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Proteomic characterization of CD34+ CD123+ stem cells from patients with acute myeloid leukemia

ABSTRACT: The comparative characterization of hematopoietic stem cells from healthy stem cell donors and patients with acute myeloid leukemia on a proteome level has the potential to reveal differentially regulated proteins which might be candidates for specific immunotherapy target molecules. Exemplarily, we analyzed the proteome of the cytosolic and the membrane fraction of CD34 and CD123 co-expressing FACS-sorted leukemic progenitors from five patients with acute myeloid leukemia employing mass spectrometry. As a reference, CD34+CD123+ normal hematopoietic progenitor cells from five healthy stem cell donors were analyzed. In this TMT 10-plex labeling based approach 2068 proteins were identified with 256 proteins differentially regulated in one or both cellular compartments. This study demonstrates the feasibility of a mass spectrometry based proteomic approach to detect differentially expressed proteins in two compartment fractions of leukemic stem cells as compared to their healthy stem cell counterparts.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Cell, Stem Cell, Blood

DISEASE(S): Acute Leukemia

SUBMITTER: Johannes R. Schmidt

LAB HEAD: Martin von Bergen

PROVIDER: PXD008378 | Pride | 2018-02-19

REPOSITORIES: Pride

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Pilot Study on Mass Spectrometry-Based Analysis of the Proteome of CD34⁺CD123⁺ Progenitor Cells for the Identification of Potential Targets for Immunotherapy in Acute Myeloid Leukemia.

Schmidt Johannes R JR Rücker-Braun Elke E Heidrich Katharina K von Bonin Malte M Stölzel Friedrich F Thiede Christian C Middeke Jan M JM Ehninger Gerhard G Bornhäuser Martin M Schetelig Johannes J Schubert Kristin K von Bergen Martin M Heidenreich Falk F

Proteomes 20180212 1

Targeting of leukemic stem cells with specific immunotherapy would be an ideal approach for the treatment of myeloid malignancies, but suitable epitopes are unknown. The comparative proteome-level characterization of hematopoietic stem and progenitor cells from healthy stem cell donors and patients with acute myeloid leukemia has the potential to reveal differentially expressed proteins which can be used as surface-markers or as proxies for affected molecular pathways. We employed mass spectrome ...[more]

PMID: 29439554

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Project description:We show the molecular and functional characterization of a novel population of lineage-negative CD34-negative (Lin- CD34-) hematopoietic stem cells (HSCs) from chronic myelogenous leukemia (CML) patients at diagnosis. Molecular caryotyping and quantitative analysis of BCR/ABL transcript demonstrated that about one third of CD34- was leukemic. CML CD34- cells showed kinetic quiescence and limited clonogenic capacity. However, stroma-dependent cultures and cytokines induced CD34 expression on some HSCs, cell cycling, acquisition of clonogenic activity and increased expression of BCR/ABL transcript. CML CD34- cells showed an engraftment rate in immunodeficient mice similar to that of CD34+ cells. Gene expression profiling revealed the down-regulation of cell cycle arrest genes together with genes involved in antigen presentation and processing, while the expression of angiogenic factors was strongly up-regulated when compared to normal counterparts. Flow cytometry analysis confirmed the significant down-regulation of HLA class I and II molecules in CML CD34-cells. Increasing doses of imatinib mesilate (IM) did not affect fusion transcript levels, BCR-ABL kinase activity and the clonogenic efficiency of CML CD34- cells as compared to leukemic CD34+cells. Thus, we identified in CML a novel CD34- leukemic stem cell subset with peculiar molecular and functional characteristics which may be a potential target for CML therapeutics. Leukemic cells were obtained from 12 chronic phase Ph+ CML patients at diagnosis and before treatment. Normal samples were leukapheresis products from 12 healthy stem cell donors receiving recombinant human granulocyte colony-stimulating factor (G-CSF; Lenograstim, Sanofi-Aventis, Milan, Italy). The protocol was approved by the ethical committee of the University Hospital and each patient/donor gave written informed consent. Hemopoietic stem/progenitor cell purification and phenotypic analyses were performed as previously described (Lemoli et al, Br J Haematol, 2003; Lemoli RM et al., Blood, 1997). Aliquots of sorted Lin-CD34-, Lin-CD34+ and Lin+CD34+ were reanalyzed by FacScan (Becton Dickinson, Franklin Lakes, NJ) to assess their purities. Total cellular RNA was extracted from 0.5x105 cells of each sample using RNeasy Micro kit (Qiagen, Valencia, CA) following the protocol supplied by the manufacturer. Disposable RNA chips (Agilent RNA 6000 Nano LabChip kit, Agilent Technologies, Waldbrunn, Germany) were used to determine the concentration and purity/integrity of RNA samples using Agilent 2100 Bioanalyzer. RNAs originating from 12 normal donors or from 12 CML patients were pooled in order to obtain at least 2 mg per sample. One-cycle target labeling assays, as well as the Affymetrix Human HG-U95Av2 GeneChip arrays hybridization, staining, and scanning, were performed, using Affymetrix standard protocols (Affymetrix, Santa Clara,CA).

			Action	DRS
	AML.msf	Msf
	AML.pep.xml	Pepxml
	AML_TMT10_plex_cytosolic_profile_NCE38.raw	Raw
	AML_TMT10_plex_membrane_profile_NCE38.raw	Raw
	SEARCH.txt	Txt