bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract B31: Combination inhibitor strategies targeting KRAS effector signaling in KRAS-mutant pancreatic cancer
Authors: Ozkan-Dagliyan Irem , Goodwin Craig M. , Bryant Kirsten L. , George Samuel D. , Lucas Kelly , Gautam Prson , Wennerberg Krister , Cox Adrienne D. , Der Channing J.
Published: June 2021

With KRAS mutations found in 95% of pancreatic ductal adenocarcinoma (PDAC), an effective anti-KRAS therapeutic strategy is anticipated to make a significant impact on the treatment of PDAC. Among the major directions currently being pursued, inhibitors of KRAS effector signaling are believed to be the most promising. However, as monotherapy, effector inhibitors have not demonstrated significant activity and concerns with normal tissue toxicity remain. Combination inhibitor strategies are considered the best approach to overcome these limitations. To identify effector inhibitor-based combinations, we applied a 525-oncology drug screen to identify combinations that enhanced the cytotoxic activity of inhibitors of the RAF-MEK-ERK and the PI3K-AKT-mTOR effector pathways. While many cytotoxic combinations were identified with RAF effector pathway inhibitors, few were identified with PI3K pathway inhibitors. The same classes of inhibitors were identified with RAF, MEK and ERK inhibitors. In addition to inhibitors of the PI3K-AKT-mTOR pathway, microtubule, HDAC and HSP90 inhibitors synergistically enhanced RAF and ERK inhibitor antitumor activity. Addressing a basis for synergy, we found that the different combinations showed enhanced loss of MYC protein, a key ERK substrate. In summary, our studies identify promising effector inhibitor-based combinations for PDAC treatment.

Citation Format: Irem Ozkan-Dagliyan, Craig M. Goodwin, Kirsten L. Bryant, Samuel D. George, Kelly Lucas, Prson Gautam, Krister Wennerberg, Adrienne D. Cox, Channing J. Der. Combination inhibitor strategies targeting KRAS effector signaling in KRAS-mutant pancreatic cancer [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 B31.
Abstract B35: Discovery of K-Ras(G12D)-targeting peptide KRpep-2d and its optimization strategy
Authors: Sakamoto Kotaro
Published: June 2021

Mutated Ras proteins play important roles in cell differentiation, proliferation, and survival of various cancers, especially mutations at residue Gly12 where G12D in K-Ras is the most frequent mutation. Although over 30 years have passed since the discovery of mutated Ras in human cancers, effective drug targeting to Ras has not been marketed. Very recently, KRpep-2d, an artificial cyclic peptide, was discovered as a first selective inhibitor to K-Ras(G12D) by phage display technology. KRpep-2d possesses low nanomol level activity in cell-free assays and shows preferable target selectivity in cell-based assay. In this presentation, I review the biochemical activity, biophysical activity, and SAR of KRpep-2d. In addition, I will discuss a strategy to brush up KRpep-2d for its therapeutic use.

Citation Format: Kotaro Sakamoto. Discovery of K-Ras(G12D)-targeting peptide KRpep-2d and its optimization strategy [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 B35.
Abstract B47: Systems-level dissection of tumor-macrophage crosstalk in ovarian cancer resistance to MEK inhibition
Authors: Wang Stephanie J. , Miller Miles A. , Lauffenburger Douglas A.
Published: June 2021

Several subtypes of ovarian cancer are associated with dysregulated MAPK signaling, including low-grade serous ovarian carcinoma (LGSOC), yet targeted inhibitors of the MAPK pathway have shown relatively limited success in clinical trials for gynecologic malignancies. Here, we investigated whether this could be explained in part by crosstalk between ovarian cancer cells and neighboring tumor-associated macrophages (TAMs) within the tumor microenvironment. Using in vitro models, human ovarian cancer cell lines were mildly protected against MEK inhibitor-induced cell killing by transwell co-culture with alternatively activated (IL-4 stimulated), but not classically activated (LPS and IFN-gamma stimulated), primary monocyte-derived macrophages. We employed multiplexed bead-based immunoassays to acquire a compendium of over 1,200 phosphoprotein, cytokine, and growth factor measurements across combinations of 3 ovarian cancer cell lines and macrophages derived from 4 healthy peripheral mononuclear blood cell (PBMC) donors. Principal components analysis (PCA) and partial least squares regression (PLSR) revealed that compensatory receptor tyrosine kinase signaling occurs in tumor cells upon MEK inhibitor treatment, and that this is amplified upon co-culture with alternatively activated macrophages. Further work will determine if targeting these tumor-intrinsic and crosstalk effects in combination with MEK inhibition represents a potential therapeutic strategy.

