Predoctoral Trainees

Casey Dorr
Mansky lab
Phone: 624-5172
E-mail: dorr0022@umn.edu

Human T-cell leukemia virus type 1 (HTLV-1) and human immunodeficiency virus type 1 (HIV-1) are both associated with cancer. HTLV-1 is the etiological agent of adult T-cell leukemia/lymphoma (ATLL). Cancers such as Kaposi's sarcoma and Non-Hodgkin lymphoma are more prevalent in HIV-infected individuals. Not only does antiretroviral therapy hinder viral infection, but it also curbs the progression of these cancers. Antiviral drug resistance is a continual problem that mandates the ongoing search for new drug targets and new antiretroviral drugs. Casey seeks to discover novel antiretroviral compounds and to determine their mechanism of action. These studies are directed towards the translation to new clinical therapies. Casey is currently focused on 1) a group of compounds derived from Betula papyrifera (white birch of Minnesota and North America) and 2) a series of novel molecules based upon these plant-derived compounds. Casey has discovered that both groups of compounds possess antiretroviral activity. Preliminary observations suggest that the compounds function by inhibiting the maturation of the Gag structural polyprotein in a manner related to that of PA-457 (Bevirimat). The long-term goal of Casey's research is to develop new antiretroviral drugs that hold retroviral replication in check and decrease the prevalence of ATLL and AIDS-associated cancers.

Justin Haworth
Bielinsky lab
Phone: 624-0460
E-mail: hawo0007@umn.edu

In the US, cancer accounts for one in every four deaths, stressing the need for a better understanding of the basic processes important for the proliferation of cancer cells, such as DNA replication. A major focus of the Bielinsky laboratory is to fully understand the role of minichromosome maintenance (Mcm) 10 in DNA replication. Critical for Mcm10's function during replication is its DNA-binding activity. The crystal structure of X. laevis Mcm10 was recently solved. Based on this structural data, residues in Mcm10 were identified that, when mutated, significantly decrease the DNA-binding activity of X. laevis Mcm10 in vitro. Justin has shown that the corresponding mutations in S. cerevisiae Mcm10 lead to defects in DNA replication, providing evidence that DNA binding is important in vivo. In addition to its DNA-binding activity, Mcm10 regulates DNA polymerase-a (pol-a), the only enzyme capable of de novo DNA synthesis in yeast and human cells. In the absence of Mcm10, the catalytic subunit of pol-a, Cdc17 in yeast, is rapidly degraded. Degradation of Cdc17 is proteasome-dependent and, more specifically, requires the ubiquitin conjugating (E2) enzyme Ubc4 and the ubiquitin ligase (E3) Not4. The regulation of pol-a by Mcm10 is conserved in human cells, suggesting that this is a functionally important pathway in the cell. Justin is currently testing the functional relevance of this pathway by deregulating Ubc4- and Not4-dependent pol-a turnover in the presence of Mcm10 in yeast cells.

Jon Larson
Largaespada lab
Phone: 626-6971
E-mail: lars0327@umn.edu

Over 40,000 primary brain tumors are diagnosed and more than 10,000 brain tumor patients die each year in the United States. These tumors affect people of all ages and are categorized by their location and cell type. Gliomas are glial cell tumors common in adults, while primitive neuroectodermal tumors (PNET) are common in children and include medulloblastoma and supratentorial PNET subtypes. Current understanding of the heterogeneous genetic basis for these tumors has provided insight into their histogenesis and pathology. However, activation of distinct signaling pathways can cause histologically similar tumor types that have dissimilar clinical outcomes, suggesting yet unknown genetic diversity. To better understand the genetic origins of brain tumors, Jon is using brain-specific Sleeping Beauty (SB) transposon-mediated insertional mutagenesis in mice. This forward genetics method directs the identification of novel genes and genetic pathways required for tumor formation. The mutagenic transposon vector T2/Onc is capable of activating oncogenes or silencing tumor suppressor genes upon integration within or near such genes. T2/Onc integration sites are subsequently identified by transposon-specific sequences and genes that are recurrently mutated by T2/Onc integrations are strong candidate cancer genes. Jon is applying this method to identify candidate brain cancer genes that cooperate with alterations in PTEN, TRP53 and NF1 regulated pathways. Jon is also using SB transposon-mediated transgenesis in the mouse brain to mimic tumor formation in vivo and further characterize candidate brain cancer genes. Together, these experiments help to better understand the genetic causes of glioma and PNET development, and lead to potential molecular therapeutic applications in human disease.

