bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract B098: A novel monoclonal antibody for a cancer stem cell marker CD133 exerts antitumor and antimetastasis activities for colon cancers
Authors: Kato Yukinari , Ohishi Tomokazu , Kawada Manabu , Kaneko Mika K
Published: March 2020

Background: Cancer stem cells contribute to tumorigenesis, chemoresistance, and metastasis. CD133 is a pentaspan membrane glycoprotein, and is used for the isolation of stem-like cells from cancer tissues. However, specific and sensitive monoclonal antibodies (mAbs), which are useful in every application, such flow cytometry, Western blot, and immunohistochemical analyses have not been established. Furthermore, antibody drugs targeting CD133 have not been established. Purpose: We aimed to develop novel anti-CD133 mAbs, which not only are useful for all applications but also exerts anti-tumor and anti-metastasis activities. Methods: We employed Cell-Based Immunization and Screening (CBIS) method for the development of anti-CD133 mAbs. The full-length CD133-overexpressed LN229 glioblastoma cells were immunized into mice, and flow cytometry was used for the first screening. Western blot and immunohistochemical screenings were further performed. Human colon cancer cell lines were used for examining the anti-tumor and the anti-metastasis activities of anti-CD133 mAbs. Results: We established a novel anti-CD133 mAb, CMab-43 (IgG2a, kappa), which demonstrated a sensitive and specific reaction against colon cancer cells in flow cytometry, Western blot, and immunohistochemical analyses. CMab-43 showed cancer-specific staining patterns in colon cancer tissues. Furthermore, CMab-43 significantly reduced tumor development of colon cancer cell xenografts, and inhibited experimental metastasis of colon cancer cells. Conclusion: CMab-43, which was developed by CBIS method, is useful for many applications and exerts anti-tumor or anti-metastasis activities. CMab-43 could be advantageous for antibody therapy against CD133-expressing colon cancers.

Citation Format: Yukinari Kato, Tomokazu Ohishi, Manabu Kawada, Mika K Kaneko. A novel monoclonal antibody for a cancer stem cell marker CD133 exerts antitumor and antimetastasis activities for colon cancers [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 B098. doi:10.1158/1535-7163.TARG-19-B098
Abstract B128: K-11706 exerts potent anti-cancer activity via suppression of GATA2/AR/cMyc signaling axis in castration resistant prostate cancer
Authors: Kaochar Salma , Rajapakshe Kimal , Robertson Matthew J. , Song Yongcheng , Cristian Coarfa , Mitsiades Nicholas
Published: March 2020

Despite recent therapeutic advances, prostate adenocarcinoma (PC) progression to lethal castration-resistant PC (CRPC) remains a major clinical problem, creating an unmet need for innovative treatment approaches. Targeted therapies for PC have mostly focused on the androgen receptor (AR) axis, while several other PC drivers, such as GATA2, c-Myc, FOXM1, CENPF, and EZH2, remain undruggable in the clinic so far. We and others have previously reported several critical functions of GATA2 in AR signaling in PC: GATA2 induces AR expression; serves as a pioneer factor for AR binding to chromatin; and also post-translationally promotes transcriptional activity of both full-length and splice-variant AR. GATA2 is often elevated in CRPC. Here, we investigated the role of GATA2 in PC cells beyond the AR signaling axis, and characterized the pharmacological potency of the GATA2 small molecule inhibitor (SMI) K-11706 against PC cells. Chromatin immunoprecipitation and RNA sequencing analysis revealed that GATA2 is a key modulator of cMyc expression, which induces the expression of FOXM1 and EZH2. We extensively profiled five CRPC cell lines following treatment with K-11706, which inhibited the proliferation and invasive behavior of PC cells, and dramatically reduced the genome-wide transcriptional activity of GATA2, AR, and cMyc, leading to downregulation of several PC drivers and AR/cMyc effector genes, notably FOXM1, EZH2, and CENPF. Transcriptional profiling and functional pathway analysis of the K-11706 transcriptomic footprint against curated databases delineated a biological network composed of genes involved in cell cycle/proliferation, stemness, metastasis and DNA repair. In vivo, K-11706 suppressed the growth of multiple cell-derived and patient-derived xenograft PC models. We propose that GATA2 is a master regulator of multiple PC drivers and a promising actionable therapeutic target in CRPC. Pharmacologic inhibition of GATA2, via a potent second-generation small molecule inhibitor is feasible and effective in PC models both in vitro and in vivo.

Citation Format: Salma Kaochar, Kimal Rajapakshe, Matthew J. Robertson, Yongcheng Song, Coarfa Cristian, Nicholas Mitsiades. K-11706 exerts potent anti-cancer activity via suppression of GATA2/AR/cMyc signaling axis in castration resistant prostate 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 B128. doi:10.1158/1535-7163.TARG-19-B128
Abstract B138: Biochemical and cell-based activities of ectonucleotidases CD39 and CD73 using a homogenous HTS formatted platform
Authors: Goueli Said , Hsiao kevin
Published: March 2020

