bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract B50: ERK MAPK inhibition enhances the immunogenicity of KRAS-mutant colorectal cancer
Authors: Ryan Meagan B. , Fece de la Cruz Ferran , Ahronian Leanne G. , Phat Sarah , Myers David T. , Shahzade Heather A. , Hong Catriona , Corcoran Ryan B.
Published: June 2021

The three RAS genes are the most commonly mutated oncogenes in cancer and are refractory to conventional therapies. Oncogenic mutation of KRAS in 44% of colorectal cancers (CRC) leads to aberrant activation of downstream effector pathways, including the ERK MAPK signaling cascade, which can lead to an immunosuppressive tumor microenvironment. Single-agent therapies targeting the ERK MAPK cascade or immune checkpoint (PD-1/PD-L1) have been mostly unsuccessful in KRAS-mutant CRC, and efforts are under way to identify effective combination strategies. We employed an unbiased RNA-seq approach to identify gene signatures significantly upregulated upon ERK MAPK pathway inhibition in a panel of KRAS-mutant CRC cell lines. We found that treatment with either the MEK inhibitor trametinib or ERK inhibitor Vx-11e induced upregulation of interferon gene signatures and JAK/STAT pathway signaling components. Inhibition of MEK or ERK also transcriptionally increased levels of MHC Class I and antigen presentation machinery. Upregulation of MHC Class I was reversed with either pharmacologic inhibition of JAK/STAT signaling or genetic knockdown of the transcription factor IRF1. We propose that combining MAPK inhibition with checkpoint immunotherapy may provide an effective treatment in KRAS-mutant CRC tumors in vivo and that potential synergy may be due to enhancing the immunogenicity of tumors through priming of interferon response pathways and antigen presentation machinery.

Citation Format: Meagan B. Ryan, Ferran Fece de la Cruz, Leanne G. Ahronian, Sarah Phat, David T. Myers, Heather A. Shahzade, Catriona Hong, Ryan B. Corcoran. ERK MAPK inhibition enhances the immunogenicity of KRAS-mutant 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 B50.
Abstract IA07: KRAS4b’s unique diffusion behavior is defined by plasma membrane and effector interactions
Authors: Goswami Debanjan , Chen De , Columbus John , Yang Yue , Lightstone Felice , Turbyville Thomas
Published: June 2021

RAS proteins are GTP-dependent switches that control and regulate signaling pathways involved in cell fate and are frequently mutated in cancer. RAS association with the plasma membrane, or with certain endomembrane compartments, is a required step for its activity. Why precisely this is so remains an open question. One possibility is that RAS is merely required for recruitment of RAF and other effectors to the membrane where it can associate with key regulatory molecules that lead to signaling activation. However, four isoforms exist in humans (HRAS, NRAS, and two splice variants, KRAS4b and KRAS4a) and their differences lie within 22 amino acids in the C-terminal hypervariable region (HVR, aa 167-189). These differences in the domain responsible for membrane association result in the recruitment and organization of RAS into distinct membrane nanodomains, and it is thought that this results in differential signaling behavior from RAS isoforms. Isoform-specific RAS nanodomains are thought to both be highly dynamic, differentially composed of a variety of lipids and proteins, and supported by interactions with cytoskeletal structures. How these domains are regulated, and how they influence isoform-specific RAS signaling, remains unclear. Here, we report the molecular mobility of RAS variants in the plasma membrane of living cancer cells using single-molecule tracking methods. Detailed analysis of tracks revealed that KRAS4b molecules exhibit confined mobility with three diffusive states in the active plasma membrane of living cells. This diffusion characteristic was unique to KRAS4b and influenced by both the hypervariable region and globular domain of the protein, compared to all the other Ras isoforms. Importantly, the occupancy of each diffusive states was altered for the oncogenic mutant of KRAS4b, suggesting that the diffusive states we observe are related to signaling events. Our working hypothesis is that the HVR of KRAS4b may be directly involved in assembling the membrane lipid and protein nanodomain necessary for KRAS4b signaling activity. In addition, using two-color single-molecule tracking studies, we are beginning to characterize the interactions of KRAS4b with its major effector RAF. From these studies, we are learning about the kinetics of RAS/RAF interactions in the membrane of living cells. Understanding the underlying principle of KRAS4b functionality on cell membranes is useful for developing novel therapeutic strategies for targeting oncogenic KRAS4b.

Citation Format: Debanjan Goswami, De Chen, John Columbus, Yue Yang, Felice Lightstone, Thomas Turbyville. KRAS4b’s unique diffusion behavior is defined by plasma membrane and effector interactions [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 IA07.
Abstract IA08: Lipid dependence of KRAS plasma membrane interactions
Authors: Hancock John F.
Published: June 2021

RAS proteins must localize to the plasma membrane (PM) in order to function. We have examined how different RAS PM anchors select and assemble highly specific phospholipids into signaling platforms called nanoclusters, how these transient lipid assemblies impact signal transmission, and in consequence how the lipid composition of the PM can operate as high-level regulator of RAS signal transmission. For example, the KRAS polybasic domain (PBD)-farnesyl membrane anchor exhibits exquisite specificity for phosphatidylserine (PtdSer) over other anionic phospholipids by virtue of defined structural dynamics of the C-terminal polybasic domain and farnesyl group. This specificity extends to PtdSer with specific combinations of lipid side chains. PtdSer binding is therefore essential for stable KRAS PM binding and the spatial organization of KRAS into nanoclusters. These and complementary results with RAC1 highlight the critical role of specific anionic PM lipids in the biologic function of small GTPases. In this context for KRAS we have now shown that the PM levels and lateral availability of PtdSer are critically dependent on sphingomyelin metabolism such that multiple perturbations of sphingomyelin content or distribution, or endolysosmal lipid trafficking, profoundly affect KRAS plasma membrane localization, nanoclustering, and function. Concordantly, directly targeting the molecular machinery that maintains PtdSer on the PM also abrogates KRAS function in multiple pancreatic cancer cell lines. These new observations suggest multiple new potential therapeutic targets to abrogate oncogenic KRAS signaling, which we are evaluating in model systems.

