Highly Phosphorylated FOXO3A Is an Adverse Prognostic Factor in Acute Myeloid Leukemia

Published OnlineFirst March 14, 2010; DOI: 10.1158/1078-0432.CCR-09-2551

Clinical Imaging, Diagnosis, Prognosis Cancer Research Highly Phosphorylated FOXO3A Is an Adverse Prognostic Factor in Acute Myeloid Leukemia

Steven M. Kornblau1, Neera Singh1, YiHua Qiu1, Wenjing Chen1, Nianxiang Zhang2, and Kevin R. Coombes2

Abstract Purpose: The Forkhead transcription factors (FOXO) are tumor suppressor genes regulating differentiation, metabolism, and apoptosis that functionally interact with signal transduction pathways shown to be deregulated and prognostic in acute myelogenous leukemia (AML). This study evaluated the level of expression and the prognostic relevance of total and phosphorylated FOXO3A protein in AML. Experimental Design: We used reverse-phase protein array methods to measure the level of total and phosphoprotein expression of FOXO3A, in leukemia-enriched protein samples from 511 newly diagnosed AML patients. Results: The expression range was similar to normal CD34+ cells and similar in blood and marrow. Levels of total FOXO3A were higher at relapse compared with diagnosis. Levels of pFOXO3A or the ratio of phospho to total (PT) were not associated with karyotpe but were higher in patients with FLT3 mutations. Higher levels of pFOXO3A or PT-FOXO3A were associated with increased proliferation evidenced by strong correlation with higher WBC, percent marrow, and blood blasts and by correlation with higher levels of Cyclins B1, D1 and D3, pGSK3, pMTOR, and pStat5. Patients with High levels of pFOXO3A or PT-FOXO3A had higher rates of primary resistance and shorter remission durations, which combine to cause an inferior survival experience (P = 0.0002). This effect was independent of cytogenetics. PT-FOXO3A was a statistically significant independent predictor in multivariate analysis. Conclusions: High levels of phosphorylation of FOXO3A is a therapeutically targetable, independent adverse prognostic factor in AML. Clin Cancer Res; 16(6); 1865–74. ©2010 AACR.

Forkhead transcription factors are a superfamily of evo- tumors and leukemias. The FOXO1 gene was identified lutionary conserved proteins that function in diverse phys- in the studies of t(2, 13)(q35;q14) and t(1, 13)(p36;q14) iologic processes including cellular differentiation, tumor chromosomal translocations found in rhabdomyosar- suppression, metabolism, cell cycle arrest, resistance, and comas, whereas the FOXO3 gene was identified at t(6;11) apoptosis (1, 2). The forkhead genes are grouped into 19 (q21;q23) chromosomal translocation from an acute mye- subclasses of FOX genes (FOXA to FOXS) based on a con- loid leukemia patient and FOXO4 at t(x;11)(q13;q23) in served 100-residue DNA binding “forkhead box” domain acute lymphoblastic leukemia (9). FOXO3A overexpression (1, 3–5). The Forkhead O transcription factor subfamily inhibits tumor growth in vitro and tumor size in vivo in (FOXO), which consists of four members FOXO1, breast cancer cells and cytoplasmic location of FOXO3A FOXO3, FOXO4, and FOXO6, is deregulated in several tu- correlated with poorer survival in patients with breast can- mor types (3). The role of FOXO proteins in tumorigenesis cer (10, 11). Genetic deletion of five FOXO alleles showed were initially identified by their involvement at sites of modest neoplastic phenotypes, whereas deletion of all of chromosomal rearrangements in humans tumors (6–8). the FOXO alleles generated thymic lymphomas and he- For example, FOXO1, FOXO3A,andFOXO4 genes were mangiomas (12). These data define FOXO proteins as bona found at chromosomal breakpoints in human soft-tissue fide tumor suppressor genes (12). Studies by Fei et al. (13) have suggested that low expres-

