified to remove domain II of the PE toxin and six T-cell epitopes. All of our immunotoxins display high affinity to human GPC3, with HN3-T20 having a KD value of 7.4 nM. A real-time cell growth inhibition assay demonstrates that a single dose of HN3-T20 at 1.6 nM is capable of inhibiting nearly all cell proliferation during the 10-day experiment. To enhance HN3-T20’s serum retention, we further engineer the HN3-T20 by adding a streptococcal albumin binding domain (ABD) and a llama single-domain antibody fragment (ALB1) specific for serum albumin. For the detection of immunotoxin in mouse serum, we develop a highly sensitive ELISA and find that HN3-ABD-T20 has a 45-fold higher serum half-life than HN3-T20 (326 min vs 7.3 minutes); consequently, HN3-ABD-T20 shows the best serum retention and remains detectible even after a 24-hour period in mice. Furthermore, addition of an albumin binding domain results in HN3-ABD-T20 mediated tumor regression in mice at a low dose (1 mg/kg). These data show that the albumin binding deimmunized immunotoxins are high potency therapeutics ready to be evaluated in clinical trials for the treatment of liver cancer.

Citation Format: Mitchell Ho, Bryan D Fleming, Tim F Greten, Ira Pastan. Immunotoxins targeting GPC3 for liver cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr CN07-02. doi:10.1158/1535-7163.TARG-19-CN07-02
Abstract CN08-02: BMP signaling in glioblastoma
Authors: Miyazono Kohei , Tanabe Ryo
Published: March 2020

Glioma-initiating cells (GICs) play central roles in the initiation, progression, recurrence, tumor heterogeneity and drug resistance of glioblastoma. Bone morphogenetic proteins (BMPs) regulate normal embryonic neural development and exert anti-tumor activities in glioblastoma. However, its mechanisms of action remain to be elucidated. Through RNA-seq analyses, we have identified several BMP-target genes in GICs, including SMAD6, Noggin (NOG), Distal-less homeobox 2 (DLX2), and Paired-related homeobox 1 (PRRX1). DLX2 promotes apoptosis and neural differentiation of GICs. We also show that the expression of PRRX1 is induced by BMP stimulation, and that PRRX1 induces decrease in the CD133-positive GIC population and inhibits in vivotumorigenic activity of GICs. Of the two spliced isoforms of PRRX1, the longer form of PRRX1, but not the shorter form, is involved in the induction of differentiation of GICs through epigenetic regulation of the CD133/PROM1gene. In addition, we show that BMP-4 represses the expression of a transmembrane protein, which is preferentially expressed in mesenchymal subtype of glioblastoma. Potential use of these proteins for diagnosis and treatment of glioblastoma will be discussed.

Citation Format: Kohei Miyazono, Ryo Tanabe. BMP signaling in glioblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr CN08-02. doi:10.1158/1535-7163.TARG-19-CN08-02
Abstract KN01: Dissecting antitumor immune response in ovarian cancer
Authors: Coukos George
Published: March 2020

In epithelial ovarian cancer (EOC), intratumoral or intraepithelial tumor infiltrating lymphocytes (TILs), i.e. T cells specifically infiltrating tumor islets, occur in approximately half of the patients and correlate with longer survival. As with many other solid tumors, despite promising results in mouse models, response of EOC to PD-1 blockade has been quite limited in the clinic, but the mechanisms underlying therapeutic failures in immunoreactive human tumors harboring TILs are not well understood. Our work has sought to answer the following key questions: a) whether ovarian TILs are tumor-specific; b) if so, what are the relevant antigens recognized by TILs; c) whether the PD-1/PD-L1 pathway is relevant; d) if so, what are the mechanisms underlying response to PD-1 blockade; and finally e) what mechanisms govern TIL recruitment and engraftment. The data presented will show that there is indeed a spontaneous and tumor-specific T-cell response in a proportion of EOC, accurately heralded by intraepithelial CD8+ TILs. Tumor-specific antigens will be discussed. Our work also shows that the PD-1/PD-L1 is a central immune checkpoint pathway in these tumors. However, only a fraction of such tumors with ieTILs respond to PD-1. We dissected the mechanisms underlying response to PD-1 blockade and therapeutic approaches to enhance response to PD-1 in human and mouse preclinical models. Therapeutic approaches to achieve effective immunotherapy in ovarian cancer will be discussed, including combination checkpoint blockade, adoptive T cell therapy and new rational combinations. Finally, successful T-cell engraftment in ovarian cancer requires tumor cell-derived CCL5 and is amplified by myeloid cell-secreted IFNg-inducible CXCL9, dependent on activation of tumor-infiltrating lymphocytes (TILs). Spontaneous, epigenetically-mediated loss or stable knockdown of ccl5 leads to CD8+ T-cell loss and immune desertification in the mouse, while forced ccl5 expression prevents loss of cxcl9 and TILs, and leads to attenuated tumor growth. Accordingly, CCL5-CXCL9 co-expression reveals immunoreactive tumors with prolonged survival and response to PD-1 blockade. The impact of homologous recombination and BRCA loss on the establishment of immunoreactive tumors will be also discussed.

