Contributions
Abstract: EP415
Type: E-Poster Presentation
Session title: Acute myeloid leukemia - Biology & Translational Research
Background
Current cytotoxic treatments for acute myeloid leukemia (AML) eliminate the bulk of proliferating cells, but leave the majority of quiescent leukemic stem cells (LSC) and chemoresistant cells unaffected. The latter, might be able to generate a subpopulation of cells with the same characteristics, promoting chemotherapy resistance. A previous in silico study performed in our laboratory identified the prolactin receptor (PRLR) as one of the main pathways implicated in AML progression and the study of its signalling pathways lead us to investigate its role in chemoresistance and AML progression.
Aims
The main objective was the identification of the role of the prolactin (PRL):PRLR axis in the transformation events associated with the initiation and maintenance of AML and its druggable potential.
Methods
The surface expression of PRLR was assessed by flow cytometry. The effect in cell viability of the natural ligand prolactin (hPRL) and its antagonist (hPRL-G129R) was studied by flow cytometry. Experiments using wildtype (PRLRwt) and a dominant negative (PRLRsh) receptor ectopically expressed and an active (PRLwt) and a dominant negative (PRLmut) ligand expressed on AML cells were performed to assess their effect on chemotherapy resistance. Gene expression analyses were done by real time quantitative PCR. To determine the role of PRLR-PRL axis in vivo, conditioned NSG mice were transplanted with RLuc-AML cells and mice were treated with hPRL or hPRL-G129R and monitored during the experiment. Next, conditioned NSG mice were transplanted with PRLwt- and PRLmut-transduced AML cells and the engraftment was analysed by flow cytometry.
Results
PRLR surface expression correlated positively with a higher resistance to Cytarabine, but not Azacytidine or Daunorubicine. The same results were observed when PRLRwt isoform was overexpressed. Nevertheless, changes in genes related to Cytarabine resistance were not observed and no differences were observed on PRLR surface expression in refractory vs non-refractory AML primary samples. On the other hand, cells transduced to secrete active PRL did not possess such resistance.
PRLRwt overexpression promoted a higher proliferative rate in comparison to parental and PRLRsh transduced cells. In addition, hPRL treatment, but not hPRL-G129R, induced proliferation only due to ectopic PRLRwt expression. hPRL treatment increased the proliferation of AML cells in comparison to hPRL-G129R treatment in vivo. What is more, mice injected with PRLmut transduced cells had a lower engraftment capacity in comparison to PRLwt injected ones in vivo and a lower clonogenicity capacity in vitro.
Conclusion
PRLR expression, but not increased levels of its ligand PRL, confers resistance to Cytarabine in vitro, but genes implicated in this process were not affected, suggesting that other mechanism should be involved.
PRL-PRLR signalling promotes proliferation signals in AML, both in vivo and in vitro. An overexpression of PRLR generates a worse AML prognosis due to its effect in proliferation, while antagonizing the receptor decreases proliferation. Interestingly, the inactive form of hPRL confers a lower clonogenicity in vitro and engraftment capacity in vivo.
In summary, PRLR and PRL have an important role in AML proliferation and prognosis, so targeting PRLR and using inactive forms of its ligand may be a useful treatment against AML.
Keyword(s): Acute myeloid leukemia, Drug resistance, Receptor tyrosine kinase, Therapy
Abstract: EP415
Type: E-Poster Presentation
Session title: Acute myeloid leukemia - Biology & Translational Research
Background
Current cytotoxic treatments for acute myeloid leukemia (AML) eliminate the bulk of proliferating cells, but leave the majority of quiescent leukemic stem cells (LSC) and chemoresistant cells unaffected. The latter, might be able to generate a subpopulation of cells with the same characteristics, promoting chemotherapy resistance. A previous in silico study performed in our laboratory identified the prolactin receptor (PRLR) as one of the main pathways implicated in AML progression and the study of its signalling pathways lead us to investigate its role in chemoresistance and AML progression.
Aims
The main objective was the identification of the role of the prolactin (PRL):PRLR axis in the transformation events associated with the initiation and maintenance of AML and its druggable potential.
Methods
The surface expression of PRLR was assessed by flow cytometry. The effect in cell viability of the natural ligand prolactin (hPRL) and its antagonist (hPRL-G129R) was studied by flow cytometry. Experiments using wildtype (PRLRwt) and a dominant negative (PRLRsh) receptor ectopically expressed and an active (PRLwt) and a dominant negative (PRLmut) ligand expressed on AML cells were performed to assess their effect on chemotherapy resistance. Gene expression analyses were done by real time quantitative PCR. To determine the role of PRLR-PRL axis in vivo, conditioned NSG mice were transplanted with RLuc-AML cells and mice were treated with hPRL or hPRL-G129R and monitored during the experiment. Next, conditioned NSG mice were transplanted with PRLwt- and PRLmut-transduced AML cells and the engraftment was analysed by flow cytometry.
Results
PRLR surface expression correlated positively with a higher resistance to Cytarabine, but not Azacytidine or Daunorubicine. The same results were observed when PRLRwt isoform was overexpressed. Nevertheless, changes in genes related to Cytarabine resistance were not observed and no differences were observed on PRLR surface expression in refractory vs non-refractory AML primary samples. On the other hand, cells transduced to secrete active PRL did not possess such resistance.
PRLRwt overexpression promoted a higher proliferative rate in comparison to parental and PRLRsh transduced cells. In addition, hPRL treatment, but not hPRL-G129R, induced proliferation only due to ectopic PRLRwt expression. hPRL treatment increased the proliferation of AML cells in comparison to hPRL-G129R treatment in vivo. What is more, mice injected with PRLmut transduced cells had a lower engraftment capacity in comparison to PRLwt injected ones in vivo and a lower clonogenicity capacity in vitro.
Conclusion
PRLR expression, but not increased levels of its ligand PRL, confers resistance to Cytarabine in vitro, but genes implicated in this process were not affected, suggesting that other mechanism should be involved.
PRL-PRLR signalling promotes proliferation signals in AML, both in vivo and in vitro. An overexpression of PRLR generates a worse AML prognosis due to its effect in proliferation, while antagonizing the receptor decreases proliferation. Interestingly, the inactive form of hPRL confers a lower clonogenicity in vitro and engraftment capacity in vivo.
In summary, PRLR and PRL have an important role in AML proliferation and prognosis, so targeting PRLR and using inactive forms of its ligand may be a useful treatment against AML.
Keyword(s): Acute myeloid leukemia, Drug resistance, Receptor tyrosine kinase, Therapy