Citation Format: Stephanie J. Wang, Miles A. Miller, Douglas A. Lauffenburger. Systems-level dissection of tumor-macrophage crosstalk in ovarian cancer resistance to MEK inhibition [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 B47.
Abstract B52: KRAS-IRF2 axis drives immune suppression and immune therapy resistance in colorectal cancer
Authors: Liao Wenting , Overman Michael , Boutin Adam , Kopetz Scott , DePinho Ron , Wang Alan
Published: June 2021

The precise biologic functions and mechanisms of oncogenic KrasG12D (Kras*) in therapeutic resistance, including immune therapy, are not understood. Here, utilizing a colorectal cancer (CRC) mouse model engineered with an inducible Kras* transgene and conditional null APC and Trp53 alleles (iKAP) that we recently established (Boutin et al., Gene & Development 2017), we observed that KRAS*-expressing invasive tumors possess a prominent immune-suppressive profile characterized by a preponderance of myeloid-derived suppressor cells (MDSCs). Integrated transcriptomic and genomic analyses identified the Interferon Regulatory Factor 2 (IRF2) gene as a key downstream target of KRAS* mediating tumor progression and immune suppression. Mechanistically, integrated transcriptomics, cytokine profiling and ChIP-seq analysis identified CXCL3 as a key direct target of IRF2, and pharmacologic inhibition of CXCL3 or its receptor CXCR2 resulted in suppression of MDSC migration and infiltration in KRAS*-expressing CRC tumors. Translational studies established that de novo anti-PD1 resistance of KRAS*-expressing tumors can be overcome by enforced IRF2 expression or by treatment with small-molecule CXCR2 antagonist SX-682. Correspondingly, CRC patient tumors with higher IRF2 expression show increased response to anti-PD1 therapy. This KRAS*-mediated immune suppressive mechanism provides a framework for patient selection and combination therapies to enhance the effectiveness of immune checkpoint blockade therapy in advanced CRC.

Citation Format: Wenting Liao, Michael Overman, Adam Boutin, Scott Kopetz, Ron DePinho, Alan Wang. KRAS-IRF2 axis drives immune suppression and immune therapy resistance in colorectal cancer [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 B52.
Abstract IA04: Targeting RAS for anticancer therapy
Authors: Campbell Sharon , Carey Leiah , Prakash Ajit , Clark Geoff , Scarry Sarah , Aube Jeff
Published: June 2021

RAS is the most frequently activated oncoprotein in human cancer. Mutations in KRAS are prevalent in pancreatic, colon, and lung cancer. Despite the need for anti-RAS therapies, there are no clinically successful targeted therapies for RAS. We will present efforts to characterize KRAS G12C covalent inhibitors and compounds that bind to the KRAS P1 pocket. In addition, the Clark lab (U. of Louisville) has identified novel small molecules that bind and directly inhibit RAS function. Uniquely, the current lead compound binds preferentially to the activated form of RAS. It blocks the interaction of RAS with downstream effectors and suppresses the transforming activity of RAS in soft agar assays. The best compounds give an IC50 of ~500 nM in vitro. The inhibitor is active in vivo and suppresses growth of RAS-driven xenografts in NRG mice. There is no detectable toxicity. We have initiated efforts to solve the structure of these inhibitors in complex with RAS, to characterize the nucleotide dependence as well as inhibition of RAS interactions with regulatory and effector proteins in vitro using X-ray crystallography in combination with NMR, biochemical, and bioactivity assays. Results from these analyses will aid in elucidating the specificity and the mechanism of action needed to optimize the inhibitors and facilitate development into a drug candidate.

Citation Format: Sharon Campbell, Leiah Carey, Ajit Prakash, Geoff Clark, Sarah Scarry, Jeff Aube. Targeting RAS for anticancer therapy [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 IA04.
Abstract IA13: Targeting redox dependencies in pancreatic cancer
Authors: Chio Iok In Christine , Oertlin Christian , Robert Francis , Chan Karina , Liberm Dana Kapellar , Santana Abram , Park Young , Larsson Ola , Pelletier Jerry , Tuveson David
Published: June 2021

Dysregulation of mRNA translation is a common feature of human cancers. As such, therapeutic agents that target components of the protein synthesis apparatus hold promise as anticancer drugs. We previously showed that protein synthesis is upregulated in Kras mutant pancreatic cancer cells in a manner that is redox-dependent (1). By modulating formation of the eIF4F complex, the PI3K/AKT and mTOR signaling pathways act as major regulators of global and mRNA-specific translation. Consistently, we observed that protein synthesis and the viability of pancreatic tumor organoids are selectively decreased by the pan-AKT inhibitor MK2206, an effect that was potentiated by co-treatment with the pro-oxidant BSO1. Treatment of KrasG12D;p53R172H;PdxCre (KPC) mice with this drug combination resulted in suppressed tumor kinetics and modest improvement in survival. Inhibition of AKT/mTOR blunts cap-dependent translation initiation but also perturbs a plethora of other cellular functions. We thus investigated more selective means of targeting the translation machinery that do not entail wholesale ablation of the AKT/mTOR pathway. The RNA helicase eIF4A initiates translation by unwinding highly structured 5′-untranslated regions (UTRs) in mRNAs. Using polysome profiling followed by deep sequencing, we found that the translation of eIF4A-sensitive mRNAs was selectively increased in Kras-mutant tumor versus normal organoids. The rocaglate, CR-13-1b, is a compound that selectively inhibits the eIF4A helicase and displayed high toxicity in Kras mutant tumor organoids, but not in normal organoids. Polysome sequencing revealed that CR131b selectively modulates translational efficiencies in tumor but not normal organoids. Interestingly, this included multiple mRNAs encoding proteins involved in mitochondrial activity and redox homeostasis. As a single agent, CR131b treatment in KPC mice effectively inhibited protein synthesis in vivo, resulting in a significant decrease in tumor kinetics in a short-term study and an improvement in survival in a longitudinal study.

Reference

1. Chio II et al. NRF2 promotes tumor maintenance by modulating mRNA translation in pancreatic cancer. Cell 2016;166:963-76, doi:10.1016/j.cell.2016.06.056.

