The B Cell Mutator AID Promotes B Lymphoid Blast Crisis and Drug-Resistance in Chronic Myeloid Leukemia.

Abstract 3274

Poster Board III-1

Chronic myeloid leukemia (CML) is induced by BCR-ABL1 and can be effectively treated for many years with Imatinib until leukemia cells acquire drug resistance through BCR-ABL1 mutations and progress into fatal B lymphoid blast crisis (LBC). Treatment of patients with CML in chronic phase with the BCR-ABL1 kinase inhibitor Imatinib leads to a five-year overall survival of ∼95%. Upon blast crisis progression, however, median overall survival decreases to less than seven months and CML blasts are frequently Imatinib-resistant due to BCR-ABL1 mutations.

Despite its clinical significance, the mechanism of progression into LBC is unknown. Here we show that LBC but not CML cells aberrantly express the B cell-specific mutator enzyme AID. During normal B cell development, AID is activated within germinal centers to initiate somatic hypermutation and class-switch recombination of immunoglobulin genes during affinity maturation of B cells. When ectopically expressed in CML cells, AID promotes widespread hypermutation and thereby promotes B lymphoid blast crisis progression. To examine whether aberrant mutational activity of Imatinib leads to Imatinib-resistance in vivo, we then labeled GFP⁺> and AID/GFP⁺> transduced CML cells with lentiviral firefly luciferase and injected them intrafemorally into sublethally irradiated NOD/SCID mice. When focal expansion of leukemic growth became evident (day 20), treatment with Imatinib (100 mg/kg twice daily) was started. By day 35, all 14 mice injected with AID/GFP-transduced CML cells had developed progressive disease as opposed to only 5 of 12 GFP-transduced control mice (P <0.02). Correspondingly, all mice injected with AID/GFP⁺> CML cells were dead at day 54, whereas about half of the mice injected with GFP⁺> CML cells were still alive 162 days post-injection (n=12; P<0.001).In a genetic loss-of-function experiment, we tested the ability of AID^+/+> and AID^-/-> BCR-ABL1-transformed leukemia cells to acquire Imatinib-resistance and BCR-ABL1 mutation. AID^+/+> but not AID^-/-> BCR-ABL1-transformed leukemia cells became Imatinib-resistant within six under cell culture conditions. Sequence analysis revealed that most of the AID^+/+> Imatinib-resistant clones had acquired in the BCR-ABL1 kinase domain that confer Imatinib-resistance in patients. From AID^-/-> leukemia cells, only one clinically relevant mutation was amplified. To determine whether AID promotes overall genetic instability in BCR-ABL1 leukemias, we measured gene copy number alterations (i.e. deletions or amplifications) in 23 primary cases of Ph⁺> ALL using a 250K NspI SNP array. Based on AID mRNA levels, Ph⁺> ALL samples were classified as either AID^high> (16 cases) or AID^low> (7 cases). SNP analysis revealed a higher frequency of gene copy number alterations in the AID^high> as compared to the AID^low> group (median 14 [range 6–50] vs. median 5 [range 2–8]; P = 0.02). Notably, deletion frequencies at the tumor suppressor genes ARF (CDKN2A) and INK4B (CDKN2B) at 9p21 were considerably higher in the presence of AID (P = 0.04).

The transcription factor PAX5 regulates both AID-gene expression and B cell lineage commitment of hematopoietic progenitor cells. Interestingly, ectopic expression of PAX5 in myeloid CML cells was sufficient to induce AID transcription and B cell lineage conversion including expression of CD19 in a small subset of PAX5/GFP⁺> but not in GFP⁺> CML cells. To determine whether PAX5 can promote Imatinib-resistance in CML, PAX5/GFP⁺> and GFP⁺ >human leukemia cells were cultured in the presence of increasing concentrations of Imatinib (0.1 μmol/L to 1.75 μmol/L). A time-dependent outgrowth of PAX5/GFP⁺ >CML cells in the presence of Imatinib was observed and sequence analysis confirmed the presence of mutations within the BCR-ABL1 kinase domain of PAX5/GFP but not in GFP-transduced CML cells. We conclude that enforced PAX5 expression in myeloid CML cells can lead to partial B cell lineage conversion, AID expression, and Imatinib-resistance via BCR-ABL1 mutation.

We demonstrate that AID expression in CML cells promotes overall genetic instability by hypermutation of tumor suppressor and DNA repair genes. Importantly, our data uncover a causative role of AID activity in the acquisition of BCR-ABL1 mutations leading to Imatinib-resistance, thus providing a rationale for the rapid development of drug resistance and blast crisis progression.