Danica Wiredja

Activation of tumor suppressor protein PP2A inhibits KRAS-driven tumor growth

Targeted cancer therapies, which act on specific cancer-associated molecular targets, are predominantly inhibitors of oncogenic kinases. While these drugs have achieved some clinical success, the inactivation of kinase signaling via stimulation of endogenous phosphatases has received minimal attention as an alternative targeted approach. Here, we have demonstrated that activation of the tumor suppressor protein phosphatase 2A (PP2A), a negative regulator of multiple oncogenic signaling proteins, is a promising therapeutic approach for the treatment of cancers. Our group previously developed a series of orally bioavailable small molecule activators of PP2A, termed SMAPs. We now report that SMAP treatment inhibited the growth of KRAS-mutant lung cancers in mouse xenografts and transgenic models. Mechanistically, we found that SMAPs act by binding to the PP2A A&agr; scaffold subunit to drive conformational changes in PP2A. These results show that PP2A can be activated in cancer cells to inhibit proliferation. Our strategy of reactivating endogenous PP2A may be applicable to the treatment of other diseases and represents an advancement toward the development of small molecule activators of tumor suppressor proteins.

Small-Molecule Activators of Protein Phosphatase 2A for the Treatment of Castration-Resistant Prostate Cancer

Primary prostate cancer is generally treatable by androgen deprivation therapy, however, later recurrences of castrate-resistant prostate cancer (CRPC) that are more difficult to treat nearly always occur due to aberrant reactivation of the androgen receptor (AR). In this study, we report that CRPC cells are particularly sensitive to the growth-inhibitory effects of reengineered tricyclic sulfonamides, a class of molecules that activate the protein phosphatase PP2A, which inhibits multiple oncogenic signaling pathways. Treatment of CRPC cells with small-molecule activators of PP2A (SMAP) in vitro decreased cellular viability and clonogenicity and induced apoptosis. SMAP treatment also induced an array of significant changes in the phosphoproteome, including most notably dephosphorylation of full-length and truncated isoforms of the AR and downregulation of its regulatory kinases in a dose-dependent and time-dependent manner. In murine xenograft models of human CRPC, the potent compound SMAP-2 exhibited efficacy comparable with enzalutamide in inhibiting tumor formation. Overall, our results provide a preclinical proof of concept for the efficacy of SMAP in AR degradation and CRPC treatment.Significance: A novel class of small-molecule activators of the tumor suppressor PP2A, a serine/threonine phosphatase that inhibits many oncogenic signaling pathways, is shown to deregulate the phosphoproteome and to destabilize the androgen receptor in advanced prostate cancer. Cancer Res; 78(8); 2065-80. ©2018 AACR.

Phosphoproteomics Profiling of Nonsmall Cell Lung Cancer Cells Treated with a Novel Phosphatase Activator

Activation of protein phosphatase 2A (PP2A) is a promising anticancer therapeutic strategy, as this tumor suppressor has the ability to coordinately downregulate multiple pathways involved in the regulation of cellular growth and proliferation. In order to understand the systems‐level perturbations mediated by PP2A activation, we carried out mass spectrometry‐based phosphoproteomic analysis of two KRAS mutated non‐small cell lung cancer (NSCLC) cell lines (A549 and H358) treated with a novel small molecule activator of PP2A (SMAP). Overall, this permitted quantification of differential signaling across over 1600 phosphoproteins and 3000 phosphosites. Kinase activity assessment and pathway enrichment implicate collective downregulation of RAS and cell cycle kinases in the case of both cell lines upon PP2A activation. However, the effects on RAS‐related signaling are attenuated for A549 compared to H358, while the effects on cell cycle‐related kinases are noticeably more prominent in A549. Network‐based analyses and validation experiments confirm these detailed differences in signaling. These studies reveal the power of phosphoproteomics studies, coupled to computational systems biology, to elucidate global patterns of phosphatase activation and understand the variations in response to PP2A activation across genetically similar NSCLC cell lines.

