T-CELL TREATMENTS FOR SOLID AND HEMATOLOGICAL TUMORS

Cell-based therapies have demonstrated potency and efficacy as cancer treatment modalities. T cells can be dichotomized by their T cell receptor (TCR) complexes where alpha/beta T cells (95% of T cells) and gamma/delta T cells (+T cells proliferated to clinically significant numbers and ROR1+ tumor cells were effectively targeted and killed by both ROR1-specific CAR+ T cell populations, although ROR1RCD137 were superior to ROR1RCD28 in clearance of leukemia xenografts in vivo. The second specific aim focused on generating bi-specific CD19-specific CAR+ gamma/delta T cells with polyclonal TCRgamma/delta repertoire on CD19+ artificial antigen presenting cells (aAPC). Enhanced cytolysis of CD19+ leukemia was observed by CAR+ gamma/delta T cells compared to CARneg gamma/delta T cells, and leukemia xenografts were significantly reduced compared to control mice in vivo. The third specific aim looked at the broad anti-tumor effects of polyclonal gamma/delta T cells expanded on aAPC without CAR+ T cells, where Vdelta1, Vdelta2, and Vdelta3 populations had naïve, effector memory, and central memory phenotypes and effector function strength in the following order: Vdelta2>Vdelta3>Vdelta1. Polyclonal gamma/delta T cells eliminated ovarian cancer xenografts in vivo and increased survival compared to control mice. Thus, translating these methodologies to clinical trials will provide cancer patients novel, safe, and effective options for their treatment.

DEFINING THE ROLE OF EGFR ACETYLATION IN CELLULAR PROCESSES: CLINICAL IMPLICATIONS

Epidermal growth factor receptor (EGFR) is a cell membrane tyrosine kinase receptor and plays a pivotal role in regulating cell growth, differentiation, cell cycle, and tumorigenesis. Deregulation of EGFR causes many diseases including cancers. Intensive investigation of EGFR alteration in human cancers has led to profound progress in developing drugs to target EGFR-mediated cancers. While exploring possible synergistic enhancement of therapeutic efficacy by combining EGFR tyrosine kinase inhibitors (TKI) with other anti-cancer agents, we observed that suberoylanilide hydroxamic acid (SAHA, a deacetylase inhibitor) enhanced TKI-induced cancer cell death, which further led us to question whether SAHA-mediated sensitization to TKI was associated with EGFR acetylation. What we know so far is that SAHA can inhibit class I and II histone deacetylases (HDACs), which could possibly preserve acetylation of underlying HDAC-targeted proteins including both histone and non-histone proteins. In addition, it has been reported that an HDAC inhibitor, TSA, enhanced EGFR phosphorylation in ovarian cancer cells. EGFR acetylation has also been reported to play a role in the regulation of EGFR endocytosis recently. These observations indicate that there might be an intrinsic correlation between acetylation and phosphorylation of EGFR. In other words, the interplay between EGFR acetylation and phosphorylation may contribute to HDAC inhibitors (HDACi)-augmented EGFR phosphorylation. In this investigation, we showed that CBP acetyltransferase acetylated EGFR in vivo. In response to EGF stimulation, CBP rapidly translocated from the nucleus to the cytoplasm. We also demonstrated protein-protein interaction between CBP and EGFR as well as the enhancement of EGFR acetylation by CBP. Moreover, EGFR acetylation enhanced EGFR tyrosine phosphorylation and augmented its association with Src kinase. Acetylation-deficient EGFR mutant (EGFR-K3R) significantly reduced the function and activity of EGFR. Furthermore, ectopic expression of EGFR-K3R mutant abrogated its ability to respond to EGF-induced cell proliferation, DNA synthesis, and anchorage-independent growth using cell-based assays and tumor growth in nude mice. In addition, we demonstrated that EGFR expression was associated with SAHA resistance in the treatment of cancer cells that overexpress EGFR. The knockdown of EGFR in MDA-MB-468 breast cancer cells could sensitize the cells to respond to SAHA. The overexpression of EGFR in SAHA-sensitive MDA-MB-453 breast cancer cells rendered the cells resistant to SAHA. Together, these findings suggest that EGFR plays an important role in SAHA resistance in breast carcinoma cells that we tested. The combination therapy of HDACi with TKI has been proposed for treating cancers with aberrant expression of EGFR. The evidence from pre-clinical or clinical trials demonstrated significant enhancement of therapeutic efficacy by using such a combination therapy. Our in vivo study also demonstrated that the combination of SAHA and TKI for the treatment of breast cancer significantly reduced tumor burden compared with either SAHA or TKI alone. The significance of our study elucidated another possible underlying molecular mechanism by which HDACi mediated sensitization to TKI. Our results unveiled a critical role of EGFR acetylation that regulates EGFR tyrosine phosphorylation and may further provide an experiment-based rationale for combinatorial targeted therapy.

