bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 697: Comparison of tumor-infiltrating lymphocytes between primary and metastatic tumors in breast cancer
Authors: Lee Miseon , Lee Hee Jin , Song In Hye , Park In Ah , Heo Sun-Hee , Kim Young-Ae , Bang Won Seon , Lee AReum , Lee Hyeonjin , Lee Heejae , Gong Gyungyub
Published: August 2018

Background: The level of tumor-infiltrating lymphocytes (TILs) and the presence of tertiary lymphoid structures (TLSs) are significant prognostic and predictive factors in primary breast cancer. However, understanding about the differences of TILs and TLSs among different metastatic sites or between primary breast tumors and metastatic sites is limited.

Methods: A total of 337 cases of metastatic breast cancer with available hematoxylin and eosin slides of metastatic sites (biopsy, n=185; operation, n=152) were included. We analyzed the percentage of TILs (defined as the percentage of invasive carcinoma stroma infiltrated by lymphocytes in 10% increments; if less than 10% of stroma was infiltrated by TILs, 1% or 5% criteria were used; all available full sections were evaluated) and the presence of TLSs (defined as the lymphoid aggregation with high endothelial venules) in primary and metastatic sites.

Results: The mean percentage of TILs in the lung (19.5%) was significantly higher than those in the liver (4.2%), brain (8.3%), and ovary (3.4%, p<0.001). The level of TILs in metastatic tumors of the lung was higher than those in primary tumors (Wilcoxon signed-rank test, p<0.001), while metastatic tumors of the liver (p=0.024) and brain (p=0.095) showed lower level of TILs than in primary tumors. TLSs were found only in the lung (27%) and liver (13%). Patients with lung metastasis (p<0.001), higher level of TILs (p=0.001), and presence of TLS (p=0.002) showed better overall survival. In patients with lung metastasis, who showed best clinical outcome, those with 10% or more of TIL had better overall survival than those with less than 10% (p=0.008).

Conclusion:. Metastatic breast tumors in the lung had more TILs than other sites and matched primary tumors. Underlying mechanism of differences of TILs among metastatic sites should be investigated.

Citation Format: Miseon Lee, Hee Jin Lee, In Hye Song, In Ah Park, Sun-Hee Heo, Young-Ae Kim, Won Seon Bang, AReum Lee, Hyeonjin Lee, Heejae Lee, Gyungyub Gong. Comparison of tumor-infiltrating lymphocytes between primary and metastatic tumors in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 697.
Abstract 742: Nonclinical safety evaluation of STRO-001, a site-specific anti-CD74 antibody-drug conjugate for the potential treatment of B-cell malignancies
Authors: Solis Willy , Almeida Venita De , Yu Abigail , Bruhns Maureen , Zawada James , Galan Adam , Matheny Shannon , Molina Arturo , Hallam Trevor , Lupher Mark
Published: August 2018

Antibody-drug conjugates (ADCs) constitute an expanding class of therapeutic molecules in preclinical and clinical development for multiple oncology indications. These antibody-based therapeutics are designed to selectively deliver cytotoxic payloads to tumor targets to improve efficacy and minimize toxicity. Cluster of differentiation 74 (CD74), a membrane-bound glycoprotein, is an antigen amenable ADC targeting because of its low expression in normal tissues and over expression in B-cell malignancies (multiple myeloma and non-Hodgkin's Lymphoma). STRO-001, a well-defined, homogenous, and site-specific ADC, is comprised of maytansinoid cytotoxic drugs conjugated to an anti-CD74 aglycosylated antibody (SP7219) through a noncleavable linker at two engineered non-natural amino acids. In an in vitro immunological activation assay, STRO-001 did not elicit cytokine production when incubated with human PBMCs for 48 hours, suggesting a low risk for immunological response in cancer patients. In a tissue cross-reactivity study with human tissues, STRO-001-specific membranous staining was detected primarily in lymphoid organs and lymphocytic infiltrates in multiple non-lymphoid tissues. In an exploratory nonclinical safety study in cynomolgus monkeys, a pharmacologically relevant model for toxicity testing, STRO-001 was tolerated at 1, 3, and 10 mg/kg, but not at 30 mg/kg, when intravenously administered on days 1 and 15 followed by a 4-week observation period. Dose-dependent and transient decreases in B-cells and monocytes were observed at tolerated doses (on-target pharmacology), with no effects on NK/T-cells. At tolerated doses, the main findings included reversible toxicities to hematopoietic and lymphoid tissues that correlated with cytopenias. Hematopoietic toxicity is considered antigen-independent, clinically manageable, and consistent with that of other ADCs with similar payloads. Toxicokinetic analysis confirmed drug exposures (total antibody, ADC, and a prominent linker-maytansine catabolite) with evidence of non-linearity suggesting target-dependent clearance. In summary, STRO-001 ADC has a favorable nonclinical safety profile and Phase I study of STRO-001 in patients with B-cell malignancies is planned.

