bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract IA01: Mechanisms of formation and progression of preleukemic stem cells
Authors: Steidl Ulrich
Published: August 2018

Relapse continues to be the most common cause of death in AML and overall cure rates remain low. Recent evidence has shown that the accumulation of stepwise genetic and epigenetic changes in HSC lead to the formation of precancerous/preleukemic stem cells (pre-LSC) that play a pivotal role not only in disease origination but also in leukemia relapse. While the existence and essentiality of such precancerous cell states has been demonstrated in mice and humans, still very little is known about the molecular mechanisms driving pre-LSC formation and progression. We have recently performed molecular studies of pre-leukemic cell states in mouse genetic models as well as primary cells from patients, and discovered new transcription factors and regulatory mechanisms in pre-LSC in MDS and AML. Specifically, we discovered critical roles for several nonclustered homeobox transcription factors in pre-LSC, and identified functionally critical and pharmacologically targetable downstream effectors. We also found that enhancer haplodeficiency and consequent minimal reduction of key transcription factors is sufficient to induce pre-LSC formation and subsequent progression to MDS and AML. Such models provide novel tools for mechanistic study of pre-LSC and their progression to overt MDS and AML, and for the development and testing of pharmacologic approaches to therapeutically interfere with these processes.

In summary, recent studies have started to shed light on preleukemic cell states as the earliest origin of MDS and AML, as well as molecular mechanisms driving their formation and progression, which provides a basis for their specific therapeutic targeting for causative treatment of MDS and AML.

Citation Format: Ulrich Steidl. Mechanisms of formation and progression of preleukemic stem cells [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr IA01.
Abstract IA10: Targeted therapies as molecular probes for comprehensive preclinical evaluation
Authors: Dawson Mark A.
Published: August 2018

Cancer biology is profoundly influenced by changes in the epigenome. Because the dynamic plasticity of the epigenome lends itself well to therapeutic manipulation, the past few years have witnessed an unprecedented investment in the development, characterization, and translation of targeted epigenetic therapies. Several of these epigenetic therapies have now transitioned into the clinical arena, and emerging evidence suggests that these drugs are well tolerated and show single-agent efficacy in a range of hematopoietic cancers. The enduring challenge for clinicians and scientists will be understanding the cellular and molecular mechanisms that may govern sensitivity and resistance to these agents. This information could then be used to design more effective combination regimes. A major limitation to our current knowledge is the fact that we are blinded to the mechanism of action of these agents in a cellular context. In this seminar, I will discuss our strategy to overcome this limitation by converting epigenetic agents into molecular probes that maintain the functional integrity of the compound but now allow the compounds to be used much like antibodies in all routine cell and molecular assays both in vitro and in vivo.

Citation Format: Mark A. Dawson. Targeted therapies as molecular probes for comprehensive preclinical evaluation [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr IA10.
Abstract IA31: Targeting immune receptor mutations in lymphoma
Authors: Wendel Hans-Guido
Published: August 2018

The HVEM (TNFRSF14) receptor gene is among the most frequently mutated genes in germinal center lymphomas. We report that loss of HVEM leads to cell-autonomous activation of B cell proliferation and drives the development of GC lymphomas in vivo. HVEM-deficient lymphoma B cells also induce a tumor-supportive microenvironment marked by exacerbated lymphoid stroma activation and increased recruitment of T follicular helper (TFH) cells. These changes result from the disruption of inhibitory cell-cell interactions between the HVEM and BTLA (B and T lymphocyte attenuator) receptors. Accordingly, administration of the HVEM ectodomain protein (solHVEM(P37-V202)) binds BTLA and restores tumor suppression. To deliver solHVEM to lymphomas in vivo, we engineered CD19-targeted chimeric antigen receptor (CAR) T cells that produce solHVEM locally and continuously. These modified CAR-T cells show enhanced therapeutic activity against xenografted lymphomas. Hence, the HVEM-BTLA axis opposes lymphoma development, and our study illustrates the use of CAR-T cells as “micropharmacies” able to deliver an anticancer protein.

Citation Format: Hans-Guido Wendel. Targeting immune receptor mutations in lymphoma [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr IA31.
Abstract PR02: Mechanisms of NT5C2 activating mutations driving thiopurine resistance in relapsed lymphoblastic leukemia
Authors: Dieck Chelsea L. , Tzoneva Gannie , Forouhar Farhad , Carpenter Zachary , Sanchez-Martin Marta , Ambesi-Impiombato Alberto , Kirschner-Schwabe Renate , Lew Scott , Seetharaman Jayaraman , Tong Liang , Ferrando Adolfo
Published: August 2018

Activating mutations in cytosolic 5'-nucleotidase II (NT5C2) drive resistance to 6-mercaptopurine (6-MP) in relapsed acute lymphoblastic leukemia (ALL). These gain-of function alleles encode proteins with increased nucleotidase activity, yet their specific molecular mechanisms are poorly understood. Allosteric activation of NT5C2 is primarily mediated by a marked conformational change in the helix A region, which reconfigures the catalytic center for substrate access and catalysis. Here we demonstrate that constitutively active NT5C2 mutant proteins (K359Q and L375F) adopt active helix A region structures in the absence of allosteric activator. Moreover, crystallographic, structural modeling, genetic, and functional antibody analyses identify a switch-off mechanism mediated by the dynamic mobilization of Asp407 in the tip region of the arm segment which, guided by positively charged residues located in the surface of a basic inter-monomeric cavity exposed upon allosteric activation, turns off NT5C2 by interacting with and disrupting the helical conformation of helix A. Loss of this regulatory mechanism in NT5C2 mutants involving the tip region of the arm segment and the inter-monomeric cavity drives increased nucleotidase activity and allosteric response. Finally, increased allosteric activation by the Q523X mutation results from loss of the NT5C2 C-terminal acidic tail segment, which promotes a closed inactive state in basal conditions. These results uncover dynamic mechanisms responsible for NT5C2 regulation targeted by chemotherapy resistance-driving NT5C2 mutations, with important implications for the development of NT5C2 inhibitor therapies.

