Supreet Agarwal

NOTCH3 Is a Prognostic Factor That Promotes Glioma Cell Proliferation, Migration and Invasion via Activation of CCND1 and EGFR

Using a GWA analysis of a comprehensive glioma specimen population, we identified whole gain of chromosome 19 as one of the major chromosomal aberrations that correlates to patients’ outcomes. Our analysis of significant loci revealed for the first time NOTCH3 as one of the most significant amplification. NOTCH3 amplification is associated with worse outcome compared to tumors with non-amplified locus. NOTCH receptors (NOTCH1-4) are key positive regulators of cell-cell interactions, angiogenesis, cell adhesion and stem cell niche development which have been shown to play critical roles in several human cancers. Our objective is to determine the molecular roles of NOTCH3 in glioma pathogenesis and aggressiveness. Here we show for the first time that NOTCH3 plays a major role in glioma cell proliferation, cell migration, invasion and apoptosis. Therefore, our study uncovers the prognostic value and the oncogenic function of NOTCH3 in gliomagenesis and supports NOTCH3 as a promising target of therapy in high grade glioma. Our studies allowed the identification of a subset of population that may benefit from GSI- or anti-NOTCH3- based therapies. This may lead to the design of novel strategies to improve therapeutic outcome of patients with glioma by establishing medical and scientific basis for personalized chemotherapies.

Tumor Derived Mutations of Protein Tyrosine Phosphatase Receptor Type K Affect Its Function and Alter Sensitivity to Chemotherapeutics in Glioma

Poor prognosis and resistance to therapy in malignant gliomas is mainly due to the highly dispersive nature of glioma cells. This dispersive characteristic results from genetic alterations in key regulators of cell migration and diffusion. A better understanding of these regulatory signals holds promise to improve overall survival and response to therapy. Using mapping arrays to screen for genomic alterations in gliomas, we recently identified alterations of the protein tyrosine phosphatase receptor type kappa gene (PTPRK) that correlate to patient outcomes. These PTPRK alterations are very relevant to glioma biology as PTPRK can directly sense cell–cell contact and is a dephosphorylation regulator of tyrosine phosphorylation signaling, which is a major driving force behind tumor development and progression. Subsequent sequencing of the full length PTPRK transcripts revealed novel PTPRK gene deletion and missense mutations in numerous glioma biopsies. PTPRK mutations were cloned and expressed in PTPRK-null malignant glioma cells. The effect of these mutations on PTPRK anti-oncogenic function and their association with response to anti-glioma therapeutics, such as temozolomide and tyrosine kinase inhibitors, was subsequently analyzed using in vitro cell-based assays. These genetic variations altered PTPRK activity and its post-translational processing. Reconstitution of wild-type PTPRK in malignant glioma cell lines suppressed cell growth and migration by inhibiting EGFR and β-catenin signaling and improved the effect of conventional therapies for glioma. However, PTPRK mutations abrogated tumor suppressive effects of wild-type PTPRK and altered sensitivity of glioma cells to chemotherapy.

Gambogic acid inhibits thioredoxin activity and induces ROS-mediated cell death in castration-resistant prostate cancer

Advanced prostate cancer (PrCa) is treated with androgen deprivation therapy, and although there is usually a significant initial response, recurrence arises as castrate resistant prostate cancer (CRPC). New approaches are needed to treat this genetically heterogeneous, phenotypically plastic disease. CRPC with combined homozygous alterations to PTEN and TP53 comprise about 30% of clinical samples. We screened eleven traditional Chinese medicines against a panel of androgen-independent Pten/Tp53 null PrCa-derived cell lines and identified gambogic acid (GA) as a highly potent growth inhibitor. Mechanistic analyses revealed that GA disrupted cellular redox homeostasis, observed as elevated reactive oxygen species (ROS), leading to apoptotic and ferroptotic death. Consistent with this, we determined that GA inhibited thioredoxin, a necessary component of cellular anti-oxidative, protein-reducing activity. In other clinically relevant models, GA displayed submicromolar, growth inhibitory activity against a number of genomically-representative, CRPC patient derived xenograft organoid cultures. Inhibition of ROS with N-acetyl-cysteine partially reversed growth inhibition in CRPC organoids, demonstrating ROS imbalance and implying that GA may have additional mechanisms of action. These data suggest that redox imbalances initiated by GA may be useful, especially in combination therapies, for treating the heterogeneity and plasticity that contributes to the therapeutic resistance of CRPC.