Authors' Affiliations: Departments of 1Stem Cell Transplantation and sion of FOXO3A expression is associated with the develop- Cellular Therapy and 2Bioinformatics and Computational Biology, The ment of ovarian tumors. More recently, studies have shown University of Texas M.D. Anderson Cancer Center, Houston, Texas the importance of FOXOs in preserving the self-renewal ca- Note: Supplementary data for this article are available at Clinical Cancer pacity of hematopoietic stem cells through undefined me- Research Online (http://clincancerres.aacrjournals.org/). chanisms (14). Studies have shown that FOXO3A Corresponding Author: Steven M. Kornblau, Section of Molecular transcriptional activity may be required to prevent B-chronic Hematology and Therapy, Unit 448, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX lymphocytic leukemia and chronic myelogenous leukemia 77030-4095. Phone: 713-794-1568; Fax: 713-794-1938; E-mail: (14, 15). [email protected]. The FOXO transcription factors are functionally inte- doi: 10.1158/1078-0432.CCR-09-2551 grated (see Fig. 1 for a summary) with several signal trans- ©2010 American Association for Cancer Research. duction pathways including many kinase pathways, e.g.,

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expression and activation status has not been reported. Translational Relevance We have previously shown that numerous signal transduction pathways that regulate FOXO and which are also reg- In this article, we show that the phosphorylation of ulated by FOXO are abnormally activated in AML with FOXO3A, a protein that interacts with numerous signal adverse prognostic consequences (36). This led us to study transduction pathways, is an adverse prognostic factor the role of FOXO3A expression and inactivation through in AML that is associated with increased proliferation, phosphorylation, in a cohort of 511 cases of AML and resistance to therapy, and shorter survival. There is ac- 21 cases of APL using reverse-phase protein array (RPPA). tive investigation into developing therapies aimed at In this study, we report a correlation between phopsho- restoring FOXO3A function by altering localization FOXO3A expression and clinical characteristics, and or inhibiting phosphorylation. Strategies designed to French-American-British classification (FAB) and overall inhibit AKT activity might counteract this effect in pa- survival in leukemia patients. tients with high phospho to total ratios. In contrast, strategies designed to drive resting cells into prolifer- Materials and Methods ation might counteract the inhibitory effect of high levels of unphosphorylated FOXO3A but would be Patient population. Peripheral blood and bone marrow unlikely to have additional effects on those that are specimens were collected from 511 patients with newly di- already highly proliferative due to high PT-FOXO3A agnosed AML and 21 with newly diagnosed acute promye- ratios. FOXO3A therefore makes an attractive target for locytic leukemia (APL) evaluated at The University of Texas therapy in AML but correct application of FOXO3A-di- M.D. Anderson Cancer Center (MDACC) between Septem- rected therapies requires defining the mechanism by ber 1999 and March 2007. Samples were acquired during which FOXO3A is inactivated to optimize efficacy. This routine diagnostic assessments in accordance with the regu- research therefore defines FOXO3A as a target and lations and protocols (Lab 01-473) approved by the Inves- guides the application of FOXO3a directed therapies. tigational Review Board of MDACC. Informed consent was obtained in accordance with the Declaration of Helinski. Samples were analyzed under and Institutional Review Board–approved laboratory protocol (Lab 05-0654). Sam- c-Jun-NH2-kinase (16), MST1 (17) AMPK (18), IkB (11), SGK (19), CDK2 (20), extracellular signal-regulated ki- ples were enriched for leukemic cells by performing ficoll nase (ERK)1/2 (10), DYRK1A (21), Caspase (22), phos- separation to yield a mononuclear fraction followed by phoinositide 3-kinase -AKT (23), and Wnt (24), which CD3/CD19 depletion to remove contaminating T and B can regulate a broad array of cellular process that include cells, if they were calculated to be >5% based on the differ- stem cell proliferation (14, 25), aging (26), and malig- ential. The samples were normalized to a concentration of 1 4 nancy (2). FOXO proteins can act not only as transcrip- ×10 cells/μL and a whole-cell lysate was prepared as previ- tional activators but also as transcriptional repressors ously described (36). A total of 387 marrow and 283 blood (12, 15, 27, 28). Their functions are tightly regulated at samples were studied from the newly diagnosed cases with multiple levels by phosphorylation, ubiquitylation, acetyla- 140 having both available. Among the 142 AML and 4 APL tion, and protein-protein interactions (29–31). Phosphory- cases that relapsed, a paired relapse sample was available for lation on threonine 24, serine 256, or serine 318 by AKT 49 of the AML and 1 of the APL cases. All but one relapse inhibits FOXO3A activity by increasing nuclear export and samples were from the marrow. Outcomes analysis for this this in turn increases proliferation (32). Similar to the reti- report is restricted to the newly diagnosed patients. The as- noblastoma tumor suppressor gene, FOXO function could sociated demographics are described in Table 1. Although therefore be lost by either diminished expression or the in- this population is typical for the MDACC referral pattern, activation by phosphorylation. FOXO transcription factors the median age of these patients (65.7 y) is older than the are therefore emerging as master signaling regulators, which national average of 58 y, and this population has a high per- control various physiologic and pathologic processes, in- centage of cases with unfavorable cytogenetics (49%) and a cluding cancer protection (15, 28, 33). This combination very high percentage with an antecedent hematologic disor- of functional activity combined with the frequency of ab- der (AHD; 40%). normal expression in malignancy makes the FOXO proteins Of the 511 AML cases, 415 patients were treated at interesting targets for therapeutic intervention. MDACC and are evaluable for outcome. Among these, 277 There is limited data available on the link between FOX- received high-dose ara-C (HDAC), 191 with an anthracy- O3A and the progression of leukemia. A previous study cline, 49 with fludarabine (FLAG, FA), 28 with clofarabine, showed that increased expression of nuclear FOXO3A 8 with other agents, and 1 with an HDAC alone. Another 35 was found to be associated with increased drug resistance received standard-dose ara-C with clofarabine (n = 33) or in leukemic cells through enhanced phosphoinositide with daunorubicin and etoposide (n = 2), whereas 8 received 3-kinase PI3K/AKT activity (34). A forthcoming publica- low-dose ara-C–based regimens. Idarubicin and troxacita- tion found that high FOXO3a mRNA levels correlated bine was used in 2 cases and VNP40101M was used for 25 with inferior survival in patients with normal karyotype cases. Demethylating or histone deacetylating agents were acute myelogenous leukemia (AML; ref. 35) but protein used alone or in combination for 45 patients. Targeted agents

