Ramzi M. Mohammad - US grants

5'nucleotidase; SCID mouse; neoplasm /cancer chemotherapy

DESCRIPTION (provided by applicant): Pancreatic neoplasia is the fifth most common cancer in the United States with >28,000 newly diagnosed cases per year with an annual mortality rate of greater than 99%. Its etiology is largely unknown and no curative treatment is presently available. In pancreatic cancer, several important survival molecules such as EGFR, NF-kB, COX-2, and Bcl-xL are highly activated. NF-kB regulates many genes involved in tumor progression, survival, angiogenesis and invasion such as Bcl-xL, COX-2, VEGF, MMP-9 and uPAR. High expression of anti-apoptotic Bcl-xL protein promotes cell survival and plays a key role in pancreatic cancer progression leading to drug resistance. Its expression was found in 90% of pancreatic cancer. The overexpression of the cyclooxygenase-2 (COX-2) enzyme in pancreatic cancer has also been shown to contribute to growth, metastasis, and chemoresistance. Therefore, inhibitions of NF-kB, Bcl-2/Bcl-xL, and COX-2 should serve as a novel treatment strategy for pancreatic cancer. Our co-investigator has designed non-peptide, drug-like, cell permeable potent small molecule inhibitors (SMI) that bind to the pocket of Bcl-2 and Bcl-xL and block/disrupt their anti-apoptotic function. These SMI were tested in an in vitro binding assay and found to be potent inhibitor of the binding of Bak BH3 peptide to Bcl-2 and Bcl-xL. We have also shown that genistein inactivates NF-kB and down regulates Bcl-xL, VEGF, MMP-9, and uPAR by transcriptional inactivation. Furthermore, the inhibition of COX-2 activity and Akt/NF-kB by celecoxib could be potentially useful in killing pancreatic cancer cells. We have found that human pancreatic cancer cell lines are notable for high expression of Bcl-XL, NF-kB, and COX-2. Therefore, we hypothesize that targeting the Bcl-2/Bcl-xL survival pathway by SMI and inactivating NF-kB-induced Bcl-2/BcI-xL/COX-2 generation by genistein and celecoxib could be novel therapeutic strategies for the treatment of pancreatic cancer. Of importance to this work is that NF-kB transcriptionally regulates the expression of Bcl-xL, hence inactivation of NF-kB by genistein and treatment with SMI will lead to decreased Bcl-xL. Moreover, the addition of celecoxib may be very useful for inducing pancreatic cancer cell death by inhibiting NF-kB as well as transcription and enzyme activity of COX-2. Our Specific Aims are: 1) To investigate the molecular mechanisms by which SMI promote apoptosis in human pancreatic cancer cell lines, 2) To determine anti-tumor activity of SMI using human pancreatic cancer xenograft model, 3) To determine whether transcriptional repression of Bcl-xL by genistein will synergize SMI-induced apoptosis in vitro and in vivo, and 4) To determine the effects of inhibition of COX-2 by celecoxib on Akt/NF-kB activation in genistein-treated and SMI-treated pancreatic cancer cells in vitro and in SCID mouse xenograft models. Targeting Bcl-2/Bcl-xL, NF-kB and COX-2 could be novel therapeutic treatment of pancreatic cancer.

