PP2A Is Required for B Cell Survival and Represents a Therapeutic Target in Acute Lymphoblastic Leukemia

Background: The Ser/Thr Protein phosphatase 2A (PP2A) attenuates activity of RAS-ERK and PI3K-AKT signaling pathways and functions as important tumor suppressor in chronic myeloid leukemia (CML) by downregulating output of oncogenic BCR-ABL1 signaling. Restoration of PP2A activity has been proposed for the treatment of CML and eradication of TKI-resistant leukemia CML-stem cells (Neviani et al., 2013). While the tumor suppressor function of PP2A was independently confirmed by multiple groups, our analyses of PP2A function during early B cell development suggested a role for PP2A to contribute to B cell survival. Therefore, we studied whether PP2A has different roles in B-lineage ALL and CML and whether these mechanistic differences are relevant for patients with B-lineage and B-lymphoid blast crisis CML.

Results: Consistent with a divergent role of PP2A in B-lineage ALL (compared to CML), we found that 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 therefore, studied the function of PP2A in a genetic mouse model for Cre-induced deletion of Ppp2r1a in BCR-ABL1 (Ph⁺) ALL. Inducible activation of Cre reduced protein expression of the targeted PP2A subunit A but also the catalytic subunit C and was paralleled by near-complete loss of PP2A phosphatase activity. Conversely, Cre-mediated deletion increased phosphorylation levels of p70^S6K and S6 ribosomal protein, which indicated elevated mTOR signaling. Acute deletion of Ppp2r1a^fl/fl in B cell-lineage ALL cells dramatically affected survival and colony formation, both of which could be rescued by overexpression of wildtype PP2A. In agreement with previous studies, however, Cre-mediated deletion had no deleterious effects in a Ppp2r1a^fl/fl CML model. To verify that the observed difference between B cell and myeloid disease was indeed lineage-dependent, we reprogrammed B cell lineage ALL cells into myeloid CML using inducible overexpression of CEBPα. As expected, CEBPα-driven myeloid lineage conversion prevented cell death upon deletion of PP2A, demonstrating that PP2A-function represents a B cell-specific vulnerability.

We next transplanted luciferase-labeled Ppp2r1a^fl/fl 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 mice died eventually from leukemia, the ALL cells isolated from the mice retained Ppp2r1a floxed alleles in all cases studied demonstrating that fatal leukemia arose from few subclones that had escaped Cre-mediated deletion of PP2A.

Interestingly, inducible deletion of PP2A caused profound imbalances of glucose metabolism in B cell lineage Ph⁺ ALL but not 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-deleted ALL cells, together with decreased anti-oxidant gene expression, increased H2AX phosphorylation and p53 expression indicated impaired balance of glycolytic flux may account for increased death of those cells. This notion was supported by a strong rescue effect of overexpression of the antioxidant catalase in PP2A-deleted cells.

The unexpected role of PP2A in Ph⁺ ALL was further validated by CRISPR-Cas9 mediated disruption of PPP2R1A in ALL xenografts derived from three patients. In addition, a PP2A specific inhibitor LB-100 (in clinical trial for solid tumors) was employed to pharmacologically inhibit PP2A activity. Low micromolar concentrations of LB-100 induced cell death in patient-derived ALL xenografts in parallel with ROS-accumulation and increased S6 and H2AX phosphorylation.

Conclusion: Cre-mediated ablation of PP2A in mouse Ph⁺ ALL cells induced rapid cell death through excessively oxidative stress but not in myeloid CML cells. We confirmed this pro-survival role of PP2A in human Ph⁺ ALL-patients derived leukemia cells through both CRISPR-Cas9 mediated genetic study and PP2A inhibition by a novel small molecule LB-100 (Jie Lu et al., 2009). These findings highlight PP2A as a therapeutic target with potential relevance in Ph⁺ ALL and B-lymphoid blast crisis progression of CML.