Lawrence B. Gardner - US grants

DESCRIPTION (Applicant's Description): This proposal is designed to provide the principal investigator, Lawrence Gardner, with the necessary scientific experience to allow for a successful transition to an independent clinician scientist. Dr. Gardner has been involved in research since high school. He graduated from the Yale University School of Medicine, after an additional year dedicated to research. Following the completion of medical school, Dr. Gardner pursued a residency in internal medicine, and then specialty training in hematology/oncology, all at The Johns Hopkins Hospital. Over the past two years Dr. Gardner has devoted over 90 percent effort to basic research in the laboratory of Dr. Chi Dang, a professor of Medicine and Oncology. During this time he has studied molecular responses to hypoxia in normal cells, and has become proficient in many standard molecular and cellular biology techniques. Drs. Gardner and Dang have made several novel observations during these studies. They have delineated the mechanism responsible for hypoxia-induced G1 cell cycle arrest, a fundamental response in normal cells. They have also identified several neoplastic cell lines that do not undergo a G1 arrest in hypoxia. In July 2000, Dr. Gardner will become an instructor in the Department of Medicine at the Johns Hopkins Medical Institution. Clinical duties, including teaching, clinic twice a month, and a month of consult attending, will occupy 20 percent of his time. His remaining effort will be dedicated to obtaining the training necessary for a gradual transition to independent status. The training proposed includes didactics, attendance at national and specialty meetings, and specific feedback from a formal committee. The research plan detailed in this application builds on the observations and skills obtained by Dr. Gardner in Dr. Dang's lab over the past two years. The investigators have determined that the key regulator of hypoxia-induced G1 arrest in normal cells is the transcriptional induction of the cyclin- dependent kinase inhibitor p27. The research project will first study the molecular signals by which normal cells induce p27 in response to hypoxia. They will then determine why some neoplastic cells do not arrest in hypoxia. Finally, using neoplastic cells that do not arrest in hypoxia, as well as methods they have developed to abrogate hypoxia-induced G1 arrest, they will determine the contribution of this G1 arrest in hypoxia's effect on genomic instability and radioprotection.

[unreadable] DESCRIPTION (provided by applicant): The differentiating erythroid cell, particularly in thalassemia, is exposed to severe cellular stresses including oxidative and hypoxic stress. Both reactive oxygen species and hypoxia lead to the phosphorylation of eIF2a, a translation factor vital for the initiation of protein synthesis, and genetic studies have demonstrated that the regulation of this phosphorylation plays an important role in erythropoiesis. We have determined that the inhibition of nonsense mediated RNA decay in hypoxic cells is dependent on eIF2a phosphorylation. Nonsense mediated RNA decay (NMD) is a multi-step pathway responsible for the degradation of 30% of all human mutated mRNAs, as well as up to 10% of normal cellular mRNAs. But because NMD has not been thought of as a regulated pathway, its activity in normal or thalassemic erythroid cells has not been closely studied, and its significance in erythropoiesis has not been determined. We hypothesize that NMD is inhibited by cellular hypoxia and reactive oxygen species in differentiating thalassemic erythroid cells. This inhibition, mediated by eIF2a phosphorylation, alters gene expression, augments the stress response, and improves the survival of these cells. We propose to 1) Determine if eIF2a is phosphorylated by reactive oxygen species in human thalassemic erythroid cells, and whether this phosphorylation is sufficient for the inhibition of NMD. Briefly, we will amplify and differentiate erythroid cells from peripheral blood, and also examine bone marrow biopsies, from control and thalassemic volunteers. These samples will be assessed for eIF2a phosphorylation status, induction of stress responsive genes, and reactive oxygen species. We will then use a variety of stresses and engineered cell lines to determine if eIF2a phosphorylation is sufficient to inhibit NMD. 2) Determine the biological significance of eIF2a phosphorylation and NMD inhibition in normal and thalassemic erythroid cells. Briefly, we will determine if eIF2a phosphorylation by reactive oxygen species or cellular hypoxia correlates with apoptosis in differentiating erythroid cells. Using expression arrays we will determine the mRNAs that are stabilized when NMD is genetically deleted in erythroid cells and/or when NMD is inhibited by hypoxia in these cells. NMD targets alternatively spliced mRNAs, and by using expression arrays that identify mRNA splice variants we will determine if these variants are enriched in hypoxic and NMD repressed erythroid cells. 3) Determine the mechanism of hypoxia-induced inhibition of nonsense mediated RNA decay by pursuing our working model, based on preliminary data, that eIF2a phosphorylation sequesters NMD targeted mRNAs to cytoplasmic stress granules, where these mRNAs cannot be degraded. Using confocal microscopy we will determine the localization of enzymes important for NMD, as well as NMD degraded mRNAs in stressed cells where eIF2a is phosphorylated. PUBLIC HEALTH RELEVANCE: The normal growth and development of red blood cells is vital to sustain health. Impairment of red cell development, in diseases such as thalassemia, may lead to a severe anemia and a dependence on transfusions. We have identified a novel mechanism that controls the stability of messenger RNA, the carrier of genetic information from DNA to proteins in a cell. Because this mechanism is regulated by cellular stresses found in the normal, and particularly thalassemic, erythroid cells, we will determine if erythroid cell survival is affected by this novel form of gene regulation, and identify the genes that are regulated by this mechanism. