Role of prolyl hyrdoxylase domain enzymes in normal and malignant haematopoiesis

Abstract

Acute Myeloid Leukaemia (AML) arises from a dysregulation in haematopoietic stem and progenitor cells (HSPCs), leading to the production of highly proliferative blast cells. Current chemotherapies can eliminate these cells but fail to eradicate the leukaemic stem cell (LSC) source. There has been increasing interest in the microenvironment in which LSCs, as well as their haematopoietic stem cell (HSC) counterparts reside, with particular focus on the hypoxic nature of the niche. As such, this led us to investigate Hypoxia Inducible Factors (HIFs) in leukaemia development where our previously published data shows that HIFs are tumour suppressors in AML. This thesis builds on this work, investigating the therapeutic potential of Prolyl Hydroxylases Domain (PHD) inhibition, which stabilises HIFs. Results show that deletion of the Phd isoforms Phd1 and Phd2 reduces leukaemic transformation and development in vitro. Additionally, when leukaemic cells deficient in Phd1 and Phd2 were transplanted into syngeneic lethally irradiated recipient mice, there was a reduction in leukaemic engraftment, resulting in an increase in disease latency when compared to the control cells. In addition, I further validated this result by using a doxycycline-inducible shRNA model to knockdown levels of Phd2 in leukaemic cells. Mirroring the results of the gene deletion study, Phd2 knockdown led to a regression of leukaemia both in vitro and in vivo. To further validate PHDs as therapeutic targets in AML, I investigated the impact of deletion of Phd1 and Phd2 specifically in the hematopoietic system using Vav-iCre. Steady state analysis of the HSPC and differentiated cell compartments found there were no significant differences when compared to control mice. Moreover, further utilising the inducible shRNA model, I acutely deleted Phd2 in vivo, discovering a decrease in the frequency and functionality of the HSC compartment, with no other discernible phenotypes. Given there were no significant detrimental effects on normal haematopoiesis, this study provides a strong rationale for studying the inhibition of Phds in combination with current chemotherapeutic regimes, aiming to provide targeted therapies for AML.