The SMURF2-YY1-C-MYC Axis in the Germinal Center Reaction and Diffuse Large B Cell Lymphoma: A Dissertation

Diffuse large B cell lymphoma (DLBCL) is the most common non-Hodgkin’s lymphoma. Patients who fail conventional therapy (~50%) have a poor prognosis and few treatment options. It is essential to understand the underlying biological processes, the progression of the disease, and utilize this information to develop new therapeutics. DLBCL patients with high C-MYC expression have a poor prognosis and new therapeutics for these patients are needed. This thesis describes work testing the hypothesis that JQ1, which can indirectly inhibit C-MYC in some tumors, can be used as an effective treatment for DLBCL. Some tumors have an unknown mechanism causing high C-MYC expression, leading me to investigate the underlying mechanisms. YY1 is a transcriptional regulator of c- Myc and has been implicated in DLBCL and as a potential regulator of the germinal center (GC) reaction. DLBCL arises from GC cells or post-GC cells. I tested the hypothesis that YY1 regulates the GC reaction. SMURF2 is an E3-ubiquitin ligase for YY1 and a tumor suppressor for DLBCL. I was interested in examining the mechanism underlying the suppression of DLBCL by SMURF2 leading to the hypothesis that SMURF2 regulates the GC. This thesis shows JQ1 leads to cell death and cellular senescence in human DLBCL cells. I conclude that BRD4 inhibition by JQ1 or derivatives could provide a new therapeutic avenue for DLBCL patients. I also show loss of YY1 perturbs the GC by decreasing the dark zone and increasing apoptosis. Finally I show modulation of SMURF2 does not affect the GC, suggesting SMURF2 utilizes a different mechanism to act as a tumor suppressor and may not modulate YY1 in the context of the GC

Smurf2 regulates hematopoietic stem cell self-renewal and aging

The age-dependent decline in the self-renewal capacity of stem cells plays a critical role in aging, but the precise mechanisms underlying this decline are not well understood. By limiting proliferative capacity, senescence is thought to play an important role in age-dependent decline of stem cell self-renewal, although direct evidence supporting this hypothesis is largely lacking. We have previously identified the E3 ubiquitin ligase Smurf2 as a critical regulator of senescence. In this study, we found that mice deficient in Smurf2 had an expanded hematopoietic stem cell (HSC) compartment in bone marrow under normal homeostatic conditions, and this expansion was associated with enhanced proliferation and reduced quiescence of HSCs. Surprisingly, increased cycling and reduced quiescence of HSCs in Smurf2-deficient mice did not lead to premature exhaustion of stem cells. Instead, HSCs in aged Smurf2-deficient mice had a significantly better repopulating capacity than aged wild-type HSCs, suggesting that decline in HSC function with age is Smurf2 dependent. Furthermore, Smurf2-deficient HSCs exhibited elevated long-term self-renewal capacity and diminished exhaustion in serial transplantation. As we found that the expression of Smurf2 was increased with age and in response to regenerative stress during serial transplantation, our findings suggest that Smurf2 plays an important role in regulating HSC self-renewal and aging

Inhibition of Bromodomain Proteins in Treatment of Diffuse Large B-cell Lymphoma

Only ~50% of patients with diffuse large B-cell lymphoma (DLBCL), the most common and aggressive subtype of non-Hodgkin’s lymphoma, enter long-term remission after standard chemotherapy, and patients who do not respond to treatment have few options. Therefore, there is a critical need for effective and targeted therapeutics for DLBCL. Recent studies highlight the incidence of increased c-MYC protein in DLBCL and the correlation between high levels of c-MYC and poor survival prognosis of DLBCL patients, suggesting that c-MYC is a compelling therapeutic target for DLBCL therapy. The small molecule JQ1 suppresses c-MYC expression through inhibition of the BET family of bromodomain proteins. We show that JQ1 efficiently inhibited cell proliferation of human DLBCL cells regardless of their molecular subtypes, suggesting a broad effect of JQ1 in DLBCL. After JQ1 treatment, initial G1 arrest in DLBCL cells was followed by either apoptosis or senescence. In DLBCL cells treated with JQ1, we found that c-MYC expression was suppressed in the context of the natural, chromosomally-translocated or an amplified gene locus. Furthermore, JQ1 treatment significantly suppressed growth of DLBCL cells engrafted subcutaneously and improved survival of mice engrafted with DLBCL cells intraperitoneally. These results demonstrate that inhibition of the BET family of bromodomain proteins, and consequently c-MYC, has the potential clinical utility in DLBCL treatment