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This is a renewal application for a multidisciplinary hematology training program at New York University School of Medicine which has been in existence for over 42 years. The program is designed to provide two- year research opportunities for 5-6 post-doctoral fellows under the guidance of a faculty of 13 mentors. The training faculty consists of academic hematologists and hematopathologists with active laboratories applying basic science methods to the study of hematologic disorders as well as mentors of the basic science departments of NYU School of Medicine. There is abundant interaction among the faculty members within the program as well as with other scientists at this institution. The clinical research at this institution has been recognized by the NIH in its designation as a Comprehensive Cancer Center and an AIDS Center. The program director's research spans both areas. New faculty members have strengthened the basic science orientation of the program. The faculty works with this framework to train physician-scientists for future work in the academic community. A common theme of all the projects is their orientation to human disease. These aims will be accomplished by training promising candidates in the design, execution and evaluation of experiments so that they may join the ranks of academic physician-scientists in basic as well as translational hematology research. RELEVANCE (See instructions): As the Nation's population is aging, it is becomes even more necessary that we train physician-scientists in hematologic disorders in basic and translational research. The training program at NYU School of Medicine will continue that mission.

DESCRIPTION (provided by applicant): The differentiating erythroid cell, particularly in thalassemia, is exposed to severe cellular stresses including oxidative and hypoxic stress. Both reactive oxygen species and hypoxia lead to the phosphorylation of eIF2a, a translation factor vital for the initiation of protein synthesis, and genetic studies have demonstrated that the regulation of this phosphorylation plays an important role in erythropoiesis. We have determined that the inhibition of nonsense mediated RNA decay in hypoxic cells is dependent on eIF2a phosphorylation. Nonsense mediated RNA decay (NMD) is a multi-step pathway responsible for the degradation of 30% of all human mutated mRNAs, as well as up to 10% of normal cellular mRNAs. But because NMD has not been thought of as a regulated pathway, its activity in normal or thalassemic erythroid cells has not been closely studied, and its significance in erythropoiesis has not been determined. We hypothesize that NMD is inhibited by cellular hypoxia and reactive oxygen species in differentiating thalassemic erythroid cells. This inhibition, mediated by eIF2a phosphorylation, alters gene expression, augments the stress response, and improves the survival of these cells. We propose to 1) Determine if eIF2a is phosphorylated by reactive oxygen species in human thalassemic erythroid cells, and whether this phosphorylation is sufficient for the inhibition of NMD. Briefly, we will amplify and differentiate erythroid cells from peripheral blood, and also examine bone marrow biopsies, from control and thalassemic volunteers. These samples will be assessed for eIF2a phosphorylation status, induction of stress responsive genes, and reactive oxygen species. We will then use a variety of stresses and engineered cell lines to determine if eIF2a phosphorylation is sufficient to inhibit NMD. 2) Determine the biological significance of eIF2a phosphorylation and NMD inhibition in normal and thalassemic erythroid cells. Briefly, we will determine if eIF2a phosphorylation by reactive oxygen species or cellular hypoxia correlates with apoptosis in differentiating erythroid cells. Using expression arrays we will determine the mRNAs that are stabilized when NMD is genetically deleted in erythroid cells and/or when NMD is inhibited by hypoxia in these cells. NMD targets alternatively spliced mRNAs, and by using expression arrays that identify mRNA splice variants we will determine if these variants are enriched in hypoxic and NMD repressed erythroid cells. 3) Determine the mechanism of hypoxia-induced inhibition of nonsense mediated RNA decay by pursuing our working model, based on preliminary data, that eIF2a phosphorylation sequesters NMD targeted mRNAs to cytoplasmic stress granules, where these mRNAs cannot be degraded. Using confocal microscopy we will determine the localization of enzymes important for NMD, as well as NMD degraded mRNAs in stressed cells where eIF2a is phosphorylated. PUBLIC HEALTH RELEVANCE: The normal growth and development of red blood cells is vital to sustain health. Impairment of red cell development, in diseases such as thalassemia, may lead to a severe anemia and a dependence on transfusions. We have identified a novel mechanism that controls the stability of messenger RNA, the carrier of genetic information from DNA to proteins in a cell. Because this mechanism is regulated by cellular stresses found in the normal, and particularly thalassemic, erythroid cells, we will determine if erythroid cell survival is affected by this novel form of gene regulation, and identify the genes that are regulated by this mechanism.

