| Summary: | Chronic myeloid leukaemia (CML) is the neoplastic expansion of haematopoietic stem/progenitor cells caused by BCR-ABL, a constitutively active tyrosine kinase. CML progresses through three stages by the modulation of the proliferation, survival and differentiation of haematopoietic progenitor cells. This is reversed by the BCR-ABL specific inhibitor STI571 (Oleevec, Imatinib). BCR-ABL mediates its oncogenic activities through the activation of different pathways, most notably through the activation of the phosphoinositide 3-kinase (PI3K) / protein kinase B (PKB/AKT) pathway. The activation of this pathway recapitulated BCR-ABL transformation of haematopoietic progenitor cells, and its inhibition resulted in proliferation arrest. apoptosis and differentiation of CML cells. A major regulator of signals emanating from the PI3K1PKB pathway is the forkhead box 0 (FOXO) family of transcription factors. FOXOs and in particular, FOX03a, are important regulators of proliferation, survival, diiTerentiation and stress resistance. Therefore, FOX03a could be a potential mediator of BCR-ABL signalling in CML cells. Indeed, FOX03a was shown to be highly phosphorylated in BCR-ABL expressing cells. This inhibitory PKB-dependent phosphorylation was abolished after STI571 treatment, leading to FOX03a activation. I-Iere, FOX03a is shown to directly bind the BIM promoter in vitro and in vivo and activate its expression through a forkhead response element (FHRE). This resulted in the induction of apoptosis that was inhibited in FOX03a- or BIM-deficient BCR-ABL expressing cells. FOX03a also activated p27':ip l expression downstream of STI571 signalling, through direct binding and recruitment of p300 co-activator. CYCLIN D2, on the other hand, is downregulated in a FOX03a-dependent fashion. This repression is mediated by FOX03a direct activation of the BCL6 repressor, BCL6 then binds eYCLIN D2 promoter leading to its repression. The regulation of p27Kipi and CYCLIN D2 is important for the regulation of proliferation ofCML cells. STl571 treatment of K562 CML cells leads to their differentiation towards the erythroid lineage. This is shown to be mediated by FOX03a direct repression of IDl. FOX03a binds the FHRE site within the IDl promoter in vivo and in vitro, leading to its downregulation. The identification of IDl as R novel direct target of FOX03a dependent repression was further investigated to understand the molecular mechanisms involved in this regulation. FOX03a regulation of IDI was achieved through the direct binding of FOX03a to inhibitor of growth Ib (INO1b) protein, a p53 binding protein. The two proteins cooperate to recruit the mSIN3A/HDACl complex to IDl promoter, leading to histone tails' deacetylation. The deacetylation of histone tails result in a closed chromatin state favouring repression. FQX03a and INO1b bind IDI promoter at the FHRE and the adjacent AT rich region,· respectively. Furthermore, a genetic interaction between H JXO]a, ING1h and If) I is shown to regulate erythroid differentiati.on of STl571-treated ((562 cells.. ING 1b is also shown to attenuate FOX03a dependent activation by delaying the activation of proliferation but not the apoptotic genes. Together. these findings provide evidence for a role for FOX03a as a tumour suppressor. regulating survival. proliferation and differentiation of CML cells. They also provide insights into the transcriptional regulation by FOX03a, and the molecular mechanisms governing target gene expression. Interestingly, this work shows a mechanistic parallel between FOXO- and p53- dependent signalling, transcriptional regulation and proteinprotein interactions. This work is important for future targeted therapy in CML, and other malignancies and disease where the PI3K1AKT/FOXO signalling pathway is deregulated.
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