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Evaluation of Insulin-mediated Regulation of AKT Signaling in Childhood Acute Lymphoblastic Leukemia

Wang, Jian, MD*; Xue, Hong-Man, PhD; Chen, Yan-Ru, PhD; Xu, Hong-Gui, MD*; Lin, Shao-Fen, MD*; Tang, Xi-Kang, MD*; Chen, Chun, PhD

Journal of Pediatric Hematology/Oncology: March 2019 - Volume 41 - Issue 2 - p 96–104
doi: 10.1097/MPH.0000000000001425
Original Articles
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SDC

Objective: Hyperglycemia increases the risk of early recurrence and high mortality in some adult blood cancers. In response to increased glucose levels, insulin is secreted, and several studies have shown that insulin-induced AKT signaling can regulate tumor cell proliferation and apoptosis. The AKT pathway is aberrantly activated in adult acute lymphoblastic leukemia (ALL), but the mechanisms underlying this activation and its impact in pediatric patients with ALL are unclear.

Materials and Methods: We evaluated the insulin-induced chemoresistance and AKT pathway activation by measuring cell proliferation, apoptosis, and other parameters in ALL cell lines (Jurkat and Reh cells), as well as in primary pediatric leukemic cell samples, after culture with insulin, the chemotherapeutic drugs daunorubicin (DNR), vincristine (VCR), and L-asparaginase (L-Asp), or anti-insulin-like growth factor-1 receptor (IGF-1R) monoclonal antibody.

Results: DNR, VCR, and L-Asp-induced toxicity in Jurkat and Reh cells was reduced in the presence of insulin. DNR promoted cell proliferation, whereas DNR, VCR, and L-Asp all reduced apoptosis in both cell lines cotreated with insulin compared with that in cell lines treated with chemotherapeutics alone (P<0.05). Furthermore, addition of an anti-IGF-1R monoclonal antibody promoted apoptosis, downregulated IGF-1R expression, and decreased the phosphorylation of AKT, P70S6K, and mTOR intracellular signaling pathway proteins in both cell lines, as well as in primary cultures (P<0.05).

Conclusions: Our results suggest that insulin-induced chemoresistance and activation of the AKT signaling pathway in pediatric ALL cells.

*Department of Pediatrics, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou

Department of Pediatrics, the Seventh Affiliated Hospital of Sun Yat-sen University, Shenzhen

Department of Pediatrics, Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai, China

Supported by National Nature Scientific Foundation of China (no. 81370625).

The authors declare no conflict of interest.

Reprints: Chun Chen, PhD, Department of Pediatrics, The Seventh Affiliated Hospital, Sun Yat-sen University, Shenzhen 518017, China (e-mail: docchunchen@126.com).

Received April 7, 2017

Accepted December 26, 2018

Acute lymphoblastic leukemia (ALL), caused by the altered differentiation of hematopoietic stem cells, is the most common pediatric malignancy. Advances in individualized therapy have significantly improved the outcomes of pediatric patients with ALL in recent decades. However, tumor recurrence, disease progression, and therapeutic drug toxicity–related mortality are still increasing.1 Inductive chemotherapy for pediatric patients with ALL commonly involves treatment with L-asparaginase (L-Asp) and glucocorticoids. However, both drugs can affect the production, release, and function of insulin and thus induce chemotherapy-related hyperglycemia, a common complication that occurs in up to 56% of pediatric patients with ALL.2,3 The studies suggest that hyperglycemia is a risk factor for early recurrence and high mortality in adult ALL and acute myeloid leukemia.4,5 However, few reports have examined the impact of chemotherapy-related hyperglycemia on the prognosis of pediatric ALL, and results from these studies are inconsistent.3,6,7 Our recent retrospective study of 159 pediatric patients with ALL compared the 5-year recurrence-free survival and 5-year overall survival of hyperglycemic and nonhyperglycemic pediatric patients with ALL undergoing inductive chemotherapy. What needs to be pointed out is that in our clinical treatment, all of 159 patients did not decrease the dosage of L-Asp and glucocorticoids in order to ensure the curative effect. We showed that the 5-year overall survival was lower in the hyperglycemic group than in the euglycemic group (83.8%±6.0% vs. 94.9%±2.4%; P=0.014), and the 5-year recurrence-free survival was significantly lower in the hyperglycemic group than in the euglycemic group (62.9%±8.7% vs. 80.2%±9.1%; P<0.001).8