CoPhosK: A Method for Comprehensive Kinase Substrate Annotation Using Co-phosphorylation Analysis

We present CoPhosK to predict kinase-substrate associations for phosphopeptide substrates detected by mass spectrometry (MS). The tool utilizes a Naïve Bayes framework with priors of known kinase-substrate associations (KSAs). Through the mining of MS data for the collective dynamic signatures of the kinases’ substrates, as revealed by correlation analysis of phosphopeptide intensity data, the tool infers KSAs in the data for the considerable body of substrates lacking such annotations. We benchmarked the tool against existing approaches for predicting KSAs that rely on static information (e.g. sequences, structures and interactions) using publically available MS data, including breast and ovarian cancer models. The benchmarking reveals that co-phosphorylation analysis can improve prediction performance when static information is available (about 35% of sites) while providing reliable predictions for the remainder, tripling the KSAs available from the experimental MS data to comprehensively and reliably characterize the landscape of kinase-substrate interactions well beyond current limitations.

Global phosphoproteomics of CCR5-tropic HIV-1 signaling reveals reprogramming of cellular protein production pathways and identifies p70-S6K1 and MK2 as HIV-responsive kinases required for optimal infection of CD4+ T cells

BackgroundViral reprogramming of host cells enhances replication and is initiated by viral interaction with the cell surface. Upon human immunodeficiency virus (HIV) binding to CD4+ T cells, a signal transduction cascade is initiated that reorganizes the actin cytoskeleton, activates transcription factors, and alters mRNA splicing pathways.MethodsWe used a quantitative mass spectrometry-based phosphoproteomic approach to investigate signal transduction cascades initiated by CCR5-tropic HIV, which accounts for virtually all transmitted viruses and the vast majority of viruses worldwide.ResultsCCR5-HIV signaling induced significant reprogramming of the actin cytoskeleton and mRNA splicing pathways, as previously described. In addition, CCR5-HIV signaling induced profound changes to the mRNA transcription, processing, translation, and post-translational modifications pathways, indicating that virtually every stage of protein production is affected. Furthermore, we identified two kinases regulated by CCR5-HIV signaling—p70-S6K1 (RPS6KB1) and MK2 (MAPKAPK2)—that were also required for optimal HIV infection of CD4+ T cells. These kinases regulate protein translation and cytoskeletal architecture, respectively, reinforcing the importance of these pathways in viral replication. Additionally, we found that blockade of CCR5 signaling by maraviroc had relatively modest effects on CCR5-HIV signaling, in agreement with reports that signaling by CCR5 is dispensable for HIV infection but in contrast to the critical effects of CXCR4 on cortical actin reorganization.ConclusionsThese results demonstrate that CCR5-tropic HIV induces significant reprogramming of host CD4+ T cell protein production pathways and identifies two novel kinases induced upon viral binding to the cell surface that are critical for HIV replication in host cells.

The KSEA App: a web-based tool for kinase activity inference from quantitative phosphoproteomics

Summary Computational characterization of differential kinase activity from phosphoproteomics datasets is critical for correctly inferring cellular circuitry and how signaling cascades are altered in drug treatment and/or disease. Kinase‐Substrate Enrichment Analysis (KSEA) offers a powerful approach to estimating changes in a kinases activity based on the collective phosphorylation changes of its identified substrates. However, KSEA has been limited to programmers who are able to implement the algorithms. Thus, to make it accessible to the larger scientific community, we present a web‐based application of this method: the KSEA App. Overall, we expect that this tool will offer a quick and user‐friendly way of generating kinase activity estimates from high‐throughput phosphoproteomics datasets. Availability and implementation the KSEA App is a free online tool: casecpb.shinyapps.io/ksea/. The source code is on GitHub: github.com/casecpb/KSEA/. The application is also available as the R package ‘KSEAapp’ on CRAN: CRAN.R‐project.org/package=KSEAapp/. Contact mark.chance@case.edu Supplementary information Supplementary data are available at Bioinformatics online.