Citation Format: George Coukos. Dissecting antitumor immune response in ovarian cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr KN01. doi:10.1158/1535-7163.TARG-19-KN01
Abstract LB-A04: Rapid non-uniform adaptation to conformation-specific KRAS G12C inhibition
Authors: Xue Jenny , Zhao Yulei , Aronowitz Jordan , Mai Trang T , Vides Alberto , Qeriqi Besnik , Kim Dongsung , Li Chuanchuan , Stanchina Elisa de , Mazutis Linas , Risso Davide , Lito Piro
Published: March 2020

KRAS GTPases are activated in one-third of cancers and KRAS G12C is the most common activating alteration in lung adenocarcinoma. KRAS G12C inhibitors are in Phase-I clinical trials and early data show only partial responses in lung cancer patients. How cancer cells bypass inhibition, to prevent maximal responses to therapy, is not understood. Because KRAS G12C cycles between an active and inactive conformation, and the inhibitors only bind to the latter, we hypothesized that isogenic cell populations respond non-uniformly. Here we studied the effect of treatment at the single cell level and showed that shortly after treatment, some cancer cells were sequestered in a quiescent state with low KRAS activity, while others reactivated KRAS to resume proliferation. By combining cell fate-specific gene expressions and results from a CRISPR-Cas9 screen, we identified that this rapid divergent response is due to new KRAS G12C produced in response to suppressed MAPK output. Upstream-acting adaptive signals, such as epidermal growth-factor receptor and aurora kinase signaling, maintain new KRAS G12C protein in its active/drug-insensitive state to restore KRAS output. Cells without these adaptive changes (or cells where they are pharmacologically inhibited) remain sensitive to drug treatment, because new KRAS G12C is either not available, or it exists in its inactive/drug-sensitive state. Combined inhibition of these adaptive signals along with KRAS G12C produced more potent antitumor effects in vivo. Our study uncovers a flexible non-uniform fitness mechanism that enables groups of cells within a population to rapidly bypass the effect of treatment. This adaptive process must be overcome to maximize the therapeutic potential of conformation-specific KRAS G12C inhibitors in the clinic.

Citation Format: Jenny Xue, Yulei Zhao, Jordan Aronowitz, Trang T Mai, Alberto Vides, Besnik Qeriqi, Dongsung Kim, Chuanchuan Li, Elisa de Stanchina, Linas Mazutis, Davide Risso, Piro Lito. Rapid non-uniform adaptation to conformation-specific KRAS G12C inhibition [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-A04. doi:10.1158/1535-7163.TARG-19-LB-A04
Abstract LB-A07: Targeting nucleotide excision repair defects in cancer
Authors: Topka Sabine , Steinsnyder Zoe , Tkachuk Kaitlyn , Ravichandran Vignesh , Madsen Mogens Winkel , Furberg-Barnes Helena , Iyer Gopakumar , Stanchina Elisa De , Joseph Vijai , Offit Kenneth
Published: March 2020