Conventional views hold that hyperglycemia promotes immunosuppression and increases the risk of infection (2.1 to 2.5 times that of euglycemic patients), reduces the overall effectiveness of chemotherapy, and decreases the clearance of leukemic minimal residual lesions, thereby impairing treatment of ALL.4,9 However, more current views suggest that the relationship between abnormal glucose metabolism and a poor prognosis of cancer is multifactorial. Recent studies showed that high concentrations of insulin and glucose promote the growth of a variety of tumor cells, and insulin reduces tumor cell apoptosis and induces drug resistance.10–13 Insulin possesses somatomedin-like properties. During hyperglycemia, hyperinsulinemia and high insulin-like growth factor (IGF) levels stimulate the AKT/mTOR signaling pathway. This pathway is also aberrantly activated in ALL,14,15 and it plays important roles in cell proliferation, antiapoptosis, and differentiation.16 Furthermore, as IGF-1 receptor (IGF-1R) promotes self-renewal of chronic myeloid leukemia cells,17 it is an attractive target for cancer treatment.

Clinical trials have confirmed that monoclonal antibodies targeting IGF-1R or other components mediating insulin signaling exhibit an antitumor activity when used alone or in combination with chemotherapeutic agents or other monoclonal antibodies.18 Consistently, IGF-1 increases the proliferation of B-cell precursor ALL cell lines, and this proliferative effect is blocked by AKT inhibitors.19 In T-cell ALL, deletion of IGF-1R results in the aberrant activation of Notch1 and PI3K/AKT pathways, leading to a decrease in the growth of T-ALL cells.16 However, there is limited literature on pediatric ALL. Here, we evaluated the response of pediatric ALL cells (primary cultures and cell lines) to insulin and the potential curative effect of a treatment with anti-IGF-1R for pediatric patients with ALL.

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MATERIALS AND METHODS

Cell Lines and Primary Patient Samples

Jurkat (T-ALL) and Reh (non-T non-B ALL) human ALL cell lines were purchased from Typical Culture Preservation Commission Cell Bank, Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI 1640 medium from Grand Island Biological Company (Gland Island, NY) supplemented with 10% heat-inactivated fetal calf serum (Grand Island Biological Company). All cells were grown at 37°C in a 5% CO2 humidified atmosphere incubator and used at a density of 2 to 3×105 cells/mL in all experiments.

Specimens from 6 individual patients (Table 1)20 were obtained from our hospital (Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China) before administration of chemotherapy, and primary leukemic cell samples were obtained for in vitro experiments. The anticoagulated (heparin-treated) blood and bone marrow samples were diluted 1:1 in phosphate-buffered saline (PBS), added to an equal volume of lymphocyte separation medium, and centrifuged at 1200g for 15 minutes at 20°C. The lymphoblast interface, in which lymphocytes are concentrated, was collected with a cannula and diluted into 4 mL in RPMI 1640, centrifuged at 1500g for 10 minutes at 20°C. The supernatant was discarded, and the cell pellet resuspended in 2 to 3 mL of RPMI 1640. The cells were cultured in growth medium as described above. The study was approved by the Ethics Committee of Sun Yat-sen Memorial Hospital, and all patients provided a written informed consent. This study was conducted in accordance with the declaration of Helsinki. This study was conducted with approval from the Ethics Committee of Sun Yat-sen University. Written informed consent was obtained from all participants’ guardians.

TABLE 1

TABLE 1

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Proliferation Analysis

Cell proliferation was measured with a CCK-8 assay (Dojindo, Kumamoto, Japan) as previously described.16,21 Approximately 2×105 cells/mL were seeded into 96-well plates and incubated with the required concentrations of drugs for 48 hours. Insulin (Novo Nordisk A/S, Novo Alle, Denmark) and DNR (Actavis Italy S.p.A., Nerviano, Italy) (20 mg per sample) stored at 4°C, and diluted to required concentrations by using sterile water. Then, 4 hours before the end of the culture, 10 μL of the CCK-8 solution was added to each well. The absorbance was read on a 96-well plate reader at a wavelength of 450 nm. Control cells were incubated with PBS instead of drugs.