The success of immunotherapy treatment in oncology ushered a new modality for treating a wide variety of cancers. However, lack of effect in some patients made it imperative to identify other pathways that are exploited by cancer cells to circumvent immune surveillance, and possibly synergize immune checkpoint treatment in those cases. It has been recently recognized that adenosine levels increased significantly in the tumor microenvironment and that adenosine/adenosine receptors play a powerful role as immunosuppressant and attenuating several effector T cell functions. The two main enzymes responsible for generating adenosine in the microenvironment are the ectonucleotidases CD39 and CD73, the former utilizes both ATP and ADP and producing AMP while the latter utilizes AMP and generates adenosine. Thus, these two enzymes combined are the major source for the bulk of adenosine produced in the microenvironment. They were shown to be validated targets in oncology leading to several clinical trials that include small molecules as well as antibodies, showing positive and encouraging results in the preclinical arena. Towards the development of novel drugs to target these enzymes, we have developed a platform that can be utilized to monitor the activities of both enzymes in vitro (biochemical) as well as in cells (cell based) assays. We have developed very sensitive and homogenous assays that enabled us to monitor the activity of both enzymes and demonstrate selectivity of known small molecule inhibitors as well as selective monoclonal antibodies. This should speed up screening for novel inhibitors leading to more effective cancer therapy

Citation Format: Said Goueli, kevin Hsiao. Biochemical and cell-based activities of ectonucleotidases CD39 and CD73 using a homogenous HTS formatted platform [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 B138. doi:10.1158/1535-7163.TARG-19-B138
Abstract C030: A new low potency DNA guanine monoalkylating ADC payload with enhanced i
n vivo tolerability
Authors: Veillard Nicolas , Andriollo Paolo , Cascio Francesco , Jackson Paul J. M. , Thurston David E.
Published: March 2020

Although five Antibody-Drug Conjugates (ADCs) have been approved and over eighty others are in development, the majority contain payloads belonging to two classes: tubulin inhibitors and DNA interactive agents. Most DNA-interactive payloads (e.g., the PBD dimers and IGNs) have potent cytotoxicity but ADCs containing them have high hydrophobicity and a narrow therapeutic window. Thus, there is interest in developing novel payloads which benefit from similar in vivo efficacy but possess lower hydrophobicity and a substantially wider Therapeutic Window (TW). The pyridinobenzodiazepines (PDDs) are a new class of sequence-selective, DNA guanine monoalkylating ADC payload which contain a polyheterocyclic chain with sufficient span to guide them to specific DNA sequences (e.g., transcription factor binding sites). The lead high potency PDD payload (FGX2-62) is active in vitro in the picomolar range in a wide panel of cell-lines, is easily conjugated and has in vivo potency at sub-mg/kg levels in ADC form. Within the PDD platform, we have now developed a lower potency DNA monoalkylator with substantially superior in vivo properties to other DNA alkylating agents. FGX20-75 is active in the low nanomolar range (e.g., 2.1 nM in the gastric cell-line SW48, 72 hour incubation), but is sufficiently hydrophilic to allow efficient conjugation at DARs > 4. The increase in loading compared to other DNA-interactive payloads compensates for the drop in potency of FGX20-75, resulting in a potent ADC with substantially increased tolerability. ADCs have been generated by conjugating a maleimide-linked analogue of FGX20-75 to the EGFR-targeting antibody Cetuximab with DAR of 4.2. The ADCs exhibit significant in vivo efficacy compared to other DNA-interactive payloads, and a substantially increased tolerability profile. A maximum tolerated dose (MTD) has not yet been reached at 35 mg/kg (single dose, CD1 mice). The favourable hydrophobicity profile of the low potency alkylator (FGX20-75) and its ease of conjugation to antibodies, along with the significant in vivo efficacy and tolerability of the ADCs produced, suggest that this low potency DNA-alkylating payload represents a promising new approach in ADC development.

Citation Format: Nicolas Veillard, Paolo Andriollo, Francesco Cascio, Paul J. M. Jackson, David E. Thurston. A new low potency DNA guanine monoalkylating ADC payload with enhanced in vivo tolerability [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 C030. doi:10.1158/1535-7163.TARG-19-C030
Abstract C040: INTH-454, a p300/CBP interactome modulator, targets multiple pathogenesis factors in solid tumors and induces strong anti-proliferative effects in vitro and in vivo as single agent
Authors: Cattori Valentino , Hentsch Bernd , Kessler Ulrich
Published: March 2020

Background: INTH-454 is a novel rationally designed ligand of the CH1/TAZ1 domain of p300/CBP, two closely related paralog transcription co-activators crucially involved in cancer cell proliferation and tumor progression. Here, we report the in vitro and in vivo characterization of INTH-454 in preclinical models of castration-resistant prostate (CRPC) and gastrointestinal (GI) cancer. Methods: The anti-proliferative effects of INTH-454 were assessed in colon, gastric and prostate cancer cells harboring wild type, mutant or no p53 protein followed by cell cycle analysis, beta-galactosidase staining and caspase activation assays. Levels of p53, acetylated p53 and p21 were determined by Western blotting. The impact of INTH-454 on ubiquitination/degradation of p53 and p21 was investigated and pharmacodynamic and anti-tumoral effects of INTH-454 were illustrated in cell line and patient derived xenograft models of prostate and GI cancer. Results: INTH-454 exhibited anti-proliferative effects at low nanomolar concentrations in all cell lines inducing strong cell cycle arrest followed by senescence or cell death. Levels of p21 were stably increased independent of p53-status and the amount of both, total ubiquitinated protein and ubiquitinated p53, was significantly decreased in treated cells. Oral administration of INTH-454 triggered strong inhibition of tumor growth in xenograft mouse models of CRPC and GI cancer up to tumor eradication at well tolerated doses. Analysis of tumor tissue by immunohistochemistry and Western blotting confirmed the in vitro results and illustrated additional pharmacodynamic effects on disease specific markers such as reduction of VEGF and PSA. Conclusions: Blockage of the CH1/TAZ1 domain of p300/CBP has a profound effect on cell cycle regulation in cancer cells. INTH-454 causes cell cycle arrest through stabilization/overexpression of p21 in a p53-dependent and -independent manner followed by either cell death or senescence and sustained anti-tumoral effects in xenograft models of prostate and gastrointestinal carcinomas.

