bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract B40: Role of mutant HRAS in growth and drug sensitivity of head and neck squamous cell cancers (HNSCC)
Authors: Javaid Sehrish , Waters Andrew M. , Nguyen Victoria V. , Der Channing J. , Cox Adrienne D.
Published: June 2021

Activating mutations in the HRAS oncogene are present in 5-11% of head and neck squamous cell cancers (HNSCC). We set out to determine if HRAS is a driver of HNSCC growth and whether pharmacologic inhibitors of HRAS membrane association and/or RAS effector signaling may have therapeutic value in these cancers. To determine the importance of HRAS, we depleted it genetically from HRAS-mutant HNSCC cell lines by shRNA and evaluated the effects on cell growth and survival. To determine the potential therapeutic value of blocking HRAS membrane association and/or signaling through the key RAS effector, ERK MAPK, we treated HNSCC cells with inhibitors of farnesyltransferase (FTIs, tipifarnib and lonafarnib) or ERK1/2 (ERKi, SCH772984), respectively. We monitored target inhibition by aqueous/detergent fractionation, gel shift and immunoblotting; quantitated cell viability by Alamar blue assay; and assessed cell cycle distribution and apoptosis by flow cytometry. We found that genetic depletion of mutant HRAS inhibited the growth of HRAS-mutant HNSCC cell lines in vitro, caused a modest G1 cell cycle arrest and robustly induced apoptosis. Since genetic depletion is not clinically feasible, we wished to identify targeted therapies suitable for this patient population. Several cancer types driven by mutant RAS are sensitive to inhibitors of the ERK pathway downstream of RAS, and HRAS is the sole RAS isoform whose activity is blocked by FTIs. We found that FTIs and ERKi each inhibited their respective targets at nanomolar range but only FTIs inhibited the growth of HRAS-mutant HNSCC in a target-dependent manner. In cell lines where FTI treatment upregulated ERK signaling, ERKi enhanced sensitivity to FTI. We established that genetic suppression of mutant HRAS blocks HNSCC growth, validating HRAS as a potential therapeutic target. HRAS-mutant HNSCC cell lines are sensitive to FTI but, surprisingly, less sensitive to ERKi. The combination of FTI and ERKi may further sensitize a subset of HRAS-mutant HNSCC. Other targeted combinations will also be discussed.

Citation Format: Sehrish Javaid, Andrew M. Waters, Victoria V. Nguyen, Channing J. Der, Adrienne D. Cox. Role of mutant HRAS in growth and drug sensitivity of head and neck squamous cell cancers (HNSCC) [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 B40.
Abstract B46: Systems modeling of Ras reveals systems mechanisms that dictate response to treatment
Authors: Stites Edward C.
Published: June 2021

A relative abundance of biochemical and mechanistic data makes the Ras signaling network an ideal “model system” for the application of systems biology approaches to cancer biology. We here describe our application of systems modeling to the previously unexplained situation where colorectal cancer (CRC) patients with a KRAS G13D mutations are more responsive to anti-EGFR agents than patients with other KRAS mutations. Our computational model of Ras signaling was developed in accordance with the well-established network architecture of RasGTP signal regulation. Measured biochemical and biophysical properties comprise the parameters of the model. The previous biochemical characterization of specific oncogenic mutants allows different oncogenic alleles to be modeled. We use the model to find the behaviors that logically follow from what is known, but which the scope and complexity of the network prevent from otherwise being inferred. We simulated anti-EGFR dose responses for the three most common Ras mutants in CRC: G12D, G12V, and G13D. Our simulations unexpectedly found KRAS G13D was considerably more responsive to anti-EGFR agents. This implies that the known biochemical differences between these mutants are sufficient to explain why KRAS G13D CRC is more sensitive to EGFR inhibition. Our model further predicted that wild-type RasGTP levels within KRAS-mutant cancers should decrease much more for KRAS G13D cancers than KRAS G12V and KRAS G12D cancers. Our analysis predicted that these variations in wild-type RasGTP levels follow from mutant-specific differences in their interaction with Ras GAPs like the tumor suppressor neurofibromin (NF1). This mechanism explains why KRAS G13D CRC, but not KRAS G12D and G12V CRC, retains a dependency upon EGFR. We have now completed experiments that confirm this systems-level mechanism that governs responsiveness to treatment.

Citation Format: Edward C. Stites. Systems modeling of Ras reveals systems mechanisms that dictate response to treatment [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 B46.
Abstract IA10: Multiscale simulations of k-Ras on lipid bilayers
Authors: Streitz Frederick H.
Published: June 2021

Simulating proteins on lipid membranes could provide unprecedented insights into cancer biology and a host of other phenomena. However, such simulations face conflicting computational constraints: reaching biologically relevant time and length scales (milliseconds and microns) requires continuum-level models but understanding the processes of interest requires molecular level detail. I will present a new type of massively parallel, multiscale simulation framework that brings together these two modeling paradigms. Using a machine learning algorithm, we couple a novel continuum model to an ensemble of molecular dynamics simulations. The result is a simulation at macro length- and time-scales that incorporates microscale precision. We demonstrate this capability with a large-scale simulation of several hundred k-Ras proteins on a realistic lipid bilayer spanning 1 μm2.

