Background
Acute myeloid leukemia (AML) is an aggressive bone marrow malignancy that arises from the somatic transformation of hematopoietic stem and progenitor cells [
1]. Cytogenetic analysis assigns about 50% of AML patients to the normal karyotype (NK-AML) [
2‐
4]. The discovery of numerous genetic alterations that are associated with NK-AML through whole-genome analyses has led to significant advances in AML research [
5]. Nucleophosmin (
NPM1) is one of the most frequently altered genes in NK-AML, accounting for one-third of AML cases (50 to 60% of adult NK-AML) [
6]. To date, > 55 unique mutations have been identified in exon 12 of the gene
NPM1, and the most common mutation is referred to as the type A mutation (NPM1-mA) with a 4-base-pair TCTG insertion at nucleotide position 960 [
7,
8]. Because of its distinctive molecular and clinical characteristics, NPM1-mutated AML has been defined as a distinct entity in the 2016 updated World Health Organization (WHO) classification of myeloid neoplasms [
9,
10]. Although the NPM1 mutation is an AML-driving lesion, this mutation alone is not sufficient to cause AML and it requires a cooperative event that aids leukemogenesis [
11].
An accumulating body of evidence indicates that the phosphoinositide-3 kinase (PI3K) pathway plays an important role in the regulation of hematopoiesis [
12]. Abnormal activation of the PI3K signaling pathway has been reported in >50% of AML cases [
13]. Activated PI3K phosphorylates the phosphatidyl-inositol bisphosphate (PIP
2) to generate phosphatidyl-inositol trisphosphate (PIP
3), and thereby facilitates recruitment and activation of the AKT protein [
14,
15]. In turn, PI3K/AKT signaling can be terminated by several phosphoinositide phosphatases that dephosphorylate phosphatidylinositol phosphate (PIP) species, i.e., the phosphatase and tensin homolog (PTEN) and src homology 2-containing inositol phosphatase (SHIP) family hydrolyze PIP
3 to generate PIP
2 [
16,
17]. Recently, Inositol polyphosphate 4-phosphatase type II (INPP4B), a new factor in the regulation of the PI3K signaling module in tumors, was observed to preferentially dephosphorylate PI (3,4) P
2 to produce PI (3) P and thereby block the activation of AKT [
18,
19]. The suppressive function of INPP4B, akin to that of PTEN, was initially identified in breast cancer [
20], and later confirmed in ovarian [
21] and prostate cancers [
22]. Interestingly, several recent reports have shown that INPP4B overexpression could be detected in other cancer contexts, such as PIK3CA-mutant breast cancer [
23] and a subset of melanoma [
24]. Notably, INPP4B was aberrantly overexpressed and emerged as an independent predictor of poor prognosis in AML patients with normal cytogenetics [
25]. To date, however, the biological role of INPP4B in NPM1-mutated AML and the molecular mechanisms by which INPP4B contributes to leukemogenesis remain unclear.
In breast and other epithelial cancers, INPP4B has been predicted to be a tumor suppressor that blocks AKT activation. However, INPP4B expression is not associated with the changes in AKT phosphorylation status in leukemia, indicating that AKT-independent mechanisms are likely at play [
25]. Serum and glucocorticoid-regulated kinase-3 (SGK3), another PI3K-dependent serine/threonine kinase, shares high structural and functional similarities with the AKT protein [
26]. However, unlike AKT, SGK3 contains a unique N-terminal phox homology (PX) domain that binds to PI (3) P, thus targeting early endosomes where SGK3 is fully activated [
27,
28]. Indeed, SGK3 emerges as an alternative downstream effector of INPP4B that diverges from canonical AKT signaling [
29]. Recent studies have indicated an association between high INPP4B expression and SGK3 phosphorylation levels in PIK3CA-mutant breast cancers and melanoma, in which INPP4B-mediated activation of SGK3 enhances cell proliferation and promotes anchorage-independent cell growth [
23,
24]. Herein, we report that INPP4B is frequently upregulated in NPM1-mutated AML, and promotes leukemia cell survival in a SGK3-dependent and AKT-independent manner. Increased INPP4B expression is partially caused by the NPM1 mutant through ERK/Ets-1 signaling. In addition, high INPP4B is associated with poor outcome in NPM1-mutated patients in our study. Previous reports and the present study suggest that INPP4B provides a survival advantage through the activation of SGK3 in NPM1-mutated leukemia cells. These findings further indicate that INPP4B might be a potential target for the treatment of NPM1-mutated AML.