Citation Format: Valentino Cattori, Bernd Hentsch, Ulrich Kessler. INTH-454, a p300/CBP interactome modulator, targets multiple pathogenesis factors in solid tumors and induces strong anti-proliferative effects in vitro and in vivo as single agent [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 C040. doi:10.1158/1535-7163.TARG-19-C040
Abstract C055: Novel imipridone ONC206 inhibits cell proliferation and induces apoptosis in uterine serous cancer through altering MAPK/AKT/AMPK signaling network and metabolic reprogramming
Authors: Yeung Chi Lam Au , Hu Wen , Zhang Li , Ferri-Borgogno Sammy , Tarapore Rohinton , Lu Karen H , Mok Samuel C
Published: March 2020

Uterine serous carcinoma (USC) is the most aggressive subtype of endometrial cancer. Patients with advanced stage USC have poor survival rates and new treatment regimens are lacking. Recent clinical studies showed that imipridones constitute a novel class of agents with antitumor activities. Among them, ONC206, a novel orally bioavailable small molecule that has recently been shown to bind G protein-coupled receptor (GPCR)-dopamine receptor D2 (DRD2) by comprehensive GPCR screening, induces apoptosis in glioblastoma cells. However, the effects of ONC206 on USC progression and the mechanism of action have not been thoroughly explored. Using both in vitro and in vivo models, and multiple USC cell lines, the effects of ONC206 on cell proliferation, cell cycle progression, and apoptosis were determined. Reverse phase protein arrays (RPPAs) and Western blot analyses were used to determine the effect of ONC206 on the expression of key proteins in various signaling networks in ONC206 treated USC cells. The results showed that ONC206 suppressed USC cell proliferation and induces apoptosis in a does dependent manner. It increased the number of USC cells in the G1 phase of the cell cycle. Luciferase labeled USC cell ARK1-bearing mice treated with 100mg/kg ONC206 had significantly lower chemiluminescent signals than those treated with the control buffer. RPPA data showed that ONC206 treated USC cells had markedly lower expression signals in p38MAPK, p-AKT, p-S6, CDKs, and multiple mitochondrial proteins associated with mitochondrial ATP synthesis including MTCO1, SDHB, and HSPD1 than the control cells did. In addition, markedly higher expression levels of p-AMPK, Bim, and cleaved caspases were also observed in ONC206 treated cells than the control did. Significantly lower ATP levels and cytochrome c oxidase activities in ONC206 treated USC cells than in control cells were demonstrated by luminescent ATP detection assay kit and the cytochrome c oxidase assay kit. These data suggest that ONC206 suppresses USC progression through inhibiting MAPK/AKT network, which subsequently leads to cell cycle arrest, metabolic reprogramming and increased apoptosis. Further studies which demonstrate the optimal dosage and the efficacy of treatment of USC using ONC206 are warranted.

Citation Format: Chi Lam Au Yeung, Wen Hu, Li Zhang, Sammy Ferri-Borgogno, Rohinton Tarapore, Karen H Lu, Samuel C Mok. Novel imipridone ONC206 inhibits cell proliferation and induces apoptosis in uterine serous cancer through altering MAPK/AKT/AMPK signaling network and metabolic reprogramming [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 C055. doi:10.1158/1535-7163.TARG-19-C055
Abstract C098: Preclinical evaluation of eribulin in a veterinary clinical trial of metastatic spontaneous hemangiosarcoma in pet dogs
Authors: Lopes Christina Kelly , Post Gerald , Lambert Lindsay , Lewis Benjamin , Allen Thaddeus David , Patel Angel , Littlefield Bruce A
Published: March 2020

Eribulin is a microtubule dynamics inhibitor that is currently in clinical use for treatment of certain patients with advanced breast cancer or liposarcoma. FDA approval for both indications was based on extension of overall survival. Based on eribulin’s known vascular remodeling effects, its activity against at least one sarcoma type, and literature reports of taxane activity in angiosarcoma, we hypothesized that eribulin may show activity against angiosarcoma. Due to a paucity of preclinical cell-based, xenograft, and PDX models for human angiosarcoma, we sought other alternatives for preclinical testing in this disease. While rare in humans, angiosarcoma is common in some breeds of domestic dogs, where it is known in veterinary parlance as hemangiosarcoma (HSA). Accordingly, we undertook a small veterinary clinical trial to evaluate eribulin against spontaneous HSA in pet dogs. Canine HSA most frequently occurs in the spleen, so splenectomy is a common first-line treatment; however, non-splenic sites also occur and these are often unresectable. In keeping with eribulin’s clinical use in the metastatic setting, we selected metastatic canine HSA, which we defined as dogs with histologically confirmed HSA with either non-resectable or post-splenectomy residual disease confirmed by imaging (PET/CT, CT). Of 19 pets initially screened, 12 dogs were imaged; 6 of these had imageable disease and thus began eribulin treatment. Eribulin was administered using the human clinical schedule (d1,8Q21), with the first cycle at 0.3 mg/m2 followed by intra-patient dose escalation to 0.6 mg/m2 after determination of tolerability. Of the 6 dogs entering treatment, 5 completed cycle 1, 3 completed cycle 2, and 2 completed the full per-protocol 3 cycles of treatment. These 2 dogs survived 121 and 179 days after initial diagnosis, contrasting with the 0.9 month (~27 days) median survival time reported by Wendelburg et al. (JAVMA, 2015) for 52 post-splenectomy canine HSA patients with residual metastases. Although limited in scope, our results with spontaneous metastatic canine HSA in pet dogs provides a preclinical foundation for further exploration of eribulin in malignancies of vascular origin in both the veterinary and human settings.