DESCRIPTION (provided by applicant): Pancreatic cancer is a deadly disease that takes two American lives every 30 minutes (annual mortality >33,000). Failure of standard chemotherapies and newly developed targeted therapies has stagnated any improvement on the overall dismal survival (<5%) indicating that newer strategies for the management of this disease are urgently needed. In line with the goals of this RFA i.e. to identify potential molecula targets, and to test new therapeutic strategies, we intend to investigate the therapeutic potential of targeting CRM-1 (chromosome region maintenance 1) by our newly developed selective inhibitors of nuclear export (SINE) in pancreatic cancer. CRM-1 is a member of the importin superfamily of nuclear transport receptors, recognizing proteins bearing a leucine-rich nuclear export sequence (NES) and is the major receptor for the export of proteins out of the nucleus. Among the target of CRM-1 is the tumor suppressor protein (TSP) prostate apoptosis response-4 (Par-4). Earlier we had proposed Par-4 as a potential therapeutic target in pancreatic cancer since downregulation of this TSP has been directly linked to poor overall survival. Nuclear exclusion of Par-4 by CRM-1 renders the cancer cells resistant to apoptosis. Our preliminary findings demonstrate that SINE (KPT-185 and clinical candidate KPT-251) can lock Par-4 in pancreatic cancer cell nucleus by inhibiting CRM-1 that leads to cancer cell selective apoptosis and anti-tumor effects. To our surprise normal cells did not respond SINEs and this emerges to be mechanistically co-related to low Par-4 expression and insignificant Par-4 phosphorylation due to low protein kinase A (PKA). Hence we hypothesize that targeted inhibition of CRM-1 is an attractive strategy to guide TSPs (specifically Par-4) in the nucleus, inducing pancreatic cancer specific apoptosis. Based on our provocative initial findings and to test our hypothesis we propose 1. To establish whether there is a direct relationship between Par-4 nuclear localization and growth inhibitory and apoptosis inducing activity of KPT-185 and KPT-251 in a panel of pancreatic cancer cell lines with differential PKA and Par-4 expression and establish whether knockdown of PKA can cause resistance to KPT-mediated killing through Par-4 and other mechanisms. 2. Assess the in vivo efficacy of KPT- 251 (clinical candidate at MTD) in animal xenograft mice models developed from pancreatic cell lines with differential Par-4 expression (high Par-4 PKA vs low Par-4 PKA). Impact: CRM-1 is a druggable target in pancreatic cancer, however, currently there is no existing drug targeting this detrimental nuclear exporter. Earlier attempts to develop CRM-1 inhibitor were not successful as exemplified by the failure of the natural product Leptomycin B in a single clinical trial due to severe toxicity. Sinc then the field has not witnessed any serious attempts to develop newer classes of CRM-1 inhibitors that could be used clinically for the treatment of pancreatic cancer. Our newly developed KPT-SINE's are highly specific, orally active drugs with excellent pharmacokinetic parameters and hold promise against deadly pancreatic cancer. PUBLIC HEALTH RELEVANCE: CRM-1 is a druggable target in pancreatic cancer, however, currently there is no existing drug targeting this detrimental nuclear exporter. Earlier attempts t develop CRM-1 inhibitor were not successful as exemplified by the failure of the natural product Leptomycin B in a single clinical trial due to severe toxicity. Since then the field has not witnessed any serious attempts to develop newer classes of CRM-1 inhibitors that could be used clinically for the treatment of pancreatic cancer. Our newly developed KPT-SINE's are highly specific, orally active drugs with excellent pharmacokinetic parameters and hold promise against deadly pancreatic cancer.

DESCRIPTION (provided by applicant): Epithelial-to-Mesenchymal Transition (EMT); a driver of tumor resistance and metastasis, is a complex mechanism that arises through an intricate cross talk between highly robust biological networks. There is minimal information on the most central genes in the networks that drive EMT primarily due to the lack of proper computational tools. To address this unmet problem, two PIs (a computational biologist and a molecular biologist) have teamed together to identify the central genes that are differentially expressed between epithelial and mesenchymal subtypes in well recognized Weinberg's EMT cell models. While these models have been the subject of differentially expressed (DE) gene analyses using the t-test and the F-test, it is not sufficient to interrogate the entire EMT phenomena due to the presence of additional genes that do not meet the DE criteria. Existing models for network analysis, co-expression analysis, and gene clustering can only provide information about a group of genes with similar behavior. However, such analysis cannot extract EMT-specific characterization of mesenchymal pathway genes; i.e. identifying the distinguishing set of mesenchymal patterns in the entire co-expressed gene groups that may be specific to EMT only. Here, we propose a network-based differential analysis model for analyzing the topological differences between two gene networks constructed from the expression data. We hypothesize that for deeper understanding of EMT a differential network analysis coupled with biological validation of the EMT associated genes in the correct models is critical. To this end, we performed comparative genomic microarrays expression investigations on Weinberg's K-ras-HMLE (Epithelial) and K-ras-HMLE-SNAIL (Mesenchymal) 4 cell lines datasets. Our analyses revealed a significant global gene expression difference between parent K-ras-HMLE and HMLE-SNAIL cells. As they are SNAIL driven EMT models, we challenged these cells with a small molecule inhibitor (SMI) against SNAIL (GN-25). Our new computational approach utilizes differential network analysis in multiple EMT models in cell culture, and in animal tumor model (to verify the influence of tumor microenvironment on EMT in situ). This will be coupled with more robust biological validation in the presence of newer network targeted drugs. Therefore, our Specific Aims are 1) Identifying EMT central genes using differential network-based algorithms and 2) Biological validation and evaluation of targeted strategies against centralized genes in the EMT networks. The identified central gene will be validated at the mRNA and protein expression level and their cause-effect relationship will be evaluated using RNA interference in the paired EMT models. In a network-driven drug design, the EMT cells will be challenged with a repertoire of small molecule drugs (identified through our chemical library screening) as single agent or in combination and verify whether drug treatments could target the central genes. Additional validation using efficacy trial of the most potent SMI or its combination in animal tumor models will fortify the clinical application of our network derived EMT targeted drugs.

