Goutham Narla - US grants

? DESCRIPTION (provided by applicant): Prostate cancer is the second leading cause of cancer-related death among males in the United States. Beyond the burden in lives affected and lost, more than 192,280 new cases of prostate cancer are projected in 2014. The need to define the genetic basis of this disease is clear. One of the hallmarks of oncogenesis is the aberrant activation of various cellular kinases and growth promoting nuclear hormone receptor signaling. Sustained oncogenic activation requires coordinate inactivation of tumor suppressor genes, such as protein phosphatases, to allow propagation of signaling. Although the inhibition of oncogenic signaling through the development of nuclear hormone antagonists (such as MDV3100) or kinase inhibitors has resulted in some therapeutic success, most exhibit modest efficacy, leading to eventual tumor resistance. The therapeutic activation of tumor suppressor genes has remained largely unexplored. We have developed a series of novel drugs that uniquely target protein phosphatases and possess favorable pharmacokinetics and no significant toxicity. Characterization of these compounds revealed their ability to simultaneously inhibit both the MYC and AR effector pathways in prostate cancer cell lines and mouse models. 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 for the treatment of prostate cancer specifically and other cancers more generally.

PROJECT SUMMARY Cancer has been a major focus of biomedical research since the war on cancer began with the National Cancer Act of 1971. Despite this focus, nearly 8 million people die of cancer each year, worldwide. To help ensure that the nations? biomedical, clinical, and behavioral cancer research workforce remains strong, we propose the development of a Cancer-focused Summer Undergraduate Research (CanSUR) Program, to recruit highly motivated undergraduate students from diverse backgrounds and foster their excitement for a career in cancer research. The CanSUR program will support 32 undergraduate scholars each year, recruited from regional and national colleges/universities for a 10-week period, to participate in a cancer research experience with members of the Case Comprehensive Cancer Center (Case CCC). Key objectives of the CanSUR Program are to: (1) provide each scholar with a summer cancer research experience in a Case CCC members lab; (2) foster an understanding of biomedical, cancer research career opportunities and (3) providing scientific enrichment and professional skills curriculum to help prepare them for the next step in a cancer research focused career. Four major components have been developed to meet these objectives, including an Intensive Week 1 Cancer Immersion Workshop, a series of interactive cancer research lectures on the fundamentals of cancer biology, current cancer therapies, cancer research techniques, cancer career paths and innovative 3-dimensional cancer education modules (called the HoloLens). We propose a novel 2- tier mentorship structure that includes both the primary mentor (lab leader), and a junior mentor, which will be the person in charge of the day-to-day training of the CanSUR scholar within the research environment. Beyond their individual research experience, we have developed a series of Scientific Enrichment components which address important issues in cancer research, such as developing professional habits, effective communication strategies, and an appreciation of how cancer impacts individuals and their families (using talks by patient advocates). Numerous cancer-focused career paths will be introduced, and guidance about how to prepare for the next step of a specific career path will be provided by graduate and medical school admissions directors, professionals who have achieved their cancer-related career goals, and current trainees. Given the importance of our objectives, we have been pledged significant institutional support and have 82 faculty and institutional leaders who focus on cancer education prepared to recruit and train outstanding undergraduates in this summer program. As such, we are well-positioned to convert highly-motivated undergraduates into the nation?s next cancer research workforce.

Project Summary/Abstract Lung cancer progression involves coordinate changes in both oncogene and tumor suppressor function. Protein Phosphatase 2A (PP2A) is a tumor suppressor that is dysregulated and deactivated in lung cancer. PP2A is inactivated through several mechanisms including somatic mutations, suppression of individual subunits, increased expression of endogenous PP2A inhibitors and changes in post-translational modifications of the C subunit. While several therapeutics have been developed to indirectly target PP2A, recent studies have uncovered small molecules such as phenothiazines that directly activate PP2A. Nevertheless, because their pronounced extrapyramidal and anti-cholinergic effects were severely dose limiting, further pursuit of phenothiazines and related dibenzazepines for treatment of cancer seems unlikely. Our lab has reengineered the tricyclic neuroleptics to decouple the CNS pharmacology from the anti-proliferative properties of this drug- class. We have developed and characterized these small molecule activators of PP2A (SMAPs) as both chemical tools to probe PP2A regulation and for the treatment of PP2A dependent diseases. Our combined structure- function studies have revealed that SMAPs bind to PP2A in a way that protects the regulatory C-terminal tail of the catalytic subunit and stabilizes the holoenzyme. Our goal is to define the SMAP induced alterations on PP2A holoenzyme composition that promote the tumor suppressive properties of PP2A. We aim to identify the tumor suppressive PP2A regulatory subunits enhanced by SMAPs in order to correlate treatment response to clinical and genetic characteristics, and to aid in the design of more selective, next-generation SMAPs. In this project, three robust aims are designed to assess how activation of PP2A using SMAPs represents an effective treatment option for patients diagnosed with NSCLC. Aim 1 will structurally and functionally characterize the physiologic and cancer-relevant posttranslational regulation of PP2A by SMAPs. We will investigate the SMAPs-specific structural modulation of the PP2A holoenzyme by various, recurrent tumor derived mutations through coupling methyl-PP2A-C antibody and immunoprecipitation-mass spectrometry based approaches to quantify post- translational modification and PP2A activity. Aim 2 will discern how PP2A A? mutations modulate the biological activity of SMAPs. We will assess the functional role of common missense mutations in lung cancer by using isogenic CRISPR/Cas9 cell lines to identify cellular phenotypes, subunit expression, SMAPs response, and biological responses in nude mice. Aim 3 will determine combination treatment strategies to promote regression of tumors in KRAS mutant lung cancer. We will examine rational treatment combinations with SMAPs based on our studies demonstrating that protein phosphatase 2A (PP2A) activity defines the response of KRAS mutant lung cancer cells across library of >200 kinase inhibitors. Collectively, these studies will reveal novel insights into the molecular basis of regulation of PP2A and functional consequences of PP2A activation/inactivation in lung cancer.

