Mary E. Murray

The Immune System in Cancer Pathogenesis: Potential Therapeutic Approaches

Interplay among immune activation and cancer pathogenesis provides the framework for a novel subspecialty known as immunooncology. In the rapidly evolving field of immunooncology, understanding the tumor-specific immune response enhances understanding of cancer resistance. This review highlights the fundamentals of incorporating precision medicine to discover new immune biomarkers and predictive signatures. Using a personalized approach may have a significant, positive impact on the use of oncolytics to better guide safer and more effective therapies.

Project Development Teams: A Novel Mechanism for Accelerating Translational Research

The trend in conducting successful biomedical research is shifting from individual academic labs to coordinated collaborative research teams. Teams of experienced investigators with a wide variety of expertise are now critical for developing and maintaining a successful, productive research program. However, assembling a team whose members have the right expertise requires a great deal of time and many resources. To assist investigators seeking such resources, the Indiana Clinical and Translational Sciences Institute (Indiana CTSI) created the Project Development Teams (PDTs) program to support translational research on and across the Indiana University–Purdue University Indianapolis, Indiana University, Purdue University, and University of Notre Dame campuses. PDTs are multidisciplinary committees of seasoned researchers who assist investigators, at any stage of research, in transforming ideas/hypotheses into well-designed translational research projects. The teams help investigators capitalize on Indiana CTSI resources by providing investigators with, as needed, mentoring and career development; protocol development; pilot funding; institutional review board, regulatory, and/or nursing support; intellectual property support; access to institutional technology; and assistance with biostatistics, bioethics, recruiting participants, data mining, engaging community health, and collaborating with other investigators. Indiana CTSI leaders have analyzed metrics, collected since the inception of the PDT program in 2008 from both investigators and team members, and found evidence strongly suggesting that the highly responsive teams have become an important one-stop venue for facilitating productive interactions between basic and clinical scientists across four campuses, have aided in advancing the careers of junior faculty, and have helped investigators successfully obtain external funds.

Abstract 3180: Preclinical validation of EZH2 as a therapeutic target in pediatric Ewing's sarcoma

Disease-free survival in relapsed Ewing9s Sarcoma Family of Tumors (ESFT) has not improved over the past 25 years. Current standard-of-care (SOC) agents result in 70% survival in patients with localized ESFT; however, for relapsed patients the survival rates remain between 15-20%. Approximately 85% of ESFTs have the chromosomal translocation t(11;22)(q24;q12) which encodes for the oncogenic EWS/FL1 fusion protein. The EWS/FL1 functions as a potent transcription factor leading to the dysregulated expression of genes that promote and maintain tumorigenesis. A major epigenetic regulator that is a downstream target of EWS/FL1 is the enhancer of Zeste Homolog 2 (EZH2). EZH2 is the catalytic component of the polycomb repressor complex 2 (PRC2). Notably, it is overexpressed in ESFT and maintains tumor oncogenicity by tri-methylating histone 3 lysine 27 (H3K27me3) to modulate gene expression. Genome and transcriptome data obtained by the Pediatric Precision Genomics Program at Riley Hospital for Children at Indiana University Health (IUH) indicate that EZH2 is expressed at high levels in ESFT biopsies. Additionally, other groups have reported that high levels of EZH2 protein in ESFT and other cancers correlate with increased chemoresistance to SOC therapy. We are testing tested the hypothesis that EZH2 contributes to chemoresistance in ESFT by regulating critical growth and survival genes. In addition, we are investigating if pharmacological inhibition of EZH2 will enhance sensitivity to the cytotoxic effects of SOC agents. Pediatric primary and relapsed ESFT cell lines and ESFT xenografts were validated for the EWS/FLI, EZH2, and H3K27me3 signatures. In vitro- and in vivo-pharmacodynamic studies of EZH2 inhibition via tazemetostat were conducted to optimize dosing effect. In ESFT cell lines (TC71, A673, CHLA-9, and CHLA-10), tazemetostat dose-response experiments indicated a significant reduction of H3K27me3 by one day post-treatment which was either sustained or completely blocked by 7-days post-treatment compared to vehicle treated (p Citation Format: Pankita H. Pandya, Barbara Bailey, Adily E. Elmi, Heather R. Bates, Courtney N. Hemenway, Anthony L. Sinn, Khadijeh Bijangi-Vishehsaraei, M. Reza Saadatzadeh, Harlan E. Shannon, Jixin Ding, Mark S. Marshall, Michael J. Ferguson, Lijun Cheng, Lang Li, Mary E. Murray, Jamie L. Renbarger, Karen E. Pollok. Preclinical validation of EZH2 as a therapeutic target in pediatric Ewing9s sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3180.