This abstract is also being presented as Poster 05.

Citation Format: Chelsea L. Dieck, Gannie Tzoneva, Farhad Forouhar, Zachary Carpenter, Marta Sanchez-Martin, Alberto Ambesi-Impiombato, Renate Kirschner-Schwabe, Scott Lew, Jayaraman Seetharaman, Liang Tong, Adolfo Ferrando. Mechanisms of NT5C2 activating mutations driving thiopurine resistance in relapsed lymphoblastic leukemia [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr PR02.
Abstract A02: Investigating the contribution of RAD51 paralog mutations to cancer development
Authors: Baldock Robert A. , Pressimone Catherine A. , Bernstein Kara A.
Published: August 2018

Homologous recombination (HR) is a high-fidelity DNA repair mechanism that utilizes a homologous template to repair double-strand breaks (DSBs). The defining step of HR is the formation of a RAD51 nucleoprotein filament, which performs the homology search and strand invasion steps. RAD51 filament formation is aided by the RAD51 paralogs, which structurally resemble RAD51. The RAD51 paralogs form sub-complexes including CX3 (RAD51C and XRCC3) and BCDX2 (RAD51B, RAD51C, RAD51D, and XRCC2).

Here we use a use a Yeast 3-hybrid assay to determine which patient-identified mutations in RAD51D impair the interaction with RAD51C and XRCC2. We further validate this interaction data in mammalian cells. Lastly, we are going to complement RAD51D and XRCC2-depleted cells with mutant versions of the proteins to determine their impact on HR as well as sensitivity to specific chemotherapeutic agents.

Citation Format: Robert A. Baldock, Catherine A. Pressimone, Kara A. Bernstein. Investigating the contribution of RAD51 paralog mutations to cancer development. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A02.
Abstract A08: A novel system determines the functional significance of ovarian tumor mutations in the homologous recombination gene RAD51C
Authors: Sullivan Meghan R. , Prakash Rohit , Mihalevic Michael J. , Baird Jared M. , Jasin Maria , Bernstein Kara A.
Published: August 2018

Homologous recombination (HR) deficiency is associated with hereditary ovarian carcinomas. HR deficiency can occur through somatic and germline mutations in BRCA1, BRCA2, and other associated genes such as RAD51C. Understanding the impact of HR gene mutations on DNA double-strand break (DSB) repair is critical for developing targeted therapeutic strategies for these patients. Although BRCA1 and BRCA2 deficiency has been well characterized, limited functional analysis has occurred in patients harboring mutations in RAD51C. RAD51C mutations are found in familial breast and ovarian cancers and in Fanconi anemia-like syndrome, FANCO. To determine which RAD51C-tumor associated mutations disrupt HR, we utilized a novel RAD51C conditional knockout breast epithelial cell line (MCF10A) generated by Dr. Maria Jasin. In collaboration with the Jasin laboratory, we complemented these cells with RAD51C and RAD51C cancer-associated mutants. Using yeast-two-hybrid and co-immunoprecipitation strategies, we identified RAD51C mutations that disrupt RAD51C protein-protein interactions. For example, we identified RAD51C mutations that impair RAD51C interaction with either its binding partner RAD51B, RAD51D, or XRCC3. We next measured RAD51C mutant cells for viability and HR proficiency at an I-SceI-induced DSB using a DR-GFP reporter assay. We identified RAD51C mutations that impact HR repair, suggesting that patients harboring these mutations would be good candidates for PARPi. Our goal is to identify specific RAD51C mutations that disrupt HR to predict which RAD51C-deficient tumors will respond to therapy.

Citation Format: Meghan R. Sullivan, Rohit Prakash, Michael J. Mihalevic, Jared M. Baird, Maria Jasin, Kara A. Bernstein. A novel system determines the functional significance of ovarian tumor mutations in the homologous recombination gene RAD51C. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A08.
Abstract A10: Fatty acid synthesis inhibitor, orlistat, potentiates cisplatin-induced toxicity in ovarian cancer cells
Authors: Houten Bennett Van , Heft-Neal Molly , Qian Wei , Roginskaya Vera
Published: August 2018

Background: Fatty acid synthase (FAS) is a key enzyme in the synthesis of fatty acids and has been shown to be upregulated in many cancers. Additional studies found that high levels of FAS correlated with worse prognosis and with chemoresistance in ovarian tumors. As such, FAS has become a promising target for antitumor therapies. The aim of this study was to gain mechanistic insights into the cytotoxic effects of fatty acid inhibition with orlistat treatment alone or in combination with cisplatin in the ovarian cancer cell line A2780.