Citation Format: Willy Solis, Venita De Almeida, Abigail Yu, Maureen Bruhns, James Zawada, Adam Galan, Shannon Matheny, Arturo Molina, Trevor Hallam, Mark Lupher. Nonclinical safety evaluation of STRO-001, a site-specific anti-CD74 antibody-drug conjugate for the potential treatment of B-cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 742.
Abstract 764: GSTP1 knockdown and inhibition impairs pancreatic ductal adenocarcinoma (PDAC) growth
Authors: Singh Rahul Raj , Reindl Katie M.
Published: August 2018

Pancreatic ductal adenocarcinoma (PDAC) is the third-leading cause of cancer-related mortalities in the Western world and continues to be a major unresolvable health problem at the start of the 21st century. Resistance to the currently available treatment options has led to development of new approaches, such as personalized medicine and immunotherapy. However, new therapeutic strategies based on the unique molecular biology and physiology of pancreatic cancer hold the greatest promise. Glutathione S-transferase pi 1 (GSTP1) is a key detoxification enzyme which metabolizes xenobiotic compounds and byproducts of cellular metabolism. GSTP1 is overexpressed in many tumors, particularly ovarian, non-small cell lung, breast, colon, and pancreas. Moreover, GSTP1 is overexpressed in drug-resistant cancer cell lines. The reasons for increased expression ratios compared to normal tissues or wild-type cell lines are not well understood.

To investigate the role of GSTP1 in PDAC pathogenicity, we generated two knockdown lines of GSTP1 in metabolically diverse PDAC cells. We showed that GSTP1 knockdown impairs the growth and proliferation of PDAC cells. Additionally, GSTP1 knockdown cells exhibit elevated reactive oxygen species (ROS) levels and a prolonged G0/G1 phase of the cell cycle. Intrigued by these results, we next examined whether pharmacological inhibition with a selective GSTP1 inhibitor, Ezatiostat (TLK199), also impaired PDAC pathogenicity. Ezatiostat is a small molecule drug and is novel glutathione analog, which selectively binds and inhibits GSTP1. Ezatiostat treatment in PDAC cells recapitulated the proliferation impairments observed with genetic inactivation of GSTP1 and elevated ROS levels. Orthotopic implantation of GSTP1 knockdown cells in athymic nude mice resulted in reduced tumor weight and volume compared to the control. The growth trajectory of the tumors was monitored via Vevo-3100 ultrasound imaging system. Our preliminary data indicate enhanced sensitivity of glycolytic cancer cells towards an ROS-inducing agent and a GSTP1 inhibitor, piperlongumine (PL). Interestingly, we have found PL is less cytotoxic to cells with reduced GSTP1 levels, indicating that PL primarily works by inhibiting GSTP1 activity. Together, these data suggest that GSTP1 knockdown and inhibition impairs the growth and survival of phenotypically diverse PDAC cells in vitro and in vivo. Moreover, GSTP1 knockdown results in elevated ROS levels and an extended G0/G1 phase of the cell cycle. With these data, we propose that GSTP1 is a novel therapeutic target for PDAC.

Citation Format: Rahul Raj Singh, Katie M. Reindl. GSTP1 knockdown and inhibition impairs pancreatic ductal adenocarcinoma (PDAC) growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 764.
Abstract 767: Identification of cell surface proteins expressed exclusively on mutant KRas-dependent cancer cells
Authors: Agrawal Vijayendra , Fang Bin , Welsh Eric A. , Koomen John , Sebti Said M.
Published: August 2018

Mutant KRas is a major contributor to human oncogenesis and confers resistance to therapy. Identification of cell surface proteins that are expressed exclusively in mutant KRas-driven tumors could lead to novel approaches for diagnosing and treating mutant KRas-dependent tumors. To this end, we used a biotinylation/mass spectrometry approach to capture cell surface proteins from mutant KRas-dependent and -independent human cancer cells. KRas was depleted from both mutant KRas-dependent and -independent cells, and the cells were subjected to cell surface protein biotinylation using EZ-Link Sulfo-NHS-SS-Biotin reagent and captured via neutravidin column. The efficiency of cell surface protein capture was confirmed by the presence of membrane proteins EGFR, IGF1R and absence of cytosolic proteins RhoGDIa in biotin-pull down fractions (western blot analysis). Mass spectrometric and proteomics analyses of biotinylated proteins yielded 20 highly probable hits with very high expression in mutant KRas-dependent cells and very low expression in mutant KRas-independent cells. Upon KRas depletion, the level of these proteins was significantly reduced in mutant KRas-dependent cells (up to 25 fold) but not in mutant KRas-independent cells. Currently, we are analyzing these 20 hits to determine if any of these are required for the survival of mutant KRas-dependent cells. Results from these studies have the potential to lead to novel approaches for diagnosing and treating mutant KRas-driven cancers.

Citation Format: Vijayendra Agrawal, Bin Fang, Eric A. Welsh, John Koomen, Said M. Sebti. Identification of cell surface proteins expressed exclusively on mutant KRas-dependent cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 767.
Abstract 792: A novel covalent reversible BTK inhibitor for B cell malignancies with enhanced selectivity and in vivo efficacy
Authors: Chen Xi , Huang Shaoqiang , Hu Fred , Xian Jun , Yang Jian , Qiang Jing , Le Meijie , Wu Yuchuan
Published: August 2018

