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Purpose: Salivary gland adenoid cystic carcinoma (ACC) has heterogeneous clinical behavior. Currently, all patients are treated uniformly, and no standard-of-care systemic therapy exists for metastatic ACC. We conducted an integrated proteogenomic analyses of ACC tumors to identify dysregulated pathways and propose a classification with therapeutic implications. Experimental Design: RNA/DNA sequencing of 54 flash-frozen salivary ACCs and reverse phase protein array (RPPA) in 38 specimens were performed, with validation by Western blotting and/or IHC. Three independent ACC cohorts were used for validation. Results: Both unbiased RNA sequencing (RNA-seq) and RPPA analysis revealed two molecular subtypes: ACC-I (37%) and ACC-II (63%). ACC-I had strong upregulation of MYC, MYC target genes, and mRNA splicing, enrichment of NOTCH-activating mutations, and dramatically worse prognosis. ACC-II exhibited upregulation of TP63 and receptor tyrosine kinases (AXL, MET, and EGFR) and less aggressive clinical course. TP63 and MYC were sufficient to assign tumors to ACC subtypes, which was validated in one independent cohort by IHC and two additional independent cohorts by RNA-seq. Furthermore, IHC staining for MYC and P63 protein levels can be used to identify ACC subtypes, enabling rapid clinical deployment to guide therapeutic decisions. Our data suggest a model in which ACC-I is driven by MYC signaling through either NOTCH mutations or direct amplification, which in turn suppress P63 signaling observed in ACC-II, producing unique therapeutic vulnerabilities for each subtype. Conclusions: Cooccurrence of multiple actionable protein/pathways alterations in each subtype indicates unique therapeutic vulnerabilities and opportunities for optimal combination therapy for this understudied and heterogeneous disease.

Purpose: This study was aimed at investigating whether the PPARγ agonist pioglitazone—given in combination with trabectedin—is able to reactivate adipocytic differentiation in myxoid liposarcoma (MLS) patient-derived xenografts, overcoming resistance to trabectedin. Experimental Design: The antitumor and biological effects of trabectedin, pioglitazone, and the combination of the two drugs were investigated in nude mice bearing well-characterized MLS xenografts representative of innate or acquired resistance against trabectedin. Pioglitazone and trabectedin were given by daily oral and weekly i.v. administrations, respectively. Molecular studies were performed by using microarrays approach, real-time PCR, and Western blotting. Results: We found that the resistance of MLS against trabectedin is associated with the lack of activation of adipogenesis. The PPARγ agonist pioglitazone reactivated adipogenesis, assessed by histologic and gene pathway analyses. Pioglitazone was well tolerated and did not increase the toxicity of trabectedin. The ability of pioglitazone to reactivate adipocytic differentiation was observed by morphologic examination, and it is consistent with the increased expression of genes such as ADIPOQ implicated in the adipogenesis process. The determination of adiponectin by Western blotting constitutes a good and reliable biomarker related to MLS adipocytic differentiation. Conclusions: The finding that the combination of pioglitazone and trabectedin induces terminal adipocytic differentiation of some MLSs with the complete pathologic response and cure of tumor-bearing mice provides a strong rationale to test the combination of trabectedin and pioglitazone in patients with MLS.

Scutellarin is a flavonoid compound that is found in Scutellaria barbata. It has been reported to exhibit anticancer and anti-inflammation activities. However, the anticancer properties of scutellarin and its molecular targets have not been investigated in esophageal squamous cell carcinoma (ESCC). In the current study, we report that scutellarin is a potential AKT inhibitor that suppresses patient-derived xenograft ESCC tumor growth. To identify possible molecular targets of scutellarin, potential candidate proteins were screened by an in vitro kinase assay and Western blotting. We found that scutellarin directly binds to the AKT1/2 proteins and inhibits activities of AKT1/2 in vitro. The AKT protein is activated in ESCC tissues and knockdown of AKT significantly suppresses growth of ESCC cells. Scutellarin significantly inhibits anchorage-dependent and independent cell growth and induces G2 phase cell-cycle arrest in ESCC cells. The inhibition of cell growth by scutellarin is dependent on the expression of the AKT protein. Notably, scutellarin strongly suppresses patient-derived xenograft ESCC tumor growth in an in vivo mouse model. Taken together, our data suggest that scutellarin is a novel AKT inhibitor that may prevent progression of ESCC.

Cancer chemoprevention by benzyl isothiocyanate (BITC), which is derived from cruciferous vegetables like garden cress, in a transgenic mouse model of breast cancer is associated with inhibition of breast cancer stem-like cells (bCSC), but the molecular regulators of this effect remain elusive. This study demonstrates a protective effect of Krüppel-like factor 4 (KLF4)-p21CIP1 axis in bCSC inhibition by BITC. Exposure of human breast cancer cells (MCF-7, MDA-MB-231, and SUM159) to plasma-achievable concentrations of BITC resulted in a robust induction of KLF4 mRNA and its protein expression as determined by qRT-PCR and Western blotting or confocal microscopy. BITC-mediated suppression of bCSC markers, including aldehyde dehydrogenase 1 activity and mammosphere frequency, was significantly augmented by transient or stable knockdown of KLF4. Western blotting and IHC revealed relatively higher levels of KLF4 protein in mammary tumor sections from BITC-treated mice in comparison with controls, but the difference was insignificant. Analysis of the breast cancer RNA-Seq data from The Cancer Genome Atlas indicated significant positive correlation between expression of KLF4 and that of p21CIP1 (CDKN1A) but not β-Catenin (CTNNB1). Knockdown of p21CIP1 protein also amplified BITC-mediated suppression of bCSC. Finally, KLF4 was recruited to the promoter of p21CIP1 as indicated by chromatin immunoprecipitation assay. These results indicate that induction of KLF4–p21CIP1 axis attenuates inhibitory effect of BITC on bCSC self-renewal. Translational implication of these findings is that breast cancer chemoprevention by BITC may be augmented with a combination regimen involving BITC and an inhibitor of KLF4.

Carboxylesterase 1c (CES1c) is responsible for linker-drug instability and poor pharmacokinetics (PK) of several antibody–drug conjugates (ADC) in mice, but not in monkeys or humans. Preclinical development of these ADCs could be improved if the PK in mice would more closely resemble that of humans and is not affected by an enzyme that is irrelevant for humans. SYD985, a HER2-targeting ADC based on trastuzumab and linker-drug vc-seco-DUBA, is also sensitive to CES1c. In the present studies, we first focused on the interaction between CES1c and SYD985 by size- exclusion chromatography, Western blotting, and LC/MS-MS analysis, using recombinant CES1c and plasma samples. Intriguingly, CES1c activity not only results in release of the active toxin DUBA but also in formation of a covalent bond between CES1c and the linker of vc-seco-DUBA. Mass spectrometric studies enabled identification of the CES1c cleavage site on the linker-drug and the structure of the CES1c adduct. To assess the in vivo impact, CES1c−/− SCID mice were generated that showed stable PK for SYD985, comparable to that in monkeys and humans. Patient-derived xenograft (PDX) studies in these mice showed enhanced efficacy compared with PDX studies in CES1c+/+ mice and provided a more accurate prediction of clinical efficacy of SYD985, hence delivering better quality data. It seems reasonable to assume that CES1c−/− SCID mice can increase quality in ADC development much broader for all ADCs that carry linker-drugs susceptible to CES1c, without the need of chemically modifying the linker-drug to specifically increase PK in mice. Mol Cancer Ther; 17(11); 2389–98. ©2018 AACR.

Purpose: This study examined the potential role of the nuclear deubiquitinating enzyme BRCA1-associated protein-1 (BAP1) in radioresistance in head and neck squamous cell cancer (HNSCC). Experimental Design: We overexpressed, knocked down, and rescued BAP1 expression in six HNSCC cell lines, three human papillomavirus (HPV)–negative and three HPV-positive, and examined the effects on radiosensitivity in vitro and in an HNSCC mouse xenograft model. Radiosensitivity was assessed by clonogenic cell survival and tumor growth delay assays; changes in protein expression were analyzed by immunofluorescence staining and Western blotting. We also analyzed The Cancer Genome Atlas HNSCC database to test for associations between BAP1 expression and outcome in patients. Results: Overexpression of BAP1 induced radioresistance in both cell lines and xenograft models; conversely, BAP1 knockdown led to increased ubiquitination of histone H2A, which has been implicated in DNA repair. We further found that BAP1 depletion suppressed the assembly of constitutive BRCA1 foci, which are associated with homologous recombination (HR), but had minimal effect on γ-H2AX foci and did not affect proteins associated with nonhomologous end joining, suggesting that BAP1 affects radiosensitivity in HNSCC by modifying HR. Finally, in patients with HNSCC, overexpression of BAP1 was associated with higher failure rates after radiotherapy. Conclusions: BAP1 can induce radioresistance in HNSCC cells, possibly via deubiquitination of H2Aub and modulation of HR, and was associated with poor outcomes in patients with HNSCC. BAP1 may be a potential therapeutic target in HNSCC. Clin Cancer Res; 24(3); 600–7. ©2017 AACR.

Objective: Pancreatic cancer is currently one of the most lethal human cancers and continues to be a major unsolved health problem in the 21st century. Since pancreatic cancer has a high metastatic potential, chemotherapy is inevitable for most pancreatic cancer patients, whereas the clinical responses to anticancer agents are not satisfied. In this work, we designed and synthesized a novel compound, termed as 6-MPA, and found that 6-MPA could improve the proteasomal degradation of ErbB2 pancreatic cancer cells. Methods: The inhibitory effects of 6-MPA on migration and invasion was measured by scratch experiment, wound healing assay and transwell experiment. Clone formation and CCK-8 assay was used to determine the cytotoxicity of 6-MPA. Real-time PCR and western blotting was used to determine the expression of indicated genes or proteins respectively. Immunoprecipitation was used to characterize protein–protein interactions. Results: 4, 8, and 16 μM of 6-MPA exhibited an inhibitory effects on the migration of SW1990 cells and the inhibition rates reached 40.2±10.1%, 58.2±11.2%, and 65.2±9.9% in vitro, respectively. RNA-seq showed that 6-MPA treatment manipulated ErbB2 level and its downstream PI3K/AKT/mTOR signaling pathways, which was verified by western blotting assay. Treatment with 6-MPA could enhance the interaction between ErbB2 and ubiquitin, and thus promote the proteasomal degradation of ErbB2. Conclusion: In this study, we obtained a novel small molecule 6-MPA, which could specifically degrade ErbB2 through the activation of protein ubiquitinination. And we found that 6-MPA possessed potent inhibitory effects on migration and invasion of pancreatic cancer cells. Therefore, our work illustrated a novel structure skeleton that could be used to target ErbB2, and potentially suppressed the metastasis of pancreatic cancer. Citation Format: Bo Zhang, Fei Teng, Nengming Lin. Targeting ErbB2 degradation via the ubiquitin–proteasome pathway to inhibit the metastasis of pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-057.

In the original version of [this article][1] ([1][2]), there were three errors in Fig. 6: in Fig. 6A, the GADPH blots were inadvertently duplicated from Fig. 5D; in Fig. 6B, the image for Aspirin/NC was a duplicate of DMSO/sh#2; and in Fig. 6B, the Aspirin/vector image was a duplicate of Aspirin/sh#

Introduction: Arginine methylation deregulation in cancer has been well studied and PRMT5 which modulates dimethylation of arginine has emerged as an attractive therapeutic strategy in various cancer types, including lung cancer, lymphoma, glioblastoma, pancreatic cancers, etc. PRMT5 is over-expressed in multiple cancers leading to repression of tumor suppressor genes and genetic studies have identified it is a validated target in lymphoma. Recently, dysregulation of the splicing machinery in cancers has been identified to be one of the therapeutic vulnerabilities for PRMT5 inhibition, especially in glioblastoma. Therefore, inhibitors selectively targeting PRMT5 could be of high clinical value, especially in cancers with defects in spliceosome machinery. Methods: Rational design and structure based drug design were used to identify novel PRMT5 inhibitors. To assess in vitro potency, flash plate based activity assay was used. Cell based activity of these inhibitors was assessed by measuring the symmetrical dimethylation of known cellular protein SmD3 by ELISA and Western blotting. Long term cell proliferation assays were used to assess the functional effect of PRMT5 inhibition. Tumor growth inhibition was measured in orthotopic glioblastoma model in mice. Results: One of the lead PRMT5 inhibitors had an in vitro potency of 3 nM in the biochemical assay which translated well in the cell based SDMA ELISA where the EC50 was Conclusion: Given the therapeutic importance of PRMT5 in glioblastoma and other lymphomas, this molecule will be extremely valuable in treating these cancers both as a standalone therapy and in combination with other standard of care agents. Citation Format: Dhanalakshmi Sivanandhan, Sridharan Rajagopal, Naveen Sadhu M, Chandru Gajendran, Saravanan Vadivelu, Natarajan Tamizharasan, Indu N. Swamy, Santhosh Viswakarma, Amir Siddiqui, Saif wahid, Mohammed Zainuddin, Rudresh G, Prashanthi Daram, Ramchandraiah Gosu, Dinesh Tiagaraj, Shivani Garapaty, Sreekala Nair, Namratha Kapoor. Selective, novel, small molecule PRMT5 inhibitors for treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1128.