Methods: A2780 cells were treated with cisplatin (0.4-12 μM) for 4 hours followed by orlistat (25-100 μM, 24 hr). Cell proliferation was measured via CyQuant assay; cell apoptotic activity was measured by both caspase 3/7 assay and flow cytometry with annexin V and propidium iodide. Fatty acid β-oxidation was then characterized using a Seahorse XF24 Extracellular Flux Analyzer. ATP levels were measured with a luminescent ATP detection kit. Finally, Western blot analyses for capsase 3 and phospho-ATM were performed.

Results: Cisplatin treatment (4 and 10 μM) of A2780 cells induced an increase in OXPHOS and steady-state levels of ATP 24 hr after damage. Orlistat (25 and 50 μM, 24 hr) decreased basal oxygen consumption and decreased steady-state ATP levels in cisplatin-treated cells. A2780 cells showed decreased proliferation and increased apoptotic activity (Caspase 3/7 and Annexin V) when treated with cisplatin followed by orlistat, as compared to either drug alone. Using the Chou Talaly method the combination index was found to be 0.73 indicating a synergistic relationship. Cisplatin increased the level of phospho-ATM that was reduced by orlistat.

Conclusions: Orlistat in combination with cisplatin treatment of A2780 ovarian tumor cells increased apoptosis compared to either treatment alone. Orlistat caused decreased oxidative phosphorylation and prevented a cisplatin-induced increase in OXPHOS and ATP. The combination of cisplatin and orlistat decreases p-ATM, suggesting decreased activation of the DNA repair pathway after cisplatin treatment. This may represent the mechanism of synergistic action of the combination treatment.

Citation Format: Bennett Van Houten, Molly Heft-Neal, Wei Qian, Vera Roginskaya. Fatty acid synthesis inhibitor, orlistat, potentiates cisplatin-induced toxicity in ovarian cancer cells. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A10.
Abstract A16: COL11A1 confers cisplatin resistance through fatty acid oxidation in ovarian cancer cells
Authors: Rada Miran , Cha Jennifer , Sage Jessica , Zhou Bo , Yang Wei , Orsulic Sandra , Cheon Dong-Joo
Published: August 2018

Cisplatin resistance is the major challenge in the treatment of ovarian cancer, and it is crucial to develop novel therapeutic strategies to block cisplatin resistance. The purpose of this study was to identify new therapeutic targets for the treatment of cisplatin-resistant ovarian cancer.

We previously identified collagen type XI alpha 1 (COL11A1) as a novel biomarker associated with cisplatin resistance in ovarian cancer. COL11A1 is expressed by cancer-associated fibroblasts (CAFs) adjacent to cancer cells as well as cisplatin-resistant ovarian cancer cell lines. Our data indicate that COL11A1 inhibits cisplatin-induced apoptosis in ovarian cancer cells, but not through altered gene expression of many of the well-established mechanisms of cisplatin resistance (i.e., transport, inactivation, DNA repair pathways). Rather, our proteomics data revealed that mitochondrial fatty acid beta-oxidation (FAO) was the most upregulated pathway by COL11A1 in ovarian cancer cells. To determine a link between COL11A1-driven cisplatin resistance and FAO, we knocked down COL11A1 in A2780CIS cisplatin-resistant ovarian cancer cells, a cell line expressing high endogenous levels of COL11A1. COL11A1 knockdown not only decreased the expression of genes involved in fatty acid beta-oxidation, such as CD36, CPT1A, ACAA2, HADHA, but also decreased fatty acid oxidation rate and increased cisplatin-induced apoptosis in A2780CIS ovarian cancer cells. In contrast, ovarian cancer cells became resistant to cisplatin-induced apoptosis and increased fatty acid beta-oxidation when exposed to COL11A1 protein or cocultured with COL11A1-overexpressing CAFs. Importantly, these cancer cells became more sensitive to the FAO inhibitor etomoxir.

Collectively, our results suggest that COL11A1 confers cisplatin resistance by increasing FAO in ovarian cancer cells. Our study uncovers FAO as a promising therapeutic target to reduce cisplatin resistance in ovarian cancer, particularly in recurrent ovarian cancers that typically express high levels of COL11A1.

Citation Format: Miran Rada, Jennifer Cha, Jessica Sage, Bo Zhou, Wei Yang, Sandra Orsulic, Dong-Joo Cheon. COL11A1 confers cisplatin resistance through fatty acid oxidation in ovarian cancer cells. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A16.
Abstract A35: Ovarian cancer stem cells subvert tumor-specific T cells by disrupting T cells’ metabolic fitness
Authors: Qian Feng , Liao Jianqun , Miliotto Anthony J. , Collins Katherine A. , Odunsi Kunle
Published: August 2018

