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The Role of Fos and Junb in the Reprogramming of Acute Myeloid Leukemia Cells

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The Role of Fos and Junb in the Reprogramming of Acute Myeloid Leukemia Cells Dickinson College Dickinson Scholar Student Honors Theses By Year Student Honors Theses 5-19-2019 The Role of Fos and JunB in the Reprogramming of Acute Myeloid Leukemia Cells Kayla Bendinelli Dickinson College Follow this and additional works at: https://scholar.dickinson.edu/student_honors Part of the Cell Biology Commons, and the Molecular Biology Commons Recommended Citation Bendinelli, Kayla, "The Role of Fos and JunB in the Reprogramming of Acute Myeloid Leukemia Cells" (2019). Dickinson College Honors Theses. Paper 321. This Honors Thesis is brought to you for free and open access by Dickinson Scholar. It has been accepted for inclusion by an authorized administrator. For more information, please contact [email protected]. 1 The role of Fos and JunB in the reprogramming of Acute Myeloid Leukemia Cells Kayla Bendinelli Submitted in partial fulfillment of the Biochemistry and Molecular Biology Honors Requirement Dr. Michael Roberts, Advisor, Committee Chair Dr. Rebecca Connor, Reader Dr. Dana Somers, Reader May 8, 2019 2 The role of Fos and JunB in the reprogramming of Acute Myeloid Leukemia cells Acute Myeloid Leukemia (AML) is the most common form of leukemia in adults and while it has a high remission rate, relapse with therapy resistance is common, indicating the need for more targeted and effective therapies. It is possible to reprogram AML cells in culture to undergo cell cycle arrest, differentiation into “normal” macrophage-like cells, and apoptosis using phorbol 12-myristate 13-acetate (PMA), a diacyl glycerol (DAG) mimic. While this is effective in “curing” leukemia in culture, PMA is too toxic to serve as a therapy in AML patients. During these PMA-induced changes, approximately 1250 genes change in expression. The goal of this study was to see if the genes Fos and JunB, which are highly upregulated post PMA treatment, are responsible for portions of the genetic reprogramming mediating these phenotypic changes. These genes are transcription factors and members of the AP-1 complex, which is known to play a role in regulating the cell cycle, differentiation and programmed cell death. In this study we show that Fos and JunB are capable of initiating specific responses by overexpressing them individually or together via transfection in the AML cell line, HL-60. 3 Title 1 Abstract 2 Table of Contents 3 Chapter 1: Introduction Acute Myeloid Leukemia 4 Activator Protein 1 (AP-1) 5 Introduction to AP-1 5 Fos Family 7 JunB 8 Role of AP-1 on morphological processes 9 Cell cycle arrest 8 Differentiation 10 Apoptosis 12 Clinical Significance 13 Summary 13 Chapter 2: The role of Fos and JunB in the Reprogramming of Acute Myeloid Leukemia Cells Abstract 15 Chapter Introduction Results Overexpression Validation 16 Target Gene Investigation 17 Cell Cycle Gene Expression 19 Assay 20 Macrophage Differentiation Gene Expression 20 Microscopy 20 Apoptosis 21 Summary 22 Chapter 3: General Discussion and Conclusion Discussion 23 Conclusion 25 Chapter 4: Tables and Figures 26 Chapter 5: Materials and Methods 50 Chapter 6: References 52 4 Chapter 1: Introduction Acute Myeloid Leukemia Acute myeloid leukemia (AML) is the most common form of adult acute leukemia with around 20,000 new cases a year1. Over the years the incidence of AML has been increasing while the mortality rate has remained relatively constant leading to the necessity for more effective therapies (Figure 1). AML is a high mortality disease, curable in 35-40% of patients younger than 60 years of age but only between 5%-15% of patients over the age of 60 2. While incidence of AML is higher in patients with a hematological disorder or who have received previous treatment including radiation for another cancer there is still no definitive cause for leukemia1. Most AML cases occur as a primary disease characterized by the proliferation of undifferentiated myeloid cells. Cases of AML are categorized into subtypes based on the morphological and genetic mutations present in the patient’s leukemic cells. The most well-established example of a primary mutation driving oncogenic mutation is the translocation of chromosomes 15 and 17 which effects the retinoic acid receptor gene and causes the development of acute promyelocytic leukemia (APL) 1. In patients there are mutational changes to the genome resulting in activation of cell proliferation and inhibition of differentiation1. The types and number of mutations present in the cancerous cells contributes to the prognosis of the patient (Table 1). As mutations accumulate in the affected myeloid stem cells a main clonal population will develop with at least one subclonal population resulting in tumor heterogeneity2. While the mutated cells continuously divide there is a loss of myeloid lineage cells, including those involved with the immune response, and the crowding out of other necessary cells including red blood cells in the 5 bloodstream. This leads to the symptoms experienced by patients