HER2 is amplified in 7% to 20% of gastric cancer (GC) and is associated with poor clinical outcome. Trastuzumab, HER2-targeting antibody, has already been successfully employed for the treatment of HER2 amplified metastatic GC patients. However, most GC patients who achieved an initial response to the trastuzumab-based regimen developed resistance within 1 year after treatment. In this study, we aimed to investigate the efficacy and biological mechanism of trastuzumab deruxtecan (T-DXd) for overcoming trastuzumab resistance in HER2 amplified GC cell lines. We established four trastuzumab resistant cell lines from HER2 amplified GC cell lines by trastuzumab dose-escalation treatment; YCC-33TR, YCC-38TR, NCI-N87TR, and SNU-216TR. Next, we compared the anti-tumor efficacy of trastuzumab and T-DXd (Daiichi Sankyo, Japan), a HER2-targeting antibody-drug conjugate (ADC), in parental and trastuzumab resistant cell lines. Drug sensitivities were determined by the inhibition rates (IR) at 100μg/ml using cell viability assay. Both in baseline and in post-treatment drug responses, expression levels of proteins such as HER2, Erk, Akt, H2A.X and cPARP were detected by western blotting. The parental cell lines were sensitive to trastuzumab, while four trastuzumab resistant (TR) cell lines demonstrated significant resistance to trastuzumab (p In summary, the established trastuzumab resistant cell lines retained high HER2 expression, and both HER2 amplified parental and trastuzumab resistant cell lines were sensitive to T-DXd. Our data indicated that T-DXd may have a potential therapeutic effect to overcome trastuzumab resistance in GC. Citation Format: Juin Park, Inhye Jeong, Sun Kyoung Kang, Seo Young Yu, Woo Sun Kwon, Tae Soo Kim, Jihyun Hwang, Hyun Cheol Chung, Sun Young Rha. Overcoming trastuzumab resistance using trastuzumab deruxtecan (T-DXd), a HER2 targeting antibody drug conjugate, in HER2 amplified gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1090.

Introduction: Epidemiological evidence suggests that saturated fatty acid has a role in prostate cancer progression; however underlying mechanisms remain unknown. Here, we conducted a comprehensive omics analysis to investigate candidate molecules associated with saturated fat-induced prostate cancer (PCa) progression. Methods: TRAMP-C2 cells were subcutaneously injected into two groups of C57BL6 mice: the lard diet (LD) group, which was fed isocaloric saturated fatty acid, and the fish oil diet (FOD) group, which was fed polyunsaturated fatty acid. We performed an oligonucleotide microarray with the Affymetrix Gene Chip Mouse Gene 2.0 ST Array and proteomic analysis using nano-liquid chromatography-tandem mass spectrometry using xenograft tumors and validated the result by western blotting and immunohistochemistry. The cell proliferation and mRNA levels of CRABP2 in two PCa cell lines cultured with mice serum were assessed by performing an MTT cell assay and quantitative real-time polymerase chain reaction. The effect of CRABP2 on survival in PCa patients was evaluated by reviewing data from The Cancer Genome Atlas database. Results: The mean body weight of the LD group tend to be higher than that of the LD group (p = 0.066). Subcutaneous tumor volume were significantly higher in the LD group than the FOD group (p = 0.044). One hundred fifty-three up- and 120 downregulated genes and 734 up- and 1514 downregulated proteins with >1.5-fold changes were identified in the LD group. Multi-omics approach showed 32 candidate molecules which have the potential to be highly associated with saturated fatty acid diet-induced prostate cancer progression. In the molecules, CRABP2 was selected as the most candidate molecule. The higher expression of CRABP2 in xenografts of the LD group was validated by western blotting and immunohistochemistry. PCa cells cultured in a medium containing LD mouse serum were associated with significantly higher cell proliferation rates and higher CRABP2 expression levels than those of PCa cells cultured in FOD. Overall survival significantly worse in the patients with altered CRABP2 than in those with unaltered CRABP2 (p < 0.001). Conclusion: Saturated fatty acid-accelerated PCa growth was enhanced by CRABP2. CRABP2 may be a key regulator of diet-induced PCa progression and a novel therapeutic target for PCa. A low saturated fat diet in males may be encouraged. Citation Format: Hiromi Sato, Shintarao Narita, Masanori Ishida, Soki Kashima, Ryohei Yamamoto, Atsushi Koizumi, Taketoshi Nara, Kazuyuki Numakura, Mitsuru Saito, Shigeru Satoh, Naoshi Dohmae, Tomonori Habuchi. Cellular retinoic acid-binding protein 2 enhances saturated fatty acid-induced prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2662.

Purpose: This study was performed to identify the detailed mechanisms by which miR-296-3p functions as a tumor suppressor to prevent lung adenocarcinoma (LADC) cell growth, metastasis, and chemoresistance. Experimental Design: The miR-296-3p expression was examined by real-time PCR and in situ hybridization. MTT, EdU incorporation, Transwell assays, and MTT cytotoxicity were respectively performed for cell proliferation, metastasis, and chemoresistance; Western blotting was performed to analyze the pathways by miR-296-3p and HDGF/DDX5 complex. The miRNA microarray and luciferase reporter assays were respectively used for the HDGF-mediated miRNAs and target genes of miR-296-3p. The ChIP, EMSA assays, and coimmunoprecipitation combined with mass spectrometry and GST pull-down were respectively designed to analyze the DNA–protein complex and HDGF/DDX5/β-catenin complex. Results: We observed that miR-296-3p not only controls cell proliferation and metastasis, but also sensitizes LADC cells to cisplatin (DDP) in vitro and in vivo. Mechanistic studies demonstrated that miR-296-3p directly targets PRKCA to suppress FAK–Ras-c–Myc signaling, thus stimulating its own expression in a feedback loop that blocks cell cycle and epithelial–mesenchymal transition (EMT) signal. Furthermore, we observed that suppression of HDGF–β-catenin–c-Myc signaling activates miR-296-3p, ultimately inhibiting the PRKCA–FAK–Ras pathway. Finally, we found that DDX5 directly interacts with HDGF and induces β-catenin–c-Myc, which suppresses miR-296-3p and further activates PRKCA–FAK–Ras, cell cycle, and EMT signaling. In clinical samples, reduced miR-296-3p is an unfavorable factor that inversely correlates with HDGF/DDX5, but not PRKCA. Conclusions: Our study provides a novel mechanism that the miR-296-3p–PRKCA–FAK–Ras–c-Myc feedback loop modulated by HDGF/DDX5/β-catenin complex attenuates cell growth, metastasis, and chemoresistance in LADC. Clin Cancer Res; 23(20); 6336–50. ©2017 AACR.

The off-target distribution of anticancer nanoparticles to fibroblasts creates a barrier to the effective treatment of desmoplastic tumors. However, we hypothesized that this nanoparticle detriment might be exploited to target the expression of secreted cytotoxic proteins from tumor-associated fibroblasts (TAF) as an anticancer strategy. In addressing this hypothesis, plasmids encoding the secretable TNF-related factor sTRAIL were loaded into lipid-coated protamine DNA complexes and administered by infusion in a murine xenograft model of human desmoplastic bladder carcinoma. Three doses were sufficient to generate approximately 70% of TAFs as sTRAIL-producing cells. sTRAIL triggered apoptosis in tumor cell nests adjacent to TAFs. Furthermore, it reverted residual fibroblasts to a quiescent state due to insufficient activation, further compromising tumor growth and remodeling the microenvironment to favor second-wave nanotherapy. We confirmed the efficacy of this strategy in an orthotopic xenograft model of human pancreatic cancer, where the desmoplastic stroma is well known to be a major barrier to the delivery of therapeutic nanoparticles. Collectively, our results offer a proof of concept for the use of nanoparticles to modify TAFs as an effective strategy to treat desmoplastic cancers. Cancer Res; 77(3); 719–31. ©2016 AACR.

Protein phosphorylation is a reversible reaction that is integral in numerous signaling cascades. Characterization of signaling cascades has been largely detected by immunoblotting with phospho-specific antibodies, which may or may not have enough specificity or affinity. Currently, a separate lysate without any phosphatase inhibitors or a separate blot is needed to determine an antibody's specificity. Here we describe a simple assay that leverages automation and quantitation with capillary electrophoresis-based immunoassay (CEIA) to assess the specificity of these antibodies with a single lysate preparation. In this study, three lysate models are used: K562 ± TNFα treatment, 50 ng/mL phorbol myristate acetate (PMA) differentiated THP-1 ± 1 μg/mL lipopolysaccharide (LPS) treatment, and cytotoxic T lymphocytes (CTL) ± 10 ng/mL PMA and 500 ng/mL ionomycin treatment. K562 cell lysates are commercially purchased whereas THP-1 lysates are generated in-house. For CTL cells, whole blood cells from a single donor are isolated and expanded with commercially available kits. Expanded CTL cells are then stimulated with PMA and ionomycin for 15 minutes. Untreated and treated lysate samples are separated and captured to the inner lumen of the capillary wall with UV activated crosslink chemistry. Cross-linked proteins are treated with lambda phosphatase for 1 hour followed by the immunoassay to investigate the specificity of antibodies against phosphorylated protein targets respective to each activated pathway using either chemiluminescent or fluorescent detection. Preliminary data suggest phospho-specific signal decreased >90% with no significant changes to the non-specific noise. The method described here eliminates the need for multiple lysate preparations or an additional blot to assess an antibody's specificity to a phosphorylated protein target. Citation Format: Daryl A. Taketa, Rainer Grant, Tufan Aydogdu. Determination of antibody's specificity towards phosphorylated protein targets with automated in-capillary enzyme treatment and immunoassay [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1436.

FLT3 mutations (mFLT3) in acute myeloid leukemia (AML) carry a poor prognosis.The mandatory co-occurrence of other driver mutations with mFLT3 leads to suboptimal clinical responses with single agent FLT3 inhibitors (FLT3i). Pre-existing or emergent RAS mutations are one mechanism of primary and secondary resistance to single agent FLT3i. Therefore development of combinatorial strategies to overcome resistance conferring co-mutations is critical. Herein we report a novel in-vitro combination of the FLT3/SYK inhibitor (FSI) TAK-659 with Proteasome inhibitor (PI) ixazomib, to co-operatively inhibit proliferation of mFLT3 and RAS or WT1 co-mutated AML blasts, which are otherwise less sensitive to Flt3i and FSI. Peripheral blood or bone marrow derived cells from patients with AML were isolated and treated in vitro with TAK-659 and Ixazomib alone or in combination. Cytosolic/nuclear protein lysates were obtained at 12 hours of drug/control treatment and western blotting performed for proteins of interest. Tritiated thymidine proliferation assays were also performed with titrated doses of each agent alone, and combination of fixed dose of one agent with titration of the second. Patient samples were annotated for mutations by Next Generation Sequencing. Similar experiments were carried out on cell lines MV411 (Flt3ITD/MLL), THP-1 and OCI-AML3 (both NRAS). Proliferation assays on a series of primary AML samples revealed an inherent dichotomy in response patterns. mFLT-3 samples with co-mutations in DNMT3A, NPM1 or tMLL were very sensitive to TAK-659 (IC50 100nM). The co-occurrence of RAS or WT1 mutations predicted for resistance to TAK-659 but interestingly sensitized cells to PI alone and in combination. In fact, presence of RAS mutations irrespective of FLT3 status predicted for sensitivity to PI (IC50 In summary, we delineate a RAS/RAC/PAK/Beta-catenin pathway to be linked to critical events in RAS or WT1 mutated AML progression and resistance. FLT3 mutated AML with these co-mutations are insensitive to single-agent activity of FLT3i or FSI alone and the addition of PI is critical to overcome them. Citation Format: Sravanti Rangaraju, Santosh Kumar Pasupuleti, Silvia Marino, Daniela Nicoleta Petrusca, Larry D. Cripe, Bhaskar Ramdas, Lakshmi Reddy Palam, Katie J. Sargent, Hamid Sayar, Heiko Konig, Reuben Kapur, David G. Roodman, Scott H. Boswell. Exploiting the proteasome to overcome RAS and WT1 mutation mediated resistance to FLT-3 inhibition in acute myeloid leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 800.

