AKT inhibitor Hu7691 induces differentiation of neuroblastoma cells

Abstract

While neuroblastoma accounts for 15% of childhood tumor-related deaths, treatments against neuroblastoma remain scarce and mainly consist of cytotoxic chemotherapeutic drugs. Currently, maintenance therapy of differentiation induction is the standard of care for neuroblastoma patients in clinical, especially high-risk patients. However, differentiation therapy is not used as a first-line treatment for neuroblastoma due to low efficacy, unclear mechanism, and few drug options. Through compound library screening, we accidently found the potential differentiation-inducing effect of AKT inhibitor Hu7691. The protein kinase B (AKT) pathway is an important signaling pathway for regulating tumorigenesis and neural differentiation, yet the relation between the AKT pathway and neuroblastoma differentiation remains unclear. Here, we reveal the anti-proliferation and neurogenesis effect of Hu7691 on multiple neuroblastoma cell lines. Further evidence including neurites outgrowth, cell cycle arrest, and differentiation mRNA marker clarified the differentiation-inducing effect of Hu7691. Meanwhile, with the introduction of other AKT inhibitors, it is now clear that multiple AKT inhibitors can induce neuroblastoma differentiation. Furthermore, silencing AKT was found to have the effect of inducing neuroblastoma differentiation. Finally, confirmation of the therapeutic effects of Hu7691 is dependent on inducing differentiation in vivo, suggesting that Hu7691 is a potential molecule against neuroblastoma. Through this study, we not only define the key role of AKT in the progression of neuroblastoma differentiation but also provide potential drugs and key targets for the application of differentiation therapies for neuroblastoma clinically.

Graphical abstract

This study not only defines the key role of AKT in the progression of neuroblastoma differentiation but also provides potential drugs and key targets for the application of differentiation therapies for neuroblastoma clinically.

1. Introduction

Neuroblastoma originates from neural crest cells and is regarded as the most fatal neuroendocrine tumor in children, accounting for 15% of all pediatric tumors-related deaths^1,2. Current therapeutics against neuroblastoma mainly consist of surgery, cytotoxic chemotherapeutics including vincristine, etoposide, cisplatin, carboplatin, cyclophosphamide, and topotecan, along with differentiation therapy. Although low and intermediate-risk groups of neuroblastoma patients benefit from the standard of care treatment, the 5-year overall survival rate of the high-risk group patients, often with relapsed and metastatic neuroblastoma, is only around 50%³. Therefore, therapeutic strategies including targeted therapies have aroused increasing attention to make up for the shortage of chemotherapy. Activating mutations or overexpression of ALK are found in about 10%–15% of neuroblastoma patients⁴. Benefiting from the success of ALK inhibitors applied to lung cancer, the most advanced clinical study against neuroblastoma is targeting ALK, especially for lorlatinib, which is currently in phase 3 clinical trial (NCT03126916). Other targeted therapies undergoing clinical trials mainly consisted of agents achieving successes on other types of tumors, which lack solid bases of research on mechanisms as well as specificity for neuroblastoma, including targeting TRK, the RAS–MAPK pathway, cell cycle-related proteins, and HDACs. Currently, immune therapy targeting GD-2 by dinutuximab is the only US Food and Drug Administration (FDA)-approved targeted therapy overcoming neuroblastoma. However, FDA also alerts the side effects of dinutuximab, including fever, hypersensitivity, capillary leak syndrome, neuropathic pain, etc⁵. Thus, target therapies for neuroblastoma confront the failure of safe and effective therapeutic interventions.

Intermediate between neural crest differentiate to the peripheral nervous system, adrenal medulla, and melanocytes⁶, neuroblastoma exhibits an impaired differentiation characteristic. Thus, the differentiation states of neuroblastoma have been listed as one of the indicators for dividing patient risk groups in the Shimada histology grading system. In addition, a group of neuroblastoma patients has a unique ability to spontaneously differentiate, which surprisingly facilitates tumor regression⁷. Taken together, the differentiation property of neuroblastoma can not only be used as markers in diagnosis but also suggest the possibilities and prospection of conducting differentiation therapy on neuroblastoma.

Since the late 1970s, the concept of differentiation therapy was proposed based on the findings that differentiation of leukemia, embryonal carcinoma, and neuroblastoma can be induced by retinoids, cyclic AMP, and cytokines, which irreversibly altered the phenotype of cancer cells through differentiation. Meanwhile, neuroblastoma possesses features of fewer mutations and druggable targets compared to other tumors⁸, leading to difficulties in developing targeting agents for neuroblastoma. Thus, there exists a much greater need for novel therapeutic strategies based on the pathological and physiological properties of neuroblastoma, such as differentiation therapy. Currently, differentiation therapy has been successfully applied in the treatment of acute promyelocytic leukemia (APL) with an overall cure rate of around 95%⁹. Given the concerted action of multiple oncogenic pathways in most solid tumors, differentiation therapy for solid tumors seems more difficult compared to leukemia¹⁰. However, in cancers with a relatively simple genetic background, such as neuroblastoma, blockage of such oncogenic pathway result in cell differentiation and loss of stemness^11,12. Nowadays, differentiation therapy with retinoids, combined with surgery and chemotherapy has proven to be beneficial for patients with high-risk neuroblastoma¹³. The clinical trial had proven that the 3-year event-free survival rate among the 130 patients assigned to receive retinoic acid was significantly higher than the 128 patients with no further therapy (P = 0.027)¹⁴. However, unlike the overwhelming benefits achieved by retinoids in APL patients, retinoids are only limited to maintenance therapy in neuroblastoma. Since the benefit of retinoids as a first-line therapeutical agent is currently unclear, new targets and more effective drugs, and more drug options are badly required. Targets associated with neuroblastoma differentiation such as PHOX2B, MYCN, and TRK have been reported. However, due to the driving factor of neuroblastoma differentiation not yet determined and inherent difficulties in developing small-molecular inhibitors targeting transcription factors, differentiation inducers limit to retinoids up to present. Collectively, insight into neuroblastoma differentiation and new differentiation therapeutic inducing agents are urgently needed for neuroblastoma patients.