The NYU Langone Medical Center experienced an unprecedented storm on October 29th, 2012, causing damage that deeply impacted our patient care, research, and educational facilities. NYULMC?s emergency power system was designed and built according to all safety codes to withstand a surge higher than the highest flood level for New York City in the past century. Superstorm Sandy obviously exceeded those levels. Although we safely evacuated over 300 patients in the midst of the storm, the extensive damage to its mechanical, electrical, and plumbing systems, required the temporary closure of our main campus. We are now in a post-disaster recovery period addressing the damage caused by the storm and assessing mitigation needs. NYU contains three basic science buildings, the Skirball Institute, the Smilow Research Center, and the Medical Science Building (MSB). The Gardner lab is located in the Smilow Research Center. However because Core laboratories, required for projects related to the grant, were located in MSB, the lab was also impacted by damage to MSB. The below-ground levels of Smilow Research Center and MSB, which included two vivarium facilities, were significantly damaged. The animal vivarium of Smilow was deemed completely Research Strategy Page 20 Principal Investigator/Program Director (Last, first, middle): Gardner, Lawrence, B unrecoverable. There was a complete loss of power, backup power, water and heat to these buildings for almost two weeks. Through the concerted efforts of literally hundreds of engineers and staff, the Smilow Research Center was re-opened by early January. However MSB sustained the most extensive damage, and repairs are ongoing. The restoration of MSB will result in a phased return of operations beginning this summer. For almost a month after the storm, the Smilow building was closed, elevators were not operational and gasoline fumes throughout the building limited the time we were able to safely visit the laboratory. However, we were able to make intermittent trips, carrying liquid nitrogen canisters to the 12th floor to try and salvage precious samples. All reagents that had been kept at -80 and -20 were lost. Most of our reagents at 4 degrees were lost, though some antibodies and restriction enzymes were still functional. Lost items include some commercial antibodies, serum, kits (e.g. glutathione, ATP, LDH), chemicals (e.g. reagents synthesized by Chembridge for our NMD inhibition experiments, detailed below), and spent several months generating, and protein lysates and RNA that were awaiting analyses. In addition, our laboratory is highly dependent on retroviruses and lentiviruses we generate. Generation of these viruses take several weeks and, depending on number of viruses placed in a cell and selection mechanism (e.g. FACS sorting or antibiotics), it can take several months to regenerate specific cell lines. Unfortunately, our manipulated growing cell lines, some of which we had were in the midst of generating over the previous several months with multiple lentiviruses were lost. Work requiring the Core laboratories (including FACS sorting, expression arrays, and mass spectroscopy) were significantly delayed by ~ 4 months. In addition, shipping and receiving were delayed, and critical experiments (detailed below) requiring radiation could not be completed While the laboratory was off limits, the PI was able to re-locate to an off-site office operated by the NYU Cancer Institute, where electricity had been restored and there was access to a computer. During this period the PI was able to continue with some work related to the grant, albeit at decreased productivity. Specifically, the PI analyzed data, completed drafts of four manuscripts related to the grant (one which is now in press, one has been accepted at MCB, one which is in Review, and one which is about to be submitted). Communication with lab personnel was initially hampered, as all email servers were non-functional for over a week after the storm. Eventually lab personnel supported by the grant assisted in bringing liquid nitrogen to the floor, but were not able to participate in experiments. Work initiated as part of a collaboration with the Rivella lab at Cornell University, which was unaffected by the storm, was continued. However, aspects of the project which required the ongoing generation of reagents from the Gardner lab could not be completed. Offers of