Xazmin Lowman
Kelekar lab
Phone: 626-2358
E-mail: lowma006@umn.edu
Noxa is a pro-apoptotic, "BH3-only" member of the Bcl-2 family that interacts with the anti-apoptotic protein Mcl-1_L through its Bcl-2 homology-3 (BH3) domain at the mitochondrial outer membrane, promoting the release of apoptogenic molecules that elicit cell death. Human Noxa is constitutively expressed in many myeloid and lymphoid malignancies, suggesting that post-translational regulatory mechanisms keep its pro-apoptotic activity in check. Aside from conferring cell survival, Mcl-1_L has been shown to regulate the cell cycle. Preliminary studies show that Noxa is both a cytosolic and a nuclear protein in hematopoietic cells and that Noxa levels may affect cell division rates. Xazmin is testing the hypothesis that Noxa, via its interaction with nuclear Mcl-1_L, promotes cell cycle progression by counteracting the role of Mcl-1_L in delaying mitotic entry. A number of nuclear proteins involved in G2/M progression and in the DNA damage response are regulated via interactions with Mcl-1, and Noxa may compete with these nuclear binding partners to advance the cell cycle. Additionally, nuclear Noxa/Mcl-1_L interactions in the nucleus may be regulated via phosphorylation of a threonine residue near the Noxa BH3 domain. The presence of a kinase consensus motif, TQ, adjacent to the BH3 domain, suggests regulation by a Class IV nuclear PI3 kinase. Xazmin is determining how Noxa exerts an influence on the cell cycle; the role of the BH3 domain and phosphorylation of T²⁷ in regulating Noxa/Mcl-1 interactions in the nucleus and how these may promote the cell cycle. The interface between apoptosis and the cell cycle is essential to preserve homeostasis and genomic integrity. By binding Mcl-1 in the nucleus as well as on the mitochondria, Noxa may ensure that the damaged cell enters mitosis when it is most vulnerable to apoptotic pressures. Deregulation of apoptosis underlies many pathological states and a protein, such as Noxa, that participates in regulating both cell division and cell death can be a potential target for a variety of therapeutic applications.

Michelle Miller
Mayo lab
Phone: 625-8611
E-mail: mill0935@umn.edu

Angiogenesis is a critical process in tumor growth and progression. Without new blood vessel growth, tumors are restricted to a size smaller than about 2 mm in diameter. The Mayo lab has designed the b-sheet-forming peptide anginex, a 33-mer that is potently anti-angiogenic both in vitro and in vivo. Subsequently, shorter partial peptide mimetic analogs of anginex were made, which use dibenzofuran as a b-turn scaffold to induce the folding of the 13 remaining amino acid residues from anginex into a short b-sheet. One of these partial peptide mimetics, 6DBF7, has been shown to inhibit tumor growth in mice better than parent anginex. Recently, yeast two hybrid assays were used to identify galectin-1 as the target for anginex, and this was confirmed by BIAcore analysis and NMR spectroscopy. The complex formed between galectin-1 and anginex interferes with angiogenically-activated endothelial cell adhesion and migration on the extracellular matrix, a process that is required for angiogenesis. When this mechanism for efficient cell migration is attenuated, endothelial cells undergo apoptosis. Michelle is using a structure-based approach to design and optimize analogs of 6DBF7 that exhibit greater binding affinity to galectin-1, thereby increasing anti-angiogenic properties and in vivo exposure. NMR spectroscopy is being used to determine the solution structure of galectin-1 bound to 6DBF7. This structural work will elucidate the protein-ligand interactions that will guide the design modifications to parent 6DBF7. In addition, Michelle is defining how galectin-1 interacts with model glycans. This will test the efficacy of newly designed and synthesized analogs of 6DBF7, using endothelial cell proliferation and migration assays.

Mariangellys Rodriguez
Potter lab
Phone: 626-7207
E-mail: rodri242@umn.edu

Hormonal factors and gain of weight during adulthood increase the risk of breast cancer. Mariangellys is elucidating the role of metabolic signaling (e.g. insulin-like growth factor) in the metabolism of dietary lipids (i.e. omega-3 fatty acids) to epoxides that may promote breast cancer progression. Mariangellys has shown that IGF-1R activation by IGF1 induces cytochrome P450 1A1 (CYP1A1) in the T47D-CO breast cancer line. CYP1A1 selectively epoxygenates eicosapentaenoic acid (EPA), an essential omega-3 fatty acid commonly found in fish oil, into the lipid 17(18)-epoxyeicosatetraenoic acid (17,18-EpETE). This EPA epoxide induces growth of the estrogen receptor positive breast cancer line MCF7 and the estrogen receptor negative line MDA-MB-231, indicating a novel role for 17,18-EpETE in breast cancer. These results suggest that IGF-1R activation induces CYP1A1, which then metabolizes EPA into 17,18-EpETE, thereby promoting breast cancer proliferation and survival. Characterization of this novel signaling pathway will advance our understanding about how diet regulated IGF1 signaling and environmental factors such as dietary omega 3 fatty acids may interact to promote breast cancer progression. These studies may also lead to the development of preventative approaches for breast cancer that intervene in this pathway.

