NTAL is associated with treatment outcome, cell proliferation and differentiation in acute promyelocytic leukemia

Non-T cell activation linker (NTAL) is a lipid raft-membrane protein expressed by normal and leukemic cells and involved in cell signaling. In acute promyelocytic leukemia (APL), NTAL depletion from lipid rafts decreases cell viability through regulation of the Akt/PI3K pathway. The role of NTAL in APL cell processes, and its association with clinical outcome, has not, however, been established. Here, we show that reduced levels of NTAL were associated with increased all-trans retinoic acid (ATRA)-induced differentiation, generation of reactive oxygen species, and mitochondrial dysfunction. Additionally, NTAL-knockdown (NTAL-KD) in APL cell lines led to activation of Ras, inhibition of Akt/mTOR pathways, and increased expression of autophagy markers, leading to an increased apoptosis rate following arsenic trioxide treatment. Furthermore, NTAL-KD in NB4 cells decreased the tumor burden in (NOD scid gamma) NSG mice, suggesting its implication in tumor growth. A retrospective analysis of NTAL expression in a cohort of patients treated with ATRA and anthracyclines, revealed that NTAL overexpression was associated with a high leukocyte count (P = 0.007) and was independently associated with shorter overall survival (Hazard Ratio: 3.6; 95% Confidence Interval: 1.17–11.28; P = 0.026). Taken together, our data highlights the importance of NTAL in APL cell survival and response to treatment.


NTAL mediates ATRA-induced differentiation and NTAL knockdown decreases cell viability and proliferation.
To explore the molecular effects of NTAL on APL cells, we first evaluated the modulation of NTAL protein levels in NB4 cells treated with different concentrations of ATRA and ATO for 48 and 72 hours. As depicted in Fig. 1A, both drugs induced a reduction in NTAL protein levels in a dose-dependent manner. We also measured NTAL mRNA expression following ATRA and ATO treatment (Fig. 1B). To investigate NTAL function, NB4 and NB4-R2 (ATRA-resistant) cells were transduced with three different shRNA sequences. Cells transduced with sequence TNRC000128292 exhibited a higher level of NTAL inhibition compared to the control (CT -cells transduced with scrambled RNA) and was chosen for further functional assays ( Supplementary  Fig. S1A).
To evaluate whether the decrease in NTAL is involved in regulating cell survival, caspase levels were measured at baseline in NTAL-KD cells and compared to the CT cells. NB4 NTAL-KD cells showed increased levels of caspase-3 and caspase-8 ( Fig. 1D) resulting in increased numbers of apoptotic cells at baseline (i.e., spontaneous apoptosis), and following ATO-treatment (Fig. 1E). In addition, induction of apoptosis in response to the Akt inhibitor, perifosine, was assessed in NB4-CT and NB4-NTAL-KD cells, alone, or in combination with zVAD (an irreversible pan-caspase inhibitor). NB4-NTAL-KD cells exhibited higher sensitivity to perifosine (with increased cleavage of caspase-8 and PARP) in comparison with NB4-CT cells, while no differences were detected with perifosine in combination with zVAD (Fig. 1F). In addition, NTAL-KD cells showed increased reactive oxygen species (ROS) levels and loss of the mitochondrial membrane potential (Fig. 1G,H and Supplementary Fig. S1C).
www.nature.com/scientificreports www.nature.com/scientificreports/ autophagy marker levels were increased in NB4 NTAL-KD cells at baseline, and further increased following the treatments described above. Compared to the CT cells, NB4 NTAL-KD cells presented lower levels of LC3-II following treatment with chloroquine and rapamycin, reinforcing the theory that NTAL participates in the regulation of  Supplementary Fig. S3A). Taken together, these data suggest that NTAL is involved in the regulation of apoptosis and autophagic flux in APL cells.