Methods
The cancer genome atlas (TCGA) gene expression data analysis
Gene expression levels and clinical information of 200 AML patients were retrieved from The Cancer Genome Atlas (TCGA,
http://www.cancergenome.nih.gov). A total of 171 samples had IlluminaGA RNA-Seq gene expression data. Clinical data and INPP4B mRNA expression data for AML samples were analyzed using the cBioPortal for Cancer Genomics. The
INPP4B mRNA expression was compared between AML cases with the NPM1 mutation (
n = 41) and those without the NPM1 mutation (
n = 130).
Patient samples
Peripheral blood samples of 36 AML patients, who had been recently diagnosed, including 22 NPM1-unmutated and 14 NPM1-mutated cases, were obtained from Southwest Hospital of the Third Military Medical University and the First Affiliated Hospital of Chongqing Medical University. Mononuclear cells were enriched by Ficoll gradient purification. The isolated mononuclear cells were used for analyses of NPM1-mA and INPP4B relative expression. Details of the Clinical characteristics of patients are provided in Table
1.
Table 1
Clinical Characteristics of Newly Diagnosed AML Patients
Sex
|
Female | | 19 |
Male | | 17 |
Total | | 36 |
Age
|
Median, years | 53.8 (26–79) | |
WBC
|
Median, ×109/L | 44 (0.3–295.0) | |
Platelets
|
Median, ×109/L | 57.3 (3.0–655.0) | |
AML FAB subtype
|
AML without maturation: M1 | | 4 |
AML with maturation: M2 | | 6 |
Acute promyelocytic leukemia: M3 | | 9 |
Acute myelomonocytic leukemia: M4 | | 7 |
Acute monoblastic or monocytic leukemia: M5 | | 9 |
Other subtype | | 1 |
Karyotype
|
Normal | | 14 |
t (8;21) | | 5 |
t (15;17) | | 6 |
inv. (16) | | 7 |
Unknown | | 4 |
Gene mutations
|
NPM1 | | 14 |
FLT3-ITD | | 10 |
WT1 | | 9 |
CBFB-MYH11 | | 5 |
Cell cultures
Human myeloid leukemia cells HL60, KG1a, K562 and THP-1 were obtained from the American Type Culture Collection (ATCC, MD, USA). The OCI-AML3 AML cells harboring NPM1-mA [
30] were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig, Germany). All cell lines were routinely cultured in RPMI 1640 medium (Gibco, MD, USA), supplemented with 10% fetal bovine serum (FBS; Gibco, MD, USA) and 1% penicillin and streptomycin (Beyotime, Shanghai, China) in a 5% CO
2 humidified incubator at 37 °C.
Reverse transcription PCR and quantitative real-time PCR
Total RNA was isolated using the TRIzol reagent (Takara, Kyoto, Japan), and transcribed into cDNA using the PrimeScript™ RT Reagent Kit (Takara, Kyoto, Japan). Quantitative real-time PCR (qRT-PCR) analysis was performed on an MJ Mini™ Gradient Thermal Cycler Real-Time PCR machine (Bio-Rad, CA, USA) with the SYBR Green reaction kit (KAPA Biosystems, MA, USA). The following primers were used for real-time amplification: INPP4B (Forward 5’-GGAAAGTGTGAGCGGAAAAG-3′ and Reverse 5′- CGAATTCGCATCCACTTATTG-3′); NPM1-mA (Forward F: 5′-TGGAGGTGGTAGCAAGGTTC-3′ and Reverse 5′-CTTCCTCC ACTGCCAGACAGA-3′); SGK3 (Forward 5′-CTGAGATCTCACCATGCAAA GAGATCACACC-3′ and Reverse 5′-GGGGCTAGCTCACAAAAATAAG TCTTCT-3′); Ets-1(Forward 5′-GTCGTGGTAAACTCGG-3′ and Reverse 5′-CAG CAGGAATGACAGG-3′); β-actin (Forward 5′-TAGTTGCGTTACACCCTTTC TTG-3′ and Reverse 5′-TGCTGTCACCTTCA CCGTTC-3′). The mRNA expression levels were analyzed using the 2- ΔΔCt method and expressed as a fold change.
