Our Current Trainees
The Cancer Biology Training Grant currently has funding for four predoctoral trainees and five postdoctoral trainees.Faculty preceptors are notified when a slot becomes available and a competitive application process is held. Applicants are evaluated and trainees selected by the training grant steering committee.
UV radiation is one of the most common sources of DNA damage. UV-induced lesions are repaired by nucleotide excision repair, but during S phase unrepaired lesions cause the replication machinery to stall. This, in turn, leads to an increase in mutations and chromosomal breakage, which ultimately become the driving force of genome rearrangements. Normal cells deal with lesions that impede the progression of replication by activating DNA damage tolerance pathways. Tolerance pathways are subdivided into two branches: error- prone translesion synthesis and error-free template switch. These pathways are triggered by the ubiquitination (Ub) of the replication clamp, proliferating cell nuclear antigen (PCNA), at lysine 164 (K164).
Mono-Ub of PCNA is catalyzed by Rad18 and activates translesion synthesis. At the expense of introducing mutations, this pathway signals for the replacement of the replicative polymerase with a low-fidelity polymerase allowing replication to proceed directly across the damaged template. This process reduces the risk of chromosomal rearrangements. Somatic inactivating mutations in these low-fidelity polymerases, as seen in xeroderma pigmentosum variant (XP-V) patients, have a strong predisposition to sunlight-induced skin cancer. A balance in translesion synthesis activity must be achieved as enhanced or diminished activity has been linked to increased tumorigenesis. An alternative pathway that is linked to poly-ubiquitination of PCNA, is template switching. Here, accurate damage bypass occurs through the use of the undamaged sister chromatid as a temporary template for replication. Ub is not the only post-translational modification (PTM) of PCNA. Sumoylation of PCNA also targets K164 and functions to inhibit unwanted homologous recombination (HR) during S phase. Currently, not much is known about these Ub and SUMO pathways in humans. I hypothesize that DNA damage tolerance in humans is completely dependent on PCNA ubiquitination, and mutations that block this PTM drive aberrant use of HR repair, leading to loss of heterozygosity (LOH).
My studies involve generating a human cell line that expresses mutant PCNA defective for ubiquitin and SUMO conjugation at K164. This will allow me to investigate what bypass mechanisms human cells utilize in the face of UV damage when tolerance pathways are abolished and how ablation of PCNA ubiquitination and sumoylation affects mutagenesis and LOH. Further, I will generate a human RAD18 knockout cell line that will be proficient in PCNA sumoylation but deficient in PCNA ubiquitination. This cell line will be utilized to investigate the role of SUMO-PCNA in DNA damage resistance and maintaining genome integrity. The molecular pathways identified will provide a basis for novel anti-cancer therapies targeted at cancer cell genome instability.
Lung cancer remains the most common cause of death from cancer in the US and there is a high need for improved therapeutic interventions. Epidermal growth factor receptor (EGFR) is a major target for the treatment of NSCLC, but only a subset of patients with EGFR mutations benefit from EGFR tyrosine kinase inhibitors (TKIs) such as erlotinib (an FDA-approved drug). Intrinsic lack of response to erlotinib and development of acquired resistance are driven by mechanisms not fully understood. This proposal seeks to develop and implement a combination therapy that will be beneficial to NSCLC patients with or without EGFR mutations. The goal is to combine erlotinib with a molecule that targets an important downstream mediator common to EGFR, and other TKIs. Signal transducer and activator of transcription (Stat3) is a key mediator of EGFR signaling and other tyrosine kinase receptors (RTKs). Targeting the EGFR/STAT3 axis in a combination therapy is an intriguing idea that could allow for a broader effect not solely dependent on EGFR mutation status.