Abstract 2194: Tyrosyl DNA phosphodiesterase I is a prognostic factor and its inhibition synergizes response to topoisomerase poisons in malignant glioma.

Malignant Glioma is the most common type of primary brain tumors in adults accounting for about 80% of all gliomas. Despite the advances in treatment and diagnosis, the prognosis of malignant glioma is still poor with a median survival less than a year for glioblastoma patients. Targeting DNA-repair mechanisms is a clinically relevant option for enhancing response to DNA-damaging anticancer agents. TDP1 has been recently discovered to be responsible for the repair of the irreversible DNA-topoisomerase I covalent complexes produced by topoisomerase I poisons. Other studies have suggested its role in the repair of the DNA damage produced by ionizing radiation and temozolomide. However, TDP1 relevance has not yet been studied in glioma. Thus the aims of this project are to identify the role of TDP1 in glioma aggressiveness and resistance to therapies. We hypothesize that high-grade gliomas possess higher TDP1 levels which are associated with lower survival and resistance to therapy. Quantitative real-time PCR analysis of TDP1 in glioma biopsies revealed a negative prognostic effect of TDP1, where the level of TDP1 mRNA was inversely correlated with patient survival. Moreover, western blot showed an overexpression of TDP1 in glioma biopsies compared to normal tissues and increased expression with higher grades. TDP1 overexpression in U87 cell lines did not induce resistance to temozolomide, topotecan, etoposide or doxorubicin. This may be related to other downstream DNA repair enzymes that need to be also modulated to fully induce drug resistance. However, TDP1 inhibition using small molecules synergizes the cytotoxicity of topotecan, etoposide, and doxorubicin, confirming thus its critical role in response to several therapeutic agents. As a conclusion, TDP1 is a new and promising prognostic factor and a modulator of therapy response in malignant glioma that may help in stratification of patients. Additionally, targeting TDP1 is a novel strategy that will widen the therapeutic options for malignant glioma where topoisomerase I and II poisons can be part of the gold standard therapy when used in combination with TDP1 inhibitors. Citation Format: Maha Al-Keilani, Supreet Agarwal, Mohammad Alqudah, Zita Sibenaller, Timothy Ryken, Mahfoud Assem. Tyrosyl DNA phosphodiesterase I is a prognostic factor and its inhibition synergizes response to topoisomerase poisons in malignant glioma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2194. doi:10.1158/1538-7445.AM2013-2194

Abstract B018: A high-throughput screen identifies HSP90 inhibitors as potent therapeutics across multiple clinically representative organoid models of advanced prostate cancer

Androgen-deprivation therapy (ADT) remains the gold-standard therapy for prostate cancer (PrCa), and although ADT is initially effective, most men progress to castrate-resistant prostate cancer (CRPC) within 2-3 years. Advanced CRPC is challenging to treat because intrinsic tumor heterogeneity and phenotypic plasticity engender short-lived responses and underlie resistance to conventional therapies. Combined PTEN/TP53 alterations represent a major genotype of advanced CRPC (25-30%) and are associated with poor clinical outcomes. Established PrCa cell lines do not accurately represent the heterogeneity of advanced CRPC, and therefore, nonbiased pharmacogenomics screens have not been done. The development of clinically representative, tractable models suitable for high-throughput target identification and validation is crucial for advancing novel CRPC therapies to the clinic. A comprehensive nonbiased high-throughput screen performed on seven cell lines derived from a genetically engineered mouse model (GEMM) of Pten/Tp53 null PrCa identified strongly active compounds, including inhibitors of PI3K/AKT/mTOR signaling, the proteasome, cell cycle regulatory proteins, heat shock proteins, DNA repair signaling, NFKB signaling, MAPK signaling, and several types of epigenetic modifiers. HSP90 inhibitors were one of the most efficacious classes of compounds in the screen, and ganetespib, a clinically used second-generation HSP90 inhibitor with a favorable safety profile, was the most potent. Although HSP90 inhibitors have yet to be successful as single agents, they have not been thoroughly investigated in clinically representative models of advanced PrCa and have shown potential as “network drugs,” prompting our investigations into their utility in polytherapy. We first validated ganetespib as a single agent, where it displayed strong activity against several GEMM-derived and LuCaP PDX-derived organoid models encompassing genotypic, phenotypic, and lineage heterogeneity. These 10 novel LuCaP PDX-derived organoids are representative of the numerous categories of CRPC, including adenocarcinomas with wild-type AR, adenocarcinomas with altered AR, adenocarcinoma with neuroendocrine features, and neuroendocrine disease. Single-agent ganetespib was also strongly inhibitory in vivo, decreasing growth of Pten/Tp53 null endogenous GEMM tumors as well as a human PDX tumor. Mechanistic interrogation of cell lines, organoids, and tumors exposed to ganetespib revealed inhibition of targets from several inter-related networks including AR and pAKT, two central and mutually compensatory growth and survival pathways for PrCa. The efficacy of ganetespib against a diverse group of CRPC organoids and the simultaneous inhibition of PrCa survival signaling suggested it may work well in combination. We performed a proof-of-principle high-throughput matrix screen on organoids derived from a Pten/Tp53 null GEMM and identified docetaxel and etoposide to be synergistic when combined with ganetespib. Preclinical in vivo studies to validate these findings are ongoing. In all, comprehensive data from multiple near-patient models suggest novel contexts for second-generation HSP90-directed intervention against a variety of CRPC genotypes and phenotypes and expand upon the potential of HSP90 inhibitors to simultaneously inhibit oncogenic signaling and compensatory resistance mechanisms. Citation Format: Keith H. Jansson, John B. Tucker, Lauren E. Stahl, John K. Simmons, Caitlyn Fuller, Michael L. Beshiri, Supreet Agarwal, Yasmine Abbey, Lei Fang, Paul G. Hynes, Alilin Aian Neil, Jacob Cawley, Ross Lake, Crystal Tran, Caitlin M. Tice, JuanJuan Yin, Xiahu Zhang, Rajarshi Guha, Shelley Hoover, R. Mark Simpson, Holly Nguyen, Eva Corey, Craig J. Thomas, David Proia, Kathleen Kelly. A high-throughput screen identifies HSP90 inhibitors as potent therapeutics across multiple clinically representative organoid models of advanced prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B018.