Progenitor cells as remote "bioreactors": Neuroprotection via modulation of the systemic inflammatory response.

Acute central nervous system (CNS) injuries such as spinal cord injury, traumatic brain injury, autoimmune encephalomyelitis, and ischemic stroke are associated with significant morbidity, mortality, and health care costs worldwide. Preliminary research has shown potential neuroprotection associated with adult tissue derived stem/progenitor cell based therapies. While initial research indicated that engraftment and transdifferentiation into neural cells could explain the observed benefit, the exact mechanism remains controversial. A second hypothesis details localized stem/progenitor cell engraftment with alteration of the loco-regional milieu; however, the limited rate of cell engraftment makes this theory less likely. There is a growing amount of preclinical data supporting the idea that, after intravenous injection, stem/progenitor cells interact with immunologic cells located in organ systems distant to the CNS, thereby altering the systemic immunologic/inflammatory response. Such distant cell "bioreactors" could modulate the observed post-injury pro-inflammatory environment and lead to neuroprotection. In this review, we discuss the current literature detailing the above mechanisms of action for adult stem/progenitor cell based therapies in the CNS.

Neuroretina specification in mouse embryos requires Six3-mediated suppression of Wnt8b in the anterior neural plate.

Retinal degeneration causes vision impairment and blindness in humans. If one day we are to harness the potential of stem cell-based cell replacement therapies to treat these conditions, it is imperative that we better understand normal retina development. Currently, the genes and mechanisms that regulate the specification of the neuroretina during vertebrate eye development remain unknown. Here, we identify sine oculis-related homeobox 3 (Six3) as a crucial player in this process in mice. In Six3 conditional-mutant mouse embryos, specification of the neuroretina was abrogated, but that of the retinal pigmented epithelium was normal. Conditional deletion of Six3 did not affect the initial development of the optic vesicle but did arrest subsequent neuroretina specification. Ectopic rostral expansion of Wnt8b expression was the major response to Six3 deletion and the leading cause for the specific lack of neuroretina, as ectopic Wnt8b expression in transgenic embryos was sufficient to suppress neuroretina specification. Using chromatin immunoprecipitation assays, we identified Six3-responsive elements in the Wnt8b locus and demonstrated that Six3 directly repressed Wnt8b expression in vivo. Our findings provide a molecular framework to the program leading to neuroretina differentiation and may be relevant for the development of novel strategies aimed at characterizing and eventually treating different abnormalities in eye formation.

ROLES FOR BRAF KINASE ACTIVATING MUTATIONS IN MELANOMA: MICROENVIRONMENTAL IMMUNOSUPPRESSION

The BRAF oncogene demonstrates a characteristic mutation (V600E) in a significant fraction of cutaneous melanomas, leading to constitutive activation of the MAP kinase pathway. This genetic lesion endows tumor cells with proliferative and survival advantages, and metastatic melanoma patients treated with the BRAF(V600E)-specific inhibitor, Vemurafenib, have shown dramatic clinical responses. Here, I show that BRAF(V600E) induces transcription of the IL-1α and IL-1β genes in both melanocytes and melanoma cell lines and that this upregulation is specifically abrogated by targeted BRAF(V600E) inhibitors. Furthermore, treatment of melanoma tumor-associated fibroblasts (TAFs) with IL-1α/β significantly enhanced the ability of TAFs to suppress the proliferation and function of melanoma antigen-specific cytotoxic T cells. IL-1α/β treatment of TAFs upregulated multiple immunosuppressive factors, including COX-2 and the PD-1 ligands PD-L1 and PD-L2. Specific BRAF(V600E) inhibitors largely abrogated the ability of melanoma cells to confer T cell-suppressive properties on TAFs. These results support a model in which BRAF(V600E) promotes immune suppression in the melanoma tumor environment through an IL-1-mediated mechanism involving resident stromal fibroblasts. Based on these findings, combination therapies involving targeted BRAF inhibition and T cell-based immunotherapies are warranted.