Postdoctoral Trainees

Gwen Dressing, Ph.D.
Lange lab
Phone: 624-1971
E-mail: dress088@umn.edu

Progesterone, acting via activation of progesterone receptors (PR), regulates rapid intracellular signaling cascades and both directly (acting as a transcription factor) and indirectly (phosphorylation of mitogen activated kinases) modifies gene expression. Progesterone and PR are required for normal mammary gland development and are also involved in the development and progression of breast cancer, yet they remain understudied relative to estrogen receptors (ER). Treatment options for ER/PR positive breast tumors include antiestrogens or aromatase inhibitors, which block the production of estrogen. Direct involvement of progesterone and PR in breast cancer may offer a valuable target for additional endocrine-based drug therapies. Progesterone, via PR, has been linked to cell cycle progression and normal breast development. Gwen is focusing on understanding the interactions between PR and cell cycle proteins including cyclins, cyclin dependent kinases and the cip/kip family of cell cycle inhibitors. The cross talk between PR and cell cycle proteins may represent therapeutic points of intervention to effectively halt breast cancer cell cycle progression and encourage cancer cell death.

Keir Fogarty, Ph.D.
Mansky lab
Phone: 624-5172
E-mail: fogarty@umn.edu

Human T-cell leukemia virus type 1 (HTLV-1) was the first human retrovirus to be discovered and is the etiological agent of adult T-cell leukemia/lymphoma (ATLL). About 5% of the 20 million HTLV-infected individuals worldwide develop ATLL. Much attention has focused on HTLV-1 pathogenesis and understanding how the virus transforms cells and induces ATLL. HTLV-1 is notorious for being very difficult to work with in cell culture, so studies directed at understanding mechanisms of replication have been limited. Basic studies of mechanisms behind how the Gag structural protein is involved in virus particle assembly and release are quite limited. In particular, very little is known regarding the early events of the virus assembly pathway (ie, Gag-Gag, Gag-RNA, and Gag-cellular protein interactions). Keir's research focuses on the application of the novel biophysical technology of fluorescence fluctuation spectroscopy (FFS) towards the study of Gag oligomerization, specifically the oligomeric state of cytosolic HTLV-1 Gag in living cells. Presently, FFS is the only available technology that has been applied to living cells that has single molecule resolution. Thus, FFS has promise for greatly facilitating mechanistic studies where single molecule quantitation is crucial. FFS's unique features are ideally suited for testing the hypothesis that distinct oligomerization states exist for Gag protein that can be influenced by viral RNA and cellular protein interactions.

Jason Mitchell, Ph.D.
Shimizu lab
Phone: 612-626-6713
E-mail: mitch490@umn.edu

The regulation of integrin mediated adhesion is critical for immunosurveillance against the development of cancer cells. The induction of T cell adhesion mechanisms allows for sustained contacts during cancer antigen recognition, leading to productive initiation of the immune response. T cell adhesion regulation is accomplished through signaling molecules binding to integrin cytoplasmic domains, which regulate cytoskeletal mediated integrin clustering and the induction of high affinity integrin conformations. The focus of Jason's research is to determine the molecular mechanisms regulating integrin activation in T cells in response to antigen mimetic signals. One such mechanism involves T cell receptor-mediated activation of the small GTPase Rap1. To determine the importance of Rap1 activation in integrin activation, Jason is using an adenovirus infection model to express mutant signaling proteins in T cell lines and mouse primary cells with genetic knockouts. These cells are then used in adhesion and soluble ligand binding assays to assess which molecular interactions are important to activate Rap1 and scaffold to integrin cytoplasimc domains to regulate T cell receptor-directed integrin activation.

Justin Taylor, Ph.D.
Jenkins lab
Phone: 626-1188
E-mail: tayl0611@umn.edu

The levels of protective antibody generated in response to vaccination are highly variable from person-to-person. For example, Hepatitis B-specific antibody levels following a full course of vaccination range from undetectable to 280,000 IU/L. The benchmark "protective" level of 10 IU/L is not achieved in 5-10% of cases, leaving these people susceptible to Hepatitis B infection, the direct cause of hepatocellular carcinoma. The mechanism underlying this tremendous variability is not understood, but is not linked to a global immunodeficiency or hyper-responsiveness. This lack of understanding can be attributed to a general lack of knowledge about the antigen-specific response that confers protection. Two antigen-specific cell types are necessary for the production of protective antibody: B cells, which produce antibody; and CD4+ helper T cells, which provide critical help to B cells. Justin hypothesizes that antibody responses are dictated by the frequency of naive antigen-specific B and/or CD4+ helper T cells. In order to test this hypothesis, the naïve frequencies of both antigen-specific lymphocyte populations involved in antibody production must be simultaneously assessed. The study of antigen-specific cells has historically been hampered by an inability to detect these rare cells, which are found at frequencies as low as 1 per 10 million leukocytes. Justin will be utilizing a recently developed enrichment strategy that has been adapted to allow for simultaneous analysis of both naïve antigen-specific CD4+ T cells and naïve antigen-specific B cell frequencies and their relation to the levels of antibody produced following a full course of hepatitis B vaccination. These studies will detect both of the antigen-specific cell types involved in antibody production prior to, and throughout the course of human vaccination. Moreover, these studies will reveal predictive markers of vaccine response.