Cancer stem cells (CSCs) are defined by their unlimited self-renewal ability and their capacity to initiate and maintain malignancy, traits that are not found in most cells that compose the tumor. These stem cells have been shown to be relatively resistant to conventional chemotherapeutic regimens and radiation. Previously, we have reported that spheroid cells derived from 3D epithelial ovarian cancer cells are enriched with cancer stem cells by in vitro and in vivo experiments. In addition, we have demonstrated that CSCs adopt unique metabolic pathways by routing glucose predominantly to anaerobic glycolysis and pentose cycle to the detriment of rerouting glucose for anabolic purposes. In the current study, we hypothesized that CSCs may contribute to the immunosuppressive microenvironment of ovarian cancer by disrupting the metabolic fitness of infiltrating T cells. We isolated fresh spheroid cells from ovarian cancer patients’ ascites and examined their effect on T-cell responses ex vivo. In addition, other than ovarian cancer, we generated more spheroid cells from pancreatic cancer and breast cancer cells. Compared to nonspheroid cells, these spheroid cells have upregulated stem cell gene expression, higher ALDH activity, and more tumor-initiating capacities in SCID mice, indicating CSCs enrichment in spheroid cells. Seahorse XF analysis revealed a glycolytic metabolic phenotype of spheroid cells with increased extracellular acidification rate (ECAR) compared to nonspheroid cells. Spheroid cells suppress the response of cocultured T cells to TCR stimulation by inhibiting T-cell activation, growth, proliferation, and function independent of cell-cell contact determined by transwell assay and supernatant incubation. After spheroid cells treatment, activated T cells metabolically shifted from energetic to quiescence phenotype, which is reversely correlated with increased glycolysis in spheroid cells, indicating that highly glycolytic spheroid cells suppress T-cell response through reprogramming T-cell metabolic status. Furthermore, we found that spheroid cells dampen T cells’ response by inhibiting Erk-AKT-mTor signaling and by activating the integrated stress response pathway. This study provides novel insights into the relationship between cancer stem cells and T cells’ response and has clinical implications for cancer therapy.

Citation Format: Feng Qian, Jianqun Liao, Anthony J. Miliotto, Katherine A. Collins, Kunle Odunsi. Ovarian cancer stem cells subvert tumor-specific T cells by disrupting T cells’ metabolic fitness. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A35.
Abstract A40: Specific mutations in the D1-D2 linker region of VCP/p97 enhance ATPase activity and confer resistance to VCP inhibitors
Authors: Bastola Prabhakar , Freudenthal Bret , Schaich Matthew , Chou Tsui-Fen , Gan Taiping , Wang Feng , Chien Jeremy
Published: August 2018

Valosin-containing protein (VCP) has been shown to be involved in various protein quality control pathways including ubiquitin proteasome system, endoplasmic reticulum-associated degradation, chromatin-associated degradation, protein aggregate processing, and autophagy. Recent studies also identified VCP as a lineage-specific essential gene in ovarian cancer. An orally bioavailable VCP inhibitor, CB-5083, is currently in phase I clinical trials because it shows therapeutic effects in multiple tumor xenograft models. However, the mechanism of resistance to CB-5083 is unknown. Here, we characterized molecular mechanism of resistance to CB-5083. Using incremental exposure to CB-5083, we established CB-5083-resistant ovarian cancer cells that showed 5-6 fold resistance in vitro compared to parental cells. Genomic and complementary DNA sequencing of the VCP coding region reveal a pattern of coselected mutations: (1) missense mutations at codon 470 in one copy resulting in increased ATPase activity and (2) nonsense or frameshift mutations at codon 606 or codon 616 in another copy causing the loss of allele-specific expression. Unbiased molecular docking studies show codon 470 as a putative binding site for CB-5083. Analysis of somatic mutations in cancer genomes from The Cancer Genome Atlas indicates that codon 616 contains hotspot mutations in VCP. Thus, identification of these mutations associated with in vitro resistance to VCP inhibitors may be useful as potential theranostic markers while screening for patients to enroll in clinical trials. VCP has emerged as a viable therapeutic target for several cancer types, and therefore targeting such hyperactive VCP mutants should aid in improving the therapeutic outcome in cancer patients.

Citation Format: Prabhakar Bastola, Bret Freudenthal, Matthew Schaich, Tsui-Fen Chou, Taiping Gan, Feng Wang, Jeremy Chien. Specific mutations in the D1-D2 linker region of VCP/p97 enhance ATPase activity and confer resistance to VCP inhibitors. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A40.
Abstract A63: Estrogen receptor signaling in fallopian tube epithelia of BRCA mutation carriers
Authors: Dodds Leah V. , Nelson Omar , Shaw Patricia , Milea Anca , Sowamber Ramlogan , George Sophia HL
Published: August 2018

Background: The most common and aggressive type of epithelial ovarian cancers (EOC) is high-grade serous carcinoma (HGSC), which accounts for 90% of ovarian cancer deaths. HGSC is the predominant histotype associated with BRCA1 and BRCA2 mutations. Prophylactic surgery in BRCA mutation carriers has implicated the fallopian tube epithelium (FTE), a hormonal responsive tissue, as the etiologic site of origin for HGSC. Estrogen and its receptors are major regulators of growth and differentiation in normal ovaries and fallopian tubes, and its mutagenic properties have been linked to ovarian carcinogenesis. Estrogen receptors (ER) are rarely mutated, amplified, or deleted in HGSC, yet only 10% of patients respond to antiestrogen treatment. TP53 mutations in the form of the p53 signature have been found in serous tubal intraepithelial carcinomas (STIC) and are ubiquitously present in patients with HGSC. We hypothesized that in the presence of dysfunctional p53, subsequent promiscuous binding of ER will yield aberrant signaling, contributing to cellular transformation.

