The Absence of Retroelement Activity Is Characteristic for Childhood Acute Leukemias and Adult Acute Lymphoblastic Leukemia

Abstract

Retroelements (RE) have been proposed as important players in cancerogenesis. Different cancer types are characterized by a different level of tumor-specific RE insertions. In previous studies, small cohorts of hematological malignancies, such as acute myeloid leukemia, multiple myeloma, and chronic lymphocytic leukemia have been characterized by a low level of RE insertional activity. Acute lymphoblastic leukemia (ALL) in adults and childhood acute leukemias have not been studied in this context. We performed a search for new RE insertions (Alu and L1) in 44 childhood ALL, 14 childhood acute myeloid leukemia, and 14 adult ALL samples using a highly sensitive NGS-based approach. First, we evaluated the method sensitivity revealing the 1% detection threshold for the proportion of cells with specific RE insertion. Following this result, we did not identify new tumor-specific RE insertions in the tested cohort of acute leukemia samples at the established level of sensitivity. Additionally, we analyzed the transcription levels of active L1 copies and found them increased. Thus, the increased transcription of active L1 copies is not sufficient for overt elevation of L1 retrotranspositional activity in leukemia.

Mobile retroelements induced by hypomethylating agents are restricted to transpose in myeloid malignancies

“…However, as we expected that novel L1 insertions could be present in minor subpopulations and the whole genome sequencing methods would have limited sensitivity to detect them, we applied a targeted-NGS-based approach with the ability to capture retrotransposition events in 1% of cells (26). This approach was previously used to demonstrate somatic retrotranspositions in brain tissue (37) and colorectal cancer (29) and the absence of RE activity in leukemia (26). Thus, we assume that the sensitivity of the NGS method was not the reason for the negative result.…”

“…A next-generation sequencing method for detecting new L1 insertions of the transpositionallyactive subfamily L1HS was used according to our previous reports (26,29). Briefly, gDNA was digested by a mixture of selected endonucleases TaqI and FspBI to generate fragments that consisted of a 3' part of L1 retroelement and its adjacent genomic region (i.e., flank).…”

“…The coordinates of the insertions in the serial samples were matched to identify insertions related to clonal propagation (i.e., compared to the corresponding pre-treatment sample). Following our previous report (26), candidate somatic L1 insertions were validated by an independent locus-specific PCR with initial gDNA as a template, and Encyclo Polymerase Mix (Evrogen) and/or Q5 High-Fidelity 2X Master Mix (New England Biolabs).…”

“…This result prompted us to proceed with detecting novel insertions using NGS. We applied a high-sensitive NGS-based protocol enabling us to detect L1 insertions down to 1% of cells (26). Candidate de novo L1 insertions for each time point (3, 7, 14, and 28 days after Aza-dC addition) were determined by comparing their genomic coordinates to the list of L1 insertion sites identified in the same cell line before treatment (day 0).…”

A Glance at Molecular Advances in Cancer Genetics: A Baffling Puzzle Still to Be Solved

“…Urazbakhtin et al and Choi et al, respectively, include more cryptic and neglected aspects of tumorigenesis, e.g., retrotranspositional activity in child and adult acute lymphloblastoid leukemia or oncogenic hotspot variants identification, such as that in GNAQ in the hepatocellular carcinoma [ 5 , 6 ].…”