Identifying novel drug therapies for acute myeloid leukaemia by targeting the interactions between leukaemia cells and bone marrow microenvironment
Dhami, Sukhraj Pal Singh
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Acute myeloid leukaemia (AML) is a malignancy caused by a block in differentiation in which aberrant leukemic stem cells drive the production of undifferentiated or partially differentiated leukemic blast cell clones. Between 10-40% of patients, however, have refractory disease or undergo relapse. The impact of the bone marrow microenvironment (BMM) is increasingly recognised as a reason for this. Hence, this thesis is aimed at understanding the interactions between leukemic cells and the BMM and to devise a therapeutic strategy to target these interactions. We have developed a functional, drug testing system that can incorporate the impact of BMM while rapidly and faithfully predicting the clinical response of the patient to cytarabine+daunorubicin (AraC+Dnr) therapy. We have developed and characterised a layered co-culture system consisting of primary AML blasts with immortalised bone marrow stromal cells (BMSCs). This BMSC-AML co-culture can predict the clinical response of AraC+Dnr therapy with very high accuracy [area under the curve (AUC=0.94)]. The advantage of this model over more complex pre-clinical AML models is its suitability to be developed into a laboratory diagnostic tool, which could greatly advance the clinical decision on treatment choice. Having established the model that mimics the BMM, we have studied its role in protecting the AML cells against cytotoxic agents such as BH3-mimetics, cytarabine and daunorubicin. We found that BMSCs induce Mcl-1 expression over Bcl-2 and/or Bcl-XL in AML cells and that inhibition of Mcl-1 with a small-molecule inhibitor, A1210477, or through repression of its expression with the cell division cycle-7 kinase/ cyclin dependent kinase 9 (CDC7/CDK9) dual-inhibitor, PHA-767491, restores sensitivity to chemotherapeutics. Importantly, the CD34+/CD38− leukemic stem cell-encompassing population was equally sensitive to this combination. These results highlight the potential of Mcl-1-repression to revert BMM-mediated drug resistance thus preventing disease relapse and ultimately improving patient survival. Next, we investigated the mechanism through which the BMM protects the FLT3-ITD mutated AML cells against tyrosine kinase inhibitors (TKIs). We found that FLT3- ITD cells do not depend on intrinsic, FLT3-driven survival signalling pathways in BMM. We also observed that exposure of BMSCs to a mild, proteostatic stress revert its ability to protect AML cells against chemotherapeutics. Importantly, we also found that proteostatic stressconditioned BMSCs themselves trigger an anti-leukemic effect, which is mediated through secreted lipids or non-protein moieties. In summary, the results presented in this thesis illustrate the role of microenvironment interactions in providing chemoresistance to malignantly transformed cells. We have also presented strategies to target these interactions for effective and novel therapies that could improve patient outcome.
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