Chronic myeloid leukaemia (CML) patients who develop blast crisis accumulate somatic mutations associated with resistance to tyrosine kinase inhibitor (TKI) therapy. To characterize mutations acquired at blast crisis we used RNA and exome sequencing for 65 CML patients, with samples at both diagnosis and blast crisis. We demonstrate that off-target V(D)J recombination is the predominant mutational mechanism in lymphoblastic CML. The V(D)J recombination pathway, including the recombination activation genes (RAG1 and RAG2) and DNTT, is an essential component of developing lymphocytes, but is normally under tight regulatory control. V(D)J recombination events have a characteristic mutational signature at break points including the recombination signal sequence motif ‘CACAGTG’, and DNTT-mediated nucleotide insertions. This mutational signature was present in 31/40 validated structural variants in patients with high RAG expression. This included mutations at genes associated with hematological malignancies such as IKZF1 deletions, IKZF1-IGK fusions, RUNX1 deletions and a recurrent HBS1L-MYB intergenic locus deletion. We find that supraphysiological, constitutive expression of the V(D)J recombination pathway is necessary for RAG-mediated events, and suggest this is a component of lymphoid blast crisis transformation as opposed to physiological lymphocyte development. Furthermore, we serendipitously observed that TKI treatment induces RAG expression, which we confirmed using cell-line models, suggestive that TKI treatment has the pleiotropic effect of exacerbating RAG expression and mutation via off-target V(D)J recombination. Finally, we show that patients with elevated expression of RAG1/2 or DNTT at diagnosis) (10 of 10) subsequently developed lymphoid blast crisis, offering new biomarkers of blast crisis progression and potential oncogenic mutation.
In conclusion, we profiled the mutational landscape of CML to identify the dominant mechanism by which structural variants are gained in CML lymphoid blast crisis, we identify new biomarkers of CML disease progression and show how TKI treatment itself can compound susceptibility to genomic mutation.