Citation Format: Frederick H. Streitz. Multiscale simulations of k-Ras on lipid bilayers [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 IA10.
Abstract IA11: Tissue- and cell-specific functions of Hras, Kras4A, and Kras4B in skin and lung cancer
Authors: Balmain Allan
Published: June 2021

Mouse models of cancer have been instrumental in the development of our understanding of the processes that drive multistage carcinogenesis from the initiation stage to metastasis. Tumors induced in mice by exposure to carcinogenic chemicals replicate many of the biologic and genetic features of multistage cancer in human populations. Skin and lung tumors initiated by chemical mutagens generally have carcinogen-specific mutations in Hras or Kras, respectively, but the reasons for this selectivity are unknown. We have used mouse models of skin and lung cancer to address some of the unanswered questions in cancer biology related to the specific functions of Hras, Kras4A, and Kras4B in cancer development in vivo. The KRAS gene, encoding two splice isoforms (KRAS4A and KRAS4B), is mutated in many lethal human tumors and has become a major target for cancer drug discovery. We show that germline deletion of either the major Kras4B or the minor Kras4A isoform in mice confers complete resistance to development of Kras mutant lung cancers. Double heterozygotes that are able to express both isoforms, but in which splicing has been uncoupled, are also tumor resistant, suggesting that coordinated expression of both splice variants is necessary for cancer initiation in vivo. KRAS4A is enriched in human tumor cells with stem cell properties, and the two splice variants show different responses to inducers of hypoxia or ER stress. Our data help to rationalize previous conflicting information on metabolic changes in KRAS mutant tumors and suggest that KRAS4A and 4B may require separate targeting in cancer stem and progenitor cells.

Citation Format: Allan Balmain. Tissue- and cell-specific functions of Hras, Kras4A, and Kras4B in skin and lung 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 IA11.
Abstract IA27: Context-dependent role of KRAS in GI malignancies
Authors: Liao Wen-Ting , Overman Michael J. , Boutin Adam , Dey Prasenjit , Zhao Di , Wang Guocan , Li Jiexi , Lan Zhengdao , Li Jun , Shang Xiaoying , Tang Ming , Jiang Shan , Ma Xingdi , Chen Peiwen , Katkhuda Riham , Korphaisarn Krittiya , Chakravarti Deepavali , Chang Qing , Zhang Jianhua , Maru Dipen M. , Maeda Dean Y. , Zebala John A. , Kopetz Scott , Wang Y. Alan , DePinho Ronald A.
Published: June 2021

Oncogenic Kras (Kras*) plays a key role in tumor maintenance and therapeutic resistance in several cancer types, although the precise biologic functions and mechanisms are not completely understood. We had previously shown in pancreatic ductal adenocarcinoma that some tumors underwent spontaneous relapse and were devoid of Kras* expression and downstream canonical MAPK signaling, instead acquiring amplification and overexpression of the transcriptional coactivator Yap1. Functional studies established the role of Yap1 and the transcriptional factor Tead2 in driving Kras*-independent tumor maintenance. In a second previous study, we developed a faithful colorectal cancer (CRC) mouse model and showed that Kras* drove invasion and maintenance in CRC. Here, utilizing this CRC mouse model, we demonstrate that KRAS* promotes a pronounced immune-suppressive profile distinguished by a predominance of myeloid-derived suppressor cells (MDSCs). We performed integrated transcriptomic and genomic analyses that identified IRF2 (Interferon Regulatory Factor 2) as a key KRAS* effector mediating immune suppression. IRF2 suppresses MDSC migration and infiltration in KRAS*-expressing CRC tumors by targeting the CXCL3/CXCR2 axis. We also observed that de novo resistance of KRAS*-expressing tumors to anti-PD1 therapy could be overcome by enforced IRF2 expression or by treatment with SX-682, a small-molecule CXCR2 antagonist. In addition, CRC patient tumors with higher IRF2 expression showed increased response to anti-PD1 therapy. This KRAS*-mediated immune-suppressive mechanism provides a framework for CRC patient selection and combination therapies to enhance the effectiveness of immune checkpoint blockade therapy in advanced disease.

Citation Format: Wen-Ting Liao, Michael J. Overman, Adam Boutin, Prasenjit Dey, Di Zhao, Guocan Wang, Jiexi Li, Zhengdao Lan, Jun Li, Xiaoying Shang, Ming Tang, Shan Jiang, Xingdi Ma, Peiwen Chen, Riham Katkhuda, Krittiya Korphaisarn, Deepavali Chakravarti, Qing Chang, Jianhua Zhang, Dipen M. Maru, Dean Y. Maeda, John A. Zebala, Scott Kopetz, Y. Alan Wang, Ronald A. DePinho. Context-dependent role of KRAS in GI malignancies [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 IA27.
Abstract PR08: Biochemical and structural analysis of the neurofibromin (NF1) protein and a potential role for protein destabilization in Rasopathy diseases
Authors: Sherekar Mukul , Han Sae-Won , Messing Simon , Drew Matthew , Ye Xiaoying , Gillette William , Juneja Puneet , O’Neill Hugh , Stanley Christopher , Ramanathan Arvind , de Val Natalia , Lorenzo Claire , McCormick Frank , Esposito Dominic
Published: June 2021

Neurofibromin is the 320-kilodalton protein product of the NF1 gene that is mutated in the Rasopathy disease neurofibromatosis type I. Defects in NF1 lead to abrogation of the GTPase-activating activity of the protein, leading to aberrant signaling through the RAS-MAPK pathway. Very little is known about the function of the majority of the NF1 protein—to date, only the GAP domain and a region containing a Sec-PH motif have detailed structural information. To better understand the role of this large protein, we have carried out a series of biochemical and biophysical studies that clearly show that full-length NF1 protein exists primarily as a dimer. Data from in vivo experiments confirmed that this dimerization also occurs in cells, and presumably has biologic implications. A model was generated from negative-stain EM data that suggests that NF1 monomers form both intermolecular contacts to form dimers and intramolecular contacts that stabilize the individual monomers. Deletion analysis permitted us to map these two interacting domains to different parts of the NF1 protein, and crosslinking studies coupled with mass spectrometry helped to identify regions of possible interaction. In addition, introduction of point mutations observed in patients with NF diseases caused a dramatic decrease in the stability of proteins both in vitro and in vivo, potentially arguing for a rationale for loss of NF1 activity by these single mutations.

