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Many malignancies are characterised by inappropriate substrate phosphorylation which causes aberrant proliferation and decreased apoptosis; this is often due to an overactive kinase e.g.  BCR-ABL or FLT-3 in myeloid malignancy, or ERK or ErbB2 in solid tumours. What is less clear is why this effect is not simply countered by normal cellular phosphatases, of which the most important is protein phosphatase 2A (PP2A). PP2A is normally inhibited by SET, (which is stabilised by a binding protein SETBP1) and cancerous inhibitor of PP2A (CIP2A). We have shown that high CIP2A levels confer a poor outcome in chronic myeloid leukaemia (similar to findings in many solid tumours), and  also that high CIP2A levels are associated with stabilisation of c-Myc and E2F1, as well as PP2A inactivation in CML. There are also data to suggest that high SETBP1 levels may confer a poor outcome in AML, presumably by PP2A inhibition via increased action of SET, though little is known of CIP2A in AML. 

To investigate if CIP2A or network related proteins could predict clinical outcome in AML patients. 

CIP2A and PP2A related proteins were investigated in 120 AML patient samples collected in the UK MRC/NCRI trials AML15, 16 and  17. These were stratified into intermediate or adverse risk, based on cytogenetics as previously defined (Grimwade et al, Blood. 1998: 92; 2322-33). Protein levels were studied in diagnostic mono-nuclear cells samples by flow cytometry.  CIP2A levels in individual samples were then defined as either high or low based on receiver-operator characteristics analysis as previously described.

Diagnostic CIP2A levels were significantly higher in younger intermediate than in adverse risk patients (p=0.004). In the intermediate risk group, overall survival was dominated by the survival from relapse. High diagnostic CIP2A was associated with high levels of c-Myc (p=0.003), inactive PP2A (PP2A^Y307, p=0.04) and activation of AKT as identified by its phosphorylation on serine 473 (AKT^S473) (p=0.0009). Interestingly, high diagnostic CIP2A was strongly associated with the presence of a FLT3-ITD mutation (p=0.009). For younger intermediate risk patients with high and low CIP2A levels, median survival from relapse censored at transplant was 56 days and 303 days respectively, and multivariate analysis adjusted for FLT3-ITD showed that a high CIP2A level predicted inferior survival from relapse (p=0.04, hazard ratio 4.02).

 

Rates of high levels of CIP2A were lower in adverse risk patients but instead we found elevated levels of SETBP1, which acts via SET as an alternative inhibitor of PP2A. In younger patients with adverse cytogenetics, high SETBP1 levels predict for an inferior overall survival   (p=0.02). High SETBP1 was also associated with secondary AML (p=0.06).

 

In univariate analysis, high levels of AKT^S473 were associated with poor survival in all patient groups. Multivariate analysis of known predictors of outcome (age, cytogenetics, white count, secondary leukaemia, FLT3-ITD) together with CIP2A and  related proteins (PP2A^Y307, SET, SETBP1, c-Myc E2F1, AKT^S473, STAT5, and E2F1) was used to derive a prognostic model for survival. This ranked high levels of AKT^S473 as the third most important predictor of outcome after age and cytogenetics. 

AKT phosphorylation at S473 is a surrogate ‘readout’ of PP2A inhibition by either CIP2A (intermediate risk) or SETBP1 (adverse risk patients). The diagnostic AKT^S473 level appears to be a novel biomarker for treatment outcome in AML. This should now be tested prospectively as part of a clinical trial.