Acute myeloid leukemia (AML) comprises a biologically and clinically heterogeneous group of aggressive disorders that occur as a consequence of a wide variety of genetic and epigenetic abnormalities in hematopoietic progenitors. Despite significant advances in the understanding of AML biology, overall survival remains poor due chiefly to the high rate of relapse after achieving complete remission, as well as primary induction chemotherapy failure. In fact, apart from the exceptions represented by a few subgroups, such as younger patients with more favorable genetic outcome, improvements in the treatment of AML have been slow.21 Therefore, it is necessary to develop new therapies aimed at molecular targets to improve clinical outcome in AML.
Protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase that negatively regulates numerous signal transduction pathways, and functions as a tumor suppressor in several cancers.3 Our group reported that PP2A inhibition is a common event in AML, and that restoration of PP2A activity induces cell growth arrest and caspase-dependent apoptosis, suggesting that PP2A inactivation plays a crucial role in AML, and confirming its potential as a therapeutic target in hematologic malignancies.4 Moreover, we found the PP2A endogenous inhibitor SET over-expressed in 28% of AML patients, where it is associated with short overall survival (OS).5 Here, we study the prevalence and the prognostic significance of the high expression of CIP2A, another specific endogenous inhibitor of PP2A, in AML.
The CIP2A protein mainly performs its role in malignant cellular growth by inhibiting PP2A activity toward the oncogenic transcription factor c-MYC,6 an oncoprotein that plays an important role in AML.7 CIP2A is expressed in very few normal tissues but it is over-expressed in most human cancer types, where it is often associated with a clinically aggressive behavior.6 Nevertheless, its role in AML is unknown. To further clarify the significance of CIP2A high expression in AML, we first investigated its prevalence using quantitative real time RT-PCR (QRT-PCR) in a series of 203 normal karyotype AML patients (NK-AML) at diagnosis. Detailed information about the molecular and statistical analyses performed is provided in the Online Supplementary Appendix. CIP2A 2- median expression in AML was 0.0044 (range 0.00012–0.59) while in bone marrow (BM) samples from healthy donors the median expression was 0.0055 (range 0.0027–0.0070). To define CIP2A high (CIP2Ahigh) expression levels, a cut-off value of 0.0072 was chosen (corresponding to CIP2A mean expression in normal BM+2SD and 75 percentile in AML patients). Surprisingly, several AML cases had low levels of CIP2A, even below normal BM. Additional studies are needed to clarify the meaning of this interesting finding. CIP2Ahigh expression showed no significant differences regarding clinical characteristics. However, correlations with molecular markers revealed a significant inverse association between CIP2A and NPM1. Patients with higher CIP2A expression showed a reduced incidence of NPM1 mutations (NPM1 mut) (30% vs. 48%; P=0.021). Patients’ characteristics classified according to CIP2A expression are shown in Online Supplementary Table S1. To date, only one study has analyzed CIP2A expression in AML.8 In this study, Wang et al. using conventional RT-PCR found that 77.4% of cases with AML expressed CIP2A (55 of 84), and confirmed their results at protein level. They classified the patients according to the French-American-British (FAB), but they provided no quantitative data. Thus, for the first time, we show CIP2Ahigh expression in a large cohort of NK-AML patients using QRT-PCR.
We next analyzed the prognostic significance of CIP2A expression in 190 cases treated with intensive chemotherapy (missing follow-up data, n=13). There were no significant differences in the rate of complete remission (CR) (75% vs. 83%), resistance (7% vs. 5%) or death during induction therapy (12% vs. 17%) between patients with CIP2Ahigh or CIP2Alow expression. The median follow-up time for survival was 20 months (range 1–122 months), and the estimated probability of overall survival (OS) at three years was 41%±4. Univariate analysis revealed that patients with CIP2Ahigh expression presented a significantly shorter OS compared with those with CIP2A expression (27% vs. 50%; P=0.0004) (Figure 1A). The median follow-up time in 147 AML patients who achieved CR was 21 months (range 0.9–120 months). Disease-free survival (DFS) at three years was 40%±4 and relapse-free survival (RFS) was 48%±5. Univariate analysis showed that patients with CIP2Ahigh expression presented a significantly shorter DFS (30% vs. 47%; P=0.0043) and RFS (46% vs. 60%; P=0.0090) compared with those with CIP2A expression (Figure 1B and C). Similar results for OS, DFS and RFS were obtained in the subgroup of patients younger than 65 years (32% vs. 58%, P=0.0017; 30% vs. 53%, P=0.0062; and 50% vs. 66%, P=0.0218, respectively).
