Acute Lymphoblastic Leukemia-Associated PAX5 Mutations Induce Aberrant B Cell Development

Abstract 10 Over 60% of pediatric B progenitor acute lymphoblastic leukemia (ALL) cases contain somatic mutations in genes regulating B cell development, with PAX5 being the most common target of mutation (>32% of cases). The transcription factor PAX5 is required for commitment and maintenance of the B lymphoid lineage. A variety of PAX5 mutations has been identified including, mono-allelic deletions, sequence mutations, internal deletions, frame-shift mutations and translocations. We have previously shown that these PAX5 mutations result in reduced transcriptional activity either as a result of haploinsufficiency or the generation of altered PAX5 isoforms with reduced DNA-binding and/or transcriptional activity. However, the direct effect of the mutations on normal B cell development remains unknown. To address this question, we assessed the ability of a series of PAX5 mutations to rescue normal B cell development in Pax5-/- bone marrow (BM) cells using a murine in vitro culture system. Whole BM or transduced cells were grown in IL-7 producing stromal-supported cultures for two weeks and then assessed for their extent of B cell differentiation using flow cytometry. Under these in vitro conditions, both Pax5+/+ and Pax5+/− BM cells differentiated to a Hardy fraction D pre-B cell stage of differentiation (CD43−/B220+/CD19+/BP1+), with only a slight decrease in the level of expression of BP1 detected in the Pax5+/− cells. By contrast, Pax5-/- cells failed to undergo significant differentiation under these in vitro growth conditions and were arrested at an early pro-B stage of development (CD43+/−/B220+/CD19−/BP1−). To assess the biological activity of the identified PAX5 mutants, we then transduced lineage-depleted BM cells from Pax5+/+, Pax5+/− and Pax5-/- mice with MSCV-based retroviral vectors expressing either wild type (WT) or mutant PAX5 followed by in vitro culture. Three classes of PAX5 mutations were assessed: DNA binding domain mutations (P80R, P34Q, and V26G), an internal deletion mutation (Δe6-8), and translocation-induced PAX5 chimeric genes (PAX5-ETV6, PAX5-FOXP1 and PAX5-ZNF521). As expected, expression of WTPAX5 resulted in full rescue of Pax5-/- cells and induced no significant effects on the ability of Pax5+/+ and +/− cells to differentiate. By contrast, PAX5 DNA-binding domain mutants resulted in only partial rescue of Pax5-/- cells, with P80R inducing B220+/CD19−/BP1−, P34Q producing B220+/CD19+ cells with weak BP1 expression, and V26G yielding CD19+/BP1+ cells with minimally reduced levels of BP1. Similarly, expression of Δe6-8 resulted in partial rescue with the expansion of B220+/CD19+/−/BP1− cells. In stark contrast, expression of the translocation encoded PAX5 fusion proteins failed to induce any evidence of rescue. Moreover, these fusion proteins induced only minimal perturbations in the ability of Pax5+/+ and +/− cells to differentiate, suggesting that these fusion proteins were weak competitive inhibitors of normal Pax5 transcriptional activity under intra-cellular conditions. To further characterize the effects of these PAX5 mutations on B cell differentiation, we next analyzed the gene expression patterns of the resultant cell populations using the Mouse Genome 430 2.0 Arrays (Affymetrix) and compared the profiles to those obtained from purified Hardy fractions from normal murine BM. The expression signatures of the Pax5-/- cells were identical to those for normal Hardy fraction A and shifted to the signature of Hardy fraction C following rescue with WTPAX5. Transduction of Pax5-/- cells with either V26G or P34Q resulted in a near complete rescue with expression signatures similar to those obtained for Hardy fractions B/C. By contrast, transduction with P80R or Δe6-8 yielded a more incomplete rescue with expression profiles that were between Hardy fractions A and B. Interestingly, a number of genes within the B cell receptor signaling pathway were altered in cells rescued by P80R and Δe6-8, including the down regulation of CD19, Btk and Blnk. In summary, our data demonstrate that leukemia-associated PAX5 mutations have a graded effect on the transcriptional network that controls normal B cell development and differentiation. Defining the differential target gene specificity of the various PAX5 mutants should provide valuable insights into the molecular mechanisms through which these genetic lesions contribute to leukemogenesis. Disclosures: No relevant conflicts of interest to declare.