MOLECULAR CHARACTERIZATION OF LEUKEMIC STEM CELLS IN CHRONIC MYELOID LEUKEMIA BY SMALL RNA SEQUENCING

RUIZ MS; BONECKER S; SANCHEZ MB; FURTADO C; KOILE D; YANKILEVICH P; ZALCBERG I; MORDOH J; BIANCHINI M

Congreso; Reunión conjunta de sociedades de biociencias; 2017

Molecular relapse after treatment discontinuation in chronic myeloid leukemia (CML) has been attributed to the persistence of leukemic stem cells (LSCs), which share multiple features with their normal counterparts (hematopoietic stem cells, HSCs). In order to contribute to their molecular characterization, we performed next-generation sequencing (NGS) of small RNAs comparing LSCs vs. HSCs in CML patients. Materials and methods: bone marrow or peripheral blood CD34+ cells from CML patients at diagnosis were sorted into a LSC-enriched (CD34+CD38-CD26+) and a HSC-enriched (CD34+CD38-CD26-) fraction. Purity was assessed by BCR-ABL1 RT-qPCR in colony-forming unit (CFU) assays. Total RNA preserving the small RNA fraction was directly isolated from sorted cells. Small RNA NGS libraries were sequenced using the Illumina HiSeq 2500 platform. MicroRNA counts and differential expression values were obtained by Chimira and GFOLD algorithms. In silico predictions of targets and pathways were performed with miRPath. Results: The protocol for cell sorting and RNA extraction from highly pure, low-abundance fractions was optimized and performed in 7 CML patients and 5 healthy donors. Given the low yield of RNA, we pooled samples from different patients or donors. LSC- vs. HSC-enriched fractions resulted in 100% vs. 8.3% of BCR-ABL1+ CFUs, respectively (mean %BCR-ABL1/ABL1: 99.7% vs. 0.95%, n=2). One hundred and twenty-three microRNAs were differentially expressed in LSC- vs. HSC-enriched fractions from CML patients. Comparison against healthy donors resulted in 19 microRNAs that clustered in potentially relevant pathways such as ECM-receptor interaction, regulation of pluripotency of stem cells, and proteoglicans in cancer. Conclusions: As far as we are concerned, this is the first report of the miRNome of highly enriched LSC and HSC fractions in CML patients by NGS, providing valuable data for the search of LSC-specific therapeutic targets.