This abstract is also being presented as Poster A03.

Citation Format: Mukul Sherekar, Sae-Won Han, Simon Messing, Matthew Drew, Xiaoying Ye, William Gillette, Puneet Juneja, Hugh O’Neill, Christopher Stanley, Arvind Ramanathan, Natalia de Val, Claire Lorenzo, Frank McCormick, Dominic Esposito. Biochemical and structural analysis of the neurofibromin (NF1) protein and a potential role for protein destabilization in Rasopathy diseases [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 PR08.
Abstract AP03: DISTINCT CELL/TISSUE SOURCES OF HIGH-GRADE SEROUS OVARIAN CANCER
Authors: Zhang Shuang , Neel Benjamin G.
Published: June 2021

The cell-of-origin of high grade serous ovarian carcinoma (HGSC) has been a focus of debate. Here, by using genetic mouse models as well as cognate organoid systems, we assessed the tumor forming capacity and properties of the fallopian tube epithelium (FTE) and ovarian surface epithelium (OSE) bearing the same oncogenic abnormalities. Combined RB family inactivation (via T121 expression) and Tp53 mutation in Pax8+ fallopian tube secretory cells causes transformation and characteristics of Serous Tubal Intraepithelial Carcinoma (STIC). This genetically engineered mouse HGSC model is faithfully recapitulated in fallopian organoids, from which serous ovarian cancer with broad abdominal metastasis is generated upon orthotropic injection. The same genetic events in Lgr5+ OSE cells organdies derived from these cells also result in an apparent neoplastic process, expressing markers of early serous carcinoma (but not Pax8), which subsequently develop into serous-like tumors. Hence, both Pax8+ fallopian tube epithelial cells and Lgr5+ ovarian surface epithelial cells can undergo similar neoplastic transformation, suggesting that HGSC might derive from distinct cell and tissue sources. Similar organoid systems can be used to rapidly model other gene combinations associated with HGSC.

Citation Format: Shuang Zhang, Benjamin G. Neel. DISTINCT CELL/TISSUE SOURCES OF 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 AP03.
Abstract AP08: DEVELOPMENT OF A MULTI-PROTEIN CLASSIFIER FOR OVARIAN CANCER DETECTION BY SIMULTANEOUS MEASUREMENT OF 92 SERUM PROTEINS ON PROSEEK MULTIPLEX ONCOLOGY II PLATES
Authors: Skubitz Amy P.N. , Boylan Kristin L.M. , Geschwind Kate , Cao Qing , Starr Timothy K. , Geller Melissa A. , Celestino Joseph , Bast Robert C. , Lu Karen H. , Koopmeiners Joseph S.
Published: June 2021

There are no FDA approved screening tools for detecting ovarian cancer in the general population. The two best known ovarian cancer biomarkers, CA125 and HE4, are neither adequately sensitive nor specific when used alone to screen the general population for early stage disease. By using a combination of protein biomarkers for screening, it may be possible to increase the sensitivity and specificity over CA125 alone. In this study, we used Proseek Multiplex Oncology II plates to simultaneously measure the expression of 92 cancer-related proteins in serum using proximity extension assays. This technology combines the sensitivity of the polymerase chain reaction with the specificity of antibody-based detection methods, allowing multiplex biomarker detection and high throughput quantification. We analyzed one microliter of serum from 61 women with advanced stage high grade serous ovarian cancer and compared the values obtained to 88 age-matched healthy women. Principle component analysis and unsupervised hierarchical clustering separated the ovarian cancer patients from the healthy, with minimal misclassification. Data from the Proseek plates for CA125 levels exhibited a strong correlation with previously measured clinical values for CA125 (correlation coefficient of 0.91). CA125 and HE4 were detected at low levels in samples from healthy women, while higher levels were observed in the ovarian cancer cases. We identified 52 proteins that differed significantly (p < 0.006) between ovarian cancer and healthy samples; several of which are novel serum biomarkers for ovarian cancer. In total, 40 proteins had an estimated area under the ROC curve of 0.70 or greater. CA125 alone achieved a sensitivity of 93.4% at a specificity of 98%. However, by adding five proteins to CA125, we increased the assay sensitivity to 98.4%, while holding the specificity fixed at 98%. Our data demonstrate that the Proseek technology can replicate the results established by conventional clinical assays for known biomarkers, identify new candidate biomarkers, and improve the sensitivity and specificity of CA125 alone.