A proportion of cancer patients carry inherited pathogenic variants or somatic alterations in the DNA helicase genes ERCC2 and ERCC3, important components of the nucleotide excision repair (NER) pathway. Cancer risk associated with mutations in nucleotide excision repair is not very well understood and there are no targeted therapies available for patients with NER gene mutations. We have recently shown that functionally significant cancer-associated mutations in ERCC3 exhibit remarkable sensitivity against the fungal sesquiterpene IlludinS. Irofulven, a derivative of IlludinS, has previously been evaluated as an antitumor agent in multiple clinical trials and has shown antitumor activity in a subset of patients. Based on the observation that ERCC3 mutant cell lines are highly sensitive to IlludinS we explored the role of Irofulven as a potential therapeutic agent to treat patients with hypomorphic mutations in nucleotide excision repair genes. We used CRISPR/Cas9 to create isogenic pairs of wildtype and mutant ERCC2 or ERCC3 cell lines and assessed in vitro and in vivo response to Irofulven. In vitro studies showed significantly increased sensitivity of the ERCC3 mutant cells towards Irofulven, when compared to commonly used drugs such as PARP inhibitors or alkylating agents. ERCC2 mutant cells showed elevated in vitro sensitivity to Irofulven even surpassing response to the current standard of care agent Cisplatin. Additionally, in vitro experiments show impaired ability of ERCC2/3 mutant cells to repair Irofulven induced DNA damage. The enhanced Irofulven sensitivity of ERCC3 mutants was replicated in vivo by xenograft experiments. Transcriptome analyses of the tumor tissues gave insights into pathways deregulated in the mutant background and revealed novel biology mediating Irofulven sensitivity suggesting potential for the development of combinatorial therapies. Similar to tumors arising in the background of homologous repair defects, mutations in nucleotide excision repair genes such as ERCC2 and ERCC3 could constitute potential biomarkers of therapeutic response. The present study provides pre-clinical evidence that a subset of tumors with mutations in nucleotide excision repair pathway genes exhibits exceptional sensitivity to Irofulven.

Citation Format: Sabine Topka, Zoe Steinsnyder, Kaitlyn Tkachuk, Vignesh Ravichandran, Mogens Winkel Madsen, Helena Furberg-Barnes, Gopakumar Iyer, Elisa De Stanchina, Vijai Joseph, Kenneth Offit. Targeting nucleotide excision repair defects in cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-A07. doi:10.1158/1535-7163.TARG-19-LB-A07
Abstract LB-A08: Novel strategy for treatment of triple-negative breast cancer by highly potent caspase-cleavable docetaxel prodrug
Authors: Cho Youngseok , Kim Harin , Chung Seung Woo , Byun Youngro
Published: March 2020

Despite the emergence of advanced therapeutics such as targeted therapy and immunotherapy in the modern oncology, cytotoxic chemotherapy still remains as the first-line treatment option in a wide range of cancers attributing to its potency. Many endeavors have been made to overcome the toxicity issues of cytotoxic chemotherapy by improving the specific delivery to the tumor, with active tumor targeting being one of the most popular approaches. However, such an approach has been challenged by the intratumor heterogeneity and the lack of valid molecular target in many types of cancer. Here, we introduce a novel albumin-binding prodrug MPD03 that could specifically deliver highly potent cytotoxin docetaxel to the tumor as an important component of chemotherapy for the treatment of triple-negative breast cancer (TNBC). MPD03 was synthesized by conjugating docetaxel to the C-terminus of the KGDEVD peptide via self-eliminating linker and introducing a maleimide group to the Lys sid chain of the peptide. MPD03 was able to bind albumin after administration via maleimide group for an extended circulation time and metabolized into docetaxel in tumor specific manner by reacting with the caspase-3 upregulated in tumor by apoptosis inducing agent, exerting a highly potent anticancer effect with good safety profile in TNBC xenograft model. PARP inhibitor olaparib was used in combination therapy with MPD03 in our strategy in terms of apoptosis inducer. Using our treatment regimen, we seek to expand our strategy to eradicate metastatic tumors.

Citation Format: Youngseok Cho, Harin Kim, Seung Woo Chung, Youngro Byun. Novel strategy for treatment of triple-negative breast cancer by highly potent caspase-cleavable docetaxel prodrug [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-A08. doi:10.1158/1535-7163.TARG-19-LB-A08
Abstract LB-A09: EED targeted PROTACs degrade EED, EZH2, and SUZ12 in the PRC2 complex
Authors: Hsu Jessie Hao-Ru , Rasmusson Timothy , Robinson James , Pachl Fiona , Read Jon , Kawatkar Sameer , O'Donovan Daniel H , Bagal Sharan , Code Erin , Rawlins Philip , Argyrou Argyrides , Tomlinson Ronald , Gao Ning , Zhu Xiahui , Chiarparin Elisabetta , Jacques Kelly , Shen Minhui , Woods Haley , Bednarski Emma , Wilson David M. , Drew Lisa , Castaldi M. Paola , Fawell Stephen , Bloecher Andrew
Published: March 2020