This proposal seeks to determine whether the anti-tumor activity of the FDA approved TKI erlotinib can be enhanced through pharmacological inhibition of STAT3. A cyclic oligonucleotide molecule with a novel mechanism that acts as a STAT3 “decoy” will be tested in combination with erlotinib in NSCLC. The “decoy” mimics the DNA consensus sequence in the promoter region of STAT3-responsive genes, causing the binding of phosphorylated STAT3 dimers and preventing their nuclear translocation. Evidence shows that STAT3 is persistently activated in 22% - 65% of NSCLC and overexpression of phosphorylated STAT3 (pSTAT3, the active form of Stat3) is associated with poor prognosis. This project could therefore be of major significance because the potential of the cyclic Stat3 decoy (CS3D) to treat NSCLC is high (given that pStat3 is overexpressed in NSCLC), and the CS3D has not yet been tested for the treatment of NSCLC.
The proposed research is relevant to public health because it will help identify lung cancer patients appropriate for treatment with a novel therapeutic that targets Stat3, an important oncogenic molecule. This proposal also will determine whether a Stat3 inhibitor can be combined with erlotinib, an FDA-approved drug for the treatment of lung cancer.
Amanda Oliveira Salzwedel
Pancreatic cancer is the fourth leading cause of cancer-related deaths in the US, and the diagnosis of 46,420 new cases of the disease are expected in 2014. Because the disease is often diagnosed in its late stage, less than 20% of diagnosed patients are eligible for surgical removal of tumors. The lack of effective treatment combined with poor early detection results in a five-year overall survival of less than 10%.
Phase II clinical trials have shown that following adjuvant therapy, combination of systemic IFN-α (IFN) with fluorouracil (5-FU), and radiation resulted in a remarkable 35% increase in the five-year survival of patients. Despite these promising results, this therapy resulted in low intratumoral levels of IFN and high patient dropout because of IFN systemic toxicity. These drawbacks have hampered the full therapeutic potential of IFN therapy, especially because IFN has many potential benefits such as chemo-radio sensitization, immune-stimulation, and direct cytotoxic effect in cancer cells. Inspired by the outstanding results reported in clinical trials, my project aims to overcome the ¬drawbacks and improve the therapeutic outcome of IFN-combined chemoradiation in pancreatic-cancer treatment by using an oncolytic adenovirus expressing IFN (OAd-IFN).
Oncolytic adenovirus (OAd) can be a powerful tool to include in combination therapy. Besides stimulating anti-tumor immunity by releasing tumor-specific peptides during oncolysis, the virus can work to express therapeutic transgenes at the tumor site. Our laboratory has developed a fiber-modified replication competent oncolytic adenovirus with enhanced selectivity and tropism to pancreatic cancer cells. This virus (OAd-IFN) has its replication controlled by the Cox-2 promoter, which is overexpressed in pancreatic cancer. We believe that restricting IFN expression to the tumor site will significantly potentiate the therapeutic effects of the cytokine resulting in higher chemo-radio sensitization, anti-tumor response stimulation, and reduced systemic IFN toxicity.
My project is based on the development of in vitro assays and in vivo models to address the therapeutic potential of OAd-IFN combination therapy. We anticipate that the use of immunocompetent syngeneic models of pancreatic cancer, as well as xenograft models with patient tissues, will result in highly translatable data regarding therapy efficacy and toxicity. As the IFN combination therapy tested in clinical trials is one of the few protocols that shows promising results in the treatment of pancreatic cancer, we expect that the development of our OAd-IFN combination therapy will become an effective therapy against the disease with higher patient tolerability.
Katelyn Goodman Paz
Chronic graft versus host disease (cGVHD) is a life-threatening, long-term complication following hematopoietic stem cell transplant (HSCT), which is currently the only curative treatment for hematological malignancies. This disease has a complex pathogenesis involving the development of germinal centers, which produce antibody producing plasma cells that are involved in antibody deposition and tissue/organ damage. The germinal center reaction is aided by the specialized CD4+ T cell subset, T follicular helper (Tfh) cells, which are found to be increased in cGVHD. Specialized T regulatory cells, T follicular regulatory (Tfr) cells, are suppressors of the germinal center reaction. Our lab is interested in these two cell subsets, specifically investigating ways to decrease the activity/frequency of the germinal center facilitating Tfh or increasing the suppressing Tfr populations to decrease cGVHD.
