Abstract
Fifty percent of AML patients relapse and exhibit poor long-term disease-free survival. This unmet clinical need is underpinned by the adherence of AML cells in the protective niche of the BM micro-environment (BMME). Therefore, disrupting this adhesion and releasing AML cells into the peripheral blood, where they are more susceptible to chemotherapy, could unlock exciting new therapeutic strategies.This thesis describes the development and optimisation of a robust, reproducible, in vitro co-culture model of the AML-BMME. The optimised BM adhesion system (BMAS) comprises of stromal (HS-5), endothelial cells (HUVEC), and osteoblasts (hFOB 1.19) in equal proportions. Using BMAS, two AML cell lines, KG1a and OCI-AML3, were found to be 77% and 72% adhesive respectively. The BMAS was initially used to establish differences in adhesion markers between adhered and non-adhered AML cells. Adhered AML cells expressed higher levels of surface CD34 using KG1a, OCI-AML3 and primary AML cells. CD38 was also higher in adhered KG1a cells and CD44 was higher in both adhered OCI-AML3 and KG1a cells, but variable in primary AML cells. Interestingly, there was a significant correlation between CD44 and CD34 levels in primary cells.
The BMAS was next shown to recapitulate the cell adhesion mediated drug resistance (CAM-DR) seen clinically as adhered AML cells were most resistant to treatment with Cytarabine. Several adhesion blocking agents were tested to reduce the number of adhered AML cells. Anti-CD44 treatment was established as the most effective in preventing AML adhesion of OCI-AML3, KG1a and primary AML cells. Combining anti-CD44 with Cytarabine significantly increased AML apoptosis in both cell lines and primary cells. Importantly, the combination of 5µM Cytarabine and 5µg/mL anti-CD44 increased apoptosis of primary AML cells more than the additive effect of either alone.
However, even at the highest dose of anti-CD44, nearly 50% of the AML cells remained adhered. These were isolated and characterised transcriptionally to identify novel druggable targets. A paired transcriptional comparison of adhered versus non-adhered anti-CD44 pre-treated AML cells identified focal adhesion (FAK) as a novel target. Defactinib (dual FAK and PYK2 inhibitor) caused a significant decrease in adhered KG1a cells, and its combination with anti-CD44 prevented adhesion better than either alone.
Taken together, the unique BMAS has revealed novel ways to target AML within the BMME and provides a platform for testing combination therapies designed to overcome CAM-DR and improve AML chemotherapeutic susceptibility. It also has the potential to explore patient heterogeneity and develop personalised therapeutic strategies.
| Date of Award | Sept 2023 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Andrea Pepper (Supervisor) & Chris Pepper (Supervisor) |