Date of Graduation

2016

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Family Medicine

Committee Chair

Laura F Gibson

Committee Co-Chair

John Barnett

Committee Member

Karen Martin

Committee Member

William P Petros

Committee Member

Linda Vona-Davis

Committee Member

Jeffrey Vos

Abstract

Acute lymphoblastic leukemia (ALL) is a hematological malignancy with approximately 6000 newly diagnosed cases every year. Although ALL is the most common malignancy in children, it can occur in patients of all ages. Great strides have been made in the treatment of ALL and remission rates are at all-time highs. However, relapse rates have remained relatively consistent, and relapse continues to be correlated with a poor prognosis in patients with ALL. As the site of origin and progression of ALL, the bone marrow microenvironment (BMM) is important in regulating tumor cell quiescence and proliferation. Of clinical relevance is the frequency with which quiescent leukemic cells survive treatment, initiate proliferation, and contribute to relapse of aggressive disease. In order to design innovative therapies, a better understanding of the mechanisms by which this regulation occurs is needed.;In order to investigate the mechanisms of BMM mediated protection, and to develop innovative targeted strategies to disrupt it, in vitro co-culture models remain critical. Classically, co-culture models that include hematopoietic cells have only investigated the tumor population as a whole, without regard for potential different phenotypes based on location of the leukemic cell relative to the adherent BMM cells. In the first study (Chapters 2 and 3), we investigated whether ALL cells have a variance in phenotype based on their spatial location within the co-culture. Utilizing bone marrow stromal cells (BMSC) and human osteoblasts (HOB) as representative elements of the BMM, in culture with ALL cells, we found that ALL cells form three distinct populations relative to the BMSC or HOB. ALL tumor cells that migrated beneath the stromal layer, referred to as phase dim (PD), were characterized by a quiescent and chemotherapy resistant phenotype. Cell labeling experiments demonstrated that the co-culture model was dynamic and that ALL cells readily transitioned between populations relative to the adherent BMM cells. Furthermore, co-culture studies using non-bone marrow derived adherent layers found that while these co-cultures supported formation of a PD population, they did not protect ALL cells from chemotherapy exposure. These results suggest that the increased chemotherapy resistance seen in the PD population is specific to the crosstalk between ALL cells and the BMSC or HOB. PD ALL cells were also seen to have an altered metabolic profile, which may contribute to their increased resistance to chemotherapy. Additionally, we provide a written and video protocol for the isolation of the three ALL populations from the stromal adherent layers. This extension of the standard co-culture model will provide researchers a more biologically relevant method to investigate resistant ALL disease in the context of BMM derived support.;In the final study (Chapter 4), data suggest that microenvironment regulation of BCL6 in leukemic cells is one factor involved in the transition between the proliferative and quiescent states of ALL. Observations utilizing Bcr-Abl negative (Ph-), positive (Ph+) ALL cell lines, and primary patient samples suggest that tumor cell BCL6 protein expression is decreased due to BMSC and HOB derived signals. Leukemic cells with decreased BCL6 are characterized by diminished proliferation, G0 accumulation, and chemotherapy resistance. Conversely, removal of ALL cells from marrow-derived stroma results in leukemic cells with increased BCL6 expression that are proliferative and sensitive to chemotherapy. Chemical inhibition or knock-down of BCL6 by shRNA in ALL cells results in diminished proliferation reminiscent of the quiescent tumor cells supported by the marrow microenvironment which are chemotherapy resistant and contribute to relapse of disease. We have developed a unique in vitro recovery model to test chemotherapy protection of tumor in this unique niche. BMSC/HOB co-cultured tumor cells are exposed to chemotherapy, and subsequently allowed to recover from drug imposed insult to determine factors important to tumor survival and repopulation. While down regulation of leukemic BCL6 leads to a quiescent phenotype, surviving leukemic cells released from microenvironment constraint have increased BCL6 expression and undergo a period of aggressive proliferation. Since many chemotherapy regimens require tumor cell proliferation for optimal efficacy, we investigated the consequences of forced BCL6 expression in leukemic cells when in the protective microenvironment niche. Data suggest that forcing leukemic cells to express BCL6 when co-cultured with BMSC or HOB sensitizes the tumor to chemotherapy induced cell death. Furthermore, pre-treatment with drugs that increase BCL6 expression such as the proteasome inhibitor MG132 or the ATM pathway inhibitor caffeine sensitize microenvironment protected ALL cells to chemotherapy treatment. These data suggest that BCL6 is one factor, modulated by microenvironment derived cues that may contribute to regulation of ALL cell cycle progression and subsequently therapeutic response.;The overall goal of the studies presented herein was to provide a platform to investigate treatment resistant ALL that is due to BMM support through the use of our in vitro co-culture model. Additionally, through the use of this model we are able to demonstrate mechanistic changes, which can lead to chemotherapy resistance such as downregulation of BCL6 in ALL subsequent to BMM interaction. These studies provide mechanistic insight that will contribute to the design of novel treatment strategies that disrupt protective microenvironment signaling, with a goal of increased chemotherapy efficacy and less intensive therapies for ALL patients. With a primary goal of reducing the frequency of relapse, a critical secondary advantage would be the reduction of long-term effects from cytotoxic therapies and potential reduction of treatment induced secondary malignancies.

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