including fatigue, shortness of breath and easy bruising. Once the genetics of a patient’s AML is determined, their course of treatment is decided. Most will undergo induction therapy which consists of infusions of the chemotherapy agents cytarabine, which inhibits DNA synthesis, and anthracycline, which forms DNA intercalations, with the goal of achieving a complete response1. Trials are being undertaken to see if high doses of daunorubicin, which also forms DNA intercalations, will have similar success but using current therapies complete remission can be achieved in 60-85% of patients under 60 years of age2. Post-remission consolidation therapy consisting of chemotherapy and potentially bone marrow cell transplantation is required to eliminate residual disease and subclonal populations that may have escaped the initial treatment2. These are high risk and toxic therapies, especially for patients over the age of 60, so more targeted therapies are needed to produce durable responses. Therapies being tested include monoclonal antibodies that work to direct chemotherapy directly to the cancerous cells and inhibitors of the Signal transducer and activator of transcription 3 (STAT3) transcription factor which is activated in many cancer patients1. These new targeted therapies focus on the genetic mutations of the cancer in hopes of selectively killing the cancer cells and sparing the patient from unnecessary side effects due to normal cell death. Activator Protein 1 Activating protein-1 (AP-1) is a transcription factor composed of a dimer complex made up of members of the Fos, Jun, ATF and Maf families and has been linked to cell proliferation, differentiation and apoptosis3. These components are typically regulated as “immediate-early” 6 response genes which are a set of genes activated by present transcription factors in response to extracellular stimuli including growth factors and stress signals4. Some members of the AP-1 complex such as the Jun family proteins are capable of forming homodimers or heterodimers, while other members such as the Fos family only form heterodimers with other AP-1 members. All of the AP-1 proteins contain a basic leucine zipper domain (bZIP) that allows the proteins to dimerize which aids in the stability of the protein5. Next to the dimerization domain the Fos and Jun proteins contain a basic DNA binding domain that can bind to a palindromic motif with the sequence 5’-TGAG/CTCA-3” (Figure 2)3,6. The individual members of the AP-1 family are regulated independently but once translated into protein they can form dimeric complexes that can activate downstream target genes that contain TPA-responsive elements (TREs), the AP-1 binding site5.Each AP-1 member’s binding domain has a different binding specificity for the target site allowing heterodimers to bind to a more diverse set of half-sites in different orientations than homodimers. Target genes contain a version of the conserved consensus sequence for AP-1 in their promoter or enhancer regions that often contain some variation resulting in different binding capacities and thus outcomes for the varying Fos-Jun complexes7. Under different conditions in different cell types, cells can contain a variety of AP-1 complexes, which are dependent on the expression levels of the individual proteins that are subject to change from outside stimuli. A stimulus can lead to an increase or decrease in a particular AP-1 gene leading to changes in the AP-1 complexes present within a cell that determine different transcriptomes8,3,7. A study done with mammary adenocarcinoma cell lines found that Fos was highly expressed in the non-invasive cells while in metastatic cells from the same tumor Fos was undetectable and Fra-1 and Fra-2 were strongly expressed8. A shift in AP-1 7 composition could be responsible for the progression of the tumor, however, this is very cell type dependent. Alternatively, a shift in AP-1 composition could assist in the reversion of the disease state. A study done on cervical cancer showed that Fos expression was high in the malignant cells and that treatment with the antioxidant pyrroline dithiocarbamate (PDTC) led to a reduction in Fos expression and an increase in c-Jun and Fra-18. Fos Family The Fos family consists of c-Fos, FosB, Fra-1 and Fra-2. These genes encode proteins with similar domains but can elicit different transcriptional responses, often depending on cell type. For example, Fos and Fra-1 can have similar effects including increased osteoblast differentiation and expression of genes involved with mammary tumor metastasis8,9. They can also show opposing functions as is the case in cervical cancer where Fra-1 is tumor suppressive and Fos tumorigenic8.The role of each of these genes is dependent on the cell type and the components of the AP-1 complex. Fos, is an important member of the Fos family and is responsive to a variety of signals. The promoter contains a cAMP-response element (CRE) as well as a serum-response element (SRE), allowing Fos expression to respond to internal changes, changes in Ca2+ and cAMP, as well as external changes including changes in growth factors and cytokines4.
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