Citation Format: John F. Hancock. Lipid dependence of KRAS plasma membrane interactions [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 IA08.
Abstract IA14: Exploiting the metabolic dependencies of mutant Kras lung tumors
Authors: Martins Carla P.
Published: June 2021

Lung adenocarcinoma (LUAD) is a major cause of cancer-related death worldwide. Kras and TP53 (p53) are the most frequently mutated genes in LUAD (~30% and ~50% of LUAD cases, respectively). Since mutations in these genes cannot be currently targeted in the clinic, alternative approaches to target these aggressive and largely therapy-resistant tumors are needed. To identify new vulnerabilities associated with these tumors, our lab is characterizing the alterations that promote the development, and enable the maintenance, of mutant Kras, p53-deficient lung tumors. Using mouse models that closely recapitulate human LUAD, we have recently shown that low- and high-grade tumors exhibit distinct genetic, transcriptional, and metabolic signatures, which in turn influence their therapeutic susceptibilities. In particular, we have shown that mutant Kras gene dosage and the type of p53 mutation present have a major impact on lung tumor metabolism in vivo, creating unique dependencies that can be exploited to selectively target specific mutant Kras lung tumor subsets. This phenotypic heterogeneity is also present in the human disease and may have contributed to the poor therapeutic responses often associated with mutant Kras LUAD. We will discuss these findings and how unique metabolic signatures can be potentially exploited to improve lung cancer targeting in the clinic.

Citation Format: Carla P. Martins. Exploiting the metabolic dependencies of mutant Kras lung 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 IA14.
Abstract IA16: The interplay between RAS/MAPK signaling and tumor immunology
Authors: Hegde Priti
Published: June 2021

The role of MAPK in regulating the expression of pathways important for immune recognition of tumors such as modulation of the antigen presenting machinery and MHC Class I expression is now well studied preclinically. The clinical experience of the combination of MAPK inhibition with checkpoint inhibition (anti-PD-L1/PD-1) in cancer, however, has been less understood. Here, we will review the clinical data for checkpoint inhibitors and correlation with outcomes in the context of KRAS mutations in lung cancer and melanoma. In addition, the clinical activity of combinations of checkpoint inhibitors with MAPK inhibitors will be reviewed for both inflamed cancers such as melanoma and noninflamed cancers such as colorectal cancer.

Citation Format: Priti Hegde. The interplay between RAS/MAPK signaling and tumor immunology [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 IA16.
Abstract IA20: Unlocked groove—developing covalent inhibitors of KRASG12C
Authors: Lipford J. Russell , Cee Victor , Lanman Brian , Saiki Anne , Rex Karen , Volak Laurie , Shimanovich Roman , Hinkle Beth
Published: June 2021

KRAS is one of the most frequently mutated oncogenes in human cancer, with KRASp.G12D, p.G12V, and p.G12C constituting the major mutational subtypes across lung, colon, and pancreatic cancers. Despite over three decades of research, indirect approaches targeting KRAS mutant cancers have largely failed to show clinical benefit, and direct approaches have been limited by the apparent “undruggable” nature of KRAS. Recently, remarkable progress has been reported for targeting KRASG12C with small molecules that bind to the shallow, highly flexible P2 pocket of KRAS and form covalent adducts with the adjacent mutant cysteine. This class of inhibitors has demonstrated robust blockade of KRAS signaling and cell viability with substantial selectivity for KRASp.G12C tumors. Clinical validation of this approach is eagerly awaited. We have used iterative covalent library design and screening to develop unique inhibitors of KRASG12C. In one series, co-crystal structures of improved hit molecules revealed that side-chain motion exposed an undescribed shallow groove that was occupied by ligand atoms. This series was optimized to generate inhibitors with promising cellular activities (cellular IC50 ~100-200 nM and selectivity >200-fold vs. non-G12C lines). To further enhance potency, selectivity, and drug-like properties, scaffold hopping was employed. After extensive optimization, potent and selective inhibitors were identified that potently suppressed KRAS-MAPK signaling (cellular phospho-ERK IC50 ~30 nM) and impaired cell viability (IC50 ~2 nM). In mouse xenograft tumor models, these inhibitors induce tumor regressions when dosed orally, once daily at a dose of 30 mg/kg. The results obtained with these inhibitors have critically informed our efforts to develop KRASG12C inhibitors suitable for clinical testing.