Cox multivariate analysis included age, sex, white blood cell count, percentage of BM blasts, FLT3-ITD, NPM1 mutations and CIP2A expression. For OS, age over 65 years (P=0.001; HR=2.423), FLT3-ITD (P=0.038; HR=1.654) and CIP2Ahigh expression (P=0.013; HR=2.017) were independent prognostic factors. For DFS, age over 65 years (P=0.042; HR=1.920), NPM1mut without FLT3-ITD (P=0.036; HR=0.560) and CIP2Ahigh expression (P=0.003; HR=2.488) were independent prognostic factors. For RFS age over 65 years (P=0.018; HR=2.497) and CIP2Ahigh expression (P=0.010; HR=2.650) were independent prognostic factors (Table 1). When only AML patients younger than 65 years were considered, FLT3-ITD and CIP2Ahigh expression were independently associated with a worse outcome for OS, DFS and RFS. NPM1mut without FLT3-ITD were associated with better clinical outcome for OS and DFS (Table 1). The clinical relevance of CIP2Ahigh expression as a prognostic marker has been established in various solid and hematologic cancers, including gastric, bladder, ovarian, tongue, hepatocellular, colon, non-small cell lung carcinoma, and chronic myeloid leukemia;6 however, the prognostic impact of this molecular marker in AML was unknown. Therefore, our results show for the first time that CIP2Ahigh expression is an independent poor prognostic factor in NK-AML. The adverse prognosis for patients with CIP2Ahigh expression was particularly significant in younger patients without NPM1 mutations. This interesting finding could be relevant because molecular markers may define subgroups of patients and help in clinical decision-making.
We next evaluated the functional role of CIP2A in AML. Western blot showed that high CIP2A protein level is a recurrent event in AML cell lines (Online Supplementary Figure S1). Afterwards, CIP2A was down-regulated using a specific siRNA as previously described9 in the AML HEL and TF1 cell lines. As previously reported in the HL-60 cell line,8 CIP2A knockdown in these cell lines decreased cell proliferation and clonogenic growth, together with c-MYC protein stability (Figure 2 and Online Supplementary Figure S2). c-MYC is frequently activated in AML and plays an important role in leukemogenesis induction.7 Our results indicate that CIP2Ahigh expression could contribute to the leukemogenic role of c-MYC in AML. Moreover, it has been reported that myeloid progenitor cells expressing c-MYC in bone marrow transplantation-transduction assays possess an intrinsic mechanism of resistance to c-MYC-induced apoptosis.10 Although we detected no changes in apoptosis when CIP2A was down-regulated (data not shown), we observed an increase of differentiation to the megakaryocytic lineage (Figure 2 and Online Supplementary Figure S2). Finally, as expected, CIP2A knockdown increased PP2A activity by nearly 30%, together with a reduction of the inactivated form of PP2A catalytic subunit (PP2Ac) in HEL cells, decreasing the activity of ERK, a PP2A target (Figure 2). Two recent studies reported that patients with CIP2A negative tumors respond significantly better to cancer therapies.6 In this regard, we and others have previously shown that PP2A activating drugs represent very promising anti-cancer molecules that can be used alone or in association with either kinase inhibitors or traditional chemotherapy in myeloid leukemias.1211 The 5-year survival rate of NK-AML patients varies from 24% to 42%, suggesting a genetic diversity within this subgroup.
Mutational and wide-genomic studies are proving very useful to refine prognosis in this AML subgroup, and it has been reported that the screening of at least ten genes: ASXL1, CEBPA, DNMT3A, FLT3, IDH1, IDH2, MLL, NPM1, PHF6, and TET2, would discriminate intermediate-risk AML patients into robust, clinically relevant risk groups.1413 However, as indicated above, the challenge in this aggressive disease is to provide potential therapeutic targets. Given this, our results identify a subgroup of patients who might benefit from future therapies with PP2A activators.3
In conclusion, we show that CIP2Ahigh expression is a recurrent event in AML where it represents a marker of reduced overall survival and a poor prognostic factor, as previously reported in other tumors. Moreover, CIP2A depletion down-regulates c-MYC, leading to a reduction of cell proliferation, and supporting the positive relationship between CIP2A and this oncogenic transcription factor in AML. Our results confirm that CIP2A behaves as an oncoprotein in AML, and suggest that it could represent a novel therapeutic target in AML.
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