Deregulation of the PRC2 components EZH2, SUZ12, and EED plays critical roles in driving aberrant hypermethylation of H3K27 and tumorigenicity of many solid and hematological malignancies. Although SAM competitive small molecule inhibitors of EZH2 show promising clinical activity in PRC2-dependent cancers, preclinical data suggests that resistance can be acquired through secondary mutations in EZH2 that abrogate drug target engagement. To address these limitations, we have designed several hetero-bifunctional PROTACs (Proteolysis Targeting Chimera) to efficiently target EED for elimination. Our EED-targeting PROTACs bind to EED (pKD= 9.02-9.27) and promote stable ternary complex formation with the E3 ubiquitin ligase. The PROTACs potently inhibit PRC2 enzyme activity (pIC50= 8.11-8.17) that results in a decrease in H3K27me3 levels in cells. Interestingly, EED-targeting PROTACs induce rapid degradation of EED, as well as its associated proteins, including EZH2 and SUZ12 in the PRC2 complex. Inhibition of the ubiquitin proteasome pathway abrogates PROTAC-mediated degradation of EED and its associated proteins. Furthermore, the EED targeting PROTACs selectively inhibit proliferation and survival of PRC2-dependent cancer cells (GI50= 49-58 nM). In summary, our data demonstrate a novel therapeutic modality in treating PRC2 dependent cancer through PROTAC mediated platform.

Citation Format: Jessie Hao-Ru Hsu, Timothy Rasmusson, James Robinson, Fiona Pachl, Jon Read, Sameer Kawatkar, Daniel H O'Donovan, Sharan Bagal, Erin Code, Philip Rawlins, Argyrides Argyrou, Ronald Tomlinson, Ning Gao, Xiahui Zhu, Elisabetta Chiarparin, Kelly Jacques, Minhui Shen, Haley Woods, Emma Bednarski, David M. Wilson, Lisa Drew, M. Paola Castaldi, Stephen Fawell, Andrew Bloecher. EED targeted PROTACs degrade EED, EZH2, and SUZ12 in the PRC2 complex [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-A09. doi:10.1158/1535-7163.TARG-19-LB-A09
Abstract LB-B02: Orthogonal pathogen challenges in tumor burdened mice reveal systemic impairment of de novo immune responses
Authors: Hiam Kamir , Allen Breanna , Spitzer Matthew
Published: March 2020

Harnessing immune defense mechanisms has revolutionized cancer therapy. However, the limited efficacy of these approaches across patients and cancer types reflects our incomplete understanding of the factors governing immune responses in cancer. Here, we use orthogonal viral and bacterial challenges in mice with preexisting malignancy to assess functionality of the immune system in cancer. The tumor burdened immune system mounted dampened peripheral T cell responses characterized by reduced proliferation and effector differentiation. Disruptions in T cell activity in vivo were not cell intrinsic but rather due to reduced responses in antigen-presenting cells (APCs). Promoting APC activation was sufficient to restore T cell responses to orthogonal infection. These results demonstrate that tumor development dynamically reshapes the function of the immune macroenvironment, arguing for a deeper understanding of the tumor-experienced systemic context for rational immunotherapeutic design.

Citation Format: Kamir Hiam, Breanna Allen, Matthew Spitzer. Orthogonal pathogen challenges in tumor burdened mice reveal systemic impairment of de novo immune responses [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-B02. doi:10.1158/1535-7163.TARG-19-LB-B02
Abstract LB-C01: The kinase polypharmacology landscape of clinical PARP inhibitors
Authors: Antolin Albert A , Ameratunga Malaka , Banerji Udai , Clarke Paul , Workman Paul , Al-Lazikani Bissan
Published: March 2020

