**6. AZD8055 treatment in combination with MEK/ERK inhibitor**

The problem that will plague single-target drugs is the cancer's ability to activate alternative survival pathways leading to drug resistance and toxicity even in the multimodality setting. In addition, activation of multiple signaling pathways effectively causes cancer cell proliferation and survival. For example, RAS and PI3KCA are concurrently activated in melanoma, lung, and colorectal cancers [42–44]. These results suggested that combination therapies targeted on multiple signaling pathways could be more effective than targeting either pathway alone. Although AZD8055 is a promising drug in NB treatment [13], AZD8055-resistant NB sublines were acquired by prolonged stepwise exposure. After incubation with AZD8055 for 4–12 weeks, two acquired AZD8055-resistant sublines proliferated stably in RPMI1640 plus 10% FBS medium in the presence of AZD8055 (3 μM) [12]. The AZD8055-resistant sublines exhibited marked resistance to AZD8055 compared to the parent cells, and IC50 of the resistant sublines was 60–100 times higher than the parent NB cell lines [12]. By cell cycle analysis, accumulation of S phase was observed, and cyclin D3 and CDK4 were upregulated in AZD8055-resistant sublines [12]. Although AZD8055 treatment inhibited MEK/ERK activation in parent cells, MEK/ERK phosphorylation was continued despite AZD8055 treatment in resistant cells [12]. The combination therapy of AZD8055 and MEK/ERK inhibitor U0126 significantly inhibited cell proliferation compared to U0126 monotherapy [12], suggesting that combination therapy can overcome AZD8055 resistance. Furthermore, in athymic mice model, AZD8055 and U0126 co-treatment was more efficient to suppress resistant NB tumor growth compared to U0126 monotherapy [12].

## **7. MK2206 treatment in combination with PDK1 inhibitor**

MK2206 treatment induced a dose-dependent inhibition of cell proliferation, with IC50 ranging from 1.22 to 4.35 μM in NB cell lines [11]. MK2206-resistant cells

**67**

**Figure 1.**

*calculated depending on the MTT results.*

*Molecular Target Therapy against Neuroblastoma DOI: http://dx.doi.org/10.5772/intechopen.81706*

higher than parent cell lines [11].

were induced by stepwise escalation of MK-2206 exposure (4–12 weeks) [11]. These cells proliferated in RPMI1640 plus 10% FBS medium in the presence of MK2206 (5 μM), while cell death was induced in parent cells [11]. IC50 of the resistant cell lines was 6–7 times higher than the parent NB cell lines [11]. Small-molecule GSK2334470 selectively inhibits 3-phosphoinositide-dependent protein kinase 1 (PDK1) with low concentration but does not suppress the activity of other protein kinases at higher concentrations [45]. Although GSK2334470 attenuated cell proliferation in both parent cells and MK2206-resistant sublines, IC50 of GSK2334470 in resistant sublines was lower than that of parent cell lines [11]. GSK2334470 induced G0-G1 accumulation of cell cycle phase distribution in parent cell lines [11]. In MK2206-resistant sublines, G0-G1 accumulation induced by GSK2334470 was

**8. Effect of NB MYCN status on susceptibility to AZD8055**

Amplification of the MYCN oncogene is the most powerful single predictor of adverse outcome of NB [46]. MYCN amplification is observed in about 20% of all NB patients and is usually associated with fatal outcome of the disease [47]. Schramm et al. demonstrated transcriptomal upregulation of mTOR-related genes by MYCN [48]. MYCN-driven NB in mice displayed activation of the mTOR pathway on the protein level, and activation of MYCN in NB cells resulted in high sensitivity toward mTOR inhibition [48]. Therefore, it is examined whether MYCN

*Effect of MYCN status on susceptibility to AZD8055. (A) MYCN gene in tested 12 NB cell lines. MYCN status was defined as previously [53]. No alteration: cells with two MYCN signals and two CEP2 signals; amplification: the number of MYCN signals is at least 10 copies greater than the control probe signals; loss/ imbalance: presence of at least two MYCN signals and increased CEP2 signals; gain: the number of MYCN signals is 1–9 copies more than CEP2 signals. (B) Fifteen NB cell lines were treated with AZD8055 at different concentrations in RPMI1640 + 10% FBS. Cell growth was evaluated as cell numbers at 72 hours. Data are expressed as the mean ± SD. IC50 (half maximal inhibitory concentration) of the 15 NB cell lines was* 

*Molecular Target Therapy against Neuroblastoma DOI: http://dx.doi.org/10.5772/intechopen.81706*