Methods: We used our previously published gene expression profiles to generate a candidate gene list, which was chosen based on the presence of known estrogen-responsive elements. We analyzed expression of 6 ER responsive genes using data collected from laser capture microdissection in normal FTE tissues. Tissue microarray analysis (TMA) was also performed on a subset of HGSC tumor samples from this cohort, staining for PR, ER, and p53, and expression was analyzed alongside their respective outcome and overall survival Finally, to mimic the in vivo environment of early carcinogenesis, FTE-normal and FTE-p53 mutant cell lines were established and treated with 100nM estradiol, an estrogen analog, to observe changes in response.

Results: Preliminary data showed that FTE-BRCA and FTE-nonBRCA seemingly look and express ER and PR proteins similarly. Underlying these morphologic similarities is a potential haploinsufficiency predisposing FTE-BRCA to cytotoxic stresses. Microarray gene expression of laser captured FTE-BRCA and FTE-nonBRCA showed varied levels of ER mRNA expression across samples (n=25) while PR transcript levels change dynamically. The data generated have facilitated the development of gene signatures and biomarkers that will predict response to antiestrogen therapy and identify patients who will benefit from hormonal therapies.

Citation Format: Leah V. Dodds, Omar Nelson, Patricia Shaw, Anca Milea, Ramlogan Sowamber, Sophia HL George. Estrogen receptor signaling in fallopian tube epithelia of BRCA mutation carriers. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A63.
Abstract A65: Combination of a thioxodihydroquinazolinone compound with cisplatin eliminates ovarian cancer stem cell-like cells (CSC-LCs) and shows preclinical potential
Authors: Ma Jing , Salamoun Joseph , Wipf Peter , Edwards Robert , Houten Bennett Van , Qian Wei
Published: August 2018

Cancer stem cell-like cells (CSC-LCs) contribute to drug resistance and recurrence of ovarian cancer. Strategies that can eradicate CSC-LCs are expected to substantially improve the outcome of ovarian cancer treatments. We have previously identified a class of thioxodihydroquinazolinone small molecules, which have strong synergistic antitumor activity with platinum drugs, the standard chemotherapeutic agents for ovarian cancer treatment. In the current study, using the activity of aldehyde dehydrogenase (ALDH) as a marker of CSC-LCs, we demonstrated that the combination of thioxodihydroquinazolinone compound 19 with cisplatin is able to deplete ALDH-high CSC-LC population in both established platinum-resistant ovarian cancer cell lines and primary ovarian cancer cells from metastatic ascites of a cisplatin-resistant patient. Compound 19 enhanced the accumulation of intracellular cisplatin in ALDH-high ovarian cancer cells. The combination of compound 19 with cisplatin was also able to reduce the sphere-forming capability of cisplatin-resistant ovarian cancer cells. Using a spheroid-based in vitro metastasis model of ovarian cancer, we demonstrated that the combination of compound 19 with cisplatin prevents spheroid ovarian cancer cells from attaching to substratum and spreading. In vivo, the treatment by the combination of compound 19 with cisplatin significantly reduced tumor burden in a cisplatin-resistant intraperitoneal ovarian cancer xenograft mouse model, as compared to cisplatin-alone treatment. Taken together, our study demonstrated that the thioxodihydroquinazolinone compounds are a new class of agents that in combination with cisplatin are capable of eliminating CSC-LCs in ovarian cancer. Further development of thioxodihydroquinazolinone small molecules may yield a more effective treatment for cisplatin-resistant metastatic ovarian cancer.

Citation Format: Jing Ma, Joseph Salamoun, Peter Wipf, Robert Edwards, Bennett Van Houten, Wei Qian. Combination of a thioxodihydroquinazolinone compound with cisplatin eliminates ovarian cancer stem cell-like cells (CSC-LCs) and shows preclinical potential. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A65.
Abstract B05: Cloning and vulnerability of intrinsically resistant subset of ovarian cancer stem cells
Authors: Chang Guoqiang , Li Jinxiu , Wang Bailiang , Yamamoto Yusuke , Mahalingam Rajasekaran , Duleba Marcin , Rao Wei , Xie Jingzhong , Wang Shan , Jazaeri Amir , Davies Peter , Torti Suzy , Draetta Giulio , Anderson Matthew , Brewer Molly , Crum Christopher , McKeon Frank , Xian Wa
Published: August 2018

High-grade ovarian cancer is extremely sensitive to chemotherapy and yet usually lethal due to recurrent disease. To address the underlying mechanism of chemotherapy resistance in this disease, we employed novel technology developed to clone normal adult epithelial stem cells to generate libraries of patient-specific, clonogenic tumor cells. These clones possess hallmarks of “cancer stem cells” including long-term self-renewal and recapitulation of tumors of appropriate histology in immunodeficient mice. Significantly, these clonogenic tumor cells possess the overall pattern of genomic alterations of the whole tumor and yet display clone-specific patterns of variation reflecting intratumor heterogeneity that is stable despite months of continuous propagation. Our analyses at the clonal level have revealed a subset that display an intrinsic, pretherapy resistance to paclitaxel and hypervariable genomics in contrast to the bulk of clones sampled from the library. These intrinsically resistant clones share genomic and gene expression profiles with those surviving paclitaxel treatment of the whole library, suggesting a role for intrinsically resistant cells in recurrent disease. Remarkably, known drugs that interfere with signaling pathways enriched in both intrinsically resistant and paclitaxel survivor clones are synthetically lethal with standard-of-care chemotherapy. The targeting of intrinsically resistant clones from patient-specific libraries of cancer stem cells offers new strategies for preempting recurrent disease.