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were used in 13, 6 received gemtuzumab ozogamicin (GO) Statistical analysis. Supercurve algorithms were used to in combination with interleukin-11, and 4 received phase generate a single value from the five serial dilutions (41). 1 agents. For the 21 APL cases, 15 received arsenic trioxide Loading control (42) and topographical normalization plus ATRA, and 6 of these also receive gemtuzumab procedures accounted for protein concentration and back- ozogamicin. Four received idarubicin and all trans reti- ground staining variations. Analysis using unbiased clus- noic acid (ATRA), one in conjunction with gemtuzumab tering, perturbation bootstrap clustering, and principle ozogamicin, and one each receive liposomal ATRA and component analysis was then done as fully described in gemtuzumab ozogamicin plus ATRA. ref. (38). A composite variable based on the ratio of RPPA method. Proteomic profiling was done on samples phospho to total FOXO3A was generated (PT-FOXO3A). from patients with AML using RPPA. The method and val- Use of the ratio allows for recognition that a high ratio in idation of the technique are fully described in previous the setting of low total levels would produce the same publications (37–39). Briefly, patient samples were biological consequence of inactivation as a high ratio in printed in five serial dilutions onto slides along with nor- the face of high levels of total FOXO3A. That information malization and expression controls. Slides were probed is not present when the results for the individual total or with a strictly validated primary antibody against total phosphor protein are analyzed. These variables were di- FOXO3A or phospho serine 318/321 (Cell Signaling) vided into thirds (low, medium, and high cohorts) based and a secondary antibody to amplify the signal, and final- on the range of expression of all 511 samples. ly a stable dye (40) is precipitated. The stained slides were Comparison of the protein levels between paired samples analyzed using the Microvigene software (Vigene Tech) to was done by performing paired t test. Association between produce quantified data. protein expression levels and categorical clinical variables

