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RAG enhances BCR-ABL1-positive leukemic cell growth through its endonuclease activity in vitro and in vivo.

ABSTRACT: BCR-ABL1 gene fusion associated with additional DNA lesions involves the pathogenesis of chronic myelogenous leukemia (CML) from a chronic phase (CP) to a blast crisis of B lymphoid (CML-LBC) lineage and BCR-ABL1⁺ acute lymphoblastic leukemia (BCR-ABL1⁺ ALL). The recombination-activating gene RAG1 and RAG2 (collectively, RAG) proteins that assemble a diverse set of antigen receptor genes during lymphocyte development are abnormally expressed in CML-LBC and BCR-ABL1⁺ ALL. However, the direct involvement of dysregulated RAG in disease progression remains unclear. Here, we generate human wild-type (WT) RAG and catalytically inactive RAG-expressing BCR-ABL1⁺ and BCR-ABL1^- cell lines, respectively, and demonstrate that BCR-ABL1 specifically collaborates with RAG recombinase to promote cell survival in vitro and in xenograft mice models. WT RAG-expressing BCR-ABL1⁺ cell lines and primary CD34⁺ bone marrow cells from CML-LBC samples maintain more double-strand breaks (DSB) compared to catalytically inactive RAG-expressing BCR-ABL1⁺ cell lines and RAG-deficient CML-CP samples, which are measured by γ-H2AX. WT RAG-expressing BCR-ABL1⁺ cells are biased to repair RAG-mediated DSB by the alternative non-homologous end joining pathway (a-NHEJ), which could contribute genomic instability through increasing the expression of a-NHEJ-related MRE11 and RAD50 proteins. As a result, RAG-expressing BCR-ABL1⁺ cells decrease sensitivity to tyrosine kinase inhibitors (TKI) by activating BCR-ABL1 signaling but independent of the levels of BCR-ABL1 expression and mutations in the BCR-ABL1 tyrosine kinase domain. These findings identify a surprising and novel role of RAG in the functional specialization of disease progression in BCR-ABL1⁺ leukemia through its endonuclease activity.

SUBMITTER: Yuan M

PROVIDER: S-EPMC8253288 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The pathogenic role of the overactivated ABL1 tyrosine kinase (TK) pathway is well recognized in some forms of BCR-ABL1 like acute lymphoblastic leukemia (ALL); TK inhibitors represent a useful therapeutic choice in these patients who respond poorly to conventional chemotherapy. Here we report a novel peptide biosensor (PABL)-ELISA assay to investigate ABL1 activity in four immortalized leukemic cell lines with different genetic background. The PABL sequence comprises an ABL1 tyrosine (Y) phosphorylation site and a targeting sequence that increases the specificity for ABL1; additional peptides (Y-site-mutated (PABL-F) and fully-phosphorylated (PPHOSPHO-ABL) biosensors) were included in the assay. After incubation with whole cell lysates, average PABL phosphorylation was significantly increased (basal vs. PABL phosphorylation: 6.84 ± 1.46% vs. 32.44 ± 3.25%, p-value < 0.0001, two-way ANOVA, Bonferroni post-test, percentages relative to PPHOSPHO-ABL in each cell line). Cell lines expressing ABL1-chimeric proteins (K562, ALL-SIL) presented the higher TK activity on PABL; a lower signal was instead observed for NALM6 and REH (p < 0.001 and p < 0.05 vs. K562, respectively). Phosphorylation was ABL1-mediated, as demonstrated by the specific inhibition of imatinib (p < 0.001 for K562, NALM6, ALL-SIL and p < 0.01 for REH) in contrast to ruxolitinib (JAK2-inhibitor), and occurred on the ABL1 Y-site, as demonstrated by PABL-F whose phosphorylation was comparable to basal levels. In order to validate this novel PABL-ELISA assay on leukemic cells isolated from patient's bone marrow aspirates, preliminary analysis on blasts derived from an adult affected by chronic myeloid leukaemia (BCR-ABL1 positive) and a child affected by ALL (BCR-ABL1 negative) were performed. Phosphorylation of PABL was specifically inhibited after the incubation of BCR-ABL1 positive cell lysates with imatinib, but not with ruxolitinib. While requiring further optimization and validation in leukemic blasts to be of clinical interest, the PABL-based ELISA assay provides a novel in vitro tool for screening both the aberrant ABL1 activity in BCR-ABL1 like ALL leukemic cells and their potential response to TK inhibitors.

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RAG enhances BCR-ABL1-positive leukemic cell growth through its endonuclease activity in vitro and in vivo.

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