Citation Format: J. Russell Lipford, Victor Cee, Brian Lanman, Anne Saiki, Karen Rex, Laurie Volak, Roman Shimanovich, Beth Hinkle. Unlocked groove—developing covalent inhibitors of KRASG12C [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 IA20.
Abstract IA21: Preventing KRAS processing
Authors: Maciag Anna , Yang Yue , Turner David , Dyba Marcin , Kumari Vandana , Smith Brian , Fan Lixin , Gysin Stephan , Wolfe Andrew , Abdelkarim Hazem , Gaponenko Vadim , Lightstone Felice , Nissley Dwight , McCormick Frank
Published: June 2021

Tipifarnib, a drug that targets HRAS through inhibiting farnesyl transferase, is in phase II clinical trials and appears to show activity in tumors harboring oncogenic HRAS mutations (https://kuraoncology.com/pipeline/#tipifarnib). Unfortunately, these drugs are not expected to be effective on KRAS cancers because KRAS, unlike HRAS, can be prenylated by geranylgeranyl transferase following farnesyl transferase inhibition. To address this, we have developed compounds that prevent farnesylation of KRAS 4B by covalent reaction with C185, the site of prenylation. These compounds bind to a pocket in the G-domain that is formed by interaction with the hypervariable region, an interaction that does not seem to occur in other RAS proteins. This existence of this pocket has been demonstrated through biochemical and biophysical analysis, including NMR and small-angle X-ray scattering. The compounds we have developed are active in cells: they prevent proliferation of MEFs driven by oncogenic KRAS proteins but do not affect MEFs supported by myristoylated KRAS G12D C185S at equivalent concentrations. We are currently optimizing these compounds for further preclinical development.

Citation Format: Anna Maciag, Yue Yang, David Turner, Marcin Dyba, Vandana Kumari, Brian Smith, Lixin Fan, Stephan Gysin, Andrew Wolfe, Hazem Abdelkarim, Vadim Gaponenko, Felice Lightstone, Dwight Nissley, Frank McCormick. Preventing KRAS processing [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 IA21.
Abstract IA28: Systematic interrogation of KRAS in cancer
Authors: Hahn William C.
Published: June 2021

To complement ongoing large-scale characterization of cancer genomes, we have developed genome-scale tools to systematically annotate the function of genes involved in cancer initiation and progression. In particular, we have dissected the signaling pathways related to one of the most frequently mutated oncogenes KRAS. Using both gain-of-function (ORFs) and loss-of-function (RNAi and CRISPR-Cas9) approaches, we have identified new components of KRAS effector pathways that are essential for the survival of KRAS driven cancers. We have also performed comprehensive genetic screens to identify genes that modulate the response of KRAS-driven cancer cell lines to clinical-grade MEK inhibitors. Together these ongoing approaches provide new insights into KRAS signaling and identify potential targets for KRAS-driven cancers.

Citation Format: William C. Hahn. Systematic interrogation of KRAS in 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 IA28.
Abstract IA29: Global proteomics reveals the context-dependence of K-Ras signaling
Authors: Haigis Kevin
Published: June 2021

KRAS is the most frequently mutated oncogene in cancer. In the absence of direct inhibitors for most K-Ras mutants, therapeutic strategies focus on targeting downstream pathways that mediate its oncogenic properties. This approach is limited, however, by an incomplete understanding of the contextual cues that modulate the signaling output of activated K-Ras. To determine how tissue context, genetic background, and KRAS allele modulate oncogenic signaling on a global level, we performed multiplexed mass spectrometry on mouse tissues expressing wild-type or mutant K-Ras. Different mutant forms of K-Ras dramatically altered the proteomes and phosphoproteomes of colons, pancreases, and spleens, even when a given mutant allele did not alter tissue homeostasis. The vast majority of changes were context specific (i.e., not conserved between tissues or KRAS alleles), leading to several unique K-Ras-regulated network architectures. We developed an approach to integrate proteomic data from mouse models with proteomic and mutational data from human cancers, identifying key nodes in the K-Ras network that are conserved between primary tumors and a model organism. Comparison of the integrated K-Ras signaling networks to genetic screening data identified several genes that are synthetic lethal with mutant K-Ras. Altogether, these studies demonstrate the extreme context-dependent plasticity of K-Ras signaling and identify noncanonical mediators of K-Ras oncogenicity that lie within the K-Ras-regulated signaling network.

Citation Format: Kevin Haigis. Global proteomics reveals the context-dependence of K-Ras 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 IA29.
Abstract PR03: Modeling the genetic heterogeneity of KRAS mutant lung adenocarcinomas for therapeutic discovery
Authors: Best Sarah A. , Ding Sheryl , Reljic Boris , Song Ji-Ying , Sutherland Kate D.
Published: June 2021

Lung adenocarcinoma (ADC) is the most common histologic subtype of lung cancer. Oncogenic driver mutations in KRAS are found in 36% of lung ADC cases and with a lack of specific KRAS inhibitors, this subset presents a significant clinical challenge. There is therefore an urgency to identify actionable co-occurring mutations as an alternative therapeutic approach. One of the most critical stress-response pathways in mammals is mediated by the transcription factor Nuclear Factor Erythroid-2-Related Factor 2 (NFE2L2/NRF2), which is negatively regulated by Kelch-like ECH-Associated Protein 1 (KEAP1). Loss-of-function mutations in KEAP1 have been identified in 19% of ADC, and often co-occur with KRAS mutations, but appear to be largely mutually exclusive with TP53 mutations. To best model these genetic subgroups of KRAS mutant lung ADC, we generated genetically engineered mouse models (GEMMs) whereby oncogenic KrasG12D was expressed either in combination with loss of p53 (KP mice) or Keap1 (KK mice). Critically, loss of Keap1 promoted KrasG12D-induced malignant transformation, which was associated with a unique metabolic program that could be exploited using pharmacologic inhibitors. Furthermore, bronchiolar epithelial cells appeared to be more sensitive than alveolar epithelial cells to transformation following Keap1 loss. Interestingly, this cell-type bias appeared to directly modulate the immune phenotype of lung tumors. Taken together, we have generated powerful in vivo models that enable us to address the relationship between tumor cells and resident immune cells to evaluate novel treatment strategies aimed at improving the long-term outcomes for patients carrying KRAS alterations.