Objectives: Olaparib is a poly ADP-ribose polymerase inhibitor (PARPi) approved for use as maintenance and treatment in advanced and recurrent ovarian cancer (OC). The imipridone ONC206, a chemical derivative of ONC201, is a bioavailable dopamine receptor D2 (DRD2) antagonist that has anti-tumorigenic effects via induction of apoptosis and activation of the integrated stress response. ONC206 exhibits differentiated receptor pharmacology, nanomolar potency, and enhanced bioavailability relative to ONC201. Both olaparib and imipridones (i.e. ONC201) are being evaluated in OC clinical trials, but have yet to be explored in combination. Thus, we sought to examine the effects of olaparib in combination with ONC206 in human OC cell lines. Methods: The OC cell lines, OVCAR3 and OVCAR5, were used. Both cell lines were treated with varying doses of ONC206 (Oncoceutics) and olaparib (MedChemExpress) alone and in combination. Cell proliferation and apoptosis were assessed by MTT and cleaved caspase assays, respectively. Synergy between olaparib and ONC206 was evaluated by the combination index (CI) method of Chou-Talalay. Reactive oxygen species (ROS) was determined by DCFDA assay. Adhesion and migration were assessed by laminin and wound healing assays, respectively. Western blotting was performed to evaluate the effects of olaparib and ONC206 on apoptotic and invasion-related proteins in both OC cell lines. Results: Both drugs inhibited OC cell proliferation in a dose-dependent manner after 72 hrs of exposure (IC50 for olaparib of 15-25 µM for both OVCAR3 and OVCAR5; IC50 1.5 µM and 0.4µM for ONC206 in OVCAR3 and OVCAR5, respectfully). Simultaneous exposure of ONC206 with olaparib resulted in a synergistic anti-proliferative effect (CI Conclusions: ONC206 in combination with olaparib resulted in synergistic anti-tumorigenic and anti-metastatic effects in OC cell lines, suggesting that this novel dual therapy may be worthy of further exploration in clinical trials in OC. Citation Format: Gabrielle M. Hawkins, Sarah E. Paraghamian, Yali Fan, Wenchaun Sun, Xin Zhang, Varun Prabhu, Joshua E. Allen, Chunxiao Zhou, Victoria L. Bae-Jump. Olaparib potentiates the anti-proliferative and anti-metastatic effects of ONC206 in ovarian cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5336.

Exosomes are small extracellular vesicles (40-150 nm in diameter) secreted by cells and initially proposed as a mechanism to eliminate cellular waste. More recently, exosomes have been reported to participate in cancer metastasis because of their ability to mediate local and distant cell-to-cell communication. Furthermore, exosomes create cancer-favored microenvironments, potentially enhancing cancer metastasis. However, it is unknown whether exosomes interact with E-selectin, an adhesion molecule expressed by vascular endothelium and shown to be involved in metastasis. Our lab has previously shown that BT-20 human breast cancer cells adhesively interact with endothelial E-selectin via several E-selectin ligands such as sialofucosylated CD44 variants, Mac-2BP, and gangliosides. Therefore, we hypothesize that exosomes secreted by the BT-20 human breast cancer cell line interact with E-selectin. To investigate this hypothesis, exosomes were generated by culturing cells in serum-depleted media and isolated by a series of ultracentrifugation steps. Molecular size analysis using dynamic light scattering revealed that the size range of the isolated particles was similar to the expected size of exosomes. To detect CD9 (an exosome marker) and E-selectin ligands, both breast cancer whole cell lysate and exosome lysate were tested by Western blot. The higher expression of CD9 in isolated particles than the whole cell lysates confirmed that the isolated particles were exosomes. When Western blots were labeled with recombinant E-selectin/Fc chimera with Ca2+, bands were detected at approximately 130 kDa, indicating the presence of E-selectin ligands. As a negative control, the divalent cation chelator EDTA was added to the primary labeling incubation. Bands were not detected in these negative control experiments, indicating no E-selectin interactions in the absence of Ca2+. Human IgG (hIgG) was also used as a negative control in Western blots and showed no signal with exosome lysates, as expected. In comparison, experiments were also performed with Hs578T breast cancer cells, a cell line that our lab has shown does not express E-selectin ligands. Exosomes isolated from Hs578T cells did not express E-selectin ligands, consistent with whole cells. Altogether, these results suggest that BT-20 human breast cancer exosomes express E-selectin ligands. Future work will focus on identifying and characterizing the exosome E-selectin ligands, as well as investigating other cancer cells that express E-selectin ligands. Ultimately, understanding the interaction between exosomes and E-selectin can lead to new diagnostic and therapeutic strategies for cancer metastasis. Citation Format: Yinan Huang, Christian A. Showalter, Monica M. Burdick. Exosomes derived from the BT-20 human breast cancer cell line express E-selectin ligands [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2626.

Cutaneous Squamous Cell Carcinoma (cSCC) is the second most common form of skin cancer, frequently arising from sun-induced mutations. Most clinical cases of cSCC are non-invasive and achieve complete cure through tumor excision. However, advanced cSCC is associated with high mortality, thus research aimed at understanding factors influencing metastasis of invasive cSCC is needed. Cancer metastasis is a multistep process, including epithelial-to-mesenchymal transition (EMT). During EMT, epithelial cells undergo a dedifferentiation process whereby they transition to a highly mobile and invasive mesenchymal cell type with metastatic capacities. Changes in expression of several proteins, including TGF-β, vimentin, and E-cadherin have been identified as a hallmark of EMT, which may be promoting cancer progression. We hypothesize that during development of cSCC, cells within the lesions are undergoing EMT that facilitates invasion into the tissues. In situ and invasive cSCC tissues were obtained from excised tumors being prepped for histological sections from Mohs surgery; once clean margins were identified, adjacent normal tissues (ANT) were obtained during suturing. For all tissues, total protein was isolated; western blots were performed to determine expression of TGF-β1,2,3, Bcl-2, E-cadherin, phosphorylated E-cadherin, and vimentin. Analysis of MMP activity for each tissue type was performed using a FRET assay. For all proteins analyzed, cSCC tissues had higher levels of expression compared to ANT, with invasive cSCC tissues demonstrating increased protein expression over in situ samples (p Citation Format: Christopher S. Pulford, Chandana K. Uppalapati, Elizabeth E. Hull, Kathryn J. Leyva. Cutaneous squamous cell carcinoma: Evidence of epithelial to mesenchymal transition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1540.

Purpose: Autophagy is a series of processes in which biomolecules are digested and recycled, and it also provides energy for the development and progression of cancer. Thus, autophagy inhibitors can be used to suppress the progression of cancer. In this study, we investigated whether decursin, which is derived from natural products, inhibits the growth and autophagy of cancer cells. Methods: The gastric cancer cell lines SNU216 and NCI-N87 were treated with or without decursin. Cell growth was evaluated by Cell-Counting Kit-8, colony formation, and BrdU assays, as well as by propidium iodide staining, and autophagic flux was evaluated by western blotting, LC3 puncta, GFP-RFP-LC3, and transmission electron microscopy. Finally, tumor spheroids and patient-derived gastric cancer organoids were used to confirm the effect of decursin in three-dimensional culture. Results: Decursin reduced the growth and anchorage-dependent viability of gastric cancer cells. LC3 cleavage and LC3-GFP puncta were increased by decursin in a time- and concentration-dependent manner. However, the increase in autophagosome formation did not lead to an increase in autophagic flux. Decursin downregulated cathepsin C (CTSC), a lysosomal protease, irrespective of cellular pH. In addition, knockdown of CTSC reduced the mRNA and protein levels of E2F3, resulting in cell-cycle arrest. Decursin also reduced cell growth in both tumor spheroids and patient-derived gastric cancer organoids. Conclusion: The results demonstrated that decursin inhibited tumor growth and progression by suppressing CTSC-mediated autophagic flux and cell-cycle progression. Therefore, decursin is a novel inhibitor of autophagic flux, with potential for the treatment of gastric cancer. Citation Format: Solbi Kim, Mina Joo, Nayoung KIM, Myung-Won Lee, Heung Jin Jeon, Hyewon Ryu, Ik-Chan Song, Hyo Jin Lee. Decursin inhibits gastric cancer cell growth and autophagic flux by suppressing cathepsin C [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4170.

Pancreatic cancer patients have limited treatment options in spite of several advanced treatment strategies. Pancreatic tumors exhibit high basal autophagy compared to other cancers. Several studies including from our lab reported that enhanced autophagy can lead to apoptosis in cancer cells. In this study, we have demonstrated that pimavanserin (PVT) suppresses pancreatic tumor growth by inducing autophagy-mediated apoptosis. Our results indicated that PVT induced apoptosis and reduced the proliferation of pancreatic cancer cells with IC50 ranging between 3-9µM after 24, 48 and 72 hours of treatment. In addition, PVT inhibited the colony formation of pancreatic cancer cells. Treatment of pancreatic cancer cells with increasing concentrations of PVT resulted in a concentration-dependent increase in autophagy as evaluated by acridine orange assay by flow cytometry. PVT induced the expression of autophagy markers ULK1, FIP200, Atg101, Beclin-1, LC3B and p62 in a concentration-dependent manner in several pancreatic cancer cells. Apoptotic effects of PVT in pancreatic cancer cells was validated by increase in cleavage of caspase3 and PARP. Oral administration of PVT suppressed BxPC3 tumor xenografts by 50% in athymic nude mice. In another in vivo experiment, PVT treatment inhibited the growth of orthotopicaly implanted PANC1 tumors by 75%. Autophagy and apoptosis was confirmed in the tumors of PVT treated mice by immunohistochemistry and western blotting. Chronic administration of PVT did not exhibit any general signs of toxicity or behavioral side effects in mice. Moreover, long-term administration of PVT did not alter the clinical chemistry parameters like ALT, AST, total serum protein, calcium and albumin. Collectively, our results indicate that PVT mediated pancreatic tumor growth suppression was associated with induction of autophagy and apoptosis. Since PVT is already available in the clinic with an established safety profile, our results will accelerate its clinical development for pancreatic cancer therapy. Citation Format: Sharavan Ramachandran, Sanjay K. Srivastava. Pimavanserin induces autophagy mediated apoptosis in pancreatic cancer and suppresses pancreatic tumor growth [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1218.

Background: IDE196 is a potent and selective PKC inhibitor targeting the novel (δ, ϵ, η, θ) and classical (α, β) PKC isoforms that has demonstrated preliminary clinical activity in patients with metastatic uveal melanoma (MUM). IDE196 is being further evaluated for safety and efficacy as a monotherapy in a Phase1/2 basket trial in non-MUM patients whose tumors harbor GNAQ/GNA11 hotspot mutations (Clinicialtrials.gov: NCT03947385). Patients with solid tumors harboring PRKC fusions or GNAQ/11 non-hotspot mutations may be eligible after preclinical validation of the activity of these fusions and mutations in non-MUM indications. PRKC gene fusions exist at very low frequencies with various gene partners across multiple indications. Hotspot mutations in GNAQ/GNA11 (codons Q209 and R183) constitutively activate the PKC pathway in 90% of MUM patients and may also occur in non-MUM indications such as colorectal cancer (CRC) and cutaneous melanoma. Furthermore, there are additional non-hotspot mutations found in these GTPases across various non-MUM indications. To help identify patients that may benefit from IDE196 in this basket trial, we assessed the sensitivity of non-MUM cell lines that have PRKC gene fusions or GNAQ/11 hotspot/non-hotspot mutations to IDE196. Methods The most prevalent fusions in patient samples listed in TCGA are the HIF1A-PRKCH (n=20) and RP11-47I22.3-PRKCH (n=3) fusions, therefore cell lines containing PRKCH fusions listed in CCLE were prioritized for cell viability screening using short (3-day) and long-term (10-day) assays after IDE196 treatment. For PD analyses, cells were treated for 3hrs with IDE196. Expression of the fusions and pharmacodynamic modulation of phospho-MARCKS was assessed using western blotting (WB). RNASeq data was used to determine fusion structure, size and whether the fusion was in-frame. Results 8/10 PRKCH fusion lines were highly resistant to IDE196 (IC50>10μM, including HIF1A-PRKCH and RP11-47I22.3-PRKCH fusion lines), whereas 2/10 showed sensitivity at high doses in the long-term viability assay (IC50≥1μM, lines with PRKCH-SPTB or VWA2-PRKCH fusions). PD assessment showed that the PKC substrate, pMARCKS, was decreased in all cell lines by IDE196 at doses similar to those seen in the MUM cell line, 92.1 (≥0.3μM). Fusion protein expression was not detectable by WB consistent with literature suggesting that PKC fusions are unstable (1). In summary, cell lines with clinically prevalent PRKCH fusions were insensitive to IDE196 despite robust PD modulation. Assessment of cell lines harboring GNAQ/GNA11 hotspot/non-hotspot mutations for IDE196 sensitivity is ongoing. (1) An-Angela N. Van, Timothy R. Baffi, Maya T. Kunkel, Corina E. Antal and Alexandra C. Newton, FASEB J. (2018); vol 32, suppl.no. 1, abstract 687.6. Citation Format: Marie Wagle, Nandini Ravindran, Carol O'Brien, Christian Frey, Julie Hambleton, Mark R. Lackner, Zineb Mounir. Preclinical evaluation of PRKC fusions and GNA11/GNAQ mutations as genetic drivers of PKC activation in non-MUM indications to support a phase 1/2 basket trial of IDE196 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4152.