MoBaS on Phosphorylation Data

Although advances in high-throughput omics technologies revolutionized our understanding of the genomic underpinnings of cancer, there are still many challenges in understanding how patients with common driver mutations may display diverging phosphoproteomic responses to the same treatment. Thus, an examination of the signaling landscape will provide essential molecular information for modeling personalized patient treatment design. However, integrative bioinformatics approaches to identify phosphoproteomics-based molecular states are in their infancy. To address this challenge, we integrated several bioinformatics tools to compare and contrast the drug-induced global signaling alterations of two KRAS mutated non-small cell lung cancer (NSCLC) cell lines, A549 and H358, treated with a novel activator of the tumor suppressor Protein Phosphatase 2A (PP2A) versus DMSO control. To identify protein subnetworks that are enriched in differentially phosphorylated proteins, we use our algorithm MoBaS which is designed to identify protein subnetworks that are enriched in disease-associated genomic variants identified by genome-wide association studies (GWAS). MoBaS takes as input a PPI network and a score for each protein indicating the proteins association with the phenotype of interest. It then identifies protein subnetworks that are (i) composed of densely interacting proteins, and (ii) enriched in proteins with high scores. MoBaS also assesses the statistical significance of the identified subnetworks using permutation tests that enable multiple hypothesis testing. Subsequently, we do kinase enrichment within the statistical significant modules and find Aurora KB as a key kinase differentially regulated between the two cell lines in response to our compound. Further corroborating this finding, Aurora KB was downregulated at the protein and mRNA levels with our treatment in A549 but not in H358. Ultimately, our approach models the diverging protein-level drug response across two similar cell lines representing NSCLC patients. Such phosphoproteomic information will potentially inform the optimal therapy regimen for each individual.

Abstract 3865: Therapeutic activation of protein phosphatase 2A for the treatment of lung cancer

PP2A is a phosphatase tumor suppressor that is dysregulated and deactivated in lung cancer. It is one of the most abundant cellular proteins and regulates the activity of numerous kinases Where achievable, restoration of PP2A function inhibits cancer progression, and notably, by the inhibition of the downstream effectors of the oncogenic kinases that initiate and drive cancer progression. In this study, we determined PP2A inactivation in human lung cancer with specific molecular genotypes and we ascertained the biological and functional consequences of PP2A reactivation. In assessing a lung cancer TMA, we identified that PP2A inactivation was correlated with poor survival and was significantly higher in patients with Kras mutations. In order to understand the therapeutic potential of restoration of PP2A activity in KRAS mutant lung cancer, our lab developed a series of small molecule activators of PP2A (SMAPs) through reverse engineering of tricyclic neuroleptic drugs. SMAP treatment of lung cancer cell lines resulted in an induction of apoptosis and decreased cell viability. Structural and biophysical studies have identified the site of drug binding and mechanism for PP2A activation by this small molecule series. Additionally, cell lines harboring drug-binding mutations were resistant to SMAP therapy as compared to wild type PP2A and EGFP control. Global phosphoproteomic analysis of SMAP treated KRAS lung cancer cell lines revealed ERK signaling as a commonly perturbed pathway in drug treated cell lines. Given the marked dephosphorylation of ERK upon treatment of cell lines with SMAPs, we overexpressed a constitutively active form of MEK (MEKDD) to blunt SMAP mediated ERK dephosphorylation to determine the relevance of ERK inactivation for the biological effects of SMAPs on cellular apoptosis. Overexpression of MEKDD resulted in a blunted apoptotic response to SMAP treatment. Single agent SMAP treatment of KRAS GEMM and xenograft mouse models of lung cancer resulted in tumor stasis, induction of tumor cell apoptosis and cell cycle arrest to comparable levels seen with a combination of AKT and MEK inhibitors. Western blotting and immunohistochemical analysis of the tumors demonstrated that SMAP treatment resulted in of ERK, AKT, and PP2A-Y307 dephosphorylation in vivo. Additionally, these compounds demonstrate favorable pharmacokinetics and show no overt toxicity. Furthermore, combination of SMAPs with kinase inhibitors further decreased tumor growth in vivo. Taken together, these findings point to therapeutic activation of PP2A as a novel strategy for the treatment of KRAS-mutant NSCLC. Citation Format: Jaya Sangodkar, Rita Tohme, Janna Kiselar, Sudeh Izadmehr, Divya Hoon, Sahar Mazhar, Abbey Perl, Danica Wiredja, Daniela Schlatzer, Shen Yao, David Kastrinsky, Neelesh Sharma, David Brautigan, Mark Chance, Alain Borczuk, Michael Ohlmeyer, Yiannis Ioannou, Goutham Narla. Therapeutic activation of protein phosphatase 2A for the treatment of lung cancer. [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 3865.