Western blotting
The cultured cells were washed and lysed in cell extraction buffer. Equal amounts of extracts were loaded into sodium dodecyl sulfate (SDS) polyacrylamide gels for electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in 5% low-fat dry milk for 3 h, and then incubated overnight at 4 °C with primary antibodies against INPP4B, p-SGK3T320, SGK3, p-AKTT308, AKT, p-ERK, ERK (Cell Signaling Technology, MA, USA); p-Ets-1, Ets-1, Flag (Bioworld Technology Inc. MN, USA); NPM1-mA (Abcam, Cambrige, UK) and β-actin (Santa Cruz Biotechnology Inc. CA, USA) as loading control. Membranes were washed in Tris-buffered saline (TBS) (10 mM Tris-HCl pH 8, 150 mM NaCl) containing 0.1% Tween 20, and then incubated with HRP-conjugated secondary antibody for 1 h, and subsequently exposed to enhanced chemiluminescence substrate (Millipore, MA, USA). Membrane blot signals were detected using the Bio-Rad Gel Imaging System on cool image workstation II (Viagene, FL, USA). Quantification of protein expression was normalized against the β-actin protein expression using imaging software.
Delivery of siRNA and cell transfection
The siRNA targeting INPP4B, SGK3, Ets-1 and control siRNA were purchased from Genechem (Shanghai, China). The OCI-AML3 cells were transfected with siRNA using the RfectPM siRNA Transfection Reagent (BaiDai, Changzhou, China) according to the manufacturer’s instructions. After 48 h of transfection, the cells were collected for qRT-PCR or western blotting analysis. The sequences of siRNA were as follows: siINPP4B1 (sense: 5’-CCAGGAGGCAUUCUUAAGATT-3′; antisense: 5’-UCUUAAGAAUGCCUCCUGGTT-3′); siINPP4B2 (sense: 5’-GCCGCAAACUGAAUGGUAUTT-3′; antisense: 5’-AUACCAUUCAGUUUGCGGCTT-3′); siSGK3 (sense: 5’-GCAGGACUAAACGAAUUCATT-3′; antisense: 5’-UGAAUUCGUUUA GUCCUGCTT-3′); siEts-1 (sense: 5’-ACUUGCUACCAUCCCGUAC-3′; antisense: 5’-GUACGGGAUGGUAGCAAGU-3′); Control (sense: 5’-UUCUUCGAACGUGUCACGUTT-3′; antisense: 5’-ACGUGACACGUUCGGAGAATT-3′).
Lentiviral vectors and cell infection
The lentivirus-based short hairpin RNA (shRNA) vectors targeting INPP4B (5’-CCATCTGAGTATCCCATCTAT-3′) and scramble lentiviral vectors were purchased from Genechem (Shanghai, China). The OCI-AML3 cells were infected with lentivirus for 48 h in the presence of 5 μg/mL polybrene (Sigma, CA, USA), after which they were subjected to 2 μg/mL puromycin selection for 7 d (Sigma, CA, USA). The puromycin-resistant cells were isolated and propagated for further analysis.
Plasmids and cell transfection
The pEAK-Flag/INPP4B and pCMV-Flag/SGK3 plasmids were purchased from Addgene (
http://www.addgene.org). The pEGFPC1-NPM1-mA, pEGFPC1-NPM1-wt and empty pEGFPC1 were kindly provided by Dr. Falini B (Institute of Hematology, University of Perugia, Perugia, Italy). All transfection experiments were performed using the xfect™ reagent (Clontech, CA, USA) according to the manufacturer’s instructions. After 48 h of transfection, the cells were collected for qRT-PCR or western blotting analysis.
Cell viability assay
Cell viability was determined by the Cell Counting Kit-8 (CCK8, Dojindo Laboratories, Japan), according to the manufacturer’s instructions. Cells were seeded into 96-well plates (Corning, NY, USA) in triplicate at a density of 5 × 103 cells per well with RPMI-1640 containing 10% FBS. The cell numbers were quantified at the indicated time points with the CCK8 (10 μl/well at 37 °C for 2 h), and the numbers of cells per well were determined by measuring absorbance at 450 nm using the microplate reader (Eon, BioTeck, CA, USA). The cell growth curves were plotted with the cell number values as the ordinate and time as the abscissa. Each experiment was performed in triplicate.
