Developing novel therapeutic strategies for the treatment of TET2-mutated acute myeloid leukaemia

Ph. D. Thesis.Acute myeloid leukaemia (AML) is a clinically heterogeneous disease driven by somatic mutations and chromosomal abnormalities. TET2 mutations occur in 7%-23% of AML, with approximately 75% monoallelic and 25% biallelic mutations. TET2 encodes a protein involved in regulating DNA demethylation and TET2-mutated AML has a significantly lower event-free survival and higher relapse rate compared to disease with wild-type TET2. As such, there is an urgent clinical need to develop novel methods to treat patients with TET2-mutated AML. Hypomethylating agents such as azacytidine that inhibits DNA methylation are now established as a therapeutic strategy for AML. This study evolved from an index patient with biallelic TET2-mutated AML, that was chemoresistant to anthracycline and cytarabine, but acutely sensitive to azacytidine monotherapy resulting in long-term morphological remission with overall survival of 850 days. The primary focus of my research is to interrogate the role of TET2 mutant allele dosage in determining AML cell response to azacytidine. Using an isogenic cell model system, we demonstrated that biallelic TET2 mutation leads to complete loss of TET2 protein and acquisition of a hypermethylated phenotype. Of note, TET2 mutant allele dosage significantly affected the azacytidine sensitivity of cells. TET2 biallelic mutants were hypersensitive to azacytidine with significantly lower proliferation in liquid media (P<0.0001) and cloning efficiency (P=0.0056) compared to their isogenic TET2 monoallelic mutants. RNA-sequencing identified ABCB1, a major azacytidine efflux transporter, to be downregulated in cells with TET2 biallelic mutation. Inhibition of ABCB1 using selective inhibitors (Tariquidar and Verapamil) increased sensitivity to azacytidine. Also, azacytidine resistant AML cell clones were developed through long-term exposure of TET2 mutated cells to azacytidine. ABCB1 is upregulated in resistant clones compared to the azacytidine sensitive cells and inhibition of ABCB1 re-sensitised the resistant clones to azacytidine. In addition, gene ontology analysis identified ribosomal pathway components to be downregulated in TET2 biallelic mutated cells compared to their isogenic TET2 monoallelic mutated counterparts. As such, the findings of this investigation suggest the potential involvement of multiple genes and pathways in sensitizing TET2 null cells to azacytidine. Finally, using ten different AML cell lines, we demonstrated that there was significant correlation between TET2 protein expression and azacytidine IC90 (R2=0.77, P=0.0008) and IC50 (R2=0.88, P<0.0001). Together, our findings argue in favour of using azacytidine for the treatment of TET2 biallelic mutated AML and highlights the importance of TET2 mutant allele dosage in developing precision medicine in AML.Newcastle University Overseas Research Scholarship (NUORS), Funds for Women Graduates (FFWG), Bloodwis