Characterization of the surfaceome of hematopoetic and leukemic stem cells to improve the development of novel therapies for leukemia.
(2017) Cole Foundation research day — Location: Montreal, Canada
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- Authors
Aubert, Léo 
- Abstract
- Background Information: Hematopoietic stem cell transplantation (HSCT) has become a potent life-saving procedure for both adults and children with various hematological malignancies, including acute myeloid leukemia (AML). Umbilical cord blood (CB) appears as one of the most attractive sources of HSCs for transplantation, due to its immature immune system and widespread availability. Unfortunately, many patients are deprived from this therapeutic strategy as the low stem cell dose in CB units results in delayed engraftment and compromises transplantation outcome. Our collaborators from Guy Sauvageau’s group, have recently discovered a small-molecule, UM171, which stimulates the expansion of CB HSCs ex vivo. Interestingly, UM171 can also promote the in vitro maintenance of leukemic stem cells (LSCs), which are considered one of the major causes of therapeutic failure in AML. However, the unavailability of specific surface markers that can prospectively identify HSCs and LSCs is still a major hurdle for the optimization of CB grafts and the development of new targeted therapies. Purpose of the study: The aim of this collaborative research project is to identify new HSC and LSC surface markers using quantitative proteomic methods. These results will help better purify and characterize HSCs and LSCs in vitro, with the final purpose of helping patients with leukemic disorders either through the optimization of HSCT or the uncovering of new therapeutic targets. Methods: To discover novel and reliable HSC and LSC surface markers, we have optimized and adapted a cutting-edge chemoproteomic approach based on the labeling of cell surface proteins with cell-impermeable biotin reagents, their subsequent purification with avidin chromatography, and quantification using label-free quantitative proteomics with liquid chromatography-tandem mass spectrometry. Results: As a proof of concept experiment, we used this quantitative proteomic approach in the hematopoietic cell line OCI-AML5 in combination with UM171-induced cell expansion. We found that UM171 promotes the upregulation of more than 35 cell surface proteins. Among them, we identified the endothelial protein C receptor (EPCR) which we recently characterized as a novel surface marker for the HSC population derived from CB expanded with UM171. To optimize this method for the analysis of CB cells, we performed a OCI-AML5 cell titration and determined the minimal number of hematopoietic cells required. We then performed a surface proteomics analysis of cell populations from UM171-expanded CB units and sorted according to levels of CD34 and EPCR. Preliminary results reveal the enrichment in more than 100 surface proteins in the CD34+EPCR+ population, and several of these proteins are currently being tested as potential new markers for HSCs. Conclusion: Altogether, these results validate the surfaceome approach for the identification of novel HSC- and LSC-specific surface molecules using CB units and primary AML specimens, respectively.
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Citations
Aubert, L., Chagraouit, J., Bordeleau, M.-E., Farès, I., Girard, S., Sauvageau, G., & Roux, P. P. (2017). Characterization of the surfaceome of hematopoetic and leukemic stem cells to improve the development of novel therapies for leukemia. Cole Foundation research day, Montreal, Canada. https://hdl.handle.net/2078.5/24900