The PI3K pathway is a key-signaling pathway necessary for T cell activation and differentiation. Changes in signaling through this pathway can result in downstream changes in T cell metabolism. T effector and Treg cell subsets have different energy needs and therefore different metabolic profiles. Effector T cells rely heavily on glycolysis to meet their high energy demands while Tregs favor fatty acid β-oxidation. The metabolism of T lymphocytes in cGVHD has not been investigated. My goal is to determine a metabolism profile of CD4+ lymphocytes in cGVHD as well as investigate the PI3K signaling pathway. GVHD remains a leading cause of mortality following cancer HCST, second only to relapse into primary disease. In a similar manner to effector T cells, cancer cells have increased reliance on glycolysis for their metabolic needs. Drugs aimed at manipulating metabolism are currently of great interest for cancer therapy, including compounds I am interested in repurposing for treatment of cGVHD to promote a Treg favorable metabolic profile. Therapeutic interventions aimed at targeting metabolism may have the capability for treating two leading causes of death following HCST, primary disease relapse and cGVHD.
Ryan Baxley, Ph.D.
The overall goal of the Bielinsky laboratory is to understand how human cells maintain a stable genome. Current evidence suggests that multiple pathways converge in a genome stability network that shields cells from chromosome breakage by either preventing DNA damage or facilitating repair. Double-strand breaks (DSBs) are a severe form of DNA damage, such that defects in DSB repair proteins predispose individuals to cancer. One crucial factor in the only error-free DSB repair pathway, homologous recombination (HR), is the breast cancer type 2 susceptibility (BRCA2) gene. Germ line mutations in BRCA2 cause hereditary breast and ovarian cancers. BRCA2 encodes a 3,418 amino acid protein with a centrally conserved domain of eight BRC repeats that bind to Rad51, a protein required to initiate HR. In addition, BRCA2 has been implicated in preserving the integrity of stalled replication forks and preventing fork collapse, which generates DSBs. While the exact role of BRCA2 at stalled forks remains unknown, the Bielinsky lab found that BRCA2 interacts with the essential replication factor minichromosome maintenance protein 10 (Mcm10). Mcm10 is part of the BRCA1-PALB2 (partner and localizer of BRCA2)-BRCA2 complex, in which Mcm10 and BRCA2 directly interact and do not require PALB2 or BRCA1 to mediate binding. Multiple genome-wide genetic screens have identified human Mcm10 as a major player in DNA damage prevention, consistent with its role as a scaffold protein that connects different fork components. Mcm10 may preserve genome integrity simply by stabilizing fork complexes. However, in the light of the interaction with BRCA2, another possibility is that Mcm10 has a direct role in recruiting BRCA2 to stalled replication forks to prevent fork collapse and chromosome breakage. Preliminary data suggests that an N-terminal coiled coil motif in Mcm10 serves as the binding surface for a subset of specific BRC repeats in BRCA2. My studies will characterize the interaction between Mcm10 and BRCA2 and investigate how cancer associated mutations in the BRCA2 BRC motifs affect this interaction. Further, I will generate a human cell line that expresses mutant Mcm10 defective for BRCA2 binding. This will allow me to investigate if Mcm10 is required for BRCA2 recruitment to stalled forks and whether this interaction plays a significant role in suppressing chromosome breakage.
Katherine Leehy, Ph.D.