We also used transmission electron microscopy to assess morphological alterations promoted by NTAL-KD at the ultrastructural level. NB4-NTAL-KD cells demonstrated a decrease in cytoplasmic vacuolization with the presence of mitochondrial degeneration ( Supplementary Fig. S3B).  Supplementary Fig. S4). Western blot analyses showed the same pattern of signaling proteins in NTAL-KD engrafted tumor models as seen in the in vitro studies (Fig. 3E). The levels of total Ras, p-p44/42 MAPK (ERK1/2-Thr202/204) and cleaved caspase-3 increased, while total and p44/42 MAPK (ERK1/2) protein remained similar in the NB4-NTAL-KD engrafted tumors samples when compared to that seen in the NB4-CT engrafted tumors samples. www.nature.com/scientificreports www.nature.com/scientificreports/ High NTAL transcript levels may predict lower overall survival in ApL patients. Using public a databank (BloodSpot) we compared the values of NTAL expression in samples from newly diagnosed APL patients (n = 54) and healthy volunteers (n = 6). NTAL expression was significantly lower in APL bone marrow (BM) samples compared with the Hematopoietic Stem-and progenitor-cells and promyelocytes from healthy volunteers (P < 0.01; Fig. 4A).
We dichotomized patients according to the median value of NTAL expression and compared the groups of APL patients with low and high NTAL expression. Baseline characteristics were similar in the two groups (Table 1), except for higher white blood cell (WBC) counts and a higher frequency of patients with hyperleukocytosis (defined as patients with equal or more than 10 × 10 9 cells/L) in the high NTAL group (P = 0.007). With a median follow-up of 76 months (1-101 months), the estimated 5-year overall survival (OS) rate was 73% (95% confidence interval, CI: 63-80%) in the entire cohort. Overall, 95/114 (83%) of APL patients achieved complete hematological remission (CHR). Of the 19 patients (17%) who failed to achieve CHR, 15 (79%) experienced early mortality (i.e., death within 30 days after diagnosis). NTAL expression had no impact on CHR achievement (P = 0.315), or on early mortality (P = 0.235). In contrast, patients with a high NTAL expression had a lower 5-year OS rate (62%, 95% CI: 46-74%) compared to patients with a low NTAL expression (84%, 95% CI: 71-91%) (hazard ratio, HR: 2.26, 95% CI: 1.02-5.01, P = 0.019; Fig. 4B). These findings were confirmed by a multivariate analysis, demonstrating that NTAL overexpression was independently associated with a shorter OS (HR: 3.6, 95% CI: 1.17-11.28, P = 0.026) ( Table 2), with modifiers of treatment outcome: age, gender, WBC, and albumin levels 13 . NTAL expression showed no association with the disease-free survival and event-free survival rates (Fig. 4C,D). www.nature.com/scientificreports www.nature.com/scientificreports/ internal validation data. The final prediction model was internally validated using a bootstrap resampling procedure with 10,000 repetitions from the original database to assess model bias. Internal validation resulted in an Area Under the Curve (0.641, 95% CI: 0.51 to 0.75) very similar to that described for the original data. The bootstrap results are depicted in Table 3. Briefly, for Overall Survival (OS) and Event Free-Survival (EFS) at different time points, the procedure yielded a mean and 95% CI virtually identical to its original match. Also, for all comparisons, the pairwise hypothesis testing showed significance (P < 0.0001) for the difference across the distributions means.

Discussion
In the present study, we show that the knockdown of NTAL reduced mTOR activation and downstream targets, and increased levels of Ras-MAPK-ERK in NB4 cells. In addition, we observed increased expression of autophagic flux and apoptosis markers in NTAL-KD cells, at baseline and following ATO-treatment. These results reinforce the importance of NTAL in cell survival, and are in agreement with our previous results showing that NTAL depletion from lipid rafts decreases cell viability through regulation of the Akt/PI3K pathway 11 . We also identified signals of mitochondrial degradation in NB4-NTAL-KD cells. ATO-induced ROS in APL cells have been shown to originate from NADPH oxidase through the upregulation of virtually all components of this mitochondrial membrane-associated enzyme complex 15 .
NTAL was also involved in the granulocytic differentiation of APL blasts treated with ATRA. The inhibition of mTOR 16  www.nature.com/scientificreports www.nature.com/scientificreports/ explain these findings. ERK and JNK activation can stimulate the autophagic removal of mitochondria during oxidative stress 19 . In vivo, the inoculation of NB4-NTAL-KD cells resulted in lower tumor growth rates in immunodeficient mice compared to controls.
