Investigating the heterogeneity of leukaemia kinase networks and the impact of the microenvironment on leukaemic cell signalling
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The tumour microenvironment plays a key role in tumour progression. In this thesis acute myeloid leukaemia (AML) was used as a model system to investigate the interplay between stromal and cancer cells. AML is a heterogeneous clonal disorder of haematopoietic undifferentiated progenitor cells or ‘blast cells’, which accumulate in the bone marrow and lead to the reduced output of crucial haematopoietic elements. Due to its heterogeneity (at least in part), treatment of the disease has not witnessed great innovation in the past 30 years. The bone marrow microenvironment (BMM) has a key role in the haematological malignancies contributing to the survival of leukaemic blasts. Relapse in AML occurs because of residual disease and evidence suggests that this resistance is facilitated through leukaemic cells ability to reside in BMM niches. To understand the precise role of the BMM in AML progression and therefore target any supportive mechanisms requires knowledge of how AML cells communicate with their microenvironment. In the work presented in this thesis I undertook a multi-proteomic approach that utilised liquid chromatography tandem mass spectrometry (LC-MS/MS) to assess the interplay between AML and BMM cell signalling. This thesis shows the results of a secretomic analysis of stromal cell lines, which identified a previously uncharacterized panel of six stromal secreted proteins (BMP-1, CSF-1, CTGF, HGF, S100-A4 and S100-A11) that support primary AML cell survival and proliferation in culture. Comparison of AML cell signalling (using global phosphoproteomic methods) following treatment with the newly identified growth factors revealed that these signalling proteins elicit multi-nodular activation of signalling networks with known anti-apoptotic activity. Consistent with the cell signalling proteomics data, cell viability studies as a function of pharmacological kinase inhibitor treatment determined that the sensitivity of AML to targeted kinase inhibitors was modulated by the supportive stromal conditioned media. To investigate heterotypic signalling between cell populations, AML/stromal cell co-cultures were designed, tested and optimised. These studies identified additional activated pathways in AML cells that were only present when AML cells had physical interaction with stroma. Complementary analysis of the stromal cells which had been first cultured with AML cells revealed that despite heterogeneity there is an emerging stromal phospho-proteomic signature that is different in BMM independent AML cells vs BMM interactive AML cells. Collectively these findings evidence the influence that the BMM can have on AML signalling. Although evidence for the influence of BMM in modulating AML resistance to standard chemotherapy exists, this study highlights specific BMM components that contribute to the ability of AML cells to circumvent current treatments based on kinase targeted drugs. These observations have implications for designing future therapies for AML.
AuthorsDokal, Arran D
- Theses