Gadd45-α and Metastatic Hepatocellular Carcinoma Cell Migration

Gadd45-alpha is a tumor suppressor protein identified by microarray analysis with a reduced expression in hepatocellular carcinoma (HCC) cell lines with high migration ability. The role of Gadd45-alpha in HCC migration was tested by stable shRNA knockdowns in a non-metastasizing murine cell line (BL185). The migration levels of the Gadd45-alpha knockdowns were significantly increased relative to the parental non-metastasizing line, indicating that decreased Gadd45-alpha expression is sufficient to promote increased cell migration

Finding Shangri-La: Limiting the Impact of Senescence on Aging

Senescence plays an important role in the age-associated decline of tissue functions. Recent studies now show that targeting senescent cells can enhance the functions of stem/progenitor cells in aged mice and extend lifespan

YY1 Regulates the Germinal Center Reaction by Inhibiting Apoptosis

The germinal center (GC) reaction produces high-affinity Abs for a robust adaptive immune response. When dysregulated, the same processes cause GC B cells to become susceptible to lymphomagenesis. It is important to understand how the GC reaction is regulated. In this study, we show that transcription factor YY1 is required to maintain a robust GC reaction in mice. Selective ablation of YY1 significantly decreased in the frequency and number of GC B cells during the GC reaction. This decrease of GC B cells was accompanied by increased apoptosis in these cells. Furthermore, we found that loss of YY1 disrupted the balance between dark zones and light zones, leading to a preferential decrease in dark zone cells. Collectively, these results indicate that YY1 plays an important role in regulating the balance between dark zone and light zone cells in GCs and between survival and death of GC B cells

Getting a Feel for Science: Adapting Existing Non-Formal Education Techniques to Conform to - Universal Design

This project implemented and tested methods to adapt Commonwealth Scientific and Industrial Research Organisation (CSIRO) science programs to accommodate Australian students with disabilities. Universal Design principles were applied to the Tyre Track activity in the Forensic Frenzy program to improve the science experience for visually impaired students. The team gathered survey and observational data, evaluated activity effectiveness, and delivered recommendations and guidelines. Statistical analysis demonstrated that non-formal education programs modified to contain multi-sensory components increase student knowledge and understanding

Inhibition of bromodomain proteins for the treatment of human diffuse large B-cell lymphoma

Purpose: Approximately 50% of patients with diffuse large B-cell lymphoma (DLBCL) enter long-term remission after standard chemotherapy. DLBCL patients who do not respond to chemotherapy have few treatment options. There remains a critical need to identify effective and targeted therapeutics for DLBCL. Experimental Design: Recent studies have highlighted the incidence of increased c-MYC protein in DLBCL and the correlation between high levels of c-MYC protein and poor survival prognosis of DLBCL patients, suggesting that c-MYC is a compelling target for DLBCL therapy. The small molecule JQ1 suppresses c-MYC expression through inhibition of the bromodomain and extra-terminal (BET) family of bromodomain proteins. We investigated whether JQ1 can inhibit proliferation of DLBCL cells in culture and xenograft models in vivo. Results: We show that JQ1 at nanomolar concentrations efficiently inhibited proliferation of human DLBCL cells in a dose-dependent manner regardless of their molecular subtypes, suggesting a broad effect of JQ1 in DLBCL. The initial G1 arrest induced by JQ1 treatment in DLBCL cells was followed by either apoptosis or senescence. The expression of c-MYC was suppressed as a result of JQ1 treatment from the natural, chromosomally-translocated or amplified loci. Furthermore, JQ1 treatment significantly suppressed growth of DLBCL cells engrafted in mice and improved survival of engrafted mice. Conclusion: Our results demonstrate that inhibition of the BET family of bromodomain proteins by JQ1 has potential clinical utility in the treatment of DLBCL