Introduction: Extracellular vesicles (EVs) contain protein and nucleic cargo that has been shown to be reflective of physiological and pathological states, or their originating cells, in many diseases including cancer. Specifically, these EV-associated biomarkers have been shown to fluctuate with the change in pathological processes. As a result, characterization of blood-based circulating EVs have been widely investigated for diagnostic applications such as disease monitoring and treatment selection. The heterogeneity of blood-based EVs present significant challenges to current rapid isolation methods due to enrichment with unwanted sub-populations, e.g. apoptotic bodies, or contamination with lipids, e.g. APOB, for both capture-based chemistry-based isolation methods. We have developed a novel lab-on-a-chip technology for isolation and on-chip characterization of EVs from blood-based matrices using an AC electrokinetics (ACE) methodology. Methods: Blood samples were collected from 10 healthy volunteers and 10 donors with known cancer diagnosis into K2EDTA tubes under IRB approved protocols. Plasma was processed from the blood and stored at -80C. 120 µL of thawed plasma was applied to the microelectrode array flow cell (ExoVerita Flex, Biological Dynamics, San Diego, CA) and the EVs were isolated on the microelectrodes. Following isolation, the flow cell was washed, the isolated material was released from the array and eluted from the flow cell. For comparison, isolation of EVs from the same amount of plasma was performed using a chemistry-based method (ExoQuick Ultra, System Biosciences, Palo Alto, CA) according to manufacturer's instructions. Following isolation, the eluates were evaluated for the presence of EV-associated biomarkers using Western blotting capillary electrophoresis and mass spectrometry (Orbitrap Fusion Lumos, TFS). Nanoparticle tracking analysis (qNano Gold, Izon Science, New Zealand) was performed to determine EV concentration and size distribution. Quantitative qRT-PCR analysis of the purified plasma Evs was performed to confirm presence of EV-bound mRNA. Results: Western Blotting demonstrated that eluates from both ACE-based methodology and chemistry-based isolation contain the expected CD9 and CD63. The ACE-isolated EVs showed a single prominent band for CD63, whereas chemistry-based isolated EVs display multiple smaller CD63-reactive species, which was suggestive of proteolysis. Mass spectrometry analysis confirmed presence of CD9 and CD81 in EVs isolated by ACE method; but not in EVs purified by chemistry-based method. APOB contamination was present in chemistry-based EVs, but not in ACE-isolated EVs. For RT-PCR experiments, EVs were purified from 5 subjects with lung cancer and 3 subjects with melanoma patient plasma using ACE method. Quantitative PCR confirmed presence of mRNA, via successful amplification of the housekeeping genes PGK1 and β-actin, in all ACE-isolated EVs. Conclusions: The novel ACE-based platform successfully demonstrated direct isolation of EVs from plasma while preserving the integrity of protein and mRNA biomarkers. The compatibility of the eluted EVs with multiple downstream technologies, such as mass spectrometry, Western blotting and qRT-PCR, may enable novel biomarker discovery and use of EV-associated biomarkers in diagnostic assays. Citation Format: Rajaram Krishnan, Jean Lewis, David Searson, Orlando Perrera, Alfred Kinana, Heath Balcer, Iryna Clark, Juan Pablo Hinestrosa. Characterization of circulating extracellular vesicles isolated from plasma of cancer subjects using novel AC electrokinetics platform [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2850.

Background The Helicobacter outer protein (HopQ) is a novel surface-located Helicobacter pylori adhesion protein that binds to various human carcinoembryonic antigen-related cell adhesion molecules (CEACAM1, 3, 5, 6). Our aim was to validate fluorescence targeting of colorectal tumors in patient derived orthotopic xenograft models with fluorescently labeled HopQ. Materials/Methods CEACAM binding protein HopQ was conjugated to near-infrared dye IR800CW (LI-COR). Western blotting was performed with various colon cancer cell lysates to determine CEACAM expression. Nude mice (n=4) received orthotopic implantation of patient-derived primary colon tumors and patient-derived colon cancer metastases. Mice were administered either 25 ug or 50 ug of HopQ-IR800CW via tail vein injection and imaged 48 hours later. Intravital images were analyzed using the Image Studio Software Small Animal Imaging Analysis to determine tumor-to-background ratio (TBR). Results Western blotting demonstrated varied expression of CEACAM binding in all 15 patient derived colon cancer lysates. Dose response intravital imaging demonstrated TBR of 1.41 when administered 25 ug of HopQ-IR800CW and TBR 3.78 when administered 50 ug of HopQ-IR800CW. Conclusion CEACAM binding protein HopQ shows promising use for fluorescence imaging of patient derived orthotopic colon cancer. HopQ may be useful for tumor detection for pre-surgical diagnosis and fluorescence-guided surgery. Citation Format: Hannah M. Hollandsworth, Siamak Amirfakhri, Filemoni Filemoni, Verena Schmitt, Gunther Wennemuth, Alexej Schmidt, Robert M. Hoffman, Bernhard B. Singer, Michael Bouvet. Anti-CEACAM binding protein for fluorescence visualization of primary colon cancer in patient-derived orthotopic xenograph mouse models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1658.

Background: Previously, we have identified aptamers through ovarian cancer (OC) tissue microfluidic platform which show high specificity to ovarian cancer tissue and cells. However, the clinical diagnostic and therapeutic roles of these aptamers are still largely unknown. Materials and Methods: Liquid chromatography-mass spectrometry (LC-MS) was used to find binding candidates of aptamer, Tx-01, from serous type ovarian cancer cells. Immunohistochemistry (IHC), clinical survival analysis, western blotting and confocal microscopic images were performed to detect the expression of Tx-01 and HSP70/ Notch1 intracellular domain (NICD) in OC tissues. The functions of Tx-01 were studied in vitro and in vivo experiments. Inhibitors treatment, nuclear protein extraction and western blotting were carried out to demonstrate the possible mechanisms of Tx-01 function. Results: High expression Tx-01 in OC tissues was correlated to poor patients' survival in serous ovarian carcinoma. In vitro experiments showed that Tx-01 has a feature of recognizing membrane-bound HSP70. In the meantime, Tx-01 inhibit HSP70 signal activation via interrupting intracellular HSP70/NICD interaction to reduce cell migration and invasion. Furthermore, Tx-01 inhibited the tumor growth in the mice experiment. Conclusion: Our data showed that Tx-01 acted as a prognostic predictor in serous OC through recognizing the membrane-bound HSP70. Also, Tx-01 reduced the tumor cell migration, invasion through blocking HSP70 signal activation. Thus, our work demonstrated that Tx-01 might be a prognostic biomarker and potential inhibitor in serous OC therapy. Citation Format: Chang-Ni Lin, Yu-Ling Liang, Pei-Ying Wu, Gwo-Bin Vincent Lee, Keng-Fu Hsu. A tissue-microfluidic derived aptamer displays prognostic and therapeutic characteristics in serous ovarian carcinoma through heat shock protein 70(HSP70) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4144.

Purpose: Extracellular vesicles (EVs) are small (50nm-800nm) membrane-enclosed structures with highly heterogeneous content. Recently, the importance of EVs in tumor progression as well as biomarkers has become apparent in many malignancies. However, there has been very limited research done to investigate EVs derived from uveal melanoma (UM). UM is the most common primary intraocular tumor in adults, and it displays a high frequency of metastases to the liver. The aim of this study was (i) to characterize the protein signatures of UM cell line-derived EVs to determine potential biomarkers, (ii) to determine EV uptake by recipient cell lines, (iii) to analyze the activation of the MAPK pathway in the recipient cells, and to examine the potential of UM EV-educated BRCA1 deficient fibroblasts (Fibro-BKO) to develop tumors in NOD-SCID mice. Methods: EVs were isolated from the conditioned media of UM cell lines (MP41, MP46, 92.1, MEL285, MEL270, OMM2.5) and normal choroidal melanocytes cells using an ultracentrifugation method. Western blots and immunogold-labelling Transmission Electron Microscopy were employed to validate various EV markers (CD63, CD81 and TSG101). Proteomic analysis by mass spectrometry was performed to determine the UM-derived EV proteome signature that could elucidate potential key players in UM metastasis. After exposure to EVs, recipient cells were analysed for the activation of downstream signaling pathways, and were inoculated in NOD-SCID mice to analyze their behaviour. Results: We observed 2069 proteins in EVs using Scaffold Viewer. FUNRich analysis revealed that 95% of these EV proteins were found in the Vesiclepedia database. Gene Ontology analysis confirmed the presence of exosomal markers (CD81, TSG101and Syntenin) as well as cell-cell adhesion-related proteins. The DAVID Functional Annotation chart revealed proteins related to endocytosis, PI3K-Akt signaling pathway and focal adhesion. In addition, we observed high hits in HSP90 and HSP70, well-known biomarkers in UM. Integrin alpha V, which are known for preparing premetastatic niches in the liver environment, were also present. Western blot results confirmed the increase in MAPK pathway in the recipient cells. Furthermore, UM EVs-educated Fibro-BKO transformed and gave rise to tumors in vivo. Conclusion: In conclusion, our study reports an essential step towards understanding protein emission through EVs from UM cells. Our analyses revealed UM EVs cargo candidates that may play a role in pre-metastatic niche formation and metastasis in specific organs, such as the liver. The in vivo study model confirmed that EVs derived from UM are capable of inducing tumorigenesis. We believe that our data pave the way to the application of liquid biopsy to the monitoring of UM-affected patients. Citation Format: Thupten Tsering, Alexander Laskaris, Mohamed Abdouh, Prisca Bustamante Alvarez, Goffredo Arena, Julia Valdemarin Burnier. Characterization of extracellular vesicles derived from uveal melanoma cell lines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6455.

G-quadruplexes are noncanonical four-stranded DNA or RNA secondary structures that have emerged as novel therapeutic targets. MYC is one of the most important oncogenes and is overexpressed in a number of human cancers. The G-quadruplex formed in the MYC oncogene promoter (MycG4) is a transcriptional silencer and amenable to small molecule targeting. Indenoisoquinolines are human topoisomerase I inhibitors with improved physicochemical and biological properties as compared to the clinically used camptothecin anticancer drugs. Three indenoisoquinolines, indotecan (LMP400), indimitecan (LMP776), and LMP744, have entered phase I clinical trials in adults with relapsed solid tumors and lymphomas. However, some indenoisoquinolines showed potent anticancer activity without being strong topoisomerase I inhibition activity, suggesting additional mechanisms of action. Herein, we demonstrate that a large number of anticancer indenoisoquinolines strongly bind and stabilize MycG4 in vitro, using fluorescence, nuclear magnetic resonance (NMR), and circular dichroism (CD) spectroscopy, as well as gel electromobility shift assay (EMSA). In addition, MycG4-interactive indenoisoquinolines lower MYC mRNA and protein levels in vivo, as shown by western blotting and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) experiments. These results indicate that targeting the MYC promoter G4 to downregulate MYC is a likely mechanism of action for the anticancer activities of these particular indenoisoquinolines. In addition, structure-activity-relationships of MycG4 recognition by indenoisoquinolines were investigated and elucidated the essential structural requirements for strong binding. The analysis of indenoisoquinoline derivatives for their MYC inhibitory, topoisomerase I inhibitory, and anticancer activity led to the discovery of a synergistic effect of MYC inhibition and topoisomerase I inhibition on anticancer activity. In conclusion, our result reveals MYC downregulation by MycG4 binding as a new mechanism of action for anticancer indenoisoquinolines. Moreover, we suggest dual targeting of MycG4 and topoisomerase I as a novel strategy for cancer therapy. Citation Format: Kaibo Wang. Dual targeting MYC G-quadruplex and topoisomerase I: Lead discovery indenoisoquinolines for cancer therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6433.

INTRODUCTION: Epithelial ovarian cancer (EOC) represents the gynecologic cancer with the highest mortality rate. Despite a high initial response to platinum (PT)-based chemotherapy, the majority of patients with advanced EOC relapses and develops chemo-resistance that results in treatment failure and poor prognosis. Therefore, understanding the mechanisms underlying PT-resistance and finding strategies to overcome them are urgently needed. METHODS: Three isogenic models of PT-resistance generated by our group from EOC cell lines (TOV-112D, OVSAHO and MDAH) (Sonego M. et al, 2017), were characterized by a proteomic approach taking advantage of two-dimensional differential in gel electrophoresis (2-D DIGE) followed by Mass Spectrometry. Proteomics data were also analyzed by the ingenuity pathway analysis (IPA) software. Synergistic anti-proliferative activity was evaluated by calculating combination indexes (CI) by the Chou-Talalay method and colony formation assay. Apoptosis was measured by flow-cytometry and by western blotting evaluation of caspase-3 and PARP cleavage. In vivo experiments were performed on xenograft model in athymic mice. RESULTS: We identified 23, 24 and 20 differentially expressed proteins in PT-resistant TOV-112D, OVSAHO and MDAH respectively, compared with parental cells. A relevant relationship between all the identified proteins in the three models was highlighted by IPA. Interestingly, eight of the identified proteins, HSP7C, HNRPL, ATPA, EFTU, PHB, HNRPK, LMNA and PHGDH, shared at least within two cell lines, are all included in one main network related with cancer. Notably, the protein chaperone HSP90 emerged as a central hub of this network and its up-regulation was observed in all the PT-resistant cell lines compared with parental counterparts. Further molecular characterization also showed overexpression of HSF1 transcription factor, a relevant HSP90 activator and client protein, in PT-resistant cells. On this bases, we evaluated the effect of cisplatin in combination with two HSP90 inhibitors (17AAG or ganetespib), both in phase II/III clinical development in cancer patients, observing synergistic anti-proliferative activity and reduced colony formation, particularly in PT-resistant cells. These data were confirmed in primary cultures from PT-Resistant ovarian cancer patients. Furthermore we confirmed for the first time in vivo the synergistic antitumor effect of cisplatin and ganetespib combination in TOV112D PT-resistant xenograft model. Increased pro-apoptotic effect and DNA-damage induction by the combination treatment compared to untreated or single agents treated tumors was confirmed both in vitro and in vivo. CONCLUSIONS: Our data suggest an innovative antitumor strategy based on the combination of platinum compounds with HSP90 inhibitors, to re-challenge PT-resistant EOC, that warrants further clinical evaluation. Citation Format: Rita Lombardi, Maura Sonego, Laura Addi, Federica Iannelli, Francesca Capone, Maria Serena Roca, Maria Rita Milone, Alice Costa, Francesca Bruzzese, Biagio Pucci, Gustavo Baldassarre, Alfredo Budillon. HSP90 identified by a proteomic approach as druggable target to reverse platinum-resistance in ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5270.