NTAL expression was significantly reduced in APL blasts when compared to normal bone marrow mononuclear cells. In a cohort of patients treated with ATRA and anthracycline-based chemotherapy, high NTAL levels were associated with high WBC counts, and this may explain the independent association with lower OS, without impacting on CHR rate 13 . Despite the progress in APL treatment, 10-15% of patients relapse after treatment with ATRA plus chemotherapy, and frequently present with ATRA resistance 20 . Arsenic can circumvent this scenario, but its cost and health surveillance agencies politics remain a significant barrier for many low-and middle-income countries. In the context of the ATRA plus chemotherapy, we have described other molecular markers that carry prognostic information [21][22][23] , suggesting that heterogeneity in APL outcomes may be higher than expected. Previously, we showed that NTAL is a primary protein involved during 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC) treatment in different leukemic cell lines 11 , not only for APL but for AML as well. Here, we looked at the role of the NTAL gene and protein expression in APL only, as a homogenous model of AML. The downregulation of NTAL led to decreased activation of Akt/PI3K and downstream pathways, impaired cell proliferation and survival, and an increased sensitivity to the primary drugs used in the clinical setting at equivalent doses applied during patient treatment. Furthermore, the increased doses of ATRA showed that the downregulation of NTAL mRNA and protein levels were both time and dose-dependent. We demonstrated that increased NTAL levels were associated with decreased OS, regardless of other risk variables. Interestingly, NTAL expression was decreased in APL samples compared to healthy controls. This could argue for the fact that NTAL is not associated with malignant transformation per se, but seems to exert its functional effects on APL blasts cells, orchestrating several biological processes that dictate a more aggressive biology. Although NTAL-KD cells exhibited a high sensitivity to ATO, recent data have shown that some prognostic markers established for conventional ATRA and chemotherapy regimens could not be reproduced in the case of treatment with ATO 24 . The potential role of Akt-inhibitor treatment, which results in NTAL degradation 11 in ATRA and ATO refractory APL cases, remains to be evaluated.
In conclusion, our results showed that reducing NTAL levels led to anti-leukemic activity in APL cells in vitro and in vivo. Our results support the notion that NTAL is involved in the cross talk between proliferation, differentiation, and autophagy in APL cells, and that high NTAL expression its associated with decreased OS in APL patients.

Methods
All methods were carried out in accordance with the approved guidelines.   www.nature.com/scientificreports www.nature.com/scientificreports/ (2 mM), and penicillin/streptomycin (Invitrogen). Mycoplasma contamination was routinely tested (once per month). The cell lines were authenticated by short tandem repeat analysis (last authentication -November 2018). Western blotting. Samples were separated by SDS-PAGE (7.5 or 10% or 12%) and electro transferred to PVDF membranes (GE Lifesciences, Pittsburgh, PA, USA). The membranes were blocked with wash buffer (25 mM Tris-HCl, pH 7.5, 0.5 M NaCl and 0.1% Tween-20) containing 5% non-fat dry milk and incubated with a primary antibody following manufacturer's instructions (see Supplementary Table S1). The following secondary antibodies were used: horseradish peroxidase-conjugated goat anti-rabbit IgG (#7074) or horse anti-mouse IgG secondary antibody (#7076) or streptavidin-HRP conjugate (#3999) (Cell Signaling). Targets were visualized with ECL Western blotting Detection Reagents (GE Lifesciences) using a CCD-Camera (Image Quant LAS 4000 mini, Uppsala, Sweden), and images were acquired using precision and/or increment mode. Densitometric analysis was performed using the ImageJ software 26 , and bands were normalized to constitutive proteins (β-actin, β-tubulin or GAPDH). Western blots were statistically analyzed by two-tailed unpaired t test.

Granulocytic differentiation induction.
Apoptosis assay. NB4 and NB4-R2 cells were seeded in 24-well plates and treated with ATO (1 µM) and vehicle (NaOH) for 24 h. Cells were then washed twice with ice cold PBS and resuspended in binding buffer containing 1 µg/ml propoidium iodide (PI) and 1 µg/ml APC-labeled annexin V (BioLegend, San Diego, CA, USA). All specimens were acquired by flow cytometry (FACSCalibur; Becton-Dickison) after incubation for 15 min at room temperature and analyzed using FlowJo software (Treestar, Inc., San Carlos, CA, USA).