Citation Format: Iok In Christine Chio, Christian Oertlin, Francis Robert, Karina Chan, Dana Kapellar Liberm, Abram Santana, Young Park, Ola Larsson, Jerry Pelletier, David Tuveson. Targeting redox dependencies in pancreatic cancer [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 IA13.
Abstract IA15: T-Cells targeting mutated RAS—clinical trials
Authors: Yang James , Hanada Kenichi , Rosenberg Steven
Published: June 2021

Durable complete regressions of metastatic cancers can be achieved by the transfer of in vitro expanded tumor-reactive T-cells. In patients with melanoma, this can be done by giving tumor-infiltrating lymphocytes (TIL) reactive with tumor-specific mutated antigens (“neoantigens”). Unfortunately, the vast majority of immunogenic neoantigens have been shown to be totally patient specific. One exception is that T cells recognizing mutated KRAS have been found in the TIL of multiple patients. In one patient with G12D-mutated colon cancer, these cells were grown and administered to that patient (after a course of preparative immunosuppression) and mediated regression of multiple pulmonary metastases. A single lesion that had lost its presenting MHC Class I allele grew and was resected, and the patient remains disease free over 2 years later. Other patients have demonstrated additional Class I and Class II presented epitopes from mutated RAS, although they have not yet been administered clinically. In an effort to develop standardized reagents to target common RAS mutations, HLA-A11 transgenic mice were immunized with peptides containing G12D and G12V RAS mutations and HLA-A11 restricted T cells isolated. Murine T-cell receptors specific for these mutated epitopes were cloned and when retrovirally introduced into human PBL, they conferred recognition of mutated RAS but not wild-type RAS. Such cells could cause regression of established palpable pancreatic cancer xenografts in NSG mice. Two clinical trials targeting the G12D and G12V RAS variants with T-cell receptor-engineered autologous PBL in any patient who is HLA-A11 are now open to enrollment. Future efforts are under way to expand the library of receptors to encompass additional RAS mutations, other HLA alleles, and other shared mutations in common driver oncogenes.

Citation Format: James Yang, Kenichi Hanada, Steven Rosenberg. T-Cells targeting mutated RAS—clinical trials [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 IA15.
Abstract IA17: Molecular mechanisms that regulate Raf kinase signaling
Authors: Morrison Deborah
Published: June 2021

Raf kinases are essential for normal Ras pathway signaling, and activating mutations in components of this pathway are associated with a variety of human cancers as well as a group of related developmental disorders known collectively as the RASopathies. Of the core pathway components, the mechanisms that modulate the Raf family kinases are by far the most complex, involving changes in subcellular localization, protein interactions, and phosphorylation/dephosphorylation events. In addition, like numerous other protein kinases, the Raf kinases can form dimers. Functional studies investigating the importance of Raf dimerization have revealed that Raf dimer formation is required for normal Ras-dependent Raf activation and for the biologic activity of many disease-associated Raf mutants. At the meeting, I will present recent work examining the regulatory differences among Raf family members and will discuss how these differences contribute to Raf-mediated disease signaling. I will also present studies investigating the effect that various therapeutic agents have on Raf regulatory interactions and will describe new approaches to disrupt Ras-Raf-MEK-ERK pathway signaling.

Citation Format: Deborah Morrison. Molecular mechanisms that regulate Raf kinase signaling [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 IA17.
Abstract IA18: Targeting K-Ras directly
Authors: Shokat Kevin
Published: June 2021

Somatic mutations in the small GTPase K-Ras are the most common activating lesions found in human cancer and are generally associated with poor response to standard therapies. Efforts to directly target this oncogene have faced difficulties due to its picomolar affinity for GTP/GDP and the absence of known allosteric regulatory sites (1). Oncogenic mutations result in functional activation of Ras family proteins by impairing GTP hydrolysis. With diminished regulation by GTPase activity, the nucleotide state of Ras becomes more dependent upon relative nucleotide affinity and concentration. This gives GTP an advantage over GDP and increases the proportion of active GTP-bound Ras. I will discuss the development of small molecules that irreversibly bind to a common oncogenic mutant, K-RasG12C (2). These compounds rely on the mutant cysteine for binding and therefore do not affect the wild-type protein (WT). Crystallographic studies reveal the formation of a new pocket that is not apparent in previous structures of Ras, beneath the effector binding switch-II region. These data provide structure-based validation of a novel allosteric regulatory site on Ras that is targetable in a mutant-specific manner.

References

1. Stephen AG, Esposito D, Bagni RK, McCormick F. Dragging Ras back in the ring. Cancer Cell 2014;25: 272–81.

2. Ostrem JM, Peters U, Sos ML, Wells JA, Shokat KM. Nature 2013;12796:503, 548–51.

Citation Format: Kevin Shokat. Targeting K-Ras directly [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 IA18.
Abstract IA25: Pancreatic cancer models and medicine
Authors: Tuveson David
Published: June 2021