Purpose: This aim of study was to investigate the clinicopathologic significance and potential role of histone methyltransferase SET8 in the progression of gastric carcinoma (GC). Experimental Design: SET8 was screened out by gene microarray in gastric cell lines with or without miR-192/-215 inhibitors or mimics. The effects of SET8 on cell proliferation, invasion, and metastasis were examined using EdU assays, invasion studies, subcutaneous tumor experiments, and tail-vein injection in nude mice. Immunohistochemical staining was performed to detect SET8 expression in GC tissues, and real-time PCR was performed to examine the correlations between miR-192/-215 and SET8. SA-β-Gal staining assays were performed to identify the occurrence of senescence. Senescence-associated proteins were examined by real-time PCR and Western blotting. Results: SET8 regulated by miR-192/-215 displayed decreased expression in GC. Inhibition of miR-192/-215 resulted in weakened proliferation, migration, and invasion in GC. The 3'UTR of SET8 was targeted by miR-192/-215, and SET8 siRNA treatment successfully rescued the biological phenomenon. SET8 was found to trigger oncogene-induced senescence (OIS) in GC in vivo and in vitro, highlighting the functional mechanism of the miR-192/-215/SET8 axis in GC. During OIS, p53 and p21 were key genetic switch points. Taken together, these data show that the miR-192/-215/SET8/p53 circuit is a key regulator of GC promotion and is highly associated with proliferation, migration, and metastasis. Conclusions: Our findings reveal that SET8 functions via the OIS-signaling pathway as a negative regulator of metastasis. This provides an experimental rationale for investigation of the OIS-signaling pathway as a potential therapeutic target in GC. Supported by: National Nature Science Foundation of China (81772592) to Z.J.; National Nature Science Foundation of China (81871969) to X.Z.; National Natural Youth Science Foundation of China (31601028) to Y.P.; Guangdong Provincial Key Laboratory of Regional Immunity and Diseases (2019B030301009) to Z.J. Citation Format: Xiaojing Zhang, Yin Peng, Fan Hu, Yuli Gao, Jin Zhe, Stephenj Meltzer. Histone methyltransferase SET8 is regulated by miR-192/-215 and induces oncogene-induced senescence via p53-dependent DNA damage in human gastric carcinoma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3834.

Introduction: Although recent studies showed that HER3 plays a key role in the pathophysiology of HER2-amplified breast cancers, its role in other cancers driven by HER2 amplification remains unknown. This study aimed to define the importance of HER3 signaling in other types of HER2-amplified cancer. Method: The expression and signaling activity of HER2, HER3, and downstream pathway proteins were studied in cell panels representing HER2-amplified cancers of the breast, bladder, colon and rectal, stomach, esophagus, lung, tongue, and endometrium along with controls lacking HER2 amplification. The time-dependent compensatory upregulation of HER3 in response to HER2 inhibitor therapy was assayed by western blotting and the apoptotic effects of HER2 inhibition were assayed by flow cytometry. The functional significance of HER3 in promoting tumorigenic growth in xenograft assays was determined in selected HER2-amplified cancer cell types by shRNA knockdown. Results: Many HER2-amplified cancers of the breast (HCC1569, HCC1954, HCC1419, UACC732, SUM190), bladder (CLS-439), stomach (N87, OE19), esophagus (TE4, KYSE410), lung (Calu3), and endometrium (USPC-ARC1, USPC-ARC2) showed HER3 activation, whereas HER2-nonamplified cancers of the same organs did not. With some exceptions, all HER2-amplified cancer cells undergo apoptosis when HER2 signaling is inhibited by lapatinib. Lapatinib induced apoptosis correlated with HER3 activation. Tumor growth was reduced upon HER3 knockdown in HER2-amplified cancers other than breast cancers, particularly cancers with constitutive HER3 phosphorylation. Significance: These results suggest that HER3 plays a functionally significant role in HER2 amplified cancers other than breast cancers, and may underlie resistance to HER2-targeting in these cancers. Combined HER2-HER3 targeting may be of value across the spectrum of different types of cancers. Citation Format: Avisek Majumder, Veronica Steri, Fernando Salangsang, Mark Moasser. The role of HER3 in HER2-amplified cancers other than breast cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-326.

Cancer phosphoproteomics can provide insights regarding kinases that can be targeted for therapeutic applications. Monitoring the phosphoproteomics in cancer is expected to play a key role in optimizing treatments with kinase inhibitors. Clinical phosphoproteomics in surgical tissues and patient-derived models has been studied intensively. However, the reported data may not accurately reflect the phosphosignaling status in patients due to the effect of ischemia occurring during surgery or changes in the characteristics of cancer cells when establishing the models. In contrast, endoscopic biopsies have an advantage for clinical phosphoproteomics because they can be rapidly cryo-preserved. We aimed to develop a highly sensitive method for phosphoproteomics in endoscopic biopsies of gastric cancer.Three tumor/normal biopsies from gastric cancer tissue were obtained by endoscopy, and subjected to our optimized phosphoproteomics. Phosphopeptides were enriched with an immobilized metal affinity chromatography, and labeled with Tandem Mass Tag reagent. Quantified phosphosites were compared between the pairs of tumor/normal biopsies within same patient. Cancer-specific activated pathways and kinases were identified by pathway enrichment analysis and kinase-substrate enrichment analysis.Our protocol enabled the identification of more than 10,000 class-1 phosphosites from endoscopic biopsies. A comparison between samples from cancer tissue and normal mucosa demonstrated differences in the phosphosignaling, including biomarkers of response to DNA damage. Finally, cancer-specific activation of DNA damage response signaling was validated by additional phosphoproteomics of other patients and western blotting of gastric cancer/normal cells. In summary, our pioneering approach will facilitate more accurate clinical phosphoproteomics in endoscopic biopsies, which can be applied to monitor the activities of therapeutic kinases and, ultimately, can be a useful tool to precision medicine. Citation Format: Jun ADACHI, Yuichi Abe, Hidekazu Hirano, Hirokazu Shoji, Daigo Gunji, Kazufumi Honda, Narikazu Boku, Takeshi Tomonaga. Comprehensive characterization of the phosphoproteome of gastric cancer from endoscopic biopsy specimens [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3772.

Metastasis is a major cause of death in epithelial cancers, largely because of a lack of effective treatments. The metastatic inhibitor MBQ-167 was developed to target the Rho GTPases Rac and Cdc42, which promote cell cycle progression, proliferation, survival, migration/invasion, and thus, metastasis. Our published data show that MBQ-167 inhibits the activation of Rac and Cdc42 with ~100 nM IC50, and subsequently inhibits their effector p21-activated kinase (PAK) activation by autophosphorylation. In metastatic cancer cells, MBQ-167 induces a loss of cell polarity, followed by detachment from the substrate, and subsequent apoptosis (anoikis). The objective herein was to determine the potential off-target effects of MBQ-167. Therefore, the effect of MBQ-167 on the triple negative breast cancer kinome was investigated using a Human Phospho-Kinase Array for 45 phospho proteins. MDA-MB-231 cells treated with MBQ-167 for 24 h at 200 nM demonstrated no difference in phosphorylation of off-target kinases. However, phospho (p)-CREB and p-c-Jun transcription factor levels increased by ~3-fold in the cells that remained attached following MBQ-167 treatment. This was confirmed by Western blot for p-CREB and p-c-Jun as well as their transcriptional targets (Cyclin D1, survivin, ZEB1). However, a time-course analysis via Western blot revealed that this increase in CREB and c-Jun activity is transient. Western blotting of the attached and detached cell populations of MDA-MB-231 cells following MBQ-167 treatment for 24, 48, or 96 h demonstrated decreased c-Jun/p-c-jun, and CREB and Jun targets in the detached cell population. Moreover, the detached cells demonstrated additional decreases in phospho forms of ERK1/2, Akt, mTOR, beta catenin and STAT3, which have all been implicated in Rac/PAK signaling; as well as reduced expression of the anti-apoptotic proteins Bcl2, BCL-XL, and MCl-1. Therefore, we posit that the initial upregulation of transcription factors in response to MBQ-167 is a transient reaction to its strong Rac/Cdc42/PAK inhibition. Prolonged treatment with MBQ-167 causes a massive deregulation of cancer drivers and pro-survival proteins to ultimately result in anoikis. We attempted to create a MBQ-167 resistant cell population by treating MDA-MB-231 cells with ascending concentrations of MBQ-167 over time, but were unable to recover any live cells. Therefore, we conclude that MBQ-167 is a direct and specific inhibitor of Rac and Cdc42 and that potential off-target effects of MBQ-167 are only temporary, where 100% of the metastatic cancer cells treated with MBQ-167 ultimately undergo apoptosis. Collectively, our data on the mechanism of action of MBQ-167 and its efficacy in vitro and in vivo in mouse models of metastatic cancer demonstrate that MBQ-167 is a viable anti-metastatic cancer inhibitor for further development as an experimental therapeutic. Citation Format: Hector M. Picon, Maria Del Mar Maldonado, Surangani F. Dharmawardhane Flanagan. Investigating potential off-target effects of the dual Rac and Cdc42 inhibitor MBQ-167 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 623.

Background: KRAS is a well-established cancer driver. Clinically successful therapeutics have not yet been developed despite its disease burden, suggesting that KRAS-driven cancers are more complicated than previously thought. Aim: Our goal is to identify the global changes in KRAS interaction patterns that occur in disease states. This is accomplished by mapping the dynamic interactome of both WT and oncogenic KRAS isoforms using the Mammalian Membrane Two-Hybrid (MaMTH) assay, which is suitable for identification of protein interactors of virtually any integral membrane or membrane-associated protein. Methods: To probe the protein-protein interactions (PPIs) of KRAS, we used the Mammalian Membrane Two-Hybrid (MaMTH) assay, a split-ubiquitin-based two-hybrid system that can be applied to any cell line. This assay is suited for PPI detection using both low- to-medium throughput array and high-throughput large-scale PPI screening. We generated stably expressing “bait”-tagged KRAS constructs in HEK293T cells that contained the MaMTH reporter system. KRAS baits, namely KRAS-WT, -G12D, -G12V, and -Q61H, were characterized using signaling assays as well as within the MaMTH system using “prey”-tagged CRAF interaction control, Western blotting, and immunofluorescence techniques. Currently, we are performing unbiased screening for PPIs of KRAS using the human ORFeome (hORFeome) library consisting of approximately 13,000 fully sequenced human ORFs. Results: We first validated compatibility of KRAS in the MaMTH assay through several means. Oncogenic KRAS variants showed increased interaction signal with CRAF (a known KRAS binding partner) compared to KRAS-WT. This corresponded with upregulated MAPK pathway activation by oncogenic KRAS, as determined from increased pERK levels via Western blotting. KRAS bait expression levels were similar across all variants, suggesting that these findings were not due to differential expression of KRAS isotypes. Additionally, we confirmed that the KRAS bait correctly localizes to the plasma membrane, consistent with previous literature. After establishing KRAS compatibility with the MaMTH assay, we have begun unbiased screening of the hORFeome against KRAS baits in the WT, G12D, G12V, and Q61H isoforms. Conclusions: We have characterized KRAS compatibility with the MaMTH assay. Currently, we are performing large screening protocols for high-throughput detection of PPIs of KRAS using MaMTH. Citation Format: Ingrid Claudia Grozavu, Jamie Snider, Anna Lyakisheva, Igor Stagljar. Mapping KRAS signaling pathways using the Mammalian-Membrane Two-Hybrid (MaMTH) assay to elucidate novel therapeutic targets [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A37.