Here, we report that the novel AKT inhibitor Hu7691, which had completed preclinical studies and was approved for clinical trials by the National Medical Products Administration (2020LP00826), can effectively and rapidly induce differentiation of neuroblastoma cells¹⁵. Moreover, we demonstrate that the inactivation of the AKT pathway is critical for the initiation of the differentiation process in neuroblastoma. Taken together, our result can not only alleviate the problem of a few drug options for neuroblastoma differentiation therapy but also suggest the correlation between differentiation and AKT, providing insight into the underlying mechanism of neuroblastoma differentiation.

2. Methods and materials

2.1. Cells culture and reagents

All cell lines’ source and culture conditions are detailed in Supporting Information (Tables S1 and S2). All cell lines were maintained at 37 °C in a humidified atmosphere containing 5% CO₂.

ATRA was purchased from Sigma and dissolved in ethanol. Hu7691 was a kind gift from Dr. Xiaowu Dong (Zhejiang University, Hangzhou, China) and was dissolved in dimethyl sulfoxide (DMSO). AZD5363 and GSK2141795 were purchased from Topscience and were dissolved in DMSO.

2.2. Cellular proliferation and apoptosis analysis of Hu7691

Different neuroblastoma cell lines were seeded in 96-well plates and kept the cell density at 20%–30%. The survival rate of Neuro2a, IMR-32, SK-N-BE(2), SK-N-DZ, CHP-126, and SK-N-SH were determined after being treated with Hu7691, AZD5363, and GSK2141795 (0, 0.625, 1.25, 2.5, 5, 10, and 20 μmol/L) for 3 days. A colorimetric SRB assay was performed to examine the cell viability. In detail, SRB (4 mg/mL) was added to a 96-well plate after various treatments, then they were incubated for 30 min. Discard the liquid in a 96-well plate and wash it with 1% acetic acid more than 5 times. A microplate reader (Thermo, Fisher Scientific, Waltham, MA, USA) was used to measure the absorbance at 515 nm. Each sample was repeated in triplicate.

Different neuroblastoma cell lines were seeded in 6-well plates and kept the cell density at 20%–30%. Cell apoptosis was detected after Neuro2a and CHP-126 were treated with Hu7691 (5 and 10 μmol/L) for 3 days with the Annexin V-FITC Apoptosis Detection Kit according to the manufacturer's instructions (BD Biosciences).

2.3. RNA extraction and quantitative real-time PCR

Total RNA was isolated from cells with Trizol reagent (Invitrogen), and cDNA was transcribed using a transcript kit (TransGen Biotech). qRT-PCR analysis was performed using the SYBR Green (Bio-Rad) method on the ABI Fast 7500 real-time PCR instrument (Perkin–Elmer Applied Biosystems). The gene expression was normalized to the expression of the gene encoding Gapdh. Sequences of the primers for qRT-PCR are shown in Supporting Information Table S3.

2.4. Western blotting

The protein samples were resolved on SDS-PAGE gels and transferred onto a polyvinylidene difluoride membrane. Membranes were blocked in 5% (w/v) non-fat milk in T-PBS (1× PBS with 0.1% (v/v) Tween-20) for 1 h at room temperature. Membranes were incubated with primary antibody diluted in T-PBS at 4 °C overnight. After being washed with T-PBS, the membrane was incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody diluted in 5% non-fat milk in T-PBS for 1 h at room temperature. The resultant bands were visualized by the Chemiluminescence Western Blotting Detection System (AI600, General Electric) on X-ray films. The main primary antibodies used are shown in Supporting Information Table S4.

2.5. Flow cytometry

Cell cycles were analyzed after staining with propidium iodide (PI) on a FACS Caliber cytometer (Becton Dickinson, San Jose, CA, USA) as previously described¹⁶.

2.6. Immunofluorescence

Neuroblastoma was seeded on the glass bottom of 24-well plates and treated as described earlier. Cells were fixed in 4% paraformaldehyde for 15 min at room temperature. After washing with iced PBS twice, cells were permeabilized with 0.4% Triton X-100 and 2% BSA for 1 h at room temperature and then incubated with rabbit polyclonal anti-β-III tubulin (1:100, Abcam) overnight at 4 °C. After washing three times, cells were incubated with Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:1000) secondary antibody for 1 h at room temperature. Nuclei were co-stained with DAPI (1:5000). Images were acquired with a Zeiss LMS 710 confocal microscope.

2.7. Gene set enrichment analysis and KEGG pathway enrichment

Gene set enrichment analysis (GSEA)¹⁷ was used to assess the enrichment of the gene sets based on the custom gene sets collection constructed from the Gene Ontology Resource database. The custom gene set collection consisted of a total of 15 biological processes related to neuroblastoma differentiation. Instead of the maximin gene set size filter being changed to 1500, other factors were set as default. Gene sets with normalized enrichment scores (NES) higher than 1, nominal p-values lower than 0.01, and FDR q-values lower than 0.25 are considered significantly enriched. Details of custom gene set collection and RNA-seq data are presented in the Supporting Information.

After setting the screening limits to genes up or down-regulated more than two-fold in all 3 AKT inhibitors groups, identified differential genes were uploaded to Metascope (https://metascape.org/gp/index.html#/main/step1) to perform KEGG pathway enrichment.

2.8. Animals and treatments

6- to 8-week-old female nude mice were purchased from GemPharmatech, Nanjing, China. Neuro2a xenograft was established via axillary inoculation in nude mice. When tumors reached an average volume of 30–45 mm³, mice were randomized into control and treated groups. Mice were orally administered (i.g.) with placebo (CMC-Na), 40 and 80 mg/kg of Hu7691, and 80 mg/kg of ATRA, respectively. The dosage and dosing frequency of each group was based on the data from preclinical trials. Tumor volume and body weight were measured every day. On the 17th day of treatment, mice were sacrificed, and tumor samples were divided into two pieces and kept at −80 °C for protein and RNA extraction.

2.9. Statistical analysis

All the data are presented as the mean ± standard deviation (SD) [except in vivo data are presented as the mean ± standard error of the mean (SEM)] from a minimum of three independent experiments. Data analysis was performed using Microsoft Excel version 16.16.23, Image J (version 1.8.0), and GraphPad Prism Software Version 8.0 (GraphPad Prism, San Diego, CA, USA). Statistical parameters including statistical analysis, statistical significance, and values are indicated in the figure legends. For statistical comparison, we performed a two-tailed Student's t-test, one-way ANOVA Tukey's post hoc analysis for multiple comparisons, and log-rank (Mantel–Cox) test. A value of P < 0.05 is considered significant (represented as ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 or not significant (n.s.).

