Final Report Summary - HSCS AND HIF (The role of hypoxia-inducible factors in human haematopoietic stem cell biology and leukaemogenesis)
Hematopoietic stem/progenitor cells (HSPCs), which reside in bone marrow niches, are exposed to low levels of oxygen and follow an oxygen gradient throughout their differentiation. Hypoxia-inducible factors (HIFs) are the main factors regulating the cell response to oxygen variation. However the role of HIF-1a and HIF-2a in human HSPCs is still unclear.
During the period of my Marie Curie grant I have studied the role of HIF factors in normal hematopoiesis and leukemia development. These are the results obtained:
1. We have shown for the first time that HIF-1a and HIF-2a subunits are expressed in freshly isolated HSPCs cord blood-derived cells.
2. To investigate the role of each of the HIF-a subunits we constructed lentiviral vectors containing small hairpin RNA (shRNA) against HIF-1a and HIF-2a subunits. We have confirmed the efficient knock down both at the mRNA and protein levels.
3. We have shown that silencing HIF-2a and to a lesser extent HIF-1a affects human short-term repopulating cells in vivo using the NSG (NOD-SCID/IL2Rgnull) xenotransplantation model. We have observed that the defect in the engraftment is not due to a homing defect.
4. To address the impact that each of the HIF-a subunits could have in the self-renewal capacity of human long-term HSPCs we have performed secondary transplantations. We have shown that knockdown (KD) of HIF-2a but not HIF-1a impairs the long-term reconstitution ability of human HSCs, demonstrating that the maintenance of human HSCs with self-renewing ability is dependent on HIF-2a.
5. To better understand the role of HIF-a in HSPCs, we knocked-down the binding partner of the HIF-a subunits, the constitutively expressed beta subunit (HIF-1beta). We have demonstrated that HIF-1beta KD in HSPCs recapitulates the HIF-2a KD phenotype, confirming the role of HIF complexes in the maintenance of human HSPCs.
6. To better understand the molecular mechanisms by which HIF-2a regulates HSPC biology, we have performed a large-scale gene expression analysis in KD cells compared to control. Gene pathway enrichment analysis identified the oxidative stress, endoplasmic reticulum (ER) stress response and apoptosis pathways as those with the largest change in gene signature. We have validated this and demonstrated that:
• HIF-2a KD increases ROS production and affects mitochondria homeostasis.
• The increase in ROS production in HIF2a KD cells induces the Endoplasmic Reticulum (ER) stress and triggers the Unfolded Protein Response (UPR) signalling pathway.
• Treatment with tunicamycin (TM), a well-known inducer of the UPR pathway, increases the percentage of apoptotic cells in shHIF2a cells compared to shCtl. This suggests that HIF-2a silenced HSPCs have significantly increased levels of ER stress and are more susceptible to apoptosis as a result.
• Finally we rescue the apoptosis caused by TM by pre-treating the cells with either NAC or tempol, two potent antioxidants, as significantly fewer apoptotic cells are observed.
7. In solid tumors, the expression of HIF-2a has been associated with poor prognosis and we therefore decided to investigate the effect of the KD of HIF-2a on human primary Acute Myeloid Leukemia (AML). We have observed that HIF-2a KD impedes the growth of human AML, both in vitro and in vivo. In addition, HIF-2a KD cells are more susceptible to apoptosis as a result of ER stress.
Conclussions
In this work, we examine the effect of the knockdown (KD) of HIF-1a and HIF-2a in human HSPCs. We observe that silencing HIF-2a, and to a lesser degree HIF-1a, impedes the repopulating capacity of HSPC-derived umbilical cord blood (UCB) in vivo. We demonstrate that HIF-2a KD HSPCs show signs of endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR) pathway, which ultimately affects the survival of the HSPCs. Moreover, we observe that the increase in the ER stress response in HIF-2a KD cells is due to an increase in reactive oxygen species (ROS) production. We also describe that cells from AML patient samples are dependent on the level of HIF-2a for their survival. Collectively, our report provides further evidence of a central role for HIF-2a in protecting HSPCs and AML cells from apoptosis induced by ER stress.