Abstract LB-281: Elucidating potential therapeutic targets in a model of Pten/Tp53 null prostate cancer using high-throughput screening technology

Prostate cancer (PCa) demonstrates significant intra and inter tumor heterogeneity, similar to many other solid tumors. The “gold standard” of therapy for metastatic prostate cancer is androgen deprivation therapy (ADT). Although ADT is initially effective, acquired resistance, termed castration resistant prostate cancer (CRPC), almost always occurs. It is believed that development of CRPC is governed by both intrinsically resistant progenitor cells and by acquired genomic mutations. Clinically advanced CRPC tumors often display loss of PTEN (40%) and aberrations in TP53 (50%), the presence of which in primary prostate cancer are associated with poor clinical prognosis. Our goal is to identify novel targeted therapies against progenitor cells harboring Pten/Tp53 mutations. To enable high throughput screening, we derived multiple cell lines from a PB-Cre4 Ptenfl/fl;Tp53fl/fl mouse model before (intact) and after (castrate) castration. The RNA-seq analyses of the 8 resultant cell lines revealed distinct clustering of intact and castrate lines as well as patterns of expression characteristic of progenitor cells. Cell lineage marker analysis showed strong KRT8 and minimal KRT5 expression. Using these 8 distinct luminal cell lines, we performed the mechanism interrogation plate (MIPe) screen, a high throughput 1,912 compound screening assay at the National Center for Advancing Translational Science (NCATS). Post-screen informatics data processing and subsequent analysis sorted out compounds that displayed strong activity (1.1 and 1.2 curve class). Multiple compounds were unique to each cell line, however, no difference in sensitivity between the two groups of intact and castrate cell types was observed. Grouping of intact and castrate cell lines together as one population identified a diverse array of 235 compounds (12% of MIPe library) with robust activity against most of the cell lines. Some of these compounds are redundant for specific targets that are key regulators of a multitude of signaling pathways, such as the heat shock protein HSP90AB1, which was targeted by 11 different compounds. In contrast, other targets, like BIRC5, were potently targeted by only one compound. A majority of the 235 compounds targeted components of signaling pathways important in PCa, including: 28 compounds targeting PI3K/AKT/mTOR signaling (AKT1, PIK3CA, mTORc 1/2), 13 compounds targeting cell cycle regulators (CDK1, CDK4), 13 compounds targeting DNA repair and replication (CHEK1, TOP2A), 6 compounds targeting NFKB signaling (IKBKB,ITK) and 5 compounds targeting MAPK signaling (MAPK8, MAP2K1). Using high throughput screening technology to sort for compounds based on potency and activity identified clinically and biologically relevant therapeutic applications for existing compounds in a novel context, Pten/Tp53 null PCa progenitor cells. Citation Format: Keith H. Jansson, John K. Simmons, Caitlyn Fuller, Supreet Agarwal, Paul G. Hynes, Lei Fang, Ross Lake, Jacob Cawley, Lauren Stahl, Xiaohu Zhang, Rajarshi Guha, Craig Thomas, Kathleen Kelly. Elucidating potential therapeutic targets in a model of Pten/Tp53 null prostate cancer using high-throughput screening technology. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-281.