Fig. 1. FOXO proteins are involved in multiple signaling pathways that control cell proliferation, apoptosis, survival, and immune functions in response to external and internal stimuli. Stimulated growth factors (insulin or insulin-like growth factor I) and their receptors (such as epidermal growth factor receptor) lead to activation of ERK (A), Akt (B) and CDK2 (C) through RAS and phosphoinositide 3-kinase–dependent phosphorylation of FOXOs. ERK and Akt-mediated phosphorylation of FOXO proteins inhibit transcription of the gene that promote apoptosis (Bim, FasL, and TRAIL) and cell cycle arrest (p27, p21, and c-myc) by exporting FOXO proteins to cytoplasm and inducing ubiquitination and proteosomal degradation. Akt-mediated phosphorylation allows FOXOs cytoplasmic retention through 14-3-3 protein binding, whereas ERK pathway activation leads to FOXO ubiquitination through MDM2 binding. Similarly, tumor necrosis factor α (TNFα) stimulates IKK, which phosphorylates FOXO and thus promote cytoplasmic retention. FOXOs phosphorylation by CDKs also leads to cytoplasmic localization. However, this activity is abolished on any DNA damage, leading to the activation of CHK1 and CHK2.

In case of any oxidative stress, FOXO proteins translocate back to the nucleus with the help of c-Jun-NH2-kinase (JNK)–dependent and MST1 phosphorylation (D) and on interaction with β-catenins.

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Table 1. Demographic and clinical characteristics of 511 newly diagnosed AML patients in the study set

All cases Treated P All Lowest 3rd Middle 3rd Highest 3rd

N 511 414 129 142 144 Gender Male/female 291:220 218:197 73:56 71:70 75:69 0.66 Age Min 15.8 17.4 27 18.8 17.39 0.32 Max 87.23 86.8 86.6 85.6 86.8 Median 65.7 64.16 64.24 65.3 63.58 FAB M0 5.7% 5.1% 3.9% 6.3% 4.9% 0.67 M1 10.8% 11.6% 8.5% 11.3% 14.6% 0.29 M2 33.1% 37.0% 44.2% 44.4% 22.9% 0.00002 M4 22.7% 23.4% 19.4% 22.5% 27.8% 0.26 M5 10.0% 9.9% 5.4% 4.2% 19.4% 0.000008 M6 5.3% 5.1% 6.2% 4.9% 4.2% 0.72 M7 2.0% 1.9% 2.3% 0.7% 2.8% 0.41 Unknown 3.3% 0.7% 0.8% 1.4% 0.0% RAEBT 7.2% 5.6% 9.3% 4.2% 3.5% 0.068 WHO* Abnormal cytogenetics 9.2% 10.4% 13.2% 11.3% 8.3% 0.57 Multilineage dysplasia 21.5% 18.8% 24.8% 18.3% 13.9% 0.16 Therapy related 14.3% 12.8% 16.3% 12.0% 10.4% 0.36 Not in Others 55.0% 58.0% 45.0% 58.5% 68.8% 0.0008 Cyto† Favorable 6.7% 8.0% 10.9% 10.6% 2.8% 0.019 Intermediate 44.0% 45.2% 37.2% 45.1% 52.1% Unfavorable 49.3% 47.1% 51.9% 44.4% 45.1% FLT3 ITD 14.9% 17.9% 7.0% 15.5% 29.9% 0.000004 D835 3.1% 3.9% 1.6% 4.2% 5.6% 0.045 Both 1.6% 1.9% 0.0% 2.8% 2.8% 0.16 Zubrod PS 3 or 4 3.3% 3.1% 1.6% 2.8% 4.9% 0.42 AHD ≥2 Mo 39.9% 37.4% 48.8% 34.5% 29.9% 0.004 Infection Yes 19.8% 22.2% 14.0% 21.1% 30.6% 0.004 WBC Median 8.8 9.9 3.55 9.2 30.1 <0.00001 Platelet Median 56 55.5 53 53.5 59 0.11 Hemoglobin Median 9.6 9.6 9.7 9.5 9.5 0.56 % marrow blast Median 46 50 38 46 70 <0.00001 % blood blast Median 18 20.5 8 20.5 48 <0.00001 Response CR NA‡ 55.8% 59.7% 59.2% 48.6% 0.11 Resistant NA 34.3% 31.8% 31.7% 38.9% 0.34 Fail NA 10.1% 8.5% 9.2% 12.5% 0.49 Relapse NA 61.5% 62.3% 57.1% 64.3% 0.64 Alive NA 25.1% 24.8% 33.8% 16.7%