Bruton's tyrosine kinase (BTK), a member of Tec family kinase, plays a pivotal role in the regulation of BCR signaling, which is important for B cell development and function. Ibrutinib, which has been approved by FDA, is an effective treatment option for multiple types of B cell neoplasms. However, the treatment associated adverse events (AEs) are one of the major concerns due to its poor kinase selectivity . Here, we present a novel covalent, but reversible BTK inhibitor, XNW-1011, which demonstrated enhanced potency, better selectivity, improved drug-like properties and superior in vivo efficacy with a favorable safety profile. XNW-1011 was developed structurally and biochemically differently from current BTK inhibitors, being more potent and highly selective. In the in vitro assay, unlike Ibrutinib, it did not inhibit EGFR, ITK, JAK3, LYN and SRC. It completely suppressed the cell proliferation of a panel of lymphoma cell lines such as TMD-8 and Rec-1. Besides, XNW-1011 inhibited cellular phosphorylation of BTK and calcium efflux, confirming the inhibition of activation of BTK in vitro. We have shown that XNW-1011 formed covalent bond with cysteine in a cysteine reactivity assay. In addition, a washout assay by Western Blot confirmed the reversibility of inhibition of XNW-1011 to BTK. XNW-1011 has a good prolife of ADME, which translated into improved in vivo PK properties over Ibrutinib (low clearance and prolonged half-life). XNW-1011 was further evaluated in several animal models such as REC-1 and TMD-8 tumor xenografts. XNW-1011 inhibited tumor growth with as low as 3.125 mpk by BID and resulted in tumor regression with 12.5 mpk by BID. Interestingly, XNW-1011 completely inhibited the tumor growth of a patient derived xenograft (PDX) with Myd88 mutation. The dose proportionality was confirmed through determination of the plasma concentration and in vivo BTK occupancy in the last day of treatment. In summary, XNW-1011 is a potent, selective, covalent reversible BTK inhibitor with improved ADME prolife and in vivo efficacy, which makes it suitable for further evaluation in clinical setting.

Citation Format: Xi Chen, Shaoqiang Huang, Fred Hu, Jun Xian, Jian Yang, Jing Qiang, Meijie Le, Yuchuan Wu. A novel covalent reversible BTK inhibitor for B cell malignancies with enhanced selectivity and in vivo efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 792.
Abstract 812: New c-KIT mutations in GISTs: Bad for good
Authors: Laurini Erik , Aulic Suzana , Marson Domenico , Fermeglia Maurizio , Riboni Roberta , Lucioni Marco , Dellera Elena , Alessiani Mario , Perfetti Vittorio , Pricl Sabrina
Published: August 2018

Gastrointestinal stromal tumors are mesenchymal tumors arising in the stomach and small bowel and more rarely in the rectum, esophagus, peritoneum and retroperitoneum. These tumors are characterized by KIT or PDGFRA mutations. KIT mutations are all in frame and lead to constitutive tyrosine kinase domain activation without ligand binding. Mutations concern four exons (9, 11, 13 and 17) but mainly exon 11.

In this work we report a case of a GIST patient with wild-type PDGFRA but bearing two mutations in KIT exon 9, i.e., the insertion mutation Y503_F504insAY and the missense mutation K509N.

The patient was treated with Imatinib at standard regimen dose (400 mg/day), and responded well.

Accordingly, in silico experiments were performed to disclose the effects exerted by these new mutations on the protein structure, its thermodynamic stability, and the ability to promote/interfere with Imatinib binding.

Direct drug binding thermodynamics and kinetics of the mutant KIT were measured using isothermal titration calorimetry. FInally, in vitro kinase assays were performed to monitor the phosphorylation status of this new mutant KIT.

The prototypical KIT mutations Δ559 and T670I were also analyzed in parallel for comparison.

To our knowledge, only AY 502-503 duplication/insertion and K509I substitution have been previously reported.

Citation Format: Erik Laurini, Suzana Aulic, Domenico Marson, Maurizio Fermeglia, Roberta Riboni, Marco Lucioni, Elena Dellera, Mario Alessiani, Vittorio Perfetti, Sabrina Pricl. New c-KIT mutations in GISTs: Bad for good [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 812.
Abstract 816: Antibody-drug conjugate targeting glypican-1 shows potent antitumor effect in pancreatic cancer
Authors: Serada Satoshi , Nishigaki Takahiko , Sugase Takahito , Saito Yurina , Takahashi Tsuyoshi , Hiramatsu Kosuke , Fujimoto Minoru , Mori Masaki , Doki Yuichiro , Naka Tetsuji
Published: August 2018

Pancreatic cancer is the most lethal malignancy; thus developing new treatment options is urgently required. We confirmed that enhanced expression of glypican-1 (GPC1) was observed in pancreatic cancer. In this study, we aimed to develop novel antibody-drug conjugate (ADC) targeting GPC1 as a new therapy for pancreatic cancer. By the immunohistochemical analysis, enhanced expression of GPC1 was observed in pancreatic cancer. Among pancreatic cancer cell lines, BxPC3 and T3M4 cells expressed high expression of GPC1 compared to SUIT2 cells. We developed new anti-GPC1 monoclonal antibody (mAb) and selected a clone having highly internalizing activity. The anti-GPC1 mAb was conjugated with cytotoxic agent monomethyl auristatin F (MMAF). Compared with the control ADC, this GPC1-ADC showed potent antitumor effect toward BxPC3 and T3M4, while little activity in SUIT2 cells. In BxPC3 xenograft model, GPC1-ADC had significant and potent tumor growth inhibition in a dose dependent manner. In addition, using patient derived tumor xenograft (PDX) model with GPC1-positive pancreatic cancer, GPC1-ADC also showed significant tumor growth inhibition. In the tumor tissue, GPC1-ADC-mediated G2/M phase cell cycle arrest was detected in GPC1-ADC treated mice compared to control-ADC treated mice. In summary, our newly developed GPC1-ADC showed significant tumor growth inhibition against not only GPC1-positive pancreatic cell line but also patient derived GPC1-positive pancreatic cancer. Our preclinical data demonstrated that targeting GPC1 by ADC is a promising therapy for patients with GPC1-positive pancreatic cancer.