Abstract 2666: Receptor protein tyrosine phosphatase type kappa is a prognostic marker, essential for regulating malignant behavior of glioma cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Malignant gliomas have a poor survival resulting from rapid growth of the tumor and extensive dispersion throughout the brain parenchyma. Discovery of tumor-specific biomarkers hold great promise in improving survival and thus call for better understanding of regulatory signals driving glioma cell migration and dispersal. Using genome-wide exploration, we have identified frequent genetic alterations at the Receptor protein tyrosine phosphatase type kappa (PTPRK) locus. These PTPRK alterations were among the top significant and very relevant to glioma biology since PTPRK is a dephosphorylation regulator of tyrosine phosphorylation (TP) signaling, a frequently altered signaling cascade in glioma as reported previously. Understanding PTPRK function will provide relevant novel insights into the molecular mechanisms underlying glioma cell growth and migration. PTPRK expression was analyzed by quantitative-RT-PCR and western blot. Mutations of PTPRK were identified by sequencing full length PTPRK transcripts from a subset of patients’ samples showing PTPRK loss of heterozygosity. Two major PTPRK variants were cloned and expressed in cells harboring altered PTPRK (U87MG cell line) to study their effect on PTPRK phosphatase activity and functionality. PTPRK locus undergoes allelic loss in glioma and contributes to cancer phenotype. It is an independent prognostic marker in glioma. Patients with altered PTPRK (23%) have shorter survival compared to those with normal PTPRK molecular status (p=0.001, Log-rank test, 14 vs. 30 months). PTPRK mRNA as well as protein levels were lower in tumor biopsies compared to non-tumor specimens. Mutational analysis revealed missense mutations in the phosphatase domain, resulting in altered PTPRK phosphatase activity, possibly due to changes in active site conformation. Further, PTPRK alterations changed expression profiles of the PTPRK protein as a result of altered post-translational processing. Reconstitution of PTPRK expression in glioma cells (U87MG cell line) harboring PTPRK deletions suppressed cell growth and migration which was alleviated by the PTPRK variants to differing extent. PTPRK knockdown via shRNA down-regulated its onco-suppressive function compared to PTPRK expressing cells. Our findings provide the first evidence of the key role of PTPRK expression in glioma and shed light on the biological consequences of mutations in the PTPRK gene which significantly alter PTPRK functionality. Biological relevance of PTPRK variants will be determined and validated on a large scale. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2666. doi:1538-7445.AM2012-2666

Abstract 1283: Chromosome 19 amplification and the consequent NOTCH and VEGF pathways over-activation are associated with malignant glioma poor outcome

Malignant gliomas are the most frequent type of primitive brain tumors. Their outcome did not improve despite new therapeutics, resulting mainly from their genetic heterogeneity and complexity. By using genome-wide explorations we have recently identified, for the first time, that chr.19 amplification correlates with a grim therapeutic outcome. The aim of our current study is to investigate the significance of the chromosome 19 genetic signature. To narrow the search for specific genes or pathways that may be responsible for this adverse outcome, we selected genes located on chromosome 19 and focused on those that were statistically significantly deregulated between tumors and non tumor tissues. We identified 41 genes which mapped to chromosome 19. The most significant genes are NOTCH3 and HIF3α. These genes belong to NOTCH and VEGF pathways and play important roles in angiogenesis. Using quantitative real time PCR, RT-PCR and immunobloting, we further analyzed the level of gene expression of critical elements of these pathways and confirmed that NOTCH3 was amplified in primary glioma cell lines that harbor gains of chr.19. This increase has been shown at both the mRNA and protein levels. Moreover, NOTCH ligand, and other constituents of the pathways (DLL3, SHC3, NOTCH4) were also activated in these particular cell lines. Further, HIF3α was also activated in our conditions whereas HIF1α, the other player of HIF transcriptional pathway, was down regulated. These over-expressions were accompanied by a parallel VEGF activation in these cell lines. This novel genetic signature may lead to critical insights into diagnosis, classification, prognosis, and prediction in improving therapeutic strategies and outcome based on the genetic makeup of the tumor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1283.