Citation Format: Amy P.N. Skubitz, Kristin L.M. Boylan, Kate Geschwind, Qing Cao, Timothy K. Starr, Melissa A. Geller, Joseph Celestino, Robert C. Bast Jr., Karen H. Lu, and Joseph S. Koopmeiners. DEVELOPMENT OF A MULTI-PROTEIN CLASSIFIER FOR OVARIAN CANCER DETECTION BY SIMULTANEOUS MEASUREMENT OF 92 SERUM PROTEINS ON PROSEEK MULTIPLEX ONCOLOGY II PLATES [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 AP08.
Abstract GMM-033: CONDITIONAL BRCA2 SWITCH IN HUMAN CELLS TO STUDY TUMOR PROGRESSION
Authors: Jimenez-Sainz J. , Jensen R.B.
Published: June 2021

The BRCA2 (Breast Cancer Susceptibility 2) gene is a caretaker of genome integrity. Germline mutations in BRCA2 predispose individuals to a high risk for ovarian and breast cancer. The BRCA2 protein plays an important role in repair of DNA double-strand breaks (DSBs) by homologous recombination. The hallmarks found in BRCA2 mutant tumors involve: genome instability including chromosome aberrations, sensitivity to cross-linking agents, presence of micronuclei, an abnormal number of centrosomes, and severe defects in homologous recombination. However, the initial steps of tumor progression in BRCA2 carriers remain elusive as the majority of cellular models are derived from tumor cell lines that have undergone prior selection in an environment of genomic instability. Here, we present the generation of isogenic, inducible BRCA2 human fallopian tube cell lines providing a model to study the acute loss of BRCA2 protein in the setting of initiation and progression towards tumorigenesis. In these otherwise normal human somatic cells, we have found that acute loss of BRCA2 impinges upon viability as well as other cellular abnormalities that may drive the accumulation and tolerance for further somatic mutations. We have discovered copy number variations modulated by crosslinking compounds in our inducible BRCA2 human fallopian tube cells. We are analyzing both the genetic and biochemical functions of BRCA2 in these processes. Our long-term goal is to elucidate the underlying molecular mechanisms that drive and sustain tumor initiation in the absence of normal BRCA2 function.

Citation Format: Jimenez-Sainz J., and Jensen R.B.. CONDITIONAL BRCA2 SWITCH IN HUMAN CELLS TO STUDY TUMOR PROGRESSION [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-033.
Abstract GMM-035: TARGETING SPSB1 SENSITIZES PLATINUM-RESISTANT OVARIAN CANCER CELLS TO CISPLATIN
Authors: Kim Mi-Kyung , Kim Hyun-Jung , Kim Yun Hwan , Kim Seung Cheo , Ju Woong
Published: June 2021

OBJECTIVE: Emergence of chemoresistance remains a critical clinical issue responsible for treatment failure in patients with ovarian cancer. The precise mechanisms underlying the development of chemoresistance are not fully understood. In our previous study using methylomic and transcriptomic analyses, SPRY domain-containing SOCS box protein 1 (SPSB1) was found to be differentially expressed between cisplatin-sensitive and –resistant ovarian cancer cells. Here, we investigated the roles of SPSB1 as a new therapeutic target for ovarian cancer cells resistant to cisplatin.

METHODS: Knockdown of SPSB1 gene was performed by transfection of SPSB1 siRNA. Ovarian cancer cell lines were cultured for 5 days and 12 days after transfection for MTT assay and colony forming assay, respectively. Protein level was analyzed by western blot analysis. Cell migration assay was performed using Boyden chamber method.

RESULTS: Knockdown of SPSB1 using RNAi method dramatically inhibited cell viability and migratory capability of ovarian cancer cells. Also, SPSB1 knockdown significantly induced CDK inhibitors, p21 and p27 and inhibited anti-apoptotic Bcl-2, indicating that SPSB1 inhibition induces cell cycle arrest and apoptosis in ovarian cancer cells.

CONCLUSION: Our results suggest that SPSB1 might be a potential therapeutic target for cisplatin-resistant ovarian cancer.

Citation Format: Mi-Kyung Kim, Hyun-Jung Kim, Yun Hwan Kim, Seung Cheol Kim, Woong Ju. TARGETING SPSB1 SENSITIZES PLATINUM-RESISTANT OVARIAN CANCER CELLS TO CISPLATIN [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-035.
Abstract GMM-037: CARBONIC ANHYDRASE 9 AND TUMOR HYPOXIA ARE ASSOCIATED WITH RECURRENCE AND RESISTANCE IN BRCA1-EXPRESSING HGSOC
Authors: Krieg Adam J. , Campbell Shawn , Mhawech-Fauceglia Paulette , Lim Jeong Youn , Pejovic Tanja
Published: June 2021

Tumor hypoxia contributes to poor outcome in multiple cancer types, in part by influencing the expression of DNA repair enzymes. The objective of this study was to examine the possible relationship between Carbonic Anhydrase 9 (CA-IX, an indicator of tumor hypoxia), BRCA1 expression (a potential indicator of competent DNA repair), and patient outcome in epithelial ovarian cancer. BRCA1 and CA-IX were evaluated by immunohistochemistry in a tissue microarray of 167 epithelial ovarian cancer (EOC) patient samples, and their respective expression correlated to other biological parameters in all patients. The correlations between BRCA1 and CA-IX expression, and clinical outcome were determined in the High Grade Serous Ovarian Carcinoma (HGSOC) samples (N = 101) on the array using Spearman's correlation and log-rank (Mantel-Cox) tests. Elevated expression of BRCA1 strongly correlated with shorter intervals for disease recurrence and mortality (median time to recurrence = 1.05 yrs vs 1.94 yrs, P <0.001; median time to mortality: 2.01 yrs vs 4.72 yrs, P <0.001). There was a significant positive correlation between CA-IX and BRCA1 expression (Spearman's r = 0.486, P < 0.001). CA-IX expression was significantly associated with recurrence-free survival (restricted to first 1500 days, P <0.01). When patients were stratified by BRCA1 expression, co-expression of CA-IX and BRCA1 correlated with the shortest recurrence-free interval (P <0.05). In complementary in vitro studies, exposure of multiple BRCA1-wild-type cell lines to hypoxia (1% O2, mimicking general tumor hypoxia) had only a minimal effect on BRCA1 expression. Additionally, in MTT viability assays, application of the hypoxia-activated pro-drug TH-302 preferentially killed cells in 1% O2 compared to standard conditions (21% O2). In contrast, the PARP inhibitor olaparib was 2-5 times less effective in hypoxic conditions than in standard growth conditions, suggesting that tumor hypoxia promotes a more resistant phenotype.