Citation Format: Satoshi Serada, Takahiko Nishigaki, Takahito Sugase, Yurina Saito, Tsuyoshi Takahashi, Kosuke Hiramatsu, Minoru Fujimoto, Masaki Mori, Yuichiro Doki, Tetsuji Naka. Antibody-drug conjugate targeting glypican-1 shows potent antitumor effect in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 816.
Abstract 817: Overcoming FGFRs-mediated drug resistance by inhibiting myristoylation of FRS2α
Authors: Cai Houjian , Li Qianjin
Published: August 2018

Fibroblast growth factor (FGF)/fibroblast growth factor receptor (FGFR) signaling facilitates tumor initiation and progression. Although there are currently approved inhibitors and agents in clinical trials targeting FGFR kinase activity that show therapeutic benefit, over-expression and/or mutations of FGFRs eventually confer drug-resistance and bypass the mechanisms of kinase inhibitors in a variety of cancer types. We hypothesize that a compound blocks the myristoylation of FRS2α, thus suppressing its interaction with FGFRs or FGFRs drug resistant mutants (FGFRsDRM) and downstream proteins. As a result, it inhibits oncogenic signaling and tumor progression mediated by FGF/FGFRs or FGFRsDRM. The goal of this study is to investigate if the novel lead compound inhibits FGF/FGFRs oncogenic signaling and overcomes FGFRsDRM mediated drug resistance. We used CRISPR/Cas9 mediated gene knockout to demonstrate that FRS2α is essential for FGF/FGFR signaling. The myristoylation of FRS2α was detected by Click chemistry. Additionally, inhibition of cell proliferation, migration, invasion assays and cell cycle analysis were analyzed in numerous cancer cells. Prostate regeneration assay and xenograft tumor models were applied for in vivo studies. We demonstrate that loss of myristoylation of FRS2α, a scaffold protein key in FGFRs signaling, inhibits FGF/FGFRs-mediated oncogenic signaling and FGF10-induced tumorigenesis. Additionally, a previously synthesized myristoyl-CoA analog, B13, which targets the activity of N-myristoyltransferases, suppresses FRS2α myristoylation, and modulates the phosphorylation and localization of FRS2α at the cytoplasmic membrane. B13 exhibited an inhibitory effect on oncogenic signaling induced by wild type FGFRs and their drug resistant mutants (FGFRsDRM). The single agent or in collaboration with an FGFR inhibitor suppressed FGF induced wild type FGFR or FGFRDRM initiated PI3K and/or MAPK signaling, subsequently inhibiting cell proliferation and migration in a variety of cancer cell types by inducing cell cycle arrest. Finally, B13 significantly inhibited growth of xenograft tumors without pathological toxicity to the liver, kidney, or lung in vivo. Our study demonstrates that B13, a myristoyl-CoA analog, inhibits FGF/FGFR-mediated oncogenic signaling and tumor progression. This study provides a novel therapeutic approach for inhibiting cancer progression mediated by FGF/FGFRs and their drug resistant mutants.

Citation Format: Houjian Cai, Qianjin Li. Overcoming FGFRs-mediated drug resistance by inhibiting myristoylation of FRS2α [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 817.
Abstract 823: Anti-melanoma effects of novel Cripto-1 CFC small peptide mimetics
Authors: Iaccarino Emanuela , Untiveros Gustavo , Sandomenico Annamaria , Caporale Andrea , Calvanese Luisa , Falcigno Lucia , D'Auria Gabriella , Ruvo Menotti , Strizzi Luigi
Published: August 2018

The diagnosis of melanoma is increasing and current therapies for advanced disease remain unsuccessful. Thus, research aimed at developing novel targeting approaches is needed. Cripto-1 (CR-1), an epidermal growth factor (EGF)-like developmental morphogen, functions as a co-receptor for the tumor growth factor (TGF)-beta related molecule, Nodal. CR-1/Nodal signaling pathways can induce cell survival, proliferation and migration of cancer cells. Studies have shown that Nodal is expressed at high levels in advanced melanoma. CR-1 has also been shown to be expressed in melanoma but limited to a small subpopulation of melanoma cells with more stem cell-like characteristics. Recent work has demonstrated that targeting Nodal in melanoma is associated with reduction of a significant population of melanoma cells. However, a residual population continues to persist. We hypothesize that interference with CR-1/Nodal receptor binding dynamics will negatively affect downstream Nodal-dependent signaling events important for cancer growth and further reduce the number of surviving melanoma cells. To test this hypothesis, we have developed small peptides that mimic CR-1 CFC binding domain and prevent CR-1 from binding to the ALK4 receptor necessary for proper CR-1 co-receptor function for Nodal. We show that in vitro treatment of human melanoma cells with our prototype CFC mimetic B3 reduced melanoma cell viability and negatively affected melanoma cell cycle progression. We also show that treatment with B3 resulted in reduction of active Smad2/3 and ERK1/2, known to be involved in downstream Nodal signaling, as a result of B3 interference with CR-1 co-receptor function. These preliminary data provide the scientific rationale for the development of anti-CR-1 and Nodal therapeutics that can synergize with or complement current treatment approaches in melanoma.