Citation Format: Guoqiang Chang, Jinxiu Li, Bailiang Wang, Yusuke Yamamoto, Rajasekaran Mahalingam, Marcin Duleba, Wei Rao, Jingzhong Xie, Shan Wang, Amir Jazaeri, Peter Davies, Suzy Torti, Giulio Draetta, Matthew Anderson, Molly Brewer, Christopher Crum, Frank McKeon, Wa Xian. Cloning and vulnerability of intrinsically resistant subset of ovarian cancer stem cells. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B05.
Abstract B09: IGF axis plays a pivotal role in the ovulation-induced malignant transformation of fallopian tube fimbrial epithelial cells
Authors: Chu Tang-Yuan , Huang Hsuan-Shun , Hsu Che-Fang , Chen Pao-Chu
Published: August 2018

Owing to uncertain tissue-of-origin and elusive etiology, high-grade serous ovarian carcinoma (HGSC) is typically diagnosed at late stages with poor prognosis. Recent studies have unveiled the fallopian tube fimbria as the major tissue of origin and incessant ovulation as the main etiology, but the mechanism of cancer initiation remains elusive. In a series of studies we have found a majority of preovulatory follicular fluids (FF) contain high level of ROS that induce DNA double-strand breaks as well as apoptosis to the fimbrial epithelial cells in vitro and in vivo. We also found that hemoglobin (Hb) in pelvis, most likely contributed by retrograde menstruation of previous cycles, reduces the ovulatory ROS level extracellularly and results in a shifting from the apoptotic to the survival fate of ROS-stressed fimbrial epithelial cells. These initiating factors in FF, although they may contribute to the TP53 mutation and other early genetic hits in the genesis of HGSC, cannot fully transform the immortalized fimbrial epithelial cell lines with loss of p53 and Rb. Here we show that IGF axis proteins, including IGFBP2- and IGFBP6-bound IGFs as well as the IGFBP-lytic enzyme PAPP-A, are abundant in ovulatory FF. Upon engaging with glycosaminoglycans (GAG) on the membrane of fimbrial epithelial cells, PAPP-A cleaves IGFBPs and releases bioactive IGF in close proximity to its receptor IGF-1R. Through the PI3K/Akt/mTOR signaling pathway, the FF-derived IGF exerts transformation effects on immortalized fimbrial epithelial cells, inducing anchorage independent growth and xenograft tumorigenesis. By contrast, much less transformation was noted when IGFBP2/6 or PAPP-A was depleted from the FF, when the GAG-docking was blocked, or when IGF-1R ormTOR was inhibited. Taken together, the serial studies have unveiled multiple cancer initiating factors in ovulation and retrograde menstruation where ROS acts as the mutagen, hemoglobin serves as the surviving rescuer, and the IGF axis acts as the main transforming signal.

Citation Format: Tang-Yuan Chu, Hsuan-Shun Huang, Che-Fang Hsu, Pao-Chu Chen. IGF axis plays a pivotal role in the ovulation-induced malignant transformation of fallopian tube fimbrial epithelial cells. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B09.
Abstract B14: IGF axis proteins are the main carcinogens in ovulatory follicular fluid: Evidences from a mammary fat pad tumorigenesis model
Authors: Huang Hsuan-Shun , Chu Sung-Chao , Chu Tang-Yuan
Published: August 2018

Ovarian high-grade serous carcinoma (HGSC) is the most devastating gynecologic cancer with elusive etiology and poor prognosis. Previous studies have disclosed that TP53 mutation is the universal and earliest genetic hit in its origin in the fallopian tube fimbriae. We and others have reported that ROS in ovulatory follicular fluid (FF) is the main mutagen that induces DNA double-strand breaks in fimbrial epithelial cells. However, ROS alone could not explain the transformation activity of FF found by our early study. In parallel studies (abstract by Chu TY et al.), we disclosed the presence of inactive IGF axis proteins in FF, including IGFBP-bonded IGFs and the GAG-free PAPP-A cleaving enzyme. Upon docking to the cell membrane at GAG sites, PAPP-A cleaves IGFBP, frees IGF to bind with IGF-1R, activates the downstream PI3K/Akt/mTOR signaling pathway, and results in the full transformation of the p53/Rb-null immortalized fimbrial epithelial cells. In this study, we established a tumorigenesis induction model in Trp53-/- mice to study the carcinogenic activity of FF. Upon repeated injection of ROS-high FF into the mammary fat pad of Trp53-/- mice, 10 of the 18 (56%) mice grew tumor around the injection site within 8 weeks. The histology showed small and mature B cell non-Hodgkin lymphoma. In contrast, no tumor growth was noted by 16 weeks when FF was injected subcutaneously or when mice were castrated. When IGF2 was depleted from the FF by monoclonal antibody, there was no tumor growth 8 weeks after the same repeated fat pad injections. By contrast, repeated injection of 100ng/ml IGF2 resulted in tumorigenesis in 33% (2/6) of the mice. The study provides in vivo evidence showing that other than ROS, IGF axis proteins are the main carcinogen in ovulatory follicular fluid.