Since the most common KRAS oncoproteins lack therapeutic antagonists, we utilized mouse and organoid models to discover new characteristics of oncogenic KRAS that could represent new therapeutic opportunities in pancreatic ductal adenocarcinoma (PDAC). Continuous oncogenic Kras expression is required in PDAC mouse models to support the characteristic desmoplastic stroma that impairs drug delivery, suppresses the immune response, and promotes tumor progression. We identified the secretion of Tgf-β and IL-1α by neoplastic cells as ligands that produce stroma by stimulating the differentiation of pancreatic stellate cells into myofibroblasts (myCAFs) and inflammatory fibroblasts (iCAFs), respectively. Mutual antagonism between these two ligands led to the spatial patterning of these two fibroblast populations, with myCAFs juxtaposed to neoplastic cells while iCAFs located more distantly. Genetic and pharmacologic perturbation of iCAFs support the role of this subpopulation in PDAC progression, nominating them as a cellular target in Kras-dependent PDAC. Secondly, taking a cell-intrinsic approach, we found that oncogenic KRAS induced BNIP3L/Nix to stimulate selective mitophagy. Nix induction restricted flux of glucose to the mitochondria and enhanced cellular redox control, and loss of Nix reverted these effects and decreased cell proliferation in vitro. Furthermore, loss of Nix markedly delayed progression of pancreatic cancer in vivo. Our results reveal the Kras-Nix mitophagy program as a new mechanism to promote aerobic glycolysis, redox homeostasis, and disease progression in KRAS-mutant pancreatic cancer, and motivate the exploration of methods to antagonize Nix pharmacologically.

Citation Format: David Tuveson. Pancreatic cancer models and medicine [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 IA25.
Abstract IA33: Development of effective therapies for neurofibromatosis type 1 and other pediatric RAS-driven tumors
Authors: Widemann Brigitte
Published: June 2021

The RAS pathway is critical for cell survival and development. More than 20% of all adult cancers and many of the most aggressive pediatric cancers are driven by sporadic mutations of the RAS family of genes. In spite of substantial efforts by academia and the pharmaceutical industry, no effective therapies targeting RAS-mutated cancers have been identified to date. RASopathies are genetic disorders characterized by germline mutations in RAS pathway genes. These syndromes, such as neurofibromatosis type 1 (NF1), Noonan syndrome (NS), cardiofaciocutaneous syndrome, and Costello syndrome (CS), have a widely variable incidence (1:1000 NS -1:300,000 CS), are usually first diagnosed in children, and exhibit a wide variety of manifestations including an increased risk of development of pediatric cancers such as sarcomas, leukemias, and CNS tumors (up to 42-fold increased risk compared to the general population). No approved medical therapies exist for RASopathies. Through the conduct of a longitudinal natural history study, preclinical studies in collaboration with extramural investigators, and early-phase trials, the NCI Pediatric Oncology Branch recently identified the MEK inhibitor selumetinib as the first active therapy for debilitating peripheral nerve sheath tumors called plexiform neurofibromas (PN) in NF1. The POB has identified and clinically characterized atypical neurofibromas (ANF) in NF1 as precursors to aggressive sarcomas called malignant peripheral nerve sheath tumors (MPNST). This has allowed for the development of targeted therapies and prevention strategies for these tumors. This example raises the possibility of developing effective therapies for manifestations of other RASopathies. Recent comprehensive molecular, epigenetic, and genomic studies have demonstrated the mechanisms of RAS pathway activation in several pediatric malignancies including rhabdomyosarcoma, relapsed neuroblastoma, gliomas, and leukemias. These studies will enable the development of clinical trials targeting these tumors more effectively.

Citation Format: Brigitte Widemann. Development of effective therapies for neurofibromatosis type 1 and other pediatric RAS-driven tumors [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 IA33.
Abstract PR05: Selective contribution of the SHOC2 phosphatase complex to ERK pathway dynamics highlights its potential as a therapeutic target
Authors: Boned del Rio Isabel , Young Lucy C. , Sari Sibel , Jones Greg G. , Bhamra Amandeep , Surinova Silvia , Rodriguez-Viciana Pablo
Published: June 2021

The ERK signaling pathway is hyperactivated in the majority of human cancers. However, ERK signaling mediates myriad physiologic responses and on-target toxicity has severely limited the clinical efficacy of current ERK pathway inhibitors. Despite the key role of RAF kinases in physiology and disease, we still lack a complete understanding of their regulation. Here we show that dephosphorylation of the conserved ‘S259’ RAF inhibitory site is a key activating step mediated by the SHOC2-MRAS-PP1 complex. Gain-of-function mutations in SHOC2, MRAS, PP1 and CRAF (clustering around S259) found in Noonan syndrome independently validate the important role of the SHOC2 complex in RAF-ERK pathway regulation. We have used CRISPR/cas9 to knock out SHOC2 and the 3 RAF isoforms in colorectal DLD-1 cancer cells. Our data show that SHOC2 is required for EGF-induced dephosphorylation of S365 BRAF, 14-3-3 and MEK dissociation from BRAF and RAF dimerization. However, whereas SHOC2 function is essential for a rapid transient phase of ERK activation in response to EGF stimulation, it is not required for a slow, sustained phase that is instead driven by palmitoylated H/N-RAS proteins and N-region phosphorylation of CRAF. Redundancy makes SHOC2 dispensable for ERK activation and proliferation in 2D whereas KRAS mutant cells rely on SHOC2 for ERK signaling under anchorage-independent/3D conditions, which thus presents a therapeutic opportunity. This study highlights a context-dependent contribution of SHOC2 to ERK pathway dynamics that is preferentially engaged by KRAS oncogenic signaling and provides a biochemical framework for how targeting the SHOC2 regulatory node would allow for selective ERK pathway inhibition, likely to provide improved therapeutic margins in the clinic.

This abstract is also being presented as Poster A32.