This abstract is also being presented as Poster B10.

Citation Format: Sarah A. Best, Sheryl Ding, Boris Reljic, Ji-Ying Song, Kate D. Sutherland. Modeling the genetic heterogeneity of KRAS mutant lung adenocarcinomas for therapeutic discovery [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 PR03.
Abstract PR04: Anti peptide-HLA (TCR-like) antibodies specific for the KRAS G12V neoantigen
Authors: Maserati Avisa , Makris Dahna , Kiefel Ben , Beasley Matt , McColl Brad , Clifton Ben
Published: June 2021

The remarkably high prevalence of Ras driver mutations in tumors of diverse origins and stages indicates that Ras neoantigens are poor substrates for HLA-mediated detection by the native immune system. Nevertheless, immunotherapies based upon tumor-infiltrating lymphocytes targeting Ras neoantigens have been reported, indicating that such mutations are subject to HLA display and immune surveillance. Antibodies capable of recognizing specific peptide-HLA complexes at the cell surface have the potential to target intracellular tumor neoantigens for therapeutic intervention. However, such reagents have proved to be challenging to develop. Using a novel antibody discovery platform in combination with a proprietary human germline scFv library, we have developed a rapid method for the isolation of antibodies against peptide-HLA complexes. Applying this technology to KRAS G12V, we have isolated two scFvs that recognize the peptide VVVGAVGVGK in complex with HLA-A*11:01. This peptide-HLA combination forms functional complexes both in vitro and at the cell surface and has been reported to elicit CTL responses in vivo. In contrast, the native peptide is a poor substrate for binding to HLA-A*11:01. Binding affinities of the two scFv binders for the target HLA complex are in the sub-100nM range. Both show highly specific, peptide-dependent recognition of the KRAS-HLA-A*11:01 complex, as demonstrated by the failure to bind complexes containing wild-type KRAS peptides or other control peptides. Alanine scanning mutagenesis reveals multiple residues within the neoepitope peptide that are critical for antibody binding, including the mutated valine residue. The extended binding footprint encompassing the neoepitope and the poor presentation of the unmutated peptide are likely to contribute to the on-target specificity of these antibodies. Incorporation of the scFvs into a bispecific T-cell engager (BiTE) format has demonstrated the simultaneous engagement of both the KRAS HLA-A*11:01 complex and the CD3 surface marker of T cells. Ongoing work is aimed to demonstrate BiTE-dependent CTL-mediated lysis of target cells displaying the KRAS HLA-A*11:01 complex. We are currently developing CAR-T cells carrying peptide-HLA specific scFvs and are screening further mutant Ras peptides in the context of other HLA-A subtypes.

This abstract is also being presented as Poster B48.

Citation Format: Avisa Maserati, Dahna Makris, Ben Kiefel, Matt Beasley, Brad McColl, Ben Clifton. Anti peptide-HLA (TCR-like) antibodies specific for the KRAS G12V neoantigen [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 PR04.
Abstract AP05: OVARIAN CANCER-ASSOCIATED RAD51D MUTATIONS WHICH IMPAIR ITS INTERACTION WITH XRCC2 RESULT IN DNA REPAIR DEFICIENCY
Authors: Baldock Robert A. , Pressimone Catherine A. , Baird Jared M. , Khodakov Anton Y. , Karpenshif Yoav , Garcin Edwige B. , Gon Stéphanie , Modesti Mauro , Bernstein Kara A.
Published: June 2021

The proficiency of ovarian cancer cells to repair DNA double-strand breaks (DSBs) by homologous recombination (HR) is a key determinant in predicting response to targeted therapies such as PARP inhibitors (PARPi). The RAD51 paralogs act downstream of BRCA1/2 to facilitate HR. Numerous epidemiological studies have linked mutations in the RAD51 paralogs with hereditary ovarian cancer predisposition. Despite their substantial links to cancer predisposition and development, RAD51 paralog function during HR has remained elusive, in part due to limitations in studying recombination events downstream of RAD51 filament formation. Here we investigate the impact of cancer-associated mutations in the RAD51 paralog, RAD51D, using yeast 2/3-hybrid assays to screen for altered protein-protein interactions. Following the identification of mutations that disrupt the interaction between RAD51D and XRCC2 in yeast, we validated the interaction by co-immunoprecipitation in human cells. Importantly, we determined the impact of these mutations on HR-proficiency using a direct-repeat recombination assay. By characterizing the impact of cancer-associated mutations in the RAD51 paralogs on HR-proficiency, we aim to develop more effective predictive models for therapeutic sensitivity and resistance in patients who harbor similar mutations in these essential genes.