3. Result

3.1. Identification of the AKT inhibitor Hu7691 as a novel differentiation-inducing agent for neuroblastoma cells

To identify new potential leads and to further relieve the problem of limited drug options for neuroblastoma differentiation therapy, we performed a structurally-diverse compound library screening for differentiation-related phenotype, which is the morphologic changes of neuroblastoma cells including filopodium formation and neurites sprouting^18,19. This compound library contains approximately 20,000 compounds, assisted by cluster analysis. A total of 24 compounds with significantly different structural skeletons, including derivatives of quinoline, purine, indole, and so on, were selected (Supporting Information Fig. S1). Additionally, ATRA was added to our screening library as a positive control due to its current clinical usage and the definite differentiation-inducing effect reported in other studies. Neuroblastoma cell Neuro2a was treated with different compounds along with ATRA for 72 h before measuring the average neurites outgrowth. Considering the differentiation effect of ATRA on neuroblastoma cannot be observed until 5–7 days in multiple studies^20,21, 7 days of ATRA treatment was also added. Several compounds demonstrated a good proliferation-inhibiting activity such as compounds L2, L5, L14, L16, and L18 (Supporting Information Fig. S2). Specifically, quantified by the average neurites' outgrowth with 2.5 μmol/L of each compound, treatment with compound L2 (also termed Hu7691) could induce neurites to sprout and elongate 3.2 times higher than control. Whereas 7 days of treatment of ATRA could only promote neurites’ outgrowth to 2.1-fold, indicating a strong and rapid differentiation-inducing potential of Hu7691 (Fig. 1A).

Figure 1.

Identification of the AKT inhibitor Hu7691 as a potential differentiation-inducing agent for neuroblastoma cells based on phenotypic screening. (A) Neuro2a cells were treated with a structurally-diverse compound library and positive control ATRA for 72 h. Relative average neurites outgrowth = Average neurites outgrowth_Treatment/Average neurites outgrowth_Control. (B) The structure of AKT inhibitor L2 (Hu7691). (C) Dose–response curves for cell viability of Hu7691 treatment in 6 neuroblastoma cell lines for 72 h (Neuro2a, IMR-32, SK-N-BE(2), SK-N-DZ, CHP-126, and SK-N-SH). Cell viability was determined by SRB; IC₅₀ was calculated in GraphPad Prism 8.0 and displayed respectively. (D) The proliferation rate of Neuro2a exposed with 3 different concentrations (1.25, 2.5, and 5 μmol/L) of Hu7691 and CHP-126 was exposed with 2 different concentrations (5 and 10 μmol/L) were evaluated by SRB, respectively. (E) Neurites outgrowth in Neuro2a, CHP-126, IMR-32, SK-N-BE(2), and SK-N-DZ after Hu7691 exposure (5, 20, 20, 10, and 10 μmol/L, respectively) for 72 h were observed with Leica microscope (100 × ). (F) Relative average neurites outgrowth was quantified after treatment of 3 different Hu7691 concentrations in Neuro2a, CHP-126, IMR-32, SK-N-BE(2), and SK-N-DZ cells. (G, H) Images of Neuro2a were obtained after treatment of 5 μmol/L of Hu7691 at different time points and 20 μmol/L of ATRA treatment at 72 h. Pictures were taken after 3 different concentrations of Hu7691 (2.5, 5, 7.5 μmol/L) and 20 μmol/L of ATRA treatment at different time points. Every picture was measured and calculated its neurites per cell, average, and maximin neurite outgrowth by Neuron J. Data are shown as mean ± SD, n = 3. Statistical significance between groups was calculated with one-way ANOVA. n.s., no significant differences, P > 0.05 (vs. Control); ∗P < 0.05 (vs. Control); ∗∗P < 0.01 (vs. Control); ∗∗∗P < 0.001 (vs. Control).

Novel AKT inhibitor Hu7691 is a clinical candidate discovered by our lab through a systematic structure–activity relationship (SAR) study of 196 compounds, the optimization of which is focused on AKT inhibiting activity, anti-tumor activity, kinase specificity, pharmacokinetic properties, human ether-a-go-go-related gene (hERG) channel blocking activity, and toxicity¹⁵. Currently, Hu7691 has completed preclinical studies, which showed a rather high oral bioavailability and significant anti-tumor efficacy of multiple human tumor xenografts and was approved for clinical trials by the National Medical Products Administration (2020LP00826) (Fig. 1B). However, given the differences between childhood cancers and adult cancers, the anti-tumor and pharmacological effects remain unclear in neuroblastoma. Thus, we took 6 neuroblastoma cell lines including MYCN amplify (Neuro2A, IMR-32, SK-N-BE(2), SK-N-DZ, CHP126), and non-amplify (SK-N-SH) to test the potential differentiation and anti-proliferation effect of Hu7691. The result shows a concentration-dependent decrease in cell viability after 72 h of treatment of Hu7691 within a tolerable concentration in vitro, which IC₅₀ values ranging between 2.73 and 18.0 μmol/L (Fig. 1C and D). Neuro2a and CHP-126 cells exhibited the highest sensitivity to Hu7691 and were therefore selected for continued study. Since the inactivation of AKT often leads to cell apoptosis²², we then determined the effect of Hu7691 on apoptosis. Interestingly, both Neuro2a and CHP-126 cells did not show any apoptosis after Hu7691 treatment at concentrations above IC₅₀ (Supporting Information Fig. S3A). Image of differentiation-like morphologic changes in various neuroblastoma cell lines further implies the differentiation effect after Hu7691 treatment (Fig. 1E). Relative neurites outgrowth to control, which refers to the degree of differentiation, showed the differentiation-like morphologic changes of neuroblastoma cell lines were concentration dependent (Fig. 1F). To further exclude other possible effect triggered by Hu7691, we compared the effect of cell death induced by standard chemotherapeutic agent vincristine (VCR), the iron death inducer RSL3, and the autophagy inducer Torin1 (Fig. S3B). Neuro2a cells were treated with VCR, RSL3, Torin1, and Hu7691 for 3 days at the concentration around its IC₅₀. PI staining results show that the Hu7691 treatment only exhibited little effect on inducing cell death, whereas the percentage of cell death was significantly upregulated after treating 3 other compounds (Fig. S3C). These results indicate that the anti-proliferation effect of Hu7691 might depend on promoting cell differentiation without inducing cell death. Thus, we analyzed and quantified the morphologic changes of Neuro2a cells, which were the most significant among other cell lines, after Hu7691 treatment by introducing 2 additional markers: the average neurites per cell and the maximum neurite outgrowth, which refer to the differentiation rate and the maturation stage of differentiation, respectively. All markers were evident after continuous treatment for 72 h with sublethal but antiproliferative concentrations of Hu7691. In contrast to the positive control ATRA, Hu7691 can rapidly and significantly promote neurite outgrowth in Neuro2a cells within 24 h (Fig. 1G and H). Therefore, our study indicates that the novel AKT inhibitor Hu7691 can obviously inhibit cell proliferation and rapidly induces differentiation-like morphologic changes in neuroblastoma cells.