Requirements for Staphylococcus aureus in vitro cellular internalization

Staphylcoccus aureus is a prokaryotic organism capable of causing numerous superficial and severe human infections. Adhesion of S. aureus to host tissues or cells is believed to be a crucial event in S. aureus infections. Subsequently, S. aureus can seed into the bloodstream resulting in metastasis of the infection. Several reports show that S. aureus can be internalized by non-professional phagocytes, a process which has been proposed to be important in S. aureus dissemination. An intracellular residence has also been proposed to provide safe harbor to reservoirs of dormant bacteria contributing to the persistence of infection. This dissertation describes an investigation into the molecular mechanisms of S. aureus internalization into both fibroblast and epithelial cells. Bacterial requirements for internalization were found to be limited to expression of proteins that bind the extracellular matrix protein fibronectin. A previously unknown fibronectin-binding region in the S. aureus fibronectin-binding protein A was discovered after showing competitive inhibition of S. aureus internalization. This novel fibronectin-binding activity is characterized. Internalization also required cell-based factors. The presence of fibronectin and cell surface receptors of the β1 integrin class, which are known to bind and internalize fibronectin, were found to be necessary for optimal internalization of S. aureus. These results led to the conclusion that fibronectin acts as a bridge between the bacterium and integrins on the host cells. The internalization process exhibits features characteristic of integrin-mediated cell migration on fibronectin-coated surfaces. Both processes involved an active form of the β1 integrin subunit and the protein tyrosine kinase Src. Finally, a Src inhibitor previously shown to be effective in reducing osteoporosis in an in vivo rat model is capable of greatly reducing S. aureus internalization. ^

GENETIC INTERACTIONS OF FACTORS THAT REGULATE ALTERNATIVE RNA SPLICING IN THE MALE GERM LINE OF DROSOPHILA

Alternative RNA splicing is a critical process that contributes variety to protein functions, and further controls cell differentiation and normal development. Although it is known that most eukaryotic genes produce multiple transcripts in which splice site selection is regulated, how RNA binding proteins cooperate to activate and repress specific splice sites is still poorly understood. In addition how the regulation of alternative splicing affects germ cell development is also not well known. In this study, Drosophila Transformer 2 (Tra2) was used as a model to explore both the mechanism of its repressive function on its own pre-mRNA splicing, and the effect of the splicing regulation on spermatogenesis in testis. Half-pint (Hfp), a protein known as splicing activator, was identified in an S2 cell-based RNAi screen as a co-repressor that functions in combination with Tra2 in the splicing repression of the M1 intron. Its repressive splicing function is found to be sequence specific and is dependent on both the weak 3’ splice site and an intronic splicing silencer within the M1 intron. In addition we found that in vivo, two forms of Hfp are expressed in a cell type specific manner. These alternative forms differ at their amino terminus affecting the presence of a region with four RS dipeptides. Using assays in Drosophila S2 cells, we determined that the alternative N terminal domain is necessary in repression. This difference is probably due to differential localization of the two isoforms in the nucleus and cytoplasm. Our in vivo studies show that both Hfp and Tra2 are required for normal spermatogenesis and cooperate in repression of M1 splicing in spermatocytes. But interestingly, Tra2 and Hfp antagonize each other’s function in regulating germline specific alternative splicing of Taf1 (TBP associated factor 1). Genetic and cytological studies showed that mutants of Hfp and Taf1 both cause similar defects in meiosis and spermatogenesis. These results suggest Hfp regulates normal spermatogenesis partially through the regulation of taf1 splicing. These observations indicate that Hfp regulates tra2 and taf1 activity and play an important role in germ cell differentiation of male flies.

Use of Echogenic Immunoliposomes for Delivery of both Drug and Stem Cells for Inhibition of Atheroma Progression

