PP2A Inhibition By CIP2A or SETBP1 Leads to Elevated Levels of AKT S473 Which Can be Used As a Biomarker of Outcome in Acute Myeloid Leukaemia

Many cells utilise reversible phosphorylation as a mechanism of post-translational modification for activating and deactivating key regulatory molecules involved in cell signalling. Many malignancies are characterised by overactive kinases e.g. BCR-ABL or FLT-3 in myeloid malignancy, or ERK or ErbB2 in solid tumours. A major phosphatase working in opposition to kinases is protein phosphatase 2A (PP2A) but why the cellular phosphatases such as PP2A simply do not counteract the overactive kinase activity is unclear. PP2A activity is regulated by inhibitor proteins SET, (which is stabilised by a binding protein SETBP1) and cancerous inhibitor of PP2A (CIP2A). In CML, we have shown that high levels of CIP2A at diagnosis is a prospective biomarker for future progression to blast crisis. High levels of CIP2A in other malignancies have also been reported to confer a poor prognosis. In CML, high CIP2A levels lead to the stabilisation of c-Myc and elevation of E2F1, as well as PP2A inactivation. In AML there are data suggesting that high SETBP1 levels may confer a poor outcome in AML, by PP2A inhibition via increased action of SET, though little is known of CIP2A in AML. The aim of the study was to investigate if CIP2A or network related proteins could predict clinical outcome in AML patients.

CIP2A and network related proteins were investigated in 119 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 cell samples by flow cytometry. The diagnostic CIP2A protein level (mean fluorescence intensity (MFI)) for all patients was calculated. This range was 0-13.69, the interquartile range was 1.47-5.11, the median was 3 and the mean was 3.5. High CIP2A patients are defined as those patients with a CIP2A level greater than or equal to 3.

In all AML samples studied high CIP2A was associated with inactive PP2A as shown by high levels of PP2A^Y307 (p=0.05), as well as elevated levels of c-Myc p=<0.0001 and E2F1 p=<0.0001, a similar finding to what we have observed in CML. We also found that AKT^S473 and STAT5 were also elevated in high CIP2A AML patients (p=0.002 and p=0.006 respectively). Unlike CML, we found in AML patients high CIP2A was associated with high levels of SET (p=<0.0001) and SETBP1 (p=0.01).

Younger intermediate AML patients had significantly higher diagnostic CIP2A levels than adverse risk patients (p=0.004). In the intermediate risk group, overall survival was dominated by the survival from relapse. 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. Multivariate analysis adjusted for FLT3-ITD showed that a high CIP2A level predicted inferior survival from relapse (p=0.04, hazard ratio 4.02). Interestingly, high diagnostic CIP2A was strongly associated with the presence of a FLT3-ITD mutation (p=0.03).

In adverse risk patients SETBP1 levels were elevated but CIP2A levels were lower. SETBP1 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). For all 119 AML patients those with detectable SETBP1 protein levels at diagnosis had an inferior overall survival (p=0.01). High SETBP1 was also associated with secondary AML.

In univariate analysis, high levels of AKTphosphorylation at seine 473 (which is inversely correlated with PP2A activity)were associated with poor survival in all patients. 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. Our data suggest that AKT^S473 levels are elevated as a result of PP2A inactivation (p=>0.0001) caused by high level of PP2A inhibitors, either CIP2A (p=0.003) or SETBP1 (p=0.03).

In summary, AKT phosphorylation at S473 is a biomarker 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 survival in AML.