Citation Format: Emanuela Iaccarino, Gustavo Untiveros, Annamaria Sandomenico, Andrea Caporale, Luisa Calvanese, Lucia Falcigno, Gabriella D'Auria, Menotti Ruvo, Luigi Strizzi. Anti-melanoma effects of novel Cripto-1 CFC small peptide mimetics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 823.
Abstract 824: Targeting epidermal growth factor receptors and urokinase-type plasminogen activator receptors for sarcoma therapy
Authors: Graef Ashley J. , Kim Jong-Hyuk , Vallera Daniel A. , Modiano Jaime F.
Published: August 2018

eBAT is a bispecific angiotoxin consisting of truncated, deimmunized Pseudomonas exotoxin fused to epidermal growth factor (EGF) and the amino terminal fragment of urokinase. This drug was designed to use epidermal growth factor receptors (EGFR) and urokinase-type plasminogen receptors (uPAR) as baits to deliver the lethal toxin into the cells, rather than as a means to interrupt signaling by the receptors. We previously showed that eBAT is remarkably safe, and that it reduced tumor burden in mice with established human rhabdomyosarcoma xenografts. It also was safe and showed a strong efficacy signal when used in the adjuvant setting in a clinical trial for dogs with splenic hemangiosarcoma. Nevertheless, the therapeutic mechanism of action of eBAT remains to be elucidated. Our goal is to identify patterns of uPAR expression in sarcomas that promote sarcoma growth and that mediate sensitivity for therapeutic activity by eBAT. We generated uPAR-knockout (KO) cells from MC1A-C1 mouse fibrosarcoma cell line using the CRISPR-Cas system to target exon 2 of the uPAR gene. The resulting cell lines D10 and H6 are clonal uPAR knockouts, whereas K02 and K06 represent mixtures where approximately 80% and 30% of the cells retain uPAR expression. uPAR expression in K02 and K06 has remained stable over numerous passages, indicating that loss of uPAR does not create selective pressure to enhance or inhibit cell growth in vitro. Furthermore, the growth kinetics of D10 and H6 are comparable to parental MC1A-C1 cells. eBAT treatment significantly inhibited proliferation of parental cells, but not of uPAR KO cells. In contrast, all of the cells were resistant to treatment with EGF- and uPA-monospecific toxins. The engraftment efficiency and tumor growth of uPAR KO cells in syngeneic hosts was substantially diminished when compared to K02 and K06 uPAR+ cells, suggesting that, even while uPAR-KO cells might be more resistant to eBAT therapy, uPAR expression appears to be necessary to create the tumor niche and/or to promote tumor growth in vivo. In our ongoing work, we are investigating how uPAR expression in stromal cells and in cells of the innate immune system influence sarcoma growth and the response to eBAT.

Citation Format: Ashley J. Graef, Jong-Hyuk Kim, Daniel A. Vallera, Jaime F. Modiano. Targeting epidermal growth factor receptors and urokinase-type plasminogen activator receptors for sarcoma therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 824.
Abstract 829: SAS1B protein: Determining whether ASTL or SAS1B has a role in tumor progression
Authors: Khokhar Zunair S. , Pires Eusebio S. , Kim Alexander J. , D'Souza Ryan
Published: August 2018

The matrix metalloproteinase Sperm Acrosomal SLLP1 Binding (SAS1B) is an ideal molecular and cellular candidate for both female contraception and for cancer therapies. SAS1B translation is restricted among adult tissues to the ovary and oocytes, and SAS1B first appears in oocytes of follicles at the primary-secondary transition and also in tumors of varied types and stages. Research conducted using SAS1B antibodies suggested that the protein is localized at the cell membrane of cancer cells and can get internalized in a classical endocytosis assay and can be killed using an immunotoxin linked onto antibodies. The current study aims at deciphering whether the ASTL gene or the SAS1B protein is important for tumor progression, invasion and metastasis through cell biology and molecular biology tools and ensuring tumor cell-killing properties via an ADC-mediated killing using biochemistry and molecular biology tools. PCR, immunohistochemistry, Immunofluorescence, and Western blots revealed that a noncancerous breast cell line, MCF10A5E, is SAS1B null and can be used to study cancer-like properties once stably transfected with the SAS1B construct. Previous work in the lab indicated that an SAS1B-ADC strategy is a viable immunotherapy that could benefit patients who have failed to respond to chemotherapy, if indeed SAS1B plays a role in cancer development and progression. The outcomes of an SAS1B-ADC in a breast cancer model will be presented.

Citation Format: Zunair S. Khokhar, Eusebio S. Pires, Alexander J. Kim, Ryan D'Souza. SAS1B protein: Determining whether ASTL or SAS1B has a role in tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 829.
Abstract 852: Specificity of PSMA-617 radiotherapy for prostate cancer
Authors: Reddy Joseph A. , Nelson Melissa , Xu LeCun , Westrick Elaine , Vetzel Marilynn , Wheeler Leroy , Felten Albert , Santhapuram Hari , Vlahov Iontcho , Leamon Christopher P.
Published: August 2018

