The role of cytokines in governing the expansion of the T315I mutation in chronic myeloid leukaemia.

Abstract

Since the introduction of imatinib, the outlook for patients with chronic myeloid leukaemia (CML) has dramatically improved. However, approximately 30-40% of patients develop intolerance or resistance to the drug and cease therapy. Among those who develop resistance, the most common cause is the development of point mutations in the kinase domain (KD) of BCR-ABL1 which impair drug binding and hence result in the loss of kinase inhibition. The T315I mutation (gatekeeper mutation) is of particular significance as it is one of the most common mutations and it is resistant to all 3 tyrosine kinase inhibitors (TKI) currently approved for therapeutic use in Australia. There is evidence to suggest that KD mutations in BCR-ABL1 may alter the biological activity of Bcr-Abl. Both in vitro and clinical studies have suggested that the T315I mutation results in greater oncogenic potential. Patients harbouring this mutation have poorer prognoses and a significantly higher rate of progression compared to other mutations. There is also ample evidence to suggest that secretion of cytokines and growth factors play a role in rendering BCR-ABL1 positive cells resistant to TKI. This thesis focuses on the role of cytokines in the resistance mechanism of cells with the T315I mutation and how this mechanism is achieved. K562-T315I cells were developed in our laboratory by exposing them to increasing levels of dasatinib over several months. HL60 cell lines were virally transduced with the BCR-ABL1p210 [p210 in superscript] and BCR-ABL1T315I [T315I in superscript] constructs. Investigations have identified that several soluble factors are preferentially secreted by cells with the T315I mutation namely, FGF-2, IL-8, MCP-1 and G-CSF. Additionally, the supernatant of K562-T315I cells also contains higher concentrations of GM-CSF and IL-6. These studies also identified that FGF-2 was able to protect K562 naïve cells from TKI-induced cell death and suggests that this occurs via activation of the MAPK and STAT5 pathways. Thus, in addition to acquiring point mutations that result in the inability of TKIs to bind Bcr-Abl, the T315I mutation also results in overexpression of FGF-2 which can confer resistance to non-mutated cells. Furthermore, in the presence of imatinib, dasatinib and nilotinib, K562-T315I cells proliferate and survive better than in the absence of a TKI. This is due to hyperactivation of the MAPK pathway whereas signalling of other pathways, JAK/STAT5 and PI3K/Akt are not increased. This phenomenon was demonstrated especially with nilotinib but studies did not indicate a cytokine mediated effect through an autocrine hypersecretion by the K562-T315I cells. Nevertheless, this finding may be one of the reasons why the T315I mutation confers a worse outcome in patients with CML if they remain on imatinib, nilotinib and dasatinib treatment.