A methylcellulose clonogenic assay was carried out to determine cell colony forming ability, by planting 1 × 103 cells per well in triplicate in a 24 well-plate, and maintaining those cells in RPMI 1640 medium containing 20% FBS at 37 °C in an incubator. Colony numbers were scored 10 d later. The colony forming units were counted using an inverted microscope.
Inhibitor treatment
The PI3K inhibitor, LY294002 (30 μM); the AKT selective inhibitor, MK-2206 (5 μM); and the mTOR inhibitor, rapamycin (5 μM) were used to treat OCI-AML3 cells for 24 h and the treated cells were harvested for western blotting. The ERK inhibitor PD98059 was used to treat OCI-AML3 cells with different concentrations (0, 10, 20 and 40 μM) for 24 h and the treated cells were harvested for qRT-PCR and western blotting. These inhibitors were purchased from Selleck Chemicals (Selleckchem, TX, USA).
PI (3,4) P2 and PI (3) P enzyme-linked immunosorbent assay (ELISA)
Cellular PI (3,4) P2 and PI (3) P were quantitated using RY-02853 Human PI (3,4) P2 ELISA kit and RY-02851 Human PI (3) P ELISA kit, respectively, obtained from Runyu Biotechnology (Shanghai, China), according to the manufacturer’s instruction. The results were recorded and analyzed using the microplate reader (Eon, BioTeck, CA, USA).
Survival analysis
Gene expression levels and clinical survival information of 153 AML patients, including 38 patients harboring NPM1 mutations, were retrieved from TCGA dataset. All patients were stratified by INPP4B expression levels into quartiles, to categorize patients into either a high cohort or low cohort. Kaplan-Meier data of AML patients and NPM1-mutated patients were used to analyze the overall survival (OS) and the three-year event free survival (EFS). Details of the clinical characteristics according to high or low INPP4B expression among NPM1-mutated patients from TCGA dataset are provided in Table
2.
Table 2
Clinical Characteristics of NPM1-mutated AML Patients with Low or High INPP4B Expression
Sex
|
Female | 15 | 6 |
Male | 14 | 3 |
Age, years
|
Median, range | 52.93 (21–81) | 50.56 (21–82) |
WBC, ×10
9
/L
|
Median, range | 61.58 (5–137) | 49.78 (1–134) |
Platelets, ×10
9
/L
|
Median, range | 53.86 (8–174) | 63.56 (11–232) |
Bone marrow blast, %
|
Median, range | 78.93 (48–98) | 70.11 (41–95) |
FAB classification
|
M0 | 0 | 1 |
M1 | 11 | 2 |
M2 | 3 | 4 |
M3 | 0 | 0 |
M4 | 10 | 0 |
M5 | 5 | 2 |
M6 | 0 | 0 |
M7 | 0 | 0 |
Cytogenetics
|
Normal | 27 | 7 |
Abnormal | 0 | 1 |
Unknown | 2 | 1 |
Cytogenetic risk
|
Favorable | 0 | 0 |
Intermediate | 28 | 7 |
Adverse | 0 | 1 |
Unknown | 1 | 1 |
Gene mutations
|
FLT3-ITD | 16 | 6 |
IDH1 | 8 | 0 |
Statistical analysis
All data were derived from three independent experiments and the results were summarized and represented as mean ± s.d. Statistical analysis was performed, using the SPSS (Version 17.0) and GraphPad (Prism 5.0) software programs. The statistical significance of differences between each group was analyzed using the unpaired Students’ t-test. The Kaplan–Meier survival data were analyzed using the long-rank test. Any p-value <0.05 was considered statistically significant.
Discussion
The
NPM1 mutations are among the most frequent genetic alterations in AML, especially in cases with a normal karyotype. However, the pathogenesis of NPM1-mutated AML has not been fully elucidated. Herein, our data demonstrate that INPP4B functions to promote leukemia cell survival in a SGK3-dependent manner, high levels of INPP4B are at least partially caused by the NPM1 mutant via ERK/Ets-1 signaling, and high INPP4B is potentially correlated with poor clinical outcome in NPM1-mutated leukemia (Fig.
7f).