Background: A significant proportion of breast cancers (BC) that express estrogen receptor α (ERα) will initially respond to antiestrogens or aromatase inhibitors. However, endocrine therapy resistance is common with progression to incurable metastases. A significant proportion (~39%) of therapy resistant tumors acquires ERα mutations which enables estrogen-independent constitutive interaction of ERα with coregulators, transcription and resistance to endocrine therapy. Drugs that specifically target and block mutant-ERα (MT ER)-coregulator driven signaling are urgently needed to treat therapy-resistant BC. Methods: Using an innovative strategy of oligobenzamide-based peptidomimetics (small synthetic organic molecules), we developed an estrogen receptor modulator ERX-41 that disrupt the interactions between MT ERα and critical coregulators. Effect of ERX-41 was evaluated using BC models that express wild type (WT) ERα (MCF-7, ZR-75) and BC models with acquired resistance (MCF-7-TamR, MCF-7-LTLT), and engineered models that express ERα mutations (MCF-7-MT ERα-D538G, MCF-7-MT ERα-Y537S, ZR-75-MT ERα-D538G, ZR-75-MT ERα-Y537S). Mechanistic studies were performed using RNA-Seq, Mass spectrometry, immunoprecipitation, Western blotting, RT-qPCR and reporter gene assays. The in vivo efficacy of ERX-41 was examined using xenograft, and patient-derived explant (PDEX) models. Results: ERX-41 exhibited broad and potent activity (IC50 between 50-125 nM) against both WT and MT ERα driven BC models in in vitro assays. ERX-41 had no activity against benign breast epithelial cells. Mechanistic studies confirmed that ERX-41 modulates multiple ERα functions including alterations in ERα transcription, down regulation of ERα and ERα-signaling. Using immunoprecipitation mass spectrometry (IPMS) analyses, we found that ERX-41 disrupts the interaction of ERα with a number of protein binding partners. RNA-Seq analyses of ERX-41 treated BC cells indicated upregulation of endoplasmic reticulum stress pathways. Furthermore, mechanistic studies confirmed that anti-proliferative effect of ERX-41 is directly related to its ability to induce endoplasmic reticulum stress and subsequent unfolded protein response. Treatment of ERX-41 decreased the proliferation (Ki-67 staining) and increased apoptosis (TUNEL staining) in PDEX models. ERX-41 showed potent activity against WT ERαand MTERα xenograft models but no effect on mouse liver or body weight. Histologic evaluation of these tumors showed dramatically decreased Ki-67 proliferation indices. Conclusions: Collectively, our data indicate that ERX-41 could effectively target both the WT and MT ERα driven signaling and could be clinically translated for the treatment of therapy-resistant BC. Supported by NIH grant CA223828. Citation Format: Ratna Vadlamudi, Suryavathi Viswanadhapalli, Xihui Liu, Tae-Kyung Lee, Gangadhara R Sareddy, Shihong Ma, Mengxing Li, Ben Ho Park, Rajeshwar R Tekmal, Jung-Mo Ahn, Ganesh V. Raj. ERX-41, a novel drug to target and block mutant-ERα-coregulator driven signaling [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-05.

The repertoire of antigens associated with the development of an autoimmune response in breast cancer has relevance to detection and treatment strategies. We have investigated the occurrence of autoantibodies associated with the development of triple-negative breast cancer (TNBC) in the before diagnosis setting and in samples collected at the time of diagnosis of TNBC. Lysate arrays containing protein fractions from the TNBC MDA-MB-231 cell line were hybridized with TNBC plasmas from the Women's Health Initiative cohort, collected before clinical diagnosis and with plasmas from matched controls. An immune response directed against spliceosome and glycolysis proteins was observed with case plasmas as previously reported in estrogen receptor+ breast cancer. Importantly, autoantibodies directed against networks involving BRCA1, TP53, and cytokeratin proteins associated with a mesenchymal/basal phenotype were distinct to TNBC before diagnosis samples. Concordant autoantibody findings were observed with mouse plasma samples collected before occurrence of palpable tumors from a C3(1)-T triple negative mouse model. Plasma samples collected at the time of diagnosis of stage II TNBC and from matched healthy controls were subjected to proteomic analysis by mass spectrometry to identify Ig-bound proteins yielding a predominance of cytokeratins, including several associated with a mesenchymal/basal phenotype among cases compared with controls. Our data provide evidence indicative of a dynamic repertoire of antigens associated with a humoral immune response reflecting disease pathogenesis in TNBC. Cancer Res; 75(16); 3246–54. ©2015 AACR.

Multiple myeloma pathogenesis is driven by the MYC oncoprotein, its dimerization with MAX, and the binding of this heterodimer to E-Boxes in the vicinity of target genes. The systemic utility of potent small molecule inhibitors of MYC-MAX dimerization was limited by poor bioavailability, rapid metabolism, and inadequate target site penetration. We hypothesized that new lipid-based MYC-MAX dimerization inhibitor prodrugs delivered via integrin-targeted nanoparticles (NP) would overcome prior shortcomings of MYC inhibitor approaches and prolong survival in a mouse model of cancer. An Sn 2 lipase-labile prodrug inhibitor of MYC-MAX dimerization (MI1-PD) was developed which decreased cell proliferation and induced apoptosis in cultured multiple myeloma cell lines alone (P < 0.05) and when incorporated into integrin-targeted lipid-encapsulated NPs (P < 0.05). Binding and efficacy of NPs closely correlated with integrin expression of the target multiple myeloma cells. Using a KaLwRij metastatic multiple myeloma mouse model, VLA-4–targeted NPs (20 nm and 200 nm) incorporating MI1-PD (D) NPs conferred significant survival benefits compared with respective NP controls, targeted (T) no-drug (ND), and untargeted (NT) control NPs (T/D 200: 46 days vs. NT/ND: 28 days, P < 0.05 and T/D 20: 52 days vs. NT/ND: 29 days, P = 0.001). The smaller particles performed better of the two sizes. Neither MI1 nor MI1-PD provided survival benefit when administered systemically as free compounds. These results demonstrate for the first time that a small molecule inhibitor of the MYC transcription factor can be an effective anticancer agent when delivered using a targeted nanotherapy approach. Mol Cancer Ther; 14(6); 1286–94. ©2015 AACR.

Background: In a recent study based on cDNA microarray data of breast cancer patients, we could show that the mRNA expression of some glycosylation enzymes involved in synthesis and trimming of N-glycansis significantly associated with metastasis and recurrence in breast cancer patients suggesting an important role of N-glycosylation in tumour progression (Milde-Langosch et al., Breast Cancer Res Treat 2014). The aim of the present study was to further investigate this issue on a protein and glycoprotein level (1.) by western blot analysis of selected glycosylation enzymes and (2.) by lectin histochemistry using lectins which bind to O- and/or N-glycans. Methods: In order to confirm the role of two selected glycosylation enzymes, ribophorin (RPN1) and mannosidase (MAN1A1) on protein level, we analyzed their expression in a cohort of 196 breast cancer samples by western blot analysis. In addition, we studied tumour cell binding of various lectins, which recognize diverse O- or N-glycans (UEA1, HPA, PNA, GNA, PHA-L), using a tissue microarray with up to 293 evaluable breast cancer samples. These results were correlated with recurrence-free survival (RFS) and overall survival (OS) and data on vascular and lymphatic invasion. Results: By western blot analysis, the negative prognostic significance of ribophorin (RPN1), a key enzyme of N-glycosylation, was corroborated on a protein level. For MAN1A1 which is involved in trimming of N-glycan structures, two isoforms with different molecular weight could be detected on western blots, showing contradictory associations with OS. Protein levels of RPN1 and total MAN1A1 showed significant correlations to the respective mRNA expression data. By lectin histochemistry, we found significant correlations of lectin binding to lymph node metastasis, vascular invasion and lymphangiosis in breast cancer samples. Binding of PHA-L and GNA (binding to N-glycans) and PNA (binding to O- and N-glycans) was significantly associated with vascular invasion, whereas PNA and PHA-L binding correlated with lymph node involvement. In addition, PNA and HPA reactivity was associated with microscopically detected lymphangiosis. PNA binding also correlated with shorter RFS and OS. Conclusion: While the role of O-glycans in breast cancer metastasis has already been described, our data point to an additional strong impact of N-glycosylation on metastasis and progression of mammary carcinomas. Citation Format: Karin Milde-Langosch, Tosca Karius, Dina Schütze, Harriet Wikman, Isabell Witzel, Leticia Oliveira-Ferrer, Volkmar Müller, Udo Schumacher. N-glycosylation changes in tumour cells are associated with vascular invasion and metastasis in breast cancer patients [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-21.

Background Insulin-like growth factors (IGF). IGFs are potent mitogens for breast epithelial cells that modulate the action of AKT, which has been reported to be associated with tamoxifen resistance. To date, however, anti-IGF strategies have proved disappointing in clinical trials We have investigated whether the IGFBP family of proteins, which modulate the activity of IGF, may play a role in tamoxifen resistance, opening up the route for alternative anti-IGF based therapies. Methodology We investigated the expression of IGF axis genes in parental and tamoxifen-resistant (TamR) MCF-7 cells using qRT-PCR. Gene and protein expression was confirmed using ELISA, Western, and ligand blotting. shRNA transfection was used to silence candidate IGF axis genes in both cell lines. Cell sensitivity 4-hydroxytamoxifen (4-HT) was investigated by WST-1 cell proliferation assay. Results IGF-IR, IGF-2R, IGFBP-2, -4 and -5 genes had the highest expression levels. IGFBP-5 was down-regulated 7-fold while IGFBP-2 was up-regulated by 2-fold in TamR v wt cells. Changes in IGFBP-5 and IGFBP-2 gene expression were mirrored in protein levels measured in conditioned media by ELISA, Western and Ligand blot. Importantly knock down of IGFBP-2 in TamR cells restored sensitivity to 4-HT suggesting a causal role for IGFBP-2 in the acquisition of tamoxifen resistance. Conclusion IGFBP-5 and IGFPB-2 are reciprocally regulated on the acquisition of tamoxifen resistance by MCF-7 cells. Preliminary studies suggest that IGFBP-2 may play a role in the development of tamoxifen resistance in vitro. Citation Format: Yousef M Hawsawi, James Beattie, Reem El-Gendy, Valerie Speirs, Christopher Twelves. The insulin like growth factor axis and development of tamoxifen resistance in breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-12-12.

Background: Telomeres are tandem repeats of sequences present at the end of the chromosomes that maintain chromosomal integrity. After repeated cell division, telomeres shorten to a critical level, triggering replicative senescence or apoptosis, which is a key determinant of cellular aging. Short telomeres also contribute to genome instability and are a hallmark of many cancers. There are several methods for estimating telomere length (TL) from extracted DNA samples. Southern blot is accurate but requires a large quantity of DNA and is expensive. qPCR is cost-effective and requires a small quantity of DNA and is therefore widely used for large-scale epidemiologic studies; however, it typically requires triplicates. We describe a novel multiplexed probe-based non-PCR method for TL measurement. Methods: A small amount of DNA (∼50 ng) is hybridized to telomere repeat sequence–specific probes (T) and a reference single gene probes (R). T and R signals are detected from a single reaction well containing the same input DNA. Branching DNA technology is used to amplify the signal, which is detected by Luminex technology. Results: The intra- and interassay CV (∼3% and ∼5%, respectively) shows the precision of the new assay and the measurements from single well correlated well with traditional single-plex qPCR run in triplicate (r = 0.7 to 0.8). The assay was also validated in an independent set of samples using Southern blot (r = 0.74). Conclusion: We describe a novel assay for TL assessment using the Luminex platform. Impact: This may offer an alternative cost-efficient way to study TL in extracted DNA samples. See all the articles in this CEBP Focus section, “Biomarkers, Biospecimens, and New Technologies in Molecular Epidemiology.” Cancer Epidemiol Biomarkers Prev; 23(12); 2667–72. ©2014 AACR.

Purpose: NAT10 (N-acetyltransferase 10) is a nucleolar protein, but may show subcellular redistribution in colorectal carcinoma. In this study, we evaluated membranous staining of NAT10 in colorectal carcinoma and its clinical implications, and explored the mechanism of regulation of NAT10 redistribution. Experimental Design: The expression and subcellular redistribution of NAT10, β-catenin, E-cadherin, and GSK-3β were evaluated by immunohistochemistry in 222 cases of colorectal carcinoma. Regulation of NAT10 and its influence on cell motility were analyzed with inhibitors of GSK-3β, transfection of wild-type or kinase-inactivated GSK-3β, or expression of various domains of NAT10, and evaluated with immunofluorescence, Western blotting, and Transwell assays. Results: NAT10 localized mainly in the nucleoli of normal tissues, and was redistributed to the membrane in cancer cells, particularly at the invasive “leading edge” of the tumor. This correlated well with nuclear accumulation of β-catenin (P < 0.001; χ2 = 68.213). In addition, NAT10 membrane staining reflected the depth of invasion and tendency to metastasize (all P values < 0.001), and was associated with a poorer prognosis (P = 0.023; χ2 = 5.161). Evaluation of the mechanism involved demonstrated that subcellular redistribution of NAT10 may result from its increased stability and nuclear export, which is brought about by inhibition of GSK-3β. Moreover, redistribution of NAT10 induces alteration of cytoskeletal dynamics and increases cancer cell motility. Conclusion: The subcellular redistribution of NAT10 can be induced by decreases in GSK-3β activity. This redistribution increases cancer cell motility, and is, thus, correlated with invasive potential and poorer clinical outcome. This finding suggests that NAT10 may be a useful prognostic marker and potential therapeutic target in colorectal carcinoma. Clin Cancer Res; 20(17); 4717–29. ©2014 AACR.