Citation Format: Robert A. Baldock, Catherine A. Pressimone, Jared M. Baird, Anton Y. Khodakov, Yoav Karpenshif, Edwige B. Garcin, Stéphanie Gon, Mauro Modesti, Kara A. Bernstein. OVARIAN CANCER-ASSOCIATED RAD51D MUTATIONS WHICH IMPAIR ITS INTERACTION WITH XRCC2 RESULT IN DNA REPAIR DEFICIENCY [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 AP05.
Abstract AP18: PRO-INVASIVE TUMOUR-STROMA INTERACTIONS: ROLE OF THE SECRETED OXIDOREDUCTASE CLIC3
Authors: Hernandez-Fernaud Juan R , Ruengeler Elena , Neilson Lisa J , Ismail Shehab , McNeish Iain , Atkinson Samuel , Lilla Sergio , Santi Alice , Valenzuela Stella M , Blyth Karen , Yin Huabing , Mazzone Massimiliano , Norman Jim C , Zanivan Sara
Published: June 2021

Cancer cells are embedded within a microenvironment populated with cancer associated fibroblasts (CAFs) with whom they establish two-way communications through paracrine factors and physical interactions. Through these interactions, CAFs play pivotal roles in cancer and have emerged as promising therapeutic target.

To unravel key contributors of CAF-tumour/stroma cell interactions, we have developed mass spectrometry (MS)-based approaches to map proteins secreted in condition medium and extracellular matrix (ECM). Through an extensive MS-proteomic comparative analysis of CAFs with their normal fibroblasts (NFs) counterpart, we have identified the chloride intracellular channel protein 3 (CLIC3), a protein previously considered an intracellular chloride channel (regulator), as one of the most upregulated proteins in CAFs and deposited in the ECM.

Secreted CLIC3 promotes invasive behaviour of endothelial cells to drive blood vessels growth and increases invasiveness of cancer cells, both in vivo and in 3D cell culture models, via activation of the tissue transglutaminase-2 (TGM2). We found that CLIC3 is a glutathione-dependent oxidoreductase that reduces TGM2 and regulates TGM2 binding to its cofactors. Finally, CLIC3 is also secreted by cancer cells, is abundant in the stromal and tumour compartments of aggressive ovarian cancers and its levels in primary tumours and omental metastases correlate with poor clinical outcome.

Our work has unraveled an unprecedented mechanism of cell invasion to be explored for targeting in ovarian cancer.

Citation Format: Juan R Hernandez-Fernaud, Elena Ruengeler, Lisa J Neilson, Shehab Ismail, Iain McNeish, Samuel Atkinson, Sergio Lilla, Alice Santi, Stella M Valenzuela, Karen Blyth, Huabing Yin, Massimiliano Mazzone, Jim C Norman and Sara Zanivan. PRO-INVASIVE TUMOUR-STROMA INTERACTIONS: ROLE OF THE SECRETED OXIDOREDUCTASE CLIC3 [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 AP18.
Abstract GMM-032: UNRAVELLING THE STEROID METABOLOME IN EPITHELIAL OVARIAN CANCER
Authors: Jeevan David N. , Arlt Wiebke , Foster Paul A. , Sundar Sudha
Published: June 2021

INTRODUCTION: Ovarian tissue possesses the capacity for steroidogenesis from as early as seven weeks gestation. Perturbations in the steroidogenic pathways have been implicated in the development and progression of epithelial ovarian carcinoma in adulthood but the evidence has been limited. Analysis of RNA-seq data from ovarian tissue now allows the genes involved in these pathways to be studied comprehensively helping direct future investigations and highlighting possible mechanisms of the disease.

METHODS: RNA-seq data from 293 serous epithelial ovarian carcinomas from The Cancer Genome Atlas were extracted and the genes involved in steroidogenesis were systematically interrogated for high and low expression. Pathway analysis was employed to confirm significant findings. Comparison was made with novel RNA-seq data from first trimester fetal ovary and healthy adult ovary.

RESULTS: Genes involved in the synthesis of androgens (HSD17B1, SRD5A1, AKR1C3, AR and STS) were all significantly upregulated in the high grade serous ovarian carcinoma group when compared to normal healthy ovary (p<0.05).

Expression of several steroidogenic genes in malignancy appeared to mimic increased fetal expression with relative quiescence noted in the healthy ovary group.

CONCLUSIONS: Transcriptome analysis shows upregulated gene expression of the androgen synthesis pathway is associated with epithelial ovarian cancer. This is supported by immunohistochemistry findings in recent studies. Investigation of the androgen pathway does remain a valid opportunity for mechanistic insight into this disease and potentially diagnostic and therapeutic exploitation.

Ovarian steroidogenic gene expression in the first trimester appears to correlate with neoplastic growth in serous ovarian cancer but the significance of this is uncertain.