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Detection of Fluorescence Intensity

Approximately 3×105 cells/mL were seeded in 24-well plates and incubated with required concentrations of drugs for 48 hours. The anti-IGF-1R monoclonal antibody was purchased from Abcam (Cambridge, UK) and stored at −20°C. After culture, cells were harvested and washed twice in PBS. The cells were stained with 5 μL of a PE-conjugated rabbit monoclonal antibody against IGF-1R (CD221, BD Biosciences, San Diego, CA) or 5 μL of isotype-matched, PE-conjugated control IgG1 antibody (BD Biosciences). Flow cytometry analysis was performed using a FACSCalibur (BD Biosciences).

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Apoptosis Analysis

Approximately 3×105 cells/mL were seeded in 24-well plates and incubated with required concentrations of drugs for 48 hours. After culture, cells were harvested and washed twice in PBS. Each cell pellet was resuspended in 100 μL of binding buffer (1×), and 5 μL annexin V fluorescein isothiocyanate (FITC) was added. After a 10-minute incubation at room temperature, an additional 400 μL of binding buffer was added for a final volume of 500 μL. Cells were stained with propidium iodide (PI, 0.6 μg/mL) immediately before measurement. Annexin V FITC and PI were purchased from BD Biosciences. Unstained and single-stained controls were included in each experiment. Flow cytometry analyses were performed using a FACSCalibur, and the data obtained were analyzed with Cell Quest software (BD Biosciences).21,22

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Western Blotting

Approximately 3×105 cells/mL were seeded in culture flasks and incubated with required concentrations of drugs for 48 hours. After culture, cells were pelleted by centrifugation and rinsed in PBS. The cell pellets were then lysed in a total of 300 μL of RIPA buffer (BestBio, Shanghai, China). DNA that was present in the lysate was sheared by sonication. All western blot analyses were performed using whole-cell lysates. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed using a standard method. Briefly, protein concentrations of the cell samples were measured using a modified Lowry method (BestBio). Equal amounts of total protein from each sample were loaded onto sodium dodecyl sulfate polyacrylamide gels. Immunoblotting was performed using nitrocellulose membranes (Merck Millipore, Darmstadt, Germany). Antibodies against phospho-AKT (Ser473), phospho-mTOR (Ser2448), and phospho-p70 S6 kinase (Thr389) were obtained from Cell Signaling Technology (Beverly, MA). A Kodak X-AR film was used to record the image generated by enhanced chemiluminescence using an ECL kit (Merck Millipore).

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Statistical Analysis

Results are presented as the mean±SD. Group comparisons were performed using a paired t test when only 2 groups were compared or 1-way analysis of variance (ANOVA) followed by the Tukey-Kramer multiple comparison test when >2 groups were compared. Differences were considered statistically significant at a value of P<0.05.

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RESULTS

DNR-induced Toxicity in Jurkat and Reh Cells is Reduced in the Presence of Insulin

In our previous study, we investigated the mRNA expression levels of IGF-1R and the insulin receptor (IR) in peripheral blood lymphocytes of pediatric patients with ALL. We found that IGF-IR plays a crucial role in the proliferation and apoptosis of ALL cells.23 Because insulin can influence the growth of ALL cells, we next explored whether IGF-1R signaling influences ALL cell chemoresistance. We used the anthracycline daunorubicin (DNR), a drug commonly used in cancer chemotherapy, to investigate the effect of insulin on ALL cell chemoresistance.

ALL cell proliferation was measured by a CCK-8 assay as described previously.16,21 Given that long-term culturing without renewal of culture medium can affect cell proliferation, we decided to use a 48-hour incubation time. The Jurkat and Reh ALL cell lines were treated with varying concentrations of DNR (0.01 to 100 nmol/L) with or without insulin (10–9 mol/L) for 48 hours. Absorbance values at 490 nm were then measured. Cotreatment with DNR and insulin significantly increased the absorbance value compared with the treatment with DNR alone at every concentration evaluated (paired t test, P<0.05; Fig. 1). These findings indicate that insulin promoted cell proliferation even in the presence of DNR.