The majority of patients with metastatic breast cancer on Trastuzumab, an anti-HER2 monoclonal antibody, generally develop resistance to the drug within a year after initiation of the treatment. Therefore, there is an unmet need for a novel agent to treat HER2 overexpressing breast cancers. Here we described a novel anti-HER2 humanized monoclonal antibody, 19H6-hu, which binds to HER2 ECD with high affinity and could inhibit the proliferation of multiple HER2-overexpressing cancer cell lines as a single agent, though, to a lesser degree when compared to Trastuzumab alone. Interestingly, when in combination with Trastuzumab, 19H6-hu exhibited much stronger potency in inhibition of BT474, N87 and SKBR3 cell growth relative to that of Perjeta in combination with Trastuzumab as well as in suppression of tumor growth in vivo when compared to Trastuzumab alone. Alanine scanning demonstrated that the antibody binds to an alpha-helix located within the domain III and in proximity to the domain IV of HER2 ECD, which is different from the known binding sites of Trastuzumab and Perjeta. Western blotting further confirmed that 19H6-hu in combination with Trastuzumab is more efficacious in blocking phosphorylation of ERK and phosphorylation of HER2 at Y1248 when compared to Trastuzumab in combination with Perjeta or applied alone. Thereby, our results highlight the functional variability of HER2 domains and provide a new insight into the design of HER2-targeting agents. Citation Format: Haomin Huang, xuesai zhang, jianhe Chen, Le Zhao, qingrou Li, zhenping zhu. A novel anti-HER2 antibody that displays superior synergetic effects in Trastuzumab mediated HER2-overexpressing tumor arrest [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 355.

Aviscumine, a recombinant mistletoe lectin I, is produced in E. coli and has been evaluated for its antitumor activities in various experimental in-vitro and in-vivo tumor models as well as in clinical trials. A phase II trial demonstrated the safety and efficacy of Aviscumine in pretreated patients with metastatic melanoma (stage IV). However, the mechanism(s) underlying the effect of Aviscumine in melanoma is inconclusive. Here, the antitumor activities and relevant mode of actions of Aviscumine were investigated in human melanoma A375, mouse melanoma Yummer and B16 cells as well as their relevant xenograft animal models. Cells were treated with Aviscumine (0-20 ng/mL) for 48 and 72 hrs, and cell proliferation was measured by MTT assay. Alteration of cell growth regulatory proteins and cell signaling proteins were determined by Reverse Phase Proteomic Array (RPPA) and validated with western blotting. Aviscumine exerted much stronger anti-proliferative activity in both A375 and Yummer cells with IC50 of 0.18 ± 0.03 ng/ml and 0.41 ± 0.06 ng/ml, respectively than that of B16 cells (IC50 15.93 ± 3.48 ng/ml). In A375 cells, Aviscumine significantly increased subG0/G1 population suggesting Aviscumine treatment led to apoptotic and necrotic cell death. Additionally, downregulation of various proteins associated with apoptotic cell death (pCDK1, pRB, MCl-1) was also observed by RPPA. Furthermore, Aviscumine significantly decreased c-Myc protein expression in a concentration-dependent manner measured by both RPPA and western blot. Interestingly, baseline c-Myc protein expression was much higher in the Aviscumine-sensitive A375 and Yummer cells than that in the Aviscumine-resistant B16 cells. Finally, the effects of Aviscumine on tumor growth were tested via subcutaneous injection (30 ng/kg, twice per week for three weeks) in mice bearing A375 or B16 melanoma. At 3 weeks, A375 tumors were markedly smaller in Aviscumune treated mice (383.8 ± 102.2 mg) than in the control-treated group (922.4 ± 296.1 mg). c-Myc protein expression was also significantly reduced (62% vs. control) in Aviscumine treated A375 tumors, as were levels of multiple downstream metabolites (pyruvate, malate, and glutamate) (p< 005). In contrast, similar to the in vitro data, Aviscumine treatment showed no tumor growth inhibitory effects in B16 tumor-bearing mice evidenced by minimum reduction on terminal tumor weight between vehicle control (1455 ± 430 mg) and Aviscumine treated group (1323 ± 270 mg). Together, these preliminary data implicate c-Myc as both a target and predictive biomarker for Aviscumine elicited antitumor activity. Citation Format: Peiying Yang, Tara Conway, Patrea Rhea, Dongmei Chen, Bo Wei, Jibin Ding, Hans Lentzen, Jennifer McQuade. Aviscumine (ME-503) suppresses the growth of melanoma by potentially targeting c-Myc pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1861.

Background: Although interfering with the actions of microtubule-associated proteins (MAP’s) is successful against ovarian epithelial carcinoma (OVCA), knowledge of the MAP’s in OVCA cells is lacking. We sought to identify cancer-specific MAP’s in OVCA cells.Methods: Four human OVCA cell lines, primary OVCA tissue, ascitic cells and metastases from 9 patients and normal ovarian tissue from six women were compared to control human and rat brain tissue, uterine myomas (benign tumors) and rat and human non-neural organs. Modified Western blotting using panels of anti-microtubule-associated protein monoclonal antibodies were used. An in-vitro brain and OVCA mixing and recovery experiment was carried out to assess possible degradation of MAP1a by OVCA. A paclitaxel-driven tubulin assembly assay was performed to determine whether the OVCA protein has microtubule-associated protein properties. The transcriptome of CSP-80 was sequenced using primer extension sequencing. Results: (1) In contradistinction to the 350 kDa MAP1a present in control brain extracts, only an 80 kDa molecule with MAP1a immune-reactivity was found in OVCA cell lines and tissues. No immune-reactivity was present in the negative controls. (2)Following the mixing experiments there was no increase of CSP-80; CSP-80 is not a degradation artifact of MAP1a. (3) The paclitaxel-driven tubulin assembly assay showed that CSP-80 bound to microtubules during tubulin polymerization. (4) No CSP-80 was identified in human or rat non-malignant tissues including normal human and rat brain or normal human ovary, placenta or uterine myomas, or normal rat liver, spleen or lung. (5) cDNA sequencing shows a 2603 bp fragment at a 5’ terminal of p80 to be homologous with the analogous segment of MAP1a. (6) Further testing showed that CSP-80 was present in other reproductive and non-reproductive carcinomas. Again, no 350 kDa MAP1a was present. Studies are underway to determine whether CSP-80 is a mutated protein or the product of post-transcriptional modification. Conclusion: CSP-80 is a novel cancer-related protein that appears to be limited to an 80 kDa sequence of the MAP1a gene, or a post-transcriptionally modified product. Since CSP-80 exhibits tubulin-binding activity, this may explain the efficacy of taxane derivatives. CSP-80 may disclose some aspects of carcinogenesis, be a valuable marker of cancer and therapeutic efficacy, and offer new avenues for cancer treatment. Citation Format: Ahmed Fadiel, Viswam S. Nair, Peter Aceto, Frederick Naftolin. Identification of cancer specific protein 80 (CSP-80), a novel 80 kDa MAP1a-like protein in ovarian carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4296.

Background: BCL-2, an anti-apoptotic protein and oncogene, has been considered one of the top 10 targets for cancer drug development. The inhibition of BCL-2 and other anti-apoptotic proteins will help to restore the apoptotic pathway. Aiming to target much diverse BCL-2 family protein dependency in solid tumors, we have developed a potent BCL-2 and BCL-xL dual inhibitor APG-1252, which is currently in phase 1 trials for solid tumors in USA, China and Australia. Mechanistically, BCL-2 and BCL-xL protect cells from cell death by sequestering pro-death BH3-only proteins like BIM. Thus, BCL-2 and BCL-xL inhibitors are designed to disrupt the protein complexes and release pro-death proteins to trigger downstream apoptotic cascade. In this study, we intended to further understand the mechanism of action of APG-1252 in vivo and factors regulating its sensitivity. To this end, we conducted trials in SCID mice carrying patient-derived xenograft (PDX) tumors, and studied target engagement. Methods and Experiments: We first selected eleven gastric and esophageal cancer PDX models that bore high levels of BCL-xL (BCL-xLhigh) but normal levels of MCL-1 (MCL-1nor) based on the gene expression profiles and the consideration of that BCL-xL may play a major role in tumorigenesis in solid tumors and MCL-1 a resistant factor for the dual inhibitor. Mice bearing PDX tumors were intravenously treated with 100 mg/kg of APG-1252 twice per week for three weeks. At the end of treatments, tumor samples were collected. An advanced ELISA system (MSD, Meco Scale Discovery) and Western blotting were performed to detect BCL-2 and BCL-xL protein complex and individual proteins. Results: 1. The MSD assays revealed dominant BCL-xL:BIM complex signals in the solid tumor PDX samples in comparison with hematological malignancy cell lines, in which BCL-2:BIM was the major complex. 2. The dual inhibitor treatment disrupted BCL-xL:BIM complex, thus BCL-xL:BIM complex may serves as a pharmacodynamic (PD) marker for target engagement. 3. BCL-2 family protein profiles showed high BCL-xL and MCL-1 levels in the solid tumor PDXs. The treatment with APG-1252 led to a significant increase in BCL-2 protein levels, while there was no major change on pro-apoptotic BH3-only proteins BIM and PUMA. 4. The more efficacious responders to the treatment exhibited a more substantial decrease in BCL-xL:BIM complex signals and low MCL-1 protein levels, whereas a much less significant decrease in BCL-xL:BIM complex and higher MCL-1 levels were found in the poor or non-responders. Conclusion: Both BCL-xL protein and BCL-xL:BIM complex are dominant in the solid tumor PDX tissues. The decrease of BCL-xL:BIM complex indicates the on-target activity after the treatment with APG-1252. Thus, the complex may be used to monitor the target engagement and, potentially, a predictive biomarker for APG-1252 efficacy. These assays may be applied to patient samples to guide the clinical trials. Citation Format: Jing Deng, Xiaojing Huang, Guoqin Zhai, Tingting Mao, Yan Yin, Douglas D. Fang, Dajun Yang, Yifan Zhai. Predicting drug sensitivity to a novel BCL-2 and BCL-xL dual inhibitor in solid tumor PDX trials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2503.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a dismal prognosis and 5 year survival rate of less than 5%. Desmoplastic reaction, characterized by the proliferation of myofibroblast-like cells (also known as activated pancreatic stellate cells (PSCs)) and the significant deposition of extracellular matrix components, such as hyaluronan, is a prominent pathological characteristic of PDAC and significantly contributes to its chemoresistance. All trans retinoic acid (ATRA) is a pleiotropic agent modulating multiple signaling pathways that renders activated PSCs quiescent. Previous work from our laboratory demonstrated various phenotypic changes in ATRA-treated PSCs and comparative gene expression microarray analysis revealed that the pentraxin-related protein 3 (PTX3) expression is down-regulated in quiescent PSCs compared to activated PSCs. Since PTX3 plays a key role in the formation of the hyaluronan-rich matrix via its interaction with tumor necrosis factor stimulated gene 6 (TSG-6) and hyaluronic acid (HA) chains, we sought to investigate the role of PTX3 in PDAC and to assess it as a potential diagnostic biomarker for patients with pancreatic cancer. Serum PTX3 concentrations were evaluated in 140 patients with PDAC and 138 controls (healthy individuals, patients with other pancreatic diseases or inflammatory conditions) by ELISA. To establish the source of PTX3 secretion we analyzed in vitro cell cultures of PSCs and cancer cell lines. PTX3 specificity was confirmed upon PTX3 siRNA analysis by western blot and qPCR. Three-dimensional organotypic cultures, KPC mice and human tissue were assessed by immunohistochemistry. Expression of TSG-6 and HA were also evaluated using immunofluorescence studies. Our results showed that patients with PDAC had significantly higher serum PTX3 concentrations than patients with other pancreatic diseases and healthy individuals. ROC curve analysis confirmed the reliability of PTX3 serum levels in diagnosing pancreatic cancer with a sensitivity and a specificity of 86%. Western blotting, qPCR and immunofluorescence validated that activated PSCs produce and secrete PTX3 and its expression was down-regulated on rendering these cells quiescent upon ATRA treatment. Organotypic models elucidated that hyaluronan is expressed and secreted by the PSCs and the analysis of human and mouse tissues demonstrated predominant PTX3 expression in the stromal compartment of PDAC. Our study identified that serum PTX3 is a sensitive and specific diagnostic biomarker for PDAC with the ability to separate malignant from other benign conditions of the pancreas. Activated PSCs are the main source of PTX3 secretion and contribute to the formation and stabilization of the PDAC extracellular matrix architecture. Citation Format: Michelle R. Goulart, Jennifer Watt, Imran Siddique, Rita Lawor, Thomas Dowe, Satyajit Bhattacharya, Tatjana Crnogorac-Jurcevic, Paola Allavena, Aldo Scarpa, Hemant M. Kocher. Pentraxin 3: A stromal derived diagnostic biomarker for pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4892.