Progesterone binds to PR (progesterone receptor) causing dimerization and localization in the nucleus where it binds to progesterone response elements (PREs) or is tethered to other transcription factors. In the normal mammary gland PR/progestins facilitates development by mediating the massive expansion of breast tissue that occurs during puberty and pregnancy to facilitate lactation. While PR is only present in 7-10% of normal breast tissue where it initiates both paracrine and autocrine signaling to enhance proliferation, it is expressed in up to 70% of breast cancers. Recent in vitro mechanistic studies by our group and others demonstrated that PR is capable of driving breast cancer progression in both the absence and presence of progestin. Activation of PR by kinase mediated phosphorylation events increases cellular proliferation and anchorage independent cell survival. Notably, breast tumors that express a phospho-PR gene signature are predicted to have a poor prognosis. Moreover, large-scale clinical trials examining hormone replacement therapies containing estrogen and progestin showed that progestins increased breast cancer risk and tumor aggressiveness in women relative to estrogens alone. Recent interest in the use of antiprogestins as therapeutics for steroid receptor positive breast cancers further emphasizes the importance of understanding the mechanisms by which PR promotes breast cancer and may prove critical to determining which patients will benefit most from targeting PR and PR associated pathways.
PR is an important mediator of breast cancer development and progression. In particular, PR is activated during cell cycling and interacts with many components of the cell cycle regulation machinery. Direct interactions between PR and cyclins A, D and E have been observed. There are a number of phosphorylation sites on PR that are induced by kinases cdk2, ck2 and MAPK that affect its transcriptional activity. In addition, many cancers upregulate MAP kinase activity and down regulate cell cycle inhibitors, which functions to further activate PR transcriptional programs. Therefore, we speculate that loss of checkpoint controls as early events in breast cancer may push PR into a hyperactive state that drives breast cancer progression.
p53 is the most commonly mutated gene in human cancer, and both loss of expression and mutations in p53 are associated with malignancy. Interestingly, mutations in p53 can have a pro-oncogenic effect as cells become resistant to apoptosis. Loss of active p53 is present in half of all breast cancers. These changes allow the cells to divide continuously without cell cycle inhibition and in the absence of normal growth factors. Interestingly, parity has been shown to protect against breast cancer in part by upregulation of p53 and p21 in luminal (SR +) mammary cells. Additionally, treatment with of anti-progestins prevents mammary tumor formation p53 null/BRAC1 mice, suggesting a potential link between DNA damage control, cell cycle regulation, and hormone action. Therefore, investigating the effects of loss/mutation of p53 on PR expression and regulation is an important step to help determine/optimize alternate breast cancer therapy.
We hypothesize that PR is a major driver of cancer progression by changes in phosphorylation status and thru interaction with cell cycle components. PR and p53, a master cell cycle regulator, act together to initiate tumor development and facilitate progression of breast cancer. My project will examine how changes in cell cycle regulation affect PR. 1) Determine the effect of p53 loss/mutation on regulation of PR isoforms. 2) Determine the effects of changes in p53 status on progestin-driven proliferation and pro-survival in ER+/PR+ breast cancer cell lines. 3) Determine how these changes effect tumors in mice with and without progestin treatment. . These experiments will determine the effects of p53 mutation in PR+ cells on tumor formation/progression and demonstrate the potential for use of anti-progestin treatments that may block hormone-dependent growth pathways in luminal (ER+/PR+) breast cancer.
Kristin Renkema, Ph.D.
Melanoma is the most aggressive skin cancer and affects 2% of the entire American population. Anti-tumor CD8 T cells can control melanoma, but developing a strong and suitable CD8 T cell response is inefficient by current methods. Here we study a novel approach for inducing tumor-specific CD8 T cells in a mouse model of melanoma.
CD8 T cells express unique T cell receptors (TCR) that recognize a peptide bound to MHC Class I (MHC-I) molecules. Tyrosine-related protein-2 (Trp2) – a melanocyte enzyme over-expressed in melanoma – encodes a peptide that can be seen by CD8 T cells. T cells that recognize such “self” peptides are typically killed in the thymus or held in check by regulatory mechanisms – however, Trp2 specific CD8 T cells arise in healthy mice (and humans), and when sufficiently activated can eliminate melanoma. However, current methods fail to induce a strong CD8 T cell response to the Trp2 peptide in mice, limiting potential for this approach in humans.