Use of Echogenic Immunoliposomes for Delivery of both Drug and Stem Cells for Inhibition of Atheroma Progression By Ali K. Naji B.S. Advisor: Dr. Melvin E. Klegerman PhD Background and significance: Echogenic liposomes can be used as drug and cell delivery vehicles that reduce atheroma progression. Vascular endothelial growth factor (VEGF) is a signal protein that induces vasculogenesis and angiogenesis. VEGF functionally induces migration and proliferation of endothelial cells and increases intracellular vascular permeability. VEGF activates angiogenic transduction factors through VEGF tyrosine kinase domains in high-affinity receptors of endothelial cells. Bevacizumab is a humanized monoclonal antibody specific for VEGF-A which was developed as an anti-tumor agent. Often, anti-VEGF agents result in regression of existing microvessels, inhibiting tumor growth and possibly causing tumor shrinkage with time. During atheroma progression neovasculation in the arterial adventitia is mediated by VEGF. Therefore, bevacizumab may be effective in inhibiting atheroma progression. Stem cells show an ability to inhibit atheroma progression. We have previously demonstrated that monocyte derived CD-34+ stem cells that can be delivered to atheroma by bifunctional-ELIP ( BF-ELIP) targeted to Intercellular Adhesion Molecule-1 (ICAM-1) and CD-34. Adhesion molecules such as ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1) are expressed by endothelial cells under inflammatory conditions. Ultrasound enhanced liposomal targeting provides a method for stem cell delivery into atheroma and encapsulated drug release. This project is designed to examine the ability of echogenic liposomes to deliver bevacizumab and stem cells to inhibit atheroma progression and neovasculation with and without ultrasound in vitro and optimize the ultrasound parameters for delivery of bevacizumab and stem cells to atheroma. V Hypotheses: Previous studies showed that endothelial cell VEGF expression may relate to atherosclerosis progression and atheroma formation in the cardiovascular system. Bevacizumab-loaded ELIP will inhibit endothelial cell VEGF expression in vitro. Bevacizumab activity can be enhanced by pulsed Doppler ultrasound treatment of BEV-ELIP. I will also test the hypothesis that the transwell culture system can serve as an in vitro model for study of US-enhanced targeted delivery of stem cells to atheroma. Monocyte preparations will serve as a source of CD34+ stem cells. Specific Aims: Induce VEGF expression using PKA and PKC activation factors to endothelial cell cultures and use western blot and ELISA techniques to detect the expressed VEGF.  Characterize the relationship between endothelial cell proliferation and VEGF expression to develop a specific EC culture based system to demonstrate BEV-ELIP activity as an anti-VEGF agent. Design a cell-based assay for in vitro assessment of ultrasound-enhanced bevacizumab release from echogenic liposomes.  Demonstrate ultrasound delivery enhancement of stem cells by applying different types of liposomes on transwell EC culture using fluorescently labeled monocytes and detect the effect on migration and attachment rate of these echogenic liposomes with and without ultrasound in vitro.

The Role of Cell Sterilization in Population Based Studies of Radiogenic Second Cancers Following Radiation Therapy

Advances in radiotherapy have generated increased interest in comparative studies of treatment techniques and their effectiveness. In this respect, pediatric patients are of specific interest because of their sensitivity to radiation induced second cancers. However, due to the rarity of childhood cancers and the long latency of second cancers, large sample sizes are unavailable for the epidemiological study of contemporary radiotherapy treatments. Additionally, when specific treatments are considered, such as proton therapy, sample sizes are further reduced due to the rareness of such treatments. We propose a method to improve statistical power in micro clinical trials. Specifically, we use a more biologically relevant quantity, cancer equivalent dose (DCE), to estimate risk instead of mean absorbed dose (DMA). Our objective was to demonstrate that when DCE is used fewer subjects are needed for clinical trials. Thus, we compared the impact of DCE vs. DMA on sample size in a virtual clinical trial that estimated risk for second cancer (SC) in the thyroid following craniospinal irradiation (CSI) of pediatric patients using protons vs. photons. Dose reconstruction, risk models, and statistical analysis were used to evaluate SC risk from therapeutic and stray radiation from CSI for 18 patients. Absorbed dose was calculated in two ways: with (1) traditional DMA and (2) with DCE. DCE and DMA values were used to estimate relative risk of SC incidence (RRCE and RRMA, respectively) after proton vs. photon CSI. Ratios of RR for proton vs. photon CSI (RRRCE and RRRMA) were then used in comparative estimations of sample size to determine the minimal number of patients needed to maintain 80% statistical power when using DCE vs. DMA. For all patients, we found that protons substantially reduced the risk of developing a second thyroid cancer when compared to photon therapy. Mean RRR values were 0.052±0.014 and 0.087±0.021 for RRRMA and RRRCE, respectively. However, we did not find that use of DCE reduced the number of patents needed for acceptable statistical power (i.e, 80%). In fact, when considerations were made for RRR values that met equipoise requirements and the need for descriptive statistics, the minimum number of patients needed for a micro-clinical trial increased from 17 using DMA to 37 using DCE. Subsequent analyses revealed that for our sample, the most influential factor in determining variations in sample size was the experimental standard deviation of estimates for RRR across the patient sample. Additionally, because the relative uncertainty in dose from proton CSI was so much larger (on the order of 2000 times larger) than the other uncertainty terms, it dominated the uncertainty in RRR. Thus, we found that use of corrections for cell sterilization, in the form of DCE, may be an important and underappreciated consideration in the design of clinical trials and radio-epidemiological studies. In addition, the accurate application of cell sterilization to thyroid dose was sensitive to variations in absorbed dose, especially for proton CSI, which may stem from errors in patient positioning, range calculation, and other aspects of treatment planning and delivery.