Background: Triple negative breast cancer (TNBC) is the most aggressive form of breast cancer with poor prognosis. Due to the frequent distant metastasis and lack of successful targeted therapies, the overall survival rate for the patients with TNBC is significantly lower than estrogen receptor positive and HER2 positive breast cancers. To identify potential druggable targets, previously, we performed a gene expression analysis and identified protein kinases that are highly expressed in ER-negative breast cancers. Through these studies, we discovered that the expression of pseudo kinase-7 (PTK-7) is highly elevated in TNBCs and correlated with p53 mutation status and disease progression. We hypothesize that high expression of PTK-7 is required for the growth and invasiveness of TNBC cells. Experimental design and methods: In this study, we analyzed the expression status of PTK-7 in TNBC and the association of PTK-7 expression with p53 status and disease prognosis using publicly available datasets. Protein levels of PTK-7 were determined through western blotting analysis. The association of p53 and PTK-7 expression was determined using Oncomine analysis. Using specific siRNAs against PTK-7, we determined whether PTK-7 is required for the growth of TNBC cells. The role of PTK-7 in migration and invasion of TNBC cells was determined using Boyden trans-well assays. Results: Analysis of publicly available datasets reveals that PTK-7 expression is elevated in TNBCs compared with normal breast and ER-positive breast cancers. High PTK-7 expression was found to be associated with p53 mutations. Knockdown of PTK-7 using siRNA significantly reduced the growth of TNBC cells in vitro. In vitrotrans-well migration and invasion studies also demonstrated that knockdown of PTK-7 significantly reduced the migration and invasion of TNBC cells. In contrast knockdown of PTK-7 did not affect the growth and invasiveness non-TNBC cells. Conclusion: Our results suggest that the expression of PTK-7, a critical regulator of growth and invasion of TNBCs. These results suggest that PTK-7 is a promising target for the treatment of women with TNBC. This work was supported by Susan G Komen Promise Grant (PB), SAB Komen grant (PB) and Young Foundation grant (PB). Citation Format: Lakshmi Reddy Bollu. High expression of PTK-7 regulates the growth and invasion of triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1784.

DNA double-strand brakes (DSBs) are a highly toxic form of DNA damage originating from many endogenous and exogenous sources. Cells employ specialized mechanisms for rapid detection and repair of these lesions. Histone H2AX phosphorylated at Ser139 (known as γ-H2AX) acts as a central platform for recruitment of other components of DSB repair. γ-H2AX assessment is currently the most widely used approach for quantitation of DSBs. However, γ-H2AX can be also induced by certain nongenotoxic stressors and during apoptosis, which can lead to false positive observations, especially in prolonged exposures. γ-H2AX undergoes RNF168-mediated K13/K15 monoubiquitination which is necessary for the recruitment of the downstream DSB repair factors. This ubiquitination event is rarely analyzed despite its functional importance. We examined formation and detection of γ-H2AX and its mono- (ub1) and diubiquitinated (ub2) forms in several human cell lines in response to mechanistically distinct inducers of DSBs and false-positive DSBs stressors. We found that γ-H2AX-ub forms were poorly detected using some common blotting procedures and antibodies. Under optimized conditions, γ-H2AX-ub1 was the predominant form accounting up to 80-90% of total γ-H2AX in primary human cells and its formation was strictly associated with the presence of DSBs. γ-H2AX and γ-H2AX-ub1 showed similar dose dependence and disappearance kinetics whereas H2AX-ub2 form was a more stable and dose-independent product. Unlike γ-H2AX, its ubiquitinated forms were not observed in the absence of DSBs (heat-shock or during apoptotic DNA cleavage). Apoptotic cells showed cleavage of the γ-H2AX-targeting E3 ubiquitin ligase RNF168. Our findings demonstrate that the ub1 form is a major fraction in the overall formation of γ-H2AX and its quantitation offers advantage in specificity for nonapoptotic DSBs. Citation Format: Michal W. Luczak, Anatoly Zhitkovich. Monoubiquitinated γ-H2AX - a more specific biomarker of DNA double-strand breaks [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1754.

MB21D2 (Mab-21 containing domain 2), a member of the Mab family, is predicted to form protein complexes with different partners, thus performs diverse functions in cells. In silico analyses using TCGA data revealed MB21D2 amplification/overexpression in various types of human cancer and correlated with poor survivals in patients with head and neck and endometrial cancer. More interestingly, a recurrent Q311E mutation was found in the Mab-21 domain critical for protein-protein interactions and downstream signaling. When expressed in oral cancer TW206 and CAL27 cells (low endogenous MB21D2), wild-type MB21D2 moderately increased cell proliferation, migration, and invasion as compared to the vector control. Significantly, the mutant variant (Q311E) showed stronger oncogenic effects on those processes and formed bigger spheres in a 3-D culture condition. Data mining, functional network predictions, and Western blotting confirmed the involvement of MB21D2 in centrosome-associated ciliogenesis via a crosstalk between PIK3CA and PKA (GSK and CREB) signaling pathways. Blockage of such interactome by specific inhibitors against PIK3CA or PKA reduced oncogenic activities mediated by MB21D2. Our data therefore suggest MB21D2 amplification/overexpression or the Q311E mutation as cancer drivers promoting cancer development through alteration of centrosome and primary cilia activity. Citation Format: Daniel Esguerra Gracilla, Praveen Kumar Korla, Louis Lai, Jim Jinn-Chyuan Sheu. MB21D2 amplification and recurrent mutation exhibit oncogenic activity through centrosome and cilia alteration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4657.

The incidence of esophageal adenocarcinoma (EAC) is rising rapidly in the US and Western countries. The development of Barrett’s esophagus (BE) and its progression to EAC have been linked to gastroesophageal reflux disease (GERD). Exposure of BE and EAC cells to acidic bile salts (ABS) in GERD conditions induces high levels of DNA damage. In this study, we investigated the role of insulin-like growth factor binding protein 2 (IGFBP2) in regulating ABS-induced DNA double-strand breaks (DSBs). Immunohistochemistry analysis demonstrated frequent overexpression of IGFBP2 in EACs (31/57). Treatment of EAC cells with ABS, to mimic GERD conditions, induced high levels of IGFBP2 expression. Knocking down endogenous IGFBP2 in FLO1 cells (with constitutive high levels of IGFBP2) led to a significant increase in DNA DSBs and apoptosis, following transient exposure (20 min) to ABS. On the other hand, overexpression of exogenous IGFBP2 in OE33 cells (with low endogenous levels of IGFBP2) had a protective effect against DSBs and apoptosis. Using western blotting and immunofluorescence assays, we found that IGFBP2 is required for ABS-induced nuclear accumulation and phosphorylation of EGFR and DNA-PKcs, which are required for DNA damage repair activity. Using co-immunoprecipitation assay, we detected IGFBP2 forms complex with EGFR and DNA-PKcs and co-localizes with EGFR and DNA-PKcs in nucleus, following acidic bile salts treatment. We also demonstrated, using cycloheximide (CHX) chase assay, that IGFBP2 promotes EGFR protein stability in response to ABS exposure. In summary, our data indicate that IGFBP2 protects EAC cells against ABS-induced DNA damage and apoptosis through stabilization and activation of EGFR - DNA-PKcs signaling axis. Citation Format: Zhangjian Zhou, Heng Lu, Shoumin Zhu, Ahmed Gomaa, Zheng Chen, Jin Yan, Wael El-Rifai, Chengxue Dang, Dunfa Peng. IGFBP2 is required to activate EGFR-DNA-PKcs pathway to protect esophageal adenocarcinoma cells from acidic bile salts-induced DNA damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 878.

To date there was no evidence of existence of the naturally circulating antibodies to checkpoint receptors in humans. LAG3 checkpoint is crucial in modulation of immune response that is altered in humans with inflammatory and malignant conditions. We have previously described novel methodology in identification of diagnostic and therapeutic antibodies (US Patent #9810694). Here we report identification of novel potentially therapeutic fully human anti-LAG3 antibodies in cancer patients. Anti-LAG3 antibody presence was evaluated in 25 individuals: 11 healthy donors (controls) and 14 cancer patients in two different laboratories blinded to clinical information. Surface plasmon resonance analysis was done using the optical biosensor BiacoreT200 where LAG3 protein was covalently immobilized on the optical chip and biosensor signals from human serum samples were analyzed. Western Blotting used recombinant LAG3 loaded on a 10% SDS PAGE followed by blotting with plasma samples followed by biotinylated anti-human IgA/IgG/IgM and IrDye 800 streptavidin. Plasma anti-LAG3 IP utilized recombinant LAG3 crosslinked to MagResyn Carboxyl beads followed by incubation with plasma, followed by a biotinylated anti human IgA/IgG/IgMand IrDye streptavidin. No anti-LAG3 antibodies were detected in control group. Among three assays there was complete correlation for presence of anti-LAG3 antibody in 3 cancer patients. One of these patients had sufficient plasma quantity and anti-LAG3 concentration (21 - 33 µg/ml) that should allow for the antibody purification and de novo sequencing. The novel fully human anti-LAG3 antibody will be identified, purified, and the sequence will be determined.To our knowledge this is the first report of checkpoint antibody presence in humans. Further study with cloning and evaluation of therapeutic properties of novel fully human anti-LAG3 antibody in xenograft models will be performed. Citation Format: Ilya Tsimafeyeu, Pavel Ershov, Leonid Kaluzhskiy, Jean-Philippe Couture, Hugo Gagnon, Vladimir Kuznetsov, Aleksei Kalachev, Alexis Ivanov, Gennady Bratslavsky. Naturally circulating anti-LAG3 antibodies exist in cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4115.

Background: Molecular alterations involving FGFR family genes (FGFR 1-4) are emerging driver events in a variety of solid tumors, mainly represented by urothelial carcinoma (UC) and intrahepatic cholangiocarcinoma (CC). Several tyrosine kinase inhibitors (TKI) are in clinical development to counteract FGFR-driven diseases, being especially active against activating gene mutations and rearrangements. Progression on these targeted agents eventually appears and the understanding of molecular mechanisms of resistance is crucial to develop novel strategies. Methods: In the MATCH-R prospective study (NCT02517892), patients with unresectable or metastatic cancer are included upon acquired resistance to targeted therapies or immunotherapy, defined as progressive disease after complete/partial response or stable disease for six months. Serial blood samples are collected and tumor biopsy is performed upon progression. Targeted NGS, CGH, WES and RNAseq are performed on the tissue samples. PDX models and patient-derived cell lines are developed to fully investigate the underlying mechanisms of resistance. Only patients receiving TKI for FGFR-mutated or -rearranged tumors were included (i.e. FGFRamplifications were excluded) in the analysis. Results: From June 2015 to November 2018, 113 patients treated with a TKI were included in the MATCH-R study, of which 17 (15%) had received an FGFR inhibitor. Tumor types and corresponding molecular aberrations were as follows: 8 CC (n=6 FGFR2-rearranged, n=1 FGFR2:C383R, n=1 FGFR3:S249C), 7 UC (n=5 FGFR3:S249C, n=1 FGFR3:R248C, n=1 FGFR3:Y373C), 1 breast (FGFR3-rearranged) and 1 ovarian (FGFR2-rearranged) cancers. Evaluable tumor biopsies were taken upon progression to treatment with erdafitinib (n=12), pemigatinib (INCB54828) (n=3) or TAS-120 (n=4). Two patients underwent multiple biopsies as progressing on sequential FGFR inhibitors. Resistance mechanisms consisted of polyclonal secondary mutations (n=5), bypass pathways activation (n=3) and the remaining nine cases are still under investigation. PDX models/patient-derived cell lines were obtained in eight cases and extensively characterized in three. Adaptive treatment with novel FGFR TKI or combinatorial strategies aiming to block the bypass pathways allowed to restore sensitivity in both cell lines (readouts: IC50 and Western Blots) and PDX (readout: median tumor growth). Novel mutations potentially implicated in resistance to FGFR TKI were characterized by infecting Ba/F3 cells with respective lentiviral vectors, as well as the inhibitory potential of the differential FGFR inhibitors. Conclusions: Novel mechanisms of resistance to FGFR inhibitors in solid tumors were identified and consequent treatment strategies allowed to regain sensitivity in both patient-derived cell lines and PDX. Updated results will be presented at the Meeting. Citation Format: Francesco Facchinetti, Rastislav Bahleda, Antoine Hollebecque, Yohann Loriot, Gonzalo Recondo, Ludovic Bigot, Ken A. Olaussen, Gilles Vassal, Stefan Michiels, Rosa L. Frias, Justine Galissant, Tony Sourisseau, Claudio Nicotra, Maud Ngo-Camus, Linda Mahjoubi, Ludovic Lacroix, Etienne Rouleau, Catherine Richon, Aurélie Abou-Lovergne, Olivier Deas, Nathalie Auger, Thierry De Baere, Frederic Deschamps, Eric Solary, Jean-Yves Scoazec, Eric Angevin, Alexander Eggermont, Fabrice André, Benjamin Besse, Jean-Paul Thiery, Jean-Charles Soria, Christophe Massard, Luc Friboulet. Mechanisms of acquired resistance to FGFR inhibitors in molecularly-selected solid tumors: A prospective cohort from the MATCH-R study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 318.