Citation Format: Christina Kelly Lopes, Gerald Post, Lindsay Lambert, Benjamin Lewis, Thaddeus David Allen, Angel Patel, Bruce A Littlefield. Preclinical evaluation of eribulin in a veterinary clinical trial of metastatic spontaneous hemangiosarcoma in pet dogs [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 C098. doi:10.1158/1535-7163.TARG-19-C098
Abstract C101: Comprehensive identification of novel therapeutic targets for treatment of PD-1 resistant solid tumors via a genome-scale CRISPR/Cas9 in vivo T-cell screen
Authors: Mercier Isabelle Le , Merkin Jason J. , Keegan Sean , Calnan Conor , Hohmann Anja F. , Colletti Nick , Fagerberg Eric , Shenker Sol , Bullock Caroline , Wrocklage Chris , Tubo Noah , Xu Tianlei , Noyes Matt , Rahal Rami , Arlauckas Sean , Morales Aria Pearlman , Stegmeier Frank , Cadzow Louise , Schlabach Mike , Kryukov Gregory , Benson Micah J.
Published: March 2020

Although immunotherapy with PD-1/PD-L1 antagonists has significantly advanced patient care, the majority of cancer patients currently do not benefit from checkpoint inhibitor therapies. To identify novel targets for the treatment of PD-1 insensitive cancers, we developed a novel Immune-CRISPRomicsTM platform that enabled genome-wide CRISPR/Cas9 screens in primary T cells in an in vivo setting. Notably, the screen identified clinically active molecules, such as PD-1 and also predicted recent clinical failures. In addition, we identified multiple targets that enhanced anti-tumor T-cell function similar to or better than PD-1 as a monotherapy. The anti-tumor activity of targets was assessed across a collection of PD-1 sensitive and PD-1 refractory syngeneic tumor models. One of the targets identified, IO-7, was found to possess robust activity across multiple PD-1 refractory models. We found that inhibition of IO-7 leads to long-term T-cell memory and prevents tumor growth upon re-challenge. Moreover, mechanistic follow-up studies demonstrate that IO-7 inhibition leads to an expansion of central memory T-cell subsets, which have been implicated in driving the durable clinical response of checkpoint inhibitors. We describe the first genome-wide in vivo T-cell CRISPR/Cas9 screen, which identified multiple therapeutic targets that present promising therapeutic opportunities for the treatment of PD-1 resistant solid tumors.

Citation Format: Isabelle Le Mercier, Jason J. Merkin, Sean Keegan, Conor Calnan, Anja F. Hohmann, Nick Colletti, Eric Fagerberg, Sol Shenker, Caroline Bullock, Chris Wrocklage, Noah Tubo, Tianlei Xu, Matt Noyes, Rami Rahal, Sean Arlauckas, Aria Pearlman Morales, Frank Stegmeier, Louise Cadzow, Mike Schlabach, Gregory Kryukov, Micah J. Benson. Comprehensive identification of novel therapeutic targets for treatment of PD-1 resistant solid tumors via a genome-scale CRISPR/Cas9 in vivo T-cell screen [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 C101. doi:10.1158/1535-7163.TARG-19-C101
Abstract C121: Long Pentraxin-3 modulates bladder cancer progression
Authors: Matarazzo Sara , Melocchi Laura , Rezzola Sara , Grillo Elisabetta , Maccarinelli Federica , Giacomini Arianna , Taranto Sara , Zammataro Luca , Cerasuolo Marianna , Bugatti Mattia , Vermi William , Presta Marco , Ronca Roberto
Published: March 2020

Bladder tumors are a diffuse type of cancer with a relatively benign prognosis when treated at early stage/low-grade stages, but with poor outcome in the muscle invasive (MIBC) form or at recurrence. Long Pentraxin-3 (PTX3) is a component of the innate immunity with pleiotropic functions in the regulation of immune response, tissue remodeling and cancer progression. PTX3 may act as an oncosuppressor in different contexts, functioning as an antagonist of the FGF/FGFR system, rewiring the immune-microenvironment or acting through mechanisms not yet fully clarified. In this study, we report for the first time that PTX3 is differentially expressed in bladder cancer (BC) biopsies and tumor cell lines during the different stages of BC progression. Taking advantage of BC cell lines representative of different tumor grades, we demonstrate that PTX3 production by tumor cells decreases along the progression from low-grade to high grade/MIBC because of promoter methylation. In vitro and in vivo data reveal that PTX3 modulation in BC cells has a significant impact on different biological features of BC growth, including cell proliferation, motility, metabolism, stemness and drug resistance. In conclusion, PTX3 exerts an oncosuppressive effect on BC progression and may represent a potential functional biomarker in BC evolution.