(Continued on the following page)

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Table 1. Demographic and clinical characteristics of 511 newly diagnosed AML patients in the study set (Cont'd)

All cases Treated P All Lowest 3rd Middle 3rd Highest 3rd

Overall survival, NA 48.7 64.85 60.14 33.714 0.0002 median, wk Remission duration, NA 45.57 41 52.9 34.7 0.12 median, wk

NOTE: The demographics of only those that were treated is shown in the second column and the distribution based on the ratio of phospho to total FOXO3A levels are shown in the next three columns. The P values refer to comparisons of the 3 PT-FOXO3A groups. Abbreviation: NA, not applicable. *Based on the 2008 classification schema. †Favorable, t(8:21) or inversion(16); intermediate, Diploid or −y; unfavorable, all others including −5, −7, +8, and t(6:9); miscellaneous changes, +21, 11q23. ‡Because not all cases were treated, outcome measurements were not applicable. were assessed in R using standard t tests, linear regression, or PT-FOXO3A by FAB classification or by cytogenetics is mixed effects linear models. Association between continu- shown in Supplementary Fig. S1A and B. Cases of monocytic ous variable and protein levels were assessed by using the leukemia (M5) had significantly higher levels of pFOXO3A Pearson and Spearman correlation and linear regression. expression and higher PT ratios (P = 0.0000008). Patients Bonferroni corrections were done to account for multiple with FAB M2 were more likely to have low PT ratios (P = statistical parameters for calculating statistical significance. 0.00002). Levels of total FOXO3A trended higher (P =0.09) The Kaplan-Meier method was used to generate the survival and levels of pFOXO3A were significantly higher (P =0.002) curves. Univariate and multivariate Cox proportional hazard in patients with intermediate and unfavorable cytogenetics. modeling was done to investigate association with survival Patients with favorable cytogenetics were significantly less with protein levels as categorized variables using the Statis- likely to have high PT-FOXO3a levels (0.017). Cases tica version 6 software (StatSoft). This data set contains with either a FLT3-ITD or D835 mutation had significantly patients treated before some more recent prognostic markers higher levels of total, (P = 0.001), phospho (P = 0.008), or were discovered (e.g., NPM1); therefore, the multivariate PT-FOXO3A (P = 0.000004; Fig. 3A). Neither showed differ- analysis did not contain all known AML prognostic markers. ent expression on the basis of gender or performance status. Total (P = 0.008) and PT-FOXO3A (P = 0.004) but not Results phospho levels were higher in patients with AHD. Levels of pFOXO3A were significantly lower in cases with a his- FOXO3A, phospho FOXO3A (pFOXO3A), and the ratio tory of prior malignancy, chemotherapy, or radiation ther- of phospho to total FOXO3A (PT-FOXO3A) protein ex- apy (P = 0.03, 0.02, and 0.01). Levels of total FOXO3A pression was analyzed in 511 newly diagnosed AML pa- were significantly lower (P = 0.0004), and pFOXO3A tients by RPPA. To determine the effect of source on (P = 0.02) and PT-FOXO3A (P = 0.004) were significantly protein levels, the expression in the 140 same day paired higher in patients with active infections. The protein levels blood and marrow samples was compared. (Fig. 2, top for total, pFOXO3A, and PT ratio were modestly but statis- row). No statistically significant differences were observed tically significantly (R < 0.30; P < 0.000001) correlated with between blood- and marrow-derived samples, thus both WBC, percent marrow blasts, percent blood blasts, and source samples were combined for the subsequent data the absolute peripheral blood blast count (Fig. 3B–D), and analysis. Compared with expression in normal CD34+ the PT ratio was correlated with CD34. Categorized levels were cells, levels of total and pFOX3A were ≤2 SDs below the strongly associated with these characteristics (see Table 1). mean in 30.3% and 17.8% of cases, respectively, and were Correlation with other proteins. This same RPPA array infrequently ≥2 SDs above the mean (7.3% and 1.6%; has been probed with 174 other antibodies and the full Fig. 2. bottom row). Among the 49 paired diagnosis/re- analysis will be published separately. Notably pFOXO3A lapse paired samples, the expression levels of pFOXO3A or PT-FOXO3a levels were significantly positively correl- did not change with disease progression, but levels of total ated with proliferation markers Cyclins B1, D1, D3, FOXO3A were significantly higher in relapsed samples GSK3, pGSK3, LYN, MTOR, pPKCδ, pPKCγ,andpStat3, (median fold change, 1.49; P = 0.0007). (R = 0.42, 0.23, 0.36, 0.39, 0.30, 0.31, 0.35, 0.33, 0.21, and FOXO3, pFOXO3A, and PT ratio expression, and clinical 0.23, all with P < 0.0000004) and negatively correlated with characteristics. Variation in the level of total, phospho, and pAKT-thr308, pRB, Yap, and pYAP (R = −0.21, −0.25, −0.35,