*Biophysical Chemistry - Advance Applications*

that insensitive group showed lower mTOR (p < 0.001) expression and lower activity of mTOR complex 1 (p = 0.013) and mTOR complex 2 (p = 0.023) [13]. Cell cycle distribution analysis of NB cell lines was performed by flow cytometry. Cell cycle was affected with an increase in G0/G1 phase in dose-dependent manner of AZD8055 [13]. Western blotting analysis revealed that Cyclin D1 and Cyclin D3 were downregulated in AZD8055-treated NB cells [13]. AZD8055 inhibited both mTOR S2448 and mTOR S2481 phosphorylation significantly in a concentration-dependent manner [13]. Phosphorylation of downstream targets of mTOR complex 1, P70S6K T389 and 4E-BP1 S65, was also inhibited by AZD8055 treatment [13]. AZD8055 inhibited mTOR complex 2 substrates, Akt S473 and Akt T308 [13]. Although phosphorylation of Akt S473 was persistently inhibited in response to AZD8055 treatment, phosphorylation of Akt at the T308 site was inhibited for only 3–6 hours. These results indicate that AZD8055 inhibits mTOR activity and its downstream proteins in vitro in NB cells. Interestingly, NB cell lines were induced autophagy by AZD8055 treatment via downregulation of Akt/mTOR signaling pathway [13]. Autophagy inhibitor, 3-methyladenine, treatment resulted in a significant decrease of the AZD8055-induced apoptosis [13]. These results suggest that AZD8055 inhibited cell growth and induced cell cycle arrest, autophagy, and apoptosis in NB cells. Moreover, NB tumor growth in athymic nude mice was significantly inhibited by AZD8055 without toxicity [13]. Taken together, our results highlight that mTOR is a promising target for NB treatment. AZD8055 might be

investigated for treatment of patients with advanced and refractory NB.

**7. MK2206 treatment in combination with PDK1 inhibitor**

MK2206 treatment induced a dose-dependent inhibition of cell proliferation, with IC50 ranging from 1.22 to 4.35 μM in NB cell lines [11]. MK2206-resistant cells

**6. AZD8055 treatment in combination with MEK/ERK inhibitor**

The problem that will plague single-target drugs is the cancer's ability to activate alternative survival pathways leading to drug resistance and toxicity even in the multimodality setting. In addition, activation of multiple signaling pathways effectively causes cancer cell proliferation and survival. For example, RAS and PI3KCA are concurrently activated in melanoma, lung, and colorectal cancers [42–44]. These results suggested that combination therapies targeted on multiple signaling pathways could be more effective than targeting either pathway alone. Although AZD8055 is a promising drug in NB treatment [13], AZD8055-resistant NB sublines were acquired by prolonged stepwise exposure. After incubation with AZD8055 for 4–12 weeks, two acquired AZD8055-resistant sublines proliferated stably in RPMI1640 plus 10% FBS medium in the presence of AZD8055 (3 μM) [12]. The AZD8055-resistant sublines exhibited marked resistance to AZD8055 compared to the parent cells, and IC50 of the resistant sublines was 60–100 times higher than the parent NB cell lines [12]. By cell cycle analysis, accumulation of S phase was observed, and cyclin D3 and CDK4 were upregulated in AZD8055-resistant sublines [12]. Although AZD8055 treatment inhibited MEK/ERK activation in parent cells, MEK/ERK phosphorylation was continued despite AZD8055 treatment in resistant cells [12]. The combination therapy of AZD8055 and MEK/ERK inhibitor U0126 significantly inhibited cell proliferation compared to U0126 monotherapy [12], suggesting that combination therapy can overcome AZD8055 resistance. Furthermore, in athymic mice model, AZD8055 and U0126 co-treatment was more efficient to suppress resistant NB tumor growth compared to U0126 monotherapy [12].

**66**

were induced by stepwise escalation of MK-2206 exposure (4–12 weeks) [11]. These cells proliferated in RPMI1640 plus 10% FBS medium in the presence of MK2206 (5 μM), while cell death was induced in parent cells [11]. IC50 of the resistant cell lines was 6–7 times higher than the parent NB cell lines [11]. Small-molecule GSK2334470 selectively inhibits 3-phosphoinositide-dependent protein kinase 1 (PDK1) with low concentration but does not suppress the activity of other protein kinases at higher concentrations [45]. Although GSK2334470 attenuated cell proliferation in both parent cells and MK2206-resistant sublines, IC50 of GSK2334470 in resistant sublines was lower than that of parent cell lines [11]. GSK2334470 induced G0-G1 accumulation of cell cycle phase distribution in parent cell lines [11]. In MK2206-resistant sublines, G0-G1 accumulation induced by GSK2334470 was higher than parent cell lines [11].
