Differential Regulation of Telomeric Complex by BCR-ABL1 Kinase in Human Cellular Models of Chronic Myeloid Leukemia—From Single Cell Analysis to Next-Generation Sequencing

Abstract

Telomeres are specialized nucleoprotein complexes, localized at the physical ends of chromosomes, that contribute to the maintenance of genome stability. One of the features of chronic myeloid leukemia (CML) cells is a reduction in telomere length which may result in increased genomic instability and progression of the disease. Aberrant telomere maintenance in CML is not fully understood and other mechanisms such as the alternative lengthening of telomeres (ALT) are involved. In this work, we employed five BCR-ABL1-positive cell lines, namely K562, KU-812, LAMA-84, MEG-A2, and MOLM-1, commonly used in the laboratories to study the link between mutation, copy number, and expression of telomere maintenance genes with the expression, copy number, and activity of BCR-ABL1. Our results demonstrated that the copy number and expression of BCR-ABL1 are crucial for telomere lengthening. We observed a correlation between BCR-ABL1 expression and telomere length as well as shelterins upregulation. Next-generation sequencing revealed pathogenic variants and copy number alterations in major tumor suppressors, such as TP53 and CDKN2A, but not in telomere-associated genes. Taken together, we showed that BCR-ABL1 kinase expression and activity play a crucial role in the maintenance of telomeres in CML cell lines. Our results may help to validate and properly interpret results obtained by many laboratories employing these in vitro models of CML.

Genetic Biomarkers in Chronic Myeloid Leukemia: What Have We Learned So Far?

“…Although no pathogenic variations were found in telomere-associated genes, they identified mutations and/or copy number variations in tumor suppressor genes, namely in TP53 (loss and mutations), CDKN2A (deletion), ATM (mutations) [46]. In line with previous studies [83], a correlation between expression of BCR-ABL1 and telomere length was found [46]. The importance of TP53 mutation or loss of function, including codon 72 polymorphism, was previously described as being preponderant for CML progression and therapy resistance [48][49][50][89][90][91][92].…”

“…Recently, FISH and Next-Generation Sequencing (NGS) allowed studying of the correlation between expression, copy number, and activity of BCR-ABL1 with the mutational status, copy number, and expression of telomere maintenance genes in five BCR-ABL1positive cell lines (K562, KU-812, LAMA-84, MEG-A2 and MOLM-1) [46]. Although no pathogenic variations were found in telomere-associated genes, they identified mutations and/or copy number variations in tumor suppressor genes, namely in TP53 (loss and mutations), CDKN2A (deletion), ATM (mutations) [46]. In line with previous studies [83], a correlation between expression of BCR-ABL1 and telomere length was found [46].…”

“…BCR-ABL1 fusion is the major therapeutic target for patients with CML and detection of changes in gene expression levels might help in the prognosis and diagnosis of CML [43]. While other genes have been reported as potential markers for prognosis and indicators of sensitivity/resistance to drugs in CML, including TGF-β, TNF-α [44], vascular endothelial growth factor A (VEGFA) [45], and 53 (TP53) [46][47][48][49][50] pathways. On the other hand, identification of genes whose expression is related with an increased probability of failure could be helpful in selecting more proper imatinib doses or drug combinations.…”

RHOA-regulated IGFBP2 promotes invasion and drives progression of BCR-ABL1 chronic myeloid leukemia

“…Our recent study of the BCR-FGFR1 fusion kinase in SCLL demonstrated a role for the GEF domain in the BCR component of the chimeric kinase in activating RHOA and influencing leukemia progression 12 . To obtain a better understanding of the role of RHOA in the development of Ph+ CML, we created knock down clones from two human, p210-expressing cell lines, K562 and KU812 29 , using a double targeting CRISPR-Cas9 approach (Figure 1A). Both cell lines were first transduced with pCDH-CMV-Nluc-P2A-copGFP-T2A-Puro to co-express luciferase and GFP, and then transduced with lentiCRISPR v2 and both the sgRNAs and Cas9 to generate the KO cell clones.…”

RAP1/TERF2IP—A Multifunctional Player in Cancer Development

“…Recently, our group determined whether the BCR/ABL1 copy number, expression, and/or activity were responsible for telomere maintenance and the deregulated expression of selected shelterin components (TRF1, TRF2, RAP1, and POT1) in widely used chronic myeloid leukemia (CML) cell lines. We found that the expression of RAP1 and POT1 at the protein level was positively correlated with the levels of expression and activity of BCR/ABL1, suggesting that BCR/ABL1 kinase expression and activity may upregulate the levels of RAP1 and POT1 and play a crucial role in the maintenance of telomeres in CML cells [ 36 ]. The differential expression of RAP1 in selected human cancers is summarized in Table 2 .…”