Recent studies suggest peptide-binding affinity for MHC-I is critical for generating strong anti-tumor CD8 T cell responses; we hypothesize that improving the stability of the Trp2-MHC-I interaction will improve priming of potent anti-melanoma CD8 T cells. “Single chain trimers” (SCT) covalently link a peptide to the MHC-I molecule: This structure is very stable yet effective primes antigen specific CD8 T cells which protect against pathogen infection. We propose using Trp2 SCT to prime anti-melanoma CD8 T cells and determining whether the resulting CD8 T cell response can control B16 melanoma cancer in mice. If Trp2 SCT improves anti-melanoma CD8 T cell responses, the approach could be developed for future application in humans. In addition, our observations may lead to future studies on functional tolerance of self-specific Trp2 specific CD8 T cells, regardless of Trp2 SCT priming efficacy.
Jamie Van Etten, Ph.D.
Prostate cancer is the most frequently diagnosed cancer in males and will account for an estimated 13% of new cancer cases this year. Nearly all prostate cancer related deaths occur in patients with metastatic disease, treatment of which relies upon androgen deprivation therapy (ADT) after failure of first-line, localized treatments that include radical prostatectomy and radiation. Such treatments serve to block androgen synthesis as well as activity of the androgen receptor (AR). AR, a steroid hormone receptor, functions as a master regulator and transcription factor in cells of prostatic origin, which depend on androgens for growth. Androgen blockade by ADT and reduction of the AR is initially successful in localized and metastatic prostate cancer; however, castration-resistant prostate cancer (CRPC) results within several years of ADT treatment due to alterations in the AR signaling axis and persistent transcriptional activity of the AR despite castrate levels of androgens. Direct alterations to the AR gene are common in CRPC and include AR amplification, gene rearrangements, and point mutations. AR rearrangements that lead to expression of truncated splice variants lacking the ligand binding domain, which can render the AR constitutively active, have been implicated in ADT resistance and progression of CRPC. For example, in the CRPC cell line 22Rv1, a 35 kb duplication occurs in the AR gene, which correlates with expression of the AR splice variant AR-V7. Expression of AR-V7 is associated with androgen-independent PCa cell growth and proliferation, and is resistant to ADT. Despite correction of the underlying gene rearrangement with TALENs, AR-V7 expression persists in 22Rv1 sublines. This suggests that multiple mechanisms are at play during the course of AR-V7 expression in CRPC. My work aims to characterize differences between pre-mRNA processing events of AR variants and full length AR, and will shed light on the mechanisms by which variants are expressed in CRPC. Importantly, these findings will provide insight into regulators of aberrant AR signaling in CRPC, which may ultimately lead to novel therapies and circumvent ADT resistance.
Kyle Williams, Ph.D.
Neurofibromatosis Type 1 (NF1) is a prevalent (~1:3,000 births) genetic disorder caused by mutations in the tumor suppressor gene NF1. Among other symptoms, NF1 predisposes individuals to formation of benign tumors (neurofibromas), which on their own can cause significant pain and mobility problems. Subsets of these tumors (~10% of patients) progress further into malignant peripheral nerve sheath tumors (MPNSTs) and are a leading cause of death among NF1 patients. Treatment options for both the benign neurofibromas and MPNSTs are extremely limited, mostly relying on surgical resection and broad-spectrum chemotherapy. There are however recent promising results (from our group and others) with drug therapies targeting signaling pathways implicated in disease progression. My current work is focused around both assessing effectiveness of modulating some of these signaling pathways and searching for novel pathways for therapeutic targeting.
Previous work from the Largaespada lab identified the Wnt/β-catenin signaling pathway as involved in NF1 related tumorigenesis. Moreover, we have shown in mouse models of NF1/PNST, human MPNST cell lines, and primary tissue samples that many tumors do in fact have increased β-catenin expression, which is often correlated with higher tumor grade.
I am working to define the extent of β-catenin de-regulation in MPNSTs and determine if modulation of β-catenin activity in vivo can impact tumor development in mouse models of the disease. In an effort to uncover new pathways for future therapeutic use, I am also conducting a variety of synthetic lethal genetic, and drug, screens that could reveal druggable targets relevant to NF1 tumor formation.