Citation Format: David N. Jeevan, Wiebke Arlt, Paul A. Foster and Sudha Sundar. UNRAVELLING THE STEROID METABOLOME IN EPITHELIAL OVARIAN CANCER [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-032.
Abstract GMM-039: BUILDING AN ORGANOID-BASED MODEL FOR OVARIAN CANCER
Authors: Lõhmussaar Kadi , Kopper Oded , Clevers Hans C.
Published: June 2021

Over the last few years several studies led to a significant change in the field of ovarian cancer and it is currently believed that the fallopian tube and not the ovary surface epithelium (OSE) is the main origin of high-grade serous ovarian cancer (HG-SOC). Nevertheless, due to the lack of unique markers and adequate model systems the relative contribution of each tissue is not yet clear and the notion that OSE has a role in HG-SOC development was not cast aside altogether. In this work we have established novel organoid systems derived from both mouse OSE and oviduct (Ovi, the equivalent of human fallopian tube). These systems recapitulate their tissue of origin and demonstrate differences in medium requirements as well as gene expression. To establish comparable tumor progression models for both OSE and Ovi we used CRISPR-Cas9 technique and targeted commonly mutated genes in ovarian cancer (Trp53, Brca1, Nf1 and Pten). Thus, we were able to establish clones with different combinations of mutations. Histological, metaphase spread and gene expression analysis of the mutated organoid clones from both OSE and Ovi demonstrated different degrees of deviation from their wild type counterpart. This deviation became more evident as the amount of introduced mutations increased. Preliminary transplantation experiments showed that only triple mutants gave rise to tumors (Trp53, Brca1 and Pten or Trp53, Brca1 and Nf1). Moreover, Ovi mutant clones were more prone to grow into tumors in comparison to OSE as no tumors have been detected in mice transplanted with the OSE equivalent clones so far. Taken together, in this study we present the first comparable Ovi/OSE research platform that enables addressing questions related to origin and early stages of HG-SOC development.

Citation Format: Kadi Lõhmussaar, Oded Kopper, Hans C. Clevers. BUILDING AN ORGANOID-BASED MODEL FOR OVARIAN CANCER [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-039.
Abstract GMM-050: IDENTIFICATION OF NOVEL CHROMOSOMAL REARRANGEMENTS IN HIGH-GRADE SEROUS OVARIAN CANCER
Authors: Sarvilinna Nanna , Pradhan Barun , Faisal Imrul , Tumiati Manuela , Alkodsi Amjad , Kauppi Liisa
Published: June 2021

High-grade serous ovarian cancer (HGS-OVCA) is the most common type of ovarian cancer. HGS-OVCA genomes are usually very complex and unstable. Many studies have addressed cytogenetic changes, and with the advent of high-throughput sequencing, complex chromosomal rearrangements. However, little is known about how gene fusions, potentially causing aberrant transcripts and protein products drive tumorigenesis, cell growth and/or metastasis in HGS-OVCA. Patients with the same histological or morphological phenotype of cancer have tumors with diverse genetic backgrounds or molecular phenotypes. There is thus an urgent need to better stratify patients and their tumors and to understand the chemotherapy and other treatment responses at the molecular level.

The purpose of this study is to find and charaterize novel recurrent chromosomal rearrangements from HGS-OVCA patient samples. We aim to identify rearrangement hotspots from genomic data. However, not only do we need to identify, characterize and stratify the genomic alterations, but also to find those changes with potentially tumorigenic functions at the RNA and protein levels.

We utilise datasets of The Cancer Genome Atlas (https://cancergenome.nih.gov/) and HERCULES Project (http://www.project-hercules.eu/) to identify rearrangement-prone chromosomal regions (putative “breakpoint hotspots”) and complex structural aberrations and variations. We concentrate on exonic aberrations seen in both datasets in at least 2 patients. The findings will be validated using two HGS-OVCA fresh-frozen tumor sample collections from Helsinki and Turku University Hospitals. Clinical data and homologous recombination (HR) score of the tumor samples (HR proficient or HR deficient; Tumiati et al. 2018) will also be analyzed.

Preliminary results of this work will be presented. We believe that combining sequencing and clinical data with state-of-the-art molecular level analyses and functional tests will deepen our understanding of HGS-OVCA biology and help to develop new anti-cancer treatment options for this “silent killer”.

Citation Format: Nanna Sarvilinna, Barun Pradhan, Imrul Faisal, Manuela Tumiati, Amjad Alkodsi, Liisa Kauppi. IDENTIFICATION OF NOVEL CHROMOSOMAL REARRANGEMENTS IN HIGH-GRADE SEROUS OVARIAN CANCER [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-050.
Abstract GMM-054: AN INVESTIGATION OF THE EFFECTS OF THE ATYPICAL PKC-ζ INHIBITOR ζ-STAT ON CLEAR CELL CARCINOMA OVARIAN CANCER PROLIFERATION AND INVASION
Authors: Smalley Tracess , Patel Rekha , Acevedo-Duncan Mildred
Published: June 2021

Ovarian cancer is the most lethal gynecological cancer diagnosis. There is no efficient screening process for ovarian cancers and the average stage of diagnosis is stage III. The most common ovarian cancer diagnosis is epithelial ovarian carcinomas, which constitute for 85-90% of prognosis; of this percent, clear cell ovarian carcinoma (CCOC) represents 5% of incidence. CCOC presents unique pathological features and has a high reoccurrence rate after treatment. An overexpression of atypical PKCs (PKC-ζ and PKC-ι/λ) has been observed in various malignant cells lines and are linked to pathways for cellular proliferation and invasion. In this investigation, ovarian cell lines (TOV21G and ES-2) were treated with the atypical PKC-ζ inhibitor ζ-Stat and assayed to determine the effects on proliferation and cellular invasion. These assays included protein quantification, cell proliferation, and wound healing. Mouse xenograph experiments were also performed to determine the effects of ζ-Stat on TOV21G tumor growth in vivo. Our data shows that ζ-Stat decreases the proliferation of clear cell carcinoma ovarian cancer cells and decreases wound healing. Our results also show that tumor growth in athymic female mice is decreased when treated with ζ-Stat and that mouse body weight is maintained. This suggests that PKC-ζ is a novel target in carcinogenesis and inhibition of this protein decreases the rate of proliferation.