CONCLUSION: The co-expression of CA-IX and BRCA1 may be a useful prognostic indicator for rapid recurrence in high grade serous ovarian cancer. Dissecting the mechanisms regulating therapeutic resistance in hypoxic ovarian cancer cells could lead to more effective treatment options for HGSOC patients.

Citation Format: Adam J. Krieg, Shawn Campbell, Paulette Mhawech-Fauceglia, Jeong Youn Lim, Tanja Pejovic. CARBONIC ANHYDRASE 9 AND TUMOR HYPOXIA ARE ASSOCIATED WITH RECURRENCE AND RESISTANCE IN BRCA1-EXPRESSING HGSOC [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-037.
Abstract GMM-057: A FUNCTIONAL HOMOLOGOUS RECOMBINATION ASSAY PREDICTS PRIMARY CHEMOTHERAPY RESPONSE AND LONG-TERM SURVIVAL IN OVARIAN CANCER PATIENTS
Authors: Tumiati Manuela , Hietanen Sakari , Hynninen Johanna , Pietilä Elina , Färkkilä Anniina , Alkodsi Amjad , Li Yilin , Lehtonen Rainer , Erkan Erdogan Pekcan , Tuominen Minna M. , Lehti Kaisa , Hautaniemi Sampsa K. , Vähärautio Anna , Grénman Seija , Carpén Olli , Kauppi Liisa
Published: June 2021

PURPOSE: Homologous recombination deficiency (HRD) correlates with platinum sensitivity in ovarian cancer patients, which clinically is the most useful predictor of sensitivity to PARPi. For this reason, there is clearly a critical need to identify homologous recombination deficiency (HRD) as early as possible. Currently, HRD is mostly diagnosed by genetic testing, which however fails to identify a large proportion of HR-deficient tumors and predict a patient's response to chemotherapy, thus we aimed to develop an ex vivo functional HRD detection test that could predict both platinum-sensitivity and patient eligibility to targeted drug treatments.

METHODS: We obtained a functional HR score by quantifying homologous recombination (HR) repair after ionizing radiation-induced DNA damage in primary ovarian cancer samples (n=32). Samples clustered in 3 categories: HR-deficient, HR-low and HR-proficient. We analysed the HR score association with platinum sensitivity and treatment response, platinum-free interval (PFI) and overall survival (OS), and compared it with other clinical parameters. In parallel, we performed whole-genome sequencing and targeted DNA-sequencing of HR genes to assess if functional HRD can be predicted by currently offered genetic screening.

RESULTS: Low HR scores predicted primary platinum sensitivity with high statistical significance (p=0.0103), associated with longer PFI (HR-deficient vs HR-proficient: 531 vs 53 days), and significantly correlated with improved OS (HR score <35 vs ≥35, hazard ratio=0.08, p=0.0116). At the genomic level, we identified a few unclear mutations in HR genes and the mutational signature associated with HRD, but, overall, genetic screening failed to predict functional HRD.

CONCLUSIONS: We developed an ex vivo assay that detects tumor functional HRD and an HR score able to predict platinum sensitivity, which holds the clinically relevant potential to become the routine companion diagnostic in the management of ovarian cancer patients.

Citation Format: Manuela Tumiati, Sakari Hietanen, Johanna Hynninen, Elina Pietilä, Anniina Färkkilä, Amjad Alkodsi, Yilin Li, Rainer Lehtonen, Erdogan Pekcan Erkan, Minna M. Tuominen, Kaisa Lehti, Sampsa K. Hautaniemi, Anna Vähärautio, Seija Grénman, Olli Carpén, and Liisa Kauppi. A FUNCTIONAL HOMOLOGOUS RECOMBINATION ASSAY PREDICTS PRIMARY CHEMOTHERAPY RESPONSE AND LONG-TERM SURVIVAL IN OVARIAN CANCER PATIENTS [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-057.
Abstract NT-105: ANTI-TUMOUR EFFECTS OF ALL-TRANS RETINOID ACID, AN ANNEXIN A2-S100A10 PATHWAY INHIBITOR ON SEROUS OVARIAN CANCER
Authors: Lokman Noor A , Ho Rachel , Gunasegaran Kavyadharshini , Bonner Wendy M , Oehler Martin K , Ricciardelli Carmela
Published: June 2021

Annexin A2 is increased in serous ovarian cancer and plays an essential role in ovarian cancer invasion and metastasis. In combination with S100A10, annexin A2 plays an important role in the plasminogen activator system regulating plasmin production. The aim of this study was to investigate the potential utility of all-trans retinoid acid (ATRA), an inhibitor of the annexin A2-S100A10 signalling pathway, as a new therapeutic against serous ovarian cancer. Survival of serous ovarian cancer cells (OVCAR-3, OV-90, & OAW28) was significantly decreased by ATRA treatment (1-5µM). ATRA (1µM) also significantly decreased proliferation (Ki67 positivity, p=0.0034), S100A10 protein levels (p=0.0273), and increased cell apoptosis (cleaved caspase-3 positivity, p=0.0024) in serous ovarian cancer tissues using an ex vivo explant assay. In OAW28 cells, reduced cell survival following ATRA treatment was associated with a reduction of S100A10 protein levels, S100A10 and annexin A2 membrane localization, plasmin generation, motility and invasion. In contrast, ATRA inhibited OV-90 cell survival and invasion but did not affect plasmin activation or S100A10 and annexin A2 membrane localization or protein levels. These findings suggest that ATRA inhibits serous ovarian cancer proliferation and invasion via both S100A10 dependant and S100A10 independent mechanisms. Our results show that ATRA has promising potential as a novel therapy against serous ovarian cancer that warrants further evaluation.

