Date of Award

Spring 2019

Project Type

Dissertation

Program or Major

Biochemistry

Degree Name

Doctor of Philosophy

First Advisor

Sherine F. Elsawa

Second Advisor

Arturo Andrade

Third Advisor

Martin Fernandez-Zapico

Abstract

The tumor microenvironment (TME) plays an important role in the initiation, progression and maintenance of cancer cells and is implicated in cancer cell resistance to therapy. In Waldenström macroglobulinemia (WM), a B-cell malignancy characterized by the overproduction of a monoclonal IgM protein, the TME plays an important role in disease biology by secreting cytokines that promote the malignant phenotype. In previous work, we have shown that the transcription factor GLI family zinc finger 2 (GLI2) regulates interleukin-6 (IL-6) secretion in the WM TME. IL-6, in turn, promotes WM growth and IgM secretion by malignant B cells. Tocilizumab/Actemra is an anti-IL-6R antibody, which can competitively block IL-6 binding to the IL-6R. In this dissertation, we investigated the efficacy of targeting the TME using Tocilizumab in a preclinical mouse model of WM that considers the role of the TME in disease biology. Furthermore, we screened for novel GLI2 target genes in the TME and identified cluster of differentiation 40 ligand as a novel GLI2 target gene in the TME. Our data shows that Tocilizumab therapy results in a reduction in tumor growth rate and IgM secretion in mice sera. In addition, there was no significant change in mice weight suggesting Tocilizumab induced no toxicities to the mice. Taken together, our data suggests that administration of Tocilizumab to tumor bearing mice, results in a significant reduction in tumor volume and IgM secretion. Therefore, the evaluation of the role of Tocilizumab alone or in combination with other therapies that target malignant cells in WM patients may provide therapeutic efficacy. As a single agent, Tocilizumab may alleviate symptoms associated with IgM. In combination, it may reduce IgM levels and slow the rate of tumor growth, thereby allowing therapeutic agents that target cancer cells survival to better induce cell death. Further screening of the role of GLI2 in the TME identified CD40 ligand (CD40L) as a novel GLI2 target gene. We provide evidence of a novel pathway controlling the transcriptional activation of CD40 ligand in bone marrow-derived stromal cells. CD40L plays an important role in normal and malignant B cell biology and we found increased Erk phosphorylation and cell growth in malignant B cells co-cultured with CD40L expressing stromal cells. Further analysis indicated that GLI2 overexpression induced increased CD40L expression, and conversely, GLI2 knockdown reduced CD40L expression. We demonstrate that GLI2 directly binds and regulates the activity of the CD40L promoter. Additionally, we found that the CCR3-PI3K-AKT signaling modulates GLI2-CD40L axis and GLI2 is required for CCR3-PI3K/AKT mediated regulation of CD40L promoter. Finally, co-culture of malignant B cells with cells stably expressing human CD40L, results in increased Erk phosphorylation and increased malignant B cell growth indicating CD40L in the TME promotes malignant B cell activation. Therefore, our studies identify a novel molecular mechanism of regulation of CD40L by the transcription factor GLI2 in the TME and suggests that targeting GLI2 may be therapeutically beneficial.

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