Citation Format: Tracess Smalley, Rekha Patel, Mildred Acevedo-Duncan. AN INVESTIGATION OF THE EFFECTS OF THE ATYPICAL PKC-ζ INHIBITOR ζ-STAT ON CLEAR CELL CARCINOMA OVARIAN CANCER PROLIFERATION AND INVASION [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-054.
Abstract NT-104: SYNERGISTIC COMBINATION OF MORTALIN-TARGETING AND P53 REACTIVATOR DRUGS FOR OVARIAN CANCER
Authors: Ramraj Satish Kumar , Benbrook Doris M.
Published: June 2021

PURPOSE: TP53 gene missense mutations represent the most common genetic defect in fallopian tube (FT) lesions and high grade serous ovarian cancer (HGSOC) and cause of drug resistance. We hypothesized that drugs with complementary p53 protein-targeted mechanisms (SHetA2 drug release of p53 from mortalin binding, PRIMA-1MET drug reactivation of p53 transcription activity) synergistically kill ovarian cancer cells without harming normal cells.

EXPERIMENTAL DESIGN: SHetA2-mediated cytoplasmic to nuclear/mitochondrial p53 translocation was studied in wildtype and mutant p53 ovarian cancer cell lines using Western blot analysis of subcellular-enriched fractions and fluorescent microscopic imaging of immunocytochemistry. Human FT epithelial cells (hFTSECs) were used as healthy controls. The p53 reactivator drugs PRIMA-1 and PRIMA-1MET were combined with SHetA2 to study drug interactions and mechanisms using Western blot, rtPCR, cytotoxicity, reactive oxygen species (ROS) and ATP assays.

RESULTS: SHetA2 induced p53 nuclear and mitochondrial accumulation in ovarian cancer cell lines, but not in hFTSECs. Both mortalin and p53 expression altered SHetA2 cytotoxicity in ovarian cancer cells. Modest effects of PRIMA-1MET on cell lines inversely correlated with SLC7A11 expression, a known biomarker of PRIMA-1MET sensitivity. SHetA2 induced SLC7A11 in the presence of wildtype or missense mutant p53, but inhibited SLC7A11 in the absence of p53. SHetA2 and PRIMA-1MET were synergistic-to-strongly synergistic in ovarian cancer cells with mutant p53, synergistic with wildtype p53, additive with null p53 and antagonistic in hFTSECs. Elevated apoptosis, p53 transcription activity and ROS in combination with reduced ATP were associated with this mutant p53-dependent synergy.

CONCLUSION: Altogether, our findings identified the SHetA2 and PRIMA-1MET combination as a promising new therapeutic strategy for the treatment of HGSOCs with missense mutant p53.

GRANT SUPPORT: Supported by the US National Cancer Institute grant R01 CA196200 and Foundation for Women's Cancer- Ovarinnovate: Ovarcome Research Excellence Award.

Citation Format: Satish Kumar Ramraj, Doris M. Benbrook. SYNERGISTIC COMBINATION OF MORTALIN-TARGETING AND P53 REACTIVATOR DRUGS FOR OVARIAN CANCER [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 NT-104.
Abstract NT-112: APTAMER CONJUGATED NANOPARTICLES FOR TARGETED DRUG DELIVERY
Authors: Thiviyanathan Varatharasa , Pi Fengmei , Zhang Hui , Li Hui , Wang Hongyu , Guo Peixuan , Sood Anil
Published: June 2021

Targeted Delivery of therapeutic drugs and imaging agents, using nanoparticles, shows great promise in diverse fields, including the diagnostics and treatment of cancer. Monoclonal antibodies (mAbs) that have been traditionally used for detecting specific proteins can also be used as targeting moieties. However, due to their intrinsic characteristics such as immunogenicity, batch-to-batch variation and instability, antibodies have several limitations for in vivo applications. Nucleic acid based aptamers are emerging as attractive alternatives for mAbs. We have developed single-stranded DNA aptamers, which can specifically recognize and bind to ovarian cancer endothelial cells. Using mass-spectroscopy based proteomics, we have identified Annexin A2 as the protein target for the selected aptamers. These aptamers can act as homing devices to deliver therapeutic drugs and/or imaging agents specifically to ovarian cancer cells. Using a highly stable, three-way junction (3WJ) motif from the phi29 packaging RNA as a scaffold, we made a multi-functional RNA nanoparticle that can harbor therapeutic drugs, imaging agent, siRNA and the targeting ligand. The aptamer-containing multi-functional RNA nanoparticle is used to deliver Doxorubicin to Annexin A2 positive ovarian cancer cells, while the control nanoparticles failed to deliver. After systemic injection in mice, the DNA/RNA hybrid nanoparticles remained intact and strongly bound to tumors with little accumulation in healthy organs 6 hour post-injection. The aptamer-3WJ-Sph1/Dox intercalates selectively enhanced toxicity to Annexin A2 positive ovarian cancer cells in vitro, demonstrating the potential of the constructed DNA/RNA hybrid nanoparticles to enhance the therapeutic efficiency of Doxorubicin at low doses for ovarian cancer treatment. The use of the aptamer-3WJ RNA nanoparticle will benefit ovarian cancer patients by reducing the side effects of cancer chemo-therapeutics and increasing its local concentration in the tumor microenvironment after systemic administration. Our ultimate goal is the development of patient-optimized aptamer-nanoparticle delivery systems for chemotherapy and molecular diagnostics imaging.