Prostate-specific membrane antigen (PSMA) is a biomarker that is overexpressed on prostate cancer as well as on the neovasculature of many non-prostate solid tumors. Lately, several small molecular PSMA binding ligands chelated to various radionuclides have been studied for imaging and treatment of prostate cancer lesions. One such clinically tested agent is PSMA-617, a DOTA derivative of a PSMA-specific targeting ligand. In our hands, radiochemical yields of PSMA-617 labeled with either 177Lu (β emitter) or 225Ac (α emitter) were greater than 97%. Binding experiments with 177Lu and 225Ac chelates of PSMA-617 and D-Glu PSMA-617 (a predicted non-PSMA binding, D-glutamic acid isomer of PSMA-617) were performed using the PSMA-positive, 22RV1 cell line. PSMA-617, was found to bind in a concentration dependent manner with very high affinity, whereas the cellular association of D-Glu PSMA-617 was found to be > 60-fold lower. The in vivo biodistribution of these two agents were investigated in BALB/c nu/nu mice bearing subcutaneous 22RV1 xenografts. Organ distribution revealed specific PSMA-617 uptake in the 22RV1 tumors and the PSMA+ kidneys, while the uptake of D-Glu PSMA-617 remained near background in both of these tissues. Different from tumor, PSMA-617 exhibited a rapid clearance from the kidneys to yield a high tumor-to-background contrast (tumor:blood = 300; tumor:muscle = 290; and tumor:kidney = 15) at 24 h post dose. Complementary antitumor activity studies were performed with 225Ac-labeled PSMA-617. Here, significant tumor growth inhibition was observed without detectable hematologic or renal toxicity to the mice. These data provide additional support for a phase 3 prospective trial currently being planned to evaluate 177Lu-PSMA-617 in patients with metastatic PSMA+ prostate cancers, along with the continued clinical investigation of 225Ac-PSMA-617 in a similar patient population.

Citation Format: Joseph A. Reddy, Melissa Nelson, LeCun Xu, Elaine Westrick, Marilynn Vetzel, Leroy Wheeler, Albert Felten, Hari Santhapuram, Iontcho Vlahov, Christopher P. Leamon. Specificity of PSMA-617 radiotherapy for prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 852.
Abstract 861: Angiopoietin-2 blocking antibodies improve tumor growth inhibition and survival in mice treated with low doses of radiation
Authors: Kallio Pauliina , Jokinen Elina , Das Suvendu , Högström Jenny , Heino Sarika , Lähde Marianne , Alitalo Kari
Published: August 2018

Almost 50 % of all cancer patients receive radiation therapy during their treatment, yet several tumor types show radiation therapy resistance that compromises treatment efficacy. Many radiation sensitizers have been tested for improvement of the outcome of radiation therapy. For example, inhibitors of VEGF and VEGF receptors were tested in attempt to normalize the tumor vasculature and to improve the outcome of radiation therapy. Here, we have analysed if antibodies blocking the angiogenic factor angiopoietin-2 could improve radiation therapy of mouse tumor isografts and human tumor xenografts in mice. To test if the anti-tumor effect of small doses of radiation can be increased by Ang2 blocking antibodies, we analysed the radiation sensitivity of subcutaneous tumor isografts in C57bl/6JRj mice, and human xenografts in NOD scid gamma mice. In several models, the combination treatment with anti-Ang2 plus radiation was superior to the monotherapies in decreasing tumor growth and increasing the lifespan of the tumor-bearing mice both in immunocompetent and immunocompromised mice. Based on our results, the anti-Ang2 blocking antibody treatment could be tested in the clinics to improve the effect of radiation therapy, especially in those cases in which an optimal radiation dose cannot be given because of the radiation sensitivity of the surrounding tissues.

Citation Format: Pauliina Kallio, Elina Jokinen, Suvendu Das, Jenny Högström, Sarika Heino, Marianne Lähde, Kari Alitalo. Angiopoietin-2 blocking antibodies improve tumor growth inhibition and survival in mice treated with low doses of radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 861.
Abstract 862: Effects of fixation and antigen retrieval on the RNA integrity number
Authors: Otali Dennis , Mullen Maitlyn , Qinghua He , Oelschlager Denise , Grizzle William E.
Published: August 2018

The effect of fixation and antigen retrieval (AR) on RNA as measured by the RNA integrity number (RIN) has not been adequately explored. RIN is an index (pseudo-measure) used to measure stability of mRNA; however, RIN is an actual measure of the stability of ribosomal RNA and not an actual measure of mRNA. RIN values range from 1 for highly degraded to 10 for intact RNA.

Most surgical specimens are fixed in 10% neutral buffered formalin (3.7% formaldehyde) because of its ability to preserve morphological detail and prevent degradation. However, formaldehyde fixation causes changes in proteins through formation of crosslinks, which masks the immunorecognition of some antigens. In immunohistochemical evaluations, antigen retrieval (AR) approaches e.g., boiling of tissue sections under pressure are used to reverse these effects of fixation.

It is believed that fresh or snap frozen specimens serve as most the suitable source of intact mRNA, DNA and other macromolecules for molecular analysis. However, more recent studies using rt-Q PCR indicate that RNA and DNA can be measured in formalin fixed paraffin embedded (FFPE) tissue samples which usually have RIN values of 3 or less. The question arises as to what is the usefulness of RIN in FFPE tissues.

This study used three cancer cell lines DU145 (prostate), MDA231 (breast) and S2VP10 (pancreas) to examine the relationship between 10% NBF fixation, RIN and AR. The first objective was to analyze whether the duration of fixation of cells in 10% NBF correlated with decreases in RIN. Three fixation times i.e., 5 minutes, 20 minutes and 120 minutes were studied. The second objective was to investigate the effects of two AR approaches on RIN. Two AR conditions at pH 6 and pH 9 were used on the same cell lines and fixation times.

