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The finely tuned regulation of haematopoietic stem and progenitor cells (HSPCs) is crucial to ensure a normal haematopoiesis. Its dysregulation can generate leukaemic stem cells (LSCs) which are difficult to eliminate with current therapies, placing a focus on identifying novel therapeutic targets to eradicate LSCs. Emerging studies have described that key oncometabolites (such as succinate and fumarate) are main inhibitors of 2-oxoglutarate dependent dioxygenases (2-OGDO), a family of enzymes that have been reported to play an important role in leukaemogenesis. Here two of these 2-OGDO, Jmjd6 (a jumonji protein associated with alternative splicing) and Phd2 (a prolyl hydroxylase that controls hypoxic response), were investigated in normal haematopoiesis and acute myeloid leukaemia (AML).

The aim of this study is to investigate the role of two 2-OGDO (Phd2 and Jmjd6) in the development and/or maintenance of AML LSCs.

 

To elucidate the role of Jmjd6 in normal haematopoiesis, we generated a Jmjd6 conditional knock-out within the haematopoietic system (Jmjd6^fl/fl;Vav-iCre) and fully characterized it. To study the role of Jmjd6 and Phd2 in leukaemogenesis, purified HSPCs from Jmjd6^fl/fl;Vav-iCre and Phd2^fl/fl;Vav-iCre mice were transduced with retrovirus expressing respectively Mll-AF9 and Meis1/Hoxa9. These transformed cells were characterized in vitro (e.g. colony forming cell assay) and their leukemic potential was assessed by transplantation. The Mll-AF9 knock-in (Mll-AF9^KI/+) transgenic model was also used to determine levels of mRNA expression.

In normal haematopoiesis studies, mice lacking Jmjd6 specifically within the haematopoietic system presented hyposplenism and reduced spleen cellularity. Although these mice have normal numbers of stem and progenitor cells (namely Lin^-Sca-1⁺c-Kit⁺ (LSK)), the distribution within this compartment is perturbed, leading to an accumulation of more committed progenitor cells. This phenotype functionally correlates with the reduced engraftment exhibited by mice transplanted with cells lacking Jmjd6 upon transplantation with either Jmjd6^fl/fl;Vav-iCre HSCs or total bone marrow (BM) when compared to controls.

Preliminary data from the leukaemic studies showed that the absence of Jmjd6 does not impact on the generation of pre-leukaemic stem cells (pre-LSC). Conversely, LSCs from the transgenic Mll-AF9^KI/+ mouse model have decreased Jmjd6 mRNA transcript levels. In vitro, Meis1/HoxA9 transformed Phd2-deficient cells showed increased proliferation and lower apoptosis however, in vivo, mice transplanted with cells lacking Phd2 developed disease later than the respective controls.

Deletion of Jmjd6 in the haematopoietic system leads to an accumulation of committed progenitor cells that have a decreased self renewal capacity and, therefore, a compromised repopulating capacity. The deletion of Jmjd6 did not affect the in vitro generation of pre-LSC cells. With regard to Phd2, it was observed a delay in AML initiation in mice transplanted with Phd2 KO pre-LSCs. Taken together, these data unravel a new role for Jmjd6 as a player in normal haematopoiesis and suggest that Phd2 may act as tumor suppressor in AML.