Current United States stem cell policy considerations and a recommendation for an alternative policy based on guidelines from existing international policies

Embryonic stem cell research is a widely debated topic in modern politics and religion. Differing views on the fetal rights conflict with the rights of an embryo. Those who believe an embryo has the same human qualities as a fetus accordingly believe embryonic stem cell research is unethical because it destroys a potential human life. However, scientists advocate the embryo does not have human qualities and should be used for valuable research in the stem cell field. Stem cell research may lead to vast developments in medical treatments, including cancer and brain conditions and injuries that are currently incurable. ^ The current stem cell policy introduced by President Bush in 2001 in an attempt to balance the moral issues with the need for scientific research has broad negative implications on the furthering of stem cell research. There is a limited diversity of available stem cell lines, there may be constitutional issues, there is an increasing disparity between the public and private research spheres, and the U.S. is struggling to maintain its scientific community. The U.S. must develop a new stem cell research policy that will balance the interest of science and public health with the moral issues that concern the public. ^ The United Kingdom allows researchers great liberty in conducting research, permitting the creation of embryos for the sole purpose of research, while Germany is equally conservative in their laws, as their policies support the philosophy that all embryos deserve the protection of full life. The United States should adopt a policy that takes the "middle ground" approach and permit research on excess embryos created for IVF purposes, rather than simply discarding those potentially valuable research tools. ^

Application of cell line based genomic predictors to predict response to targeted therapies in breast cancer

Cancer cell lines can be treated with a drug and the molecular comparison of responders and non-responders may yield potential predictors that could be tested in the clinic. It is a bioinformatics challenge to apply the cell line-derived multivariable response predictors to patients who respond to therapy. Using the gene expression data from 23 breast cancer cell lines, I developed three predictors of dasatinib sensitivity by selecting differentially expressed genes and applying different classification algorithms. The performance of these predictors on independent cell lines with known dasatinib response was tested. The predictor based on weighted voting method has the best overall performance. It correctly predicted dasatinib sensitivity in 11 out of 12 (92%) breast and 17 out of 23 (74%) lung cancer cell lines. These predictors were then applied to the gene expression data from 133 breast cancer patients in an attempt to predict how the patients might respond to dasatinib therapy. Two predictors identified 13 patients in common to be dasatinib sensitive. Sixty two percent of these cases are triple negative (ER-negative, HER2-negative and PR-negative) and 76% are double negative. The result is consistent with the findings from other studies, which identified a target population for dasatinib treatment to be triple negative or basal breast cancer subtype. In conclusion, we think that the cell line-derived dasatinib classifiers can be applied to the human patients. ^

A movement from traditional malarial chemoprophylaxis to the use of intermittent preventive treatment as a method of protection for children diagnosed with sickle-cell disease: A proposal for consideration based on a systematic review of the literature

Approximately 200,000 African children are born with sickle-cell anemia each year. Research has shown that individuals with hemoglobin disorders, particularly sickle-cell anemia, have increased susceptibility to contracting malaria. Currently it is recommended that patients diagnosed with sickle-cell anemia undergo malaria chemoprophylaxis in order to decrease their chances of malarial infection. However, studies have shown that routine administration of these drugs increases the risk of drug resistance and could possibly impair the development of naturally acquired immunity. Clinical trials have shown intermittent preventive treatment (IPT) to be an effective method of protection against malaria. The objective of this report was to review previously conducted clinical trials that study the effects of intermittent preventive treatment on malaria and anemia in infants and children. Based on the review, implications for its appropriateness as a protective measure against malaria for infants and children diagnosed with sickle-cell disease were provided.^ The 18 studies reviewed were randomized controlled trials that focused on IPT’s effect on malaria (7 studies), anemia (1 study), or both (8 studies). In addition to these 16, one study looks at IPT’s effect on molecular resistance to malaria, and another study is a follow-up to a study in order to review IPT’s potential to cause a rebound effect. The 18 th study in this review specifically looks at IPT’s protective efficacy in children with SCA. The studies in this report were restricted to randomized controlled trials that have been performed from 2000 to 2010. Reports on anemia were included to illustrate possible added benefits of the use of IPT specific to burdens associated with SCA other than malaria susceptibility. The outcomes of these studies address several issues of concern involving the administration of IPT: protective efficacy (in reference to age, seasonal versus perennial malaria regions, and overall effectiveness against malaria and anemia), drug resistance, drug rebound effect, drug side-effects, and long-term effects. Overall, these showed that IPT has a significant level of protective efficacy against malaria and/or anemia in children. More specifically, the IPT study evaluating children diagnosed with sickle-cell anemia proved IPT to be a more effective method of protection than traditional chemoprophylaxis. ^