Socio-economic impact
Defining the role of HIF in HSPCs provides for the first time a molecular link between the hypoxic niche and quiescent HSPCs and helps to understand how HSPCs are maintained in their niches. Nowadays it is starting to be accepted that leukemic stem cells (LSCs) contribute to tumor progression through preferential resistance to radiation and chemotherapy. Therefore the elucidation of new molecular regulators essential for the normal stem cell biology that might be deregulated in LSCs is crucial for the development of anti-cancer therapies
During the period of my Marie Curie grant I have studied the role of HIF factors in normal hematopoiesis and leukemia development. These are the results obtained:
1. We have shown for the first time that HIF-1a and HIF-2a subunits are expressed in freshly isolated HSPCs cord blood-derived cells.
2. To investigate the role of each of the HIF-a subunits we constructed lentiviral vectors containing small hairpin RNA (shRNA) against HIF-1a and HIF-2a subunits. We have confirmed the efficient knock down both at the mRNA and protein levels.
3. We have shown that silencing HIF-2a and to a lesser extent HIF-1a affects human short-term repopulating cells in vivo using the NSG (NOD-SCID/IL2Rgnull) xenotransplantation model. We have observed that the defect in the engraftment is not due to a homing defect.
4. To address the impact that each of the HIF-a subunits could have in the self-renewal capacity of human long-term HSPCs we have performed secondary transplantations. We have shown that knockdown (KD) of HIF-2a but not HIF-1a impairs the long-term reconstitution ability of human HSCs, demonstrating that the maintenance of human HSCs with self-renewing ability is dependent on HIF-2a.
5. To better understand the role of HIF-a in HSPCs, we knocked-down the binding partner of the HIF-a subunits, the constitutively expressed beta subunit (HIF-1beta). We have demonstrated that HIF-1beta KD in HSPCs recapitulates the HIF-2a KD phenotype, confirming the role of HIF complexes in the maintenance of human HSPCs.
6. To better understand the molecular mechanisms by which HIF-2a regulates HSPC biology, we have performed a large-scale gene expression analysis in KD cells compared to control. Gene pathway enrichment analysis identified the oxidative stress, endoplasmic reticulum (ER) stress response and apoptosis pathways as those with the largest change in gene signature. We have validated this and demonstrated that:
• HIF-2a KD increases ROS production and affects mitochondria homeostasis.
• The increase in ROS production in HIF2a KD cells induces the Endoplasmic Reticulum (ER) stress and triggers the Unfolded Protein Response (UPR) signalling pathway.
• Treatment with tunicamycin (TM), a well-known inducer of the UPR pathway, increases the percentage of apoptotic cells in shHIF2a cells compared to shCtl. This suggests that HIF-2a silenced HSPCs have significantly increased levels of ER stress and are more susceptible to apoptosis as a result.
• Finally we rescue the apoptosis caused by TM by pre-treating the cells with either NAC or tempol, two potent antioxidants, as significantly fewer apoptotic cells are observed.
7. In solid tumors, the expression of HIF-2a has been associated with poor prognosis and we therefore decided to investigate the effect of the KD of HIF-2a on human primary Acute Myeloid Leukemia (AML). We have observed that HIF-2a KD impedes the growth of human AML, both in vitro and in vivo. In addition, HIF-2a KD cells are more susceptible to apoptosis as a result of ER stress.
Conclussions
In this work, we examine the effect of the knockdown (KD) of HIF-1a and HIF-2a in human HSPCs. We observe that silencing HIF-2a, and to a lesser degree HIF-1a, impedes the repopulating capacity of HSPC-derived umbilical cord blood (UCB) in vivo. We demonstrate that HIF-2a KD HSPCs show signs of endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR) pathway, which ultimately affects the survival of the HSPCs. Moreover, we observe that the increase in the ER stress response in HIF-2a KD cells is due to an increase in reactive oxygen species (ROS) production. We also describe that cells from AML patient samples are dependent on the level of HIF-2a for their survival. Collectively, our report provides further evidence of a central role for HIF-2a in protecting HSPCs and AML cells from apoptosis induced by ER stress.
Socio-economic impact
Defining the role of HIF in HSPCs provides for the first time a molecular link between the hypoxic niche and quiescent HSPCs and helps to understand how HSPCs are maintained in their niches. Nowadays it is starting to be accepted that leukemic stem cells (LSCs) contribute to tumor progression through preferential resistance to radiation and chemotherapy. Therefore the elucidation of new molecular regulators essential for the normal stem cell biology that might be deregulated in LSCs is crucial for the development of anti-cancer therapies