Citation Format: Isabel Boned del Rio, Lucy C. Young, Sibel Sari, Greg G. Jones, Amandeep Bhamra, Silvia Surinova, Pablo Rodriguez-Viciana. Selective contribution of the SHOC2 phosphatase complex to ERK pathway dynamics highlights its potential as a therapeutic target [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 PR05.
Abstract AP02: EXPOSING THE LOCAL FACTORS THAT CONTRIBUTE TO OVARIAN COLONIZATION OF FALLOPIAN TUBE-DERIVED TUMORS
Authors: Burdette Joanna E. , Zink Katherine , Dean Matthew , Sanchez Laura M.
Published: June 2021

High grade serous cancer (HGSC) originates in the fallopian tube and typically invade and colonize the ovary as part of peritoneal metastasis. The purpose of this study was to define the factors that contribute to ovarian specific metastasis of fallopian tube HGSC. While protein factors have been explored that facilitate migration and invasion of fallopian tube cell to the ovary, the small molecule communication between the tissues during metastasis remains largely unexplored. Due to the proximity of ovarian primary metastasis, an experimental design was optimized using imaging mass spectrometry to capture the spatial composition of small molecules uniquely expressed when fallopian tube-derived tumor cells were grown in the microenvironment of the ovary as a model of primary metastasis. The observed mass-to-charge ratios (m/z's) that were induced specifically in co-culture represent small molecules that may contribute to the metastasis of HGSOC selectively to the ovary. Human fallopian tube epithelial HGSOC and tumorigenic murine oviductal epithelial cells, but not normal cell types, repeatedly induced a signal from the ovary at m/z 170. This signal was identified as norepinephrine, which was confirmed to simulate invasion of ovarian cancer cells lacking wildtype p53. Norepinephrine has an established role in ovarian cancer metastasis to secondary sites and the use of beta-blockers in women is associated with increased survival. The use of imaging mass spectrometry also revealed a series of new molecules that are specifically enhanced from the ovary or the fallopian tube during co-culture. These molecules may reveal pathways that contribute to metastasis and biological targets for therapeutic intervention to block ovarian metastasis of fallopian tube-derived HGSOC. The developed mass spectrometry method can be adapted to other mammalian-based model systems for investigation of untargeted metabolomics that facilitate metastasis.

Citation Format: Joanna E. Burdette, Katherine Zink, Matthew Dean, and Laura M. Sanchez. EXPOSING THE LOCAL FACTORS THAT CONTRIBUTE TO OVARIAN COLONIZATION OF FALLOPIAN TUBE-DERIVED TUMORS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr AP02.
Abstract AP13: GENOME-SCALE CRISPR KNOCKOUT SCREEN IDENTIFIES TIGAR AS A MODIFIER OF PARP INHIBITOR SENSITIVITY
Authors: Fang Pingping , Minn Kay , Chien Jeremy
Published: June 2021

A key challenge is to understand the mechanisms that contribute to “BRCAness” and hence sensitivity to poly (ADP-ribose) polymerase (PARP) inhibitors. Using the sensitivity to PARP inhibitor olaparib as a surrogate for “BRCAness”, we performed a CRISPR/Cas9-based genome-scale loss-of-function screen and identified TIGAR as a modifier of olaparib response. TIGAR knockdown increases the level of intracellular reactive oxygen species, induces more DNA damage after olaparib treatment, and causes “BRCAness” by downregulating BRCA1 and genes in the Fanconi anemia pathway. Further, TIGAR knockdown induces senescence, reduces spheroid tumor cell growth, and enhances the cytotoxic effect of olaparib. Finally, TIGAR is amplified in ovarian cancer and a higher expression of TIGAR is associated with poor overall survival in high-grade serous ovarian cancer. Our study shows that TIGAR knockdown causes extensive transcriptional reprogramming and enhances sensitivity to PARP inhibitor, demonstrating that TIGAR is a relevant therapeutic target for treating ovarian cancer.

Citation Format: Pingping Fang, Kay Minn, Jeremy Chien. GENOME-SCALE CRISPR KNOCKOUT SCREEN IDENTIFIES TIGAR AS A MODIFIER OF PARP INHIBITOR SENSITIVITY [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr AP13.
Abstract AP30: BRCA1 INTRON RETENTION GENERATES TRUNCATED PROTEINS THAT AVOID BRCT MUTATION MISFOLDING AND PROMOTE PARP INHIBITOR RESISTANCE
Authors: Wang Yifan , Bernhardy Andrea J. , Nacson Joseph , Krais John J. , Tan Yin-Fei , Slifker Michael , Peri Suraj , Johnson Neil
Published: June 2021

INTRODUCTION: BRCA1 BRCT domain mutations result in protein structural defects, and consequently misfolded proteins are subject to proteasomal degradation. Loss of BRCA1 protein activity results in sensitivity to PARP inhibitor (PARPi) therapy.

EXPERIMENTAL PROCEDURES: In this study, we examined PARPi resistance using the SNU-251 human endometrial ovarian cancer cell line. SNU-251 cells harbor a nonsense mutation c.5444G>A (E1815X) in BRCA1 exon 23; that disrupts BRCT domain folding. Cells were cultured in the presence of increasing concentrations of the PARPi rucaparib until resistant cells emerged. RT-PCR, Western blotting and mass spectrometry were used to measure BRCA1 mRNA and protein levels.

