PF399 GENE EXPRESSION PROFILING OF CD26+ LEUKEMIC STEM CELL POPULATION FROM CML PATIENTS

Background: Nowadays, chronic myeloid leukemia (CML) has become a well manageable disease and majority of the patients achieve remission on tyrosine kinase inhibitor (TKI) treatment. However, the disease usually shows a low‐level persistence during therapy that arises from putative leukemic stem cells (LSC), which are, despite BCR‐ABL1 positivity, resistant to TKI treatment. LSC can be identified and isolated based on a surface expression of specific surface markers of which CD26 is perhaps the best described with high positive correlation with the occurrence of BCR‐ABL1 . Aims: Our aim was to compare gene expression (GE) profiles of CD26+ LSC, CD26‐ HSC and CD38+ progenitor cells (PC) from CML patients, and identify potential novel CML LSC markers or disrupted pathways. Methods: Bone marrow (BM) cells were obtained from 18 CML patients and stained for the following surface markers – CD45, CD34, CD38 and CD26. Cells were FACS sorted into 3 populations – LSC (CD34+CD38‐CD26+), HSC (CD34+CD38‐CD26‐) and PC (CD34+CD38+). cDNA samples from 5 patients were pre‐amplified with Ovation Pico WTA System V2 whole transcriptome amplification kit (NuGEN) and analysed on SurePrint G3 human GE 8x60k microarray (Agilent). Gene expression data validation was performed on the same cell populations collected from a novel extended cohort of 13 CML patients on Wafergen SmartChip real‐time PCR system. Results: Analysis of GE microarray data identified over 272 differentially expressed genes (160 upregulated and 112 downregulated) between CD26‐ HSC and CD26+ LSC, and over 1330 differentially expressed genes (531 upregulated and 799 downregulated) between CD26‐ HSC and CD38+ progenitors. (P ≤ 0.05; fold change ≥ 1). The subset of the most deregulated genes between populations was validated on an extended cohort of different CML patients using SmartChip real‐time PCR, showing high correlation between both systems (R 2 LSC  = 0.808; R 2 PC  = 0.913) and supporting the results of GE microarray analysis. Validated candidate genes (n = 15), consisting of those involved in important pathways (e.g. IL1, mTOR signalling ‐ TAB2, PELI2, MYCBP2) or associated with oncogenic transformation (e.g. MYB, RAD51), will be further studied in functional assays. Of highest interest are the top deregulated genes histidine decarboxylase (HDC) and immunoglobulin J chain (IGJ), whose expression was previously reported deregulated in BCR‐ABL1 positive cells or CML stem cells. Both HDC and IGJ exhibited opposite expression pattern in CD26+ LSC and CD38+ PC subpopulations compared to CD26‐ HSC (FC HDC  = −2.8 vs 4.3; FC IGJ  = 2.8 vs −2.8; FC ‐ fold diference in expression). Further, we identified CD69 (early lymphocyte activation antigen) as a gene specifically upregulated in CD26 + LSC, whose expression was also shown to be elevated in cell lines expressing BCR‐ABL1 . Currently, we are analysing its potential as a specific surface marker. Summary/Conclusion: In summary, we identified consistent changes of gene expression in CD26+ LSC as well as CD38+ progenitor populations, with only 3% gene overlap showing specificity of these patterns for selected populations. These results were successfully verified using high‐throughput real‐time PCR. The involvement of identified candidate genes and pathways, which could provide important insights into the CML stem cell biology, are currently evaluated. Supported by MUNI/A/1105/2018, by Ministry of Health of the Czech Republic, grant nr. 17–30397A, and by institutional support of Faculty of Medicine, MU.