3.2. AKT inhibitor Hu7691 drives the neuronal differentiation of neuroblastoma cells

Since Hu7691 is found to significantly promote the formation of long neurite-like protrusions, we then tried to determine the relationship between morphological changes and neural differentiation in neuroblastoma cell lines. Hu7691-induced sprouting of long neurite-like protrusion was positive for β-III tubulin, a neuronal differentiation marker, determined by immunofluorescence staining²³. The result demonstrates that the number of cells with neurites outgrowth was significantly higher after treatment with Hu7691 compared with untreated, whereas the differentiation-like morphological changes were inconspicuous after 3 days of treatment of ATRA in Neuro2a, CHP-126, and SK-N-BE(2) (Fig. 2A). Cell cycle study demonstrated that Hu7691 treatment arrested G0/G1 cell cycle up to around 70%, a common phenomenon in neuroblastoma differentiation²⁴ (Fig. 2B). Particularly, the G0/G1 cell cycle arrest was the most significant in Neuro2a cells which accumulated from 47.1% to 73.4% (Fig. 2C). Neuro2a cells were further tested for 2 proteins, Cyclin D1 and Cyclin E1, which will both accumulate in the G0/G1 phase, to further confirm the changes in cell cycle after Hu7691 treatment. The Western blot result shows a time-dependent accumulation of Cyclin D1 and E1 after Hu7691 treatment within 3 days (Fig. 2D).

Figure 2.

Hu7691 can significantly promote the differentiation of neuroblastoma. (A) Immunofluorescence images of Neuro2a, CHP-126, and SK-N-BE(2) after being treated with a relatively low or high concentration of Hu769 and 20 μmol/L of ATRA for 72 h and stained with β-III tubulin (green) and counterstained with 40,6-diamidino-2-phenylindole (DAPI, blue). White arrows point out neurites. Scale bar, 25 μm. (B) Shifting in the cell cycle of Neuro2a, CHP-126, and SK-N-BE(2) caused by Hu7691 treatment was detected by PI staining. (C) PI staining diagram of untreated and 10 μmol/L of Hu7691 treatment in Neuro2a cells. (D) Western blotting detected the expression of Cyclin D1 and Cyclin E1 after 5 μmol/L of Hu7691 treatment from different time points. (E) Relative mRNA levels of neural differentiation marker Eno2, Tubb3, and Rbfox3 were detected by qRT-PCR after 3 different concentrations of Hu7691 (2.5, 5, and 7.5 μmol/L) and 20 μmol/L of ATRA treatment. (F) Western blotting detected the expression of NSE and SYNGR1 after 3 different concentrations of Hu7691 (2.5, 5, and 7.5 μmol/L) and 20 μmol/L of ATRA treatment. (G) Morphologic changes of 8 different NB PDCs (NB-2, 7p, 18, 22, 50, 51, 58, and 61) after 20 μmol/L Hu7691 exposure were acquired with a Leica microscope (100×). Data are shown as mean ± SD, n ≥ 3. Statistical significance between groups was calculated with one-way ANOVA. n.s., no significant differences, P > 0.05 (vs. Control); ∗P < 0.05 (vs. Control); ∗∗P < 0.01 (vs. Control); ∗∗∗P < 0.001 (vs. Control).

We next examined the effects of Hu7691-induced neuroblastoma differentiation by detecting the mRNA changes of primary neural differentiation markers Gap43²⁵. After 3 days of Hu7691 treatment, our data show that GAP43 was elevated in all 5 cell lines after Hu7691 treatment, whereas the effect was relatively weaker by ATRA (Supporting Information Fig. S4). Neuro2a was selected as the main cell line for subsequent experiments because of its obvious changes in morphology, cell cycle, and mRNA marker. The mRNA levels of Eno2²⁶, Tubb3, and Rbfox3²⁷, common markers of neural differentiation in Neuro2a were detected. Up-regulation appeared in all differentiation markers after incubation with Hu7691, and more significant changes were observed in advanced differentiation markers such as Eno2 and Rbfox3, indicating the ability of Hu7691 on inducing advanced differentiation, whereas ATRA treatment showed a similar result in cell morphology and cell cycle study (Fig. 2E). Finally, we detected 2 neural maturation markers, NSE encoded by Eno2 and SYNGR1 which modulates the localization of synaptophysin into synaptic-like microvesicles and plays a role in synaptic-like microvesicle maturation, to determine the differentiation effect²⁸. The Western blotting result demonstrates moderately increased levels of NSE and a significantly upregulated expression of SYNGR1 (Fig. 2F).

Due to the increasing clinical trials combining differentiation-induction and other therapeutics²⁹ (NCT01183429 and NCT03033303), and to highlight the advantages of Hu7691, we compared Hu7691 to the combination of traditional chemotherapy (etoposide and cisplatin) and ATRA. The selection of ATRA and Hu7691's concentrations was based primarily on the results of their C_max (C_max = 2.158 μmol/L), whereas chemotherapy agents were selected with a minimum effect on cell survival to highlight the co-administration effect. Data show that compared to the combination of ATRA and chemotherapy agents, similar effects were detected with Hu7691 alone (Supporting Information Fig. S5A). Importantly, the SRB assay shows that the combination of Hu7691 and chemotherapy agents could have a better anti-proliferation effect than a single agent, further indicating its advantages (Fig. S5B). Finally, the result of clone formation ability demonstrates that Hu7691 singlehandedly overwhelms the combination of chemotherapy agents and ATRA indicating the potential of replacing ATRA clinically (Fig. S5C). These data not only show the potential of effective single-agent differentiation therapy of Hu7691 but also highlight its clinical value in combination with chemotherapy.