Akt hypo-phosphorylation and phosphorylation. NB4 cells (CT and NTAL-KD) were maintained
in culture in serum-free medium overnight (16-18 h). Cells were then stimulated with myeloid growth factors (10 ng/ml hr-IL-3 or 10 ng/ml hr-GM-CSF, 10 ng/ml hr-G-CSF, 50 ng/ml hr-SCF) (PeproTech, Mexico City, Mexico). Aliquots were removed after 15 min of stimulation and assayed by antibody array. pathScan intracellular signaling array kit. The PathScan Intracellular signaling array kit containing fixed antibodies against phosphorylated proteins by the chemiluminescent sandwich ELISA format was used according to manufacturer's instructions (#7323, Cell Signaling). Images were analyzed with LI-COR Image Studio v4.0 analysis software by loading the image as a gray scale picture. Each protein array dot was selected manually, and an average intensity was calculated for each protein. Normalization within one stimulation experiment was done by subtracting the intensity of the negative control dot from each value. For comparison of different conditions, sets were normalized so that the positive controls had nearly equal intensities.
www.nature.com/scientificreports www.nature.com/scientificreports/ Active ras detection assay. Active Ras protein in NB4 (CT and NTAL-KD) cells was evaluated using (#8821, Cell Signaling) according to manufacturer's instructions. NB4 (CT or NTAL-KD) cells lysates (500 µg), treated with GTPγS (for a final concentration of 0.1 mM) or GDP (for a final concentration of 1 mM) to respectively activate or inactivate RAS, and non-treated were incubated with GST-Raf1-RBD and gluthatione-agarose resin to pool-down active Ras. Input and pull-down samples were separated by SDS-PAGE and western blotting analysis against Ras. transmission electron microscopy. The NB4 (CT and NTAL-KD) cells were treated overnight or not with 10 µM chloroquine. The cells were fixed in 2% glutaraldehyde in 0.1 M sodium phosphate buffer at pH 7.4 (PB) for 2 h. The specimens were post-fixed in PB buffer containing 1% OsO 4 for 2 h, and dehydrated in an ethanol series (30%, 50%, 70%, 80%, 95%, and 100%), for 10 min each, followed by 2 times with propylene oxide, for 5 min each. They were then infiltrated with propylene oxide and epoxy resin (V/V = 1:1), embedded with EPON 812 epoxy resin, DDSA, DMP-30, and MNA resin, and then aggregated for 24-48 h at 60 °C. Ultra-thin sections (60-70 nm) were cut with a diamond knife and stained with uranyl acetate and lead citrate. Sections were examined with a TEM (Jeol, Jem 100cx, Tokyo, Japan). Pictures were taken and converted to digital files (Hamamatsu, ORCA-HR Amtv542, Hamamatsu City, Japan).

Animal xenograft studies.
All animal studies were approved by the Animal Ethics Committee of the tumor engraftment in murine model. week-old NSG mice were maintained receiving NUVITAL (autoclavable rodents' pellets) and water (autoclaved) ad libitum, under a 12/12 light/dark cycle, at 23 °C environmental temperature and 55% relative humidity. Mice were injected subcutaneously with 1 × patients, treatment protocol, ethics approval, and consent to participate. To validate our gene expression data, we analyze the NTAL transcript levels (Probe, #211768_at) in normal bone marrow mononuclear cells (BMMC), purified promyelocytes and CD34 + cells and newly diagnosed APL samples available at the BloodSpot databank (http://servers.binf.ku.dk/bloodspot/). Details about the cohort design and gene expression evaluation are described published elsewhere 27 .
A total of 114 consecutive patients with newly diagnosed APL who were enrolled in the IC-APL study were included. Details regarding diagnosis, classification, and treatment protocol are published elsewhere 14 .