Polypharmacology plays an important role in defining response and adverse effects of drugs. For some mechanisms, experimentally mapping polypharmacology is commonplace, although often this is done within the same protein class. Four PARP inhibitors have been approved by the FDA as cancer therapeutics, yet a precise mechanistic rationale to guide clinicians on which to choose for a particular patient is lacking. The four drugs have largely similar PARP family inhibition profiles, but several differences at the molecular and clinical level have been reported that remain poorly understood. Here, we report the first comprehensive characterization of the off-target kinase landscape of four FDA-approved PARP drugs. We demonstrate that all four PARP inhibitors have a unique polypharmacological profile across the kinome. Niraparib and rucaparib inhibit DYRK1s, CDK16 and PIM3 at clinically achievable, submicromolar concentrations. The most potently inhibited off-targets are CDK16 for rucaparib (IC50 = 381 nM) and DYRK1B for niraparib (IC50 = 254 nM). These kinases represent the most potently inhibited off-targets of PARP inhibitors identified to date and should be investigated further to clarify their potential implications for efficacy and safety in the clinic.

Citation Format: Albert A Antolin, Malaka Ameratunga, Udai Banerji, Paul Clarke, Paul Workman, Bissan Al-Lazikani. The kinase polypharmacology landscape of clinical PARP inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-C01. doi:10.1158/1535-7163.TARG-19-LB-C01
Abstract LB-C11: Targeting metastatic prostate cancer with a multi-functional therapy approach
Authors: Gordon Ryan , Zhang Limin , Pattanayak Abhinandan , Li Wenqi , Bergan Raymond
Published: March 2020

Prostate cancer (PCa) cells move throughout the body, hone to and destroy the bone microenvironment, grow and thereby cause death. Our historical inability to target cell movement has precluded our realization of multifunctional therapy wherein each of these processes is targeted. Our group has recently shown that inhibition of intracellular activation of Raf1 with the small molecule therapeutic, KBU2046, permits for the first time selective inhibition of cell motility. Here we demonstrate that simultaneous disruption of multiple distinct functions that drive progression of PCa to induce death results in advanced disease control. Using an orthotopic murine model of human PCa metastasis, KBU2046 combined with docetaxel achieves sustained anti-tumor action and improved inhibition of metastasis, compared to monotherapy. KBU2046 does not interfere with androgen deprivation or androgen receptor antagonist mediated hormone therapy in either in vitro or in vivo models. Cell movement is necessary for osteoclast-mediated bone degradation. KBU2046 inhibits Raf1 and its downstream activation of MEK1/2 and ERK1/2 in osteoclasts, inhibiting cytoskeleton rearrangement, resorptive cavity formation and bone destruction in vitro, with improved effects observed when the bone microenvironment is chemically modified by pretreatment with zoledronic acid. Using a murine cardiac injection model of human PCa bone destruction quantified by computed tomography, KBU2046 plus zoledronic acid exhibited improved inhibitory efficacy, compared to monotherapy. The combined disruption of pathways that drive cell movement, honing to the bone and growth constitutes a multi-functional targeting strategy that provides advanced disease control.

Citation Format: Ryan Gordon, Limin Zhang, Abhinandan Pattanayak, Wenqi Li, Raymond Bergan. Targeting metastatic prostate cancer with a multi-functional therapy approach [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-C11. doi:10.1158/1535-7163.TARG-19-LB-C11
Abstract LB-C15: EpCAM-high liver cancer stem cells resist natural killer cell-mediated cytotoxicity by upregulating CEACAM1
Authors: Sung Pil Soo , Yoon Seung Kew
Published: March 2020

Natural killer (NK) cells are innate lymphoid cells with potent cytolytic functions that defend the host against microbial infections and tumours. Here, we investigated the cytotoxic effects of NK cells on epithelial cell adhesion molecule (EpCAM)-expressing liver cancer stem cells (CSCs). The frequency of recurrence after curative resection was higher in patients with peritumoural EpCAM expression than patients with pantumoural or negative EpCAM expression. Co-culture experiments demonstrated that EpCAMhigh Huh-7 cells resisted NK cell-mediated cytotoxicity. Silencing carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), which is overexpressed on the surface of EpCAMhigh Huh-7 cells and other hepatoma cells or primary hepatocytes, restored cytotoxicity of NK cells against EpCAMhigh Huh-7 cells. Neutralization of CEACAM1 on the NK cells also enhanced the killing of Huh-7 cells, suggesting CEACAM1 expressed on the NK cells transmits inhibitory signals into the NK cells. EpCAMhigh Hepa1-6 cells formed larger tumours with higher CEACAM1 expression levels in vivo. Neutralization of CEACAM1 in Hepa1-6 xenograft mouse model resulted in more activated phenotype of tumour-infiltrating NK cells and delayed growth of the tumour. Finally, CEACAM1 expression positively correlated with EpCAM expression in human HCC tissues and the serum CEACAM1 levels were also significantly higher in patients with EpCAM+ HCC. Moreover, analyses with TCGA dataset revealed that EpCAM expression shows positive correlation with CEACAM1 expression in various types of cancers. In conclusion, our data demonstrated that EpCAMhigh liver CSCs resist NK cell-mediated cytotoxicity by upregulating CEACAM1 expression.