Abstract 1281: Genomic structure variation in large screening for pediatric sarcoma therapy

Complex chromosomal aberrations such as amplification and deletion of DNA copy number are frequently seen in sarcoma. Fifty-five DNA structure variation has been listed as standard clinical diagnosis for sarcoma by standard of National Comprehensive Cancer Network (NCCN). However, copy number variation (CNV) as a biomarker of drug treatment for pediatrics sarcoma is still unclear, especially for relapsed and high recurrent patients of pediatric sarcoma. The paper aims to detect the prognosis biomarkers for rhabdomyosarcoma, Ewing9s sarcoma (ES), and osteosarcoma based on copy number variation for 128 FDA-approved cancer drugs systematically. The 182 copy number variation (CNV) profiles from clinical sarcoma patients across three types of sarcoma, including osteosarcoma, rhabdomyosarcoma and ES, are observed. Cox survival regression model is used to select significant cancer-related CNVs systematically by correlation with overall survival analysis. 532 significant common CNVs and 782 specific CNVs for different types of sarcoma are observed by p-value Citation Format: Lijun Cheng, Pooja Chandra, Limei Wang, Karen Pollok, Pankita Pandya, Mary Murray, Jacquelyn Carter, Michael Ferguson, Mohammad Reza, Mashall Mark, Lang Li, Jamie Renbarger. Genomic structure variation in large screening for pediatric sarcoma therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1281.

Abstract A49: Differential expression of the complement regulatory protein CD55 in wildtype and mutant p53 Glioblastoma and Ewing's Sarcoma

Background/Objectives: Solid tumors such as Glioblastoma (GBM) and Ewing9s Sarcoma result in relapse or refractory disease due the tumor9s ability to develop resistance to anti-cancer therapies. One mechanism by which solid tumors such as GBM and Ewing9s Sarcoma, circumvent the immuno-editing process and result in resistance to treatments is by the up-regulation of membrane-bound complement regulatory proteins (mCRPs) CD46, CD55, and CD59. While it has been reported that mCRPs are up-regulated in pediatric liquid tumors and influence the efficacy of monoclonal antibody treatments, regulation of mCRP expression in solid tumors has not been explored in detail. To delineate potential mechanisms regulating expression of mCRPs, we first screened wildtype (wt-p53) or mutant p53 solid tumor cell lines for mCRP expression. Our preliminary data suggest that p53 status correlates with CD55 but not CD46 or CD59 expression. These studies may serve to be the foundation for potentially recognizing the mCRPs as immune biomarkers in pediatric and adult solid tumors, ultimately, resulting in the development of novel immunotherapies for improved clinical outcomes and patient care. Design/Methods: Adult GBM and pediatric Ewing9s Sarcoma cell lines that are wildtype p53 (GBM10 and CHLA-9) or mutant p53 (GBM-43 and CHLA-10) were exploited for in vitro studies. The GBM-43 was generated from a primary GBM while the GBM-10 from a patient with recurrent GBM. The paired Ewing9s Sarcoma cell lines, CHLA-9 and CHLA-10, were generated from the same patient at primary diagnosis and at relapse respectively. Western blot and qPCR were used to confirm expression and mutational status of p53. mCRP expression was evaluated using RT-PCR and flow cytometry. Results: All cancer cell lines expressed p53 to varying degrees. No significant difference was observed in the expression of CD46 and CD59 among the wt-p53 and mutant p53 glioblastoma cell lines. Mutant p53 cell lines from glioblastoma and Ewing9s sarcoma had increased CD55 transcript levels compared to their wildtype counterpart cell lines where the transcripts were undetectable. In addition, flow cytometry data revealed increased expression of the mCRP, CD55, in mutant p53 glioblastoma (GBM-43) versus wt-p53 (GBM-10) cells (p Conclusion: These preliminary findings highlight the importance of further investigating the role of mCRPs on wt-p53 and mutant p53 cell lines. Studies are in progress to determine if expression of CD55 is regulated by p53 status. Understanding how this critical mCRP is regulated in solid tumors will be important for immune biomarker development as well as useful in guiding the use of antibody-based therapeutic approaches in solid tumors. Citation Format: Pankita H. Pandya, Reza Saadatzadeh, Jixin Ding, Barbara Bailey, Sydney Ross, Mary E. Murray, Jamie L. Renbarger, Karen E. Pollok. Differential expression of the complement regulatory protein CD55 in wildtype and mutant p53 Glioblastoma and Ewing9s Sarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A49.