RESULTS: PARPi resistant cell lines all demonstrated elevated BRCA1 protein levels that could be detected with N- but not C-terminal specific antibodies. Notably, the gel migration and molecular weight of BRCA1 was different from the mutation-induced stop codon expected size. Mass spectrometric analyses identified BRCA1 peptides encoded by exons 2-15; however, despite harboring a mutation in exon 23, no peptides encoded by exons 16-24 were present in SNU-251 cells. RT-PCR analyses showed that SNU-251 cells translated protein from exon 15 and into intron 15, terminating at an intron 15 generated stop codon. We overexpressed BRCA1 cDNA that harbored stop codons located in either the BRCT domain or in intron 15. BRCT mutation containing constructs had undetectable protein levels due to protein misfolding, and cells were highly PARPi sensitive. In contrast, cells expressing the BRCA1 intron 15 encoded stop codon had robust protein expression, demonstrated RAD51 foci and chemotherapy resistance in vitro and in vivo.

CONCLUSIONS: In summary, we discovered that BRCA1 intron translation generates new stop codons resulting in loss of the entire BRCT domain. BRCTless proteins avoid mutant protein folding problems and promote residual DNA repair and chemotherapy resistance.

Citation Format: Yifan Wang, Andrea J. Bernhardy, Joseph Nacson, John J. Krais, Yin-Fei Tan, Michael Slifker, Suraj Peri and Neil Johnson. BRCA1 INTRON RETENTION GENERATES TRUNCATED PROTEINS THAT AVOID BRCT MUTATION MISFOLDING AND PROMOTE PARP INHIBITOR RESISTANCE [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr AP30.
Abstract DP-006: THE EFFECT OF AGE AND OCP USE ON THE INCIDENCE OF PRE-CANCEROUS P53 LESIONS AND THE DEVELOPMENT OF HIGH GRADE SEROUS OVARIAN CARCINOMA
Authors: Greening Kendall , Karnezis Anthony , Cochrane Dawn , Hanley Gillian , Huntsman David
Published: June 2021

BACKGROUND: High grade serous ovarian cancer (HGSOC) accounts for >70% of ovarian cancer related deaths and is the most common ovarian cancer histotype, most originating from pre-cancerous p53 lesions in the fallopian tube (FT) fimbria. Use of oral contraceptive pills (OCPs) for 5 years or more is associated with >40% reduction in risk of HGSOC, but the mechanism is unknown. We hypothesize that OCP use reduces the incidence of p53 lesions. Our preliminary data show higher incidence of p53 lesions in post- compared to pre-menopausal women, therefore we aim quantify p53 lesions in post-menopausal women who previously did or did not use OCPs. This will provide insight into the protective effects of OCPs against HGSOC.

PRELIMINARY RESULTS: We determined the presence of p53 lesions by immunohistochemistry (IHC) in FT of women up to 40 years old (n=27) and >60 years old (n=24) who underwent salpingectomies for non-cancer reasons. p53 lesions were identified in 3/27 cases of the younger cohort (11%) and in 10/24 of the older cohort (42%). Thus, we conclude an increased incidence of p53 lesions in older compared to younger women.

PROPOSED DESIGN: IHC for p53 will be performed on FT fimbria of women >55 years old who received salpingectomy/hysterectomy for non-cancer reasons. Based on an assumed reduction in p53 lesions of 35% in women who used OCPs for 5 years or more compared to non-users (25 vs. 42%), analysis of 190 cases from each group will provide >80% power (p<0.05). Cases will be identified through Population Data BC and blind analysis by tp53 IHC will be performed at the Vancouver General Hospital. Post-menopausal status will be confirmed by endometrium histology and data flowed back to Pop Data BC to compare to OCP data.

CONCLUSION: Our preliminary study found that 42% of post menopausal women had p53 lesions, informing this study design. The study registered through this abstract will be the first to examine the impact of OCPs on the earliest known precursors of HGSOC.

Citation Format: Kendall Greening, Anthony Karnezis, Dawn Cochrane, Gillian Hanley, David Huntsman. THE EFFECT OF AGE AND OCP USE ON THE INCIDENCE OF PRE-CANCEROUS P53 LESIONS AND THE DEVELOPMENT OF HIGH GRADE SEROUS OVARIAN CARCINOMA [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr DP-006.
Abstract DP-010: INTRAPERITONEAL CHEMOTHERAPY USE AND OUTCOMES: EXPLORATION OF UPMC OVARIAN CANCER REGISTRY DATA
Authors: S Adambekov , S Wang , S Taylor , R Buckanovich , L Coffman , RP Edwards , F Linkov
Published: June 2021

BACKGROUND: Ovarian cancer (OC) is the deadliest gynecologic cancer. Despite randomized clinical trials showing improved survival for patients receiving intraperitoneal chemotherapy (IP), IP has not received a wide use outside of specialty hospitals. The aim of this study was to explore the impact of IP on OC patient survival and to evaluate whether treatment facility type impacted the outcomes.

METHODS: Detailed demographic and clinical information on OC patients (N=2924) who underwent treatment in UPMC facilities between 2000-2016 was obtained from the UPMC Cancer Registry. Duplicate records, rare, grade 1, and non-epithelial tumors were excluded. Kaplan-Meier plots were constructed to compare 10-year survival rates based on the chemotherapy type (IP vs. no-IP), cancer grade, cancer stage, surgery type, neoadjuvant and treatment facility (specialized vs. community). Multivariable Gray's models and logistic regression model were fitted to evaluate the effect of different factors on survival and tumor recurrence respectively. Two tailed P-values <0.05 were considered significant. R software package was used to analyze the data.