? DESCRIPTION (provided by applicant): Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease in urgent need of newer molecularly targeted drugs. Aberrations in the Kras oncogene for long have been appreciated to be a major driver of this disease. Ras genes code for a set of proteins that are instrumental in cellular signaling, and when mutated, permit uncontrolled cellular proliferation in PDAC. Even though, the Ras signaling network has been well understood, however this knowledge could not be translated into developing new cancer drugs. This is primarily because Ras proteins lack the ideal binding pockets that usually serve as attractive targets for small molecule drugs. To overcome this scientific challenge, newer targets, either from the Ras structure itself, or from critical direct interacting partners or downstream effectors of Kras (here the p21 activated kinase 4/PAK4) need to be urgently exploited. The PAK family members are key effectors downstream of Ras, which act as regulatory switches that control critical cellular processes, leading to tumor aggressiveness. Recently, studies have shown amplification of PAK4 gene in large PDAC patient cohorts. Our investigations in gemcitabine (GEM) resistant PDAC models showed a very strong correlation between PAK4 over-expression and drug resistance. Therefore we hypothesize that PAK4 protein is an attractive druggable candidate in the elusive Ras pathway and its inhibition will overcome GEM resistance by suppressing Kras mediated proliferative signaling in PDAC. Earlier unsuccessful attempts to target PAK4 (tested in non-pancreatic models) resulted in the development of a Type I ATP competitive inhibitor PF-03798309 that was prematurely discontinued based on a single clinical trial in view of its undesirable pharmacokinetic characteristics due to excessive drug efflux through multi-drug resistance proteins (MDRs). Since then there have been no serious attempts to develop newer and superior inhibitors against this elusive protein, and thus there is a void in our knowledge in relation to PAK4 inhibitors. Filling this scientific void we have developed the first in class Type II allosteric modulators of PAK4 that show selective activity in resistant pancreatic cancer. Most importantly, unlike PF-03798309, our Type II PAK4 allosteric modulators are not substrates to multi-drug resistance (MDR) proteins. In this highly translational proposal, the utility of our novel PAK4 inhibitors against resistant PDAC will be delineated. These studies will help in the understanding of PAK4 dependent resistance mechanisms in PDAC. Our specific aims are: Aim-1: Demonstrate that PAK4 is a diagnostic and therapeutic biomarker for resistant PDAC. Aim-2: Evaluate the impact on tumor growth of PAK4 inhibition in orthotopic and well recognized pancreatic cancer transgenic [KrasG12D/+; LSL-Trp53 R172H/+; Pdx-1-Cre] animal models. Impact: Our newly discovered Type II PAK4 allosteric modulators show activity against therapy resistant PDAC. The outcome of our proposed pre-clinical studies will enable us to have a focused design, toxicity and efficacy testing of PAK4 allosteric modulators in PDAC.