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Fig. 2. Distribution of expression of FOXO3A and phosphoFOXO3A in AML based on source, disease status, and relative to normal CD34+ cells. Top, the ratio of expression between 140 paired blood and marrow specimens is shown. There was no significant difference between the two sources of leukemic blasts. Middle, the ratio of expression between 49 paired diagnosis and relapse specimens is shown. FOXO3A levels were significantly higher in relapse compared with diagnosis, but pFOXO3A levels did not differ. Bottom, comparison of expression distribution histograms of 511 AML samples (gray) relative to 10 normal CD34+ samples (black) is shown. Both FOXO3A and pFOXO3A showed expression levels that were significantly below that of the normal CD34+ cells. and −0.39, all P < 0.0000001). PT-FOXO levels were also O3A or PT-FOXO3A remission rates were similar for those strongly correlated with apoptosis proteins Bax, BAK, and with low or medium levels (∼60%) and were significantly inactivated Bad-pSerine136 (R = 0.25, 0.24, and 0.22; all higher compared with patients in the highest third (49%, P P < 0.000001). A list of all proteins with R ≥ 0.2 is shown = 0.001). Relapse rates were not associated with total, p-FOX- in Supplementary Fig. S2. O3a, or PT-FOXO3a levels (P = 0.64). Higher levels of pFOX- FOXO3A expression, overall survival, and remission dura- O3A and PT-FOXO3A were both highly prognostic adverse tion. For outcome analysis, the values were categorized into markers for overall survival (P = 0.0002 for both) with a me- thirds (low, medium, and high) based on the range of ex- dian survival of 60 and 64 weeks for low and middle com- pression in all 511 cases. A higher proportion of those with pared with 33 weeks for high PT-FOXO3A (Fig. 4A), but total low levels were not treated at MDACC, making the size of the FOXO3A was not (P = 0.34). This was also true among pa- “low” cohort smaller than the other two. Total FOXO3A le- tients treated with more traditional HDAC-based therapies vels were not prognostic of remission attainment. For pFOX- (n = 285; P = 0.0007) or those treated with nontraditional