FIGURE 1

FIGURE 1

We next determined the effect of insulin on cell apoptosis in the presence of DNR at the highest concentration of 100 nmol/L to explore the maximum difference between the 2 groups. Three experimental groups (treatment with insulin or DNR, and combination treatment) were set up, and cells were cultured for 48 hours. After staining with annexin V/PI to determine the proportion of apoptotic cells by flow cytometry, the control and insulin groups revealed a physiological apoptotic percentage of ∼5% in Jurkat cells. By contrast, the proportion of apoptotic cells after addition of both insulin and DNR was significantly decreased compared with that in cells treated with DNR alone (15.98%±2.23% vs. 20.25%±2.36%; P<0.05; Fig. 2A). The same trend was observed in Reh cells (25.29±4.61 vs. 32.69±4.52, P<0.05; Fig. 2D). Similar to the above experiments, we also added another chemotherapeutic drug, vincristine (VCR; 0.5 ng/mL), or L-Asp (5 units/mL) in the culture of both cell lines. The results show that after addition of insulin, the percentage of cell apoptosis induced by the 2 chemotherapeutics was obviously decreased in both cell lines (P<0.05; Figs. 2B, C, E, F). These results show that toxicity induced by these 3 chemotherapeutics is reduced in the presence of insulin.

FIGURE 2

FIGURE 2

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Phospho-AKT Expression Level in ALL Cells Increases With Insulin Addition in a Dose-dependent Manner

Western blotting analysis was used to measure the protein expression levels of phospho-AKT in Reh cells, which were cultured with varying concentrations of insulin (10−11 to 10−7 mol/L) for 48 hours. The levels of phospho-AKT increased as the concentration of insulin increased (Fig. 3).

FIGURE 3

FIGURE 3

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Neutralization of IGF-1R Decreases its Availability on the Surface of ALL Cells

Insulin and IGFs are key regulators of energy metabolism and growth.24 Moreover, IGF-1R expression correlates with tumor growth and is a marker of a poor prognosis in several cancer types.25 We hypothesized that insulin could promote proliferation and inhibit apoptosis of leukemic cells by binding to IGF-1R and activating AKT/mTOR signaling in pediatric ALL. Therefore, we used the anti-IGF-1R monoclonal antibody ab16890, which binds specifically and with high affinity to human IGF-1R, to prevent IGF-1R ligand binding and activation of downstream AKT/mTOR signaling. The ALL cell lines Reh and Jurkat and 6 consecutive primary leukemic cell samples from patients with ALL (patients 1 to 6) were evaluated.

We first investigated whether levels of available IGF-1R were altered by treatment with ab16890 at different concentrations. The 2 cell lines were treated with increasing concentrations (0, 0.001, 0.01, 0.1, 1, and 10 μg/mL) of ab16890 for 48 hours. Because of the limited resources in bone marrow from patients with ALL, the effects of only 1 concentration (1 μg/mL) of ab16890 were evaluated in patient primary samples 1 to 3. Mouse anti-human IGF-1R conjugated to PE was then added to the cell samples, and flow cytometry was performed to measure the average fluorescence intensity of each sample compared with that of isotope controls (cells treated with control isotype-matched antibody), and the fluorescence index (FI) formula {FI=[(inspected sample—isotype control)/isotype control]}26 was used to calculate the FI and evaluate IGF-1R expression. Treatment of Jurkat cells with increasing concentrations of ab16890 for 48 hours reduced the FI in a dose-dependent manner (Fig. 4A, from 3.41±0.43, 2.50±0.20, 1.65±0.17, 0.90±0.09, and 0.89±0.20 at 10 μg/mL ab16890 compared with the control group, 6.37±1.10). Similar results were obtained using Reh cells (Fig. 4B, from 8.26±0.19, 7.61±0.24, 6.86±0.38, 6.26±0.13, and 5.00±0.65 at 10 μg/mL ab16890 compared with the control group, 10.85±0.35). Addition of 1 μg/mL ab16890 to patient cells also resulted in a decreased fluorescence intensity compared with control cells (Fig. 4C). These results indicate that the anti-IGF-1R monoclonal antibody ab16890 decreases the availability of IGF-1R on the surface of ALL cells.

