Activation-Induced Cytidine Deaminase Accelerates Clonal Evolution of BCR-ABL1-Driven B Cell Lineage Acute Lymphoblastic Leukemia.

Abstract 181

Activation-Induced Cytidine Deaminase (AID) is required for somatic hypermutation and immunoglobulin (Ig) class switch recombination in germinal center (GC) B cells. Occasionally, AID can target non-Ig genes and thereby promote GC B cell lymphomagenesis. We recently demonstrated that the oncogenic BCR-ABL1 kinase induces aberrant expression of AID in pre-B acute lymphoblastic leukemia (ALL). Compared to other ALL subtypes, BCR-ABL1 ALL is considered high risk and is characterized by a high degree of genetic instability. Because aberrant mutational activity of AID is associated with malignant transformation in B cell lymphoma, we sought to determine whether aberrant AID expression contributes to clonal evolution and genetic instability in Ph⁺ ALL. To investigate the function of AID expression in Ph+ ALL, we established a genetic loss-of-function model for Ph⁺ ALL: Bone marrow cells from AID^−/− mice and AID^+/+ controls were transformed by retroviral transduction with BCR-ABL1 under B lymphoid culture conditions and subsequently injected into lethally irradiated congenic recipients. Mice transplanted with AID^−/−BCR-ABL1 ALL had prolonged median survival as compared to mice transplanted with leukemia cells generated from AID^+/+ bone marrow (AID^−/− 34 days (n=18) vs AID^+/+ 13 days (n=21); p<0.0001). In secondary and tertiary transplant experiments, however, the difference between AID^−/− and AID^+/+BCR-ABL1 ALL narrowed as determined by a decreasing hazard ratio (from 25.5 in the primary transplant to 5.1 in the secondary and 2.9 in the tertiary transplantation). These findings suggest that aberrant AID expression accelerates clonal evolution of Ph+ ALL, but AID-independent factors exist that are sufficient for transformation.

In support of enzymatic activity of AID in BCR-ABL1-transformed ALL cells, we observed that aberrant somatic hypermutation of non-immunoglobulin genes in these leukemias was largely dependent on AID: mutations in the known hypermutation target genes Pax5 and Rhoh were increased in AID^+/+ but not AID^−/−BCR-ABL1 ALL cells. Mutations in the first intron of Rhoh as observed here are relevant because they interfere with Rhoh transcription. Indeed, we found that Rhoh mRNA levels are significantly higher in AID^−/− compared to AID^+/+BCR-ABL1 ALL cells. Rhoh is a hematopoietic specific GTPase that negatively regulates Rac-mediated signaling downstream of the oncogenic BCR-ABL1 kinase. AID-dependent mutation and transcriptional inactivation of Rhoh in BCR-ABL1 ALL therefore likely augments oncogenic BCR-ABL1 signaling.

Consistent with a causative role of AID in genetic instability, AID^−/− leukemia had a lower frequency of amplifications (17⁺2 vs 45⁺7; p=0.002) and deletions (11⁺2 vs 40⁺7; p=0.003) as compared to AID^+/+ leukemias. AID^−/− and AID^+/+ ALL cells showed a markedly distinct gene expression pattern with 2,365 differentially expressed genes (p=0.003; FDR 0.05). A detailed analysis of these differences in gene expression revealed that AID^−/−BCR-ABL1 ALL cells failed to downregulate a number of tumor suppressor genes including p53, Rhoh, Cdkn1a (p21), and Blnk (SLP65). AID-dependent downregulation of p53 in BCR-ABL1 ALL cells is of particular importance, because previous work demonstrated that transcriptional repression of p53 in normal GC B cells is required to make these cells permissive to high levels of AID expression. AID-induced DNA damage would otherwise activate p53 and rapidly induce apoptosis.

Compared to AID-deficient BCR-ABL1 ALL, AID^+/+BCR-ABL1 ALL cells are more resistant to Imatinib-treatment. However, acquisition of BCR-ABL1 kinase domain mutations does not appear to be the main cause of drug-resistance in this experiment, since only one relevant mutation was amplified from AID^+/+ ALL cells (no mutations in AID^−/− ALL cells). We conclude that AID accelerates clonal evolution in BCR-ABL1 ALL by enhancing genetic instability, aberrant somatic hypermutation, and by negative regulation of tumor suppressor genes.