Citation Format: Pil Soo Sung, Seung Kew Yoon. EpCAM-high liver cancer stem cells resist natural killer cell-mediated cytotoxicity by upregulating CEACAM1 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-C15. doi:10.1158/1535-7163.TARG-19-LB-C15
Abstract PL03-01: Targeting DNA Repair and Defective DNA repair
Authors: Bono Johann de
Published: March 2020

Genomic instability and defective DNA repair (DDR) are key hallmarks of many currently incurable malignancies, with these often associating with poorer outcomes and more aggressive disease. DDR supports tumor evolution but can generate genomic alterations leading to neoantigen generation from single nucleotide alterations and genomic rearrangements/fusions, which may lead to tumor sculpting by immune responses and tumor cell adaptations to abrogate anticancer immune responses through not only immune checkpoint expression but also the secretion of cytokines and chemokines that impact immune cell migration and function. Multiple studies have now shown that DDR can be a vulnerability, both through synthetic lethal strategies with drugs like PARP and ATR inhibition and DNA damaging agents, as well as through strategies targeting the immune response as best exemplified by mismatch repair defects and PD-1 targeting and more recently emerging evidence on CDK12 alterations. This presentation will focus on the therapeutic targeting of tumors with DNA repair defects, synthetic lethal strategies, and the challenges of developing anticancer drugs targeting DNA repair.

Citation Format: Johann de Bono. Targeting DNA Repair and Defective DNA repair [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr PL03-01. doi:10.1158/1535-7163.TARG-19-PL03-01
Abstract PL03-02: Exploiting DNA replication stress for cancer therapies
Authors: Halazonetis Thanos
Published: March 2020

A feature of most cancers is genomic instability. We and others have proposed a model to explain the presence of such genomic instability. Briefly, we proposed that oncogenes, which are activated in practically all cancers and drive cell proliferation, induce DNA replication stress. The presence of DNA replication stress leads to the formation of DNA double-strand breaks, which, when incorrectly repaired, lead to genomic instability. DNA replication stress is a somewhat non-specific term used to indicate that cells have difficulties to replicate their DNA. Inhibitors of DNA polymerases slow down DNA replication and induce DNA replication stress. However, in this case, the replisome, i.e. the proteins that replicate the DNA, remains intact and DNA double-strand breaks are not formed. Oncogenes induce a different type of DNA replication stress, as select replisomes are disrupted, leaving the DNA “naked” and susceptible to DNA double-strand break formation. Recent results from a number of laboratories have provided great insights on how oncogenes induce DNA replication stress. Specifically, it has been found that oncogenes induce the firing of novel DNA replication origins. These oncogene-induced origins are present within transcribed genes. When these origins fire and DNA replication begins, the replisomes collide with the transcription machinery. These aberrant collisions lead to disruption of the replisome. Disrupted, aka as collapsed, replisomes need to be repaired for DNA replication to restart. Errors in this repair lead to DNA double-strand breaks and genomic instability. The main pathway for repair of collapsed DNA replication forks appears to be break-induced replication. This pathway is used very infrequently by normal cells and, as such, certain proteins that function in this pathway are not essential for normal development. However, their inactivation suppresses cancer development, because cancer cells rely on break-induced replication to repair their collapsed DNA replication forks. It follows that proteins that function in break-induced replication may be good targets for the development of effective, non-toxic cancer therapies.