FIGURE 4

FIGURE 4

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Neutralization of IGF-1R Promotes Apoptosis of ALL Cells

We next explored the effect of IGF-1R neutralization on apoptosis of ALL cells. Cells were treated with the anti-IGF-1R monoclonal antibody ab16890 as described above. After 48 hours of culture, each cell sample was stained with FITC-annexin V and PI, and flow cytometry was used to evaluate the proportion of apoptotic (annexin V-binding) ALL cells. In Jurkat cells (Fig. 5A), the control cells exhibited a physiological apoptotic percentage of ∼5%, whereas a gradual increase in ab16890 concentration led to a corresponding increase in the percentage of apoptotic cells (from 13.73%±1.31%, 15.64%±0.24%, 22.94%±1.97%, 34.77%±4.82%, and 44.67%±8.12% at 10 μg/mL ab16890 compared with the control group, 4.80%±0.22%). Similar results were obtained using Reh cells (Fig. 5B, from 11.35%±1.27%, 17.53%±3.48%, 25.26%±6.61%, 41.37%±2.52%, and 51.60%±5.94% at 10 μg/mL ab16890 compared with the control group, 4.51%±1.20%). In patient samples, addition of 1 μg/mL ab16890 also significantly increased the percentage of apoptotic cells (Fig. 5C). Taken together, these results indicate that the anti-IGF-1R monoclonal antibody ab16890 induces ALL cell apoptosis in a dose-dependent manner, as well as apoptosis of cells from patients with ALL.

FIGURE 5

FIGURE 5

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Neutralization of IGF-1R Blocks Phosphorylation of AKT Pathway Proteins

We next evaluated the effect of insulin and 1 μg/mL ab16890 on the levels of 3 phosphorylated proteins (phospho-AKT, phospho-P70S6K, and phospho-mTOR) in Jurkat and Reh cells, as well as patients 4 to 6, by western blotting. After 48 hours of culture, total protein was extracted from control, insulin-treated, and insulin plus ab16890-treated samples. In contrast to the control sample, treatment with insulin stimulated the expression of phospho-AKT, phospho-P70S6K, and phospho-mTOR in both cell types and cells obtained from 3 patients. Meanwhile, addition of ab16890 attenuated the insulin-induced phosphorylation of these 3 proteins (Fig. 6). These results indicate that the anti-IGF-1R monoclonal antibody ab16890 blocks IGF-1R-induced activation of the AKT pathway.

FIGURE 6

FIGURE 6

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DISCUSSION

The association between chemotherapy-related hyperglycemia and pediatric ALL prognosis is still controversial, although hyperinsulinemia and subsequent increase in IGF-1 levels promote the growth of solid tumors.27–29 Furthermore, the IGF signaling system plays crucial roles by regulating multiple cellular pathways and influencing tumor initiation and progression.30 The IGF system comprises ligands (insulin, IGF-1, and IGF-2), receptors (IGF-1R and IGF-2R) distributed across the cell surface, and at least 6 binding proteins insulin-like growth factor binding proteins that regulate the growth and differentiation of most organs in the body.31 Beyond these physiological functions, experimental, clinical, and epidemiological studies have found that IGF signaling pathways can trigger a phenotypically normal cell to express neoplastic traits through induction of biochemical and molecular processes.30 Furthermore, changes in the physiological conditions that control the balance between expression and activity of the IGF axis can lead to malignancy.