In addition, to more accurately predict the effect of Hu7691 on neuroblastoma in the clinic, we then tested the anti-proliferating and differentiation-inducing effect of Hu7691 on patient-derived neuroblastoma cells (neuroblastoma PDCs). SRB assay results show that a high concentration of Hu7691 can more effectively inhibit the proliferation of all neuroblastoma PDCs than ATRA treatment (Supporting Information Fig. S6). The differentiation-like morphology changes of multiple neuroblastoma PDCs also indicated the potential differentiation-inducing effect of Hu7691 (Fig. 2G). Taken together, the results of cell morphology, cell cycle, and neural differentiation marker demonstrate that, in contrast to ATRA, Hu7691 is significantly rapid and efficient in inducing differentiation of neuroblastoma in both cell lines and PDCs.

3.3. AKT inhibitors AZD5363 and GSK2141795 can also promote neuroblastoma differentiation

To explicit the Hu7691-induced differentiation of Neuro2a cells is not unique, we further introduced other competitive AKT inhibitors AZD5363³⁰ and GSK2141795³¹, which have been reported to effectively prevent substrate phosphorylation by AKT (Fig. 3A). SRB assay shows that GSK2141795 and Hu7691 have a close average IC₅₀ for the inhibition of neuroblastoma cell proliferation, 2.95 and 11.28 μmol/L, respectively, whereas AZD5363 shows a relatively weak effect (Fig. 3B). Similarities between the results of the anti-proliferation effect and the morphological data after treatment of 3 AKT inhibitors in multiple neuroblastoma cell lines (Supporting Information Fig. S7) and the tendency of promoting neurites outgrowth (Fig. 3C and D) were found. In addition, up-regulation of neural advanced differentiation marker Eno2 and Rbfox3 by both AZD5363 and GSK2141795 indicated a similar mechanism of all 3 AKT inhibitors in inducing neuroblastoma differentiation (Fig. 3E). However, given the optimization of Hu7691 based on limited anti-tumor activity and toxicity of AZD5363 and GSK2141795, we believe that Hu7691 holds better promises in further clinical application.

Figure 3.

Other AKT inhibitors can also induce neuroblastoma to differentiate. (A) The structure of AKT competitive inhibitors AZD5363 and GSK2141795. (B) Dose–response curves for cell viability of AZD5363 and GSK2141795 treatment in 4 neuroblastoma cell lines for 72 h (Neuro2a, SK-N-BE(2), SK-N-DZ, CHP-126). Cell viability was determined by SRB; IC₅₀ was calculated in GraphPad Prism 8.0 and displayed respectively. (C, D) Images of Neuro2a were obtained after treatment of 5 μmol/L of Hu7691 at different time points and 20 μmol/L of ATRA treatment at 72 h. Pictures were taken after 3 different concentrations of Hu7691 (2.5, 5, and 7.5 μmol/L) and 20 μmol/L of ATRA treatment at different time points. Every picture was measured and calculated its neurites per cell, average, and maximin neurite outgrowth by Neuron J. (E) Relative mRNA level of Eno2 and Rbfox3 was detected by qRT-PCR after 3 different concentrations of AZD5363 (2.5, 5, and 7.5 μmol/L) and GSK2141795 (1.25, 2.5, and 5 μmol/L) treatment. Data are shown as mean ± SD, n = 3. Statistical significance between groups was calculated with one-way ANOVA. n.s., no significant differences, P > 0.05 (vs. Control); ∗P < 0.05 (vs. Control); ∗∗P < 0.01 (vs. Control); ∗∗∗P < 0.001 (vs. Control).

To further explore the downstream network changes underlying the differentiation induction of Neuro2a cells in response to 3 different AKT inhibitors, we performed gene expression analysis by RNA-seq. Samples were collected after treating Neuro2a cells of all 3 AKT inhibitors for 48 h. Gene expression differed significantly between the Hu7691 treated and control groups. Several genes implicated in neuronal differentiation and favorable prognoses, such as neuron-specific enolase (Eno2), early growth response 2 (Egr2), and brain-derived neurotrophic factor (Bdnf), were upregulated after Hu7691 treatment (Fig. 4A). In addition, gene set enrichment analysis (GSEA) was performed. GSEA analysis comparing AZD5363, GSK2141795, and Hu7691 to control was conducted separately but showed similar results (Supporting Information Table S5). In consideration of the similarities of the 3 AKT inhibitors, the “AKTi treated” group consisted of the RNA-seq results from all 3 AKT inhibitors. By using the limitation of |normalized enrichment score| > 1, P-value < 0.05, and false discovery rate < 25%, enrichment results demonstrate the upregulation of gene sets involved in neurogenesis, neuron differentiation, and development after treating 3 different AKT inhibitors (Fig. 4B). Moreover, Genes up- or down-regulated more than 2-fold in all 3 AKT inhibitors groups were selected for further analysis. 1086 genes were found to have been significantly altered, among which 86 were up-regulated and 27 were down-regulated in a total of 113 genes in all 3 groups (Fig. 4C and E). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of these 113 genes found several differentiation-related pathways, including glycosaminoglycan degradation, Hedgehog pathway, NOD-like receptor pathway, and TGF-β signaling pathway (Fig. 4D). In summary, our results further demonstrate the differentiation-promoting effect of 3 AKT inhibitors, and similarities between them indicate the critical role of AKT in regulating the differentiation of neuroblastoma cells.

Figure 4.

