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

Karen H Martin

Committee Member

Ivan Martinez

Committee Member

William Petros

Committee Member

Michael J Ruppert

Committee Member

Linda Vona-Davis

Abstract

While modern acute lymphoblastic leukemia (ALL) treatment regimens have dramatically improved the survival of ALL patients, disease relapse still occurs in approximately 20% of cases. ALL relapse is, in part, due to the inability of traditional chemotherapeutic agents to efficiently target quiescent, non-dividing tumor cells. The number of viable remaining leukemia cells in patients after initial treatment, termed minimal residual disease (MRD), is directly correlated with likelihood of disease relapse. This reflects the proliferative potential of these refractory cells, consistent with the ability of their healthy hematopoietic progenitor pro- and pre-B cell counterparts. Developing novel therapies to combat refractory disease is essential in the continued battle of eradicating this cancer based on the aggressive nature of relapsed ALL that is much more challenging to effectively treat than disease at initial diagnosis. Many features of malignant immature B-lineage cells, as noted above, are shared with normal B cell progenitors, including their ability to respond to diverse signals from the bone marrow microenvironment that culminate on regulation of cell cycle progression and survival. Bone marrow derived cues from supportive stromal and osteoblast cells influence many elements of both steady state hematopoiesis and hematopoietic tumor cell phenotype through modulation of pro- and pre-B cell gene expression profiles.;Developing a system that models interactions between bone marrow niche cells and leukemia is essential in supporting discovery of mediators of bone marrow protection of ALL. Although in vivo animal modeling can be superior to standard cell culture, disadvantages exist including expense, time expenditure, and the inability to perform efficient high-throughput experimentation. In Chapters II and III, we describe an in vitro co-culture model in which both ALL cell lines and primary patient samples uniquely interact with bone marrow derived stromal cells (BMSC) or human osteoblasts (HOB). ALL cells in this model form distinct subpopulations, including one subset that buries beneath the adherent bone marrow niche cell monolayer, termed "phase dim" (PD) ALL. They exhibit a unique phenotype characterized by altered metabolism, distinct protein expression profiles, increased quiescence, and pronounced chemotherapy resistance. The PD ALL cell bone marrow derived protection is specific to bone marrow niche cells, compared to adherent cells from other anatomical locations suggesting biologically relevant features have been retained in this model that is, by design, simplified to allow targeted investigation. From these observations, along with the ability to isolate this clinically relevant ALL subpopulation, studies focused on the PD subpopulation of tumor cells may more efficiently inform pre-clinical design and the investigation of MRD and relapse that arises from bone marrow supported leukemia cells compared to models that include the entire tumor population.;Utilizing the model outlined above, our laboratory focused on bone marrow niche regulated ALL miRNAs as a master regulator of phenotype that had not been previously evaluated in ALL as a potential pathway through which the marrow niche may impact on tumor cell biology. miRNAs define one regulatory class of small non-coding RNAs that have been shown to be increasingly important in diverse settings of malignancy. Global alterations in miRNA profiles were observed in ALL cells following exposure to BMSC or HOB. Two miRNAs of interest, miR-221 and miR-222, were reduced in ALL cells co-cultured with bone marrow niche cells coincident with an increase in CDKN1B (p27) protein levels. Increased p27 protein in ALL cells exposed to BMSC or HOB is consistent with accumulation of tumor cells in the G0 phase of cell cycle and resistance to chemotherapy induced death described in Chapters II and III. Constitutive expression of miR-221 in ALL cells was partially able to overcome the protective nature of the bone marrow niche and render cells more susceptible to chemotherapy exposure. Traditional chemotherapy, with the addition of miR-221/222 manipulation, could be an intriguing addition to a clinician's arsenal of treatment regimens for ALL with the potential to eradicate resistant disease or potentially decrease the intensity of agents required if sensitivity of tumor cells could be improved.;The overall objective of these studies was to increase our understanding of bone marrow regulated chemotherapy resistant ALL by utilizing our well characterized in vitro co-culture model. Through this model system we identified a subset of bone marrow niche associated ALL cells that were quiescent and highly resistant to chemotherapy exposure, partially due to modulation of miR-221/222. From these studies, we hope to contribute to the design of novel treatment strategies for aggressive and resistant ALL, with an emphasis on improving the outcome of patients with disease with poor prognosis. Ideally, both a better response during initial treatment, as well as more efficacious therapy when relapse does occur can be achieved by better understanding the molecular basis of niche supported tumor cell survival. As a very common site of metastatic disease, the broader application of this work is the potential relevance to additional tumor types that find sanctuary from therapy-induced death in the same marrow niches that support normal hematopoiesis and hematopoietic disease.

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