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.

Keywords: acute leukemia; mobile elements; retroelements; tumor-specific insertions.

Figure 1

Principle of the method. gDNA was digested by either TaqI + FspBI (for L1), or FspBI + Csp6I (for Alu) and ligated to a stem-loop adapter (pink). Retroelement-specific primers (yellow and green arrows) were used for selective amplification of 3′ L1 or 5′ Alu flanking sequences (flanks). Indexing PCR introduced the sample barcodes and oligonucleotides that were necessary for Illumina sequencing (i5 and i7). dL1—part of an L1 element, dAlu—part of an Alu element, UMI—Unique Molecular Identifier.

Figure 2

Validation of candidate tumor-specific insertions. (A). For L1, a 3′-L1-specific primer (yellow arrow) was used in combination with GSP-R primer (genomic locus-specific primer reverse, black) corresponding to the unique genomic 3′ flank of each L1 insertion. GSP-R and GSP-F primers were used to amplify an empty allele. (B). For Alu, an additional reaction with 5′-Alu-specific (green arrow) and GSP-F primers was performed. (C). The expected results of the PCR confirming tumor-specific insertion of L1 (left) or Alu (right).

Figure 3

Transcription of active L1 copies in leukemia, colorectal cancer, and normal bone marrow samples. Each dot indicates the number of RNA-Seq reads that were mapped to the potentially traspositionally-competent L1HS (left) or L1PA2 (right) from L1base. BM—normal bone marrow, chALL—childhood acute lymphoblastic leukemia, ALL-CS—chALL samples from the present study, CLL—chronic lymphocytic leukemia, CRC—colorectal cancer with (CRC-T) or without (CRC-F) new tumor-specific insertions. Adjusted p-value (Padj), * p < 0.05, ** p < 0.01, *** p < 0.001, ns—non-significant.