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therapies (e.g., demethylating or histone deacetylating ics (P = 0.02) and this effect was predominant among agents, gemtuzumab ozogamicin or VNP40101M; n = 72; those with a FLT3 abnormality. High levels of pFOXO3A P = 0.06). Neither total (P = 0.83), pFOXO3A (P = 0.21), were adverse for those with favorable (P = 0.015) or un- or PT-FOXO3A (P = 0.11) were prognostic of remission du- favorable cytogenetics (P = 0.008) ration (P = 0.83; Fig. 4B), although for all three, those with For APL, there were eight, six, and seven patients with medium levels had the longest median remission duration. low, medium, or high PT-FOXO3A. All but one achieved Identical results are observed if the variables are split at the complete remission (CR) (the lone failure presented with medianinsteadofintothirds. a WBC of 180K and had medium PT-FOXO3A). Four have The effect of total, pFOXO3A, and PT-FOXO3A within relapsed, one with middle, and three with high PT-FOX- different cytogenetic groups was evaluated as well. A O3A. Although the numbers are small, this suggests that high PT-FOXO3A level was adverse for patients with in- high PT-FOXO3A may predict for relapse in APL (P = 0.09). termediate (P = 0.034) and unfavorable (P = 0.024) cy- PT-FOXO3A level is an independent predictor of outcome togenetics with a similar but not significant trend noted in multivariate analysis. A Cox proportional hazard model for those with favorable cytogenetics. This was indepen- was done evaluating for factors that were independent pre- dent of the presence or absence of a FLT3-ITD or D835 dictors of overall survival. Starting with 16 variables that mutation. In agreement with the findings of Santamaria were univariate predictors in this data set, or that have tra- et al. (35), we observed that high levels of total FOX- ditionally been prognostic (e.g., performance status, ante- O3A was adverse for cases with intermediate cytogenet- cedent hematologic disorder), a stepwise analysis was

Fig. 3. Correlation of phospho to total FOXO3A levels with (A) FLT3 ITD mutation status, (B) percentage bone marrow blasts, (C) WBC count, and (D) absolute peripheral blood blast count. Black box, 24 to 75 percentile levels; white box, the median level. Bars, 2 SDs. Outliers (O) and extremes (*) are shown. The results of the Kruskal-Wallis and F tests are shown in the other box.

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conducted until only significant (P < 0.05) variables re- were similar in blood and marrow blasts. Levels of total mained. This was followed by sequential add back of all FOX3A but not pFOXO3A were higher at relapse compared previously removed variables, one by one, until a final with diagnosis. Higher levels of pFOXO3A or the PT ratio model with only significant variables remained. The final were observed in FAB M5 and lower levels in FAB M2. model contained six variables, age, cytogenetics (favorable, Levels of p-FOXO3A or PT-FOXO3A were higher among intermediate, unfavorable) FLT3 mutation, albumin, PT- patients with FLT3 mutations, substantiating observations FOXO3A,andgender,asshowninTable2.Performance in FLT3-ITD+ cell lines (43), but otherwise were not asso- status likely was not prognostic as only 3% of cases had a ciated with specific cytogenetic abnormalities. Higher Zubrod PS of 3 or 4. This shows that the PT ratio of FOX- levels of p-FOXO3A or PT-FOXO3A were clearly associated O3A is an independent predictor of outcome in AML. with a higher proliferative potential as seen by the strong correlation between inactivated (phosphorylated) FOXO3A Discussion with higher WBC, percent marrow, and blood blasts. This was reinforced by the association of higher p-FOXO3A or In the present study, total and phosphoprotein expres- PT-FOXO3A levels with higher levels of other proteins as- sion of FOXO3A was assessed by RPPA in AML patients. Ex- sociated with proliferation including Cyclins B1, D1 and D3, pression was generally similar to, or at the lower end of the pGSK3, pMTOR, and pStat5 as well as by negative correla- range of expression seen in normal CD34+ cells, and levels tions with pRB. Paradoxically levels were inversely correlated

Fig. 4. Kaplan Meier curves for overall survival (top) and remission duration (bottom) for the ratio of phospho to total FOXO3A are shown.

