Rajgopal Govindarajan, Ph.D. - US grants

DESCRIPTION (provided by applicant): Despite superior cytotoxicity against early-stage pancreatic tumors, gemcitabine is practically ineffective in late-stage tumors due to the invasive growth characteristics and apoptotic resistance of tumor cells. Evidence in literature suggest that epigenetic alterations (e.g. histone and DNA hypermethylation) may play vital roles in silencing tumor suppressor genes, allowing tumor cells to clonally expand and resist cytotoxicity. Our long-term goal is to improve the chemotherapeutic management of pancreatic cancer. The overall objective of this R03 grant application is to test the concept that the reactivation of epigenetically silenced tumor suppressor genes can augment chemosensitization in pancreatic cancer. Specifically, it is our central hypothesis that a mechanism-based structure optimization of a novel histone-demethylation agent, 3'deazaneplanocin-A (DZnep), in combination with gemcitabine will enact a superior cytotoxic response in pancreatic cancer cells. This hypothesis is based on our preliminary data that show transport-dependent augmentation of gemcitabine chemosensitization by DZnep in cultured pancreatic cancer cells. The rationale underlying the proposed research is that proof of synthetic demethylating agents improving gemcitabine chemosensitivity will provide an experimental basis to continue investigating the potential of epigenetic therapy for pancreatic cancer. The central hypothesis will be tested by pursuing three specific aims. Specific aim 1 will determine the cellular transport and activation mechanisms of DZnep in pancreatic cancer. The working hypothesis for aim 1 is that the demethylating and chemosensitizing abilities of hydrophilic DZnep will be rate-limited by a carrier-mediated transport process. This hypothesis is based on our preliminary data that show a significant reduction in DZnep-induced responses when nucleoside transport activity was inhibited. Specific aim 2 will generate and test a battery of DZnep acyl derivatives in vitro. The working hypothesis for aim 2 is that N4-substituted fatty acid amide derivatives will increase the lipophilicity of DZnep and allow it to bypass the transport requirement for intracellular activation. Our hypothesis is based on our preliminary studies showing the acyl prodrugs of another nucleoside (troxacitabine) bypassing the transport requirement. Specific aim 3 will determine the in vivo anti-cancer efficacies of DZnep prodrug-gemcitabine combinations. Our working hypothesis is that the combined treatment of a DZnep prodrug with gemcitabine in mice carrying pancreatic cancer xenografts will exhibit superior chemotherapeutic efficacy compared with gemcitabine treatment alone. This hypothesis is based on the extrapolation of preliminary in vitro results obtained with DZnep and gemcitabine combination schedules in cell culture. The contribution from these studies will be significant because successful demonstration of a superior epigenetic-chemotherapeutic regimen is likely to replace the less efficacious gemcitabine monotherapy practice. The proposed work is innovative because it addresses for the first time the possibility of a nucleoside-based epigenetic-chemotherapeutic therapy in pancreatic cancer. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because pancreatic cancer is a dire problem in the US with an estimated death of about 35,000 people annually. Since gemcitabine monotherapy is not very effective, epigenetic agents in combination with standard chemotherapeutic care will act as a superior treatment regimen necessary for improving survival in pancreatic cancer patients.

DESCRIPTION (provided by applicant): Twenty one exclusive mutations in the human equilibrative nucleoside transporter-3 (hENT3), a nucleoside transporter predominantly localized in mitochondria cause a spectrum of human genetic disorders with wide-ranging skin and musculoskeletal disease manifestations (e.g. scleroderma, hypertrichosis, hallus valgus, short stature, etc.). Although there are intriguing similarities between the hENT3-spectrum disorders and mitochondrial disorders, the mechanistic involvement of hENT3 in the initiation, progression, and perhaps in the intervention of these disorders is not understood. Our long-term goal is to identify the molecular pathogeneses of hENT3 spectrum disorders. The overall objective of this R03 grant application is to prove/disprove the concept that abnormalities of mitochondrial physiology are responsible for hENT3 spectrum disorders. Specifically, it is our central hypothesis that interference with mitochondrial nucleoside transport is the molecular explanation for the physiological, biochemical, and clinical manifestations of hENT3-spectrum disorders. This hypothesis is based on preliminary data which show that all hENT3 disease mutations severely impair mitochondrial transport of nucleosides, mitochondrial localization, and/or the stability of hENT3 protein. The rationale underlying the proposed research is that proof in mice that dysfunctional mitochondrial nucleoside transport is the root cause of hENT3-spectrum disorders would provide an experimental model in which to subsequently investigate, in depth, the molecular pathogeneses and potential remedies for these diseases. This central hypothesis will be tested by pursuing two specific aims. Specific aim 1 will determine the role of ENT3 in mitochondrial path physiology. The working hypothesis is that ENT3 will significantly influence in vitro and vivo mouse mito- chondrial functions. The hypothesis is based on our own preliminary data which identify the reduction of mito- chondrial nucleoside transport in hENT3 spectrum syndromes. Cells derived from human disease patients and mENT3 KO mice will be utilized to evaluate mitochondrial path physiology. Specific aim 2 will determine the occurrence and rescue of pathologic changes in mENT3 KO mice. The working hypothesis is that the abnormalities discovered in mENT3 KO mice will closely mimic those seen in hENT3 disorders and that the restoration of mitochondrial transport functions will help in reversal of disease pathology. The hypothesis is based on the similarities between the clinical manifestations of hENT3 and mitochondrial disorders and on the spatial synchrony of mENT3 developmental expression. Comparisons of hENT3 disorder manifestations with the mouse-equivalent of those manifestations will characterize the mouse model. Our studies will validate a crucial disease model and test the concept that interference with mitochondrial nucleoside transport could result in the types of abnormalities seen in hENT3 disorders. This contribution would be significant because the resultant model would enable subsequent mechanistic investigations. The proposed research is innovative because it would enable applicability to multiple skin and musculoskeletal disorders.