All procedures were approved by the Ethics Committee of the Faculdade de Medicina de Ribeirão Preto -USP, and by the National Commission of Ethics in Research, National Health Council, Ministry of Health (Conep) (registry: 12920; process number: 13496/2005; CAAE: 155.0.004.000-05). Informed consent was obtained from all patients and approved by the Research Ethics Board, as described in the Ethics approval and consent to participate section. All methods were carried out in accordance with the approved guidelines and the Declaration of Helsinki.
Gene expression profile of NTAL. All samples used for gene expression analyses were obtained at diagnosis from bone marrow aspirates and were processed according to standard techniques. Following total RNA extraction, real-time quantitative polymerase chain reaction (RQ-PCR) assays with sample-derived cDNA were performed in duplicate on MicroAmp optical 96-well plates using a 7500 Real-Time PCR System (Applied BioSystems, Foster City, CA, USA) with the GAPDH and ACTB Standard Kit as endogenous controls. NTAL gene expression was determined by real-time reverse transcriptase polymerase chain reaction using TaqMan Gene Expression Assay (Hs00247916_m1, Applied BioSystems), following the manufacturer's instructions. The gene expression of NTAL was calculated relative to a reference cDNA (NB4 cell line, a human acute promyelocytic leukemia cell line, positive for PML/RARA fusion gene) and set at 1. Of importance, the same reference cDNA was used as an internal control throughout all experiments to ensure that the results would be fully comparable among experiments. The gene expression values of NTAL were calculated as relative quantification using the ∆Ct method and expressing the results as 2-ΔΔCt, in which ΔΔCt = ΔCt patients − ΔCt NB4 cell line .
Bootstrap with replacement analysis. In order to cross-validate our findings, we have performed a non-parametric bootstrap procedure with 10 000 resamplings of the original cohort allowing replacement 28 . The function calculated a given survival (e.g., overall survival or disease-free survival) in three different time points (12-months, 2-years, and 5-years) for both categories (low and high NTAL expression), and it also estimated their respective 95% confidence interval computing the bias-corrected and accelerated (BCa) bootstrap interval. We also had tested the hypothesis that the mean of both bootstrap sampling distributions was different using either www.nature.com/scientificreports www.nature.com/scientificreports/ a Welch two sample t-test when the distributions were normal in a QQ plot inspection or using a Wilcoxon rank-sum test when not normal. Statistical analysis. Patient baseline characteristics were reported descriptively. With the use of survival ROC curve analysis 29 , we dichotomized patients according to the median value of NTAL expression (low expression, <0.17; high expression ≥0.17). All P values were two-sided with a significance level of 0.05. In order to cross-validate our findings, we performed a non-parametric bootstrap procedure with 10,000 resamplings of the original cohort allowing replacement 30,31 .
Univariable and multivariable proportional hazards regression analysis associated with interactions between the variables were performed for potential prognostic factors for overall survival (OS), event-free survival (EFS) and disease-free survival (DFS). OS was defined as the time from diagnosis to death from any cause; those alive or lost to follow-up were censored at the date last known alive. Early mortality was defined as death occurring within 30 days from diagnosis. For patients who achieved CR, DFS was defined as the time from CR achievement to the first adverse event: relapse, development of secondary malignancy, or death from any cause, whichever occurred first. EFS was defined as the time from the initiation of induction therapy to disease relapse, development of secondary malignancy, or death from any cause, whichever occurred first. Patients who were alive without disease relapse or secondary malignancy were censored at the time they were last seen alive and disease-free. Potential prognostic factors examined in multivariable regression analysis were age at diagnosis, gender, white blood cell (WBC) counts, platelet counts, hemoglobin levels, coagulopathy, FAB classification (M3 or M3v), PML breakpoint, creatinine, albumin, uric acid, and fibrinogen and FLT3-ITD mutation (with allele ratio evaluation). Linearity assumption for all continuous variables was examined using restricted cubic spline estimates of the relationship between the continuous variable and log relative hazard/risk. All P values were two sided with a significance level of 0.05.
All calculations were performed using Stata Statistic/Data Analysis version 14.1 (Stata Corporation, USA), statistical package for the social sciences (SPSS) 19.0 and R 3.3.2 (The CRAN project, www.r-project.org) software.

Data availability
The data used and analyzed during this study are available from the corresponding author on request.