Citation Format: Sara Matarazzo, Laura Melocchi, Sara Rezzola, Elisabetta Grillo, Federica Maccarinelli, Arianna Giacomini, Sara Taranto, Luca Zammataro, Marianna Cerasuolo, Mattia Bugatti, William Vermi, Marco Presta, Roberto Ronca. Long Pentraxin-3 modulates bladder cancer progression [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 C121. doi:10.1158/1535-7163.TARG-19-C121
Abstract CN05-02: Next generation strategies to overcome adaptive resistance to MAPK-directed therapies
Authors: Poulikakos Poulikos
Published: March 2020

Adaptive resistance remains a major challenge limiting the clinical effectiveness of inhibitors of oncogenic MAPK signaling (RAF and MEK inhibitors), there is thus a pressing need for compounds and combinatorial strategies that would potently and durably suppress MAPK signaling in the tumor, while retaining a broad therapeutic window. A common mechanism of adaptive resistance is mediated by feedback-induced RTKs and RAS activation by inhibitor that results in incomplete MAPK inhibition in the tumor. SHP2 (PTPN11) is a non-receptor tyrosine phosphatase and scaffold protein that mediates signal transduction downstream of multiple RTKs promoting RAS activation. Targeting SHP2 provides the opportunity to overcome adaptive resistance by co-targeting MAPK signaling and feedback-induced RTK-mediated RAS activation. Targeting SHP2 using a selective allosteric SHP2 inhibitor, in combination with inhibitors of components of MAPK signaling, prevents adaptive resistance and is broadly effective in Triple Negative Breast Cancer models and tumors with RAS mutations at G12, whereas tumors with RAS(G13D) or RAS(Q61X) mutations were resistant. This indicates that different RAS mutant proteins vary considerably in the dependence of their activity on upstream RTK/SHP2 signaling, suggesting hitherto unappreciated differences in their biochemical properties in cells. In each tumor that was sensitive to combined treatment, p(Y542)SHP2 induction was observed in response to MAPK signaling inhibition. In addition, a subset of BRAF(V600E) tumors was identified that were resistant to the combined treatment. In such tumors, FGFR signaling was found to drive feedback-induced RAS activation independently of SHP2, indicating additional combinatorial pharmacologic approaches for this subset of tumors. Thus, understanding mechanisms of adaptive resistance and response to pharmacologic perturbations of oncogenic signaling enables the rational design of more effective combinatorial strategies for the treatment of MAPK-driven cancers.

Citation Format: Poulikos Poulikakos. Next generation strategies to overcome adaptive resistance to MAPK-directed 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 CN05-02. doi:10.1158/1535-7163.TARG-19-CN05-02
Abstract LB-A03: Molecular systems-guided imaging of multicellular in situ adaptation to targeted therapy in BRAF-mutant cancer
Authors: Wang Stephanie , Weissleder Ralph , Lauffenburger Douglas , Miller Miles
Published: March 2020

Adaptive signaling through receptor tyrosine kinases (RTKs) has been highly implicated in the resistance to drugs targeting constitutive RAS/RAF/MEK/ERK signaling, particularly in malignancies with constitutive V600 BRAF mutation. However, it has been difficult to systematically understand how such RTK-mediated bypass signaling is influenced by dynamic and localized interactions between tumor cells and neighboring stromal and immune populations. Here we present a pipeline that combines single-cell and bulk transcriptomic analysis from patients, with systems-level mapping of phosphosignaling pathways in co-cultures, to guide the longitudinal in vivo confocal (intravital) microscopy of adaptive bypass signaling in mouse models of BRAF-mutant cancer. This pipeline prioritizes key myeloid populations and phosphosignaling activities (including of ERK) to image in vivo. Intravital microscopy reveals how therapy-induced myeloid accumulation simultaneously shapes the local activity of multiple co-regulated signaling pathways in neighboring cancer cells, in a RTK-dependent manner. Overall, this work offers an integrative approach to quantitating multiple single-cell signaling pathway activities in the tumor microenvironment, and directly images the dynamics with which known cancer-cell autonomous mechanisms of targeted therapy resistance can be locally amplified by neighboring immune cell populations.

Citation Format: Stephanie Wang, Ralph Weissleder, Douglas Lauffenburger, Miles Miller. Molecular systems-guided imaging of multicellular in situ adaptation to targeted therapy in BRAF-mutant 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-A03. doi:10.1158/1535-7163.TARG-19-LB-A03
Abstract A11: Combined Src and EGFR inhibition targets STAT3 to induce stromal remodeling and increase drug delivery to improve survival in a mouse model of pancreatic cancer
Authors: Dosch Austin R. , Dai Xizi , Reyzer Michelle L. , Kwon Deukwoo , Caprioli Richard , Merchant Nipun B. , Nagathihalli Nagaraj S.
Published: March 2020