DESCRIPTION (provided by applicant): Pancreatic cancer is a highly aggressive malignancy that is resistant to most chemotherapeutic agents. Nucleoside analogs are currently used as the treatment of choice despite their suboptimal benefits. Our long-term goal is to improve the chemotherapeutic management of pancreatic cancer. The overall objective of this application is to elucidate the role of miR-663 and -4787 in pancreatic cancer chemoresistance. We hypothesize that the restoration of miR-663 and -4787, miRs epigenetically silenced in pancreatic cancer, will target a TGF-?et-7 axis to potentiate the nucleoside analog chemotherapeutic efficacy. This research has important translational significance because it will enable further clinical studies to improve pancreatic cancer patient sensitivity to therapy. Guided by strong preliminary data, the central hypothesis will be tested by pursuing three specific aims. Specific aim 1 will determine the epigenetic regulation of miR-663 and -4787 in pancreatic cancer. The working hypothesis states that miR-663a and -4787-5p are silenced in pancreatic cancer consequent to histone methylation-dependent chromatin changes. In this aim, the tumor chromatin landscape will be investigated by a) studying the epigenetic silencing marks in the promoter regions of miR-663 and -4787, b) identifying epigenetic protein complexes regulating miR-663 and -4787, and c) evaluating the cancer control consequences after manipulation of epigenetic marks and histone writers by knockdown and pharmacological approaches. Specific aim 2 will elucidate the mechanism of miR-663 and -4787-induced chemosensitization. The working hypothesis is that the chemosensitization activities of miR-663 and -4787 are mediated by a reduction in epithelial-mesenchymal transition (EMT)-driven drug resistance via a TGF-let-7 axis. In this aim, the detailed biochemical mechanisms of miR-663 and -4787 regulation of TGF-?expression and let-7 subtypes' maturation will be investigated to understand their role in EMT-directed chemoresistance. In addition, the impact of miR-663 and -4787 in the innate and acquired nucleoside analog resistance will be determined. Specific aim 3 will evaluate the in vivo efficacy of miR-663 and -4787 in pancreatic tumor growth control. The working hypothesis is that the tumor delivery of miR-663 and -4787 will potentiate gemcitabine efficacy in mice carrying orthotopic pancreatic cancer xenografts. Overexpression and knockdown of miR-663 and -4787, novel nucleoside analog-oligonucleotide (miR) nanoparticle formulations and hydrodynamic delivery to the mouse tumors and pharmacokinetics will be evaluated. This approach offers a substantive innovation in the field of pancreatic cancer chemotherapy by directing co-delivery of a novel epigenetic chemoresistance inhibitor with the gemcitabine chemotherapeutic for optimized cancer cell destruction. The proposed research is significant because it directly addresses the overriding cause of nucleoside analog treatment failure in pancreatic cancer patients, chemoresistance, and advances an alternate epigenetic-chemotherapeutic combination approach to preclinical testing to overcome the limitations of current chemotherapy.

