Divergent Lineage-Specific Functions of PP2A in Chronic Phase CML and Lymphoid Blast Crisis

Background & Hypothesis: Protein phosphatase 2A (PP2A) is a Ser/Thr phosphatase and functions as negative regulator of Akt- and Erk-signaling. PP2A attenuates downstream signaling of BCR-ABL1 and thereby functions as important tumor suppressor in chronic myeloid leukemia (CML). PP2A-activating drugs have been proposed for the treatment of CML and eradication of tyrosine kinase inhibitor resistant leukemia stem cells in CML (Neviani et al., 2013). Studying Cre-mediated ablation of PP2A in mouse models for BCR-ABL1-driven leukemia including myeloid CML in chronic phase (CML-CP), B lymphoid blast crisis (LBC) and B cell lineage Ph⁺ ALL, we were able to provide genetic evidence for this treatment concept in CML. Seemingly paradoxically, however, inducible ablation of PP2A in B cell lineage LBC and Ph⁺ ALL cells has the opposite effect and induced rapid cell death. These findings highlight fundamental differences in the regulation of Akt- and Erk-signaling in myeloid lineage and B cell lineage cells and have implications for specific therapy concepts that distinguish between chronic phase CML and lymphoid blast crisis/ Ph⁺ ALL.

Results: Interestingly, PP2A protein levels are significantly higher in B cell lineage Ph⁺ ALL and CML lymphoid blast crisis (LBC) compared to chronic phase CML. To explore the function of PP2A during early B cell development, we deleted the α isoform of the PP2A subunit A in pro-B cells by introducing Mb1-Cre into Ppp2r1a^fl/fl mice. Analysis of bone marrow from Ppp2r1a^fl/flMb1-Cre (PP2A-cko) mice showed obvious B cell development block at the pre-B cell receptor checkpoint, although V(D) recombination and RAG1/2 activity were intact in PP2A-cko pre-B cells. Since high mRNA levels of PP2A subunits at the time of diagnosis predict poor outcome of children (COG P9906; n=207) and adults (ECOG 2993; n=215) with ALL, we studied the function of PP2A in mouse models for B cell lineage ALL including Ph⁺ ALL and CML lymphoid blast crisis (LBC). After Cre-induced deletion of Ppp2r1a, we observed reduced protein expression of both PP2A subunit A and subunit C accompanied with decreased PP2A phosphatase. The PP2A-cKO ALL cells showed normal proliferation but significantly impaired colony formation capability and increased apoptosis, both of which could be rescued by overexpression of wildtype PP2A in PP2A-cKO ALL cells. We next transplanted luciferase expressing PP2A-KO ALL cells into recipient mice and monitored cell growth and leukemia progression. Cre-mediated deletion significantly prolonged overall survival of recipient mice that were transplanted with Ppp2r1a^fl/fl ALL cells. Although all of those mice died eventually from leukemia, the ALL cells isolated from the mice retained Ppp2r1a floxed alleles suggesting that these mice died from clones that had escaped Cre-mediated deletion of PP2A. Cre-mediated deletion increased phosphorylation levels of Stat5 and Erk, resulted in accumulation of p53 and increased output of the PI3K-AKT signaling pathway as measured by increased phosphorylation of FoxO factors, p70^S6K and S6 ribosomal protein.

Based on the involvement of PI3K-AKT in glucose metabolism, we measured the effects of inducible PP2A-deletion on glycolytic function in CML and Ph⁺ ALL/LBC cells. Interestingly, inducible deletion of PP2A induced profound imbalances of glucose metabolism in B cell lineage Ph⁺ ALL/LBC but not myeloid lineage CML-like cells. Upon PP2A-deletion, ALL cells showed higher glycolytic flux shunted into lactate rather than NADPH production. Lower NADPH/NADP ratio and higher reactive oxygen species level in PP2A-KO ALL cells indicated impaired balance of glycolytic flux and may account for increased apoptosis of those cells. This notion was supported by a strong rescue effect of overexpression of the antioxidant catalase in PP2A-cKO cells. In agreement with reported tumor suppressor role of PP2A in CML cells (Neviani et al., 2005), we observed that CEBPα-driven lineage conversion of B cell lineage Ph⁺ ALL/LBC into not myeloid lineage.

Conclusion: Here we reported the requirement of PP2A in normal B cell development and lineage-specific oncogenic role of PP2A in BCR-ABL1-driven leukemia. Divergent roles of PP2A in Ph⁺ ALL/LBC and myeloid lineage CML may lead to future insights into mechanisms of LBC-transformation of CML and lineage-specific requirement of BCR-ABL1-transformation.