Abstract 4592: Complement regulatory protein expression in solid tumors: implications for resistance to antibody-mediated immunotherapy

Background: Resistance to anti-cancer therapies results in relapsed/refractory disease of Glioblastoma (GBM) and Ewing’s Sarcoma. Up-regulation of membrane-bound complement regulatory proteins (mCRPs) CD46, CD55, and CD59 can enable solid tumors to confer resistance to antibody-mediated immunotherapy by preventing complement and antibody-dependent cytotoxicity. mCRPs’ inhibitory role in monoclonal antibody treatments for liquid tumors have been reported, but their role and regulation in solid tumors has not been explored. In the context of refractory tumors, others have reported that vascular endothelial growth factor-A (VEGF-A) can induce mCRP expression in endothelial cells. Notably, p53 mutational status induces VEGF-A and its receptor (VEGFR2) in breast cancer cell lines. To investigate potential links among p53 status, VEGF-A, and mCRP, we screened wildtype (wt-p53) and mutant p53 solid tumor cell lines for mCRP expression and VEGF-A secretion. Our data suggest that p53 mutational status is associated with expression of CD55 and VEGF-A secretion. These studies provide foundation for potentially recognizing mCRPs as immune biomarkers in solid tumors, ultimately, resulting in development of novel immunotherapies for improved clinical outcomes. Methods: Pediatric Ewing’s sarcoma (CHLA9 and CHLA10) and adult GBM (GBM10 and GBM43) cell lines differing in p53 status were selected for in vitro studies. GBM43 originates from a primary GBM, while GBM10 is from a recurrent GBM patient. Ewing’s Sarcoma cell lines, CHLA9 and CHLA10, were generated from the same patient at primary diagnosis and at relapse respectively. Western blot, and sequencing confirmed the expression and p53 mutational status. mCRP expression was evaluated using RT-PCR and flow cytometry. Milliplex platform assessed VEGF-A expression in cell supernatants. Results: Whole genome sequencing data confirmed p53 mutations in all cell lines. CHLA9 and GBM10 harbor wt-p53. CHLA10 cells have p53 deletion and GBM43 cells have a F270C p53 mutation in both alleles CD55 transcripts were undetected in wt-p53 lines (CHLA9 and GBM10), but CD55 transcripts were increased in mutant/deleted p53 lines (CHLA10 and GBM43). Flow cytometry data show increased CD55 expression in mutant p53 glioblastoma (GBM43) versus wt-p53 (GBM10) cells (p Conclusion: These findings highlight the importance of further investigating role of VEGF-A in regulating mCRPs in wt-p53 versus mutant p53 solid tumor cell lines. Elucidating mechanisms for mCRP regulation is critical for immune biomarker development and in facilitating the use of antibody-based therapeutic approaches for solid tumors. Citation Format: Pankita Hemant Pandya, M. R. Saadatzadeh, Jixin Ding, Barbara Bailey, Sydney Ross, Khadijeh Bijangi-Vishehsaraei, Mary E. Murray, Karen E. Pollok, Jamie L. Renbarger. Complement regulatory protein expression in solid tumors: implications for resistance to antibody-mediated immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4592. doi:10.1158/1538-7445.AM2017-4592