Other AKT inhibitors can also induce neuroblastoma to differentiate and activate multiple differentiation-associated pathways. (A) Volcano plot showing differentially expressed genes in Hu7691-treated versus control groups. Neuronal differentiation genes Eno2, Egr2, and Bdnf were marked (red circles). (B) GSEA indicates enrichment and upregulation of genes involved in positive regulation of neuron differentiation, neurogenesis, generation of neurons, and neuron projection development after treatment of Hu7691, AZD5363, and GSK2141795. (C) After 48 h of treatment of AZD5363, GSK2141795, and Hu7691 (5, 2.5, and 5 μmol/L, respectively), differential genes were overlaid. (D) Overlaid genes were analyzed by KEGG pathway enrichment analysis and displayed. (E) The top 20 up-regulated and 10 down-regulated genes are shown as a heatmap. Nom p-Val, normalize p-value. FDR, false discovery rate. NES, normalized enrichment score.

3.4. Only panAKT knockdown can promote the differentiation of neuroblastoma cells

As the downstream of receptor tyrosine kinase (RTKs), the previous report showed that AKT was involved in cytokine-mediated embryonic cell proliferation, migration, and differentiation³². Given that all 3 AKT inhibitors show the differentiation induction effect on neuroblastoma cells, we then asked whether AKT kinase activity contributed to this inhibitors-induced differentiation of neuroblastoma. Firstly, we found that 3 AKT inhibitors effectively inhibited the phosphorylation of AKT substrates, including PRAS40 and GSK-3β, further indicating the relation between AKT and neuroblastoma differentiation, whereas the phosphorylation of AKT increases due to the feedback loop of the AKT pathway³² (Fig. 5A).

Figure 5.

Only knock-downing panAKT can promote the differentiation of neuroblastoma cells. (A) Western blotting of AKT and its downstream protein: mTOR and GSK-3β as well as their phosphorylated state including p-FOXO1 and p-PRAS40 after being treated with different concentrations of Hu7691, AZD5363, and GSK2141795 (0, 2.5, 5, and 10 μmol/L, respectively). (B, C) Neuro2a cells were infected with lentivirus-vector, lentivirus-shAKT1 (shAKT1#1, shAKT1#2), lentivirus-shAKT2 (shAKT2#1, shAKT2#2) or lentivirus-shAKT3 (shAKT3#1, shAKT3#2) for 7 days. In the meantime, Neuro2a was treated with 5 μmol/L of Hu7691 for 3 days. Then, pictures of neurites were captured and analyzed by Neuron J. Statistical differences shown in green font were compared with the Hu7691 group. (D) Neuro2a cells were infected with a lentivirus-vector or co-infected with lentivirus-shAKT1 (shAKT1#2), lentivirus-shAKT2 (shAKT2#2), and lentivirus-shAKT3 (shAKT3#1) as shpanAKT for 72 h. The protein level of AKT1/2/3 and p-AKT were demonstrated by Western blot. (E, F) Neuro2a cells were infected with lentivirus-shpanAKT for 7 days. In the meantime, Neuro2a was treated with 5 μmol/L of Hu7691 for 3 days. Then, pictures of neurites were captured and analyzed by Neuron J. Statistical differences shown in green font were compared with the Hu7691 group. (G) Relative mRNA levels of Gap43 and Eno2 were detected by qRT-PCR after Neuro2a cells infected with lentivirus-vector or co-infected with lentivirus-shAKT, sequence same as before, for 7 days. (H) Western blotting detected the expression of NSE and SYNGR1 after being infected with lentivirus-shpanAKT for 7 days. (I, J) CHP-126 cells were infected with lentivirus-vector or lentivirus-shpanAKT for 7 days. In the meantime, CHP-126 was treated with 10 μmol/L of Hu7691 for 3 days. Then, pictures of neurites were captured and analyzed by Neuron J. (K) Relative mRNA levels of GAP43, ENO2, and RBFOX3 were detected by qRT-PCR after CHP-126 cells were infected with lentivirus-vector or lentivirus-shpanAKT, sequence same as before, for 7 days. Statistical differences shown in green font were compared with the Hu7691 group. Data are shown as mean ± SD, n ≥ 3. Statistical significance between groups was calculated by one-way ANOVA. n.s., no significant differences, P > 0.05 (vs. shCtrl); ∗P < 0.05 (vs. shCtrl); ∗∗P < 0.01 (vs. shCtrl); ∗∗∗P < 0.001 (vs. shCtrl); ^#P < 0.05 (vs. Hu7691 group); ^###P < 0.001 (vs. Hu7691 group).

With the emerging evidence of distinct functions of different AKT isozymes (AKT1, AKT2, and AKT3)^33,34, the function of AKT isozymes in neuroblastoma remains unclear. To further explore the differences between 3 AKT isozymes, Neuro2a cells were infected with two different shRNAs targeting each isoform of AKT, separately to distinguish their effects on inducing differentiation (Supporting Information Fig. S8A). shRNA-mediated down-regulation of different AKT isozymes shows a limited effect on sprouting neurites of Neuro2a cells compared to Hu7691 treatment (Fig. 5B and C). In consideration of the potential compensatory effects of different AKT isozymes in the regulation of neuroblastoma differentiation, we then co-infected Neuro2a cells with shRNAs targeting all 3 isoforms of AKT as shpanAKT (Fig. 5D). The results show that shpanAKT was able to induce significantly differentiation-like morphological changes compared to single AKT silence, which effect was like the relatively poor selectivity between AKT isozymes of AKT inhibitors (Fig. 5E and F). We then introduced primary and advanced neural differentiation markers, Gap43 and Eno2, respectively, to determine the shRNA-associated differentiation of Neuro2a. Corresponding to the morphological changes, Gap43 up-regulation was detected in a few single AKT isozyme knockdowns, whereas Eno2 was up-regulated only by shpanAKT (Fig. 5G). Additionally, the result of protein levels of NSE and SYNGR1 also demonstrates that shpanAKT led to the differentiation of Neuro2a cells (Fig. 5H).

In addition to Neuro2a, we also examined the differentiation effect of CHP-126 after shpanAKT (Fig. S8B). Similar results in cell morphology were observed after shpanAKT which the average neurites outgrowth had no significant differences from the Hu7691 treatment (Fig. 5I and J). Differentiation markers such as GAP43, ENO2, and RBFOX3 were also upregulated after shpanAKT (Fig. 5K). Taken together, due to the overlaying function of all AKT isozymes, we showed that panAKT kinase inhibition has a critical influence on the differentiation of neuroblastoma.