Recent large-scale epidemiological studies suggest that elevated insulin and IGF-1 levels in the circulation are associated with an increased risk of several cancers.30 Feng et al11 defined a 3-dimensional relationship between cell proliferation, insulin, and glucose, demonstrating that high glucose levels promote the growth of human breast and pancreatic cancer cells in an insulin-dependent manner. They also found that high insulin and glucose concentrations promoted chemoresistance to doxorubicin and gemcitabine in breast cancer and pancreatic cancer cells, respectively. Consistently, our current experiments revealed that DNR-induced toxicity in Jurkat and Reh cells is reduced in the presence of insulin. In addition, the proportion of DNR, VCR, or L-Asp-treated cells undergoing apoptosis was decreased following addition of insulin. These results suggest that high levels of circulating insulin may compromise the efficacy of chemotherapy. These findings parallel results from our clinical research on the effects of chemotherapy-related hyperglycemia on the poor prognosis of pediatric ALL. We also confirmed a relationship between insulin and the AKT pathway, with increased levels of phospho-AKT with increasing concentrations of insulin.

The biological actions of the IGFs are mainly regulated by IGF-1R, which is a transmembrane tyrosine kinase similar to the insulin receptor (INSR).32–34 However, INSR signaling is predominately associated with metabolic actions, whereas IGF-1R signaling primarily regulates growth activities.35 Importantly, IGF-1R has antiapoptotic effects and, as such, plays a key role in oncogenic transformation,36 as well as in the occurrence and development of several human malignancies as shown by preclinical observations,37,38 thus rendering the IGF axis an attractive oncological therapeutic target. Consistently, preclinical data have shown that IGF-1R-targeting drug candidates possess potential antitumor activity.39 To date, >30 drugs in laboratory studies and over 60 phases I to III clinical trials have evaluated the value of IGF-1R-targeting in the cancer therapy field.40 For example, a recent analysis revealed that IGF-1 expression correlates with poor prognosis in breast cancer treated with anti-IGF-1R inhibitors.41 In addition, another related study found that of 41 different human breast cancer cell lines tested, only 7 were sensitive to the growth-inhibitory effects of an anti-IGF-1R antibody.42 Two main factors cited as predictive for anti-IGF-1R treatment are the expression of IGF-1R and the growth-stimulatory effects of IGF-1. Similarly, another study has reported the prognostic significance of IGF-1R expression in tumors.43

Targeting receptors with a monoclonal antibody is a method frequently used to block metabolic pathways in clinical investigations. Therefore, we propose that using neutralizing antibodies to block IGF signaling could have potent antitumor activity. Our previous study compared IGF-1R levels in peripheral blood samples by reverse transcription-polymerase chain reaction from 43 children with ALL and 14 healthy children and showed significantly increased levels of IGF-1R mRNA in children with ALL in all diagnosis, relapse, and hyperglycemia groups compared with that in healthy children. Presently, we demonstrate that IGF-1R is expressed on the surface of Jurkat and Reh cell lines, as well as in samples from patients with ALL. Meanwhile, addition of the anti-IGF-1R monoclonal antibody ab16890 downregulated the expression of IGF-1R. Furthermore, ab16890 promoted apoptosis and paralleled the effects of IGF-1R expression. We next confirmed that changes in the AKT signaling pathway resulted from the reduced IGF-1R signaling, and we identified reduced levels of phospho-AKT, phospho-P70S6K, and phospho-mTOR. These findings indicate that the anti-IGF-1R monoclonal antibody promotes apoptosis in 2 ALL cells lines and in patient samples through its action on IGF-1R. This action likely involves inhibition of IGF-1R ligand binding, thus blocking AKT signaling and downstream biological effects.

Our results show that DNR, VCR, or L-Asp-induced toxicity in Jurkat and Reh cells is reduced in the presence of insulin. Moreover, we propose that identification of a subset of patients with high levels of IGF-1R expression can predict the responsiveness to IGF-1R-targeted therapy, which may significantly improve the success rate of treatment in children with ALL. Therefore, the IGF axis represents a promising target for treatment of pediatric ALL. While similar studies have been conducted for other tumors, our study utilized primary leukemic cell samples and is therefore more representative of the true clinical conditions of pediatric ALL. We also anticipate that further basic and clinical research will achieve better therapeutic outcomes in the future.

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Keywords:

child acute lymphoblastic leukemia; chemotherapy-related hyperglycemia; insulin; insulin-like growth factor-1 receptor; anti-IGF-1R monoclonal antibody

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