It has been known that INPP4B is a phosphoinositide phosphatase with pleiotropic functions in various cellular processes [
36]. As a novel factor in the PI3K signaling pathway, INPP4B has been found to play a tumor suppressive role in prostate, breast, and ovarian cancers, and possibly in leukemia [
37,
38]. However, unexpected findings from recent reports indicate that INPP4B function might be more complicated than previously thought [
39]. In the present study, we analyzed
INPP4B mRNA levels in NPM1-mutated AML patients from the dataset of TCGA and further detected INPP4B expression in NPM1-mutated AML primary blasts and NPM1-mA positive OCI-AML3 cells. These results revealed relative overexpression of INPP4B in NPM1-mutated AML. The overexpression of INPP4B first became evident after the analysis of gene expression in leukemic blasts from BCR/ABL-positive pediatric acute lymphoblastic leukemia [
40]. High INPP4B expression was recently reported in a subset of AML patients with lower response rates to chemotherapy and shorter survival [
41]. Next, we explored the biological effects of INPP4B overexpression in NPM1-mutated leukemia. We observed that knockdown of
INPP4B resulted in inhibition of OCI-AML3 cell proliferation in vitro, and conversely, recovered
INPP4B could rescue this inhibitory effect
, which confirms the gain-of-function of
INPP4B in NPM1-mutated AML. Our findings were consistent with those of a previous study, in which reported that the introduction of
INPP4B conferred a significant increase in the colony forming potential in OCI-AML3 cells [
41]. Moreover, Rijal et al. [
25] reported that ectopic overexpression of
INPP4B enhanced leukemic resistance to cytosine arabinoside (Ara-C). These findings and our data imply that the tumor suppressor gene
INPP4B plays a potential oncogenic role in NPM1-mutated leukemia. In a future study, the function of INPP4B in mouse knock-in models that mimic human NPM1-mutated AML are worthy to be further investigated.
The INPP4B protein is a phosphoinositide phosphatase and acts as an important regulator in PI3K pathway-associated cancer [
42]. In the present study, we found that introduction of INPP4B elevated SGK3 phosphorylation and knockdown of INPP4B reduced SGK3 phosphorylation in OCI-AML3 cells. These results are consistent with those of another report, in which the expression of INPP4B leads to enhanced SGK3 activation in PIK3CA-mutated breast cancer cells [
23]. Because AKT is the canonical downstream effector of INPP4B in the PI3K pathway and INPP4B has the seemingly paradoxical role in AKT activation in a variety of different types of cancers [
42], we tested whether INPP4B was involved in the regulation of phosphorylated AKT in leukemia cells. Surprisingly, our data demonstrate that loss or gain of INPP4B did not appear to affect AKT activation in NPM1-mutated leukemia cells. These results are consistent with those of a previous report of Rijal et al. [
25], who reported no correlation between endogenous INPP4B protein levels and AKT phosphorylation status in AML. Next, we treated OCI-AML3 cells with PI3K pathway inhibitors and found that the PI3K inhibitor, LY294002 (but not the AKT-selective inhibitor, MK-2206 and the mTOR inhibitor, rapamycin) markedly reduced the p-SGK3 levels. These findings and our data indicate that INPP4B mediates activation of the PI3K downstream factor, SGK3, but not AKT in NPM1-mutated leukemia. It is well known that INPP4B preferentially hydrolyzes PI (3,4) P
2 to produce PI (3) P through its lipid phosphatase activity [
39]. Importantly, SGK3 phosphorylation and subsequent activation are dependent on binding to the PI (3) P [
43]. In our study, we observed that introduction of exogenous INPP4B increased PI (3) P levels in OCI-AML3 cell. These results reveal that enhanced activation of SGK3 mediated by INPP4B might be due to the accumulation of PI (3) P in NPM1-mutated AML. Of note, INPP4B possesses both lipid and protein phosphatase activity [
44], which remains possible that INPP4B also regulates phosphorylated SGK3 status through its protein phosphatase activity. Recently, Lopez et al. [
44] has reported that K846M INPP4B mutant lacks lipid phosphatase activity but retains protein phosphatase activity. In the future study, we will further investigate the effects of INPP4B phosphatase activity on SGK3 using the K846M INPP4B mutant.