RESULTS: The final sample consisted from 1840 patients (250 IP and 1590 no-IP). IP chemotherapy was used only in 14% of OC patients and was associated with improved long-term survival. Similarly, cases reported by specialty treatment facilities (Magee and Passavant) had better survival compared to other hospitals. Multivariable Gray's model showed that IP and younger age were significantly associated with lower hazard of death, whereas higher cancer stage is significantly associated with higher hazard of death. Multivariable logistic regression showed that IP is not significantly associated with recurrence after adjusting for median income in the zip code of patient residence and cancer stage.

CONCLUSIONS: These findings demonstrate enhanced long-term survival of patients treated with IP therapy at specialty centers. Increasing IP therapy use in clinical practice for OC patient treatment may be important strategy to improve OC outcomes.

Citation Format: Adambekov S, Wang S, Taylor S, Buckanovich R, Coffman L, Edwards RP, Linkov F. INTRAPERITONEAL CHEMOTHERAPY USE AND OUTCOMES: EXPLORATION OF UPMC OVARIAN CANCER REGISTRY DATA [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr DP-010.
Abstract GMM-028: VALIDATION OF RUNX3 AND CAMKIINα HYPERMETHYLATION AS PROGNOSTIC MARKER FOR OVARIAN CARCINOMA
Authors: Heinze Karolin , Jansen Lars , Oliveira-Ferrer Leticia , Schmalfeldt Barbara , Dürst Matthias , Runnebaum Ingo B , Häfner Norman
Published: June 2021

INTRODUCTION: Ovarian cancer is still the most lethal gynecological malignancy with a low five-years-overall survival rate. Except for BRCA1/2 mutations there is no predictive nor prognostic marker available indicating whether a patient is in need and eligible for personalized therapy. In a previous genome-wide array study we identified different methylation patterns in type II epithelial ovarian cancer patients with either good or poor outcome (PFS < vs. > 3 years). Using methylation-specific PCR (MSP) five marker candidates showed high prognostic value whereby the marker combination RUNX3/CaMKIINα was the most promising one [Häfner et al., 2015]. Moreover, in vitro experiments provided evidence that a transcript variant of RUNX3 can mediate Cisplatin sensitivity, further underlining the clinical relevance of this gene [Heinze et al., 2018].

METHODS: In this study we aim to validate the previous described prognostic value of the five candidates, especially of RUNX3 and CaMKIINα via MSP. Therefore, 100 fresh-frozen primary type II EOC samples from two different surgical centers were tested. Furthermore, previously analyzed samples were retested to gain insight on the marker distributions throughout the tumor and possible heterogeneity. The DNA was isolated via phenol/chloroform or QiaAmp Kit, converted to bisulfite DNA and tested via optimized MSP. All samples had similar clinical parameter (histology, FIGO grade, chemotherapy).

RESULTS AND DISCUSSION: By re-testing a subset of EOC samples previous results could been confirmed. Additionally, the methylation pattern was similar after renewed sectioning for DNA extraction pointing towards little tumor heterogeneity in the analyzed samples. Interestingly the methylation rate is not as high as expected from the initial analyses. Statistical analysis of the data is currently ongoing.

REFERENCES

Häfner N, Steinbach D, Jansen L, Diebolder H, Dürst M, Runnebaum IB. RUNX3 and CAMK2N1 hypermethylation as prognostic marker for epithelial ovarian cancer, Int J Cancer. 2016

Heinze K, Kritsch D, Mosig AS, Dürst M, Häfner N, Runnebaum IB. Functional Analyses of RUNX3 and CaMKIINα in Ovarian Cancer Cell Lines Reveal Tumor-Suppressive Functions for CaMKIINα and Dichotomous Roles for RUNX3 Transcript Variants, Int J Mol Sci. 2018

Citation Format: Karolin Heinze, Lars Jansen, Leticia Oliveira-Ferrer, Barbara Schmalfeldt, Matthias Dürst, Ingo B Runnebaum, Norman Häfner. VALIDATION OF RUNX3 AND CAMKIINα HYPERMETHYLATION AS PROGNOSTIC MARKER FOR OVARIAN CARCINOMA [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-028.
Abstract GMM-041: OVARIAN CANCER CELLS INDUCE A FEEDBACK RESPONSE TO PLATINUM-BASED CHEMOTHERAPY BY ACTIVATING AN ONCOGENIC RSK-EPHA2-GPRC5A SIGNALING SWITCH
Authors: Moyano-Galceran Lidia , Turunen Siri Pauliina , Pietilä Elina , Alkasalias Twana , Corvigno Sara , Hjerpe Elisabet , Joneborg Ulrika , Bulanova Daria , Eriksson Mina , Dahlstrand Hanna , Carlson Joseph , Lehti Kaisa
Published: June 2021

