title: Investigating the Role of Monocyte Derived Macrophages in the Tumour Microenvironment of Chronic Lymphocytic Leukaemia and Evaluating their Function in Mediating Responses to Therapeutic Antibodies
Creators: name: Burgess, Melinda L
Publisher: name: Griffith University
Year: 2024
Subjects: subject: CLL
subject: macrophage
subject: tumour microenvironment

description: Chronic lymphocytic leukaemia (CLL) is the most common adult leukaemia and is characterised by the accumulation of mature malignant B cells. Despite having defects in cell death, CLL cells undergo spontaneous apoptosis when cultured in vitro which indicates a strong reliance on the tumour microenvironment (TME) for survival. The CLL TME is a complex and bidirectional collaboration between leukaemic cells and supportive cells, soluble factors and pro-survival signalling events which provide protection from therapeutic agents in addition to promoting tumour cell survival. Further understanding of the mechanisms controlling microenvironmental influences could lead to more effective therapeutic strategies. There have been numerous significant advancements in treatment options for CLL, including antibody-based therapies and small molecule inhibitors, some of which directly alter TME interactions. Despite having durable responses, these therapies are still compromised by resistance and CLL remains an incurable disease. Better understanding of microenvironmental processes contributing to resistance will inform new treatment strategies. This thesis identifies resistance mechanisms to antibody-based therapies in current use in CLL by examining the role of the CLL microenvironment and identifies approaches to reinstate sensitivity to existing therapeutic antibody strategies. The manuscripts presented in this thesis gave rise to several original discoveries. The early papers address challenges to reproduce the disease in vitro by developing a simple long-term culture system which recapitulates the in vivo CLL microenvironment. This in vitro system can be utilised to examine soluble factors and cell phenotypes which affect in vitro CLL cell survival and can be applied to a diverse range of experimental applications. This culture system led to the identification of two novel chemokines, CCL2 and CXCL2 which enhanced CLL cell survival, primarily in a paracrine manner. This longterm culture system also established the novel role the homing marker CD62L, plays in providing pro-survival stimuli in the CLL TME. My studies showed, for the first time, that blocking CD62L promoted CLL cell survival in vitro and provided a rationale for combining CD62L modulation with standard chemotherapy approaches. Additionally, I showed that CD62L expression was profoundly overexpressed on CLL cells located within proliferation centres (bone marrow and lymph nodes), which are known to be resistant to apoptosis due to niche specific pro-survival signals. Thus, CD62L is a pathologically meaningful, and novel, pro-survival effector that may represent a potential therapeutic target in CLL using antibody-based approaches. Furthermore, this work led to the filing of a provision patent. The next chapters (Chapters 3 - 5) examine TME mediated antibody resistance in CLL. Whilst monocyte derived cells (MDCs) are a major constituent of the CLL microenvironment, they are also critical immune effectors. In this thesis, I demonstrate that MDCs derived from CLL patients with stable disease can elicit superior cytotoxic responses to antibody therapies than those from patients with progressive disease. These changes in cytotoxic responses were not attributed to substantive differences in transcriptomes but more likely associated with post receptor binding signalling events. My studies demonstrate that antibody resistance is, in part, attributable to a change in phenotype of MDCs responsible for mediating antibody-dependent tumour cell killing which correlates with reduced antibody-dependent cell-mediated cytotoxicity and antibody-dependent phagocytosis responses due to the loss of Fc gamma receptor (FcγR)-dependent activation signalling relative to FcγRIIB-dependent inhibitory pathways. Defects in FcγR signalling is actionable and can be manipulated with inhibitors of FcγRIIB signalling to reverse antibody resistance in primary cultures of patient derived CLL cells. The final chapter focuses on exploiting MDC mediated antibody resistance by pharmacologically manipulating the phenotype of CLL MDCs. My studies demonstrate that Class IIa histone deacetylases (HDACs), namely, HDAC7, regulates both FcγRdependent and FcγR-independent phagocytosis in CLL MDCs via a previously unknown, yet direct modulation of BTK acetylation and phosphorylation. Moreover, HDAC7 contributes to muted phagocytic responses commonly associated with MDCs derived from patients with progressive, relapsed, or refractory disease. This is an actionable pathology and suggests that HDAC7-specific inhibitors could be used to improve responses to therapeutic antibodies and to enhance the innate immune response in CLL patients by restoration of phagocytic responses to both opsonised and non-opsonised targets. Overall, this thesis led to the development of a novel and versatile in vitro culture system which can be utilised to study the complex interplay controlling the CLL TME. Using this model, I was able to define several novel therapeutic targets that could be exploited to induce sensitivity to existing therapeutic antibodies and chemotherapeutics. Moreover, my studies identified novel effector pathways that contribute to CLL survival, the main pathological defect of CLL disease. Principally, this thesis discusses the complexity of macrophage mediated antibody resistance in depth and endeavors to provide therapeutic intervention strategies which can reverse antibody mediated resistance, which remains a significant clinically relevant challenge in CLL.