Considering the activation of SGK3 in NPM1-mutated leukemia cells, we assessed the role of SGK3 in INPP4B-mediated cell proliferation. The results from our experiments revealed that depletion of SGK3 led to significant inhibition of cell proliferation. We also performed a rescue assay and found that ectopic expression of SGK3 could reverse shINPP4B-induced inhibition of cell proliferation. Collectively, these findings support the hypothesis that INPP4B activates SGK3 signaling, to promote cell proliferation in NPM1-mutated leukemia. Several studies have demonstrated that SGK3 contributes to INPP4B-mediated cell proliferation in colon cancer [
33] and a subset of melanomas cells [
24]. In addition, INPP4B has been known to activate SGK3 and drive tumorigenesis in a subset of breast cancers with low levels of AKT [
23]. Recently, a study has reported that INPP4B dephosphorylates tumor suppressor PTEN through its protein phosphatase activity and subsequent degradation of PTEN, thereby promotes cell proliferation of colon cancer [
33]. Thus, in addition to SGK3, the other potential mechanisms underlying oncogenic role of INPP4B in NPM1-mutated leukemia cells needs to be determined.
Because INPP4B is aberrantly expressed in NPM1-mutated AML, the question is whether high expression of INPP4B in leukemic cells correlates with NPM1 mutations. We investigated the effects of NPM1 mutation on INPP4B expression. The results revealed that enforced expression of NPM1-mA increased
INPP4B mRNA and protein levels, whereas knockdown of NPM1-mA had the opposite effect. As INPP4B protein expression seems largely correlated with its mRNA expression in NPM1-mutated leukemia cells (Fig.
5a-d), it is likely that INPP4B is elevated by transcriptional mechanisms. We next searched for the specific transcription factors involved in this process. A recent paper has identified that the increased expression of INPP4B is due to transcriptional upregulation mediated by the transcription factor Ets-1 in colon cancer cells [
33]. In the present study, we found that siRNA-mediated knockdown of Ets-1 significantly downregulated
INPP4B mRNA and protein levels in OCI-AML3 cells. It has been well documented that ERK activation is specifically required for the transcriptional function of Ets-1 [
45]. Moreover, our previous study has verified that ERK signaling is continuously activated by NPM1-mA [
35]. In this study, we treated OCI-AML3 cells with the ERK inhibitor, PD98059, and found that this treatment inhibited Ets-1 phosphorylation and further reduced the INPP4B levels in a dose-dependent manner. More notably, the loss of NPM1-mA weakened ERK/Ets-1 signaling and then reduced INPP4B protein levels. These results indicate that INPP4B is partially upregulated by NPM1-mA via ERK/Ets1 signaling. Previous studies have reported that AML cases carrying NPM1 mutations are generally associated with other common mutations (FLT3-ITD or DNMT3A) [
46,
47]. Recently, a study from TCGA Research Network showed the complex relationships of cooperation or mutual exclusivity among these mutations related to AML [
48]. Future studies will determine whether there is any association between the presence of other mutations and the level of INPP4B in NPM1-mutated patients. In addition, NPM1 is a nucleolar phosphoprotein and its phosphorylation status alters its functions. Previous study has shown that NPM1 could be dephosphorylated on Thr199 by the Ser/Thr protein phosphatase PP1β in response to DNA damage [
49]. Considering the fact that INPP4B has protein tyrosine phosphatase activity [
44] and Ser/Thr phosphatase activity [
33], whether NPM1 might be a substrate of the INPP4B protein in leukemia remains to be clarified.
Next, we also observed that knockdown of NPM1-mA significantly inhibited cell proliferation in OCI-AML3 cells and the introduction of INPP4B successfully reversed this inhibitory effect. These observations indicate that NPM1-mA enhanced INPP4B expression and promoted cell survival in AML. We have previously identified the crucial role of NPM1 mutations in the invasion phenotype [
35], cell differentiation block [
32], and autophagic activity [
50] in AML. Finally, we evaluated the clinical significance of INPP4B in NPM1-mutated AML cases derived from the TCGA dataset. High levels of INPP4B delineated a poorer prognosis in AML patients. Importantly, NPM1-mutated patients with high INPP4B tended to have shorter survival outcome. Our results support this observation, by showing that high INPP4B levels are associated with a poor outcome in AML among six independent gene expression datasets [
41].