Citation Format: Hsuan-Shun Huang, Sung-Chao Chu, Tang-Yuan Chu. IGF axis proteins are the main carcinogens in ovulatory follicular fluid: Evidences from a mammary fat pad tumorigenesis model. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B14.
Abstract B39: The origins of endometriosis-associated cancers
Authors: Cochrane Dawn , Tessier-Cloutier Basile , Lawrence Katherine , Nazeran Tayyebeh , Karnezis Anthony , Salamanca Clara , Lee Timothy , Cheng Angela , McAlpine Jessica , Hoang Lien , Gilks Blake , Huntsman David
Published: August 2018

Both clear cell ovarian carcinoma (CCOC) and endometrioid ovarian carcinoma (ENOC) are associated with ovarian endometriotic cysts, which is believed to be the precursor lesion of these cancers. Women with endometriotic cysts have up to a 3-fold increased risk of developing CCOC and ENOC. It is perplexing that these two clinically distinct histotypes of ovarian cancer arise from the same precursor lesion. We have performed whole-genome sequencing of ovarian cancer histotypes and found that while some genomic features are more common to one histotype than the other, there is not a single feature that is unique to either histotype. Lacking genomic evidence that could explain the differences between these histotypes, we hypothesized that these cancers arise from distinct cells of origin within endometrial tissue, and the cellular context accounts for their differences. We performed global proteomic analysis of ovarian cancer histotypes and identified CTH as a marker for CCOC. Upon examination of normal Müllerian tissues, we found that CTH is highly expressed in the ciliated cells of endometrium (both ectopic endometrium and endometriosis) and of the fallopian tube, with very little expression in the secretory cells of these tissues. We also find that other ciliated cell markers are expressed in CCOC, whereas endometrial secretory cell markers are expressed in ENOC. We propose a new model of CCOC and ENOC histogenesis wherein ENOC is derived from cells of secretory cell lineage whereas CCOC is derived from, or shares similarities to, cells of ciliated cell lineage. There remain, however, many unanswered questions. For example, while CCOC and ENOC occur at roughly equal prevalence, ciliated cells of the endometrium are rare compared to secretory cells. Cells in the endometriotic cyst are exposed to factors such as inflammation and reactive oxygen species, which could influence differentiation of endometrial progenitor cells into the secretory or ciliated cell lineage. To test factors that promote ciliated cell differentiation in normal endometrium, we treated organoid cultures of normal endometrium with IL-6 and Notch pathway modulators. We propose that ovarian cancer histotypes arise from different cells of origin and that the biology of the normal cells will be partly responsible for determining the phenotype of the cancers.

Citation Format: Dawn Cochrane, Basile Tessier-Cloutier, Katherine Lawrence, Tayyebeh Nazeran, Anthony Karnezis, Clara Salamanca, Timothy Lee, Angela Cheng, Jessica McAlpine, Lien Hoang, Blake Gilks, David Huntsman. The origins of endometriosis-associated cancers. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B39.
Abstract B44: Early loss of monoubiquitylation of H2B alters key metabolic and immune signaling pathways promoting the progression of high-grade serous ovarian cancer
Authors: Hooda Jagmohan , Novak Marian , MacDuffie Emily , Song Melissa , Lester Jenny , Parkash Vinita , Mills Gordon B. , Karlan Beth Y. , Oren Moshe , Drapkin Ronny
Published: August 2018

High-grade serous ovarian carcinoma (HGSOC) is the most common form of ovarian cancer, accounting for over 70% of all cases. HGSOC is frequently complicated by the inevitable development of therapeutic resistance, and thus remains an incurable disease. Recent insights supporting the fallopian tube epithelium (FTE) as a point of origin for HGSOC and the serous tubal intraepithelial carcinoma (STIC) as the precursor lesion for a majority of HGSOCs provide the necessary context to study the mechanisms that drive the development and progression of HGSOC. Understanding these key molecular processes is essential to inform the development of next-generation therapies. In this study we investigated the role of the ubiquitin ligase RNF20 and histone H2B monoubiquitylation (H2Bub1) in serous tumorigenesis. H2Bub1, catalyzed largely by RNF20, is an epigenetic mark with tumor-suppressor properties. The loss of RNF20/H2Bub1 has been linked with cancer progression. We used immunohistochemistry to characterize the expression of H2Bub1 in HGSOC and FTE precursors. We found that H2Bub1 is lost or downregulated in a large proportion of STICs and HGSOC tumors, implicating RNF20/H2Bub1 loss as an early event in serous tumorigenesis. Consistent with our findings, analysis of TCGA data shows that the majority of HGSOCs exhibit heterozygous loss of RNF20. Functionally, we demonstrated that shRNA-mediated knockdown of RNF20, with concomitant loss of H2Bub1, was sufficient to increase cell migration and clonogenic growth of immortalized FTE cells. To understand the mechanisms underlying these effects, we performed RNA-seq and proteomics profiling (RPPA) in RNF20 knockdown cells. Major changes were observed in a number of structural, metabolic (e.g., glutamine pathway), and cytokines and inflammatory signaling pathways, providing early mechanistic insights for the observed oncogenic phenotypes. In summary, our study identifies that loss of H2Bub1 is an early epigenetic event in HGSOC that rewires glutamine metabolism and certain inflammatory pathways, which may represent unique therapeutic opportunities.