Citation Format: Noor A Lokman,Rachel Ho, Kavyadharshini Gunasegaran, Wendy M Bonner , Martin K Oehler, Carmela Ricciardelli. ANTI-TUMOUR EFFECTS OF ALL-TRANS RETINOID ACID, AN ANNEXIN A2-S100A10 PATHWAY INHIBITOR ON 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 NT-105.
Abstract TMIM-072: COMBINATIONAL TARGETING OF HDAC6 AND PD-1/PD-L1 IMMUNE CHECKPOINT IN ARID1A-MUTATED OVARIAN CANCERS
Authors: Fatkhutdinov Nail , Fukumoto Takeshi , Zhang Rugang
Published: June 2021

Ovarian clear cell carcinoma (OCCC) typically has a low response rate to platinum-based standard of care, and has no effective therapy for advanced stage disease. About 50% OCCCs harbor a mutation in ARID1A, a subunit of a SWI/SNF chromatin remodeling complex. We have previously demonstrated the dependence of ARID1A-mutated OCCCs on HDAC6 activity. In addition, ARID1A inactivation impairs mismatch repair, thus sensitizing ARID1A-mutated tumors to checkpoint blockade. However, anti-PD-L1 treatment only marginally improved the survival of ARID1A-deficient mice. We sought to determine whether combinational approach will improve efficacy of checkpoint blockade therapy in ARID1A-mutated OCCC. We combined HDAC6 inhibitor ACY1215 and PD-1/PD-L1 immune checkpoint blockade utilizing Arid1a-/-/Pik3caH1047R genetic ovarian clear cell model. Combination further prolonged mice survival as compared to single treatments and observed effects correlated with an increase in the activity of anti-tumor cytotoxic T-cells. In addition, we observed increase in dendritic cell infiltration following HDAC6 inhibition, suggesting of an increase in antigen presentation. Our findings indicate that combination of HDAC6 inhibition with PD-L1 checkpoint blockade could be a potential strategy for ARID1A-mutated OCCCs, not only eliminating ARID1A-mutated tumor cells but also restoring anti-tumor immunity.

Citation Format: Nail Fatkhutdinov, Takeshi Fukumoto, and Rugang Zhang. COMBINATIONAL TARGETING OF HDAC6 AND PD-1/PD-L1 IMMUNE CHECKPOINT IN ARID1A-MUTATED OVARIAN CANCERS [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 TMIM-072.
Abstract TMIM-083: A TWO-STEP TARGETING STRATEGY UTILIZING GLYCOENGINEERED MESENCHYMAL STEM CELLS FOR OVARIAN CANCER
Authors: Layek Buddhadev , Shetty Mihir , Starr Tim , Prabha Swayam
Published: June 2021

Current tumor targeted drug delivery systems suffer from a lack of selectivity for tumor cells. We propose a two-step tumor targeting strategy to improve the delivery of therapeutic agents to metastatic ovarian cancer. The two-step targeting approach relies on the introduction of non-natural targets (azide functional groups) in the tumor tissue, followed by the delivery of drug-loaded polymeric nanoparticles that are surface modified (with dibenzyl cyclooctyne; DBCO) and have high affinity for these synthetic targets. The objective of the present study was to determine the in vivo efficacy of two-step targeting in a clinically-realistic, patient-derived xenograft (PDX) model of ovarian cancer. The DBCO functionalized, paclitaxel (PTX)-loaded nanoparticles (16.2% w/w PTX; diameter 320 ± 10 nm; and zeta potential -11.5 ± 1.3 mV) were formulated using the FDA approved, biodegradable and biocompatible polymer, poly (DL-lactide-co-glycolide) (PLGA), by emulsion-solvent evaporation method. Glycoengineered mesenchymal stem cells (MSC-Az) were generated by culturing MSCs in N-azidoacetylmannosamine-tetraacylated supplemented media without affecting their viability or tumor homing properties. Anticancer efficacy of MSC-Az mediated two-step targeting strategy was evaluated in female athymic nude mice bearing PDX tumors. Mice with generation 2 tumors were randomly divided into four groups receiving intravenous injection of saline, PTX solution, DBCO-PTX nanoparticles, and intravenous injection of MSC-Az followed by intravenous injection of DBCO-PTX nanoparticles (MSC-Az + DBCO-PTX). We observed significantly reduced tumor growth (p <0.05) and improved survival in the group receiving MSC-Az + DBCO-PTX compared to all other groups. Furthermore, immunohistochemical analysis of the tumor tissues demonstrated that the animals treated with MSC-Az + DBCO-PTX had extensive necrosis compared to other groups, suggesting the potential of MSCs for improved tumor targeting and inhibition. In summary, our results demonstrate the potential of two-step tumor targeting strategy to enhance tumor specific delivery of conventional chemotherapeutic drugs resulting in significantly improved anti–cancer efficacy.