Citation Format: Varatharasa Thiviyanathan, Fengmei Pi, Hui Zhang, Hui Li , Hongyu Wang, Peixuan Guo, Anil Sood. APTAMER CONJUGATED NANOPARTICLES FOR TARGETED DRUG DELIVERY [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 NT-112.
Abstract NT-114: CATALYTIC SUBUNITS SWITCH DRIVES RESISTANCE TO EZH2 INHIBITORS IN ARID1A-MUTATED CELLS
Authors: Wu Shuai , Fatkhutdinov Nail , Fukumoto Takeshi , Bitler Benjamin G. , Park Pyoung Hwa , Kossenkov Andrew V. , Trizzino Marco , Gardini Alessandro , Speicher David W. , Zhang Rugang
Published: June 2021

The SWI/SNF chromatin remodeling complex is altered in ~20% of human cancers. ARID1A, a component of the SWI/SNF chromatin-remodeling complex, is the most frequently mutated epigenetic regulator in human cancers. Inactivation of the SWI/SNF complex is synthetically lethal with inhibition of EZH2 activity. EZH2 inhibitors are entering clinical trials for specific tumor types with SWI/SNF mutations. However, mechanisms of de novo or acquired resistance to EZH2 inhibitors in cancers with inactivating SWI/SNF mutations are unknown. Here we show that the switch of the SWI/SNF catalytic subunits from SMARCA4 to SMARCA2 drives resistance to EZH2 inhibitors in ARID1A-mutated ovarian cancer cells. SMARCA4 decease dominates over SMARCA2 increase in the switch. SMARCA4 loss leads to suppression of apoptotic pathways through upregulating anti-apoptotic genes such as BCL2 in the EZH2 inhibitor resistant cells. EZH2 inhibitor resistant ARID1A-mutated cells are hypersensitive to BCL2 inhibitors such as ABT263. ABT263 is sufficient to overcome resistance to EZH2 inhibitor and synergistic with EZH2 inhibitor in vivo in ARID1A-inactivted ovarian tumour mouse models. Together, these data establish that the switch of the SWI/SNF catalytic subunits from SMARCA4 to SMARCA2 underlies the acquired resistance to EZH2 inhibitors and BCL2 inhibition alone or in combination with EZH2 inhibition represents a novel strategy to overcome and/or prevent EZH2 inhibitor resistance in ARID1A-mutated cancers. Given that the SWI/SNF subunits are among the most frequently mutated genes in human cancers and EZH2 inhibitors are in the clinical trials for tumor with mutations in the SWI/SNF complex, we expect our findings to have far-reaching implications for developing cancer epigenetic therapeutics.

Citation Format: Shuai Wu, Nail Fatkhutdinov, Takeshi Fukumoto, Benjamin G. Bitler, Pyoung Hwa Park, Andrew V. Kossenkov, Marco Trizzino, Alessandro Gardini, David W. Speicher, Rugang Zhang,. CATALYTIC SUBUNITS SWITCH DRIVES RESISTANCE TO EZH2 INHIBITORS IN ARID1A-MUTATED CELLS [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 NT-114.
Abstract NT-121: AN ANTI-AMPHIREGULIN ANTIBODY AS POTENTIAL TREATMENT FOR OVARIAN CANCER
Authors: Zmora Einav , Lindzen Moshit , Yarden Yosef
Published: June 2021

The ErbB family of receptors consists of four receptors, which bind with eleven ligands. These EGF-like ligands can be classified according to their affinity to EGFR: EGF, TGFα, HB-EGF and betacellulin are considered high affinity ligands, whereas AREG, EREG and EPGN are considered low affinity ligands. The literature reports that AREG is a type II cytokine which plays a role in inflammation, its expression is known to contribute to tissue repair and regeneration due to its ability to promote cell survival and proliferation. Furthermore, AREG is expressed by activated Th2 cells, mast cells, eosinophils, basophils and M1 macrophages.

Profiling the repertoire of EGF-like ligands in ascites fluids collected from 43 ovarian cancer patients, we found that 86% expressed high levels of AREG. These findings lead us to generate an anti-AREG antibody active against both human and murine AREG. Efficacy of this antibody was tested in several in vivo experiments using anti-AERG alone or in combination with chemotherapy (cisplatin). A murine anti-EGFR antibody was used as a positive control. Our results showed a significant prolongation of the lifespan of mice treated with an anti-AREG antibody, postponing the advancement of the disease compared to the control untreated group, or the groups treated with an anti-EGFR antibody or with chemotherapy only.

One of our next goals is to establish a model that is completely murine. We are using immunocompetent C57 black female mice, the ID8 cell line which is an ovarian cancer mouse cell line and our antibody, which blocks the murine form of AREG. We hope that in this way we would be able to evaluate the involvement of the immune system. The knowledge we will gain about the efficacy and toxicity of an anti-AREG approach may not be available from humanized models.

Citation Format: Einav Zmora, Moshit Lindzen, Yosef Yarden. AN ANTI-AMPHIREGULIN ANTIBODY AS POTENTIAL TREATMENT FOR OVARIAN CANCER [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 NT-121.