3.5. Hu7691 arrests tumor growth and induces differentiation in neuroblastoma xenograft mice

Finally, we investigated the therapeutic effects of Hu7691 on neuroblastoma in vivo. Neuro2a xenograft, established via axillary inoculation in nude mice, were treated with 40 or 80 mg/kg Hu7691, or 80 mg/kg of ATRA daily by oral administration for 17 days. The dosing frequency of each group was administered 5 times a week to minimize toxicity. The average volume of vehicle-treated tumors increased continuously, reaching an average volume at the end of the experiment of 2416 ± 432 mm³ compared with 1079 ± 318 mm³ for the treatment of 80 mg/kg Hu7691 (Fig. 6A and B). Moreover, significantly reduced tumor weight (47.7%) and obvious therapeutic activity, as indicated by a relative tumor volume (RTV) T/C value of 54.6% (determined by RTV Treatment/RTV control × 100) in the Hu7691 treatment group were observed (Fig. 6C and D). Importantly, all mice gained weight during the experiments indicating tolerable toxicity of this dosing regimen (Fig. 6E).

Figure 6.

Hu7691 arrests tumor growth and induces differentiation in Neuro2a xenograft models. (A) Neuro2a xenograft was established via axillary inoculation in nude mice (n = 8). Images of Neuro2a xenograft tumors were captured on the 17th day of treatment. (B) Tumor volumes were measured daily, and growth curves were drowned as mean ± SEM. Mice were orally administered (i.g.) with placebo (CMC-Na), 40 and 80 mg/kg of Hu7691, and 80 mg/kg of ATRA after xenograft establishment. The dosing frequency of each group was administered 5 times a week. The black arrow pointed out no oral administrated time point. (C) Neuro2a xenograft tumors were weighed on the 17th day. (D) Effects of Hu7691 and ATRA were analyzed by RTV and T/C (%). RTV, relative tumor volume; T/C (%) = RTV_Treatment/RTV_Control × 100. (E) Body weight was measured daily, and growth curves were drowned as mean ± SEM. (F) Neuro2a xenograft tumors were ground for further use. Western blot of AKT and GSK-3β, and their phosphorylated state in Neuro2a xenograft tumors, respectively. (G) Neuro2a xenograft tumors were sliced and stained with β-III tubulin and NSE antibodies. (H) Neuro2a xenograft tumors were ground and neuroblastoma differentiation markers, Gap43 and Tubb3, were detected by qRT-PCR. Grow curve of Neuro2a xenograft volume was shown as mean ± SEM, n = 8. Other data are shown as mean ± SD, n = 8. Statistical significance between groups was calculated by one-way ANOVA. n.s., no significant differences, P > 0.05 (vs. Control); ∗P < 0.05 (vs. Control); ∗∗P < 0.01 (vs. Control); ∗∗∗P < 0.001 (vs. Control).

To further confirm the therapeutic effect of Hu7691 in vivo, we began by testing the AKT pathway changes. The phosphorylation levels of AKT in Neuro2a xenograft were dramatically increased after Hu7691 treatment, and a slight decrease of p-GSK-3β was also detected, indicating AKT inhibition (Fig. 6F). After confirming the efficacy of Hu7691 in vivo, we observed that differentiation markers, Gap43, and Tubb3, were around 10 times higher in 80 mg/kg Hu7691 groups compared to untreated (Fig. 6H), suggesting its strong differentiation effect in vivo. Fluorescent staining of xenograft with both β-III tubulin and NSE demonstrated that 80 mg/kg Hu7691 exerted significant upregulation of both markers further confirming the in vivo differentiation effect (Fig. 6G). Whereas the relatively weak effect of ATRA treatment on the differentiation marker might explain its limited in vivo antitumor effect. Collectively, these data demonstrated a good therapeutic effect of Hu7691 in Neuro2a xenograft mice.

4. Discussion

Since it was not until the recent decade that pediatric solid tumors including neuroblastoma raised serious concerns, there currently remains a severe lack of targets and corresponding inhibiting agents which intervenes with the development of therapeutical strategies for neuroblastoma patients. In this study, we reported for the first time that the novel AKT inhibitor Hu7691 can inhibit neuroblastoma proliferation while effectively inducing cell differentiation in vitro and in vivo. Thus, our data characterize AKT as a pivotal target and provide a potential molecule for differentiation therapy.

Clinical evidence shows that the activation of the AKT signal pathway, due to overexpression or activating mutations of proteins upstream of AKT such as PTEN, ALK, and PI3K³⁵, is closely related to the poor prognosis of neuroblastoma³⁶. AKT activation is found in 50%–60% of all primary neuroblastoma samples indicating that targeting AKT may display selective antitumor effects. Most important of all, our discovery of promoting cell differentiation by AKT inhibition reveals the underlying biological effect mediated by AKT in the progression of neuroblastoma. Given that the standard of care for neuroblastoma patients includes applying differentiation therapy and our results of the antitumor effect of Hu7691 within the tolerable concentration in vitro and in vivo, we are convinced that inhibition of AKT is more clinically feasible attributed to its selectivity, potency, and effectiveness in inducing neuroblastoma differentiation.

Therefore, targeting AKT in neuroblastoma is an attractive anticancer strategy and has been developed³⁷. The most clinically advanced AKT inhibitor is MK-2206, an allosteric inhibitor, which binds to the motif and interacts with both the pleckstrin homology and catalytic domains to prevent the translocation and activation of AKT³⁸. Classical AKT competitive inhibitors including ATP and kinase domain competitive inhibitors such as AZD5363³⁰ and GSK2141795 have also been developed. However, due to limited anti-tumor activity and low bioavailability of several AKT inhibitors monotherapy across a range of tumor types, attention has focused on combination trials and molecule optimization³⁷. In our previous study¹⁵, the novel AKT inhibitor Hu7691, discovered by our lab after a thorough SAR study and toxicity optimization, is currently under phase I clinical trials with indications of multiple solid tumors and more tolerable side effects. Based on the differentiation effect of 3 AKT inhibitors and AKT knockdown, we clarified that the differentiation of neuroblastoma cells depends on AKT repression. From the viewpoint of inducing differentiation, Hu7691 and GSK2141795 exhibited better effects than AZD5363, while in terms of the clinical application, GSK2141795 exhibited a higher frequency of treatment-related adverse events including diarrhea (64%), nausea (61%), and fatigue (49%)³⁹ than the other two. Thus, although both AZD5363 and GSK2141795 possess the ability to induce neuroblastoma differentiation, Hu7691 still exhibits the best prospect. Taken together, we proposed a brand-new agent for differentiation therapy. In addition, a high correlation between AKT inhibition and neuroblastoma differentiation was also discovered.

