B-Lymphocyte Development Is Impaired by FLT3/ITD Signaling in Knock-in Mice Because of Defective Non-Homologous End-Joining.

Abstract 184

Constitutive activation of FLT3 by internal tandem duplication (ITD) mutations is one of the most common molecular alterations found in up to 35% of acute myeloid leukemia (AML) and 3% of acute lymphoid leukemia (ALL). Its roles in leukemic transformation of normal hematopoietic stem/ progenitor cells (HSPCs) are not yet fully understood. We have generated a FLT3/ITD knock-in mouse model in which mice heterozygous for a FLT3/ITD mutation develop myeloid proliferation and a block in B-lymphocyte development at the Pro-B stage. To investigate the mechanisms for this block in lymphoid development, we studied V(D)J recombination in the B-lineage cells from the bone marrow (BM) of FLT3/ITD mice. The ligation-mediated PCR assay showed that Pro-B cells from FLT3/ITD mice accumulated high levels of signal-end recombination intermediates, generated by DNA cleavage at the DJ_H recombination signal sequences (RSSs), with a concomitant decrease in the frequency of completed DJ_H rearrangements when compared to wild-type littermate controls. These observations are consistent with decreased repair of double strand breaks (DSBs) introduced by the V(D)J recombinase in B cell progenitors from FLT3/ITD mice, resulting in impaired development beyond the Pro-B stage.

The classical Ku and DNA-PK-dependent non-homologous end-joining (NHEJ) pathway is required for rejoining DSBs during V(D)J recombination. In vivo NHEJ assays demonstrate that BM cells, including Pro-B cells, with FLT3/ITD mutations have a lower efficiency and decreased accuracy of repair of DSBs, suggesting a defect in the classical NHEJ pathway. Quantitative RT-PCR and Western blotting analysis of Pro-B cells from FLT3/ITD mice demonstrate greatly reduced expression of Ku70/Ku86 and Ligase IV, key components of the the classical NHEJ pathway, confirming the idea that Classical NHEJ is defective. We and others have recently demonstrated that alternative (ALT) NHEJ pathway(s) compensate for deficiencies in classical NHEJ. Components of this pathway include DNA ligase III/XRCC1 and poly (ADP) ribose polymerase 1 (PARP1). Indeed, we show by Western blotting analysis that steady state levels of ALT NHEJ components DNA ligase III and PARP1 are increased in FLT3/ITD cells, compared with wild-type controls.

These data suggest that during the process of DJ_H recombination in Pro-B cells from FLT3/ITD mice, impairment of the classical NHEJ pathway decreases the ability of cells to complete post-cleavage DSB ligation, resulting in failure to complete DJ_H recombination with a subsequent block of Pro-B cell maturation. With the classical NHEJ pathway impaired, BM cells from mice with FLT3/ITD mutations appear to repair DSBs through the highly error-prone ALT NHEJ pathway. The resultant increased genomic instability might underlie an important mechanism for the leukemic transformation of normal HSPCs by FLT3/ITD mutations and explain the poor prognosis of AML patients with this mutation.