Project Summary / Abstract Neuroendocrine tumors (NETs) are incurable, clinically challenging malignancies that are rising in incidence. Although tumors grow slowly, they progress relentlessly and lack effective therapies once they become metastatic. Many patients with advanced NETs will die from their disease. Greater understanding of mechanisms driving NET progression and metastasis is needed to inform new therapies and improve patient outcomes. We discovered a RABL6A-PP2A pathway that promotes pancreatic NET (PNET) pathogenesis. RABL6A (RAB-like GTPase) is upregulated in patient PNETs and is required for PNET proliferation and survival. RABL6A acts through multiple mechanisms that are only partly defined, including inhibition of tumor suppressors (p27, RB1) and activation of oncogenic pathways (MYC, AKT-mTOR). A common regulator of all those factors is protein phosphatase 2A (PP2A), a powerful tumor suppressor whose role in NETs has not been explored. We found that RABL6A activates AKT-mTOR signaling in PNETs by inhibiting PP2A. In turn, RABL6A is down regulated by PP2A although the molecular mechanism by which these two proteins inhibit each other's function is unknown. Excitingly, specific `small molecule activators of PP2A' (called SMAPs) suppress PNET cell proliferation and survival in a RABL6A-dependent manner and abolish tumor growth in vivo. These findings support a novel strategy for PNET therapy involving PP2A reactivation. However, the role of RABL6A and PP2A in NET progression is only partly understood and their importance in NET metastasis is not known. This multi-PI study draws upon the collective knowledge of both PIs and their unique expertise / resources in NETs (Quelle) and PP2A (Narla) to test the central hypothesis that the RABL6A-PP2A pathway is a critical driver of NET progression and response to targeted therapies. Aim 1 will determine the mechanisms of RABL6A-PP2A reciprocal regulation. Aim 2 will define the roles and interdependence of RABL6A and PP2A in NET progression and metastasis. Aim 3 will establish the importance of the RABL6A- PP2A alterations in NET pathogenesis and the efficacy of pathway targeted combination therapies. This project will provide new insights into molecular events driving NET progression and metastasis while establishing the efficacy of promising NET therapeutics, thus addressing a critical gap in NET research that may ultimately improve patient outcomes. Moreover, this work builds upon an emerging strategy for anticancer therapy (i.e., pharmacological reactivation of PP2A), and may have broad cancer relevance beyond NETs given the importance of RABL6A overexpression and PP2A inactivation in other tumor types.

Abstract: Protein phosphorylation is the most frequent and best studied post-translational modification. In general, kinases activate signaling pathways, whereas phosphatases behave as the ?off-switch? of those paths. In many human diseases this balance is skewed toward kinase hyperactivation and/or phosphatase suppression. The enhanced activity of hyperphosphorylation is a commonality in many diseases as proliferation (in the case of cancer) in unchecked with phosphatase suppression, leading to rapid cell proliferation and tumor growth. Similarly, in the case of Alzheimer?s Disease, the kinase-to-phosphatase activity ratio is skewed, such that the microtubule tau gains a net increase in phosphorylation. Phosphorylated tau aggregates into fibrils that are the hallmark of Alzheimer?s Disease and other neurological disorders including Parkinson?s Disease. Because kinase activity is commonly increased in many diseases, including cancer and Alzheimer?s Disease, kinase inhibitors have recently been developed as therapeutic approaches. Unfortunately, the redundancy in kinases in regulating signaling pathways, an inability to selectively target unique kinases, and the toxicity associated with this approach, have limited progress in the development of kinase inhibitors and their use in the clinic. Theoretically, a phosphatase activator should have a similar therapeutic potential in correcting the kinase:phosphatase activity in disease states. However, the development of ?activators? is generally much more difficult than developing an ?inhibitor?. Therefore, phosphatases, including those in the PP2A family, has long been seen as ?undruggable? targets in the pharmaceutical industry. This paradigm has recently been overcome with new strategies as we, and others, have developed small molecules that stabilize phosphatase complexes to reactivate their activity and to combat disease. In this supplemental proposal, we seek to expand the novel strategy we have successfully developed in reactivating PP2A phosphatases in combating cancer, to an analogous strategy in reactivating PP2A phosphatases to combat Alzheimer?s Disease.