assistance were received from others in the NYC Hematology community that were unaffected by the storm, including Jim Bieker from Mount Sinai. After the laboratory reopened in early January, all members of the laboratory (supported and not-supported by the grant) worked tireless to restore the laboratory, including testing and discarding destroyed reagents, sterilizing tissue culture hoods and incubators, re-ordering reagents, and regenerating cell lines which were destroyed. Experiments that were interrupted by the laboratory were re-initiated. It is estimated that in addition to the cost of reagents, approximately 2 month of ongoing studies were lost when the storm hit, the lab was not habitable for 1.0 months, and it required approximately 3 months to restore the lab (environment, reorder and generate commercial reagents, and re-generate cell lines), totaling 6 months. In addition, it took several more months to restore full efficiency. We still consider ourselves relatively lucky, since with the exception of one new chemical that had to be re-synthesized, our ruined reagents could be purchased commercially and/or regenerated within a few months, and we did not have long-term ongoing experiments (including mice who were generated) that perished in the storm. As of March, the laboratory is fully operational, at full productivity, and making good progress of continuing the experiments delineated in our initial proposal and revised in our yearly progress reports. Our productivity over the last four years of the grant is strong, resulting in 12 publications (9 as corresponding author), and have one article under review.

Nonsense mediated RNA decay (NMD) is a mechanism to rapidly degrade select mRNAs. Recent studies have found that the UPF1 gene, required for NMD, is strikingly mutated and inactivated in >80% of adenosquamous pancreatic cancer (ASPC), a particularly aggressive form of pancreatic cancer. We have determined that UPF1 mutations in pancreatic cancer result in decreased UPF1 expression. Other mutations recently reported to inactivate NMD are found in pancreatic ductal adenocarcinoma, and we have reported that many of the stresses commonly found in pancreatic cancer repress NMD activity. NMD inhibition promotes the growth of transformed cells in soft agar, subcutaneous explants, and in an orthotopic pancreatic transplant model. Our overall goal is to better understand how NMD inhibition augments tumor growth and explore how we can exploit NMD inhibition for therapeutic gain in pancreatic cancer. RNA stability screens, RNAseq, and metabolomics screens have identified Notch signaling and Glycolysis as NMD regulated pathways. Both Notch signaling and Glycolysis play an important role in pancreatic cancer in general, and recent pancreatic cancer molecular classification studies indicate that these two pathways are particularly active in ASPC, where NMD is typically genetically inactivated. Importantly these pathways can also be targeted. In Aim 1 we will identify the mechanism and significance of NMD inhibition on Notch activation in pancreatic cancer. Based on our preliminary data we hypothesize that reduced NMD inhibition expression stabilizes Notch ligands and receptors, and the activation of Notch signaling represses e-cadherin expression to play a key role in metastases and chemo-resistance. However we also hypothesize that NMD inhibited pancreatic cancers will be particularly susceptible to Notch inhibitors. In Aim 2 we will determine how reduced NMD inhibition regulates metabolic pathways and exploit this for therapeutic gain. Based on our preliminary data, we hypothesize that NMD inhibition stabilizes alternatively spliced transcripts encoding members of the mitochondrial respiration system, and this activates glycolysis and the pentose phosphate shunt. The activation of these pathways should render tumors with UPF1 mutations more sensitive to clinically available mitochondrial inhibitors and other metabolic inhibitors, as indicated by preliminary focused shRNA synthetic lethality screens. For both Aims we will use a variety of in vitro cell biology, biochemical, and molecular techniques. We will validate our in vitro findings with unique ASPC tissue arrays, as well as a novel genetically engineered mouse in which we can temporally down-regulate UPF1 expression in pancreas, and can thus faithfully model the consequences of UPF1 mutations found in ASPC.