Abstract A38: Therapeutic targeting of oncogenic KRAS signaling using a novel small molecule agonist of the PP2A tumor suppressor gene

Metastatic non-small cell lung cancer (NSCLC) is the most common cause of cancer death. Cytotoxic chemotherapy has historically been the mainstay of therapy but is associated with only modest improvements in patient survival. Over the past decade, a better understanding of the pathogenesis of NSCLC, coupled with high throughput genomic technologies applied to patient tumor samples, has led to a molecular classification of NSCLC and a new generation of “precision” therapies. However, the most common recurrent oncogenomic mutation driving the growth of NSCLC, mutant KRAS, accounting for ∼25% of patients with advanced NSCLC, remains without an effective targeted therapy. Mutations in KRAS lead to downstream signaling through ERK, as well as cross talk with the PI3K-Akt pathway, the latter of which is amplified in the presence of inhibition of ERK pathway signaling alone. These findings likely explain, at least in part, why targeting ERK pathway signaling alone in NSCLC has been largely unsuccessful in the clinic, and suggest that coordinate inhibition of both ERK and Akt is necessary for optimal therapy. Approaches to inhibit both of these pathways simultaneously with co-administration of two small molecular kinase inhibitors has shown some promise, but has been limited by both “off-target” treatment-limiting side effects and suboptimal coordinate inhibition of both Akt and ERK signaling. Thus, novel therapies, are critically needed, to improve the lives of patients suffering from KRAS driven lung cancers and while oncogenic kinases have proven to be successful targets for cancer treatment, the therapeutic targeting of phosphatases, the key negative regulators of these same pathways, has remained largely unexplored. Starting with the observation that tricyclic neuroleptic drugs exert anticancer effects in xenograft models, we employed combinatorial chemistry to reverse engineer these drugs into a series of novel compounds that retain the anti-proliferative effects but are devoid of the dose-limiting effects on the central nervous system. We have demonstrated these agents exert potent anti-proliferative effects in both cell culture and in vivo lung cancer models and these effects are functionally linked with simultaneous inhibition of both PI3K-Akt and MAPK signaling. Importantly, these agents that have favorable pharmaceutic properties directly bind and activate the serine/threonine phosphatase 2A (PP2A) and we call these novel first-in-class agents Small Molecule Activators of PP2A (SMAPs). A critical role for PP2A as a tumor suppressor has previously been established, and inhibition and loss-of-function changes in PP2A occur in human lung cancers. Furthermore, protein phosphatase 2A (PP2A) accounts for the majority of cellular serine/threonine phosphatase activity, and its dominant and best defined targets are protein kinases and oncogenic proteins including ERK and AKT. Here we demonstrate for the first time the development and validation of a first-in-class orally bioavailable pharmacological agent that can directly bind and activate PP2A driving coordinate inhibition of both the MAPK and AKT effector pathways in cell culture and both xenograft and genetically engineered mouse models (GEMM) of human lung cancer. Global phosphoproteomic analysis of SMAP treated KRAS lung cancer cell lines reveals ERK signaling as the only commonly perturbed pathway in drug treated cell lines. Single agent SMAP treatment of KRAS GEMM and xenograft mouse models of lung cancer resulted in tumor stasis, induction of tumor cell apoptosis and cell cycle arrest to comparable levels seen with a combination of AKT and MEK inhibitors. Additionally, the compounds demonstrate favorable pharmacokinetics and show no overt toxicity. Taken together, these findings point to therapeutic activation of PP2A as a novel strategy for the treatment of advanced KRAS-mutant NSCLC. While research and clinical effort has largely focused on development of inhibitors of oncogenic kinases, the identification of small molecule activators of tumor suppressor proteins has remained elusive. Activation of such proteins could offer the opportunity to identify novel synergistic strategies for the treatment of a number of cancer types. Nevertheless, translation of a PP2A activation strategy into clinical medicine has required pharmaceutically tractable agents for development. Our studies represent a first step into that new territory and highlight the potential for the development of small molecule activators of other protein phosphatases and tumor suppressor proteins. Citation Format: Jaya Sangodkar, Sahar Mazhar, Danica Wiredja, Giridharan Gokulrangan, Daniela Schlatzer, David Kastrinsky, Analisa Difeo, Shen Yao, Sudeh Izadmehr, Neelesh Sharma, Yiannis Ioannou, Michael Ohlmeyer, Goutham Narla. Therapeutic targeting of oncogenic KRAS signaling using a novel small molecule agonist of the PP2A tumor suppressor gene. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A38. doi: 10.1158/1557-3125.RASONC14-A38

