MuLV-related endogenous retroviral elements and Flt3 participate in aberrant end-joining events that promote B-cell leukemogenesis

Abstract

During V(D)J recombination of immunoglobulin genes, p53 and nonhomologous end-joining (NHEJ) suppress aberrant rejoining of DNA double-strand breaks induced by recombinase-activating genes (Rags)-1/2, thus maintaining genomic stability and limiting malignant transformation during B-cell development. However, Rag deficiency does not prevent B-cell leukemogenesis in p53/NHEJ mutant mice, revealing that p53 and NHEJ also suppress Rag-independent mechanisms of B-cell leukemogenesis. Using several cytogenomic approaches, we identified a novel class of activating mutations in Fms-like tyrosine kinase 3 (Flt3), a receptor tyrosine kinase important for normal hematopoiesis in Rag/p53/NHEJ triple-mutant (TM) B-cell leukemias. These mutant Flt3 alleles were created by complex genomic rearrangements with Moloney leukemia virus (MuLV)-related endogenous retroviral (ERV) elements, generating ERV-Flt3 fusion genes encoding an N-terminally truncated mutant form of Flt3 (trFlt3) that was transcribed from ERV long terminal repeats. trFlt3 protein lacked most of the Flt3 extracellular domain and induced ligand-independent STAT5 phosphorylation and proliferation of hematopoietic progenitor cells. Furthermore, expression of trFlt3 in p53/NHEJ mutant hematopoietic progenitor cells promoted development of clinically aggressive B-cell leukemia. Thus, repetitive MuLV-related ERV sequences can participate in aberrant end-joining events that promote development of aggressive B-cell leukemia.

B cell acute lymphoblastic leukemia cells mediate RANK-RANKL–dependent bone destruction

“…Furthermore, cytokines serve critical roles in early B cell development and function. Flt3 was the most highly enriched cytokineencoding gene in the TM cells [log 2 Fc (fold change) = −5.39 and FDR-adjusted q = 4.20 × 10 −9 ], in line with our previous finding that somatic Flt3 mutation causes activation and aberrant expression in TM B-ALL (40). Among other cytokine-encoding genes overexpressed in TM relative to DM B-ALL, we focused by Tnfsf11 (Rankl) encoding RANKL (log 2 Fc = −3.46 and FDR-adjusted q = 4.81 × 10 −4 ), due to its critical role in bone remodeling.…”

“…In previous studies, we identified an ectopically expressed Flt3 mutation that promotes early B cell leukemogenesis in triple mutant (TM) mice (40). The TM mice harbor mutations that arrest B cell development (Rag2 −/− ) and disrupt DNA repair (Prkdc scid/scid ) and DNA damage checkpoints (p53 −/− ) (41).…”

“…The DM and TM mouse colonies were maintained on acidified water, and mice were monitored daily for labored breathing or peripheral lymphadenopathy indicative of systemic leukemia. At the first sign of morbidity, mice were euthanized, and single-cell suspensions were isolated from lymphoid tissues and used immediately (40). Cells were also viably frozen in 90% fetal calf serum (FCS) with 10% (v/v) dimethyl sulfoxide (DMSO), stored in liquid nitrogen, and later used for engraftment of B6.Ly5.1.Rag2 −/− recipient mice.…”

Advances in understanding the pathogenesis of CNS acute lymphoblastic leukaemia and potential for therapy

“…CNS BCP-ALL was observed in mice triple mutant for RAG1/2, the non-homologous end joining repair and TP53 (Gladdy et al, 2003). Further molecular studies of this unique mouse model revealed a molecular rearrangement that results in the activation of FLT3 (Johnson et al, 2014). They further showed that this activation was essential for the CNS phenotype.…”

PiggyBac transposon-based polyadenylation-signal trap for genome-wide mutagenesis in mice

“…Animal models with genetic mutations are commonly used to determine gene function. Numerous methods for generating mutations, including X-rays, chemical mutagenesis, retroviral transfection, and transgenic technology, have been used in gene function research 11 12 13 14 ; however, these methods do not produce stable expression, frequently affect multiple genes or lead to chromosomal rearrangements, and cannot provide molecular markers to allow collection of mutation information 12 .…”