Lack of durable response to cytotoxic chemotherapy is a major contributor to the dismal outcomes seen in pancreatic ductal adenocarcinoma (PDAC). Extensive tumor desmoplasia and poor vascular supply are two predominant characteristics that hinder the delivery of chemotherapeutic drugs into PDAC tumors and mediate resistance to therapy. Previously, we have shown that STAT3 is a key biomarker of therapeutic resistance to gemcitabine treatment in PDAC, which can be overcome by combined inhibition of the Src kinase and epidermal growth factor receptor (EGFR) pathways. Although it is well established that concurrent EGFR and Src inhibition exert these anti-eoplastic properties through inhibiting mitogenic pathways in tumor cells directly, the influence of this combined therapy on stromal constituents in PDAC tumors remains unknown. In the present study, we demonstrate in both orthotopic tumor xenografts and Ptf1acre/+;LSL-KrasG12D/+;Tgfbr2flox/flox (PKT) mouse models that concurrent EGFR and Src inhibition abrogates STAT3 activation, resulting in stromal remodeling through the depletion of tumor collagen content, increasing microvessel density, and preventing tissue fibrosis in vivo. Furthermore, these stromal changes resulting from combined pathway inhibition produce a direct increase in intratumoral gemcitabine levels as determined by MALDI-IMS and improve overall survival in PKT mice treated with combined EGFR and Src inhibition with gemcitabine. As a phase 1 clinical trial utilizing concurrent EGFR and Src inhibition in combination with gemcitabine has recently concluded, these data provide translational insight into the novel mechanism of action of this regimen and expand our understanding into the phenomenon of stromal-mediated therapeutic resistance.

Citation Format: Austin R. Dosch, Xizi Dai, Michelle L. Reyzer, Deukwoo Kwon, Richard Caprioli, Nipun B. Merchant, Nagaraj S. Nagathihalli. Combined Src and EGFR inhibition targets STAT3 to induce stromal remodeling and increase drug delivery to improve survival in a mouse model of 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 A11.
Abstract A34: Suppression of dystroglycan function is a hallmark of acinar-to-ductal metaplasia and favors the development of neoplasias and PDAC
Authors: Huang Ge , Lanciault Christian , Sears Rosalie , Muschler John
Published: March 2020

The basement membrane (BM) is a predominant microenvironmental factor of the precancerous exocrine pancreas, and evidence suggests that the BM functions not only as a barrier to tumor cell invasion but also as an active tumor-suppressing signaling substrate. However, the molecular underpinnings of such a mechanism have not been resolved. Here we explore the expression and function of the prominent BM receptor dystroglycan (DG) in the normal pancreas and pancreatic disease. We show that DG is highly expressed in the acinar compartment of the normal pancreas; however, there is a strong suppression of DG expression with acinar-to-ductal metaplasia (ADM) in chronic pancreatitis and in all stages of pancreatic cancer evolution, from ADM to neoplasias to adenocarcinoma. The conditional knockout of DG in the murine pancreas produced no evidence of developmental or functional deficiency. However, deletion of DG expression in the context of oncogenic Kras expression (p48-Cre;KrasG12D) led to a clear acceleration of disease, including accelerated PanIN formation and an increased incidence of adenocarcinoma and metastases. Disease acceleration was accompanied by neoplasias with a strongly cystic morphology. These data establish DG as a potent suppressor of pancreatic neoplasias and cancers, and one that is muted in chronic pancreatitis and at the earliest stages of oncogenic transformation. We conclude that DG is a key mediator of the tumor-suppressing functions of the normal BM, and that maintenance or restoration of DG function is a potential target for the delay or prevention of pancreatic cancer evolution.

Citation Format: Ge Huang, Christian Lanciault, Rosalie Sears, John Muschler. Suppression of dystroglycan function is a hallmark of acinar-to-ductal metaplasia and favors the development of neoplasias and PDAC [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 A34.
Abstract A41: Cancer-associated fibroblast (CAF) specific biomarkers in pancreatic ductal adenocarcinoma (PDAC): Transcriptomic and molecular insight
Authors: Putri Jayarani F. , Kumagai Yutaro , Miyazaki Yoshihiro , Oda T. , Kida Yasuyuki S.
Published: March 2020

Pancreatic ductal adenocarcinoma (PDAC) is one of malignancy-related death with poor prognosis. The late diagnosis due to nonspecific symptoms, highly aggressive nature, intrinsic resistance to chemotherapeutic, and the lack of effective therapeutic are the main causes of poor prognosis in PDAC. This cancer type comprises up to 90% cellular component of desmoplastic stroma that holds a major role in drug resistance and malignancy. The complexity of the genome is another leading cause of molecular alteration in PDAC. Hence, the urge to find the prognosis marker for PDAC is extremely needed for the new therapeutic regimens. In this study we performed RNA-seq analysis from MSC in single and co-cultured conditions (in vitro), MSC and Xeno-MSC (in vivo), and patient-derived CAF. Read alignment and junction mapping was accomplished using HISAT2 and cufflinks respectively and followed by map reads using UCSC hg38 reference genome annotation. Data were expressed as fragments per kilobase of exon per million fragments mapped (FPKM). Three out of eight clusters on heatmap were selected based on their expression pattern across all the samples. Approximately 900 transcripts from selected clusters were classified into three major protein classes: transcription factors, kinases, and membrane proteins. Remarkably, several genes on selected clusters have been studied in CAF, giving proof of the fidelity of our data analysis. Prompted to get more specific CAF-related genes, we put more parameters such us: unexpressed in normal pancreatic, relatively low expression in pancreatic cancer, ≥2 folds increase of CAF mRNA expression to normal or pancreatic cancer, and have not studied in PDAC. We found 16 transmembrane protein-coding genes that are potentially involved in CAF remodeling. To gain a better understanding of tumor-CAF interaction from these candidate genes, immunostaining using paraffin sections from tumor-bearing mice model together with αSMA/IL6 and protein analysis was carried out. We sought the downstream pathways as well to discover novel regulatory networks and causal relationship between CAF and pancreatic cancer. Our study addresses the extent to which factor(s) can reset stromal alteration and elucidate the role of transmembrane protein during CAF reprogramming.