Abstract 1616: Phosphoprotein enrichment pathway analysis tools for studying cancer signaling

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Cancer development and progression is driven by dysregulated signaling transduction, which is predominantly regulated by serine, threonine, and tyrosine phosphorylation. Consequently, MS-based phosphoproteomics, which can identify and quantitate thousands of phosphopeptides, provides a powerful quantitative tool to detect and comprehensively catalog alterations in cancer signaling cascades that cannot be captured by analysis of DNA or RNA alone. Unfortunately, the complexity of the datasets generated by these advanced proteomic methodologies are still not matched with well-established or fully tested bioinformatics tools to extract critical pathways from these high-dimensional phosphoprotein data. Here, we present two approaches for identifying pathways with altered phosphorylation patterns: Protein Set Enrichment Analysis (PSEA) and Protein Interaction Enrichment Network Analysis (PIENA). Development of these tools was inspired by two published methods utilizing gene expression datasets to identify disrupted gene sets: Gene Set Enrichment Analysis (GSEA) and Gene Interaction Enrichment Network Analysis (GIENA), respectively. PSEA, through pathway scoring, extracts pathways that show a uniform directional phosphorylation change through all protein members of that set. As in the traditional GSEA method, an enrichment score is calculated for each pathway based on intensity values from all detected phosphopeptides. The second method, PIENA, looks for notable differences in phosphorylation between phosphopeptide pairs. Ultimately, this approach identifies pathways enriched with altered phosphoprotein interactions. Both methods have been used to identify changes in signaling states for two lung adenocarcinoma cell lines, A549 and H358, treated with either control or a novel phosphatase activator with anti-cancer properties. Both PSEA and PIENA identify pathways classically implicated in cancer, including p53, mitosis and cell cycle progression, insulin secretion, immune regulation, and RAS signaling cascades. Furthermore, the ERK pathway is consistently found to be significantly disrupted in both A549 and H358. This corroborates biological findings that this novel drug has activity against KRAS mutated cell lines. Although future cellular experiments will help to confirm the detailed mechanism of this drugs effects, our current results demonstrate the advantage of these new pathway identification tools in providing unbiased and global identification of key drivers of signaling. Citation Format: Danica Wiredja, Yu Liu, Daniela Schlatzer, Giridharan Gokulrangan, Goutham Narla, Mark Chance. Phosphoprotein enrichment pathway analysis tools for studying cancer signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1616. doi:10.1158/1538-7445.AM2014-1616