PAX5-Mediated Lineage Conversion and Expression of AID Accelerates Clonal Evolution and Initiates Darwinian Selection of BCR-ABL1-Mutants in Chronic Myeloid Leukemia.

Abstract

Chronic Myeloid Leukemia (CML) is typically derived from the myeloid lineage. CML cells in terminal blast crisis, however, often exhibit a B lymphoid phenotype. The reasons for progression into blast crisis and myeloid/B lymphoid lineage switch are largely unknown. Studying expression of the B cell-specific mutator enzyme AID in BCR-ABL1-transformed leukemias, we found that AID is expressed in BCR-ABL1-positive B cell lineage ALL and B lymphoid blast crisis CML but not chronic phase or myeloid blast crisis CML. Studying five primary cases of mixed lineage bast crisis CML, AID expression was only found in the sorted B cell lineage but not myeloid lineage subclones. This pattern of AID expression correlated with a high frequency of DNA single-strand breaks within the tumor suppressor genes CDKN2A and CDKN2B, which were found in B cell lineage but not myeloid lineage subclones of blast crisis CML. Interestingly, the expression pattern or AID parallels the frequency of mutations within the BCR-ABL1 kinase domain that confer resistance to the BCR-ABL1 kinase inhibitor STI571. To investigate whether AID introduces point mutations within the BCR-ABL1 kinase domain that confer resistance to STI571, we transduced eight AID-negative CML lines with retroviral expression constructs either encoding AID/GFP or GFP alone. Within 16 days of treatment with gradually increasing concentrations of STI571, all eight AID/GFP-transduced CML lines but only one GFP-transduced CML line acquired drug resistance. Single drug resistant cells were sorted and sequence analysis confirmed that clinically relevant mutations E255K and T315I were acquired in all cases. In one cell line (K562), drug resistance and mutations were pre-existing and were not introduced by AID. Given that transcriptional activation of AID requires the B cell lineage-specific transcription factor PAX5, we transduced five CML cell lines with retroviral expression vectors encoding either PAX5/GFP or GFP alone and incubated them with STI571 at gradually rising concentrations. Outgrowth of drug-resistant subclones was observed in one of five cell lines. This outgrowth was accompanied with lineage conversion of the drug-resistant cells including cell surface expression of CD19 and upregulation of PAX5-target genes BLNK, CD79A and AID. Subsequent sequence analysis of the BCR-ABL1 kinase domain confirmed that PAX5-mediated lineage conversion has led to the acquisition of relevant BCR-ABL1 kinase mutations, likely owing to PAX5-mediated de novo expression of the mutator enzyme AID. In the four other CML cell lines, PAX5-target genes BLNK, CD79A and AID were also upregulated. However, ectopic expression of PAX5 in these cell lines did not result in CD19 surface expression and no relevant mutations were found in the BCR-ABL1 kinase domain. These findings suggest that full lineage conversion is needed to induce a, presumably AID-dependent, mutator phenotype in CML cells. We propose that B cell lineage conversion in lymphoid blast crisis CML including aberrant expression of AID accelerates the clonal evolution and induces Darwinian selection of drug-resistant mutants in CML.