For each cell line and treatment i.e., no fixation, or fixation at different times (with and without AR), RNA was extracted and RIN analyzed using the Agilent 2100 Bioanalyzer. RIN values obtained from the Agilent 2100 Bioanalyzer were statistically analyzed. T-tests show that the large decreases to about 3 in RIN values after fixation (5, 20 and 120 minutes) are statistically significant for all cell lines. All p-values are less than 0.0001. This study confirms that fixation of cells in 10% NBF lowers RIN to about 3 the value observed for fixed tissues. RIN did not vary significantly with time of fixation in 10% NBF and RIN did not change from the fixed values after either condition of AR. Thus RIN is not a useful measure of RNA stability in 10% NBF fixed tissue.

Citation Format: Dennis Otali, Maitlyn Mullen, He Qinghua, Denise Oelschlager, William E. Grizzle. Effects of fixation and antigen retrieval on the RNA integrity number [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 862.
Abstract 877: Sigma 2 ligands kill pancreatic cancer cells via disruption of lysosomal cholesterol transport
Authors: Jin Linda X. , Vangveravong Suwanna , Sankpal Narendra V. , Cullinan Darren R. , Goedegebuure Peter , Spitzer Dirk , Hawkins William G.
Published: August 2018

Introduction: The sigma 2 receptor is overexpressed in pancreatic cancer and has been an attractive target for development of cancer selective therapies. However, the mechanism of sigma 2 mediated cancer cell death remains elusive. The sigma 2 receptor has been recently identified as TMEM97, a lysosomal/endoplasmic reticulum protein involved in regulation of cholesterol export in conjunction with NPC1.

Methods: Uptake of SW43 in pancreatic cancer cells was visualized using a NBD fluorophore conjugated ligand. Intracellular cholesterol staining was visualized using filipin staining. Protein expression was quantified by both Western blot and RT qPCR. Cell death was assayed in triplicate using Titer-Glo viability assay.

Results: We hypothesized that sigma 2 ligands may kill pancreatic cancer cells through disruption of lysosomal cholesterol export. Using ASPC1 pancreatic cancer cells, we first demonstrate that sigma 2 ligand SW43 is rapidly taken up and targets to endo-lysosomal compartments, and that treatment with SW43 causes a rapid and dose dependent sequestration of cholesterol in lysosomal compartments as visualized by filipin cholesterol staining. This correlated to an increase in TMEM97 and corresponding decrease in NPC1 expression following SW43 treatment. We next demonstrate that this cholesterol sequestration phenotype is required for cell death, and might be related to the free amine side chain of SW43, as its N-phthalimide derivative fails to induce both lysosomal cholesterol sequestration and cell death despite high doses of ligand used. Lysosomal cholesterol sequestration from sigma 2 ligand treatment sensitizes cells to further targeting of cholesterol metabolism. Using low doses of simvastatin and SW43, we demonstrate that targeting both cholesterol export and cholesterol synthesis displays significant synergism in killing ASPC1 pancreatic cancer cells.

Conclusions: These findings provide further insight into how the sigma 2 receptor/ligand interaction mediates cancer cell death and provides the basis for rationally combined targeted therapy regimens to be tested in vivo.

This work was supported by NIH R01CA163764 and NIH T32CA009621-26A1 and the SUS Foundation Resident Research Award.

Citation Format: Linda X. Jin, Suwanna Vangveravong, Narendra V. Sankpal, Darren R. Cullinan, Peter Goedegebuure, Dirk Spitzer, William G. Hawkins. Sigma 2 ligands kill pancreatic cancer cells via disruption of lysosomal cholesterol transport [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 877.
Abstract 915: AKT inhibition sensitizes TNBC cells with acquired drug resistance to UM-164
Authors: Merrill Nathan M. , Lachacz Eric J. , Gilani Rabia A. , Bao LiWei , Wu Zhi Fen , Merajver Sofia D. , Soellner Matthew B.
Published: August 2018

One of the major clinical challenges in breast cancer treatment is the rapid development of resistance to targeted therapies. In light of this, there is significant interest in identifying markers of drug resistance. In particular, markers of drug resistance are sought that can be targeted to overcome acquired drug resistance. To this end, we have used reverse phase protein array (RPPA) analysis to identify differences in protein expression levels between cell lines that are sensitive or have acquired resistance to UM-164, a mechanistically novel, dual c-Src/p38 kinase inhibitor with potent anti-TNBC activity. UM-164 resistant cells acquire increased resistance also to many diverse chemotherapeutics and targeted agents relative to their drug-sensitive counterparts. We analyzed RPPA data for both UM0164-sensitive and UM-164-resistant cells and identified an increase in phospho-AKT, phospho-ERK, and phospho-FAK levels in the resistant cells. Using inhibitors of each upregulated pathway, we found that combination of UM-164 with MK2206, an inhibitor of AKT, resulted in an increased sensitization of cells to treatment with UM-164. Furthermore, formal Chou-Talalay synergy analyses demonstrate that AKT inhibitors + UM-164 are effectively synergistic in reducing the proliferation of drug-resistant cells. We further evaluated this combination treatment in mice, combining UM-164 and MK2206 in a xenograft of UM-164-resistant cells, showing significant anti-tumor efficacy. Finally, we show that the combination treatment resulted in increased cell death in ex vivo cultures of patient derived TNBC materials. Together, we have demonstrated that hyperactivation of AKT leads to UM-164 resistance and AKT inhibitors can be used to re-sensitize TNBC cells to UM-164. These results demonstrate that RPPA analyses are a valuable tool for the systematic and unbiased identification of truly actionable markers of therapeutic resistance.