Citation Format: Buddhadev Layek, Mihir Shetty, Tim Starr, Swayam Prabha. A TWO-STEP TARGETING STRATEGY UTILIZING GLYCOENGINEERED MESENCHYMAL STEM CELLS 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 TMIM-083.
Abstract A37: Engineering armored TCR-modified T cells to enhance anti-tumor efficacy
Authors: Drakes Dylan J. , Rafiq Sarwish , Purdon Terence , Brentjens Renier
Published: June 2021

Clinical trials utilizing T cells genetically engineered to express a tumor-targeted T-cell receptor (TCR) illustrate the promise of this therapy in treating cancer patients. However, there are obstacles that must be overcome to improve the efficacy of TCR gene therapy. Two separate signals are needed to completely activate T cells and drive T-cell proliferation and differentiation. Co-stimulatory molecules present on the surface of cancer cells can become downregulated as an escape mechanism, thus depriving the T cells of complete activation and leading to T-cell apoptosis or anergy. This project aims to armor tumor-associated antigen-specific T cells with proinflammatory cytokines, specifically IL-12 and IL-18. We hypothesize that this genetic modification will provide an alternate form of co-stimulation that will augment antitumor efficacy. Tumor-associated antigen specific pmel-1 T cells transduced with retroviral constructs were shown to be more functional through increased secretion of IFNγ when co-cultured with antigen-positive tumor cells. Additionally, the armored pmel T cells were able to significantly enhance lysis of the B16F10 melanoma cell line in vitro. Subsequently, the in vivo efficacy of the armored T cells was assessed in a syngeneic, immune-competent B16F10 melanoma mouse model. The armored pmel T cells were confirmed to significantly delay the B16F10 tumor progression and enhance the survival of these mice in comparison to control pmel T cells. These results provide future insight into applying the concept of armored TCR T cells to a therapeutically relevant human tumor target to enhance TCR-modified T-cell antitumor responses.

Citation Format: Dylan J. Drakes, Sarwish Rafiq, Terence Purdon, Renier Brentjens. Engineering armored TCR-modified T cells to enhance anti-tumor efficacy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A37.
Abstract A46: Degradation of hyaluronan from the tumor extracellular matrix increases infiltration of CD8+ T cells and natural killer cells in an orthotopic pancreatic tumor model
Authors: Lee Jisook , Kimbler Trevor , Thompson Curtis , Thompson Benjamin J.
Published: June 2021

T-cell migration and infiltration in solid tumors is a critical step for maximizing the efficacy of many cancer immunotherapies. Hyaluronan (HA) is a glycosaminoglycan that accumulates in the extracellular matrix (ECM) of a range of solid tumors, including pancreatic ductal adenocarcinoma (PDA). PEGylated recombinant human hyaluronidase PH20, pegvorhyaluronidase alfa (PEGPH20; PVHA), is a novel, first-in-class biologic that enzymatically degrades HA in the tumor microenvironment (TME) and is in clinical development in combination with chemotherapy for the treatment of HA-accumulating tumors. Previously, in a murine PDA tumor model, we demonstrated that accumulation of HA restricts T-cell infiltration and promotes immunosuppression. In the present study, we hypothesized that degradation of HA by PEGPH20 would increase access of CD8+ T cells to the tumor. To test this hypothesis, Pan02 murine PDA cells were engineered to overexpress hyaluronan synthase 3 (Pan02-HAS3) and were implanted orthotopically into the pancreas. In Pan02-HAS3 tumors, HA accumulation was 53-fold higher (2606 ng/mg) compared with Pan02 parental tumors (50 ng/mg) as determined by HA ELISA. Degradation of HA in Pan02-HAS3 tumors with intravenous PEGPH20 as a single agent inhibited tumor growth by 58% at a dose of 0.0375 mg/kg and 73% at a dose of 1 mg/kg. Flow cytometric analysis of tumor-infiltrating immune cells in PEGPH20-treated tumors demonstrated a significant increase in the numbers of cytotoxic CD8+ tumor-infiltrating lymphocytes and natural killer (NK) cells versus control tumors. However, no changes in CD8+ T-cell proliferation were detected, as measured by Ki67 expression or bromodeoxyuridine (BrdU) incorporation. Degradation of HA in the TME was also associated with an increased frequency of CD103+ dendritic cells and a decreased frequency of immunosuppressive CD206+ tumor-associated macrophages. These data support prior reports that accumulation of HA in the TME restricts T-cell infiltration and promotes immunosuppression, and that PEGPH20 treatment may facilitate access of CD8+ T cells and NK cells into tumors. These findings are consistent with prior studies and support the ongoing clinical evaluation of combining PEGPH20 with immune checkpoint inhibitors to improve antitumor efficacy in HA-accumulating tumors.

Citation Format: Jisook Lee, Trevor Kimbler, Curtis Thompson, Benjamin J. Thompson. Degradation of hyaluronan from the tumor extracellular matrix increases infiltration of CD8+ T cells and natural killer cells in an orthotopic pancreatic tumor model [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A46.
Abstract A79: Myeloid-lymphatic endothelial progenitors significantly contribute to lymph node metastasis in clinical breast cancer
Authors: Volk-Draper Lisa , Patel Radhika , Bhattarai Nihit , Yang Jie , Ran Sophia
Published: June 2021

Introduction: Lymph node (LN) metastasis, a key prognostic factor for poor survival of breast cancer (BC) patients, directly depends on lymphangiogenesis, i.e., formation of new lymphatic vessels. Using orthotopic BC models, we previously showed that lymphangiogenesis is strongly promoted by tumor-mobilized myeloid-derived lymphatic endothelial cell (LEC) progenitors (M-LECP) and that M-LECP density correlates with lymphatic metastasis. The goal of this study was to examine the density and a prometastatic role of M-LECP in clinical human BC.