Citation Format: Jayarani F. Putri, Yutaro Kumagai, Yoshihiro Miyazaki, T. Oda, Yasuyuki S. Kida. Cancer-associated fibroblast (CAF) specific biomarkers in pancreatic ductal adenocarcinoma (PDAC): Transcriptomic and molecular insight [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 A41.
Abstract A53: Meflin-positive cancer-associated fibroblasts inhibit pancreatic carcinogenesis
Authors: Mizutani Yasuyuki , Kobayashi Hiroki , Enomoto Atsushi , Takahashi Masahide
Published: March 2020

Cancer-associated fibroblasts (CAFs) constitute a major component of the tumor microenvironment. Recent observations in genetically engineered mouse models and clinical studies have suggested that there may exist at least two functionally different populations of CAFs, i.e., cancer-promoting CAFs (pCAFs) and cancer-restraining CAFs (rCAFs). Although various pCAF markers have been identified, the identity of rCAFs remains unknown because of the lack of rCAF-specific marker(s). In the present study, we found that Meflin, a glycosylphosphatidylinositol-anchored protein that is a marker of mesenchymal stromal/stem cells and maintains their undifferentiated state, is expressed by pancreatic stellate cells that are a source of CAFs in pancreatic ductal adenocarcinoma (PDAC). Analysis of human PDAC tissues revealed that the infiltration of Meflin-positive CAFs correlated with favorable patient outcome. Consistent herewith, Meflin deficiency led to significant tumor progression with poorly differentiated histology in a PDAC mouse model. Similarly, genetic ablation of Meflin-positive CAFs resulted in poor differentiation of tumors in a syngeneic transplantation model. Conversely, delivery of a Meflin-expressing lentivirus into the tumor stroma or overexpression of Meflin in CAFs suppressed the growth of xenograft tumors. Lineage tracing revealed that Meflin-positive cells gave rise to α-smooth muscle actin-positive CAFs that are positive or negative for Meflin, suggesting a mechanism for generating CAF heterogeneity. Mechanistically, Meflin interacts with lysyl oxidase to inhibit its collagen crosslinking activity, and Meflin deficiency resulted in straightened and wide stromal collagen fibers that represent a signature for aggressive tumors. Together, the data suggest that Meflin is a marker of rCAFs that suppress PDAC progression.

Citation Format: Yasuyuki Mizutani, Hiroki Kobayashi, Atsushi Enomoto, Masahide Takahashi. Meflin-positive cancer-associated fibroblasts inhibit pancreatic carcinogenesis [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 A53.
Abstract B23: Bioadhesive nanoparticles as a delivery vehicle in pancreatic cancer epithelial cells and pancreatic fibroblasts
Authors: Hasan Nesrin M. , Suh Hee Won , Saltzman Mark , Ahuja Nita
Published: March 2020

Pancreatic cancer, referred to as pancreatic ductal adenocarcinoma (PDAC), is characterized by high lethality and limited treatment options. PDAC is generally diagnosed at a later stage and is resistant to chemotherapy and radiation therapy. Therefore, there is an urgent unmet need for novel treatment options to improve the survival of patients. PDAC is characterized by dense solid tumors that hinder drug penetration, and novel treatment technologies should be utilized to improve drug access to cancer cells. Nanoparticle-based compounds have been shown to improve site specific targeting, intracellular penetration, and circulation time and therefore can be used for therapeutic delivery to solid tumors. In our study, we performed in vitro characterization studies of non-bioadhesive nanoparticles (NNPs) and bioadhesive nanoparticles (BNPs) in pancreatic cancer epithelial cells and pancreatic fibroblasts. NNPs are polylactic acid-hyperbranched polyglycerol (PLA-HPG) nanoparticles and BNPs are obtained through conversion of HPG to an aldehyde-rich corona, which increases the bioadhesive property through formation of linkages with proteins. The aim of our study was to characterize the internalization of nanoparticles in vitro in different pancreatic cell lines. We incubated pancreatic cancer epithelial cells (PANC-1 and MIA PaCa-2) and pancreatic fibroblasts with fluorescent DiO-loaded NNPs and BNPs to assess internalization. Both DiO-NNPs and DiO-BNPs were internalized by PANC-1, MIA PaCa-2, and pancreatic fibroblasts as determined by both flow cytometry and fluorescence microscopy. The nanoparticles were internalized in a dose- and treatment time-dependent manner. Although both nanoparticles were internalized, due to their increased bioadhesive properties BNPs had 1.7-fold higher internalization compared to NNPs. Viability of the cells following DiO-NNPs and DiO-BNPs treatment was assessed through both acridine orange staining and flow cytometry analysis. DiO-NNPs and DiO-BNPs treatments did not affect the viability of the cells. Similar results were obtained with DiI-loaded nanoparticles. In summary, our study shows that nanoparticles can be internalized by pancreatic cancer epithelial cells and pancreatic fibroblasts and do not cause any loss of viability. Our results demonstrate the potential applicability of nanoparticles, especially bioadhesive nanoparticles, for synergistic therapeutic targeting of different cell types in pancreatic cancer to improve treatment outcomes.