Citation Format: Nathan M. Merrill, Eric J. Lachacz, Rabia A. Gilani, LiWei Bao, Zhi Fen Wu, Sofia D. Merajver, Matthew B. Soellner. AKT inhibition sensitizes TNBC cells with acquired drug resistance to UM-164 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 915.
Abstract 916: A screen to target EGFR (C797S) in osimertinib-resistant lung cancer identifies brigatinib-based combinations that synergistically inhibit cell growth
Authors: Martin Matthew J. , Floc'h Nicolas , Michaloglou Chrysiis , Finlay M Raymond , Ward Richard A. , Smith Paul D. , Cross Darren A.
Published: August 2018

Up to 20% of non small-cell lung cancers are driven by activating mutations in EGFR (EGFRm). While EGFR tyrosine kinase inhibitors (TKIs) have proven highly effective in the clinic (e.g. gefitinib in first line EGFRm and osimertinib in T790M post-TKI resistance), acquired resistance mechanisms present an obstacle to curing EGFR-driven disease. To-date, approximately 20% of osimertinib-resistant cases in the 2nd line setting are EGFRm/T790M/C797S, and there is currently no approved therapy for these “triple-mutant” (TM) patients. We have obsrerved that the ALK inhibitor brigatinib shows moderate, equipotent activity against both EGFR-TM and the single activating mutant. We used this information to screen a panel of compounds to identify brigatinib-based combinations that could effectively target EGFR-TM-expressing cells. Among the most potent combination partners were selumetinib and other inhibitors of the MAP kinase pathway. These agents were able to synergistically impair growth of TM cells when combined with brigatinib in vitro, and accordingly show prolonged suppression of downstream pathway biomarkers in combination- vs. brigatinib monotherapy-treated cells. Further, we have shown effective combinations can dramatically enhance apoptotic cell death compared to brigatinib treatment alone. Critically, neither monotherapy treatment with effective combination partners, nor combinations based on osimertinib showed growth inhibition in the EGFR triple-mutant setting. Taken together, these data suggest novel therapeutic strategies for patients in this critical area of unmet need that warrant further preclinical testing.

Citation Format: Matthew J. Martin, Nicolas Floc'h, Chrysiis Michaloglou, M Raymond Finlay, Richard A. Ward, Paul D. Smith, Darren A. Cross. A screen to target EGFR (C797S) in osimertinib-resistant lung cancer identifies brigatinib-based combinations that synergistically inhibit cell growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 916.
Abstract 96: Novel inverse functioning molecules, GSN as metastatic promoter and OAS2 as metastatic repressor in colorectal adenocarcinoma
Authors: Kim Jin Cheon , Ha Je Jin , Tak Ka Hee , Roh Seon Ae , Kim Chan Wook , Yoon Yong Sik , Lee Jong Lyul , Kim Seon-Kyu , Kwon Ee Hong , Cho Dong-Hyung , Kim Seon-Young , Kim Yong Sung
Published: August 2018

Background LVI and PNI, recognized as major histological traces of metastatic CRC, have been recognized as a category I prognostic factor representing aggressive CRC. The primary aim of our study was to find out molecules associated with LVI and PNI and to examine their biological behavior with their prognostic significance.

Methods LVI- and PNI-associated molecules were identified and verified by utilizing (1) RNA-seq and selecting 117 genes by differential expression values in primary cancer samples of 130 patients with and without systemic recurrence (n = 72 and 58) (2) association analysis to extract six genes associated with LVI or PNI (3) assessment of biological property measuring proliferation, invasion/migration, epithelial-mesenchymal transition, and autophagy flux (4) verification of disease-free survival using public data sets of gene

Results GSN-overexpressing LoVo cells showed more than 2-fold greater invasion compared with control cells 5 (p < 0.001 - 0.005), whereas OAS2-overexpressing RKO cells showed a significantly reduced invasion compared with control cells (p < 0.001 - 0.005). GSN repressed expressions of E-cadherin, β-catenin, claudin-1, and snail, whereas it enhanced N-cadherin and ZEB1 expressions. Reverse findings were noticeably identified in OAS2-overexpression. Interestingly, expressions of multiple autophagy-related proteins including ATG5-12, ATG6/BECN1, ATG7, and ATG101 were decreased in GSN-overexpressing Lovo cells. In addition, accumulation of p62, a well-known substrate of autophagic degradation, suggested that an autophagic flux was also reduced in the GSN-overexpressing Lovo cells. In contrast to GSN overexpression, reverse findings of autophagy flux was identified in OAS2-overexpressing RKO cells. The 5-year RFS rates were significantly greater in the GSN underexpression group than in the overexpression group (73.6% and 64.7%, p = 0.038), whereas patients with OAS2 overexpression presented a greater RFS rate than those with underexpression (73.4% and 63.7%, p = 0.01).

Conclusion GSN and OAS2, as a recurrence promoter and suppressor, respectively, suggest their potential value as recurrence predictor or therapeutic targets in future clinical validation studies.

Citation Format: Jin Cheon Kim, Je Jin Ha, Ka Hee Tak, Seon Ae Roh, Chan Wook Kim, Yong Sik Yoon, Jong Lyul Lee, Seon-Kyu Kim, Ee Hong Kwon, Dong-Hyung Cho, Seon-Young Kim, Yong Sung Kim. Novel inverse functioning molecules, GSN as metastatic promoter and OAS2 as metastatic repressor in colorectal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 96.