Methods: Circulating CD14+ cells from BC patients (N=25) and healthy donors (N=24) were analyzed for expression of lymphatic-specific markers. Specimens from BC patients (N=104) along with normal mammary tissues (N=6) were co-stained with antibodies against myeloid and lymphatic markers including CD68, CD14, CD18 as well as LYVE1, podoplanin, and VEGFR-3. Sections were also double stained to identify LYVE1+ cells expressing lymphoid, M2-type macrophage, immunosuppressive, and stem cell markers. Densities of tumor CD68+ macrophages, LYVE1+ vessels, and CD68+/LYVE1+ double-stained M-LECP were quantified and correlated with node status and other parameters. Prometastatic potential of experimentally produced mouse M-LECP was determined by injecting in vitro differentiated myeloid-lymphatic cells into mice bearing syngeneic EMT6 breast tumors.

Results: 92% of BC patients had monocytes expressing at least one lymphatic marker compared to 2% of healthy donors. More than 70% of breast tumors contained M-LECP with density ranging from 2 to 50 cells per high-power field. Tumor-recruited M-LECP expressed specific markers of myeloid but not lymphoid lineages as well as M2-type, immunosuppressive and stem cell markers. Tumors with high density (>20 cells/field) of M-LECP were significantly more likely to belong to aggressive subgroups (HER2+ and TNBC) and present with lymph node metastasis (P-value <0.02).

Conclusion: These data show for the first time that human breast tumors recruit significant amounts of immature myeloid cells with immunosuppressive and lymphatic-promoting properties. Such cells are largely absent in normal breast tissues. Statistical analyses indicate that M-LECP can significantly contribute to formation of new lymphatic vessels and promote metastasis.

Citation Format: Lisa Volk-Draper, Radhika Patel, Nihit Bhattarai, Jie Yang, Sophia Ran. Myeloid-lymphatic endothelial progenitors significantly contribute to lymph node metastasis in clinical breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A79.
Abstract A93: Development of blocking antibodies to human CD47 with lower side effects on RBCs and platelets
Authors: Zhang Richard , Lin Haishan
Published: June 2021

CD47 is a major immune “checkpoint” molecule for macrophage activation. It is highly expressed on many types of cancers, including human acute myeloid leukemia (AML), non-Hodgkin’s lymphoma (NHL), and solid tumors such as small-cell lung cancer (SCLC). Binding of CD47 to its receptor, SIRPα, on macrophages leads to inhibition of macrophage activation and phagocytosis. CD47 overexpression is considered to be one of the mechanisms cancer cells employ to evade immune surveillance and correlates with poor prognosis in cancers. Several strategies have been used to block CD47 binding to SIRPα, including anti-CD47 blocking antibodies and SIRPα Fc proteins. However, since CD47 is also expressed on normal cells, a major concern is that these anti-CD47 antibodies may also induce platelet aggregation and red blood cell hemagglutination. In addition, since anti-CD47 antibodies bind to many different cell types in the peripheral circulation, the half-life for these antibody drugs could be lower. Here we report the generation and characterization of a panel of high-affinity anti-human CD47 blocking antibodies. The lead antibodies bound to human CD47 with sub-nanomolar affinities and displayed a range of activities in blocking CD47/SIRPα interaction. These antibodies showed robust activities in stimulating macrophage-mediated phagocytosis of cancer cells. Importantly, our antibodies did not induce red blood cell hemagglutination in in vitro assays. One of the lead antibodies demonstrates pH- dependent binding to human CD47. It has lower binding affinity to human CD47 at neutral pH but higher binding affinity to CD47 at more acidic pH, suggesting that this antibody may preferentially bind to CD47 in tumor microenvironment. Currently we are evaluating these anti-CD47 antibodies in preclinical efficacy and safety models.

Citation Format: Richard Zhang, Haishan Lin. Development of blocking antibodies to human CD47 with lower side effects on RBCs and platelets [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A93.
Abstract A95: A genome-wide CRISPR screen identifies novel ligands for the Siglec family of glyco-immune checkpoint receptors
Authors: Wisnovsky Simon , Bertozzi Carolyn
Published: June 2021

Cancer cells are frequently characterized by extensive remodeling of their cell surface glycocalyx, the complex pattern of sugar modifications that decorate cell surface proteins. In particular, cancer cells often overexpress an unusually high density of sialic acid glycans on their plasma membrane. This modification shields tumors from immune detection through engagement of inhibitory Siglec (Sialic acid-binding immunoglobulin-type lectin) receptors, which are widely expressed in innate immune cells. Blockade of these Siglec-based immune checkpoints has shown the potential to increase immune activity against highly sialylated cancer cells. The specific glycosylated ligands that serve as counter-receptors for Siglecs, however, are not well characterized. Here we describe a strategy for better defining these immune checkpoints using a CRISPR-based genetic screening approach. A library of gRNAs was screened using FACS to identify genes whose ablation significantly reduced cell-surface binding of recombinantly expressed, fluorescently labeled Siglecs. We thereby generated a list of genes with putative roles in driving the cell-surface expression of sialic acid-containing Siglec ligands. Our screen correctly identified known genes involved in sialic acid biosynthesis, validating the specificity of this approach. Additionally, this screen revealed that knockdown of a single heavily O-glycosylated protein plasma membrane protein dramatically reduced binding of Siglec7, the Siglec receptor expressed specifically in NK cells. Follow-up work has shown that Siglec7 binds to this ligand with high affinity and that genetic blockade of this interaction potentiates NK cell killing activity in vitro. This study has thus defined a novel and specific glyco-immune interaction with potential significance in driving tumor escape from innate immune surveillance.

Citation Format: Simon Wisnovsky, Carolyn Bertozzi. A genome-wide CRISPR screen identifies novel ligands for the Siglec family of glyco-immune checkpoint receptors [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A95.