Polo-like kinase 1 (PLK1) is an essential cell cycle mitotic kinase component that plays an important role in cell cycle progression and has been reported to be involved in various cancers, including neuroblastoma (NB). PLK1 also regulates G2/M transition, chromosomal segregation, spindle assembly maturation, and mitotic exit. NB is an early embryonic-stage heterogeneous solid tumor and accounts for 15% of all pediatric cancer-related deaths. Therefore, we aimed to develop a targeting strategy for PLK1 by repurposing HMN-214 in NB. HMN-214 is a prodrug of HMN-176 and is known to selectively interfere with PLK1 function. In the present study, we performed the transcriptomic analysis of a large cohort of primary NB patient samples and revealed that PLK1 expression is inversely correlated with the overall survival of NB patients. Additionally, we found that PLK1 strongly correlates with NB disease and stage progression. HMN-214 significantly inhibited NB proliferation and colony formation in both MYCN-amplified and -nonamplified cell lines in a dose-dependent manner. Furthermore, HMN-214 induces apoptosis and significantly obstructs the cell cycle at the G2/M phase in NB cells by inhibiting multiple cell-cycle-related genes, such as PLK1, WEE1, CDK1, CDK2, Cyclin B1, CHK1, and CHK2. HMN-214 significantly inhibits cell cycle regulator CDK1 and the phosphorylation and activation of PLK1 in NB. In the NB 3D spheroid tumor model, HMN-214 significantly and in a dose-dependent manner inhibits spheroid tumor mass and growth. Overall, our study highlights that targeting PLK1 using HMN-214 is a novel therapeutic approach for NB.
Introduction High‐riskneuroblastoma (NB) is an aggressive pediatric tumor which develops from the extracranial sympathetic nervous system and accounts for almost 15% of all childhood cancer‐related deaths. Despite recent advancements in therapeutic approaches, the overall long‐term survival rate for NB patients is still less than 50%. Drug‐ resistance, metastasis, tumor relapse, and drug‐related toxicities mandate the development of less toxic and more effective novel therapeutic approaches for treating NB patients. Survivin is known to be involved in controlling cell division and apoptosis and belongs to the inhibitor of apoptosis protein family. Oncogenic activation of survivin has been reported in different cancers, including NB. We hypothesized that inhibiting surviving protein by using a specific small molecule inhibitor will inhibit NB cell proliferation, induce apoptosis, and sensitize tumor cells to chemotherapies. Methods NB patient datasets were analyzed using publicly available R2 Genomic analysis and visualization platform. Different NB cell lines, either MYCN‐amplified NGP, IMR‐32, LAN‐5 or, MYCN non‐amplified SH‐SY5Y, SK‐N‐AS, CHLA‐255 were used in the present study. Cell‐Titer AQueous One Solution was used to perform cell proliferation assays as per the manufacturer's instructions. Clonogenic assays were performed using crystal violet dye. Apoptosis and cell cycle assays were performed using eBioscience Annexin V Apoptosis Detection Kit and Click‐iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit respectively, using Attune NxT Flow Cytometer. In vitro tumorigenic studies were performed by using Corning 3D spheroid microplate system. All the assays were performed at least three times with replicates. Results In the present study, we analyzed genomic datasets of 1135 NB patients and found that high expression of survivin coding gene BIRC5 strongly correlates with poor overall and event‐free survival of NB patients. More aggressive tumors have significantly higher BIRC5 levels. To understand the effects of inhibiting survivin on NB growth, we used a specific small molecule inhibitor and performed cell proliferation and clonogenic assays. Results revealed that inhibition of survivin significantly inhibit NB proliferation and colony formation in all cell lines tested. Furthermore, survivin inhibition significantly induce apoptosis in NB cells and block the cell cycle progression. In vitro 3D spheroidal assays that recapitulate the in vivo tumor growth showed the survivin inhibitor significantly inhibits spheroid growth and induce apoptosis in a dose dependent manner. Conclusion Overall, our results suggest that survivin promotes the oncogenic potential of NB. Inhibition of survivin by using specific small molecule inhibitor is a novel therapeutic approach for NB. In our future efforts, we will combine the survivin inhibitor with chemotherapy drugs such as doxorubicin to develop a dual therapeutic approach for NB.
Neuroblastoma is a highly heterogenous pediatric tumor that develops during the early embryonic stages, and accounts for almost 15% of pediatric cancer related deaths. Despite advanced and intensive therapeutic approaches that combine surgery, radiation, and chemotherapy, high-risk neuroblastoma (NB) patients' long-term survival is still less than 50%. Tumor metastasis, relapse, drug-resistance, and treatment related toxicities mandate the development of novel therapeutic approaches to treat NB patients. MAPK/ERK pathway plays an important oncogenic role in different types of cancers, including NB. Activation of the MAPK/ERK pathway is known to induce cancer cell proliferation, progression, metabolism, and resistance to drug-induced apoptosis. In the present study, we investigated the effect of a small-molecule MEK inhibitor that targets MEK1 and MEK2 to block MAPK/ERK pathway in NB. Cytotoxicity assays using Cell-Titer One solution in different MYCN-amplified (NGP, LAN-5, CHLA-255-MYCN) and MYCN-non-amplified (SH-SY5Y, SK-N-AS, CHLA-255) NB cell lines show that MEK inhibitor significantly reduce the NB cell proliferation in a dose-dependent manner. We further performed clonogenic assays and 3D spheroidal assays to mimic the NB tumor growth, and our results show that MEK inhibitor significantly inhibit NB colony formation, inhibit 3D spheroidal tumor size by inducing cell death in 3D spheroidal tumors, in comparison to controls. Additionally, MEK inhibitor in a dose-dependent manner induces apoptosis and blocks cell cycle progression in NB cells in comparison to control, as determined by Annexin V apoptosis assays and Click-iT EdU cell proliferation assay respectively. Furthermore, gene expression analysis show that MEK inhibitor significantly reduce the mRNA expression of specific MAPK/ERK pathway targets such as MEK1, MEK2, and ERK2. Western blot assays further confirm the efficacy of MEK inhibitor in blocking MAPK/ERK pathway by significantly inhibiting the phosphorylation of key pathway proteins. Taken together, our results highlight that: a) MAPK/ERK pathway is implicated in NB growth, and b) direct targeting of MAPK/ERK signaling pathway by using a novel small molecule MEK inhibitor inhibit NB proliferation and 3D tumor growth. We will further combine this MEK inhibitor with current chemotherapy drugs to develop effective therapeutic approaches for NB patients. Citation Format: Rameswari Chilamakuri, Bharti Sharma, Danielle C. Rouse, Saurabh Agarwal. Direct targeting of MAPK/ERK signaling pathway is a novel therapeutic approach for high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1346.
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