DESCRIPTION (provided by applicant): Chemoresistance to nucleoside analog drugs (e.g., gemcitabine) is one of the main underlying reasons for the extremely poor prognostic state of pancreatic cancer. While the pivotal roles of cellular nucleoside transporters (NTs) and intercellular gap junctional connexins (Cxs) in determining tumor exposure of nucleoside drugs have begun to emerge, their functional interplay in determining nucleoside analog chemosensitivity and their potential in predicting response to chemotherapy remain unknown. Our long-term goal is to improve the chemotherapeutic management of pancreatic cancer. The overall objective of this R15 application is to investigate NT-Cx interplay in pancreatic cancer chemotherapy. The central hypothesis is that the type of NT-Cx combination expressed will dictate chemosensitivity and that the specific combination(s) can then be targeted to increase anti-tumor efficacy. This hypothesis has been formulated on the basis of the continuum of studies on NTs and Cxs and the recent discovery of non-genetic functional alterations in NT and Cx subtypes in pancreatic tumors. The rationale underlying the proposed research is that understanding the precise determinants of nucleoside analog sensitivity will help in the preselection of patients suitable for this type of therapy and the identification of clinical strategies to increase efficacy in poor responders. Specific aim 1 will define the role of NT-Cx combinations in nucleoside analog chemosensitivity. The working hypothesis is that gemcitabine cytotoxicity will be superior in cells expressing the concentrative NT 1 (hCNT1)-Cx32 combination, and that chemoresistance due to the loss of one component can be effectively compensated by the other. This hypothesis is based on the expression and permeation characteristics of NTs and Cxs in pancreatic tumors. Specific aim 2 will determine the regulators governing NT-Cx interplay to improve chemosensitivity. The working hypothesis is that the favorable manipulation of NT-Cx combinations to increase drug exposure can be achieved by manipulating cadherins or microRNAs. This hypothesis is based on the applicant's studies showing cadherin control of Cx assembly and microRNAs as putative regulators of hCNT1. Specific aim 3 will investigate the in vivo predictability of nucleoside analog response using NT-Cx combinations. The working hypothesis is that a composite index comprising of specific NTs and Cxs will better predict in vivo gemcitabine response than with the existing single index (i.e., equilibrative NT 1 (hENT1) alone). This hypothesis is based on the extrapolation of preliminary in vitro results obtained in cultured pancreatic cancer cells. The anticipated outcomes of this work are the delineation of NT-Cx interplay in nucleoside analog chemosensitivity, strategies to improve NT-Cx-mediated drug targeting, and evaluation of clinical measures for predicting chemotherapeutic responses in pancreatic cancer subtypes. This approach is innovative because it focuses on a comprehensive index for judging chemotherapeutic response. This contribution is significant because it will enable subsequent translational studies that are expected to improve treatment and survival outcomes in pancreatic cancer patients.

PROJECT SUMMARY The nucleoside reverse transcriptase inhibitors (NRTIs) have potent activities against HIV, but their therapeutic benefit in patients undergoing NRTI therapies is limited by significant adverse drug reactions (ADR), resulting in poor patient compliance and compromised drug efficacy. Our group has recently described an indispensable role of a lysosomal nucleoside transporter ENT3 in lysosomal homeostasis via deletion of ENT3 in mice. Intriguingly, ENT3 KO mice manifest clinical phenotypes closely resembling NRTI ADR. The overall objective of this application is to evaluate ENT3-loss driven lysosomal toxicity as a putative mechanism involved in the chronic adverse sequelae of NRTIs. The central hypothesis of this proposal is that NRTIs that do not interfere with ENT3-supported lysosomal homeostasis or the inclusion of lysosomal signaling agents will minimize the occurrence of NRTI ADR. Aim 1 will evaluate the strategies to avoid NRTI toxicity without compromising drug efficacy. Our working hypothesis for this aim is that disruption of interaction between ENT3 and NRTIs or the inclusion of pharmacological agonists of lysosomal-autophagy pathway will mitigate the onset and severity of NRTI ADR. The preliminary studies that demonstrate the involvement of the cell surface NRTI transporters (e.g., ENT1, CNT) and not the lysosomal ENT3 for NRTI efficacy, the misregulation of the AMPK and mTOR signaling axis in the Ent3-/- mice, and the functional rescue of multi-organ dysfunction in Ent3-/- mice using a pharmacological AMPK activator AICAR; all support this aim. Aim 2 will elucidate the mechanism(s) of occurrence of NRTI-specific ADR signs. Our working hypothesis for this aim is that NRTIs, when present at clinically relevant blood concentrations, will inhibit the ENT3-regulated adult stem cell functions resulting in disruption of tissue repair and regeneration. In addition, we hypothesize that NRTIs will differentially impact the ENT3 function in adult tissues to bring distinct inflammatory, metabolic and degenerative changes that coupled with stem cell alterations, will explain the clinically observed NRTI ADR signs. The preliminary studies that demonstrate the transport of many ADR-producing NRTIs by ENT3, the inhibition of lysosomal adenosine transport by NRTIs and the perturbation of lysosomal recycling of adenosine in Ent3-/- mice leading to adult stem cell exhaustion, tissue inflammation and degeneration, and breaches of mesodermal tissue integrity, which taken together supports this aim. The project will utilize biochemical and molecular approaches, novel ENT3 probes, newly generated ENT3 mouse models, metabolomics, tissue engineering, pharmacophore modeling, synthetic and screening procedures and PKPD to accomplish the goals. The successful completion of the project will provide new insights into the mechanisms of occurrence of NRTI ADR and may have translational benefit for optimizing treatments (such as long-term efficacy, adherence, tolerability, etc.) in patients undergoing NRTI therapies.