Given the importance of the differentiation state for the prognosis of neuroblastoma patients and the favorable outcome after differentiation therapy administration, using retinoids as maintenance therapy in neuroblastoma is efficient and well-established, and even shows potential as first-line therapy. However, only the specific pathogenesis of APL driven by fusion protein PML/RARα provides the rationale for the dramatic success of retinoids as a targeted therapy⁴⁰. In the absence of the molecular target of retinoids, combined with the problems of effective blood concentration and considerably more complex crosstalk between oncogenic pathways, it seems difficult to implement differentiation therapy on other malignancies as it did in APL¹⁰. It takes a longer onset time (ranging from 5 to 28 days) and higher concentration to trigger differentiation in vivo and in vitro compared to APL^20,21,41, 42, 43, 44, 45, 46. Interestingly, research showed that the activation of the AKT pathway was essential for the differentiation of neuroblastoma induces by retinoids⁴⁷, which causes a conflicting relationship between the widely regarded tumor-promoting mechanism of AKT and the favorable outcome of differentiation therapy in neuroblastoma. The side effect of the activated AKT pathway by retinoids might weaken its differentiation-inducing effects. Therefore, the mechanisms underlying the AKT inhibition or Hu7691-induced differentiation warrant further investigation.

AKT is known for its multiple functions in controlling essential intracellular pathways. During fetal development, transmembrane molecules, such as FGF receptors, PDGF receptors, or integrins, activate AKT to control embryonic cell proliferation, migration, differentiation, and cell fate decisions⁴⁸. While many studies in mouse embryos have implicated AKT signaling in the differentiation of several neural crest derivatives, information on the pathological conditions caused by excessively activated AKT during the process of neural crest differentiation had remained relatively scarce. Studies showed that down-regulation of N-Myc, a key driver of neuroblastoma tumorigenesis and progression, can induce neuroblastoma differentiation⁴⁹, and phosphorylate of N-Myc by GSK-3β, the substrate of AKT, can destabilize N-Myc⁵⁰. Our data revealed that phosphorylation GSK-3β and N-Myc were simultaneously down-regulated after treatment of Hu7691 (Supporting Information Fig. S9), indicating a potential mechanism. However, since significant morphological changes in both multiple MYCN amplified and non-amplified neuroblastoma cells were observed, we lack evidence to prove that neuroblastoma differentiation induced by Hu7691 is dependent on N-myc. In response to the blockage of the AKT pathway, mTORC2, regulated by S6K, can reboot AKT activity by phosphorylating AKT at Ser473^51,52. Interestingly, our result shows that pronounced feedback phosphorylation of AKT caused by AKT inhibitors was seemingly related to the effects of differentiation induction. Meanwhile, mTORC2 can modulate the function of the N-Myc downstream-regulated gene (NDRG) family protein by phosphorylation, whereas NDRG2 and NDRG4 have regulatory roles in neuronal differentiation, synapse formation, and axonal survival⁵³. Taken together, we advance the hypothesis that mTORC2 is hyperactivated in response to agents and promotes neuroblastoma differentiation through a non-AKT-dependent pathway. Further studies are needed to clarify the above assumptions about whether Hu7691 affects N-Myc or mTORC2. Importantly, our findings reveal a previously unrecognized relation between AKT inhibition and differentiation regulation of neuroblastoma.

The neural crest, which consists of multipotent stem cells, gives rise to a plethora of differentiated cells, including cells of the peripheral nervous system, adrenal medulla, and melanocytes during migration⁶. Because of the versatility of neural crest cells, little is known about the cell fate determination of neuroblastoma. Interestingly, RNA-seq array data highlighted hypertrophic cardiomyopathy (HCM), which was associated with neural crest-derived cardiomyocytes⁵⁴, indicating the possible differentiation direction of neuroblastoma. Additionally, numerous studies have reported that glycosaminoglycans, such as hyaluronic acid, chondroitin sulfate, and heparan sulfate, are involved in neural stem cell proliferation and differentiation, neural development, regulation of glioma development, and other differentiation-related processes55, 56, 57. Research shows that the hedgehog pathway is involved in the differentiation of neural crest cells into glia, neurons, melanocytes, myofibroblasts, and chondrocytes, whereas the disruption of the hedgehog pathway results in embryonic lethality or severe craniofacial malformations in mice, indicating the necessity of hedgehog pathway in neural differentiation⁵⁸. These data give us a glimpse into the vast network of neuroblastoma differentiation.

5. Conclusions

Currently, the molecular mechanisms that regulate the progression of neuroblastoma differentiation remain unclear. Rather than being dependent on AKT in the previous studies, our data demonstrated that neuroblastoma differentiation is driven by AKT inhibition. In addition, we present a novel molecule Hu7691 with lower toxicity and a suitable pharmacokinetic profile. Taken together, our data gained insight into the mechanism of neuroblastoma differentiation, and Hu7691 may open a new therapeutic opportunity for differentiation therapy for neuroblastoma patients.

Acknowledgments

We thank Prof. Xiaowu Dong for gifting a structurally diverse compound library. This work was supported by the National Natural Science Foundation of China (No. U20A20137), the Zhejiang Provincial Natural Science Foundation of China (No. LD21H310001), and the Fundamental Research Funds for the Central Universities (No. 2021XZZX037, China).

Author contributions

Meidan Ying, Shaowei Bing, and Senfeng Xiang conceived the study and analyzed data; Meidan Ying and Shaowei Bing wrote the manuscript; Yilong Wang and Zhonghai Guan provided patient-derived neuroblastoma cells for further investigation; Shaowei Bing, Senfeng Xiang, Zhimei Xia, and Aixiao Xu performed the experiments; Jinxin Che and Xiaowu Dong provided structurally diverse compound library and Hu7691; Ji Cao, Bo Yang, Jinhu Wang, and Qiaojun He conceived the experiments and helped organize the paper.

Conflicts of interest

The authors declare that they have no conflict of interest.

Appendix A. Supplementary data

The following is the Supplementary data to this article.