Citation Format: Jagmohan Hooda, Marian Novak, Emily MacDuffie, Melissa Song, Jenny Lester, Vinita Parkash, Gordon B. Mills, Beth Y. Karlan, Moshe Oren, Ronny Drapkin. Early loss of monoubiquitylation of H2B alters key metabolic and immune signaling pathways promoting the progression of high-grade serous ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B44.
Abstract B46: DICER1 and FOXL2 mutations correlate with clinicopathologic features of ovarian Sertoli-Leydig cell tumors
Authors: Karnezis Anthony N. , Wang Yemin , Magrill Jamie , Keul Jacqueline , Kommoss Stefan , Tessier-Cloutier Basile , Proctor Lily , Schmidt Dietmar , Gilks C Blake , Huntsman David G. , Kommoss Friedrich
Published: August 2018

Sertoli-Leydig cell tumors are rare mixed ovarian sex cord-stromal neoplasms consisting of a mixture of Sertoli cells, Leydig cells, and occasionally heterologous elements or retiform differentiation. The only known recurrent genetic abnormality is mutations in DICER1, with rare mutations reported in FOXL2. We set out to determine the DICER1 and FOXL2 mutation status in a series of 51 SLCTs with the goal of establishing a molecular classifier using mutation status and clinicopathologic features. Five tumors were well differentiated, 39 moderately differentiated, and 7 poorly differentiated. Nine tumors had heterologous elements (8 epithelial, one mesenchymal), and four showed retiform differentiation; all of these were moderately differentiated. DICER1 mutations were identified in 16/45 of successfully genotyped tumors (36%, 15 moderately differentiated, 1 poorly differentiated), including 7 with heterologous elements and 3 of 4 with retiform differentiation. FOXL2 c.402C>G mutation was identified in 10/51 tumors (20%, 8 moderately differentiated, 2 poorly differentiated). DICER1 and FOXL2 mutations were mutually exclusive. Median age for the entire cohort was 44 years (range 15-90). Patients with DICER1 mutations were younger (median 23.5 years, range 15-62) than patients with neither DICER1 nor FOXL2 mutation (median 41.5 years, range 16-74). Tumors with FOXL2 mutation occurred exclusively in postmenopausal patients (median 78 years, range 54-90), whereas 11/13 tumors with either heterologous elements (median 26 years, range 16-62) or retiform differentiation (median 38 years, range 18-57) occurred in premenopausal patients. Our data suggest the existence of at least three molecular subtypes of SLCT: DICER1 mutant (younger, moderately/poorly differentiated, including heterologous elements or retiform differentiation), FOXL2 mutant (postmenopausal, moderately/poorly differentiated, no heterologous elements or retiform differentiation), and DICER1/FOXL2 wild-type (no specific age, no heterologous elements, including all well-differentiated tumors). These findings suggest that well-differentiated and moderately/poorly differentiated SLCTs may be different histomolecular entities with different etiologies.

Citation Format: Anthony N. Karnezis, Yemin Wang, Jamie Magrill, Jacqueline Keul, Stefan Kommoss, Basile Tessier-Cloutier, Lily Proctor, Dietmar Schmidt, C Blake Gilks, David G. Huntsman, Friedrich Kommoss. DICER1 and FOXL2 mutations correlate with clinicopathologic features of ovarian Sertoli-Leydig cell tumors. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B46.
Abstract B47: Rgnef (p190RhoGEF/Arhgef28) loss impairs ovarian tumor metastatic growth
Authors: Kleinschmidt Elizabeth G. , Tancioni Isabelle , Taylor Kristin , Osterman-Diaz Carlos , Miller Nichol L. G. , Jiang Shulin , Schlaepfer David D.
Published: August 2018

Increased activation of FAK and Rho GTPase regulatory pathways is associated with shorter survival in patients with high-grade serous ovarian carcinoma (HGSOC). However, the key mediators of these signaling events in HGSOC remain unknown. We have previously shown that the Rho guanine nucleotide exchange factor Rgnef (also named p190RhoGEF or Arhgef28) activates FAK as well as RhoA GTPase in mouse fibroblasts. Here we find that Rgnef protein expression is elevated in Stage 3-4 HGSOC and that Rgnef knockout prevents ovarian tumor spheroid growth in vitro and in vivo. We isolated ascites-associated murine ID8 cells to generate a more aggressive cell line, termed ID8-IP. ID8-IP cells exhibit higher Rgnef and FAK expression than parental ID8 cells. CRISPR/Cas9-induced knockout of Rgnef in ID8-IP cells does not alter 2D growth as compared to Rgnef-KO cells re-expressing GFP-Rgnef. However, Rgnef loss significantly decreased growth as spheroids in a 3D Matrigel-on-Top assay. At five weeks following intraperitoneal injection into syngeneic C57Bl/6 mice, ID8-IP Rgnef-KO cells form significantly fewer ascites-associated cells, as well as fewer metastatic lesions on the peritoneal wall. Notably, ID8-IP Rgnef-KO cells display lower FAK and ERK1/2 phosphorylation. These studies demonstrate that Rgnef loss impedes metastatic growth in the peritoneal cavity, and suggest that an integrin-mediated Rgnef-FAK linkage facilitates ovarian tumor metastasis.

Citation Format: Elizabeth G. Kleinschmidt, Isabelle Tancioni, Kristin Taylor, Carlos Osterman-Diaz, Nichol L. G. Miller, Shulin Jiang, David D. Schlaepfer. Rgnef (p190RhoGEF/Arhgef28) loss impairs ovarian tumor metastatic growth. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B47.