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for leukemic blasts that survive therapy, leading to higher Table 2. Multivariate analysis, final model rates of resistance and more rapid leukemic recurrence. When there are high levels of unphosphorylated FOXO3A Variable β Exponent β Wald P statistic present, it contributes to resistance of stem cells, possibly by maintaining them in a resting state, thereby enabling some Age (y) 0.03 1.04 65.66 <0.000001 to survive and eventually repopulate the marrow with leu- Cytogenetics −0.657 0.518 37.67 <0.000001 kemia. The first is a more rapid effect, the second more de- Albumin −0.375 0.687 21.54 0.000003 layed. There is active investigation into developing therapies FLT3-abnormal 0.184 1.201 7.846 0.005 aimed at restoring FOXO3A function by altering localiza- PT-FOXO3A 0.191 1.21 6.98 0.008 tion or inhibiting phosphorylation (46–48). Strategies de- Gender 0.261 1.298 4.705 0.03 signed to inhibit AKT activity might counteract this effect in patients with high PT ratios but would not be expected with PT-AKT-thr308 levels. Based on current understand- to affect the second mechanism. In contrast, strategies de- ing that phosphorylation leads to cytoplasmic localization signed to drive resting cells into proliferation (G or granu- and inactivation of FOXO3A, these findings are consistent locyte macrophage colony-stimulating factor, CXCR4 with the idea that increased proliferation results when antagonists) might counteract the inhibitory effect of high FOXO3A is inactivated in AML. levels of unphosphorylated FOXO3A but would be unlikely Although there is compelling evidence for a role for the to have additional effects on those that are already highly FOX proteins in malignancy, the prognostic implications of proliferative due to high PT-FOXO3A ratios. FOXO3A their expression and activation has not been extensively therefore makes an attractive target for therapy in AML studied (2). For FOXO3A, divergent observations are re- but correct application of FOXO3A-directed therapies may ported; high mRNA was adverse in diploid AML (35); high require defining the mechanism by which FOXO3A is inac- total FOXO3A protein levels were favorable in lymphoma tivated to optimize efficacy. (44); and low protein expression was adverse in ovarian We evaluated phosphorylation on a single site, but differ- and hepatocellular cancer (13, 45). The prognostic implica- ent kinases achieve inactivation by phosphorylating different tions of phosphorylation has not been previously reported, sites on FOXO3A (e.g., AKT on T32, S253 and S315 and ERK κ and the activation state imparted by phosphorylation may on S294, S344 and S425, and I K on S644; ref. 48). A more account for these opposite findings. We found that high le- comprehensive analysis of FOXO3A phosphorylation might vels of phosphorylation of FOXO3A was an adverse prog- detect additional phosphorylated cases and improve the κ nostic factor for overall survival. These patients had higher prognostic discrimination. Recent reports suggest that I K rates of primary resistance and shorter remission durations phosphorylation is important in AML (49). However, there that combine to cause an inferior survival experience. This is likely to be significant overlap as we have previously effect was independent of cytogenetics as high p-FOXO3A shown that simultaneous activation of multiple signal trans- or PT-FOXO3A was prognostic within individual cytogenet- duction pathways is a common event in AML (36). In this ic groups, and remained a statistically significant data set, increased pAKT and pERK2 was strongly associated P independent variable in multivariate analysis along with cy- with increased FOXO3A ( < 0.00001; data not shown). In togenetics. Validation of this finding in a data set with other summary, phosphorylation of FOXO3A is an adverse prognostic factors such as NPM1 requires future research. prognostic factor in AML that is associated with increased Although not statistically significant, the remission duration proliferation, resistance to therapy, and shorter survival. of patients with low levels of p-FOXO3A or PT-FOXO3A was shorter than patients with middle ratios and their sur- Disclosure of Potential Conflicts of Interest vival experience diverged after 104 weeks. These patients No potential conflicts of interest were disclosed. would be expected to have more FOX3A in the nucleus and this could lead to protection of leukemic stem cells Acknowledgments from chemotherapy and ultimately to relapse. This would be consistent with the finding of Hui et al. (34) that in- The costs of publication of this article were defrayed in part by the creased expression of nuclear FOXO3A was associated with payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to increased drug resistance in leukemic cells. This would im- indicate this fact. ply that FOXO levels can influence responsiveness in two manners. When inactivated by phosphorylation, prolifera- Received 09/18/2009; revised 12/16/2009; accepted 01/12/2010; tion is increased and this creates a repopulation advantage published OnlineFirst 03/09/2010.

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1874 Clin Cancer Res; 16(6) March 15, 2010 Clinical Cancer Research

Highly Phosphorylated FOXO3A Is an Adverse Prognostic Factor in Acute Myeloid Leukemia

Steven M. Kornblau, Neera Singh, YiHua Qiu, et al.

Clin Cancer Res 2010;16:1865-1874. Published OnlineFirst March 14, 2010.

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