Citation Format: Nesrin M. Hasan, Hee Won Suh, Mark Saltzman, Nita Ahuja. Bioadhesive nanoparticles as a delivery vehicle in pancreatic cancer epithelial cells and pancreatic fibroblasts [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 B23.
Abstract B44: Clinical utility of semiquantitative evaluation of progesterone receptor immunohistochemistry in neuroendocrine tumors of the pancreas
Authors: Purushothaman Sonya , Michele Simona De , Low Dorothy , Remotti Helen E.
Published: March 2020

Purpose: Progesterone receptor (PR) is expressed strongly in a subset of neuroendocrine cells in the pancreas. Tamoxifen has long been used in adjuvant endocrine therapy of steroid hormone receptor–positive breast cancer, and currently clinical trials are assessing its use in pancreatic neuroendocrine tumors (NET) that show variable PR expression. Prior studies have reported receptor status as “positive” or “negative,” using 1% or 10% positive nuclei as a cutoff. In this study, we assessed PR immunohistochemical expression in a cohort of NET and other pancreatic nonexocrine carcinomas. Theoretically, tumors with highest PR expression are more likely to respond to adjuvant tamoxifen.

Experimental Design: Immunohistochemical staining for PR was performed on tissue microarrays of 54 well-differentiated neuroendocrine tumors of the pancreas and 8 poorly differentiated carcinomas of the pancreas, including 4 acinar cell carcinomas, 2 neuroendocrine carcinomas, and 2 small-cell neuroendocrine carcinomas. A minimum of 4 separate 2-mm tumor cores were sampled on each case.

Results: PR expression in non-neoplastic pancreatic islets was used for positive control. PR staining was evaluated using a semiquantitative H-score method. Positive PR staining (defined as greater than 1%) was identified in 61% (33 of 54 cases) of well-differentiated NET (mean H-score 100) and 0% (0 of 8 cases) of poorly differentiated carcinomas of the pancreas. Of the 33 PR positive cases of well-differentiated NET, 3 cases had less than 10% nuclear staining (mean H-score 7), 8 cases had 10% to less than 50% nuclear staining (mean H-score 44), and 22 cases had 50% or greater nuclear staining (mean H-score 229).

Conclusions: Based on these findings, we suggest analysis of NET tumor PR expression should include a quantitative or semiquantitative score rather than dichotomized immunohistochemical evaluation of hormone receptors, particularly in the evaluation of patients’ response to clinical trials with tamoxifen. High-grade endocrine and acinar carcinomas showed no PR expression and are less likely to derive benefit from adjuvant endocrine therapy.

Citation Format: Sonya Purushothaman, Simona De Michele, Dorothy Low, Helen E. Remotti. Clinical utility of semiquantitative evaluation of progesterone receptor immunohistochemistry in neuroendocrine tumors of the pancreas [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 B44.
Abstract C28: KMT2D mediates TGF-β-induced epithelial-to-mesenchymal transition to promote more aggressive pancreatic cancer
Authors: Kim Hong S. , Lu Shuang , Cao Yubo , Yi Zhujun , Jeyarajan Sivakumar , Zhao Lili , Bedi Karan , Narayanan Ishwarya V. , Paulsen Michelle T. , Ljungman Mats , Shi Jiaqi
Published: March 2020

Pancreatic ductal adenocarcinoma (PDAC) is predicted to be the second leading cause of cancer-related death in the United States by 2030. Surgery is the only potential curative treatment for patients with PDAC. However, less than 20% of patients are operable due to early metastasis. The mechanism of metastasis in PDAC is not fully understood. Recent studies failed to find metastasis-specific genetic mutations in PDAC, suggesting that aberrant epigenetic regulation may be one of the crucial mechanisms. Lysine-specific histone methyltransferase 2D (KMT2D) is one of the most frequently mutated epigenetic genes in human cancers, including PDAC. TCGA database analysis showed that KMT2D mutation is associated with poor survival in PDAC patients. However, the role of KMT2D in PDAC development remains elusive. Using human pancreatic tissue microarray containing both primary and metastatic PDAC tissues, we have found that the expression of KMT2D is specifically and significantly suppressed in metastatic PDAC, suggesting an important role of KMT2D in metastasis. Furthermore, treating PDAC cells with TGF-β resulted in decrease in KMT2D expression. In order to investigate the role of KMT2D in PDAC, we knocked out KMT2D (KMT2D KO) expression using CRISPR/Cas9 system in PDAC cell lines. We performed a novel Bru-seq analysis to investigate the effect of KMT2D on global nascent RNA transcript profile. Interestingly, epithelial-to-mesenchymal transition (EMT) pathway was one of the pathways that were highly upregulated upon KMT2D loss. RNA and protein assays confirmed that KMT2D KO cells had decreased epithelial marker, E-cadherin, and increased mesenchymal markers, vimentin and Snail. KMT2D KO cells also displayed enhanced ability to migrate and invade. Orthotopic xenograft mouse model using KMT2D KO cells showed less differentiated and more mesenchymal tumor cell morphology and larger primary and metastatic tumors compared to control cells. Taken together, loss of KMT2D in PDAC cells mediates TGF-β-induced EMT and enhances tumor growth and metastatic ability. These findings link epigenetic regulation to TGF-β signaling in PDAC and lay the foundation for designing novel therapeutic strategies.

Citation Format: Hong S. Kim, Shuang Lu, Yubo Cao, Zhujun Yi, Sivakumar Jeyarajan, Lili Zhao, Karan Bedi, Ishwarya V. Narayanan, Michelle T. Paulsen, Mats Ljungman, Jiaqi Shi. KMT2D mediates TGF-β-induced epithelial-to-mesenchymal transition to promote more aggressive 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 C28.