Despite increased understanding of chemotherapy resistance, the effects of the chemotherapy itself on the oncogenic cancer cell properties and adaptive resistance mechanisms of the therapy-escaping metastatic cancer cells remain largely undefined. In this study, we show that the platinum-based chemotherapeutic drugs cisplatin and carboplatin induce a switch from the canonical EphA2 receptor tyrosine kinase activity to the stemness and invasion-associated, ligand independent signaling via EphA2 S897 phosphorylation. In patient-derived high grade serous ovarian cancer (HGSOC) cells and established ovarian cancer cell lines, the chemotherapy treatment led to a significant increase in the ratio between S897 and Y588 phosphorylation of EphA2 in conjunction with GPRC5A protein upregulation and beta1-integrin activation. Notably, high protein levels of GPRC5A were significantly associated to poor survival in patients with metastatic HGSOC, revealing a previously unknown association of this EphA2 interacting receptor with the clinical HGSOC outcome. Moreover, pharmacological inhibition of RSK serine-threonine kinases blocked both the EphA2 S897 phosphorylation and the GPRC5A induction coincident with markedly increased cisplatin and carboplatin sensitivity of the HGSOC cells ex vivo. These results identify a novel mechanism of chemotherapy-induced adaptive resistance via the RSK-EphA2-GPRC5A oncogenic signaling axis in HGSOC.

Citation Format: Lidia Moyano-Galceran, Siri Pauliina Turunen, Elina Pietilä, Twana Alkasalias, Sara Corvigno, Elisabet Hjerpe, Ulrika Joneborg, Daria Bulanova, Mina Eriksson, Hanna Dahlstrand, Joseph Carlson, Kaisa Lehti. OVARIAN CANCER CELLS INDUCE A FEEDBACK RESPONSE TO PLATINUM-BASED CHEMOTHERAPY BY ACTIVATING AN ONCOGENIC RSK-EPHA2-GPRC5A SIGNALING SWITCH [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-041.
Abstract GMM-044: IDENTIFICATION OF A NOVEL CANDIDATE BIOMARKER FOR DIAGNOSIS AND THERAPEUTIC OVARIAN CANCER TARGET
Authors: Park Kyoungsook , Son Chaeyeon , Song Sang Yong
Published: June 2021

Ovarian cancer is the most fatal gynecological cancer with high mortality. It still has no reliable diagnostic marker and effective treatment due to its high drug resistance and high recurrence. To develop a novel biomarker for diagnosis and therapeutic target of intractable ovarian cancer, we selected for an upregulated gene in serous ovarian tumors compared with normal ovary counterparts by microarray analysis and confirmed its elevated mRNA expression in ovarian tumors by RT-PCR. Consistent with its high mRNA level, tumor-specific preferential high expression of candidate protein was detected in the cytoplasm of the ovarian tumor tissues by immunohistochemistry. To our surprise, indirect immunofluorescence staining revealed an unexpected cytoplasmic subcellular localization of a candidate protein. Furthermore, forced expression of a candidate gene in cultured cells with no detectable level showed a marked inhibition of cell proliferation. To address these unexpected findings, we set out to identify a candidate interacting protein by yeast 2-hybrid screening to explore a novel function and its underlying mechanism. Among them, one of cell death associated protein was identified as a novel interacting partner of a candidate protein and their physical interaction in mammalian cells was confirmed by co-immunoprecipitation assay and confocal microscopy. Further domain mapping analysis should reveal critical domains for their interaction and would provide fundamental and pivotal information for a novel function and an underlying molecular mechanism of a candidate gene. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2018R1D1A1B07047640).

Citation Format: Kyoungsook Park, Chaeyeon Son, Sang Yong Song. IDENTIFICATION OF A NOVEL CANDIDATE BIOMARKER FOR DIAGNOSIS AND THERAPEUTIC OVARIAN CANCER TARGET [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-044.
Abstract GMM-052: EVIDENCE FOR APOBEC MUTAGENESIS IN CLEAR CELL OVARIAN CARCINOMA
Authors: Serebrenik Artur S. , Jarvis Matthew C. , Argyris Prokopios P. , Brown William B. , Harris Reuben S.
Published: June 2021

Clear cell ovarian carcinoma (CCOC) accounts for 1-12% of ovarian epithelial cancers and, notwithstanding early diagnosis, often has poor clinical outcomes. CCOC genomic sequences show strong cytosine to thymine (C-to-T) transition mutation biases (Wang et al., 2017, Nature Genetics; Maru et al., 2017, Gynecologic Oncology; Shibuya et al., 2018, Genes Chromosomes Cancer). Many of these mutations occur in APOBEC signature motifs (5'-TCA or TCT), implicating at least one of the nine APOBEC DNA cytosine deaminase family members in CCOC mutation and evolution. Three of the nine family members associate with cancer mutagenesis, but only one, APOBEC3B (A3B), correlates in multiple cancer types with worse clinical outcomes including overall and metastasis-free survival.

Here, we further investigate the specific involvement of APOBEC mutagenesis in CCOC. First, we show that tumors positive for APOBEC signature mutations associate with poor clinical outcomes in comparison to those that do not. Second, we perform a comprehensive analysis of mutation signatures from new whole genome sequences and APOBEC expression profiles from tumor and normal tissues, including RTqPCR for mRNA quantification and immunohistochemistry to distinguish APOBEC protein expression in tumor cells from surrounding connective tissue and immune cells. Our results indicate a significant role for APOBEC mutagenesis in CCOC warranting further studies including evaluation of APOBEC as a prognostic biomarker.

Citation Format: Artur S. Serebrenik, Matthew C. Jarvis, Prokopios P. Argyris, William B. Brown, Reuben S. Harris. EVIDENCE FOR APOBEC MUTAGENESIS IN CLEAR CELL OVARIAN CARCINOMA [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-052.