Citation Format: Thanos Halazonetis. Exploiting DNA replication stress for cancer therapies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr PL03-02. doi:10.1158/1535-7163.TARG-19-PL03-02
Abstract PL04-02: Extracellular vesicles as opportunities for integrative or focused liquid biopsy studies
Authors: Welsh Joshua , Aldape Kenneth , Jones Jennifer
Published: March 2020

Background: For patients who present with or develop progressive, metastatic cancer, new immunotherapies offer significant hope of remission or cure. However, many of the most effective immunotherapies and cancer vaccines are often effective in fewer than 50% of patients treated, and assessment of treatment responses is only feasible after multiple weeks or months of treatment, a period of time during which other more effective treatment options or combinations might have been selected instead for the non-responding patients. Therefore, improved methods for monitoring treatment responses, especially early during treatment, are needed. Extracellular Vesicles (EVs), are continuously released and carry surface receptors and RNA/DNA cargo related to the state of their cell of origin. Thus, EVs may enable near-real-time clinical blood and/or urine tests for early screening, risk stratification, and monitoring of cancer immunotherapies. Methods: To extract the information that is carried by EVs, our lab has developed a first-in-class pipeline to characterize EV heterogeneity and provide high-sensitivity quantification of informative EV subsets by combining multiplex assays with high-resolution, single EV flow cytometric methods together into a Multiplex-to-Single EV Analysis (Mt-SEA) pipeline. Results: In preliminary data from ongoing clinical trials, we have identified EV subset changes that correspond to responses to treatment. Moreover, as with other complex biological systems, we find that it is possible to interrogate several dimensions of EV cargo to further improve the utility and impact of this approach. The methods and tools that we have developed are being made available for use in the wider research community. Conclusions: We hypothesize that identification of sensitive and specific EV-associated biomarkers for responses to immunotherapies would significantly advance efforts to improve treatment outcomes and quality of life for advanced cancer patients. The Mt-SEA Pipeline of methods developed by the NCI Translational Nanobiology lab establishes a foundation for interrogating different genetic and molecular profiles in tumor-derived and immune cell-derived EVs in urine and/or blood within 1-2 weeks of the start of treatment.

Citation Format: Joshua Welsh, Kenneth Aldape, Jennifer Jones. Extracellular vesicles as opportunities for integrative or focused liquid biopsy studies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr PL04-02. doi:10.1158/1535-7163.TARG-19-PL04-02
Abstract A19: The role of metaplastic tuft cell chemosensory signaling in pancreatic cancer
Authors: Hoffman Megan T. , The Stephanie , Salas-Escabillas Daniel J. , DelGiorno Kathleen E. , Bednar Filip , Margolskee Robert F. , Rao Arvind , Crawford Howard C.
Published: March 2020

Pancreatic ductal adenocarcinoma (PDA) is the third most common cause of cancer death in the US with a 9% 5-year survival rate. PDA is characterized by a robust stromal reaction, a large portion of which is composed of infiltrating immune cells and supported by fibroblasts. This fibroinflammatory response promotes both the initiation and progression of PDA. Key to this progression is the crosstalk between the tumor epithelium and immune compartment. Metaplastic tuft cells (MTCs) are a subset of the neoplastic epithelium derived by the transdifferentiation of acinar cells. Tuft cells have been characterized to have a “sense-and-respond” role in luminal surfaces throughout the body, using the taste chemosensory system to promote an inflammatory response in infected and damaged tissues. Among the pancreatic epithelium, MTCs uniquely express components of the gustation sensory system including taste receptors, TRPM5 and αGustducin, as well as an array of inflammatory cytokines, suggesting they are capable of promoting an immune response to extracellular challenges. To explore this hypothesis, we ablated αGustducin, a key protein in the chemosensory cascade, in a model of pancreatitis-associated tumorigenesis. We found that neoplasia in the GNAT3-/-; KrasG12D; PTF1aCreERT mice initiates and progresses more rapidly, a difference associated with alteration in immune cell subtypes. To further quantitate changes in the immune response, we performed mass cytometry (CyTOF), as a way to explore changes in cell number and type, and single-cell sequencing, to more deeply probe the unique gene signatures of cells within the tumor stroma. These large-scale analyses suggest substantial changes in the number of T-cell and myeloid cell populations, as well as alterations of fibroblasts and myeloid cell polarity. We conclude that MTCs use chemosensory signaling to promote the formation of a tumor-suppressive microenvironment.

Citation Format: Megan T. Hoffman, Stephanie The, Daniel J. Salas-Escabillas, Kathleen E. DelGiorno, Filip Bednar, Robert F. Margolskee, Arvind Rao, Howard C. Crawford. The role of metaplastic tuft cell chemosensory signaling in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A19.