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Myeloid cells and therapy resistance in Chronic Lymphocytic Leukemia

Yazdanparast, Haniyeh

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Abstract

Chronic lymphocytic leukemia (CLL) is characterized by an accumulation of mature malignant CD5+CD19+ B cells. Survival and proliferation of these B cells are highly dependent on the tumor microenvironment, which comprises monocyte-derived nurse-like cells (NLCs) as key players that possess tumor-supportive properties and resemble tumor-associated macrophages (TAMs). One of the main goals of this thesis was to understand and target the tumor supportive properties of myeloid cells in CLL. Colony Stimulating Factor 1 Receptor (CSF-1R) is expressed on cells of the myeloid lineage and known to be important for TAM differentiation in solid tumors. Targeting CSF-1R using the monoclonal antibody TG3003, reduced NLC numbers in CLL cocultures, a well-accepted in vitro model mimicking the lymph node microenvironment in CLL, slightly reduced the expression of factors with functional relevance in the CLL tumor microenvironment (e.g. CCL2, sCD14), and significantly reduced NLC-mediated survival support for primary CLL cells in vitro. Further, a potential therapeutic effect of TG3003 was investigated in a preclinical model of CLL by adoptively transferring splenocytes from leukemic Eμ-TCL-1 mice into C57BL/6 mice (TCL-1 AT) with a humanized CSF-1R gene. TG3003 treatment resulted in reduced monocyte numbers with a stronger effect on Ly6Clow monocytes, a subset that is highly enriched in CLL patients. The observed reduction did, however, not lead to a complete normalization of monocytic cell subsets. In conventional dendritic cells (cDCs), the expression levels of surface molecules required for adhesion, co-stimulation of T cells, and antigen presentation remained at low levels in leukemic mice after treatment with TG3003. In line with this, adaptive immunity with respect to T cells was also not altered. Finally, TG3003 treatment did not lead to an improvement of disease outcome in the TCL-1 AT model, possibly due to insufficient changes in the immune status of treated mice. The second focus of this thesis was a comprehensive investigation of the myeloid tumor microenvironment in the TCL-1 AT model and under the influence of Ibrutinib, a Bruton’s tyrosine kinase (BTK) inhibitor that blocks B-cell receptor (BCR) signaling, and thereby efficiently controls and inhibits CLL development in patients. As BTK is also expressed in myeloid cells, an indirect activity of Ibrutinib on the CLL tumor microenvironment was hypothesized. Among other findings, this study revealed that Ibrutinib is capable of inhibiting the enrichment of the CLL-associated Ly6Clow monocyte subset and normalizing several CLL-induced molecules associated with activation of cellular growth and proliferation, inflammation and immune suppression (e.g. PECAM-1, TREM-1, PD-L1). Ibrutinib further enhanced the CLL-associated tolerogenic/immature immunophenotype of cDCs (e.g. expression of PD-L1). Thus, Ibrutinib’s effect on the myeloid tumor microenvironment in CLL is of opposing nature, thereby inaugurating room for improvement with respect to therapy. Exploring mechanisms of Ibrutinib resistance in the TCL-1 AT model was the third aim of the current work. Even though Ibrutinib represents a paradigm shift in the treatment of CLL, a growing number of resistant patients with highly aggressive disease are identified, whereby the underlying mechanism of resistance is so far only partly understood. Despite an initial response to Ibrutinib, continuous treatment of TCL-1 AT mice revealed a loss of therapeutic efficacy after several weeks. This observation was accompanied by an increase of proliferating leukemic cells in the blood and lymphoid organs and phenotypic alterations of Ibrutinib-resistant leukemia cells including deregulation of proteins that are involved in the BCR signaling. Re-transplantation of therapy-resistant tumor cells led to uncontrolled leukemia development under re-applied Ibrutinib treatment, demonstrating acquired tumor microenvironment-independent and cell-intrinsic resistance of these tumors. Whole exome and RNA sequencing of tumor cells revealed the absence of genetic mutations in Btk and its target molecule Plc2. Analysis of transcriptional changes identified several gene sets and biological complexes (e.g. Bcl-2 family and NF-B complex) that are significantly different in Ibrutinib-resistant and –sensitive cells and which will be the focus of future investigations. In summary, the current thesis illuminates several tumor-promoting as well as dysfunctional features of myeloid cells in CLL and under Ibrutinib treatment. These findings may serve as a basis for the design of specific therapeutic strategies targeting myeloid cells, and their rational combination, for example with existing tumor-targeting drugs, which such as Ibrutinib might harbor also non-beneficial effects on the tumor microenvironment. The identification of target molecules in leukemic cells that have become resistant to Ibrutinib and are highly independent of their tumor microenvironment launches novel therapeutic modalities for patients that relapse under Ibrutinib treatment.

Item Type: Dissertation
Supervisor: Umansky, Prof. Dr. Viktor
Date of thesis defense: 6 March 2018
Date Deposited: 18 May 2018 06:59
Date: 2018
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
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